Cancer Letters

Cancer Letters

Volume 433, 1 October 2018, Pages 43-52
Cancer Letters

Original Articles
HOTAIR is a REST-regulated lncRNA that promotes neuroendocrine differentiation in castration resistant prostate cancer

https://doi.org/10.1016/j.canlet.2018.06.029Get rights and content

Highlights

  • LncRNA HOTAIR is a novel target of REST.

  • HOTAIR is upregulated in CRPC during progression.

  • HOTAIR is a novel driver for NED of PCa cells.

Abstract

Long non-coding RNAs (lncRNAs) are emerging as novel diagnostic markers of prostate cancer (PCa) and new determinants of castration-resistant PCa (CRPC), an aggressive and metastatic form of PCa. In addition to androgen receptor (AR) signaling, neuroendocrine differentiation (NED) is associated with CRPC. Recent reports demonstrate that the downregulation of repressor element-1 silencing transcription factor (REST) protein is a key step in NED of PCa cells. Here, we report HOTAIR as a novel REST-repressed lncRNA that is upregulated in NED PCa cells and in CRPC. HOTAIR overexpression is sufficient to induce, whereas knockdown of HOTAIR suppressed NED of PCa cells. Gene ontology (GO) analysis of differentially expressed genes under HOTAIR overexpression and in CRPC versus benign prostatic hyperplasia (BPH) suggests that HOTAIR may participate in PCa progression. Taken together, our results provide the first evidence of lncRNA HOTAIR as a driver for NED of PCa cells.

Introduction

Prostate cancer (PCa) is one of the major causes of cancer mortality in male populations worldwide [56]. It is first manifested as an androgen-dependent cancer that can be successfully treated with androgen-deprivation therapy (ADT) by either surgical or chemical castration strategies. Unfortunately castration-resistant prostate cancer (CRPC) eventually develops a few years after remission following ADT. Initial studies of CRPC demonstrated that resumption of androgen receptor (AR) signaling is crucial for cancer recurrence [21,30,43,55]. This prompted the development of improved AR antagonists. However, a subset of PCa reliant on survival mechanisms divergent from AR signaling has been reported and increasing evidence has demonstrated CRPC is in positive association with neuroendocrine differentiation (NED) [32,54]. Clinical observations also showed that NED cells are significantly increased in CRPC patients after ADT treatment [38], and neuroendocrine PCa (NEPC) encompasses a complex spectrum of phenotypes from prostate adenocarcinoma with NE features to pure small-cell carcinoma [2].

PCa cells undergoing NED show features similar to NE cells and so-called neuroendocrine-like (NE-like) cells. Various NED inducers for PCa cells in in vitro culture systems have been identified. These include androgen deprivation [6,53,60], IL-6 [7,14], cAMP [12] and hypoxia [36,37]. IL-6 is particularly relevant, as clinical studies have demonstrated that the serum levels of IL-6 are higher in androgen-independent PCa patients [1,18]. NE-like cells are terminally differentiated, non-proliferative, and AR-negative cells that are highly resistant to apoptosis induced by chemotherapy [27]. Consistent with this, the presence of NE-like cells is correlated with a poor prognosis of CRPC [32,54]. Identifying new molecular mechanisms underlying NED holds promise to improve current diagnostics and therapeutics for CRPC.

The recent development of next-generation sequencing (NGS) technology revealed the presence of large amounts of long non-coding RNAs (lncRNAs) throughout the human genome. LncRNAs are RNA transcripts longer than 200 nucleotides (nts), polyadenylated, spliced and devoid of protein-coding potential [19,45], although some recent evidence shows that certain lncRNAs translate small functional peptides [42]. Emerging evidence suggests that lncRNAs are able to regulate multiple biological processes through transcriptional [16], post-transcriptional [25,61], and epigenetic [22,29,57] mechanisms and therefore play important roles in both normal development and disease progression [3]. Most importantly, dysregulation of lncRNAs expression has been linked to tumorigenesis [26]. LncRNAs are generally expressed at a lower level compared with mRNAs, display a higher tissue-specific expression pattern, and lack evolutionary conservation across species [15]. Therefore, functional analysis of lncRNAs in cancer is largely dependent on clinical studies.

