The Response to Sulforaphane in Metastatic Prostate Cancer Cells Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/b5644v17w

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  • Sulforaphane is an isothiocyanate derived from cruciferous vegetables and has been under investigation as a cancer chemopreventive agent for over two decades. The compound is well tolerated and has been shown to slow cancer progression in several different pre-clinical models of carcinogenesis, such as of the lung, breast and colon. Recent work has suggested that sulforaphane may also slow cancer growth and progression in a pre-clinical model of prostate carcinogenesis. Prostate cancer accounted for approximately 14% of all new cancer diagnoses in 2014, making it the most commonly diagnosed type of cancer. Developing sulforaphane as a cancer therapeutic agent could provide an inexpensive and safe new treatment option to delay prostate cancer onset and to reduce the number of new diagnoses for this common cancer. In addition, gaining a mechanistic understanding of how sulforaphane slows prostate cancer progression could identify new protein targets for therapeutic development. Previous work utilizing human prostate cancer cell lines has identified many proteins and signaling pathways that respond to sulforaphane and have subsequently been proposed as contributors to prostate cancer suppression by sulforaphane (i.e. mechanistic targets). However, a majority of previous investigations charactering the response to sulforaphane in prostate cancer cells utilize sulforaphane at concentrations above what any prostate cancer cell will experience in vivo and/or for treatment periods that are not consistent with what we know about sulforaphane pharmacokinetics, tissue distribution and elimination. There is therefore a critical need to characterize the response to sulforaphane in prostate cancer cells under conditions that adhere to in vivo concentration and pharmacokinetics if we are to understand how sulforaphane may slow prostate cancer progression. To address this issue we carried out a series of in vitro investigations under conditions to mimic an in vivo exposure and explored the potential contribution of two cellular processes on sulforaphane-mediated suppression, chromatin regulation and autophagy. Previous research has suggested that sulforaphane stimulates the turnover of the chromatin-modifying enzymes class I histone deacetylase 3 and DNA-methyltransferases. These enzymes modify chromatin in protein complexes that include the enzymes that control histone methylation, suggesting that changes in histone deacetylase and DNA-methyltransferase protein levels may influence histone methylation levels. We therefore tested whether sulforaphane treatment leads to changes in global histone methylation levels. We found that sulforaphane leads to a transient decrease in histone H3 lysine 9 trimethylation levels, a mark that is controlled by the enzyme SUV39H1. Sulforaphane treatment led to posttranslational modification of SUV39H1 that coincided with its dissociation from chromatin. These novel findings suggest histone methylation may have a role in the cellular response to sulforaphane. Several investigations have suggested that autophagy is involved in influencing the cellular response to sulforaphane in prostate cancer cells in vitro and in vivo; however, previous work has not specifically addressed autophagic flux. In addition, sulforaphane has been proposed to decrease the level of histone deacetylase 6 (HDAC6) and HDAC6 has recently be shown to be required for efficient autophagic flux in mouse embryonic fibroblasts. We therefore tested whether sulforaphane or HDAC6-inhibition decreases autophagic flux in prostate cancer cells. We found that sulforaphane stimulates autophagic flux in metastatic prostate cancer cells at high concentrations. An assessment of HDAC6-interacting proteins in LNCaP metastatic prostate cancer cells showed HDAC6 interacts with proteins associated with autophagy. However, HDAC6-inhibition with the small molecule tubacin did not influence autophagic flux, suggesting metastatic prostate cancer cells do not require HDAC6 activity for autophagy. Changes in gene transcription in response to sulforaphane have been used to infer the outcome of sulforaphane treatment. However, sulforaphane can lead to a rapid decrease in global protein synthesis in some prostate cancer cell lines. This suggests that using gene transcription to infer the outcome of sulforaphane treatment may be misleading if transcripts are not efficiently translated into proteins. To address this issue we applied proteomics to directly assess the global protein profile of LNCaP cells in response to sulforaphane. We found that sulforaphane does not lead to a global remodeling of the proteome following sulforaphane treatment in this cell type. Proteomics did, however, identify proteins that have not previously been implicated in LNCaP cell maintenance (e.g. TRIAP1). We show that TRIAP1 influences LNCaP cell proliferation, and thus show that proteomics can be used to identify novel candidate therapeutic targets in metastatic prostate cancer cells. The central findings from this dissertation work suggest that sulforaphane does not directly influence key outcomes (e.g. apoptosis, autophagy) that have been associated with sulforaphane treatment in metastatic prostate cancer cells when treatment concentrations and exposure times are made to conform to in vivo sulforaphane pharmacokinetics. This is important in understanding how sulforaphane may lead to prostate cancer suppression because it suggests the involvement of important in vivo factors that mediate suppressive activity. Future in vivo work that builds off the results presented in this dissertation will be important for the further development of sulforaphane as a prostate cancer therapeutic agent.
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