PRMT5 promotes chemotherapy-induced neuroendocrine differentiation in NSCLC.

BACKGROUND: In response to therapeutic treatments, cancer cells can exhibit a variety of resistance phenotypes including neuroendocrine differentiation (NED). NED is a process by which cancer cells can transdifferentiate into neuroendocrine-like cells in response to treatments, and is now widely accepted as a key mechanism of acquired therapy resistance. Recent clinical evidence has suggested that non-small cell lung cancer (NSCLC) can also transform into small cell lung cancer (SCLC) in patients treated with EGFR inhibitors. However, whether chemotherapy induces NED to confer therapy resistance in NSCLC remains unknown. METHODS: We evaluated whether NSCLC cells can undergo NED in response to chemotherapeutic agents etoposide and cisplatin. By Knock-down of PRMT5 or pharmacological inhibition of PRMT5 to identify its role in the NED process. RESULTS: We observed that both etoposide and cisplatin can induce NED in multiple NSCLC cell lines. Mechanistically, we identified protein arginine methyltransferase 5 (PRMT5) as a critical mediator of chemotherapy-induced NED. Significantly, the knock-down of PRMT5 or pharmacological inhibition of PRMT5 suppressed the induction of NED and increased the sensitivity to chemotherapy. CONCLUSION: Taken together, our results suggest that targeting PRMT5 may be explored as a chemosensitization approach by inhibiting chemotherapy-induced NED.

JNJ-64619178 radiosensitizes and suppresses fractionated ionizing radiation-induced neuroendocrine differentiation (NED) in prostate cancer.

Background: Radiation therapy (RT) is a standard treatment regimen for locally advanced prostate cancer; however, its failure results in tumor recurrence, metastasis, and cancer-related death. The recurrence of cancer after radiotherapy is one of the major challenges in prostate cancer treatment. Despite overall cure rate of 93.3% initially, prostate cancer relapse in 20-30% patients after radiation therapy. Cancer cells acquire radioresistance upon fractionated ionizing radiation (FIR) treatment, eventually undergo neuroendocrine differentiation (NED) and transform into neuroendocrine-like cells, a mechanism involved in acquiring resistance to radiation therapy. Radiosensitizers are agents that inhibit the repair of radiation-induced DNA damage. Protein arginine methyltransferase 5 (PRMT5) gets upregulated upon ionizing radiation treatment and epigenetically activates DNA damage repair genes in prostate cancer cells. In this study, we targeted PRMT5 with JNJ-64619178 and assessed its effect on DNA damage repair gene activation, radiosensitization, and FIR-induced NED in prostate cancer. Methods: γH2AX foci analysis was performed to evaluate the DNA damage repair after radiation therapy. RT-qPCR and western blot were carried out to analyze the expression of DNA damage repair genes. Clonogenic assay was conducted to find out the surviving fraction after radiation therapy. NED was targeted with JNJ-64619178 in androgen receptor (AR) positive and negative prostate cancer cells undergoing FIR treatment. Results: JNJ-64619178 inhibits DNA damage repair in prostate cancer cells independent of their AR status. JNJ-64619178 impairs the repair of ionizing radiation-induced damaged DNA by transcriptionally inhibiting the DNA damage repair gene expression and radiosensitizes prostate, glioblastoma and lung cancer cell line. It targets NED induced by FIR in prostate cancer cells. Conclusion: JNJ-64619178 can radiosensitize and suppress NED induced by FIR in prostate cancer cells and can be a potential radiosensitizer for prostate cancer treatment.

Reprogramming landscape highlighted by dynamic transcriptomes in therapy-induced neuroendocrine differentiation.

