Vivek K. Kashyap

Ormeloxifene suppresses prostate tumor growth and metastatic phenotypes via inhibition of oncogenic β-catenin signaling and EMT progression

Ormeloxifene is a clinically approved selective estrogen receptor modulator, which has also shown excellent anticancer activity, thus it can be an ideal repurposing pharmacophore. Herein, we report therapeutic effects of ormeloxifene on prostate cancer and elucidate a novel molecular mechanism of its anticancer activity. Ormeloxifene treatment inhibited epithelial-to-mesenchymal transition (EMT) process as evident by repression of N-cadherin, Slug, Snail, vimentin, MMPs (MMP2 and MMP3), β-catenin/TCF-4 transcriptional activity, and induced the expression of pGSK3β. In molecular docking analysis, ormeloxifene showed proficient docking with β-catenin and GSK3β. In addition, ormeloxifene induced apoptosis, inhibited growth and metastatic potential of prostate cancer cells and arrested cell cycle in G0–G1 phase via modulation of cell-cycle regulatory proteins (inhibition of Mcl-1, cyclin D1, and CDK4 and induction of p21 and p27). In functional assays, ormeloxifene remarkably reduced tumorigenic, migratory, and invasive potential of prostate cancer cells. In addition, ormeloxifene treatment significantly (P < 0.01) regressed the prostate tumor growth in the xenograft mouse model while administered through intraperitoneal route (250 μg/mouse, three times a week). These molecular effects of ormeloxifene were also observed in excised tumor tissues as shown by immunohistochemistry analysis. Our results, for the first time, demonstrate repurposing potential of ormeloxifene as an anticancer drug for the treatment of advanced stage metastatic prostate cancer through a novel molecular mechanism involving β-catenin and EMT pathway. Mol Cancer Ther; 16(10); 2267–80. ©2017 AACR.

Antibody-Drug Conjugates for Cancer Therapy: Chemistry to Clinical Implications

Chemotherapy is one of the major therapeutic options for cancer treatment. Chemotherapy is often associated with a low therapeutic window due to its poor specificity towards tumor cells/tissues. Antibody-drug conjugate (ADC) technology may provide a potentially new therapeutic solution for cancer treatment. ADC technology uses an antibody-mediated delivery of cytotoxic drugs to the tumors in a targeted manner, while sparing normal cells. Such a targeted approach can improve the tumor-to-normal tissue selectivity and specificity in chemotherapy. Considering its importance in cancer treatment, we aim to review recent efforts for the design and development of ADCs. ADCs are mainly composed of an antibody, a cytotoxic payload, and a linker, which can offer selectivity against tumors, anti-cancer activity, and stability in systemic circulation. Therefore, we have reviewed recent updates and principal considerations behind ADC designs, which are not only based on the identification of target antigen, cytotoxic drug, and linker, but also on the drug-linker chemistry and conjugation site at the antibody. Our review focuses on site-specific conjugation methods for producing homogenous ADCs with constant drug-antibody ratio (DAR) in order to tackle several drawbacks that exists in conventional conjugation methods.

MUC13 contributes to rewiring of glucose metabolism in pancreatic cancer

Pancreatic tumors are rewired for high-glucose metabolism and typically present with exceptionally poor prognosis. Recently, we have shown that MUC13, which is highly expressed in pancreatic tumors, promotes tumor progression via modulation of HER2 receptor tyrosine kinase activity. Herein, we investigate a novel, MUC13-mediated molecular mechanism responsible for higher glucose metabolism in pancreatic tumors. Our results demonstrate that MUC13 expression leads to the activation/nuclear translocation of NF-κB p65 and phosphorylation of IκB, which in turn upregulates the expression of important proteins (Glut-1, c-Myc, and Bcl-2) that are involved in glucose metabolism. MUC13 functionally interacts and stabilizes Glut-1 to instigate downstream events responsible for higher glucose uptake in pancreatic cancer cells. Altered MUC13 expression by overexpression and knockdown techniques effectively modulated glucose uptake, lactate secretion, and metastatic phenotypes in pancreatic cancer cells. NF-κB inhibitor, Sulfasalazine, abrogates the MUC13 and Glut-1 interaction, and attenuates events associated with MUC13-induced glucose metabolism. Pancreatic ductal adenocarcinoma (PDAC) patient tissue samples also show a positive correlation between the expression of these two proteins. These results delineate how MUC13 rewire aberrant glucose metabolism to enhance aggressiveness of pancreatic cancer and revealed a novel mechanism to develop newer therapeutic strategies for this exceptionally difficult cancer.

