Identification of an anaplastic subtype of prostate cancer amenable to therapies targeting SP1 or translation elongation.

Histopathological heterogeneity is a hallmark of prostate cancer (PCa). Using spatial and parallel single-nucleus transcriptomics, we report an androgen receptor (AR)-positive but neuroendocrine-null primary PCa subtype with morphologic and molecular characteristics of small cell carcinoma. Such small cell-like PCa (SCLPC) is clinically aggressive with low AR, but high stemness and proliferation, activity. Molecular characterization prioritizes protein translation, represented by up-regulation of many ribosomal protein genes, and SP1, a transcriptional factor that drives SCLPC phenotype and overexpresses in castration-resistant PCa (CRPC), as two potential therapeutic targets in AR-indifferent CRPC. An SP1-specific inhibitor, plicamycin, effectively suppresses CRPC growth in vivo. Homoharringtonine, a Food And Drug Administration-approved translation elongation inhibitor, impedes CRPC progression in preclinical models and patients with CRPC. We construct an SCLPC-specific signature capable of stratifying patients for drug selectivity. Our studies reveal the existence of SCLPC in admixed PCa pathology, which may mediate tumor relapse, and establish SP1 and translation elongation as actionable therapeutic targets for CRPC.

A Stapled Peptide Inhibitor Targeting the Binding Interface of N6-Adenosine-Methyltransferase Subunits METTL3 and METTL14 for Cancer Therapy.

METTL3, a primary methyltransferase catalyzing the RNA N6-methyladenosine (m6A) modification, has been identified as an oncogene in several cancer types and thus nominated as a potentially effective target for therapeutic inhibition. However, current options using this strategy are limited. In this study, we targeted protein-protein interactions at the METTL3-METTL14 binding interface to inhibit complex formation and subsequent catalysis of the RNA m6A modification. Among candidate peptides, RM3 exhibited the highest anti-cancer potency, inhibiting METTL3 activity while also facilitating its proteasomal degradation. We then designed a stapled peptide inhibitor (RSM3) with enhanced peptide stability and formation of the α-helical secondary structure required for METTL3 interaction. Functional and transcriptomic analysis in vivo indicated that RSM3 induced upregulation of programmed cell death-related genes while inhibiting cancer-promoting signals. Furthermore, tumor growth was significantly suppressed while apoptosis was enhanced upon RSM3 treatment, accompanied by increased METTL3 degradation, and reduced global RNA methylation levels in two in vivo tumor models. This peptide inhibitor thus exploits a mechanism distinct from other small-molecule competitive inhibitors to inhibit oncogenic METTL3 activity. Our findings collectively highlight the potential of targeting METTL3 in cancer therapies through peptide-based inhibition of complex formation and proteolytic degradation.

An integrated ceRNA network identifies miR-375 as an upregulated miRNA playing a tumor suppressive role in aggressive prostate cancer.

Prostate cancer (PCa) remains a significant cause of morbidity and mortality among men worldwide. A number of genes have been implicated in prostate tumorigenesis, but the mechanisms underlying their dysregulation are still incompletely understood. Evidence has established the competing endogenous RNA (ceRNA) theory as a novel regulatory mechanism for post-transcriptional alterations. Yet, a comprehensive characterization of ceRNA network in PCa lacks. Here we utilize stringent in-silico methods to construct a large ceRNA network across different PCa stages, and provide experimental demonstration for the competing regulation among protumorigenic SEC23A, PHTF2, and their corresponding ceRNA pairs. Using machine learning, we establish a ceRNA-based signature (ceRNA_sig) predictive of androgen receptor (AR) activity, tumor aggressiveness, and patient outcomes. Importantly, we identify miR-375 as a key node in PCa ceRNA network, which is upregulated in PCa relative to normal tissues. Forced expression of miR-375 significantly inhibits, while its inhibition promotes, aggressive behaviors of both AR+ and AR- PCa cells in vitro and in vivo. Mechanistically, we show that miR-375 predominantly targets genes possessing oncogenic roles (e.g., proliferation, DNA repair, and metastasis), and thus release targets with tumor suppressive functions. This action model well clarifies why an upregulated miRNA plays a tumor suppressive role in PCa. Together, our study provides new insights into understanding of transcriptomic aberrations during PCa evolution, and nominates miR-375 as a potential therapeutic target for combating aggressive PCa.

