Impact of interdisciplinary Translational Research in developing innovative therapeutic strategies in Clinical Pharmacy Science

Abstract

Uterine Fibroid (UF) is by far the most common benign tumor arising in the myometrium (MM) of reproductive age women. Estrogen and β-catenin signaling are significant for this tumor growth. However, the mechanisms underlying disease pathogenesis are largely unknown. UF is known to be associated with DNA damage and genomic instability, since mutations in mediator complex subunit 12 (MED12) was present in about 80% of tested tumors. Hypovitaminosis D is a known risk factor for UFs, especially among Black women. Consequently, Vitamin D3 has been shown to effectively inhibit UF phenotype. However, its mechanisms remain unclear. Unfortunately, there are limited number of treatment options available especially for safe long-term treatment for UFs. Thus, deep understanding of the mechanisms underlying UF etiology will help to develop new therapies for UFs. Ulipristal acetate (UPA) treatment has shown superior effect compared to placebo in both European and American clinical trials. However, the American trial, which included black patients with known higher Vitamin D3 deficiency risk, showed lower rate of amenorrhea responders and insignificant UF size reduction unlike the European trial. Aim of the work: First, we explored the link between nuclear β-catenin and UF phenotype, β-catenin crosstalk with estrogen and histone deacetylases (HDACs) and explored the effect of HDAC inhibitors (HDACis) as potential novel epigenetic therapy against UF. Secondly, we explored whether Vitamin D3 can ameliorate UFs tumor progression via recovering the damaged DNA repair system. Lastly, we investigated whether combining Vitamin D3 with UPA can enhance UPA potency on UF phenotype. Patients and methods: Human UF and adjacent MM tissues have been collected from over 80 consented women with UFs undergoing surgery. Primary cells as well as stem cells have been isolated from fresh tissues, while the rest of tissues have been snap frozen for molecular analysis (protein and gene expression) or fixed in formalin for subsequent histological and immunostaining. Isolated primary cells, stem cells as well as cell lines from UF and MM were exposed to different treatments based on the research question and aim of the work including HDACis, vitamin D3 and UPA, followed by several techniques for protein expression measurement including western blot, ELISA, Immunofluorescence, while gene expression was measured using qRT-PCR and custom designed PCR arrays. Additionally, apoptosis, cell cycle and cell proliferation assays have been performed. Genes code for β-catenin and vitamin D receptor (VDR) have been knocked down (KD) in UF and MM cell lines respectively using lentiviral particles to confirm their roles in UF pathogenesis. Results: First, UF tissues/cells showed significantly higher expression of nuclear β-catenin, CyclinD1, C-Myc, and HDACs (1, 2, 3, 8) compared to matched MM. Estradiol induced β-catenin nuclear-translocation and consequently its responsive genes in both MM and UF cells, while Estrogen receptor antagonist reversed this induction effect. Treatment with β-catenin or HDAC inhibitors showed a dose dependent growth inhibition, while Wnt3a treatment increased proliferation compared to control. Chemical inhibition of β-catenin decreased CyclinD1 and c-Myc expression levels, while β-catenin activation increased same markers expression. Genetic KD of CTNNB1 resulted in marked decrease in β-catenin, CyclinD1 and C-Myc expression. While treatment of UF cells with HDACis decreased nuclear β-catenin, CyclinD1 and C-Myc expression. Moreover, HDACis induced UF cells apoptosis and cell cycle arrest. Secondly, Unrepaired DNA double-strand breaks (DSBs) was accumulated along with VDR expression decrease in UFs compared to normal MM. Where the RNA and protein levels of key DNA repair members belonging to DNA DSB sensors (MRE11, NBS1, RAD50), mediators and effectors (CHECK2, BRCA1, RAD51) were downregulated in UFs compared with myometrial cells. VDR KD induced DSB accumulation and DNA damage response (DDR) defects in myometrial cells. Using the DNA damage PCR array, many additional DNA repair genes were downregulated in VDR KD cells. Interestingly, VitaminD3 treatment (100 nM) of UF cells significantly decreased DNA damage and restored DDR concomitant with VDR induction. Notably, the PCR array demonstrated that among 75 downregulated genes after VDR KD, 67 (89.3%) were upregulated after vitaminD3 treatment. These studies provided compelling evidence for a dysfunctional DNA repair system in UFs. Gain- and loss-of-function analyses demonstrated novel link between DNA damage and vitaminD3/VDR axis in UFs. Lastly, UPA/VitaminD3 combinations induced significant dose and time dependent growth inhibitory effects on UF cells compared to untreated cells at 2 and 4 days (P˂0.05). Importantly, VitaminD3/UPA combination significantly reduced cells proliferation as compared to UPA at 2, 4, 6 and 8 days (P˂0.05). Combination treatment significantly decreased protein expression of proliferation markers Ki-67, PCNA and CyclinD1 by more than 50% compared to UPA alone (P˂0.05) along with significant increase in apoptosis. Combination treatment resulted in 2-fold decrease in protein levels of extracellular matrix markers Collagen-1 and Fibronectin besides pro-fibrogenic cytokine TGFβ3 (P˂0.05). Moreover, it significantly decreased mRNA expression and the production of pro-inflammatory cytokines Interleukins (ILs) 6, 8, 1α and 1β compared to UPA alone (P˂0.05). Finally, mRNA expression of angiogenesis related genes was reduced in response to VitaminD3 addition. Conclusion: β-catenin nuclear-translocation contributes to UF phenotype, estradiol and HDAC modulate β-catenin signaling. Moreover, vitamin D3 suppresses the UF phenotype through orchestrated targeting at multiple molecules involved in DNA repair pathways, thus offering novel mechanistic insights into the clinical effectiveness of vitamin D3 on UFs treatment. Finally, combination of vitamin D3 with UPA exhibits additional anti-UF effects, therefore might offer more favorable clinical option especially for women of color who frequently suffer from Vitamin D deficiency such as black women.