Shantibhusan Senapati

Recent Trends in Antibody-based Oncologic Imaging

Antibodies, with their unmatched ability for selective binding to any target, are considered as potentially the most specific probes for imaging. Their clinical utility, however, has been limited chiefly due to their slow clearance from the circulation, longer retention in non-targeted tissues and the extensive optimization required for each antibody-tracer. The development of newer contrast agents, combined with improved conjugation strategies and novel engineered forms of antibodies (diabodies, minibodies, single chain variable fragments, and nanobodies), have triggered a new wave of antibody-based imaging approaches. Apart from their conventional use with nuclear imaging probes, antibodies and their modified forms are increasingly being employed with non-radioisotopic contrast agents (MRI and ultrasound) as well as newer imaging modalities, such as quantum dots, near infra red (NIR) probes, nanoshells and surface enhanced Raman spectroscopy (SERS). The review article discusses new developments in the usage of antibodies and their modified forms in conjunction with probes of various imaging modalities such as nuclear imaging, optical imaging, ultrasound, MRI, SERS and nanoshells in preclinical and clinical studies on the diagnosis, prognosis and therapeutic responses of cancer.

Mucin-interacting proteins: from function to therapeutics

Mucins are high molecular weight glycoproteins that are involved in regulating diverse cellular activities both in normal and pathological conditions. Mucin activity and localization is mediated by several molecular mechanisms, including discrete interactions with other proteins. An understanding of the biochemistry behind the known interactions between mucins and other proteins, coupled with an appreciation of their pathophysiological significance, can lend insight into the development of novel therapeutic agents. Indeed, a recent study demonstrated that a cell permeable inhibitor, PMIP, that disrupts the MUC1-EGFR interaction, is effective in killing breast cancer cells in vitro and in tumor models.

Clinical potential of mucins in diagnosis, prognosis, and therapy of ovarian cancer

Knowledge of mucins and their multiple roles in various normal and pathological processes has improved greatly in the past two decades. Mucins belong to a family of glycoproteins characterised by densely O-glycosylated repetitive domains and expressed by various surface epithelial cells. Altered expression of mucins is present in various diseases, including cancer. Ovarian cancer is the sixth most common cancer worldwide and the seventh leading cause of cancer-related deaths in women. The most common ovarian cancer is epithelial ovarian carcinoma, which is characterised by few early symptoms, widespread peritoneal dissemination, and ascites at advanced stages that result in poor prognosis. After diagnosis, 5 year survival is only 35-45%. Therefore, improved strategies for early diagnosis and treatment are needed. Because of the surface epithelial origin of epithelial ovarian cancer, mucins are obvious biomolecules for investigation as markers for early diagnosis and as therapeutic targets. We discuss the potential role and clinical usefulness of mucins in early diagnosis, prognosis, and treatment of ovarian cancer.

Overexpression of macrophage inhibitory cytokine-1 induces metastasis of human prostate cancer cells through the FAK-RhoA signaling pathway.

An elevated level of macrophage inhibitory cytokine-1 (MIC-1) is reported in the sera of patients with metastatic prostate cancer compared with that of benign diseases and healthy adults. We investigated the mechanistic role of MIC-1 overexpression in the metastasis of prostate cancer cells. Our study showed a progressive increase in secretory MIC-1 production correlated with the increase in the metastatic potential of PC-3 and LNPCa prostate cancer metastatic variants. Further, the in vitro studies using ‘loss-’ and ‘gain’-of-function approaches showed that ectopic overexpression of MIC-1 (PC-3-MIC-1) and forced downregulation of MIC-1(PC-3M-siMIC-1) enhanced and reduced the motility and invasiveness of these cells, respectively. Supporting our in vitro observations, all the mice orthotopically implanted with PC-3-MIC-1 cells developed metastasis compared with none in the PC-3-vector group. Our results showed that MIC-1 overexpression was associated with apparent changes in actin organization. In addition, an enhanced phosphorylation of focal adhesion kinase (FAK) and guanosine-5′-triphosphate (GTP)-bound RhoA was also seen; however, no significant change was observed in total FAK and RhoA levels in the PC-3-MIC-1 cells. Altogether, our findings show that MIC-1 has a role in prostate cancer metastasis, in part, by promoting the motility of these cells. Activation of the FAK–RhoA signaling pathway is involved in MIC-1-mediated actin reorganization, and thus, leads to an increase in the motility of prostate cancer cells.