Metastatic and locally advanced prostate cancer is treated by pharmacological targeting of androgen synthesis and androgen response via androgen signaling inhibitors (ASI), most of which target the androgen receptor (AR). However, ASI therapy invariably fails after 1-2 years. Emerging clinical evidence indicates that in response to ASI therapy, the AR-positive prostatic adenocarcinoma can transdifferentiate into AR-negative neuroendocrine prostate cancer (NEPC) in 17-25 % treated patients, likely through a process called neuroendocrine differentiation (NED). Despite high clinical incidence, the epigenetic pathways underlying NED and ASI therapy-induced NED remain unclear. By utilizing a combinatorial single cell and bulk mRNA sequencing workflow, we demonstrate in a time-resolved manner that following AR inhibition with enzalutamide, prostate cancer cells exhibit immediate loss of canonical AR signaling activity and simultaneous morphological change from epithelial to NE-like (NEL) morphology, followed by activation of specific neuroendocrine (NE)-associated transcriptional programs. Additionally, we observed that activation of NE-associated pathways occurs prior to complete repression of epithelial or canonical AR pathways, a phenomenon also observed clinically via heterogenous AR status in clinical samples. Our model indicates that, mechanistically, ASI therapy induces NED with initial morphological change followed by deactivation of canonical AR target genes and subsequent de-repression of NE-associated target genes, while retaining AR expression and transcriptional shift towards non-canonical AR activity. Coupled with scRNA-seq and CUT&RUN analysis, our model system can provide a platform for screening of potential therapeutic agents that may prevent ASI-induced NED or reverse the NED process.

Discovery and Biological Characterization of PRMT5:MEP50 Protein-Protein Interaction Inhibitors.

Protein arginine methyltransferase 5 (PRMT5) is a master epigenetic regulator and an extensively validated therapeutic target in multiple cancers. Notably, PRMT5 is the only PRMT that requires an obligate cofactor, methylosome protein 50 (MEP50), to function. We developed compound 17, a novel small-molecule PRMT5:MEP50 protein-protein interaction (PPI) inhibitor, after initial virtual screen hit identification and analogue refinement. Molecular docking indicated that compound 17 targets PRMT5:MEP50 PPI by displacing the MEP50 W54 burial into a hydrophobic pocket of the PRMT5 TIM barrel. In vitro analysis indicates IC50 < 500 nM for prostate and lung cancer cells with selective, specific inhibition of PRMT5:MEP50 substrate methylation and target gene expression, and RNA-seq analysis suggests that compound 17 may dysregulate TGF-ß signaling. Compound 17 provides a proof of concept in targeting PRMT5:MEP50 PPI, as opposed to catalytic targeting, as a novel mechanism of action and supports further preclinical development of inhibitors in this class.

Decoding regulatory associations of G-quadruplex with epigenetic and transcriptomic functional components.

G-quadruplex (G4) has been previously observed to be associated with gene expression. In this study, we performed integrative analysis on G4 multi-omics data from in-silicon prediction and ChIP-seq in human genome. Potential G4 sites were classified into three distinguished groups, such as one group of high-confidence G4-forming locations (G4-II) and groups only containing either ChIP-seq detected G4s (G4-I) or predicted G4 motif candidates (G4-III). We explored the associations of different-confidence G4 groups with other epigenetic regulatory elements, including CpG islands, chromatin status, enhancers, super-enhancers, G4 locations compared to the genes, and DNA methylation. Our elastic net regression model revealed that G4 structures could correlate with gene expression in two opposite ways depending on their locations to the genes as well as G4-forming DNA strand. Some transcription factors were identified to be over-represented with G4 emergence. The motif analysis discovered distinct consensus sequences enriched in the G4 feet, the flanking regions of two groups of G4s. We found high GC content in the feet of high-confidence G4s (G4-II) when compared to high TA content in solely predicted G4 feet of G4-III. Overall, we uncovered the comprehensive associations of G4 formations or predictions with other epigenetic and transcriptional elements which potentially coordinate gene transcription.

Advanced Functions Embedded in the Second Version of Database, Global Evaluation of SARS-CoV-2/hCoV-19 Sequences 2.