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.

YY1 complex in M2 macrophage promotes prostate cancer progression by upregulating IL-6.

BACKGROUND: Tumor-associated macrophages are mainly polarized into the M2 phenotype, remodeling the tumor microenvironment and promoting tumor progression by secreting various cytokines. METHODS: Tissue microarray consisting of prostate cancer (PCa), normal prostate, and lymph node metastatic samples from patients with PCa were stained with Yin Yang 1 (YY1) and CD163. Transgenic mice overexpressing YY1 were constructed to observe PCa tumorigenesis. Furthermore, in vivo and in vitro experiments, including CRISPR-Cas9 knock-out, RNA sequencing, chromatin immunoprecipitation (ChIP) sequencing, and liquid-liquid phase separation (LLPS) assays, were performed to investigate the role and mechanism of YY1 in M2 macrophages and PCa tumor microenvironment. RESULTS: YY1 was highly expressed in M2 macrophages in PCa and was associated with poorer clinical outcomes. The proportion of tumor-infiltrated M2 macrophages increased in transgenic mice overexpressing YY1. In contrast, the proliferation and activity of anti-tumoral T lymphocytes were suppressed. Treatment targeting YY1 on M2 macrophages using an M2-targeting peptide-modified liposome carrier suppressed PCa cell lung metastasis and generated synergistic anti-tumoral effects with PD-1 blockade. IL-4/STAT6 pathway regulated YY1, and YY1 increased the macrophage-induced PCa progression by upregulating IL-6. Furthermore, by conducting H3K27ac-ChIP-seq in M2 macrophages and THP-1, we found that thousands of enhancers were gained during M2 macrophage polarization, and these M2-specific enhancers were enriched in YY1 ChIP-seq signals. In addition, an M2-specific IL-6 enhancer upregulated IL-6 expression through long-range chromatin interaction with IL-6 promoter in M2 macrophages. During M2 macrophage polarization, YY1 formed an LLPS, in which p300, p65, and CEBPB acted as transcriptional cofactors. CONCLUSIONS: Phase separation of the YY1 complex in M2 macrophages upregulated IL-6 by promoting IL-6 enhancer-promoter interactions, thereby increasing PCa progression.

Bone marrow-confined IL-6 signaling mediates the progression of myelodysplastic syndromes to acute myeloid leukemia.

Myelodysplastic syndromes (MDS) are age-related myeloid neoplasms with increased risk of progression to acute myeloid leukemia (AML). The mechanisms of transformation of MDS to AML are poorly understood, especially in relation to the aging microenvironment. We previously established an mDia1/miR-146a double knockout (DKO) mouse model phenocopying MDS. These mice develop age-related pancytopenia with oversecretion of proinflammatory cytokines. Here, we found that most of the DKO mice underwent leukemic transformation at 12-14 months of age. These mice showed myeloblast replacement of fibrotic bone marrow and widespread leukemic infiltration. Strikingly, depletion of IL-6 in these mice largely rescued the leukemic transformation and markedly extended survival. Single-cell RNA sequencing analyses revealed that DKO leukemic mice had increased monocytic blasts that were reduced with IL-6 knockout. We further revealed that the levels of surface and soluble IL-6 receptor (IL-6R) in the bone marrow were significantly increased in high-risk MDS patients. Similarly, IL-6R was also highly expressed in older DKO mice. Blocking of IL-6 signaling significantly ameliorated AML progression in the DKO model and clonogenicity of CD34-positive cells from MDS patients. Our study establishes a mouse model of progression of age-related MDS to AML and indicates the clinical significance of targeting IL-6 signaling in treating high-risk MDS.

A m6Avalue predictive of prostate cancer stemness, tumor immune landscape and immunotherapy response.

