LncRNA-SERB promotes vasculogenic mimicry (VM) formation and tumor metastasis in renal cell carcinoma.

A growing body of evidence shows that vasculogenic mimicry (VM) is closely related to the invasion and metastasis of many tumor cells. Although the estrogen receptor (ER) can promote initiation and progression of renal cell carcinoma (RCC), how the downstream biomolecules are involved, and the detailed mechanisms of how ER expression is elevated in RCC remain to be further elucidated. Here, we discovered that long noncoding RNA (LncRNA)-SERB is highly expressed in tumor cells of RCC patients. We used multiple RCC cells and an in vivo mouse model for our study, and results indicated that LncRNA-SERB could boost RCC VM formation and cell invasion in vitro and in vivo. Although a previous report showed that ERß can affect the VM formation in RCC, it is unclear which factor could upregulate ERß. This is the first study to show LncRNA-SERB can be the upstream regulator of ERß to control RCC progression. Mechanistically, LncRNA-SERB may increase ERß via binding to the promoter area, and ERß functions through transcriptional regulation of zinc finger E-box binding homeobox 1 (ZEB1) to regulate VM formation. These results suggest that LncRNA-SERB promotes RCC cell VM formation and invasion by upregulating the ERß/ZEB1 axis and that therapeutic targeting of this newly identified pathway may better inhibit RCC progression.

The androgen receptor in bladder cancer.

Bladder cancer is the ninth most common cancer worldwide with a striking sex-based difference in incidence. Emerging evidence indicates that the androgen receptor (AR) might promote the development, progression and recurrence of bladder cancer, contributing to the observed sex differences. Targeting androgen-AR signalling has promise as potential therapy for bladder cancer and helps to suppress progression of this disease. In addition, the identification of a new membrane AR and AR-regulated non-coding RNAs has important implications for bladder cancer treatment. The success of human clinical trials of targeted-AR therapies will help in the development of improved treatments for patients with bladder cancer.

Targeting the radiation-induced ARv7-mediated circNHS/miR-512-5p/XRCC5 signaling with Quercetin increases prostate cancer radiosensitivity.

BACKGROUND: Radiation therapy (RT) with androgen deprivation therapy (ADT) is an effective therapy to suppress the locally advanced prostate cancer (PCa). However, we unexpectedly found that RT could also induce the androgen receptor splice variant 7 (ARv7) expression to decrease the radiosensitivity. METHODS: The study was designed to target ARv7 expression with Quercetin or ARv7-shRNA that leads to enhancing and increasing the radiation sensitivity to better suppress the PCa that involved the modulation of the circNHS/miR-512-5p/XRCC5 signaling. RESULTS: Mechanism studies revealed that RT-induced ARv7 may function via altering the circNHS/miR-512-5p/XRCC5 signaling to decrease the radiosensitivity. Results from preclinical studies using multiple in vitro cell lines and in vivo mouse models concluded that combining RT with the small molecule of Quercetin to target full-length AR and ARv7 could lead to better efficacy to suppress PCa progression. CONCLUSION: Together, these results suggest that ARv7 may play key roles to alter the PCa radiosensitivity, and targeting this newly identified ARv7 mediated circNHS/miR-512-5p/XRCC5 signaling with Quercetin may help physicians to develop a novel RT to better suppress the progression of PCa.

Targeting the androgen receptor to enhance NK cell killing efficacy in bladder cancer by modulating ADAR2/circ_0001005/PD-L1 signaling.

Although androgen receptor (AR) can influence bladder cancer (BCa) initiation and progression, its impact on tumor immune escape remains unclear. Here, we found that targeting AR could enhance natural killer (NK) cell tumor-killing efficacy by decreasing PD-L1 expression. Both antiandrogen treatment and AR knockdown effectively reduced membrane PD-LI expression to facilitate NK cell-mediated BCa cell killing by downregulating circ_0001005. Mechanistically, AR upregulated circRNA circ_0001005 expression via the RNA-editing gene ADAR2. circ_0001005 competitively sponged the miRNA miR-200a-3p to promote PD-L1 expression. A preclinical BCa xenograft mouse model further confirmed this newly identified signaling using the small molecule circ_0001005-shRNA to improve NK cell killing of BCa tumor cells. Collectively, these results suggest that targeting the newly identified ADAR2/circ_0001005/miR-200a-3p/PD-L1 pathway to impact antitumor immunity may suppress progression and boost immunotherapeutic efficacy in BCa.

