Loss of LCMT1 and biased protein phosphatase 2A heterotrimerization drive prostate cancer progression and therapy resistance.

Loss of the tumor suppressive activity of the protein phosphatase 2A (PP2A) is associated with cancer, but the underlying molecular mechanisms are unclear. PP2A holoenzyme comprises a heterodimeric core, a scaffolding A subunit and a catalytic C subunit, and one of over 20 distinct substrate-directing regulatory B subunits. Methylation of the C subunit regulates PP2A heterotrimerization, affecting B subunit binding and substrate specificity. Here, we report that the leucine carboxy methyltransferase (LCMT1), which methylates the L309 residue of the C subunit, acts as a suppressor of androgen receptor (AR) addicted prostate cancer (PCa). Decreased methyl-PP2A-C levels in prostate tumors is associated with biochemical recurrence and metastasis. Silencing LCMT1 increases AR activity and promotes castration-resistant prostate cancer growth. LCMT1-dependent methyl-sensitive AB56αCme heterotrimers target AR and its critical coactivator MED1 for dephosphorylation, resulting in the eviction of the AR-MED1 complex from chromatin and loss of target gene expression. Mechanistically, LCMT1 is regulated by S6K1-mediated phosphorylation-induced degradation requiring the ß-TRCP, leading to acquired resistance to anti-androgens. Finally, feedforward stabilization of LCMT1 by small molecule activator of phosphatase (SMAP) results in attenuation of AR-signaling and tumor growth inhibition in anti-androgen refractory PCa. These findings highlight methyl-PP2A-C as a prognostic marker and that the loss of LCMT1 is a major determinant in AR-addicted PCa, suggesting therapeutic potential for AR degraders or PP2A modulators in prostate cancer treatment.

Complications and Peri-operative Characteristics of Flaps for Reconstruction of Defects after Ablation of Squamous Cell Carcinoma of the Floor of the Mouth.

OBJECTIVE: To determine the appropriate method to use to repair defects after ablation of squamous cell carcinoma (SCC) of the floor of the mouth (FOM). METHODS: A retrospective review of 119 patients who underwent surgical resections of SCC of the FOM and flap reconstructions was conducted. A Student t test was used to examine the statistical differences in operative time, length of hospital stay and complications among groups with different reconstructions. RESULTS: Advanced-stage patients were repaired with more free flaps than local pedicled flaps that provided more reconstructions for small-to-medium defects. The most common recipient complication was wound dehiscence, and patients in the anterolateral thigh flap group developed a greater number of overall recipient site complications compared with those in other groups. Patients undergoing local flap reconstructions had shorter operative times compared with those with free flap reconstructions. CONCLUSION: In contrast to a radial forearm free flap as a more appropriate reconstruction for defects involving the tongue, an anterolateral thigh flap was better suited for defects with dead spaces. A fibular flap was appropriate for massive complex defects involving the mandible, FOM and tongue. A pectoralis major musculocutaneous flap provided the last line of reconstruction for patients with relapsed SCC or high-risk factors for microsurgical reconstructions.

Oncofusion-driven de novo enhancer assembly promotes malignancy in Ewing sarcoma via aberrant expression of the stereociliary protein LOXHD1.

Ewing sarcoma (EwS) is a highly aggressive tumor of bone and soft tissues that mostly affects children and adolescents. The pathognomonic oncofusion EWSR1::FLI1 transcription factor drives EwS by orchestrating an oncogenic transcription program through de novo enhancers. By integrative analysis of thousands of transcriptomes representing pan-cancer cell lines, primary cancers, metastasis, and normal tissues, we identify a 32-gene signature (ESS32 [Ewing Sarcoma Specific 32]) that stratifies EwS from pan-cancer. Among the ESS32, LOXHD1, encoding a stereociliary protein, is the most highly expressed gene through an alternative transcription start site. Deletion or silencing of EWSR1::FLI1 bound upstream de novo enhancer results in loss of the LOXHD1 short isoform, altering EWSR1::FLI1 and HIF1α pathway genes and resulting in decreased proliferation/invasion of EwS cells. These observations implicate LOXHD1 as a biomarker and a determinant of EwS metastasis and suggest new avenues for developing LOXHD1-targeted drugs or cellular therapies for this deadly disease.

Targeting androgen regulation of TMPRSS2 and ACE2 as a therapeutic strategy to combat COVID-19.