Tannic acid induces endoplasmic reticulum stress-mediated apoptosis in prostate cancer

Endoplasmic reticulum (ER) stress is an intriguing target with significant clinical importance in chemotherapy. Interference with ER functions can lead to the accumulation of unfolded proteins, as detected by transmembrane sensors that instigate the unfolded protein response (UPR). Therefore, controlling induced UPR via ER stress with natural compounds could be a novel therapeutic strategy for the management of prostate cancer. Tannic acid (a naturally occurring polyphenol) was used to examine the ER stress mediated UPR pathway in prostate cancer cells. Tannic acid treatment inhibited the growth, clonogenic, invasive, and migratory potential of prostate cancer cells. Tannic acid demonstrated activation of ER stress response (Protein kinase R-like endoplasmic reticulum kinase (PERK) and inositol requiring enzyme 1 (IRE1)) and altered its regulatory proteins (ATF4, Bip, and PDI) expression. Tannic acid treatment affirmed upregulation of apoptosis-associated markers (Bak, Bim, cleaved caspase 3, and cleaved PARP), while downregulation of pro-survival proteins (Bcl-2 and Bcl-xL). Tannic acid exhibited elevated G1 population, due to increase in p18INK4C and p21WAF1/CIP1 expression, while cyclin D1 expression was inhibited. Reduction of MMP2 and MMP9, and reinstated E-cadherin signifies the anti-metastatic potential of this compound. Altogether, these results demonstrate that tannic acid can promote apoptosis via the ER stress mediated UPR pathway, indicating a potential candidate for cancer treatment.

miRNA-205 Nanoformulation Sensitizes Prostate Cancer Cells to Chemotherapy

The therapeutic application of microRNA(s) in the field of cancer has generated significant attention in research. Previous studies have shown that miR-205 negatively regulates prostate cancer cell proliferation, metastasis, and drug resistance. However, the delivery of miR-205 is an unmet clinical need. Thus, the development of a viable nanoparticle platform to deliver miR-205 is highly sought. A novel magnetic nanoparticle (MNP)-based nanoplatform composed of an iron oxide core with poly(ethyleneimine)-poly(ethylene glycol) layer(s) was developed. An optimized nanoplatform composition was confirmed by examining the binding profiles of MNPs with miR-205 using agarose gel and fluorescence methods. The novel formulation was applied to prostate cancer cells for evaluating cellular uptake, miR-205 delivery, and anticancer, antimetastasis, and chemosensitization potentials against docetaxel treatment. The improved uptake and efficacy of formulations were studied with confocal imaging, flow cytometry, proliferation, clonogenicity, Western blot, q-RT-PCR, and chemosensitization assays. Our findings demonstrated that the miR-205 nanoplatform induces significant apoptosis and enhancing chemotherapeutic effects in prostate cancer cells. Overall, these study results provide a strong proof-of-concept for a novel nonviral-based nanoparticle protocol for effective microRNA delivery to prostate cancer cells.

Abstract LB-400: Tannic acid induces prostate cancer cell death via unfolded protein response (UPR) and modulation of CHOP

