*3.2. Role of AC in Pathological Conditions*

Lysosome architecture involves the basic unit, SL. Both Farber disease (FD) and spinal muscular atrophy with progressive myoclonic epilepsy (SMA-PME) are rare lysosomal storage disorders. They are caused by a missense mutation in the *ASAH1* gene resulting in the absence or decrease in the AC enzyme. There have been fewer than 200 cases of FD and SMA-PME. With no cure for AC deficiency, gene therapy and enzyme replacement therapy is under development [21]. *Asah1* knockout in mice results in embryonic lethality [22]. Examples of AC inhibitors are shown in Figure 3. All the inhibitors exhibit a polar head group with a lipophilic tail. The calculated logP is congruent with the observation that these molecules are more lipophilic. AC is implicated in several cancers, as discussed below.

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**Figure 3.** Examples of AC inhibitors.

## 3.2.1. Prostate Cancer (PC)

The role of AC in tumor proliferation and resistance to treatment has been studied in depth in regard to PC. PC cells develop resistance by upregulating AC. Clonogenicity and cytotoxicity assays confirm that radio sensitization can be established by the genetic downregulation of AC with small interfering RNA (siRNA). Also, the radiation-induced AC upregulation creates cross-resistance to chemotherapy. The use of the small molecule AC inhibitor, LCL385, sensitizes primary prostatic carcinoma cell line (PPC-1) cells to radiation [23]. In addition, the same cell line preferentially upregulates AC, resulting in increased radiation resistance and proliferation. The use of an AC inhibitor, LCL521, acts as a radiosensitizer preventing relapse [24]. This was validated with immunohistochemical studies in these tissues, where higher levels of AC were observed after radiation treatment failure than in irradiation-naïve cancer, intraepithelial neoplasia, or benign tissue.

In addition, AC inhibition prevented relapse in an animal xenograft model by producing radio sensitization. Higher IHC expression of AC in primary PCs is associated with a more advanced disease stage. In a model derived from PC-3 cells, the highly tumorigenic, metastatic, and chemo-resistant PC-3/Mc clone expressed higher levels of AC than the non-metastatic PC-3/S clone. Stable knockdown of *ASAH1* in PC-3/Mc cells resulted in an accumulation of Cer, a reduction in clonogenic potential, and the inhibition of tumorigenesis and lung metastases [25]. Treatment of DU-145 cells with the AC inhibitor LCL204, a lysosomotropic analog of B13, induces apoptosis in a cathepsin-dependent manner. Upon treatment with this inhibitor, destabilization of membranous lysosomes and the release of lysosomal proteases into the cytosol lead to mitochondria depolarization and caspase activation, resulting in apoptosis [26]. Both PC-3 and DU 145 are hormone-refractory human PC cell lines. In both of these cell lines, combination treatment with fenretinide (4-HPR), a ceramide-generating anticancer agent, and DM102, a novel synthetic AC inhibitor, resulted in a considerable decrease a cell viability and the combination therapy was more effective than either treatment alone. In the PC-3 cell line, the treatment induces apoptosis through ROS generation. However, another AC inhibitor, *N*-oleoylethanolamine (NOE), in combination with 4-HPR, does not result in synergistic activity [27]. In another study using PPC1 and DU145 PC cells, AC overexpression was shown to result in increased lysosomal density and autophagy and increased expression of the motor protein KIF5B, contributing to lysosomal stability. AC overexpression, in addition to increasing radiation and chemotherapy resistance, increases stress resistance. Although increased lysosomal density in these cells makes them more sensitive to therapeutic agents targeting lysosomes, the overexpression of AC provides a new therapeutic target [28].

