The long non-coding RNA PCGEM1 is regulated by androgen receptor activity in vivo
- Abhijit Parolia1, 2, 3,
- Francesco Crea2, 3, 5,
- Hui Xue2, 3,
- Yuwei Wang2, 3,
- Fan Mo3,
- Varune Rohan Ramnarine3,
- Hui Hsuan Liu2,
- Dong Lin2, 3, 5,
- Nur Ridzwan Nur Saidy1, 2,
- Pier-Luc Clermont2, 4,
- Hongwei Cheng2, 3,
- Colin Collins3,
- Yuzhuo Wang†2, 3Email author and
- Cheryl D Helgason†2, 6Email author
© Parolia et al.; licensee BioMed Central. 2015
Received: 18 November 2014
Accepted: 5 February 2015
Published: 21 February 2015
Long non-coding RNAs (lncRNAs) can orchestrate oncogenic or tumor-suppressive functions in cancer biology. Accordingly, PCGEM1 and PRNCR1 were implicated in progression of prostate cancer (PCa) as transcriptional co-regulators of the androgen receptor (AR). However, these findings were recently refuted asserting that neither gene physically binds to the AR. Despite evidence for differing AR transcriptional programs in vivo and in vitro, studies investigating AR-regulation of these genes hitherto have only been conducted in vitro. Here, we further examine the relevance of PCGEM1 and PRNCR1 in PCa, and their relationship with AR signaling, using patient-derived xenograft models.
RNA sequencing of two distinct androgen-dependent models shows PCGEM1 to be considerably expressed, while PRNCR1 showed scant basal expression. PCGEM1 was sharply down-regulated following castration and up-regulated upon AR activation in vivo. However, we found no parallel evidence following AR stimulation in vitro. A PCGEM1-associated gene expression signature (PES) was significantly repressed in response to androgen ablation therapy and in hormone-refractory versus hormone-naïve PCa patients. Furthermore, we found PCGEM1 was uniformly distributed in PCa cell nucleus and cytoplasm which remained unaltered upon AR transcriptional activation. PCGEM1 was up-regulated in primary PCa but not in metastasized PCa. Accordingly, the PES was significantly down-regulated in advanced and higher grade PCa patients from multiple independent studies.
Our results demonstrate PCGEM1 as an in vivo androgen-regulated transcript with potential nuclear and/or cytoplasmic function(s). Importantly, the clinical expression profile of PCGEM1 implicates it in the early stages of PCa warranting further research in this direction.
In recent years, long non-coding RNAs (lncRNAs) have emerged as major contributors to cellular homeostasis as well as initiation and progression of numerous diseases , including prostate cancer (PCa) . The latest GENCODE v7 project annotated 14,880 human lncRNA transcripts with only a few characterized to date . Of the lncRNAs functionally validated in various human malignancies, a majority have been identified as constituents of oncogenic or tumor suppressive pathways [4,5]. Some prominent lncRNAs implicated in prostate carcinogenesis and its progression include prostate cancer associated transcript 1 (PCAT1) , second chromosome locus associated with prostate 1 (SChLAP1) , prostate cancer associated 3 (PCA3) , prostate cancer gene expression marker 1 (PCGEM1; aka PCAT9)  and prostate cancer associated non-coding RNA 1 (PRNCR1; aka PCAT8) . Notably, SChLAP1 has been extensively validated in the clinics as a biomarker of aggressive PCa  and PCA3 is currently used in diagnostic tests . Recently, we described PCAT18 (aka Loc728606, Linc01092) as a mediator of metastatic progression based on expression profiling of our patient-derived PCa xenograft models from the Living Tumor Laboratory (LTL) . PCGEM1, a highly prostate-specific transcript, was one of the first oncogenic lncRNAs to be described in PCa . Subsequently, its over-expression was reported to attenuate the apoptotic response  and also promote cell proliferation and colony formation . On the other hand, PRNCR1 is not as well investigated, although its knockdown reportedly inhibits cell viability . Recently, both of these lncRNAs occupied center stage due to their labeling as androgen receptor (AR)-interacting genes  – a claim now disputed .
AR is a ligand-responsive regulatory protein that mediates the effector functions of androgenic hormones in PCa. It is well established that sustained AR activity is indispensable for PCa cell survival and disease progression, even following androgen-deprivation therapy [18-20]. This “AR addiction” has led to many studies investigating genes serving as conduits for aberrant restoration of AR activity in recurrent tumors as potential therapeutic targets. In this regard, Srikantan et al. described PCGEM1 as an AR regulated gene. Later on, PCGEM1 acting in complicity with PRNCR1 was shown to physically bind to the AR, thereby facilitating its ligand-independent transcriptional activity in castration resistant PCa (CRPC) . In contrast, a recent publication indicated that neither PCGEM1 nor PRNCR1 interacted with the AR to render androgen-independence, and that both genes had no prognostic relevance in PCa . Furthermore, the latter study found no evidence of PCGEM1 and PRNCR1 transcripts being AR regulated.
