Polymerase II-associated Factor Research Articles

Abstract 1794: PAF1 reprograms metabolism in pancreatic cancer by interacting with HIF1α

Abstract Background: Reprogrammed cellular metabolism has been refocused on in the past decade, as many cancer cells rewire various metabolic pathways to facilitate their survival, unlimited cell growth, and division. 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 in normal cells; however, its overexpression has been implicated in promoting pancreatic tumorigenesis and metastasis through epigenetic regulation, chemoresistance, radioresistance and maintenance of cancer stem cells. While most of these studies have provided evidence of the multifunctional nature of PAF1/PD2 in PDAC progression, no study has investigated the role of PAF1/PD2 during the metabolic rewiring of cancer. Methods: In this study, we sought to examine the role of PAF1/PD2 in the metabolic rewiring of cancer cells in pancreatic ductal adenocarcinoma (PDAC) and decipher how PAF1/PD2 mediates this metabolic reprogramming in PDAC. Pancreatic cancer cell lines were transfected with shRNAs to knock down PAF1/PD2. Metabolic genes regulated by PAF1/PD2 were identified by qPCR and western blot upon PAF1/PD2 depletion. Metabolic assays were performed to investigate the role of PAF1/PD2. Immunoprecipitations identified proteins that interact with PAF1/PD2. Confocal microscopy confirmed the co-localization of PAF1/PD2 with its protein partners. We performed chromatin immunoprecipitation (ChIP) - Polymerase chain reaction to confirm the binding of the PAF1 sub-complex to its target gene. Results: Our results showed that pancreatic cancer cells depleted PAF1/PD2 downregulate genes involved in aerobic glycolysis compared to control cells. Also, the lactate release assay indicated that more lactate was produced in control cells than in cells with PAF1/PD2 knockdown. Interestingly, we identified that HIF1α interacts with PAF1, specifically in pancreatic cancer cells. PAF1 and HIF1α co-localization was observed in pancreatic cancer cell lines. We also observed that the PAF1/PD2- HIF1α complex bound to the promoter region of LDHA to regulate the expression of LDHA in pancreatic cancer cells, reprogramming the metabolism to utilize the aerobic glycolysis pathway preferentially. Conclusions: In conclusion, our study shows that PAF1/PD2 rewires the metabolism of PDAC by interacting with HIF1 α to regulate the expression of LDHA, which is the rate-limiting step of aerobic glycolysis. Citation Format: Ayoola O. Ogunleye, Neelanjana Gayen, Saravanakumar Marimuthu, Rama Krishna Nimmakayala, Sanchita Rauth, Zahraa Alsafwani, Jesse L. Cox, Surinder K. Batra, Moorthy P. Ponnusamy. PAF1 reprograms metabolism in pancreatic cancer by interacting with HIF1α [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1794.

LEO1 Is Required for Efficient Entry into Quiescence, Control of H3K9 Methylation and Gene Expression in Human Fibroblasts.

(1) Background: The LEO1 (Left open reading frame 1) protein is a conserved subunit of the PAF1C complex (RNA polymerase II-associated factor 1 complex). PAF1C has well-established mechanistic functions in elongation of transcription and RNA processing. We previously showed, in fission yeast, that LEO1 controls histone H3K9 methylation levels by affecting the turnover of histone H3 in chromatin, and that it is essential for the proper regulation of gene expression during cellular quiescence. Human fibroblasts enter a reversible quiescence state upon serum deprivation in the growth media. Here we investigate the function of LEO1 in human fibroblasts. (2) Methods: We knocked out the LEO1 gene using CRISPR/Cas9 methodology in human fibroblasts and verified that the LEO1 protein was undetectable by Western blot. We characterized the phenotype of the ΔLEO1 knockout cells with FACS analysis and cell growth assays. We used RNA-sequencing using spike-in controls to measure gene expression and spike-in controlled ChIP-sequencing experiments to measure the histone modification H3K9me2 genome-wide. (3) Results: Gene expression levels are altered in quiescent cells, however factors controlling chromatin and gene expression changes in quiescent human cells are largely unknown. The ΔLEO1 knockout fibroblasts are viable but have reduced metabolic activity compared to wild-type cells. ΔLEO1 cells showed a slower entry into quiescence and a different morphology compared to wild-type cells. Gene expression was generally reduced in quiescent wild-type cells. The downregulated genes included genes involved in cell proliferation. A small number of genes were upregulated in quiescent wild-type cells including several genes involved in ERK1/ERK2 and Wnt signaling. In quiescent ΔLEO1 cells, many genes were mis-regulated compared to wild-type cells. This included genes involved in Calcium ion transport and cell morphogenesis. Finally, spike-in controlled ChIP-sequencing experiments demonstrated that the histone modification H3K9me2 levels are globally increased in quiescent ΔLEO1 cells. (4) Conclusions: Thus, LEO1 is important for proper entry into cellular quiescence, control of H3K9me2 levels, and gene expression in human fibroblasts.

