**Cysteine Cathepsins Inhibition A**ff**ects Their Expression and Human Renal Cancer Cell Phenotype**

**Magdalena Rudzi ´nska 1,**† **, Alessandro Parodi 1,**† **, Valentina D. Maslova <sup>2</sup> , Yuri M. Efremov <sup>3</sup> , Neonila V. Gorokhovets <sup>1</sup> , Vladimir A. Makarov <sup>1</sup> , Vasily A. Popkov <sup>4</sup> , Andrey V. Golovin 1,2,5 , Evgeni Y. Zernii 1,4 and Andrey A. Zamyatnin Jr. 1,4,\***


Received: 6 April 2020; Accepted: 19 May 2020; Published: 21 May 2020

**Abstract:** Renal cancer would greatly benefit from new therapeutic strategies since, in advanced stages, it is refractory to classical chemotherapeutic approaches. In this context, lysosomal protease cysteine cathepsins may represent new pharmacological targets. In renal cancer, they are characterized by a higher expression, and they were shown to play a role in its aggressiveness and spreading. Traditional studies in the field were focused on understanding the therapeutic potentialities of cysteine cathepsin inhibition, while the direct impact of such therapeutics on the expression of these enzymes was often overlooked. In this work, we engineered two fluoromethyl ketone-based peptides with inhibitory activity against cathepsins to evaluate their potential anticancer activity and impact on the lysosomal compartment in human renal cancer. Molecular modeling and biochemical assays confirmed the inhibitory properties of the peptides against cysteine cathepsin B and L. Different cell biology experiments demonstrated that the peptides could affect renal cancer cell migration and organization in colonies and spheroids, while increasing their adhesion to biological substrates. Finally, these peptide inhibitors modulated the expression of LAMP1, enhanced the expression of E-cadherin, and altered cathepsin expression. In conclusion, the inhibition of cysteine cathepsins by the peptides was beneficial in terms of cancer aggressiveness; however, they could affect the overall expression of these proteases.

**Keywords:** cysteine cathepsins; cysteine cathepsin inhibitors; lysosome; renal cancer

#### **1. Introduction**

Cysteine cathepsins (Cts) are lysosomal proteases belonging to the C1 family of papain-like enzymes. They are responsible for the degradation and turnover of cellular [1] and extracellular [2] proteins, covering an essential role in maintaining cell and tissue homeostasis. Different enzymes with endo-, exo-, and endo/exopeptidase activity [3] compose the Cts family. Their proteolytic properties rely on a residue of cysteine in their active site, while other cathepsins are characterized by the presence of aspartic acid or serine amino acid [4]. Cathepsin expression is dysregulated in many pathological conditions, including cancer [5], and their overexpression is traditionally associated with the acquisition of a more aggressive tumor phenotype [6].

Cts, in particular, were shown to play a pivotal role in cancer invasiveness [7], tumor cell communication [8], apoptosis [9], and autophagy [10]. Considering that the Cts family includes 11 different members, only a few of them were tested extensively as tumor pharmacological targets, and, despite the overall scientific opinion, current data regarding the positive or negative contribution of Cts to cancer disease are contradictory [4,11–16]. For example, in the lysosomes, they can contribute to the proper function of autophagy, rescuing the cells from exogenous and endogenous stress [17]. In contrast, when they were found free in the cell cytoplasm, they could induce apoptosis through a caspase-independent mechanism of cellular death [9,18]. The role of Cts in cancer disease was shown to depend on their activation state [19] and cellular location [20].

Nevertheless, conclusive progress in the field is hampered by potential redundant and compensatory activities [21] both within the different members of the Cts family [21] and between other cathepsins/proteases [22]. Most of the research performed by far, unveiled the role of single Cts in cancer disease, with few further considerations on how the investigated inhibitors, the genetic silencers, or modifications affected the overall biology of the cells and the expression of the targeted enzymes. These considerations are pivotal to the correct designing of more effective pharmacological interventions and evaluate their long term effects.

To address these questions, we designed two small fluoromethyl ketone (FMK)-containing peptides to broadly inhibit the activity against these enzymes. The designing of these inhibitors was inspired by a well-known substrate of the papain-like cysteine protease Triticain-α, derived from *Triticum aestivum* (wheat) [23]. This peptide consists of four amino acids Acetyl-Pro-Leu-Val-Gln (Ac-PLVQ), while the inhibitor sequences are Acetyl-Pro-Leu-Val-Glu-FMK (Ac-PLVE-FMK) and Acetyl-Val-Leu-Pro-Glu-FMK (Ac-VLPE-FMK) (Figure 1a). The working mechanism of both inhibitors should be the same as other selective irreversible cysteine proteinase inhibitors like Z-VAD-FMK (Figure 1b) [24], as well as other FMK-containing drugs. In this class of inhibitors, the FMK group forms a covalent bond with the catalytic cysteine, with the fluorine ion leaving [25,26]. As a model of tumor disease, we chose human renal cancer since this pathology showed aberrant expression of some members of the Cts family [27]. Additionally, further progress in kidney tumor disease depends on the discovery of new targetable markers [28] since when the diagnosis is performed at an advanced stage, the survival rate is low [29], and traditional chemotherapy is ineffective [30]. Investigation on Cts inhibition in this cancer model is rare, and to our knowledge, no comprehensive study in relation to phenotypic and enzymatic alterations has ever been performed. In this work, we tested the biological impact of our inhibitory peptides on the biology of human renal cancer cells, with a focus on the overall lysosomal compartment. We demonstrated that the general inhibition of Cts over longer time periods does not affect the cell proliferation rate. Still, it can affect the overall biology of human renal cancer cells, as well as impacting on the overall Cts expression.

biology of human renal cancer cells, as well as impacting on the overall Cts expression.

*Cancers* **2020**, *12*, x 2 of 20

by the presence of aspartic acid or serine amino acid [4]. Cathepsin expression is dysregulated in many pathological conditions, including cancer [5], and their overexpression is traditionally

Cts, in particular, were shown to play a pivotal role in cancer invasiveness [7], tumor cell communication [8], apoptosis [9], and autophagy [10]. Considering that the Cts family includes 11 different members, only a few of them were tested extensively as tumor pharmacological targets, and, despite the overall scientific opinion, current data regarding the positive or negative contribution of Cts to cancer disease are contradictory [4,11–16]. For example, in the lysosomes, they can contribute to the proper function of autophagy, rescuing the cells from exogenous and endogenous stress [17]. In contrast, when they were found free in the cell cytoplasm, they could induce apoptosis through a caspase-independent mechanism of cellular death [9,18]. The role of Cts in cancer disease was shown to depend on their activation state [19] and cellular location [20].

Nevertheless, conclusive progress in the field is hampered by potential redundant and compensatory activities [21] both within the different members of the Cts family [21] and between other cathepsins/proteases [22]. Most of the research performed by far, unveiled the role of single Cts in cancer disease, with few further considerations on how the investigated inhibitors, the genetic silencers, or modifications affected the overall biology of the cells and the expression of the targeted enzymes. These considerations are pivotal to the correct designing of more effective pharmacological

To address these questions, we designed two small fluoromethyl ketone (FMK)-containing peptides to broadly inhibit the activity against these enzymes. The designing of these inhibitors was inspired by a well-known substrate of the papain-like cysteine protease Triticain-α, derived from *Triticum aestivum* (wheat) [23]. This peptide consists of four amino acids Acetyl-Pro-Leu-Val-Gln (Ac-PLVQ), while the inhibitor sequences are Acetyl-Pro-Leu-Val-Glu-FMK (Ac-PLVE-FMK) and Acetyl-Val-Leu-Pro-Glu-FMK (Ac-VLPE-FMK) (Figure 1a). The working mechanism of both inhibitors should be the same as other selective irreversible cysteine proteinase inhibitors like Z-VAD-FMK (Figure 1b) [24], as well as other FMK-containing drugs. In this class of inhibitors, the FMK group forms a covalent bond with the catalytic cysteine, with the fluorine ion leaving [25,26]. As a model of tumor disease, we chose human renal cancer since this pathology showed aberrant expression of some members of the Cts family [27]. Additionally, further progress in kidney tumor disease depends on the discovery of new targetable markers [28] since when the diagnosis is performed at an advanced stage, the survival rate is low [29], and traditional chemotherapy is ineffective [30]. Investigation on Cts inhibition in this cancer model is rare, and to our knowledge, no comprehensive study in relation to phenotypic and enzymatic alterations has ever been performed. In this work, we tested the biological impact of our inhibitory peptides on the biology of human renal cancer cells, with a focus on the overall lysosomal compartment. We demonstrated that the general inhibition of

associated with the acquisition of a more aggressive tumor phenotype [6].

interventions and evaluate their long term effects.

**Figure 1.** The structure and working mechanism of Ac-PLVE- fluoromethyl ketone (FMK) and Ac-VLPE-FMK inhibitors: (**a**) Inhibitory peptides structure and (**b**) their working mechanism based on Z-VAD-FMK inhibitor.

#### **2. Results**

#### *2.1. Computational Modeling of the Peptide Inhibitory Properties on Cts Activity*

A docking simulation to predict the interactions of the inhibitors with the binding site of the Cts was performed with the protein-ligand docking software PLANTS [31]. Both Ac-PLVE-FMK and Ac-VLPE-FMK were docked into CtsB, L, and W active sites at pH 4.5, 6.5, and 7.2 since pH can influence the protein interactions through the protonation of the ionizable residues [32]. CtsB and L were chosen as enzymatic models because they have endo and endo/exopeptidase activity, respectively, addressing all the Cts proteolytic mechanisms. They were also shown to play a pivotal role in renal cancer malignancy, and their overexpression was associated with a more aggressive cancer phenotype [33–35]. On the other hand, CtsW represents a poorly investigated protease in renal cancer. In contrast, in other investigations, it was shown to preferentially locate in the endothelial reticulum [36], and it is evolutionarily distinguished from CtsB and L [37], representing, therefore, optimal negative control for our research.

Fifty poses per binding site for each ligand were obtained. The analysis revealed that our inhibitors did not bind with the proteases in the pre-reaction state with the fluorine atom of the FMK group in a 3.5 Å radius from the HD2 hydrogen atom of the catalytic histidine and with the carbon atom of the fluoromethyl group in a 3.5 Å radius from the SG atom of the catalytic cysteine. The non-covalent binding energy of the peptides to the proteases is substantially lower than the energy of one covalent bond. Thus, the conformation suitable for covalent bonding may be far from the optimal peptide position in the non-covalent mode. To find non-covalent interactions potentially leading to covalent bond formation, we analyzed the crystal structure of Z-VAD-FMK, covalently bonded to the cysteine protease *Marasmius oreades agglutinin* (PDB id 5D61). In this case, the oxygen atom of the FMK group interacts via hydrogen bonds with the oxyanion hole of the enzyme, formed by the catalytic cysteine backbone N and NE1 atom of Trp-208. For further docking simulations, we added a distance constraint between the oxygen atom of the FMK group of our inhibitors and two hydrogen atoms from the oxyanion holes in the Cts structures. This adjustment allowed for obtaining poses close to the pre-reaction state for both inhibitors. The resulting poses demonstrated that Ac-PLVE-FMK tends to occupy S2 binding sites with either Val or Leu residues, depending on the backbone conformation. The C-terminal Glu residue fitted in the groove around site S1'. However, the N-terminal residue did not bind in S3 or S4 sites and laid closer to the protein surface. Ac-VLPE-FMK instead, tended to bind in S1'-S2′ sites of Cts with its N-terminal residues' sidechains. Thus, both inhibitors should occupy mostly hydrophobic substrate-binding pockets by Val and Leu side chains. Representative docking of the peptides with the CtsB, L, and W at pH 4.5 is shown in Figure 2a–c. According to the docking score, both inhibitors are less likely to bind in the pre-reaction state, capable of covalent bond formation with CtsW confirming its potential role as a negative control (Figure 2d). Ac-VLPE-FMK also has a weaker binding with all Cts than Ac-PLVE-FMK, possibly due to the lack of conformation variability around Pro residue. *Cancers* **2020**, *12*, x 4 of 20

**Figure 2.** Binding poses of designed inhibitors to cysteine cathepsins (Cts). Docking of the peptides in (**a**) CtsB,(**b**) CtsL, (**c**) CtsW (pH 4.5). Hydrogen atoms are omitted for better depiction. Catalytic Cysteine and Histidine are shown in thick sticks. Inhibitors are shown in purple or green. S1-S3 and S1' binding pockets are labeled. (**d**) Binding scores (PLANTS Chemplp energy units) for Ac-PLVE-FMK and Ac-VLPE-FMK docking results at different pH levels with and without distance constraints. **Figure 2.** Binding poses of designed inhibitors to cysteine cathepsins (Cts). Docking of the peptides in (**a**) CtsB,(**b**) CtsL, (**c**) CtsW (pH 4.5). Hydrogen atoms are omitted for better depiction. Catalytic Cysteine and Histidine are shown in thick sticks. Inhibitors are shown in purple or green. S1-S3 and S1' binding pockets are labeled. (**d**) Binding scores (PLANTS Chemplp energy units) for Ac-PLVE-FMK and Ac-VLPE-FMK docking results at different pH levels with and without distance constraints.

