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Our understanding of breast cancer and its therapeutic approach has improved greatly due to the advancement of molecular biology in recent years. Clinically, breast cancers are characterized into three basic types based on their immunohistochemical properties. They are triple-negative breast cancer, estrogen receptor (ER) and progesterone receptor (PR)-positive-HR positive breast cancer, and human epidermal growth factor receptor 2 (HER2)-positive breast cancer. Even though these subtypes have been characterized, assessment of a breast cancer's receptor status is still widely used to determine whether or not a targeted therapy could be applied. Moreover, drug resistance is common in all breast cancer types despite the different treatment modalities applied. The development of resistance to different therapeutics is not mutually exclusive. It seems that tumor could be resistant to multiple treatment strategies, such as being both chemoresistant and monoclonal antibody resistant. However, the underlying mechanisms are complicated and need further investigation. In this chapter, we aim to provide a brief review of the different types of breast cancer and their respective treatment strategies. We also review the possible mechanisms of potential drug resistance associated with each treatment type. We believe that a better understanding of the drug resistance mechanisms can lead to a more effective and efficient therapeutic success.

The role of lymphocyte antigen 6 complex, locus K (LY6K) in breast cancer has been studied, whereas the epigenetic control of LY6K transcription is not fully understood. Here, we report that breast cancer patients with increased LY6K expression had shorter disease-free and overall survival than the patients with low levels of LY6K by multivariate analysis. LY6K also was upregulated in breast cancer patients with distant metastases than those without distant metastases, downregulating E-cadherin expression. Furthermore, xenograft tumor volumes from LY6K knockdown nude mice were reduced than those of mice treated with control lentivirus. Interestingly, LY6K has a CpG island (CGI) around the transcription start site and non-CGI in its promoter, called a CGI shore. LY6K expression was inversely correlated with methylation in not only CGI but CGI shore, which are associated with histone modifications. Additionally, LY6K methylation was increased by the PAX3 transcription factor due to the SNP242 mutation in LY6K CGI shore. Taken together, breast cancer risk and metastasis were significantly associated with not only LY6K expression, but also methylation of CGI shore which induced by SNP242 mutation. Our results suggest that an understanding epigenetic mechanism of the LY6K gene may be useful to diagnose carcinogenic risk and predict outcomes of patients with metastatic breast cancer.

Estrogen receptor-alpha (ERα) is a clinically important therapeutic target for breast cancer. However, tumors that lose ERα are less responsive to anti-estrogens such as tamoxifen. MicroRNAs (miRNAs) are small RNAs that regulate expression of their target gene and dysregulations of miRNA has been identified in many diseases including human cancer. However, only a few miRNAs associated with tamoxifen resistance has been reported. In this study, we found that lymphocyte antigen 6 complex (LY6K), which is a member of the Ly-6/μPAR superfamily and related to breast cancer progression and metastasis, is inversely correlated with ERα expression. We, for the first time, found miRNAs involved in the regulatory molecular mechanism between ERα and LY6K and related to tamoxifen susceptibility in breast cancer. miR-192-5p, induced by LY6K, downregulates ERα directly and induced tamoxifen resistance in ERα-positive breast cancer cells. In addition, re-expression of ERα in ERα-negative breast cancer cells increased miR-500a-3p expression and directly inhibits LY6K expression. Ectopic expression of miR-500a-3p sensitized ERα-negative cells to tamoxifen by increasing apoptosis. Finally, we observed an inverse correlation between LY6K and ERα in primary breast cancer samples. We found that patients with recurrence showed high expression of miR-192-5p after tamoxifen treatments. In addition, expression of miR-500a-3p was significantly correlated to survival outcome. As miRNAs involved in the regulatory mechanism between LY6K and ERα can affect tamoxifen resistance, downregulating miR-192-5p or re-expressing miR-500a-3p could be a potential therapeutic approach for treating tamoxifen resistant patients., Competing Interests: Authors declare no conflicts of interest.

