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Combined genomic and phenotype screening reveals secretory factor SPINK1 as an invasion and survival factor associated with patient prognosis in breast cancer.

Soon WW, Miller LD, Black MA, Dalmasso C, Chan XB, Pang B, Ong CW, Salto-Tellez M, Desai KV, Liu ET - EMBO Mol Med (2011)

Bottom Line: In ER- cases, the prognostic effect did not reach statistical significance.Intriguingly, these anti-apoptotic effects of SPINK1 were abrogated by mutations of its protease inhibition domain.Because SPINK1 effects are abrogated by neutralizing antibodies, we suggest that SPINK1 is a viable potential therapeutic target in breast cancer.

View Article: PubMed Central - PubMed

Affiliation: Cancer Biology and Pharmacology, Genome Institute of Singapore.

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Related in: MedlinePlus

SPINK1 induces cellular invasion in vitro and in vivoCell invasion assay with MDA-MB-231 cells. Loss of invasion upon SPINK1 siRNA treatment, and rescued with SPINK1-CM.Effect of SPINK1-CM on invasion in two breast cell lines. The total number of cells that invaded was measured after 24 h (MB231) or 48 h (BT549) in response to SPINK1-CM and vector control CM.Effect of SF9spink1CM and immunoneutralization on MDA-MB-231 cells. (White bars—SF9vecCM; black bars—SF9spink1CM diluted 10×; checkered bars—CM with addition of neutralizing antibody against SPINK1).Invasion of MDA-MB-231 treated with WT SPINK1 and mutant SPINK1. Number of cells invaded were measured 24 h after assay setup. (*p < 0.005; **p < 0.05).
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fig10: SPINK1 induces cellular invasion in vitro and in vivoCell invasion assay with MDA-MB-231 cells. Loss of invasion upon SPINK1 siRNA treatment, and rescued with SPINK1-CM.Effect of SPINK1-CM on invasion in two breast cell lines. The total number of cells that invaded was measured after 24 h (MB231) or 48 h (BT549) in response to SPINK1-CM and vector control CM.Effect of SF9spink1CM and immunoneutralization on MDA-MB-231 cells. (White bars—SF9vecCM; black bars—SF9spink1CM diluted 10×; checkered bars—CM with addition of neutralizing antibody against SPINK1).Invasion of MDA-MB-231 treated with WT SPINK1 and mutant SPINK1. Number of cells invaded were measured 24 h after assay setup. (*p < 0.005; **p < 0.05).

Mentions: As part of our cellular screen for SPINK1 induced phenotypes, we had found that knockdown of SPINK1 attenuated invasion of breast cancer cell lines and that this effect could be rescued using SPINK-CM but not by Vec-CM (Figs S9 and 10A of Supporting information). We extended this analysis using recombinant SPINK1 and found that in two invasive breast cancer cell lines, MDA-MB-231 and BT549, SPINK1-CM significantly increased invasion by 2–3 fold (Fig 10B). Non-invasive MCF10A and MCF-7 cells do not invade in 24 h. After 48 h, these cells show a relative increase in invasion, however, the total number of cells was extremely low (Fig S10 of Supporting information). Importantly, we found that induction of cellular invasion in MDA MB 231 and BT-549 cells could be specifically immuno-neutralized by anti-SPINK1 antibodies (Fig 10C, Fig S11 of Supporting information). We then tested whether the invasive property of SPINK1 was due to its activity as a trypsin/serine protease inhibitor. We exposed K18YCM to MDA-MB-231 cells, and found that K18YCM retained the ability to induce cellular invasion like wild-type (WT) SPINK1-CM, suggesting that active protease inhibitor function is not necessary (Fig 10D). Similar effects were observed in BT549 cells (data not shown). Thus, the anti-apoptotic and the pro-invasive activity of SPINK1 are separable by its protease inhibitor function.


Combined genomic and phenotype screening reveals secretory factor SPINK1 as an invasion and survival factor associated with patient prognosis in breast cancer.

Soon WW, Miller LD, Black MA, Dalmasso C, Chan XB, Pang B, Ong CW, Salto-Tellez M, Desai KV, Liu ET - EMBO Mol Med (2011)

SPINK1 induces cellular invasion in vitro and in vivoCell invasion assay with MDA-MB-231 cells. Loss of invasion upon SPINK1 siRNA treatment, and rescued with SPINK1-CM.Effect of SPINK1-CM on invasion in two breast cell lines. The total number of cells that invaded was measured after 24 h (MB231) or 48 h (BT549) in response to SPINK1-CM and vector control CM.Effect of SF9spink1CM and immunoneutralization on MDA-MB-231 cells. (White bars—SF9vecCM; black bars—SF9spink1CM diluted 10×; checkered bars—CM with addition of neutralizing antibody against SPINK1).Invasion of MDA-MB-231 treated with WT SPINK1 and mutant SPINK1. Number of cells invaded were measured 24 h after assay setup. (*p < 0.005; **p < 0.05).
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Related In: Results  -  Collection

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fig10: SPINK1 induces cellular invasion in vitro and in vivoCell invasion assay with MDA-MB-231 cells. Loss of invasion upon SPINK1 siRNA treatment, and rescued with SPINK1-CM.Effect of SPINK1-CM on invasion in two breast cell lines. The total number of cells that invaded was measured after 24 h (MB231) or 48 h (BT549) in response to SPINK1-CM and vector control CM.Effect of SF9spink1CM and immunoneutralization on MDA-MB-231 cells. (White bars—SF9vecCM; black bars—SF9spink1CM diluted 10×; checkered bars—CM with addition of neutralizing antibody against SPINK1).Invasion of MDA-MB-231 treated with WT SPINK1 and mutant SPINK1. Number of cells invaded were measured 24 h after assay setup. (*p < 0.005; **p < 0.05).
Mentions: As part of our cellular screen for SPINK1 induced phenotypes, we had found that knockdown of SPINK1 attenuated invasion of breast cancer cell lines and that this effect could be rescued using SPINK-CM but not by Vec-CM (Figs S9 and 10A of Supporting information). We extended this analysis using recombinant SPINK1 and found that in two invasive breast cancer cell lines, MDA-MB-231 and BT549, SPINK1-CM significantly increased invasion by 2–3 fold (Fig 10B). Non-invasive MCF10A and MCF-7 cells do not invade in 24 h. After 48 h, these cells show a relative increase in invasion, however, the total number of cells was extremely low (Fig S10 of Supporting information). Importantly, we found that induction of cellular invasion in MDA MB 231 and BT-549 cells could be specifically immuno-neutralized by anti-SPINK1 antibodies (Fig 10C, Fig S11 of Supporting information). We then tested whether the invasive property of SPINK1 was due to its activity as a trypsin/serine protease inhibitor. We exposed K18YCM to MDA-MB-231 cells, and found that K18YCM retained the ability to induce cellular invasion like wild-type (WT) SPINK1-CM, suggesting that active protease inhibitor function is not necessary (Fig 10D). Similar effects were observed in BT549 cells (data not shown). Thus, the anti-apoptotic and the pro-invasive activity of SPINK1 are separable by its protease inhibitor function.

Bottom Line: In ER- cases, the prognostic effect did not reach statistical significance.Intriguingly, these anti-apoptotic effects of SPINK1 were abrogated by mutations of its protease inhibition domain.Because SPINK1 effects are abrogated by neutralizing antibodies, we suggest that SPINK1 is a viable potential therapeutic target in breast cancer.

View Article: PubMed Central - PubMed

Affiliation: Cancer Biology and Pharmacology, Genome Institute of Singapore.

Show MeSH
Related in: MedlinePlus