Xuesen Dong

Consensus PP1 binding motifs regulate transcriptional corepression and alternative RNA splicing activities of the steroid receptor coregulators, p54nrb and PSF

Originally identified as essential pre-mRNA splicing factors, non-POU-domain-containing, octamer binding protein (p54nrb) and PTB-associated RNA splicing factor (PSF) are also steroid receptor corepressors. The mechanisms by which p54nrb and PSF regulate gene transcription remain unclear. Both p54nrb and PSF contain protein phosphatase 1 (PP1) consensus binding RVxF motifs, suggesting that PP1 may regulate phosphorylation status of p54nrb and PSF and thus their function in gene transcription. In this report, we demonstrated that PP1 forms a protein complex with both p54nrb and PSF. PP1 interacts directly with the RVxF motif only in p54nrb, but not in PSF. Association with PP1 results in dephosphorylation of both p54nrb and PSF in vivo and the loss of their transcriptional corepressor activities. Using the CD44 minigene as a reporter, we showed that PP1 regulates p54nrb and PSF alternative splicing activities that determine exon skipping vs. inclusion in the final mature RNA for translation. In addition, changes in transcriptional corepression and RNA splicing activities of p54nrb and PSF are correlated with alterations in protein interactions of p54nrb and PSF with transcriptional corepressors such as Sin3A and histone deacetylase 1, and RNA splicing factors such as U1A and U2AF. Furthermore, we demonstrated a novel function of the RVxF motif within PSF that enhances its corepression and RNA splicing activities independent of PP1. We conclude that the RVxF motifs play an important role in controlling the multifunctional properties of p54nrb and PSF in the regulation of gene transcription.

Transcriptional Regulation of Human Myometrium and the Onset of Labor

ignificant progress has been made in understanding the mechanisms of parturition in animal species, but identification of the pathways leading to human labor remains obscure. In most animal species progesterone contributes to the maintenance of quiescence during pregnancy, and withdrawal of this progesterone effect, manifest as a decline in maternal circulating progesterone levels, generally precedes the onset oflabor at term. However, it is clear that there is no such fall in plasma or tissue progesterone levels in women. A central paradox of human obstetrical research for more than 50 years has therefore been how the processes of uterine activation and stimulation can occur in the absence of systemic progesterone withdrawal. An explanation has come from the suggestion that in women the birth process at term and preterm is affected

UGT2B17 Expedites Progression of Castration-Resistant Prostate Cancers by Promoting Ligand-Independent AR Signaling

Castration-resistant prostate cancer (CRPC) is characterized by a shift in androgen receptor (AR) signaling from androgen-dependent to androgen (ligand)-independent. UDP-glucuronosyltransferase 2B17 (UGT2B17) is a key enzyme that maintains androgen homeostasis by catabolizing AR agonists into inactive forms. Although enhanced UGT2B17 expression by antiandrogens has been reported in androgen-dependent prostate cancer, its roles in regulating AR signaling transformation and CRPC progression remain unknown. In this study, we show that higher UGT2B17 protein expression in prostate tumors is associated with higher Gleason score, metastasis, and CRPC progression. UGT2B17 expression and activity were higher in androgen-independent compared to androgen-dependent cell lines. UGT2B17 stimulated cancer cell proliferation, invasion, and xenograft progression to CRPC after prolonged androgen deprivation. Gene microarray analysis indicated that UGT2B17 suppressed androgen-dependent AR transcriptional activity and enhanced of ligand-independent transcriptional activity at genes associated with cell mitosis. These UGT2B17 actions were mainly mediated by activation of the c-Src kinase. In CRPC tumors, UGT2B17 expression was associated positively with c-Src activation. These results indicate that UGT2B17 expedites CRPC progression by enhancing ligand-independent AR signaling to activate cell mitosis in cancer cells. Cancer Res; 76(22); 6701-11. ©2016 AACR.

