The Interaction of PI3K Inhibition with Homologous Recombination Repair in Triple Negative Breast Cancer Cells

- Purpose: Aberrant activation of the phosphatidylinositol 3'-kinase (PI3K)-Akt signaling pathway is observed in many types of human cancer including triple negative breast cancer (TNBC). Additionally, dysregulation in the homologous recombination (HR)-dependent DNA-repair is associated with TNBC phenotype due to BRCA1/2 mutations or HR deficiency. Therefore, the hypothesis of this study was to evaluate the association of PI3K inhibition with HR pathway in TNBC in terms of BRCA1 mutation status. Methods: To examine the potential therapeutic effect of LY294002, an inhibitor of PI3K, on TNBC cell lines with known BRCA1 status, WST-1, annexin V, cell cycle analysis and AO/EB staining were performed. Additionally, RT-PCR and immunofluorescence analysis was used to explore the interaction between the inhibition of PI3K and HR functionality. Results: The findings showed that LY294002 could significantly inhibited the proliferation of TNBC cells. Furthermore, the suppression of PI3K resulted in HR impairment by BRCA1 and RAD51 downregulation and apoptotic cell death by the induction of DNA damage and BAX overexpression. Therefore, LY294002 was more effective in BRCA1 -deficient TNBC cells. Conclusions: Consequently, targeted therapies based on the interaction of PI3K inhibition with BRCA1 mutations or HR deficiency in TNBC may be a promising strategy for the treatment of patients with TNBC. _______________________________________________________________________________________


INTRODUCTION
Triple-negative breast cancer (TNBC) is a sub-type of breast cancer with a worst prognosis among all subtypes of breast cancer. Treatment of TNBC is based on the using of different chemotherapeutic agents for neoadjuvant or adjuvant therapy. However, there is no effective and molecularly targeted treatment options for TNBC due to heterogeneity of disease (1)(2)(3)(4). Therefore, there is urgently needed to identify the potential molecular target for TNBC therapy.
Mutations in the BRCA1/2 genes, play a crucial role in the homologous recombination (HR) mechanism to repair of double-strand DNA break (dsDNA), are strongly associated with TNBC. Several studies have identified that the prevalence of the BRCA1/2 mutation in unselected TNBC patients for age and family history is between 9.4 and 18.2% in the literature (4)(5)(6)(7). Additionally, there is increasing evidence that further genes involved in HR pathway are associated with the TNBC phenotype (8)(9)(10). Thus, therapeutic approaches targeting BRCA mutation and/or HR deficiency status are more effective for developing of successful treatment strategies for TNBC patients.
The phosphatidylinositol 3-kinase (PI3K)/ AKT-pathway is a crucial intracellular signaling pathway in cell growth, proliferation and metabolism and over-activation of the pathway promotes cancer cell proliferation, invasion and metastasis (11,12). TNBC exhibits aberrant activation of PI3K pathway through different mechanism including loss of PTEN, overexpression of EGFR and mutations in PIK3CA gene. Additionally, an interaction between PI3K and HR pathway is an essential role in DNA repair through replication fork stabilization under physiological conditions. Therefore, the PI3K/AKT pathway represents therapeutic potential in TNBC patients (13)(14)(15).
In this context, we investigated for the first time that the interaction of PI3K inhibition by the PI3K inhibitor, LY294002 with HR pathway in TNBC, in vitro. For this purpose, the therapeutic potential of LY294002 in two of the TNBC cell lines (HCC1937  WST-1 Analysis LY294002 was purchased from Biovision (San Francisco, CA, USA) and stock solution of LY294002 was prepared by DMSO and stored at -20ºC. In briefly, cells were seeded in 96-well plates. After 24 and 48 hours of incubation with LY294002 (0-20 µM), WST-1 dye (Biovision, San Francisco, CA, USA) was added into each well and incubated with 30 min in the dark. Then, cell viability was determined by microplate reader at 450 nm (Allsheng, China).