Epidemiological data showing increased severity and mortality of COVID-19 in men suggests a potential role for androgen in SARS-CoV-2 infection. Here, we present evidence for the transcriptional regulation of SARS-CoV-2 host cell receptor ACE2 and TMPRSS2 by androgen in mouse and human cells. Additionally, we demonstrate the endogenous interaction between TMPRSS2 and ACE2 in human cells and validate ACE2 as a TMPRSS2 substrate. Furthermore, camostat-a TMPRSS2 inhibitor-blocked the cleavage of pseudotype SARS-CoV-2 surface Spike without disrupting TMPRSS2-ACE2 interaction, thus providing evidence for the first time of a direct role of TMPRSS2 in priming the SARS-CoV-2 Spike, required for viral fusion to the host cell. Importantly, androgen-deprivation, anti-androgens, or camostat attenuated the SARS-CoV-2 S-mediated cellular entry. Together, our data provide a strong rationale for clinical evaluations of TMPRSS2 inhibitors and androgen-deprivation therapy/androgen receptor antagonists alone or in combination with antiviral drugs as early as clinically possible to prevent COVID-19 progression.

Author Correction: LRIG1 is a pleiotropic androgen receptor-regulated feedback tumor suppressor in prostate cancer.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

LRIG1 is a pleiotropic androgen receptor-regulated feedback tumor suppressor in prostate cancer.

LRIG1 has been reported to be a tumor suppressor in gastrointestinal tract and epidermis. However, little is known about the expression, regulation and biological functions of LRIG1 in prostate cancer (PCa). We find that LRIG1 is overexpressed in PCa, but its expression correlates with better patient survival. Functional studies reveal strong tumor-suppressive functions of LRIG1 in both AR+ and AR- xenograft models, and transgenic expression of LRIG1 inhibits tumor development in Hi-Myc and TRAMP models. LRIG1 also inhibits castration-resistant PCa and exhibits therapeutic efficacy in pre-established tumors. We further show that 1) AR directly transactivates LRIG1 through binding to several AR-binding sites in LRIG1 locus, and 2) LRIG1 dampens ERBB expression in a cell type-dependent manner and inhibits ERBB2-driven tumor growth. Collectively, our study indicates that LRIG1 represents a pleiotropic AR-regulated feedback tumor suppressor that functions to restrict oncogenic signaling from AR, Myc, ERBBs, and, likely, other oncogenic drivers.

CDK7 Inhibition Suppresses Castration-Resistant Prostate Cancer through MED1 Inactivation.

Metastatic castration-resistant prostate cancer (CRPC) is a fatal disease, primarily resulting from the transcriptional addiction driven by androgen receptor (AR). First-line CRPC treatments typically target AR signaling, but are rapidly bypassed, resulting in only a modest survival benefit with antiandrogens. Therapeutic approaches that more effectively block the AR-transcriptional axis are urgently needed. Here, we investigated the molecular mechanism underlying the association between the transcriptional coactivator MED1 and AR as a vulnerability in AR-driven CRPC. MED1 undergoes CDK7-dependent phosphorylation at T1457 and physically engages AR at superenhancer sites, and is essential for AR-mediated transcription. In addition, a CDK7-specific inhibitor, THZ1, blunts AR-dependent neoplastic growth by blocking AR/MED1 corecruitment genome-wide, as well as reverses the hyperphosphorylated MED1-associated enzalutamide-resistant phenotype. In vivo, THZ1 induces tumor regression of AR-amplified human CRPC in a xenograft mouse model. Together, we demonstrate that CDK7 inhibition selectively targets MED1-mediated, AR-dependent oncogenic transcriptional amplification, thus representing a potential new approach for the treatment of CRPC. SIGNIFICANCE: Potent inhibition of AR signaling is critical to treat CRPC. This study uncovers a driver role for CDK7 in regulating AR-mediated transcription through phosphorylation of MED1, thus revealing a therapeutically targetable potential vulnerability in AR-addicted CRPC.See related commentary by Russo et al., p. 1490.This article is highlighted in the In This Issue feature, p. 1469.

Linking prostate cancer cell AR heterogeneity to distinct castration and enzalutamide responses.