#### *2.2. Assessment of Peptide Inhibitory Properties on Cts Activity via Biochemical Assays 2.2. Assessment of Peptide Inhibitory Properties on Cts Activity via Biochemical Assays*

Ac-PLVE-FMK and Ac-VLPE-FMK inhibitory properties were evaluated against human recombinant CtsL and B. These recombinant enzymes were expressed in *E. coli* via plasmid transformation and further purification, using nickel-nitrilotriacetic acid (Ni-NTA) sepharose. Gel zymography assay was used to provide a preliminary evaluation of the ability of these recombinant proteins to degrade the gelatin substrate previously embedded in the gel (Supplementary Figure S1). Following Coomassie staining, the Cts were detectable as single bands. As expected, a translucent area was evident, due to the substrate digestion in the proximity of the proteins. Then, the recombinant proteins were tested for their ability to digest the Triticain-α substrate Ac-PLVQ via fluorescent protease activity assay, previously optimized by our group [23]. This probe was conjugated with the fluorogenic chromophore 7-amino-4-methylcoumarin (Ac-PLVQ-AMC). After proteolytic cleavage, it emits a detectable fluorescent signal. Ac-PLVE-FMK and Ac-VLPE-FMK inhibitory properties were evaluated against human recombinant CtsL and B. These recombinant enzymes were expressed in *E. coli* via plasmid transformation and further purification, using nickel-nitrilotriacetic acid (Ni-NTA) sepharose. Gel zymography assay was used to provide a preliminary evaluation of the ability of these recombinant proteins to degrade the gelatin substrate previously embedded in the gel (Supplementary Figure S1). Following Coomassie staining, the Cts were detectable as single bands. As expected, a translucent area was evident, due to the substrate digestion in the proximity of the proteins. Then, the recombinant proteins were tested for their ability to digest the Triticain-α substrate Ac-PLVQ via fluorescent protease activity assay, previously optimized by our group [23]. This probe was conjugated with the fluorogenic chromophore 7-amino-4-methylcoumarin (Ac-PLVQ-AMC). After proteolytic cleavage, it emits a detectable fluorescent signal.

Both human recombinant CtsB and L (20 nM each) were able to cleave the probe Ac-PLVQ-AMC (50 μM) and increase the fluorescent signal detection (red line). In the same experimental conditions, performed in the presence of Ac-PLVE-FMK or Ac-VLPE-FMK (2 μM), the intensity of the fluorescent signal was significantly affected (Supplementary Figure S2—blue and green line, respectively). Both human recombinant CtsB and L (20 nM each) were able to cleave the probe Ac-PLVQ-AMC (50 µM) and increase the fluorescent signal detection (red line). In the same experimental conditions, performed in the presence of Ac-PLVE-FMK or Ac-VLPE-FMK (2 µM), the intensity of the fluorescent signal was significantly affected (Supplementary Figure S2—blue and green line, respectively).

To evaluate the inhibitory properties of the peptides directly on the human renal cancer cells, we firstly assessed the impact of Ac-PLVE-FMK and Ac-VLPE-FMK on the cell viability of 769-P and A498 cells to determine the working concentrations for further experiments. The peptides were easily dispersed in water and administered at increasing doses to both cell lines for 72 h (Figure 3a,b). The inhibitors showed cytostatic properties on both cell lines only within 48 h of treatment when all the concentrations used negatively impacted on cell proliferation, in particular in the case of 769-P cells. However, after 72 h of incubation, cell viability increased, reaching values not significantly different from the control cells. Further experiments were performed using a concentration of 20 μM. To understand if the peptides could affect the activity of caspase proteases, we tested their effect on cell viability in combination with the chemotherapeutic paclitaxel (PXT) in both the cell lines. The cell line A498 did not result in high mortality even when the cells were treated with PXT alone (data not shown), confirming previously published data [38]. However, in the case of 769-P cells, the ability of To evaluate the inhibitory properties of the peptides directly on the human renal cancer cells, we firstly assessed the impact of Ac-PLVE-FMK and Ac-VLPE-FMK on the cell viability of 769-P and A498 cells to determine the working concentrations for further experiments. The peptides were easily dispersed in water and administered at increasing doses to both cell lines for 72 h (Figure 3a,b). The inhibitors showed cytostatic properties on both cell lines only within 48 h of treatment when all the concentrations used negatively impacted on cell proliferation, in particular in the case of 769-P cells. However, after 72 h of incubation, cell viability increased, reaching values not significantly different from the control cells. Further experiments were performed using a concentration of 20 µM. To understand if the peptides could affect the activity of caspase proteases, we tested their effect on cell viability in combination with the chemotherapeutic paclitaxel (PXT) in both the cell lines. The cell line A498 did not result in high mortality even when the cells were treated with PXT alone (data not shown),

on renal cancer cell proteolytic activity against the fluorogenic probe Ac-PLVQ-AMC. In this case,

confirming previously published data [38]. However, in the case of 769-P cells, the ability of the PXT to kill cancer cells was evident after 72 h of treatment at a concentration of 100 nM. When the treatment with PXT was performed in combination with the peptides (20 µM), no significant differences were observed (Supplementary Figure S3). Next, we evaluated the effect of the peptides on renal cancer cell proteolytic activity against the fluorogenic probe Ac-PLVQ-AMC. In this case, 769-p and A498 cells were treated for 30 min with Ac-PLVE-FMK or Ac-VLPE-FMK and were then exposed for 10 min to the substrate Ac-PLVQ-AMC prior fluorescence microscopy analysis (Figure 3c,d). As depicted by the pictures, both the peptides were effective in inhibiting the generation of the fluorescence derived from the cleavage of the substrate in both the cell lines and fluorimetric analysis confirmed that they significantly inhibited the probe degradation. Overall, these data demonstrate that the peptides do not considerably interfere with cell viability and that their inhibitory properties do not affect the proteolytic activity of the proteases that are involved in cell apoptosis; however, they can effectively interfere with the cellular Cts. *Cancers* **2020**, *12*, x 5 of 20 769-p and A498 cells were treated for 30 min with Ac-PLVE-FMK or Ac-VLPE-FMK and were then exposed for 10 min to the substrate Ac-PLVQ-AMC prior fluorescence microscopy analysis (Figure 3c,d). As depicted by the pictures, both the peptides were effective in inhibiting the generation of the fluorescence derived from the cleavage of the substrate in both the cell lines and fluorimetric analysis confirmed that they significantly inhibited the probe degradation. Overall, these data demonstrate that the peptides do not considerably interfere with cell viability and that their inhibitory properties do not affect the proteolytic activity of the proteases that are involved in cell apoptosis; however, they can effectively interfere with the cellular Cts.

**Figure 3.** The effect of the peptide inhibitors on human 769-P and A498 renal cancer cell proliferation and proteolytic activity. (**a**) 769-P and (**b**) A498 cells were treated with increasing doses of Ac-PLVE-FMK (red bars) and Ac-VLPE-FMK (blue bars) (2.5–250 μM). Cell proliferation was measured after **Figure 3.** The effect of the peptide inhibitors on human 769-P and A498 renal cancer cell proliferation and proteolytic activity. (**a**) 769-P and (**b**) A498 cells were treated with increasing doses of Ac-PLVE-FMK (red bars) and Ac-VLPE-FMK (blue bars) (2.5–250 µM). Cell proliferation was measured after 24, 48,

24, 48, and 72 h via MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Data represent the mean (±S.D.) of at least three independent experiments, each performed in triplicate. (**c**)

for 30 min prior incubation with the fluorescent substrate Ac-PLVQ-AMC for 10 min. Data are expressed as mean (±S.D.), and significance was calculated through one-way ANOVA followed by

Dunnet's test. \* = *p* < 0.05, \*\* = *p* < 0.01, \*\*\* = *p* < 0.001.

and 72 h via MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Data represent the mean (±S.D.) of at least three independent experiments, each performed in triplicate. (**c**) Fluorescence microscopy evaluation and quantification of the cell proteolytic activity in 769-P and (**d**) A498 cells towards the fluorogenic substrate Ac-PLVQ-AMC in the absence or presence of Ac-PLVE-FMK and Ac-VLPE-FMK. The cells were seeded in a 96- well plate and after exposure to the peptides for 30 min prior incubation with the fluorescent substrate Ac-PLVQ-AMC for 10 min. Data are expressed as mean (±S.D.), and significance was calculated through one-way ANOVA followed by Dunnet's test. \* = *p* < 0.05, \*\* = *p* < 0.01, \*\*\* = *p* < 0.001.

#### *2.3. E*ff*ect of the Inhibitory Peptides on Human Tumor Cell Biology*

The effective inhibition of specific members of the Cts family was associated with changes in cancer cell properties, such as a decrease in cell invasiveness and migration properties in different tumors, including renal cancer cell lines [39–41]. For this reason, various tests were used to evaluate potential changes in cancer cell biology following treatment with the inhibitory peptides. Firstly, we assessed the ability of renal cancer cells to generate colonies. 769-P and A498 cells were treated with the inhibitors for a total of 2 days (20 µM) before being seeded at very low confluency in 10 cm diameter dishes. After cell seeding, the treatment with the peptides was prolonged for an additional 10 days until colony formation was detectable, and identification and quantification of the colonies were performed through Crystal Violet staining. Even though in the case of A498, the colonies were smaller than with 769P cells, both the inhibitors were effective in significantly decreasing colony numbers, as shown in Figure 4a,b. Next, we evaluated the ability of the inhibitory peptides to affect spheroids formation. The cells were treated for 48 h with the Cts inhibitors (20 µM) before being seeded in Matrigel-coated 96-well plates for an additional 7 days. Additionally, in this case, A498 spheroid size and number were smaller than 769-P cells, and the presence of the inhibitors negatively affected the total number of spheroids in both the cell lines (Figure 4c,d), reaching highly significant decreasing values in 769P cells. It is worth noting that, compared to untreated cells, the peptides decreased spheroid size, while increasing their circularity in both the cell lines (Supplementary Figure 4a,b). Finally, we evaluated the ability of the inhibitors to contrast the motility of the cells in a classical scratch healing assay. Human renal cancer cells were seeded and grown until they reached the confluency when a gap was artificially created with a 200 µM tip. As shown in Figure 4e,f, the inhibitors were efficient in a similar fashion in decreasing the gap closure velocity, both at 8 and 24 h after the formation of the scratch. In light of the registered changes in cell phenotype, we hypothesized that the inhibition of Cts could have impacted on cell adhesion to biological substrates. Human renal cancer cells were treated for 48 h before assessing their adhesion properties on collagen IV and Matrigel. Both the peptides induced an increase in cell adhesion on both the biological substrates (Figure 5a,b), and these properties were accompanied by changes in cell stiffness, evaluated through atomic force microscopy analysis (Supplementary Figure S5a,b). Overall, these data demonstrated that the peptides affected renal cancer cell biology.

