Sanjeev K. Srivastava

Bio: Sanjeev K. Srivastava is an academic researcher from University of South Alabama. The author has contributed to research in topics: Pancreatic cancer & Cancer. The author has an hindex of 30, co-authored 71 publications receiving 2481 citations. Previous affiliations of Sanjeev K. Srivastava include Semmelweis University & Sanjay Gandhi Post Graduate Institute of Medical Sciences.

Comparative analysis of exosome isolation methods using culture supernatant for optimum yield, purity and downstream applications.

Abstract: Exosomes have received significant attention for their role in pathobiological processes and are being explored as a tool for disease diagnosis and management. Consequently, various isolation methods based on different principles have been developed for exosome isolation. Here we compared the efficacy of four kits from Invitrogen, 101Bio, Wako and iZON along with conventional ultracentrifugation-based method for exosome yield, purity and quality. Cell culture supernatant was used as an abundant source of exosomes, and exosome quantity, size-distribution, zeta-potential, marker-expression and RNA/protein quality were determined. The Invitrogen kit gave the highest yield but the preparation showed broader size-distribution likely due to microvesicle co-precipitation and had the least dispersion stability. Other preparations showed <150 nm size range and good stability. Preparation from iZON column; however, had a broader size-distribution in the lower size range suggestive of some impurities of non-vesicular aggregates. RNA quality from all preparations was comparable; however, proteins from Invitrogen method-based exosomal preparation showed polyethylene glycol (PEG) contamination in mass spectrometry. Chemical impurities from the precipitant could also be the cause of toxicity of Invitrogen method-based exosomal preparation in biological growth measurement assay. Together, these findings should serve as a guide to choose and further optimize exosome isolation methods for their desired downstream applications.

CXCL12–CXCR4 signalling axis confers gemcitabine resistance to pancreatic cancer cells: a novel target for therapy

Abstract: CXCL12–CXCR4 signalling axis confers gemcitabine resistance to pancreatic cancer cells: a novel target for therapy

Exosomes confer chemoresistance to pancreatic cancer cells by promoting ROS detoxification and miR-155-mediated suppression of key gemcitabine-metabolising enzyme, DCK.

Abstract: Exosomes confer chemoresistance to pancreatic cancer cells by promoting ROS detoxification and miR-155-mediated suppression of key gemcitabine-metabolising enzyme, DCK

MicroRNA-150 directly targets MUC4 and suppresses growth and malignant behavior of pancreatic cancer cells

Abstract: Pancreaticcancer (PC)has theworstprognosisamongallcancersdue to its late diagnosis and lack of effective therapies. Therefore,identification of novel gene targets, which are differentially ex-pressed in PC and functionally involved in malignant phenotypes,is critical to achieve early diagnosis and development of effectivetherapeutic strategies. We have shown previously that MUC4,an aberrantly overexpressed transmembrane mucin, promotesgrowth, invasion and metastasis of PC cells, thus underscoringits potential as a clinical target. Here, we report a novel micro-RNA(miRNA)-mediatedmechanismunderlyingaberrantexpres-sion of MUC4 in PC. We demonstrate that the 3# untranslatedregion of MUC4 contains a highly conserved miRNA-150 (miR-150) binding motif and its direct interaction with miR-150 down-regulates endogenous MUC4 protein levels. We also show thatmiR-150-mediated MUC4 downregulation is associated witha concomitant decrease in human epidermal growth factor recep-tor 2 and its phosphorylated form, leading to reduced activationof downstream signaling. Furthermore, our findings demonstratethat miR-150 overexpression inhibits growth, clonogenicity, mi-gration and invasion and enhances intercellular adhesion in PCcells. Finally, our data reveal a downregulated expression of miR-150 in malignant pancreatic tissues, which is inversely associatedwith MUC4 protein levels. Altogether, these findings establishmiR-150 as a novel regulator of MUC4 and a tumor suppressormiRNA in PC.IntroductionPancreatic cancer (PC) is a highly lethal malignancy and has the worstprognosis among all cancers. Currently, it is the fourth leading causeof cancer-related deaths in the USA (1). The collective median sur-vival for all patients with PC is 2–8 months, and only 1–4% of allpatients survive 5 years after diagnosis (2). Such a grim prognosis ofPC is explained by the fact that at the time of diagnosis, majority ofpatients have already developed an aggressive form of the disease thuslimiting the potential for therapeutic intervention (3). Even smalladenocarcinoma of pancreas at diagnosis is genetically advancedand carries numerous genetic and epigenetic aberrations that co-operatively act to confer aggressive malignant phenotypes (4,5).Recent years have witnessed important advances in our understandingof the molecular progression of PC, and several important targets havebeen identified and experimentally tested for their functional participa-tion in the disease processes (4,6,7).MUC4 is a high-molecular weight glycoprotein that belongs to thefamily of membrane-bound mucins (8). It is overexpressed in pancre-atic adenocarcinomas and tumor cell lines while remains undetectablein the normal pancreas (9). Expression analysis of MUC4 in increas-ing grade pancreatic intraepithelial neoplasias and malignant lesionsdemonstrated a positive correlation of MUC4 with disease progres-sion (10). Importantly, in our earlier studies, we have shown a patho-genic role of MUC4 in promoting pancreatic tumor growth andmetastasis (11,12). Furthermore, aberrant MUC4 expression is alsoreported in other malignancies indicating its clinical relevance asa target for therapeutic intervention (8,13). However, there is still littleknown about the molecular mechanisms that regulate MUC4 expres-sion and whose perturbation ultimately leads to its aberrant expressionduring cancer initiation and progression (8,14).Recently, a novel class of endogenous small non-coding gene reg-ulatory RNAs, termed as microRNAs (miRNAs or miRs), has gainedsignificant attention (15). These small molecules exert their regulatoryeffects by base pairing with partially complementary messengerRNAs (mRNAs) and function by two mechanisms: degrading targetmRNA or inhibiting their translation (16,17). It is now well estab-lished that miRNAs play critical roles in the development of cancer byaltering the expression of oncogenes and tumor suppressor genes(15,16). In the present study, we have investigated the role of micro-RNA-150 (miR-150) in the regulation of MUC4 expression in PCcells. Our findings demonstrate that 3# untranslated region (UTR)of MUC4 contains putative binding site for miR-150, which is highlyconserved across several mammalian species. Furthermore, we exper-imentally show that miR-150 directly targets the 3# UTR of MUC4 tosuppress its expression. Downregulation of MUC4 by miR-150 alsoleads to a concomitant decrease in human epidermal growth factorreceptor 2 (HER2), an interacting partner of MUC4 (18), and itsphosphorylated form leading to reduced activation of downstreamsignaling molecules. Our findings also demonstrate that miR-150overexpression leads to reduced growth, clonogenicity, migrationand invasion in PC cells. Finally, our data reveal a discordant expres-sion of MUC4 at the transcript and protein levels, which is inverselyassociated with miR-150 expression in malignant clinical specimens.Altogether, our study characterizes a novel miRNA-mediated mech-anism of MUC4 regulation and suggests tumor suppressive actions ofmiR-150 in PC cells.Materials and methods

