Aging represses lung tumorigenesis and alters tumor suppression.

Most cancers are diagnosed in persons over the age of sixty, but little is known about how age impacts tumorigenesis. While aging is accompanied by mutation accumulation - widely understood to contribute to cancer risk - it is also associated with numerous other cellular and molecular changes likely to impact tumorigenesis. Moreover, cancer incidence decreases in the oldest part of the population, suggesting that very old age may reduce carcinogenesis. Here we show that aging represses tumor initiation and growth in genetically engineered mouse models of human lung cancer. Moreover, aging dampens the impact of inactivating many, but not all, tumor suppressor genes with the impact of inactivating PTEN, a negative regulator of the PI3K/AKT pathway, weakened to a disproportionate extent. Single-cell transcriptomic analysis revealed that neoplastic cells from tumors in old mice retain many age-related transcriptomic changes, showing that age has an enduring impact that persists through oncogenic transformation. Furthermore, the consequences of PTEN inactivation were strikingly age-dependent, with PTEN deficiency reducing signatures of aging in cancer cells and the tumor microenvironment. Our findings suggest that the relationship between age and lung cancer incidence may reflect an integration of the competing effects of driver mutation accumulation and tumor suppressive effects of aging.

Efficient and multiplexed somatic genome editing with Cas12a mice.

Somatic genome editing in mouse models has increased our understanding of the in vivo effects of genetic alterations in areas ranging from neuroscience to cancer biology and beyond. However, existing models are limited in their ability to create multiple targeted edits. Thus, our understanding of the complex genetic interactions that underlie development, homeostasis, and disease remains incomplete. Cas12a is an RNA-guided endonuclease with unique attributes that enable simple targeting of multiple genes with crRNA arrays containing tandem guides. To accelerate and expand the generation of complex genotypes in somatic cells, we generated transgenic mice with Cre-regulated and constitutive expression of enhanced Acidaminococcus sp. Cas12a (enAsCas12a). In these mice, enAsCas12a-mediated somatic genome editing robustly generated compound genotypes, as exemplified by the initiation of diverse cancer types driven by homozygous inactivation of trios of tumor suppressor genes. We further integrated these modular crRNA arrays with clonal barcoding to quantify the size and number of tumors with each array, as well as the efficiency of each crRNA. These Cas12a alleles will enable the rapid generation of disease models and broadly facilitate the high-throughput investigation of coincident genomic alterations in somatic cells in vivo .

High-Throughput Identification, Modeling, and Analysis of Cancer Driver Genes In Vivo.

The vast number of genomic and molecular alterations in cancer pose a substantial challenge to uncovering the mechanisms of tumorigenesis and identifying therapeutic targets. High-throughput functional genomic methods in genetically engineered mouse models allow for rapid and systematic investigation of cancer driver genes. In this review, we discuss the basic concepts and tools for multiplexed investigation of functionally important cancer genes in vivo using autochthonous cancer models. Furthermore, we highlight emerging technical advances in the field, potential opportunities for future investigation, and outline a vision for integrating multiplexed genetic perturbations with detailed molecular analyses to advance our understanding of the genetic and molecular basis of cancer.

Combinatorial Inactivation of Tumor Suppressors Efficiently Initiates Lung Adenocarcinoma with Therapeutic Vulnerabilities.

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 that have improved 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 using quantitative autochthonous mouse model systems to uncover the genetic and biochemical changes that enable efficient lung tumor initiation in the absence of oncogene alterations. Generation of hundreds of diverse combinations of tumor suppressor alterations demonstrates that inactivation of suppressors of the RAS and PI3K pathways drives the development of oncogene-negative lung adenocarcinoma. Human genomic data and histology identified RAS/MAPK and PI3K pathway activation as a common feature of an event in oncogene-negative human lung adenocarcinomas. These Onc-negativeRAS/PI3K tumors and related cell lines are vulnerable to pharmacologic inhibition of these signaling axes. These results transform our understanding of this prevalent yet understudied subtype of lung adenocarcinoma. SIGNIFICANCE: To address the large fraction of lung adenocarcinomas lacking mutations in proto-oncogenes for which targeted therapies are unavailable, this work uncovers driver pathways of oncogene-negative lung adenocarcinomas and demonstrates their therapeutic vulnerabilities.

Reduction in O-glycome induces differentially glycosylated CD44 to promote stemness and metastasis in pancreatic cancer.

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.

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.

Pancreatic Tumor Microenvironment Factor Promotes Cancer Stemness via SPP1-CD44 Axis.

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.

Proteasomal Regulation of Mammalian SPT16 in Controlling Transcription.

FACT (facilitates chromatin transcription), an essential and evolutionarily conserved heterodimer from yeast to humans, controls transcription and is found to be upregulated in various cancers. However, the basis for such upregulation is not clearly understood. Our recent results deciphering a new ubiquitin-proteasome system regulation of the FACT subunit SPT16 in orchestrating transcription in yeast hint at the involvement of the proteasome in controlling FACT in humans, with a link to cancer. To test this, we carried out experiments in human embryonic kidney (HEK293) cells, which revealed that human SPT16 undergoes ubiquitylation and that its abundance is increased following inhibition of the proteolytic activity of the proteasome, thus implying proteasomal regulation of human SPT16. Furthermore, we find that the increased abundance/expression of SPT16 in HEK293 cells alters the transcription of genes, including ones associated with cancer, and that the proteasomal degradation of SPT16 is impaired in kidney cancer (Caki-2) cells to upregulate SPT16. Like human SPT16, murine SPT16 in C2C12 cells also undergoes ubiquitylation and proteasomal degradation to regulate transcription. Collectively, our results reveal a proteasomal regulation of mammalian SPT16, with physiological relevance in controlling transcription, and implicate such proteasomal control in the upregulation of SPT16 in cancer.

