BMP-6 promotes E-cadherin expression through repressing δEF1 in breast cancer cells

<p>Abstract</p> <p>Background</p> <p>Bone morphogenetic protein-6 (BMP-6) is critically involved in many developmental processes. Recent studies indicate that BMP-6 is closely related to tumor differentiation and metastasis.</p> <p>Methods</p> <p>Quantitative RT-PCR was used to determine the expression of BMP-6, E-cadherin, and δEF1 at the mRNA level in MCF-7 and MDA-MB-231 breast cancer cells, as well as in 16 breast cancer specimens. Immunoblot analysis was used to measure the expression of δEF1 at the protein level in δEF1-overexpressing and δEF1-interfered MDA-MB-231 cells. Luciferase assay was used to determine the rhBMP-6 or δEF1 driven transcriptional activity of the E-cadherin promoter in MDA-MB-231 cells. Quantitative CHIP assay was used to detect the direct association of δEF1 with the E-cadherin proximal promoter in MDA-MB-231 cells.</p> <p>Results</p> <p>MCF-7 breast cancer cells, an ER⁺cell line that expressed high levels of BMP-6 and E-cadherin exhibited very low levels of δEF1 transcript. In contrast, MDA-MB-231 cells, an ER^-cell line had significantly reduced BMP-6 and E-cadherin mRNA levels, suggesting an inverse correlation between BMP-6/E-cadherin and δEF1. To determine if the same relationship exists in human tumors, we examined tissue samples of breast cancer from human subjects. In 16 breast cancer specimens, the inverse correlation between BMP-6/E-cadherin and δEF1 was observed in both ER⁺cases (4 of 8 cases) and ER^-cases (7 of 8 cases). Further, we found that BMP-6 inhibited δEF1 transcription, resulting in an up-regulation of E-cadherin mRNA expression. This is consistent with our analysis of the E-cadherin promoter demonstrating that BMP-6 was a potent transcriptional activator. Interestingly, ectopic expression of δEF1 was able to block BMP-6-induced transactivation of E-cadherin, whereas RNA interference-mediated down-regulation of endogenous δEF1 in breast cancer cells abolished E-cadherin transactivation by BMP-6. In addition to down-regulating the expression of δEF1, BMP-6 also physically dislodged δEF1 from E-cadherin promoter to allow the activation of E-cadherin transcription.</p> <p>Conclusion</p> <p>We conclude that repression of δEF1 plays a key role in mediating BMP-6-induced transcriptional activation of E-cadherin in breast cancer cells. Consistent with the fact that higher level of δEF1 expression is associated with more invasive phenotype of breast cancer cells, our collective data suggests that δEF1 is likely the switch through which BMP-6 restores E-cadherin-mediated cell-to-cell adhesion and prevents breast cancer metastasis.</p

A Proof-Of-Principle Study of Epigenetic Therapy Added to Neoadjuvant Doxorubicin Cyclophosphamide for Locally Advanced Breast Cancer

BACKGROUND: Aberrant DNA methylation and histone deacetylation participate in cancer development and progression; hence, their reversal by inhibitors of DNA methylation and histone deacetylases (HDACs) is at present undergoing clinical testing in cancer therapy. As epigenetic alterations are common to breast cancer, in this proof-of-concept study demethylating hydralazine, plus the HDAC inhibitor magnesium valproate, were added to neoadjuvant doxorubicin and cyclophosphamide in locally advanced breast cancer to assess their safety and biological efficacy. METHODOLOGY: This was a single-arm interventional trial on breast cancer patients (ClinicalTrials.gov Identifier: NCT00395655). After signing informed consent, patients were typed for acetylator phenotype and then treated with hydralazine at 182 mg for rapid-, or 83 mg for slow-acetylators, and magnesium valproate at 30 mg/kg, starting from day –7 until chemotherapy ended, the latter consisting of four cycles of doxorubicin 60 mg/m(2) and cyclophosphamide 600 mg/m(2) every 21 days. Core-needle biopsies were taken from primary breast tumors at diagnosis and at day 8 of treatment with hydralazine and valproate. MAIN FINDINGS: 16 patients were included and received treatment as planned. All were evaluated for clinical response and toxicity and 15 for pathological response. Treatment was well-tolerated. The most common toxicity was drowsiness grades 1–2. Five (31%) patients had clinical CR and eight (50%) PR for an ORR of 81%. No patient progressed. One of 15 operated patients (6.6%) had pathological CR and 70% had residual disease <3 cm. There was a statistically significant decrease in global 5(m)C content and HDAC activity. Hydralazine and magnesium valproate up- and down-regulated at least 3-fold, 1,091 and 89 genes, respectively. CONCLUSIONS: Hydralazine and magnesium valproate produce DNA demethylation, HDAC inhibition, and gene reactivation in primary tumors. Doxorubicin and cyclophosphamide treatment is safe, well-tolerated, and appears to increase the efficacy of chemotherapy. A randomized phase III study is ongoing to support the efficacy of so-called epigenetic or transcriptional cancer therapy

