Prostate Cancer Progression Relies on the Mitotic Kinase Citron Kinase.

Prostate cancer remains the second leading cause of cancer death in men in Western cultures. A deeper understanding of the mechanisms by which prostate cancer cells divide to support tumor growth could help devise strategies to overcome treatment resistance and improve survival. Here, we identified that the mitotic AGC family protein kinase citron kinase (CIT) is a pivotal regulator of prostate cancer growth that mediates prostate cancer cell interphase progression. Increased CIT expression correlated with prostate cancer growth induction and aggressive prostate cancer progression, and CIT was overexpressed in prostate cancer compared with benign prostate tissue. CIT overexpression was controlled by an E2F2-Skp2-p27 signaling axis and conferred resistance to androgen-targeted treatment strategies. The effects of CIT relied entirely on its kinase activity. Conversely, CIT silencing inhibited the growth of cell lines and xenografts representing different stages of prostate cancer progression and treatment resistance but did not affect benign epithelial prostate cells or nonprostatic normal cells, indicating a potential therapeutic window for CIT inhibition. CIT kinase activity was identified as druggable and was potently inhibited by the multikinase inhibitor OTS-167, which decreased the proliferation of treatment-resistant prostate cancer cells and patient-derived organoids. Isolation of the in vivo CIT substrates identified proteins involved in diverse cellular functions ranging from proliferation to alternative splicing events that are enriched in treatment-resistant prostate cancer. These findings provide insights into the regulation of aggressive prostate cancer cell behavior by CIT and identify CIT as a functionally diverse and druggable driver of prostate cancer progression. SIGNIFICANCE: The poorly characterized protein kinase citron kinase is a therapeutic target in prostate cancer that drives tumor growth by regulating diverse substrates, which control several hallmarks of aggressive prostate cancer progression. See related commentary by Mishra et al., p. 4008.

Protein Kinase N1 control of androgen-responsive serum response factor action provides rationale for novel prostate cancer treatment strategy.

Sustained reliance on androgen receptor (AR) after failure of AR-targeting androgen deprivation therapy (ADT) prevents effective treatment of castration-recurrent (CR) prostate cancer (CaP). Interfering with the molecular machinery by which AR drives CaP progression may be an alternative therapeutic strategy but its feasibility remains to be tested. Here, we explore targeting the mechanism by which AR, via RhoA, conveys androgen-responsiveness to serum response factor (SRF), which controls aggressive CaP behavior and is maintained in CR-CaP. Following a siRNA screen and candidate gene approach, RNA-Seq studies confirmed that the RhoA effector Protein Kinase N1 (PKN1) transduces androgen-responsiveness to SRF. Androgen treatment induced SRF-PKN1 interaction, and PKN1 knockdown or overexpression severely impaired or stimulated, respectively, androgen regulation of SRF target genes. PKN1 overexpression occurred during clinical CR-CaP progression, and hastened CaP growth and shortened CR-CaP survival in orthotopic CaP xenografts. PKN1's effects on SRF relied on its kinase domain. The multikinase inhibitor lestaurtinib inhibited PKN1 action and preferentially affected androgen regulation of SRF over direct AR target genes. In a CR-CaP patient-derived xenograft, expression of SRF target genes was maintained while AR target gene expression declined and proliferative gene expression increased. PKN1 inhibition decreased viability of CaP cells before and after ADT. In patient-derived CaP explants, lestaurtinib increased AR target gene expression but did not significantly alter SRF target gene or proliferative gene expression. These results provide proof-of-principle for selective forms of ADT that preferentially target different fractions of AR's transcriptional output to inhibit CaP growth.

A comprehensive analysis of coregulator recruitment, androgen receptor function and gene expression in prostate cancer.

Standard treatment for metastatic prostate cancer (CaP) prevents ligand-activation of androgen receptor (AR). Despite initial remission, CaP progresses while relying on AR. AR transcriptional output controls CaP behavior and is an alternative therapeutic target, but its molecular regulation is poorly understood. Here, we show that action of activated AR partitions into fractions that are controlled preferentially by different coregulators. In a 452-AR-target gene panel, each of 18 clinically relevant coregulators mediates androgen-responsiveness of 0-57% genes and acts as a coactivator or corepressor in a gene-specific manner. Selectivity in coregulator-dependent AR action is reflected in differential AR binding site composition and involvement with CaP biology and progression. Isolation of a novel transcriptional mechanism in which WDR77 unites the actions of AR and p53, the major genomic drivers of lethal CaP, to control cell cycle progression provides proof-of-principle for treatment via selective interference with AR action by exploiting AR dependence on coregulators.