#### 3.2.2. Head- and Neck Cancer (HNC)

AC was overexpressed in four of six primary tumor tissues and six of nine cell lines in HNC. AC also contributed to decreased cisplatin sensitivity. Pharmacological (with N-oleoyl-ethanolamine) or genetic (with short hairpin RNA) inhibition of AC enhanced cisplatin-induced HNC cell death by increasing Cer and activating other proapoptotic proteins [29]. In mouse squamous cell carcinoma SCCVII, the inhibition of AC with the small molecule inhibitor LCL521 significantly decreased the survivability after photodynamic therapy by effectively restricting regulatory T lymphocytes and myeloid-derived suppressor cell activity [30]. The combination of photodynamic therapy and dasatinib also decreases SCCVII cell survivability by decreasing AC, leading to increased Cer activating caspase-3-induced apoptosis [31]. In SCC-1, overexpression of AC increased the resistance to Fas-induced cell death, which was reversible using specific AC siRNA. The AC inhibitor LCL 204 sensitizes HNSCC cell lines to Fas-induced apoptosis both in vitro and in a xenograft model in vivo, providing an option for combination therapy [32].

#### 3.2.3. Melanoma

The largest organ of the human body, the skin, has a very complex structure with four main layers. SLs are found throughout each layer, and maintain the functions of this organ. AC expression is significantly higher in normal human melanocytes and proliferative melanoma cell lines, compared with other skin cells (keratinocytes and fibroblasts) and nonmelanoma cancer cells. Melanoma cells exhibit lower amounts of Cer by downregulating the de novo synthesis pathway. The AC inhibitor ARN14988 in combination with 5-Fluoro Uracil increases cytotoxicity in the proliferative melanoma cell lines by increasing Cer and reducing S1P levels [33]. In human A375 melanoma cells, dacarbazine (DTIC) decreases ACdase activity through reactive-oxygen-species-dependent activation of cathepsin Bmediated degradation of the enzyme. Downregulating AC expression increased Cer level and sensitivity to DTIC, providing a therapeutic tool for the treatment of metastatic melanoma [34]. The deletion of *ASAH1* in human A375 melanoma cells using CRISPR-Cas9-mediated gene editing showed a significantly greater accumulation of long-chain saturated Cers that are substrates for AC in *ASAH1*-null cells. The cells lose the ability to undergo self-renewal [35]. Our hypothesis on this observation correlates with Cer flux and its function.

#### 3.2.4. Myeloid Leukemia

Primary acute myeloid leukemia (AML) cells have a higher expression of AC, which is essential for AML blast survival. In AML cell lines, increased levels of AC induced a higher expression of antiapoptotic Mcl-1 protein, increased S1P, and decreased Cer. Treatment with the AC inhibitor, LCL204, induces Cer accumulation and decreases Mcl-1. The overall survival of C57BL/6 mice engrafted with leukemic C1498 cells increased significantly with LCL204 treatment, while the treatment significantly decreased the disease burden in NSG mice engrafted with primary human AML cells [36]. IFN regulatory factor 8 (IRF8) is a key transcription factor for myeloid cell differentiation. Without IRF8, hematopoietic cells in human myeloid leukemia patients rapidly proliferate and remain undifferentiated. Thus, acting as a tumor suppressor by promoting cell differentiation, IRF8 expression is frequently lost in myeloid leukemias. One of the repressive transcriptional targets of IRF8 is AC; consequently, as IRF8 is lost, AC expression increases, solidifying its role as a tumor suppressor. In chronic myelogenous leukemia (CML), overexpression of IRF8 repressed AC expression, resulting in C16 Cer accumulation and increased sensitivity of CML cells to FasL-induced apoptosis. AC expression is significantly higher in cells derived from IRF8-deficient mice. In these cells, inhibition of AC activity or application of exogenous Cer sensitizes the cells to FasL-induced apoptosis [37].

#### 3.2.5. Non-Small Cell Lung Cancer (NSCLC)

In NSCLC cells with acquired resistance to ChoKα inhibitors, these cells also display increased levels of *ASAH1*. Inhibition of AC synergistically sensitizes lung cancer cells to the antiproliferative effect of ChoKα inhibitors, which opens up a new therapeutic option for combinatorial treatments of ChoKα inhibitors and AC inhibitors [38].

