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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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SPINK1 expression in normal breast and breast tumoursSPINK1 expression was negligible in 10 normal breast cores (panels a–h).This panel shows representative cores from the commercial breast TMA. As shown, invasive ductal carcinomas were positive but displayed various levels of SPINK1 (panels a–h).SPINK1 nuclear staining was largely restricted to the breast tumour cells (green arrow) as compared to adjacent normal cells (red arrow).Localization of SPINK1 in vitro. MDA-MB-231 cells were treated with SPINK1-CM and uptake of SPINK1 (if any) was studied using two antibodies. SPINK1 staining observed in cells after 12–24 h of SPINK1 treatment, recapitulating staining observed in primary tumours (Blue: DAPI; Green: anti-SPINK1 antibody; Red: anti-His antibody).
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fig03: SPINK1 expression in normal breast and breast tumoursSPINK1 expression was negligible in 10 normal breast cores (panels a–h).This panel shows representative cores from the commercial breast TMA. As shown, invasive ductal carcinomas were positive but displayed various levels of SPINK1 (panels a–h).SPINK1 nuclear staining was largely restricted to the breast tumour cells (green arrow) as compared to adjacent normal cells (red arrow).Localization of SPINK1 in vitro. MDA-MB-231 cells were treated with SPINK1-CM and uptake of SPINK1 (if any) was studied using two antibodies. SPINK1 staining observed in cells after 12–24 h of SPINK1 treatment, recapitulating staining observed in primary tumours (Blue: DAPI; Green: anti-SPINK1 antibody; Red: anti-His antibody).

Mentions: To investigate the localization of the SPINK1 protein in both benign and malignant breast tissues, we immunostained 98 clinically annotated cores on a tissue microarray (TMA) using an anti-SPINK1 antibody. Normal pancreas served as a positive control and specificity of the staining was evaluated by pre-neutralizing the antibody with recombinant SPINK1 protein (Fig S3 of Supporting information). The expression of SPINK1 was negligible in normal breast tissue, but most tumours were immunoreactive (95%; Fig 3, panels A–C). This suggested that SPINK1 was probably expressed early in tumour development and continued to be present in advanced disease. Of the 98 cores stained, 81 breast cancer samples were annotated for patient survival data. Analysis (see Materials and Methods Section) showed that high SPINK1 protein levels correlated with overall lower disease-free survival. Upon sub-division of patients into ER+ and ER− cohorts, this correlation was most significant in the subset of ER+ breast cancer cases (Fig 4), supporting our initial findings that the SPINK1 transcript displayed positive association with poor prognosis predominantly with ER+ cases. Moreover, SPINK1 expression did not associate with grade, age, ethnicity, stage and cERBB2 (Table S3 of Supporting information). Interestingly, SPINK1 subcellular localization seemed to vary across tumour tissues, with some expressing SPINK1 in the cytoplasm and others in the nuclei. To confirm that this nuclear localization is not an artefact of the immunohistochemical staining of paraffin processed tissue, we performed immunofluorescence on MDA-MB-231 cells treated with soluble recombinant SPINK1 bearing a 6Xhis tag. Using an antibody against the tag as well as an antibody against SPINK1, we confirmed that exogenously applied SPINK1 localized in the nucleus within 24 h of application (Fig 3D).


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 expression in normal breast and breast tumoursSPINK1 expression was negligible in 10 normal breast cores (panels a–h).This panel shows representative cores from the commercial breast TMA. As shown, invasive ductal carcinomas were positive but displayed various levels of SPINK1 (panels a–h).SPINK1 nuclear staining was largely restricted to the breast tumour cells (green arrow) as compared to adjacent normal cells (red arrow).Localization of SPINK1 in vitro. MDA-MB-231 cells were treated with SPINK1-CM and uptake of SPINK1 (if any) was studied using two antibodies. SPINK1 staining observed in cells after 12–24 h of SPINK1 treatment, recapitulating staining observed in primary tumours (Blue: DAPI; Green: anti-SPINK1 antibody; Red: anti-His antibody).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3377086&req=5

fig03: SPINK1 expression in normal breast and breast tumoursSPINK1 expression was negligible in 10 normal breast cores (panels a–h).This panel shows representative cores from the commercial breast TMA. As shown, invasive ductal carcinomas were positive but displayed various levels of SPINK1 (panels a–h).SPINK1 nuclear staining was largely restricted to the breast tumour cells (green arrow) as compared to adjacent normal cells (red arrow).Localization of SPINK1 in vitro. MDA-MB-231 cells were treated with SPINK1-CM and uptake of SPINK1 (if any) was studied using two antibodies. SPINK1 staining observed in cells after 12–24 h of SPINK1 treatment, recapitulating staining observed in primary tumours (Blue: DAPI; Green: anti-SPINK1 antibody; Red: anti-His antibody).
Mentions: To investigate the localization of the SPINK1 protein in both benign and malignant breast tissues, we immunostained 98 clinically annotated cores on a tissue microarray (TMA) using an anti-SPINK1 antibody. Normal pancreas served as a positive control and specificity of the staining was evaluated by pre-neutralizing the antibody with recombinant SPINK1 protein (Fig S3 of Supporting information). The expression of SPINK1 was negligible in normal breast tissue, but most tumours were immunoreactive (95%; Fig 3, panels A–C). This suggested that SPINK1 was probably expressed early in tumour development and continued to be present in advanced disease. Of the 98 cores stained, 81 breast cancer samples were annotated for patient survival data. Analysis (see Materials and Methods Section) showed that high SPINK1 protein levels correlated with overall lower disease-free survival. Upon sub-division of patients into ER+ and ER− cohorts, this correlation was most significant in the subset of ER+ breast cancer cases (Fig 4), supporting our initial findings that the SPINK1 transcript displayed positive association with poor prognosis predominantly with ER+ cases. Moreover, SPINK1 expression did not associate with grade, age, ethnicity, stage and cERBB2 (Table S3 of Supporting information). Interestingly, SPINK1 subcellular localization seemed to vary across tumour tissues, with some expressing SPINK1 in the cytoplasm and others in the nuclei. To confirm that this nuclear localization is not an artefact of the immunohistochemical staining of paraffin processed tissue, we performed immunofluorescence on MDA-MB-231 cells treated with soluble recombinant SPINK1 bearing a 6Xhis tag. Using an antibody against the tag as well as an antibody against SPINK1, we confirmed that exogenously applied SPINK1 localized in the nucleus within 24 h of application (Fig 3D).

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