MUC4 down-regulation reverses chemoresistance of pancreatic cancer stem/progenitor cells and their progenies

The present study was undertaken to estimate the therapeutic benefit to down-regulate the MUC4 mucin for reversing chemoresistance of pancreatic cancer (PC) stem/progenitor cells and their progenies. The results have revealed that MUC4 mucin is overexpressed in CD133(+) and CD133(-) pancreatic cells (PCs) detected in patients adenocarcinoma tissues while no significant expression was seen in normal pancreatic tissues. The gain- and loss-of-function analyses have indicated that the overexpression of MUC4 in PC lines is associated with a higher resistance to the anti-proliferative, anti-invasive and apoptotic effects induced by gemcitabine. Importantly, the treatment of the MUC4-overexpressing CD18/HPAF-Src cells with gemcitabine resulted in an enrichment of the side population (SP) cells expressing CD133 while the total PC cells including non-SP cells detected in MUC4 knockdown CD18/HPAF-shMUC4 cells were responsive to the cytotoxic effects induced by gemcitabine. These data suggest that the MUC4 down-regulation may constitute a potential therapeutic strategy for improving the efficacy of gemcitabine to eradicate the total PC cell mass, and thereby preventing disease relapse.

Novel INTeraction of MUC4 and galectin: potential pathobiological implications for metastasis in lethal pancreatic cancer.

Purpose: Several studies have reported aberrant expression of MUC4 in pancreatic cancer (PC), which is associated with tumorigenicity and metastasis. Mechanisms through which MUC4 promote metastasis of PC cells to distant organs are poorly defined. Experimental Design: Identification of MUC4–galectin-3 interaction and its effect on the adhesion of cancer cells to endothelial cells were done by immunoprecipitation and cell–cell adhesion assays, respectively. Serum galectin-3 level for normal and PC patients were evaluated through ELISA. Results: In the present study, we have provided clinical evidence that the level of galectin-3 is significantly elevated in the sera of PC patients with metastatic disease compared with patients without metastasis (P = 0.04) and healthy controls (P = 0.00001). Importantly, for the first time, we demonstrate that MUC4 present on the surface of circulating PC cells plays a significant role in the transient and reversible attachment (docking) of circulating tumor cells to the surface of endothelial cells. Further, exogenous galectin-3 at concentrations similar to that found in the sera of PC patients interacts with MUC4 via surface glycans such as T antigens, which results in the clustering of MUC4 on the cell surface and a stronger attachment (locking) of circulating tumor cells to the endothelium. Conclusions: Altogether, these findings suggest that PC cell-associated MUC4 helps in the docking of tumor cells on the endothelial surface. During cancer progression, MUC4–galectin-3 interaction–mediated clustering of MUC4 may expose the surface adhesion molecules, which in turn promotes a stronger attachment (locking) of tumor cells to the endothelial surface. Clin Cancer Res; 17(2); 267–74. ©2010 AACR. Clin Cancer Res; 17(2); 267–74. ©2010 AACR.

RNA Polymerase II Associated Factor 1/PD2 Maintains Self-Renewal by Its Interaction with Oct3/4 in Mouse Embryonic Stem Cells

Embryonic stem cells (ESCs) maintain self‐renewal while ensuring a rapid response to differentiation signals, but the exact mechanism of this process remains unknown. PD2 is the human homolog of the RNA polymerase II‐associated factor 1 (Paf1). The Paf1/PD2 is a member of the human PAF complex that consists of four other subunits, hCdc73, hLeo1, hCtr9, and hSki8, and is involved in the regulation of transcriptional elongation and further downstream events. Here, we show that Paf1/PD2 is overexpressed in mouse ESCs and is involved in the maintenance of mouse ESCs. The Paf1/PD2 knockdown and knockout ESCs grown under self‐renewal conditions express substantially reduced levels of self‐renewal regulators, including Oct3/4, SOX2, Nanog, and Shh. We observed that the level of Paf1/PD2 expression is much higher in self‐renewing mouse embryonic carcinoma cells than in the differentiating cells. Knockout of Paf1/PD2 altered ESC phenotype by increasing apoptosis and decreasing the percentage of cells in S‐phase of the cell cycle. Interestingly, we found that the key genes that regulate endodermal differentiation (Gata4, Gata6, and Fgf8) are induced in the Paf1/PD2 heterozygous knockout ESCs. This suggests that Paf1/PD2 plays a specific role in regulating early commitment of ESCs to endodermal differentiation. Furthermore, for the first time, we showed that Paf1/PD2 protein interacts with Oct3/4 and RNA polymerase II, and through this interaction Paf1/PD2 may regulate Oct3/4‐mediated gene expression. Thus, the Paf1/PD2 protein is a newly discovered element of the interconnected regulatory network that maintains the self‐renewal of mouse ESCs. STEM CELLS 2009;27:3001–3011