Apoptotic Analysis
For Annexin V analysis, cells were trypsinized and washed with PBS after 48 h of incubation with different concentrations of LY294002. Then, cells were stained with Muse TM Annexin V & Dead Cell Assay kit (Millipore, Germany) and incubated for 30 minutes in the dark condition. For cell cycle analysis, cells were fixed in ice-cold 70% ethanol in PBS and kept at -20°C for at least three hours. Afterwards, cells were centrifuged and stained with Muse TM Cell Cycle Kit (Merck Millipore, Germany) for 30 minutes in the dark condition. Finally, each experiment was analyzed at Muse TM Cell Analyzer (Merck Millipore, Germany) (n=3).
Acridine Orange (AO) and Ethidium Bromide (EB) Staining Following different concentration of LY294002 exposure, cells were washed with PBS and fixed with 4% paraformaldehyde. Afterwards, cells were stained with AO/EB for 30 minutes. After washing twice with PBS, all images were recorded by a EVOS FL Cell Imaging System (Thermo Fisher Scientific, Waltham, MA, USA).

Gene expression
High Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Waltham, MA, USA was used for cDNA synthesis after total RNA isolation with an E.Z.N.A. Total RNA Kit (Omega Bio-Tek, Norcross, GA). RT-PCR was performed using the Step One Plus TM Real-Time PCR (Applied Biosystems, Foster City, CA) to detect changes in the H2AFX, BRCA1, RAD51, BCL-2 and BAX gene expression levels. ACTB was used as an internal normalization control. Differentially expressed genes were analyzed by Qiagen software tool.

Immunofluorescence Analysis
Immunofluorescence analysis of γH2AX, BRCA1 and RAD51 was conducted on HCC1937 and MDA-MB-231 cells after 48 h of exposure to LY294002. After incubation, cells were fixed with 4% paraformaldehyde, permeabilized and blocked with 0.1% Triton X-100 and 5% goat serum in 1× PBS medium for 1 hour. Subsequently, cells were incubated with anti-mouse γH2AX, BRCA1 and RAD51 primer antibodies at a dilution of 1:250, 1:200 and 1:10, respectively for two hours. Then, anti-mouse Alexa Fluor 488 goat IgG was diluted 1:1000 and incubated for 60 minutes. Finally, DAPI was used for visualization of nuclei. Images were acquired using an EVOS FL Cell Imaging System (Thermo Fisher Scientific, Waltham, MA, USA).

STATISTICAL ANALYSIS
One-way ANOVA with Post-hoc Tukey test and two-way repeated measures ANOVA were performed by using SPSS 22.0 (SPSS Inc., Chicago, IL, USA) for multiple comparison. All experiments were repeated at least 3 times and the obtained data presented as the mean ± standard deviation. P<0.05 was considered significant.

Cytotoxic effect of LY294002, PI3K inhibitor on TNBC cell lines
To determine the effects of LY294002 on cell proliferation, we carried out WST-1 assay. As shown in Figure 1, we determined that LY294002 significantly inhibited the proliferation of TNBC cells (p<0.05). Exposure to 10 μM of LY294002 led to 52.9 ± 0.3% and 22.4 ± 4.4% cell death in HCC1937 and MDA-MB-231 cells, respectively for 48 h. Additionally, a considerable inhibition (55.5 ± 1.7% and 41.8 ± 2.6%) was observed at 20 μM for 48 h in TNBC cells (HCC1937 and MDA-MB-231 cells, respectively, p<0.01). Therefore, HCC1937 cells were more sensitive to LY294002 compared with MDA-MB-231 cells. On the other hand, treatment with 10 and 20 µM of LY294002 resulted in a decrease (75.6 ± 2.7% and 65.6 ± 5.3%, respectively) the viability of MCF-10A cells for 48 h.
Furthermore, a two-way repeated measures ANOVA analysis was performed to evaluate the differences between the anti-proliferative effect of LY294002 and cell type compared to control cells. As shown in Table 1   To further confirm LY294002-induced cell death, the differences in cell cycle phase distribution were assessed (Figure 3). Incubation of TNBC and MCF-10A cells with LY294002 led to a remarkable increase of the G0/G1 peak (p<0.01). Furthermore, a dose dependent increase in G0/G1 phase was detected after exposure to LY294002 in TNBC cells irrespective of BRCA1 mutation status. However, the percentage of G0/G1 phase in HCC1937 cells was remarkably higher than that in MDA-MB-231 cells.    Cell morphological changes following incubation with LY294002 LY294002 treated cells were stained with AO/EB to observe and validate LY294002-induced apoptotic cell death (Figure 4). In general, TNBC and MCF-10A cells exhibited fragmented green nucleus as sign of early apoptotic cells following incubation with LY294002 compared to each control group. Additionally, some yellow/green dots of condensed nuclei as well as intracytoplasmic vacuoles were observed in MDA-MB-231 cells at 20 µM of LY294002 due to late apoptosis. On the other hand, LY294002 induced cell shrinkage and rounding morphology in MCF-10A cells at the highest concentration.

