Suprit Gupta

Guggulsterone decreases proliferation and metastatic behavior of pancreatic cancer cells by modulating JAK/STAT and Src/FAK signaling.

Inadequate efficacy, high toxicity and drug resistance associated with existing chemotherapeutic agents mandate a need for novel therapeutic strategies for highly aggressive Pancreatic Cancer (PC). Guggulsterone (GS) exhibits potent anti-proliferative effects against various cancer cells and has emerged as an attractive candidate for use in complementary or preventive cancer therapies. However, the knowledge regarding the therapeutic potential of GS in PC is still limited and needs to be explored. We studied the effect of GS on PC cell growth, motility and invasion and elucidated the molecular mechanisms associated with its anti-tumor effects. Treatment of Capan1 and CD18/HPAF PC cells with GS resulted in dose- and time-dependent growth inhibition and decreased colony formation. Further, GS treatment induced apoptosis and cell cycle arrest as assessed by Annexin-V assay and FACS analysis. Increased apoptosis following GS treatment was accompanied with Bad dephosphorylation and its translocation to the mitochondria, increased Caspase-3 activation, decreased Cyclin D1, Bcl-2 and xIAP expression. Additionally, GS treatment decreased motility and invasion of PC cells by disrupting cytoskeletal organization, inhibiting activation of FAK and Src signaling and decreased MMP9 expression. More importantly, GS treatment decreased mucin MUC4 expression in Capan1 and CD18/HPAF cells through transcriptional regulation by inhibiting Jak/STAT pathway. In conclusion, our results support the utility of GS as a potential therapeutic agent for lethal PC.

Unbiased analysis of pancreatic cancer radiation resistance reveals cholesterol biosynthesis as a novel target for radiosensitisation

Background:Despite its promise as a highly useful therapy for pancreatic cancer (PC), the addition of external beam radiation therapy to PC treatment has shown varying success in clinical trials. Understanding PC radioresistance and discovery of methods to sensitise PC to radiation will increase patient survival and improve quality of life. In this study, we identified PC radioresistance-associated pathways using global, unbiased techniques.Methods:Radioresistant cells were generated by sequential irradiation and recovery, and global genome cDNA microarray analysis was performed to identify differentially expressed genes in radiosensitive and radioresistant cells. Ingenuity pathway analysis was performed to discover cellular pathways and functions associated with differential radioresponse and identify potential small-molecule inhibitors for radiosensitisation. The expression of FDPS, one of the most differentially expressed genes, was determined in human PC tissues by IHC and the impact of its pharmacological inhibition with zoledronic acid (ZOL, Zometa) on radiosensitivity was determined by colony-forming assays. The radiosensitising effect of Zol in vivo was determined using allograft transplantation mouse model.Results:Microarray analysis indicated that 11 genes (FDPS, ACAT2, AG2, CLDN7, DHCR7, ELFN2, FASN, SC4MOL, SIX6, SLC12A2, and SQLE) were consistently associated with radioresistance in the cell lines, a majority of which are involved in cholesterol biosynthesis. We demonstrated that knockdown of farnesyl diphosphate synthase (FDPS), a branchpoint enzyme of the cholesterol synthesis pathway, radiosensitised PC cells. FDPS was significantly overexpressed in human PC tumour tissues compared with healthy pancreas samples. Also, pharmacologic inhibition of FDPS by ZOL radiosensitised PC cell lines, with a radiation enhancement ratio between 1.26 and 1.5. Further, ZOL treatment resulted in radiosensitisation of PC tumours in an allograft mouse model.Conclusions:Unbiased pathway analysis of radioresistance allowed for the discovery of novel pathways associated with resistance to ionising radiation in PC. Specifically, our analysis indicates the importance of the cholesterol synthesis pathway in PC radioresistance. Further, a novel radiosensitiser, ZOL, showed promising results and warrants further study into the universality of these findings in PC, as well as the true potential of this drug as a clinical radiosensitiser.

MUC16 contributes to the metastasis of pancreatic ductal adenocarcinoma through focal adhesion mediated signaling mechanism.

