*2.4. Rad51 Expression Is Substantially Reduced in AKT-Inhibited Cancer Cells*

Given that attenuation of DNA damage repair in AKT-inhibited cancer cells might be due to the decreased efficacy of homology-mediated repair of DNA DSBs, we analyzed the expression of Rad51 recombinase, known as a key protein involved in DSB repair. For this purpose, we performed a quantitative analysis of Rad51 expression in cancer cells treated with Dox and MK-2206 by a similar way as shown on Figure 4. As expected, no difference in Rad51 expression was found between control (Figure 6A) and MK-2206-treated cells (Figure 6B). When Dox-treated cells were washed out (to exclude a permanent DNA damage), we found a substantial increase of Rad51 intensity in cancer cells (Figure 6C). Strikingly, intensity of Rad51-mediated fluorescence substantially decreased when MK-2206 was introduced in cell culture after Dox washout (Figure 6D). This was observed for GIST T-1R cells (Figure 6) and STS (data not shown), thereby illustrating the possible mechanism of MK-2206-induced sensitization of cancer cells to Topo II inhibitors. These findings were in consistency with WB data illustrating a substantial decrease of Rad51 expression in AKT-inhibited cancer cells (Figure 5). *Int. J. Mol. Sci.* **2020**, *21*, x FOR PEER REVIEW 7 of 21

**Figure 3.** Inhibition of AKT-signaling attenuates DNA double-stand break (DSB) repair*.* GIST T-1R cells treated with DMSO (negative control) and Dox 0.5 μg/mL for 2 h. After the drugs were washed out, cancer cells were further cultured in absence (Dox washout) or presence of MK-2206 (5 μM), an AKT inhibitor (Dox washout + MK-2206) for 8 h. **(A)** representative images of comets from the experimental settings shown above (Scale bars = 100 μm). **(B)** graphic depiction of the calculated tail moment (TM) and olive tail moment (OTM) from alkaline comet assay shown in Figure 3A. Columns, mean of at least three independent experiments with a minimum of 50 cells counted per each **Figure 3.** Inhibition of AKT-signaling attenuates DNA double-stand break (DSB) repair. GIST T-1R cells treated with DMSO (negative control) and Dox 0.5 µg/mL for 2 h. After the drugs were washed out, cancer cells were further cultured in absence (Dox washout) or presence of MK-2206 (5 µM), an AKT inhibitor (Dox washout + MK-2206) for 8 h. (**A**) representative images of comets from the experimental settings shown above (Scale bars = 100 µm). (**B**) graphic depiction of the calculated tail moment (TM) and olive tail moment (OTM) from alkaline comet assay shown in Figure 3A. Columns, mean of at least three independent experiments with a minimum of 50 cells counted per each experiment; bars, SE. \* *p* < 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.001.

experiment; bars, SE. \* *p* < 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.001.

**Figure 4.** Inhibition of AKT-signaling delays the kinetics of γ-H2AX decline in Dox-treated GIST T-1R cells (representative experiment). Left panel-cancer cells were treated with DMSO (negative control) (**A**), MK-2206, a selective AKT inhibitor (5 μM) (**B)**, and Dox (0.5 μg/mL) for 2 h. The cells were further washed from the compounds indicated above and cultured for 8 h in absence (Dox **Figure 4.** Inhibition of AKT-signaling delays the kinetics of γ-H2AX decline in Dox-treated GIST T-1R cells (representative experiment). Left panel-cancer cells were treated with DMSO (negative control) (**A**), MK-2206, a selective AKT inhibitor (5 µM) (**B**), and Dox (0.5 µg/mL) for 2 h. The cells were further washed from the compounds indicated above and cultured for 8 h in absence (Dox washout) (**C**) or presence of MK-2206 (Dox washout + MK-2206) (**D**). Right panel-histograms illustrating the intensity of γ-H2AX-specific fluorescence at the single-nucleus level. GIST T-1R cells were grown on slides for 24 h and treated with Dox and MK-2206 as indicated above. Cells were fixed with paraformaldehyde and stained with DAPI (blue) and γ-H2AX-specific antibody (red). The intensity of γ-H2AX-specific fluorescence was measured for each nucleus (DAPI) and calculated automatically. All images were acquired by GE Cytell imager as shown in Section 4.10.

