Prostate Cancer Tumorigenesis Research Articles

PDZRN4 suppresses tumorigenesis and androgen therapy-resistance in prostate cancer.

Background: PDZRN4 (PDZ domain-containing RING finger 4), a member of the LNX (ligand of numb protein-X) family that regulates the levels of NUMB, plays a critical role in suppressing the proliferation and invasion of hormone-related malignant tumours. There are few studies on the role of PDZRN4 in the pathogenesis of prostate cancer (PCa). We aimed to examine whether PDZRN4 regulates the growth and development of PCa.Methods: Cell transduction and Western blotting were used to establish and confirm PDZRN4 knock down in PC cells. Using the MTT, wound healing, transwell migration, and animal experiments, we explored the biological function of PDZRN4 knockdown (PDZRN4-kd) cells. Via PCR and immunohistochemistry, the mRNA and protein expression of PDZRN4 was examined in PC cells and tissues.Results: Hormone-dependent (LNCap) and hormone-independent (DU145, PC3, and C4-2) PC lines were transfected with lentivirus carrying PDZRN4 shRNA. The Western blotting results showed that the expression of PDZRN4 was stably downregulated in PDZRN4 knockdown (PDZRN4-kd) cells. The proliferation, invasion and migration of PDZRN4-kd cells were dramatically increased in vivo. To explore the expression of PDZRN4 in prostate cancer samples, we analysed TCGA data and found that PDZRN4 was negatively correlated with the development of PC. PDZRN4 levels were downregulated by androgen deprivation in hormone-sensitive cells. Moreover, PDZRN4 failed to induce proliferation in DU145 cells with androgen deprivation.Conclusions: PDZRN4 is a functional suppressor of prostate cancer growth and development and is a potential target of biochemical therapy in hormone-resistant PC.

E2F transcription factor 2-activated DLEU2 contributes to prostate tumorigenesis by upregulating serum and glucocorticoid-induced protein kinase 1

Long noncoding RNAs (lncRNAs) participate in biological processes in multiple types of tumors. However, the regulatory patterns of lncRNAs in prostate cancer remain largely unclear. Here, we evaluated the expression and roles of the lncRNA DLEU2 in prostate cancer. Our results showed that DLEU2 was upregulated in advanced prostate cancer tissues. Patients with prostate cancer displaying high expression of DLEU2 had a poor prognosis. Moreover, we demonstrated that overexpression of DLEU2 facilitated the proliferation, migration, and invasion of prostate cancer in vitro. Mechanistically, DLEU2 promoted serum and glucocorticoid-induced protein kinase 1 (SGK1) expression by acting as an miR-582-5p sponge, and the transcription of DLEU2 was activated by the dysregulation of E2F transcription factor 2 (E2F2) expression in prostate cancer. Furthermore, knockdown of DLEU2 attenuated prostate cancer tumorigenesis in vivo. Notably, these findings suggested that E2F2-activated DLEU2 may function as a competing endogenous RNA to facilitate prostate cancer progression by targeting the miR-582-5p/SGK1 axis.

Melatonin decreases androgen-sensitive prostate cancer growth by suppressing SENP1 expression.

BackgroundMelatonin is a hormone naturally produced by the pineal gland in the brain. In addition to modulating circadian rhythms, it has pleiotropic biological effects including antioxidant, immunomodulatory, and anti-cancer effects. Herein, we report that melatonin has the ability to decrease the growth and metastasis of androgen-dependent prostate cancer.MethodsTo evaluate the anti-cancer effect of melatonin on androgen-sensitive prostate cancer in vitro or in vivo, the effects of cell proliferation, apoptosis, migration and invasion were analyzed by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), colony formation, flow cytometry, Transwell assay, and immunohistochemistry (IHC), respectively. Next, the interaction between androgen receptor (AR) and SUMO specific protease 1 (SENP1) was detected by real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and western blotting, and confirmed by luciferase reporter assay. Furthermore, the Small Ubiquitin-like Modifier (SUMO) proteins are a group of small proteins that are covalently attached to and detached from other proteins in cells to modify their function. (SUMOylation) of histone deacetylases 1 (HDAC1) was measured by proximity ligation assay (PLA).ResultsThe treatment of melatonin cripples the transcriptional activity of AR, which is essential for the growth of the androgen-dependent prostate cancer cell, LNCaP. The lower activity of AR was dependent on melatonin induced SUMOylation of HDAC1, which has been established as a key factor for the transcriptional activity of AR. Mechanistically, the effect of melatonin on AR was due to the decreased SENP1 protein level and the subsequent increased HDAC1 SUMOylation level. The overexpression of SENP1 abrogated the anti-cancer ability of melatonin on LNCaP cells.ConclusionsThese findings indicate that melatonin is a suppressor of androgen-dependent prostate cancer tumorigenesis.