*Cancers* **2020**, *12*, x 6 of 20

The effective inhibition of specific members of the Cts family was associated with changes in cancer cell properties, such as a decrease in cell invasiveness and migration properties in different tumors, including renal cancer cell lines [39–41]. For this reason, various tests were used to evaluate potential changes in cancer cell biology following treatment with the inhibitory peptides. Firstly, we assessed the ability of renal cancer cells to generate colonies. 769-P and A498 cells were treated with the inhibitors for a total of 2 days (20 μM) before being seeded at very low confluency in 10 cm diameter dishes. After cell seeding, the treatment with the peptides was prolonged for an additional 10 days until colony formation was detectable, and identification and quantification of the colonies were performed through Crystal Violet staining. Even though in the case of A498, the colonies were smaller than with 769P cells, both the inhibitors were effective in significantly decreasing colony numbers, as shown in Figure 4a,b. Next, we evaluated the ability of the inhibitory peptides to affect spheroids formation. The cells were treated for 48 h with the Cts inhibitors (20 μM) before being seeded in Matrigel-coated 96-well plates for an additional 7 days. Additionally, in this case, A498 spheroid size and number were smaller than 769-P cells, and the presence of the inhibitors negatively affected the total number of spheroids in both the cell lines (Figure 4c,d), reaching highly significant decreasing values in 769P cells. It is worth noting that, compared to untreated cells, the peptides decreased spheroid size, while increasing their circularity in both the cell lines (Supplementary Figure 4a,b). Finally, we evaluated the ability of the inhibitors to contrast the motility of the cells in a classical scratch healing assay. Human renal cancer cells were seeded and grown until they reached the confluency when a gap was artificially created with a 200 μM tip. As shown in Figure 4e,f, the inhibitors were efficient in a similar fashion in decreasing the gap closure velocity, both at 8 and 24 h after the formation of the scratch. In light of the registered changes in cell phenotype, we hypothesized that the inhibition of Cts could have impacted on cell adhesion to biological substrates. Human renal cancer cells were treated for 48 h before assessing their adhesion properties on collagen IV and Matrigel. Both the peptides induced an increase in cell adhesion on both the biological substrates (Figure 5a,b), and these properties were accompanied by changes in cell stiffness,

*2.3. Effect of the Inhibitory Peptides on Human Tumor Cell Biology* 

**Figure 4.** Effect of the peptides on renal cancer cell biology: colony formation assay of (**a**) 769-P and (**b**) A498 under treatment with Ac-PLVE-FMK and Ac-VLPE-FMKI. The cells were treated for 48 h with 20 μM inhibitors and seeded into 10 culture dishes (10 cm diameter) at low confluency where they grew with or without the peptides for an additional 10 days. Representative images of the colony formation assay and quantitative data analysis are shown in the graph. (**c**) 769P and (**d**) A498 spheroid formation evaluated after 7 days of culture on Matrigel coated dishes. The graph is showing the **Figure 4.** Effect of the peptides on renal cancer cell biology: colony formation assay of (**a**) 769-P and (**b**) A498 under treatment with Ac-PLVE-FMK and Ac-VLPE-FMKI. The cells were treated for 48 h with 20 µM inhibitors and seeded into 10 culture dishes (10 cm diameter) at low confluency where they grew with or without the peptides for an additional 10 days. Representative images of the colony formation assay and quantitative data analysis are shown in the graph. (**c**) 769P and (**d**) A498 spheroid formation evaluated after 7 days of culture on Matrigel coated dishes. The graph is showing the number of spheroids. (**e**) 769-p and (**f**) A498 scratch assay. The graph shows the cell migration rate. All pictures were taken under 10× magnification. Data are expressed as mean (±S.D.), and significance was calculated through one-way ANOVA followed by Dunnet's test. \* = *p* < 0.05, \*\* = *p* < 0.01, \*\*\* = *p* < 0.001. *Cancers* **2020**, *12*, x 7 of 20 number of spheroids. (**e**) 769-p and (**f**) A498 scratch assay. The graph shows the cell migration rate. All pictures were taken under 10× magnification. Data are expressed as mean (±S.D.), and significance was calculated through one-way ANOVA followed by Dunnet's test. \* = *p* < 0.05, \*\* = *p* < 0.01, \*\*\* = *p* < 0.001.

**Figure 5.** Effect of Cts inhibitors on renal cancer cell adhesion on biological coatings**:** (**a**) adhesion 769- P and (**b**) A498 cells to collagen IV and Matrigel after treatment with the inhibitors. Data are expressed as mean (±S.D.), and significance was calculated through one-way ANOVA followed by Dunnet's test. \* = *p* < 0.05, \*\* = *p* < 0.01, \*\*\* = *p* < 0.001. **Figure 5.** Effect of Cts inhibitors on renal cancer cell adhesion on biological coatings: (**a**) adhesion 769-P and (**b**) A498 cells to collagen IV and Matrigel after treatment with the inhibitors. Data are expressed as mean (±S.D.), and significance was calculated through one-way ANOVA followed by Dunnet's test. \* = *p* < 0.05, \*\* = *p* < 0.01, \*\*\* = *p* < 0.001.

#### *2.4. Effect of the Inhibitory Peptides on E-Cadherin and SNAIL1 2.4. E*ff*ect of the Inhibitory Peptides on E-Cadherin and SNAIL1*

be seen in Supplementary Figure S7).

The collected data indicated that Ac-PLVE-FMK and Ac-VLPE-FMK could affect the overall renal cancer cell phenotype. These properties could be the result of the modulation of the effectors controlling EMT, as previously shown in other tumor models [40,42–44]. To test this hypothesis, the The collected data indicated that Ac-PLVE-FMK and Ac-VLPE-FMK could affect the overall renal cancer cell phenotype. These properties could be the result of the modulation of the effectors controlling EMT, as previously shown in other tumor models [40,42–44]. To test this hypothesis, the cells were

that E-cadherin showed the highest increase after treatment, compared to untreated CTRL.

These markers are critical players in the control of EMT, and they are associated with opposite effects on the cancer cell phenotype. While E-cadherin is considered a marker of differentiation, working against EMT (favoring mesenchymal-epithelial transition, as known as MET), SNAIL1 is associated with the acquisition of an undifferentiated phenotype. Both the peptides increased the protein expression of E-cadherin in the first 48 h in both the cell lines (Figure 6). At 72 h, the values of E-cadherin further increased in 769P cells, while decreased in A498 cells. On the other hand, SNAIL1 increased significantly at 72 h only in the case of 769P cells treated with AC-PLVE-FMK, while dropped to values more similar to the control upon treatment with Ac-VLPE-FMK and in A498 cells with both the peptides. More importantly, in both the cell lines, E-cadherin and SNAIL1 followed a similar trend in response to the treatments. Taken together, these data demonstrated that the inhibitory peptides could affect the cell phenotype, involving the genes that control EMT. These phenomena could be at the base of the observed cell adhesion and motility properties, considering

treated with the inhibitors for 24, 48, and 72 h by changing the media every day, and they were tested for E-cadherin and SNAIL1 expression (Figure 6; detail information of western blots can be seen in *Cancers* Supplementary Figure S7). **2020**, *12*, x 8 of 20

**Figure 6.** Effects of the peptides on E-cadherin and SNAIL1 protein expression: (**a**,**b**) protein expression of E-cadherin and SNAIL1 in 769-P and (**c**,**d**) A498 cells after 24, 48, and 72 h of treatment with the peptides. Data are represented as mean ± SD of at least three replicates. Significance was calculated through one-way ANOVA, followed by Dunnet's test. **Figure 6.** Effects of the peptides on E-cadherin and SNAIL1 protein expression: (**a**,**b**) protein expression of E-cadherin and SNAIL1 in 769-P and (**c**,**d**) A498 cells after 24, 48, and 72 h of treatment with the peptides. Data are represented as mean ± SD of at least three replicates. Significance was calculated through one-way ANOVA, followed by Dunnet's test.

*2.5. Impact of the Peptides on Lysosomal Biology*  To evaluate the potential changes to the lysosomal compartment, we measured the expression of Lysosome Associated Membrane Protein 1 (LAMP-1) over 72 h of treatment. This protein is usually associated with the lysosomal membrane, and it is universally recognized as a marker of these organelles. Western blotting analysis demonstrated a very different effect of the peptides on the modulation of this marker in the two cell lines. In 769-P cells, both the inhibitors significantly increased the expression of this lysosomal biomarker at all the considered time points, reaching significant peaks at 48 h of treatment and generally increasing this marker in all the considered time points. Additionally, Ac-VLPE-FMK demonstrated to be more efficient than Ac-PLVE-FMK in increasing the expression of LAMP-1 (Figure 7a). These data were corroborated by confocal microscopy analysis, confirming that at 48 h, both the peptides increased LAMP-1 expression (Figure 7b), while at 72 h, the content of LAMP-1 decreased towards control levels. On the other hand, LAMP-1 protein expression in A498 cells was significantly affected during the first 48 h, and it was These markers are critical players in the control of EMT, and they are associated with opposite effects on the cancer cell phenotype. While E-cadherin is considered a marker of differentiation, working against EMT (favoring mesenchymal-epithelial transition, as known as MET), SNAIL1 is associated with the acquisition of an undifferentiated phenotype. Both the peptides increased the protein expression of E-cadherin in the first 48 h in both the cell lines (Figure 6). At 72 h, the values of E-cadherin further increased in 769P cells, while decreased in A498 cells. On the other hand, SNAIL1 increased significantly at 72 h only in the case of 769P cells treated with AC-PLVE-FMK, while dropped to values more similar to the control upon treatment with Ac-VLPE-FMK and in A498 cells with both the peptides. More importantly, in both the cell lines, E-cadherin and SNAIL1 followed a similar trend in response to the treatments. Taken together, these data demonstrated that the inhibitory peptides could affect the cell phenotype, involving the genes that control EMT. These phenomena could be at the base of the observed cell adhesion and motility properties, considering that E-cadherin showed the highest increase after treatment, compared to untreated CTRL.

#### characterized by a substantial recovery at 72 h, reaching values slightly higher than CTRL levels *2.5. Impact of the Peptides on Lysosomal Biology*

(Figure 7c,d). A similar trend in both the cell lines was observed by analyzing the endolysosomal compartment integrity through Neutral red assay (Supplementary Figure S6) and LysoTracker red fluorescence measurement where we registered slightly increasing and decreasing values only at 48 h of treatment for 769-P and A498 cells, respectively. To evaluate the potential changes to the lysosomal compartment, we measured the expression of Lysosome Associated Membrane Protein 1 (LAMP-1) over 72 h of treatment. This protein is usually associated with the lysosomal membrane, and it is universally recognized as a marker of these organelles. Western blotting analysis demonstrated a very different effect of the peptides on the modulation of this marker in the two cell lines. In 769-P cells, both the inhibitors significantly increased the expression of this lysosomal biomarker at all the considered time points, reaching significant peaks at 48 h of treatment and generally increasing this marker in all the considered

cells.

time points. Additionally, Ac-VLPE-FMK demonstrated to be more efficient than Ac-PLVE-FMK in increasing the expression of LAMP-1 (Figure 7a). These data were corroborated by confocal microscopy analysis, confirming that at 48 h, both the peptides increased LAMP-1 expression (Figure 7b), while at 72 h, the content of LAMP-1 decreased towards control levels. On the other hand, LAMP-1 protein expression in A498 cells was significantly affected during the first 48 h, and it was characterized by a substantial recovery at 72 h, reaching values slightly higher than CTRL levels (Figure 7c,d). A similar trend in both the cell lines was observed by analyzing the endolysosomal compartment integrity through Neutral red assay (Supplementary Figure S6) and LysoTracker red fluorescence measurement where we registered slightly increasing and decreasing values only at 48 h of treatment for 769-P and A498 cells, respectively. *Cancers* **2020**, *12*, x 9 of 20

**Figure 7.** Effect of the peptides on LAMP-1 protein expression: (**a**) Western blotting analysis of LAMP-1 after treatment with PLVE and VLPE (20 μM) for 24, 48, and 72 h in 769-P cells. (**b**) Confocal microscopy evaluation of LAMP-1 expression after 48 and 72 h of treatment with PLVE and VLPE (20 μM) in 769-P cells. (**c**) Western blotting analysis of LAMP-1 after treatment with PLVE and VLPE (20 μM) for 24, 48, and 72 h in A498 cells. (**d**) Confocal microscopy evaluation of LAMP-1 expression after 48 and 72 h of treatment with PLVE and VLPE (20 μM) in A498 cells. Data are represented as mean ± SD of at least three replicates. Significance was calculated through one-way ANOVA followed by Dunnet's test. \* = *p* < 0.05, \*\* = *p* < 0.01, \*\*\* = *p* < 0.001. **Figure 7.** Effect of the peptides on LAMP-1 protein expression: (**a**) Western blotting analysis of LAMP-1 after treatment with PLVE and VLPE (20 µM) for 24, 48, and 72 h in 769-P cells. (**b**) Confocal microscopy evaluation of LAMP-1 expression after 48 and 72 h of treatment with PLVE and VLPE (20 µM) in 769-P cells. (**c**) Western blotting analysis of LAMP-1 after treatment with PLVE and VLPE (20 µM) for 24, 48, and 72 h in A498 cells. (**d**) Confocal microscopy evaluation of LAMP-1 expression after 48 and 72 h of treatment with PLVE and VLPE (20 µM) in A498 cells. Data are represented as mean ± SD of at least three replicates. Significance was calculated through one-way ANOVA followed by Dunnet's test. \* = *p* < 0.05, \*\* = *p* < 0.01, \*\*\* = *p* < 0.001.

Next, we evaluated the protein expression of CtsB, L, and W (Figure 8). In both the cell lines, the Cts protein expression was similar. In particular, CtsB and L increased significantly at the later time points of treatment with both the peptides (Figure 8a–d). In contrast, in the case of CtsW, the peptides negatively affected the protein expression in 769-P cells, while no particular differences were detected in A498 cells (Figure 8e,f). Other tested Cts were modulated in their protein expression similarly to CtsB and L. Overall, these experiments indicated that the peptides could affect the biology of the lysosomal compartment and the expression of Cts. The only exception to this rule was represented by CtsW, which did not show a high affinity for the peptides, and its expression was negatively affected by the peptides only in 769-P cells, while no substantial differences were registered in A498 Next, we evaluated the protein expression of CtsB, L, and W (Figure 8). In both the cell lines, the Cts protein expression was similar. In particular, CtsB and L increased significantly at the later time points of treatment with both the peptides (Figure 8a–d). In contrast, in the case of CtsW, the peptides negatively affected the protein expression in 769-P cells, while no particular differences were detected in A498 cells (Figure 8e,f). Other tested Cts were modulated in their protein expression similarly to CtsB and L. Overall, these experiments indicated that the peptides could affect the biology of the lysosomal compartment and the expression of Cts. The only exception to this rule was represented by CtsW, which did not show a high affinity for the peptides, and its expression was negatively affected by the peptides only in 769-P cells, while no substantial differences were registered in A498 cells.