with paraformaldehyde and stained with DAPI (blue) and γ-H2AX-specific antibody (red). The intensity of γ-H2AX-specific fluorescence was measured for each nucleus (DAPI) and calculated

automatically. All images were acquired by GE Cytell imager as shown in Section 4.10.

washout) (**C**) or presence of MK-2206 (Dox washout + MK-2206) (**D**). Right panel-histograms

**Figure 5.** An impact of AKT inhibition on γ-H2AX and Rad51 recombinase in cancer cells. Immunoblot analysis was performed on RD rhabdomyosarcoma (**A**), U2-OS osteosarcoma (**B**), HT-1080 fibrosarcoma (**C**), GIST T-1R (**D**), GIST 430 (**E**), SK-LMS-1 leiomyosarcoma and (**F**) treated with DMSO (control), Dox (0.25 g/mL), MK-2206 (5 M) alone and in combination (e.g., Dox + MK-2206) for 48–72 **Figure 5.** An impact of AKT inhibition on γ-H2AX and Rad51 recombinase in cancer cells. Immunoblot analysis was performed on RD rhabdomyosarcoma (**A**), U2-OS osteosarcoma (**B**), HT-1080 fibrosarcoma (**C**), GIST T-1R (**D**), GIST 430 (**E**), SK-LMS-1 leiomyosarcoma and (**F**) treated with DMSO (control), Dox (0.25 g/mL), MK-2206 (5 M) alone and in combination (e.g., Dox + MK-2206) for 48–72 h. Actin was used as a loading control.

All together this data illustrates that inhibition of AKT-signaling in STS and GIST sensitizes them to Dox due to the inhibition of DNA damage repair. The impact of AKT-signaling on Rad51 expression was also assessed by immunofluorescence staining. Non-treated GIST T-1R cells exhibited a low level of γ-H2AX/Rad51 foci, whereas vast majority of tumor cells became γ-H2AX/Rad51-foci positive after Dox treatment (Figure 7A).

h. Actin was used as a loading control.

*2.4. Rad51 Expression is Substantially Reduced in AKT-Inhibited Cancer Cells*  Given that attenuation of DNA damage repair in AKT-inhibited cancer cells might be due to the decreased efficacy of homology-mediated repair of DNA DSBs, we analyzed the expression of Rad51 recombinase, known as a key protein involved in DSB repair. For this purpose, we performed a quantitative analysis of Rad51 expression in cancer cells treated with Dox and MK-2206 by a similar way as shown on Figure 4. As expected, no difference in Rad51 expression was found between control (Figure 6A) and MK-2206-treated cells (Figure 6B). When Dox-treated cells were washed out (to Strikingly, a substantial decrease of Rad51 foci-positive cells was found when cancer cells were treated with Dox in combination with MK-2206 (Figure 7A,B). Moreover, inhibition of AKT-signaling attenuated co-localization between Rad51 and γ-H2AX foci after Dox treatment (Figure 7C, bottom panel), thus suggesting a failure in the recruitment of Rad51 to DSBs in AKT-inhibited tumor cells after Dox treatment. Similar results were observed for STS cells lines, including RD rhabdomyosarcoma cells, as shown in Figure S7. Indeed, the majority of Dox-treated cells exhibited high co-localization between Rad51 and γ-H2AX foci, whereas inhibition of AKT-signaling dramatically reduced the number of cells with co-localized proteins indicated above.

exclude a permanent DNA damage), we found a substantial increase of Rad51 intensity in cancer cells (Figure 6C). Strikingly, intensity of Rad51-mediated fluorescence substantially decreased when MK-2206 was introduced in cell culture after Dox washout (Figure 6D). This was observed for GIST T-1R cells (Figure 6) and STS (data not shown), thereby illustrating the possible mechanism of MK-2206-induced sensitization of cancer cells to Topo II inhibitors. These findings were in consistency with WB data illustrating a substantial decrease of Rad51 expression in AKT-inhibited cancer cells (Figure 5). Of note, increased number of Rad51 foci in Dox-treated cells was in contrast to the substantial decrease of Rad51 expression observed in most of cancer cells utilized in present study, as shown in Figure 4. These might be due to the differences in the methodologies utilized for these assays. Indeed, RIPA buffer which is commonly utilized for WB analysis might be not effective in extracting of the chromatin-bound proteins, whereas the increased number of Rad51 nuclear foci observed during immunofluorescence staining mainly represents Rad51 associated with chromatin. To confirm this possibility, we examined the distribution of Rad51 in different subcellular fractions in GIST/STS cells treated with Dox alone or in presence of AKT inhibitor, as well. The results showed that while in control cells Rad51 was evenly distributed between Triton X-100 soluble (i.e., cytoplasmic and soluble nuclear) and DNAase-soluble fractions (i.e., chromatin-associated), inhibition of AKT-signaling with MK-2206 induced its re-distribution from the chromatin, whereas Dox brought a pronounced immobilization of the protein in the DNAase-soluble fraction (Figure S8). Strikingly, inhibition of AKT signaling in Dox-treated cells attenuated recruitment of Rad51 in chromatin-associated fraction, thereby revealing