The MUC gene family: Their role in the diagnosis and prognosis of gastric cancer

Early diagnosis of gastric cancer and its differential diagnosis from other non-malignant gastric diseases like gastritis is still a major clinical problem. Most patients are asymptomatic in the early stages of gastric cancer, and there is no reliable marker available for the early and specific diagnosis of gastric cancer. Many attempts have been made to define the biological profile of gastric cancer to improve the chances of its early diagnosis, prognosis and treatment. Several studies have shown the aberrant expression profile of mucins in different malignancies, suggesting that mucins have a great potential to be used as a diagnostic and prognostic marker in gastric cancer. In this review, we have briefly described the different types of gastric adenocarcinomas and the progression of gastric cancer. Furthermore, the role of mucins and their related carbohydrate epitope is discussed in the normal stomach and in the diagnosis and prognosis of gastric adenocarcinomas.

Role of MUC4–NIDO domain in the MUC4-mediated metastasis of pancreatic cancer cells

MUC4 is a large transmembrane type I glycoprotein that is overexpressed in pancreatic cancer (PC) and has been shown to be associated with its progression and metastasis. However, the exact cellular and molecular mechanism(s) through which MUC4 promotes metastasis of PC cells has been sparsely studied. Here we showed that the nidogen-like (NIDO) domain of MUC4, which is similar to the G1-domain present in the nidogen or entactin (an extracellular matrix protein), contributes to the protein–protein interaction property of MUC4. By this interaction, MUC4 promotes breaching of basement membrane (BM) integrity, and spreading of cancer cells. These observations are corroborated with the data from our study using an engineered MUC4 protein without the NIDO domain, which was ectopically expressed in the MiaPaCa PC cells, lacking endogenous MUC4 and nidogen protein. The in vitro studies demonstrated an enhanced invasiveness of MiaPaCa cells expressing MUC4 (MiaPaCa–MUC4) compared with vector-transfected cells (MiaPaCa–Vec; P=0.003) or cells expressing MUC4 without the NIDO domain (MiaPaCa–MUC4–NIDOΔ; P=0.03). However, the absence of NIDO-domain has no significant role on cell growth and motility (P=0.93). In the in vivo studies, all the mice orthotopically implanted with MiPaCa-MUC4 cells developed metastasis to the liver as compared with MiaPaCa–Vec or the MiaPaCa–MUC4–NIDOΔ group, hence, supporting our in vitro observations. Additionally, a reduced binding (P=0.0004) of MiaPaCa–MUC4–NIDOΔ cells to the fibulin-2 coated plates compared with MiaPaCa–MUC4 cells indicated a possible interaction between the MUC4–NIDO domain and fibulin-2, a nidogen-interacting protein. Furthermore, in PC tissue samples, MUC4 colocalized with the fibulin-2 present in the BM. Altogether, our findings demonstrate that the MUC4–NIDO domain significantly contributes to the MUC4-mediated metastasis of PC cells. This may be partly due to the interaction between the MUC4–NIDO domain and fibulin-2.

Expression of intestinal MUC17 membrane-bound mucin in inflammatory and neoplastic diseases of the colon

Aim To determine the cellular location and expression of MUC17 mucin in specimens of normal, inflamed and neoplastic colon. Methods Immunohistochemical analysis of human surgical resection specimens (n=106) was performed with a specific antibody to the MUC17 apomucin protein. A semi-quantitative scoring system was used to measure MUC17 expression. In various colon cancer cell lines, the MUC17 expression was examined by immunoblot analysis and normal RT-PCR. Results MUC17 was highly expressed on the surface epithelium and crypts of colonic mucosa. In contrast, the expression of MUC17 was significantly decreased in colonic mucosa of chronic ulcerative colitis (p<0.0001) and ischaemic colitis (p=0.003). Similarly, MUC17 expression was decreased in hyperplastic polyps (p=0.0003), tubular and tubulovillous adenomas (p<0.0001) and colon cancers (p<0.0001). Furthermore, of eight different colon cancer cell lines, MUC17 expression was only detected in LS174T and LS180 cells. Conclusion Results indicate that the potential protective effects of this membrane-bound mucin are primarily or secondarily diminished in inflammatory and neoplastic conditions. Further research is needed to determine the specific role of MUC17 in the pathogenesis of these conditions.