Notably, all published data on the relationship between AR and PCGEM1/PRNCR1 hitherto have been derived from in vitro experiments, using androgen-sensitive LNCaP cells [9,17]. There is substantial evidence that transcriptional regulation of genes by a transcription factor is highly dynamic and cellular context-specific . In this light, AR was recently demonstrated to induce varied and distinct AR transcriptional programs in patient tumor tissue as opposed to PCa cell lines . This puts in doubt the suitability of cell line models for studying the transcriptional activity of the AR. In view of this, we set out to specifically investigate whether PCGEM1 and PRNCR1 are relevant in PCa and/or regulated by AR using our LTL patient-derived xenograft PCa models. The LTL has established a large panel of patient-derived PCa xenograft models that, unlike cell lines, retain key biological properties of the original malignancy, including histopathology, genomic profile, cellular heterogeneity, and invasive and metastatic ability .
Findings and discussion
As a first step, we profiled our two AR+/androgen dependent PCa xenograft models – LTL-331 and LTL-313B – for expression of both lncRNAs using RNA Sequencing. While PCGEM1 was considerably expressed (>500 FPKM; fragments per kilobase of exon per million fragments mapped), the expression of PRNCR1 was <8 FPKM in both models (Additional file 1: Table S1). Such scant expression of PRNCR1 raises serious questions about its biological relevance in AR-dependent PCa. Notably, this negligible expression of PRNCR1 is in accordance with a more extensive clinical dataset that was recently published . Together, these data weaken the claim made by Yang et al.  that PRNCR1 plays a vital role in directing the transcriptional activity of AR. We, therefore, focused solely on PCGEM1 for the remainder of our study.
AR regulates expression of PCGEM1 in vivo
AR activation does not alter the uniform sub-cellular localization of PCGEM1
It was previously suggested that a direct AR-PCGEM1 interaction regulates AR’s transcriptional activity . In line with this mechanistic model, PCGEM1’s suspected function as a transcriptional co-regulator would imply it to be predominantly contained in the nucleus, in particular when the AR is transcriptionally active. However, sub-cellular localization of PCGEM1 had never been investigated. Addressing this issue, we performed cellular fractionation of PCa patient-derived xenograft cells to isolate the nuclear and cytoplasmic RNA fractions, and quantified gene expression.
PCGEM1 is implicated in early stages of PCa
PCGEM1 -associated expression signature in PCa patient samples
PCa vs. Other Neoplasms
PCa vs. Normal Prostate
1.88E-10 - 2.00E-03
14.5 - 4.5
Metastatic PCa vs. Primary PCa
6.69E-13 - 5.00E-03
27.0 - 4.5
High Gleason PCa vs. Low Gleason PCa
8.01E-11 - 6.00E-3
16.8 - 5.0
Hormone-Refractory PCa vs. Hormone-naïve PCa
Reoccurrence at 3 or 5 years
1.45E-06 - 4.52E-04
8.4 - 5.1
Dead at 3 or 5 years
4.65E-07 - 9.02E-04
A review of literature also reveals that PCGEM1 is up-regulated in normal prostate epithelial cells of men with a family history of PCa  and certain single-nucleotide polymorphisms in the PCGEM1 gene are associated with a higher risk of PCa development in Chinese men . Together, these findings support the testable hypothesis that PCGEM1 is more important in the early stages, possibly initiation, of primary PCa. Interestingly, the PCGEM1 signature was also under-expressed in hormone-refractory PCa relative to hormone-naïve PCa (Table 1); and the pathway analysis revealed PES to be most significantly down-regulated (p = 5.47E-14, odds ratio = 121.4) in patients receiving androgen-deprivation therapy (Additional file 1: Table S3) lending further support to in vivo AR-regulation of PCGEM1.
Our work brings in light an even more pertinent concern with exploring AR transcriptional activity using cell line models. Recently, cell line models in accordance with their metastatic origin were demonstrated to exhibit the AR regulatory program that is active in metastatic advanced PCa as opposed to primary PCa . The AR regulatory activity in cultured cell line models (including LNCaP) had a greater overlap to CRPC (31% overlap) than untreated primary PCa (merely 3% overlap) (see ). Besides the absent cell microenvironment, this describes further limitations of cell line models for investigating AR-regulation, in particular for genes with a clinical expression profile akin to that of PCGEM1. Simultaneously, it underscores the importance of using clinically relevant models that best represent the original malignancy. With mounting evidence for lncRNAs implicated in carcinogenesis and cancer progression, we regard lncRNAs as essential in decrypting cancer cell biology. Our study corroborates the irrelevance of PRNCR1 in PCa, and confirms PCGEM1 as an in vivo AR-regulated transcript as well as rationalizes its oncogenic involvement in early stages of the disease.
This work was financially supported by Canadian Cancer Society Research Institute, grant number: 701097 (CDH); Canadian Institutes of Health Research, grant numbers: 102604–1, 119991–1, 123449–1 (YZW); Prostate Cancer Canada (YZW); Terry Fox Research Institute, grant number: 116129–1 (YZW); Michael Smith Foundation for Health Research Fellowship, number: 5629 (FC); and BC Prostate Cancer Foundation “grant-in-aid” award (FC).
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