Cholesterol Sulfotransferase, SULT2B1b, is a Modulator of Cell Growth Properties, AR Activity, and Sensitivity to Death Ligand TNF-Alpha in Prostate Cancer Cells

Renee Elizabeth Vickman, Purdue University

Abstract

Prostate cancer is the most commonly diagnosed non-cutaneous cancer and third leading cause of cancer death in American men. Cholesterol is known to accumulate in prostate lesions and has been linked to prostate cancer incidence and progression. A modified form of cholesterol, cholesterol sulfate (CS), has also been demonstrated to accumulate in human prostate adenocarcinoma and precancerous prostatic intraepithelial neoplasia (PIN) lesions compared with normal regions of the same tissue sample. CS is the primary sulfonation product of cholesterol sulfotransferase (SULT2B1b), and it was hypothesized that SULT2B1b-mediated production of CS provides a growth advantage to prostate cancer cells. To address this, prostate cancer cell lines with RNAi-mediated knockdown (KD) of SULT2B1b were used to assess the impact on cell growth and survival. Data herein demonstrate that SULT2B1b KD results in decreased cell growth/viability, induces cell death in several prostate cancer cell lines, and also significantly reduces androgen receptor (AR) activity and expression. However, attempts to rescue AR expression and activity in SULT2B1b KD cells did not overcome the cell death phenotype. Single-cell mRNA sequencing (scRNAseq) analysis comparing SULT2B1b KD versus Control KD LNCaP cells validated these results, confirming downregulation of multiple AR-regulated genes and discovering alterations within numerous intracellular pathways involved in cell death phenotypes. This analysis also identified over 2,000 differentially expressed (DE) genes altered in SULT2B1b KD cells compared to Control KD cells. Interestingly, tumor necrosis factor (TNF) and NF-κB were predicted as upstream regulators of a subset of these genes, and TNF expression was determined to be upregulated in SULT2B1b KD cells. In LNCaP cells, NF-κB activity was significantly increased in SULT2B1b KD cells in a TNF-dependent manner. However, studies investigating whether increased TNF expression was involved in SULT2B1b-mediated cell death did not support that cell death was initiated by TNF. Further studies demonstrated that SULT2B1b KD significantly increased sensitivity to exogenous TNF in both TNF-sensitive LNCaP cells and TNF-resistant C4-2 cells. Conversely, SULT2B1b overexpression in LNCaP cells reduced sensitivity to exogenous TNF. These data support that SULT2B1b plays a role in the sensitivity status of prostate cancer cells to TNF-mediated apoptosis. In LNCaP cells, enhanced sensitivity to TNF in SULT2B1b KD cells could be due to significant upregulation of TNF-receptor associated death domain (TRADD), but no significant alterations of TRADD were observed in C4-2. Thus, a mechanism other than NF-κB activation or TRADD upregulation may be involved in the acquired sensitivity of SULT2B1b KD cells to TNF in the advanced prostate cancer model, C4-2 cells. Together, these findings provide evidence that SULT2B1b is a novel regulator of AR activity, cell growth, and sensitivity to TNF in prostate cancer cells and should be further investigated for therapeutic potential. Ongoing studies are being conducted to determine if SULT2B1b KD inhibits growth of other advanced and drug-resistant prostate cancer cell lines, demonstrate how AR is modulated by SULT2B1b and whether availability of AR co-regulators are involved, as well as reveal if SULT2B1b modulates expression and/or activity of AR variants. These studies will provide greater insight to the biological function of SULT2B1b and hopefully identify novel therapeutic targets for prostate cancer patients.

Degree

Ph.D.

Advisors

Ratliff, Purdue University.

Subject Area

Biology|Molecular biology|Oncology

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