R01 Grant CA129038 Research Articles

Pimavanserin tartrate: A potential drug for pancreatic cancer therapy in future

Despite major advances in cancer treatment, pancreatic cancer is still incurable and the treatment outcomes are limited. In the current study, we evaluated the anti‐cancer effects of pimavanserin tartrate (PVT), a drug used for the treatment of Parkinson's disease psychosis. Our observations indicated that, PVT significantly suppressed the proliferation of pancreatic cancer cells by inducing apoptosis without exerting any cytotoxic effects in normal human pancreatic ductal epithelial (HPDE‐6) cells. The colony forming ability of pancreatic cancer cells was significantly inhibited with PVT treatment. Anti‐proliferative and apoptosis inducing effects of PVT were mediated by the inhibition of pAkt (Ser473), Akt, Gli1, Oct‐4, SOX‐2, NANOG and c‐Myc. Akt and Oct‐4 inhibition by PVT treatment was further validated by immunofluorescence analysis. Apoptotic effects of PVT was confirmed by increase in cleavage of caspase‐3 and PARP. Moreover, PVT inhibited the formation of tumorspheres in PANC1 pancreatic cancer cells. Pharmacologically inhibiting or genetically knocking out Akt or Gli1 enhanced the growth suppressive effects of PVT in pancreatic cancer cells. Subsequently, we evaluated the effects of PVT in gemcitabine‐resistant cells. Our results demonstrated that, PVT reduced the survival of MIAPaCa2 gemcitabine‐resistant cells in a concentration and time‐dependent manner. Further mechanistic analysis indicated that, PVT suppressed the phosphorylation of Akt at Ser 473 and inhibited the expression of Gli1, Oct‐4 and c‐Myc in MIAPaCa2 gemcitabine‐resistant cells. Moreover, PVT increased the cleavage of caspase‐3 and PARP as an indicator of apoptosis. Oral administration of PVT suppressed BxPC3 tumor xenografts by 50% in athymic nude mice. In another in vivo experiment, PVT treatment inhibited the growth of orthotopically implanted PANC1 tumors by 75%. Chronic administration of PVT did not exhibit any general signs of toxicity or behavioral side effects in mice. Taken together, our results indicate that pancreatic tumor growth suppression by PVT is orchestrated by inhibition of Akt/Gli1 signaling. Since PVT is already available in the clinic with an established safety profile, our results will accelerate its clinical development for the treatment of patients with pancreatic cancer.Support or Funding InformationSupported in part by R01 grant CA129038, awarded to (S.K.S.) by the National Cancer Institute, Syngenta fellowship grant awarded by Syngenta pharmaceuticals.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract 3518: Piperlongumine, a novel treatment option to overcome BRAF inhibitor resistance in melanoma

Abstract Tumor cell metabolism is a hallmark of chemoresistance and its role has been well-defined in BRAF inhibitor resistance in melanoma. Melanoma cells are highly proliferative and are shown to develop addiction towards the metabolic pathways like glycolysis and mitochondrial respiration to meet energy demands. Melanoma cells predominantly utilize cytosolic aerobic glycolysis or Warburg effect to develop resistance towards BRAF inhibitors (vemurafenib and dabrafenib). Our observations indicated that vemurafenib resistant cells possessed 1.5 fold higher extra-cellular acidification rate (ECAR), an indicator of glycolysis when compared to sensitive cells. We also observed that STAT3, a known regulator of glycolysis was up-regulated in A375 and SKMEL28 BRAF inhibitor resistant cells when compared to the parental cell line. In addition, LDH-A, a key glycolytic enzyme was significantly overexpressed in A375 vemurafenib resistant cells when compared to its parental counterpart. Consequently, we observed that vemurafenib and dabrafenib treatment induces the expression of STAT3 in A375 and SKMEL-28 melanoma cells. Our results showed that piperlongumine inhibits the survival of A375 vemurafenib resistant (A375VR) melanoma cells. Our observations indicated that the combination of piperlongumine with vemurafenib and dabrafenib shows synergistic effects by suppressing 75% - 85% of cell survival when compared to vemurafenib/dabrafenib and piperlongumine (25% - 45%) treatment alone in A375 and SKMEL-28 BRAF inhibitor resistant melanoma cells. Moreover, the combination of piperlongumine with vemurafenib or dabrafenib induced 2-8 fold apoptosis when compared to vemurafenib/dabrafenib and piperlongumine treatment alone in A375 and SKMEL-28 BRAF inhibitor resistant melanoma cells. Piperlongumine treatment reduced the ECAR (glycolysis) in a concentration dependent manner after 12h of treatment in A375VR cells and suppressed nearly 50% of ECAR parameters glycolysis and glycolytic capacity. Treatment of A375VR cells with piperlongumine for 48h inhibited the expression of LDH-A and its upstream regulators pSTAT3 (Y705), β-catenin, c-Myc and HIF-1α as evaluated by western blot. In addition, oral administration of piperlongumine and vemurafenib combination suppressed 86% of tumor growth when compared to vemurafenib treatment. Taken together, our study thus indicate that piperlongumine potentiates the effects of BRAF inhibitors by inhibiting Warburg effect and its upstream regulators. [Supported in part by R01 grant CA129038, awarded to (S.K.S) by the National Cancer Institute. Citation Format: Sharavan Ramachandran, Neel Fofaria, Sanjay Srivastava. Piperlongumine, a novel treatment option to overcome BRAF inhibitor resistance in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3518.

