Two decades after the discovery of the first animal microRNA (miRNA), the number of miRNAs in animal genomes remains a vexing question. Here, we report findings from analyzing 1,323 short RNA sequencing samples (RNA-seq) from 13 different human tissue types. Using stringent thresholding criteria, we identified 3,707 statistically significant novel mature miRNAs at a false discovery rate of ≤0.05 arising from 3,494 novel precursors; 91.5% of these novel miRNAs were identified independently in 10 or more of the processed samples. Analysis of these novel miRNAs revealed tissue-specific dependencies and a commensurate low Jaccard similarity index in intertissue comparisons. Of these novel miRNAs, 1,657 (45%) were identified in 43 datasets that were generated by cross-linking followed by Argonaute immunoprecipitation and sequencing (Ago CLIP-seq) and represented 3 of the 13 tissues, indicating that these miRNAs are active in the RNA interference pathway. Moreover, experimental investigation through stemloop PCR of a random collection of newly discovered miRNAs in 12 cell lines representing 5 tissues confirmed their presence and tissue dependence. Among the newly identified miRNAs are many novel miRNA clusters, new members of known miRNA clusters, previously unreported products from uncharacterized arms of miRNA precursors, and previously unrecognized paralogues of functionally important miRNA families (e.g., miR-15/107). Examination of the sequence conservation across vertebrate and invertebrate organisms showed 56.7% of the newly discovered miRNAs to be human-specific whereas the majority (94.4%) are primate lineage-specific. Our findings suggest that the repertoire of human miRNAs is far more extensive than currently represented by public repositories and that there is a significant number of lineage-and/or tissue-specific miRNAs that are uncharacterized. SignificanceMicroRNAs (miRNAs) are small ∼22-nt RNAs that are important regulators of posttranscriptional gene expression. Since their initial discovery, they have been shown to be involved in many cellular processes, and their misexpression is associated with disease etiology. Currently, nearly 2,800 human miRNAs are annotated in public repositories. A key question in miRNA research is how many miRNAs are harbored by the human genome. To answer this question, we examined 1,323 short RNA sequence samples and identified 3,707 novel miRNAs, many of which are human-specific and tissue-specific. Our findings suggest that the human genome expresses a greater number of miRNAs than has previously been appreciated and that many more miRNA molecules may play key roles in disease etiology.

Retinoblastoma (RB; encoded by RB1) is a tumor suppressor that is frequently disrupted in tumorigenesis and acts in multiple cell types to suppress cell cycle progression. The role of RB in tumor progression, however, is poorly defined. Here, we have identified a critical role for RB in protecting against tumor progression through regulation of targets distinct from cell cycle control. In analyses of human prostate cancer samples, RB loss was infrequently observed in primary disease and was predominantly associated with transition to the incurable, castration-resistant state. Further analyses revealed that loss of the RB1 locus may be a major mechanism of RB disruption and that loss of RB function was associated with poor clinical outcome. Modeling of RB dysfunction in vitro and in vivo revealed that RB controlled nuclear receptor networks critical for tumor progression and that it did so via E2F transcription factor 1-mediated regulation of androgen receptor (AR) expression and output. Through this pathway, RB depletion induced unchecked AR activity that underpinned therapeutic bypass and tumor progression. In agreement with these findings, disruption of the RB/E2F/nuclear receptor axis was frequently observed in the transition to therapy resistance in human disease. Together, these data reveal what we believe to be a new paradigm for RB function in controlling prostate tumor progression and lethal tumor phenotypes. IntroductionRetinoblastoma (RB; encoded by RB1), a tumor suppressor protein, is a critical negative regulator of tumor development. RB prevents tumorigenesis by suppressing cell cycle progression (1). However, the role of RB in tumor progression is poorly understood, and the clinical importance of RB loss during this process has not been well considered. Here, we identified a clinically relevant function for RB in tumor progression, manifest through control of hormone signaling networks.The function of RB in cell cycle control has been well described (1). Conditions favoring cell cycle arrest induce RB hypophosphorylation and activation. Active RB binds to promoters of genes required for S-phase entry (e.g., CCNA2 and MCM7) and, through association with the SWI/SNF complex and corepressor molecules (e.g., Sin3B), elicits transcriptional corepression. Many RB target genes are positively regulated by activator E2F transcription factors, supporting the current model that RB acts by suppressing E2F-mediated transcriptional activation. Indeed, the minimal transcriptional repression and tumor suppression domain of RB contains the E2F binding motif. E2F-independent functions of RB have been identified (2), but the contribution of these functions to tumor suppression is uncertain. Thus, contemporary views of RB suggest that the protein prevents cell cycle deregulation and tumor development through suppression of activator E2Fs.

