Vindhya Palagani

Inhibition of hedgehog signaling attenuates carcinogenesis in vitro and increases necrosis of cholangiocellular carcinoma

The Hedgehog signaling pathway plays a pivotal role during embryonic development, stem cell maintenance, and wound healing. Hedgehog signaling also is deregulated in many cancers. However, the role of this signaling pathway in the carcinogenesis of cholangiocarcinoma (CCC) is still unknown. In this study, we investigated the effects of Hedgehog inhibition by cyclopamine and 5E1 in cultured human CCC cell lines and in vivo using a xenograft mouse model. We also investigated the involvement of Hedgehog in epithelial to mesenchymal transition (EMT), migration, and CCC tumor growth. Sonic hedgehog (Shh) ligand was highly expressed in 89% of human CCC tissues and in CCC cell lines. Cyclopamine and 5E1 treatments effectively inhibited cell proliferation, migration, and invasion by down‐regulating the Hedgehog target genes glioblastoma 1 and glioblastoma 2. In vitro and in vivo, we detected an increase in epithelial marker, E‐cadherin, after Hedgehog inhibition. In addition, we saw an increase in necrotic areas and a decrease in mitotic figures in cyclopamine and 5E1‐treated CCC xenograft tumors. Conclusion: This study supports the presence of autocrine Hedgehog signaling in human CCC, where CCC cells produce and respond to Shh ligand. Blocking the Hedgehog pathway inhibited EMT and decreased the viability of CCC cells. In addition, cyclopamine and 5E1 inhibited the growth of CCC xenograft tumors. (HEPATOLOGY 2013)

Epithelial Mesenchymal Transition and Pancreatic Tumor Initiating CD44+/EpCAM+ Cells Are Inhibited by γ-Secretase Inhibitor IX

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease with a high rate of metastasis. Recent studies have indicated that the Notch signalling pathway is important in PDAC initiation and maintenance, although the specific cell biological roles of the pathway remain to be established. Here we sought to examine this question in established pancreatic cancer cell lines using the γ-secretase inhibitor IX (GSI IX) to inactivate Notch. Based on the known roles of Notch in development and stem cell biology, we focused on effects on epithelial mesenchymal transition (EMT) and on pancreatic tumor initiating CD44+/EpCAM+ cells. We analyzed the effect of the GSI IX on growth and epithelial plasticity of human pancreatic cancer cell lines, and on the tumorigenicity of pancreatic tumor initiating CD44+/EpCAM+ cells. Notably, apoptosis was induced after GSI IX treatment and EMT markers were selectively targeted. Furthermore, under GSI IX treatment, decline in the growth of pancreatic tumor initiating CD44+/EpCAM+ cells was observed in vitro and in a xenograft mouse model. This study demonstrates a central role of Notch signalling pathway in pancreatic cancer pathogenesis and identifies an effective approach to inhibit selectively EMT and suppress tumorigenesis by eliminating pancreatic tumor initiating CD44+/EpCAM+ cells.

Combined inhibition of Notch and JAK/STAT is superior to monotherapies and impairs pancreatic cancer progression

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease with a high rate of metastasis. Recent studies have indicated that Notch and janus kinase 2 (JAK2)/signal transducers and activators of transcription 3 (STAT3) signaling pathways are both important for the initiation and progression of PDAC. The purpose of this study was to determine the outcome of targeting these two tumor signaling pathways simultaneously both in vitro and in vivo. We assessed the combinational effects of the γ-secretase inhibitor IX (GSI IX) and JAK2 inhibitor (AG-490) on growth and epithelial plasticity of human pancreatic cancer cell lines, and in a genetically engineered mouse model (Pdx1-Cre, LSL-KrasG12D, p53(lox/+)) of PDAC. Dual treatment with GSI IX and AG-490 significantly impaired cell proliferation, migration, invasion, soft agar growth and apoptosis when compared with monotherapies. Most importantly, combinational treatment significantly attenuates tumor progression in vivo and suppresses conversion from acinar-ductal-metaplasia to PDAC. Our results suggest that targeting Notch and JAK2/STAT3 signaling pathways simultaneously is superior to single inhibitions, supporting combined treatment by GSI IX and AG-490 as a potential therapeutic approach for PDAC. However, the study design limits the direct transfer into the clinic and the impact of dual treatment in patients with PDAC remains still to be determined.