*Cancers* **2020**, *12*, x 10 of 20

**Figure 8.** Effect of the peptides on CtsB, L, and W expression: (**a**) protein expression of CtsB in 769-P and (**b**) A498 cells after 24, 48, and 72 h of treatment with Ac-PLVE-FMK and Ac-VLPE-FMK. (**c**) protein expression of CtsL in 769-P and (**d**) A498 cells after 24, 48, and 72 h of treatment with Ac-PLVE-FMK and Ac-VLPE-FMK. (**e**) Protein expression of CtsB in 769-P and (**f**) A498 cells after 24, 48, and 72 h of treatment with Ac-PLVE-FMK and Ac-VLPE-FMK. Data are represented as mean ± S.D. of at least three replicates. Significance was calculated through one-way ANOVA followed by **Figure 8.** Effect of the peptides on CtsB, L, and W expression: (**a**) protein expression of CtsB in 769-P and (**b**) A498 cells after 24, 48, and 72 h of treatment with Ac-PLVE-FMK and Ac-VLPE-FMK. (**c**) protein expression of CtsL in 769-P and (**d**) A498 cells after 24, 48, and 72 h of treatment with Ac-PLVE-FMK and Ac-VLPE-FMK. (**e**) Protein expression of CtsB in 769-P and (**f**) A498 cells after 24, 48, and 72 h of treatment with Ac-PLVE-FMK and Ac-VLPE-FMK. Data are represented as mean ± S.D. of at least three replicates. Significance was calculated through one-way ANOVA followed by Dunnet's test.

#### **3. Discussion**

Dunnet's test.

**3. Discussion**  Cts are universally considered as key players in maintaining proteostasis regulation, and their proteolytic activity is traditionally associated with the lysosomes. However, their sphere of action is not limited just to the lumen of these organelles. They were also detected in the cell cytoplasm [45], nucleus [46], and when secreted, in the extracellular space [46], regulating important processes like autophagy [47], apoptosis [48], gene expression [49], cell signaling [50], and angiogenesis [51]. In this scenario, a potential contribution of Cts in tumor disease is practically obvious, and the first pieces of evidence regarding their role in cancer were published in the late 80s [52]. In the case of renal cancer, the upregulation of CtsB was shown to decrease three- and five-year patient survival rates [33], and CtsK was shown to be overexpressed in renal cell carcinoma patients with Xp11 translocation [53]. Despite detailed data regarding the role of specific Cts in tumor disease, this family of proteases counts 11 members with redundant and compensatory activities, as shown in autoimmune diseases [54] and or thyroglobulin processing [55], respectively. For this reason, strategies aimed at specifically inhibiting the activity of a single Cts member could result in a low impact on cancer cell biology, as well as a limited understanding of the role of these proteases in tumor disease. More importantly, current literature generally did not focus on understanding the impact of the Cts inhibitors on the overall expression of these proteases. In this context, the investigation performed in renal cancer represents an additional novelty since specific studies in the field are very rare in this disease model. Cts are universally considered as key players in maintaining proteostasis regulation, and their proteolytic activity is traditionally associated with the lysosomes. However, their sphere of action is not limited just to the lumen of these organelles. They were also detected in the cell cytoplasm [45], nucleus [46], and when secreted, in the extracellular space [46], regulating important processes like autophagy [47], apoptosis [48], gene expression [49], cell signaling [50], and angiogenesis [51]. In this scenario, a potential contribution of Cts in tumor disease is practically obvious, and the first pieces of evidence regarding their role in cancer were published in the late 80s [52]. In the case of renal cancer, the upregulation of CtsB was shown to decrease three- and five-year patient survival rates [33], and CtsK was shown to be overexpressed in renal cell carcinoma patients with Xp11 translocation [53]. Despite detailed data regarding the role of specific Cts in tumor disease, this family of proteases counts 11 members with redundant and compensatory activities, as shown in autoimmune diseases [54] and or thyroglobulin processing [55], respectively. For this reason, strategies aimed at specifically inhibiting the activity of a single Cts member could result in a low impact on cancer cell biology, as well as a limited understanding of the role of these proteases in tumor disease. More importantly, current literature generally did not focus on understanding the impact of the Cts inhibitors on the overall expression of these proteases. In this context, the investigation performed in renal cancer represents an additional novelty since specific studies in the field are very rare in this disease model.

In this work, we designed peptide-based inhibitors that could universally inhibit the action of Cts. Ac-PLVE-FMK and Ac-VLPE-FMK derive from the well-known substrate of the Triticain-α cysteine protease Ac-PLVQ, which was extensively used by our group in previous work to define the activity of this enzyme through fluorimetric assay [23]. The proposed inhibition mechanism is based on FMK-containing drugs, which are known as selective and efficient cysteine protease inhibitors [56]. Molecular docking is a powerful computational approach commonly employed to predict the binding poses and affinities of various ligands to macromolecules. Ligand conformations, obtained in the docking procedure, allow for estimating the interactions required for successful binding and In this work, we designed peptide-based inhibitors that could universally inhibit the action of Cts. Ac-PLVE-FMK and Ac-VLPE-FMK derive from the well-known substrate of the Triticain-α cysteine protease Ac-PLVQ, which was extensively used by our group in previous work to define the activity of this enzyme through fluorimetric assay [23]. The proposed inhibition mechanism is based on FMK-containing drugs, which are known as selective and efficient cysteine protease inhibitors [56]. Molecular docking is a powerful computational approach commonly employed to predict the binding poses and affinities of various ligands to macromolecules. Ligand conformations, obtained in the docking procedure, allow for estimating the interactions required for successful binding

and provide insights for further improvements in ligand design. However, this method has limitations, since it does not allow for predicting the occurrence of covalent bonds between macromolecules and ligands [57]. In our case, both Ac-PLVE-FMK and Ac-VLPE-FMK were designed to bind the catalytic cysteine in the Cts covalently; however, the docking model can only estimate the interactions between the peptides and the Cts in the pre-reaction binding.

In the simulation, Ac-PLVE-FMK and Ac-VLPE-FMK tend to occupy the S2 binding site of Cts with aliphatic side chains and form hydrogen bonds by the peptide backbone atoms of the respective residues. However, the N-terminal amino acids of the peptides tend to form fewer contacts with the Cts proteins. Thus, the designed inhibitor molecules can be further improved by the rational design of their N-terminus. According to the docking results, Ac-VLPE-FMK appears to be a weaker binder to all the Cts we considered (see Figure 2d), since the Pro residue hinders the movement of this peptide, thereby reducing the possibility of its proper pre-reaction binding. Thus, Ac-PLVE-FMK theoretically represents a more promising target for further improvement.

Both peptides showed a similar efficiency in inhibiting the activity of recombinant human CtsB and L. More importantly, they demonstrated pronounced inhibitory properties in vitro directly on the cells, implying their ability to penetrate the cell membrane with moderate cytostatic effects only registered during the first 48 h of treatment. Previous work performed with the multi-Cts inhibitor E64 on pancreatic cells showed only a moderated cytotoxic effect, which reached a plateau phase after 48 and 72 h [58]. In general, Cts inhibitors cannot be considered very potent cytostatic molecules. However, as demonstrated in other works, they could increase chemotherapy efficacy [59], even though when used in combination with PXT our peptides did not increase cell toxicity in a significant way. We exclude, however, that the peptides lost their potency over time because the treatments were administered afresh every day. Therefore, we conclude that our peptides do not have a significant impact on renal cancer cell viability.

On the other hand, Cts activity was shown as a modulator of invasive properties, including cellular adhesion [8], anchorage-independent growth [60], colony formation [61], and motility [44] of cells. Our data support this evidence in both the human renal cancer cell lines tested with the peptides, decreasing their ability to migrate in a scratch assay, while increasing their adhesion to biological substrates. It is important to note that differences in cell spreading, associated with more potent cellular adhesive force, can be accompanied by decreased cell migration [62,63]. Additionally, the peptides inhibited colony and spheroid formation, phenomena that can be favored by Cts activity [44,64]. These effects could be a result of an increased E-cadherin expression, a protein involved in cell adhesion, and considered as a marker of differentiation during MET, which increased upon treatment with both the peptides, reaching significant levels at the later time points. A previous work [65] demonstrated that in renal cancer spheroid formation, a down-regulation of E-cadherin occurs, highlighting its potential contribution to the detected anti-spheroid and colony formation properties shown by our peptides.

Interestingly, the inhibitory proteolytic properties of our peptides impacted on Cts homeostasis. We observed different variations in the expression of LAMP-1. At 48 h of treatment with both the peptides, LAMP-1 increased in 769-P cells, while it decreased in A498 cells. However, at 72 h, both the cell lines were characterized by an increase in LAMP-1 expression, even though it did not reach significant levels. These data were corroborated by further fluorescent microscopy analysis as well as by the evaluation of the endosomal compartment integrity performed through neutral red assay and LysoTracker Red. An increase in LAMP-1 expression after CtsB and L knock out was similarly registered in mouse embryonic fibroblast cells [66] and bone marrow-derived macrophages [67].

More importantly, the peptides affected Cts turnover by inducing two different trends in their expression. The CtsB and L expression increased over time after treatment with the peptides in both the cell lines. On the other hand, CtsW protein expression was very stable, and in the case of 769-P cells, it decreased. We can speculate that in the case of CtsB and L, the proteolytic inhibition, induced by the peptides, was counterbalanced by an over-expression of these proteins. This rule was not to apply to CtsW that probably follows other mechanisms of expression regulation [37]. In addition, our data demonstrated a weaker interaction of the inhibitors in the case of the CtsW active site. Compared to other Cts, CtsW was shown to be significantly localized in the endoplasmic reticulum of immune cells [36], and this evidence could form the basis of its differential regulation.

From the pharmacological standpoint, the development of these inhibitors could provide new avenues of research to develop targeted therapies aimed at inhibiting cancer cell proteostasis while impacting their overall phenotype since they showed to affect cell migration and increasing adhesion and expression of E-Cadherin.

Future work is required to take into consideration the potential side effects of this treatment strategy and its impact on cancer biology in vivo, evaluating the peptides' synergistic effects with current chemotherapeutics, as well as revealing their effects on renal cancer spreading. In addition, more insights are necessary to evaluate their overall effect on the endolysosomal compartment stability, integrity, function (i.e., autophagy) as well on the cell metabolism. On the other hand, the generation of new Cts inhibitors could provide fundamental insights into understanding lysosomal biology and lysosomal-related conditions. In particular, more evidence is necessary to unveil the role of the inhibitors in regulating Cts expression as well as lysosomal turnover, as previously demonstrated by other works [58].

#### **4. Materials and Methods**

#### *4.1. Docking Studies*

Crystal structures of CtsB (6AY2) and L (2XU4) were obtained from the PDB databank. The CtsW structure was predicted using Modeller [68,69]. CtsB was used as a template structure. All protein structures were protonated with PROPKA at PDB2PQR server at pH 4.5, 6.5, and 7.2 [69]. Ligand structures were built and optimized in the GAFF force field using Avogadro [70,71].

Docking was performed using PLANTS [72]. The Chemplp scoring function was used in combination with search speed 1. The binding center was set at the SG atom of catalytic cysteine of all considered Cts. Both catalytic cysteine and histidine were set as flexible. When docking with constraints, simple distance constraints between the oxygen atom in the FMK group and atoms H in the catalytic cysteine or amine hydrogens of Gln-23/19/20 in CtsB/L/W were used. The constraint was applied for the distance between 1 and 3 Å. For each ligand, five poses per run were obtained. Ten runs per pH per protein per ligand were made.

#### *4.2. Protein Expression and Purification*

Total RNA extract from retinoblastoma J79 cells was used to obtain cDNA. A pair of oligonucleotides (TATACATATGCGGAGCAGGCCCTCTTTC and CTCGAGTTAGATCTTTTCCCAGTACTG) was used for the amplification of DNA fragment containing CtsB, the product of which was ligated into pET15b (Merck Millipore, Billerica, MA, USA) using NdeI and XhoI. DNA fragment containing CtsL was amplified using a pair of oligonucleotides (TATAGCTAGCACTCTAACATTTGATCACAGTTT and ATTAAGCTTTCACACAGTGGGGTAGCTG) and ligated into pET28a (+) (Merck Millipore, Billerica, MA, USA) using NheI and HindIII. After the transformation of obtained vectors into Rosetta gammy B(DE3) cells (Merck Millipore, Billerica, MA, USA), these *E. coli* strains were used for the expression of 6His-tagged CathB or CathL using a procedure described by Gorokhovets et al. (2017) for the expression of 6His-tagged papain-like cysteine protease triticain-α [23]. CtsB or CtsL from the insoluble fraction were purified using Ni-NTA sepharose and then refolded using the methods described in detail for protease triticain-α in Gorokhovets et al. (2017).

