Longjiang Shao

Activation of NF-kB by TMPRSS2/ERG fusion isoforms through Toll-like receptor-4

The TMPRSS2/ERG (T/E) fusion gene is present and thought to be an oncogenic driver of approximately half of all prostate cancers. Fusion of the androgen-regulated TMPRSS2 promoter to the ERG oncogene results in constitutive high level expression of ERG which promotes prostate cancer invasion and proliferation. Here, we report the characterization of multiple alternatively spliced T/E fusion gene isoforms which have differential effects on invasion and proliferation. We found that T/E fusion gene isoforms differentially increase NF-κB-mediated transcription, which may explain in part the differences in biological activities of the T/E fusion isoforms. This increased activity is due to phosphorylation of NF-κB p65 on Ser536. Tissue microarray immunochemistry revealed that p65 phospho-Ser536 is present in the majority of prostate cancers where it is associated with ERG protein expression. The T/E fusion gene isoforms differentially increase expression of a number of NF-κB associated genes including PAR1, CCL2, FOS, TLR3, and TLR4 (Toll-like receptor). TLR4 activation is known to promote p65 Ser536 phosphorylation and knockdown of TLR4 with shRNA decreases Ser536 phosphorylation in T/E fusion gene expressing cells. TLR4 can be activated by proteins in the tumor microenvironment and lipopolysacharide from Gram (-) bacteria. Our findings suggest that bacterial infection of the prostate and/or endogenous microenvironment proteins may promote progression of high-grade prostatic intraepithelial neoplasia and/or prostate cancers that express the T/E fusion gene, where the NF-κB pathway might be targeted as a rational therapeutic approach.

Highly specific targeting of the TMPRSS2/ERG fusion gene using liposomal nanovectors

Purpose: The TMPRSS2/ERG (T/E) fusion gene is present in half of all prostate cancer tumors. Fusion of the oncogenic ERG gene with the androgen-regulated TMPRSS2 gene promoter results in expression of fusion mRNAs in prostate cancer cells. The junction of theTMPRSS2- and ERG-derived portions of the fusion mRNA constitutes a cancer-specific target in cells containing the T/E fusion gene. Targeting the most common alternatively spliced fusion gene mRNA junctional isoforms in vivo using siRNAs in liposomal nanovectors may potentially be a novel, low-toxicity treatment for prostate cancer. Experimental Design: We designed and optimized siRNAs targeting the two most common T/E fusion gene mRNA junctional isoforms (type III or type VI). Specificity of siRNAs was assessed by transient co-transfection in vitro. To test their ability to inhibit growth of prostate cancer cells expressing these fusion gene isoforms in vivo, specific siRNAs in liposomal nanovectors were used to treat mice bearing orthotopic or subcutaneous xenograft tumors expressing the targeted fusion isoforms. Results: The targeting siRNAs were both potent and highly specific in vitro. In vivo they significantly inhibited tumor growth. The degree of growth inhibition was variable and was correlated with the extent of fusion gene knockdown. The growth inhibition was associated with marked inhibition of angiogenesis and, to a lesser degree, proliferation and a marked increase in apoptosis of tumor cells. No toxicity was observed. Conclusions: Targeting the T/E fusion junction in vivo with specific siRNAs delivered via liposomal nanovectors is a promising therapy for men with prostate cancer. Clin Cancer Res; 18(24); 6648–57. ©2012 AACR.

Targeting Fibroblast Growth Factor Receptor Signaling Inhibits Prostate Cancer Progression

