title: Characterisation and Role of SLFN5 in Castration Resistant Prostate Cancer
Creators: name: Rafael, Sánchez Martínez
Publisher: name: University of Glasgow
Year: 2020
Subjects: subject: SLFN5, Schlafen 5, Prostate Cancer, Castration Resistant Prostate Cancer, CRPC, LAT1

description: Castration resistant prostate cancer (CRPC) is a currently incurable form of prostate cancer. While varying in different countries, prostate cancer recurrence typically manifests clinically around 12-24 months after androgen deprivation therapy, signifying disease treatment resistance1. Tumour heterogeneity at the molecular level driving CRPC is a major challenge for developing effective treatment. In order to address this diversity, an unbiased SILAC-based proteomic study was performed using three different in vivo CRPC models. Three matched pairs of hormone naïve and respective isogenic castration resistant cell models (namely LNCaP/LNCaP AI, CWR22/22Rv1, VCAP/VCAPCR cells) were optimised to generate orthotopic xenografts (orthografts) in CD8 nude mice. Pair-wise analysis identified Schlafen 5 (SLFN5) protein to be highly expressed in all three CRPC orthografts. SLFN5 belongs to the relatively understudied Schlafen family of genes and has not been implicated in prostate cancer. SLFN5 has been shown to have both pro- or anti- tumorigenic depending on the tissue type, and it has been described to be able to regulate cell growth and migration. In this thesis, I tested the hypothesis that SLFN5 is mechanistically required for the development and maintenance of CRPC, and further explored the molecular basis involved. In vitro analysis revealed enhanced SLFN5 expression following suppression androgen receptor in LNCaP cells, and suppression of SLFN5 after addition of androgens to the castration resistant cells LNCaP AI and 22Rv1. I further investigated the clinical relevance of SLFN5 expression in a clinical cohort of prostate cancer including CRPC samples. SLFN5 immunoreactivity was predominantly nuclear and significantly upregulated in CRPC. SLFN5 expression positively correlated with high Gleason score, PSA recurrence and risk of premature. CRISPR/Cas9 technology was utilised to create SLFN5 knockout LNCaP AI and 22Rv1 CRPC cell clones. LNCaP AI SLFN5 KO cells have reduced migratory and in vitro growth capabilities. Furthermore, the growth of the SLFN5 KO orthografts was significantly impaired in in vivo androgen deprived conditions. Transcriptomic analysis of 22Rv1 KO tumours and 22Rv1 KO cells from androgen-depleted condition was carried out. 64 genes were significantly altered (p &lt; 0.05) in both in vitro and in vivo conditions, among which 52 genes were downregulated, raising the possibility that these downregulated genes may be positively regulated by SLFN5 in CRPC. Of note, the transcripts of both components of the LAT1 amino acid transporter were severely reduced, SLC7a5 and SLC3a2. In addition, proteomic analysis of 22Rv1 KO tumours in castrated mice revealed significant downregulation of 38 proteins, two of which are the aforementioned LAT1 components. LAT1 expression in prostate cancer is transcriptionally regulated by MYC and ATF4. Transient silencing of ATF4 in 22Rv1 cells impaired the expression of several SLFN5 regulated genes, including SLC7a5 and SLC3a2. Further analysis discovered that predicted ATF4 binding sequences existed with high confidence in the proximity of 15 out of the 26 analysed SLFN5 KO affected genes and that SLFN5 and ATF4 interacted closely in the nucleus of 22Rv1 cells. LAT1 mediates cellular intake of essential amino acid (EAA) and functions as part of the amino acid sensing machinery to activate the mTOR pathway for anabolism and growth during carcinogenesis. Metabolomic analysis of androgen-depleted 22Rv1 SLFN5 KO cells identified severely reduced levels of six amino acids, including 4 EAA: leucine, isoleucine, methionine and lysine. Western blot analysis highlighted impaired growth machinery due to SLFN5 KO with reduced phosphorylation of mTOR targets (P70S6K, rpS6, 4EBP1) signifying suppressed mTOR activity. Furthermore, increased levels of the autophagy marker LC3-II were detected as well. These results signalled that the growth machinery of the 22Rv1 KO cells was impaired and could explain the reduced tumour growth observed in the metabolically straining context of in vivo castration. Importantly, forced expression of LAT1 in 22Rv1 SLFN5 KO cells rescued mTOR activation. Collectively, for the first time, data presented in this thesis reveal an important role of SLFN5 in CRPC by regulating LAT1 expression to control mTOR mediated signalling required for cancer growth.