Usually, the function of estrogen receptor alpha (ERalpha) could be silenced by ERalpha gene promoter hypermethylation. However, frequency of ERalpha promoter methylation and the clinicopathological characteristics of ERalpha methylation in Chinese women with sporadic breast cancer are unknown. The aim of this study was to determine the methylation status of ERalpha promoter and its possible correlation with clinicopathological features in a series of 138 sporadic breast cancers in Chinese women. ER1, ER3, ER4, and ER5 primers were used for methylation-specific polymerase chain reaction (MSP) to analyze the CpG methylation of promoter region of ERalpha gene. In general, we found that ERalpha was methylated in 60.1% (83/138) tumors, including 57 of 69 ERalpha negative tumors (82.6%, P < 0.00001). Specifically within each region the methylation percentage of ER1, ER3, ER4 and ER5 were 34.8%, 35.5%, 39.1%, and 36.9% respectively. The degree of methylation at four CpG sites was higher in breast cancer compared with benign breast hyperplasia (P < 0.00001). In addition, the levels of ERalpha protein expression diminished with the frequency of ERalpha methylation (P < 0.0001, r = -0.469), the probability of methylation was increased for cases with ERalpha and PgR negativity (P < 0.00001). Our preliminary findings demonstrate, for what we believe to be the first time, that ERalpha methylation occurs in high frequency and is one of the mechanisms of ERalpha expression silence in a subset of sporadic breast cancers from Chinese women. Epigenetic alteration of the ERalpha gene may play an important role in the pathogenesis of breast cancer.

Fanconi anemia complementation group F protein (FANCF) is a key factor, which maintains the function of FA/BRCA, a DNA damage response pathway. However, the functional role of FANCF in breast cancer has not been elucidated. We performed a specific FANCF-shRNA knockdown of endogenous FANCF in vitro. Cell viability was measured with a CCK-8 assay. DNA damage was assessed with an alkaline comet assay. Apoptosis, cell cycle, and drug accumulation were measured by flow cytometry. The expression levels of protein were determined by Western blot using specific antibodies. Based on these results, we used cell migration and invasion assays to demonstrate a crucial role for FANCF in those processes. FANCF shRNA effectively inhibited expression of FANCF. We found that proliferation of FANCF knockdown breast cancer cells (MCF-7 and MDA-MB-435S) was significantly inhibited, with cell cycle arrest in the S phase, induction of apoptosis, and DNA fragmentation. Inhibition of FANCF also resulted in decreased cell migration and invasion. In addition, FANCF knockdown enhanced sensitivity to doxorubicin in breast cancer cells. These results suggest that FANCF may be a potential target for molecular, therapeutic intervention in breast cancer.

Cancer stem cells (CSCs) have been suggested to serve an important role in tumor recurrence and metastasis in breast cancer. The hedgehog (Hh) signaling pathway is essential for the maintenance of breast CSCs. The present study used immunohistochemistry to investigate the expression of Patched (PTCH) and Gli1, which are the main components of the Hh signaling pathway, as well as the expression of cluster of differentiation (CD)44/CD24, which are markers for breast CSCs, in 266 patients with breast cancer. The combined expression of PTCH and Gli1 was significantly associated with larger tumors (>2.0 cm; P=0.001), lymph node metastasis (P=0.003), invasive lobular carcinoma (P=0.016) and Grade II‑III tumors (P<0.001). In addition, PTCH and Gli1 expression was associated with lymph node metastasis (P=0.005 and P=0.001) and Grade II‑II tumors (P=0.020 and P=0.033) in breast cancer patients with the CD44+/CD24‑ phenotype. The expression of PTCH and Gli1 was also associated with significantly shorter overall survival and disease‑free survival (DFS) in breast cancer patients with the CD44+/CD24‑ phenotype. Multivariate Cox regression analysis demonstrated that PTCH expression and the CD44+/CD24‑ phenotype were independent prognostic factors for decreased DFS in patients with breast cancer. These findings suggest that the Hh signaling pathway in breast CSCs may contribute to the poor outcome of patients with breast cancer.

This study investigated the expression of ALDH1A1 and β-catenin in breast cancer patients, and analyzed the correlation of their combined expression with clinicopathological features, chemotherapeutic responses, and prognosis of breast cancer patients. In total 276 human breast cancer tissues and 80 benign hyperplasia tissues were included. The expression of ALDH1A1 and β-catenin was examined using tissue microarray-based immunohistochemistry. ROC curve analysis was performed to determine an optimal cut-off score for the expression of ALDH1A1 and β-catenin, based on the survival status of breast cancer patients. Survival probabilities were estimated by the Kaplan-Meier method. ALDH1A1 expression was higher, but β-catenin showed no significant difference in breast cancer samples compared to controls. Compared with the membrane expression of β-catenin [β-catenin(m)], the cytoplasmic expression of β-catenin [β-catenin(c)] occurred significantly more frequently in breast cancer with the high expression of ALDH1A1 [ALDH1A1(high)] than in breast cancer with the low expression of ALDH1A1 [ALDH1A1(low)] (P=0.014). The expression level of ALDH1A was significantly higher in β-catenin(c) breast cancer than in β-catenin(m) breast cancer (P=0.020). ALDH1A1(high) expression or β-catenin(c) expression alone was associated with lymph node metastasis, and worse clinical outcome in breast cancer patients, especially in patients receiving cyclophosphamide treatment. Combined expression of ALDH1A1(high) and β-catenin(c) was associated with lymph node metastasis, poor outcome, and resistance to cyclophosphamide treatment. β-catenin may regulate ALDH1A1 expression in a subtype of breast cancer with ALDH1A1(high) and β-catenin(c) expression. ALDH1A1(high) and β-catenin

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