Abstract 4422: Capsaicin suppresses pancreatic tumor growth by inhibiting tumor cell metabolism

Abstract Tumor cell metabolism is considered to be a hallmark of tumorigenic progression. Pancreatic cancer cells are shown to develop addiction towards the metabolic pathways like glycolysis and mitochondrial respiration to meet energy demands. Pancreatic tumor cells predominantly utilize cytosolic aerobic glycolysis for energy production. In our previous studies, we have reported that capsaicin treatment inhibits the survival of AsPC-1 and BxPC-3 pancreatic cancer cell lines. In this study, we evaluated the effect of capsaicin on glycolysis and mitochondrial respiration in pancreatic cancer cells. Our results showed that capsaicin treatment reduced the extra-cellular acidification rate (glycolysis) and oxygen consumption rate (mitochondrial respiration) in AsPC-1 and BxPC-3 cells in a concentration-dependent manner after 24h of treatment. Capsaicin treatment caused 86% and 55% down-regulation of glycolysis process in AsPC-1and BxPC-3 cells respectively. Our results also indicate the inhibition of glycolytic capacity and glycolytic reserve by capsaicin treatment indicating the potential of capsaicin to inhibit glycolysis. In addition, capsaicin treatment reduced 98% of basal oxygen consumption rate and ATP production in AsPC-1 cells. Furthermore, capsaicin treatment inhibited spare respiratory capacity & proton leak in a concentration dependent manner in AsPC-1 cells. Treatment of AsPC-1 and BxPC-3 with capsaicin for 48h also inhibited the expression of LDH-A and its upstream regulators such as HIF-1α, pSTAT3 (Y705) and EGFR as evaluated by Western blot. Our study thus indicate that capsaicin suppresses pancreatic tumor growth by down-regulating glycolysis and mitochondrial respiration. Further mechanistic studies are in progress. [Supported in part by R01 grant CA129038, awarded to (S.K.S.) by the National Cancer Institute]. Citation Format: Sharavan Ramachandran, Sanjay K. Srivastava. Capsaicin suppresses pancreatic tumor growth by inhibiting tumor cell metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4422. doi:10.1158/1538-7445.AM2017-4422

Abstract 1666: Immune consequences of penfluridol treatment associated with inhibition of glioblastoma tumor growth

