Remya Raja

Gallbladder cancer (GBC) is a rare malignancy, associated with poor disease prognosis with a 5-year survival of only 20%. This has been attributed to late presentation of the disease, lack of early diagnostic markers and limited efficacy of therapeutic interventions. Elucidation of molecular events in GBC can contribute to better management of the disease by aiding in the identification of therapeutic targets. To identify aberrantly activated signaling events in GBC, tandem mass tag-based quantitative phosphoproteomic analysis of five GBC cell lines was carried out. Proline-rich Akt substrate 40 kDa (PRAS40) was one of the proteins found to be hyperphosphorylated in all the invasive GBC cell lines. Tissue microarray-based immunohistochemical labeling of phospho-PRAS40 (T246) revealed moderate to strong staining in 77% of the primary gallbladder adenocarcinoma cases. Regulation of PRAS40 activity by inhibiting its upstream kinase PIM1 resulted in a significant decrease in cell proliferation, colony forming and invasive ability of GBC cells. Our results support the role of PRAS40 phosphorylation in GBC cell survival and aggressiveness. This study also elucidates phospho-PRAS40 as a clinical marker in GBC and the role of PIM1 as a therapeutic target in GBC.

Cigarette smoking remains the leading cause of non-small cell lung carcinoma. Studies involving acute exposure of smoke on lung cells revealed induction of pre- cancerous state in lung cells. Recently few studies have reported the chronic effect of cigarette smoke in inducing cellular transformation. Yet no systemic study has been performed to understand the molecular alterations in lung cells due to cigarette smoke. Hence it is both important and necessary to study the chronic effect of cigarette smoke in a temporal setting to understand the molecular alterations. In this study, we carried out TMT based proteomic profiling of lung cells which were exposed to cigarette smoke condensate (CSC) for upto 12 months. We identified 2621 proteins in total, of which 145, 114, 87, 169 and 671 proteins were differentially expressed (p<0.05, 1.5 fold) in 2nd, 4th, 6th, 8th and 12th month respectively.Pathway analysis revealed enrichment of xenobiotic metabolism signaling for the first 8 months of smoke treatment, whereas continued exposure of smoke for 12 months revealed mitochondrial reprogramming in cells which includes dysregulation of oxidative phosphorylation machinery leading to enhanced reactive oxygen species and higher expression of enzymes involved in tricarboxylic acid cycle (TCA). In addition, chronic exposure of smoke led to overexpression of enzymes involved in glutamine metabolism, fatty acid degradation and lactate synthesis. This could possibly explain the availability of alternative source of carbon in TCA cycle apart from glycolytic pyruvate. Our data indicates that chronic exposure to cigarette smoke induces metabolic transformation in cells to support growth and survival. Cigarette smoking remains the leading cause of non-small cell lung carcinoma. Studies involving acute exposure of smoke on lung cells revealed induction of pre- cancerous state in lung cells. Recently few studies have reported the chronic effect of cigarette smoke in inducing cellular transformation. Yet no systemic study has been performed to understand the molecular alterations in lung cells due to cigarette smoke. Hence it is both important and necessary to study the chronic effect of cigarette smoke in a temporal setting to understand the molecular alterations. In this study, we carried out TMT based proteomic profiling of lung cells which were exposed to cigarette smoke condensate (CSC) for upto 12 months. We identified 2621 proteins in total, of which 145, 114, 87, 169 and 671 proteins were differentially expressed (p<0.05, 1.5 fold) in 2nd, 4th, 6th, 8th and 12th month respectively.Pathway analysis revealed enrichment of xenobiotic metabolism signaling for the first 8 months of smoke treatment, whereas continued exposure of smoke for 12 months revealed mitochondrial reprogramming in cells which includes dysregulation of oxidative phosphorylation machinery leading to enhanced reactive oxygen species and higher expression of enzymes involved in tricarboxylic acid cycle (TCA). In addition, chronic exposure of smoke led to overexpression of enzymes involved in glutamine metabolism, fatty acid degradation and lactate synthesis. This could possibly explain the availability of alternative source of carbon in TCA cycle apart from glycolytic pyruvate. Our data indicates that chronic exposure to cigarette smoke induces metabolic transformation in cells to support growth and survival.

Poor prognosis in gallbladder cancer is due to late presentation of the disease, lack of reliable biomarkers for early diagnosis and limited targeted therapies. Early diagnostic markers and novel therapeutic targets can significantly improve clinical management of gallbladder cancer. Proteomic analysis of four gallbladder cancer cell lines based on the invasive property (non-invasive to highly invasive) was carried out using the isobaric tags for relative and absolute quantitation labeling-based quantitative proteomic approach. The expression of macrophage migration inhibitory factor was analysed in gallbladder adenocarcinoma tissues using immunohistochemistry. In vitro cellular assays were carried out in a panel of gallbladder cancer cell lines using MIF inhibitors, ISO-1 and 4-IPP or its specific siRNA. The quantitative proteomic experiment led to the identification of 3,653 proteins, among which 654 were found to be overexpressed and 387 were downregulated in the invasive cell lines (OCUG-1, NOZ and GB-d1) compared to the non-invasive cell line, TGBC24TKB. Among these, macrophage migration inhibitory factor (MIF) was observed to be highly overexpressed in two of the invasive cell lines. MIF is a pleiotropic proinflammatory cytokine that plays a causative role in multiple diseases, including cancer. MIF has been reported to play a central role in tumor cell proliferation and invasion in several cancers. Immunohistochemical labeling of tumor tissue microarrays for MIF expression revealed that it was overexpressed in 21 of 29 gallbladder adenocarcinoma cases. Silencing/inhibition of MIF using siRNA and/or MIF antagonists resulted in a significant decrease in cell viability, colony forming ability and invasive property of the gallbladder cancer cells. Our findings support the role of MIF in tumor aggressiveness and suggest its potential application as a therapeutic target for gallbladder cancer.