Simo Huang

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.

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.

A rapid, automated surface protein profiling of single circulating exosomes in human blood

Circulating exosomes provide a promising approach to assess novel and dynamic biomarkers in human disease, due to their stability, accessibility and representation of molecules from source cells. However, this potential has been stymied by lack of approaches for molecular profiling of individual exosomes, which have a diameter of 30–150 nm. Here we report a rapid analysis approach to evaluate heterogeneous surface protein expression in single circulating exosomes from human blood. Our studies show a differential CD47 expression in blood-derived individual circulating exosomes that is correlated with breast cancer status, demonstrating a great potential of individual exosome profiles in biomarker discovery. The sensitive and high throughput platform of single exosome analysis can also be applied to characterizing exosomes derived from other patient fluids.

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.

miR-206 Inhibits Stemness and Metastasis of Breast Cancer by Targeting MKL1/IL11 Pathway

Purpose: Effective targeting of cancer stem cells is necessary and important for eradicating cancer and reducing metastasis-related mortality. Understanding of cancer stemness-related signaling pathways at the molecular level will help control cancer and stop metastasis in the clinic. Experimental Design: By analyzing miRNA profiles and functions in cancer development, we aimed to identify regulators of breast tumor stemness and metastasis in human xenograft models in vivo and examined their effects on self-renewal and invasion of breast cancer cells in vitro. To discover the direct targets and essential signaling pathways responsible for miRNA functions in breast cancer progression, we performed microarray analysis and target gene prediction in combination with functional studies on candidate genes (overexpression rescues and pheno-copying knockdowns). Results: In this study, we report that hsa-miR-206 suppresses breast tumor stemness and metastasis by inhibiting both self-renewal and invasion. We identified that among the candidate targets, twinfilin (TWF1) rescues the miR-206 phenotype in invasion by enhancing the actin cytoskeleton dynamics and the activity of the mesenchymal lineage transcription factors, megakaryoblastic leukemia (translocation) 1 (MKL1), and serum response factor (SRF). MKL1 and SRF were further demonstrated to promote the expression of IL11, which is essential for miR-206s function in inhibiting both invasion and stemness of breast cancer. Conclusions: The identification of the miR-206/TWF1/MKL1-SRF/IL11 signaling pathway sheds lights on the understanding of breast cancer initiation and progression, unveils new therapeutic targets, and facilitates innovative drug development to control cancer and block metastasis. Clin Cancer Res; 23(4); 1091–103. ©2016 AACR.

Abstract 5152: Cancer stem cell targeted exosomes for the treatment of metastatic breast cancer

Introduction: Therapy resistance and metastasis remain the two biggest challenges of breast cancer that result in a majority of cancer deaths. Cancer stem cells (CSCs) are a subpopulation of cancer cells that can initiate new tumors, remain refractory to conventional therapies, and spread throughout the body. In order to effectively block metastasis, the present study aims to develop novel therapeutics that specifically target breast CSCs and spare normal cells. While nanomedicine represents one of the promising future directions, it is necessary to overcome the shortcomings of synthetic nanoparticles, including bio-instability and off-target toxicity. Here, we aim to bio-engineer the naturally produced cell-secreted small vesicles, exosomes (~100 nm diameter), for a novel therapeutic delivery system. Methods: Exosomes from the human mesenchymal stem cells (MSCs) were isolated by differential ultracentrifugation, and characterized by multiple approaches including transmission EM, Immunoblotting and immunofluorescence assays to confirm the size, shape and presence of exosomal markers CD63, CD81 and LAMP2B. Exosomes were engineered to express a peptide ligand on its surface to specifically recognize and target CSCs, and the presence of the ligand was confirmed by western blot, flow cytometry (FCM) and a super resolution confocal laser scanning microscopy (CLSM, Leica SP8). The CSC targeting efficiency of the exosomes was evaluated in vitro by FCM and CLSM. Further, the exosomes were loaded with microRNAs (miR-200, miR-30c), as one of the key factors that regulate breast CSC functions, by following a novel transfection technology. Results: The bio-engineered exosomes specifically target and internalize into the CSCs compared to normal cells with absent expression of the ligand-binding protein. The developed exosomes can effectively deliver candidate miRNAs into the recipient cells and knock down the genes important for functional CSCs. The in vivo targeting of CSCs and therapeutic applications of the exosomes using patient-derived human-in-mouse breast tumor xenograft models are underway. Conclusion: It is expected that exosome-based miRNA therapeutics will have high biocompatibility, low toxicity, high specificity, and promising efficiency in preclinical and clinical applications to reduce breast cancer metastasis and improve patient survival. Citation Format: Golam Kibria, Katelyn E. Lee, Erika K. Ramos, Simo Huang, Clifford V. Harding, Jan Lotvall, Huiping Liu. Cancer stem cell targeted exosomes for the treatment of metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5152. doi:10.1158/1538-7445.AM2017-5152

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

Abstract 4198: Transcriptional regulation of breast cancer microRNAs by GATA3 and TFAP2A

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Metastasis remains a major obstacle in the successful treatment of breast cancer because it is responsible for an estimated ninety percent of breast cancer related deaths. An emerging field in the understanding of breast cancer metastasis is the study of microRNAs (miRNAs) which are short nucleotide sequences shown to be involved in the regulation of cell self-renewal, epithelial-to-mesenchymal transition (EMT) and cancer progression [1]. Our previous data suggested that miR-200 and other miRNAs regulate EMT and breast cancer metastasis [2]. Given the significant roles of miRNAs and their modulated levels in cancer, it is important to understand their regulatory mechanisms. We hypothesize that miRNAs are transcriptionally regulated in breast cancer. Our preliminary data with clinical breast cancer specimens suggests correlative relationships between transcription factors TFAP2A and GATA3 with mir-200a and other miRNAs. The goal of this project is to determine whether TFAP2A and GATA3 regulate these miRNA levels in breast cancer cells. Using quantitative real-time PCR, the miRNA levels were measured in MDA-MB 231 breast cancer cells with modulated expression of GATA3 or TFAP2A. The results show that overexpression of GATA3 in MDA-MB 231 cancer cells significantly increased the expression of miRNAs aberrantly downregulated in ER- breast tumors, such as miR-138. Chromatin-immunoprecipitation and reporter luciferase assays will be used to determine whether the regulation of miRNAs by GATA3 and TFAP2A is a direct transcriptional regulation. 1. Mani, S.A. et al., 2008. The epithelial-mesenchymal transition generates cells with properties of stem cells. Cell 133, 70415. 2. Park, S.M. et al., 2008. The miR-200 family determines the epithelial phenotype of cancer cells by targeting the E-cadherin repressors ZEB1 and ZEB2. Genes Dev 22, 894-907. 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 4198. doi:1538-7445.AM2012-4198