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

Abstract

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

“…First, this work only considers the effects of giant obscurins on the mechanobiological response of MCF10A breast epithelial cells to matrix stiffness. These cells were chosen due to the mounting evidence our labs have found regarding the role of giant obscurins in breast tumor formation and metastasis [ 10 – 12 , 26 , 52 ]. The OBSCN gene was identified as one of 189 “candidate cancer genes” in both breast and colorectal cancers due to its high mutational frequency [ 24 ], and therefore future studies could examine whether obscurins play a similar role in promoting metastasis and mechanosensing in the context of colorectal cancer.…”

“…Depletion of giant obscurins from non-tumorigenic MCF10A breast epithelial cells promotes apoptotic resistance [ 10 ], disrupts adherens junctions, increases cell migration and invasion in vitro , and potentiates tumorigenicity and metastasis in vivo [ 12 ]. These alterations are attributed to the critical role of obscurins in cell cytoskeletal organization and dynamics [ 11 , 12 , 26 ]. The cell cytoskeleton is largely regulated by the family of RhoGTPases, including RhoA, which has been implicated in the regulation of cell mechanosensitivity in microenvironments of varying stiffness [ 27 , 28 ].…”

Thick Filament Protein Network, Functions, and Disease Association

“…Loss of obscurins from breast epithelium results in increased activation of the PI3K cascade contributing to enhanced tumorigenicity and metastasis, suggesting that obscurins act upstream of the PI3K cascade regulating its activation (436, 516, 517). Given that the PI3K pathway is a major driver of growth and proliferation in multiple tissues, it is highly likely that obscurins modulate the activity of PI3K in cardiac and skeletal muscles, too.…”

Obscure functions: the location–function relationship of obscurins

“…Two other variants in OBSCN, V2161D and D5966N, which map to Ig domain 21, with unknown function, and the PH domain of obscurins, respectively, were also identified in separate DCM patients (Marston et al 2015). In breast epithelial cells, the obscurin PH domain interacts with PI3K to mediate the activation of the PI3K pathway (Shriver et al 2016). It is likely that the D5966N variant within the PH domain of obscurin impacts the activation of the PI3K pathway, leading to the pathogenesis of DCM.…”