PHF5A promotes esophageal squamous cell carcinoma progression via stabilizing VEGFA.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is the main subtype of esophageal cancer. Current therapeutic effect is far from satisfaction. Hence, identifying susceptible genes and potential targets is necessary for therapy of ESCC patients. METHODS: Plant homeodomain (PHD)-finger domain protein 5 A (PHF5A) expression in ESCC tissues was examined by immunohistochemistry. RNA interference was used for in vitro loss-of-function experiments. In vivo assay was performed using xenograft mice model by subcutaneous injection. Besides, microarray assay and co-immunoprecipitation experiments were used to study the potential downstream molecules of PHF5A in ESCC. The molecular mechanism between PHF5A and vascular endothelial growth factor A (VEGFA) was explored by a series of ubiquitination related assays. RESULTS: We found that PHF5A was highly expressed in ESCC tissues compared to normal tissues and that was correlated with poor prognosis of ESCC. Loss-of-function experiments revealed that PHF5A silence remarkably inhibited cell proliferation, migration, and induced apoptosis as well as cell cycle arrest. Consistently, in vivo assay demonstrated that PHF5A deficiency was able to attenuate tumor growth. Furthermore, molecular studies showed that PHF5A silencing promoted VEGFA ubiquitination by interacting with MDM2, thereby regulating VEGFA protein expression. Subsequently, in rescue experiments, our data suggested that ESCC cell viability and migration promoted by PHF5A were dependent on intact VEGFA. Finally, PI3K/AKT signaling rescue was able to alleviate shPHF5A-mediated cell apoptosis and cell cycle arrest. CONCLUSION: PHF5A is a tumor promoter in ESCC, which is dependent on VEGFA and PI3K/AKT signaling. PHF5A might serve as a potential therapeutic target for ESCC treatment.

Spliceosome component PHD finger 5A is essential for early B lymphopoiesis.

The spliceosome, a multi-megadalton ribonucleoprotein complex, is essential for pre-mRNA splicing in the nucleus and ensuring genomic stability. Its precise and dynamic assembly is pivotal for its function. Spliceosome malfunctions can lead to developmental abnormalities and potentially contribute to tumorigenesis. The specific role of the spliceosome in B cell development is poorly understood. Here, we reveal that the spliceosomal U2 snRNP component PHD finger protein 5A (Phf5a) is vital for early B cell development. Loss of Phf5a results in pronounced defects in B cell development, causing an arrest at the transition from pre-pro-B to early pro-B cell stage in the bone marrow of mutant mice. Phf5a-deficient B cells exhibit impaired immunoglobulin heavy (IgH) chain expression due to defective V-to-DJ gene rearrangement. Mechanistically, our findings suggest that Phf5a facilitates IgH gene rearrangement by regulating the activity of recombination-activating gene endonuclease and influencing chromatin interactions at the Igh locus.

Plant homeodomain-finger protein 5A: A key player in cancer progression.

PHF5A is a member of the zinc-finger proteins. To advance knowledge on their role in carcinogenesis, data from experimental studies, animal models and clinical studies in different tumorigenesis have been reviewed. Furthermore, PHF5A as an oncogenic function, is frequently high expressed in tumor cells and a potential prognostic marker for different cancers. PHF5A is implicated in the regulation of cancer cell proliferation, invasion, migration and metastasis. Knockdown of PHF5A prevented the invasion and metastasis of tumor cells. Here, the role of PHF5A in different cancers and their possible mechanism in relation to recent literature is reviewed and discussed. There is an open promising perspective to their therapeutic management for different cancer types.

PHF5A as a new OncoTarget and therapeutic prospects.

