Kaitlin E. Swanson

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.

Annexin A6 modifies muscular dystrophy by mediating sarcolemmal repair

Significance Many forms of muscular dystrophy produce muscle weakness through injury to skeletal muscle myofibers and specifically disruption of the muscle plasma membrane. Using a mouse model of muscular dystrophy in a genetically diverse background, a genome-wide scan for genetic modifiers was undertaken. A modifier locus that altered plasma membrane leak was interrogated, and a splice site variant in Anxa6, encoding annexin A6, was identified. The Anxa6 splice site produces a truncated annexin A6 protein. The truncated annexin A6 protein was found to inhibit membrane repair by disrupting the formation of the normal annexin A6-rich cap and repair zone. These data demonstrate annexin A6s role in muscle membrane leak and repair in muscular dystrophy. Many monogenic disorders, including the muscular dystrophies, display phenotypic variability despite the same disease-causing mutation. To identify genetic modifiers of muscular dystrophy and its associated cardiomyopathy, we used quantitative trait locus mapping and whole genome sequencing in a mouse model. This approach uncovered a modifier locus on chromosome 11 associated with sarcolemmal membrane damage and heart mass. Whole genome and RNA sequencing identified Anxa6, encoding annexin A6, as a modifier gene. A synonymous variant in exon 11 creates a cryptic splice donor, resulting in a truncated annexin A6 protein called ANXA6N32. Live cell imaging showed that annexin A6 orchestrates a repair zone and cap at the site of membrane disruption. In contrast, ANXA6N32 dramatically disrupted the annexin A6-rich cap and the associated repair zone, permitting membrane leak. Anxa6 is a modifier of muscular dystrophy and membrane repair after injury.

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.

An actin-dependent annexin complex mediates plasma membrane repair in muscle

Demonbreun et al. visualized muscle membrane repair in real time after laser-induced microdamage. Annexin proteins were observed to form a repair cap at the site of injury, supporting a shoulder-like structure containing EHD1, EHD2, dysferlin, and MG53.

EHD1 mediates vesicle trafficking required for normal muscle growth and transverse tubule development.

EHD proteins have been implicated in intracellular trafficking, especially endocytic recycling, where they mediate receptor and lipid recycling back to the plasma membrane. Additionally, EHDs help regulate cytoskeletal reorganization and induce tubule formation. It was previously shown that EHD proteins bind directly to the C2 domains in myoferlin, a protein that regulates myoblast fusion. Loss of myoferlin impairs normal myoblast fusion leading to smaller muscles in vivo but the intracellular pathways perturbed by loss of myoferlin function are not well known. We now characterized muscle development in EHD1-null mice. EHD1-null myoblasts display defective receptor recycling and mislocalization of key muscle proteins, including caveolin-3 and Fer1L5, a related ferlin protein homologous to myoferlin. Additionally, EHD1-null myoblast fusion is reduced. We found that loss of EHD1 leads to smaller muscles and myofibers in vivo. In wildtype skeletal muscle EHD1 localizes to the transverse tubule (T-tubule), and loss of EHD1 results in overgrowth of T-tubules with excess vesicle accumulation in skeletal muscle. We provide evidence that tubule formation in myoblasts relies on a functional EHD1 ATPase domain. Moreover, we extended our studies to show EHD1 regulates BIN1 induced tubule formation. These data, taken together and with the known interaction between EHD and ferlin proteins, suggests that the EHD proteins coordinate growth and development likely through mediating vesicle recycling and the ability to reorganize the cytoskeleton.

Enhanced Muscular Dystrophy from Loss of Dysferlin Is Accompanied by Impaired Annexin A6 Translocation after Sarcolemmal Disruption

Dysferlin is a membrane-associated protein implicated in membrane resealing; loss of dysferlin leads to muscular dystrophy. We examined the same loss-of-function Dysf mutation in two different mouse strains, 129T2/SvEmsJ (Dysf(129)) and C57BL/6J (Dysf(B6)). Although there are many genetic differences between these two strains, we focused on polymorphisms in Anxa6 because these variants were previously associated with modifying a pathologically distinct form of muscular dystrophy and increased the production of a truncated annexin A6 protein. Dysferlin deficiency in the C57BL/6J background was associated with increased Evans Blue dye uptake into muscle and increased serum creatine kinase compared to the 129T2/SvEmsJ background. In the C57BL/6J background, dysferlin loss was associated with enhanced pathologic severity, characterized by decreased mean fiber cross-sectional area, increased internalized nuclei, and increased fibrosis, compared to that in Dysf(129) mice. Macrophage infiltrate was also increased in Dysf(B6) muscle. High-resolution imaging of live myofibers demonstrated that fibers from Dysf(B6) mice displayed reduced translocation of full-length annexin A6 to the site of laser-induced sarcolemmal disruption compared to Dysf(129) myofibers, and impaired translocation of annexin A6 associated with impaired resealing of the sarcolemma. These results provide one mechanism by which the C57BL/6J background intensifies dysferlinopathy, giving rise to a more severe form of muscular dystrophy in the Dysf(B6) mouse model through increased membrane leak and inflammation.

Eps 15 Homology Domain (EHD)-1 Remodels Transverse Tubules in Skeletal Muscle

We previously showed that Eps15 homology domain-containing 1 (EHD1) interacts with ferlin proteins to regulate endocytic recycling. Myoblasts from Ehd1-null mice were found to have defective recycling, myoblast fusion, and consequently smaller muscles. When expressed in C2C12 cells, an ATPase dead-EHD1 was found to interfere with BIN1/amphiphysin 2. We now extended those findings by examining Ehd1-heterozygous mice since these mice survive to maturity in normal Mendelian numbers and provide a ready source of mature muscle. We found that heterozygosity of EHD1 was sufficient to produce ectopic and excessive T-tubules, including large intracellular aggregates that contained BIN1. The disorganized T-tubule structures in Ehd1-heterozygous muscle were accompanied by marked elevation of the T-tubule-associated protein DHPR and reduction of the triad linker protein junctophilin 2, reflecting defective triads. Consistent with this, Ehd1-heterozygous muscle had reduced force production. Introduction of ATPase dead-EHD1 into mature muscle fibers was sufficient to induce ectopic T-tubule formation, seen as large BIN1 positive structures throughout the muscle. Ehd1-heterozygous mice were found to have strikingly elevated serum creatine kinase and smaller myofibers, but did not display findings of muscular dystrophy. These data indicate that EHD1 regulates the maintenance of T-tubules through its interaction with BIN1 and links T-tubules defects with elevated creatine kinase and myopathy.

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 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