#### *4.3. Gelatin Zymography*

A 5× non-reducing loading buffer (0.05% bromophenol blue, 10% SDS, 1.5 M Tris, 50% glycerol) was added to all recombinant proteins: CtsL and B and prior to loading. Then, the proteins were

resolved by 12% SDS-polyacrylamide gels containing 0.2% gelatin at 4 ◦C. Gels were removed, and enzymes were refolded for four washes in 2.5% Triton-X100, 15 min each. Next, the gels were washed twice and incubated in activating buffer (NaAc, pH 4.8, 1 mM EDTA, and 20 mM L-cysteine hydrochloride monohydrate) for 24 h at 37 ◦C. In the morning, the gels were fixed for 1 h in 50% methanol with 10% acetic acid and then stained for 1 h in Coomassie (10% acetic acid, 25% isopropanol, 4.5% Coomassie Blue). The gels were destained in 10% isopropanol and 10% acetic acid and scanned using the Bio-Rad ChemiDoc MP Imaging System.

#### *4.4. Cathepsin Inhibitors*

Specific inhibitors for cysteine Cts were developed with the help of computer-graphic modeling, based on the structure of the proteins. The two peptides, Ac-PLVE-FMK and Ac-VLPE-FMK were selected as the specific inhibitors, that provide a binding affinity to Cts and can block their activity. The inhibitors were synthesized by Pepmic (Pepmic Suzhou Jiangsu, China).

#### *4.5. Enzymatic Kinetic Studies*

The activity of recombinant CtsL and B was detected by the hydrolysis of the fluorogenic substrate Ac-Pro-Leu-Val-Gln- 7-amino-4-methylcoumarin (AMC) (Pepmic Suzhou Jiangsu, China). A total of 20 nM of each protein was mixed in a 96-well plate with 0.1 M sodium acetic buffer (100 mM NaCl, 0.5% DMSO, 0.6 mM EDTA pH 4.6) in the presence or absence of cysteine Cts inhibitor at a final concentration of 2 µM. The substrate was added to a final concentration of 50 µM, and its hydrolysis was continuously measured for 12 min using a CLARIOstar® Plus plate reader (BMG Labtech Ortenberg Baden-Württemberg, Germany) at excitation and emission wavelengths of 353 and 442 nm, respectively.

#### *4.6. Cell Culture*

The human renal cancer cell line lines 786-P, A498 were obtained from Dr. Vadim Pokrovsky (purchased from American Type Culture Collection). The cells were cultured in RPMI 1640, supplemented with 10% fetal bovine serum and 1% mixture of antibiotics penicillin-streptomycin (all from Gibco, Waltham, MA, USA) at 5% CO<sup>2</sup> and 37 ◦C in a humidified chamber. Cells were grown to confluence and harvested by trypsinization, using a 0.25 mg/mL trypsin/EDTA solution (ThermoFisher, Carlsbad, CA, USA) and resuspended in the fresh culture medium. Viable cells were enumerated on the Countess II FL Automated Cell Counter (ThermoFisher, Waltham, MA, USA), following Trypan Blue staining. The cell lines were tested for mycoplasma contamination regularly, using the Molecular Probes™ MycoFluor Mycoplasma Detection Kit (ThermoFisher, Waltham, MA, USA).

#### *4.7. MTT*

The cell number was evaluated by counting viable cells using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) colorimetric assay. A total of 2 × 10<sup>4</sup> cells/well were seeded on independent 96-well plates for each time point (0, 24, 48, and 72 h), with five replicates and treated with two inhibitors (2.5–250 µM). Then, 10 µL of the MTT reagent was added to each well, and cells were incubated for another 5 h. Next, the absorbance value was measured using a CLARIOstar® Plus plate reader (BMG Labtech, Ortenberg, Germany) at 490 nm. Triplicate wells were assayed, and S.D.s were determined.

#### *4.8. RNA Extraction and cDNA Synthesis*

Total RNA was extracted from cells using the Total RNA isolation kit (Evrogen, Moscow, Russia). Complementary DNA (cDNA) was transcribed from mRNA using a cDNA synthesis kit (Evrogen, Moscow, Russia), according to the manufacturer's protocols. For RT reaction 1 µg of total RNA

was used with optical density OD260/OD280 1.7-2.0 measured with NanoDrop One (ThermoFisher, Waltham, MA, USA).

#### *4.9. Western Blot Analysis*

Cells were seeded in RPMI containing 10% FBS and cultured for 24 h. Next, 30 µM of inhibitors were added to the culture medium and incubated for 24, 48, and 72 h. Control cells were treated with 0.01% DMSO. At all time points the samples were lysed in 50 mM Tri-HCl (pH 8.0), 100 mM NaCl, 0.5% NP-40, 1% Triton X-100, 1× protease inhibitor cocktail (ThermoFisher, Waltham, MA, USA). The 50 µg of protein lysates were separated by electrophoresis on a 12% SDS-PAGE gel and transferred to the PFDF membranes. The expression of cysteine Cts, LAMP-1, E-cadherin, and SNAIL1 were identified by a reaction with specific primary antibodies (CtsB-Ab190077, Abcam, UK; CtsL-Ab95154, Abcam, UK; CtsW Ab191083; LAMP-1- Ab24170, Abcam, UK; SNAIL1 Ab216347, Abcam, UK and e-cadherin-612131, BD Sciences Franklin Lakes, NJ, USA) which were resuspended in 5% non-fat milk in PBST (all Cts 1:3000, LAMP-1, SNAIL1 and E-cadherin 1:1000) and incubated O/N. The next day, the membranes were washed three times with PBST and incubated for 1 h with secondary antibodies (P-GAR Iss (Goat pAb to rabbit IgG (HRP), Abcam, UK or Rabbit Ab to mouse, Abcam, UK; both 1:5000) in 5% non-fat milk in PBST. After an additional wash (three times with PBS), reactive bands were detected by chemiluminescence (Bio-Rad, Irvine, CA, USA). As a loading control, the membranes were incubated with a polyclonal anti-tubulin antibody (1:5000; Ab52866, Abcam, UK) identically.

#### *4.10. Immunofluorescence Staining*

The cells were treated for 48 and 72 h, then were fixed in 4% PFA/PBS for 15 min and permeabilized in 0.25% Triton®X-100 for 10 min. After blocking the non-specific sites in 2% BSA/PBS-T, the immunofluorescence was performed overnight with primary antibody anti-LAMP-1 (1:100, Abcam, Eugene, OR, USA) incubation, followed by incubation with the appropriate fluorophore-labeled secondary antibody Donkey anti-Rabbit IgG (H+L) ReadyProbes™, Alexa Fluor 488 (1:500; ThermoFisher, USA). The cells were then counterstained with nuclear dye DAPI and visualized under a fluorescent and/or confocal microscope (Olympus BX51, Shinjuku, Tokyo, Japan, and AxioObserver Z1, Zeiss, Oberkochen, Germany) using oil-immersion lenses.

#### *4.11. AFM Measurements*

Before the AFM experiments, the 50,000 cells were seeded per dish and treated for 48 and 72 h with inhibitors. The AFM measurements were performed at 37 ◦C, using a commercial atomic force microscope Bioscope Resolve AFM (Bruker, Billerica, MA, USA) combined with an inverted optical microscope (Carl Zeiss, Ulm, Germany). The PeakForce QNM-Live Cell cantilevers (PFQNM-LC-A-CAL, Bruker AFM Probes, USA) with a pre-calibrated spring constant (in a range of 0.06–0.08 N/m) and a 70 nm tip radius was used. The deflection sensitivity (nm/V) was calibrated from the thermal using the pre-calibrated value of the spring constant. The nanomechanical maps were acquired in the force volume mode with a typical map size of 80 × 80 microns and 40 × 40 measurement points [73]. For the force curves, a vertical ramp distance was 3 µm, a vertical piezo speed was 183 µm/s, and the trigger force was 0.5–1 nN. The Young's modulus (E) was calculated by fitting the force curves with the Hertz model with a bottom-effect correction [73,74].

#### *4.12. Scratch Assay*

The cells were treated for 48 h with Cts inhibitors in a six-well plate. At experimental time zero, a scratch of culture monolayer was made in each well using a pipette tip. The monolayers were washed with PBS to remove detached cells and cell debris and next refilled with growth medium, including Cts inhibitors. The wells were imaged at time zero and again 6 and 24 h later. Using ImageJ, a measurement was taken for how much the denuded area had filled after 6 and 24 h.

#### *4.13. Colony-Forming Assay*

769-P and A498 cells were treated 48 h with 30 µM Ac-PLVE-FMK or Ac-VLPE-FMK, next calculated, and 300 cells were placed on 10 cm plates. Cells were maintained in the completed medium with inhibitors for the 10 days, then fixed with 4% paraformaldehyde and stained with 0.4% Crystal Violet solution, finally photographed.

#### *4.14. Spheroids Formation Assay*

The 769-P cells were treated for 48 h with Cts inhibitors and next suspended in 2% Matrigel in the total medium containing 30 µM Ac-PLVE-FMK or Ac-VLPE-FMK. The 100 prepared cells were seeded in 96-well microplates on top of 50 µL Matrigel (Corning, NY, USA) and incubated for 6 days. The formed spheroids were imaged under an Olympus IX71 microscope, and their number, size, and circularity were measured using ImageJ software. Each experiment had two replicates and was repeated three times.

#### *4.15. Adhesion*

The 96-well plates were coated with either 15 µg/mL collagen IV (Imtek, Moscow, Russia) or Matrigel 0.5% in RPMI-1640 (Corning, NY, USA) and stored at 4 ◦C O/N. On the day of the assay, plates were washed twice with PBS and 40,000 cells/well were seeded and incubated for 50 min at 37 ◦C. Adherent cells were fixed and stained with 0.2% crystal violet/10% ethanol and read at 485 nm on a microplate reader. All the experiments were performed in triplicate.

#### *4.16. LysoTracker Red Fluorescence Measurement*

First, 1.5 × 10<sup>4</sup> 769-P or A498 cells were seeded in 96-well plates in full medium. The day after the cells were treated with the peptides for 3 consecutive days to establish 24, 48, and 72 h groups of treatment. LysoTracker Red fluorescence intensity was measured via microplate reader following the protocols described in [75] and applying Ex/Em = 570/600. The cells were incubated with 75 nM of LysoTracker red for 1 h.

#### *4.17. Statistical Analysis*

All experiments were repeated at least three times. Data are reported as mean ± SD. Data were analyzed using one-way ANOVA, followed by Dunnet's test (GraphPad, Prism 6.00 for Windows, Graf Pad software, San Diego, CA, USA). The *p*-value of <0.05 was considered statistically significant. with \*, similarly *p*< 0.01 with \*\* and *p*< 0.001 with \*\*\*.

#### **5. Conclusions**

Recently, it has been recognized that the pathogenic function of Cts in cancerogenesis is far more complicated than initially conceived [76]. Experimental studies have shown that many Cts are overexpressed in different tumor types, frustrating every attempt to precisely correlate the role of single Cts with the disease development [77]. In this work, we generated two novel peptides with wide-ranging inhibitory properties towards Cts that could provide new resources to develop new treatments. In particular, they could improve current treatments for conditions such as renal cancer that is resistant to standard chemotherapeutic approaches and could benefit from novel targeted therapies [30,78]. Despite their low cytostatic power, these small inhibitors demonstrated broad inhibiting properties, high membrane permeability, minimal toxicity, and above all, a significant impact on cancer cell phenotype.

Our data demonstrated that the peptides could inhibit Cts activity in two different human renal cancer cell lines impacting their motility, anchorage-independent growth, colony formation, and their adhesion. More importantly, this strategy affected Cts expression, and this evidence should be taken into consideration when similar treatment strategies are designed for cancer and other diseases.

