Chika Nwachukwu

MicroRNA-30c inhibits human breast tumour chemotherapy resistance by regulating TWF1 and IL-11.

Chemotherapy resistance frequently drives tumour progression. However, the underlying molecular mechanisms are poorly characterized. Epithelial-to-mesenchymal transition has been shown to correlate with therapy resistance, but the functional link and signalling pathways remain to be elucidated. Here we report that microRNA-30c, a human breast tumour prognostic marker, has a pivotal role in chemoresistance by a direct targeting of the actin-binding protein twinfilin 1, which promotes epithelial-to-mesenchymal transition. An interleukin-6 family member, interleukin-11 is identified as a secondary target of twinfilin 1 in the microRNA-30c signalling pathway. Expression of microRNA-30c inversely correlates with interleukin-11 expression in primary breast tumours and low interleukin-11 correlates with relapse-free survival in breast cancer patients. Our study demonstrates that microRNA-30c is transcriptionally regulated by GATA3 in breast tumours. Identification of a novel microRNA-mediated pathway that regulates chemoresistance in breast cancer will facilitate the development of novel therapeutic strategies.

Basal-like Breast cancer DNA copy number losses identify genes involved in genomic instability, response to therapy, and patient survival

Breast cancer is a heterogeneous disease with known expression-defined tumor subtypes. DNA copy number studies have suggested that tumors within gene expression subtypes share similar DNA Copy number aberrations (CNA) and that CNA can be used to further sub-divide expression classes. To gain further insights into the etiologies of the intrinsic subtypes, we classified tumors according to gene expression subtype and next identified subtype-associated CNA using a novel method called SWITCHdna, using a training set of 180 tumors and a validation set of 359 tumors. Fisher’s exact tests, Chi-square approximations, and Wilcoxon rank-sum tests were performed to evaluate differences in CNA by subtype. To assess the functional significance of loss of a specific chromosomal region, individual genes were knocked down by shRNA and drug sensitivity, and DNA repair foci assays performed. Most tumor subtypes exhibited specific CNA. The Basal-like subtype was the most distinct with common losses of the regions containing RB1, BRCA1, INPP4B, and the greatest overall genomic instability. One Basal-like subtype-associated CNA was loss of 5q11–35, which contains at least three genes important for BRCA1-dependent DNA repair (RAD17, RAD50, and RAP80); these genes were predominantly lost as a pair, or all three simultaneously. Loss of two or three of these genes was associated with significantly increased genomic instability and poor patient survival. RNAi knockdown of RAD17, or RAD17/RAD50, in immortalized human mammary epithelial cell lines caused increased sensitivity to a PARP inhibitor and carboplatin, and inhibited BRCA1 foci formation in response to DNA damage. These data suggest a possible genetic cause for genomic instability in Basal-like breast cancers and a biological rationale for the use of DNA repair inhibitor related therapeutics in this breast cancer subtype.

MicroRNA-30c Targets Cytoskeleton Genes Involved in Breast Cancer Cell Invasion

Metastasis remains a significant challenge in treating cancer. A better understanding of the molecular mechanisms underlying metastasis is needed to develop more effective treatments. Here, we show that human breast tumor biomarker miR-30c regulates invasion by targeting the cytoskeleton network genes encoding twinfilin 1 (TWF1) and vimentin (VIM). Both VIM and TWF1 have been shown to regulate epithelial-to-mesenchymal transition. Similar to TWF1, VIM also regulates F-actin formation, a key component of cellular transition to a more invasive mesenchymal phenotype. To further characterize the role of the TWF1 pathway in breast cancer, we found that IL-11 is an important target of TWF1 that regulates breast cancer cell invasion and STAT3 phosphorylation. The miR-30c-VIM/TWF1 signaling cascade is also associated with clinical outcome in breast cancer patients.

CpG island methylation affects accessibility of the proximal BRCA1 promoter to transcription factors.