LINC01207 promotes prostate cancer progression by sponging miR-1182 to upregulate AKT3.

Prostate cancer (PC) is recognized as a common malignancy in male patients. Long non-coding RNA (lncRNA) has been implicated in the development of PC. Recently, long intergenic non-protein coding RNA 1207 (LINC01207) has been reported to regulate the carcinogenesis of multiple cancer types. However, its role in the progression of PC remains to be determined. The aim of the present study was to investigate the expression profile, clinicopathological implication and molecular mechanism of action of LINC01207 in the progression of PC. LINC01207 expression levels were compared between PC tumor and paired normal tissue samples from The Cancer Genome Atlas. The expression of LINC01207 was further analyzed in PC cell lines and a normal prostatic cell line. The role of LINC01207 in proliferation, migration and invasion of PC cells was examined using small interfering RNA-mediated silencing. Western blot analysis was used to investigate the changes in protein levels underlying the mechanism of action of LINC01207. The role of LINC01207 in tumorigenesis was evaluated in a xenograft model. LINC01207 was upregulated in PC tumor samples from TCGA data compared with paired normal tissue. LINC01207 expression was significantly increased in PC cells and tumor tissues compared with in normal prostate cells (RWPE1) and normal prostate tissues, respectively. Furthermore, LINC01207 silencing inhibited PC cell proliferation and colony formation and induced apoptosis. Mechanistic experiments showed that LINC01207 promoted carcinogenesis by sponging miR-1182 to regulate the protein levels of AKT3 in PC cell lines. Thus, the findings of the present study indicated that LINC01207 might play a role in the tumorigenesis of PC and may serve as a therapeutic target for PC treatment.

Comprehensive analysis of the functional and prognostic value of E2F transcription factors in human prostate cancer through data mining and experimental validation.

BackgroundA growing body of evidence shows that E2F transcription factors play a significant role in the tumorigenesis of prostate cancer. However, their functional and prognostic value has not been fully illustrated. Therefore, we used bioinformatics methods to further analyze the possible roles of E2F transcription factors in the development and progression of prostate cancer.MethodsWe explored the expression levels of E2F transcription factors using data from The Cancer Genome Atlas (TCGA) and Oncomine database in paired and unpaired samples. The clinical correlation and prognostic value of E2F transcription factors were assessed. Using the R package “pROC”, we judged the diagnostic value of E2F transcription factors. The online website tool cBioPortal was also employed to find possible gene alterations of E2F transcription factors in samples from TCGA. The R package “clusterprofiler” was used to conduct functional analysis. Moreover, we also used the Tumor Immune Estimation Resource to search for the associations between E2F transcription factors and the infiltration levels of 6 kinds of immune cells. Finally, quantitative real-time polymerase chain reaction (PCR) was conducted to validate the expression levels of E2F transcription factors in human paired prostate tissues.ResultsE2F1/2/3/5 messenger RNA (mRNA) expression levels were higher in prostate cancer tissues than in normal tissues, while E2F4 and E2F6 mRNA expression levels were lower (P<0.05). All E2F transcription factors were associated with clinical parameters. Kaplan-Meier analysis revealed that E2F1/4/6/8 were notably associated with the overall survival of patients with prostate cancer (P<0.05). Receiver operating characteristic (ROC) curve results showed that except for E2F7, the other E2F transcription factors had diagnostic value for prostate cancer (P<0.05). We further found close associations between E2F transcription factors and the infiltration levels of immune cells. The results of quantitative real-time PCR were consistent with those from public databases.ConclusionsE2F transcription factor family members are differentially expressed in prostate cancer and are significantly related to the prognosis of patients, suggesting that they may be adopted as biomarkers for prognosis prediction and the treatment of prostate cancer.

Knockdown of microRNA-214-3p Promotes Tumor Growth and Epithelial-Mesenchymal Transition in Prostate Cancer.