The Global Evaluation of SARS-CoV-2/hCoV-19 Sequences 2 (GESS v2 https://shiny.ph.iu.edu/GESS_v2/) is an updated version of GESS, which has offered a handy query platform to analyze single-nucleotide variants (SNVs) on millions of high coverages and high-quality severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) complete genomes provided by the Global Initiative on Sharing Avian Influenza Data (GISAID). Including the tools in the first version, the GESS v2 is embedded with new functions, which allow users to search SNVs, given the viral nucleotide or amino acid sequence. The GESS v2 helps users to identify SNVs or SARS-CoV-2 lineages enriched in countries of user's interest and show the migration path of a selected lineage on a world map during specific time periods chosen by the users. In addition, the GESS v2 can recognize the dynamic variations of newly emerging SNVs in each month to help users monitor SNVs, which will potentially become dominant soon. More importantly, multiple sets of analyzed results about SNVs can be downloaded directly from the GESS v2 by which users can conduct their own independent research. With these significant updates, the GESS v2 will continue to serve as a public open platform for researchers to explore SARS-CoV-2 evolutionary patterns from the perspectives of the prevalence and impact of SNVs.

Targeting Protein Arginine Methyltransferase 5 Suppresses Radiation-induced Neuroendocrine Differentiation and Sensitizes Prostate Cancer Cells to Radiation.

Prostate cancer remains the second leading cause of cancer death among American men. Radiotherapy is a potentially curative treatment for localized prostate cancer, and failure to control localized disease contributes to the majority of prostate cancer deaths. Neuroendocrine differentiation (NED) in prostate cancer, a process by which prostate adenocarcinoma cells transdifferentiate into neuroendocrine-like (NE-like) cells, is an emerging mechanism of resistance to cancer therapies and contributes to disease progression. NED also occurs in response to treatment to promote the development of treatment-induced neuroendocrine prostate cancer (NEPC), a highly aggressive and terminal stage disease. We previously demonstrated that by mimicking clinical radiotherapy protocol, fractionated ionizing radiation (FIR) induces prostate cancer cells to undergo NED in vitro and in vivo. Here, we performed transcriptomic analysis and confirmed that FIR-induced NE-like cells share some features of clinical NEPC, suggesting that FIR-induced NED represents a clinically relevant model. Furthermore, we demonstrated that protein arginine methyltransferase 5 (PRMT5), a master epigenetic regulator of the DNA damage response and a putative oncogene in prostate cancer, along with its cofactors pICln and MEP50, mediate FIR-induced NED. Knockdown of PRMT5, pICln, or MEP50 during FIR-induced NED and sensitized prostate cancer cells to radiation. Significantly, PRMT5 knockdown in prostate cancer xenograft tumors in mice during FIR prevented NED, enhanced tumor killing, significantly reduced and delayed tumor recurrence, and prolonged overall survival. Collectively, our results demonstrate that PRMT5 promotes FIR-induced NED and suggests that targeting PRMT5 may be a novel and effective radiosensitization approach for prostate cancer radiotherapy.

PRMT5: a putative oncogene and therapeutic target in prostate cancer.

Protein arginine methyltransferase 5 (PRMT5) was discovered two decades ago. The first decade focused on the biochemical characterization of PRMT5 as a regulator of many cellular processes in a healthy organism. However, over the past decade, evidence has accumulated to suggest that PRMT5 may function as an oncogene in multiple cancers via both epigenetic and non-epigenetic mechanisms. In this review, we focus on recent progress made in prostate cancer, including the role of PRMT5 in the androgen receptor (AR) expression and signaling and DNA damage response, particularly DNA double-strand break repair. We also discuss how PRMT5-interacting proteins that are considered PRMT5 cofactors may cooperate with PRMT5 to regulate PRMT5 activity and target gene expression, and how PRMT5 can interact with other epigenetic regulators implicated in prostate cancer development and progression. Finally, we suggest that targeting PRMT5 may be employed to develop multiple therapeutic approaches to enhance the treatment of prostate cancer.

Hyperexcitability and Pharmacological Responsiveness of Cortical Neurons Derived from Human iPSCs Carrying Epilepsy-Associated Sodium Channel Nav1.2-L1342P Genetic Variant.