MUC16, a heavily glycosylated type-I transmembrane mucin is overexpressed in several cancers including pancreatic ductal adenocarcinoma (PDAC). Previously, we have shown that MUC16 is significantly overexpressed in human PDAC tissues. However, the functional consequences and its role in PDAC is poorly understood. Here, we show that MUC16 knockdown decreases PDAC cell proliferation, colony formation and migration in vitro. Also, MUC16 knockdown decreases the tumor formation and metastasis in orthotopic xenograft mouse model. Mechanistically, immunoprecipitation and immunofluorescence analyses confirms MUC16 interaction with galectin-3 and mesothelin in PDAC cells. Adhesion assay displayed decreased cell attachment of MUC16 knockdown cells with recombinant galectin-1 and galectin-3 protein. Further, CRISPR/Cas9-mediated MUC16 knockout cells show decreased tumor-associated carbohydrate antigens (T and Tn) in PDAC cells. Importantly, carbohydrate antigens were decreased in the region that corresponds to MUC16 and suggests for the decreased MUC16-galectin interactions. Co-immunoprecipitation also revealed a novel interaction between MUC16 and FAK in PDAC cells. Interestingly, we observed decreased expression of mesenchymal and increased expression of epithelial markers in MUC16-silenced cells. Additionally, MUC16 loss showed a decreased FAK-mediated Akt and ERK/MAPK activation. Altogether, these findings suggest that MUC16-focal adhesion signaling may play a critical role in facilitating PDAC growth and metastasis.

Afatinib radiosensitizes head and neck squamous cell carcinoma cells by targeting cancer stem cells

The dismal prognosis of locally advanced and metastatic squamous cell carcinoma of the head and neck (HNSCC) is primarily due to the development of resistance to chemoradiation therapy (CRT). Deregulation of Epidermal Growth Factor Receptor (EGFR) signaling is involved in HNSCC pathogenesis by regulating cell survival, cancer stem cells (CSCs), and resistance to CRT. Here we investigated the radiosensitizing activity of the pan-EGFR inhibitor afatinib in HNSCC in vitro and in vivo. Our results showed strong antiproliferative effects of afatinib in HNSCC SCC1 and SCC10B cells, compared to immortalized normal oral epithelial cells MOE1a and MOE1b. Comparative analysis revealed stronger antitumor effects with afatinib than observed with erlotinib. Furthermore, afatinib enhanced in vitro radiosensitivity of SCC1 and SCC10B cells by inducing mesenchymal to epithelial transition, G1 cell cycle arrest, and the attenuating ionizing radiation (IR)-induced activation of DNA double strand break repair (DSB) ATM/ATR/CHK2/BRCA1 pathway. Our studies also revealed the effect of afatinib on tumor sphere- and colony-forming capabilities of cancer stem cells (CSCs), and decreased IR-induced CSC population in SCC1 and SCC10B cells. Furthermore, we observed that a combination of afatinib with IR significantly reduced SCC1 xenograft tumors (median weight of 168.25 ± 20.85 mg; p = 0.05) compared to afatinib (280.07 ± 20.54 mg) or IR alone (324.91 ± 28.08 mg). Immunohistochemical analysis of SCC1 tumor xenografts demonstrated downregulation of the expression of IR-induced pEGFR1, ALDH1 and upregulation of phosphorylated γH2AX by afatinib. Overall, afatinib reduces tumorigenicity and radiosensitizes HNSCC cells. It holds promise for future clinical development as a novel radiosensitizer by improving CSC eradication.

Emerging Trends for Radioimmunotherapy in Solid Tumors

Due to its ability to target both known and occult lesions, radioimmunotherapy (RIT) is an attractive therapeutic modality for solid tumors. Poor tumor uptake and undesirable pharmacokinetics, however, have precluded the administration of radioimmunoconjugates at therapeutically relevant doses thereby limiting the clinical utility of RIT. In solid tumors, efficacy of RIT is further compromised by heterogeneities in blood flow, tumor stroma, expression of target antigens and radioresistance. As a result significant efforts have been invested toward developing strategies to overcome these impediments. Further, there is an emerging interest in exploiting short-range, high energy α-particle emitting radionuclides for the eradication of minimal residual and micrometastatic disease. As a result several modalities for localized therapy and models of minimal disease have been developed for preclinical evaluation. This review provides a brief update on the recent efforts toward improving the efficacy of RIT for solid tumors, and development of RIT strategies for minimal disease associated with solid tumors. Further, some of promising approaches to improve tumor targeting, which showed promise in the past, but have now been ignored are also discussed.

Antibody Labeling with Radioiodine and Radiometals

Antibodies have been conjugated to radionuclides for various in vitro and in vivo applications. Radiolabeled antibodies have been used in clinics and research for diagnostic applications both in vitro as reagents in bioassays and in vivo as imaging agents. Further, radiolabeled antibodies are used as direct therapeutic agents for cancer radioimmunotherapy or as tracers for studying the pharmacokinetics and biodistribution of therapeutic antibodies. Antibodies are labeled with radiohalogens or radiometals, and the choice of candidate radionuclides for a given application is dictated by their emission range and half-life. The conjugation chemistry for the coupling of MAbs with the radiometals requires a chelator, whereas radiohalogens can be incorporated directly in the antibody backbone. In this chapter, we describe the commonly used methods for radiolabeling and characterizing the antibodies most commonly used radiohalogens (125I/131I) and radiometals (177Lu/99mTc).