Abstract Glioblastoma is the most common and lethal brain tumor associated with only 12% median survival rate of patients. Despite the development of advanced surgical, radiation or use of combinations of anti-cancer drugs, treatment for glioblastoma patients is still a challenge. The major contributing factor in glioblastoma progression and resistive nature is its ability to evade the immune surveillance. Hence, modulating the immune system in glioblastoma tumors could be an important strategy for anticancer therapeutics. Penfluridol, an antipsychotic drug has been shown to have anti-cancer properties in our recently published studies. The present study evaluates the immune response of penfluridol in glioblastoma tumors. Our results demonstrated that penfluridol treatment significantly suppressed glioblastoma tumor growth. Our current results demonstrated about 72% suppression of myeloid derived suppressor cells (MDSCs) with penfluridol treatment in mouse bearing U87MG glioblastoma tumors. MDSCs are known to increase regulatory T cells (Treg), which are immunosuppressive in nature and suppresses M1 macrophages that are tumor suppressive in nature. Our results also showed suppression of regulatory T cells as well as elevation of M1 macrophages with penfluridol treatment by 58% and 57% respectively. Decrease in CCL4 as well as IFNγ with penfluridol treatment was also observed indicating decrease in overall tumor inflammation. This is the first report demonstrating immune modulations by penfluridol treatment associated with glioblastoma tumor growth suppression prompting further investigation to establish penfluridol as a treatment option for glioblastoma patients. [Studies supported in part by R01 grant CA129038, awarded by National Cancer Institute, NIH] Citation Format: Alok Ranjan, Nehal Gupta, Sharavan Ramachandran, Stephen Wright, Sanjay Srivastava. Immune consequences of penfluridol treatment associated with inhibition of glioblastoma tumor growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1666. doi:10.1158/1538-7445.AM2017-1666

WE-FG-207A-03: Low-Dose Cone-Beam Breast CT: Physics and Technology Development

WE-FG-207A-03: Low-Dose Cone-Beam Breast CT: Physics and Technology Development

WE-FG-207A-02: Why We Need Breast CT? - Clinical Perspective

WE-FG-207A-02: Why We Need Breast CT? - Clinical Perspective

WE-FG-207A-04: Performance Characteristics of Photon-Counting Breast CT

WE-FG-207A-04: Performance Characteristics of Photon-Counting Breast CT

WE-FG-207A-00: Advances in Dedicated Breast CT

WE-FG-207A-00: Advances in Dedicated Breast CT

WE-FG-207A-01: Introduction to Dedicated Breast CT - Early Studies

WE-FG-207A-01: Introduction to Dedicated Breast CT - Early Studies

WE-FG-207A-05: Dedicated Breast CT as a Diagnostic Imaging Tool: Physics and Clinical Feasibility

WE-FG-207A-05: Dedicated Breast CT as a Diagnostic Imaging Tool: Physics and Clinical Feasibility

Abstract 5504: Penfluridol suppresses triple negative breast cancer metastasis to brain by inhibiting α6β4 integrins

Abstract Breast cancer is the second leading cause of cancer related deaths in the United States. Breast tumor metastasis to brain is primary cause of deaths. Brain metastasis of triple negative breast cancer cells (TNBC) is highly resistant to current therapies and is a cause of reduced survival rates in patients. In the current study, we evaluated the anti-cancer effects of penfluridol, a first generation antipsychotic drug against three different highly aggressive TNBC cell line. The IC50 of penfluridol was around 6 μM in 4T1, MDA-MB-231 and HCC-1806, breast cancer cells respectively. Our result showed that the expression of integrinβ4, integrinα6, Fak, Paxillin, Rac1/2/3 and ROCK1 was significantly reduced after treatment with penfluridol for 24 hours. Penfluridol treatment induced significant apoptosis in 4T1, MDA-MB-231 and HCC-1806 cells as exhibited by cleavage of caspase 3. Interestingly, integrin signaling is known to play significant role in breast cancer metastasis, and integrinα6β4 are overexpressed in breast tumors. Integrinα6 and β4 were silenced in breast cancer cells using shRNA and siRNA respectively. Silencing of integrinα6β4 resulted in apoptosis which was further increased by penfluridol treatment. On the other hand, activation of integrins by TGFβ treatment reduced the induction of apoptosis by penfluridol, confirming the role of integrins. Efficacy of penfluridol was evaluated in three in vivo experiments. In the first experiment, 4T1, breast cancer cells were implanted in the mammary fat pads of female mice and the mice were treated with 10 mg/kg penfluridol by oral gavage everyday till day 27, once the tumor size reached 50 mm3. Penfluridol treatment suppressed breast tumor growth by 50% as compared to control. In the second experiment, 4T1 luciferase transfected breast cancer cells were injected intracranially into the brain. The growth of 4T1-luc cells was monitored non-invasively using IVIS caliper and the mice started getting treatment with 10 mg/kg penfluridol by oral gavage the very next day of tumor cell injection. Our result demonstrated that the tumor growth in the brain of penfluridol treated mice was reduced by 85% as compared to control. Furthermore, anti-metastatic effect of penfluridol was determined by intracardiac injection of 4T1-luc breast tumor cells into the left ventricle of mouse heart. Once breast cancer cells lodged in the brain, mice were treated with 10 mg/kg penfluridol for 12 days. Our results showed that penfluridol treatment suppressed the growth of metastatic breast cancer cells in brain by 90%. The metastatic tumors from penfluridol treated mice exhibited reduced expression of integrins. Taken together, our result indicate that penfluridol is effective in reducing the growth of primary TNBC tumor as well as metastatic growth in brain by inhibiting integrin signaling. [Supported in part by R01 grant CA129038, awarded by National Cancer Institute, NIH]. Citation Format: Alok Ranjan, Parul Gupta, Sanjay Srivastava. Penfluridol suppresses triple negative breast cancer metastasis to brain by inhibiting α6β4 integrins. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5504. doi:10.1158/1538-7445.AM2015-5504