Knockdown of PAF1 reduces cervical cancer cell proliferation, migration and invasion via retarding FLOT2-mediated MEK/ERK1/2 pathway

ABSTRACT Cervical cancer (CC) is a very usual reproductive malignant tumor in women. RNA polymerase II-associated factor 1 (PAF1) and flotillin-2 (FLOT2) both have been discovered to key participators in cancers’ progression. However, the effects of PAF1/FLOT2 axis on CC development have not been probed. In this study, PAF1 and FLOT2 exhibited higher expression, and silencing of PAF1 down-regulated FLOT2 expression in CC. In addition, the regulatory effects of PAF1 suppression on CC progression were reversed after FLOT2 overexpression. Next, inhibition of PAF1 slowed the tumor growth in vivo through modulating FLOT2. Besides, down-regulation of PAF1 reduced FLOT2 expression to retard the MEK/ERK1/2 pathway. In conclusion, knockdown of PAF1 suppressed CC progression via retarding FLOT2-mediated MEK/ERK1/2 pathway. Our findings illustrated that the PAF1/FLOT2 axis may be useful bio-targets for CC treatment.

Aberrant accumulation of Kras-dependent pervasive transcripts during tumor progression renders cancer cells dependent on PAF1 expression

KRAS is the most commonly mutated oncogene in human cancer, and mutant KRAS is responsible for over 90% of pancreatic ductal adenocarcinoma (PDAC), the most lethal cancer. Here, we show that RNA polymerase II-associated factor 1 complex (PAF1C) is specifically required for survival of PDAC but not normal adult pancreatic cells. We show that PAF1C maintains cancer cell genomic stability by restraining overaccumulation of enhancer RNAs (eRNAs) and promoter upstream transcripts (PROMPTs) driven by mutant Kras. Loss of PAF1C leads to cancer-specific lengthening and accumulation of pervasive transcripts on chromatin and concomitant aberrant R-loop formation and DNA damage, which, in turn, trigger cell death. We go on to demonstrate that the global transcriptional hyperactivation driven by Kras signaling during tumorigenesis underlies the specific demand for PAF1C by cancer cells. Our work provides insights into how enhancer transcription hyperactivation causes general transcription factor addiction during tumorigenesis.

CNOT4 suppresses nonsmall cell lung cancer progression by promoting the degradation of PAF1.

CCR4-NOT transcription complex subunit 4 (CNOT4) and RNA polymerase II-associated factor, homolog (Saccharomyces cerevisiae) (PAF1) are implicated in nonsmall cell lung cancer (NSCLC). However, the molecular mechanism of their interaction in NSCLC progression is unknown. The expression of PAF1 and CNOT4 in human NSCLC tissues was detected by quantitative polymerase chain reaction. A549 cells that stably expressed CNOT4 and/or PAF1 were established. Western blot analysis and co-immunoprecipitation experiments were performed to reveal the interaction between CNOT4 and PAF1. Proliferation, migration, epithelial-mesenchymal transition (EMT), and colony formation assays were performed to determine the effect of CNOT4-PAF1 axis on NSCLC metastasis and stemness. Xenograft mouse tumor model was established, and tumor progression, EMT, and stemness were evaluated. It was found that CNOT4 expression was downregulated, whereas PAF1 expression was upregulated in human NSCLC tissues. CNOT4 facilitated the ubiquitination and degradation of PAF1 via the 26S proteasome. CNOT4 overexpression inhibited NSCLC progression, whereas PAF1 overexpression enhanced the proliferation, migration, and stemness of NSCLC, both in vitro and in vivo. Our results suggest that CNOT4-PAF1 axis modulates NSCLC metastasis and stemness, and may serve as potential therapeutic targets for lung cancer treatment.

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.

