Approximately 5%–10% of breast cancers are due to genetic predisposition caused by germline mutations; the most commonly tested genes are BRCA1 and BRCA2 mutations. Some mutations are unique to one family and others are recurrent; the spectrum of BRCA1/BRCA2 mutations varies depending on the geographical origins, populations or ethnic groups. In this review, we compiled data from 11 participating Asian countries (Bangladesh, Mainland China, Hong Kong SAR, Indonesia, Japan, Korea, Malaysia, Philippines, Singapore, Thailand and Vietnam), and from ethnic Asians residing in Canada and the USA. We have additionally conducted a literature review to include other Asian countries mainly in Central and Western Asia. We present the current pathogenic mutation spectrum of BRCA1/BRCA2 genes in patients with breast cancer in various Asian populations. Understanding BRCA1/BRCA2 mutations in Asians will help provide better risk assessment and clinical management of breast cancer.

BackgroundTriple-negative breast cancer (TNBC) remains a poor prognostic factor for breast cancer since no effective targeted therapy is readily available. Our previous studies confirmed miR-199a-5p is a TNBC-specific circulating biomarker, however, its functional roles in breast cancer is largely unknown. Thus, we investigated the functional implication of miR-199a-5p in TNBC and its potential underlying mechanisms.MethodsMTT assay was performed to investigate the cell proliferation after transient transfection of miR-199a-5p in MDA-MB-231 cell line, followed by cell cycle analysis. Transwell invasion assay and wound healing assay were used to study the invasion and migration ability respectively. To further investigate the stemness-related characteristics of miR-199a-5p in breast cancer cells, single-cell clonogenic assay and aldehyde dehydrogenase (ALDH) assay were performed. 32 normal and 100 breast cancer patients’ plasma were recruited to identify the potential circulating markers by qPCR.ResultsCell proliferation assay revealed significant inhibition after miR-199a-5p ectopic expression (p < 0.0001), as a result of decreased S phase (p = 0.0284), increased G0/G1 phase (p = 0.0260) and apoptosis (p = 0.0374). Invasiveness (p = 0.0005) and wound healing ability were also decreased upon miR-199a-5p overexpression. It significantly altered EMT-related genes expression, namely CDH1, ZEB1 and TWIST. Single-cell clonogenic assay showed decreased colonies in miR-199a-5p (p = 0.0182). Significant downregulation (p = 0.0088) and inhibited activity (p = 0.0390) of ALDH was observed in miR-199a-5p. ALDH1A3, which is the dominant isoform of ALDH, is significantly upregulated in breast cancer plasma especially in TNBC (p = 0.0248). PIK3CD was identified as a potential downstream target of miR-199a-5p.ConclusionsTaken together, we unraveled, for the first time, the tumor-suppressive role of miR-199a-5p in TNBC, which attributed to EMT and cancer stemness properties, providing a novel therapeutic options towards this aggressive disease.

Around 80% of mutations in the PTEN gene have been reported to be associated with diseases such as Cowden syndrome, which is an autosomal dominant disorder associated with an increased risk of developing breast, thyroid, and endometrial neoplasms. Recent studies have also demonstrated that KILLIN, which is located proximally to PTEN, shares the same transcription start site, and is assumed to be regulated by the same promoter, but is transcribed in the opposite direction. In this regard, we postulate that there may be a connection between KILLIN/PTEN genes and breast and thyroid cancers. Using real-time quantitative polymerase chain reaction (qPCR), we found that expression of KILLIN, but not PTEN, was significantly decreased in 23 Chinese women with a personal history of breast and thyroid cancer or a personal history of breast cancer and a family history of thyroid cancer, or vice versa, and at least two persons in the family with thyroid cancer or at a young age <40 years, when compared with healthy controls (P<0.0001). No PTEN mutations were found in these 23 patients. We then developed a simple methylation-sensitive restriction enzyme digestion followed by real-time quantitative assay to quantify plasma methylated KILLIN/PTEN DNA in these patients. Plasma levels of methylated KILLIN/PTEN DNA were significantly increased in these patients when compared with healthy controls (P<0.05). This study shows that plasma methylated KILLIN/PTEN DNA was significantly elevated, suggesting hypermethylation of the KILLIN/PTEN promoter in breast and thyroid cancer patients.