Simple SummaryProstate Cancer is the second leading cause of cancer-related deaths in the United States. In this study, we analyzed a molecule known as a microRNA, which regulates the expression of genes. microRNAs are involved in processes related to cancer onset and progression. Abnormal expression of microRNAs can promote prostate cancer. This study showed that knockdown of microRNA miR-214-3p enhanced the progression and of prostate cancer. In addition, miR-214 regulated the expression of many genes. These results are useful to better understand the function of miR-214-3p in prostate cancer and can be a useful target in the treatment of the disease.Abnormal expression of microRNA miR-214-3p (miR-214) is associated with multiple cancers. In this study, we assessed the effects of CRISPR/Cas9 mediated miR-214 depletion in prostate cancer (PCa) cells and the underlying mechanisms. Knockdown of miR-214 promoted PCa cell proliferation, invasion, migration, epithelial-mesenchymal transition (EMT), and increased resistance to anoikis, a key feature of PCa cells that undergo metastasis. The reintroduction of miR-214 in miR-214 knockdown cells reversed these effects and significantly suppressed cell proliferation, migration, and invasion. These in vitro studies are consistent with the role of miR-214 as a tumor suppressor. Moreover, miR-214 knockout increased tumor growth in PCa xenografts in nude mice supporting its anti-oncogenic role in PCa. Knockdown of miR-214 increased the expression of its target protein, Protein Tyrosine Kinase 6 (PTK6), a kinase shown to promote oncogenic signaling and tumorigenesis in PCa. In addition, miR-214 modulated EMT as exhibited by differential regulation of E-Cadherin, N-Cadherin, and Vimentin both in vitro and in vivo. RNA-seq analysis of miR-214 knockdown cells revealed altered gene expression related to PCa tumor growth pathways, including EMT and metastasis. Collectively, our findings reveal that miR-214 is a key regulator of PCa oncogenesis and is a potential novel therapeutic target for the treatment of the disease.

Transcription factor p53-mediated activation of miR-519d-3p and downregulation of E2F1 attenuates prostate cancer growth and metastasis.

Prostate cancer (PCa) is a commonly diagnosed malignancy in men. The transcription factor p53, a well-known cancer suppressor, has been extensively analyzed in the progression of many tumor types, but its involvement in PCa remains not fully understood. Hence, this study aims to explore the possible molecular mechanism underlying p53 in the growth and metastasis of PCa. Based on bioinformatics analysis findings of GEPIA and starBase databases, p53 was demonstrated to be involved in the development of PCa by transcriptionally activating microRNA-519d-3p (miR-519d-3p) expression to suppress the expression of E2F transcription factor 1 (E2F1) and CD147. In order to verify this finding, clinically-obtained PCa tumor tissues were enrolled and commercially-purchased PCa cell lines were used to detect the cell viability, cycle, and apoptosis, as well as invasion and migration by CCK-8, flow cytometry, and Transwell assays respectively. The results of clinical tissue experiments and in vitro cell experiments showed that miR-519d-3p and p53 were poorly-expressed in PCa tissues and cell lines, while E2F1 was highly-expressed. Overexpression of miR-519d-3p led to inhibited PCa cell proliferation, invasion and migration, and p53 overexpression was found to promote miR-519d-3p expression to suppress the malignant characteristics of PCa cells, while the additional E2F1 overexpression restored the malignant traits. Moreover, ChIP analysis and dual-luciferase reporter assay confirmed the interactions among p53, miR-519d-3p, and E2F1. Mechanistically, it was found that p53 transcriptionally activated miR-519d-3p to suppress E2F1 expression. Finally, the in vitro results were further validated by in vivo experiments, which showed that miR-519d-3p prevents tumorigenesis and lymph node metastasis of PCa in nude mice via negatively regulation of E2F1 and CD147. Taken together, the findings uncover that the transcription factor p53 could upregulate miR-519d-3p expression to directly suppress the expression of E2F1, thus inhibiting PCa growth and metastasis. It highlights a novel therapeutic strategy against PCa based on the p53/miR-519d-3p/E2F1 regulatory pathway.

Evaluation of G2013 (α-L-guluronic acid) efficacy on PC-3 cells through inhibiting the expression of inflammatory factors.