Estrogen and G protein-coupled estrogen receptor accelerate the progression of benign prostatic hyperplasia by inducing prostatic fibrosis.

Benign prostatic hyperplasia (BPH) is the most common and progressive urological disease in elderly men worldwide. Epidemiological studies have suggested that the speed of disease progression varies among individuals, while the pathophysiological mechanisms of accelerated clinical progression in some BPH patients remain to be elucidated. In this study, we defined patients with BPH as belonging to the accelerated progressive group (transurethral resection of the prostate [TURP] surgery at ≤50 years old), normal-speed progressive group (TURP surgery at ≥70 years old), or non-progressive group (age ≤50 years old without BPH-related surgery). We enrolled prostate specimens from the three groups of patients and compared these tissues to determine the histopathological characteristics and molecular mechanisms underlying BPH patients with accelerated progression. We found that the main histopathological characteristics of accelerated progressive BPH tissues were increased stromal components and prostatic fibrosis, which were accompanied by higher myofibroblast accumulation and collagen deposition. Mechanism dissection demonstrated that these accelerated progressive BPH tissues have higher expression of the CYP19 and G protein-coupled estrogen receptor (GPER) with higher estrogen biosynthesis. Estrogen functions via GPER/Gαi signaling to modulate the EGFR/ERK and HIF-1α/TGF-ß1 signaling to increase prostatic stromal cell proliferation and prostatic stromal fibrosis. The increased stromal components and prostatic fibrosis may accelerate the clinical progression of BPH. Targeting this newly identified CYP19/estrogen/GPER/Gαi signaling axis may facilitate the development of novel personalized therapeutics to better suppress the progression of BPH.

Targeting circDGKD Intercepts TKI's Effects on Up-Regulation of Estrogen Receptor ß and Vasculogenic Mimicry in Renal Cell Carcinoma.

Vasculogenic mimicry (VM) has been reported as an alternative channel to increase tumor nutrient supplies and accelerate tumor progression, and is associated with poor survival prognosis in multiple cancers, including renal cell carcinoma (RCC). The currently used anti-angiogenic treatment for metastatic RCC, sunitinib, a tyrosine kinase inhibitor (TKI), has been reported to induce VM formation. Previously we identified that the estrogen receptor ß (ERß) functions as an oncogenic factor to promote RCC progression, supported by the analytic results from The Cancer Genome Atlas (TCGA) database. We have also found evidence that sunitinib induces RCC VM formation by up-regulating ERß expression. In this study, we further demonstrated that treatment with sunitinib, as well as axitinib, another TKI, could induce ERß expression in RCC cell lines. Clinical clear cell RCC (ccRCC) patients with higher ERß expression are more likely to be found VE-cadherin positive and VM positive. Mechanism dissection showed that TKI- induced ERß transcriptionally up-regulates the circular RNA of DGKD (circDGKD, hsa_circ_0058763), which enhances VE-cadherin expression by sponging the microRNA miR-125-5p family. Targeting circDGKD intercepts sunitinib-pretreatment-induced RCC VM formation, reduces metastases and improves survival in an experimental orthotopic animal model. Targeting ERß/circDGKD signals may improve the TKI efficacy and provide novel combination therapies for metastatic RCC.

Estrogen receptor beta increases clear cell renal cell carcinoma stem cell phenotype via altering the circPHACTR4/miR-34b-5p/c-Myc signaling.

Early clinical studies indicated that estrogen receptor beta (ERß) might play key roles to impact the progression of clear cell renal cell carcinoma (ccRCC). The detailed molecular mechanisms, however, remain unclear. Here, we found ERß could increase the cancer stem cell (CSC) population via altering the circPHACTR4/miR-34b-5p/c-Myc signaling. Mechanism dissection revealed that ERß could suppress circular RNA PHACTR4 (circPHACTR4) expression via direct binding to the estrogen response elements (EREs) on the 5' promoter region of its host gene, phosphatase and actin regulator 4 (PHACTR4) to decrease miR-34b-5p expression. The decreased miRNA-34b-5p could then increase c-Myc mRNA translation via targeting its 3' untranslated region (3' UTR). The in vivo mouse model with subcutaneous xenografts of ccRCC cells also validated the in vitro data. Importantly, analysis results from ccRCC TCGA database and our clinical data further confirmed the above in vitro/in vivo data. Together, these results suggest that ERß may increase CSC population in ccRCC via altering ERß/circPHACTR4/miR-34b-5p/c-Myc signaling and that targeting this newly identified signal pathway may help physicians to better suppress ccRCC progression.