HoxA13 Regulates Phenotype Regionalization of Human Pregnant Myometrium

CONTEXTnBipedalism separates humans from most other animal species, but results in significant physiologic challenges, particularly with respect to the maintenance of pregnancy and induction of parturition. A contracted lower uterine segment (LUS) and a relaxed uterine fundal myometrium (FUN) during pregnancy are required to prevent pressure on the cervix from the fetal head due to gravity. With the onset of labor, this regionalization of myometrial function must be reversed, allowing descent of the fetus, dilation of the cervix, and expulsion of the fetus through the birth canal. However, the molecular mechanisms remain unclear.nnnOBJECTIVE AND DESIGNnThis study sought to identify phenotypic regionalization of LUS and FUN during pregnancy, RNA sequencing was performed to analyze the human myometrial transcriptome. Real-time PCR and immunoblotting were applied to validate sequencing results. Cell contraction/adhesion assays and gene microarrays were used to study the cellular functions of the identified genes.nnnRESULTSnHomeobox A13 (HoxA13), prostacyclin synthase (PTGIS), and periostin (POSTN) genes are more highly expressed in LUS than FUN of nonlaboring, but not laboring, myometrial cells at term. HoxA13 up-regulates transcription of PTGIS and POSTN genes. Elevated HoxA13 expression enhances myometrial cell contractility and cell-cell adhesion. Gene microarray studies show that HoxA13-regulated genes are associated with immune response, gap junction/cell adhesion, and pregnancy.nnnCONCLUSIONnThe LUS expresses higher levels of HoxA13, PTGIS, and POSTN, and is more contractile than the FUN at term prior to labor. This pregnancy-maintaining regionalization of myometrial function may be mediated by HoxA13.

Histone demethylase JMJD1A promotes alternative splicing of AR variant 7 (AR-V7) in prostate cancer cells

Significance Formation of androgen receptor splicing variant 7 (AR-V7), a constitutively active form of AR, plays a key role in the resistance of prostate cancer to hormone therapy. However, the mechanisms that regulate AR-V7 generation are poorly understood. Here, we identified a new role for histone demethylase JMJD1A (Jumonji domain containing 1A) in the formation of AR-V7 in prostate cancer cells. We found that JMJD1A facilitated recruitment of a splicing factor, heterogeneous nuclear ribonucleoprotein F, for alternative splicing and generation of AR-V7. The findings suggest that targeting JMJD1A may provide new therapeutic opportunity for prostate cancer. Formation of the androgen receptor splicing variant 7 (AR-V7) is one of the major mechanisms by which resistance of prostate cancer to androgen deprivation therapy occurs. The histone demethylase JMJD1A (Jumonji domain containing 1A) functions as a key coactivator for AR by epigenetic regulation of H3K9 methylation marks. Here, we describe a role for JMJD1A in AR-V7 expression. While JMJD1A knockdown had no effect on full-length AR (AR-FL), it reduced AR-V7 levels in prostate cancer cells. Reexpression of AR-V7 in the JMJD1A-knockdown cells elevated expression of select AR targets and partially rescued prostate cancer cell growth in vitro and in vivo. The AR-V7 protein level correlated positively with JMJD1A in a subset of human prostate cancer specimens. Mechanistically, we found that JMJD1A promoted alternative splicing of AR-V7 through heterogeneous nuclear ribonucleoprotein F (HNRNPF), a splicing factor known to regulate exon inclusion. Knockdown of JMJD1A or HNRNPF inhibited splicing of AR-V7, but not AR-FL, in a minigene reporter assay. JMJD1A was found to interact with and promote the recruitment of HNRNPF to a cryptic exon 3b on AR pre-mRNA for the generation of AR-V7. Taken together, the role of JMJD1A in AR-FL coactivation and AR-V7 alternative splicing highlights JMJD1A as a potentially promising target for prostate cancer therapy.

Development of Neuroendocrine Prostate Cancers by the Ser/Arg Repetitive Matrix 4-Mediated RNA Splicing Network