#### 3.2.6. Breast Cancer

Higher expression of AC has been observed in ER-positive and luminal-A-like breast cancer. High expression of AC in invasive breast cancer is associated with improved prognosis and reduced incidence of recurrence in preinvasive ductal carcinoma in situ (DCIS) [39]. The effect of AC inhibitors (DM102 and NOE) in combination with C6-Cer (C6-cer), a cell-permeable analog of Cer, has been studied in three different breast cancer cell lines MDA-MB-231, MCF-7, and BT-474 cells. Although as single agents, both C6-cer and DM102 are moderately cytotoxic, their combination induced synergistic decreases in viability. In MDA-MB-231 cells, apoptosis is induced by caspase 3/7 activation and poly (ADP-ribose) polymerase (PARP) cleavage. In the same cell line, C6-cer/DM102 increases ROS levels and results in mitochondrial membrane depolarization. Furthermore, the C6 cer/DM102 combination is antagonistic in BT-474 cells, suggesting different molecular mechanisms being cell-type-specific. AC expression is correlated to the human epidermal growth factor receptor 2 (HER2) status [40]. Another study shows that an AC inhibitor, ceranib-2, induces apoptosis in MDA-MB-231 and MCF-7 by activating stress-activated protein kinase/c-Jun N-terminal kinase and mitogen-activated protein kinase apoptotic pathways by inhibiting the antiapoptotic pathway [41]. Ceranib-2 exhibits similar effects in PC cell lines too [42].

#### 3.2.7. Ovarian Cancer (OC)

In an immunohistochemical analysis study of 112 OC patients, low AC expression has been correlated with tumor progression. This analysis contradicts the concept of AC being a cancer progression promoter, suggesting AC is involved in alternative pathways in different cancers, which requires further investigation [43].

#### 3.2.8. Hepatobiliary Cancers

AC is downregulated using the chemotherapeutic agent daunorubicin in human (HepG2) and mouse (Hepa1c17) hepatoma cell lines, as well as in primary cells from murine liver tumors, but not in cultured mice. Genetic (small interfering RNA) or pharmacological inhibition of AC with *N*-oleoylethanolamine (NOE) sensitized the cell lines to daunorubicininduced cell death, preceded by structural mitochondrial changes, stimulation of reactive oxygen species generations and cytochrome *c* release followed by caspase-3 activation. In vivo siRNA treatment targeting AC reduced tumor growth in liver tumor xenografts of HepG2 cells and enhanced daunorubicin therapy, providing a potential therapeutic target for liver cancer [44].

The key chemotherapeutic agent in pancreatic cancer, gemcitabine, exhibits different efficacy due to polymorphism in the expression of enzymes that regulate its metabolism [41,45]. Deoxycytidine kinase (dCK), which phosphorylates gemcitabine, activates the drug, while cytidine deaminase (CDA) inactivates gemcitabine by deamination [6]. In MIA PaCa-2 and PANC-1 pancreatic cancer cell lines, the novel Cer analog, AL6, inhibits cell growth, induces apoptosis, and synergistically enhances the cytotoxic activity of gemcitabine. AL6 also increases the gene expression of the gemcitabine-activating enzyme deoxycytidine kinase (dCK), improving the efficacy of gemcitabine. This study suggests the use of AL6 and gemcitabine combination therapy for pancreatic cancer [45].

#### 3.2.9. Colon Cancer

Colorectal adenocarcinoma tissues have higher *ASAH1* expressions when compared with the adjacent normal colonic mucosa. In HCT116 colon cancer cells, there is an inverse correlation between the AC expression and the p53 functional activity. Inhibition of AC using carmofur in HCT116 CELLS significantly increased the antiproliferative, proapoptotic, antimigratory, and anticlonogenic effects of oxaplatin [46]. In the human colon cancer cell line, the AC inhibitor ceranib-2 increases apoptosis by increasing *ASAH1* mRNA expression and reducing *TNFR1* expression [47].
