Arokia Priyanka Vaz

Emerging Role of Mucins in Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is an important and complex phenomenon that determines the aggressiveness of cancer cells. The morphological transformation of cancerous cells is accompanied by various cellular processes such as alterations in cell-cell adhesion, cell matrix degradation, down regulation of epithelial marker Ecadherin and upregulation of mesenchymal markers N-cadherin and Vimentin. Besides these markers several other important tumor antigens/mucins are also involved in the EMT process. Mainly high molecular weight glycoproteins such as mucin molecules (MUC1, MUC4 and MUC16) play a major role in the cellular transformation and signaling alteration in EMT process. In addition to these factors, EMT may be an essential process triggering the emergence or expansion of the CSC population, which slowly results in the initiation of tumor at metastatic sites. Furthermore, mucins have been demonstrated to be involved in the EMT process and also in the enrichment of cancer stem cell population. Mucin mediated EMT is very complex since the key components of tumor microenvironment are also regulating mucin molecules. In this review, we have discussed all the aforementioned factors and their mechanistic involvement for EMT process.

Cancer stem cells and therapeutic targets: an emerging field for cancer treatment

Recent paradigm in the field of cancer defines its origin from a small population of fast-growing cells known as cancer stem cells (CSCs), and they are mainly responsible for disease aggressiveness, drug resistance, and tumor relapse. The existence of CSCs has been proven in different types of cancer and possesses characteristic expression of a wide array of cell surface markers specific to the type of cancer. CSCs have been isolated and enriched using several surface markers in different cancer types. Self-renewal, drug resistance, and the ability to transition from epithelial to mesenchymal phenotype are the major features attributed to this fraction of mutated stem cells. The CSC hypothesis proposes that these CSCs mimic stem cells by sharing similar pathways, such as Wnt, SHH, Notch, and others. Further, the niche, which in this case is the tumor microenvironment, plays a very important role in the maintenance of CSCs. Altogether, this emerging field of research on CSCs is expected to unveil answers to the most difficult issues of one of the most dreadful diseases called cancer.

Novel role of pancreatic differentiation 2 in facilitating self-renewal and drug resistance of pancreatic cancer stem cells

Background:Cancer stem cells (CSCs) contribute towards disease aggressiveness and drug resistance. Specific identification of CSC maintenance genes and targeting can improve the efficiency of currently available treatment modalities. Pancreatic differentiation 2 (PD2) has a major role in the self-renewal of mouse embryonic stem cells. In the present study, we investigated the role of PD2 in pancreatic CSCs.Methods:Characterisation of CSCs and non-CSCs from mouse models, pancreatic cancer cells and human tissues by CSC and self-renewal marker analysis using confocal assay. Effect of PD2 knockdown in CSCs (after gemcitabine treatment) was studied by immunoblot and apoptosis assays.Results:A subpopulation of cells displayed PD2 overexpression in mouse (KrasG12D; Pdx1-Cre and KrasG12D; Trp53R172H/+; Pdx1-Cre) and human pancreatic tumours, which co-express CSC markers. Cancer stem cells exhibited elevated expression of PD2 and self-renewal markers, such as Oct3/4, Shh and β-catenin. Gemcitabine treatment maintained the CSC population with simultaneous maintenance of PD2 and CSC marker expression. Knockdown of PD2 in CSCs resulted in reduced viability of cells and enhanced apoptosis along with abrogated expression of CD133 and MDR2.Conclusions:Our results suggest that PD2 is a novel CSC maintenance protein, loss of which renders the CSCs more susceptible to drug-induced cell death.

Overexpression of PD2 leads to increased tumorigenicity and metastasis in pancreatic ductal adenocarcinoma

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.

hPaf1/PD2 interacts with OCT3/4 to promote self-renewal of ovarian cancer stem cells

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.

PD2/PAF1 at the Crossroads of the Cancer Network

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.

Abstract 2495: hPaf1/PD2 interacts with OCT3/4 in maintenance of the self-renewal process of ovarian cancer stem cells

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.