PAF1 cooperates with YAP1 in metaplastic ducts to promote pancreatic cancer

Acinar-to-ductal metaplasia (ADM) is a precursor lesion of pancreatic ductal adenocarcinoma (PDAC); however, the regulators of the ADM-mediated PDAC development and its targeting are poorly understood. RNA polymerase II-associated factor 1 (PAF1) maintains cancer stem cells leading to the aggressiveness of PDAC. In this study, we investigated whether PAF1 is required for the YAP1-mediated PDAC development and whether CA3 and verteporfin, small molecule inhibitors of YAP1/TEAD transcriptional activity, diminish pancreatic cancer (PC) cell growth by targeting the PAF1/YAP1 axis. Here, we demonstrated that PAF1 co-expresses and interacts with YAP1 specifically in metaplastic ducts of mouse cerulein- or KrasG12D-induced ADM and human PDAC but not in the normal pancreas. PAF1 knockdown (KD) reduced SOX9 in PC cells, and the PC cells showed elevated PAF1/YAP1 complex recruitment to the promoter of SOX9. The PAF1 KD reduced the 8xTEAD and SOX9 promoter-luciferase reporter activities in the mouse KC (KrasG12D; Pdx-1 Cre) cells and human PC cells, indicating that the PAF1 is required for the YAP1-mediated development of ADM and PC. Moreover, treatment with CA3 or verteporfin reduced the expressions of PAF1, YAP1, TEAD4, and SOX9 and decreased colony formation and stemness in KC and PC cells. CA3 treatment also reduced the viability and proliferation of PC cells and diminished the duct-like structures in KC acinar explants. CA3 or verteporfin treatment decreased the recruitment of the PAF1/YAP1 complex to the SOX9 promoter in PC cells and reduced the 8xTEAD and SOX9 promoter-luciferase reporter activities in KC and PC cells. Overall, PAF1 cooperates with YAP1 during ADM and PC development, and verteporfin and CA3 inhibit ADM and PC cell growth by targeting the PAF1/YAP1/SOX9 axis in vitro and ex vivo models. This study identified a regulatory axis of PDAC initiation and its targeting, paving the way for developing targeted therapeutic strategies for pancreatic cancer patients.

UBR5 targets tumor suppressor CDC73 proteolytically to promote aggressive breast cancer

UBR5, a HECT-domain E3 ubiquitin ligase, is an attractive therapeutic target for aggressive breast cancers. Defining the substrates of UBR5 is crucial for scientific understanding and clinical intervention. Here, we demonstrate that CDC73, a component of the RNA polymerase II-associated factor 1 complex, is a key substrate that impedes UBR5’s profound tumorigenic and metastatic activities in triple-negative breast cancer (TNBC) via mechanisms of regulating the expression of β-catenin and E-cadherin, tumor cell apoptosis and CD8+ T cell infiltration. Expression of CDC73 is also negatively associated with the progression of breast cancer patients. Moreover, we show that UBR5 destabilizes CDC73 by polyubiquitination at Lys243, Lys247, and Lys257 in a non-canonical manner that is dependent on the non-phosphorylation state of CDC73 at Ser465. CDC73 could serve as a molecular switch to modulate UBR5’s pro-tumor activities and may provide a potential approach to developing breast cancer therapeutic interventions.

The PAF1 complex cell autonomously promotes oogenesis in Caenorhabditis elegans.

The RNA polymerase II‐associated factor 1 complex (PAF1C) is a protein complex that consists of LEO1, RTF1, PAF1, CDC73, and CTR9, and has been shown to be involved in RNA polymerase II‐mediated transcriptional and chromatin regulation. Although it has been shown to regulate a variety of biological processes, the precise role of the PAF1C during germ line development has not been clarified. In this study, we found that reduction in the function of the PAF1C components, LEO‐1, RTFO‐1, PAFO‐1, CDC‐73, and CTR‐9, in Caenorhabditis elegans affects oogenesis. Defects in oogenesis were also confirmed using an oocyte maturation marker, OMA‐1::GFP. While four to five OMA‐1::GFP‐positive oocytes were observed in wild‐type animals, their numbers were significantly decreased in pafo‐1 mutant and leo‐1(RNAi), pafo‐1(RNAi), and cdc‐73(RNAi) animals. Expression of a functional PAFO‐1::mCherry transgene in the germline significantly rescued the oogenesis‐defective phenotype of the pafo‐1 mutants, suggesting that expression of the PAF1C in germ cells is required for oogenesis. Notably, overexpression of OMA‐1::GFP partially rescued the oogenesis defect in the pafo‐1 mutants. Based on our findings, we propose that the PAF1C promotes oogenesis in a cell‐autonomous manner by positively regulating the expression of genes involved in oocyte maturation.

