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Background Several reports have shown the role of glycosylation in pancreatic cancer (PC), but a global systematic screening of specific glycosyltransferases (glycoTs) in its progression remains unknown. Methods We demonstrate a rigorous top-down approach using TCGA-based RNA-Seq analysis, multi-step validation using RT-qPCR, immunoblots and immunohistochemistry. We identified six unique glycoTs (B3GNT3, B4GALNT3, FUT3, FUT6, GCNT3 and MGAT3) in PC pathogenesis and studied their function using CRISPR/Cas9-based KD systems. Results Serial metastatic in vitro models using T3M4 and HPAF/CD18, generated in house, exhibited decreases in B3GNT3, FUT3 and GCNT3 expression on increasing metastatic potential. Immunohistochemistry identified clinical significance for GCNT3, B4GALNT3 and MGAT3 in PC. Furthermore, the effects of B3GNT3, FUT3, GCNT3 and MGAT3 were shown on proliferation, migration, EMT and stem cell markers in CD18 cell line. Talniflumate, GCNT3 inhibitor, reduced colony formation and migration in T3M4 and CD18 cells. Moreover, we found that loss of GCNT3 suppresses PC progression and metastasis by downregulating cell cycle genes and β-catenin/MUC4 axis. For GCNT3, proteomics revealed downregulation of MUC5AC, MUC1, MUC5B including many other proteins. Conclusions Collectively, we demonstrate a critical role of O- and N-linked glycoTs in PC progression and delineate the mechanism encompassing the role of GCNT3 in PC.

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.

Lung cancer is the leading cause of cancer death worldwide, with lung adenocarcinoma being the most common subtype. Many oncogenes and tumor suppressor genes are altered in this cancer type and the discovery of oncogene mutations has led to the development of targeted therapies and better clinical outcomes. However, a large fraction of lung adenocarcinomas lacks mutations in known oncogenes, and the genesis and treatment of these oncogene-negative tumors remain enigmatic. Here, we perform iterative in vivo functional screens with quantitative autochthonous mouse model systems to uncover the genetic and biochemical changes that enable efficient lung tumor initiation in the absence of oncogene alterations. Through the generation of hundreds of diverse combinations of tumor suppressor alterations, we demonstrate that inactivation of suppressors of the RAS and PI3K pathways drive the development of oncogene- negative lung adenocarcinoma. Pathway-level human genomic data analysis and histology identified RAS/MAPK and PI3K pathway activation as a common event in oncogene-negative human lung adenocarcinomas. We demonstrate that oncogene-negative tumors and cell lines with activated RAS/MAPK and PI3K pathways are vulnerable to pharmacological inhibition of these signaling axes. Collectively, these results transform our understanding of this prevalent yet understudied subtype of lung adenocarcinoma. STATEMENT OF SIGNIFICANCEMany lung adenocarcinomas do not have mutations in known proto-oncogenes, and as a result, targeted therapies are unavailable for treating these patients. Here, we uncover driver pathways in a subset of these oncogene-negative lung adenocarcinomas and demonstrate the therapeutic value of inhibiting these pathways.

The Ras family of GTPases is involved in cell proliferation, cell survival, and angiogenesis. It is upregulated in several cancers, including pancreatic cancer (PC) and leads to uncontrolled growth and aggressiveness. PC is well known to be a lethal disease with poor prognosis, plagued by limited therapeutic modalities. MicroRNAs (miRNAs), which are short non-coding RNA molecules, have recently emerged as regulators of signaling networks and have shown potential to target pathway components for therapeutic use in several malignancies. K-Ras mutations are widespread in PC cases (90%), with mutations detectable as early as pancreatic intraepithelial neoplasias and in later metastatic stages alike; therefore, these mutations in K-Ras are obvious drivers and potential targets for PC therapy. Several K-Ras targeting miRNAs have lately been discovered, and many of them have shown promise in combating pancreatic tumor growth in vitro and in mouse models. However, the field of miRNA therapy is still in its infancy, and miRNA mimics or anti-miRNA oligonucleotides that target Ras pathway have thus far not been evaluated in PC patients. In this review, we summarize the role of several miRNAs that regulate oncogenic K-Ras signaling in PC, with their prospective roles as therapeutic agents for targeting K-Ras pathway.