Given multiple treatment strategies for prostate cancer, its mortality rate is still high; therefore, novel treatment strategies seem necessary. G2013 or α-L-guluronic acid is a new patented drug with immunomodulatory and anti-inflammatory properties. This study aimed to evaluate the property of G2013 on inflammatory molecules involved in tumorigenesis of prostate cancer. MTT assay was used to assess the effect of the drug on the proliferation of PC-3 cells. Expression of interleukin 8 (IL-8), Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), myeloid differentiation factor 88 (MYD-88), cyclooxygenase 2 (COX-2), matrix metalloproteinase-2 (MMP-2), and MMP-9genes were studied in the PC-3 cells treated with 25 (low dose) or 50 (high dose) µg/mL of G2013 for 24h using quantitative real-time polymerase chain reaction (qRT-PCR) technique. Protein expression of NF-κB and protein activities of MMP-2 and MMP-9 were assayed using flow cytometry and gelatin zymography, respectively. The expression of COX-2 (p=0.007 at low dose), MMP-2 (p=0.023 at low dose, p=0.002 at high dose), NF-κB (p=0.004 at low dose) and IL-8 (p<0.0001 in both doses) genes, NF-κB protein (p<0.0001 in both doses), and MMP-2 activity (p<0.0001 in both doses) were significantly reduced in the presence of G2013 as compared to the control group. Cancer cell proliferation was also inhibited under 10-500µg/mL G2013 treatment. Our results revealed that G2013has the potential to inhibit PC-3 cell proliferation and reduce the expression of tumour-promoting mediators, COX-2, MMP-2, NF-κB, and IL-8 involved in the progression and metastasis of prostate cancer.

Dissecting multiple roles of SUMOylation in prostate cancer.

Protein modification with small ubiquitin-like modifiers (SUMOs) plays dual roles in prostate cancer (PCa) tumorigenesis and development. Any intermediary of the SUMO conjugation cycle going awry may forfeit the balance between tumorigenic potential and anticancer effects. Deregulated SUMOylation on the androgen receptor and oncoproteins also takes part in this pathological process, as exemplified by STAT3/NF-κB and tumor suppressors such as PTEN and p53. Here, we outline recent developments and discoveries of SUMOylation in PCa and present an overview of its multiple roles in PCa tumorigenesis/promotion and suppression, while elucidating its potential as a therapeutic target for PCa.

Solute carrier family 16 member 5 downregulation and its methylation might serve as a prognostic indicator of prostate cancer.

Prostate cancer (PCa), characterized by high invasion, metastasis, and recurrence, is the most prevalent malignant tumor in men worldwide. A clear understanding of the underlying molecular mechanisms and their role during PCa tumorigenesis can help develop prognostic and targeted therapies. We analyzed datasets from public databases, including the Cancer Genome Atlas (TCGA) and Oncomine and Gene Expression Profiling Interactive Analysis for differential expression of solute carrier family 16 member 5 (SLC16A5). We further investigated its relationship with clinical stage, pathological grade, and prognosis of PCa. The promoter methylation level of SLC16A5 in PCa was also investigated by UALCAN. We also utilized datasets from UCSC Xena to explore the prognostic role of SLC16A5 methylation levels and CpG site. Correlations between SLC16A5 and immune infiltration were discovered through TIMER. We observed significantly lower levels of SLC16A5 mRNA in PCa relative to normal tissues across six datasets from Oncomine database (p< .001) and 498 cases from TCGA database (p<.0001). SLC16A5 is strongly negatively regulated by its DNA methylation, with a Spearman of -0.81 and Pearson of -0.80 (p <.001). The aberrant SLC16A5 expression resulted in a significant relationship with clinical stage, pathological grade, and lower SLC16A5 mRNA expression, and its hypermethylation was related to a poorer PCa prognosis. SLC16A5 acted as an important factor for PCa diagnosis, with an AUC of 0.9038 (95% CI: 0.8597-0.9479; p<.0001). Besides, the aberrant SLC16A5 expression revealed close correlations with multiple immune cells. Overall, these results indicate that decreased SLC16A5 expression might be a potential biomarker for determining prognosis and immune infiltration in PCa. The positive SLC16A5 modulation might be a promising therapeutic target for PCa.