Purpose: Extensive correlative studies in human prostate cancer as well as studies in vitro and in mouse models indicate that fibroblast growth factor receptor (FGFR) signaling plays an important role in prostate cancer progression. In this study, we used a probe compound for an FGFR inhibitor, which potently inhibits FGFR-1–3 and significantly inhibits FGFR-4. The purpose of this study is to determine whether targeting FGFR signaling from all four FGFRs will have in vitro activities consistent with inhibition of tumor progression and will inhibit tumor progression in vivo. Experimental Design: Effects of AZ8010 on FGFR signaling and invasion were analyzed using immortalized normal prostate epithelial (PNT1a) cells and PNT1a overexpressing FGFR-1 or FGFR-4. The effect of AZ8010 on invasion and proliferation in vitro was also evaluated in prostate cancer cell lines. Finally, the impact of AZ8010 on tumor progression in vivo was evaluated using a VCaP xenograft model. Results: AZ8010 completely inhibits FGFR-1 and significantly inhibits FGFR-4 signaling at 100 nmol/L, which is an achievable in vivo concentration. This results in marked inhibition of extracellular signal–regulated kinase (ERK) phosphorylation and invasion in PNT1a cells expressing FGFR-1 and FGFR-4 and all prostate cancer cell lines tested. Treatment in vivo completely inhibited VCaP tumor growth and significantly inhibited angiogenesis and proliferation and increased cell death in treated tumors. This was associated with marked inhibition of ERK phosphorylation in treated tumors. Conclusions: Targeting FGFR signaling is a promising new approach to treating aggressive prostate cancer. Clin Cancer Res; 18(14); 3880–8. ©2012 AACR.

Celastrol suppresses tumor cell growth through targeting an AR-ERG-NF-κB pathway in TMPRSS2/ERG fusion gene expressing prostate cancer.

The TMPRSS2/ERG (T/E) fusion gene is present in the majority of all prostate cancers (PCa). We have shown previously that NF-kB signaling is highly activated in these T/E fusion expressing cells via phosphorylation of NF-kB p65 Ser536 (p536). We therefore hypothesize that targeting NF-kB signaling may be an efficacious approach for the subgroup of PCas that carry T/E fusions. Celastrol is a well known NF-kB inhibitor, and thus may inhibit T/E fusion expressing PCa cell growth. We therefore evaluated Celastrol’s effects in vitro and in vivo in VCaP cells, which express the T/E fusion gene. VCaP cells were treated with different concentrations of Celastrol and growth inhibition and target expression were evaluated. To test its ability to inhibit growth in vivo, 0.5 mg/kg Celastrol was used to treat mice bearing subcutaneous VCaP xenograft tumors. Our results show Celastrol can significantly inhibit the growth of T/E fusion expressing PCa cells both in vitro and in vivo through targeting three critical signaling pathways: AR, ERG and NF-kB in these cells. When mice received 0.5 mg/kg Celastrol for 4 times/week, significant growth inhibition was seen with no obvious toxicity or significant weight loss. Therefore, Celastrol is a promising candidate drug for T/E fusion expressing PCa. Our findings provide a novel strategy for the targeted therapy which may benefit the more than half of PCa patients who have T/E fusion expressing PCas.

MNX1 is oncogenically upregulated in African-American prostate cancer

Incidence and mortality rates for prostate cancer are higher in African-American (AA) men than in European-American (EA) men, but the biologic basis for this disparity is unclear. We carried out a detailed analysis of gene expression changes in prostate cancer compared with their matched benign tissues in a cohort of AA men and compared them with existing data from EA men. In this manner, we identified MNX1 as a novel oncogene upregulated to a relatively greater degree in prostate cancer from AA men. Androgen and AKT signaling play a central role in the pathogenesis of prostate cancer and we found that both of these signaling pathways increased MNX1 expression. MNX1 in turn upregulated lipid synthesis by stimulating expression of SREBP1 and fatty acid synthetase. Our results define MNX1 as a novel targetable oncogene increased in AA prostate cancer that is associated with aggressive disease. Cancer Res; 76(21); 6290-8. ©2016 AACR.

The Prostate Cancer TMPRSS2:ERG Fusion Synergizes With the Vitamin D Receptor (VDR) to Induce CYP24A1 Expression-Limiting VDR Signaling

