FAK activity protects nucleostemin in facilitating breast cancer spheroid and tumor growth

Abstract

IntroductionFocal adhesion kinase (FAK) controls cell growth and survival downstream of integrin-matrix receptors. Upon adhesion loss or FAK inhibition, FAK can translocate to the nucleus. The nucleolus is a non-membrane nuclear structure that regulates ribosome biogenesis and cell proliferation. Nucleostemin (NS), a nucleolar-localized protein, modulates cell cycle progression, stemness, and three-dimensional tumor spheroid formation. The signaling pathways that regulate NS levels in tumors remain undefined.MethodsHuman breast carcinoma cells were evaluated for growth in culture (adherent and anchorage-independent spheroid) and as orthotopic tumors. FAK signaling was evaluated by pharmacological FAK inhibitor addition (PF-271, IC50 ~ 0.1 μM) and by small hairpin RNA (shRNA) knockdown followed by re-expression of FAK wildtype (WT) or a kinase-dead (KD, K454R) FAK point mutant. Immunoblotting was used to evaluate FAK, NS, nucleolar phosphoprotein B23, and nucleolin levels. Total and phosphospecific antibody imunoblotting were used to detect changes in FAK, Akt kinase (Akt also known as protein kinase B), and 4E-binding protein 1 (4E-BP1) phosphorylation, a translation repressor protein and target of the mammalian target of rapamycin (mTOR) complex. Immunohistochemical, co-immunoprecipitation, and cellular fractionation analyses were used to evaluate FAK association with nucleoli.ResultsPharmacological (0.1 μM PF-271) or genetic inhibition of FAK activity prevents MDA-MB-231 and 4T1L breast carcinoma growth as spheroids and as orthotopic tumors. FAK inhibition triggers proteasome-mediated decreased NS levels but no changes in other nucleolar proteins such as B23 (nucleophosmin) or nucleolin. Active FAK was associated with purified nucleoli of anchorage-independent cells and present within nucleoli of human invasive ductal carcinoma tumor samples. FAK co-immunoprecipitated with B23 that binds NS and a complex between FAK, NS, Akt, and mTOR was detected. Constitutively-active Akt kinase promoted tumor spheroid growth, stabilized NS levels, and promoted pS65 4E-BP1 phosphorylation in the presence of inhibited FAK. Rapamycin lowered NS levels and inhibited pS65 4E-BP1 phosphorylation in cells with activated Akt-mTOR signaling.ConclusionsFAK signaling occurs in the nucleolus, active FAK protects NS, and Akt-mTOR pathway regulates NS protein stability needed for breast carcinoma spheroid and tumor growth.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-015-0551-x) contains supplementary material, which is available to authorized users.

CXCR1/2 pathways in paclitaxel-induced neuropathic pain

“…Consistent with reparixin pharmacological effects, p-FAK activation in breast cancer cells was previously reported to be implicated in anchorage-independent growth [55] and in breast cancer tumorigenesis and progression [56–60] whereas in sensory neurons β1-integrin activity and FAK phosphorylation at tyrosine 397 (FAKpY397) are linked to neuronal polarization as well as neurite outgrowth and branching [61]. …”

Three-dimensional culture systems in cancer research: Focus on tumor spheroid model

“…Integrin αVβ3, overexpressed at the invasive front of malignant melanoma cells and angiogenic blood vessels, increases metastatic potential of melanoma cells (Felding-Habermann et al , 1992; Brooks et al , 1994). The role of focal adhesion kinase (FAK) has been observed in promoting anchorage-independent growth of breast cancer spheroids (Tancioni et al , 2015). Hence, culturing cells in laminin rich ECM (lr-ECM), ECM rich natural hydrogels, and synthetic hydrogels with integrin binding sites may provide the framework needed to study the ECM cues and their role in cancer progression.…”

The roles of nuclear focal adhesion kinase (FAK) on Cancer: a focused review

“…Tancioni et al inhibited FAK activity and led to a decrease in proteasome-mediated NS levels. They found the mechanism of FAK in nucleoli by which active FAK protected the NS from proteasomal degradation and Akt-mTOR pathway regulated the stability of NS in breast cancer cells [81]. In general, activated FAK in the nucleus controls a variety of transcription factors leading to changes in gene regulation.…”