To understand the mechanism of transcriptional down-regulation of BRCA1 by promoter methylation, we screened 51 breast cancer cell lines and identified HCC38 as another BRCA1 promoter-methylated cell line in addition to UACC3199. There was low expression of BRCA1 mRNA and BRCA1 protein in both cell lines as measured by quantitative RT-PCR and western blot analysis. After transient treatment with 5-aza-2′-deoxycytidine (5-aza-CdR) and trichostatin A (TSA), re-expression of BRCA1 mRNA and BRCA1 protein was detected in UACC3199 cells, but not in HCC38 cells. Another demethylating agent, zebularine, did not induce BRCA1 re-expression in either cell line. To test the hypothesis that methylation of CpG sites may affect accessibility of the BRCA1 promoter to transcription factors and consequently cause down-regulation of BRCA1, we analyzed the binding of four transcription factors (CTCF, Sp1, E2F1 and E2F6) to the BRCA1 promoter using chromatin immunoprecipitation assay (ChIP) and quantitative PCR. CTCF and E2F1 were enriched at the unmethylated BRCA1 promoter in MCF-7 cells. In contrast, these two transcription factors were not enriched at the methylated BRCA1 promoter in UACC3199 and HCC38 cells. Following demethylating drug treatment, E2F1 was enriched at the BRCA1 promoter in the demethylated UACC3199 cells. This indicates that reduced accessibility of transcription factors to the methylated promoter is one of the mechanisms for down-regulation of BRCA1 in heavily methylated cancer cells.

Differentiation and Loss of Malignant Character of Spontaneous Pulmonary Metastases in Patient-Derived Breast Cancer Models

Patient-derived human-in-mouse xenograft models of breast cancer (PDX models) that exhibit spontaneous lung metastases offer a potentially powerful model of cancer metastasis. In this study, we evaluated the malignant character of lung micrometastases that emerge in such models after orthotopic implantation of human breast tumor cells into the mouse mammary fat pad. Interestingly, relative to the parental primary breast tumors, the lung metastasis (met)-derived mammary tumors exhibited a slower growth rate and a reduced metastatic potential with a more differentiated epithelial status. Epigenetic correlates were determined by gene array analyses. Lung met-derived tumors displayed differential expression of negative regulators of cell proliferation and metabolism and positive regulators of mammary epithelial differentiation. Clinically, this signature correlated with breast tumor subtypes. We identified hsa-miR-138 (miR-138) as a novel regulator of invasion and epithelial-mesenchymal transition in breast cancer cells, acting by directly targeting the polycomb epigenetic regulator EZH2. Mechanistic investigations showed that GATA3 transcriptionally controlled miR-138 levels in lung metastases. Notably, the miR-138 activity signature served as a novel independent prognostic marker for patient survival beyond traditional pathologic variables, intrinsic subtypes, or a proliferation gene signature. Our results highlight the loss of malignant character in some lung micrometastatic lesions and the epigenetic regulation of this phenotype.

Molecular Subtype-Specific Expression of MicroRNA-29c in Breast Cancer Is Associated with CpG Dinucleotide Methylation of the Promoter

Basal-like breast cancer is a molecularly distinct subtype of breast cancer that is highly aggressive and has a poor prognosis. MicroRNA-29c (miR-29c) has been shown to be significantly down-regulated in basal-like breast tumors and to be involved in cell invasion and sensitivity to chemotherapy. However, little is known about the genetic and regulatory factors contributing to the altered expression of miR-29c in basal-like breast cancer. We here report that epigenetic modifications at the miR-29c promoter, rather than copy number variation of the gene, may drive the lower expression of miR-29c in basal-like breast cancer. Bisulfite sequencing of CpG sites in the miR-29c promoter region showed higher methylation in basal-like breast cancer cell lines compared to luminal subtype cells with a significant inverse correlation between expression and methylation of miR-29c. Analysis of primary breast tumors using The Cancer Genome Atlas (TCGA) dataset confirmed significantly higher levels of methylation of the promoter in basal-like breast tumors compared to all other subtypes. Furthermore, inhibition of CpG methylation with 5-aza-CdR increases miR-29c expression in basal-like breast cancer cells. Flourescent In Situ Hybridization (FISH) revealed chromosomal abnormalities at miR-29c loci in breast cancer cell lines, but with no correlation between copy number variation and expression of miR-29c. Our data demonstrated that dysregulation of miR-29c in basal-like breast cancer cells may be in part driven by methylation at CpG sites. Epigenetic control of the miR-29c promoter by epigenetic modifiers may provide a potential therapeutic target to overcome the aggressive behavior of these cancers.