PHF5A (PHD-finger domain protein 5A) is a highly conserved protein comprised of 110 amino acids that belong to PHD zinc finger proteins and is ubiquitously expressed in entire eukaryotic nuclei from yeast to man. PHF5A is an essential component of the SF3B splicing complex regulating protein-protein or protein-DNA interactions; particularly involved in pre-mRNA splicing. Besides its basic spliceosome-associated attributes encompassing the regulation of alternative splicing of specific genes, PHF5A also plays a pivotal role in cell cycle regulation and morphological development of cells along with their differentiation into particular tissues/organs, DNA damage repair, maintenance of pluripotent embryonic stem cells (CSCs) embryogenesis and regulation of chromatin-mediated transcription. Presently identification of spliceosome and non-spliceosome-associated attributes of PHF5A needs great attention based on its key involvement in the pathogenesis of cancer malignancies including the prognosis of lung adenocarcinoma, endometrial adenocarcinoma, breast, and colorectal cancer. PHF5A is an essential splicing factor or cofactor actively participating as an oncogenic protein in tumorigenesis via activation of downstream signaling pathway attributed to its regulation of dysregulated splicing or abnormal alternative splicing of targeted genes. Further, the participation of PHF5A in regulating the growth of cancer stem cells might not be ignored. The current review briefly overviews the structural and functional attributes of PHF5A along with its hitherto described role in the propagation of cancer malignancies and its future concern as a potential therapeutic target for cancer management/treatment.

PHF5A regulates the expression of the DOCK5 variant to promote HNSCC progression through p38 MAPK activation.

BACKGROUND: Previously, we identified an oncogenic splicing variant of DOCK5 in head and neck squamous cell carcinoma (HNSCC); however, the mechanism for the generation of this specific DOCK5 variant remains unknown. This study aims to explore the potential spliceosome genes involved in the production of the DOCK5 variant and validate its role in regulating the progression of HNSCC. METHODS: The differentially expressed spliceosome genes involved in the DOCK5 variant were analysed in The Cancer Genome Atlas (TCGA), and the correlation between the DOCK5 variant and the potential spliceosome gene PHF5A was verified by qRT-PCR. The expression of PHF5A was detected in HNSCC cells, TCGA data and a separate primary tumour cohort. The functional role of PHF5A was examined using CCK-8, colony formation, cell scratch and Transwell invasion assays in vitro and validated in vivo in xenograft models of HNSCC. Western blot analysis was used to explore the potential mechanism of PHF5A in HNSCC. RESULTS: PHF5A was one of the top upregulated spliceosome genes in TCGA HNSCC samples with highly expressed DOCK5 variants. Knockdown or overexpression of PHF5A in HNSCC cells correspondingly altered the level of the DOCK5 variant. PHF5A was highly expressed in tumour cells and tissues and correlated with a worse prognosis of HNSCC. Loss- and gain-of-function experiments demonstrated that PHF5A could promote the proliferation, migration and invasion of HNSCC cells in vitro and in vivo. Moreover, PHF5A inhibition reversed the oncogenic effect of the DOCK5 variant in HNSCC. Western blot analysis showed that PHF5A activated the p38 MAPK pathway, and inhibition of p38 MAPK further reversed the effect of PHF5A on the proliferation, migration and invasion of HNSCC cells. CONCLUSION: PHF5A regulates the alternative splicing of DOCK5 to promote HNSCC progression through p38 MAPK activation, which provides potential therapeutic implications for HNSCC patients.

[PHF5A Promotes Proliferation and Migration of Non-Small Cell Lung Cancer 
by Regulating of PI3K/AKT Pathway].