Effect of LY294002 on HR-related genes expression level
The expression levels of H2AFX, BRCA1 and RAD51 in TNBC and control cell lines were also identified by RT-PCR and the findings were summarized in Figure 5A. LY294002 significantly down-regulated BRCA1 and RAD51 expression levels whereas the overexpression of H2AFX was determined in TNBC and control cell lines. However, the relative BRCA1 and RAD51 mRNA expression levels were significantly higher in BRCA1 proficient MDA-MB-231 cells than BRCA1 deficient HCC1937 cells. On the other hand, H2AFX expression was significantly increased in HCC1937 cells compared with MDA-MB-231 cells (p<0.01). Furthermore, H2AFX mRNA level was expressed at higher levels in MCF-10A cells (p<0.01).
Furthermore, the expression level of two genes (BCL-2 and BAX) associated with cell death were also determined to validate LY294002induced apoptotic cell death in Figure 5B.
LY294002 dose-dependently increased BAX expression and decreased BCL-2 expression in TNBC cells (p<0.01). Therefore, inhibition of PI3K by LY294002 resulted in DNA damage, suppression of HR and caused apoptotic cell death in TNBC cells. Evaluation of HR-related protein expression by immunofluorescence The foci formation of γH2AX and the localization of BRCA1 and RAD51 in response to 20 µM of LY294002 were examined. As shown in Figure 6, we observed that there was a significant increase in the number of γH2AX foci upon LY294002 treatment in TNBC cells. However, HCC1937 cells exhibited higher levels of γH2AX foci than MDA-MB-231 cells in response to 20 µM of LY294002. Besides, both TNBC cells showed a low level of cytoplasmic staining for RAD51. However, BRCA1-deficient HCC1937 cells were unable to form BRCA1 foci compared to MDA-MB-231 cells. Therefore, higher of cytoplasmic BRCA1 and RAD51 expressions were observed in MDA-MB-231 cells.