BRCA1 methylation contributes to the triple-negative breast cancer phenotype.

Abstract #4050 Triple-negative breast cancers are tumors characterized by their lack of hormone receptors (ER and PR) and HER2. They are the most aggressive form and account for 10–17% of all breast carcinomas. A subgroup of triple negative tumors with a basal-like phenotype share morphological features and similar gene expression profiles with tumors from BRCA1 mutation carriers. It has recently been shown that inactivation of BRCA1 in breast epithelial stem cells restricts subsequent progenitor cells to a basal cell phenotype and promotes expansion of ER negative cells (Liu et al 2008, PNAS). While mechanisms of BRCA1 inactivation in sporadic tumors are not completely elucidated, methylation of the BRCA1 promoter is one important mechanism that contributes to loss of BRCA1 expression in sporadic breast cancer. We hypothesize that inactivation of BRCA1 by epigenetic mechanisms such as promoter methylation contributes to the triple negative phenotype.
 Materials and Methods : Using a combination of methylation specific PCR and Immunohistochemistry, 120 primary breast cancers were analyzed for methylation of the BRCA1 promoter and protein expression of ER, PR and HER2. All tumors with negative staining for ER, PR and HER2 were classified as triple negative and all others were classified as non triple-negative.
 Results: We were able to classify 111 of the 120 (%) tumors by IHC. We found that 30 out of 111 (27%) tumors were ER, PR, and HER2 negative (triple negative). BRCA1 methylation was detected in 14 out of 30 (47%) triple negative tumors, compared with 10 out of 81 (12%) other tumor phenotypes (p Conclusion : These data suggest that BRCA1 inactivation by mutation or methylation may in part drive breast tumor progression toward the triple negative phenotype. Ongoing work will evaluate epigenetic mechanisms regulating gene expression in triple negative breast cancers
 This abstract is supported by Breast Cancer Research Foundation and the Lee Jeans Entertainment Industry Fund. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4050.

Abstract 5337: MicroRNA-30c targets cytoskeleton genes involved in breast cancer cell invasion.

Metastasis remains a significant challenge in treating cancer. MicroRNAs have emerged as important epigenetic regulators of various cellular processes during cancer development and progression. The goal of this study was to characterize signaling pathways for miRNA biomarkers that regulate breast cancer metastasis. Here we show that human breast tumor biomarker miR-30c regulates invasion by targeting the cytoskeleton network genes encoding Twinfilin 1 (TWF1) and Vimentin (VIM). Both VIM and TWF1 have been shown to regulate epithelial-to-mesenchymal transition (EMT). Similar to TWF1, VIM also regulates F-actin formation, a key component of cellular transition to a more invasive mesenchymal phenotype. To further characterize the role of the TWF1 pathway in breast cancer, we found that IL-11 is an important target of TWF1 that regulates breast cancer cell invasion and STAT3 phosphorylation. This miR-30c VIM/TWF1-IL11-pSTAT3 pathway will expedite the development of targeting strategies to prevent and treat breast tumor progression. Citation Format: Jessica Bockhorn, Kathy Yee, Ya-Fang Chang, Aleix Prat, Dezheng Huo, Chika Nwachukwu, Rachel Dalton, Simo Huang, Kaitlin E. Swanson, Charles M. Perou, Olufunmilayo I. Olufunmilayo, Michael F. Clarke, Huiping Liu, Geoffrey Greene. MicroRNA-30c targets cytoskeleton genes involved in breast cancer cell invasion. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5337. doi:10.1158/1538-7445.AM2013-5337

Abstract 832: MicroRNA-30c inhibits human breast tumor chemo-resistance by regulating twinfinlin-1 (TWF1) and IL-11.

