Xiaohui Tan

miR-638 mediated regulation of BRCA1 affects DNA repair and sensitivity to UV and cisplatin in triple-negative breast cancer

IntroductionTriple-negative breast cancer (TNBC) represents 15 to 20% of all types of breast cancer; however, it accounts for a large number of metastatic cases and deaths, and there is still no effective treatment. The deregulation of microRNAs (miRNAs) in breast cancer has been widely reported. We previously identified that miR-638 was one of the most deregulated miRNAs in breast cancer progression. Bioinformatics analysis revealed that miR-638 directly targets BRCA1. The aim of this study was to investigate the role of miR-638 in breast cancer prognosis and treatment.MethodsFormalin-fixed, paraffin-embedded (FFPE) breast cancer samples were microdissected into normal epithelial and invasive ductal carcinoma (IDC) cells, and total RNA was isolated. Several breast cancer cell lines were used for the functional analysis. miR-638 target genes were identified by TARGETSCAN-VERT 6.2 and miRanda. The expression of miR-638 and its target genes was analyzed by real-time qRT-PCR and Western blotting. Dual-luciferase reporter assay was employed to confirm the specificity of miR-638 target genes. The biological function of miR-638 was analyzed by MTT chemosensitivity, matrigel invasion and host cell reactivation assays.ResultsThe expression of miR-638 was decreased in IDC tissue samples compared to their adjacent normal controls. The decreased miR-638 expression was more prevalent in non-TNBC compared with TNBC cases. miR-638 expression was significantly downregulated in breast cancer cell lines compared to the immortalized MCF-10A epithelial cells. BRCA1 was predicted as one of the direct targets of miR-638, which was subsequently confirmed by dual-luciferase reporter assay. Forced expression of miR-638 resulted in a significantly reduced proliferation rate as well as decreased invasive ability in TNBC cells. Furthermore, miR-638 overexpression increased sensitivity to DNA-damaging agents, ultraviolet (UV) and cisplatin, but not to 5-fluorouracil (5-FU) and epirubicin exposure in TNBC cells. Host cell reactivation assays showed that miR-638 reduced DNA repair capability in post UV/cisplatin-exposed TNBC cells. The reduced proliferation, invasive ability, and DNA repair capabilities are associated with downregulated BRCA1 expression.ConclusionsOur findings suggest that miR-638 plays an important role in TNBC progression via BRCA1 deregulation. Therefore, miR-638 might serve as a potential prognostic biomarker and therapeutic target for breast cancer.

miR-671-5p inhibits epithelial-to-mesenchymal transition by downregulating FOXM1 expression in breast cancer

MicroRNA (miRNA) dysfunction is associated with a variety of human diseases, including cancer. Our previous study showed that miR-671-5p was deregulated throughout breast cancer progression. Here, we report for the first time that miR-671-5p is a tumor-suppressor miRNA in breast tumorigenesis. We found that expression of miR-671-5p was decreased significantly in invasive ductal carcinoma (IDC) compared to normal in microdissected formalin-fixed, paraffin-embedded (FFPE) tissues. Forkhead Box M1 (FOXM1), an oncogenic transcription factor, was predicted as one of the direct targets of miR-671-5p, which was subsequently confirmed by luciferase assays. Forced expression of miR-671-5p in breast cancer cell lines downregulated FOXM1 expression, and attenuated the proliferation and invasion in breast cancer cell lines. Notably, overexpression of miR-671-5p resulted in a shift from epithelial-to-mesenchymal transition (EMT) to mesenchymal-to-epithelial transition (MET) phenotypes in MDA-MB-231 breast cancer cells and induced S-phase arrest. Moreover, miR-671-5p sensitized breast cancer cells to cisplatin, 5-fluorouracil (5-FU) and epirubicin exposure. Host cell reactivation (HCR) assays showed that miR-671-5p reduces DNA repair capability in post-drug exposed breast cancer cells. cDNA microarray data revealed that differentially expressed genes when miR-671-5p was transfected are associated with cell proliferation, invasion, cell cycle, and EMT. These data indicate that miR-671-5p functions as a tumor suppressor miRNA in breast cancer by directly targeting FOXM1. Hence, miR-671-5p may serve as a novel therapeutic target for breast cancer management.

