Marey Shriver

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.

Loss of giant obscurins from breast epithelium promotes epithelial-to-mesenchymal transition, tumorigenicity and metastasis.

Obscurins, encoded by the single OBSCN gene, are giant cytoskeletal proteins with structural and regulatory roles. The OBSCN gene is highly mutated in different types of cancers. Loss of giant obscurins from breast epithelial cells confers them with a survival and growth advantage, following exposure to DNA-damaging agents. Here we demonstrate that the expression levels and subcellular distribution of giant obscurins are altered in human breast cancer biopsies compared with matched normal samples. Stable clones of non-tumorigenic MCF10A cells lacking giant obscurins fail to form adhesion junctions, undergo epithelial-to-mesenchymal transition and generate >100-μm mammospheres bearing markers of cancer-initiating cells. Obscurin-knockdown MCF10A cells display markedly increased motility as a sheet in 2-dimensional (2D) substrata and individually in confined spaces and invasion in 3D matrices. In line with these observations, actin filaments redistribute to extending filopodia where they exhibit increased dynamics. MCF10A cells that stably express the K-Ras oncogene and obscurin short hairpin RNA (shRNA), but not scramble control shRNA, exhibit increased primary tumor formation and lung colonization after subcutaneous and tail vein injections, respectively. Collectively, our findings reveal that loss of giant obscurins from breast epithelium results in disruption of the cell–cell contacts and acquisition of a mesenchymal phenotype that leads to enhanced tumorigenesis, migration and invasiveness in vitro and in vivo.

Giant obscurins regulate the PI3K cascade in breast epithelial cells via direct binding to the PI3K/p85 regulatory subunit

Obscurins are a family of giant cytoskeletal proteins, originally identified in striated muscles where they have structural and regulatory roles. We recently showed that obscurins are abundantly expressed in normal breast epithelial cells where they play tumor and metastasis suppressing roles, but are nearly lost from advanced stage breast cancer biopsies. Consistent with this, loss of giant obscurins from breast epithelial cells results in enhanced survival and growth, epithelial to mesenchymal transition (EMT), and increased cell migration and invasion in vitro and in vivo. In the current study, we demonstrate that loss of giant obscurins from breast epithelial cells is associated with significantly increased phosphorylation and subsequent activation of the PI3K signaling cascade, including activation of AKT, a key regulator of tumorigenesis and metastasis. Pharmacological and molecular inhibition of the PI3K pathway in obscurin-depleted breast epithelial cells results in reversal of EMT, (re)formation of cell-cell junctions, diminished mammosphere formation, and decreased cell migration and invasion. Co-immunoprecipitation, pull-down, and surface plasmon resonance assays revealed that obscurins are in a complex with the PI3K/p85 regulatory subunit, and that their association is direct and mediated by the obscurin-PH domain and the PI3K/p85-SH3 domain with a KD of ∼50 nM. We therefore postulate that giant obscurins act upstream of the PI3K cascade in normal breast epithelial cells, regulating its activation through binding to the PI3K/p85 regulatory subunit.

Loss of giant obscurins alters breast epithelial cell mechanosensing of matrix stiffness

Obscurins are a family of RhoGEF-containing proteins with tumor and metastasis suppressing roles in breast epithelium. Downregulation of giant obscurins in normal breast epithelial cells leads to reduced levels of active RhoA and of its downstream effectors. Herein, we elucidate how depletion of giant obscurins affects the response of breast epithelial cells to changes in the mechanical properties of the microenvironment. We find that knockdown of obscurins increases cell morphodynamics, migration speed, and diffusivity on polyacrylamide gels of ≥ 1 kPa, presumably by decreasing focal adhesion area and density as well as cell traction forces. Depletion of obscurins also increases cell mechanosensitivity on soft (0.4–4 kPa) surfaces. Similar to downregulation of obscurins, pharmacological inhibition of Rho kinase in breast epithelial cells increases migration and morphodynamics, suggesting that suppression of Rho kinase activity following obscurin knockdown can account for alterations in morphodynamics and migration. In contrast, inhibition of myosin light chain kinase reduces morphodynamics and migration, suggesting that temporal changes in cell shape are required for efficient migration. Collectively, downregulation of giant obscurins facilitates cell migration through heterogeneous microenvironments of varying stiffness by altering cell mechanobiology.Obscurins are a family of RhoGEF-containing proteins with tumor and metastasis suppressing roles in breast epithelium. Downregulation of giant obscurins in normal breast epithelial cells leads to reduced levels of active RhoA and of its downstream effectors. Herein, we elucidate how depletion of giant obscurins affects the response of breast epithelial cells to changes in the mechanical properties of the microenvironment. We find that knockdown of obscurins increases cell morphodynamics, migration speed, and diffusivity on polyacrylamide gels of ≥ 1 kPa, presumably by decreasing focal adhesion area and density as well as cell traction forces. Depletion of obscurins also increases cell mechanosensitivity on soft (0.4-4 kPa) surfaces. Similar to downregulation of obscurins, pharmacological inhibition of Rho kinase in breast epithelial cells increases migration and morphodynamics, suggesting that suppression of Rho kinase activity following obscurin knockdown can account for alterations in morphodynamics and migration. In contrast, inhibition of myosin light chain kinase reduces morphodynamics and migration, suggesting that temporal changes in cell shape are required for efficient migration. Collectively, downregulation of giant obscurins facilitates cell migration through heterogeneous microenvironments of varying stiffness by altering cell mechanobiology.

Correction: Obscurins: Goliaths and Davids Take over Non-Muscle Tissues

[This corrects the article DOI: 10.1371/journal.pone.0088162.].

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 297: Loss of giant obscurins expression in breast epithelium disrupts epithelial junctions and promotes cell motility and invasion.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Obscurin A (∼720kDa) and B (∼870kDa) are giant multidomain proteins, encoded by the single OBSCN gene, originally shown to play important roles in the structural organization and contractile activity of striated muscles. Early studies have indicated that the OBSCN gene is highly mutated in different types of cancers. In light of this observation, our laboratory set forth to examine the expression profile and roles of giant obscurins in normal and cancer breast tissue. Obscurins are readily expressed in breast epithelial cells, but their expression is dramatically diminished in breast cancer cells. Down-regulation of obscurin A and B using small hairpin RNAs (shRNAs) in non-tumorigenic MCF10A breast epithelial cells resulted in decreased protein expression of β-catenin and E-cadherin, which are major components of adherens junctions and have been heavily implicated in the formation and metastasis of breast tumors. Specifically, β-catenin exhibited a preferential nuclear accumulation accompanied by a concomitant loss from cell-cell junctions. Additionally, loss of obscurins led to alterations in the expression levels and localization of multiple proteins associated with epithelial to mesenchymal transition (EMT). Obscurin-deficient MCF10A cells showed significantly increased motility in 2-Dimensional (2-D) substrata and confined spaces, and invasion through a matrigel coated chamber. Consistent with this, actin filaments re-distributed to extending filopodia-like protrusions where they exhibited increased dynamics. Taken together, our findings indicate that loss of giant obscurins from breast epithelium results in disruption of cell contacts and acquisition of a mesenchymal phenotype that leads to enhanced migration and invasiveness. Citation Format: Marey Shriver, Kimberly Stroka, Konstantinos Konstantopoulos, Aikaterini Kontrogianni-Konstantopoulos. Loss of giant obscurins expression in breast epithelium disrupts epithelial junctions and promotes cell motility and 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 297. doi:10.1158/1538-7445.AM2013-297