Chemotherapy resistance remains a challenging problem in the clinic and the underlying molecular mechanisms are poorly characterized. We hypothesize that epithelial-to-mesenchymal transition (EMT) is involved in therapy resistance and cancer progression, but the functional link and signalling pathways need to be elucidated. Our work discovered that miR-30c, a human breast tumour prognostic marker, plays a pivotal role in chemo-resistance and apoptosis by a direct targeting of TWF1, which encodes an actin-binding protein and promotes EMT. We also identified IL-11 as a secondary target of TWF1 in the miR-30c signalling pathway. Expression of miR-30c inversely correlated with TWF1 and IL-11 levels in primary breast tumours and low IL-11 associated with relapse-free survival in breast cancer patients. Furthermore, our study demonstrates that miR-30c is transcriptionally regulated by GATA3 in breast tumours. Identification of a novel miRNA-mediated pathway that regulates chemo-resistance and apoptosis in breast cancer will facilitate the development of novel therapeutic strategies. This study was supported in part by The University of Chicago Women9s Board (J.B.) and Chicago Fellows Program (H.L.), DOD W81XWH-09-1-0331, NIH K12 CA139160-02, NCI K99 CA160638-01A1, CTSA UL1 RR024999 (H.L.), Segal Fund and Ludwig Fund (G.L.G.). Citation Format: Huiping Liu, Jessica Bockhorn, Rachel Dalton, Chika Nwachukwu, Simo Huang, Aleix Prat, Kathy Yee, Ya-Fang Chang, Dezheng Huo, Jun Lu, Eileen Dolan, Charles M. Perou, Olufunmilayo I. Olopade, Michael F. Clarke, Geoffrey Greene. MicroRNA-30c inhibits human breast tumor chemo-resistance by regulating twinfinlin-1 (TWF1) and IL-11. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 832. doi:10.1158/1538-7445.AM2013-832

Abstract 3331: MicroRNAs regulate breast cancer stem cells and spontaneous metastases in orthotopic xenograft models

To examine the role of microRNAs (miRNAs) in breast cancer progression, we profiled miRNA and gene expression in both clinical breast tumors and human-in-mouse breast tumor models, where breast cancer stem cells (BCSCs) contribute to spontaneous metastasis. CD44+ cells from both primary tumors and lung metastases were highly enriched for tumor initiating cells. Based on the miRNA profile analyses, we identified a limited number of miRNAs that are differentially expressed in metastatic triple-negative breast tumors and regulate BCSCs and tumor invasion in vitro. To facilitate miRNA functional studies in vivo, we also developed tumor imaging approaches by transducing BCSCs with optical reporter fusion genes (Luc2-eGFP or -tdTomato), which enabled both bioluminescence imaging (BLI) and FACS-based analysis and sorting. With non-invasive BLI approaches, as few as 10 cells of stably labeled BCSCs can be tracked in vivo. When optical reporters are expressed along with miRNA precursors or inhibitors, the effects of introduced miRNA candidates can be evaluated by selective imaging of labeled tumor cells, thereby eliminating the noise of unlabeled cells. Using this model system and imaging technology, we have screened and identified miRNAs that regulate BCSCs and metastatic CSCs (MCSCs) by targeting polycomb repressors (BMI1 and the PC2 components) and cytoskeleton genes (TWF1 and VIM). Clinical studies demonstrated that the expression of candidate miRNAs was associated with and regulated by GATA3, suggesting a transcriptional regulation of aberrantly expressed miRNAs in breast tumors. The GATA3-miRNA-target genes signaling pathway was also strongly associated with relapse-free survival of breast cancer patients, indicating the clinical importance of the miRNA-gene network in breast cancer. Supported in part by the University of Chicago Women9s Board Fellowship (J.B.), NIH T90 Fellowship DK070103-05, DOD Postdoctoral Fellowship W81XWH-09-1-0331, and Chicago Fellows Program and CTSA UL1 RR024999 at The University of Chicago (H.L.), University of Chicago Cancer Research Center Pilot Research Fund, UCMC/Northshore Collaborative Research Award and the Virginia and D.K. Ludwig Fund (G.L.G and H.L). NIH R01 and Breast Cancer Research Foundation (M.F.C. and H.L.). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3331. doi:1538-7445.AM2012-3331