Polymerase II-Associated Factor 1 Complex-Regulated FLOWERING LOCUS C-Clade Genes Repress Flowering in Response to Chilling.

RNA polymerase II–associated factor 1 complex (PAF1C) regulates the transition from the vegetative to the reproductive phase primarily by modulating the expression of FLOWERING LOCUS C (FLC) and FLOWERING LOCUS M [FLM, also known as MADS AFFECTING FLOWERING1 (MAF1)] at standard growth temperatures. However, the role of PAF1C in the regulation of flowering time at chilling temperatures (i.e., cold temperatures that are above freezing) and whether PAF1C affects other FLC-clade genes (MAF2–MAF5) remains unknown. Here, we showed that Arabidopsis thaliana mutants of any of the six known genes that encode components of PAF1C [CELL DIVISION CYCLE73/PLANT HOMOLOGOUS TO PARAFIBROMIN, VERNALIZATION INDEPENDENCE2 (VIP2)/EARLY FLOWERING7 (ELF7), VIP3, VIP4, VIP5, and VIP6/ELF8] showed temperature-insensitive early flowering across a broad temperature range (10°C–27°C). Flowering of PAF1C-deficient mutants at 10°C was even earlier than that in flc, flm, and flc flm mutants, suggesting that PAF1C regulates additional factors. Indeed, RNA sequencing (RNA-Seq) of PAF1C-deficient mutants revealed downregulation of MAF2–MAF5 in addition to FLC and FLM at both 10 and 23°C. Consistent with the reduced expression of FLC and the FLC-clade members FLM/MAF1 and MAF2–MAF5, chromatin immunoprecipitation (ChIP)-quantitative PCR assays showed reduced levels of the permissive epigenetic modification H3K4me3/H3K36me3 and increased levels of the repressive modification H3K27me3 at their chromatin. Knocking down MAF2–MAF5 using artificial microRNAs (amiRNAs) in the flc flm background (35S::amiR-MAF2–5 flc flm) resulted in significantly earlier flowering than flc flm mutants and even earlier than short vegetative phase (svp) mutants at 10°C. Wild-type seedlings showed higher accumulation of FLC and FLC-clade gene transcripts at 10°C compared to 23°C. Our yeast two-hybrid assays and in vivo co-immunoprecipitation (Co-IP) analyses revealed that MAF2–MAF5 directly interact with the prominent floral repressor SVP. Late flowering caused by SVP overexpression was almost completely suppressed by the elf7 and vip4 mutations, suggesting that SVP-mediated floral repression required a functional PAF1C. Taken together, our results showed that PAF1C regulates the transcription of FLC and FLC-clade genes to modulate temperature-responsive flowering at a broad range of temperatures and that the interaction between SVP and these FLC-clade proteins is important for floral repression.

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.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Molecular characterization of the wheat putative proline-rich protein TaELF7 and its involvement in the negative regulation of Arabidopsis flowering

Late stages of floret development, such as booting, heading, and anthesis stages, are important steps for determining grain setting and for filling in wheat. Herein, we report the molecular function of Triticum aestivum ELF7 encoding RNA polymerase II-associated factor 1 (PAF1), which may act as a negative regulator in floret development and anthesis stages. Among the six TaELF7-like genes isolated from wheat, TaELF7 like1-A and TaELF7 like2-B showed contrasting expression levels during the late stage of floret development stages, with observation of decreased expression level of TaELF7 like1-A compared to that of TaELF7 like2-B. The full-length TaELF7 like1-A has a 1038-bp open reading frame that contains a proline-rich domain in the N-terminal region and a nuclear localization signal domain in the C-terminal region. TaELF7 like1-A was found to be localized in the nucleus in both tobacco and wheat. Direct interaction of TaELF7 with the RING-type E3 ligase TaHUB2 was confirmed using a yeast two-hybrid system, an in vitro pull-down assay, and a bimolecular fluorescence complementation assay. The flowering time was delayed in TaELF7-overexpressing plants compared to that in the control plants. Expression levels of few floral repressor genes were markedly increased in TaELF7-overexpressing Arabidopsis plants.