BACKGROUND: There have been many significant advances in the diagnosis and treatment of non-small cell lung cancer (NSCLC). However, the mechanism underlying the progression of NSCLC is still not clear. Plant homodomain finger-like domain-containing protein 5A (PHF5A) plays an important role in processes of chromatin remodeling, morphological development of tissues and organs and maintenance of stem cell pluripotency. This study aims to investigate the role of PHF5A in the proliferation and migration of NSCLC. METHODS: A549 and PC-9 PHF5A overexpression cell lines were constructed. PHF5A expression was decreased in H292 and H1299 cells by using siRNA. Flow cytometry was used to detect the cell cycle. MTT assay and clone formation assay were used to examine the proliferative ability of NSCLC, while migration assay and wound healing assay were performed to evaluate the ability of migration. Western blot analysis was used to measure the expressions of PI3K, p-AKT and the associated downstream factors. RESULTS: Up-regulation of PHF5A in A549 and PC-9 cells increased the proliferation rate, while down-regulation of PHF5A in H292 and H1299 cells inhibited the proliferation rate at 24 h, 48 h and 72 h (P<0.05). The metastatic ability was elevated in the PHF5A-overexpresion groups, while reduced in the PHF5A-down-regulation group (P<0.05). In addition, reduced expression of PHF5A induced cell cycle arrest at G1/S phase (P<0.05). Furthermore, decreased expression of PHF5A reduced the expression levels of PI3K, phosphorylation of AKT, c-Myc (P<0.05) and elevated the expression of p21 (P<0.05). CONCLUSIONS: These results demonstrated that PHF5A may play an important role in progression of NSCLC by regulating the PI3K/AKT signaling pathway.

PHF5A facilitates the development and progression of gastric cancer through SKP2-mediated stabilization of FOS.

BACKGROUND: Gastric cancer (GC) is the fifth most common cancer and the third most common cause of cancer death worldwide. Plant homeodomain (PHD)-finger domain protein PHF5A has been demonstrated to play a promoting role in a variety of cancers. This study aimed to clarify the role of PHF5A in the progression of GC and its potential mechanism of action. METHODS: Immunohistochemical staining experiments were performed based on tissues from clinical GC patients to reveal PHF5A expression. A series of functional experiments in vitro and in vivo were used to clarify the role of PHF5A in GC. RESULTS: Clinically, PHF5A was abundantly expressed in GC and existed clinical value indicating poor prognosis. In addition, GC cells with knockdown of PHF5A expression showed slowed proliferation, enhanced sensitivity to apoptosis and inhibition of migration. Mechanically, knockdown of PHF5A led to decreased protein stability of FOS, which was mediated ubiquitination of E3 ubiquitin ligase S-phase kinase-associated protein 2 (SKP2). Moreover, downregulation of FOS attenuated the promotion of PHF5A overexpression on GC cells. Consistently, Pladienolide B (PHF5A inhibitor) treatment reversed the induction of PHF5A overexpression on the malignant phenotypes and tumor formation of GC cells. CONCLUSION: Knockdown of PHF5A inhibited the progression of GC through SKP2-mediated ubiquitination of FOS, which may be a promising candidate target with potential therapeutic value.

PHF5A Promotes Proliferation and Migration of Non-Small Cell Lung Cancer by Regulating of PI3K/AKT Pathway / 中国肺癌杂志

BACKGROUND@#There have been many significant advances in the diagnosis and treatment of non-small cell lung cancer (NSCLC). However, the mechanism underlying the progression of NSCLC is still not clear. Plant homodomain finger-like domain-containing protein 5A (PHF5A) plays an important role in processes of chromatin remodeling, morphological development of tissues and organs and maintenance of stem cell pluripotency. This study aims to investigate the role of PHF5A in the proliferation and migration of NSCLC.@*METHODS@#A549 and PC-9 PHF5A overexpression cell lines were constructed. PHF5A expression was decreased in H292 and H1299 cells by using siRNA. Flow cytometry was used to detect the cell cycle. MTT assay and clone formation assay were used to examine the proliferative ability of NSCLC, while migration assay and wound healing assay were performed to evaluate the ability of migration. Western blot analysis was used to measure the expressions of PI3K, p-AKT and the associated downstream factors.@*RESULTS@#Up-regulation of PHF5A in A549 and PC-9 cells increased the proliferation rate, while down-regulation of PHF5A in H292 and H1299 cells inhibited the proliferation rate at 24 h, 48 h and 72 h (P<0.05). The metastatic ability was elevated in the PHF5A-overexpresion groups, while reduced in the PHF5A-down-regulation group (P<0.05). In addition, reduced expression of PHF5A induced cell cycle arrest at G1/S phase (P<0.05). Furthermore, decreased expression of PHF5A reduced the expression levels of PI3K, phosphorylation of AKT, c-Myc (P<0.05) and elevated the expression of p21 (P<0.05).@*CONCLUSIONS@#These results demonstrated that PHF5A may play an important role in progression of NSCLC by regulating the PI3K/AKT signaling pathway.