Aberrant protein glycosylation has been shown to have a significant contribution in aggressive cancer, including pancreatic cancer (PC). Emerging evidence has implicated the involvement of cancer stem cells (CSCs) in PC aggressiveness; however, the contribution of glycosylation on self-renewal properties and maintenance of CSC is understudied. Here, using several in vitro and in vivo models lacking C1GALT1 expression, we identified the role of aberrant O-glycosylation in stemness properties and aggressive PC metastasis. A loss in C1GALT1 was found to result in the truncation of O-glycosylation on several glycoproteins with an enrichment of Tn carbohydrate antigen. Mapping of Tn-bearing glycoproteins in C1GALT1 KO cells identified significant Tn enrichment on CSC glycoprotein CD44. Notably, a loss of C1GALT1 in PC cells was found to enhance CSC features (side population-SP, ALDH1+, and tumorspheres) and self-renewal markers NANOG, SOX9, and KLF4. Furthermore, a loss of CD44 in existing C1GALT1 KO cells decreased NANOG expression and CSC features. We determined that O-glycosylation of CD44 activates ERK/NF-kB signaling, which results in increased NANOG expression in PC cells that facilitated the alteration of CSC features, suggesting that NANOG is essential for PC stemness. Finally, we identified that loss of C1GALT1 expression was found to augment tumorigenic and metastatic potential, while an additional loss of CD44 in these cells reversed the effects. Overall, our results identified that truncation of O-glycans on CD44 increases NANOG activation that mediates increased CSC activation.

Background & Aims Tumor-microenvironment factors and cancer stem cells (CSCs) play a critical role in the aggressiveness of pancreatic cancer (PC). However, the degree to which tumor-microenvironment factors promote stemness remains unexplored. Here, we examined whether cancer-associated fibroblasts (CAFs) promote CSC features in PC. Methods PC cells were treated long-term (30, 60, and 90 days) with conditioned media (CM)-derived from normal human fibroblasts (NFs) and CAFs. The stemness features of tumorsphere formation and stemness populations, along with CSCs markers, were analyzed using 2-dimensional and 3-dimensional sodium alginate bead-based co-culture models. Immunohistochemistry and immunofluorescence staining were performed for CSCs and fibroblast markers in autochthonous KrasG12D/+; Trp53R172H/+; Pdx1-Cre mice and human pancreatic tumors. Polymerase chain reaction array and gene knockdown were performed to identify the mechanism of stemness enrichment. Results Long-term treatment of PC cells with CAF-CM enriched stemness, as indicated by significantly higher CD44+, ALDH+, and AF+ populations in PC cells. Increased tumorsphere formation and elevated CSC, self-renewal, and drug-resistance markers in CAF-CM–treated PC cells were observed. In addition, CAFs co-cultured with PC cells in the 3-dimensional model showed a substantial increase in stemness features. CD44 and α–smooth muscle actin were positively correlated and their expressions progressively increased from the early to late stages of KrasG12D/+; Trp53R172H/+; Pdx1-Cre mouse and human pancreatic tumors. Osteopontin/secreted phosphoprotein 1 was identified as the top differentially overexpressed gene in CAF-CM–treated PC cells and knockdown of osteopontin/secreted phosphoprotein 1 significantly reduced stemness characteristics in CAF-CM–treated PC cells. Conclusions Our data uncovered novel insight into the interplay between CAF and enrichment of stemness population through the osteopontin/secreted phosphoprotein 1–CD44 axis in PC.

