1. IntroductionProstate cancer (PCa) constituted 27% of new cancer cases in US men throughout 2021, ranking at number one in this cohort [1]. The treatment backbone for advanced PCa is androgen deprivation therapy (ADT). Androgen deprivation reduces serum androgens to castrate levels, thereby inhibiting androgen receptor (AR) signaling and PCa proliferation. While initially successful, up to 20% of patients become resistant to ADT and progress to so-called, metastatic CRPC within 5 years of treatment [2]. Metastatic CRPC is characterized by substantial morbidity, with a median survival of only 24–36 months despite registration of multiple second-line therapeutic approaches [3].Despite prolonged exposure to castrate conditions, PCa tumors continue to rely on AR signaling [4]. Gene amplification, expression of AR splice variants, de novo steroidogenesis and non-canonical signaling can all sustain AR signaling, promoting survival under castrate conditions via transcriptional control of proliferation and cell death [5,6]. Modulation of the AR transcriptional program by noncoding RNAs has also been shown to confer resistance to ADT [7,8]. In particular, lncRNAs have been found to support AR transcription, drive therapy resistance, and promote proliferation in PCa [7,9,10].For example, the lncRNA CCAT1 functions as a scaffold for the AR-transcription factor complex and promotes the expression of AR target genes under castrate conditions [9]. The expression of androgen receptor splice variant 7 (ARv7), a major driver of resistance to AR-targeted therapy, is dependent on the presence of lncRNA MALAT1 [7,11]. Additionally, the lncRNA PCA3 promotes PCa cell proliferation by suppressing the expression of tumor-suppressor p53 via the control of chromatin organization [10]. Together, lncRNAs support PCa progression under ADT through numerous molecular mechanisms. Investigation of novel lncRNAs upregulated under castrate conditions could yield new avenues for the development of future therapies and biomarkers.

In this study, an RNA-sequencing dataset containing CRPC, HSPC and benign prostate hyperplasia (BPH) samples was analyzed to identify lncRNAs upregulated in CRPC. Following semi-quantitative PCR (qPCR) validation with an expanded cohort, the lncRNA NAALADL2-AS2 (hereafter named AS2) was selected for in-depth functional characterization. Tissue-specific expression of AS2 was studied using The Cancer Genome Atlas (TCGA) and the Genotype-Tissue Expression project (GTEx) databases. Various hormone modulation and RNA interference models were used to identify drivers of AS2 expression. The role of AS2 on survival and apoptosis was assessed via AS2 knockdown models. Quantitation of AS2 transcripts in nuclear and cytoplasmic cell fractions, and RNA fluorescent in situ hybridization (RNA-FISH) were used to localize AS2 in the cell. Finally, transcriptome and pathway analyses of AS2 knockdown models were used to identify AS2 target genes and pathways.

3. DiscussionCastration resistance is the leading cause of PCa-related death. Sustained androgen signaling under ADT is a hallmark in the progression from HSPC to CRPC. Low levels of remaining androgens are likely the primary drivers of progression under ADT [21]. In fact, tumor dihydrotestosterone (DHT) levels have been shown to remain at 25% of pre-ADT levels 6 months into treatment [21]. In an effort to tackle this issue, ADT in combination with CYP17A inhibitors or AR antagonists are now standard of care in most western countries. Although modestly successful, there is room for improvement [22]. By directing research efforts towards unraveling the survival mechanisms employed by primary PCa cells under ADT, future lives may be spared. Clinical trials have largely mobilized around more potent AR antagonists, inhibitors of de novo steroidogenesis and more recently targeting AR splice variants as novel AR targeted therapies (NCT03123978, NCT02807805, NCT02566772, NCT03888612). Unfortunately, less than 1% of primary PCa patients are ARv7 positive. This number increases marginally to 3% of first line CRPC patients [23]. However, approximately 20% of all PCa patients will develop castration resistance within 5 years of their diagnosis [24].It has been shown recently that noncoding RNAs (ncRNAs) constitute a major part of a cell’s transcriptional regulation. In the nucleus, ncRNAs can modulate transcription by scaffolding with other RNAs and proteins to guide or block access to chromatin, interact with active transcriptional sites, alter splicing, or stabilize messenger RNA (mRNA) [18]. For example, the lncRNA PCGEM1 has been found to aid AR binding to the promoter regions of AR-regulated genes under ADT [25], while lncRNA PCA3 suppresses the function of the tumor suppressor p53, of which the inactivation is central to PCa tumor progression [10,26]. Another example is MALAT1, which alters splicing to produce therapy resistant splice variant ARv7 [7]. Noncoding RNAs can form an integral part of a cancer cell’s adaptive response when exposed to a threat like ADT.In this study, the lncRNA AS2 was found to be upregulated in CRPC tumor biopsies when compared to low and high-grade PCa and benign prostatic tissue. In silico analysis using TCGA data suggest that AS2 may be overexpressed in a number of other malignancies. In HSPC, AS2 likely functions as a driver for progression, as patients with high expression of AS2 have a shorter time to progression when compared to patients with low AS2 expression. A previous publication identified the AS2 overlapping protein coding NAALADL2 gene as an oncogene in PCa [27], although the function of NAALADL2 remains elusive. We found that NAALADL2 and its antisense transcript, AS2, are co-expressed. Being an antisense lncRNA, we hypothesized that AS2 functions as an mRNA stabilizer of NAALADL2 [14]. Yet, knockdown