Ubiquitylation of MYC couples transcription elongation with double-strand break repair at active promoters

The MYC oncoprotein globally affects the function of RNA polymerase II (RNAPII). The ability of MYC to promote transcription elongation depends on its ubiquitylation. Here, we show that MYC and PAF1c (polymerase II-associated factor 1 complex) interact directly and mutually enhance each other's association with active promoters. PAF1c is rapidly transferred from MYC onto RNAPII. This transfer is driven by the HUWE1 ubiquitin ligase and is required for MYC-dependent transcription elongation. MYC and HUWE1 promote histone H2B ubiquitylation, which alters chromatin structure both for transcription elongation and double-strand break repair. Consistently, MYC suppresses double-strand break accumulation in active genes in a strictly PAF1c-dependent manner. Depletion of PAF1c causes transcription-dependent accumulation of double-strand breaks, despite widespread repair-associated DNA synthesis. Our data show that the transfer of PAF1c from MYC onto RNAPII efficiently couples transcription elongation with double-strand break repair to maintain the genomic integrity of MYC-driven tumor cells.

Histone Monoubiquitination in Chromatin Remodelling: Focus on the Histone H2B Interactome and Cancer.

Simple SummaryPost-translational modifications (PTM) of histone tails represent epigenomic regulation of the chromatin landscape, influencing gene expression and the response to DNA damage. This review focusses on cancer-associated roles of ubiquitin as a histone PTM, specifically in conjunction with an E3 ubiquitin ligase cascade that results in the addition of a single ubiquitin (monoubiquitination) to histone H2B at lysine 120 (H2Bub1). H2Bub1 has roles in chromatin accessibility important for transcriptional elongation, the DNA damage response, cellular proliferation and developmental transitions, including in stem cell plasticity. It has been implicated in inflammation and tumour progression, with examples of its loss associated with a worse prognosis for patients with some cancers. Many factors involved in the H2Bub1 interactome are well known cancer-associated proteins, including p53, BRCA1 and components of the SWI/SNF remodelling complex. Increased knowledge of H2Bub1 and its interactome offers new opportunities for therapeutic targeting of malignancy.Chromatin remodelling is a major mechanism by which cells control fundamental processes including gene expression, the DNA damage response (DDR) and ensuring the genomic plasticity required by stem cells to enable differentiation. The post-translational modification of histone H2B resulting in addition of a single ubiquitin, in humans at lysine 120 (K120; H2Bub1) and in yeast at K123, has key roles in transcriptional elongation associated with the RNA polymerase II-associated factor 1 complex (PAF1C) and in the DDR. H2Bub1 itself has been described as having tumour suppressive roles and a number of cancer-related proteins and/or complexes are recognised as part of the H2Bub1 interactome. These include the RING finger E3 ubiquitin ligases RNF20, RNF40 and BRCA1, the guardian of the genome p53, the PAF1C member CDC73, subunits of the switch/sucrose non-fermenting (SWI/SNF) chromatin remodelling complex and histone methyltransferase complexes DOT1L and COMPASS, as well as multiple deubiquitinases including USP22 and USP44. While globally depleted in many primary human malignancies, including breast, lung and colorectal cancer, H2Bub1 is selectively enriched at the coding region of certain highly expressed genes, including at p53 target genes in response to DNA damage, functioning to exercise transcriptional control of these loci. This review draws together extensive literature to cement a significant role for H2Bub1 in a range of human malignancies and discusses the interplay between key cancer-related proteins and H2Bub1-associated chromatin remodelling.

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

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. 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. 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). 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.

Abstract 3679: A novel mechanism for pancreatic cancer stem cell maintenance: PAF1C-independent role of PAF1

