1.ZEB1 Induces ER-α Promoter Hypermethylation And Confers Antiestrogen Resistance In Breast Cancer 2.The Role And Mechanisim Of A Potent VEGFR2 Inhibitor YLL545 In Breast Cancer Angiogenesis And Growth

Breast cancer is a estrogen dependent tumor. Estrogen regulates a variety of physiological responses in mammary gland tissues, and is well known to be involved in breast cancer development and progression. Estrogen exerts its biological action through binding to estrogen receptor-α(ER-α). Antiestrogen resistance is a major obstacle to endocrine therapy for breast cancers. As a indispensable therapy, antiestrogen can significantly improve the prognosis of breast cancer patient. However, the de novo resistanc and acquired resistance to antiestrogen restrict its application in breast cancer therapy. Given this, elucidating the mechanism of antiestrgen resistance will greatly improve the anti-estrogen therapy in breast cancer.Although reduced expression of ER-α is a known contributing factor to antiestrogen resistance, the mechanism of ER-α downregulation in antiestrogen resistance is still not fully understood. In this study, we report that ZEB1 recruit the transmethylase DNMT3 B and histone deacetylase HDAC1 to the ER-α promoter to induce its hypermethylation and thus downregulates the expression of ER-α, which contributes to the antiestrogen resistance.By database on line, we find a Cp G island in ER-α promoter and demonstrate it may be a differentially methylated region(DMR) by Ch IP assay. Then, we verify that ZEB1 can reuglate the metylation of ER-α promoter by MSP and BSP assay in SC/sh ZEB1-MDA-MB-231 and MCS/ZEB1-MCF-7 cell lines.Functionally, ZEB1 overexpression downregulates ER-α expression and subsequently attenuates cell growth inhibition in response to antiestrogen treatment, such as tamoxifen and fulvestrant. At the molecular level, ZEB1 represses ER-α transcription by forming a ZEB1/DNMT3B/HDAC1 complex on its promoter, leading to DNA hypermethylation and silencing of ER-α. Notably, depletion of ZEB1 by RNA interference causes ER-α promoter demethylation, restores ER-α expression, and elevates the responsiveness of breast cancer cells to antiestrogen treatment.Through the study of specimens from a large cohort of subjects with breast cancer, we found a strong inverse correlation between ZEB1 and ER-α protein expression. Moreover, in ER-α-positive subjects, breast tumors that highly expressed ZEB1 exhibits DNA hypermethylation of ER-α promoter. Using a nude mouse xenograft model, we further confirm that downregulation of ZEB1 expression restores the responsiveness of breast cancer cells to antiestrogen therapy in vivo.In conclusion, our findings suggest that ZEB1 is a crucial determinant of resistance to antiestrogen therapies in breast cancer. Tumor angiogenesis plays an important role in cancer invasion and metastasis, which are the main causes of death in patients with malignant tumors. Vascular endothelial growth factor(VEGF) signal pathway is a key regulator in tumor angiogenesis. Vascular endothelial growth factor receptor 2(VEGFR2) inhibitors, including sorafenib and sunitinib, as efficient antiangiogenic agents, have been applied in cancer treatment. However, most of these anticancer drugs have poor effecacy and unexpected side effects. The discovery of novel VEGFR2 inhibitors with lower toxicity is still needed.In the present study, taking sorafenib as the lead compound, we firstly performed rational medicinal chemistry methods to construct 25 derivatives. Secondly, we identified that YLL545, as a novel, potent VEGFR2 inhibitor, and found that it significantly inhibited tansgenic zebrafish(Fli-1:EGFP) embryonic angiogenesis.Then, treatment with YLL545 demonstrated a marked repressive effect on the proliferation, migration, invasion, and tube formation of human umbilical vascular endothelial cells(HUVECs) in vitro. Matrigel plug assays in mice further revealed that YLL545 inhibited angiogenesis in vivo. All results showed that YLL545 had higher potency relative to sorafenib.Nextly, YLL545 inhibited VEGF-induced phosphorylation of VEGFR2 and activation of its downstream signaling regulators, such as phospho-ERK, phospho-STAT3 and phospho-m TOR in HUVECs. In addition to VEGFR2 blockade, we identified that YLL545 exhibited regulatory effect on obvious candidates involved in tumor angiogenesis, such as FN1,TEK,ENG,THBS1 and ITGAV.Importantly, YLL545 also inhibited proliferation and induced apoptosis of breast cancer cells in vitro and in vivo. However, YLL545 did not impair the growth of human normal epithelial cell. Administration of 50 mg/kg/d YLL545 resulted in more than 50% inhibition of human tumor xenograft growth in BALB/c nude mice.Taken together, our observations suggest that YLL545 might be a promising drug candidate in anticancer treatment, based on its pivotal role in inhibiting breast cancer angiogenesis and growth.