Human cyclin D1 is expressed as two isoforms derived by alternate RNA splicing, termed D1a and D1b, which differ for the inclusion of intron 4 in the D1b mRNA. Both isoforms are frequently upregulated in human cancers, but cyclin D1b displays relatively higher oncogenic potential. The splicing factors that regulate alternative splicing of cyclin D1b remain unknown despite the likelihood that they contribute to cyclin D1 oncogenicity. In this study, we report that Sam68, an RNA-binding protein frequently overexpressed in prostate cancer cells, enhances splicing of cyclin D1b and supports its expression in prostate cancer cells. Chromatin immunoprecipitation and RNA coimmunoprecipitation experiments showed that Sam68 is recruited to the human CCND1 gene encoding cyclin D1 and that it binds to cyclin D1 mRNA. Transient overexpression and RNAi knockdown experiments indicated that Sam68 acts to enhance endogenous expression of cyclin D1b. Minigene reporter assays showed that Sam68 directly affected alternative splicing of CCND1 message, with a preference for the A870 allele that is known to favor cyclin D1b splicing. Sam68 interacted with the proximal region of intron 4, and its binding correlated inversely with recruitment of the spliceosomal component U1-70K. Sam68-mediated splicing was modulated by signal transduction pathways that elicit phosphorylation of Sam68 and regulate its affinity for CCND1 intron 4. Notably, Sam68 expression positively correlates with levels of cyclin D1b, but not D1a, in human prostate carcinomas. Our results identify Sam68 as the first splicing factor to affect CCND1 alternative splicing in prostate cancer cells, and suggest that increased levels of Sam68 may stimulate cyclin D1b expression in human prostate cancers.

Purpose: Alternative CCND1 splicing results in cyclin D1b, which has specialized, protumorigenic functions in prostate not shared by the cyclin D1a (full length) isoform. Here, the frequency, tumor relevance, and mechanisms controlling cyclin D1b were challenged. Experimental Design: First, relative expression of both cyclin D1 isoforms was determined in prostate adenocarcinomas. Second, relevance of the androgen axis was determined. Third, minigenes were created to interrogate the role of the G/A870 polymorphism (within the splice site), and findings were validated in primary tissue. Fourth, the effect of G/A870 on cancer risk was assessed in two large case-control studies.Results: Cyclin D1b is induced in tumors, and a significant subset expressed this isoform in the absence of detectable cyclin D1a. Accordingly, the isoforms showed noncorrelated expression patterns, and hormone status did not alter splicing. Whereas G/ A870 was not independently predictive of cancer risk, A870 predisposed for transcript-b production in cells and in normal prostate. The influence of A870 on overall transcript-b levels was relieved in tumors, indicating that aberrations in tumorigenesis likely alter the influence of the polymorphism. Conclusions: These studies reveal that cyclin D1b is specifically elevated in prostate tumorigenesis. Cyclin D1b expression patterns are distinct from that observed with cyclin D1a. The A870 allele predisposes for transcript-b production in a context-specific manner. Although A870 does not independently predict cancer risk, tumor cells can bypass the influence of the polymorphism. These findings have major implications for the analyses of D-cyclin function in the prostate and provide the foundation for future studies directed at identifying potential modifiers of the G/A870 polymorphism. (Clin Cancer Res 2009;15(17):5338-49)

The cyclin/cyclin-dependent kinase (CDK)/retinoblastoma (RB)-axis is a critical modulator of cell cycle entry and is aberrant in many human cancers. New nodes of therapeutic intervention are needed that can delay or combat the onset of malignancies. The antitumor properties and mechanistic functions of PD-0332991 (PD; a potent and selective CDK4/6 inhibitor) were investigated using human prostate cancer (PCa) models and primary tumors. PD significantly impaired the capacity of PCa cells to proliferate by promoting a robust G1-arrest. Accordingly, key regulators of the G1-S cell cycle transition were modulated including G1 cyclins D, E and A. Subsequent investigation demonstrated the ability of PD to function in the presence of existing hormone-based regimens and to cooperate with ionizing radiation to further suppress cellular growth. Importantly, it was determined that PD is a critical mediator of PD action. The anti-proliferative impact of CDK4/6 inhibition was revealed through reduced proliferation and delayed growth using PCa cell xenografts. Finally, first-in-field effects of PD on proliferation were observed in primary human prostatectomy tumor tissue explants. This study shows that selective CDK4/6 inhibition, using PD either as a single-agent or in combination, hinders key proliferative pathways necessary for disease progression and that RB status is a critical prognostic determinant for therapeutic efficacy. Combined, these pre-clinical findings identify selective targeting of CDK4/6 as a bona fide therapeutic target in both early stage and advanced PCa and underscore the benefit of personalized medicine to enhance treatment response.