Intratumoral and Intertumoral Genomic Heterogeneity of Multifocal Localized Prostate Cancer Impacts Molecular Classifications and Genomic Prognosticators.

BACKGROUND: Next-generation sequencing is revealing genomic heterogeneity in localized prostate cancer (CaP). Incomplete sampling of CaP multiclonality has limited the implications for molecular subtyping, stratification, and systemic treatment. OBJECTIVE: To determine the impact of genomic and transcriptomic diversity within and among intraprostatic CaP foci on CaP molecular taxonomy, predictors of progression, and actionable therapeutic targets. DESIGN, SETTING, AND PARTICIPANTS: Four consecutive patients with clinically localized National Comprehensive Cancer Network intermediate- or high-risk CaP who did not receive neoadjuvant therapy underwent radical prostatectomy at Roswell Park Cancer Institute in June-July 2014. Presurgical information on CaP content and a customized tissue procurement procedure were used to isolate nonmicroscopic and noncontiguous CaP foci in radical prostatectomy specimens. Three cores were obtained from the index lesion and one core from smaller lesions. RNA and DNA were extracted simultaneously from 26 cores with ≥90% CaP content and analyzed using whole-exome sequencing, single-nucleotide polymorphism arrays, and RNA sequencing. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Somatic mutations, copy number alternations, gene expression, gene fusions, and phylogeny were defined. The impact of genomic alterations on CaP molecular classification, gene sets measured in Oncotype DX, Prolaris, and Decipher assays, and androgen receptor activity among CaP cores was determined. RESULTS AND LIMITATIONS: There was considerable variability in genomic alterations among CaP cores, and between RNA- and DNA-based platforms. Heterogeneity was found in molecular grouping of individual CaP foci and the activity of gene sets underlying the assays for risk stratification and androgen receptor activity, and was validated in independent genomic data sets. Determination of the implications for clinical decision-making requires follow-up studies. CONCLUSIONS: Genomic make-up varies widely among CaP foci, so care should be taken when making treatment decisions based on a single biopsy or index lesions. PATIENT SUMMARY: We examined the molecular composition of individual cancers in a patient's prostate. We found a lot of genetic diversity among these cancers, and concluded that information from a single cancer biopsy is not sufficient to guide treatment decisions.

GRM1 is An Androgen-Regulated Gene and its Expression Correlates with Prostate Cancer Progression in Pre-Clinical Models.

PURPOSE: We recently demonstrated that glutamate receptor GRM1 was expressed at high levels in castration-resistant prostate cancer (CR-PCa) tissues and cells. Herein, we determined the relationship between GRM1 and AR, PSA, and tumor growth, remission, and recurrence in preclinical PCa models. The effect of alterations in GRM1 expression was also investigated on PCa cell growth, migration and invasion. EXPERIMENTAL DESIGN: We used quantitative gene expression and immunohistochemistry to define the temporal association between GRM1 expression and AR, PSA, and tumor growth during CR progression in CWR22 (n = 59) and LuCaP 35 (n = 12) PCa xenografts. The effect of alterations in GRM1 expression levels on growth, migration, and invasion was investigated in GRM1-overexpressed or -silenced PCa cell lines. The effect of DHT on GRM1 expression was determined in the presence or absence of the antiandrogen bicalutamide. RESULTS: We found that GRM1 transcript and tissue expression directly correlated with growth and AR and PSA expression in hormone-sensitive (HS), castrated, and CR tumor xenografts. GRM1 overexpression or silencing directly correlated with PCa cell proliferation, migration, and invasion. DHT increased GRM1 expression via an AR-dependent manner in HS- and CR-PCa cell lines. CONCLUSIONS: This is a first report of GRM1 as an androgen and AR-target gene. GRM1 expression directly correlated with tumor growth, regression, and recurrence and may contribute to CR-progression of PCa in preclinical models. Further studies are needed to define the utility of GRM1 as a druggable target or biomarker for PCa.

Characterization of fibroblast-free CWR-R1ca castration-recurrent prostate cancer cell line.

