Tumor cell migration screen identifies SRPK1 as breast cancer metastasis determinant
Wies van Roosmalen, Sylvia E. Le Dévédec, Ofra Golani, Marcel Smid, Irina Pulyakhina, Annemieke M. Timmermans, Maxime P. Look, Di Zi, Chantal Pont, Marjo de Graauw, Suha Naffar-Abu-Amara, Catherine Kirsanova, Gabriella Rustici, Peter A.C. ‘t Hoen, John W.M. Martens, John A. Foekens, Benjamin Geiger, Bob van de Water
Wies van Roosmalen, Sylvia E. Le Dévédec, Ofra Golani, Marcel Smid, Irina Pulyakhina, Annemieke M. Timmermans, Maxime P. Look, Di Zi, Chantal Pont, Marjo de Graauw, Suha Naffar-Abu-Amara, Catherine Kirsanova, Gabriella Rustici, Peter A.C. ‘t Hoen, John W.M. Martens, John A. Foekens, Benjamin Geiger, Bob van de Water
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Research Article Oncology

Tumor cell migration screen identifies SRPK1 as breast cancer metastasis determinant

Abstract

Tumor cell migration is a key process for cancer cell dissemination and metastasis that is controlled by signal-mediated cytoskeletal and cell matrix adhesion remodeling. Using a phagokinetic track assay with migratory H1299 cells, we performed an siRNA screen of almost 1,500 genes encoding kinases/phosphatases and adhesome- and migration-related proteins to identify genes that affect tumor cell migration speed and persistence. Thirty candidate genes that altered cell migration were validated in live tumor cell migration assays. Eight were associated with metastasis-free survival in breast cancer patients, with integrin β3–binding protein (ITGB3BP), MAP3K8, NIMA-related kinase (NEK2), and SHC-transforming protein 1 (SHC1) being the most predictive. Examination of genes that modulate migration indicated that SRPK1, encoding the splicing factor kinase SRSF protein kinase 1, is relevant to breast cancer outcomes, as it was highly expressed in basal breast cancer. Furthermore, high SRPK1 expression correlated with poor breast cancer disease outcome and preferential metastasis to the lungs and brain. In 2 independent murine models of breast tumor metastasis, stable shRNA-based SRPK1 knockdown suppressed metastasis to distant organs, including lung, liver, and spleen, and inhibited focal adhesion reorganization. Our study provides comprehensive information on the molecular determinants of tumor cell migration and suggests that SRPK1 has potential as a drug target for limiting breast cancer metastasis.

Authors

Wies van Roosmalen, Sylvia E. Le Dévédec, Ofra Golani, Marcel Smid, Irina Pulyakhina, Annemieke M. Timmermans, Maxime P. Look, Di Zi, Chantal Pont, Marjo de Graauw, Suha Naffar-Abu-Amara, Catherine Kirsanova, Gabriella Rustici, Peter A.C. ‘t Hoen, John W.M. Martens, John A. Foekens, Benjamin Geiger, Bob van de Water

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Figure 2

Validated high-confidence hits affect cell migration in association with modulated FA dynamics.

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Validated high-confidence hits affect cell migration in association with...
(A) Sixty-four selected genes were tested in a deconvolution PKT screen with 4 single siRNAs per gene. Validated high-confidence genes (confirmed effect in primary screen in at least 3 of 4 single siRNAs and SMARTpool) are highlighted in yellow (increased compared with control) or blue (decreased). (B) Hits for each parameter were selected and tested in a live cell imaging–based migration assay. Stills from a representative movie chosen from 2 independent experiments with each 3 replicates per siRNA are shown. Scale bar: 100 μm. (C) FA dynamics was studied using TIRF imaging of H1299 cells expressing GFP-paxillin (PXN). In each experiment (mock n = 12, siGFP n = 10, siMEK2 n = 3, siSRPK1 n = 12, and siIKBKE n = 2) at least 10 cells were imaged, and representative images are shown. Decreased cell migration after knockdown of SRPK1 and MAP2K2 was related to increased stabilization of FA as indicated by the presence of “white” FAs in the time color overlay. Knockdown of IKBKE, affecting minor axis, does not affect cell migration per se, but resulted in increased cell area, reflecting the increase in minor axis in the PKT screen. Scale bar: 20 μm.