While the use of next-generation androgen receptor pathway inhibition (ARPI) therapy has significantly increased the survival of patients with metastatic prostate adenocarcinoma (AdPC), several groups have reported a treatment-resistant mechanism, whereby cancer cells can become androgen receptor (AR) indifferent and gain a neuroendocrine (NE)-like phenotype. This subtype of castration-resistant prostate cancer has been termed “treatment-induced castration-resistant neuroendocrine prostate cancer” (CRPC-NE). Recent reports indicate that the overall genomic landscapes of castration-resistant tumors with AdPC phenotypes and CRPC-NE are not significantly altered. However, CRPC-NE tumors have been found to contain a NE-specific pattern throughout their epigenome and splicing transcriptome, which are significantly modified. The molecular mechanisms by which CRPC-NE develops remain unclear, but several factors have been implicated in the progression of the disease. Recently, Ser/Arg repetitive matrix 4 (SRRM4), a neuronal-specific RNA splicing factor that is upregulated in CRPC-NE tumors, has been shown to establish a CRPC-NE-unique splicing transcriptome, to induce a NE-like morphology in AdPC cells, and, most importantly, to transform AdPC cells into CRPC-NE xenografts under ARPI. Moreover, the SRRM4-targeted splicing genes are highly enriched in various neuronal processes, suggesting their roles in facilitating a CRPC-NE program. This article will address the importance of SRRM4-mediated alternative RNA splicing in reprogramming translated proteins to facilitate NE differentiation, survival, and proliferation of cells to establish CRPC-NE tumors. In addition, we will discuss the potential roles of SRRM4 in conjunction with other known pathways and factors important for CRPC-NE development, such as the AR pathway, TP53 and RB1 genes, the FOXA family of proteins, and environmental factors. This study aims to explore the multifaceted functions of SRRM4 and SRRM4-mediated splicing in driving a CRPC-NE program as a coping mechanism for therapy resistance, as well as define future SRRM4-targeted therapeutic approaches for treating CRPC-NE or mitigating its development.

Tyrosine Residues Regulate Multiple Nuclear Functions of P54nrb

The non‐POU‐domain‐containing octamer binding protein (NONO; also known as p54nrb) has various nuclear functions ranging from transcription, RNA splicing, DNA synthesis and repair. Although tyrosine phosphorylation has been proposed to account for the multi‐functional properties of p54nrb, direct evidence on p54nrb as a phosphotyrosine protein remains unclear. To investigate the tyrosine phosphorylation status of p54nrb, we performed site‐directed mutagenesis on the five tyrosine residues of p54nrb, replacing the tyrosine residues with phenylalanine or alanine, and immunoblotted for tyrosine phosphorylation. We then preceded with luciferase reporter assays, RNA splicing minigene assays, co‐immunoprecipitation, and confocal microscopy to study the function of p54nrb tyrosine residues on transcription, RNA splicing, protein–protein interaction, and cellular localization. We found that p54nrb was not phosphorylated at tyrosine residues. Rather, it has non‐specific binding affinity to anti‐phosphotyrosine antibodies. However, replacement of tyrosine with phenylalanine altered p54nrb activities in transcription co‐repression and RNA splicing in gene context‐dependent fashions by means of differential regulation of p54nrb protein association with its interacting partners and co‐regulators of transcription and splicing. These results demonstrate that tyrosine residues, regardless of phosphorylation status, are important for p54nrb function. J. Cell. Physiol. 232: 852–861, 2017.

SRRM4 gene expression correlates with neuroendocrine prostate cancer

Neuroendocrine prostate cancer (NEPC) is an aggressive subtype of castrate‐resistant prostate cancer characterized by poor patient outcome. Whole transcriptome sequencing analyses identified a NEPC‐specific RNA splicing program that is predominantly controlled by the SRRM4 gene, suggesting that SRRM4 drives NEPC development. However, whether SRRM4 expression in patients may aid pathologists in diagnosing NEPC and predicting patient survival remains to be determined. In this study, we have applied RNA in situ hybridization and immunohistochemistry assays to measure the expressions of SRRM4, NEPC markers (SYP, CD56, and CHGA), and adenocarcinoma (AdPC) markers (AR, PSA) in a series of tissue microarrays constructed from castrate‐resistant prostate tumors, treatment‐naïve tumors collected from radical prostatectomy, and tumors treated with neoadjuvant hormonal therapy (NHT) for 0‐12 months. Three pathologists also independently evaluated tumor histology and NEPC marker status. Here, we report that SRRM4 in castrate‐resistant tumors is highly expressed in NEPC, strongly correlated with SYP, CD56, and CHGA expressions (Pearson correlation ru2009=u20090.883, 0.675, and 0.881; Pu2009<u20090.0001) and negatively correlated with AR and PSA expressions (Pearson correlation ru2009=u2009−0.544 and −0.310; Pu2009<u20090.05). Overall survival is 12.3 months for patients with SRRM4 positive tumors, comparing to 23 months for patients with SRRM4 negative tumors. In treatment‐naïve AdPC, low SRRM4 expression is detected in ∼16% tumor cores. It correlates with SYP and CHGA expressions, but not Gleason scores. AdPC treated with >7 month NHT has significantly higher SRRM4 expression. Based on these findings, we conclude that SRRM4 expression in castrate‐resistant tumors is highly correlated with NEPC and poor patient survival. It may serve as a diagnosis and prognosis biomarker of NEPC.

