Srustidhar Das

University of Nebraska Medical Center, Omaha, Nebraska, United States

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Publications (16)114.67 Total impact

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    ABSTRACT: MUC16 (CA125) is a type-I transmembrane glycoprotein that is up-regulated in multiple cancers including pancreatic cancer (PC). However, the existence and role of carboxyl-terminal MUC16 generated following its cleavage in PC is unknown. Our previous study using a systematic dual-epitope tagged domain deletion approach of carboxyl-terminal MUC16 has demonstrated the generation of a 17-kDa cleaved MUC16 (MUC16-Cter). Here, we demonstrate the functional significance of MUC16-Cter in PC using the dual-epitope tagged version (N-terminal FLAG- and C-terminal HA-tag) of 114 carboxyl-terminal residues of MUC16 (F114HA). In vitro analyses using F114HA transfected MiaPaCa-2 and T3M4 cells showed enhanced proliferation, motility and increased accumulation of cells in the G2/M phase with apoptosis resistance, a feature associated with cancer stem cells (CSCs). This was supported by enrichment of ALDH+ CSCs along with enhanced drug-resistance. Mechanistically, we demonstrate a novel function of MUC16-Cter that promotes nuclear translocation of JAK2 resulting in phosphorylation of Histone-3 up-regulating stemness-specific genes LMO2 and NANOG. Jak2 dependence was demonstrated using Jak2+/+ and Jak2-/- cells. Using eGFP-Luciferase labeled cells, we demonstrate enhanced tumorigenic and metastatic potential of MUC16-Cter in vivo. Taken together, we demonstrate that MUC16-Cter mediated enrichment of CSCs is partly responsible for tumorigenic, metastatic and drug-resistant properties of PC cells.
    Oncotarget 01/2015; · 6.63 Impact Factor
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    ABSTRACT: Pancreatic cancer (PC) is characterized by aberrant overexpression of mucins that contribute to its pathogenesis. Although the inflammatory cytokines contribute to mucin overexpression, the mucin profile of PC is markedly distinct from that of normal or inflamed pancreas. We postulated that de novo expression of various mucins in PC involves chromatin modifications. Analysis of chromatin modifying enzymes by PCR array identified differential expression of NCOA3 in MUC4-expressing PC cell lines. Immunohistochemistry analysis in tumor tissues from patients and spontaneous mouse models, and microarray analysis following the knockdown of NCOA3 were performed to elucidate its role in mucin regulation and overall impact on PC. Silencing of NCOA3 in PC cell lines resulted in significant downregulation of two most differentially expressed mucins in PC, MUC4 and MUC1 (P<0.01). Immunohistochemistry analysis in PC tissues and metastatic lesions established an association between NCOA3 and mucin (MUC1 and MUC4) expression. Spontaneous mouse model of PC (K-rasG12D; Pdx-1cre) showed early expression of Ncoa3 during pre-neoplastic lesions. Mechanistically, NCOA3 knockdown abrogated retinoic acid-mediated MUC4 upregulation by restricting MUC4 promoter accessibility as demonstrated by micrococcus nuclease digestion (P<0.05) and chromatin immuno-precipitation analysis. NCOA3 also created pro-inflammatory conditions by upregulating chemokines like CXCL1, 2, 5 and CCL20 (P<0.001). AKT, ubiquitin C, ERK1/2 and NF-κB occupied dominant nodes in the networks significantly modulated after NCOA3 silencing. In addition, NCOA3 stabilized mucins post translationally through fucosylation by FUT8, as the knockdown of FUT8 resulted in the downregulation of MUC4 and MUC1 at protein levels.Oncogene advance online publication, 22 December 2014; doi:10.1038/onc.2014.409.
    Oncogene 12/2014; · 8.56 Impact Factor
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    ABSTRACT: Amyloid precursor-like protein 2 (APLP2) is aberrantly expressed in pancreatic cancer. Here we showed that APLP2 is increased in pancreatic cancer metastases, particularly in metastatic lesions found in the diaphragm and intestine. Examination of matched human primary tumor-liver metastasis pairs showed that 38.1% of the patients had positive APLP2 expression in both the primary tumor and the corresponding liver metastasis. Stable knock-down of APLP2 expression (with inducible shRNA) in pancreatic cancer cells reduced the ability of these cells to migrate and invade. Loss of APLP2 decreased cortical actin and increased intracellular actin filaments in pancreatic cancer cells. Down-regulation of APLP2 decreased the weight and metastasis of orthotopically transplanted pancreatic tumors in nude mice.
