Connectivity mapping-based identification of pharmacological inhibitor targeting HDAC6 in aggressive pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) remains highly lethal due to limited therapeutic options and expensive/burdensome drug discovery processes. Utilizing genomic-data-driven Connectivity Mapping (CMAP) to identify a drug closer to real-world PC targeting may improve pancreatic cancer (PC) patient outcomes. Initially, we mapped CMAP data to gene expression from 106 PC patients, identifying nine negatively connected drugs. These drugs were further narrowed down using a similar analysis for PC cell lines, human tumoroids, and patient-derived xenografts datasets, where ISOX emerged as the most potent agent to target PC. We used human and mouse syngeneic PC cells, human and mouse tumoroids, and in vivo mice to assess the ability of ISOX alone and in combination with 5FU to inhibit tumor growth. Global transcriptomic and pathway analysis of the ISOX-LINCS signature identified HDAC 6/cMyc as the target axis for ISOX. Specifically, we discovered that genetic and pharmacological targeting of HDAC 6 affected non-histone protein cMyc acetylation, leading to cMyc instability, thereby disrupting PC growth and metastasis by affecting cancer stemness. Finally, KrasG12D harboring tumoroids and mice responded effectively against ISOX and 5FU treatment by enhancing survival and controlling metastasis incidence. Overall, our data validate ISOX as a new drug to treat advanced PC patients without toxicity to normal cells. Our study supports the clinical utility of ISOX along with 5FU in future PC clinical trials.

Engineering an ACE2-Derived Fragment as a Decoy for Novel SARS-CoV-2 Virus.

Entry inhibitors are an important resource in the response against emerging pathogens like the novel SARS-CoV-2, which enters human cells via interaction between the surface spike glycoprotein and the cellular membrane receptor angiotensin-converting enzyme 2 (ACE2). Using a combination of comparative structural analyses of the binding surface of the spike to ACE2, docking experiments, and molecular dynamics simulations, we identified a stable fragment of ACE2 that binds to the spike, is soluble, and is not predicted to bind to its physiological ligand angiotensin II. From this fragment we computationally designed and experimentally validated a smaller, stable peptide that disrupts ACE2-spike interaction at nanomolar concentrations, suggesting its potential use as a decoy that could interfere with viral binding by competition.

Reprogramming of tissue metabolism during cancer metastasis.

Cancer is a systemic disease that involves malignant cell-intrinsic and -extrinsic metabolic adaptations. Most studies have tended to focus on elucidating the metabolic vulnerabilities in the primary tumor microenvironment, leaving the metastatic microenvironment less explored. In this opinion article, we discuss the current understanding of the metabolic crosstalk between the cancer cells and the tumor microenvironment, both at local and systemic levels. We explore the possible influence of the primary tumor secretome to metabolically and epigenetically rewire the nonmalignant distant organs during prometastatic niche formation and successful metastatic colonization by the cancer cells. In an attempt to understand the process of prometastatic niche formation, we have speculated how cancer may hijack the inherent regenerative propensity of tissue parenchyma during metastatic colonization.

Elevated PAF1-RAD52 axis confers chemoresistance to human cancers.

Cisplatin- and gemcitabine-based chemotherapeutics represent a mainstay of cancer therapy for most solid tumors; however, resistance limits their curative potential. Here, we identify RNA polymerase II-associated factor 1 (PAF1) as a common driver of cisplatin and gemcitabine resistance in human cancers (ovarian, lung, and pancreas). Mechanistically, cisplatin- and gemcitabine-resistant cells show enhanced DNA repair, which is inhibited by PAF1 silencing. We demonstrate an increased interaction of PAF1 with RAD52 in resistant cells. Targeting the PAF1 and RAD52 axis combined with cisplatin or gemcitabine strongly diminishes the survival potential of resistant cells. Overall, this study shows clinical evidence that the expression of PAF1 contributes to chemotherapy resistance and worse clinical outcome for lethal cancers.