*Cancers* **2020**, *12*, 1310

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2072-6694/12/5/1310/s1, Figure S1: Detection of recombinant human CtsL and B activity through gelatin zymography assay. Recombinant purified human CtsL and B were loaded on the gelatin-gel zymogram. The Cts were activated through incubation of gel in an activating buffer (pH 4.8). Further staining with Coomassie brilliant blue G-250 showed the induction of proteolytic activity as bright white bands on dark background in correspondence with the bands of the enzymes, Figure S2. Determination of the peptide inhibitory properties against human purified CtsB and L. The activities of human recombinant CtsB and L, were measured via fluorimetric analysis by exploiting the fluorogenic properties of the Triticain-α substrate Ac-PLVQ-AMC. In the assay, the 20 nM of recombinant human CtsL and CtsB were incubated with 50 µM substrate without Cts inhibitors (red line) and with Ac-PLVE-FMK and Ac-VLPE-FMK (blue and green line, respectively) at a concentration of 2 µM. Fluorescence was measured as relative as relative fluorescence unit (RFU), Figure S3. Combined effect of PXT and the inhibitors on 769-P proliferation: 769-P cells were treated with 100 nM of PXT alone or in combination with 20 µM of Ac-PLVE and Ac-VLPE and cell proliferation was measured after 24, 48, and 72 h via MTT assay. Data represent the mean (± S.D.) of three independent experiments, each performed in triplicate, Figure S4. Spheroids size and circularity: (**a**,**b**) size and circularity of 769-P and (**c**,**d**) A498 cells upon treatment with Ac-PLVE-FMK and Ac-VLPE-FMK. Data represent the mean (±S.D.) of three independent experiments, each performed in triplicate, Figure S5. Effect of Cts inhibitory peptides on cell stiffness: representative optical phase contrast image of cells (first row) and stiffness maps determined by indentation (Young's modulus) of 769-P cells (second row) with and without treatment with PLVE and VLPE. Calibration bars represent 25 µm. The graph shows the values of Young's modulus of all analyzed samples, data are expressed as mean (± S.D.) and significance was calculated through one-way ANOVA followed by Dunnet's test. \* = *p* < 0.05, \*\* = *p* < 0.01, Figure S6. The effect of the peptide inhibitors on human 769-P and A498 renal cancer cell on lysosomes integrity. (**a**) 769-P and (**b**) A498 cells were treated with increasing doses of Ac-PLVE-FMK (red bars) and Ac-VLPE-FMK (blue bars) (2.5–250 µM). NR uptake was measured after 24, 48, and 72 h via MTT assay. (**c**) LysoTracker red evaluation in 769-P and (**d**) A498 cells after 24, 48, and 72 h with the inhibitors (20 µM). Data represent the mean (± S.D.) of at least three independent experiments, each performed in triplicate, Figure S7. Uncropped western blots.

**Author Contributions:** Conceptualization, A.A.Z., M.R. and A.P.; methodology, M.R., A.P., Y.M.E., N.V.G., V.A.M., E.Y.Z.; software, V.D.M., A.V.G.; validation A.A.Z., A.P., M.R.; formal analysis, A.P., M.R.; investigation, M.R., A.P., V.D.M., A.V.G., A.A.Z.; resources, A.A.Z.; data curation, M.R., A.P.; writing—original draft preparation, A.P., M.R.; writing—review and editing, A.P., M.R., A.A.Z., V.D.M., A.V.G., Y.M.E., E.Y.Z.; visualization, V.A.P., M.R.; supervision, A.A.Z.; project administration, A.A.Z.; funding acquisition, A.A.Z. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Russian Science Foundation (grant # 16-15-10410).

**Acknowledgments:** We thank Peter S. Timashev for fruitful discussions in relation to this paper.

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**


© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Review* **Therapeutic Targeting of Autophagy for Renal Cell Carcinoma Therapy**

## **Trace M. Jones, Jennifer S. Carew and Ste**ff**an T. Nawrocki \***

Division of Translational and Regenerative Medicine, Department of Medicine and The University of Arizona Cancer Center, Tucson, AZ 85724, USA; tracejones@email.arizona.edu (T.M.J.); jcarew@email.arizona.edu (J.S.C.) **\*** Correspondence: snawrocki@email.arizona.edu; Tel.: +1-520-626-7395

Received: 25 March 2020; Accepted: 3 May 2020; Published: 7 May 2020

**Abstract:** Kidney cancer is the 7th most prevalent form of cancer in the United States with the vast majority of cases being classified as renal cell carcinoma (RCC). Multiple targeted therapies have been developed to treat RCC, but efficacy and resistance remain a challenge. In recent years, the modulation of autophagy has been shown to augment the cytotoxicity of approved RCC therapeutics and overcome drug resistance. Inhibition of autophagy blocks a key nutrient recycling process that cancer cells utilize for cell survival following periods of stress including chemotherapeutic treatment. Classic autophagy inhibitors such as chloroquine and hydroxychloroquine have been introduced into phase I/II clinical trials, while more experimental compounds are moving forward in preclinical development. Here we examine the current state and future directions of targeting autophagy to improve the efficacy of RCC therapeutics.

**Keywords:** renal cell carcinoma; autophagy; hydroxychloroquine; chloroquine; ROC-325

## **1. Introduction**

It is estimated that over 73,000 new cases of renal cancer will be diagnosed in the United States this year, with upwards of 14,000 individuals succumbing to their disease [1]. The most common malignancy of the kidney is renal cell carcinoma (RCC), which accounts for 85% of cases [2]. RCC can be divided into three distinct histological subtypes. Clear cell RCC (ccRCC) is the predominant subtype (~75%), with papillary RCC (PRCC) and chromophobe RCC (ChRCC) accounting for ~20% and ~5% of cases, respectively [3]. Disease stage at the time of diagnosis is the most important factor when considering the best course of treatment. Localized disease, generally TNM stage I or II, has a positive prognosis with a 5-year relative survival rate of 92.6% [4]. Localized neoplasms can be effectively treated with either a partial or radical nephrectomy, depending on the location of the primary mass [5]. After successful surgery, patients are often simply surveyed for signs of recurrence. It is estimated that 20–30% of patients who have undergone a successful nephrectomy will experience a recurrence, often presenting between one to three years following surgery [6]. Following a relapse, patients often undergo treatment with chemotherapy or immunotherapy depending on their histologic subtype. Patients presenting with regionally or distantly invasive tumors have a less favorable prognosis. A nephrectomy is still the primary first line treatment. However, patients are often administered chemotherapy, immunotherapy, or enrolled in a clinical trial in order to manage metastases and tumors that are surgically unresectable [5].

Given that metastatic, relapsed, and surgically unresectable tumors must be treated by systemic chemotherapy or immunotherapy, great interest has been shown in the past decade in developing targeted therapeutics for RCC. The most commonly mutated gene in RCC is the *von Hippel-Lindau (VHL)* tumor suppressor gene. Approximately 50% of RCC cases contain a mutation in this gene, with an additional 20% of cases presenting with a hypermethylated gene [7]. The VHL protein is an E3 clinic (Figure 1).

ubiquitin ligase that controls the conjugation of ubiquitin molecules onto hypoxia-inducible factors (HIFs), proteins that are vital to the cellular hypoxia response pathway. Upon ubiquitylation, HIFs are processed and degraded through the ubiquitin proteasome pathway. Without a functional copy of *VHL*, HIFs are free to translocate to the nucleus and activate transcription of HIF responsive genes. A few of these HIF responsive genes code for vascular endothelial growth factor (VEGF), platelet-derived growth factor B (PDGF-B), transforming growth factor alpha (TGFα), and glucose transporter 1 (GLUT1) [7]. The overexpression of these factors is often a driving force in RCC tumorigenesis. In addition to *VHL*, genes involved in the mammalian target of rapamycin (mTOR) pathway are mutated in 28% of RCC cases [8,9]. These include genes encoding for phosphatidylinositol-3-kinase (PI3K), phosphatase and tensin homolog (PTEN), protein kinase B (AKT), and mTOR itself. These frequent mutation profiles provide the rationale for therapeutically targeting various receptor tyrosine kinases (RTKs) and downstream effector proteins currently being developed and used in the clinic (Figure 1). A hallmark of cancer is evasion of the immune response [11]. Cancer cells are capable of evading immune surveillance by expressing various signals that act as "off" switches to T-cells and natural killer (NK) cells. The most well-characterized of these signals are cytotoxic T-lymphocyte associated protein 4 (CTLA-4) and programmed cell death ligand 1 (PD-L1). When these surface proteins come in contact with the appropriate receptor on T-cells, they effectively trick the lymphocyte into recognizing the cancer cell as normal self-cells. Given this, an immense amount of energy has been dedicated to developing monoclonal antibody therapies to block the binding of cancer cell expressed PD-L1 and CTLA-4 allowing the immune cells to recognize the tumor cells as a foreign entity. These immune checkpoint inhibitor therapies enable the immune system to both eliminate tumor cells and also develop a lasting immune response. A persistent remission state is observed in two thirds of patients who experience an initial response to these therapies [12]. Importantly, immune checkpoint inhibitors have demonstrated significant efficacy in patients with RCC.

of new and more effective treatment options for the relapsed/refractory patient population.

Although targeted tyrosine kinase and mTOR inhibitors are effective first-line treatment options, many, if not all, cases of RCC will eventually become resistant to these drugs. The median time to a

*Cancers* **2020**, *12*, x 2 of 16

an additional 20% of cases presenting with a hypermethylated gene [7]. The VHL protein is an E3 ubiquitin ligase that controls the conjugation of ubiquitin molecules onto hypoxia-inducible factors (HIFs), proteins that are vital to the cellular hypoxia response pathway. Upon ubiquitylation, HIFs are processed and degraded through the ubiquitin proteasome pathway. Without a functional copy of *VHL*, HIFs are free to translocate to the nucleus and activate transcription of HIF responsive genes. A few of these HIF responsive genes code for vascular endothelial growth factor (VEGF), plateletderived growth factor B (PDGF-B), transforming growth factor alpha (TGF), and glucose transporter 1 (GLUT1) [7]. The overexpression of these factors is often a driving force in RCC tumorigenesis. In addition to *VHL*, genes involved in the mammalian target of rapamycin (mTOR) pathway are mutated in 28% of RCC cases [8,9]. These include genes encoding for phosphatidylinositol-3-kinase (PI3K), phosphatase and tensin homolog (PTEN), protein kinase B (AKT), and mTOR itself. These frequent mutation profiles provide the rationale for therapeutically targeting various receptor tyrosine kinases (RTKs) and downstream effector proteins currently being developed and used in the

**Figure 1.** Federal Drug Administration (FDA) approved agents to treat renal cell carcinoma (RCC). Various kinase and mammalian target of rapamycin (mTOR) inhibitors are amongst the most common drugs used, however, immune checkpoint inhibitors are becoming a mainstay of RCC **Figure 1.** Federal Drug Administration (FDA) approved agents to treat renal cell carcinoma (RCC). Various kinase and mammalian target of rapamycin (mTOR) inhibitors are amongst the most common drugs used, however, immune checkpoint inhibitors are becoming a mainstay of RCC treatment.

treatment. Although targeted tyrosine kinase and mTOR inhibitors are effective first-line treatment options, many, if not all, cases of RCC will eventually become resistant to these drugs. The median time to a resistant tumor phenotype is 6–15 months depending on the therapeutic regimen [10]. A better understanding of the mechanistic drivers of drug resistance in RCC will facilitate the development of new and more effective treatment options for the relapsed/refractory patient population.

A hallmark of cancer is evasion of the immune response [11]. Cancer cells are capable of evading immune surveillance by expressing various signals that act as "off" switches to T-cells and natural killer (NK) cells. The most well-characterized of these signals are cytotoxic T-lymphocyte associated protein 4 (CTLA-4) and programmed cell death ligand 1 (PD-L1). When these surface proteins come in contact with the appropriate receptor on T-cells, they effectively trick the lymphocyte into recognizing the cancer cell as normal self-cells. Given this, an immense amount of energy has been dedicated to developing monoclonal antibody therapies to block the binding of cancer cell expressed PD-L1 and CTLA-4 allowing the immune cells to recognize the tumor cells as a foreign entity. These immune checkpoint inhibitor therapies enable the immune system to both eliminate tumor cells and also develop a lasting immune response. A persistent remission state is observed in two thirds of patients who experience an initial response to these therapies [12]. Importantly, immune checkpoint inhibitors have demonstrated significant efficacy in patients with RCC.

#### **2. Targeted Therapeutics for RCC**

For multiple decades, the standard therapy for RCC patients was a regimen of cytokines. While more effective than traditional chemotherapy options, interferon-alpha, and interleukin-2 as single agents or in combination yielded low response rates in patients with the combination generating an 18.6% response rate [13]. In addition, cytokine therapy was often associated with severe adverse effects and the incidence of comorbidities was high. With the advent of targeted therapies for cancer patients came an influx of approved therapeutics for RCC patients. There are now a multitude of Federal Drug Administration (FDA)-approved targeted treatments for RCC. The target-specific therapies can roughly be broken down into three distinct categories: small molecule kinase inhibitors, mTOR inhibitors, and monoclonal antibodies. The monoclonal antibodies frequently used to treat advanced RCC can be further classified as immune checkpoint inhibitors and non-immunomodulatory antibodies. A listing of targeted therapies approved for use in RCC can be found in Table 1.