Integrative genome-wide expression analysis revealed many lncRNAs are dysregulated in PCa [46,47]. These PCa-expressed lncRNAs, such as PCAT-1 [46] and PCGEM1 [24,44,52] may play an essential role in cancer progression and possibly serve as biomarkers of the disease. One promising example is prostate cancer gene 3 (PCA3), a lncRNA that was recently approved by the FDA for diagnosis of PCa [13,34,39]. Following this concept, lncRNAs significantly overexpressed in CRPC may be used as biomarkers for CRPC [48]. In particular, HOX antisense intergenic RNA (HOTAIR), a lncRNA that is suppressed by AR and significantly upregulated in CRPC, has been shown to enhance proliferation and invasion of castration-resistant cells by maintaining AR activity in an androgen-independent manner [62]. Identifying lncRNAs that associated with NED, a survival mechanism distinct from AR-signaling, may hold great potential as combined biomarkers in monitoring CRPC.

Recent reports, including ours, identified repressor element-1 (RE-1) silencing transcription factor (REST) as a key repressor for NED of PCa cells induced by different stimuli, including androgen deprivation [32,53], hypoxia [36,37], and IL-6 treatment [7,64]. We therefore performed a transcriptome analysis in castration-resistant human PCa CWR22Rv1 cells overexpressing REST. Interestingly, HOTAIR was identified among the top 15 most highly expressed lncRNAs that are downregulated by REST overexpression. Surprisingly, RNA sequencing (RNA-seq) in combination with real-time reverse transcription and quantitative PCR (RT-qPCR) analysis showed that HOTAIR was the only lncRNA among the fifteen found to be upregulated in CRPC tissues when compared with benign prostatic hyperplasia (BPH). We provide experimental evidence to show that HOTAIR plays a pivotal role in NED of PCa cells. To our knowledge, this is the first report demonstrating HOTAIR as a neuroendocrine (NE)-related lncRNA. Taken together with previous reports, this suggests that HOTAIR has multifunctional roles in mediating PCa progression into the castration-resistant stage.

Section snippets

Cell line authentication

CWR22Rv1 was generated by Dr. Jake Jacobberger at Case Western Reserve University and maintained at Dr. Hsing-Jien Kung's lab. LNCaP, LNCaP-TR, CWR22Rv1, CWR22Rv1-TR and PC3 cells were obtained from Dr. Kung's lab in 2011. LNCaP-TR-HOTAIR and LNCaP-TR-shHOTAIR were generated using LNCaP-TR in our lab during 2012–2013. CWR22Rv1-TR-REST and CWR22Rv1-TR-shHOTAIR cells were generated using CWR22Rv1-TR in our lab in 2013–2014. All cell lines have been authenticated by the Biosafety Committee of

Identification of REST repressed lncRNAs in the human castration-resistant PCa cell line CWR22Rv1

REST is a transcription repressor that suppresses NED-related genes and is downregulated in NE-like cells. Our previous report [8] demonstrated that the endogenous level of REST is low in CWR22Rv1 cells, a PCa cell line derived from the relapsed CWR22 xenograft after castration, and which displays a NE phenotype. In addition, overexpression of REST in CWR22Rv1 reduced its NED characteristics [8]. Based on these prior observations, CWR22Rv1 cells were used to define the lncRNA transcriptome

Discussion

Increasing evidence has shown that lncRNAs are not transcription noise but instead function in diverse cellular contexts [20]. Many lncRNAs are now identified as new players in cancer initiation and progression [63] and can be used as biomarkers or therapeutic targets. The most promising example for employment of a lncRNA as a biomarker for PCa prognosis is PCA3, a lncRNA that has been recently approved by the FDA for clinical usage [13]. Thus, identification of CRPC associated lncRNAs may be

Acknowledgments

The work was supported by grants to P.C.C. (MOST, Taiwan (101-2320-B-010-047-MY3 and 104-2320-B-010-038) and Yen Tjing Ling Medical Foundation, Taiwan (CI-107-18)), grants to H.J.K (MOST, Taiwan (105-2320-B-038 -071 -MY3 and 106-2314-B-038 -093)), and grants to T.P.L (MOST, Taiwan 106-2314-B-075 -053 -MY3, Taipei Veterans General Hospital, Taiwan (V107C-195) and Yen Tjing Ling Medical Foundation, Taiwan (CI-106-17)). This work was financially supported by the “Cancer Progression Research

Author contribution

Yi-Ting Chang, Jui-Ting Tang, Yun-Li Luo, Chia-Pei Yang and Ting-Yu Cheng performed experiments and contributed to study design and data analysis. Dr. Shih-Yen Lu, Dr. Ching-Hsin Chang, Dr. Tzu-Ping Lin and Dr. Chin-Chen Pan provided the PCa specimens and interpretation of IHC staining results. Dr. Tze-Tze Liu supported high-throughput genome sequencing and big data analysis. Dr. Mel Campbell, Dr. Chi-Hung Lin and Dr. Hsing-Jein Kung contributed to study design, discussion and writing of the

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