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

Marey Shriver,Colin Paul,Janelle Geist,Tessa Seale,Aikaterini Kontrogianni-Konstantopoulos,Saravanakumar Marimuthu,Konstantinos Konstantopoulos

doi:10.18632/oncotarget.9985

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.

Highlights

Obscurins are a family of giant, cytoskeletal proteins originally identified in striated muscles, where they play important roles in their structural organization and contractile activity [1, 2]
Immunoblotting analysis revealed a significant increase in the levels of the phosphorylated forms of major components of the Phosphoinositide-3 kinase (PI3K) pathway in MCF10A obscurinknockdown cells compared to controls (Figure 1A)
We detected a considerable increase in the amounts of phosphorylated PI3K at tyrosine-458, a phospho-site that has been reported to track with the activation levels of the enzyme [12], PDK1, a downstream target of PI3K, at serine-241 that renders the enzyme catalytically active [13, 14], AKT, a direct target of PDK1, at threonine-308 and serine-473 indicating its maximal activation [15, 16], and GSK3β, a downstream target of AKT, at serine-9 leading to its inactivation that promotes cell cycle progression through stabilization of cyclin D1 [17]

Summary

INTRODUCTION

Obscurins are a family of giant, cytoskeletal proteins originally identified in striated muscles, where they play important roles in their structural organization and contractile activity [1, 2]. Obscurindepleted non-tumorigenic breast epithelial MCF10A cells exhibit a growth advantage under anchorage-independent conditions, form mammospheres enriched with markers of stemness, extend microtentacles, and undergo epithelial to mesenchymal transition (EMT) resulting in disruption of adherens junctions, and enhanced motility and invasion in vitro [9, 10] Consistent with these major alterations, depletion of giant obscurins from MCF10A cells expressing an active form of the K-Ras oncogene results in primary and metastatic tumor formation in subcutaneous and lung metastasis in vivo models, respectively [9]. Taken together, these findings indicate that giant obscurins act as tumor and metastasis suppressors in normal breast epithelium. Our findings indicate that giant obscurins act upstream of the PI3K pathway in breast epithelial cells contributing to its regulation

RESULTS

DISCUSSION

MATERIALS AND METHODS