Objectives: Endoplasmic reticulum (ER) is an intricate organelle that is crucial for cellular function and survival. Cellular environments that interfere with ER functioning can lead to the accumulation of unfolded proteins, which are sensed by transmembrane sensors that instigate the unfolded protein response (UPR) to reinstate ER proteostasis. When the UPR is perturbed or not sufficient to deal with the stress conditions, apoptotic cell death is ensued. Often, prostate tumor cells (PrCa) are exposed to intrinsic and external factors that alter protein homeostasis producing endoplasmic reticulum (ER) stress. IRE1, PERK, and ATF6 are key signaling stems of the UPR during ER stress. At molecular level, dimerization of PERK kinase phosphorylates eIF2α, resulting in translation attenuation. This selectively enhances translation of the ATF4 transcription factor. Also, activated IRE1 stimulates the nuclear translocation of XBP1. Both ATF4 and XBP1 induces expression of UPR genes. The UPR stimulates and protects tumor cells against stressful conditions within the tumor microenvironment. Therapeutic intervention on this aspect ER Stress mediated apoptosis is much needed. Here, we examined the mechanistic role of tannic acid (TA) through novel targeting inhibition of UPR via modulation of ER stress in PrCa cell death. Methods:In vitro therapeutic perspective of tannic acid was evaluated using clinically relevant human prostate cancer cell line models (C4-2, PC3 and DU145) through cell proliferation and clonogenic assays. The anti-metastatic potential of TA in PrCa cells was determined using invasive and migration studies. The phase arrest and concomitant apoptosis in PrCa cells were examined using Propidium Iodide based flow cytometric studies. TA induced ER stress-mediated UPR cellular apoptotic activity was confirmed using Western blot, real-time studies, and nuclear distortion studies. Results: Protein profiling results have showed yin approach of inhibition ER stress during TA treatments with the inhibition of CHOP protein. Also, we observed TA treated PrCa cells was able to induce ER stress response proteins, such as PERK and IRE1. Its downstream signaling events include the induction of XBP1, EIF2α expression key mediators by alteration of Bcl2/Bax ratio from shifting survival towards apoptosis. With this line we assessed the expression profile of apoptosis-associated markers such as Bid and Bim (upregulated), and Bcl-2 and Bcl-xL which ratifies the induction of apoptosis during treatments. TA exhibited prominent growth arrest at G1 phase and increase of concentration led to elevated sub G1, annotating the increase of apoptotic cells. The expression of P18 and P21 were increased during TA treatments and cyclin D1 expression was inhibited supporting the G1 phase cell arrest. The expression of MMP2 and MMP9 were reduced, signifying the superior anti-metastatic ability of this compound. Cell proliferation studies showed a dose-dependent inhibitory profile of TA. Distinct results of anti-migratory and anti-metastatic effects of TA was observed during treatments. Conclusion: This study suggests that TA can efficiently induce UPR and promote ER stress signaling in cells that renders apoptosis in PrCa cells by the over expression of CHOP. We believe this novel therapeutic paradigm have clinical significance in advanced PrCa. Citation Format: Elham Hatami, Prashanth Kumar Bhusetty Nagesh, Pallabita Chowdhury, Vivek K. Kashyap, Sheema Khan, Bilal Hafeez, Meena Jaggi, Subhash C. Chauhan, Murali M. Yallapu. Tannic acid induces prostate cancer cell death via unfolded protein response (UPR) and modulation of CHOP [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 LB-400.

Abstract 3216: Attenuation of pancreatic tumor growth by a small molecule tubulin inhibitor

Introduction: Pancreatic cancer (PanCa) is one of the most fatal cancers and is ranked as the fourth common cause of cancer-related deaths among both men and women in the US. The management of PanCa is exceptionally difficult due to the extremely poor response to available chemotherapeutic drugs. Microtubules are dynamic structures composed of α-β-tubulin heterodimers that are essential in cell division and are important targets for several clinical drugs (paclitaxel, docetaxel and vinblastine). However, clinical use of these tubulin-targeting drugs have toxicity and drug resistance issues in cancer patients. Thus, identification of more potent non-toxic inhibitors of β-tubulin is urgently required for cancer therapy purposes. In this study, we have identified a synthetic compound (ABI-231) which is a potent inhibitor of β-tubulin and evaluated its therapeutic efficacy against PanCa in vitro, and in vivo model systems. Methods: ABI-231 ((2-(1H-indol-3-yl)-1H-imidazol-4-yl) (3, 4, 5-trimethoxyphenyl)) - methanone was synthesized and characterized in our department. Effect of ABI-231 on proliferation, migration and invasion of human PanCa cells (ASPC1, HPAFII, and PANC1) was performed by in vitro functional assays (MTS, wound healing, and Boyden chamber migrations). Effect of ABI-231 on the expression of β-tubulin isoforms was determined and compared with other clinical inhibitors of β-tubulin by Western blot, and qRT-PCR. Moreover, the effect of ABI-231 on the expression of β-tubulin III in PanCa cells was determined by confocal microscopy. Therapeutic efficacy of ABI-231 against PanCa was evaluated in an ectopic xenograft mouse model. Results: ABI-231 treatment inhibited cell proliferation, invasion, migration and colony formation abilities of PanCa cells in a dose-dependent manner (1-100 nM) compared to vehicle treated group. Aberrant expression of β-tubulin III is involved in aggressiveness and drug resistance of various type of cancers including PanCa. ABI-231 effectively inhibited the protein levels and mRNA expression of total β-tubulin (TBB), TBB1, TBB2c, TBB3 and TBB4 in PanCa cells via destabilization. Our confocal microscopy results further showed inhibition of β-tubulin in ABI-231 treated PanCa cells. Upregulation of micro RNA 200c (miR-200c) has been shown to inhibit the expression of β-tubulin III in cancer cells. ABI-231 treatment of PanCa cells showed significant (p Conclusion: Taken together, our results suggest that ABI-231 is a potent β-tubulin inhibitor and chemotherapeutic agent which could be used for the treatment of pancreatic cancer. Citation Format: Vivek K. Kashyap, Bilal B. Hafeez, Qinghui Wang, Saini Setua, Andrew Massey, Aditya Ganju, Murali M. Yallapu, Duane D. Miller, Wei Li, Meena Jaggi, Subhash C. Chauhan. Attenuation of pancreatic tumor growth by a small molecule tubulin inhibitor [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 3216. doi:10.1158/1538-7445.AM2017-3216