Abstract 1104: Novel specific RTK targeting of EGFR/FAK axis in glioblastoma invasion

Background: Glioblastoma (GBM) is the most aggressive primary brain tumor with a median survival rate of 14.6 months. Currently, the first-line treatment includes surgical resection, chemoradiation, and adjuvant chemotherapy with temozolomide. However, GBM recurs most often within 6.9 months. Receptor tyrosine kinases are dysregulated in GBM, with epidermal growth factor receptor (EGFR) representing 57.4% of the deleted/mutated GBM. In addition, 30 - 40% of GBM patients with EGFR amplification carry an oncogenic gene rearrangement EGFR variant III (EGFRvIII) which is constitutively active. Yet most EGFR inhibitors have shown very little clinical efficacy in GBM. Methods: Afatinib, blood-brain barrier penetrant pan-EGFR inhibitor, covalently binds and irreversibly inhibits signaling from EGFR. Afatinib also persistently inhibits ErbB homo and hetero-dimers. Using GBM cell lines U87MG and U87MG transfected with wild type EGFR, EGFRvIII and EGFRvIII with dead kinase domain, we evaluated the efficacy of afatinib alone and in combination with temozolomide. Results: Afatinib treatment resulted in a dose dependent decrease in the proliferation of U87MG cells transfected with EGFRvIII. The IC50 value for this cell line is 2µM (afatinib). 50µM temozolomide inhibited cell proliferation by 50%. We evaluated the combinational efficacy of IC25 of both. Afatinib effectively blocked EGFR signaling even 72 hours after treatment in the U87MG cell line transfected with either wild type EGFR or EGFRvIII. This was evidenced by decreased phosphorylation of EGFR (Tyr 1068) and its downstream signaling. Focal adhesion kinase (FAK) signaling in EGFRvIII expressing cells, largely responsible for the invasiveness of GBM, was abolished by afatinib. Furthermore, treatment with afatinib and temozolomide significantly decreased the in vitro tumorigenicity (anchorage dependent growth - colony formation assay as well as anchorage independent growth - soft agar assay) of EGFRvIII expressing GBM cells. Conclusion: Altogether, these results support synergistic efficacy of afatinib and temozolomide in EGFRvIII expressing GBM. Citation Format: Raghupathy Vengoji, Satyanarayana Rachagani, Suprit Gupta, Kavita Mallya, Maneesh Jain, Moorthy Ponnusamy, Surinder Batra, Nicole Shonka. Novel specific RTK targeting of EGFR/FAK axis in glioblastoma invasion [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 1104. doi:10.1158/1538-7445.AM2017-1104

Abstract 1629: Novel interaction of MUC16 with FAK activate EMT process and metastasis of pancreatic ductal adenocarcinoma

Proceedings: AACR 107th Annual Meeting 2016; April 16-20, 2016; New Orleans, LA Introduction: MUC16 is a heavily glycosylated, type I transmembrane mucin, which is over expressed in different cancers. We have previously shown that significant overexpression of MUC16 in human PDAC tissues with disease progression compared to normal pancreas. However, the functional consequences of MUC16 and their role in PDAC is poorly understood. Based on this our hypothesis is that MUC16 can drive pancreatic cancer metastasis through FAK-mediated Akt and ERK/MAPK signaling activation and altering EMT markers. Methods: We have developed MUC16 knockdown Capan1 and Colo-357 PDAC cells to study the functional impacts. Congenic cell survival, soft-agar colony formation, and trans-well chamber assays were performed to determine the in vitro tumorigenicity. Orthotopic implantation was carried out using capan-1 and colo-357 PDAC cells to determine the oncogenic and metastatic potential of MUC16. Binding assay was performed to determine the cell adhesion property of MUC16 in colo-357 cells. The physical interaction between MUC16 and mesothelin, galectin-3 and FAK were evaluated by confocal and immunoprecipitation analysis. Immunoblot analyses were performed to determine the downstream signaling in MUC16 knockdown cells. Results: MUC16 knockdown in capan-1 and colo-357 PDAC cell lines resulted in significantly decreased cell proliferation (P<0.05), colony formation (P<0.01), and migration (P<0.01) in vitro. Further, MUC16 knockdown capan-1 and colo-357 cells significantly decreases the tumor formation (P<0.05) and metastasis (liver P<0.05, spleen P<0.001, intestinal wall P<0.01, diaphragm P<0.01 and peritoneum P<0.001) in orthotopic xenograft mouse model. Adhesion assay displays decreased cell attachment of MUC16 knockdown cells with recombinant galectin-1 and galectin-3 proteins. Immunoprecipitation and immunofluorescence studies confirmed that MUC16 interaction with mesothelin and galectin-3 in PDAC cells. Co-immunoprecipitation revealed a novel interaction between MUC16 and FAK in PDAC cells. Interestingly, we observed decreased expression of mesenchymal markers (N-cadherin and Zeb1) and increased expression of epithelial markers (E-cadherin and CK18) in MUC16 silenced PDAC cells, correlating with the decrease in metastasis. Moreover, MUC16 knockdown show decreased FAK-mediated Akt and ERK/MAPK activation in PDAC cells. Conclusion: Overall our study concludes that MUC16 interacts with FAK leads to the activation of EMT markers for enhancing pancreatic cancer metastasis. Citation Format: Sakthivel Muniyan, Dhanya Haridas, Satyanarayana Rachagani, Imayavaramban Lakshmanan, Suprit Gupta, Seema Chugh, Parthasarathy Seshacharyulu, Moorthy P. Ponnusamy, Surinder K. Batra. Novel interaction of MUC16 with FAK activate EMT process and metastasis of pancreatic ductal adenocarcinoma. [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 1629.