    Oncotarget 12/2014; · 6.63 Impact Factor
  • Srustidhar Das, Surinder K Batra
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    ABSTRACT: Pancreatic cancer, often considered a metastatic disease at the time of clinical diagnosis due to lack of any reliable early diagnostic marker(s), is refractory to conventional chemo- and radiotherapy and has a dismal 5-year survival rate of only 6%. Although surgical removal of the primary tumor is considered to be curative, the 5-year survival rate is no more than 20% even in patients with clear resection margins (R0). The recurrence of local and metastatic disease (primarily liver metastasis) post resection is considered to be the leading cause of mortality in these patients. In addition, instances of metastatic disease without any local recurrence post resection have also been observed. Cancer metastasis is the primary cause of mortality in cancer patients and is classically viewed as a late event during the progression of the disease, which is supported by the genetic studies used to understand the evolution of pancreatic cancer. However, this view has recently been challenged by studies using mathematical modeling and genetically labeled mouse models of pancreatic cancer to understand the dynamics of tumor cell dissemination and epithelial to mesenchymal transition (EMT) of tumor cells well before the primary tumor is formed. Given that EMT is a hallmark process that initiates the metastatic seeding of cancer cells and the dismal prognosis of pancreatic cancer patients even after efficient removal of the primary tumor (99.9%), an early dissemination hypothesis of cancer cells cannot be undermined. In this review, we will discuss the current views regarding pancreatic cancer metastasis with particular emphasis on the epithelial to mesenchymal transition, its influence on the selection of patients for surgical resection and the therapeutic intervention.
    Current Pharmaceutical Design 12/2014; 21(10). · 3.29 Impact Factor
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    ABSTRACT: A major problem for structural characterization of membrane proteins, such as connexins, by nuclear magnetic resonance (NMR) occurs at the initial step of the process, the production of sufficient amounts of protein. This occurs because proteins must be expressed in minimal based media. Here, we describe an expression system for membrane proteins that significantly improves yield by addressing two common problems, cell toxicity caused by protein translation and codon bias between genomes. This work provides researchers with a cost-effective tool for NMR and other biophysical studies, to use when faced with little-to-no expression of eukaryotic membrane proteins in Escherichia coli expression systems.
    Frontiers in Pharmacology 08/2013; 4:106.
    This article is viewable in ResearchGate's enriched format
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    ABSTRACT: INTRODUCTION:: Altered expression of MUC4 plays an oncogenic role in various cancers, including pancreatic, ovarian, and breast. This study evaluates the expression and role of MUC4 in non-small-cell lung cancer (NSCLC). METHODS:: We used a paired system of MUC4-expressing (H292) and MUC4-nonexpressing (A549) NSCLC cell lines to analyze MUC4-dependent changes in growth rate, migration, and invasion using these sublines. We also evaluated the alterations of several tumor suppressor, proliferation, and metastasis markers with altered MUC4 expression. Furthermore, the association of MUC4 expression (by immunohistochemistry) in lung cancer samples with patient survival was evaluated. RESULTS:: MUC4-expressing lung cancer cells demonstrated a less proliferative and metastatic phenotype. Up-regulation of p53 in MUC4-expressing lung cancer cells led to the accumulation of cells at the G2/M phase of cell cycle progression. MUC4 expression attenuated Akt activation and decreased the expression of Cyclins D1 and E, but increased the expression of p21 and p27. MUC4 expression abrogated cancer cell migration and invasion by altering N- & E-cadherin expression and FAK phosphorylation. A decrease in MUC4 expression was observed with increasing tumor stage (mean composite score: stage I, 2.4; stage II, 1.8; stage III, 1.4; and metastatic, 1.2; p = 0.0093). Maximal MUC4 expression was associated with a better overall survival (p = 0.042). CONCLUSION:: MUC4 plays a tumor-suppressor role in NSCLC by altering p53 expression in NSCLC. Decrease in MUC4 expression in advanced tumor stages also seems to confirm the novel protective function of MUC4 in NSCLC.