Identification of Ovarian Cancer Stem Cells: Molecular Pathways

Ovarian cancer is one of the major causes of cancer-related deaths among the female reproductive tract cancers worldwide. The foremost problem with ovarian cancer is in its heterogeneity, different subtypes and drug resistance. Moreover, the different types of ovarian cancer, such as endometrioid, serous, clear cell and mucinous appends to the complexity of this cancer. These tumors contain a small population of cells known as cancer stem cells (CSCs), which have the capacity to self-renew and are highly drug resistant, leading to tumor relapse. CSCs have attained tremendous attention in the last decade and have been shown to be a major target in various cancers including ovarian cancer. Understanding the molecular pathways implicated by ovarian CSCs is crucial to combat the residual disease following chemotherapy and combinational therapies. Several markers, such as CD24, CD44, CD117, CD133 and ALDH1, are found to be highly expressed on the surface of ovarian CSCs and can be used as a target as well as to battle ovarian cancer. This book chapter focuses on CSCs and their associate molecules and molecular pathways involved in the maintenance of ovarian CSCs, specific targets and the novel therapeutic implications of ovarian CSCs.

Abstract 271: Overexpression of hPaf1/PD2 in pancreatic cancer stem cells results in stemness and drug resistance: a novel target for pancreatic cancer therapy.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Cancer stem cells (CSCs) or side-population (SP) constitute a sub-population of cells in a tumor which are responsible for the maintenance of several cancers, including pancreatic cancer (PC). In the process of identification of a CSC maintenance marker for PC, we have focused on the human polymerase association factor 1 (hPaf1) or pancreatic differentiation 2 (PD2) due to its critical role in self-renewal of mouse embryonic stem cells (mESCs) through the interaction with Oct3/4, a gatekeeper of pluripotency and self-renewal for ESCs. Furthermore, PD2 has been found to be highly overexpressed in poorly differentiated PC compared to well-differentiated PC due to gene amplification in the 19q13 locus. Additionally, ectopic overexpression of PD2 led to the oncogenic transformation of NIH3T3 immortalized cells. An essential property of stem cells is its ability to self-renew, which is also an important property of CSCs. Based on the aforementioned work, we hypothesize that PD2 is involved in the maintenance of self-renewal and drug resistance of pancreatic CSCs. To test our hypothesis, SP and Non-SP were isolated from pancreatic tumors as well as from cell lines using Hoechst 33342 exclusion assay followed by FACS analysis. SP and NSP were characterized using both in vitro and in vivo tumorigenicity assays. In vitro assays such as tumorsphere assay revealed that SP maintained its colony formation and formed large tumorspheres whereas the NSP cells were dispersed and formed smaller spheres. Both confocal and western bolt analysis revealed that SP overexpressed PD2 as well as CSC markers (CD133, ALDH1) and self-renewal markers (Shh and β-catenin). In vivo assays such as subcutaneous and orthotopic injection of 5000 SP and NSP cells into athymic nude mice were performed. SP injected mice produced a larger tumor as compared to NSP injected mice. Gemcitabine treatment enriched the SP cells thereby increasing PD2 expression compared to NSP cells. Furthermore, we carried out transient knockdown of PD2 by specific siRNA against PD2 in pancreatic CSCs and subsequently treated with gemcitabine which resulted in reduced viability thereby indicating a novel role of PD2 in CSC mediated drug resistance. Also, it was further corroborated by the downregulation of CD133 and multidrug resistance gene 2 expressions in SP. Further, analysis of PD2 expression along with CSC markers in PC progression mouse models revealed co-expression of PD2 and CSC markers in specific cells. Altogether, our results propose a novel role of PD2 in CSC maintenance as well as in drug resistance, which could be anticipated to be developed as a novel targeted therapy against pancreatic CSCs. Citation Format: Arokia Priyanka Vaz, Moorthy P. Ponnusamy, Satyanarayana Rachagani, Parama Dey, Surinder Batra. Overexpression of hPaf1/PD2 in pancreatic cancer stem cells results in stemness and drug resistance: a novel target for pancreatic cancer therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 271. doi:10.1158/1538-7445.AM2013-271