BACKGROUND: The previously established CWR-R1 cell line has been used as an in vitro model representing castration-recurrent prostate cancer. Microscopic observation of subconfluent cells demonstrated two distinct cellular morphologies: polygonal closely aggregated epithelial cells surrounded by bipolar fibroblastic cells with long processes. This study sought to establish and characterize a fibroblast-free derivative of the CWR-R1 cell line. METHODS: The CWR-R1ca cell line was established from CWR-R1 cells by removing fibroblasts using multiple cycles of short-term trypsinization, cloning, and pooling single-cell colonies. Authentication of fibroblast-free CWR-R1ca cells was demonstrated by analyzing the expression of cytodifferentiation and prostate-associated markers, DNA and cytogenetic profiling, and growth pattern in the absence or presence of androgen. RESULTS: CWR-R1ca is an androgen-sensitive cell line that expresses the androgen receptor (AR) and its splice variant 7 and the luminal epithelia markers, CK-8, CK-18, and c-Met. CWR-R1fb fibroblasts isolated from CWR-R1 cells express AR, hepatocyte growth factor-α, and mouse ß-actin but not AR-V7 or epithelial markers. Cytogenetic analysis of CWR-R1ca cells revealed a hyperdiploid male with numerical gains in chromosomes 1, 7, 8, 10, 11, and 12, deletion of one chromosome 2 allele, structural abnormalities that include der(1)t(1:4), der(4)t(2:4), der(10)t(4:10), and an unbalanced reciprocal translocation between chromosome 6 and 14. DNA-profiling revealed that CWR-R1ca cells had significant short-tandem repeat marker homology with CWR22Pc and CWR22Rv1 cell lines, which indicated lineage derivation from CWR22 prostate cancer xenografts. CWR-R1ca cells were responsive to the growth stimulatory effects of dihydrotestosterone (DHT) in the femtomolar range. CONCLUSION: This study establishes CWR-R1ca cells as a fibroblast-free derivative of the castration-recurrent CWR-R1 cell line. Prostate 76:1067-1077, 2016. © 2016 Wiley Periodicals, Inc.

Regulators of Androgen Action Resource: a one-stop shop for the comprehensive study of androgen receptor action.

Androgen receptor (AR) is a ligand-activated transcription factor that is the main target for treatment of non-organ-confined prostate cancer (CaP). Failure of life-prolonging AR-targeting androgen deprivation therapy is due to flexibility in steroidogenic pathways that control intracrine androgen levels and variability in the AR transcriptional output. Androgen biosynthesis enzymes, androgen transporters and AR-associated coregulators are attractive novel CaP treatment targets. These proteins, however, are characterized by multiple transcript variants and isoforms, are subject to genomic alterations, and are differentially expressed among CaPs. Determining their therapeutic potential requires evaluation of extensive, diverse datasets that are dispersed over multiple databases, websites and literature reports. Mining and integrating these datasets are cumbersome, time-consuming tasks and provide only snapshots of relevant information. To overcome this impediment to effective, efficient study of AR and potential drug targets, we developed the Regulators of Androgen Action Resource (RAAR), a non-redundant, curated and user-friendly searchable web interface. RAAR centralizes information on gene function, clinical relevance, and resources for 55 genes that encode proteins involved in biosynthesis, metabolism and transport of androgens and for 274 AR-associated coregulator genes. Data in RAAR are organized in two levels: (i) Information pertaining to production of androgens is contained in a 'pre-receptor level' database, and coregulator gene information is provided in a 'post-receptor level' database, and (ii) an 'other resources' database contains links to additional databases that are complementary to and useful to pursue further the information provided in RAAR. For each of its 329 entries, RAAR provides access to more than 20 well-curated publicly available databases, and thus, access to thousands of data points. Hyperlinks provide direct access to gene-specific entries in the respective database(s). RAAR is a novel, freely available resource that provides fast, reliable and easy access to integrated information that is needed to develop alternative CaP therapies. Database URL: http://www.lerner.ccf.org/cancerbio/heemers/RAAR/search/.

Tryptophan Scanning Reveals Dense Packing of Connexin Transmembrane Domains in Gap Junction Channels Composed of Connexin32.