Muc4 loss mitigates epidermal growth factor receptor activity essential for PDAC tumorigenesis.

Mucin4 (MUC4) appears early during pancreatic intraepithelial neoplasia-1 (PanIN1), coinciding with the expression of epidermal growth factor receptor-1 (EGFR). The EGFR signaling is required for the onset of Kras-driven pancreatic ductal adenocarcinoma (PDAC); however, the players and mechanisms involved in sustained EGFR signaling in early PanIN lesions remain elusive. We generated a unique Esai-CRISPR-based Muc4 conditional knockout murine model to evaluate its effect on PDAC pathology. The Muc4 depletion in the autochthonous murine model carrying K-ras and p53 mutations (K-rasG12D; TP53R172H; Pdx-1cre, KPC) to generate the KPCM4-/- murine model showed a significant delay in the PanIN lesion formation with a significant reduction (p < 0.01) in EGFR (Y1068) and ERK1/2 (T202/Y204) phosphorylation. Further, a significant decrease (p < 0.01) in Sox9 expression in PanIN lesions of KPCM4-/- mice suggested the impairment of acinar-to-ductal metaplasia in Muc4-depleted cells. The biochemical analyses demonstrated that MUC4, through its juxtamembrane EGF-like domains, interacts with the EGFR ectodomain, and its cytoplasmic tail prevents EGFR ubiquitination and subsequent proteasomal degradation upon ligand stimulation, leading to sustained downstream oncogenic signaling. Targeting the MUC4 and EGFR interacting interface provides a promising strategy to improve the efficacy of EGFR-targeted therapies in PDAC and other MUC4-expressing malignancies.

Malondialdehyde-Acetaldehyde Extracellular Matrix Protein Adducts Attenuate Unfolded Protein Response During Alcohol and Smoking-Induced Pancreatitis.

BACKGROUND & AIMS: Epidemiological studies have established alcohol and smoking as independent risk factors for recurrent acute pancreatitis and chronic pancreatitis. However, the molecular players responsible for the progressive loss of pancreatic parenchyma and fibroinflammatory response are poorly characterized. METHODS: Tandem mass tag-based proteomic and bioinformatics analyses were performed on the pancreata of mice exposed to alcohol, cigarette smoke, or a combination of alcohol and cigarette smoke. Biochemical, immunohistochemistry, and transcriptome analyses were performed on the pancreatic tissues and primary acinar cells treated with cerulein in combination with ethanol (50 mmol/L) and cigarette smoke extract (40 µg/mL) for the mechanistic studies. RESULTS: A unique alteration in the pancreatic proteome was observed in mice exposed chronically to the combination of alcohol and cigarette smoke (56.5%) compared with cigarette smoke (21%) or alcohol (17%) alone. The formation of toxic metabolites (P < .001) and attenuated unfolded protein response (P < .04) were the significantly altered pathways on combined exposure. The extracellular matrix (ECM) proteins showed stable malondialdehyde-acetaldehyde (MAA) adducts in the pancreata of the combination group and chronic pancreatitis patients with a history of smoking and alcohol consumption. Interestingly, MAA-ECM adducts significantly suppressed expression of X-box-binding protein-1, leading to acinar cell death in the presence of alcohol and smoking. The stable MAA-ECM adducts persist even after alcohol and smoking cessation, and significantly delay pancreatic regeneration by abrogating the expression of cyclin-dependent kinases (CDK7 and CDK5) and regeneration markers. CONCLUSIONS: The combined alcohol and smoking generate stable MAA-ECM adducts that increase endoplasmic reticulum stress and acinar cell death due to attenuated unfolded protein response and suppress expression of cell cycle regulators. Targeting aldehyde adducts might provide a novel therapeutic strategy for the management of recurrent acute pancreatitis and chronic pancreatitis.

Chemokine-mucinome interplay in shaping the heterogeneous tumor microenvironment of pancreatic cancer.