MicroRNA-21 Down-regulates Rb1 Expression by Targeting PDCD4 in Retinoblastoma

Retinoblastoma (RB) is a childrens ocular cancer caused by mutated retinoblastoma 1 (Rb1) gene on both alleles. Rb1 and other related genes could be regulated by microRNAs (miRNA) via complementarily pairing with their target sites. MicroRNA-21 (miR-21) possesses the oncogenic potential to target several tumor suppressor genes, including PDCD4, and regulates tumor progression and metastasis. However, the mechanism of how miR-21 regulates PDCD4 is poorly understood in RB. We investigated the expression of miRNAs in RB cell lines and identified that miR-21 is one of the most deregulated miRNAs in RB. Using qRT-PCR, we verified the expression level of several miRNAs identified by independent microarray assays, and analyzed miRNA expression patterns in three RB cell lines, including Weri-Rb1, Y79 and RB355. We found that miR-19b, -21, -26a, -195 and -222 were highly expressed in all three cell lines, suggesting their potential role in RB tumorigenesis. Using the TargetScan program, we identified a list of potential target genes of these miRNAs, of which PDCD4 is one the targets of miR-21. In this study, we focused on the regulatory mechanism of miR-21 on PDCD4 in RB. We demonstrated an inverse correlation between miR-21 and PDCD4 expression in Weri-Rb1 and Y79 cells. These data suggest that miR-21 down-regulates Rb1 by targeting PDCD4 tumor suppressor. Therefore, miR-21 could serve as a therapeutic target for retinoblastoma.

Clinical Significance of a Point Mutation in DNA Polymerase Beta (POLB) Gene in Gastric Cancer.

Gastric cancer (GC) is a major cause of global cancer mortality. Genetic variations in DNA repair genes can modulate DNA repair capability and, consequently, have been associated with risk of developing cancer. We have previously identified a T to C point mutation at nucleotide 889 (T889C) in DNA polymerase beta (POLB) gene, a key enzyme involved in base excision repair in primary GCs. The purpose of this study was to evaluate the mutation and expression of POLB in a larger cohort and to identify possible prognostic roles of the POLB alterations in GC. Primary GC specimens and their matched normal adjacent tissues were collected at the time of surgery. DNA, RNA and protein samples were isolated from GC specimens and cell lines. Mutations were detected by PCR-RFLP/DHPLC and sequencing analysis. POLB gene expression was examined by RT-PCR, tissue microarray, Western blotting and immunofluorescence assays. The function of the mutation was evaluated by chemosensitivity, MTT, Transwell matrigel invasion and host cell reactivation assays. The T889C mutation was detected in 18 (10.17%) of 177 GC patients. And the T889C mutation was associated with POLB overexpression, lymph nodes metastases and poor tumor differentiation. In addition, patients with- the mutation had significantly shorter survival time than those without-, following postoperative chemotherapy. Furthermore, cell lines with T889C mutation in POLB gene were more resistant to the treatment of 5-fluorouracil, cisplatin and epirubicin than those with wild type POLB. Forced expression of POLB gene with T889C mutation resulted in enhanced cell proliferation, invasion and resistance to anticancer drugs, along with increased DNA repair capability. These results suggest that POLB gene with T889C mutation in surgically resected primary gastric tissues may be clinically useful for predicting responsiveness to chemotherapy in patients with GC. The POLB gene alteration may serve as a prognostic biomarker for GC.

May Circulating microRNAs be Gastric Cancer Diagnostic Biomarkers

Gastric cancer (GC) is the third leading cause of cancer-related deaths. More than 80% of the diagnosis was made at the advanced stages of the disease, highlighting the urgent demand for novel biomarkers that can be used for early detection. Recently, a number of studies suggest that circulating microRNAs (miRNAs) could be potential biomarkers for GC diagnosis. Cancer-related circulating miRNAs, as well as tissue miRNAs, provide a hopeful prospect of detecting GC at early stages, and the prospective participation of miRNAs in biomarker development will enhance the sensitivity and specificity of diagnostic tests for GC. As miRNAs in blood are stable, their potential value as diagnostic biomarkers in GC has been explored over the past few years. However, due to the inconsistent or sometimes conflicting reports, large-scale prospective studies are needed to validate their potential applicability in GC diagnosis. This review summarizes the current development about potential miRNA biomarkers for GC diagnosis and the obstacles hindering their clinical usage.

Beta protein 1 homeoprotein induces cell growth and estrogen-independent tumorigenesis by binding to the estrogen receptor in breast cancer.