Pancreatic differentiation 2 (PD2)/RNA polymerase II–associated factor 1 (PAF1) is the core subunit of the human PAF1 complex (PAF1C) that regulates the promoter-proximal pausing of RNA polymerase II as well as transcription elongation and mRNA processing and coordinates events in mRNA stability and quality control. As an integral part of its transcription-regulatory function, PD2/PAF1 plays a role in posttranslational histone covalent modifications as well as regulates expression of critical genes of the cell-cycle machinery. PD2/PAF1 alone, and as a part of PAF1C, provides distinct roles in the maintenance of self-renewal of embryonic stem cells and cancer stem cells, and in lineage differentiation. Thus, PD2/PAF1 malfunction or its altered abundance is likely to affect normal cellular functions, leading to disease states. Indeed, PD2/PAF1 is found to be upregulated in poorly differentiated pancreatic cancer cells and has the capacity for neoplastic transformation when ectopically expressed in mouse fibroblast cells. Likewise, PD2/PAF1 is upregulated in pancreatic and ovarian cancer stem cells. Here, we concisely describe multifaceted roles of PD2/PAF1 associated with oncogenic transformation and implicate PD2/PAF1 as an attractive target for therapeutic development to combat malignancy. Cancer Res; 78(2); 313–9. ©2018 AACR.

Background & Aims It is not clear how pancreatic cancer stem cells (CSCs) are regulated, resulting in ineffective treatments for pancreatic cancer. PAF1, a RNA polymerase II-associated factor 1 complex (PAF1C) component, maintains pluripotency of stem cells, by unclear mechanisms, and is a marker of CSCs. We investigated mechanisms by which PAF1 maintains CSCs and contributes to development of pancreatic tumors. Methods Pancreatic cancer cell lines were engineered to knockdown PAF1 using inducible small hairpin RNAs. These cells were grown as orthotopic tumors in athymic nude mice and PAF1 knockdown was induced by administration of doxycycline in drinking water. Tumor growth and metastasis were monitored via IVIS imaging. CSCs were isolated from pancreatic cancer cell populations using flow cytometry and characterized by tumor sphere formation, tumor formation in nude mice, and expression of CSC markers. Isolated CSCs were depleted of PAF1 using the CRISPR/Cas9 system. PAF1-regulated genes in CSCs were identified via RNA-seq and PCR array analyses of cells with PAF1 knockdown. Proteins that interact with PAF1 in CSCs were identified by immunoprecipitations and mass spectrometry. We performed chromatin immunoprecipitation sequencing of CSCs to confirm the binding of the PAF1 sub-complex to target genes. Results Pancreatic cancer cells depleted of PAF1 formed smaller and fewer tumor spheres in culture and orthotopic tumors and metastases in mice. Isolated CSCs depleted of PAF1 downregulated markers of self-renewal (NANOG, SOX9, and β-CATENIN), of CSCs (CD44v6, and ALDH1), and the metastasis-associated gene signature, compared to CSCs without knockdown of PAF1. The role of PAF1 in CSC maintenance was independent of its RNA polymerase II-associated factor 1 complex component identity. We identified DDX3 and PHF5A as proteins that interact with PAF1 in CSCs and demonstrated that the PAF1–PHF5A–DDX3 sub-complex bound to the promoter region of Nanog, whose product regulates genes that control stemness. Levels of the PAF1–DDX3 and PAF1–PHF5A were increased and co-localized in human pancreatic tumor specimens, human pancreatic tumor-derived organoids, and organoids derived from tumors of KPC mice, compared with controls. Binding of DDX3 and PAF1 to the Nanog promoter, and the self-renewal capacity of CSCs, were decreased in cells incubated with the DDX3 inhibitor RK-33. CSCs depleted of PAF1 downregulated genes that regulate stem cell features (Flot2, Taz, Epcam, Erbb2, Foxp1, Abcc5, Ddr1, Muc1, Pecam1, Notch3, Aldh1a3, Foxa2, Plat, and Lif). Conclusions In pancreatic CSCs, PAF1 interacts with DDX3 and PHF5A to regulate expression of NANOG and other genes that regulate stemness. Knockdown of PAF1 reduces the ability of orthotopic pancreatic tumors to develop and progress in mice and their numbers of CSCs. Strategies to target the PAF1–PHF5A–DDX3 complex might be developed to slow or inhibit progression of pancreatic cancer.