One of the properties of human breast cancer cells is cancer stemness, which is characterized by self-renewal capability and drug resistance. Protein kinase D1 (PRKD1) functions as a key regulator of many cellular processes and is downregulated in invasive breast cancer cells. In this study, we found that PRKD1 was upregulated in MCF-7-ADR human breast cancer cells characterized by drug resistance. Additionally, we discovered that PRKD1 expression was negatively regulated by miR-34a binding to the PRKD1 3'-UTR. PRKD1 expression increased following performance of a tumorsphere formation assay in MCF-7-ADR cells. We also found that reduction of PRKD1 by ectopic miR-34a expression or PRKD1 siRNA treatment resulted in suppressed self-renewal ability in breast cancer stem cells. Furthermore, we confirmed that the PRKD1 inhibitor CRT0066101 reduced phosphorylated PKD/PKCμ, leading to suppression of breast cancer stemness through GSK3/β-catenin signaling. PRKD1 inhibition also influenced apoptosis initiation in MCF-7-ADR cells. Tumors from nude mice treated with miR-34a or CRT0066101 showed suppressed tumor growth, proliferation, and induced apoptosis. These results provide evidence that regulation of PRKD1, a novel miR-34a target, contributes to overcoming cancer stemness and drug resistance in human breast cancer.

Human breast cancers include cancer stem cell populations as well as nontumorigenic cancer cells. Breast cancer stem cells have self-renewal capability and are resistant to conventional chemotherapy. miRNAs regulate the expression of many target genes; therefore, dysregulation of miRNAs has been associated with the pathogenesis of human diseases, including cancer. However, a role for miRNA dysregulation in stemness and drug resistance has yet to be identified. Members of the miR34 family are reportedly tumor-suppressor miRNAs and are associated with various human cancers. Our results confirm that miR34a expression was downregulated in MCF7/ADR cells compared with MCF7 cells. We hypothesized that this reduction was due to the p53 (TP53) mutation in MCF7/ADR cells. In this study, we found that primary and mature miR34a were suppressed by treatment with p53 RNAi or the dominant-negative p53 mutant in MCF7 cells. Ectopic miR34a expression reduced cancer stem cell properties and increased sensitivity to doxorubicin treatment by directly targeting NOTCH1. Furthermore, tumors from nude mice treated with miR34a were significantly smaller compared with those of mice treated with control lentivirus. Our research suggests that the ectopic expression of miR34a represents a novel therapeutic approach in chemoresistant breast cancer treatment., (©2014 American Association for Cancer Research.)

Estrogen receptor α (ERα) plays a crucial role in estrogen-mediated signaling pathways and exerts its action as a nuclear transcription factor. Binding of the ligand-activated ERα to the estrogen response element (ERE) is a central part of ERα-associated signal transduction pathways and its aberrant modulation is associated with many disease conditions. Human glutathione S-transferase P1-1 (GSTP) functions as an enzyme in conjugation reactions in drug metabolism and as a regulator of kinase signaling pathways. It is overexpressed in tumors following chemotherapy and has been associated with a poor prognosis in breast cancer. In this study, a novel regulatory function of GSTP has been proposed in which GSTP modulates ERE-mediated ERα signaling events. Ectopic expression of GSTP was able to induce the ERα and ERE-mediated transcriptional activities in ERα-positive but GSTP-negative MCF7 human breast cancer cells. This inductive effect of GSTP on the ERE-transcription activity was diminished when the cells express a mutated form of the enzyme or are treated with a GSTP-specific chemical inhibitor. It was found that GSTP inhibited the expression of the receptor interacting protein 140 (RIP140), a negative regulator of ERα transcription, at both mRNA and protein levels. Our study suggests a novel non-enzymatic role of GSTP which plays a significant role in regulating the classical ERα signaling pathways via modification of transcription cofactors such as RIP140., (Copyright © 2014 Elsevier Inc. All rights reserved.)

LY6K is a cancer biomarker and a therapeutic target that induces invasion and metastasis. However, the molecular mechanisms that determine human LY6K transcription are completely unknown. To elucidate the mechanisms involved in human LY6K gene regulation and expression, multiple cis-elements were predicted using TRANSFAC software, and the LY6K regulatory region was identified using the luciferase assay in the human LY6K gene promoter. We performed ChIP, EMSA, and supershift assays to investigate the transcription factor activity on the LY6K promoter, and the effect of a SNP and CpG site methylation on AP-1 transcription factor binding affinity. AP-1 and the CREB transcription factor bound to LY6K promoter within -550/-1, which was essential for LY6K expression, but only the AP-1 heterodimer, JunD, and Fra-1, modulates LY6K gene transcriptional level. A decrease in LY6K was associated with the SNP242 C allele, a polymorphic G/C-SNP at the 242 nucleotide in the LY6K promoter region (rs2585175), or methylation of the CpG site, which was closely located with the AP-1 site by interfering with binding of the AP-1 transcription factor to the LY6K promoter. Our findings reveal an important role for AP-1 activation in promoting LY6K gene expression that regulates cell mobility of breast cancer cells, whereas the SNP242 C allele or methylation of the CpG site may reduce the risk of invasion or metastasis by interfering AP-1 activation.

