Combined genomic and phenotype screening reveals secretory factor SPINK1 as an invasion and survival factor associated with patient prognosis in breast cancer.
Bottom Line: In ER- cases, the prognostic effect did not reach statistical significance.However, down-regulation of SPINK1 resulted in cell death.Intriguingly, these anti-apoptotic effects of SPINK1 were abrogated by mutations of its protease inhibition domain.
Affiliation: Cancer Biology and Pharmacology, Genome Institute of Singapore.Show MeSH
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Mentions: To determine the specificity of our SPINK1 siRNA constructs by a phenotypic rescue, we generated two sources of recombinant SPINK1 by over-expressing SPINK1 in MCF-7 and SF9 insect cells (Fig S5, panels A–C of Supporting information). Conditioned media (CM) was collected from both sources (referred to as SPINK1 CM and SF9spink1 CM), as well as from cells transfected with empty vector as controls (vector-CM and SF9vecCM, respectively). Treatment of C2siRNA cells with SPINK1-CM resulted in 5-bromodeoxyuridine (BrDU) incorporation showing that these cells survived and could subsequently divide (Fig 6). The control Vec-CM was unable to rescue this cell death. This confirms the specificity of the siRNA used as well as the functionality of the recombinant SPINK1. Interestingly, neither MCF-7 cells over-expressing SPINK1 (MCF-7 SPINK1OE) nor various breast cancer cell lines treated with SPINK1-CM showed increased proliferation (Fig S6 of Supporting information). This further supports the notion that SPINK1 is probably an important survival factor rather than a potent growth inducer in breast cancer.
Affiliation: Cancer Biology and Pharmacology, Genome Institute of Singapore.