Peptide degrader-based targeting of METTL3/14 improves immunotherapy response in cutaneous melanoma.

METTL3 has emerged as a promising therapeutic target in cancer treatment, although its oncogenic functions in melanoma development and potential for therapeutic targeting drug have not been fully explored. In this study, we define the oncogenic role of METTL3 in melanoma development and progression. Building on this insight, we examine our recently designed peptide inhibitor RM3, which targets the binding interface of METTL3/14 complex for disruption and subsequent ubiquitin-mediated proteasomal degradation via the E3 ligase STUB1. RM3 treatment reduces proliferation, migration, and invasion, and induces apoptosis in melanoma cells in vitro and in vivo. Subsequent transcriptomic analysis identified changes in immuno-related genes following RM3-mediated suppression of METTL3/14 N6-methyladenosine (m6A) methyltransferase activity, suggesting a potential for interaction with immunotherapy. A combination treatment of RM3 with anti-PD-1 antibody results in significantly higher beneficial tumor response in vivo, with a good safety profile. Collectively, these findings not only delineate the oncogenic role of METTL3 in melanoma but also showcase RM3, acting as a peptide degrader, as a novel and promising strategy for melanoma treatment.

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.

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.

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.

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.

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.

Inhibition of PRMT5 moderately suppresses prostate cancer growth in vivo but enhances its response to immunotherapy.

Protein arginine methylation is a common post-translational modification (PTM) catalyzed by nine protein arginine methyltransferases (PRMTs). As the major symmetric arginine methyltransferase that methylates both histone and non-histone substrates, PRMT5 plays key roles in a number of biological processes critical for development and tumorigenesis. PRMT5 overexpression has been reported in multiple cancer types including prostate cancer (PCa), but the exact biological and mechanistic understanding of PRMT5 in aggressive PCa remains ill-defined. Here, we show that PRMT5 is upregulated in PCa, correlates with worse patient survival, promotes corrupted RNA splicing, and functionally cooperates with an array of pro-tumorigenic pathways to enhance oncogenesis. PRMT5 inhibition via either genetic knockdown or pharmacological inhibition reduces stemness with paralleled differentiation and arrests cell cycle progression without causing appreciable apoptosis. Strikingly, the severity of antitumor effect of PRMT5 inhibition correlates with disease aggressiveness, with AR+ PCa being less affected. Molecular characterization pinpoints MYC, but not (or at least to a lesser degree) AR, as the main partner of PRMT5 to form a positive feedback loop to exacerbate malignancy in both AR+ and AR- PCa cells. Inspired by the surprising finding that PRMT5 negatively correlates with tumor immune infiltration and transcriptionally suppresses an immune-gene program, we further show that although PRMT5 inhibitor (PRMT5i) EPZ015666 or anti-PD-1 immunotherapy alone exhibits limited antitumor effects, combination of PRMT5i with anti-PD-1 displays superior efficacy in inhibiting castration-resistant PCa (CRPC) in vivo. Finally, to expand the potential use of PRMT5i through a synthetic lethality concept, we also perform a global CRISPR/Cas9 knockout screen to unravel that many clinical-grade drugs of known oncogenic pathways can be repurposed to target CRPC when used in combination with PRMT5i at low doses. Collectively, our findings establish a rationale to exploit PRMT5i in combination with immunotherapy or other targeted therapies to treat aggressive PCa.

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.

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.

Arginine and glutamate-rich 1 (ARGLU1) interacts with mediator subunit 1 (MED1) and is required for estrogen receptor-mediated gene transcription and breast cancer cell growth.