Abstract IA30: Comorbidity factors associated with human papillomavirus infectivity: Implications in cervical cancer health disparity

Objective: High-risk strains of human papillomavirus (HPV), HPV E6/E7 cause cervical cancer (CxCa). Certain underserved populations in the United States, such as American Indian and African American women disproportionately suffer from CxCa compared to their Caucasian counter parts. However, precise etiology and comorbidity factors associated with CxCa health disparity are not fully uncovered. Understanding of these factors at molecular level will entail developing novel strategies to reduce this health disparity. In this study, we have investigated the molecular interplay existing between various comorbidity factors, namely, smoking, alcohol and HIV co-infection on the HPV infectivity which are primarily known for the progression of CxCa. Method: In order to define a molecular association of smoking, alcohol and HIV co-infection with CxCa, Caski and SiHa (HPV infected cervical cancer cells) cells were treated with a smoking carcinogens Benzo[a]Pyrene (BaP) or alcohol (EthOH) or both for different time periods. Effects of these treatment was analyzed on cell proliferation, clonogenicity, cell migration, cell cycle and the expression of HPV E6/E7 was determined by qRT-PCR, immunoblotting and confocal microscopy. The effect of HIV co-infection on the expression of HPV E6/E7 was also investigated by incubating CxCa cells with conditioned medium derived from HIV infected U937 monocytic cells (U1). Additionally, we examined effect of these cofactors on the expression enzymes associated with cellular oxidative stress using immunoblotting and confocal microscopy analyses. Results: Our results show that the exposure of BaP or EthOH or their combination enhances the expression of HPV E6/E7 oncogenes. Additionally, cells treated with BaP and EthOH alone or in combination show higher oncogenic phenotypes as evident by increased cell proliferation, clonogenicity and cell migration and invasion. These cofactors in presence of HIV co-infection also augment the expression of HPVE6/E7 oncogenes. Exposure of these cofactors alter cellular oxidative stress via modulation of the expression of PRDX6 enzyme. Interestingly, curcumin and its nanoparticle formulation (Nano-Cur) effectively inhibit BaP/EthOH induced expression of E6/E7 oncogenes, tumorigenic characteristics of CxCa cells and induce apoptosis. Conclusions: The study suggests a molecular link between smoking, alcohol and HIV infection with HPV infectivity and their potential association with CxCa health disparity. These events however, can be effectively attenuated by curcumin/nano-curcumin treatment, implying its role in CxCa prevention/treatment. This provides hope for developing a feasible approach to effectively reduce CxCa health disparity among underserved populations. Citation Format: Vivek K. Kashyap, Sheema Khan, Mohammad Sikander, Diane M. Maher, Samtosh K. Kumar, Namita Sinha, Murali M. Yallapu, Nadeem Zafar, Meena Jaggi, Subhash C. Chauhan. Comorbidity factors associated with human papillomavirus infectivity: Implications in cervical cancer health disparity. [abstract]. In: Proceedings of the Ninth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2016 Sep 25-28; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2017;26(2 Suppl):Abstract nr IA30.

Abstract 2208: Novel nanoparticle formulation of Plumbagin for pancreatic cancer treatment