Interactions between the actin filament capping and severing protein gelsolin and the molecular chaperone CCT: evidence for nonclassical substrate interactions

CCT is a member of the chaperonin family of molecular chaperones and consists of eight distinct subunit species which occupy fixed positions within the chaperonin rings. The activity of CCT is closely linked to the integrity of the cytoskeleton as newly synthesized actin and tubulin monomers are dependent upon CCT to reach their native conformations. Furthermore, an additional role for CCT involving interactions with assembling/assembled microfilaments and microtubules is emerging. CCT is also known to interact with other proteins, only some of which will be genuine folding substrates. Here, we identify the actin filament remodeling protein gelsolin as a CCT-binding partner, and although it does not behave as a classical folding substrate, gelsolin binds to CCT with a degree of specificity. In cultured cells, the levels of CCT monomers affect levels of gelsolin, suggesting an additional link between CCT and the actin cytoskeleton that is mediated via the actin filament severing and capping protein gelsolin

Neuroprotection by the histone deacetylase inhibitor trichostatin A in a model of lipopolysaccharide-sensitised neonatal hypoxic-ischaemic brain injury

<p>Abstract</p> <p>Background</p> <p>Perinatal brain injury is complex and often associated with both inflammation and hypoxia-ischaemia (HI). In adult inflammatory brain injury models, therapies to increase acetylation are efficacious in reducing inflammation and cerebral injury. Our aim in the present study was to examine the neuropathological and functional effects of the histone deacetylase inhibitor (HDACi) trichostatin A (TSA) in a model of neonatal lipopolysaccharide (LPS)-sensitised HI. We hypothesised that, by decreasing inflammation, TSA would improve injury and behavioural outcome. Furthermore, TSA’s effects on oligodendrocyte development, which is acetylation-dependent, were investigated.</p> <p>Methods</p> <p>On postnatal day 8 (P8), male and female mice were exposed to LPS together with or without TSA. On P9 (14 hours after LPS), mice were exposed to HI (50 minutes at 10% O₂). Neuropathology was assessed at 24 hours, 5 days and 27 days post-LPS/HI via immunohistochemistry and/or Western blot analysis for markers of grey matter (microtubule-associated protein 2), white matter (myelin basic protein) and cell death (activated caspase-3). Effects of TSA on LPS or LPS/HI-induced inflammation (cytokines and microglia number) were assessed by Luminex assay and immunohistochemistry. Expression of acetylation-dependent oligodendrocyte maturational corepressors was assessed with quantitative PCR 6 hours after LPS and at 24 hours and 27 days post-LPS/HI. Animal behaviour was monitored with the open-field and trace fear-conditioning paradigms at 25 days post-LPS/HI to identify functional implications of changes in neuropathology associated with TSA treatment.</p> <p>Results</p> <p>TSA induced increased Ac-H4 in females only after LPS exposure. Also only in females, TSA reduced grey matter and white matter injury at 5 days post-LPS/HI. Treatment altered animal behaviour in the open field and improved learning in the fear-conditioning test in females compared with LPS/HI-only females at 25 days post-HI. None of the inflammatory mechanisms assessed that are known to mediate neuroprotection by HDACi in adults correlated with improved outcome in TSA-treated neonatal females. Oligodendrocyte maturation was not different between the LPS-only and LPS + TSA-treated mice before or after exposure to HI.</p> <p>Conclusions</p> <p>Hyperacetylation with TSA is neuroprotective in the female neonatal mouse following LPS/HI and correlates with improved learning long-term. TSA appears to exert neuroprotection via mechanisms unique to the neonate. Deciphering the effects of age, sex and inflammatory sensitisation in the cerebral response to HDACi is key to furthering the potential of hyperacetylation as a viable neuroprotectant. TSA did not impair oligodendrocyte maturation, which increases the possible clinical relevance of this strategy.</p