Estrogen receptor is a nuclear receptor superfamily member of transcriptional activators that regulate gene expression by recruiting diverese transcriptional coregulators. The Mediator complex is a central transcriptional coactivator complex that acts as a bridge between transcriptional activators and RNA polymerase II. MED1 (Mediator subunit 1) is the key Mediator subunit that directly interacts with estrogen receptor to mediate its functions both in vitro and in vivo. Interestingly, our previous biochemical analyses indicated that MED1 exists only in a subpopulation of the Mediator complex that is enriched with a number of distinct Mediator subunits and RNA polymerase II. Here, we report ARGLU1 as a MED1/Mediator-associated protein. We found that ARGLU1 (arginine and glutamate rich 1) not only colocalizes with MED1 in the nucleus, but also directly interacts with a far C-terminal region of MED1. Reporter assays indicate that ARGLU1 is able to cooperate with MED1 to regulate estrogen receptor-mediated gene transcription. Importantly, ARGLU1 is recruited, in a ligand-dependent manner, to endogenous estrogen receptor target gene promoters and is required for their expression. Furthermore, by ChIP-reChIP assay, we confirm that ARGLU1 and MED1 colocalize on the same estrogen receptor target gene promoter upon estrogen induction. Moreover, we found that depletion of ARGLU1 significantly impairs the growth, as well as anchorage-dependent and -independent colony formation of breast cancer cells. Taken together, these results establish ARGLU1 as a new MED1-interacting protein required for estrogen-dependent gene transcription and breast cancer cell growth.

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.

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.

Regulation of maternal gene expression by MEK/MAPK and MPF signaling in porcine oocytes during in vitro meiotic maturation.

Mitogen-activated protein kinase (MAPK) and maturation/M phase promoting factor (MPF) play crucial roles in oocyte meiotic maturation in mammals. However, the underlying molecular mechanisms have not been addressed. In this study, the effects of the MEK/MAPK pathway inhibitor U0126 and the MPF inhibitor roscovitine on meiotic maturation and maternal gene expression in pig cumulus-oocyte complexes (COCs) and denuded oocytes (DOs) were investigated. Both inhibitors can reversibly block the resumption of meiosis in pig oocytes. COCs or DOs initially cultured in drug-free medium for 24 h and then transferred to medium containing U0126 showed normal progress to the Metaphase II stage (MII); (90.38 vs. 92.16% control group). In contrast, roscovitine treatment from 24 to 44 h significantly inhibited maturation of COCs and DOs. To explore the underlying molecular mechanisms, expression patterns and polyadenylation states of five representative maternal transcripts, cyclin B1, Cdc2, C-mos, GDF9 and BMP15, were examined by real-time PCR and poly(A)-test PCR (PAT assay). U0126 treatment resulted in aberrant expression of Cdc2 and GDF9, while roscovitine significantly maintained all five maternal transcripts at very high levels in treated COCs compared with the control group. The polyadenylation of these mRNAs increased as well. Furthermore, the experiments were repeated in DOs, and the results also indicated that both Cdc2 and GDF9 showed significantly higher expression in both mRNA and polyadenylation levels in the drug treatment groups. Together, these results provide the first demonstration in a mammalian system that MAPK and MPF play important roles in regulation of maternal gene expression during oocyte maturation.

Ndc80 regulates meiotic spindle organization, chromosome alignment, and cell cycle progression in mouse oocytes.

Ndc80 (called Hec1 in human), the core component of the Ndc80 complex, is involved in regulation of both kinetochore-microtubule interactions and the spindle assembly checkpoint in mitosis; however, its role in meiosis remains unclear. Here, we report Ndc80 expression, localization, and possible functions in mouse oocyte meiosis. Ndc80 mRNA levels gradually increased during meiosis. Immunofluorescent staining showed that Ndc80 was restricted to the germinal vesicle and associated with spindle microtubules from the Pro-MI to MII stages. Ndc80 was localized on microtubules and asters in the cytoplasm after taxol treatment, while Ndc80 staining was diffuse after disruption of microtubules by nocodazole treatment, confirming its microtubule localization. Disruption of Ndc80 function by either siRNA injection or antibody injection resulted in severe chromosome misalignment, spindle disruption, and precocious polar body extrusion. Our data show a unique localization pattern of Ndc80 in mouse oocytes and suggest that Ndc80 may be required for chromosome alignment and spindle organization, and may regulate spindle checkpoint activity during mouse oocyte meiosis.