// Arokia Priyanka Vaz 1 , Shonali Deb 1 , Satyanarayana Rachagani 1 , Parama Dey 1 , Sakthivel Muniyan 1 , Imayavaramban Lakshmanan 1 , Saswati Karmakar 1 , Lynette Smith 2 , Sonny Johansson 3 , Subodh Lele 3 , Michel Ouellette 1, 4 , Moorthy P. Ponnusamy 1, 5 , Surinder K. Batra 1, 3, 5 1 Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE, USA 2 Department of Biostatistics, University of Nebraska Medical Center, Omaha, NE, USA 3 Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE, USA 4 Department of Internal Medicine, Division of Gastroenterology and Hepatology, University of Nebraska Medical Center, Omaha, NE, USA 5 Eppley Institute for Research in Cancer and Allied Disease, University of Nebraska Medical Center, Omaha, NE, USA Correspondence to: Surinder K. Batra, e-mail: sbatra@unmc.edu Moorthy P. Ponnusamy, e-mail: mpalanim@unmc.edu Keywords: PD2, CSC, c-Myc, PDAC Received: August 12, 2015 Accepted: November 16, 2015 Published: December 12, 2015 ABSTRACT Pancreatic differentiation 2 (PD2), an important subunit of the human PAF complex, was identified after differential screening analysis of 19q13 amplicon, and its overexpression induces oncogenic transformation of NIH3T3 cells, hence raising the possibility of a role for PD2 in tumorigenesis and metastasis. To test this hypothesis, we analyzed here the functional role and clinical significance of PD2 in pancreatic ductal adenocarcinoma (PDAC) and its pathogenesis. Using immunohistochemical analysis, we found that PD2 is detected in the acini but not in the ducts in the normal pancreas. In human PDAC specimens, PD2 was instead primarily detected in the ducts (12/48 patients 25%; p -value < 0.0001), thereby showing that PDAC correlates with increased ductal expression of PD2. Consistently, PD2 expression was increased in telomerase-immortalized human pancreatic ductal cells (HPNE cells) modified to express the HPV16 E6 and E7 proteins, whose respective functions are to block p53 and RB. In addition, ectopic expression of PD2 in PDAC cells (Capan-1 and SW1990) led to increased clonogenicity and migration in vitro , and tumor growth and metastasis in vivo . Interestingly, PD2 overexpression also resulted in enrichment of cancer stem cells (CSCs) and upregulation of oncogenes such as c-Myc and cell cycle progression marker, cyclin D1. Taken together, our results support that PD2 is overexpressed in the ducts of PDAC tissues, and results in tumorigenesis and metastasis via upregulation of oncogenes such as c-Myc and cyclin hence D1 implicating PD2 upregulation in pancreatic oncogenesis with targeted therapeutic potential.