**Table 1.** FDA-approved treatments for RCC.

Abbreviations: VEGF—vascular endothelial growth factor; PDGF—platelet-derived growth factor; VEGFR—vascular endothelial growth factor receptor; MET—tyrosine-protein kinase Met; FLT3—fms like tyrosine kinase 3; TIE-2—angiopoietin-1 receptor; AXL—AXL receptor tyrosine kinase; TRKB—tropomyosin receptor kinase B; EGFR—epidermal growth factor receptor; RAF—rapidly accelerated fibrosarcoma; FKBP-12—FK506 binding protein 12; mTOR—mammalian target of rapamycin; PD-L1—programmed death ligand 1; CTLA4—cytotoxic t-lymphocyte associated protein 4; PD-1—programmed cell death protein 1; PFS—progression free survival.

Due to the frequent inactivation of VHL and subsequent overexpression of HIF1a, RCC often presents as a highly vascularized tumor type. Hence, many of the small molecule therapeutics approved for use in RCC target various effectors in the angiogenesis pathway (VEGF, VEGFR). The goal of these drugs is to abrogate the formation of new blood vessels in the tumor microenvironment via growth factor withdrawal, which deprives the cancer cells of oxygen and nutrients that are needed to fuel their growth and survival. Many of these therapies provide an initial response. However, oxygen deprivation and nutrient withdrawal activates various stress response pathways in the cancer cell. Autophagy is one such stress response, and allows for survival during periods of therapeutic insult.

#### **3. Autophagy**

#### *3.1. Molecular Mechanisms of Autophagy*

Autophagy is a catabolic process by which cells internally break down and recycle cellular components through non-specific, lysosome-mediated degradation. Autophagy is highly conserved in eukaryotes and is a vital mechanism to mediate cellular stress and damage that results from hypoxia and starvation, as well as therapeutic intervention. Mammalian target of rapamycin complex 1 (mTORC1) is often regarded as the master regulatory kinase of cellular metabolism. Activation of mTORC1 is generally thought of as a pro-proliferation signal. mTORC1 activity can be stimulated from activated upstream tyrosine kinases such as PDGFR and VEGFR, often through the phosphoinositide 3 kinase (PI3K)/ protein kinase B (AKT) pathway. This mechanism of mTORC1 activation is especially important in RCC, given the genes encoding the proteins involved are frequently mutated. Importantly, activated mTORC1 is responsible for adding an inhibitory phosphate at Serine 757 to the Unc-51-like kinase (ULK1) complex, which prevents the initiation of autophagy [30]. Both direct and indirect inhibition of mTORC1 leads to potent activation of the autophagy inducer ULK1, which in turn promotes activity of the Beclin1-Vacuolar protein sorting 34 (VPS34) complex. The Beclin1-VPS34 complex is vital to the nucleation of the premature phagophore.

Maturation of the phagophore into a complete autophagosome involves elongation of the vesicle's lipid membrane. This process is regulated by a complex containing autophagy-related 12, autophagy-related 5 and autophagy-related 16L (ATG12-ATG5-ATG16L). Another crucial protein involved in the elongation of the autophagosome membrane is microtubule-associated protein 1A/1B light chain 3 (LC3). Cytosolic LC3 is referred to as LC3-I. LC3-I is conjugated in a ubiquitin-like fashion to phosphatidylethanolamines (PE) on the autophagosome membrane by the autophagy protein, autophagy-related 7 (ATG7). It must be mentioned that autophagy related 4B (ATG4B), a widely conserved cysteine protease, must first make a specific cleavage to allow conjugation of LC3 [31]. PE-associated LC3 protein is referred to as LC3-II, and is often considered a reliable marker of autophagosome formation and autophagy. LC3-II is vital for cargo recruitment as it binds Sequestosome-1 (p62) to the autophagosome membrane. p62 is responsible for binding misfolded proteins or dysfunctional organelles and subsequently delivering them to the autophagosome for degradation [32]. Finally, the mature autophagosome, with its contents localized and completely enclosed will fuse with the lysosome. The fusion of membranes will release the cargo of the autophagosome into the lysosome where the acidic pH, as well as various enzymes will facilitate their degradation. After degradation has occurred, the remaining molecules are released back into the cytoplasm where they can be used as building blocks for new proteins, organelles, or energy sources. We will specifically focus on the role of autophagy in RCC pathogenesis and its involvement related to emerging targeted therapies in RCC. For a more extensive review of the molecular machinery and regulation of autophagy, refer to the following articles [33–35].

#### *3.2. Targeting Autophagy to Improve RCC Therapeutic Outcome*

Autophagy is an essential lysosomal degradation process that can be used by cancer cells to generate alternative sources of energy via nutrient recycling under stress conditions [36,37]. Although many studies have demonstrated that autophagy may function as a mechanism of tumor suppression through the degradation of defective pre-malignant cells, significant data indicates a key role for autophagic degradation in the maintenance of energy balance under stress conditions including nutrient deprivation and hypoxia [38]. Futhermore, autophagy has emerged as an important mechanism of resistance to radiation, conventional chemotherapy, and targeted anticancer agents due to its ability to enhance the stress tolerance of malignant cells [39–43]. Collectively, these data support a role for autophagy as a promoter of drug resistance and cancer progression as well as a target for therapeutic inhibition. Importantly, several new studies demonstrate that alterations in the autophagy pathway may be particularly relevant for patients with RCC and impact overall survival [44,45].

RCC cell lines inherently exhibit an elevated basal level of autophagy. One study found that across many RCC cell lines, 30–60% of growing cells display prominent LC3-II puncta [46]. This compares to just 1–5% of cells in normal primary kidney cell cultures. Autophagy has been shown to counteract growth factor and nutrient withdrawal and maintain cell viability under stress conditions [47]. Importantly, inhibiting autophagy in RCC increases the efficacy of many therapeutic strategies. Sorafenib, a general RTK inhibitor, shows a significant increase in activity when combined with autophagy inhibitors [48]. The efficacy of AKT/mTOR inhibition is also significantly augmented through the use of a variety of autophagy inhibitors [49]. It has been demonstrated that RCC cells that have adopted an aggressive metastatic phenotype also rely on an increase in cellular autophagic flux. These highly aggressive cells can be rendered sensitive to the mTOR inhibitor temsirolimus by chloroquine, an anti-malarial drug that inhibits autophagy [50]. Successful enhancement of therapeutic efficacy via in vitro autophagy inhibition has provided a solid foundation for the development and clinical testing of autophagy inhibitors in RCC.

#### **4. Inhibition of Autophagy for Therapy of RCC**

#### *4.1. Chloroquine and Hydroxychloroquine*

Chloroquine (CQ) and hydroxychloroquine (HCQ) are quinone-containing compounds that have been used to combat malaria for decades. These compounds work via their accumulation in and subsequent deacidification of lysosomes [51]. This deacidification disrupts autophagy as the low pH of lysosomes is a necessity in degrading the cargo of the autophagosome. CQ and HCQ have been repurposed to pharmacologically target autophagy in a broad range of cancer types for over a decade [52]. To date, CQ and HCQ are the only autophagy inhibitors to be evaluated in clinical trials. A number of clinical trials involving the use of CQ or HCQ alone or in combination with standard of care agents for the treatment of many different malignancies are ongoing and completed. However, limited clinical studies have evaluated HCQ in patients with RCC (Table 2).



A recent study in RCC combined the mTOR inhibitor, everolimus, with twice daily doses of HCQ in a metastatic patient population refractory to at least one prior treatment [53]. No dose-limiting toxicities (DLTs) were attributed to the HCQ in the phase I portion of the trial. The median progression-free survival (PFS) for the patient population was 6.3 months, an improvement over the median PFS of 4 months, which was observed in the clinical testing of everolimus alone [21]. A separate phase I trial sought to elucidate the toxicity and efficacy of combining HCQ with an AKT inhibitor, MK-2206, in a multitude of advanced solid tumors [55]. Patients were administered 135–200 mg of MK-2206 weekly in combination with 200–600 mg HCQ twice a day. 31 of the 35 patients enrolled were taken off treatment due to relapsed or progressive disease. In addition, 94% of participants experienced an adverse event (AE) attributed to treatment with MK-2206, while only 13% experienced an AE from the HCQ. Due to high toxicity and the low enrollment on the study, no anti-tumor activity data could be interpreted. One phase I trial has provided an exciting preliminary example of the power of HCQ in combination with vorinostat, an FDA approved histone deacetylase (HDAC) inhibitor [54]. This trial included patients with a variety of advanced solid tumors who had failed conventional therapy. Of these patients, a single person presented with advanced RCC. This particular patient had failed seven previous lines of therapy. A durable, partial response was obtained with a regimen of 400 mg vorinostat and 400 mg HCQ, administered daily. This response was maintained for more than 50 three-week cycles of the drug combination. This remarkable result in an RCC patient has sparked follow-up studies to evaluate tumor characteristics that may be indicative of a positive response to concurrent HDAC and autophagy inhibition.

#### *4.2. Lucanthone*

In addition to HCQ and CQ, several other agents used for non-cancer indications that disrupt lysosomal function have been repurposed as autophagy inhibitors for cancer therapy [41]. Lucanthone has been used as an anti-schistosome agent for many years and is also being developed as an anticancer agent due to its inhibitory effects on topoisomerase II and AP endonuclease (APE1). In cell culture experiments, lucanthone demonstrated lysosomal disruption and inhibition of autophagy [56]. In addition, strong pro-apoptotic effects were evident in various breast cancer cell lines and the lysosomal protease, cathepsin D, was shown to be an important mediator for the apoptotic effects of lucanthone. The chemical structure of lucanthone has provided clues to the construction of novel, lysosome-targeting agents.

#### *4.3. ROC-325*

While the clinical benefits of adjuvant CQ and HCQ treatment have not been fully elucidated, a substantial amount of funding and effort has been put forth to develop more efficient autophagy inhibitors. Initial results stemming from the use of CQ or HCQ have indicated that while these drugs partially block the degradation of cellular components in the lysosome, the compounds may not be potent enough to completely shut off the autophagy degradation. Thus, it is essential to generate new and more potent autophagy inhibitors that may improve clinical efficacy. A complete listing of next generation autophagy inhibitors discussed in this review, as well as the cancer types they have been explored in, can be found in Table 3.

ROC-325 is a water-soluble, small molecule developed by our group that shows significantly higher efficacy than HCQ [57–60]. ROC-325 is a dimeric compound that was designed to contain core motifs of HCQ as well as lucanthone. Much like HCQ, ROC-325 targets the late stages of autophagy. We have shown that ROC-325 does not affect the formation of autophagosomes but rather accumulates in and deacidifies the lysosome. In vitro treatment with ROC-325 shows stabilization of LC3-II and p62, indicators that are consistent with autophagy inhibition. In addition, treatment with ROC-325 results in near-complete loss of acridine orange fluorescence, a strong marker for lysosome membrane permeability and lysosome deacidification. ROC-325 reduced cell viability in RCC cell lines at much

lower concentrations than HCQ, with half maximal inhibitory concentration (IC50) values of 2–10 µM vs. 50–100 µM, respectively.


**Table 3.** Selected agents that inhibit autophagy.

Abbreviations: PPT1—palmitoyl-protein thioesterase 1; VPS34—vacuolar protein sorting 34; ULK1—unc51-like-kinase 1; ATG4B—autophagy related 4B; AML—acute myeloid leukemia; RCC—renal cell carcinoma; GBM—glioblastoma.

In vivo experiments also demonstrated the improved anticancer activity of ROC-325 over HCQ. Mice treated with orally-administered ROC-325 displayed significantly decreased 786-O RCC xenograft burden when compared to both control and HCQ-treated mice. Importantly, no significant toxicities were observed in mice treated with ROC-325. Tumors taken from each group were analyzed using immunohistochemistry (IHC). Tumors treated with ROC-325 showed elevated levels of LC3-II, p62, and cleaved caspase-3, thereby confirming in vitro findings. Further study of ROC-325 especially in combination with conventional and targeted therapy is warranted.

#### *4.4. STF-62247*

STF-62247 is an experimental agent that was first discovered over a decade ago. This particular compound was identified to have potent cytotoxic effects in VHL-deficient cancer cells, but very little efficacy in wild-type (WT) VHL cells [45]. Due to this selective anti-tumor activity, this compound is a potentially exciting therapeutic option for RCC. The exact mechanism by which STF-62247 acts is not fully understood. STF-62247 is believed to induce autophagy in cancer cells, as treatment produces large, cytoplasmic vacuoles in both WT-VHL and VHL-deficient cells. However, VHL-deficient cells contain much larger vacuoles and show significantly brighter acridine orange staining [45]. This indicates that VHL-deficient cells form large, highly acidic vesicles in response to STF-62247. Upon further investigation, it was shown that Golgi vesicle trafficking proteins played a pivotal role in sensitizing cells to the compound. However, the mechanistic links between VHL and autophagy were not fully elucidated.