Pancreatic cancer (PC) is exemplified by a complex immune-suppressive, fibrotic tumor microenvironment (TME), and aberrant expression of mucins. The constant crosstalk between cancer cells, cancer-associated fibroblasts (CAFs), and the immune cells mediated by the soluble factors and inflammatory mediators including cytokines, chemokines, reactive oxygen species (ROS) promote the dynamic temporal switch towards an immune-escape phenotype in the neoplastic cells and its microenvironment that bolsters disease progression. Chemokines have been studied in PC pathogenesis, albeit poorly in the context of mucins, tumor glycocalyx, and TME heterogeneity (CAFs and immune cells). With correlative analysis from PC patients' transcriptome data, support from available literature, and scientific arguments-based speculative extrapolations in terms of disease pathogenesis, we have summarized in this review a comprehensive understanding of chemokine-mucinome interplay during stromal modulation and immune-suppression in PC. Future studies should focus on deciphering the complexities of chemokine-mediated control of glycocalyx maturation, immune infiltration, and CAF-associated immune suppression. Knowledge extracted from such studies will be beneficial to mechanistically correlate the mucin-chemokine abundance in serum versus pancreatic tumors of patients, which may aid in prognostication and stratification of PC patients for immunotherapy.

Mucin 5AC-Mediated CD44/ITGB1 Clustering Mobilizes Adipose-Derived Mesenchymal Stem Cells to Modulate Pancreatic Cancer Stromal Heterogeneity.

BACKGROUND & AIMS: Secreted mucin 5AC (MUC5AC) promotes pancreatic cancer (PC) progression and chemoresistance, suggesting its clinical association with poor prognosis. RNA sequencing analysis from the autochthonous pancreatic tumors showed a significant stromal alteration on genetic ablation of Muc5ac. Previously, depletion or targeting the stromal fibroblasts showed an ambiguous effect on PC pathogenesis. Hence, identifying the molecular players and mechanisms driving fibroblast heterogeneity is critical for improved clinical outcomes. METHODS: Autochthonous murine models of PC (KrasG12D, Pdx1-Cre [KC] and KrasG12D, Pdx1-Cre, Muc5ac-/- [KCM]) and co-implanted allografts of murine PC cell lines (Muc5ac wild-type and CRISPR/Cas knockout) with adipose-derived mesenchymal stem cells (AD-MSCs) were used to assess the role of Muc5ac in stromal heterogeneity. Proliferation, migration, and surface expression of cell-adhesion markers on AD-MSCs were measured using live-cell imaging and flow cytometry. MUC5AC-interactome was investigated using mass-spectrometry and enzyme-linked immunosorbent assay. RESULTS: The KCM tumors showed a significant decrease in the expression of α-smooth muscle actin and fibronectin compared with histology-matched KC tumors. Our study showed that MUC5AC, carrying tumor secretome, gets enriched in the adipose tissues of tumor-bearing mice and patients with PC, promoting CD44/CD29 (integrin-ß1) clustering that leads to Rac1 activation and migration of AD-MSCs. Furthermore, treatment with KC-derived serum enhanced proliferation and migration of AD-MSCs, which was abolished on Muc5ac-depletion or pharmacologic inhibition of CXCR2 and Rac1, respectively. The AD-MSCs significantly contribute toward α-smooth muscle actin-positive cancer-associated fibroblasts population in Muc5ac-dependent manner, as suggested by autochthonous tumors, co-implantation xenografts, and patient tumors. CONCLUSION: MUC5AC, secreted during PC progression, enriches in adipose and enhances the mobilization of AD-MSCs. On recruitment to pancreatic tumors, AD-MSCs proliferate and contribute towards stromal heterogeneity.

Mucin 5AC Serves as the Nexus for ß-Catenin/c-Myc Interplay to Promote Glutamine Dependency During Pancreatic Cancer Chemoresistance.