P12 - Influence of patient-derived fibroblasts on prostate cancer tumorigenesis in-vitro

P12 - Influence of patient-derived fibroblasts on prostate cancer tumorigenesis in-vitro

MP33-12 THE POTENTIAL ROLE OF CTLH COMPLEX IN THE PROSTATE CANCER

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology I (MP33)1 Sep 2021MP33-12 THE POTENTIAL ROLE OF CTLH COMPLEX IN THE PROSTATE CANCER Mario Scarpa, Marta De Menna, Eugenio Zoni, Sofia Karkampouna, and Marianna Kruithof-de Julio Mario ScarpaMario Scarpa More articles by this author , Marta De MennaMarta De Menna More articles by this author , Eugenio ZoniEugenio Zoni More articles by this author , Sofia KarkampounaSofia Karkampouna More articles by this author , and Marianna Kruithof-de JulioMarianna Kruithof-de Julio More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002042.12AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Prostate cancer (PCa) is the most frequent malignancy detected in men and the second leading cause of cancer-related male death in Europe. Current markers frequently fail in providing an effective patient stratification. Unfortunately, lethal cases receive new therapies only when they have failed local and hormonal treatment. This project is focused on the role, in PCa progression, of Ran Binding Protein 9 (RanBP9), a key component of the mammalian macromolecular complex CTLH E3-ubiquitin ligase. This complex is characterized by well conserved CTLH domain and in DU145 cell lines its ubiquitin ligase activity increases cell radiosensitivity. All these evidences suggest that the CTLH Complex, through RanBP9, is potentially involved in the PCa tumorigenesis. METHODS: To elucidate the biological mechanism and function of CTLH in the PCa, we performed in vitro analyses using prostate cancer cell lines as: PC3, LNCaP and C42B. To study the role of the CTLH complex in vivo we are using PDXs BM18 (androgen dependent), LAPC9 (androgen-independent) and GEMM for PCa: Nkx3.1CreERT2/+; Pten flox/flox; R26LSL-YFP (NP) and Nkx3.1CreERT2/+; Pten flox/flox; KRas LSLG12D/+; R26 LSL-YFP (NPK). In particular NP and NPK animals mimic different stages of PCa tumorigenesis. After tamoxifen treatment NP animals will develop high grade PIN lesions while the NPK animals will develop prostate adenocarcinoma with lung and liver metastasis. RESULTS: The levels of RanBP9 were measured on a TMA (Tissue Micro Array) of 201 High-Risk PCa patients. It revealed that higher levels of RanBP9 was associated with PSA progression. Also, in the TMA cells expressing NKX3.1 were low compare a higher number of cells expressing RanBP9. In the GEMM models NP and NPK, RanBP9 expression is higher in the more aggressive phenotype (NP primary vs NPK primary and metastasis). Interesting, the level of RanBP9 expression were particularly higher in NPK metastasis vs NPK primary. RanBP9 levels were measured in PDXs LAPC9 and BM18 in both intact and castrated setting. Interestingly, levels of RanBP9 in both PDXs model were affected by castration. Currently, to further explore the relationship between RanBP9 and the AR axis we are generating RanBP9-KD stable clones in PC3 (AR-) and LNCaP (AR+) cell lines. CONCLUSIONS: Our results suggested that RanBP9 expression is correlated with PCa progression. The modulation of the RanBP9 expression is probably regulated by the presence of testosterone due its downregulation following castration beyond the androgen-dependent or -independent profile of PCa. Source of Funding: KFS 4960-02-2020 © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e611-e611 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Mario Scarpa More articles by this author Marta De Menna More articles by this author Eugenio Zoni More articles by this author Sofia Karkampouna More articles by this author Marianna Kruithof-de Julio More articles by this author Expand All Advertisement Loading ...

MiR-1207-5p targets PYCR1 to inhibit the progression of prostate cancer

Prostate cancer, the most common non-cutaneous male cancer, is a public health problem with a third prevalence worldwide. PYCR1 and miR-1207-5p dysregulations were found in cancer progression. Our study aims to reveal the biological role of miR-1207-5p-PYCR1 axis in prostate cancer progression. First, we investigated the expression of miR-1207-5p in prostate cancer tissues and cell lines by RT-qPCR. Next, we confirmed miR-1207-5p targeting PYCR1 by luciferase assay. CCK-8 assay, BrdU assay, flow cytometry, and tanswell assay were applied for examining cell proliferation, apoptosis, and invasion in prostate cancer cells, respectively. In the present study, decreased miR-1207-5p expression was obviously observed in prostate cancer tissues and cells. Upregulation of miR-1207-5p hampered cellular proliferation and invasion, while enhanced cellular apoptosis. In addition, upregulation of PYCR1 elevated cell proliferation and invasion, but repressed apoptosis of prostate cancer cells. Moreover, miR-1207-5p inhibited the expression of PYCR1 to repress prostate cancer tumorigenesis. MiR-1207-5p inhibited the expression of PYCR1 to repress the progression of prostate cancer by inhibiting cell growth and elevating cell apoptosis. Overall, our study clarifies the biological role of miR-1207-5p-PYCR1 axis in prostate cancer progression, which might be effective biomarkers for clinical treatment of prostate cancer.