Abstract 2679: Overexpression of Mcl-1 confers resistance to BRAFV600E inhibitors alone and in combination with MEK1/2 inhibitors in melanoma

Abstract Melanoma harboring BRAF mutations frequently develop resistance to BRAF inhibitors, limiting the impact of treatment. The most prevalent mechanisms of acquired resistance appear to reactivate MAPK pathway. Furthermore, relatively little is known about the determinants of de novo resistance. Here, we establish such a mechanism of resistance and subsequently identified a suitable drug combination to overcome the resistance. Single treatment of BRAF mutant melanoma cell lines with vemurafenib or dabrafenib (BRAF inhibitors) alone or in combination with trametinib (MEK1/2 inhibitor) resulted in overexpression of Mcl-1. Overexpression of Mcl-1 in A375 and SK-MEL-28 by transfection completely blocked BRAF and MEK1/2 inhibitor-mediated inhibition of cell survival. Melanoma cells resistant to BRAF inhibitors showed massive expression of Mcl-1 as compared to respective sensitive cell lines. Silencing of Mcl-1 using siRNA completely sensitized resistant melanoma cells to growth suppression by BRAF inhibitors. In vivo, vemurafenib resistant A375 xenografts implanted in athymic nude mice showed substantial tumor growth inhibition when treated with a combination of vemurafenib and Mcl-1 inhibitor or siRNA. Immunohistochemistry and western blot analyses demonstrated enhanced expression of Mcl-1 and activation of ERK1/2 in vemurafenib-resistant tumors whereas level of Mcl-1 or p-ERK1/2 was diminished in the tumors of mice treated with either of the combination. Biopsied tumors from the patients treated with or resistant to BRAF inhibitors revealed overexpression of Mcl-1. These results suggest that the combination of BRAF inhibitors with Mcl-1 inhibitor may have therapeutic advantage to melanoma patients with acquired resistance to BRAF inhibitors alone or in combination with MEK1/2 inhibitors. [Supported in part by R01 grant CA129038 (to S.K.S) awarded by the National Cancer Institute]. Citation Format: Neel M. Fofaria, Dennie T. Frederick, Ryan J. Sullivan, Keith T. Flaherty, Sanjay K. Srivastava. Overexpression of Mcl-1 confers resistance to BRAFV600E inhibitors alone and in combination with MEK1/2 inhibitors in melanoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2679. doi:10.1158/1538-7445.AM2015-2679

Abstract 3565: Gli2 induces melanoma tumor growth and progression: A critical role of c-Myc