BACKGROUND & AIMS: A major clinical challenge for patients with pancreatic cancer (PC) is metabolic adaptation. Neoplastic cells harboring molecular perturbations suffice for their increased anabolic demand and nucleotide biosynthesis to acquire chemoresistance. The mucin 5AC expressed de novo in malignant pancreas promotes cancer cell stemness and is significantly associated with poor patient survival. Identification of MUC5AC-associated drivers of chemoresistance through metabolic alterations may facilitate the sculpting of a new combinatorial regimen. METHODS: The contributions of MUC5AC to glutaminolysis and gemcitabine resistance were examined by The Cancer Genome Atlas data analysis, RNA sequencing, and immunohistochemistry analysis on pancreatic tissues of KrasG12D;Pdx1-Cre (KC) and KrasG12D;Pdx1-Cre;Muc5ac-/- mice. These were followed by metabolite flux assays as well as biochemical and xenograft studies on MUC5AC-depleted human and murine PC cells. Murine and human pancreatic 3-dimensional tumoroids were used to evaluate the efficacy of gemcitabine in combination with ß-catenin and glutaminolysis inhibitors. RESULTS: Transcriptional analysis showed that high MUC5AC-expressing human and autochthonous murine PC tumors exhibit higher resistance to gemcitabine because of enhanced glutamine use and nucleotide biosynthesis. Gemcitabine treatment led to MUC5AC overexpression, resulting in disruption of E-cadherin/ß-catenin junctions and the nuclear translocation of ß-catenin, which increased c-Myc expression, with a concomitant rise in glutamine uptake and glutamate release. MUC5AC depletion and glutamine deprivation sensitized human PC cells to gemcitabine, which was obviated by glutamine replenishment in MUC5AC-expressing cells. Coadministration of ß-catenin and glutaminolysis inhibitors with gemcitabine abrogated the MUC5AC-mediated resistance in murine and human tumoroids. CONCLUSIONS: The MUC5AC/ß-catenin/c-Myc axis increases the uptake and use of glutamine in PC cells, and cotargeting this axis along with gemcitabine may improve therapeutic efficacy in PC.

SUMO Modification of PAF1/PD2 Enables PML Interaction and Promotes Radiation Resistance in Pancreatic Ductal Adenocarcinoma.

RNA polymerase II-associated factor 1 (PAF1)/pancreatic differentiation 2 (PD2) is a core subunit of the human PAF1 complex (PAF1C) that regulates the RNA polymerase II function during transcriptional elongation. PAF1/PD2 has also been linked to the oncogenesis of pancreatic ductal adenocarcinoma (PDAC). Here, we report that PAF1/PD2 undergoes posttranslational modification (PTM) through SUMOylation, enhancing the radiation resistance of PDAC cells. We identified that PAF1/PD2 is preferentially modified by small ubiquitin-related modifier 1 (SUMO 1), and mutating the residues (K)-150 and 154 by site-directed mutagenesis reduces the SUMOylation. Interestingly, PAF1/PD2 was found to directly interact with the promyelocytic leukemia (PML) protein in response to radiation, and inhibition of PAF1/PD2 SUMOylation at K-150/154 affects its interaction with PML. Our results demonstrate that SUMOylation of PAF1/PD2 increased in the radiated pancreatic cancer cells. Furthermore, inhibition of SUMOylation or PML reduces the cell growth and proliferation of PDAC cells after radiation treatment. These results suggest that SUMOylation of PAF1/PD2 interacts with PTM for PDAC cell survival. Furthermore, abolishing the SUMOylation in PDAC cells enhances the effectiveness of radiotherapy. Overall, our results demonstrate a novel PTM and PAF1/PD2 interaction through SUMOylation, and inhibiting the SUMOylation of PAF1/PD2 enhance the therapeutic efficacy for PDAC.

Mucins reprogram stemness, metabolism and promote chemoresistance during cancer progression.