// Saswati Karmakar 1, * , Parthasarathy Seshacharyulu 1, * , Imayavaramban Lakshmanan 1 , Arokia P. Vaz 1 , Seema Chugh 1 , Yuri M. Sheinin 3 , Sidharth Mahapatra 4 , Surinder K. Batra 1, 2 , Moorthy P. Ponnusamy 1, 2 1 Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE, USA 2 Fred and Pamela Buffett Cancer Center, Eppley Institute for Research in Cancer and Allied Disease, University of Nebraska Medical Center, Omaha, NE, USA 3 Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE, USA 4 Department of Pediatrics, University of Nebraska Medical Center, Omaha, NE, USA * These authors contributed equally to this work Correspondence to: Moorthy P. Ponnusamy, email: mpalanim@unmc.edu Surinder K. Batra, email: sbatra@unmc.edu Keywords: hPaf1/PD2, CSC, ovarian cancer, OCT3/4, self-renewal Received: November 18, 2016 Accepted: January 11, 2017 Published: January 20, 2017 ABSTRACT Cancer stem cells (CSCs), which mediate drug resistance and disease recurrence in several cancers, are therapeutically relevant to ovarian cancer (OC), wherein approximately 80% of patients manifest with tumor recurrence. While there are several markers for ovarian CSCs (OCSCs), the mechanism for their self-renewal maintenance by unique driver/markers is poorly understood. Here, we evaluated the role of hPaf1/PD2, a core component of RNA Polymerase II-Associated Factor (PAF) complex, in self-renewal of OCSCs through marker and functional analyses, including CRISPR/Cas9-silencing of hPaf1/PD2 in OCSCs and provided a possible mechanism for maintenance of OCSCs. Expression of hPaf1/PD2 showed moderate to intense staining in 32.4% of human OC tissues, whereas 67.6% demonstrated basal expression by immunohistochemistry analysis, implying that the minor proportion of cells overexpressing hPaf1/PD2 could be putative OCSCs. Isolated OCSCs showed higher expression of hPaf1/PD2 along with established CSC and self-renewal markers. Knockdown of hPaf1/PD2 in OCSCs resulted in a significant downregulation of CSC and self-renewal markers, and impairment of in vitro tumor sphere ( P < 0.05) and colony formation ( P = 0.013). Co-immunoprecipitation revealed that OCT3/4 specifically interacts with hPaf1/PD2, and not with other PAF components (Ctr9, Leo1, Parafibromin) in OCSCs, suggesting a complex-independent role for hPaf1/PD2 in OCSC maintenance. Moreover, there was a significant overexpression and co-localization of hPaf1/PD2 with OCT3/4 in OC tissues compared to normal ovary tissues. Our results indicate that hPaf1/PD2 is overexpressed in OCSCs and maintains the self-renewal of OCSCs through its interaction with OCT3/4; thus, hPaf1/PD2 may be a potential therapeutic target to overcome tumor relapse in OC.

Background & Aims Cigarette smoking is a major risk factor for pancreatic cancer. Aggressive pancreatic tumors contain cancer cells with stem cell features. We investigated whether cigarette smoke induces stem cell features in pancreatic cancer cells. Methods KrasG12D; Pdx1-Cre mice were exposed to cigarette smoke or clean air (controls) for up to 20 weeks; pancreata were collected and analyzed by histology, quantitative reverse transcription polymerase chain reaction, and confocal immunofluorescence microscopy. HPNE and Capan1 cells were exposed to cigarette smoke extract (CSE), nicotine and nicotine-derived carcinogens (NNN or NNK), or clean air (controls) for 80 days and evaluated for stem cell markers and features using flow cytometry–based autofluorescence, sphere formation, and immunoblot assays. Proteins were knocked down in cells with small interfering RNAs. We performed RNA sequencing analyses of CSE-exposed cells. We used chromatin immunoprecipitation assays to confirm the binding of FOS-like 1, AP-1 transcription factor subunit (FOSL1) to RNA polymerase II-associated factor (PAF1) promoter. We obtained pancreatic ductal adenocarcinoma (PDAC) and matched nontumor tissues (n = 15) and performed immunohistochemical analyses. Results Chronic exposure of HPNE and Capan1 cells to CSE caused them to increase markers of stem cells, including autofluorescence and sphere formation, compared with control cells. These cells increased expression of ABCG2, SOX9, and PAF1, via cholinergic receptor nicotinic alpha 7 subunit (CHRNA7) signaling to mitogen-activated protein kinase 1 and FOSL1. CSE-exposed pancreatic cells with knockdown of PAF1 did not show stem cell features. Exposure of cells to NNN and NNK led to increased expression of CHRNA7, FOSL1, and PAF1 along with stem cell features. Pancreata from KrasG12D; Pdx1-Cre mice exposed to cigarette smoke had increased levels of PAF1 mRNA and protein, compared with control mice, as well as increased expression of SOX9. Levels of PAF1 and FOSL1 were increased in PDAC tissues, especially those from smokers, compared with nontumor pancreatic tissue. CSE exposure increased expression of PHD-finger protein 5A, a pluripotent transcription factor and its interaction with PAF1. Conclusions Exposure to cigarette smoke activates stem cell features of pancreatic cells, via CHRNA7 signaling and FOSL1 activation of PAF1 expression. Levels of PAF1 are increased in pancreatic tumors of humans and mice with chronic cigarette smoke exposure.