the attenuation of Rad51-mediated repair mechanisms. In contrast to Rad51, expression of pRPA (Ser 4/8) in DNA-soluble fraction did not differ between GIST T-1R cells treated with Dox alone or in presence with AKT inhibitor, thereby suggesting that processing of DSBs in AKT-inhibited cells remained unchanged. *Int. J. Mol. Sci.* **2020**, *21*, x FOR PEER REVIEW 10 of 21

**Figure 6.** Inhibition of AKT-signaling attenuates Rad51 expression in Dox-treated cells (representative experiment). (**A**–**D**) GIST T-1R cells were treated similarly as shown for Figure 4. Left panel—cells were fixed with paraformaldehyde and stained with DAPI (blue) to outline the nucleus and Rad51 specific antibody (red). The intensity of Rad51-specific fluorescence was assayed for each nucleus (DAPI) and quantified automatically. Right panel—histograms illustrating the intensity of Rad51 specific fluorescence at the single-nucleus level. All images were acquired by GE Cytell imager as shown in "Materials and methods". **Figure 6.** Inhibition of AKT-signaling attenuates Rad51 expression in Dox-treated cells (representative experiment). (**A**–**D**) GIST T-1R cells were treated similarly as shown for Figure 4. Left panel—cells were fixed with paraformaldehyde and stained with DAPI (blue) to outline the nucleus and Rad51-specific antibody (red). The intensity of Rad51-specific fluorescence was assayed for each nucleus (DAPI) and quantified automatically. Right panel—histograms illustrating the intensity of Rad51-specific fluorescence at the single-nucleus level. All images were acquired by GE Cytell imager as shown in "Materials and methods".

The impact of AKT-signaling on Rad51 expression was also assessed by immunofluorescence staining. Non-treated GIST T-1R cells exhibited a low level of γ-H2AX/Rad51 foci, whereas vast majority of tumor cells became γ-H2AX/Rad51-foci positive after Dox treatment (Figure 7A).

Strikingly, a substantial decrease of Rad51 foci-positive cells was found when cancer cells were treated with Dox in combination with MK-2206 (Figure 7A,B). Moreover, inhibition of AKT-signaling attenuated co-localization between Rad51 and γ-H2AX foci after Dox treatment (Figure 7C, bottom panel), thus suggesting a failure in the recruitment of Rad51 to DSBs in AKT-inhibited tumor cells after Dox treatment. Similar results were observed for STS cells lines, including RD rhabdomyosarcoma cells, as shown in Figure S7. Indeed, the majority of Dox-treated cells exhibited