Pancreatic cancer (PanCa) is one of the most fatal of all cancers and is ranked as the fourth most common cause of cancer related deaths among both men and women in the US. The management of PanCa, is exceptionally difficult due to the extremely poor response to available therapeutic modalities. Highly desmoplastic microenvironment in pancreatic tumor causes suboptimal drug delivery and increases chemo-resistance. Plumbagin (PL), a naturally occurring napthoquinone derived from the root of Plumbago zeylanica L., has showed potent cancer preventive and therapeutic activity against variety of cancers. However, the clinical translation of PL has been significantly hampered due to its toxicity and suboptimal bioavailability. To address these clinically relevant issues, we have developed and characterized a novel PL-loaded magnetic nanoparticle (MNP-PL) formulation. This MNP-PL formulation was prepared using Magnetic nanoparticles (MNPs) composed of an iron oxide core which has distinct advantages in i) bio/hemo-compatibility, ii) biodegradation, iii) higher drug loading capacity and iv) improved bioavailability. Our novel MNP-PL formulation provided average size of 125 nm in dynamic light scattering (DLS) and exhibited -9.42 to -10.79 mV zeta potential with an outstanding PL loading efficiency. We have evaluated anti-cancer potential of MNP-PL formulation in human PanCa cells (HPAF-II, AsPc1 and Panc-1). We first performed MTS and colony formation assays to determine the effects of free PL and MNP-PL formulation on growth of PanCa cells. In this experiment, cells were treated with various concentrations of free PL (1-15 μM) or MNP-PL (1-15 μM) for 24 hrs. Results exhibited efficient internalization of the MNP-PL formulation in a dose-dependent manner. As a result, the MNP-PL formulation showed four fold dose advantage over free PL. IC50 of free PL was recorded 10 μM which was significantly reduced to 2.5 μM in MNP-PL. MNP-PL also showed four fold inhibition in colony formation compared to free PL. MNP-PL treatment more efficiently inhibited oncogenic CXCL12/CXCR4 signaling pathway in both PanCa and patient derived stromal fibroblast cells. MNP-PL treatment also showed decreased expression of CXCR4 protein levels in PanCa cells. Moreover, MNP-PL treatment inhibited stromal derived factor 1 (SDF-1)/CXCL12 expression in stromal fibroblasts. These results suggest that our novel MNP-PL formulation has more anti-cancer potential than free PL against PanCa. In conclusion, MNP-PL formulation may reduce the toxicity and improve the bioavailability of free PL and could be used for the treatment of PanCa. Citation Format: Bilal B. Hafeez, Vivek K. Kashyap, Vijayakumar N. Boya, Aditya Ganju, Mohammad Sikander, Murali M. Yallapu, Meena Jaggi, Subhash C. Chauhan. Novel nanoparticle formulation of Plumbagin for pancreatic cancer treatment. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2208.

Abstract 1745: Comorbidity factors associated with human papillomavirus infectivity: Implications in cervical cancer health disparity

Objective: High-risk strains of human papillomavirus (HPV), HPV E6/E7 cause cervical cancer (CxCa). Certain underserved populations in the United States, such as American Indian and African American women disproportionately suffer from CxCa compared to their Caucasian counter parts. However, precise etiology and comorbidity factors associated with CxCa health disparity are not fully uncovered. Understanding of these factors at molecular level will entail developing novel strategies to reduce this health disparity. In this study, we have investigated the molecular interplay existing between various comorbidity factors, namely, smoking, alcohol and HIV co-infection on the HPV infectivity which are primarily known for the progression of CxCa. Method: In order to define a molecular association of smoking, alcohol and HIV co-infection with CxCa, Caski and SiHa (HPV infected cervical cancer cells) cells were treated with a smoking carcinogens Benzo[a]Pyrene (BaP) or alcohol (EthOH) or both for different time periods. Effects of these treatment was analyzed on cell proliferation, clonogenicity, cell migration, cell cycle and the expression of HPV E6/E7 was determined by qRT-PCR, immunoblotting and confocal microscopy. The effect of HIV co-infection on the expression of HPV E6/E7 was also investigated by incubating CxCa cells with conditioned medium derived from HIV infected U937 monocytic cells (U1). Additionally, we examined effect of these cofactors on the expression enzymes associated with cellular oxidative stress using immunoblotting and confocal microscopy analyses. Result: Our results show that the exposure of BaP or EthOH or their combination enhances the expression of HPV E6/E7 oncogenes. Additionally, cells treated with BaP and EthOH alone or in combination show higher oncogenic phenotypes as evident by increased cell proliferation, clonogenicity and cell migration andinvasion. These cofactors in presence of HIV co-infection also augment the expression of HPVE6/E7 oncogenes. Exposure of these cofactors alter cellular oxidative stress via modulation of the expression of PRDX6 enzyme. Interestingly, curcumin and its nanoparticle formulation (Nano-Cur) effectively inhibits BaP/EthOH induced expression of E6/E7 oncogenes, growth, migration of CxCa cells and induces apoptosis. Conclusions: The study suggests a molecular link between smoking, alcohol and HIV infection with HPV infectivity and their potential association with CxCa health disparity. These events however, can be effectively attenuated by curcumin/nano-curcumin treatment, implying its role in CxCa prevention/treatment. This provides hope for developing a feasible approach to effectively reduce CxCa health disparity among underserved populations. Citation Format: Vivek K. Kashyap, Sheema Khan, Mohammad Sikander, Diane M. Maher, Santosh Kumar, Namita Sinha, Murali M. Yallapu, Nadeem Zafar, Meena Jaggi, Subhash C. Chauhan. Comorbidity factors associated with human papillomavirus infectivity: Implications in cervical cancer health disparity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1745.