    Journal of thoracic oncology: official publication of the International Association for the Study of Lung Cancer 01/2013; · 4.55 Impact Factor
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    ABSTRACT: MUC16 (CA125) belongs to a family of high-molecular weight O-glycosylated proteins known as mucins. While MUC16 is well known as a biomarker in ovarian cancer, its expression pattern in pancreatic cancer (PC), the fourth leading cause of cancer related deaths in the United States, remains unknown. The aim of our study was to analyze the expression of MUC16 during the initiation, progression and metastasis of PC for possible implication in PC diagnosis, prognosis and therapy. In this study, a microarray containing tissues from healthy and PC patients was used to investigate the differential protein expression of MUC16 in PC. MUC16 mRNA levels were also measured by RT-PCR in the normal human pancreatic, pancreatitis, and PC tissues. To investigate its expression pattern during PC metastasis, tissue samples from the primary pancreatic tumor and metastases (from the same patient) in the lymph nodes, liver, lung and omentum from Stage IV PC patients were analyzed. To determine its association in the initiation of PC, tissues from PC patients containing pre-neoplastic lesions of varying grades were stained for MUC16. Finally, MUC16 expression was analyzed in 18 human PC cell lines. MUC16 is not expressed in the normal pancreatic ducts and is strongly upregulated in PC and detected in pancreatitis tissue. It is first detected in the high-grade pre-neoplastic lesions preceding invasive adenocarcinoma, suggesting that its upregulation is a late event during the initiation of this disease. MUC16 expression appears to be stronger in metastatic lesions when compared to the primary tumor, suggesting a role in PC metastasis. We have also identified PC cell lines that express MUC16, which can be used in future studies to elucidate its functional role in PC. Altogether, our results reveal that MUC16 expression is significantly increased in PC and could play a potential role in the progression of this disease.
    PLoS ONE 10/2011; 6(10):e26839. · 3.53 Impact Factor
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    ABSTRACT: The membrane-bound mucin MUC17 (mouse homolog Muc3) is highly expressed on the apical surface of intestinal epithelia and is thought to play a role in epithelial restitution and protection. Therefore, we hypothesized that MUC17 has a role in protection of the intestinal mucosa against luminal pathogens. Human intestinal cell lines were transfected by electroporation (Caco-2 and HT 29/19A) and by retroviral expression vector (LS174T, a cell line with high levels of MUC17 expression) using MUC17 siRNA. Transepithelial electrical resistance, permeability, tight-junction protein expression, adhesion, and invasion in response to enteroinvasive Escherichia coli (EIEC) were measured in all cell lines. In some experiments, the effect of the addition of exogenous purified crude mucin or recombinant Muc3 cysteine-rich domain protein (Muc3 CRD1-L-CRD2) as preventative or protective treatment was tested. Reduction of endogenous MUC17 is associated with increased permeability, inducible nitric oxide synthase and cyclooxygenase 2 induction, and enhanced bacterial invasion in response to EIEC exposure. Bacterial adhesion is not affected. Exogenous mucin (Muc3) and recombinant Muc3CRD treatment had a small but significant effect in attenuating the effects of EIEC infection. In conclusion, these data suggest that both native and exogenous MUC17 play a role in attachment and invasion of EIEC in colonic cell lines and in maintaining epithelial barrier function.
    AJP Gastrointestinal and Liver Physiology 03/2011; 300(6):G1144-55. · 3.65 Impact Factor
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    ABSTRACT: To determine the cellular location and expression of MUC17 mucin in specimens of normal, inflamed and neoplastic colon. 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. 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. 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.
    Journal of clinical pathology 08/2010; 63(8):702-7. · 2.55 Impact Factor
  • Gastroenterology 05/2010; 138(5). · 12.82 Impact Factor
