Inhibiting LSD1 restores vitamin D responsiveness in castration resistant prostate cancer
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Prostate cancer (PCa) has the second highest mortality rate of cancers for American men. Localized PCa is frequently cured, but 20-30% of patients will experience disease recurrence. These patients are treated with androgen deprivation therapy, but invariably the disease will progress to castration resistant prostate cancer (CRPC). Current treatments of CRPC have a median survival of a few years; consequently complementary therapeutic approaches are urgently needed. Vitamin D3 is a secosteroid hormone that binds the vitamin D3 receptor (VDR) and has a potent antiproliferative effect on prostate epithelial cells, but a reduced response on advanced PCa cells. Lysine Specific Demethylase 1 (LSD1/KDM1A) is a demethylating enzyme that targets histone and non-histone proteins. LSD1 and VDR protein levels are elevated in advanced PCa and CRPC in-vivo in TRAMP mice and LSD1 is a dual co-regulator for VDR. We therefore hypothesized that LSD1 contributes to loss of sensitivity to vitamin D3 in CRPC cells, combined activation of VDR and inhibition of LSD1 can restore vitamin D3 sensitivity in CRPC. In order to examine the impact of VDR activation on 84 genes involved with carcinogenesis and tumorigenesis in advanced PCa treated BC1A cell line for 24h at 100 nM 1,25D3. 12 of the 84 genes were differentially expressed upon 1,25D3 treatment. 9 of the 12 DEGs have opposite expression patterns between primary and metastatic PCa datasets, suggesting that 1,25D3 could help prevent the progression of PCa as an adjuvant or chemopreventive drug. The anti-proliferative effect of VDR activation and LSD1 inhibition in C4-2 cells was evaluated by treating C4-2 cells for 96h at 100 nM of 1,25D3 and 45 µM of the LSD1 antagonist Bizine, and by treating C4-2 cells stably transfected with shLSD1-01 for 96h at 100 nM 1,25D3. 1,25D3 + Bizine, and 1,25D3 + shLSD1-01 inhibit proliferation more than the sum of the inhibition of cell growth by the individual treatments by 8.7% and 6.1% respectively, which suggests a potential additive effect of the combinational therapies. The combinational therapy’s ability to inhibit cell growth in a potentially additive manner suggests that LSD1 inhibition restores partial vitamin D3 responsiveness in CRPC cells at the transcriptional and phenotypical level. RNAseq analysis was performed on 4 conditions treated for 4h (shCTR + vehicle, shCTR + 100nM 1,25D3, shLSD1 + vehicle, shLSD1 + 100nM 1,25D3) to understand the genome-wide transcriptional changes upon modulating LSD1 and activating VDR. Genome wide transcriptomic data supported previous results that LSD1 acts as a co-repressor and co-activator for VDR. The differentially expressed transcripts from the shLSD1 + 1,25D3 v shCTR + 1,25D3 comparison and the shCTR + 1,25D3 v shCTR comparison belong to different protein classes and pathways, which suggest that they have two separate functions and different ways through which they regulate the cell. The combinational therapy and 1,25D3 treatment differentially regulating the cell suggests that the combinational therapy doesn’t combine the transcriptional effects of LSD1 inhibition and VDR action, but instead LSD1’s co-regulation of VDR alters their combined transcriptional regulation of VDR target genes. The VDR and LSD1 transcriptome upon VDR activation and LSD1 inhibition was profiled by integrating RNAseq data from C4-2 cells and The Cancer Genome Atlas PCa dataset. Two genes DNA binding 1 (ID1) and Ubiquitin-Conjugation Enzyme E2C (UBE2C), were differentially expressed in the RNAseq and TCGA datasets. The different response of UBE2C compared to ID1 to 1,25D3 treatment and LSD1 inhibition suggests that LSD1 inhibition modulates the effect of 1,25D3 treatment in a locus specific manner. We conclude that although vitamin D3 regulates key mediators of PCa progression in advanced PCa, the suppressed VDR action in CRPC requires vitamin D3 to be utilized in combination with a drug such as Bizine to partially restore its responsiveness to vitamin D3. Restoring responsiveness to vitamin D3 allows a lower dose of vitamin D3 to be utilized to achieve the same anti-cancer effects, which increases the potential for vitamin D3 to be effective in vivo before dose limiting hypercalcemia is induced.