Recent advances in organoid development and applications in disease modeling.

An improved understanding of stem cell niches, organogenesis, and disease models has paved the way for developing a three-dimensional (3D) organoid culture system. Organoid cultures can be derived from primary tissues (single cells or tissue subunits), adult stem cells (ASCs), induced pluripotent stem cells (iPSCs), or embryonic stem cells (ESCs). As a significant technological breakthrough, 3D organoid models offer a promising approach for understanding the complexities of human diseases ranging from the mechanistic investigation of disease pathogenesis to therapy. Here, we discuss the recent applications, advantages, and limitations of organoids as in vitro models for studying metabolomics, drug development, infectious diseases, and the gut microbiome. We further discuss the use of organoids in cancer modeling using high throughput sequencing approaches.

RNA Polymerase II-Associated Factor 1 Regulates Stem Cell Features of Pancreatic Cancer Cells, Independently of the PAF1 Complex, via Interactions With PHF5A and DDX3.

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 ß-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.

Correction: Global analysis of human glycosyltransferases reveals novel targets for pancreatic cancer pathogenesis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Global analysis of human glycosyltransferases reveals novel targets for pancreatic cancer pathogenesis.

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 ß-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.

MicroRNA regulation of K-Ras in pancreatic cancer and opportunities for therapeutic intervention.

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.

Novel role of O-glycosyltransferases GALNT3 and B3GNT3 in the self-renewal of pancreatic cancer stem cells.

BACKGROUND: Glycosylation plays a critical role in the aggressiveness of pancreatic cancer (PC). Emerging evidences indicate significant involvement of cancer stem cells (CSCs) in PC aggressiveness. However, the importance of glycosylation in pancreatic cancer stem cells (PCSCs) is yet to be addressed. Hence, we evaluated the potential role of glycosylation in maintenance of stemness of PCSCs. METHODS: Effect of glycosylation specific inhibitors on growth and PCSCs of PC cells was assessed by MTT assay and Side Population (SP) analysis. Isolated PCSCs/SP were characterized using molecular and functional assays. Expression of tumor-associated carbohydrate antigens (TACAs) was analyzed in PCSCs by western blotting. Effect of tunicamycin on PCSCs was analyzed by tumorsphere, clonogenicity, migration assay and immunoblotting for CSCs markers. The differential expression of glycogenes in PCSCs compared to non-CSCs were determined by RT-qPCR, immunoblotting and immunofluorescence. Co-expression of GALNT3 and B3GNT3 with CD44v6 was assessed in progression stages of KrasG12D; Pdx-1-Cre (KC) and KrasG12D; p53R172H; Pdx-1-Cre (KPC) tumors by immunofluorescence. Transient and CRISPR/Cas9 silencing of GALNT3 and B3GNT3 was performed to examine their effect on CSCs maintenance. RESULTS: Inhibition of glycosylation decreased growth and CSCs/SP in PC cells. PCSCs overexpressed CSC markers (CD44v6, ESA, SOX2, SOX9 and ABCG2), exhibited global expressional variation of TACAs and showed higher self-renewal potential. Specifically, N-glycosylation inhibition, significantly decreased tumorsphere formation, migration, and clonogenicity of PCSCs, as well as hypo-glycosylated CD44v6 and ESA. Of note, glycosyltransferases (GFs), GALNT3 and B3GNT3, were significantly overexpressed in PCSCs and co-expressed with CD44v6 at advanced PDAC stages in KC and KPC tumors. Further, GALNT3 and B3GNT3 knockdown led to a decrease in the expression of cell surface markers (CD44v6 and ESA) and self-renewal markers (SOX2 and OCT3/4) in PCSCs. Interestingly, CD44v6 was modified with sialyl Lewis a in PCSCs. Finally, CRISPR/Cas9-mediated GALNT3 KO significantly decreased self-renewal, clonogenicity, and migratory capacity in PCSCs. CONCLUSIONS: Taken together, for the first time, our study showed the importance of glycosylation in mediating growth, stemness, and maintenance of PCSCs. These results indicate that elevated GALNT3 and B3GNT3 expression in PCSCs regulate stemness through modulating CSC markers.

Cigarette Smoke Induces Stem Cell Features of Pancreatic Cancer Cells via PAF1.

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.

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.

Ubiquitin-Proteasome System Regulation of an Evolutionarily Conserved RNA Polymerase II-Associated Factor 1 Involved in Pancreatic Oncogenesis.

The evolutionarily conserved RNA polymerase II-associated factor 1 (Paf1) from yeast to humans regulates transcription and associated processes, and thus, malfunctions and/or misregulations of Paf1 are associated with cellular pathologies. Indeed, Paf1 (also known as PD2 or pancreatic differentiation 2) is found to be upregulated in poorly differentiated cancer cells, and such upregulation is involved in cellular transformation or oncogenesis. However, the basis for Paf1 upregulation in these cells remains largely unknown. In light of this, we have tested here the idea that the ubiquitin-proteasome system (UPS) regulates the cellular abundance of Paf1. In this direction, we analyzed the role of UPS in regulation of Paf1's abundance in yeast. We find that Paf1 undergoes ubiquitylation and is degraded by the 26S proteasome in yeast, thus deciphering UPS regulation of an evolutionarily conserved factor, Paf1, involved in various cellular processes at the crossroads of the cancer networks. Likewise, Paf1 undergoes proteasomal degradation in well-differentiated, but not poorly differentiated, pancreatic cancer cells, hence pointing to the UPS in upregulation of Paf1 in poorly differentiated cancers. Collectively, our results reveal UPS regulation of Paf1 and suggest downregulation of UPS in elevating Paf1's abundance in poorly differentiated cancers.

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.

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.