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.

Molecular characterization and polyadenylation-regulated expression of cyclin B1 and Cdc2 in porcine oocytes and early parthenotes.

Meiotic maturation of mammalian oocytes is controlled by the maturation/M-phase promotion factor (MPF), a complex of Cdc2 kinase and cyclin B protein. To better understand the molecular mechanism of oocyte maturation, we characterized porcine cyclin B1 and Cdc2 genes, both of which are widely expressed in pig tissues. We further analyzed their expression profiles during in vitro maturation of pig oocyte and early embryonic development at both the mRNA and protein level. Two isoforms of cyclin B1, comprising the same open reading frame but differing in 3'-UTR length, were identified. Cyclin B1 transcripts was up-regulated after 30 hr of maturation, while Cdc2 mRNA levels were unchanged during maturation except for a sharp decline at 44 hr. Cyclin B1 protein synthesis increased with oocyte maturation. Cdc2 protein expression was relatively low during 0-18 hr, followed by a higher level of expression up to 44 hr of maturation. Poly(A)-test PCR clearly revealed that both cyclin B1 isoforms underwent cytoplasmic polyadenylation starting around 18-24 hr during maturation, while a substantial de-adenylation and degradation of Cdc2 isoforms were observed in metaphase II oocytes and during embryo development after parthenogenetic activation. Porcine MII oocytes derived from small follicles (< or = 3 mm) and bad quality 2-cell parthenotes showed lower developmental competence and lower levels of cyclin B1 protein, and Cdc2 mRNA or both gene mRNAs, respectively, compared to their control counterparts. These results suggested that cyclin B1 was regulated posttranscriptionally by cytoplasmic polyadenylation during porcine oocyte maturation. Further, the decreased expression of maternal cyclin B1 and Cdc2 at the mRNA or protein level in developmentally incompetent oocytes and embryos was responsible for, at least in part, a profound defect in further embryonic development.

Involvement of ER-calreticulin-Ca2+ signaling in the regulation of porcine oocyte meiotic maturation and maternal gene expression.

Calcium is one of the most ubiquitous signaling molecules, and controls a wide variety of cellular processes. It is mainly stored in the endoplasmic reticulum (ER), bound to lumenal proteins. Calreticulin is the major Ca(2+)-binding chaperone in oocytes, and is integral to numerous cellular functions. To better understand the role of the ER- calreticulin-Ca(2+) pathway in oocyte maturation and early embryogenesis, we characterized the porcine calreticulin gene and investigated its expression profile during oocyte maturation and early embryonic development. Calreticulin was widely expressed in pig tissues and its transcripts were downregulated during maturation, especially at 44 hr, and were undetectable at the blastocyst stage. We also investigated the effect of increased cytosolic Ca(2+) induced by the Ca(2+)-ATPase inhibitor, cyclopiazonic acid (CPA), on pig oocyte maturation and maternal gene expression. CPA at 10 microM did not inhibit germinal vesicle breakdown, but did result in the arrest of 38.6% oocytes at or before the MI stage. In addition, expression of the maternal genes C-mos, BMP15, GDF9, and Cyclin B1 was significantly increased in CPA-treated MII oocytes compared with control groups. These data were supported by the results of poly(A)-test PCR, which revealed that the cyclin B1 short isoform (CB-S), GDF9, and C-mos underwent more intensive polyadenylation modification in CPA-treated oocytes than control oocytes, suggesting that polyadenylation may influence Ca(2+)-modulated changes in gene expression. Furthermore, CPA treatment decreased the percentage of four-cell parthenotes that developed into blastocysts, suggesting the need for functional SR/ER Ca(2+)-ATPase pumps or Ca(2+) signals during early embryo development after zygotic genome activation. Together, these data indicate that ER-calreticulin-associated Ca(2+) homeostasis plays a role in oocyte and embryo development, and that alterations in maternal gene expression may contribute to the underlying molecular mechanism, at least partially, via polyadenylation.

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.