With the wide adoption of genomic sequencing in children having seizures, an increasing number of SCN2A genetic variants have been revealed as genetic causes of epilepsy. Voltage-gated sodium channel Nav1.2, encoded by gene SCN2A, is predominantly expressed in the pyramidal excitatory neurons and supports action potential (AP) firing. One recurrent SCN2A genetic variant is L1342P, which was identified in multiple patients with epileptic encephalopathy and intractable seizures. However, the mechanism underlying L1342P-mediated seizures and the pharmacogenetics of this variant in human neurons remain unknown. To understand the core phenotypes of the L1342P variant in human neurons, we took advantage of a reference human-induced pluripotent stem cell (hiPSC) line from a male donor, in which L1342P was introduced by CRISPR/Cas9-mediated genome editing. Using patch-clamping and microelectrode array (MEA) recordings, we revealed that cortical neurons derived from hiPSCs carrying heterozygous L1342P variant have significantly increased intrinsic excitability, higher sodium current density, and enhanced bursting and synchronous network firing, suggesting hyperexcitability phenotypes. Interestingly, L1342P neuronal culture displayed a degree of resistance to the anticonvulsant medication phenytoin, which recapitulated aspects of clinical observation of patients carrying the L1342P variant. In contrast, phrixotoxin-3 (PTx3), a Nav1.2 isoform-specific blocker, can potently alleviate spontaneous and chemically-induced hyperexcitability of neurons carrying the L1342P variant. Our results reveal a possible pathogenic underpinning of Nav1.2-L1342P mediated epileptic seizures and demonstrate the utility of genome-edited hiPSCs as an in vitro platform to advance personalized phenotyping and drug discovery.SIGNIFICANCE STATEMENT A mounting number of SCN2A genetic variants have been identified from patients with epilepsy, but how SCN2A variants affect the function of human neurons contributing to seizures is still elusive. This study investigated the functional consequences of a recurring SCN2A variant (L1342P) using human iPSC-derived neurons and revealed both intrinsic and network hyperexcitability of neurons carrying a mutant Nav1.2 channel. Importantly, this study recapitulated elements of clinical observations of drug-resistant features of the L1342P variant, and provided a platform for in vitro drug testing. Our study sheds light on cellular mechanism of seizures resulting from a recurring Nav1.2 variant, and helps to advance personalized drug discovery to treat patients carrying pathogenic SCN2A variant.

Updated SARS-CoV-2 single nucleotide variants and mortality association.

By analyzing newly collected SARS-CoV-2 genomes and comparing them with our previous study about SARS-CoV-2 single nucleotide variants (SNVs) before June 2020, we found that the SNV clustering had changed remarkably since June 2020. Apart from that the group of SNVs became dominant, which is represented by two nonsynonymous mutations A23403G (S:D614G) and C14408T (ORF1ab:P4715L), a few emerging groups of SNVs were recognized with sharply increased monthly incidence ratios of up to 70% in November 2020. Further investigation revealed sets of SNVs specific to patients' ages and/or gender, or strongly associated with mortality. Our logistic regression model explored features contributing to mortality status, including three critical SNVs, G25088T(S:V1176F), T27484C (ORF7a:L31L), and T25A (upstream of ORF1ab), ages above 40 years old, and the male gender. The protein structure analysis indicated that the emerging subgroups of nonsynonymous SNVs and the mortality-related ones were located on the protein surface area. The clashes in protein structure introduced by these mutations might in turn affect the viral pathogenesis through the alteration of protein conformation, leading to a difference in transmission and virulence. Particularly, we explored the fact that nonsynonymous SNVs tended to occur in intrinsic disordered regions of Spike and ORF1ab to significantly increase hydrophobicity, suggesting a potential role in the change of protein folding related to immune evasion.

Loss of smarcad1a accelerates tumorigenesis of malignant peripheral nerve sheath tumors in zebrafish.

