Zhenyu Xu

Transient receptor potential channel TRPC5 is essential for P-glycoprotein induction in drug-resistant cancer cells

An attractive strategy to overcome multidrug resistance in cancer chemotherapy is to suppress P-glycoprotein (P-gp), which is a pump overproduced in cancer cells to remove cytotoxic drugs from cells. In the present study, a Ca2+-permeable channel TRPC5 was found to be overproduced together with P-gp in adriamycin-resistant breast cancer cell line MCF-7/ADM. Suppressing TRPC5 activity/expression reduced the P-gp induction and caused a remarkable reversal of adriamycin resistance in MCF-7/ADM. In an athymic nude mouse model of adriamycin-resistant human breast tumor, suppressing TRPC5 decreased the growth of tumor xenografts. Nuclear factor of activated T cells isoform c3 (NFATc3) was the transcriptional factor that links the TRPC5 activity to P-gp production. Together, we demonstrated an essential role of TRPC5–NFATc3–P-gp signaling cascade in P-gp induction in drug-resistant cancer cells.

ERRα augments HIF‐1 signalling by directly interacting with HIF‐1α in normoxic and hypoxic prostate cancer cells

Adaptation of cancer cells to a hypoxic microenvironment is important for their facilitated malignant growth and advanced development. One major mechanism mediating the hypoxic response involves up‐regulation of hypoxia‐inducible factor 1 (HIF‐1) expression, which controls reprogramming of energy metabolism and angiogenesis. Oestrogen‐related receptor‐α (ERRα) is a pivotal regulator of cellular energy metabolism and many biosynthetic pathways, and has also been proposed to be an important factor promoting the Warburg effect in advanced cancer. We and others have previously shown that ERRα expression is increased in prostate cancer and is also a prognostic marker. Here we show that ERRα is oncogenic in prostate cancer and also a key hypoxic growth regulator. ERRα‐over‐expressing prostate cancer cells were more resistant to hypoxia and showed enhanced HIF‐1α protein expression and HIF‐1 signalling. These effects could also be observed in ERRα‐over‐expressing cells grown under normoxia, suggesting that ERRα could function to pre‐adapt cancer cells to meet hypoxia stress. Immunoprecipitation and FRET assays indicated that ERRα could physically interact with HIF‐1α via its AF‐2 domain. A ubiquitination assay showed that this ERRα–HIF‐1α interaction could inhibit ubiquitination of HIF‐1α and thus reduce its degradation. Such ERRα–HIF‐1α interaction could be attenuated by XCT790, an ERRα‐specific inverse agonist, resulting in reduced HIF‐1α levels. In summary, we show that ERRα can promote the hypoxic growth adaptation of prostate cancer cells via a protective interaction with HIF‐1α, suggesting ERRα as a potential therapeutic target for cancer treatment. Copyright

Orphan nuclear receptor TLX functions as a potent suppressor of oncogene-induced senescence in prostate cancer via its transcriptional co-regulation of the CDKN1A (p21WAF1/CIP1) and SIRT1 genes

Oncogene‐induced senescence is an important tumour‐suppressing mechanism to prevent both premalignant transformation and cancer progression. Overcoming this process is a critical step in early cancer development. The druggable orphan nuclear receptor TLX (NR2E1) is characterized as an important regulator of neural stem cells and is also implicated in the development of some brain tumours. However, its exact functional roles in cancer growth regulation still remain unclear. Here we report that TLX can act as a promoter of tumourigenesis in prostate cancer by suppressing oncogene‐induced senescence. We determined that TLX exhibited an increased expression in high‐grade prostate cancer tissues and many prostate cancer cell lines. Functional studies revealed that TLX could perform an oncogenic function in prostate cancer cells, as its knockdown triggered cellular senescence and cell growth arrest in vitro and in vivo, whereas its over‐expression promoted the malignant growth of prostate cancer cells. Furthermore, enhancement of TLX activity, by either ectopic expression or ligand stimulation, could potently prevent doxorubicin‐induced senescence in prostate cancer cells and also allow prostatic epithelial cells to escape oncogene‐induced senescence induced either by activated oncogene H‐RasG12V or knockdown of tumour suppressor PTEN, via a mechanism of direct but differential transcriptional regulation of two senescence‐associated genes, repression of CDKN1A and transactivation of SIRT1. Together, our present study shows, for the first time, that TLX may play an important role in prostate carcinogenesis through its suppression of oncogene‐induced senescence, and also suggests that targeting the senescence‐regulatory TLX is of potential therapeutic significance in prostate cancer. Copyright