Mucins are high-molecular-weight glycoproteins dysregulated in aggressive cancers. The role of mucins in disease progression, tumor proliferation, and chemotherapy resistance has been studied extensively. This article provides a comprehensive review of mucin's function as a physical barrier and the implication of mucin overexpression in impeded drug delivery to solid tumors. Mucins regulate the epithelial to mesenchymal transition (EMT) of cancer cells via several canonical and non-canonical oncogenic signaling pathways. Furthermore, mucins play an extensive role in enriching and maintaining the cancer stem cell (CSC) population, thereby sustaining the self-renewing and chemoresistant cellular pool in the bulk tumor. It has recently been demonstrated that mucins regulate the metabolic reprogramming during oncogenesis and cancer progression, which account for tumor cell survival, proliferation, and drug-resistance. This review article focuses on delineating mucin's role in oncogenic signaling and aberrant regulation of gene expressions, culminating in CSC maintenance, metabolic rewiring, and development of chemoresistance, tumor progression, and metastasis.

The Current Landscape of Antibody-based Therapies in Solid Malignancies.

Over the past three decades, monoclonal antibodies (mAbs) have revolutionized the landscape of cancer therapy. Still, this benefit remains restricted to a small proportion of patients due to moderate response rates and resistance emergence. The field has started to embrace better mAb-based formats with advancements in molecular and protein engineering technologies. The development of a therapeutic mAb with long-lasting clinical impact demands a prodigious understanding of target antigen, effective mechanism of action, gene engineering technologies, complex interplay between tumor and host immune system, and biomarkers for prediction of clinical response. This review discusses the various approaches used by mAbs for tumor targeting and mechanisms of therapeutic resistance that is not only caused by the heterogeneity of tumor antigen, but also the resistance imposed by tumor microenvironment (TME), including inefficient delivery to the tumor, alteration of effector functions in the TME, and Fc-gamma receptor expression diversity and polymorphism. Further, this article provides a perspective on potential strategies to overcome these barriers and how diagnostic and prognostic biomarkers are being used in predicting response to mAb-based therapies. Overall, understanding these interdependent parameters can improve the current mAb-based formulations and develop novel mAb-based therapeutics for achieving durable clinical outcomes in a large subset of patients.

Secretory Mucin 5AC Promotes Neoplastic Progression by Augmenting KLF4-Mediated Pancreatic Cancer Cell Stemness.

Secreted mucin 5AC (MUC5AC) is the most abundantly overexpressed member of the mucin family during early pancreatic intraepithelial neoplasia stage I (PanIN-I) of pancreatic cancer. To comprehend the contribution of Muc5ac in pancreatic cancer pathology, we genetically ablated it in an autochthonous murine model (KrasG12D; Pdx-1cre, KC), which mirrors the early stages of pancreatic cancer development. Neoplastic onset and the PanIN lesion progression were significantly delayed in Muc5ac knockout (KrasG12D; Pdx-1 cre; Muc5ac-/-, KCM) animals with a 50% reduction in PanIN-2 and 70% reduction in PanIN-3 lesions compared with KC at 50 weeks of age. High-throughput RNA-sequencing analysis from pancreatic tissues of KCM animals revealed a significant decrease in cancer stem cell (CSC) markers Aldh1a1, Klf4, EpCAM, and CD133. Furthermore, the silencing of MUC5AC in human pancreatic cancer cells reduced their tumorigenic propensity, as indicated by a significant decline in tumor formation frequency by limiting dilution assay upon subcutaneous administration. The contribution of MUC5AC in CSC maintenance was corroborated by a significant decrease in tumor burden upon orthotopic implantation of MUC5AC-depleted pancreatic cancer cells. Mechanistically, MUC5AC potentiated oncogenic signaling through integrin αvß5, pSrc (Y416), and pSTAT3 (Y705). Phosphorylated STAT3, in turn, upregulated Klf4 expression, thereby enriching the self-renewing CSC population. A strong positive correlation of Muc5ac with Klf4 and pSTAT3 in the PanIN lesions of KC mouse pancreas reinforces the crucial involvement of MUC5AC in bolstering the CSC-associated tumorigenic properties of Kras-induced metaplastic cells, which leads to pancreatic cancer onset and progression. SIGNIFICANCE: This study elucidates that de novo expression of MUC5AC promotes cancer cell stemness during Kras-driven pancreatic tumorigenesis and can be targeted for development of a novel therapeutic regimen.

