Simultaneous Inhibition of SRC and STAT3 Induces an Apoptotic Response in Prostate Cancer Cells

Prostate cancer is the most frequently diagnosed non-cutaneous cancer and the second leading cause of cancer-related deaths in American men. Recent studies have suggested that sarcoma inducing kinase (SRC) and its downstream signaling targets are implicated in prostate cancer, but the mechanisms behind how SRC inhibition induces apoptosis are still poorly understood. This study focused specifically on the interactions between SRC and its one of its downstream targets, Signal transducer and activator of transcription 3 (STAT3), in prostate cancer cells. Contrary to studies on the SRC/STAT3 pathway, western blotting showed that SRC inhibition had minimal effects on phosphorlyated-STAT3 levels in DU145 prostate cancer cells. Simultaneous inhibition of both SRC and STAT3 through PP2 (inhibits SRC expression) and STAT3 siRNA, respectively, led to more distinct poly (ADP-ribose) polymerase 1 (PARP-1) cleavage, a hallmark indicator of apoptosis. qRT-PCR analysis showed a two-fold and three-fold decrease between simultaneous versus exclusive treatments in levels of induced myeloid cell leukemia (MCL-1), a pro-survival gene. Together, these findings suggest that the inhibition of STAT3 through SRC is ineffective and that the independent inhibition of STAT3 induces a stronger apoptotic response. Additionally, the study suggests that the simultaneous inhibition of SRC and STAT3 may be a novel and promising treatment for prostate cancer. Introduction Prostate cancer affects 35% of all American men1,2, and is the most frequently diagnosed non-cutaneous cancer in males. Men who develop androgenindependent prostate cancer (AIPC) are at a loss for any established effective treatments; however, recent studies have suggested that sarcoma inducing kinase (SRC) is a possible target in treating AIPC cells3,4. It has been shown that a subset of patients with AIPC, who exhibit an increase in SRC activity during the transition Simultaneous Inhibition of SRC and STAT3 Induces an Apoptotic Response in Prostate Cancer Cells Sherman Leung1,3, Elizabeth Duval2, and Olga Timofeeva4 Student1, Teacher2: Science, Mathematics, and Computer Science Magnet Program, Montgomery Blair High School 51 East Boulevard, Silver Spring, MD, 20901 Intern3, Mentor/Assistant Professor4: Lombardi Comprehensive Cancer Center, Georgetown University 3800 Reservoir Road, N.W. Washington, DC 20007 ACCELERATED ARTICLE of prostate cancer to an androgen-independent state, have a poorer prognosis and a reduced overall survival3. When deprived of androgen simulation, AIPC cells develop the ability to survive and thrive by upregulating oncogenic pathways where tyrosine kinases like SRC play a crucial role3,4. SRC inhibitors, currently in phase III of clinical trials, have been suggested as a viable and promising treatment option for patients with AIPC4; yet the mechanism by which SRC inhibitors trigger cancer cell death and prevent metastasis is still not completely understood. Signal transducer and activator of transcription 3 (STAT3), a downstream phosphorylation target of SRC4, has been identified as a substrate of v-src (viral src) necessary for enabling v-src induced adhesionindependence and malignant transformation5. As a transcription factor, STAT3 activates the expression of pro-survival genes (e.g. MCL-1, BCL2, BCL-xL) and genes that drive the cell cycle (e.g. c-MYC, cyclin D1) by binding to their promoters6. This results in increased survival and proliferation of cancer cells6,7. Significantly, SRC has been shown to induce cell transformation through STAT3-mediated gene regulation7. Recent research has suggested that the knockdown of STAT3 expression via RNA interference may be effective in inhibiting growth of prostate cancer cells8; yet, the mechanism by which STAT3 interacts with SRC in prostate cancer has not been investigated. The initial aim of this study was to investigate the results of SRC inhibition. It was expected that SRC inhibitors would slow cancer growth and induce apoptosis through the inhibition of STAT3. However, western blotting and a MTT assay showed that the inhibition of SRC kinase with PP2, a commonly used Src family kinase inhibitor9, induces growth inhibition in DU145 prostate cancer cells without inhibiting STAT3 phosphorylation. Stemming from this observation, the study focused on the functional interactions between SRC and STAT3 for prostate cancer cell survival, and whether the simultaneous inhibition of both STAT3 and SRC could induce a stronger apoptotic response in prostate cancer cells. Materials and Methods Transfection and qRT-PCR: DU145 cells (ATCC) were