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.

Human RNA polymerase II-association factor 1 (hPaf1/PD2) regulates histone methylation and chromatin remodeling in pancreatic cancer.

Change in gene expression associated with pancreatic cancer could be attributed to the variation in histone posttranslational modifications leading to subsequent remodeling of the chromatin template during transcription. However, the interconnected network of molecules involved in regulating such processes remains elusive. hPaf1/PD2, a subunit of the human PAF-complex, involved in the regulation of transcriptional elongation has oncogenic potential. Our study explores the possibility that regulation of histone methylation by hPaf1 can contribute towards alteration in gene expression by nucleosomal rearrangement. Here, we show that knockdown of hPaf1/PD2 leads to decreased di- and tri-methylation at histone H3 lysine 4 residues in pancreatic cancer cells. Interestingly, hPaf1/PD2 colocalizes with MLL1 (Mixed Lineage Leukemia 1), a histone methyltransferase that methylates H3K4 residues. Also, a reduction in hPaf1 level resulted in reduced MLL1 expression and a corresponding decrease in the level of CHD1 (Chromohelicase DNA-binding protein 1), an ATPase dependent chromatin remodeling enzyme that specifically binds to H3K4 di and trimethyl marks. hPaf1/PD2 was also found to interact and colocalize with CHD1 in both cytoplasmic and nuclear extracts of pancreatic cancer cells. Further, reduced level of CHD1 localization in the nucleus in hPaf1/PD2 Knockdown cells could be rescued by ectopic expression of hPaf1/PD2. Micrococcal nuclease digestion showed an altered chromatin structure in hPaf1/PD2-KD cells. Overall, our results suggest that hPaf1/PD2 in association with MLL1 regulates methylation of H3K4 residues, as well as interacts and regulates nuclear shuttling of chromatin remodeling protein CHD1, facilitating its function in pancreatic cancer cells.

High gene expression of semaphorin 5A in pancreatic cancer is associated with tumor growth, invasion and metastasis.

Semaphorin 5A (SEMA5A) is an axonal regulator molecule, which belongs to the Semaphorin family of proteins. Previously, we identified SEMA5A as a putative marker for aggressive pancreatic tumors. However, the expression, localization and functional significance of SEMA5A in pancreatic tumors remain unclear. In our study, we hypothesized that SEMA5A expression modulates pancreatic tumor growth and metastasis. We analyzed the constitutive expression and localization of SEMA5A in patient pancreatic tumors (n = 33) and unmatched normal pancreatic (n = 8) tissues and human pancreatic cancer cell lines (n = 16) with different histopathological characteristics. We observed significantly higher expression of SEMA5A protein expression (p < 0.05) in human pancreatic tumor tissue samples compared to normal pancreatic tissues. Similarly, the pancreatic cancer cell lines with higher tumorigenic and metastatic potentials as xenografts in nude mice expressed higher levels of SEMA5A mRNA compared to those with lower tumorigenic and metastatic potentials. Furthermore, we examined the functional role of SEMA5A in pancreatic tumor growth and invasion. Ectopic expression of mouse full-length Sema5A in Panc1 (SEMA5A negative) cells significantly (p < 0.05) enhanced tumorigenesis, growth and metastasis in vivo as well as proliferation, invasiveness and homotypic aggregation in vitro. Together, these data demonstrate that the expression of SEMA5A in pancreatic cancer cells regulates tumorigenesis, growth, invasion and metastasis, and it also suggests a novel target for diagnosis and treatment of pancreatic cancer.

RNA polymerase II associated factor 1/PD2 maintains self-renewal by its interaction with Oct3/4 in mouse embryonic stem cells.