Abstract Gli2 is one of the key transcription factors in sonic hedgehog (Shh) pathway. Aberrant activation of Gli2 has been reported in several malignancies. However, the role of Gli2 in melanoma progression is unclear. Our results showed that knock down of Gli2 in melanoma cells inhibited the proliferation as well as induced apoptosis in melanoma cells in vitro, which was associated with down-regulation of c-Myc. Melanoma cells with stable Gli2 knockdown demonstrated diminished tumor growth when injected subcutaneously in athymic nude mice. In addition, Gli2 inhibitors GANT58 and GANT61 significantly inhibited the growth of melanoma cell lines. Furthermore, oral administration of 5mg/kg piplartine (PL), an alkaloid inhibited the growth of melanoma tumor xenograft by 84% as compared to control. The tumor suppression by PL was associated with inhibition of Gli2 and c-Myc as observed by western blot and immunohistochemistry analysis. The concentration of PL achieved in plasma was 8.5μM whereas in tumor was 6μmol/gm as analyzed by LC-MS/MS. Interestingly, the IC50 of PL at 48 hours was around 3-4μM in a panel of 15 melanoma cells lines, correlating well with the concentrations achieved in vivo. Melanoma cells treated with 2.5, 5 and 7.5μM PL for 48 hours induced significant apoptosis, with concomitant inhibition of Gli2 as well as c-Myc. Gli2 overexpression and treatment with Shh ligand significantly blocked PL mediated inhibition of cell proliferation and induction of apoptosis, establishing the role of Gli2 in PL-mediated growth suppression of melanoma cells. PL inhibited nuclear translocation as well as DNA binding of Gli2 as observed by analysis of Gli2 in nuclear fraction and Chromatin Immunoprecipitation (ChIP) assay, indicating the importance of Gli2 transcriptional activity in melanoma cell proliferation. In conclusion, our results provide convincing evidence that Gli2 mediated induction of c-Myc plays an important in melanoma tumor growth and subsequently establishes that PL mediated inhibition of melanoma cells in vitro and in vivo is associated with inhibition of Gli2. [Supported in part by R01 grant CA129038 (to S.K.S) awarded by the National Cancer Institute]. Citation Format: Neel Manoj Fofaria, Sanjay K. Srivastava. Gli2 induces melanoma tumor growth and progression: A critical role of c-Myc. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3565. doi:10.1158/1538-7445.AM2015-3565

Abstract 2256: GLI2 inhibition suppresses metastasis of HER2+ breast cancer

Abstract Metastasis of breast cancer is a significant problem majorly in HER2 positive cancer, as there are no therapeutic targets known. Due to inherent complexity of the process, underlying mechanisms of metastasis are largely unknown. We have shown previously that HER2 overexpression enhances the metastatic potential of breast cancer cells. The results from our previous studies showed that a 22 fold increase in HER2 expression in MDA-MB-231 cells lead to several orders of magnitude increase in the metastasis of the MDA-MB-231 cells. However, HER2 inhibition alone did not significantly suppress metastasis. Hence we investigated the molecular changes induced by HER2 expression that may promote metastasis of breast cancer cells. Interestingly, we observed an increased expression of GLI2 in the metastatic tumors formed by HER2+ cells in the mice lungs. GLI2 is a transcription factor of sonic hedgehog pathway and aberrant signaling of this pathway has been implicated in several cancers. In addition, GLI2 is overexpressed in breast tumors and associated with poor prognosis in breast cancer patients. GLI2 was therefore silenced using shRNA to study its role in metastasis of MDA-MB-231 high HER2 (HH) breast cancer cells. We achieved about 66% knock down of GLI2 expression using GLI2 shRNA. Our results showed that silencing of GLI2 inhibited the migration of HH cells by more than 60%. Our results also demonstrated over 80% inhibition of the invasion of (HH) cells with silenced GLI2. Furthermore, HH luciferase expressing cells were transfected with GLI2 shRNA or scrambled shRNA and equal numbers of live cells were injected intravenously in athymic nude mice. The trajectory and growth of control and GLI2 silenced MDA-MB-231 HH cells were monitored by non-invasive IVIS imaging system. A drastic reduction of luminescence was observed in the lungs of mice injected with GLI2 silenced HH cells as compared to mice injected with control HH cells, indicating inhibition of metastasis. Taken together our results establish GLI2 as an important target for therapy in HER2+ metastatic breast cancer. Further mechanistic studies are in progress. [Supported in part by R01 grant CA129038 awarded to S.K.S. by the National Cancer Institute] Citation Format: Parul Gupta, Alok Ranjan, Neel M. Fofaria, Sanjay K. Srivastava. GLI2 inhibition suppresses metastasis of HER2+ breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2256. doi:10.1158/1538-7445.AM2015-2256

Abstract 1233: Inhibition of STAT-3 by piperlongumine induces anoikis, prevents tumor formation in pancreatic cancer in vitro and in vivo