Tryptophan was substituted for residues in all four transmembrane domains of connexin32. Function was assayed using dual cell two-electrode voltage clamp after expression in Xenopus oocytes. Tryptophan substitution was poorly tolerated in all domains, with the greatest impact in TM1 and TM4. For instance, in TM1, 15 substitutions were made, six abolished coupling and five others significantly reduced function. Only TM2 and TM3 included a distinct helical face that lacked sensitivity to tryptophan substitution. Results were visualized on a comparative model of Cx32 hemichannel. In this model, a region midway through the membrane appears highly sensitive to tryptophan substitution and includes residues Arg-32, Ile-33, Met-34, and Val-35. In the modeled channel, pore-facing regions of TM1 and TM2 were highly sensitive to tryptophan substitution, whereas the lipid-facing regions of TM3 and TM4 were variably tolerant. Residues facing a putative intracellular water pocket (the IC pocket) were also highly sensitive to tryptophan substitution. Although future studies will be required to separate trafficking-defective mutants from those that alter channel function, a subset of interactions important for voltage gating was identified. Interactions important for voltage gating occurred mainly in the mid-region of the channel and focused on TM1. To determine whether results could be extrapolated to other connexins, TM1 of Cx43 was scanned revealing similar but not identical sensitivity to TM1 of Cx32.

Analysis of trafficking, stability and function of human connexin 26 gap junction channels with deafness-causing mutations in the fourth transmembrane helix.

Human Connexin26 gene mutations cause hearing loss. These hereditary mutations are the leading cause of childhood deafness worldwide. Mutations in gap junction proteins (connexins) can impair intercellular communication by eliminating protein synthesis, mis-trafficking, or inducing channels that fail to dock or have aberrant function. We previously identified a new class of mutants that form non-functional gap junction channels and hemichannels (connexons) by disrupting packing and inter-helix interactions. Here we analyzed fourteen point mutations in the fourth transmembrane helix of connexin26 (Cx26) that cause non-syndromic hearing loss. Eight mutations caused mis-trafficking (K188R, F191L, V198M, S199F, G200R, I203K, L205P, T208P). Of the remaining six that formed gap junctions in mammalian cells, M195T and A197S formed stable hemichannels after isolation with a baculovirus/Sf9 protein purification system, while C202F, I203T, L205V and N206S formed hemichannels with varying degrees of instability. The function of all six gap junction-forming mutants was further assessed through measurement of dye coupling in mammalian cells and junctional conductance in paired Xenopus oocytes. Dye coupling between cell pairs was reduced by varying degrees for all six mutants. In homotypic oocyte pairings, only A197S induced measurable conductance. In heterotypic pairings with wild-type Cx26, five of the six mutants formed functional gap junction channels, albeit with reduced efficiency. None of the mutants displayed significant alterations in sensitivity to transjunctional voltage or induced conductive hemichannels in single oocytes. Intra-hemichannel interactions between mutant and wild-type proteins were assessed in rescue experiments using baculovirus expression in Sf9 insect cells. Of the four unstable mutations (C202F, I203T, L205V, N206S) only C202F and N206S formed stable hemichannels when co-expressed with wild-type Cx26. Stable M195T hemichannels displayed an increased tendency to aggregate. Thus, mutations in TM4 cause a range of phenotypes of dysfunctional gap junction channels that are discussed within the context of the X-ray crystallographic structure.

Tryptophan scanning mutagenesis of the first transmembrane domain of the innexin Shaking-B(Lethal).

The channel proteins of gap junctions are encoded by two distinct gene families, connexins, which are exclusive to chordates, and innexins/pannexins, which are found throughout the animal kingdom. Although the relationship between the primary structure and function of the vertebrate connexins has been relatively well studied, there are, to our knowledge, no structure-function analyses of invertebrate innexins. In the first such study, we have used tryptophan scanning to probe the first transmembrane domain (M1) of the Drosophila innexin Shaking-B(Lethal), which is a component of rectifying electrical synapses in the Giant Fiber escape neural circuit. Tryptophan was substituted sequentially for 16 amino acids within M1 of Shaking-B(Lethal). Tryptophan insertion at every fourth residue (H27, T31, L35, and S39) disrupted gap junction function. The distribution of these sites is consistent with helical secondary structure and identifies the face of M1 involved in helix-helix interactions. Tryptophan substitution at several sites in M1 altered channel properties in a variety of ways. Changes in sensitivity to transjunctional voltage (Vj) were common and one mutation (S39W) induced sensitivity to transmembrane voltage (Vm). In addition, several mutations induced hemichannel activity. These changes are similar to those observed after substitutions within the transmembrane domains of connexins.