Malignant peripheral nerve sheath tumors (MPNSTs) are a type of sarcoma that generally originates from Schwann cells. The prognosis for this type of malignancy is relatively poor due to complicated genetic alterations and the lack of specific targeted therapy. Chromosome fragment 4q22-23 is frequently deleted in MPNSTs and other human tumors, suggesting tumor suppressor genes may reside in this region. Here, we provide evidence that SMARCAD1, a known chromatin remodeler, is a novel tumor suppressor gene located in 4q22-23. We identified two human homologous smarcad1 genes (smarcad1a and smarcad1b) in zebrafish, and both genes share overlapping expression patterns during embryonic development. We demonstrated that two smarcad1a loss-of-function mutants, sa1299 and p403, can accelerate MPNST tumorigenesis in the tp53 mutant background, suggesting smarcad1a is a bona fide tumor suppressor gene for MPNSTs. Moreover, we found that DNA double-strand break (DSB) repair might be compromised in both mutants compared to wildtype zebrafish, as indicated by pH2AX, a DNA DSB marker. In addition, both SMARCAD1 gene knockdown and overexpression in human cells were able to inhibit tumor growth and displayed similar DSB repair responses, suggesting proper SMARCAD1 gene expression level or gene dosage is critical for cell growth. Given that mutations of SMARCAD1 sensitize cells to poly ADP ribose polymerase inhibitors in yeast and the human U2OS osteosarcoma cell line, the identification of SMARCAD1 as a novel tumor suppressor gene might contribute to the development of new cancer therapies for MPNSTs.

GESS: a database of global evaluation of SARS-CoV-2/hCoV-19 sequences.

The COVID-19 outbreak has become a global emergency since December 2019. Analysis of SARS-CoV-2 sequences can uncover single nucleotide variants (SNVs) and corresponding evolution patterns. The Global Evaluation of SARS-CoV-2/hCoV-19 Sequences (GESS, https://wan-bioinfo.shinyapps.io/GESS/) is a resource to provide comprehensive analysis results based on tens of thousands of high-coverage and high-quality SARS-CoV-2 complete genomes. The database allows user to browse, search and download SNVs at any individual or multiple SARS-CoV-2 genomic positions, or within a chosen genomic region or protein, or in certain country/area of interest. GESS reveals geographical distributions of SNVs around the world and across the states of USA, while exhibiting time-dependent patterns for SNV occurrences which reflect development of SARS-CoV-2 genomes. For each month, the top 100 SNVs that were firstly identified world-widely can be retrieved. GESS also explores SNVs occurring simultaneously with specific SNVs of user's interests. Furthermore, the database can be of great help to calibrate mutation rates and identify conserved genome regions. Taken together, GESS is a powerful resource and tool to monitor SARS-CoV-2 migration and evolution according to featured genomic variations. It provides potential directive information for prevalence prediction, related public health policy making, and vaccine designs.

Protein Arginine Methyltransferase 5 Promotes pICln-Dependent Androgen Receptor Transcription in Castration-Resistant Prostate Cancer.

The majority of advanced prostate cancer therapies aim to inhibit androgen receptor (AR) signaling. However, AR reactivation inevitably drives disease progression to castration-resistant prostate cancer (CRPC). Here we demonstrate that protein arginine methyltransferase 5 (PRMT5) functions as an epigenetic activator of AR transcription in CRPC, requiring cooperation with a methylosome subunit pICln. In vitro and in xenograft tumors in mice, targeting PRMT5 or pICln suppressed growth of CRPC cells. Full-length AR and AR-V7 transcription activation required both PRMT5 and pICln but not MEP50. This activation of transcription was accompanied by PRMT5-mediated symmetric dimethylation of H4R3 at the proximal AR promoter. Further, knockdown of PRMT5 abolished the binding of pICln (but not vice versa) to the AR proximal promoter region, suggesting that PRMT5 recruits pICln to the AR promoter to activate AR transcription. Differential gene expression analysis in 22Rv1 cells confirmed that PRMT5 and pICln both regulate the androgen signaling pathway. In addition, PRMT5 and pICln protein expression positively correlated with AR and AR-V7 protein expression in CRPC tissues and their expression was highly correlated at the mRNA level across multiple publicly available CRPC datasets. Our results suggest that targeting PRMT5 or pICln may be explored as a novel therapy for CRPC treatment by suppressing expression of AR and AR splice variants to circumvent AR reactivation. SIGNIFICANCE: This study provides evidence that targeting PRMT5 can eliminate expression of AR and can be explored as a novel therapeutic approach to treat metastatic hormone-naïve and castration-resistant prostate cancer.