Ion channel TRPM8 promotes hypoxic growth of prostate cancer cells via an O2‐independent and RACK1‐mediated mechanism of HIF‐1α stabilization

The growth adaptation of cancer cells to a hypoxic tumour microenvironment is mostly regulated by hypoxia‐induced transcription factor HIF‐1. HIF‐1 transcriptional activity is strictly controlled by protein levels of the HIF‐1α subunit, which is tightly regulated by a well‐characterized O2‐dependent ubiquitin ligase–proteasomal degradation pathway. The cold‐sensitive Ca2+ channel protein TRPM8 exhibits increased expression in advanced prostate cancer. However, its exact functional roles in prostate cancer growth regulation are unclear and controversial. In this work, we show that TRPM8 promotes in vitro hypoxic growth capacities, drug resistance, and in vivo tumourigenicity, accompanied with enhanced HIF‐1α protein levels. These effects are further potentiated by TRPM8 agonists but suppressed by TRPM8 gene knockdown and blocking with antagonists or TRPM8 antibody. TRPM8‐induced suppression of HIF‐1α ubiquitination and enhanced HIF‐1 transactivation were attenuated by forced RACK1 expression and TRPM8 overexpression reduced phospho‐RACK1 levels, thus affecting its dimerization status, and promoted RACK1 binding to HIF‐1α and calcineurin. These data indicate that TRPM8‐induced increase of HIF‐1α protein in hypoxia‐ or normoxia‐exposed prostate cancer cells was mediated through a newly characterized Ca2+‐dependent but O2‐independent mechanism involving binding of RACK1 to HIF‐1α and RACK1‐mediated ubiquitination of HIF‐1α. Collectively, our study not only provides a mechanistic insight into how TRPM8 promotes the hypoxic growth adaptation of cancer cells via its promotion of RACK1‐mediated stabilization of HIF‐1α but also suggests a potential therapeutic strategy for prostate cancer by targeting TRPM8.Copyright

Increased expression of activated endothelial nitric oxide synthase contributes to antiandrogen resistance in prostate cancer cells by suppressing androgen receptor transactivation

Development of antiandrogen-resistance in advanced prostate cancer involves multiple androgen receptor (AR)-dependent and -independent pathways. Here, we demonstrated that endothelial nitric oxide synthase (eNOS) exhibited an overexpression pattern in hormone-refractory prostate cancer and several models of advanced hormone-resistant prostate cancer. We further established a novel in vitro model of antiandrogen-resistant prostate cancer (LNCaP-BC) by long-term bicalutamide treatment. Besides antiandrogen-resistant and other enhanced malignant growth phenotypes, LNCaP-BC cells exhibited an increased activated eNOS expression and NO production, and suppressed AR transactivation status. Treatment with a NOS inhibitor L-NAME could re-sensitize the growth response to bicalutamide and enhance the AR transactivation in LNCaP-BC cells. Together, our present findings indicate that increased NO production by acquired increased expression of activated eNOS could contribute to the antiandrogen-resistant growth of prostate cancer cells, via a mechanism of NO-mediated suppression of AR activity, and also targeting eNOS could be a potential therapeutic strategy for antiandrogen-resistant prostate cancer.

Collapsin response mediator protein-1 (CRMP1) acts as an invasion and metastasis suppressor of prostate cancer via its suppression of epithelial–mesenchymal transition and remodeling of actin cytoskeleton organization

The cancer cells can acquire migration and invasion capacities during the metastasis process through the developmental regulatory program epithelial–mesenchymal-transition (EMT), and through its reverse process mesenchymal–epithelial transition cancer cells can recolonize at distant metastatic sites. Among the multifaceted effects exerted by this program, reorganization of actin cytoskeleton is the key mechanical drive for the invasive properties gained by cancer cells. Collapsin response mediator protein-1 (CRMP1) is a cytosolic phosphoprotein and originally characterized as the mediator of semaphorin 3A signaling involved in axon differentiation during neural development. Here we report that CRMP1 can act as a suppressor of tumorigenicity and metastasis in prostate cancer cells. We demonstrated that CRMP1 exhibited a decreased expression pattern in high-grade prostate cancer tissues and many prostate cancer cell lines, and its downregulation in cancer cells was attributed to histone deacetylation and direct repression of its gene by the EMT regulator Snail. Functional analyses revealed that CRMP1 suppressed EMT in prostate cancer cells, as its knockdown could trigger EMT and enhance in vitro invasion capacity, whereas its overexpression could inhibit EMT and suppress both in vitro invasion and in vivo metastasis capacities of prostate cancer cells. Moreover, CRMP1 overexpression could significantly confer resistance to EMT induced by Snail or transforming growth factor-β1 in prostatic epithelial cells and prostate cancer cells. Finally, we demonstrated that CRMP1 could associate with actin and WAVE1, an activator of actin nucleation complex Arp2/3, and also its knockdown could stabilize F-actin and trigger the formation of stress fibers in prostate cancer cells. Together, our study shows that CRMP1 acts an EMT and metastasis suppressor in prostate cancer cells via its regulation of actin polymerization and also suggests that targeting the CRMP1-actin signaling in actin organization could be a potential strategy for management of prostate cancer metastasis.