A novel mechanism of SRRM4 in promoting neuroendocrine prostate cancer development via a pluripotency gene network

Background Prostate adenocarcinoma (AdPC) cells can undergo lineage switching to neuroendocrine cells and develop into therapy-resistant neuroendocrine prostate cancer (NEPC). While genomic/epigenetic alterations are shown to induce neuroendocrine differentiation via an intermediate stem-like state, RNA splicing factor SRRM4 can transform AdPC cells into NEPC xenografts through a direct neuroendocrine transdifferentiation mechanism. Whether SRRM4 can also regulate a stem-cell gene network for NEPC development remains unclear. Methods Multiple AdPC cell models were transduced by lentiviral vectors encoding SRRM4. SRRM4-mediated RNA splicing and neuroendocrine differentiation of cells and xenografts were determined by qPCR, immunoblotting, and immunohistochemistry. Cell morphology, proliferation, and colony formation rates were also studied. SRRM4 transcriptome in the DU145 cell model was profiled by AmpliSeq and analyzed by gene enrichment studies. Findings SRRM4 induces an overall NEPC-specific RNA splicing program in multiple cell models but creates heterogeneous transcriptomes. SRRM4-transduced DU145 cells present the most dramatic neuronal morphological changes, accelerated cell proliferation, and enhanced resistance to apoptosis. The derived xenografts show classic phenotypes similar to clinical NEPC. Whole transcriptome analyses further reveal that SRRM4 induces a pluripotency gene network consisting of the stem-cell differentiation gene, SOX2. While SRRM4 overexpression enhances SOX2 expression in both time- and dose-dependent manners in DU145 cells, RNA depletion of SOX2 compromises SRRM4-mediated stimulation of pluripotency genes. More importantly, this SRRM4-SOX2 axis is present in a subset of NEPC patient cohorts, patient-derived xenografts, and clinically relevant transgenic mouse models. Interpretation We report a novel mechanism by which SRRM4 drives NEPC progression via a pluripotency gene network. Fund Canadian Institutes of Health Research, National Nature Science Foundation of China, and China Scholar Council.

Molecular model for neuroendocrine prostate cancer progression.

Prostate cancer (PCa) is the most common form of cancer in men in the developed world and the second leading cause of cancer‐related deaths. While advanced PCa is initially controlled with hormonal therapies targeting the androgen receptor (AR) pathway, recurrence occurs because of the emergence of lethal castration‐resistant PCa (CRPC). Despite newer AR pathway inhibitors that prolong survival, resistance still emerges, most often with rising PSA levels indicative of AR‐driven activity, but increasingly as non‐AR‐driven cancer. Treatment resistance mechanisms include AR‐signalling pathway alterations, AR‐signalling bypass mechanisms, and AR‐independent clonal evolution. The latter mechanism can lead to the emergence of neuroendocrine prostate cancer (NEPC), an aggressive lethal subtype of PCa. The incidence of treatment‐induced NEPC is rising because of the widespread use of more potent AR pathway inhibitors. This comprehensive review of major NEPC drivers and facilitators defines three coordinated processes contributing to NEPC progression. Specifically, castration‐resistant adenocarcinoma cells gain lineage plasticity under selective pressures of potent AR suppression to transform into AR‐independent tumour cells. In concert, neuroendocrine (NE)‐specific transdifferentiation factors induce NE lineage of these PCa cells, which, with support of increased proliferation factors, contributes to clonal expansion and tumour repopulation into NEPC. We examine the roles of each of the major NEPC contributors during the disease progression and identify potential therapeutic opportunities for targeted therapies.