Expression of Beta Protein 1 (BP1), a homeotic transcription factor, increases during breast cancer progression and may be associated with tumor aggressiveness. In our present work, we investigate the influence of BP1 on breast tumor formation and size in vitro and in vivo. Cells overexpressing BP1 showed higher viability when grown in the absence of serum (p < 0.05), greater invasive potential (p < 0.05) and formed larger colonies (p < 0.004) compared with the controls. To determine the influence of BP1 overexpression on tumor characteristics, MCF-7 cells transfected with either empty vector (V1) or overexpressor plasmids (O2 and O4) were injected into the fat pads of athymic nude mice. Tumors grew larger in mice receiving O2 or O4 cells than in mice receiving V1 cells. Moreover, BP1 mRNA expression levels were positively correlated with tumor size in patients (p = 0.01). Interestingly, 20% of mice injected with O2 or O4 cells developed tumors in the absence of estrogen, while no mice receiving V1 cells developed tumors. Several mechanisms of estrogen independent tumor formation related to BP1 were established. These data are consistent with the fact that expression of breast cancer anti-estrogen resistance 1 (BCAR1) was increased in O2 compared to V1 cells (p < 0.01). Importantly, O2 cells exhibited increased proliferation when treated with tamoxifen, while V1 cells showed growth inhibition. Overall, BP1 overexpresssion in MCF-7 breast cancer cells leads to increased cell growth, estrogen-independent tumor formation, and increased proliferation. These findings suggest that BP1 may be an important biomarker and therapeutic target in ER positive breast cancer.

microRNA biomarkers in colorectal cancer liver metastasis

Liver metastasis is a primary factor of prognosis and long-term survival for patients diagnosed with colorectal cancer (CRC). Colorectal cancer liver metastasis (CRCLM), is a complex biological process involving multiple factors and steps, and its mechanisms are yet to be discovered. In recent years, small noncoding RNAs, especially microRNAs (miRNAs) have been proven to play an important role in tumorigenesis, progression and metastasis in a variety of cancers, including CRC. Increasing evidence suggests that miRNAs, including those from exosomes secreted by tumor cells in circulation, could be used as promising biomarkers in early cancer detection, treatment, and prognosis. In this review, we focus on the functional roles and clinical applications of miRNAs, especially those from circulating exosomes secreted by tumor cells related to CRCLM.

A Point Mutation in DNA Polymerase β (POLB) Gene Is Associated with Increased Progesterone Receptor (PR) Expression and Intraperitoneal Metastasis in Gastric Cancer

Increased expression of progesterone receptor (PR) has been reported in gastric cancer (GC). We have previously identified a functional T889C point mutation in DNA polymerase beta (POLB), a DNA repair gene in GC. To provide a detailed analysis of molecular changes associated with the mutation, human cDNA microarrays focusing on 18 signal transduction pathways were used to analyze differential gene expression profiles between GC tissues with T889C mutant in POLB gene and those with wild type. Among the differentially expressed genes, notably, PR was one of the significantly up-regulated genes in T889C mutant POLB tissues, which were subsequently confirmed in POLB gene transfected AGS cell line. Interestingly, patients with T889C mutation and PR positivity were associated with higher incidence of intraperitoneal metastasis (IM). In vitro studies indicate that PR expression was upregulated in AGS cell line when transfected with T889C mutant expression vector. Cotransfection of T889C mutant allele and PR gene induced cell migration in the cell line. These data demonstrated that T889C mutation-associated PR overexpression results in increased IM. Therefore, T889C mutation-associated PR overexpression may serve as a biomarker for an adverse prognosis for human GC.

Abstract 1072: miR-671-5p and miR-638 serve as novel biomarkers for early breast cancer detection