Abstract 4191: Alterations in endothelin axis during pancreatic acinar to ductal metaplasia

Background: Acinar-ductal-metaplasia (ADM) is one the earliest recognizable alterations in the tumorigenesis of pancreatic ductal adenocarcinoma (PDAC). Inflammation-induced ADM, which is typically reversible upon removal of inflammatory insult, becomes irreversible in the presence of oncogenic K-Ras and progresses to Pancreatic Intraepithelial neoplastic lesions (PanINs) and subsequently PDAC. Endothelin (ET) axis comprising of endothelin isoforms (ET-1, ET-2 and ET-3) and two receptors A (ET A R) and B (ET B R), has been demonstrated to contribute to the pathobiology of pancreatitis and its components exhibit aberrant overexpression in pancreatic cancer PDAC. However, the expression patterns of ET axis in oncogene-associated early lesions remain unknown. We hypothesize that alterations in ET axis contribute to oncogene associated irreversible ADM. Thus, we studied expression pattern of ET axis in the pancreatic inflammation in the presence and absence of oncogenic KRas and in preneoplastic lesions. Methods: Expression of ET-1, ET A R and ET B R was analyzed in murine models of preneoplastic lesions [KC model: (Pdx1-Cre, Kras G12D) ) by IHC. To determine the changes in ET axis during ADM, wild type (WT) and KC mutant mice were treated with cerulein to induce pancreatitis and tissues collected at day 0, 2, 7 and 21 days post treatment were analyzed for mRNA and Immunofluorescence analysis. Expression pattern of ET axis components was also determined in the pancreas of KC and WT animals following exposure to cigarette smoke. Results: A progressive increase in the expression of ET-1, ET A R and ET B R was observed in the PanIN lesions in KC mice. In mice with WT Kras, cerulein treatment resulted in a notable increase in the expression of ET-1 and ET A R at day 2, while the levels of ET B R increased marginally; however a recovery to basal levels was observed for all three molecules by day 7. In contrast, significant increase (p A R and ET B R transcripts was observed following cerulein treatment and these levels continued to remain high even at 21 days post-trauma in KC mice. The changes in the expression ET A R and ET B R were corroborated by confocal microscopy. Cerulein treatment resulted in increased expression of ET A R and ET B R in acinar compartment in both WT and KC mice as indicated by their co-localization with amylase. In KC mice, cerulein induced acinar-to-ductal metaplasia and the resulting CK19 positive ductal components continued to express increased levels of both receptors with distinct magnitude and kinetics. Similarly, in the smoking models, ET-axis components exhibited a more robust and sustained overexpression in KC mice as compared to the WT. Conclusions: The expression of oncogenic Kras G12D results in an enhanced and sustained activation of ET-axis following inflammatory insults in the pancreatic tissues, suggesting its possible role in tumor initiation and progression. Citation Format: Suprit Gupta, Satyanarayana Rachagani, Sushil Kumar, Kavita Mallya, Surinder Kumar Batra, Maneesh Jain. Alterations in endothelin axis during pancreatic acinar to ductal metaplasia. [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 4191.

Erratum: MUC4 regulates cellular senescence in head and neck squamous cell carcinoma through p16/Rb pathway (Oncogene (2015) 34:2814 DOI:10.1038/onc.2015.116)

Correction to: Oncogene (2015) 34, 1698–1708; doi:10.1038/onc.2014.102; published online 21 April 2014 The authors wish to add H Farghaly, originally included in the Acknowledgements, to the authorship of this article, published online 21 April 2014. Dr Farghaly was omitted from the byline at final submission due to communication impediments outside the authors’ control during the manuscript review period which were shortly thereafter resolved.