**Figure 7.** Blockage of Akt signaling pathway disrupts involvement of the Rad51 protein in the repair of DNA DSBs in GIST T-1R cells. (**A**) immunofluorescence staining of GIST T-1R cells for γ-H2AX and Rad51. The cells were cultured in the presence of DMSO (control), MK-2206 (5 μM for 48 h), Dox (0.25 μg/mL for 4 h) or pretreated with MK-2206 (5 μM) for 48 h prior Dox treatment. DAPI staining (blue) was used to outline the nucleus. Scale bars = 20 μm. (**B**) graph depicting the number of GIST T-1R cells positive for Rad51 foci after Dox treatment alone or in presence of MK-2206 from three independent experiments. Cells treated with DMSO (control) and MK-2206 were used as the negative controls. \* *p* < 0.05. (**C**) graph showing co-localization of Rad51 foci with γ-H2AX foci in GIST T1-R **Figure 7.** Blockage of Akt signaling pathway disrupts involvement of the Rad51 protein in the repair of DNA DSBs in GIST T-1R cells. (**A**) immunofluorescence staining of GIST T-1R cells for γ-H2AX and Rad51. The cells were cultured in the presence of DMSO (control), MK-2206 (5 µM for 48 h), Dox (0.25 µg/mL for 4 h) or pretreated with MK-2206 (5 µM) for 48 h prior Dox treatment. DAPI staining (blue) was used to outline the nucleus. Scale bars = 20 µm. (**B**) graph depicting the number of GIST T-1R cells positive for Rad51 foci after Dox treatment alone or in presence of MK-2206 from three independent experiments. Cells treated with DMSO (control) and MK-2206 were used as the negative controls. \* *p* < 0.05. (**C**) graph showing co-localization of Rad51 foci with γ-H2AX foci in GIST T1-R cells after Dox treatment alone or in presence of MK-2206 from three independent experiments. \* *p* < 0.05. (**D**) distribution of γ-H2AX and Rad51 foci in the nucleus in a single-cell level. GIST T-1R cells were pretreated with DMSO (control) or MK-2206 (5 µM) for 48 h prior Dox treatment (0.25 µg/mL for four hours). DAPI staining (blue) was used to outline the nucleus. Scale bars = 10 µm.

#### *2.5. AKT Impacts Rad51 Stability in GIST and STS Cell Lines*

Given that AKT inhibition has a negative impact of Rad51 levels in the number of Rad51 foci in Dox-treated GIST and STS, we further examined the molecular mechanisms responsible of this phenomenon.

First, RT-PCR data illustrated very small, and statistically insignificant differences between Rad51 mRNA transcripts in control and MK-2206 treated GIST T-1R cells. Similarly, no differences in Rad51 *mRNA* levels were observed between non-treated and MK-2206-treated SK-LMS-1 leiomyosarcoma and RD rhabdomyosarcoma cell lines, thereby revealing that inhibition of AKT-signaling has no impact on *Rad51* transcription in GIST and STS (Figure S9).

Next, we examined whether low levels of Rad51 in AKT-inhibited cells were due to increased turnover of the protein via ubiquitin-mediated proteasomal pathway. To test this possibility, we initially inhibited synthesis of the new protein by cycloheximide (CHX) and compared Rad51 levels in control and MK-2206-treated cells. Indeed, we observed the substantial decrease of half-life of Rad51 in GIST cells treated with CHX in presence of MK-2206, thereby suggesting about the rapid turnover of protein in AKT-inhibited cancer cells (Figure 8, upper and middle panels). Next, we found that MG-132, a 26S proteasome inhibitor effectively restored Rad51 levels in AKT-inhibited cells, thereby revealing an increase proteasome-mediated degradation of Rad51 in MK-2006-treated cells (Figure 8, bottom panel). *Int. J. Mol. Sci.* **2020**, *21*, x FOR PEER REVIEW 13 of 21

**Figure 8.** AKT is required for regulation of Rad51 stability*.* (**A**) the half-life of Rad51 was determined by cycloheximide (CHX) chase analysis. GIST T-1R cells were treated with MK-2206 (5 μM) or DMSO (control), followed by treatment with 10 μg/mL cycloheximide for 0, 3, 6, 9, 12, 15, 18 and 24 h. Immunoblotting for Rad51 and actin was performed on samples from all time points. To examine an impact of AKT-inhibition on proteasomal degradation of Rad51, cells were treated with MK-2206 and CHX in presence of MG-132 (2 μM) and were subjected for immunoblotting for Rad51 and actin as a loading control. (**B**) densitometric analysis of Rad51 in CHX chase analysis indicated above; bars, SD. \* *p* < 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.001; \*\*\*\* *p* < 0.0001. (**C**) GIST T-1R cell lysates were immunoprecipitated with total (left) or phosphorylated at Ser473 (right) AKT Abs and immunoblotted with Rad51 Abs to demonstrate endogenous complex formation. A whole cell lysate (WCL) was included. pAKT expression is shown by arrows. Actin was used as a loading control. For immunoprecipitation **Figure 8.** AKT is required for regulation of Rad51 stability. (**A**) the half-life of Rad51 was determined by cycloheximide (CHX) chase analysis. GIST T-1R cells were treated with MK-2206 (5 µM) or DMSO (control), followed by treatment with 10 µg/mL cycloheximide for 0, 3, 6, 9, 12, 15, 18 and 24 h. Immunoblotting for Rad51 and actin was performed on samples from all time points. To examine an impact of AKT-inhibition on proteasomal degradation of Rad51, cells were treated with MK-2206 and CHX in presence of MG-132 (2 µM) and were subjected for immunoblotting for Rad51 and actin as a loading control. (**B**) densitometric analysis of Rad51 in CHX chase analysis indicated above; bars, SD. \* *p* < 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.001; \*\*\*\* *p* < 0.0001. (**C**) GIST T-1R cell lysates were immunoprecipitated with total (left) or phosphorylated at Ser473 (right) AKT Abs and immunoblotted with Rad51 Abs to demonstrate endogenous complex formation. A whole cell lysate (WCL) was included. pAKT expression is shown by arrows. Actin was used as a loading control. For immunoprecipitation experiments cells were treated with Dox (1 µg/mL) and MK-2206 (10 µM).