Breast cancer progression involves stepwise transition from atypical ductal hyperplasia (ADH), ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC). Percutaneous core needle biopsy (CNB) is the standard procedure after an abnormal mammography finding. However, a CNB diagnosis with either ADH or DCIS is often non-conclusive. A definitive diagnosis relies on surgical excision for further pathological analysis to differentiate simple ADH (sADH) from ADH coexisted with advanced lesions such as DCIS and/or IDC (cADH). Therefore, development of reliable molecular biomarkers is essential to avoid unnecessary surgical excision. microRNAs (miRNAs) are single-stranded non-coding RNAs that play an important role in breast cancer progression. A series of miRNAs that are differentially expressed during stepwise transition of breast carcinogenesis were identified earlier, of which miR-638 and miR-671-5p were functionally characterized. The expression of these miRNAs was decreased gradually from ADH, DCIS, to IDC. We hypothesize that these miRNAs may serve as valuable biomarkers following abnormal mammogram and CNB procedure. Using an improved microdissection protocol, we isolated normal, hyperplasia, DCIS, and/or IDC lesions from FFPE tissue of CNB. We also collected patients’ blood samples before CNB procedure. In addition, we employed the Human 21T breast epithelial cell lines, which were originally derived from the same patient diagnosed with metastatic breast cancer, including H16N2 (normal mammary epithelial), 21PT (ADH), 21NT (DCIS) and 21MT-1 (IDC) for in vitro model study. Real-time qRT-PCR assay was performed for examination of miRNA expression. Cell proliferation and invasion capability were examined by MTT and Transwell assays, respectively after transfection of candidate miRNAs. EMT markers were evaluated by Western blot and Immunofluorescence assays. In clinical samples, we found a synergistic expression pattern between miR-671-5p and miR-638 in cADH but not in sADH lesions. Interestingly, decreased miR-671-5p expression was detected in cADHs, but not in sADHs in both FFPE and serum samples. To explore the potential function of the two miRNAs in the transition from sADH to cADH, we performed further analysis in the Human 21T breast epithelial cell lines. Forced expression of miR-671-5p significantly inhibited cell proliferation in H16N2, 21PT, 21NT and 21MT-1 series cell lines. Overexpression of miR-671-5p attenuated invasion in 21PT, 21NT, and 21MT-1 cell lines but not in H16N2 cell line. Further, miR-671-5p overexpression resulted in significant suppression of mesenchymal marker, vimentin, and promoted the expression of epithelial marker, E-cadherin, in 21PT, 21NT, and 21MT-1cell lines. Our data suggest that miR-671-5p and miR-638 expression in serum and/or CNB tissue may server as a novel companion screening tool following an abnormal mammogram and a subsequent ADH diagnosis by CNB. Citation Format: Xiaohui Tan, Woojin Lee, Xiaoling Wu, Weaam Alshenawy, Danielle Soberman, Katayoon Rezaei, Sana Tabbara, Christine Teal, Robert S. Siegel, Rachel F. Brem, Sidney W. Fu. miR-671-5p and miR-638 serve as novel biomarkers for early breast cancer detection. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1072.

Abstract 3062: miR-671-5p promotes epithelial-to-mesenchymal transition by downregulating FOXM1 expression in breast cancer

microRNA (miRNA) dysfunction is associated with a variety of human diseases including cancer. Our previous study showed that miR-671-5p was deregulated during breast cancer progression. We aim to decipher the functional mechanism of miR-671-5p in breast cancer. We found that expression of miR-671-5p was decreased significantly in ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC) compared to normal and atypical ductal hyperplasia (ADH) in the microdissected formalin-fixed, paraffin-embedded (FFPE) tissues. Forkhead Box M1 (FOXM1), as an oncogenic transcription factor, was predicted as one of the direct targets of miR-671-5p, which was subsequently confirmed by luciferase assays. Forced expression of miR-671-5p in breast cancer cell lines downregulated FOXM1 expression, and attenuated the proliferation and invasion in breast cancer cell lines. Notably, overexpression of miR-671-5p resulted in a shift from epithelial-to-mesenchymal transition (EMT) to mesenchymal-to-epithelial transition (MET) phenotypes in MDA-MB-231 breast cancer cells and induced S-phase arrest. Moreover, miR-671-5p sensitized breast cancer cells to cisplatin, 5-fluorouracil (5-FU) and epirubicin exposure. Host cell reactivation (HCR) assays showed that miR-671-5p reduces DNA repair capability in post-drug exposed breast cancer cells. These data indicates that miR-671-5p functions as a tumor suppressor miRNA by directly targeting FOXM1 in breast cancer. Hence, miR-671-5p may serve as a novel therapeutic target for breast cancer management. Citation Format: Xiaohui Tan, Yebo Fu, Liang Chen, Shejuan An, Woojin Lee, Yinglei Lai, Katayoon Rezaei, Sana Tabbara, Christine B. Teal, Yan-gao Man, Robert Siegel, Rachel F. Brem, Sidney W. Fu. miR-671-5p promotes epithelial-to-mesenchymal transition by downregulating FOXM1 expression in breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3062. doi:10.1158/1538-7445.AM2015-3062