PHF5A promotes colorectal cancerprogression by alternative splicing of TEAD2.

Dysregulated alternative splicing (AS) plays critical roles in driving cancer progression, and the underlying mechanisms remain largely unknown. Here, we demonstrated that PHF5A, a component of U2 small nuclear ribonucleoproteins, was frequently upregulated in colorectal cancer (CRC) samples and associated with poor prognosis. PHF5A promoted proliferation and metastasis of CRC cells in vitro and in vivo. Transcriptomic analysis identified PHF5A-regulated AS targets and pathways. Particularly, PHF5A induced TEAD2 exon 2 inclusion to activate YAP signaling, and interference of TEAD2-L partially reversed the PHF5A-mediated tumor progression. Pharmacological inhibition of PHF5A using pladienolide B had potent antitumor activity. Collectively, these data revealed the oncogenic role of PHF5A in CRC through regulating AS and established PHF5A as potential therapeutic target.

SIRT7-Induced PHF5A Decrotonylation Regulates Aging Progress Through Alternative Splicing-Mediated Downregulation of CDK2.

Dysregulation of protein posttranslational modification (PTM) can lead to a variety of pathological processes, such as abnormal sperm development, malignant tumorigenesis, depression, and aging process. SIRT7 is a NAD+-dependent protein deacetylase. Besides known deacetylation, SIRT7 may also have the capacity to remove other acylation. However, the roles of SIRT7-induced other deacylation in aging are still largely unknown. Here, we found that the expression of SIRT7 was significantly increased in senescent fibroblasts and aged tissues. Knockdown or overexpression of SIRT7 can inhibit or promote fibroblast senescence. Knockdown of SIRT7 led to increased pan-lysine crotonylation (Kcr) levels in senescent fibroblasts. Using modern mass spectrometry (MS) technology, we identified 5,149 Kcr sites across 1,541 proteins in senescent fibroblasts, and providing the largest crotonylome dataset to date in senescent cells. Specifically, among the identified proteins, we found SIRT7 decrotonylated PHF5A, an alternative splicing (AS) factor, at K25. Decrotonylation of PHF5A K25 contributed to decreased CDK2 expression by retained intron (RI)-induced abnormal AS, thereby accelerating fibroblast senescence, and supporting a key role of PHF5A K25 decrotonylation in aging. Collectively, our data revealed the molecular mechanism of SIRT7-induced k25 decrotonylation of PHF5A regulating aging and provide new ideas and molecular targets for drug intervention in cellular aging and the treatment of aging-related diseases, and indicating that protein crotonylation has important implications in the regulation of aging progress.

Enterotoxigenic Bacteroidesfragilis Promotes Intestinal Inflammation and Malignancy by Inhibiting Exosome-Packaged miR-149-3p.