of AS2 did not affect NAALADL2 expression levels. Cell viability, however, was markedly decreased upon AS2 knockdown, which was accompanied by the increased induction of apoptosis. It seems that AS2 functions to promote tumor cell survival. Transcriptome analysis of LAPC-4 cells with AS2 knockdown identified 333 DEGs, the majority of which were upregulated. Amongst those upregulated DEGs, CDKN1A, GZMM, and FOSB are known modulators of apoptosis. In cancer cell lines, overexpressed CDKN1A has been found to enhance cell death responses following DNA-damage induced by cisplatin [28,29]. Furthermore, CDKN1A is transcriptionally controlled by tumor suppressor TP53, which controls key biological pathways related to cell cycle control and apoptosis. As shown in our pathway analysis, TP53-related pathways were significantly deregulated. Interestingly, amongst the few downregulated DEGs, ZC3H12C mapped to several TP53-related pathways. By modulating the expression of TP53-signaling genes, AS2 could suppress apoptosis and promote the survival of PCa cells. Independent of TP53 signaling, GZMM and FOSB are known to promote apoptosis by caspase activation and as part of the AP-1 transcription factor complex, respectively [30,31]. Increased FOSB expression has been shown to be pro-apoptotic in MCF7 cells [32]. By modulating glycogen metabolism, AS2 may also indirectly promote survival in PCa cells [33]. Amongst the AS2-modulated genes there were three enzymes involved with glycogen and carbohydrate metabolism, namely PYGM, ENO, and LCT. Glycogen is an important source of energy for cancer cells under stressful and hypoxic conditions [34]. In this light, ADT-induced AS2 expression could preserve glycogen stores by the downregulation of glycolytic enzymes such as PYGM. In turn, increased glycogen storage may promote survival of ADT-stressed PCa cells. This hypothesis finds credence in a study from Schnier et al., who found that androgen deprivation led to glycogen accumulation in hormone-sensitive LNCaP cells, but not in hormone-insensitive PC3 cells [35]. In line with our findings, AS2 expression increases in LNCaP cells exposed to ADT. Future studies could help elucidate the role of AS2 in the context of ADT, glycogen homeostasis and PCa cell survival.Over 50% of all PCa tumors bare an amplification in the 3q26.31-32 locus, a genetic region on chromosome 3 known for its high density of oncogenes including NAALADL2 and TBL1XR1. Copy number (CN) gains of these genes are associated with their increased expression, consistent with the mechanism of self-regulating expression. NAALADL2 is co-expressed with various AR-target genes while its genetic neighbor, TBL1XR, is a known AR co-activator [15]. This prevalent genetic alteration could also be a key driver of AS2 expression. Indeed, we found AS2 to be androgen regulated, but not via direct control of the AR or two of its major co-factors, GATA2 and FOXA1. GATA2 remains to be a candidate of interest due to its association with NAALADL2 expression and its binding motifs at the NAALADL2 and AS2 promoters. With this in mind, one could expect a positive association between CNs of NAALADL2 and TBL1XR1 and AS2 expression. Yet, amongst 6 PCa cell lines characterized by the CCLE, DU145 and PC3 score highest on CNs for this region but are essentially negative for AS2 expression. By focusing on TFs which are negatively regulated by androgens with binding sites near the AS2 promoter, future studies may identify the drivers of AS2, and accordingly NAALADL2, expression. To summarize, we found AS2 to be androgen regulated, promote tumor cell survival, and modulate the expression of genes involved with apoptosis and glycogen metabolism. Together, AS2 could provide HSPC cells with an adaptive response to ADT by preventing apoptosis and promoting survival.Due to remaining androgens under primary ADT and sustained AR signaling, androgen receptor signaling inhibitors (ARSIs) are used to enhance castration in first line CRPC therapy [36,37]. Successive lines of AR-targeted therapy induce an evolutionary selection for androgen-insensitive cells. In fact, clonal expansion of these cells has been shown to drive resistance to enzalutamide and abiraterone, two commonly used ARSIs [38]. Under the pressure of ARSIs, castration resistant cells gain an adaptive advantage. In this scenario, high AS2 levels may help to identify patients with largely androgen-driven tumors and sensitivity to ARSIs. Vice versa, patients with low AS2 levels might benefit from alternative treatments. In this light, castration resistant LAPC-4-CR and LNCaP-CR show decreased expression of AS2 when compared to their parental lines, suggesting a redundant role for AS2 in a castration resistant setting [16]. This could also explain the perceived discordance of AS2 levels in CRPC tissue and CRPC-like cell lines. In the context of CRPC tissue samples, AS2 expression is increased due to the exposure to first-line ADT. In contrast, CRPC-like cell lines are continuously cultured under hormone deprived conditions, akin to successive lines of AR-targeted therapy. Having adequately adapted to these conditions, AS2 expression is likely to become redundant. As such, increased AS2 expression constitutes part of an acute adaptive response for survival under ADT, a response which is not required for adequately adapted castration resistant cells. This hypothesis is supported by two of our recent publications. Here we found high plasma-AS2 levels in CRPC patients treated with ARSIs to be favorable for progression free survival and overall survival, while low AS2 levels were unfavorable [39,40]. These findings support the clinical utility of AS2 as a liquid biopsy-based biomarker in CRPC. Future clinical trials should evaluate the potential of AS2 as a liquid-biopsy based biomarker in the hormone sensitive setting.