plated in two sets of 6-well plates (2 * 105 per well) and then transfected with TransIT-TKO (Mirus Bio), using GAPDH siRNA, STAT3 siRNA-1, and STAT3 siRNA-2 (Invitrogen). After 24 hours of incubation, PP2 (Calbiochem) was added to one plate, at a final concentration of 5mM, in each set and all plates were incubated for a total of 48 hours. RNA was then extracted and purified using the RNeasyMiniKit (Qiagen). RNA concentration was measured using Thermo Scientific’s NanoDrop 2000. cDNA was made using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) and Bio-Rad’s iCycler Thermal Cycler with the following reaction conditions: 25°C for 10 mins, 37°C for 2 hours, 85°C for 5 mins, and 4°C for incubation. qRT-PCR was then performed using MCL-1 primers in MicroAmp fast Optical 96well reaction plates using the 7900HT Fast Real-Time Sherman Leung, Elizabeth Duval, and Olga Timofeeva Page 2 of 6 PCR System (Applied Biosystems). Protein Extraction and Western Blotting: After aspirating medium and washing DU145 cells with 1x phosphate buffered saline, a solution containing Tween-20 (TPBS), 100 μL of 1x Lysis Buffer (Millipore), Phosphatase Inhibitor Cocktail 2 (Sigma), and 1 protease inhibitor cocktail tablet (Roche) was added to each well. The cells were transferred to 1.5 mL tubes and kept on ice for 1530 mins (vortexing every 5 mins). They were then centrifuged at max speed for 10-15 mins at 4°C to remove cell debris. Protein concentration was measured via the Bio-Rad protein assay. Protein samples were then denatured with 2x sample buffer (Invitrogen) at 95°C for 5 mins and loaded into Tricine gels (Novex) that were placed into the XCellSureLock Mini-Cell electrophoresis system (Invitrogen). Immobilion-P Membranes (Millipore) were soaked in methanol for 1 min. The gels, support pads, gel blotting paper (Whatman), and the transfer membrane were all washed in 1x transfer buffer(ThermoFisher Scientific) before being stacked inside the Owl VEP-2 Mini Tank Electroblotting System (ThermoFisher Scientific). Membranes were retrieved and washed with TPBS and blocked with 5% non-fat milk for 1 hour at RT*. Membranes were incubated with anti-phosphorylated STAT3 and anti-PARP-1 primary antibodies (Cell Signaling Technology) in 1x TPBS (1:1000) overnight at 4°C. Membranes were then washed three times with TPBS for 5 mins each. Secondary donkey antirabbit antibodies (ThermoFisher Scientific) (1:10,000) were incubated with the membranes for 1 hour at RT. Membranes were washed again and prepared with Western Lightning Plus-ECL. Antibody–epitope binding was detected using AFP Imaging MiniMedical Automatic Film Processors. After films were processed, membranes were blocked again with milk and allowed to incubate with primary antibodies for total STAT3 (1:1000) and b-actin (1:5000) and a secondary donkey anti-rabbit antibody for STAT3 and donkey anti-mouse antibody for b-actin (1:5000). MTT assay: 10 μL of MTT (Roche) was added to each well of the 96-well plate and put into a Water Jacketed Co2 Incubator (ThermoFisher Scientific) for 4 hours. After 50 μL of SDS (Roche) was added to each well to dissolve the crystals, the absorbance was measured using a Beckman DU-64 plate reader. In order to assess the effectiveness of simultaneous inhibition, DU145 cells were transfected with STAT3 siRNA to knockdown STAT3 expression in addition to PP2 treatment. DU145 cells with the simultaneous inhibition of SRC and STAT3 exhibited more distinct Poly (ADP-Ribose) Polymerase-1 (PARP-1) cleavage, an indicator of apoptosis10. To investigate the mechanisms behind this cooperation between STAT3 and SRC inhibition, levels of induced myeloid leukemia cell differentiation Mcl-1 (MCL-1), a downstream target of STAT36, and a pro-survival gene11,12, were measured through qRT-PCR analysis. It has been shown that a decrease in MCL-1 levels induces apoptosis in cancer cells11. DU145 cells with both treatments led to 3-fold and 2 fold decreases in MCL-1 expression when compared to the exclusive treatments of STAT3 and SRC, respectively. This data suggests that an increase in apoptosis from the combination of both treatments might be due to a decrease in MCL-1 levels. Results of the study show that the simultaneous inhibition of SRC and STAT3 in DU145 prostate cancer cells is more effective in inducing the apoptotic responses of PARP-1 cleavage and lowered MCL-1 expression when compared to the exclusive inhibition of either SRC or STAT3. This study suggests that the simultaneous inhibition of SRC and STAT3 may prove to be a novel, effective therapeutic strategy for the treatment of prostate cancer patients. *(RT) = Room Temperature Sherman Leung, Elizabeth Duval, and Olga Timofeeva Page 3 of 6