The molecular mechanisms underpinning prostate cancer (PCa) progression are incompletely understood, and precise stratification of aggressive primary PCa (pri-PCa) from indolent ones poses a major clinical challenge. Here, we comprehensively dissect, genomically and transcriptomically, the m6A (N 6-methyladenosine) pathway as a whole in PCa. Expression, but not the genomic alteration, repertoire of the full set of 24 m6A regulators at the population level successfully stratifies pri-PCa into three m6A clusters with distinct molecular and clinical features. These three m6A modification patterns closely correlate with androgen receptor signaling, stemness, proliferation and tumor immunogenicity of cancer cells, and stroma activity and immune landscape of tumor microenvironment (TME). We observe a discrepancy between a potentially higher neoantigen production and a deficiency in antigen presentation processes in aggressive PCa, offering insights into the failure of immunotherapy. Identification of PCa-specific m6A phenotype-associated genes provides a basis for construction of m6Avalue to measure m6A methylation patterns in individual patients. Tumors with lower m6Avalue are relatively indolent with abundant immune cell infiltration and stroma activity. Interestingly, m6Avalue separates PCa TME into fibrotic and nonfibrotic phenotypes (instead of previously reported immune-proficient or -desert phenotypes in other cancer types). Significantly, m6Avalue can be used to predict drug response and clinical immunotherapy efficacy in both castration-resistant PCa and other cancer types. Therefore, our study establishes m6A methylation modification pattern as a determinant in PCa progression via impacting cancer cell aggressiveness and TME remodeling.

Integrins regulate stemness in solid tumor: an emerging therapeutic target.

Integrins are the adhesion molecules and transmembrane receptors that consist of α and ß subunits. After binding to extracellular matrix components, integrins trigger intracellular signaling and regulate a wide spectrum of cellular functions, including cell survival, proliferation, differentiation and migration. Since the pattern of integrins expression is a key determinant of cell behavior in response to microenvironmental cues, deregulation of integrins caused by various mechanisms has been causally linked to cancer development and progression in several solid tumor types. In this review, we discuss the integrin signalosome with a highlight of a few key pro-oncogenic pathways elicited by integrins, and uncover the mutational and transcriptomic landscape of integrin-encoding genes across human cancers. In addition, we focus on the integrin-mediated control of cancer stem cell and tumor stemness in general, such as tumor initiation, epithelial plasticity, organotropic metastasis and drug resistance. With insights into how integrins contribute to the stem-like functions, we now gain better understanding of the integrin signalosome, which will greatly assist novel therapeutic development and more precise clinical decisions.

Genetically engineered oncolytic bacteria as drug delivery systems for targeted cancer theranostics.

Drug delivery systems based on genetically engineered oncolytic bacteria have properties that cannot be achieved by traditional therapeutic interventions. Thus, they have attracted considerable attention in cancer therapies. Attenuated bacteria can specifically target and actively penetrate tumor tissues and play an important role in cancer suppression as the "factories" of diverse anticancer drugs. Over the past decades, several bacterial strains including Salmonella and Clostridium have been shown to effectively retard tumor growth and metastasis, and thus improve survival in preclinical models or clinical cases. In this review, we summarize the unique properties of oncolytic bacteria and their anticancer mechanisms and highlight the particular advantages compared with traditional strategies. With the current research progress, we demonstrate the potential value of oncolytic bacteria-based drug delivery systems for clinical applications. In addition, we discuss novel strategies of cancer therapies integrating oncolytic bacteria, which will provide hope to further improve and standardize the current regimens in the near future.

RBM38 in cancer: role and mechanism.

Cancer is the second leading cause of death globally. Abnormity in gene expression regulation characterizes the trajectory of tumor development and progression. RNA-binding proteins (RBPs) are widely dysregulated, and thus implicated, in numerous human cancers. RBPs mainly regulate gene expression post-transcriptionally, but emerging studies suggest that many RBPs can impact transcription by acting on chromatin as transcription factors (TFs) or cofactors. Here, we review the evidence that RBM38, an intensively studied RBP, frequently plays a tumor-suppressive role in multiple human cancer types. Genetic studies in mice deficient in RBM38 on different p53 status also establish RBM38 as a tumor suppressor (TS). By uncovering a spectrum of transcripts bound by RBM38, we discuss the diversity in its mechanisms of action in distinct biological contexts. Examination of the genomic features and expression pattern of RBM38 in human tissues reveals that it is generally lost but rarely mutated, in cancers. By assessing future trends in the study of RBM38 in cancer, we signify the possibility of targeting RBM38 and its related pathways as therapeutic strategies against cancer.

The spliceosome as a new therapeutic vulnerability in aggressive prostate cancer.

