Fulvestrant is a selective estrogen receptor downregulator (SERD) and highly effective antagonist to hormone-sensitive breast cancers following failure of previous tamoxifen or aromatase inhibitor therapies. However, after prolonged fulvestrant therapy, acquired resistance eventually occurs in the majority of breast cancer patients, due to poorly understood mechanisms. To examine a possible role(s) of aberrantly expressed microRNAs (miRNAs) in acquired fulvestrant resistance, we compared antiestrogen-resistant and -sensitive breast cancer cells, revealing the over-expression of miR-221/222 in the SERD-resistant cell lines. Fulvestrant treatment of estradiol (E2)- and fulvestrant-sensitive MCF7 cells resulted in increased expression of endogenous miR-221/222. Ectopic upregulation of miR-221/222 in estrogen receptor-α (ERα)-positive cell lines counteracted the effects of E2 depletion or fulvestrant-induced cell death, thus also conferring hormone-independent growth and fulvestrant resistance. In cells with acquired resistance to fulvestrant, miR-221/222 expression was essential for cell growth and cell cycle progression. To identify possible miR-221/222 targets, miR-221- or miR-222- induced alterations in global gene expression profiles and target gene expression at distinct time points were determined, revealing that miR-221/222 overexpression resulted in deregulation of multiple oncogenic signaling pathways previously associated with drug resistance. Activation of β-catenin by miR-221/222 contributed to estrogen-independent growth and fulvestrant resistance, whereas TGF-β-mediated growth inhibition was repressed by the two miRNAs. This first in-depth investigation into the role of miR-221/222 in acquired fulvestrant resistance, a clinically important problem, demonstrates that these two ‘oncomirs’ may represent promising therapeutic targets for treating hormone-independent, SERD-resistant breast cancer.

Purpose N6-methyladenosine (m6A), the most abundant mRNA modification in mammals, is involved in various biological processes. KIAA1429 is an important methyltransferase participating in m6A modification. However, the role of KIAA1429 in hepatocellular carcinoma (HCC) is still not well understood. Here, we aimed to investigate the function of KIAA1429 and its corresponding regulation mechanisms in HCC. Patients and methods HCC-related genes were analyzed by clinical and expression data of HCC patients in The Cancer Genome Atlas (TCGA) database. Expression of KIAA1429 was verified by quantitative reverse-transcription PCR, and interference efficiency was obtained using small interfering RNA (siRNA). Cell proliferation, migration, and invasion were assessed by cell counting kit-8 and transwell assays, and the m6A modification was detected by methylated RNA immunoprecipitation-PCR (MeRIP-PCR). Results We found a difference in the expression of KIAA1429 between HCC and normal hepatic tissues by analyzing data from the TCGA database. Comparing HCC cell lines (HepG2, Huh-7, HepG2.2.15) with normal hepatic cells (HL-7702), we observed an identically significant difference in KIAA1429 expression. KIAA1429 significantly enhanced proliferation, migration, and invasion of HepG2 cells. Moreover, Kyoto Encyclopedia of Genes and Genomes functional enrichment analysis and correlation analysis revealed a significant negative correlation between KIAA1429 and ID2. In the subsequent MeRIP-PCR assay, downregulation of KIAA1429 inhibited m6A modification of ID2 mRNA. Conclusion KIAA1429 facilitated migration and invasion of HCC by inhibiting ID2 via upregulating m6A modification of ID2 mRNA.

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Caveolin-1 (Cav1) is an integral membrane, scaffolding protein found in plasma membrane invaginations (caveolae). Cav1 regulates multiple cancer-associated processes. In breast cancer, a tumor suppressive role for Cav1 has been suggested; however, Cav1 is frequently overexpressed in aggressive breast cancer subtypes, suggesting an oncogenic function in advanced-stage disease. To further delineate Cav1 function in breast cancer progression, we evaluated its expression levels among a panel of cell lines representing a spectrum of breast cancer phenotypes. In basal-like (the most aggressive BC subtype) breast cancer cells, Cav1 was consistently upregulated, and positively correlated with increased cell proliferation, anchorage-independent growth, and migration and invasion. To identify mechanisms of Cav1 gene regulation, we compared DNA methylation levels within promoter ‘CpG islands' (CGIs) with ‘CGI shores', recently described regions that flank CGIs with less CG-density. Integration of genome-wide DNA methylation profiles (‘methylomes') with Cav1 expression in 30 breast cancer cell lines showed that differential methylation of CGI shores, but not CGIs, significantly regulated Cav1 expression. In breast cancer cell lines having low Cav1 expression (despite promoter CGI hypomethylation), we found that treatment with a DNA methyltransferase inhibitor induced Cav1 expression via CGI shore demethylation. In addition, further methylome assessments revealed that breast cancer aggressiveness associated with Cav1 CGI shore methylation levels, with shore hypermethylation in minimally aggressive, luminal breast cancer cells and shore hypomethylation in highly aggressive, basal-like cells. Cav1 CGI shore methylation was also observed in human breast tumors, and overall survival rates of breast cancer patients lacking estrogen receptor α (ERα) negatively correlated with Cav1 expression. Based on this first study of Cav1 (a potential oncogene) CGI shore methylation, we suggest this phenomenon may represent a new prognostic marker for ERα-negative, basal-like breast cancer.