Sunitinib increases the cancer stem cells and vasculogenic mimicry formation via modulating the lncRNA-ECVSR/ERß/Hif2-α signaling.

Sunitinib is the first-line drug for treating renal cell carcinoma (RCC), and it functions mainly through inhibition of tumor angiogenesis. However, the patients may become insensitive or develop resistance toward sunitinib treatment, but the underlying mechanisms have not yet been fully elucidated. Herein, it was found that sunitinib could have adverse effects of promoting RCC progression by increasing vascular mimicry (VM) formation of RCC cells. Mechanism dissection revealed that sunitinib can increase the expression of a long non-coding RNA (lncRNA), lncRNA-ECVSR, thereby enhancing the stability of estrogen receptor ß (ERß) mRNA. Subsequently, the increased ERß expression can then function via transcriptional up-regulation of Hif2-α. Notably, sunitinib-increased lncRNA-ECVSR/ERß/Hif2-α signaling resulted in an increased cancer stem cell (CSC) phenotype, thereby promoting VM formation. Furthermore, the sunitinib/lncRNA-ECVSR-increased ERß expression can transcriptionally regulate lncRNA-ECVSR expression via a positive-feedback loop. Supportively, preclinical studies using RCC mouse xenografts demonstrated that combining sunitinib with the small molecule anti-estrogen PHTPP can increase sunitinib efficacy with reduced VM formation. Collectively, the findings of this study may aid in the development of potential biomarker(s) and novel therapies to better monitor and suppress RCC progression.

R-2HG downregulates ERα to inhibit cholangiocarcinoma via the FTO/m6A-methylated ERα/miR16-5p/YAP1 signal pathway.

Isocitrate dehydrogenase (IDH) mutations increase (R)-2-hydroxyglutarate (R-2HG) production; however, functional mechanisms of R-2HG in regulating cholangiocarcinoma (CCA) development remain to be further investigated. We first applied the CRISPR-Cas9 gene-editing system to create IDH1R132H-mutated CCA cells. Interestingly, our data showed that R-2HG could function through downregulating estrogen receptor alpha (ERα) and Yes-associated protein 1 (YAP1) pathways to decrease CCA growth. Detailed mechanistic studies revealed that R-2HG could target and degrade the fat mass and obesity-associated protein (FTO), the first identified mRNA demethylase. This reduced FTO can increase the N 6-methyladenosine (m6A) to methylate the mRNA of ERα, and consequently decrease protein translation of the ERα. Further mechanistic studies revealed that ERα could transcriptionally suppress miR-16-5p expression, which could then increase YAP1 expression due to the reduced miR-16-5p binding to the 3' UTR of YAP1. Furthermore, data from the pre-clinical animal model with implantation of IDH1R132H QBC939 cells demonstrated that R-2HG generated by the IDH1 mutation could downregulate ERα and YAP1 to suppress CCA tumor growth. Taken together, our new findings suggested that IDH1 mutation-induced R-2HG could suppress CCA growth via regulating the FTO/m6A-methylated ERα/miR16-5p/YAP1 signaling pathway. Upregulating R-2HG or downregulating the ERα signal by short hairpin RNA ERα (shERα) or antiestrogen could be effective strategies to inhibit CCA.

Targeting androgen receptor (AR) with antiandrogen Enzalutamide increases prostate cancer cell invasion yet decreases bladder cancer cell invasion via differentially altering the AR/circRNA-ARC1/miR-125b-2-3p or miR-4736/PPARγ/MMP-9 signals.

