Nicole A. Perry

Obscurins: Goliaths and Davids take over non-muscle tissues.

Obscurins comprise a family of proteins originally identified in striated muscles, where they play essential roles in myofibrillogenesis, cytoskeletal organization, and Ca2+ homeostasis. They are encoded by the single OBSCN gene, and are composed of tandem adhesion domains and signaling motifs. To date, two giant obscurin isoforms have been described in detail that differ only at the extreme COOH-terminus; while obscurin-A (∼720 kDa) contains a non-modular COOH-terminus that harbors binding sites for the adaptor proteins ankyrins, obscurin-B (∼870 kDa) contains two COOH-terminal serine-threonine kinase domains preceded by adhesion motifs. Besides the two known giant obscurins, a thorough search of transcript databases suggests that complex alternative splicing of the obscurin transcript results in the generation of additional giant as well as small isoforms with molecular masses ranging between ∼50–970 kDa. These novel isoforms share common domains with the characterized isoforms, but also contain unique regions. Using a panel of highly specific antibodies directed against epitopes spanning the entire length of giant obscurins, we employed western blotting and immunohistochemistry to perform a systematic and comprehensive characterization of the expression profile of obscurins in muscle and non-muscle tissues. Our studies demonstrate for the first time that obscurins are not restricted to striated muscles, but are abundantly expressed in several tissues and organs including brain, skin, kidney, liver, spleen, and lung. While some obscurin isoforms are ubiquitously expressed, others are preferentially present in specific tissues and organs. Moreover, obscurins are present in select structures and cell types where they assume nuclear, cytosolic, and membrane distributions. Given the ubiquitous expression of some obscurins, along with the preferential expression of others, it becomes apparent that obscurins may play common and unique roles, respectively, in the regulation and maintenance of cell homeostasis in various tissues and organs throughout the body.

Loss of giant obscurins promotes breast epithelial cell survival through apoptotic resistance

Obscurins (∼70–870 kDa), encoded by the single OBSCN gene, are cytoskeletal proteins originally identified in striated muscles with structural and regulatory roles. Recently, analysis of 13,023 genes in breast and colorectal cancers identified OBSCN as one of the most frequently mutated genes, implicating it in cancer formation. Herein we studied the expression profile of obscurins in breast, colon, and skin cancer cell lines and their involvement in cell survival. Immunoblot analysis demonstrated significant reduction of obscurin proteins in cancer cells, resulting from decreased mRNA levels and/or the presence of mutant transcripts. In normal epithelium, obscurins localize in cytoplasmic puncta, the cell membrane, and the nucleus. Accordingly, subcellular fractionation demonstrated the presence of 2 novel nuclear isoforms of ∼110 and ∼120 kDa. Nontumorigenic MCF10A breast epithelial cells stably transduced with shRNAs targeting giant obscurins exhibited increased viability (∼30%) and reduced apoptosis (∼20%) following exposure to the DNA‐damaging agent etoposide. Quantitative RT‐PCR further indicated that the antiapoptotic genes BAG4 and HAX1 were up‐regulated (1.5‐ and 1.4‐fold, respectively), whereas initiator caspase‐9 and death caspase‐3 transcripts were down‐regulated (0.8‐ and 0.6‐fold, respectively). Our findings are the first to pinpoint critical roles for obscurins in cancer development by contributing to the regulation of cell survival.—Perry, N. A., Shriver, M., Mameza, M. G., Grabias, B., Balzer, E., Kontrogianni‐Konstantopoulos, A. Loss of giant obscurins promotes breast epithelial cell survival through apoptotic resistance. FASEB J. 26, 2764–2775 (2012). www.fasebj.org

Obscurins: Unassuming giants enter the spotlight

Discovered about a decade ago, obscurin (∼720 kDa) is a member of a family of giant proteins expressed in striated muscle that are essential for normal muscle function. Much of what we understand about obscurin stems from its functions in cardiac and skeletal muscle. However, recent evidence has indicated that variants of obscurin (“obscurins”) are expressed in diverse cell types, where they contribute to distinct cellular processes. Dysfunction or abrogation of obscurins has also been implicated in the development of several pathological conditions, including cardiac hypertrophy and cancer. Herein, we present an overview of obscurins with an emphasis on novel findings that demonstrate their heretofore‐unsuspected importance in cell signaling and disease progression.