Differential mutation spectrum and immune landscape in African Americans versus Whites: A possible determinant to health disparity in head and neck cancer.

African Americans (AA) with Head and Neck Squamous Cell Carcinoma (HNSCC) have a worse disease prognosis than White patients despite adjusting for socio-economic factors, suggesting the potential biological contribution. Therefore, we investigated the genomic and immunological components that drive the differential tumor biology among race. We utilized the cancer genome atlas and cancer digital archive of HNSCC patients (1992-2013) for our study. We found that AA patients with HNSCC had a higher frequency of mutation compared to Whites in the key driver genes-P53, FAT1, CASP8 and HRAS. AA tumors also exhibited lower intratumoral infiltration of effector immune cells (CD8+, γδT, resting memory CD4+ and activated memory CD4+ T cells) with shorter survival than Whites. Unsupervised hierarchical clustering of differentially expressed genes demonstrated distinct gene clusters between AA and White patients with unique signaling pathway enrichments. Connectivity map analysis identified drugs (Neratinib and Selumetinib) that target aberrant PI3K/RAS/MEK signaling and may reduce racial disparity in therapy response.

Alcohol and Smoking Mediated Modulations in Adaptive Immunity in Pancreatitis.

Pancreatitis is a condition of pancreatic inflammation driven by injury to the pancreatic parenchyma. The extent of acinar insult, intensity, and type of immune response determines the severity of the disease. Smoking, alcohol and autoimmune pancreatitis are some of the predominant risk factors that increase the risk of pancreatitis by differentially influencing the adaptive immune system. The overall decrease in peripheral lymphocyte (T-, B- and (natural killer T-) NKT-cell) count and increased infiltration into the damaged pancreatic tissue highlight the contribution of adaptive immunity in the disease pathology. Smoking and alcohol modulate the responsiveness and apoptosis of T- and B-cells during pancreatic insult. Acute pancreatitis worsens with smoking and alcohol, leading to the development of systemic inflammatory response syndrome and compensatory anti-inflammatory response syndrome, suggesting the critical role of adaptive immunity in fatal outcomes such as multiple organ dysfunction. The presence of CD4+ and CD8+ T-lymphocytes and perforin-expressing cells in the fibrotic tissue in chronic pancreatitis modulate the severity of the disease. Due to their important role in altering the severity of the disease, attempts to target adaptive immune mediators will be critical for the development of novel therapeutic interventions.

Unraveling mucin domains in cancer and metastasis: when protectors become predators.

A dynamic mucosal layer shields the epithelial cells lining the body cavities and is made up of high molecular weight, heavily glycosylated, multidomain proteins called mucins. Mucins, broadly grouped into transmembrane and secreted mucins, are the first responders to any mechanical or chemical insult to the epithelia and help maintain tissue homeostasis. However, their intrinsic properties to protect and repair the epithelia are exploited during oncogenic processes, where mucins are metamorphosed to aid the tumor cells in their malignant journey. Diverse domains, like the variable number tandem repeats (VNTR), sea urchin sperm protein enterokinase and agrin (SEA), adhesion-associated domain (AMOP), nidogen-like domain (NIDO), epidermal growth factor-like domain (EGF), and von Willebrand factor type D domain (vWD) on mucins, including MUC1, MUC4, MUC5AC, MUC5B, and MUC16, have been shown to facilitate cell-to-cell and cell-to-matrix interactions, and cell-autonomous signaling to promote tumorigenesis and distant dissemination of tumor cells. Several obstacles have limited the study of mucins, including technical difficulties in working with these huge glycoproteins, the dearth of scientific tools, and lack of animal models; thus, the tissue-dependent and domain-specific roles of mucins during mucosal protection, chronic inflammation, tumorigenesis, and hematological dissemination of malignant cells are still unclear. Future studies should try to integrate information on the rheological, molecular, and biological characteristics of mucins to comprehensively delineate their pathophysiological role and evaluate their suitability as targets in future diagnostic and therapeutic strategies.