Peroxiredoxin 5 (Prx5) is a member of a family consisting of six antioxidant enzymes. Prx5 is ubiquitously expressed in various tissues including mitochondria and peroxisomes, implying that Prx5 functions as a regulator of the cellular oxidation state. Prx5 plays a critical role in protecting cells from oxidative stress by inhibiting the accumulation of reactive oxygen species and cell death. Overexpression of Prx5 in mammary tissue is associated with poor prognosis of breast cancer. The present study was conducted to elucidate the regulatory mechanisms of Prx5 gene expression and the physiological relevance of Prx5 in breast cancer cell survival. Analysis of the promoter region of the Prx5 gene and reporter gene assays revealed the promoter region critical for Prx5 gene regulation to which the novel negative transcription regulator, GATA1 binds in human breast cancer cell lines. GATA1 was further confirmed as a potential transcription factor for Prx5 gene expression in another set of transfection assays using a reporter gene construct that contained a mutated GATA1 binding site. Direct binding of GATA1 to the Prx5 promoter was demonstrated using chromatin immunoprecipitation and electrophoretic mobility shift assays. Finally, functions of Prx5 associated with apoptosis were examined in two human breast cancer cell lines displaying high levels of either Prx5 or GATA1 expression. Knockdown of GATA1 led to increased expression of Prx5 and inhibition of apoptosis. Our data suggest that Prx5 may protect cells from oxidative stress-mediated apoptosis in a GATA1-regulated manner.

Tumor-initiating cells (T-ICs), a subpopulation of cancer cells with stem cell-like properties, are related to tumor relapse and metastasis. Our previous studies identified a distinct profile of microRNA (miRNA) expression in breast T-ICs (BT-ICs), and the dysregulated miRNAs contribute to the self-renewal and tumorigenesis of these cells. However, the underlying mechanisms for miRNA dysregulation in BT-ICs remain obscure. In the present study, we demonstrated that the expression and function of miR-34c were reduced in the BT-ICs of MCF-7 and SK-3rd cells, a breast cancer cell line enriched for BT-ICs. Ectopic expression of miR-34c reduced the self-renewal of BT-ICs, inhibited epithelial-mesenchymal transition, and suppressed migration of the tumor cells via silencing target gene Notch4. Furthermore, we identified a single hypermethylated CpG site in the promoter region of miR-34c gene that contributed to transcriptional repression of miR-34c in BT-ICs by reducing DNA binding activities of Sp1. Therefore, miR-34c reduction in BT-ICs induced by a single hypermethylated CpG site in the promoter region promotes self-renewal and epithelial-mesenchymal transition of BT-ICs.

The over-expression of urokinase receptor in breast cancer cells results in increased tumor invasion, growth and metastasis. As LY-6K belongs to the Ly6/uPAR superfamily, we assessed the association of LY-6K with metastasis in breast cancer cell lines and how the LY-6K gene mediates cancer cell metastasis. The migration ability of cells in which expression of LY-6K gene is up- or down-regulated was investigated by transfecting LY-6K naked DNA and treating LY-6K siRNA. Assays of wound healing, migration and invasion were performed. The assays demonstrated that cell migration significantly increased when LY-6K gene is over-expressed or LY-6K protein is abnormally expressed in breast cancer cells. In addition, LY-6K caused cancer cell metastasis and it is linked to the Ras/ERK signaling pathway. Our results suggest that LY-6K meaningfully participates in breast cancer cell metastasis by influencing cell migration and invasion through the Ras/ERK pathway.

A full-length cDNA was identified using one STS sequence containing an SNP (single nucleotide polymorphism) derived from genomic DNAs of breast cancer patients using a variety of bioinformatics tools. The cDNA encodes LY-6K, a novel member protein of the Ly-6/uPAR superfamily. It has been annotated as a target antigen for the HNSCC (head-and neck squamous cell carcinoma). We isolated the LY-6K gene from genomic DNAs obtained from breast cancer patients through large scale, case-control-screening. We performed northern blot hybridization and semi-quantitative RT-PCR on a human multiple-tissue mRNA blot from several breast cancer patients. We investigated the expression level of the LY-6K gene in human breast cancer, and compared this to expression in human normal breast tissue. We found that LY-6K was more highly expressed in the mRNA of breast tumors compared to its expression in normal breast tissue. These results suggest that LY-6K is not only a target antigen for HNSCC but also a significant new molecular marker for diagnosis and gene therapy in patients with breast cancer.