Genetic Spectrum and Distinct Evolution Patterns of SARS-CoV-2.

Four signature groups of frequently occurred single-nucleotide variants (SNVs) were identified in over twenty-eight thousand high-quality and high-coverage SARS-CoV-2 complete genome sequences, representing different viral strains. Some SNVs predominated but were mutually exclusively presented in patients from different countries and areas. These major SNV signatures exhibited distinguishable evolution patterns over time. A few hundred patients were detected with multiple viral strain-representing mutations simultaneously, which may stand for possible co-infection or potential homogenous recombination of SARS-CoV-2 in environment or within the viral host. Interestingly nucleotide substitutions among SARS-CoV-2 genomes tended to switch between bat RaTG13 coronavirus sequence and Wuhan-Hu-1 genome, indicating the higher genetic instability or tolerance of mutations on those sites or suggesting that major viral strains might exist between Wuhan-Hu-1 and RaTG13 coronavirus.

Genetic spectrum and distinct evolution patterns of SARS-CoV-2.

Four signature groups of frequently occurred single-nucleotide variants (SNVs) were identified in over twenty-eight thousand high-quality and high-coverage SARS-CoV-2 complete genome sequences, representing different viral strains. Some SNVs predominated but were mutually exclusively presented in patients from different countries and areas. These major SNV signatures exhibited distinguishable evolution patterns over time. A few hundred patients were detected with multiple viral strain-representing mutations simultaneously, which may stand for possible co-infection or potential homogenous recombination of SARS-CoV-2 in environment or within the viral host. Interestingly nucleotide substitutions among SARS-CoV-2 genomes tended to switch between bat RaTG13 coronavirus sequence and Wuhan-Hu-1 genome, indicating the higher genetic instability or tolerance of mutations on those sites or suggesting that major viral strains might exist between Wuhan-Hu-1 and RaTG13 coronavirus.

PRMT5-TRIM21 interaction regulates the senescence of osteosarcoma cells by targeting the TXNIP/p21 axis.

Osteosarcoma (OS) is the most common bone malignancy in adolescents and has poor clinical outcomes. Protein arginine methyltransferase 5 (PRMT5) has recently been shown to be aberrantly expressed in various cancers, yet its role in OS remains elusive. Here, we found that PRMT5 was overexpressed in OS and its overexpression predicted poor clinical outcomes. PRMT5 knockdown significantly triggered pronounced senescence in OS cells, as evidenced by the increase in senescence-associated ß-galactosidase (SA-ß-gal)-stained cells, induction of p21 expression, and upregulation of senescence-associated secretory phenotype (SASP) gene expression. In addition, we found that PRMT5 plays a key role in regulating DNA damaging agents-induced OS cell senescence, possibly, via affecting the repair of DNA damage. Furthermore, we found that TXNIP acts as a key factor mediating PRMT5 depletion-induced DNA damage and cellular senescence. Mechanistically, TRIM21, which interacts with PRMT5, was essential for the regulation of TXNIP/p21 expression. In summary, we propose a model in which PRMT5, by interaction with TRIM21, plays a key role in regulating the TXNIP/p21 axis during senescence in OS cells. The present findings suggest that PRMT5 overexpression in OS cells might confer resistance to chemotherapy and that targeting the PRMT5/TRIM21/TXNIP signaling may enhance the therapeutic efficacy in OS.

PRMT5 Cooperates with pICln to Function as a Master Epigenetic Activator of DNA Double-Strand Break Repair Genes.

DNA double-strand break (DSB) repair is critical for cell survival and genome integrity. Upon recognition of DSBs, repair proteins are transiently upregulated to facilitate repair through homologous recombination (HR) or non-homologous end joining (NHEJ). We present evidence that PRMT5 cooperates with pICln to function as a master epigenetic activator of DNA damage response (DDR) genes involved in HR, NHEJ, and G2 arrest (including RAD51, BRCA1, and BRCA2) to upregulate gene expression upon DNA damage. Contrary to the predominant role of PRMT5 as an epigenetic repressor, our results demonstrate that PRMT5 and pICln can activate gene expression, potentially independent of PRMT5's obligate cofactor MEP50. Targeting PRMT5 or pICln hinders repair of DSBs in multiple cancer cell lines, and both PRMT5 and pICln expression positively correlates with DDR genes across 32 clinical cancer datasets. Thus, targeting PRMT5 or pICln may be explored in combination with radiation or chemotherapy for cancer treatment.