Western blotting with Rad51 antibody to detect the complexes. As shown in Figure 8B, specific coimmunoprecipitation was detected between Rad51 and pAkt (but not total Akt) after Dox treatment, thus confirming the interaction with endogenous proteins and reveling physical interaction between activated Akt and Rad51 after DNA damage. Strikingly, inhibition of AKT signaling in Dox-treated cells abolished this interaction. As expected, negative results were also observed in control (non-

Given the functional link between Akt and Rad51, we hypothesized that they might be in a

experiments cells were treated with Dox (1 μg/mL) and MK-2206 (10 μM).

treated cells) and cells exposed to MK-2206.

Given the functional link between Akt and Rad51, we hypothesized that they might be in a complex together. For this purpose, we examined the interaction between endogenous Akt/pAkt and Rad51. Lysates from GIST T-1R cells were immunoprecipitated for Akt or pAkt Ser473, followed by Western blotting with Rad51 antibody to detect the complexes. As shown in Figure 8B, specific co-immunoprecipitation was detected between Rad51 and pAkt (but not total Akt) after Dox treatment, thus confirming the interaction with endogenous proteins and reveling physical interaction between activated Akt and Rad51 after DNA damage. Strikingly, inhibition of AKT signaling in Dox-treated cells abolished this interaction. As expected, negative results were also observed in control (non-treated cells) and cells exposed to MK-2206.

All together, these results suggest that AKT inhibition in tumor cells leads to Rad51 down-regulation via ubiquitin-mediated proteasome pathway.

Collectively, our data illustrates that inhibition of AKT pathway in GIST and STS attenuates homology-mediated DNA repair and sensitizes tumor cells to the DNA-damaging agents, such as doxorubicin.

#### **3. Discussion**

It is well-known that an efficiency of most commonly used anticancer agents, including the classic genotoxic chemotherapeutic drugs and ionizing radiation, is due to their DNA-damaging properties which in turn triggers apoptotic cell death of cancer cells harboring unrepairable DNA lesions. Apoptosis induced by DNA lesions mainly results from double-strand breaks and stalled replication forks, which activates DNA damage checkpoints networks consisting of DNA damage sensors, signal transducers and effector pathways. The central sensors are composed of the PI3K-related kinases and include ataxia telangiectasia mutated (ATM), ataxia telangiectasia and Rad3-related (ATR) and DNA-dependent protein kinase (DNA-PK). Upon activation by DSBs and DNA replication blocks, PI3Ks indicated above activate specific substrates that mediate replication fork stability, cell cycle arrest, DNA repair and apoptosis.

The accumulating evidence implicated PI3Ks and AKT are tightly coregulated in both checkpoint response and DNA DSB repair. This reflected, predominantly, the regulatory role of AKT in NHEJ-mediated DNA repair. Indeed, AKT and DNA-PK were shown to activate each other to induce and maintain an effective NHEJ-mediated DNA repair. For example, Stronach et al. demonstrated that AKT relocates to the nucleus of cisplatin-resistant cancer cells where it is phosphorylated specifically on S473 by DNA-PK, and this activation inhibits cisplatin-mediated apoptosis. Importantly, direct interaction between DNA-PK and AKT in cisplatin-resistant but not sensitive cells was revealed by immunoprecipitation [25]. The regulatory role of DNA-PK in Akt-S473 phosphorylation was also shown for glioblastoma cells in response to IR [26]. The role of AKT in DNA-PKcs-dependent DNA DSB repair has been extensively studied by Toulany M. with co-authors. In particular, they showed that inhibition of AKT1 effectively sensitized non-small cell lung cancer cell lines to IR by inhibiting DNA-PKcs-dependent DNA DSB repair [27]. They also found that AKT (predominantly Akt1) mediated DNA-PKcs autophosphorylation at S2056 that is required for efficient DNA-DSB repair and release of DNA-PKcs from the DNA damage sites induced by IR. Akt1 played a critical role in formation of the functional complex to DNA duplex ends marked by Ku dimers [28]. Lastly, they found that Akt1 and Akt3, but not Akt2, interact with DNA-PKcs in K-Ras mutant cells and stimulate DSB repair, thereby protecting cancer cell against IR [29].