Background: Docetaxel remains the first-line therapeutic intervention for metastatic and castration-resistant (CR) prostate cancer (PCa). However, the therapeutic efficacy is limited due to unresponsiveness, toxicity, and drug-resistance. The availability of additional therapies increases the PCa patient survival modestly, but the development of cross-resistance limit the therapeutic efficacy. Hence, there is a need to understand the mechanisms of resistance and identify a novel target for the better management of CR PCa patients. Methods: Docetaxel resistant 22Rv1, LNCaP C-81, and PC3 PCa cells were established, and its resistance phenotype was determined by cell growth inhibition (MTT assay), apoptosis markers (western blot), tumor sphere assay, drug efflux property (SP analysis by FACS), auto fluorescence (FACS). qRT-PCR, western blot, and confocal microscopic analysis were also performed to confirm the drug-resistant marker phenotype. Additional experiments were performed to determine the underlying molecular mechanisms of docetaxel resistance. Results: The docetaxel resistant PCa cells grew well at the higher concentration of docetaxel (120, 15 and 50 nM of docetaxel, respectively) and did not result in apoptosis as measured by cPARP and caspase-3 cleavage. Docetaxel resistant PCa cells confer cross-resistance to second-generation chemotherapeutic agent cabazitaxel and show altered cell proliferation and invasion. Blocking by the ABCB1 specific inhibitor enhances docetaxel-induced cell death on par with parental cells. Side population analysis by flow cytometry confirms the acquired drug efflux property. Side population, autofluorescence and tumor sphere analyses confirmed the drug-resistance and stem-like cell phenotype. qRT-PCR, western blot and confocal microscopy show the abundant expression levels of the drug transporters, ABCB1 and ABCG2. Further, the docetaxel resistant PCa cells show higher stem cell network proteins such as PAF1, POU5F1, NANOG and SOX9 expression levels compared to age-matched control cells. Conversely, Tet-inducible PAF1 knockdown reduces embryonic stem cell network proteins and reverses docetaxel-resistance phenotype. Conclusions: Collectively, our study suggests that the stem cell factors such as PAF1 play a major role in docetaxel resistance and aggressiveness to PCa cells. Understanding the associated mechanisms and targeting these factors could lead a better management approach for CRPCa patients. Citation Format: Sakthivel Muniyan, Rama Krishna Nimmakayala, Saswati Karmakar, Satyanarayana Rachagani, Jawed A. Siddiqui, Parthasarathy Seshacharyulu, Ming-Fong Lin, Kaustubh Datta, Moorthy P. Ponnusamy, Surinder K. Batra. Role of polymerase II associated factor 1, PAF1, in docetaxel resistant prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5897.

MUC5AC is a secretory mucin aberrantly expressed in various cancers. In lung cancer, MUC5AC is overexpressed in both primary and metastatic lesions; however, its functional role is not well understood. The present study was aimed at evaluating mechanistic role of MUC5AC on metastasis of lung cancer cells. Clinically, the overexpression of MUC5AC was observed in lung cancer patient tissues and was associated with poor survival. In addition, the overexpression of Muc5ac was also observed in genetically engineered mouse lung adenocarcinoma tissues (Kras(G12D); Trp53(R172H/+); AdCre) in comparison with normal lung tissues. Our functional studies showed that MUC5AC knockdown resulted in significantly decreased migration in two lung cancer cell lines (A549 and H1437) as compared with scramble cells. Expression of integrins (α5, β1, β3, β4 and β5) was decreased in MUC5AC knockdown cells. As both integrins and MUC5AC have a von Willebrand factor domain, we assessed for possible interaction of MUC5AC and integrins in lung cancer cells. MUC5AC strongly interacted only with integrin β4. The co-localization of MUC5AC and integrin β4 was observed both in A549 lung cancer cells as well as genetically engineered mouse adenocarcinoma tissues. Activated integrins recruit focal adhesion kinase (FAK) that mediates metastatic downstream signaling pathways. Phosphorylation of FAK (Y397) was decreased in MUC5AC knockdown cells. MUC5AC/integrin β4/FAK-mediated lung cancer cell migration was confirmed through experiments utilizing a phosphorylation (Y397)-specific FAK inhibitor. In conclusion, overexpression of MUC5AC is a poor prognostic marker in lung cancer. MUC5AC interacts with integrin β4 that mediates phosphorylation of FAK at Y397 leading to lung cancer cell migration.