BACKGROUND & AIMS: Enterotoxigenic Bacteroides fragilis (ETBF) is strongly associated with the occurrence of inflammatory bowel disease (IBD), colitis-associated colorectal cancer, and colorectal cancer (CRC). However, the mechanism of ETBF-induced intestinal inflammation and tumorigenesis remains unclear. METHODS: microRNA sequencing was used to detect the differentially expressed microRNAs in both ETBF-treated cells and exosomes derived from ETBF-inoculated cells. Cell Counting Kit 8 assays were used to evaluate the effect of ETBF and exosomes on CRC cell proliferation. The biological role and mechanism of ETBF-mediated miR-149-3p in colitis and colon carcinogenesis were determined both in vitro and in vivo. RESULTS: ETBF promoted CRC cell proliferation by down-regulating miR-149-3p both in vitro and in vivo. ETBF-down-regulated miR-149-3p depended on METTL14-mediated N6-methyladenosine methylation. As the target gene of miR-149-3p, PHF5A transactivated SOD2 through regulating KAT2A messenger RNA alternative splicing after ETBF treatment in CRC cells. miR-149-3p could be released in exosomes and mediated intercellular communication by modulating T-helper type 17 cell differentiation. The level of plasma exosomal miR-149-3p was gradually decreased from healthy control individuals to patients with IBD and CRC. miR-149-3p, existing in plasma exosomes, negatively correlated with the abundance of ETBF in patients with IBD and CRC. CONCLUSIONS: Exosomal miR-149-3p derived from ETBF-treated cells facilitated T-helper type 17 cell differentiation. ETBF-induced colorectal carcinogenesis depended on down-regulating miR-149-3p and further promoting PHF5A-mediated RNA alternative splicing of KAT2A in CRC cells. Targeting the ETBF/miR-149-3p pathway presents a promising approach to treat patients with intestinal inflammation and CRC with a high amount of ETBF.

Phf5a regulates DNA repair in class switch recombination via p400 and histone H2A variant deposition.

Antibody class switch recombination (CSR) is a locus-specific genomic rearrangement mediated by switch (S) region transcription, activation-induced cytidine deaminase (AID)-induced DNA breaks, and their resolution by non-homologous end joining (NHEJ)-mediated DNA repair. Due to the complex nature of the recombination process, numerous cofactors are intimately involved, making it important to identify rate-limiting factors that impact on DNA breaking and/or repair. Using an siRNA-based loss-of-function screen of genes predicted to encode PHD zinc-finger-motif proteins, we identify the splicing factor Phf5a/Sf3b14b as a novel modulator of the DNA repair step of CSR. Loss of Phf5a severely impairs AID-induced recombination, but does not perturb DNA breaks and somatic hypermutation. Phf5a regulates NHEJ-dependent DNA repair by preserving chromatin integrity to elicit optimal DNA damage response and subsequent recruitment of NHEJ factors at the S region. Phf5a stabilizes the p400 histone chaperone complex at the locus, which in turn promotes deposition of H2A variant such as H2AX and H2A.Z that are critical for the early DNA damage response and NHEJ, respectively. Depletion of Phf5a or p400 blocks the repair of both AID- and I-SceI-induced DNA double-strand breaks, supporting an important contribution of this axis to programmed as well as aberrant recombination.

The SF3b Complex is an Integral Component of the Spliceosome and Targeted by Natural Product-Based Inhibitors.

In this chapter, the essential role of the SF3b multi-protein complex will be discussed in the context of the overall spliceosome. SF3b is critical during spliceosome assembly for recognition of the branch point (BP) adenosine and, by de facto, selection of the 3' splice site. This complex is highly dynamic, undergoing significant conformational changes upon loading of the branch duplex RNA and in its relative positioning during spliceosomal remodeling from the A, pre-B, B, Bact and B* complexes. Ultimately, during the spliceosome activation phase, SF3b must be displaced to unmask the branch point adenosine for the first splicing reaction to occur. In certain cancers, such as the hematological malignancies CML, CLL and MDS, the SF3b subunit SF3B1 is frequently mutated. Recent studies suggest these mutations lead to inappropriate branch point selection and mis-splicing events that appear to be drivers of disease. Finally, the SF3b complex is the target for at least three different classes of natural product-based inhibitors. These inhibitors bind in the BP adenosine-binding pocket and demonstrate a pre-mRNA competitive mechanism of action resulting in either intron retention or exon skipping. These compounds are extremely useful as chemical probes to isolate and characterize early stages of spliceosome assembly. They are also being explored preclinically and clinically as possible agents for hematological cancers.