Androgen-deprivation therapy (ADT) via targeting androgens/androgen receptor (AR) signals may suppress cell proliferation in both prostate cancer (PCa) and bladder cancer (BCa), yet its impact on the cell invasion of these two urological cancers remains unclear. Here we found targeting androgens/AR with either the recently developed antiandrogen Enzalutamide (Enz) or AR-shRNAs led to increase PCa cell invasion, yet decrease BCa cell invasion. Mechanistic dissection revealed that suppressing androgens/AR signals could result in differential alterations of the selective circular RNAs (circRNAs) as a result of differential endogenous AR transcription. A negative autoregulation in PCa, yet a positive autoregulation in BCa, as a result of differential binding of AR to different androgen-response elements (AREs) and a discriminating histone H3K4 methylation, likely contributes to this outcome between these two urological tumors. Further mechanistic studies indicated that AR-encoded circRNA-ARC1 might sponge/alter the availability of the miRNAs miR-125b-2-3p and/or miR-4736, to impact the metastasis-related PPARγ/MMP-9 signals to alter the PCa vs. BCa cell invasion. The preclinical study using the in vivo mouse model confirms in vitro cell lines data, showing that Enz treatment could increase PCa metastasis, which can be suppressed after suppressing circRNA-ARC1 with sh-circRNA-ARC1. Together, these in vitro/in vivo results demonstrate that antiandrogen therapy with Enz via targeting AR may lead to either increase PCa cell invasion or decrease BCa cell invasion. Targeting these newly identified AR/circRNA-ARC1/miR-125b-2-3p and/or miR-4736/PPARγ/MMP-9 signals may help in the development of new therapies to better suppress the Enz-altered PCa vs. BCa metastasis.

Androgen receptor promotes renal cell carcinoma (RCC) vasculogenic mimicry (VM) via altering TWIST1 nonsense-mediated decay through lncRNA-TANAR.

While the androgen receptor (AR) may influence the progression of clear cell renal cell carcinoma (ccRCC), its role to impact vasculogenic mimicry (VM) to alter the ccRCC progression and metastasis remains obscure. Here, we demonstrated that elevated AR expression was positively correlated with tumor-originated vasculogenesis in ccRCC patients. Consistently, in vitro research revealed AR promoted VM formation in ccRCC cell lines via modulating lncRNA-TANAR/TWIST1 signals. Mechanism dissection showed that AR could increase lncRNA-TANAR (TANAR) expression through binding to the androgen response elements (AREs) located in its promoter region. Moreover, we found that TANAR could impede nonsense-mediated mRNA decay (NMD) of TWIST1 mRNA by direct interaction with TWIST1 5'UTR. A preclinical study using in vivo mouse model with orthotopic xenografts of ccRCC cells further confirmed the in vitro data. Together, these results illustrated that AR-mediated TANAR signals might play a crucial role in ccRCC VM formation and metastasis, and targeting this newly identified AR/TANAR/TWIST1 signaling may help in the development of a novel anti-angiogenesis therapy to better suppress the ccRCC progression.

ASC-J9® suppresses prostate cancer cell proliferation and invasion via altering the ATF3-PTK2 signaling.

BACKGROUND: Early studies indicated that ASC-J9®, an androgen receptor (AR) degradation enhancer, could suppress the prostate cancer (PCa) progression. Here we found ASC-J9® could also suppress the PCa progression via an AR-independent mechanism, which might involve modulating the tumor suppressor ATF3 expression. METHODS: The lentiviral system was used to modify gene expression in C4-2, CWR22Rv1 and PC-3 cells. Western blot and Immunohistochemistry were used to detect protein expression. MTT and Transwell assays were used to test the proliferation and invasion ability. RESULTS: ASC-J9® can suppress PCa cell proliferation and invasion in both PCa C4-2 and CWR22Rv1 cells via altering the ATF3 expression. Further mechanistic studies reveal that ASC-J9® can increase the ATF3 expression via decreasing Glutamate-cysteine ligase catalytic (GCLC) subunit expression, which can then lead to decrease the PTK2 expression. Human clinical studies further linked the ATF3 expression to the PCa progression. Preclinical studies using in vivo mouse model also proved ASC-J9® could suppress AR-independent PCa cell invasion, which could be reversed after suppressing ATF3. CONCLUSIONS: ASC-J9® can function via altering ATF3/PTK2 signaling to suppress the PCa progression in an AR-independent manner.