Expression of androgen receptor (AR) in prostate cancer (PCa) is heterogeneous but the functional significance of AR heterogeneity remains unclear. Screening ~200 castration-resistant PCa (CRPC) cores and whole-mount sections (from 89 patients) reveals 3 AR expression patterns: nuclear (nuc-AR), mixed nuclear/cytoplasmic (nuc/cyto-AR), and low/no expression (AR-/lo). Xenograft modeling demonstrates that AR+ CRPC is enzalutamide-sensitive but AR-/lo CRPC is resistant. Genome editing-derived AR+ and AR-knockout LNCaP cell clones exhibit distinct biological and tumorigenic properties and contrasting responses to enzalutamide. RNA-Seq and biochemical analyses, coupled with experimental combinatorial therapy, identify BCL-2 as a critical therapeutic target and provide proof-of-concept therapeutic regimens for both AR+/hi and AR-/lo CRPC. Our study links AR expression heterogeneity to distinct castration/enzalutamide responses and has important implications in understanding the cellular basis of prostate tumor responses to AR-targeting therapies and in facilitating development of novel therapeutics to target AR-/lo PCa cells/clones.

MicroRNA-141 suppresses prostate cancer stem cells and metastasis by targeting a cohort of pro-metastasis genes.

MicroRNAs play important roles in regulating tumour development, progression and metastasis. Here we show that one of the miR-200 family members, miR-141, is under-expressed in several prostate cancer (PCa) stem/progenitor cell populations in both xenograft and primary patient tumours. Enforced expression of miR-141 in CD44+ and bulk PCa cells inhibits cancer stem cell properties including holoclone and sphere formation, as well as invasion, and suppresses tumour regeneration and metastasis. Moreover, miR-141 expression enforces a strong epithelial phenotype with a partial loss of mesenchymal phenotype. Whole-genome RNA sequencing uncovers novel miR-141-regulated molecular targets in PCa cells including the Rho GTPase family members (for example, CDC42, CDC42EP3, RAC1 and ARPC5) and stem cell molecules CD44 and EZH2, all of which are validated as direct and functionally relevant targets of miR-141. Our results suggest that miR-141 employs multiple mechanisms to obstruct tumour growth and metastasis.

Systematic dissection of phenotypic, functional, and tumorigenic heterogeneity of human prostate cancer cells.

Human cancers are heterogeneous containing stem-like cancer cells operationally defined as cancer stem cells (CSCs) that possess great tumor-initiating and long-term tumor-propagating properties. In this study, we systematically dissect the phenotypic, functional and tumorigenic heterogeneity in human prostate cancer (PCa) using xenograft models and >70 patient tumor samples. In the first part, we further investigate the PSA-/lo PCa cell population, which we have recently shown to harbor self-renewing long-term tumor-propagating cells and present several novel findings. We show that discordant AR and PSA expression in both untreated and castration-resistant PCa (CRPC) results in AR+PSA+, AR+PSA-, AR-PSA-, and AR-PSA+ subtypes of PCa cells that manifest differential sensitivities to therapeutics. We further demonstrate that castration leads to a great enrichment of PSA-/lo PCa cells in both xenograft tumors and CRPC samples and systemic androgen levels dynamically regulate the relative abundance of PSA+ versus PSA-/lo PCa cells that impacts the kinetics of tumor growth. We also present evidence that the PSA-/lo PCa cells possess distinct epigenetic profiles. As the PSA-/lo PCa cell population is heterogeneous, in the second part, we employ two PSA- (Du145 and PC3) and two PSA+ (LAPC9 and LAPC4) PCa models as well as patient tumor cells to further dissect the clonogenic and tumorigenic subsets. We report that different PCa models possess distinct tumorigenic subpopulations that both commonly and uniquely express important signaling pathways that could represent therapeutic targets. Our results have important implications in understanding PCa cell heterogeneity, response to clinical therapeutics, and cellular mechanisms underlying CRPC.

Androgen receptor and prostate cancer stem cells: biological mechanisms and clinical implications.

Prostate cancer (PCa) contains phenotypically and functionally distinct cells, and this cellular heterogeneity poses clinical challenges as the distinct cell types likely respond differently to various therapies. Clonal evolution, driven by genetic instability, and intraclonal cancer cell diversification, driven by cancer stem cells (CSCs), together create tumor cell heterogeneity. In this review, we first discuss PCa stem cells (PCSCs) and heterogeneity of androgen receptor (AR) expression in primary, metastatic, and treatment-failed PCa. Based on literature reports and our own studies, we hypothesize that, whereas PCSCs in primary and untreated tumors and models are mainly AR(-), PCSCs in CRPCs could be either AR(+) or AR(-/lo). We illustrate the potential mechanisms AR(+) and AR(-) PCSCs may employ to propagate PCa at the population level, mediate therapy resistance, and metastasize. As a result, targeting AR alone may not achieve long-lasting therapeutic efficacy. Elucidating the roles of AR and PCSCs should provide important clues to designing novel personalized combinatorial therapeutic protocols targeting both AR(+) and AR(-) PCa cells.