Alternative splicing (AS) analysis across the entire spectrum of human prostate cancer evolution reveals the unexpected findings that intron retention is a hallmark of stemness and tumor aggressiveness, and androgen receptor controls a splicing program distinct from its transcriptional regulation. Importantly, twisted activity of the spliceosome causing abnormal AS landscape represents a therapeutic vulnerability in aggressive prostate cancer.

Intron retention is a hallmark and spliceosome represents a therapeutic vulnerability in aggressive prostate cancer.

The role of dysregulation of mRNA alternative splicing (AS) in the development and progression of solid tumors remains to be defined. Here we describe the first comprehensive AS landscape in the spectrum of human prostate cancer (PCa) evolution. We find that the severity of splicing dysregulation correlates with disease progression and establish intron retention as a hallmark of PCa stemness and aggressiveness. Systematic interrogation of 274 splicing-regulatory genes (SRGs) uncovers prevalent genomic copy number variations (CNVs), leading to mis-expression of ~68% of SRGs during PCa development and progression. Consequently, many SRGs are prognostic. Surprisingly, androgen receptor controls a splicing program distinct from its transcriptional regulation. The spliceosome modulator, E7107, reverses cancer aggressiveness and inhibits castration-resistant PCa (CRPC) in xenograft and autochthonous PCa models. Altogether, our studies establish aberrant AS landscape caused by dysregulated SRGs as a hallmark of PCa aggressiveness and the spliceosome as a therapeutic vulnerability for CRPC.

The DNA Methylation Status of Wnt and Tgfß Signals Is a Key Factor on Functional Regulation of Skeletal Muscle Satellite Cell Development.

DNA methylation is an important form of epigenetic regulation that can regulate the expression of genes and the development of tissues. Muscle satellite cells play an important role in skeletal muscle development and regeneration. Therefore, the DNA methylation status of genes in satellite cells is important in the regulation of the development of skeletal muscle. This study systematically investigated the changes of genome-wide DNA methylation in satellite cells during skeletal muscle development. According to the MeDIP-Seq data, 52,809-123,317 peaks were obtained for each sample, covering 0.70-1.79% of the genome. The number of reads and peaks was highest in the intron regions followed by the CDS regions. A total of 96,609 DMRs were identified between any two time points. Among them 6198 DMRs were annotated into the gene promoter regions, corresponding to 4726 DMGs. By combining the MeDIP-Seq and RNA-Seq data, a total of 202 overlap genes were obtained between DMGs and DEGs. GO and Pathway analysis revealed that the overlap genes were mainly involved in 128 biological processes and 23 pathways. Among the biological processes, terms related to regulation of cell proliferation and Wnt signaling pathway were significantly different. Gene-gene interaction analysis showed that Wnt5a, Wnt9a, and Tgfß1 were the key nodes in the network. Furthermore, the expression level of Wnt5a, Wnt9a, and Tgfß1 genes could be influenced by the methylation status of promoter region during skeletal muscle development. These results indicated that the Wnt and Tgfß signaling pathways may play an important role in functional regulation of satellite cells, and the DNA methylation status of Wnt and Tgfß signals is a key regulatory factor during skeletal muscle development. This study provided new insights into the effects of genome-wide methylation on the function of satellite cells.

Quantification of allelic differential expression using a simple Fluorescence primer PCR-RFLP-based method.

Allelic differential expression (ADE) is common in diploid organisms, and is often the key reason for specific phenotype variations. Thus, ADE detection is important for identification of major genes and causal mutations. To date, sensitive and simple methods to detect ADE are still lacking. In this study, we have developed an accurate, simple, and sensitive method, named fluorescence primer PCR-RFLP quantitative method (fPCR-RFLP), for ADE analysis. This method involves two rounds of PCR amplification using a pair of primers, one of which is double-labeled with an overhang 6-FAM. The two alleles are then separated by RFLP and quantified by fluorescence density. fPCR-RFLP could precisely distinguish ADE cross a range of 1- to 32-fold differences. Using this method, we verified PLAG1 and KIT, two candidate genes related to growth rate and immune response traits of pigs, to be ADE both at different developmental stages and in different tissues. Our data demonstrates that fPCR-RFLP is an accurate and sensitive method for detecting ADE on both DNA and RNA level. Therefore, this powerful tool provides a way to analyze mutations that cause ADE.