Preclinical studies using cisplatin/carboplatin to restore the Enzalutamide sensitivity via degrading the androgen receptor splicing variant 7 (ARv7) to further suppress Enzalutamide resistant prostate cancer.

The FDA-approved anti-androgen Enzalutamide (Enz) has been used successfully as the last line therapy to extend castration-resistant prostate cancer (CRPC) patients' survival by an extra 4.8 months. However, CRPC patients eventually develop Enz-resistance that may involve the induction of the androgen receptor (AR) splicing variant ARv7. Here we found that Cisplatin (Cis) or Carboplatin, currently used in chemotherapy/radiation therapy to suppress tumor progression, could restore the Enz sensitivity in multiple Enz-resistant (EnzR) CRPC cells via directly degrading/suppressing the ARv7. Combining Cis or Carboplatin with Enz therapy can also delay the development of Enz-resistance in CRPC C4-2 cells. Mechanism dissection found that Cis or Carboplatin might decrease the ARv7 expression via multiple mechanisms including targeting the lncRNA-Malat1/SF2 RNA splicing complex and increasing ARv7 degradation via altering ubiquitination. Preclinical studies using in vivo mouse model with implanted EnzR1-C4-2 cells also demonstrated that Cis plus Enz therapy resulted in better suppression of EnzR CRPC progression than Enz treatment alone. These results not only unveil the previously unrecognized Cis mechanism to degrade ARv7 via targeting the Malat1/SF2 complex and ubiquitination signals, it may also provide a novel and ready therapy to further suppress the EnzR CRPC progression in the near future.

Preclinical studies show using enzalutamide is less effective in docetaxel-pretreated than in docetaxel-naïve prostate cancer cells.

Anti-androgen therapy with Enzalutamide (Enz) has been used as a therapy for castration resistant prostate cancer (CRPC) patients after development of resistance to chemotherapy with Docetaxel (Doc). The potential impacts of Doc-chemotherapy on the subsequent Enz treatment, however, remain unclear. Here we found the overall survival rate of patients that received Enz was significantly less in patients that received prior Doc-chemotherapy than those who had not. In vitro studies from 3 established Doc resistant CRPC (DocRPC) cell lines are consistent with the clinical findings showing DocRPC patients had decreased Enz-sensitivity as well as accelerated development of Enz-resistance via enhanced androgen receptor (AR) splicing variant 7 (ARv7) expression. Mechanism dissection found that Doc treatment might increase the generation of ARv7 via altering the MALAT1-SF2 RNA splicing complex. Preclinical studies using in vivo mouse models and in vitro cell lines proved that targeting the MALAT1/SF2/ARv7 axis with small molecules, including siMALAT1, shSF2, and shARv7 or ARv7 degradation enhancers: Cisplatin or ASC-J9®, can restore/increase the Enz sensitivity to further suppress DocRPC cell growth. Therefore, combined therapy of Doc-chemotherapy with anti-ARv7 therapy, including Cisplatin or ASC-J9®, may be developed to increase the efficacy of Enz to further suppress DocRPC in patients.

Targeting the radiation-induced TR4 nuclear receptor-mediated QKI/circZEB1/miR-141-3p/ZEB1 signaling increases prostate cancer radiosensitivity.

Early studies indicated that the testicular nuclear receptor 4 (TR4) might play key roles in altering prostate cancer (PCa) progression; however, its ability to alter PCa radiosensitivity remains unclear. Here, we found that suppressing TR4 expression promoted radiosensitivity and better suppressed PCa by modulating the protein quaking (QKI)/circZEB1/miR-141-3p/ZEB1 signaling pathway. Mechanism dissection studies revealed that TR4 could transcriptionally increase the RNA-binding protein QKI to increase circZEB1 levels, which then sponges the miR-141-3p to increase the expression of its host gene ZEB1. Preclinical studies with an in vivo mouse model further proved that combining radiation therapy (RT) with metformin promoted radiosensitivity to suppress PCa progression. Together, these results suggest that TR4 may play key roles in altering PCa radiosensitivity and show that targeting this newly identified TR4-mediated QKI/circZEB1/miR-141-3p/ZEB1 signaling pathway may help in the development of a novel RT to better suppress the progression of PCa.