BCSCs (breast cancer stem cells) have been shown to be resistant to chemotherapy. However, the mechanisms underlying BCSC-mediated chemoresistance remain poorly understood. The Hh (Hedgehog) pathway is important in the stemness maintenance of CSCs. Nonetheless, it is unknown whether the Hh pathway is involved in BCSC-mediated chemoresistance. In the present study, we cultured breast cancer MCF-7 cells in suspension in serum-free medium to obtain BCSC-enriched MCF-7 MS (MCF-7 mammosphere) cells. We showed that MCF-7 MS cells are sensitive to salinomycin, but not paclitaxel, distinct from parent MCF-7 cells. The expression of the critical components of Hh pathway, i.e., PTCH (Patched), SMO (Smoothened), Gli1 and Gli2, was significantly up-regulated in MCF-7 MS cells; salinomycin, but not paclitaxel, treatment caused a remarkable decrease in expression of those genes in MCF-7 MS cells, but not in MCF-7 cells. Salinomycin, but not paclitaxel, increased apoptosis, decreased the migration capacity of MCF-7 MS cells, accompanied by a decreased expression of c-Myc, Bcl-2 and Snail, the target genes of the Hh pathway. The salinomycin-induced cytotoxic effect could be blocked by Shh (Sonic Hedgehog)-mediated Hh signalling activation. Inhibition of the Hh pathway by cyclopamine could sensitize MCF-7 MS cells to paclitaxel. In addition, salinomycin, but not paclitaxel, significantly reduced the tumour growth, accompanied by decreased expression of PTCH, SMO, Gli1 and Gli2 in xenograft tumours. Furthermore, the expression of SMO and Gli1 was positively correlated with the expression of CD44+ / CD24-, and the expression of SMO and Gli1 in CD44+ / CD24- tissues was associated with a significantly shorter OS (overall survival) and DFS (disease-free survival) in breast cancer patients receiving chemotherapy.

Altered DNA methylation of tumor suppressor gene promoters plays a role in human carcinogenesis and DNA methyltransferases (DNMTs) are responsible for it. This study aimed to determine aberrant expression of DNMT1, DNMT3a, and DNMT3b in benign and malignant ovarian tumor tissues for their association with clinicopathological significance and prognostic value. A total of 142 ovarian cancers and 44 benign ovarian tumors were recruited for immunohistochemical analysis of their expression. The data showed that expression of DNMT1, DNMT3a, and DNMT3b was observed in 76 (53.5%), 92 (64.8%) and 79 (55.6%) of 142 cases of ovarian cancer tissues, respectively. Of the serious tumors, DNMT3a protein expression was significantly higher than that in benign tumor samples (P = 0.001); DNMT3b was marginally significant down regulated in ovarian cancers compared to that of the benign tumors (P = 0.054); DNMT1 expression has no statistical difference between ovarian cancers and benign tumor tissues (P = 0.837). Of the mucious tumors, the expression of DNMT3a, DNMT3b, and DNMT1 was not different between malignant and benign tumors. Moreover, DNMT1 expression was associated with DNMT3b expression (P = 0.020, r = 0.195). DNMT1 expression was associated with age of the patients, menopause status, and tumor localization, while DNMT3a expression was associated with histological types and serum CA125 levels and DNMT3b expression was associated with lymph node metastasis. In addition, patients with DNMT1 or DNMT3b expression had a trend of better survival than those with negative expression. Co-expression of DNMT1 and DNMT3b was significantly associated with better overall survival (P = 0.014). The data from this study provided the first evidence for differential expression of DNMTs proteins in ovarian cancer tissues and their associations with clinicopathological and survival data in sporadic ovarian cancer patients.

The breast cancer susceptibility gene 1 (BRCA1) inactivation in sporadic epithelial ovarian carcinoma (EOC) is common and low BRCA1 expression is associated with promoter hypermethylation. The clinical validation of BRCA1 methylation as a prognostic marker in EOC remains unresolved. The aim of the present study was to determine the aberrant promoter methylation of BRCA1 in benign and malignant ovarian tumor tissues, to establish the association with the clinicopathological significance and the prognostic value. Additionally, the contribution of DNA methyltransferase (DNMT) expression to BRCA1 promoter hypermethylation was determined. The rate of BRCA1 methylation was observed to be 35.2% (50/142) in the EOCs; however, no methylation (0/32) was observed in the benign tumors. BRCA1 methylation was significantly associated with the downregulation of BRCA1 expression (P<0.001) and the frequency of BRCA1 methylation was greater in the carcinomas of patients whose tumor was bilateral than that of patients with a unilateral carcinoma (P=0.015). BRCA1 methylation was significantly associated with the preoperative serum carbohydrate antigen-125 level (P=0.013), improved overall survival (P=0.005) and disease-free survival (P=0.007). In addition, a significant correlation was observed between the co-expression of DNMTs and the methylation status of BRCA1. Thus, the present study provided support for BRCA1 promoter hypermethylation as a prognostic marker for survival in sporadic EOC, and co-expression of DNMTs was observed to contribute to BRCA1 promoter hypermethylation.

Breast cancer stem cells (BCSCs) are believed to be responsible for tumor chemoresistance, recurrence, and metastasis formation. Salinomycin (SAL), a carboxylic polyether ionophore, has been reported to act as a selective breast CSC inhibitor. However, the molecular mechanisms underlying SAL-induced cytotoxicity on BCSCs remain unclear. The Hedgehog (Hh) signaling pathway plays an important role in CSC maintenance and carcinogenesis. Here, we investigated whether SAL induces cytotoxicity on BCSCs through targeting Hh pathway. In the present study, we cultured breast cancer MCF-7 cells in suspension in serum-free medium to obtain breast CSC-enriched MCF-7 mammospheres (MCF-7 MS). MCF-7 MS cells possessed typical BCSC properties, such as CD44+CD24-/low phenotype, high expression of OCT4 (a stem cell marker), increased colony-forming ability, strong migration and invasion capabilities, differentiation potential, and strong tumorigenicity in xenografted mice. SAL exhibited selective cytotoxicity to MCF-7 MS cells relative to MCF-7 cells. The Hh pathway was highly activated in BCSC-enriched MCF-7 MS cells and SAL inhibited Hh signaling activation by downregulating the expression of critical components of the Hh pathway such as PTCH, SMO, Gli1, and Gli2, and subsequently repressing the expression of their essential downstream targets including C-myc, Bcl-2, and Snail (but not cyclin D1). Conversely, Shh-induced Hh signaling activation could largely reverse SAL-mediated inhibitory effects. These findings suggest that SAL-induced selective cytotoxicity against MCF-7 MS cells is associated with the inhibition of Hh signaling activation and the expression of downstream targets and the Hh pathway is an important player and a possible drug target in the pathogenesis of BCSCs.

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.