Abstract Metastasis of pancreatic cancer is a major challenge leading to the failure of its therapy. Hence, novel strategies to prevent metastasis are of prime importance. Anoikis is an anchorage independent cell death. Resistance to anoikis is one of the key features of a metastatic cell. In the current study, we show induction of anoikis by piperlongumine, a component of black pepper, through inhibition of STAT-3. Treatment of various pancreatic cancer cell lines under low attachment conditions with piperlongumine reduced the anoikis resistance in a concentration-dependent manner. The decrease in anoikis resistance was associated with decrease in the expression of STAT-3 and its phosphorylation at Y705. Significant down-regulation of Bcl-2 and Mcl-1, which are under the transcriptional regulation of STAT-3, were also observed by piperlongumine treatment indicating the role of STAT-3 in anoikis. Concentration-dependent cleavage of poly (ADP-ribose) polymerase (PARP) was observed by piperlongumine treatment in these cells. IL-6 treatment not only increased the phophorylation of STAT-3 at Y05 but also abrogated the anoikis induced by piperlongumine in Panc-1 and HPAC cells. Silencing STAT-3 by shSTAT-3 sensitized BxPc-3 and Panc-1 cells to piperlongumine treatment. On the other hand, over-expression of STAT-3 in Panc-1 cells conferred resistance to anoikis induced by piperlongumine. STAT-3 -/- BxPc-3 cells were highly sensitive to anoikis upon piperlongumine treatment. Finally, our in vivo studies established that pancreatic cancer cells treated with piperlongumine in suspension condition lost their potential to form tumors in xenograft model. On the other hand, untreated cells in suspension conditions formed aggressive tumors. Moreover, cells treated with PL in suspension conditions completely failed to metastasize when injected intravenously in mice whereas the untreated cells extensively metastasized in lungs and liver. The presence of metastatic tumors was detected by bioluminescence imaging as well as immunohistochemistry. Taken together, our study suggests that the decrease in anoikis resistance induced by piperlongumine was mediated by STAT-3 inhibition which resulted in reduced tumorigenic as well as metastatic potential of pancreatic cancer cells. Notably, STAT-3 has been shown to be constitutively upregulated in pancreatic cancer rendering it resistant to chemotherapy and potential to metastasize. [Supported in part by R01 grant CA129038 (to S.K.S) awarded by the National Cancer Institute]. Citation Format: Neel Manoj Fofaria, Sanjay K. Srivastava. Inhibition of STAT-3 by piperlongumine induces anoikis, prevents tumor formation in pancreatic cancer in vitro and in vivo. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1233. doi:10.1158/1538-7445.AM2014-1233

Abstract 2090: Novel role of STAT-3 in anoikis resistance and metastasis in melanoma cells in vitro and in vivo

Abstract Anoikis is an anchorage-independent cell death. Resistance to anoikis is one of the key features of metastatic cells. The role of STAT-3 in anoikis resistance in various melanoma cells was analyzed in the current study. Significant proportion of SK-MEL-28, SK-MEL-2, MeWo, SK-MEL-5 and B16-F0 cells resisted anoikis when grown under anchorage-independent conditions. Cells that resisted anoikis were observed to a have higher rate of migration and invasion as compared to the cells grown under anchorage dependent conditions. The anoikis resistant cells also exhibited significantly higher expression and phosphorylation of STAT-3 at Tyr 705 than the cells that were attached to the basement membrane. Treatment of these cells with IL-6, a cytokine which phosphorylates STAT-3, prevented the induction of anoikis. STAT-3 inhibitors AG490 and PL induced anoikis in a concentration-dependent manner in anoikis resistant cells, whereas IL-6 blocked anoikis. Over-expression of STAT-3 by transfection in SK-MEL-28 and SK-MEL-2 cells significantly increased the anoikis resistance, confirming the role of STAT-3 in anoikis resistance. On the other hand, silencing STAT-3 in SK-MEL-28, SK-MEL-2, SK-MEL-5 and MeWo cells decreased the potential of cancer cells to resist anoikis. Furthermore, STAT-3 (-/-) melanoma cells were more sensitive to anoikis, as compared to cells overexpressing STAT-3 (+/+) cells. STAT-3 (-/-) SK-MEL-28 cells and the SK-MEL-28 cells that were treated with PL completely failed to develop tumors when injected subcutaneously in immune-compromised mice. Moreover, these cells also failed to metastasize when injected intravenously. On the other hand, anoikis resistant wild type cells not only formed aggressive tumors subcutaneously but also highly metastasized when injected intravenously. The metastasis was confirmed by H & E staining. In summary, our results establish STAT-3 as a critical player that renders anoikis resistance to pancreatic cancer cells and enhance their metastasis potential. [Supported in part by R01 grant CA129038 (to S.K.S) awarded by the National Cancer Institute]. Citation Format: Neel Manoj Fofaria, Sanjay K. Srivastava. Novel role of STAT-3 in anoikis resistance and metastasis in melanoma cells in vitro and in vivo. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2090. doi:10.1158/1538-7445.AM2014-2090