Embryonic stem cells (ESCs) maintain self-renewal while ensuring a rapid response to differentiation signals, but the exact mechanism of this process remains unknown. PD2 is the human homolog of the RNA polymerase II-associated factor 1 (Paf1). The Paf1/PD2 is a member of the human PAF complex that consists of four other subunits, hCdc73, hLeo1, hCtr9, and hSki8, and is involved in the regulation of transcriptional elongation and further downstream events. Here, we show that Paf1/PD2 is overexpressed in mouse ESCs and is involved in the maintenance of mouse ESCs. The Paf1/PD2 knockdown and knockout ESCs grown under self-renewal conditions express substantially reduced levels of self-renewal regulators, including Oct3/4, SOX2, Nanog, and Shh. We observed that the level of Paf1/PD2 expression is much higher in self-renewing mouse embryonic carcinoma cells than in the differentiating cells. Knockout of Paf1/PD2 altered ESC phenotype by increasing apoptosis and decreasing the percentage of cells in S-phase of the cell cycle. Interestingly, we found that the key genes that regulate endodermal differentiation (Gata4, Gata6, and Fgf8) are induced in the Paf1/PD2 heterozygous knockout ESCs. This suggests that Paf1/PD2 plays a specific role in regulating early commitment of ESCs to endodermal differentiation. Furthermore, for the first time, we showed that Paf1/PD2 protein interacts with Oct3/4 and RNA polymerase II, and through this interaction Paf1/PD2 may regulate Oct3/4-mediated gene expression. Thus, the Paf1/PD2 protein is a newly discovered element of the interconnected regulatory network that maintains the self-renewal of mouse ESCs.

The human RNA polymerase II-associated factor 1 (hPaf1): a new regulator of cell-cycle progression.

BACKGROUND: The human PAF (hPAF) complex is part of the RNA polymerase II transcription apparatus and regulates multiple steps in gene expression. Further, the yeast homolog of hPaf1 has a role in regulating the expression of a subset of genes involved in the cell-cycle. We therefore investigated the role of hPaf1 during progression of the cell-cycle. METHODOLOGY/FINDINGS: Herein, we report that the expression of hPaf1, a subunit of the hPAF complex, increases with cell-cycle progression and is regulated in a cell-cycle dependant manner. hPaf1 specifically regulates a subclass of genes directly implicated in cell-cycle progression during G1/S, S/G2, and G2/M. In prophase, hPaf1 aligns in filament-like structures, whereas in metaphase it is present within the pole forming a crown-like structure, surrounding the centrosomes. Moreover, hPaf1 is degraded during the metaphase to anaphase transition. In the nucleus, hPaf1 regulates the expression of cyclins A1, A2, D1, E1, B1, and Cdk1. In addition, expression of hPaf1 delays DNA replication but favors the G2/M transition, in part through microtubule assembly and mitotic spindle formation. CONCLUSION/SIGNIFICANCE: Our results identify hPaf1 and the hPAF complex as key regulators of cell-cycle progression. Mutation or loss of stoichiometry of at least one of the members may potentially lead to cancer development.

Human PAF complexes in endocrine tumors and pancreatic cancer.

The human RNA polymerase II-associated factor (hPAF) complex is comprised of five subunits that include hPaf1, parafibromin, hLeo1, hCtr9 and hSki8. This multifaceted complex was first identified in yeast (yPAF) and subsequently in Drosophila and humans. Recent advances in the study on hPAF have revealed various functions of the complex in humans that are similar to yPAF, including efficient transcription elongation, mRNA quality control and cell cycle regulation. A major component of the hPAF complex, hPaf1, is amplified and overexpressed in pancreatic cancer. The parafibromin subunit of the hPAF complex is a product of the hereditary hyperparathyroidism type 2 (HRPT-2) tumor-suppressor gene, which is mutated in the germ line of hyperparathyroidism-jaw tumor patients. This review evaluates the role of the hPAF complex and its individual subunits in endocrine and pancreatic cancers. It focuses on the functions of the hPAF complex and its individual subunits and dysregulation of the complex, thus providing an insight into its potential involvement in the development of endocrine cancers and other tumor types.