Bouhamdani et al. recently confirmed these findings in RCC and also noted that while VHL-proficient cells also form large vacuoles, they are capable of resolving them within 48 hours of treatment [44]. Interestingly, in this study, STF-62247 was not shown to induce autophagy, but rather blocked the late stages of autophagy. No known upstream markers of increased autophagy were shown to be affected by the drug and inhibiting the vacuolar H<sup>+</sup> ATPase pump with bafilomycin A1 (BAF) showed very little, if any, additive stabilization of LC3-II or p62 when combined with STF-62247. These results cast doubt on the idea that STF-62247 is inducing autophagy. This also suggests that STF-62247 is potentially obstructing a similar stage of autophagy as BAF, indicating that it is a late-stage autophagy inhibitor, much like HCQ, CQ and ROC-325. However, much of this work is still controversial as STF-62247 has been shown to induce an autophagy-dependent cell death response in multiple malignancy types regardless of VHL status [61–63]. More data is needed in order for STF-62247 to be effectively transitioned into the clinical setting.

#### *4.5. Lys05, DQ661, and DC661*

Lys05 is a lysosome-disrupting, water-soluble salt of the compound Lys01. Lys01 consists of a pair of aminoquinolines, the major motif of CQ, connected by a methylamine-containing spacer [64,65]. Much like the previously discussed compounds, Lys01 produces an accumulation of LC3-II at a much lower dosage than CQ or HCQ. LN229 cells containing a green fluorescent protein tagged LC3 protein (GFP-LC3) treated with Lys01 display localization of LC3 to autophagic vesicles with 10x greater potency than HCQ, and electron microscopy confirms the presence of large autophagic vesicles in cells treated with Lys01. The anticancer profile of Lys01 is greater than that of HCQ when tested across colon cancer, glioma, and melanoma cell lines, with IC50 values ranging from 4–8 µM compared to 15–42 µM with HCQ. In vivo studies exhibited moderate, single agent, antitumor activity against orthotopically injected 1205Lu melanoma xenografts. Using HPLC tandem mass spectrometry, Lys05 was shown to accumulate in tumors in vivo at a much greater concentration than HCQ.

In addition to Lys05, two second-generation compounds have been developed—DQ661 and DC661 [66,67]. Interestingly, all these compounds were recently reported to block the lysosomal enzyme palmitoyl-protein thioesterase 1 (PPT1), which plays a key role in palmitoylation-mediated intracellular protein trafficking. These preclinical results imply that Lys05, DQ661, and DC661 have the potential to combat many different types of tumors. However, investigation of these compounds for RCC therapy has not been tested. It is worth noting that each of these lysosome-disrupting compounds has a similar mechanism of action, but contain unique chemical motifs. These novel structures will most likely lead to toxicity differences when clinical testing is initiated. This, in turn, could prove to be fundamental to approval and widespread therapeutic use.

#### *4.6. VPS34 Inhibitors*

The compounds discussed thus far act on the most distal component of the autophagy pathway, the lysosome. The lysosome acts as the common end point to this cellular process. The upstream, initiating machineries of autophagy show a great amount of complexity and have proven quite difficult to target. Nonetheless, multiple research groups are developing molecules to inhibit these proteins.

VPS34 is a PI3K lipid kinase family member, responsible for the addition of a phosphate group at the 3 position of the inositol ring of phosphatidylinositol (PI) on cellular membranes. This lipid phosphorylation is a vital step in the initiation and elongation of autophagosome membranes. While many pan-PI3K inhibitors currently exist, a few compounds have been developed to specifically target VPS34, including SAR405 and SB02024. SAR405 is a small molecule that targets the ATP binding pocket of VPS34 with high affinity [68,69]. In vitro work with this compound shows a reduction in autophagosome formation in GFP-LC3 tagged HeLa cells. In addition, concomitant treatment with everolimus in RCC cell lines eliminates the enhanced autophagic flux seen with mTOR inhibition. SAR405 also significantly enhanced the anticancer activity of everolimus in these same RCC cells. These preliminary findings have opened the door for combining VPS34 inhibitors with current standard-of-care therapeutics in RCC. SB02024, another recently discovered VPS34 inhibitor, shows significant anticancer activity when combined with standard RTK inhibitors, sunitinib and erlotinib, in breast cancer cell lines [70]. While this particular compound has not been tested in RCC, these findings highlight the potential of SB02024 to be paired with standard of care RCC agents.

#### *4.7. ULK1 Inhibitors*

UNC-51-like kinase 1 (ULK1) is a proximal serine/threonine kinase that is responsible for autophagy initiation. ULK1 acts as one of the key signaling regulators linking mTORC1, 5' adenosine monophosphate-activated protein kinase (AMPK), starvation, and autophagy. Due to its vital role in integrating cellular stress signals, successful inhibition of ULK1 is an attractive therapeutic strategy. SBI-0206965 was developed in 2015 as a selective ULK1 inhibitor [71]. This compound displayed potent inhibition of ULK1 activity with an in vitro IC50 of 108 nM. Treatment of A549 human lung cancer cells in vitro with a combination of SBI-0206965 and the mTOR inhibitor AZD8055 led to a significantly enhanced apoptotic response. This result further supports the rationale of inhibiting autophagy to augment the activity of other target-specific molecules. More recently, two closely related compounds, ULK-100 and ULK-101, have also been developed as more potent inhibitors of ULK1 [72]. Martin et al. demonstrated that these two small molecules show increased antitumor activity when combined with nutrient starvation in non-small-cell lung cancer cell lines. All three of these novel compounds have the potential for therapeutic impact in RCC. However, their specific efficacy in RCC models has not yet been evaluated. *Cancers* **2020**, *12*, x 9 of 16 potent inhibition of ULK1 activity with an in vitro IC50 of 108 nM. Treatment of A549 human lung cancer cells in vitro with a combination of SBI-0206965 and the mTOR inhibitor AZD8055 led to a significantly enhanced apoptotic response. This result further supports the rationale of inhibiting autophagy to augment the activity of other target-specific molecules. More recently, two closely related compounds, ULK-100 and ULK-101, have also been developed as more potent inhibitors of ULK1 [72]. Martin et al*.* demonstrated that these two small molecules show increased antitumor activity when combined with nutrient starvation in non-small-cell lung cancer cell lines. All three of these novel compounds have the potential for therapeutic impact in RCC. However, their specific efficacy in RCC models has not yet been evaluated.

#### *4.8. ATG4B Inhibitors 4.8. ATG4B Inhibitors*

ATG4B is a cysteine protease that activates LC3 for lipidation and recent studies suggest that it may be another promising target to inhibit autophagy upstream of the lysosome. Consistent with this idea, several ATG4B inhibitors have been developed including FMK-9a, NSC185058, and S130 [73–76]. NSC185058 and S130 have demonstrated significant in vivo activity against osteosarcoma and colon tumors, respectively [74,75]. Additionally, NSC185058 treatment decreased glioma xenograft growth in mice and augmented the antitumor efficacy of radiation therapy [76]. Collectively, these studies demonstrate that inhibiting ATG4B may be a suitable anti-autophagy target for the treatment of various cancers. However, these compounds remain in the earliest stages of preclinical development (Figure 2). ATG4B is a cysteine protease that activates LC3 for lipidation and recent studies suggest that it may be another promising target to inhibit autophagy upstream of the lysosome. Consistent with this idea, several ATG4B inhibitors have been developed including FMK-9a, NSC185058, and S130 [73– 76]. NSC185058 and S130 have demonstrated significant in vivo activity against osteosarcoma and colon tumors, respectively [74,75]. Additionally, NSC185058 treatment decreased glioma xenograft growth in mice and augmented the antitumor efficacy of radiation therapy [76]. Collectively, these studies demonstrate that inhibiting ATG4B may be a suitable anti-autophagy target for the treatment of various cancers. However, these compounds remain in the earliest stages of preclinical development (Figure 2).

**Figure 2.** Selected agents that target autophagy at different points in the pathway. Hydroxychloroquine, chloroquine, and ROC-325 are amongst the compounds that target the lysosome. Compounds such as SBI-0206965 and SAR405 are being developed to inhibit autophagy factors near the proximal end of the cascade. **Figure 2.** Selected agents that target autophagy at different points in the pathway. Hydroxychloroquine, chloroquine, and ROC-325 are amongst the compounds that target the lysosome. Compounds such as SBI-0206965 and SAR405 are being developed to inhibit autophagy factors near the proximal end of the cascade.

#### **5. Immune Checkpoint Inhibitor Therapy and Autophagy 5. Immune Checkpoint Inhibitor Therapy and Autophagy**

The clinical efficacy of novel immune checkpoint inhibitors has been variable. Through further research and testing, cancer types are now generally thought of as "immune hot" and "immune cold". Tumors that have significant infiltration of immune cells are considered "hot" and are generally The clinical efficacy of novel immune checkpoint inhibitors has been variable. Through further research and testing, cancer types are now generally thought of as "immune hot" and "immune cold". Tumors that have significant infiltration of immune cells are considered "hot" and are generally

responsive to immune checkpoint inhibitor therapy. The greatest immune checkpoint inhibitor

responsive to immune checkpoint inhibitor therapy. The greatest immune checkpoint inhibitor therapy success to date has been the treatment of metastatic melanoma with a monoclonal antibody-targeting CTLA-4, Ipilimumab [77]. The FDA has now approved six different immune checkpoint inhibitors in a variety of cancer types. This number only stands to grow with over 200 active clinical trials involving immune checkpoint inhibitor therapies.

RCC is characterized as being responsive to immune checkpoint inhibitor therapy. As previously mentioned, cytokine therapy, a precursor to modern day immunotherapy, was long used as the primary treatment for advanced RCC. Nivolumab, a monoclonal antibody-targeting PD-1, was approved for use in RCC in 2015 after demonstrating improved progression-free survival (4.6 months vs. 4.4 months) over everolimus in a phase III clinical trial [26]. While more traditional small molecule therapy options, such as kinase inhibitors will remain a mainstay in RCC treatment, immunomodulatory therapies are becoming increasingly common as frontline and adjuvant therapies [78,79].

The connection between autophagy and immune cell activation is not well established, particularly in RCC. However, preliminary findings in different cancer models have provided conflicting findings. Early work has suggested that autophagy inhibition can interfere with hematopoiesis and systemic immunity indicating that combination autophagy and immune checkpoint inhibitor therapy may not be beneficial [80–82]. However, recent studies demonstrate that autophagy inhibition does not block T-cell activity [83–86]. Treatment of subcutaneous B16 melanoma xenografts with CQ in immunocompetent mice has provided evidence that autophagy inhibition promotes macrophage phenotype switching from an alternatively activated (M2) to a classically activated (M1) state [87]. This switch in macrophage phenotype gave rise to an increase in CD3+/CD8+ tumor-infiltrating lymphocytes as well as increased interferon gamma (IFNγ) expression, a key marker of cytotoxic T-lymphocyte (CTL) activation. Importantly, the antitumor effects of CQ in vivo were completely reversed in T-cell deficient mice, confirming that the activity of CQ was indeed a product of immunomodulation in this particular model. Recent work has also shown that HCQ, when delivered to E.G7-OVA murine lymphoma xenografts via nanoparticle vaccination, is capable of enhancing tumor cell major histocompatibility complex (MHC)-I antigen presentation, a key event in CTL activation [88]. A significant increase in the production of IFNγ was also observed in this model. Both of these recent studies highlight a potential relationship between autophagy inhibition and responsiveness to immune checkpoint inhibitor therapy, but additional studies are needed to better understand this interaction. Furthermore, the potential benefit of dual immune checkpoint and autophagy inhibitor therapy to RCC remains to be determined.

#### **6. Conclusions**

Two decades ago, patients diagnosed with advanced, metastatic, or surgically unresectable RCC had very few approved therapeutic options. However, significant research efforts have resulted in the development of numerous targeted agents and immune-related therapies for the treatment of RCC. Despite this success, patients that are refractory to these treatments or develop drug resistance continues to be a major clinical issue. Autophagy has now been established as a key mechanism by which cancer cells are capable of surviving periods of therapy-induced stress leading to drug resistance. This provides the rationale for the development and testing of autophagy-modulating compounds to use in conjunction with the ever-expanding list of approved RCC treatments. While HCQ has demonstrated some promising activity in combination with standard agents in clinical trials, its effectiveness appears to be limited by a variety of factors. Considering this, there is a need for new and more potent autophagy inhibitors that can be tested in clinical trials. Additional information is also required to determine the differences between upstream and lysosomal autophagy targeting in regards to therapeutic efficacy. The development and classification of compounds targeting autophagy is an ongoing process, but one can hope that a breakthrough is on the horizon.

**Author Contributions:** Conceptualization, writing—original draft preparation, writing—review and editing; T.M.J., J.S.C., S.T.N. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was supported by grants from the National Cancer Institute (R01CA190789 to S.T.N. and R01CA172443 to J.S.C.) and the University of Arizona Cancer Center Support Grant P30CA023074.

**Conflicts of Interest:** The authors declare no conflict of interest.

## **Abbreviations**

The following abbreviations are used in this manuscript:


#### **References**


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