IL1-Induced JAK/STAT Signaling Is Antagonized by TGFβ to Shape CAF Heterogeneity in Pancreatic Ductal Adenocarcinoma.

Abstract: Pancreatic ductal adenocarcinoma (PDAC) is poorly responsive to therapies and histologically contains a paucity of neoplastic cells embedded within a dense desmoplastic stroma. Within the stroma, cancer-associated fibroblasts (CAFs) secrete tropic factors and extracellular matrix components, and have been implicated in PDAC progression and chemotherapy resistance. We recently identified two distinct CAF subtypes characterized by either myofibroblastic or inflammatory phenotypes; however, the mechanisms underlying their diversity and their roles in PDAC remain unknown. Here, we use organoid and mouse models to identify TGF-beta and IL-1 as tumor-secreted ligands that promote CAF heterogeneity. We show that IL-1 induces LIF expression and downstream JAK/STAT activation to generate inflammatory CAFs, and demonstrate that TGF-beta antagonizes this process by downregulating IL-1R1 expression and promoting differentiation into myofibroblasts. Our results provide a mechanism through which distinct fibroblast niches are established in the PDAC microenvironment and illuminate strategies to selectively target CAFs that support tumor growth.

Epigenetic modulators, modifiers and mediators in cancer aetiology and progression

Abstract: This year is the tenth anniversary of the publication in this journal of a model suggesting the existence of 'tumour progenitor genes'. These genes are epigenetically disrupted at the earliest stages of malignancies, even before mutations, and thus cause altered differentiation throughout tumour evolution. The past decade of discovery in cancer epigenetics has revealed a number of similarities between cancer genes and stem cell reprogramming genes, widespread mutations in epigenetic regulators, and the part played by chromatin structure in cellular plasticity in both development and cancer. In the light of these discoveries, we suggest here a framework for cancer epigenetics involving three types of genes: 'epigenetic mediators', corresponding to the tumour progenitor genes suggested earlier; 'epigenetic modifiers' of the mediators, which are frequently mutated in cancer; and 'epigenetic modulators' upstream of the modifiers, which are responsive to changes in the cellular environment and often linked to the nuclear architecture. We suggest that this classification is helpful in framing new diagnostic and therapeutic approaches to cancer.

Fibroblast heterogeneity in the cancer wound

Abstract: Fibroblasts regulate the structure and function of healthy tissues, participate transiently in tissue repair after acute inflammation, and assume an aberrant stimulatory role during chronic inflammatory states including cancer. Such cancer-associated fibroblasts (CAFs) modulate the tumor microenvironment and influence the behavior of neoplastic cells in either a tumor-promoting or tumor-inhibiting manner. These pleiotropic functions highlight the inherent plasticity of fibroblasts and may provide new avenues to understand and therapeutically intervene in malignancies. We discuss the emerging themes of CAF biology in the context of tumorigenesis and therapy.

The let-7 microrna reduces tumor growth in mouse models of lung cancer

Abstract: LB-194 Lung cancer is the most prevalent form of cancer worldwide and accounts for the most cancer deaths. MicroRNAs (miRNAs) are small, non-protein coding RNAs that have recently emerged as important regulators of gene expression and direct proper cellular growth, differentiation and cell death - all mechanisms that go awry in cancer. The let-7 miRNA is postulated to function as a tumor suppressor gene in a variety of human tissues, particularly in the lung, by negatively regulating the post-transcriptional expression of multiple oncogenes including RAS, MYC, and HMGA2, as well as other cell cycle progression genes. Here we have used both in vitro and in vivo approaches to show that let-7 directly represses cancer growth in the lung. We show that let-7 inhibits the growth of multiple human lung cancer cell lines in culture, as well as the growth of lung cancer cell xenografts in immunodeficient mice. Using the established Lox-Stop-Lox K-ras mouse lung cancer model, we find that intranasal let-7 administration can reduce tumor formation in vivo in the lungs of animals expressing a G12D activating mutation for the K-ras oncogene. These findings support the notion that let-7 functions as a tumor suppressor in the lung and indicates that this miRNA could be used as a therapeutic agent to treat lung cancer.