Abstract 1385: Orphan nuclear receptor estrogen-related receptor alpha (ERRα) characterized as a novel transcriptional regulator of the oncogenic fusion gene TMPRSS2:ERG in prostate cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The expression of androgen-responsive TMPRSS2:ERG fusion gene, which results in aberrant expression of the oncogenic transcription factor ERG under the regulation of androgen receptor (AR), is characterized as the predominant factor responsible for the initiation and also advanced progression of prostate cancer (PCa). However, its upstream transcriptional regulation in androgen-independent PCa is still not fully understood. Recent studies show that the orphan nuclear receptor estrogen-related receptor alpha (ERRα/NR3B1) plays certain regulatory roles in the advanced progression of prostate cancer, such as its cross-talk with AR signaling. Based on this, we hypothesize that besides AR, ERRα could also regulate or target to the promoter of fusion gene TMPRSS2:ERG in prostate cancer cells. Here we showed that treatment with an ERRα-specific inverse agonist (XCT790) or knockdown of ERRα could decrease the TMPRSS2:ERG expression in AR-positive VCaP prostate cancer cells. Besides, the expression levels of TMPRSS2:ERG could be elevated by ectopic ERRα expression, with further elevation by co-expression with a genetically modified ERRα-specific coactivator PGC1α(2X9), in AR-negative NCI-H660 prostate cancer cells. By ChIP and luciferase reporter analysis, three putative ERRα transactivation DNA binding sites were identified in the promoter region of TMPRSS2:ERG fusion gene. Besides, in vitro phenotype characterization studies showed that ectopic expression of TMPRSS2:ERG, using an ERRα-responsive and TMPRSS2:ERG fusion gene promoter-driven expression plasmid in AR-negative prostatic cell lines, including PC-3 prostate cancer cells and BPH-1 immortalized prostatic epithelial cells, could promote their invasion capacity. Moreover, both ERRα and TMPRSS2:ERG showed elevated expressions in a xenograft model of castration-resistant prostate cancer cells (VCaP-CRPC). Besides, luciferase reporter assays showed that both TMPRSS2:ERG promoter and ERRα-binding element (ERRE) transcriptional activities were elevated in VCaP-CRPC cells as compared to the VCaP parental cells; and their transcriptional activities could be suppressed by XCT790 or sh-ERRα. Based on these results, we conclude that ERRα could directly transactivate the TMPRSS2:ERG fusion gene in prostate cancer and may contribute to its transactivation in AR-negative prostate cancer cells and the advanced castration-resistant prostate cancer. This study is supported by a General Research Fund from the Research Grants Council of Hong Kong (Project code: 2140629). Citation Format: Zhenyu Xu, Shan Yu, Franky Leung Chan. Orphan nuclear receptor estrogen-related receptor alpha (ERRα) characterized as a novel transcriptional regulator of the oncogenic fusion gene TMPRSS2:ERG in prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1385. doi:10.1158/1538-7445.AM2014-1385

Orphan nuclear receptor TLX contributes to androgen insensitivity in castration-resistant prostate cancer via its repression of androgen receptor transcription