MiR‑184 delays cell proliferation, migration and invasion in prostate cancer by directly suppressing DLX1

A number of previous studies have reported that dysregulated miR-184 expression is associated with the development of cancer. The aim of the present study was to investigate the role of miR-184 in prostate cancer (PC) and the mechanism underlying its effects. Data from human tumor tissue samples were collected from The CEancer Genome Atlas to determine the expression levels of miR-184 and DLX1. The miR-184 mimic and pcDNA3.1-DLX1 plasmid were utilized to induce overexpression of miR-184 and DLX1 in Du145 cells, respectively. Cell Counting Kit-8, wound healing and Transwell assays were performed to examine the effects of miR-184 on the aggressiveness of PC cells. Dual-luciferase reporter gene assay was used to investigate the association between miR-184 and DLX1, and reverse transcription-quantitative PCR and western blot analyses were utilized to determine the mRNA and protein levels. miR-184 expression was found to be downregulated whereas DLX1 was upregulated in PC tissues compared with normal prostate tissues. Cell propagation, migration and invasion were all inhibited by miR-184 upregulation in Du145 cells. Dual luciferase reporter assay confirmed the association between miR-184 and DLX1. The inhibitory effect of miR-184 mimic on cell behaviors was reversed by upregulation of DLX1. These findings suggest that miR-184 plays a beneficial role in suppressing the tumorigenesis of PC by directly targeting DLX1, and it may represent a potential therapeutic strategy for PC.

Antithetic hTERT Regulation by Androgens in Prostate Cancer Cells: hTERT Inhibition Is Mediated by the ING1 and ING2 Tumor Suppressors.

Simple SummaryThe expression of the catalytic subunit of the human telomerase reverse transcriptase subunit (hTERT) is hormonally controlled. Androgen treatment suppresses the hTERT expression at a transcriptional level in prostate cancer cells. Here, we identified the responsive promoter element that mediates the androgen receptor induced transrepression of hTERT. The negative androgen response element (nARE) is identified as 62 bp located in the core promoter of hTERT. Chromatin immunoprecipitations indicate an androgen-dependent recruitment of the androgen receptor (AR) ING1 and ING2 to the hTERT promoter. Interestingly, the androgen-induced transrepression is mediated by the class II tumor suppressors inhibitor of growth 1 and 2, namely ING1 and ING2, respectively.The human telomerase is a key factor during tumorigenesis in prostate cancer (PCa). The androgen receptor (AR) is a key drug target controlling PCa growth and regulates hTERT expression, but is described to either inhibit or to activate. Here, we reveal that androgens repress and activate hTERT expression in a concentration-dependent manner. Physiological low androgen levels activate, while, notably, supraphysiological androgen levels (SAL), used in bipolar androgen therapy (BAT), repress hTERT expression. We confirmed the SAL-mediated gene repression of hTERT in PCa cell lines, native human PCa samples derived from patients treated ex vivo, as well as in cancer spheroids derived from androgen-dependent or castration resistant PCa (CRPC) cells. Interestingly, chromatin immuno-precipitation (ChIP) combined with functional assays revealed a positive (pARE) and a negative androgen response element (nARE). The nARE was narrowed down to 63 bp in the hTERT core promoter region. AR and tumor suppressors, inhibitor of growth 1 and 2 (ING1 and ING2, respectively), are androgen-dependently recruited. Mechanistically, knockdown indicates that ING1 and ING2 mediate AR-regulated transrepression. Thus, our data suggest an oppositional, biphasic function of AR to control the hTERT expression, while the inhibition of hTERT by androgens is mediated by the AR co-repressors ING1 and ING2.

RNA expression differences in prostate tumors and tumor-adjacent stroma between Black and White Americans.