CHD4 mediates proliferation and migration of non-small cell lung cancer via the RhoA/ROCK pathway by regulating PHF5A.

BACKGROUND: Chromodomain helicase DNA-binding protein 4 (CHD4) has been shown to contribute to DNA repair and cell cycle promotion; however, its roles in cancer initiation and progression remain largely unknown. This study aimed to demonstrate the role of CHD4 in the development of non-small cell lung cancer (NSCLC) and determine the potential mechanisms of action. METHODS: By using immunohistochemistry, the expression levels were evaluated in both cancer and non-cancerous tissues. Subsequently, CHD4 knockdown and overexpression strategies were employed to investigate the effects of CHD4 on cell proliferation, migration, along with the growth and formation of tumors in a xenografts mouse model. The protein expression levels of CHD4, PHF5A and ROCK/RhoA markers were determined by Western blot analysis. RESULTS: Compared with non-cancerous tissues, CHD4 was overexpressed in cancer tissues and CHD4 expression levels were closely related to clinical parameters of NSCLC patients. In H292 and PC-9 cell lines, CHD4 overexpression could promote the proliferative and migratory potential of NSCLC cells. Furthermore, down-regulation of CHD4 could reduce the proliferative and migratory ability in A549 and H1299 cell lines. Meanwhile, knockdown of CHD4 could decrease the tumorigenicity in nude mice. Finally, we demonstrated that one of the mechanisms underlying the promotive effect of CHD4 on NSCLC proliferation and migration may be through its interaction with PHD finger protein 5A (PHF5A) and subsequent activation of the RhoA/ROCK signaling pathway. CONCLUSIONS: CHD4, which is highly expressed in cancer tissue, could be an independent prognostic factor for NSCLC patients. CHD4 plays an important role in regulating the proliferative and migratory abilities of NSCLC via likely the RhoA/ROCK pathway by regulating PHF5A.

Acetylation of PHF5A Modulates Stress Responses and Colorectal Carcinogenesis through Alternative Splicing-Mediated Upregulation of KDM3A.

Alternative pre-mRNA-splicing-induced post-transcriptional gene expression regulation is one of the pathways for tumors maintaining proliferation rates accompanying the malignant phenotype under stress. Here, we uncover a list of hyperacetylated proteins in the context of acutely reduced Acetyl-CoA levels under nutrient starvation. PHF5A, a component of U2 snRNPs, can be acetylated at lysine 29 in response to multiple cellular stresses, which is dependent on p300. PHF5A acetylation strengthens the interaction among U2 snRNPs and affects global pre-mRNA splicing pattern and extensive gene expression. PHF5A hyperacetylation-induced alternative splicing stabilizes KDM3A mRNA and promotes its protein expression. Pathologically, PHF5A K29 hyperacetylation and KDM3A upregulation axis are correlated with poor prognosis of colon cancer. Our findings uncover a mechanism of an anti-stress pathway through which acetylation on PHF5A promotes the cancer cells' capacity for stress resistance and consequently contributes to colon carcinogenesis.

PHD finger protein 5A promoted lung adenocarcinoma progression via alternative splicing.