DISCUSSION
Here, the association of PI3K inhibition with HR repair mechanism has been investigated in TNBC. The findings suggest that the suppression of PI3K leads to impair BRCA1 and RAD51 mRNA and protein expression in TNBC cells. PI3K inhibition can result in increased expression of γ-H2AX, as a marker of DNA damage, apoptotic cell death and G0/G1 phase arrest. Additionally, LY294002, an inhibitor of PI3K, is more effective in BRCA1deficient TNBC cells. Thus, the therapeutic potential of PI3K inhibitors is closely linked with HR impairment in TNBC.
The overexpression of PI3K/AKT/mTOR pathway is associated with poor clinical outcomes of patients with breast cancer. Thus, the inhibition of PI3K pathway is a potential therapeutic target for treating breast cancer (16)(17)(18). In this context, the efficacy of PI3K/AKT/mTOR pathway inhibitors in the treatment of advanced solid tumors including lung, pancreatic, breast and ovarian cancer etc. have been investigated in preclinical and clinical studies (19)(20)(21)(22)(23).
TNBC is a highly aggressive heterogeneous subtype of breast cancer with a higher rates of distant recurrence (1)(2)(3)(4). Previous studies have revealed that TNBC patients showed a higher prevalence of PTEN and PI3K mutations and overactivation of mTOR pathway compared to other subtypes of breast cancer (18,(24)(25)(26). Thus, pan-PI3K inhibitors (buparlisib, pictilisib, AR245408, taselisib, and PX-866) and isoform-specific PI3K inhibitors (alpelisib) have been extensively studied in the treatment of breast cancer subtypes (13,27,28). Moreover, alpelisib has been approved by The US Food and Drug Administration (FDA) for the treatment of postmenopausal women and men patients with HR+, HER2-, PIK3CA mutated advanced or metastatic breast cancer.
LY294002, a reversible inhibitor of PI3K as a derivative of quercetin, inhibits certain mammalian PI3Ks including mTOR (mammalian target of rapamycin), DNA-PK (DNA-dependent protein kinase), other protein kinases (CK2 (casein kinase 2) and Pim-1) (29,30). Previous studies have shown that LY294002 sensitizes cerulenininduced apoptotic cell death, inhibits BCRPmediated drug resistance and increases GSK-3β activity by adenovirus-mediated gene transfer of PTEN in different types of breast cancer cell lines (MCF-7 and MBA-MB468 cells) (31)(32)(33). In the current study, LY294002 inhibits the proliferation of TNBC cells irrespective of BRCA1 mutation status and induces apoptotic cell death and cell cycle arrest at G0/G1 phase. However, the inhibition of PI3K is more profound in BRCA1 mutant TNBC cell line.
Recent studies have also revealed that PI3K/Akt inhibition is associated with impairment of DNA repair in different types of cancer cells including glioblastoma, endometrial, ovarian and pancreatic cancer (34)(35)(36)(37). Park et al. (2017) state that HS-173 PI3K inhibitor enhances radiotherapy by suppressing the DNA damage-repair pathway in pancreatic cancer cell (34). Additionally, studies of Philip et al. (2017) and Wang et al (2016) demonstrate that BKM-120 treatment is effectively blocked RAD51 foci formation and cause downregulation of BRCA in PTEN mutated endometrial cancer cells and PIK3CA mutated ovarian cancer cells, respectively and the dual inhibition of PI3K and PARP by PARP inhibitors (talazoparib and olaparib) may be an effective therapeutic approach for endometrial and ovarian cancer (35,36). Furthermore, Ibrahim et al. (2012) show that PI3K inhibition by BKM-120 induces DNA damage, downregulation of BRCA1/2, and combined inhibition of PI3K and PARP indicates significant antitumor activity in BRCA-wild-type TNBC cell lines (MDA-MB-468, MDA-MB-231, HCC70, HCC1143, and BT20) and TNBC patientderived primary tumor xenograft (38). In the present study, the suppression of PI3K by LY94002 is accompanied by downregulation of BRCA1 and RAD51 gene and protein expression and promotes the accumulation of γH2AX in TNBC. However, treatment with LY294002 leads to considerably reduced the expression levels of BRCA1 and RAD51, but increased the level of H2AFX mRNA expression and γH2AX foci formation and thus, LY294002 causes more apoptotic cell death in BRCA1-deficient TNBC cells. The apoptotic effect of LY294002 is correlated with increased the expression level of BAX and reduced the level of BCL-2.
Consequently, these results suggest that a significant link between PI3K inhibition and HR mechanism in TNBC. Thus, PI3K/AKT targeting by PI3K inhibitors may be potential therapeutic implications for TNBC patients with BRCA mutations or HR deficiency. However, further molecular investigations are needed to identify the efficacy of LY294002 in DNA repair mechanisms including HR, non-homologous end-joining (NHEJ), microhomology-mediated end-joining (MMEJ), alternative NHEJ (alt-NHEJ), synthetic lethality, "BRCAness", and the Fanconi anemia signaling network (39).
Additionally, the combination treatment of LY294002 with especially PARP inhibitors and comparison of the effectiveness of LY294002 with other PI3K inhibitors in TNBC are further investigated by in vitro and in vivo.