The KDM5 inhibitor PBIT reduces proliferation of castration-resistant prostate cancer cells via cell cycle arrest and the induction of senescence.

The compound 2-4(4-methylphenyl)-1,2-benzisothiazol-3(2H)-one (PBIT) is an inhibitor of the KDM5 family of lysine-specific histone demethylases that has been suggested as a lead compound for cancer therapy. The goal of this study was to explore the effects of PBIT within human prostate cancers. Micromolar concentrations of PBIT altered proliferation of castration-sensitive LNCaP and castration-resistant C4-2B, LNCaP-MDV3100 and PC-3 human prostate cancer cell lines. We then characterized the mechanism underlying the anti-proliferative effects of PBIT within the C4-2B and PC-3 cell lines. Data from Cell Death ELISAs suggest that PBIT does not induce apoptosis within C4-2B or PC-3 cells. However, PBIT did increase the amount of senescence associated beta-galactosidase. PBIT also altered cell cycle progression and increased protein levels of the cell cycle protein p21. PC-3 and C4-2B cells express varying amounts of KDM5A, KDM5B, and KDM5C, the therapeutic targets of PBIT. siRNA-mediated knockdown studies suggest that inhibition of multiple KDM5 isoforms contribute to the anti-proliferative effect of PBIT. Furthermore, combination treatments involving PBIT and the PPARγ agonist 15-deoxy-Δ-12, 14 -prostaglandin J2 (15d-PGJ2) also reduced PC-3 cell proliferation. Together, these data strongly suggest that PBIT significantly reduces the proliferation of prostate cancers via a mechanism that involves cell cycle arrest and senescence.

PRC2-mediated repression of SMARCA2 predicts EZH2 inhibitor activity in SWI/SNF mutant tumors.

Subunits of the SWI/SNF chromatin remodeling complex are frequently mutated in human cancers leading to epigenetic dependencies that are therapeutically targetable. The dependency on the polycomb repressive complex (PRC2) and EZH2 represents one such vulnerability in tumors with mutations in the SWI/SNF complex subunit, SNF5; however, whether this vulnerability extends to other SWI/SNF subunit mutations is not well understood. Here we show that a subset of cancers harboring mutations in the SWI/SNF ATPase, SMARCA4, is sensitive to EZH2 inhibition. EZH2 inhibition results in a heterogenous phenotypic response characterized by senescence and/or apoptosis in different models, and also leads to tumor growth inhibition in vivo. Lower expression of the SMARCA2 paralog was associated with cellular sensitivity to EZH2 inhibition in SMARCA4 mutant cancer models, independent of tissue derivation. SMARCA2 is suppressed by PRC2 in sensitive models, and induced SMARCA2 expression can compensate for SMARCA4 and antagonize PRC2 targets. The induction of SMARCA2 in response to EZH2 inhibition is required for apoptosis, but not for growth arrest, through a mechanism involving the derepression of the lysomal protease cathepsin B. Expression of SMARCA2 also delineates EZH2 inhibitor sensitivity for other SWI/SNF complex subunit mutant tumors, including SNF5 and ARID1A mutant cancers. Our data support monitoring SMARCA2 expression as a predictive biomarker for EZH2-targeted therapies in the context of SWI/SNF mutant cancers.

Hypoxia-Inducible Expression of Annexin A6 Enhances the Resistance of Triple-Negative Breast Cancer Cells to EGFR and AR Antagonists.