Identification of Novel Adenylyl Cyclase 5 (AC5) Signaling Networks in D1 and D2 Medium Spiny Neurons using Bimolecular Fluorescence Complementation Screening.

Adenylyl cyclase type 5 (AC5), as the principal isoform expressed in striatal medium spiny neurons (MSNs), is essential for the integration of both stimulatory and inhibitory midbrain signals that initiate from dopaminergic G protein-coupled receptor (GPCR) activation. The spatial and temporal control of cAMP signaling is dependent upon the composition of local regulatory protein networks. However, there is little understanding of how adenylyl cyclase protein interaction networks adapt to the multifarious pressures of integrating acute versus chronic and inhibitory vs. stimulatory receptor signaling in striatal MSNs. Here, we presented the development of a novel bimolecular fluorescence complementation (BiFC)-based protein-protein interaction screening methodology to further identify and characterize elements important for homeostatic control of dopamine-modulated AC5 signaling in a neuronal model cell line and striatal MSNs. We identified two novel AC5 modulators: the protein phosphatase 2A (PP2A) catalytic subunit (PPP2CB) and the intracellular trafficking associated protein-NSF (N-ethylmaleimide-sensitive factor) attachment protein alpha (NAPA). The effects of genetic knockdown (KD) of each gene were evaluated in several cellular models, including D1- and D2-dopamine receptor-expressing MSNs from CAMPER mice. The knockdown of PPP2CB was associated with a reduction in acute and sensitized adenylyl cyclase activity, implicating PP2A is an important and persistent regulator of adenylyl cyclase activity. In contrast, the effects of NAPA knockdown were more nuanced and appeared to involve an activity-dependent protein interaction network. Taken together, these data represent a novel screening method and workflow for the identification and validation of adenylyl cyclase protein-protein interaction networks under diverse cAMP signaling paradigms.

Cholesterol Sulfotransferase SULT2B1b Modulates Sensitivity to Death Receptor Ligand TNFα in Castration-Resistant Prostate Cancer.

Cholesterol sulfotransferase, SULT2B1b, has been demonstrated to modulate both androgen receptor activity and cell growth properties. However, the mechanism(s) by which SULT2B1b alters these properties within prostate cancer cells has not been described. Furthermore, specific advantages of SULT2B1b expression in prostate cancer cells are not understood. In these studies, single-cell mRNA sequencing was conducted to compare the transcriptomes of SULT2B1b knockdown (KD) versus Control KD LNCaP cells. Over 2,000 differentially expressed genes were identified along with alterations in numerous canonical pathways, including the death receptor signaling pathway. The studies herein demonstrate that SULT2B1b KD increases TNFα expression in prostate cancer cells and results in NF-κB activation in a TNF-dependent manner. More importantly, SULT2B1b KD significantly enhances TNF-mediated apoptosis in both TNF-sensitive LNCaP cells and TNF-resistant C4-2 cells. Overexpression of SULT2B1b in LNCaP cells also decreases sensitivity to TNF-mediated cell death, suggesting that SULT2B1b modulates pathways dictating the TNF sensitivity capacity of prostate cancer cells. Probing human prostate cancer patient datasets further supports this work by providing evidence that SULT2B1b expression is inversely correlated with TNF-related genes, including TNF, CD40LG, FADD, and NFKB1. Together, these data provide evidence that SULT2B1b expression in prostate cancer cells enhances resistance to TNF and may provide a growth advantage. In addition, targeting SULT2B1b may induce an enhanced therapeutic response to TNF treatment in advanced prostate cancer. IMPLICATIONS: These data suggest that SULT2B1b expression enhances resistance to TNF and may promote prostate cancer.