Prostate cancer (PCa) in Black Americans (BA) is diagnosed at an earlier median age and a more advanced stage than PCa in White Americans (WA). Tumor-adjacent stroma (TAS) plays a critical role in tumorigenesis of prostate cancer. We examined RNA expression in both tumor and TAS of BA compared to WA. After evaluating the geographical ancestry of each sample, preliminary analysis of our own RNA-seq data of 7 BA and 7 WA TAS revealed 1706 downregulated and 1844 upregulated genes in BA relative to WA PCa patients (padj < 0.05). An assessment of published RNA-seq data of clinically matched tumor-enriched tissues from 15 BA and 30 WA patients revealed 932 upregulated and 476 downregulated genes in BA relative to WA (padj < 0.05). When TAS and tumor epithelial cohorts were compared for the top 2500 statistically significant genes, immune responses were downregulated in BA vs WA TAS, while T cell-exhaustion pathways and the immune checkpoint gene CTLA4 were upregulated in BA vs WA tumors. We found fewer activated dendritic cells in tumor and more CD8 T-cells in TAS of BA versus WA PCa patients. Further characterization of these differences in the immune response of PCa patients of distinct geographical ancestry could help to improve diagnostics, prognostics, and therapy.

Insights into homeobox B9: a propeller for metastasis in dormant prostate cancer progenitor cells

BackgroundMetastasis is the major cause of treatment failure and cancer-related deaths in prostate cancer (PCa) patients. Our previous study demonstrated that a CD44+ subpopulation isolated from PCa cells or tumours possesses both stem cell properties and metastatic potential, serving as metastatic prostate cancer stem cells (mPCSCs) in PCa metastasis. However, the underlying mechanisms remain unknown.MethodsIn this study, we established PCa models via the orthotopic and subcutaneous implantation of different human PCa cancer cell lines, and compared the metastatic efficacy, after which process function analysis of target genes was pinpointed.ResultsSeveral novel differentially expressed genes (DEGs) between orthotopic and ectopic tumours were identified. Among them, human homeobox B9 (HOXB9) transcription factor was found to be essential for PCa metastasis, as evidenced by the diminished number of lung metastatic foci derived from orthotopic implantation with HOXB9-deficient CWR22 cells, compared with the control. In addition, HOXB9 protein expression was upregulated in PCa tissues, compared with paracancer and benign prostate hyperplasia tissues. It was also positively correlated with Gleason scores. Gain- and loss-of-function assays showed that HOXB9 altered the expression of various tumour metastasis- and cancer stem cell (CSC) growth-related genes in a transforming growth factor beta (TGFβ)-dependent manner. Moreover, HOXB9 was overexpressed in an ALDH+CD44+CXCR4+CD24+ subpopulation of PCa cells that exhibited enhanced TGFβ-dependent tumorigenic and metastatic abilities, compared with other isogenic PCa cells. This suggests that HOXB9 may contribute to PCa tumorigenesis and metastasis via TGFβ signalling. Of note, ALDH+CD44+CXCR4+CD24+-PCa cells exhibited resistance to castration and antiandrogen therapy and were present in human PCa tissues.ConclusionTaken together, our study identified HOXB9 as a critical regulator of metastatic mPCSC behaviour. This occurs through altering the expression of a panel of CSC growth- and invasion/metastasis-related genes via TGFβ signalling. Thus, targeting HOXB9 is a potential novel therapeutic PCa treatment strategy.

Abstract 1152: Targeted inhibition of SIRT6 by engineered exosomes suppressed tumorigenesis and metastasis in prostate cancer

Abstract The great majority of prostate cancer occurrence and development are driven by androgen receptor signaling, which has led to androgen dependent therapy (ADT) as the backbone of systemic therapy for advanced prostate cancer patients until now. Although ADT has improved clinical outcome in patients with castration-sensitive disease and the next-generation androgen receptor pathway inhibitors (ARPIs) like enzalutamide are commonly used in advanced prostate cancer patients, the cross-resistance between AR-targeted drugs significantly limits the therapeutic effect. Besides, early use and potentially long exposure to ARPIs preferentially lead to the evolvement of the alternative downstream signaling instead of AR pathway. Hence, targeting the AR fails to develop inescapable drug resistance, and no effective approach is available for targeting metastatic and castration-resistant prostate cancer (mCRPC), which is a major clinical challenge in prostate cancer treatment. Undoubtedly, new targets and therapeutic strategies are needed to improve the clinical outcomes. Sirt6 belongs to the Sirtuin family proteins, and as a nuclear protein, regulates diverse physiological functions like aging and metabolism. Silencing SIRT6 could inhibit prostate cancer proliferation, and in this study, we further exploited this phenomenon. We found that expression of SIRT6 was positively related with the Gleason score of the patients' prostate tissue sample and knockdown the expression of SIRT6 in prostate cancer cell lines significantly reduced migration and invasion both in vitro and in vivo. Furthermore, SIRT6 deficiency upregulated the expression of E-cadherin and downregulated the expression of N-cadherin and Vimentin, suggesting its role in regulation of epithelial-mesenchymal transition (EMT). Silencing SIRT6 inhibited both the proliferation and metastasis in prostate cancer, indicating the therapeutic value of targeting SIRT6 in the treatment of metastatic PCa. We then tested the therapeutic value of SIRT6 in prostate cancer in vivo by using aptamer-modified exosomes carrying SIRT6 siRNA. To achieve this goal, the cholesterol-covalent E3 aptamer was inserted into the membrane of 293T-derived exosomes to target specifically the prostate cancer cells, followed by SIRT6-siRNA loading into the modified exosomes with an electroporation apparatus. The engineered exosomes suppressed the proliferation and metastasis in subcutaneously and orthotopically xenograft mice tumor models, and confirmed that SIRT6 could be considered as a selective and novel target in high-grade prostate cancer. Citation Format: Qing Han, Qian Reuben Xie, Fan Li, Yirui Cheng, Tingyu Wu, Jianjun Sha, Weiliang Xia. Targeted inhibition of SIRT6 by engineered exosomes suppressed tumorigenesis and metastasis in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1152.