The metastatic castration-resistant prostate cancer (CRPC) is a lethal form of prostate cancer, in which the expression of androgen receptor (AR) is highly heterogeneous. Indeed, lower AR expression and attenuated AR signature activity is shown in CRPC tissues, especially in the subset of neuroendocrine prostate cancer (NEPC) and prostate cancer stem-like cells (PCSCs). However, the significance of AR downregulation in androgen insensitivity and de-differentiation of tumor cells in CRPC is poorly understood and much neglected. Our previous study shows that the orphan nuclear receptor TLX (NR2E1), which is upregulated in prostate cancer, plays an oncogenic role in prostate carcinogenesis by suppressing oncogene-induced senescence. In the present study, we further established that TLX exhibited an increased expression in metastatic CRPC. Further analyses showed that overexpression of TLX could confer resistance to androgen deprivation and anti-androgen in androgen-dependent prostate cancer cells in vitro and in vivo, whereas knockdown of endogenous TLX could potentiate the sensitivity to androgen deprivation and anti-androgen in prostate cancer cells. Our study revealed that the TLX-induced resistance to androgen deprivation and anti-androgen was mediated through its direct suppression of AR gene transcription and signaling in both androgen-stimulated and -unstimulated prostate cancer cells. We also characterized that TLX could bind directly to AR promoter and repress AR transcription by recruitment of histone modifiers, including HDAC1, HDAC3, and LSD1. Together, our present study shows, for the first time, that TLX can contribute to androgen insensitivity in CRPC via repression of AR gene transcription and signaling, and also implicates that targeting the druggable TLX may have a potential therapeutic significance in CRPC management, particularly in NEPC and PCSCs.

Nuclear receptor ERRα and transcription factor ERG form a reciprocal loop in the regulation of TMPRSS2:ERG fusion gene in prostate cancer

The TMPRSS2:ERG (T:E) fusion gene is generally believed to be mainly regulated by the activated androgen receptor (AR) signaling in androgen-dependent prostate cancer. However, its persistent expression in castration-resistant and neuroendocrine prostate cancers implies that other transcription factors might also regulate its expression. Here, we showed that up-regulation of nuclear receptor estrogen-related receptor alpha (ERRα) was closely associated with the oncogenic transcription factor ERG expression in prostate cancer, and their increased coexpression patterns were closely associated with high Gleason scores and metastasis in patients. Both ERRα and ERG exhibited a positive expression correlation in a castration-resistant prostate cancer (CRPC) xenograft model VCaP-CRPC. We showed that ERRα could directly transactivate T:E fusion gene in both AR-positive and -negative prostate cancer cells via both ERR-binding element- and AR-binding element-dependent manners. Ectopic T:E expression under ERRα regulation could promote both in vitro invasion and in vivo metastasis capacities of AR-negative prostatic cells. Intriguingly, ERG expressed by the T:E fusion could also transactivate the ERRα (ESRRA) gene. Hereby, ERRα and ERG can synergistically regulate each other and form a reciprocal regulatory loop to promote the advanced growth of prostate cancer. Inhibition of ERRα activity by ERRα inverse agonist could suppress T:E expression in prostate cancer cells, implicating that targeting ERRα could be a potential therapeutic strategy for treating the aggressive T:E-positive prostate cancer.

Nuclear Receptor LRH-1 Functions to Promote Castration-Resistant Growth of Prostate Cancer via Its Promotion of Intratumoral Androgen Biosynthesis

Targeting of steroidogenic enzymes (e.g., abiraterone acetate targeting CYP17A1) has been developed as a novel therapeutic strategy against metastatic castration-resistant prostate cancer (CRPC). However, resistance to steroidal inhibitors inevitably develops in patients, the mechanisms of which remain largely unknown. Liver receptor homolog-1 (LRH-1, NR5A2) is a nuclear receptor, originally characterized as an important regulator of some liver-specific metabolic genes. Here, we report that LRH-1, which exhibited an increased expression pattern in high-grade prostate cancer and CRPC xenograft models, functions to promote de novo androgen biosynthesis via its direct transactivation of several key steroidogenic enzyme genes, elevating intratumoral androgen levels and reactivating AR signaling in CRPC xenografts as well as abiraterone-treated CRPC tumors. Pharmacologic inhibition of LRH-1 activity attenuated LRH-1-mediated androgen deprivation and anti-androgen resistance of prostate cancer cells. Our findings not only demonstrate the significant role of LRH-1 in the promotion of intratumoral androgen biosynthesis in CRPC via its direct transcriptional control of steroidogenesis, but also suggest targeting LRH-1 could be a potential therapeutic strategy for CRPC management.Significance: These findings not only demonstrate the significant role of the nuclear receptor LRH-1 in the promotion of intratumoral androgen biosynthesis in CRPC via its direct transcriptional control of steroidogenesis, but also suggest targeting LRH-1 could be a potential therapeutic strategy for CRPC management. Cancer Res; 78(9); 2205-18. ©2018 AACR.