Objectives: Triple-negative breast cancer (TNBC) accounts for 15-20% of all breast cancer, and is characterized by the absence of estrogen receptor, progesterone receptor and human epidermal growth factor receptor. The lack of targeted therapy is a major obstacle for treating against this aggressive subtype, hence the search of specific biomarkers may use as potential diagnostic marker and perhaps a therapeutic target for TNBC. Methods: Plasma samples from patients with TNBC and non-TNBC were recruited for miRNA profiling. By using miRCURY LNA array containing 730 miRNAs, we compared the differential miRNA expressions in plasma of patient with TNBC (n=5) and non-TNBC (n=5), as well as healthy controls (n=5). Selected miRNAs were further validated in an independent cohort (n=250) using real-time RT-PCR. Functional study of miRNA was also carried out in the cell lines. Results: Microarray data revealed miR-16, miR-21 and miR-199a-5p have differentially expression between TNBC and non-TNBC. We found that miR-16, miR-21 and miR-199a-5p were underexpressed in TNBC cases when compared with healthy controls. Moreover, post-operative expression levels of miR-16, miR-21 and miR-199a-5p were significantly increased than that of pre-operative plasma of TNBC. We then stratified the patients by TNM stage and associate with miRNA expression level. Plasma miR-199a-5p expression in TNBC patients had significant difference when compared with healthy controls (stage I, p&lt;0.002; stage II, p&lt;0.001; stage III, p&lt;0.011). Transfection of miR-199a-5p mimic significantly reduced cell proliferation and restored the epithelial-mesenchymal transition (EMT) markers (E-cadherin, ZEB1 and TWIST2). Conclusions: Our data implicate that miR-199a-5p is a potential marker for discriminating TNBC cases and non-TNBC cases. The association of miR-199a-5p with EMT markers uncovers an important pathway in metastatic breast cancer. This study provides insights for the potential use of miRNA as diagnostic marker and targeted therapy for the treatment of TNBC. Citation Format: Vivian Y. Shin, Man T. Siu, John C. Ho, Ava Kwong. MiR-199a-5p is a biomarker for and regulator of epithelial-mesenchymal transition in triple-negative breast cancer patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 531. doi:10.1158/1538-7445.AM2014-531

Background: BRCA1 mutations are highly susceptible to familial breast and ovarian cancer, as well as the more aggressive triple-negative breast cancer (TNBC). However, DNA repair defects caused by BRCA dysfunction were reported to have better response to platinum-based chemotherapy and PARP inhibitors. Increasing evidence suggest the roles of miRNAs in regulating BRCA1 functions, with emphasis on cancer progression and therapeutic sensitivity. We previously identified miR-199a-3p as a potential TNBC biomarker, and BRCA1 is the putative downstream target based on in silico prediction. In this study, we aim to investigate the functionalities of miR-199a-3p in TNBC. Methods: The correlation between BRCA1 and miR-199a-3p was investigated through expression analysis of 32 TNBC tumor specimens, and further confirmed by dual-luciferase reporter assay. The functionalities of miR-199a-3p in TNBC cells were assessed by MTT cell proliferation, migration, and clonogenic assays. The effects of miR-199a-3p on cisplatin and PARP inhibitor veliparib sensitivities were determined by MTT assays, followed by apoptosis and cell cycle analyses. Results: An inverse correlation between miR-199a-3p and BRCA1 expressions was observed in TNBC tumor specimens. miR-199a-3p was demonstrated to directly target BRCA1, and caused downregulation of BRCA1 protein expression, and suppressed luciferase activity mediated by BRCA1-3’-UTR in reporter assays. Overexpression of miR-199a-3p in TNBC cell lines (MDA-MB-231 and MDA-MB-468) was shown to inhibit cell proliferation, migration, and clonogenic ability, suggesting tumor-suppressive functions. On the other hand, cisplatin treatment induced BRCA1 expression, concurrent with reduced miR-199a-3p, in TNBC cells. Restoration of miR-199a-3p expression sensitized the cells to cisplatin and PARP inhibitor veliparib. Ectopic expression of miR-199a-3p also suppressed aldehyde dehydrogenase (ALDH)-positive stem cell-like population in cisplatin-resistant TNBC cells. Conclusions: Our results suggest that miR-199a-3p is involved in TNBC progression and chemotherapeutic sensitivity, at least partially through regulation of BRCA1. Citation Format: John C. Ho, Vivian Y. Shin, Jiawei Chen, Man T. Siu, Isabella Cheuk, Ava Kwong. miR-199a-3p induces BRCA1 dysfunction in triple-negative breast cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A046.