Abstract 3259: Giant obscurins: Novel tumor and metastasis suppressors in breast cancer

Obscurins, encoded by the single OBSCN gene, are giant cytoskeletal proteins containing tandem adhesion and signaling domains, including an active RhoGEF motif that directly binds and activates RhoA. The OBSCN gene is highly mutated in breast cancer resulting in a 2-fold reduction of its mRNA levels. Consistent with this, obscurin proteins are nearly lost from breast cancer cell lines and human biopsies, independently of their hormonal status or molecular differentiation. Loss of giant obscurins from breast epithelial cells confers them with a survival and growth advantage, following exposure to current chemotherapies. Moreover, obscurin-depleted breast epithelial cells fail to form adhesion junctions, undergo epithelial-to-mesenchymal transition and generate primary and secondary mammospheres bearing markers of cancer-initiating cells. In line with these phenotypic alterations, obscurin-deficient cells display markedly increased motility as a sheet in 2-dimensional (2D) substrata and individually in confined spaces, and invasion in 3D matrices. More importantly, loss of giant obscurins from breast epithelial cells promotes primary tumor formation and lung colonization in vivo. Obscurin depletion leads to >50% reduction in RhoA activity, as well as decreased phosphorylation of RhoA effectors, including myosin light chain phosphatase, myosin light chain, lim kinase, and cofilin, in both attached and suspended cells. These molecular alterations manifest as decreased actomyosin contractility, allowing suspended cells to escape detachment-induced apoptosis. Moreover, ∼40% of obscurin-depleted cells extend microtentacles, tubulin-based projections that mediate the attachment of circulating tumor cells to endothelium, an advantage that persists even after paclitaxel exposure. Collectively, our findings reveal that loss of giant obscurins from breast epithelium results in disruption of cell-cell contacts and acquisition of a mesenchymal phenotype that leads to enhanced tumorigenesis, migration and invasiveness in vitro and in vivo by affecting RhoA-mediated processes. Moreover, our data suggest that loss of obscurins may represent a substantial selective advantage for breast epithelial cells during metastasis, and that treatment with paclitaxel may exacerbate this advantage by preferentially allowing obscurin-deficient, stem-like cells to attach to the endothelium of distant sites, a first step towards colonizing metastatic tumors. Citation Format: Aikaterini Kontrogianni-Konstantopoulos, Marey Shriver, Nicole Perry, Kimberly Stroka, Michele Vitole, David Huso, Stuart Martin, Konstantinos Konstantopoulos. Giant obscurins: Novel tumor and metastasis suppressors 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 3259. doi:10.1158/1538-7445.AM2015-3259

Abstract 517: Ectopic expression of an obscurin signaling cassette decreases migration and invasion of metastatic breast cancer cells.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC The obscurins (∼70-820 kDa) are a family of proteins expressed from the 150 kb OBSCN gene, located on human chromosome 1. Sequencing analysis of breast and colorectal cancers, as well as melanoma and glioblastoma, revealed that obscurins are mutated at a substantial frequency and these mutations may be driving tumor formation. Our work has recently demonstrated that obscurins are present in normal breast, skin, and colon cells, but absent in their cancerous counterparts. Downregulation of obscurins’ expression is sufficient to allow non-tumorigenic breast epithelial cells to evade apoptosis induced by etoposide. Furthermore, absence of obscurins allows increased migration and invasion of breast epithelial cells. Remarkably though, we observed that ectopic expression of a fragment of obscurin composed of tandem src homology 3-Rho guanine exchange factor-pleckstrin homology (SH3-RhoGEF-PH) motifs was sufficient to decrease monolayer migration and invasion of metastatic breast cancer MDA-MB-231 cells, which are naturally deficient in obscurins, by >20% and >70%, respectively. Based on our observations that 1) absence of obscurins allows increased migration and invasion of breast cells, and 2) expression of an obscurin cassette mitigates this migratory and invasive behavior of metastatic breast cancer cells, we postulate that obscurins may act as metastasis suppressors in breast epithelium. We have therefore begun to investigate how the tripartite obscurin fragment, SH3-RhoGEF-PH, can influence the behavior of the cytoskeleton and therefore affect migration and invasion. We have found that the RhoGEF domain directly induces guanine nucleotide exchange in RhoA, but not Rac1 or Cdc42. This activity may be allosterically regulated by the adjacent SH3 domain, which interacts with the RhoGEF domain in a yeast two-hybrid assay. Furthermore, the PH domain binds to 3’-phosphorylated inositol lipid headgroups, providing a mechanism for the obscurins to respond to signals generated at the plasma membrane. Together, this evidence suggests that the tripartite signaling cassette can recapitulate the full-length obscurins’ metastasis-suppressing activity, and may merit development as a therapy for advanced breast cancer. Citation Format: Nicole A. Perry, Aikaterini Kontrogianni-Konstantopoulos. Ectopic expression of an obscurin signaling cassette decreases migration and invasion of metastatic breast cancer cells. [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 517. doi:10.1158/1538-7445.AM2013-517