MNX1 Is Oncogenically Upregulated in African-American Prostate Cancer

Abstract

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.

Figure 1. Expression array analysis of genes known to be altered in PCa and MNX1

Fold change (cancer versus benign) of specific gene expression in PCa tissues from African-American men and for same genes in the Taylor dataset for EA men.

Figure 2. Expression of MNX1 in PCa

A. MNX1 mRNA levels in EA and AA PCa and benign tissues by Q-RT-PCR. Asterisks indicate statistically significant differences between cancer and benign tissues. Mean +/−SEM; * p<.05; *** p<.001 by t-test B. Examples of Oncomine datasets with increased expression of MNX1 relative to benign tissues; p value shown in bar and whisker plot. C. Western blot on MNX1 from PCa and matched benign tissues from AA men. β-actin is a loading control. D. Expression of MNX1 mRNA in primary prostatic epithelial cells (PrEC) and PCa cell lines.

Figure 3. Biological effects of MNX1 on PCa cells

A. Proliferation of DU145 cells overexpressing MNX1 versus vector controls. B. Proliferation of LNCaP cells with knockdown of MNX1 using two different shRNAs versus scrambled control. C. Invasion through Matrigel of DU145 cells overexpressing MNX1 versus vector controls and LNCaP cells with knockdown of MNX1 using two different shRNAs versus scrambled control. D. Final tumor weight of xenograft tumors using DU145 cells overexpressing MNX1 versus vector controls and LNCaP cells with knockdown of MNX1 using an shRNA versus scrambled control. For A–D, mean +/− SEM is shown; * p<.05; **

Figure 4. Control of expression of MNX1 by androgens

LNCaP cells were incubated in charcoal stripped serum (CSS) for 24, 48 or 72 hrs. Two plates of cells were treated with 10nM R1881 after 48 hrs in CSS; cells were collected 24 hrs after treatment. Western blot with anti-MNX1 antibody and anti β-actin is shown. In a separate experiment cells were treated with ezalutamide for 24 hrs or vehicle only (CON). Cells were also placed in CSS for 72 hrs or placed in CSS for 48 hrs and then treated with R1881 for 24 hrs as an additional set on controls.

Figure 5. Control of expression of MNX1 by AKT

A. LNCaP cells were treated with 500nM AZD5363 for 24 hrs and cell lysates prepared. Western blot for MNX1, P-AKT (S473) and P-S6 were performed. Actin is a loading control. Note increased AKT phosphorylation is due to feedback induced by inhibition of AKT kinase activity. B. LNCaP cells were treated with either 20 uM LY294002 or 1uM rapamycin or vehicle only for 24 hrs and cell lysates prepared. Western blot for MNX1, P-AKT (S473) and P-S6 were performed. Actin is a loading control.

Figure 6. MNX1 regulates lipid synthesis

A. Expression of MNX1, SREBF1, and FASN in DU145 overexpressing MNX1 or LNCaP with MNX1 knockdown using two different shRNAs versus controls. Data is expressed as percent of vector control for DU145 for each gene. Means of duplicate or triplicate determinations are shown. B. Palmitic acid content of LNCaP xenograft tumors versus scrambled controls (see Fig 3D); *** p<.001 by t-test. C. SREBP1 and FASN mRNA expression in AA benign prostate and PCa tissues. Mean +/− SEM is shown. ** p<.01; *** p<.001; Mann Whitney. D. Correlation between SREBP1 or FASN with MNX1 mRNA in AA PCa by Pearson Product Moment. Correlation coefficient and p-value are shown.