It has been previously demonstrated that the cyclin D1b splice variant harbors differential oncogenic activities and likely serves specialized functions in the prostate that promote development and/or progression of cancer (14
). Despite these observations, little is known concerning the factors that control cyclin D1b production. The present study is the first to examine expression profiles for each cyclin D1 isoform and to address the mechanisms of cyclin D1b production in PCa. Consistent with our previous report, a large number of prostatic adenocarcinomas show mis-localization or are devoid of detectable cyclin D1a (30
). However, a significant fraction of tumors express cyclin D1b, which showed distinct expression profiles not correlated to detected cyclin D1a. Notably, cyclin D1b was significantly enhanced in PCa as compared to non-neoplastic tissue. Thus, the cyclin D1b isoform appears to be specifically induced in a significant fraction of prostate cancers. Functional studies demonstrated that transcript-b
levels are refractory to androgen or AR activation, but the G/A870 polymorphism is a critical effector of cyclin D1b production. Subsequent analyses in human specimens showed that while the A-allele predicts for enhanced overall transcript-b
levels in prostate tissue, G/A870 is not independently predictive of PCa risk. Parallel studies revealed that the requirement of the A-allele for enhanced transcript-b
expression is relieved in tumor tissue. Together, these data demonstrate that the cyclin D1b isoform is predominantly associated with tumorigenesis in PCa, and implicate both the polymorphism and modifiers thereof as effectors of the cyclin D1b oncogenic variant.
Only recently have the cyclin D1 isoforms been examined in human disease, and current observations suggest that cyclin D1b regulation is under stringent but tissue-specific control. First, induction of both isoforms has been documented in primary breast carcinomas (17
). These observations are distinct from what was observed in PCa, wherein no significant induction in cyclin D1a was observed in neoplastic disease. In breast cancer models, anti-estrogen therapies enhanced cyclin D1b production, while cyclin D1a was reduced (17
). In contrast, neither cyclin D1 transcript in the current study was influenced by anti-androgen therapy, suggesting that unique distinctions exist between these two hormone-dependent cancers. Second, like the current study, the majority of primary esophageal carcinomas expressed cyclin D1b (9
). Subsequent studies revealed esophageal tumor-derived mutations that increased the oncogenic capacity of cyclin D1a in a manner synonymous to cyclin D1b (58
). Finally, increased cyclin D1b (not cyclin D1a) has been associated with histologic grade in non-small cell lung carcinoma patients (31
). Importantly, cyclin D1b correlated with poor survival and was found to be an independent risk factor for lung cancer development. These collective studies (summarized in Supplementary Fig. S6
) highlight the importance of examining cyclin D1b levels and demonstrate the need to discern the contributing isoform in tumorigenesis. Moreover, previous studies examining cyclin D1 used reagents that can potentially detect either isoform (13
). Nonetheless, it is apparent from the present findings that cyclin D1b induction is characteristic of a subset of PCa, and ongoing studies will address the impact of this induction on clinical outcome.
Based on these observations, it is imperative to define the mechanism of cyclin D1b production. In addition to alternative splicing, it is likely that multiple mechanisms exist to regulate cyclin D1b production, including: gene activation (17
), translational control (29
), transcriptional elongation (19
), and mRNA stability (62
). In breast cancer, both cyclin D1 isoforms are regulated at the transcriptional level through the actions of the estrogen receptor (49
). This is in marked contrast to PCa cells, wherein both transcripts were unchanged by manipulation of the AR pathway. By contrast, mTOR-mediated enhancement of translation appears to be required for cyclin D1b production, which has been established for cyclin D1a (29
). Thus, current therapies directed at ablation of AR activity are not likely to alter alternative splicing, but may assist in preventing translation of transcript-b
Based on these findings, critical questions were addressed with regard to the induction of transcript-b
and resultant cyclin D1b protein production. The data herein show that the CCND1
G/A870 polymorphism is a potent effector of this event. Using novel CCND1
minigenes that harbor a single base change (G- or A-allele), it was evident that the A-allele predisposes for cyclin D1b production in cells. To our knowledge, this is the first report to demonstrate that the A-allele, in isolation, influences cyclin D1b production. However, a noted caveat is that the minigenes lack most of the 3’UTR and natural polyadenylation site which could potentially contribute to transcript stability (62
) which could potentially influence the relative levels of cyclin D1a and cyclin D1b. Nonetheless, analyses of non-neoplastic prostate tissue showed that transcript-b
levels were enhanced in tissue with the AA-genotype, consistent with the minigene analyses and additional tumor types (54
). However, functional studies demonstrate that the G-allele can also produce cyclin D1b, supporting the hypothesis that the A-allele is not universally required for transcript-b
or cyclin D1b production. Future studies will be directed at factors that influence the A-allele effect, and on the importance of the transcript b:a
ratio. Collectively, these data indicate that the A-allele predisposes for transcript-b
and cyclin D1b production in PCa, but that additional factors may influence the alternative splicing of cyclin D1.