The retinoblastoma tumor suppressor protein (RB), a critical mediator of cell cycle progression, is functionally inactivated in the majority of human cancers, including prostatic adenocarcinoma. The importance of RB tumor suppressor function in this disease is evident because 25% to 50% of prostatic adenocarcinomas harbor aberrations in RB pathway. However, no previous studies challenged the consequence of RB inactivation on tumor cell proliferation or therapeutic response. Here, we show that RB depletion facilitates deregulation of specific E2F target genes, but does not confer a significant proliferative advantage in the presence of androgen. However, RB-deficient cells failed to elicit a cytostatic response (compared with RB proficient isogenic controls) when challenged with androgen ablation, AR antagonist, or combined androgen blockade. These data indicate that RB deficiency can facilitate bypass of first-line hormonal therapies used to treat prostate cancer. Given the established effect of RB on DNA damage checkpoints, these studies were then extended to determine the impact of RB depletion on the response to cytotoxic agents used to treat advanced disease. In this context, RB-deficient prostate cancer cells showed enhanced susceptibility to cell death induced by only a selected subset of cytotoxic agents (antimicrotubule agents and a topoisomerase inhibitor). Combined, these data indicate that RB depletion dramatically alters the cellular response to therapeutic intervention in prostate cancer cells and suggest that RB status could potentially be developed as a marker for effectively directing therapy. [Cancer Res 2007;67(13):6192-203]

Aberrant expression of cyclin D1 protein is a common feature of breast cancer. However, the CCND1 gene encodes two gene products, cyclin D1a and cyclin D1b, which have discrete mechanisms of regulation and impact on cell behavior. A polymorphism at nucleotide 870 in the CCND1 gene, rs603965, influences the relative production of the encoded proteins and can impart increased risk for tumor development. Here, the impact of both the G/A870 polymorphism and cyclin D1b protein production on breast cancer risk, disease phenotype and patient outcome was analysed. In a large multiethnic case–control study, the G/A870 polymorphism conferred no significant risk for breast cancer overall or by stage or estrogen receptor (ER) status. However, the cyclin D1b protein was found to be upregulated in breast cancer, independent of cyclin D1a levels, and exhibited heterogeneous levels in breast cancer specimens. High cyclin D1a expression inversely correlated with the Ki67 proliferation marker and was not associated with clinical outcome. In contrast, elevated cyclin D1b expression was independently associated with adverse outcomes, including recurrence, distant metastasis and decreased survival. Interestingly, cyclin D1b was particularly associated with poor outcome in the context of ER-negative breast cancer. Thus, specific cyclin D1 isoforms are associated with discrete forms of breast cancer and high cyclin D1b protein levels hold prognostic potential.

Cyclin D1 is a critical regulator of androgen-dependent transcription and cell cycle progression in prostate cancer cells. Despite the influence of D-type cyclins on prostate cancer proliferation, few studies have examined the expression of cyclin D1 in localised tumours or challenged its relevance to disease progression. Cyclin D1 status was characterised using immunohistochemistry in 38 non-neoplastic prostate samples, 138 primary human prostate carcinomas, and three lymph node metastatic specimens. Relevance of cyclin D1 to preoperative prostate-specific antigen (PSA) levels, Ki-67 index, and p21 Cip1 status was also examined. Cyclin D1-positive phenotype was increased in primary carcinoma compared to non-neoplastic tissue, and was evident in all lymph node metastases cases. Interestingly, at least three distinct localisation patterns were observed in the cyclin D1-positive cohort, wherein cytoplasmic localisation was identified in a large fraction, and this pattern was predominant in lower grade tumours. Relevance of altered cyclin D1 status was observed, wherein cyclin D1-positive tumours were associated with low preoperative PSA levels, consistent with in vitro reports that cyclin D1 may alter the expression of this tumour marker. Moreover, tumours with predominantly cytoplasmic cyclin D1 showed the lowest Ki-67 index, whereas nuclear cyclin D1 was associated with higher grade, elevated Ki-67, and increased nuclear p21 Cip1 . These data demonstrate that differential cyclin D1 status may influence clinicopathological parameters, and reveal new insight as to the regulation and potential consequence of cyclin D1 expression in prostate cancer.