A number of preclinical studies have shown that the activation of the vitamin D receptor (VDR) reduces prostate cancer (PCa) cell and tumor growth. The majority of human PCas express a transmembrane protease serine 2 (TMPRSS2):erythroblast transformation-specific (ETS) fusion gene, but most preclinical studies have been performed in PCa models lacking TMPRSS2:ETS in part due to the limited availability of model systems expressing endogenous TMPRSS2:ETS. The level of the active metabolite of vitamin D, 1α,25-dihydroxyvitamin D3 (1,25D), is controlled in part by VDR-dependent induction of cytochrome P450, family 24, subfamily 1, polypeptide1 (CYP24A1), which metabolizes 1,25D to an inactive form. Because ETS factors can cooperate with VDR to induce rat CYP24A1, we tested whether TMPRSS2:ETS would cause aberrant induction of human CYP24A1 limiting the activity of VDR. In TMPRSS2:ETS positive VCaP cells, depletion of TMPRSS2:ETS substantially reduced 1,25D-mediated CYP24A1 induction. Artificial expression of the type VI+72 TMPRSS2:ETS isoform in LNCaP cells synergized with 1,25D to greatly increase CYP24A1 expression. Thus, one of the early effects of TMPRSS2:ETS in prostate cells is likely a reduction in intracellular 1,25D, which may lead to increased proliferation. Next, we tested the net effect of VDR action in TMPRSS2:ETS containing PCa tumors in vivo. Unlike previous animal studies performed on PCa tumors lacking TMPRSS2:ETS, EB1089 (seocalcitol) (a less calcemic analog of 1,25D) did not inhibit the growth of TMPRSS2:ETS containing VCaP tumors in vivo, suggesting that the presence of TMPRSS2:ETS may limit the growth inhibitory actions of VDR. Our findings suggest that patients with TMPRSS2:ETS negative tumors may be more responsive to VDR-mediated growth inhibition and that TMPRSS2:ETS status should be considered in future clinical trials.

RGS12 is a novel tumor suppressor gene in African American prostate cancer that represses AKT and MNX1 expression.

African American (AA) men exhibit a relatively high incidence and mortality due to prostate cancer even after adjustment for socioeconomic factors, but the biological basis for this disparity is unclear. Here, we identify a novel region on chromosome 4p16.3 that is lost selectively in AA prostate cancer. The negative regulator of G-protein signaling RGS12 was defined as the target of 4p16.3 deletions, although it has not been implicated previously as a tumor-suppressor gene. RGS12 transcript levels were relatively reduced in AA prostate cancer, and prostate cancer cell lines showed decreased RGS12 expression relative to benign prostate epithelial cells. Notably, RGS12 exhibited potent tumor-suppressor activity in prostate cancer and prostate epithelial cell lines in vitro and in vivo We found that RGS12 expression correlated negatively with the oncogene MNX1 and regulated its expression in vitro and in vivo Further, MNX1 was regulated by AKT activity, and RGS12 expression decreased total and activated AKT levels. Our findings identify RGS12 as a candidate tumor-suppressor gene in AA prostate cancer, which acts by decreasing expression of AKT and MNX1, establishing a novel oncogenic axis in this disparate disease setting. Cancer Res; 77(16); 4247-57. ©2017 AACR.

miR-33a is a tumor suppressor microRNA that is decreased in prostate cancer

Prostate cancer is one of the most frequently diagnosed neoplasms among men worldwide. MicroRNAs (miRNAs) are involved in numerous important cellular processes including proliferation, differentiation and apoptosis. They have been found to be aberrantly expressed in many types of human cancers. They can act as either tumor suppressors or oncogenes, and changes in their levels are associated with tumor initiation, progression and metastasis. miR-33a is an intronic miRNA embedded within SREBF2 that has been reported to have tumor suppressive properties in some cancers but has not been examined in prostate cancer. SREBF2 increases cholesterol and lipid levels both directly and via miR-33a action. The levels of SREBF2 and miR-33a are correlated in normal tissues by co-transcription from the same gene locus. Paradoxically, SREBF2 has been reported to be increased in prostate cancer, which would be predicted to increase miR-33a levels potentially leading to tumor suppression. We show here that miR-33a has tumor suppressive activities and is decreased in prostate cancer. The decreased miR-33a increases mRNA for the PIM1 oncogene and multiple genes in the lipid β-oxidation pathway. Levels of miR-33a are not correlated with SREBF2 levels, implying posttranscriptional regulation of its expression in prostate cancer.Prostate cancer is one of the most frequently diagnosed neoplasms among men worldwide. MicroRNAs (miRNAs) are involved in numerous important cellular processes including proliferation, differentiation and apoptosis. They have been found to be aberrantly expressed in many types of human cancers. They can act as either tumor suppressors or oncogenes, and changes in their levels are associated with tumor initiation, progression and metastasis. miR-33a is an intronic miRNA embedded within SREBF2 that has been reported to have tumor suppressive properties in some cancers but has not been examined in prostate cancer. SREBF2 increases cholesterol and lipid levels both directly and via miR-33a action. The levels of SREBF2 and miR-33a are correlated in normal tissues by co-transcription from the same gene locus. Paradoxically, SREBF2 has been reported to be increased in prostate cancer, which would be predicted to increase miR-33a levels potentially leading to tumor suppression. We show here that miR-33a has tumor suppressive activities and is decreased in prostate cancer. The decreased miR-33a increases mRNA for the PIM1 oncogene and multiple genes in the lipid β-oxidation pathway. Levels of miR-33a are not correlated with SREBF2 levels, implying posttranscriptional regulation of its expression in prostate cancer.