An association between the A-allele and increased PCa risk (63
) has been suggested in prior studies of limited size (<300 cases and <300 controls) and composition. Consistent with the supposition that the A-allele is not sufficient for transcript-b
production, two population-based PCa studies (>3,000 cases and >3,000 controls) determined no independent risk association. These analyses had 80% power to detect effects as low as 1.11 and 1.17 for the A-allele, assuming a minor allelic frequency (MAF) of 40% (which is the average MAF in the multiethnic sample), and a log-additive or dominant effect on risk, respectively. However, assessment of luteinizing hormone (LH) levels in the controls with the AA-genotype were modestly elevated (12%, p=0.02) compared to controls with the G-allele, while other hormones (testosterone, estradiol, SHBG, DHEAS, androstenedione, androstanediol glucuronide, and prolactin) were not significantly different (data not shown). Nonetheless, the consistent lack of a significant association of the polymorphism to PCa risk, in all populations, further supports that this variant is not an independent marker.
Intriguingly, despite the relevance of the A-allele for overall transcript-b
production in non-neoplastic prostate tissue, this requirement was alleviated in tumor tissue. These observations suggest a provocative hypothesis that tumor-specific alterations may bypass or synergize with the G/A870 polymorphism to enhance total transcript-b
production. While the data herein clearly indicate the importance of the polymorphism, a number of additional factors have been suggested to influence cyclin D1b production. First, recent observations in Ewing's sarcoma cell lines and tumors suggests that chromosomal alterations between the Ewing's sarcoma oncogene and the ets
family transcription factor (FLI1) result in up-regulation of cyclin D1b through alteration of transcript elongation (18
). Interestingly, chromosomal translocations of other ets
family transcription factors are frequently observed in PCa (e.g., TMPRSS2:ERG (65
)). Therefore, it will be of importance to characterize the role of these fusions on cyclin D1b production. Second, loss of Brahma (Brm), the core ATPase of the SWI/SNF-chromatin-remodeling complex, significantly increased transcript-b
expression in colorectal cells (67
). Moreover, Brm is frequently reduced or lost in human PCa, and correlates with enhanced cellular proliferation (68
). Finally, recent observations show that AR is alternatively spliced, thus indicating that aberrant splicing of several critical effectors of AR activity and cellular proliferation occur in PCa (69
). It will be intriguing whether the mechanisms that underlie the alternative splicing events are linked.
In summary, the data herein demonstrate that cyclin D1b is specifically enhanced in PCa, thus showing a distinct expression profile from the cyclin D1a isoform. Accordingly, expression of the two isoforms is likely differentially regulated, and unlike breast cancer, the transcription and splicing of CCND1
(a or b) is independent of hormone. Rather, the A870 polymorphic allele was identified as a significant effector of cyclin D1b production, but additional events in prostate tumorigenesis may alleviate the influence of the A-allele. Together, these studies highlight a unique mechanism of cyclin D1b regulation, thereby providing the basis for future studies directed at identifying modifiers of the G/A870 polymorphism and the oncogenic consequence of cyclin D1b in PCa.
Clinical and Translational Relevance
This study has marked clinical and translational implications: First, these data reveal for the first time that cyclin D1b is specifically induced in prostate cancer. As cyclin D1b has distinct functions from cyclin D1a, this has important implications for interpretation of biomarker and functional analyses. Second, previous studies tentatively linked the G/A870 polymorphism to cancer risk and cyclin D1b, but until now these postulates remained untested. Here, case-control studies showed that G/A870 is not independently predictive of risk. Third, although not sufficient for risk, functional studies identified A870 as a critical modulator of cyclin D1b in model systems and human prostate specimens. The A870 requirement is bypassed in cancer, indicating that tumor-specific events circumvent or cooperate with A870. Together, these findings identify cyclin D1b markedly enhanced in prostate cancer and reveal the impact of G/A870 for cyclin D1b production and cancer risk.