Abstract 2393: The systematic evaluation of the oncogenic lncRNA LINC00963 using a CRISPRScan technique

Abstract Long non-coding RNAs (LncRNAs) have been found to play important roles in maintaining and promoting cellular proliferation in cancerous cells. One such lncRNA, LINC00963 has been found to promote cellular proliferation, migration and tumorigenesis in prostate, ovarian, breast and non-small cell lung (NSCLC) cancer. In this study, we aim to identify important functional regions of this lncRNA with a view to identify unique regions for targeted drug therapy. In order to achieve this, we sought to investigate this lncRNA in the A549 NSCLC cell line. We found that LINC00963 was highly expressed and when targeted with either siRNAs or antisense oligonucleotides, cells rapidly undergo apoptosis. Using cell death as a proxy for loss-of-function, we characterized LINC00963 at the genomic level using a CRIPSRScan approach. CRISPRScan is a novel way of determining lncRNA functional domains through the introduction of local mutations. These mutations could potentially disrupt lncRNA function, which for LINC00963 would lead to apoptosis of the cell. We created a unique lentivirus library comprised of 1784 single guide RNAs spanning the length of LINC00963 in order to precisely identify regions of functional importance. Upon transduction in a stably expressing Cas9-A549 cell line, we collected cellular fractions over time, and using high-throughput sequencing (NextSeq, Illumina), evaluated the depletion of these sgRNAs as a proxy for their importance in LINC00963 function. The identification of these key genomic regions that may render LINC00963 non-functional may offer new lncRNA target sites for anti-cancer therapies in the future. Additionally, the utilization of an in-depth CRISPR-Cas9 approach to determine important lncRNA domains provides a novel and precise way in which to evaluate lncRNA function. Citation Format: Roslyn M. Ray, Lu Lang, Sheela P. Pokharel, Denis O'Meally, Tristan A. Scott, Chun-Wei Chen, Kevin V. Morris. The systematic evaluation of the oncogenic lncRNA LINC00963 using a CRISPRScan technique [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2393.

REX-1 Represses RASSF1a and Activates the MEK/ERK Pathway to Promote Tumorigenesis in Prostate Cancer.

Epigenetics play an important role in the pathogenesis of prostate cancer; it is urgent to investigate vital transcription factors in methylation regulation with the aim to develop novel treatment strategies targeting prostate cancer. As a member of the zinc finger protein family, REX-1 (reduced expression-1) is a transcription factor that has been reported to be closely linked to the development of several cancers. So far, the expression level and precise function of REX-1 in prostate cancer remain largely unknown. Here, we show that REX-1 was overexpressed in prostate cancer clinical tissues, and its expression level was closely correlated with patient prognosis. REX-1 affected prostate tumor growth in vivo by MEK/ERK phosphorylation. Mechanistic studies indicated that REX-1 recruited DNMT3b (DNA methyltransferase 3b), inhibited the transcription of RASSF1a (RAS association domain family 1a), and further modulated methylation of RASSF1a promoter. Intervention of the REX-1/DNMT3b/RASSF1a axis may shed light on the development of novel therapeutic approaches for prostate cancer treatment. IMPLICATIONS: REX1 overexpression recruits DNMT3b and downregulates RASSF1a by promoter methylation, suggesting that epigenetic intervention may contribute to prostate cancer treatment.