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    ABSTRACT: Pancreatic cancer is one of the most lethal cancers in the world, as it continues to be resistant to any therapeutic approaches. The high molecular weight glycoprotein mucin 4 (MUC4) is aberrantly expressed in pancreatic cancer and contributes to the regulation of differentiation, proliferation, metastasis, and the chemoresistance of pancreatic cancer cells. The absence of its expression in the normal pancreatic ductal cells makes MUC4 a promising target for novel cancer therapeutics. Natural products have been widely investigated as potential candidates in cancer therapies, and thymoquinone (TQ), extracted from the seeds of Nigella sativa, has shown excellent antineoplastic properties in some systems. In the present study, we evaluated the effect of TQ on pancreatic cancer cells and specifically investigated its effect on MUC4 expression. The MUC4-expressing pancreatic cancer cells FG/COLO357 and CD18/HPAF were incubated with TQ, and in vitro functional assays were done. The results obtained indicate that treatment with TQ downregulated MUC4 expression through the proteasomal pathway and induced apoptosis in pancreatic cancer cells by the activation of c-Jun NH(2)-terminal kinase and p38 mitogen-activated protein kinase pathways. In agreement with previous studies, the decrease in MUC4 expression correlated with an increase in apoptosis, decreased motility, and decreased migration of pancreatic cancer cells. MUC4 transient silencing studies showed that c-Jun NH(2)-terminal kinase and p38 mitogen-activated protein kinase pathways are activated in pancreatic cancer cells, indicating that the activation of these pathways by TQ is directly related to the MUC4 downregulation induced by the drug. Overall, TQ has potential for the development of novel therapies against pancreatic cancer.
    Molecular Cancer Therapeutics 05/2010; 9(5):1419-31. · 5.60 Impact Factor
  • Gastroenterology 05/2010; 138(5). · 12.82 Impact Factor
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    ABSTRACT: The membrane-bound mucins, MUC17 (human) and Muc3 (mouse), are highly expressed on the apical surface of intestinal epithelia and are thought to be cytoprotective. The extracellular regions of these mucins contain EGF-like Cys-rich segments (CRD1 and CRD2) connected by an intervening linker domain (L). The purpose of this study was to determine the functional activity of human MUC17 membrane-bound mucin. METHODS: Endogenous MUC17 was inhibited in LS174T colon cells by stable transfection of a small hairpin RNA targeting MUC17 (LSsi cells). The effect of recombinant MUC17-CRD1-L-CRD2 protein on migration, apoptosis, and experimental colitis was determined. RESULTS: Reduced MUC17 expression in LSsi cells was associated with visibly reduced cell aggregation, reduced cell-cell adherence, and reduced cell migration, but no change in tumorigenicity. LSsi cells also demonstrated a 3.7-fold increase in apoptosis rates compared with control cells following treatment with etoposide. Exposure of colonic cell lines to exogenous recombinant MUC17-CRD1-L-CRD2 protein significantly increased cell migration and inhibited apoptosis. As a marker of biologic activity, MUC17-CRD1-L-CRD2 proteins stimulate ERK phosphorylation in colonic cell lines; and inhibition of ERK phosphorylation reduced the anti-apoptosis and migratory effect of MUC17-CRD1-L-CRD2. Finally, mice treated with MUC17-CRD1-L-CRD2 protein given per rectum demonstrated accelerated healing in acetic acid and dextran sodium sulfate induced colitis in vivo. These data indicate that both native MUC17 and the exogenous recombinant cysteine-rich domain of MUC17 play a role in diverse cellular mechanisms related to cell restitution, and suggest a potential role for MUC17-CRD1-L-CRD2 recombinant protein in the treatment of mucosal inflammatory diseases.
    The international journal of biochemistry & cell biology 03/2010; 42(6):996-1006. · 4.89 Impact Factor
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    ABSTRACT: 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.
    Trends in Biochemical Sciences 11/2009; 35(4):236-45. · 13.52 Impact Factor
  • Gastroenterology 05/2009; 136(5). · 12.82 Impact Factor
  • Gastroenterology 05/2009; 136(5). · 12.82 Impact Factor

Publication Stats

108 Citations
114.67 Total Impact Points

Institutions

  • 2010–2015
    • University of Nebraska Medical Center
      • Department of Pathology and Microbiology
      Omaha, Nebraska, United States
  • 2009–2010
    • University of Nebraska at Omaha
      • Department of Biochemistry and Molecular Biology
      Omaha, Nebraska, United States