Mechanistic and Functional Shades of Mucins and Associated Glycans in Colon Cancer.

Mucus serves as the chief protective barrier against pathogenic and mechanical insults in respiratory, gastrointestinal, and urogenital tracts. Altered mucin expression, the major component of mucus, in conjunction with differential glycosylation has been strongly associated with both benign and malignant pathologies of colon. Mucins and their associated glycans arbitrate their impact sterically as well as mechanically by altering molecular and microbial spectrum during pathogenesis. Mucin expression in normal and pathological conditions is regulated by nonspecific (dietary factors and gut microbiota) and specific (epigenetic and transcriptional) modulators. Further, recent studies highlight the impact of altering mucin glycome (cancer-associated carbohydrate antigens including Tn, Sialyl-Tn, Sialyl-Lew A, and Sialyl-Lewis X) on host immunomodulation, antitumor immunity, as well as gut microbiota. In light of emerging literature, the present review article digs into the impact of structural organization and of expressional and glycosylation alteration of mucin family members on benign and malignant pathologies of colorectal cancer.

Molecular implications of MUC5AC-CD44 axis in colorectal cancer progression and chemoresistance.

BACKGROUND: Differential expression of mucins has been associated with several cancers including colorectal cancer (CRC). In normal physiological conditions, secretory mucin MUC5AC is not expressed in the colonic mucosa, whereas its aberrant expression is observed during development of colon cancer and its precursor lesions. To date, the molecular mechanism of MUC5AC in CRC progression and drug resistance remains obscure. METHODS: MUC5AC expression was determined in colon tissue microarray by immunohistochemistry. A RNA interference and CRISPR/Cas9-mediated system was used to knockdown/knockout the MUC5AC in CRC cell lines to delineate its role in CRC tumorigenesis using in vitro functional assays and in vivo (sub-cutaneous and colon orthotopic) mouse models. Finally, CRC cell lines and xenograft models were used to identify the mechanism of action of MUC5AC. RESULTS: Overexpression of MUC5AC is observed in CRC patient tissues and cell lines. MUC5AC expression resulted in enhanced cell invasion and migration, and decreased apoptosis of CRC cells. MUC5AC interacted with CD44 physically, which was accompanied by the activation of Src signaling. Further, the presence of MUC5AC resulted in enhanced tumorigenesis and appearance of metastatic lesions in orthotopic mouse model. Additionally, up-regulation of MUC5AC resulted in resistance to 5-fluorouracil (5-FU) and oxaliplatin, and its knockout increased sensitivity to these drugs. Finally, we observed that up-regulation of MUC5AC conferred resistance to 5-FU through down-regulation of p53 and its target gene p21 and up-regulation of ß-catenin and its target genes CD44 and Lgr5. CONCLUSION: Our findings suggest that differential expression of secretory mucin MUC5AC results in enhanced tumorigenesis and also confers chemoresistance via CD44/ß-catenin/p53/p21 signaling.

Trefoil factor(s) and CA19.9: A promising panel for early detection of pancreatic cancer.