Gene expression profiling of porcine skeletal muscle satellite cells after poly(I:C) stimulation.

Porcine satellite cells (PSCs) play a vital role in the construction, development and self-renewal of skeletal muscle. In this study, PSCs were exposed to poly(I:C) stimulation to mimic viral infection during the proliferation and differentiation phases at 0, 12, 24 and 48 hours (h) of the stimulation. The untreated and treated PSCs were analyzed by the RNA-Seq technology. There were 88, 119, 104 and 95 genes being differentially expressed in 0 h vs 12 h treated, 12 h vs 24 h treated, 0 h vs 24 h treated and 24 h vs 48 h untreated comparison libraries, respectively. The GO terms analysis results showed that during the proliferation phase of treated PSCs, the up-regulated genes related to the immune system were highly expressed. In addition, the gene expressions associated with muscle structure development in response to growth factor emerged during the differentiation phase of untreated PSCs. The biological pathways associated with Influenza A, Toll-like receptor and chemokine signaling were revealed in PSCs following poly(I:C) stimulation. The differentially expressed genes were confirmed by quantitative real-time PCR. These findings expanded our understanding of gene expressions and signaling pathways about the infiltrated mechanism of the virus into PSCs.

Prostate Luminal Progenitor Cells in Development and Cancer.

Prostate cancer (PCa) has a predominantly luminal phenotype. Basal cells were previously identified as a cell of origin for PCa, but increasing evidence implicates luminal cells as a preferred cell of origin for PCa, as well as key drivers of tumor development and progression. Prostate luminal cells are understudied compared with basal cells. In this review, we describe the contribution of prostate luminal progenitor (LP) cells to luminal cell development and their role in prostate development, androgen-mediated regeneration of castrated prostate, and tumorigenesis. We also discuss the potential value of LP transcriptomics to identify new targets and therapies to treat aggressive PCa. Finally, we propose future research directions focusing on molecular mechanisms underlying LP cell biology and heterogeneity in normal and diseased prostate.

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.

Cancer stem cells: Regulation programs, immunological properties and immunotherapy.

It is becoming increasingly clear that virtually all types of human cancers harbor a small population of stem-like cancer cells (i.e., cancer stem cells, CSCs). These CSCs preexist in primary tumors, can self-renew and are more tolerant of standard treatments, such as antimitotic and molecularly targeted agents, most of which preferentially eliminate differentiated and proliferating cancer cells. CSCs are therefore postulated as the root of therapy resistance, relapse and metastasis. Aside from surgery, radiation, and chemotherapy, immunotherapy is now established as the fourth pillar in the therapeutic armamentarium for patients with cancer, especially late-stage and advanced cancers. A better understanding of CSC immunological properties should lead to development of novel immunologic approaches targeting CSCs, which, in turn, may help prevent tumor recurrence and eliminate residual diseases. Here, with a focus on CSCs in solid tumors, we review CSC regulation programs and recent transcriptomics-based immunological profiling data specific to CSCs. By highlighting CSC antigens that could potentially be immunogenic, we further discuss how CSCs can be targeted immunologically.

Histone 2B-GFP Label-Retaining Prostate Luminal Cells Possess Progenitor Cell Properties and Are Intrinsically Resistant to Castration.

The existence of slow-cycling luminal cells in the prostate has been suggested, but their identity and functional properties remain unknown. Using a bigenic mouse model to earmark, isolate, and characterize the quiescent stem-like cells, we identify a label-retaining cell (LRC) population in the luminal cell layer as luminal progenitors. Molecular and biological characterizations show that these luminal LRCs are significantly enriched in the mouse proximal prostate, exhibit relative dormancy, display bipotency in both in vitro and in vivo assays, and express a stem/progenitor gene signature with resemblance to aggressive prostate cancer. Importantly, these LRCs, compared with bulk luminal cells, maintain a lower level of androgen receptor (AR) expression and are less androgen dependent and also castration resistant in vivo. Finally, analysis of phenotypic markers reveals heterogeneity within the luminal progenitor cell pool. Our study establishes luminal LRCs as progenitors that may serve as a cellular origin for castration-resistant prostate cancer.