Abstract Background: Cancer stem cells (CSCs), a minor subset of cancer cells, mediate aggressiveness, metastasis, and drug resistance of many cancers including the deadly pancreatic cancer (PC). Molecular governance of pancreatic CSCs remains elusive, mandating the need for their better molecular characterization to improve management of PC. PD2 (Pancreatic Differentiation 2) or RNA Polymerase II-Associated Factor 1 (Paf1) is a core component of human PAF1 complex (PAF1C), which regulates transcription elongation and mRNA processing. Human PAF1C consists of 5 members: PAF1, LEO1, CDC73, CTR9 and SKI8. Recently, PAF1 has emerged as a novel pancreatic CSC marker that enhances tumorigenic and metastatic potential of PC. Paf1 also maintains the self-renewal of mouse embryonic stem cells and ovarian CSCs via its interaction with OCT3/4, a major regulator of pluripotency. However, the mechanistic role of PAF1 in CSC maintenance and CSC-mediated PC pathogenesis is poorly understood. Hypothesis: We hypothesize that “PAF1 forms a sub-complex exclusive of PAF1C, wherein PAF1 functions as the master-regulator for maintaining pancreatic CSCs by regulating stem cell gene signature”. Experimental Design: PAF1 knockdown (KD) was used to identify its targets and role in tumorigenesis. PAF1 sub-complex in CSCs was identified using immunoprecipitation (IP) and mass spectrometry (MS). ChIP-Seq was performed to confirm binding of PAF1 on its target genes. Results: Inducible KD of PAF1 led to a significant reduction in tumor burden from orthotopically implanted human PC cells in athymic nude mice, indicating its role in pancreatic tumorigenesis. CSCs from different PC cell lines demonstrated higher expression of PAF1 along with CSC markers and PAF1 KD caused a significant decrease in CSC and self-renewal markers analyzed through Western blotting and immunofluorescence. Reciprocal co-IP and MS revealed that PAF1 interacted with Phf5a, DDX3, and hnRNP-K in CSCs. PAF1 KD in CSCs showed significant down-regulation of tumorigenic and stemness maintenance genes by RNA-Seq and PCR array analysis. PAF1 along with its binding partners occupied Nanog promoter in pancreatic CSCs. Further, KD of PAF1 in CSCs did not affect the expression of other PAF1C components, whereas individual KD of single PAF1C components decreased other remaining PAF1C members including PAF1. Moreover, other PAF1C components did not interact with Phf5a, a PHD-fold harboring nuclear protein in CSCs, suggesting that PAF1 forms a sub-complex with Phf5a, independent of PAF1C that functions in CSC maintenance. Depletion of Paf1 from mouse pancreas since birth using a CRISPR/Cas9-based conditional Paf1 knockout mouse model severely affected exocrine pancreas development with peri-ductal sclerosis and inflammation. Conclusion: Altogether, PAF1 functions as the master-regulator for CSC maintenance by forming a sub-complex, which regulates CSC-network genes in PC. Citation Format: Saswati Karmakar, Sanchita Rauth, Rama Krishna Nimmakayala, Srikanth Barkeer, Mohd W. Nasser, Satyanarayana Rachagani, Dario Ghersi, Moorthy P. Ponnusamy, Surinder K. Batra. A novel mechanism for pancreatic cancer stem cell maintenance: PAF1C-independent role of PAF1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3679.

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

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. 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. 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 miceexposed 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. 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.

Fine‐Tuning of an Evolutionarily Conserved Histone Chaperone, FACT, by Ubiquitin‐Proteasome System, and its Targeting to the Active Gene by mRNA Capping Machinery to Regulate Transcriptional Elongation

Facilitates Chromatin Transcription (FACT), an evolutionarily conserved histone chaperone involved in multiple cellular processes, is upregulated in cancers, and its downregulation is associated with cellular death. However, it is not clearly understood how FACT is fine‐tuned for normal cellular functions. Here, we have used a variety of biochemical, genetic, and molecular biological methodologies to understand regulation of FACT and its abundance. We find that the FACT subunit, Spt16, is ubiquitylated by San1 (an E3 ubiquitin ligase) and degraded by the 26S proteasome. Enhanced abundance of Spt16 in the absence of San1 impairs transcriptional elongation. Likewise, decreased abundance of Spt16 also reduces transcription. Thus, an optimal level of Spt16 is required for efficient transcriptional elongation, which is maintained by San1 via ubiquitylation and proteasomal degradation. Consistently, San1 associates with the coding sequences of active genes to regulate Spt16's abundance. Further, our results reveal that enhanced abundance of Spt16 in the absence of San1 impairs chromatin reassembly at the coding sequence, similar to the results seen following inactivation of Spt16. Efficient chromatin reassembly enhances the fidelity of transcriptional elongation. These results demonstrate for the first time the fine‐tuning of FACT by ubiquitin proteasome system in promoting chromatin reassembly in the wake of elongating RNA polymerase II and transcriptional elongation. Further, it is not clear how FACT is targeted to the active gene. Our recent results reveal that FACT is targeted to the active gene by an mRNA capping enzyme, Cet1, independently of its mRNA capping activity. Subsequently, FACT enhances the release of paused RNA polymerase II from promoter proximal site via Paf1 complex (RNA polymerase II‐associated factor 1 complex) to promote transcriptional elongation. Collectively, these results shed much light on the regulation of FACT and its abundance in controlling transcription, thus providing novel regulatory mechanisms of gene expression.Support or Funding InformationNIH 1R15GM088798‐01 and 2R15GM088798‐02This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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.