Background: Ovarian cancer (OC) is the most lethal gynecological malignancy among women with more than 85% of the patients manifesting tumor recurrence. Emerging evidence suggests that a small population of cells within the tumor - the ‘cancer stem cells (CSCs)’ is capable to giving rise to the entire histopathology of the tumor and is responsible for mediating drug resistance, recurrence, and disease aggressiveness. Previously, hPaf1(human RNA polymerase II associated factor1)/PD2 (Pancreatic Differentiation2) - a core component of RNA polymerase II associated factor (PAF) complex, was shown to be overexpressed in pancreatic CSCs and involved in the maintenance of mouse ESCs. Hence, we hypothesized that hPaf1 is involved in the maintenance of self-renewal property of ovarian CSCs (OCSCs). In this study, we investigated the functional role of hPaf1 in OCSCs which has not been explored before. Methods: Expression of hPaf1, cancer stem cell marker ESA, and self-renewal protein OCT3/4 was analyzed using confocal microscopy on OC tissue array. Side population (SP) was isolated from two OC cell lines OVCAR3 and A2780 by Hoechst staining using flow-cytometer, and characterized using tumor sphere assay. Immunoblotting was performed on characterized SP and NSP (non-SP) for OCSC and self-renewal markers. Transient knockdown of hPaf1 in SP was performed to understand how hPaf1 affects the CSC phenotype through immunoblotting, confocal microscopy, colony formation assay, and tumor sphere formation assay. To determine the interaction between hPaf1 and OCT 3/4, reciprocal co-immunoprecipitation was performed in SP/OCSCs. Results: There was a significant overexpression and considerable co-localization of hPaf1/PD2 with ESA and OCT3/4 in OC tissues compared to normal ovary tissues. SP from OVCAR3 formed larger and greater number of tumor spheres compared to NSP cells. Moreover, SP isolated from OVCAR3 and A2780 showed higher expression of hPaf1 along with CSC markers (CD44, CD133, ESA, CD24), and self- renewal proteins (β-CATENIN, SOX2, OCT3/4, SHH, and HER2). Transient knockdown of hPaf1 resulted in a significant decrease in expression of CSC markers and self-renewal proteins. In addition, functional characteristics of CSCs such as in vitro tumor formation capacity in non-adherent media and colony formation ability were impaired upon knockdown of hPaf1 suggesting that hPaf1 is involved in maintenance of the CSC phenotype. We also observed that hPaf1 physically interacts with OCT3/4 in OVCAR3 SP cells which provides a mechanism for the maintenance of OCSCs by hPaf1. Conclusion: Altogether, hPaf1/PD2 is overexpressed in OCSCs and its knockdown resulted in loss of OCSC phenotype. Moreover, hPaf1 is responsible for the maintenance of OCSCs through its interaction with OCT3/4. Hence, therapies that are able to abrogate hPaf1 mediated self-renewal of OCSCs represent potential therapeutic avenues to overcome tumor relapse in OC. Citation Format: Saswati Karmakar, Parthasarathy Seshacharyulu, Arokia Priyanka Vaz, Imayavaramban Lakshmanan, Moorthy Palanimuthu Ponnusamy, Surinder Kumar Batra. hPaf1/PD2 interacts with OCT3/4 in maintenance of the self-renewal process of ovarian cancer stem cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2495.