Physiological changes such as hypoxia in the tumor microenvironment (TME) endow cancer cells with malignant properties, leading to tumor recurrence and rapid progression. Here, we assessed the effect of hypoxia (1% Oxygen) on the tumor suppressor Annexin A6 (AnxA6) and the response of triple-negative breast cancer (TNBC) cells to epidermal growth factor receptor (EGFR) and androgen receptor (AR) targeted therapies. We demonstrate that brief exposure of TNBC cells to hypoxia (within 24 h) is associated with down regulation of AnxA6 while > 24 h exposure cell type dependently stimulated the expression of AnxA6. Hypoxia depicted by the expression and stability of HIF-1/2α led to up regulation of the HIF target genes SLC2A1, PGK1 as well as AR and the AR target genes FABP-4 and PPAR-γ, but the cellular levels of AnxA6 protein decreased under prolonged hypoxia. Down regulation of AnxA6 in TNBC cells inhibited, while AnxA6 over expression enhanced the expression and cellular levels of HIF-1/2α, SLC2A1 and PGK1. RNAi mediated inhibition of hypoxia induced AnxA6 expression also strongly inhibited glucose uptake and ROS production in AnxA6 expressing TNBC cells. Using a luciferase reporter assay, we confirm that short-term exposure of cells to hypoxia inhibits while prolonged exposure of cells to hypoxia enhances AnxA6 promoter activity in HEK293T cells. Compared to cells cultured under normoxia, TNBC cells were more resistant to lapatinib under hypoxic conditions, and the downregulation of AnxA6 sensitized the cells to EGFR as well as AR antagonists. These data suggest that AnxA6 is a hypoxia inducible gene and that targeting AnxA6 upregulation may be beneficial in overcoming TNBC resistance to EGFR and/or AR targeted therapies.

Partial Rescue of Ocular Pigment Cells and Structure by Inducible Ectopic Expression of Mitf-M in MITF-Deficient Mice.

Purpose: Complete deficiency of microphthalmia transcription factor (MITF) in Mitfmi-vga9/mi-vga9 mice is associated with microphthalmia, retinal dysplasia, and albinism. We investigated the ability of dopachrome tautomerase (DCT) promoter-mediated inducible ectopic expression of Mitf-M to rescue these phenotypic abnormalities. Methods: A new mouse line was created with doxycycline-inducible ectopic Mitf-M expression on an Mitf-deficient Mitfmi-vga9 background (DMV mouse). Adult DMV mice were phenotypically characterized and tissues were collected for histology, immunohistochemistry, and evaluation of Mitf, pigmentary genes, and retinal pigment epithelium (RPE) gene expression. Results: Ectopic Mitf-M expression was specifically induced in the eyes, but was not detected in the skin of DMV mice. Inducible expression of Mitf-M partially rescued the microphthalmia, RPE structure, and pigmentation as well as a subset of the choroidal and iris melanocytes but not cutaneous melanocytes. RPE function and vision were not restored in the DMV mice. Conclusions: Ectopic expression of Mitf-M during development of Mitf-deficient mice is capable of partially rescuing ocular and retinal structures and uveal melanocytes. These findings provide novel information about the roles of Mitf isoforms in the development of mouse eyes.

Variation in the Zinc Finger of PRDM9 is Associated with the Absence of Recombination along Nondisjoined Chromosomes 21 of Maternal Origin.

Variation in the zinc finger-binding domain (ZFBD) of the protein PR Domain-Containing Protein 9 (PRDM9) is associated with altered placement of recombination in the human genome. As both the absence and altered placement of recombination are observed among chromosomes 21 that nondisjoin, we genotyped the PRDM9 ZFBD among mothers of children with Trisomy 21 in efforts to determine if variation within this region is associated with the recombination-related risk for chromosome 21 nondisjunction (NDJ). In our approach, PCR was used to amplify the ZFBD of PRDM9 and products were then subjected to bi-directional Sanger sequencing. DNA sequencing reads were aligned and compared to the sequence of the PRDM9 alleles previously identified. Chi-Square analysis was used to compare allele frequencies between cases (N=235, mothers of children with maternally-derived Trisomy 21) and controls (N=48, fathers of children with maternally-derived Trisomy 21). Results of our analysis showed that the frequency of PRDM9 ZF minor alleles is significantly increased among women displaying NDJ of chromosome 21 and no recombination on 21q (p=0.02). Even more, when compared to those for the PRDM9 major A-allele, these minor alleles displayed fewer predicted binding sites on 21q. These findings suggest that allelic variation in the ZF of PRDM9 may play a role in the risk for chromosome 21 NDJ by leading to reduced recombination on 21q.