In contrast to NHEJ, the data illustrating the regulatory role of AKT in homology-mediated DNA repair (HR) is controversial. For example, Mueck K. with co-authors demonstrated that regulatory role of Akt1 in Rad51 foci formation in IR-treated NSCLC cells and recruitment of Rad51 recombinase to γ-H2AX foci, a well-known marker of DNA DSBs. This was in a concordance with a significant decrease of Rad51 protein in the nucleus of irradiated cells exhibiting AKT knockdown (AKT-KD). Lastly, the increased number of BRCA1 foci in AKT-KD cells exposed to IR also illustrated the impaired HR repair which was revealed by using SceI-based GFP-reporter assay [30]. In contrast, Plo I. with co-authors observed the opposite effect of AKT in homology-mediated DNA repair. For example, AKT silencing restored formation of IR-induced BRCA1 foci in breast cancer cells, whereas HR-related proteins (e.g., BRCA1 and Rad51) sequestered in the cytoplasm upon activation of AKT1. Important, this was observed in tumor cell lines and biopsies from AKT-high sporadic breast cancers, thereby illustrating that AKT1 inhibits homologous recombination in breast cancer cells in vitro and in vivo [31]. Similarly, negative regulatory role of Akt on homology-mediated repair was shown for BRCA1-deficient breast cancer cells. In particular, Akt1 promoted chromosome instability in Brca1-deficent cells by impairing nuclear localization of Chk1 and disrupting its interaction with Rad51, thereby leading to attenuation of homology-mediated repair [32].

Activation of AKT-mediated pathway is well-documented for the soft tissue sarcomas and can be used as independent prognostic factor for tumor recurrence, overall survival (OS) and disease-free survival (DFS) [12,33,34]. Similarly, activation of PI3-kinase/AKT pathway was well-documented for GIST cell lines and patient samples and played an important role for was critical to survival in IM-resistant GIST [1–3,7], thereby illustrating a rationale for combination therapy (e.g., of imatinib mesylate and AKT inhibitor) for patients with GIST [8].

Despite the sensitivity of STS and (in less extent) GIST to DNA-damaging agents was shown in multiple reports [20–23], however, to date, a little is known about the role of AKT in DNA DSB repair (in particular HR) in STS and GIST. Given that inhibition of FGF-signaling in GIST effectively sensitized them to Topo II inhibitors via attenuating HR-mediated DNA DSB repair [35], we sought to examine the downstream signaling pathways responsible for this phenomenon. We present here the novel data illustrating that AKT- but not a MEK-signaling pathway regulates an efficiency of homology-mediated DNA damage repair in STS and GIST. In particular, phosphorylated (i.e., activated) form of endogenous Akt physically interacts with Rad51 recombinase after DNA damage (Figure 8C). This was in consistency with previous data illustrating that Akt might be physically associated with various DNA DSBs repair proteins. For example, Toulany M. with the colleagues demonstrated that Akt1 phosphorylated at Ser472/Ser473 interacts with activated (i.e., phosphorylated at T2609) DNA-PKcs in various cell lines including NSCLC cells A549 and H460. Of note, this interaction was found shortly (3–5 min) of post IR exposure (4Gy), thereby illustrating an important regulatory role of Akt1 in DDR signaling [27]. The detailed analysis of this interaction demonstrated that Akt1 interacts with DNA-PKs through its C-terminal domain, stimulates autophosphorylation of DNA-PKcs at S2056, promotes its kinase activity and accumulation of DNA-PKcs at DNA-DSBs [28]. Similarly, by utilizing the proximity ligation assay, Sahlberg S. with co-authors demonstrated that phosphorylated forms of Akt and DNA-PKcs also interact with each other in IR-exposed colon cancer cell lines, whereas Akt deficiency significantly impaired the rejoining of radiation-induced DNA double strand breaks and sensitized colon cancer cells to IR [36]. Next, we found that AKT regulates Rad51 protein stability via proteasome-dependent pathway (Figure 8A,B) and MK-2206-induced inhibition of AKT-signaling has a strong impact of Rad51 expression, especially after Dox-induced DNA damage. Again, our data is in a close consistency with the data published recently by Mueck C. with co-authors illustrating that knockdown of Akt1 significantly reduced the amount of Rad51 protein in the nuclear fraction of irradiated non-small ling cancer cells [30]. We also observed that AKT signaling plays a regulatory role in the recruitment of Rad51 to DNA damage sites which was evidenced by a substantial decrease of number of residual Rad51 foci in Dox-treated tumor cells (Figure 7). As a result, inhibition of AKT-signaling pathway effectively sensitized STS and GIST to DNA-damaging agent, doxorubicin, and decreased tumor cell viability due by enhancing of apoptotic cell death (Figures 1 and 2, Figures S3 and S4).

