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Kindlin-3 enhances breast cancer progression and metastasis by activating Twist-mediated angiogenesis.

ABSTRACT: The FERM domain containing protein Kindlin-3 has been recognized as a major regulator of integrin function in hematopoietic cells, but its role in neoplasia is totally unknown. We have examined the relationship between Kindlin-3 and breast cancer in mouse models and human tissues. Human breast tumors showed a ∼7-fold elevation in Kindlin-3 mRNA compared with nonneoplastic tissue by quantitative polymerase chain reaction. Kindlin-3 overexpression in a breast cancer cell line increased primary tumor growth and lung metastasis by 2.5- and 3-fold, respectively, when implanted into mice compared with cells expressing vector alone. Mechanistically, the Kindlin-3-overexpressing cells displayed a 2.2-fold increase in vascular endothelial growth factor (VEGF) secretion and enhanced β1 integrin activation. Increased VEGF secretion resulted from enhanced production of Twist, a transcription factor that promotes tumor angiogenesis. Knockdown of Twist diminished VEGF production, and knockdown of β1 integrins diminished Twist and VEGF production by Kindlin-3-overexpressing cells, while nontargeting small interfering RNA had no effect on expression of these gene products. Thus, Kindlin-3 influences breast cancer progression by influencing the crosstalk between β1 integrins and Twist to increase VEGF production. This signaling cascade enhances breast cancer cell invasion and tumor angiogenesis and metastasis.

SUBMITTER: Sossey-Alaoui K

PROVIDER: S-EPMC3986835 | biostudies-literature | 2014 May

REPOSITORIES: biostudies-literature

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Kindlin-3 enhances breast cancer progression and metastasis by activating Twist-mediated angiogenesis.

Sossey-Alaoui Khalid K Pluskota Elzbieta E Davuluri Gangarao G Bialkowska Katarzyna K Das Mitali M Szpak Dorota D Lindner Daniel J DJ Downs-Kelly Erinn E Thompson Cheryl L CL Plow Edward F EF

FASEB journal : official publication of the Federation of American Societies for Experimental Biology 20140127 5

The FERM domain containing protein Kindlin-3 has been recognized as a major regulator of integrin function in hematopoietic cells, but its role in neoplasia is totally unknown. We have examined the relationship between Kindlin-3 and breast cancer in mouse models and human tissues. Human breast tumors showed a ∼7-fold elevation in Kindlin-3 mRNA compared with nonneoplastic tissue by quantitative polymerase chain reaction. Kindlin-3 overexpression in a breast cancer cell line increased primary tum ...[more]

PMID: 24469992

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Project description:A trans-membrane receptor tyrosine kinase, cMET, belonging to the MET proto-oncogene family, is responsible for cancer metastasis and angiogenesis. But not much is known about the role of cMET in growth and progression of cancer stem cells (CSCs). Earlier studies have shown that Quinacrine (QC), a bioactive agent, has anti-CSCs activity. Here, the role of QC in deregulation of cMET-mediated metastasis and angiogenesis has been systematically evaluated in vitro in highly metastatic breast CSCs (mBCSCs), ex vivo in patient-derived breast cancer stem cells (PDBCSCs) and in vivo in xenograft mice model systems. Cell proliferation, migration, invasion and representative metastasis markers were upregulated in cMET-overexpressed cells and QC exposure inhibited these processes in both mBCSCs and PDBCSCs. Interestingly, metastasis was significantly inhibited by QC in cMET-overexpressed cells but comparatively lesser significant alteration of the process was noted in cMET-silenced cells. Increase in vascularization (in in ovo CAM assay), and cell-cell tube formation (in HUVECs), and enhanced MMP9 and MMP2 enzymatic activities (in gelatin zymography) were noted after cMET overexpression but these processes got reversed after cMET knockdown or QC treatment in cMET-overexpressed cells. QC inhibited angiogenesis significantly in cMET-overexpressed cells, but lesser significant change was observed in cMET-silenced cells. Reduction in tumor volume and decreased expression of metastatic and angiogenic markers were also noted in xenograft mice after QC treatment. Furthermore, QC inhibited cMET activity by dephosphorylation of its tyrosine residues (Y1234 and Y1356) and downregulation of its downstream cascade. Thus, QC inhibited the cMET-mediated metastasis and angiogenesis in in vitro, in ovo, in vivo and ex vivo model systems. Ligand (HGF) binding leads to receptor dimerization and phosphorylation of tyrosine kinase domain of cMET. This activates the cMET signaling cascade. The representative downstream metastasis and angiogenesis-related proteins get upregulated and induce the metastasis and angiogenesis process. But after the QC treatment, cMET get dephosphorylated and inactivated. As a result, the downstream signaling proteins of cMET along with the other representative metastatic and angiogenic factors get downregulated. These lead to inhibition of cMET-mediated metastasis and angiogenesis. (Created with BioRender.com).

Project description:Kindlin-2, an adapter protein, is dysregulated in various human cancers, including triple-negative breast cancer (TNBC), where it drives tumor progression and metastasis by influencing several cancer hallmarks. One well-established role of Kindlin-2 involves the regulation of integrin signaling, achieved by directly binding to the cytoplasmic tail of the integrin β subunit. In this study, we present novel insights into Kindlin-2's involvement in stabilizing the β1-Integrin:TGF-β type 1 receptor (TβRI) complexes, acting as a physical bridge that links β1-Integrin to TβRI. Loss of Kindlin-2 results in the degradation of this protein complex, leading to the inhibition of downstream oncogenic pathways. We used a diverse range of in vitro assays, including CRISPR/Cas9 gene editing, cell migration, 3D-tumorsphere formation and invasion, solid binding, co-immunoprecipitation, cell adhesion and spreading assays, as well as western blot and flow cytometry analyses, utilizing MDA-MB-231 and 4T1 TNBC cell lines. Additionally, preclinical in vivo mouse models of TNBC tumor progression and metastasis were employed to substantiate our findings. Our studies established the direct interaction between Kindlin-2 and β1-Integrin and between Kindlin-2 and TβRI. Disruption of these interactions, via CRISPR/Cas9-mediated knockout of Kindlin-2, led to the degradation of β1-Integrin and TβRI, resulting in the inhibition of oncogenic pathways downstream of both proteins, subsequently hindering tumor growth and metastasis. Treatment of Kindlin-2-deficient cells with the proteasome inhibitor MG-132 restored the expression of both β1-Integrin and TβRI. Furthermore, the rescue of Kindlin-2 expression reinstated their oncogenic activities in vitro and in vivo, while Kindlin-2 lacking domains involved in the interaction of Kindlin-2 with β1-Integrin or TβRI did not. This study identifies a novel function of Kindlin-2 in stabilizing the β1-Integrin:TβRI complexes and regulating their downstream oncogenic signaling. The translational implications of these findings are substantial, potentially unveiling new therapeutically targeted pathways crucial for the treatment of TNBC tumors.

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Kindlin-3 enhances breast cancer progression and metastasis by activating Twist-mediated angiogenesis.

Publications

Kindlin-3 enhances breast cancer progression and metastasis by activating Twist-mediated angiogenesis.

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