Alternative splicing (AS) and the regulation of AS by splicing factors play critical roles in cancer. Plant homeodomain (PHD)-finger domain protein PHF5A, a critical splicing factor involved in AS, has been demonstrated to play an oncogenic role in glioblastoma multiforme and breast cancer, but its biological function in lung cancer remains unclear. In the present study, we systematically analyzed the biological function and clinical relevance of PHF5A in non-small cell lung cancer (NSCLC). We found that PHF5A was significantly upregulated in NSCLC tumors compared with normal tissues in both TCGA data set and tissue microarrays. Upregulation of PHF5A was negatively correlated to the overall survival (OS) of lung adenocarcinoma (LUAD) patients. Loss-of-function and gain-of-function experiments confirmed that PHF5A functioned as an oncoprotein by promoting LUAD cell proliferation, migration and invasion, inducing G0/G1 cell cycle progression and inhibiting cisplatin-induced apoptosis. RNA-seq analysis identified many essential genes whose AS was dysregulated by PHF5A, including cell cycle-associated genes such as SKP2, CHEK2, ATR and apoptosis-associated genes such as API5 and BCL2L13. Additionally, pladienolide, a small molecular inhibitor of PHF5A, inhibited LUAD cell proliferation in a dose-dependent manner and induced AS changes similar to PHF5A knockdown. In conclusion, we validated that PHF5A played an oncogenic role via AS in LUAD and suggested that PHF5A might serve as a potential drug target with a promising anticancer therapeutic effect.

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.

PHD-finger domain protein 5A functions as a novel oncoprotein in lung adenocarcinoma.

BACKGROUND: PHD-finger domain protein 5A (PHF5A) is a highly conserved small transcriptional regulator also involved in pre-mRNA splicing; however, its biological functions and molecular mechanisms in non-small cell lung cancer (NSCLC) have not yet been investigated. The purpose of this study was to determine the functional relevance and therapeutic potential of PHF5A in lung adenocarcinoma (LAC). METHODS: The expression of PHF5A in LAC tissues and adjacent non-tumor (ANT) tissues was investigated using immunohistochemistry of a tissue microarray, qRT-PCR, western blot and bioinformatics. The function of PHF5A was determined using several in vitro assays and also in vivo assay by lentiviral vector-mediated PHF5A depletion in LAC cell lines. RESULTS: PHF5A was highly upregulated in LAC tissues compared with the ANT counterparts, and closely associated with tumor progression and poor patient prognosis. These results were further confirmed by findings of the TCGA database. Moreover, functional studies demonstrated that PHF5A knockdown not only resulted in reduced cell proliferation, increased cell apoptosis, and cell cycle arrest, but also suppressed migration and invasion in LAC cells. PHF5A silencing was also found to inhibit LAC tumor growth in nude mice. Microarray and bioinformatics analyses revealed that PHF5A depletion led to dysregulation of multiple tumor signaling pathways; selected factors in key signaling pathways were verified in vitro. CONCLUSIONS: The data suggest for the first time that PHF5A is an oncoprotein that contributes to LAC progression by regulating multiple signaling pathways, and may constitute a prognostic factor and potential new therapeutic target in NSCLC.

Opposing functions of H2BK120 ubiquitylation and H3K79 methylation in the regulation of pluripotency by the Paf1 complex.

Maintenance of stem cell plasticity is determined by the ability to balance opposing forces that control gene expression. Regulation of transcriptional networks, signaling cues and chromatin-modifying mechanisms constitute crucial determinants of tissue equilibrium. Histone modifications can affect chromatin compaction, therefore co-transcriptional events that influence their deposition determine the propensities toward quiescence, self-renewal, or cell specification. The Paf1 complex (Paf1C) is a critical regulator of RNA PolII elongation that controls gene expression and deposition of histone modifications, however few studies have focused on its role affecting stem cell fate decisions. Here we delineate the functions of Paf1C in pluripotency and characterize its impact in deposition of H2B ubiquitylation (H2BK120-ub) and H3K79 methylation (H3K79me), 2 fundamental histone marks that shape transcriptional regulation. We identify that H2BK120-ub is increased in the absence of Paf1C on its embryonic stem cell targets, in sharp contrast to H3K79me, suggesting opposite functions in the maintenance of self-renewal. Furthermore, we found that core pluripotency genes are characterized by a dual gain of H2BK120-ub and loss of H3K79me on their gene bodies. Our findings elucidate molecular mechanisms of cellular adaptation and reveal novel functions of Paf1C in the regulation of the self-renewal network.