BACKGROUND: Trefoil factors (TFF1, TFF2, and TFF3) are small secretory molecules that recently have gained significant attention in multiple studies as an integral component of pancreatic cancer (PC) subtype-specific gene signature. Here, we comprehensively investigated the diagnostic potential of all the member of trefoil family, i.e., TFF1, TFF2, and TFF3 in combination with CA19.9 for detection of PC. METHODS: Trefoil factors (TFFs) gene expression was analyzed in publicly available cancer genome datasets, followed by assessment of their expression in genetically engineered spontaneous mouse model (GEM) of PC (KrasG12D; Pdx1-Cre (KC)) and in human tissue microarray consisting of normal pancreas adjacent to tumor (NAT), precursor lesions (PanIN), and various pathological grades of PC by immunohistochemistry (IHC). Serum TFFs and CA19.9 levels were evaluated via ELISA in comprehensive sample set (n = 362) comprised of independent training and validation sets each containing benign controls (BC), chronic pancreatitis (CP), and various stages of PC. Univariate and multivariate logistic regression and receiver operating characteristic curves (ROC) were used to examine their diagnostic potential both alone and in combination with CA19.9. FINDINGS: The publicly available datasets and expression analysis revealed significant increased expression of TFF1, TFF2, and TFF3 in human PanINs and PC tissues. Assessment of KC mouse model also suggested upregulated expression of TFFs in PanIN lesions and early stage of PC. In serum analyses studies, TFF1 and TFF2 were significantly elevated in early stages of PC in comparison to benign and CP control group while significant elevation in TFF3 levels were observed in CP group with no further elevation in its level in early stage PC group. In receiver operating curve (ROC) analyses, combination of TFFs with CA19.9 emerged as promising panel for discriminating early stage of PC (EPC) from BC (AUCTFF1+TFF2+TFF3+CA19.9 = 0.93) as well as CP (AUCTFF1+TFF2+TFF3+CA19.9 = 0.93). Notably, at 90% specificity (desired for blood-based biomarker panel), TFFs combination improved CA19.9 sensitivity by 10% and 25% to differentiate EPC from BC and CP respectively. In an independent blinded validation set, the combination of TFFs and CA19.9 (AUCTFF1+TFF2+TFF3+CA19.9 = 0.82) also improved the overall efficacy of CA19.9 (AUCCA19.9 = 0.66) to differentiate EPC from CP proving unique biomarker capabilities of TFFs to distinguish early stage of this deadly lethal disease. INTERPRETATION: In silico, tissue and serum analyses validated significantly increased level of all TFFs in precursor lesions and early stages of PC. The combination of TFFs enhanced sensitivity and specificity of CA19.9 to discriminate early stage of PC from benign control and chronic pancreatitis groups.

Systems Biology Approach to Identify Novel Genomic Determinants for Pancreatic Cancer Pathogenesis.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy with a 5-year survival rate of <8%. Its dismal prognosis stems from inefficient therapeutic modalities owing to the lack of understanding about pancreatic cancer pathogenesis. Considering the molecular complexity and heterogeneity of PDAC, identification of novel molecular contributors involved in PDAC onset and progression using global "omics" analysis will pave the way to improved strategies for disease prevention and therapeutic targeting. Meta-analysis of multiple miRNA microarray datasets containing healthy controls (HC), chronic pancreatitis (CP) and PDAC cases, identified 13 miRNAs involved in the progression of PDAC. These miRNAs showed dysregulation in both tissue as well as blood samples, along with progressive decrease in expression from HC to CP to PDAC. Gene-miRNA interaction analysis further elucidated 5 miRNAs (29a/b, 27a, 130b and 148a) that are significantly downregulated in conjunction with concomitant upregulation of their target genes throughout PDAC progression. Among these, miRNA-29a/b targeted genes were found to be most significantly altered in comparative profiling of HC, CP and PDAC, indicating its involvement in malignant evolution. Further, pathway analysis suggested direct involvement of miRNA-29a/b in downregulating the key pathways associated with PDAC development and metastasis including focal adhesion signaling and extracellular matrix organization. Our systems biology data analysis, in combination with real-time PCR validation indicates direct functional involvement of miRNA-29a in PDAC progression and is a potential prognostic marker and therapeutic candidate for patients with progressive disease.