Abstract 1046: Molecular link between HER2 and epithelial-to-mesenchymal transition in breast cancer

Abstract Breast cancer is a second most lethal form of cancer. Metastasis is responsible for about 90% of cancer- related deaths. Metastasis is a multi-step process and epithelial to mesenchymal transition (EMT) is the initiating step. HER2 is overexpressed in about 25-30% of breast cancer patients and responsible for poor prognosis. Our results show a novel role of HER2 in inducing EMT in breast cancer cells. To delineate the mechanism of EMT and role of HER2, we used MDA-MB-231, MCF-7 breast cancer cells with their syngeneic variants having stable HER2 overexpression (referred to as high HER2 or HH cells) and 4T-1 cells. The Boyden's chamber assay showed that HH cells were more invasive than the parent cells. The higher invasive potential of cells was accompanied with increased expression of mesenchymal markers like N-cadherin, along with suppression of epithelial markers like Cytokeratin-18. HER2 overexpression was also associated with enhanced expression of transcription factors like SNAIL and ZEB-1 that orchestrate the process of EMT. It is known that TGFβ is a master regulator of EMT process in cancer cells. Interestingly, HH cells exhibited higher levels of TGFβ protein and mRNA levels along with increased expression of SMADs relative to the parent cell lines. SMADs are the classical downstream effector molecules for TGFβ. Hence these results indicate that HER2 overexpression modulates TGFβ signaling to promote the process of EMT. In addition, induction of SNAIL, TGFβ and SMAD4 by heregulin treatment further established the role of HER2 in EMT process. To confirm the requirement of HER2 in the maintenance of mesenchymal traits, we silenced HER2 in MDA-MB-231 and MDA-MB-231 (HH) cells. HER2 silencing suppressed the invasion and mesenchymal markers indicating a direct connection of EMT with HER2 in breast cancer cells. Taken together our results for the first time provides a molecular evidence for the regulation of EMT by HER2 through TGFβ signaling. Further mechanistic and pre-clinical in vivo studies are under progress. [Supported in part by R01 grant CA129038 (to S.K.S) awarded by the National Cancer Institute]. Citation Format: Parul Gupta, Sanjay K. Srivastava. Molecular link between HER2 and epithelial-to-mesenchymal transition in breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1046. doi:10.1158/1538-7445.AM2014-1046

Abstract 1966: A unique cross-talk between HER2 and sonic hedgehog signaling promotes anoikis resistance in breast cancer cells

Abstract Metastatic cancer cells possess a unique capacity to survive without appropriate signaling from the extracellular matrix (ECM). The epithelial cells surrounded by proper ECM within tissues are designed to grow and differentiate whereas epithelial cells lacking suitable EMC are destined to anoikis, a form of apoptosis. The metastatic cancer cells resist anoikis to survive under the absence of ECM. Current study identifies a novel interplay between HER2 and sonic hedgehog signaling in promoting anoikis resistance. MDA-MB-231 cells exhibited an up-regulation of HER2 expression in cells cultured under anchorage- independent conditions as compared to adherent cells. Similar phenomenon was also observed in 4T-1 cells. The survival of MDA-MB-231 cells with stable HER2 overexpression (HH) was significantly enhanced when cultured under anchorage-independent conditions. However, the enhanced survival of MDA-MB-231 (HH) was reduced when HER2 was silenced using shRNA. These observations indicate the obligatory role of HER2 in anoikis resistance. Furthermore cells grown under anchorage-independent conditions induced the expression of sonic hedgehog (SHH). Increase in SHH was accompanied with enhanced expressions of PTCH2 and GLI2, the receptor and subsequent transcription factors of the sonic hedgehog signaling. Our results further showed that the induction of SHH was significantly higher in MDA-MB-231 (HH) cells relative to parent cells when cultured under anchorage-independent conditions, suggesting a correlation between HER2 and SHH signaling. Treatment of MDA-MB-231 and 4T-1 cells with cyclopamine (SHH signaling inhibitor) reduced the survival of cells under anchorage-independent conditions. Interestingly, cyclopamine treatment also inhibited the induction of HER2 during anchorage-independent culture conditions. Taken together, our study suggests a SHH-mediated induction of HER2 to promote anoikis resistance in breast cancer cells. Further mechanistic and in vivo studies are under progress. [Supported in part by R01 grant CA129038 (to S.K.S) awarded by the National Cancer Institute]. Citation Format: Parul Gupta, Sanjay K. Srivastava. A unique cross-talk between HER2 and sonic hedgehog signaling promotes anoikis resistance in breast cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1966. doi:10.1158/1538-7445.AM2014-1966