Collectively, our data illustrates that overactivation of AKT-signaling pathway in STS and GIST might serve as a prospective molecular target to enhance cytotoxicity of DNA-topoisomerase II inhibitors against STS and GIST. This is in consistency with several reports illustrating a perspective role of PI3K/AKT/mTOR inhibitors to sensitize STS to standard chemotherapeutic regimens, including Topo II inhibitors. For example, Babichev Y. with colleagues demonstrated a high potency of PI3K vs. mTOR inhibitors used in combination of doxorubicin against LMS in vitro and in the leiomyosarcoma xenografts [37]. Similarly, AKT inhibition by pentacyclic triterpene ursolic acid (UA) effectively sensitized human STS cell lines in vitro to Dox treatment and induced their apoptosis [38]. Moreover, phosphorylation of AKT which was observed in STS cell lines after Dox treatment (10 M) illustrates that activation of AKT signaling represents a compensatory mechanism counteracting the cytotoxic and anti-proliferative effects of Dox [38]. This, in turn, illustrates a rationale of AKT inhibition in cancer cells treated with Dox. Our study effectively supplements these findings and illustrates the molecular mechanism responsible for this phenomenon. We show here for the first time that inhibition of AKT pathway in Dox-treated STS and GIST reduces Rad51 levels and inhibits recruitment of Rad51 recombinase to sites of DNA DSBs after Dox treatment, thereby attenuating the homology-mediated repair mechanisms. This was evidenced by substantial decrease of Rad51 foci that were co-localized with γ-H2AX when cancer cells were treated with Dox in presence of AKT inhibitor (Figure 7 and Figure S7). Moreover, data obtained from fractionation experiment revealed the substantially decrease in Rad51 level in chromatin fraction when cancer cells were treated with Dox in presence of MK-2206 but not with Dox alone (Figure S8). Thus, AKT inhibition may have a synergistic effect with Dox, standard chemotherapy for several types of STS due to inhibition of DNA DSB repair via attenuation of recruitment of Rad51 recombinase to the DNA damage lesions. Of note, we did not observe the enhanced phosphorylation of AKT in STS and GIST after Dox-induced DNA damage (Figure 2). This might be due to enhanced basal level of expression of phosphorylated form of AKT in STS and GIST cells and also in consistency with previous findings, illustrating that Dox treatment enhances AKT phosphorylation in sarcoma cell lines when was used in a high dose (10M), whereas no increase of AKT phosphorylation was observed when the cells were treated with 1M of Dox [38], which was similar to our experimental conditions. Of note, the effect of inhibition of AKT signaling might be not limited to sensitization of STS to the Topo II inhibitors. For example, MK-2206 and eribulin, a microtubule dynamics inhibitor, synergistically inhibited STS cell growth in vitro and in vivo, also revealing a rationale for the development of an AKT inhibitor in combination with eribulin for therapy of patients with STS [39]. Similarly to Dox-treated STS, expression of phosphorylated AKT significantly increased in eribulin-treated STS, thereby revealing AKT phosphorylation a common compensatory mechanism counteracting the cytotoxic and anti-proliferative effects of chemotherapeutic agents used for therapy of patients with STS. Thus, future studies with inhibitors targeting these pathways are warranted.