Background: Resveratrol is a naturally occurring polyphenolic compound in Polygonum cuspidatum and is well known for its antitumor effect by targeting several cancer-related inflammatory pathways. However, limited data is available on the effect of resveratrol in the tumor microenvironment (TME). Tumor-associated macrophages (TAMs) are the most abundant leukocyte population within the tumors and are associated with poor prognosis in over 80% of breast cancer cases. This study aimed to investigate the effect of resveratrol on TAM polarization in breast cancer progression. Methods: The antitumor effect of resveratrol was examined in MDA-MB-231(MB231), cisplatin resistance MDA-MB-231 (cisR) cells, and T47D by cell proliferation assay using MTT. Gene expressions of M1 and M2 markers in macrophages derived from THP-1 cells were examined by qPCR and immunofluorescence staining after treated with tumor-conditioned medium (TCM) with or without resveratrol (25μM). Cytokine profile of TCM was examined by Qiagen Multi-Analyte ELISArray and expression of cytokines in TCM was validated by qPCR and ELISA. The effect of resveratrol and its downstream targets on macrophage polarization were investigated by supplementing resveratrol with interferon gamma (IFN-γ, 20ng/ml) and lipopolysaccharide (LPS, 100ng/ml) for M1 macrophage polarization or IL-4 (20ng/ml) and IL-13 (20ng/ml)for M2 macrophage polarization. NOD/SCID mice were used to investigate the potential use of resveratrol in treating breast cancer and chemotherapy as complementary treatment in vivo. Results: Our preliminary data demonstrated that resveratrol significantly reduced cell proliferation and enhanced chemosensitivity in MB231, cisR, and T47D cells. Resveratrol inhibited CD163 (M2 marker) and increased CXCL10 (M1 marker) expression. In addition, resveratrol decreased exogenous IL-6 levels in TCM, thus inhibited breast cancer cell proliferation by promoting M1/M2 macrophage polarization ratio and supressing IL-6/pSTAT3 pathway. Combined use of resveratrol and cisplatin significantly decreased tumor size in vivo. Conclusion: Our data demonstrated that resveratrol enhanced chemosensitivity in vivo and in vitro by promoting M1/M2 macrophage polarization ratio and supressing STAT3 activation through reducing exogenous IL-6 levels. This study revealed a novel mechanism on resveratrol-mediated macrophage polarization on breast cancer progression. Correspondence: akwong@asiabreastregistry.com Citation Format: Isabella Wai Yin Cheuk, Jiawei Chen, Man Ting Siu, Sau Wing Lam, Jue Wang, Vivian Yvonne Shin, Ava Kwong. Resveratrol enhanced chemosensitivity by reversing macrophage polarization in breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-06-16.

Background: Presence of cancer stem-like cells (CSCs) is the main obstacle for poor treatment response and mortality in breast cancer patients. Prostaglandin E receptors have been reported to play a role in epithelial-mesenchymal transition (EMT) and metastasis, however, the contribution on cancer stem cell compartment remains unstudied. Methods: Human xenograft breast cancer model was used to study the expression of EP receptors during cancer development. Construction of stable EP2-expression cells was used to study tumorigenesis and characterization of EP2 receptor. Functional role of EP2 receptor on cell proliferation, flow cyometry, invasion and EMT gene expression array were performed in transfected cells. Expression of EP2 receptor was compared in primary tumor tissues by immunostaining and real-time PCR. Results: EP2 receptor was predominantly expressed in animal tissues during cancer development, as well as in human primary tumor tissues. In mouse xenograft model, MB-231-EP2 clone developed a more aggressive tumor with a larger tumor size and showed a significant increase in cancer stem cell marker aldehyde hydrogenase (ALDH1) expression. In vitro study, MB-231-EP2 clone increased colony formation capacity and S-phase entry by the regulation of E-cadherin, TWIST1 and ALDH1. Importantly, we found that Twist1 expression level was higher in breast cancer patients than healthy controls and was associated with ALDH1 expression. Conclusions: These findings implicated that EP2 receptor was crucial to nurture CSC phenotype and promote tumorigenesis in breast cancer. Blocking of EP2 might be a potential therapeutic strategy to improve treatment response for breast cancer patients. Citation Format: Vivian Y Shin, Man T Siu, John C Ho, Isabella Cheuk, Jiawei Chen, Ava Kwong. Prostaglandin E receptor 2 (EP2) regulates breast cancer stem-cell like property and promotes epithelial-mesenchymal transition [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-07-07.