Abstract 1251: Repurposing antipsychotic drug Penfluridol for cancer treatment

Abstract Repurposing of old drugs as new anti-cancer drugs is important as it can save time and cost of drug development. Penfluridol is a first generation, highly potent antipsychotic drug. In this study, we determined the anticancer effects of Penfluridol in pancreatic cancer cells. Penfluridol treatment inhibited the growth of Panc-1, BxPC-3 and AsPC-1 pancreatic cancer cells in a concentration-dependent manner. Panc-1 , BxPC-3 and AsPC-1 cells treated with Penfluridol exhibited apoptosis as evaluated by Annexin/FITC assay and cleavage of caspase-3 and PARP. Our results showed that Penfluridol treatment induced ER stress in pancreatic cancer cells through the up regulation of ER stress markers like Bip/Grp78, CHOP and IRE. Penfluridol treatment also induced autophagy in pancreatic cancer cells as observed by acridine orange assay. Western blot analysis of Panc-1, BxPC-3 and AsPC-1 cells treated with Penfluridol exhibited up regulation of autophagy markers like LC3B, and p62. Microscopic analysis revealed punctate LC3B after Penfluridol treatment in pancreatic cancer cells confirming autophagy. Further mechanistic studies are in progress to establish the role of ER stress and autophagy in pancreatic cancer cells and correlate it with cell growth suppression. [Supported in part by R01 grant CA129038 awarded by NIH.] Citation Format: Alok Ranjan, Sanjay K. Srivastava. Repurposing antipsychotic drug Penfluridol for cancer treatment. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1251. doi:10.1158/1538-7445.AM2014-1251

Abstract 4115: GS-19, a novel GSK inhibitor suppresses the growth of pancreatic cancer cells by inhibiting EGFR/AKT/STAT-3 signaling

Abstract Pancreatic cancer remains the fourth leading cause of cancer related deaths in the United States. Therefore, identification of novel strategies to control and treat pancreatic cancer is of great importance. GS-19 is a novel compound recently found to be a GSK inhibitor. In this study, we determined the anti-cancer effects of GS-19 in pancreatic cancer cells. GS-19 treatment inhibited the growth of Panc-1 and BxPC-3 cells in a concentration-dependent manner. Both the cell lines treated with GS-19 exhibited G-1 cell cycle arrest as well as apoptosis. Western blot analysis of both BxPC-3 and Panc-1 cells treated with GS-19 inhibited the activation (phosphorylation) and expression of key survival signaling proteins such as EGFR, STAT3 and AKT. The apoptosis induced by GS-19 was associated with cleavage of caspase-3 and PARP. Taken together, our results suggest that GS-19 suppress pancreatic cancer cell growth by inhibiting EGFR/AKT/STAT-3 signaling. However, further mechanistic studies are in progress to establish the role of EGFR/AKT/STAT-3 signaling in pancreatic cancer cells. [The studies in part were supported by R01 grant CA129038 awarded by NIH to S.K.S.] Citation Format: Alok Ranjan, Ashlee Birkenfeld, Jianyu Lu, Duy Hua, Sanjay K. Srivastava. GS-19, a novel GSK inhibitor suppresses the growth of pancreatic cancer cells by inhibiting EGFR/AKT/STAT-3 signaling. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4115. doi:10.1158/1538-7445.AM2014-4115