Combination treatment of prostate cancer with FGF receptor and AKT kinase inhibitors

Activation of the PI3K/AKT pathway occurs in the vast majority of advanced prostate cancers (PCas). Activation of fibroblast growth factor receptor (FGFR) signaling occurs in a wide variety of malignancies, including PCa. RNA-Seq of castration resistant PCa revealed expression of multiple FGFR signaling components compatible with FGFR signaling in all cases, with multiple FGF ligands expressed in 90% of cases. Immunohistochemistry confirmed FGFR signaling in the majority of xenografts and advanced PCas. AZD5363, an AKT kinase inhibitor and AZD4547, a FGFR kinase inhibitor are under active clinical development. We therefore sought to determine if these two drugs have additive effects in PCa models. The effect of both agents, singly and in combination was evaluated in a variety of PCa cell lines in vitro and in vivo. All cell lines tested responded to both drugs with decreased invasion, soft agar colony formation and growth in vivo, with additive effects seen with combination treatment. Activation of the FGFR, AKT, ERK and STAT3 pathways was examined in treated cells. AZD5363 inhibited AKT signaling and increased FGFR1 signaling, which partially compensated for decreased AKT kinase activity. While AZD4547 could effectively block the ERK pathway, combination treatment was needed to completely block STAT3 activation. Thus combination treatment with AKT and FGFR kinase inhibitors have additive effects on malignant phenotypes in vitro and in vivo by inhibiting multiple signaling pathways and mitigating the compensatory upregulation of FGFR signaling induced by AKT kinase inhibition. Our studies suggest that co-targeting these pathways may be efficacious in advanced PCa.

RET signaling in prostate cancer

Purpose: Large diameter perineural prostate cancer is associated with poor outcomes. GDNF, with its coreceptor GFRα1, binds RET and activates downstream pro-oncogenic signaling. Because both GDNF and GFRα1 are secreted by nerves, we examined the role of RET signaling in prostate cancer. Experimental Design: Expression of RET, GDNF, and/or GFRα1 was assessed. The impact of RET signaling on proliferation, invasion and soft agar colony formation, perineural invasion, and growth in vivo was determined. Cellular signaling downstream of RET was examined by Western blotting. Results: RET is expressed in all prostate cancer cell lines. GFRα1 is only expressed in 22Rv1 cells, which is the only line that responds to exogenous GDNF. In contrast, all cell lines respond to GDNF plus GFRα1. Conditioned medium from dorsal root ganglia contains secreted GFRα1 and promotes transformation-related phenotypes, which can be blocked by anti-GFRα1 antibody. Perineural invasion in the dorsal root ganglion assay is inhibited by anti-GFRα antibody and RET knockdown. In vivo, knockdown of RET inhibits tumor growth. RET signaling activates ERK or AKT signaling depending on context, but phosphorylation of p70S6 kinase is markedly increased in all cases. Knockdown of p70S6 kinase markedly decreases RET induced transformed phenotypes. Finally, RET is expressed in 18% of adenocarcinomas and all three small-cell carcinomas examined. Conclusions: RET promotes transformation associated phenotypes, including perineural invasion in prostate cancer via activation of p70S6 kinase. GFRα1, which is secreted by nerves, is a limiting factor for RET signaling, creating a perineural niche where RET signaling can occur. Clin Cancer Res; 23(16); 4885–96. ©2017 AACR.