Activation of Notch Signaling Is Required for Cholangiocarcinoma Progression and Is Enhanced by Inactivation of p53 In Vivo

Cholangiocacinoma (CC) is a cancer disease with rising incidence. Notch signaling has been shown to be deregulated in many cancers. However, the role of this signaling pathway in the carcinogenesis of CC is still not fully explored. In this study, we investigated the effects of Notch inhibition by γ-secretase inhibitor IX (GSI IX) in cultured human CC cell lines and we established a transgenic mouse model with liver specific expression of the intracellular domain of Notch (Notch-ICD) and inactivation of tumor suppressor p53. GSI IX treatment effectively impaired cell proliferation, migration, invasion, epithelial to mesenchymal transition and growth of softagar colonies. In vivo overexpression of Notch-ICD together with an inactivation of p53 significantly increased tumor burden and showed CC characteristics. Conclusion: Our study highlights the importance of Notch signaling in the tumorigenesis of CC and demonstrates that additional inactivation of p53 in vivo.

Capsaicin treatment attenuates cholangiocarcinoma carcinogenesis.

Capsaicin, the most abundant pungent molecule produced by pepper plants, represents an important ingredient in spicy foods consumed throughout the world. Studies have shown that capsaicin can relieve inflammation and has anti-proliferative effects on various human malignancies. Cholangiocarcinoma (CC) is a cancer disease with rising incidence. The prognosis remains dismal with little advance in treatment. The aim of the present study is to explore the anti-tumor activity of capsaicin in cultured human CC cell lines. Capsaicin effectively impaired cell proliferation, migration, invasion, epithelial to mesenchymal transition and growth of softagar colonies. Further, we show that capsaicin treatment of CC cells regulates the Hedgehog signaling pathway. Conclusion: Our results provide a basis for capsaicin to improve the prognosis of CCs in vivo and present new insights into the effectiveness and mode of action of capsaicin.

Islet microarchitecture and glucose transporter expression of the pancreas of the marmoset monkey display similarities to the human.

The common marmoset New World monkey (Callithrix jacchus), is a primate model with great potential for scientific research, including research on diabetes. However, in opposite to Rhesus and Java monkeys (Macaca mulatta and Macaca fascicularis) little is known about the marmosets islet microarchitecture, glucose transporter and pancreatic marker gene expression. In this work we analyze differences and similarities in size, shape, cellular composition and intra-islet topography between the common marmoset and the human endocrine pancreas. Different sized, circular and a-circular shaped islets of the common marmoset and human display α-cells in the whole islet organ leading to a ribbon-like islet type. The number of islets was significantly higher in the common marmoset compared with humans. However, the area of insulin-producing cells was significantly higher in the human pancreas. Intra-islet distribution pattern of δ- and β-cells was similar in both species. The morphology of the exocrine pancreas regarding acinar and ductal cells was quite similar as confirmed by ultrastructural analysis. Additionally the ultrastructure of secretory granules from α-, δ- and β-cells of human and non-human primate pancreas showed the same characteristics. Molecular analysis showed the presence of endocrine pancreatic marker genes like PMCA2, NCX1, SUR1, KIR6.2, MAFA, NGN3 and PDX1 also expressed in the human. For the first time we could show presence of Glut 5 and 9 transporters in addition to the low abundance transporter Glut2 and the highly expressed Glut1 glucose transporter. We propose that Callithrix jacchus displays a new animal model for diabetes research and regenerative medicine.

P81 CAPSAICIN TREATMENT TARGETS HEDGEHOG SIGNALING AND ATTENUATES HUMAN CHOLANGIOCARCINOMA CARCINOGENESIS

P80 ANGIOGENESIS POLYMORPHISMS PROFILE IN THE PREDICTION OF CLINICAL OUTCOME OF ADVANCED HCC PATIENTS RECEIVING SORAFENIB: COMBINED ANALYSIS OF VEGF AND HIF-1a. THE ALICE-2 STUDY L. Faloppi, M. Scartozzi, M. D’Anzeo, M. Bianconi, G. Svegliati Baroni, N. Silvestris, A. Casadei Gardini, G. Masi, R. Giampieri, M. Del Prete, S. De Minicis, A. Mandolesi, A. Benedetti, S. Cascinu. Department of Medical Oncology, Translational Oncology Unit, Department of Gastroenterology, Universita Politecnica delle Marche, Ancona, IRCCS – Ospedale Oncologico, Istituto Tumori ‘Giovanni Paolo II’, Bari, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (I.R.S.T.), Meldola, Azienda Ospedaliera Universitaria Pisana, Pisa, Institute of Pathological Anathomy, Universita Politecnica delle Marche, Ancona, Italy E-mail: lucafaloppi@gmail.com