Limits...
Asporin Is a Fibroblast-Derived TGF-β1 Inhibitor and a Tumor Suppressor Associated with Good Prognosis in Breast Cancer.

Maris P, Blomme A, Palacios AP, Costanza B, Bellahcène A, Bianchi E, Gofflot S, Drion P, Trombino GE, Di Valentin E, Cusumano PG, Maweja S, Jerusalem G, Delvenne P, Lifrange E, Castronovo V, Turtoi A - PLoS Med. (2015)

Bottom Line: In two in vivo murine models of TNBC, we observed that tumors expressing asporin exhibit significantly reduced growth (2-fold; p = 0.01) and metastatic properties (3-fold; p = 0.045).Survival analysis, based on gene expression (n = 375; 25-y follow-up), confirmed that low asporin levels are associated with a reduced likelihood of survival (hazard ratio = 0.58; 95% CI 0.37-0.91; p = 0.017).High asporin expression is significantly associated with less aggressive tumors, stratifying patients according to the clinical outcome.

View Article: PubMed Central - PubMed

Affiliation: Metastasis Research Laboratory, GIGA-Cancer, University of Liège, Liège, Belgium.

ABSTRACT

Background: Breast cancer is a leading malignancy affecting the female population worldwide. Most morbidity is caused by metastases that remain incurable to date. TGF-β1 has been identified as a key driving force behind metastatic breast cancer, with promising therapeutic implications.

Methods and findings: Employing immunohistochemistry (IHC) analysis, we report, to our knowledge for the first time, that asporin is overexpressed in the stroma of most human breast cancers and is not expressed in normal breast tissue. In vitro, asporin is secreted by breast fibroblasts upon exposure to conditioned medium from some but not all human breast cancer cells. While hormone receptor (HR) positive cells cause strong asporin expression, triple-negative breast cancer (TNBC) cells suppress it. Further, our findings show that soluble IL-1β, secreted by TNBC cells, is responsible for inhibiting asporin in normal and cancer-associated fibroblasts. Using recombinant protein, as well as a synthetic peptide fragment, we demonstrate the ability of asporin to inhibit TGF-β1-mediated SMAD2 phosphorylation, epithelial to mesenchymal transition, and stemness in breast cancer cells. In two in vivo murine models of TNBC, we observed that tumors expressing asporin exhibit significantly reduced growth (2-fold; p = 0.01) and metastatic properties (3-fold; p = 0.045). A retrospective IHC study performed on human breast carcinoma (n = 180) demonstrates that asporin expression is lowest in TNBC and HER2+ tumors, while HR+ tumors have significantly higher asporin expression (4-fold; p = 0.001). Assessment of asporin expression and patient outcome (n = 60; 10-y follow-up) shows that low protein levels in the primary breast lesion significantly delineate patients with bad outcome regardless of the tumor HR status (area under the curve = 0.87; 95% CI 0.78-0.96; p = 0.0001). Survival analysis, based on gene expression (n = 375; 25-y follow-up), confirmed that low asporin levels are associated with a reduced likelihood of survival (hazard ratio = 0.58; 95% CI 0.37-0.91; p = 0.017). Although these data highlight the potential of asporin to serve as a prognostic marker, confirmation of the clinical value would require a prospective study on a much larger patient cohort.

Conclusions: Our data show that asporin is a stroma-derived inhibitor of TGF-β1 and a tumor suppressor in breast cancer. High asporin expression is significantly associated with less aggressive tumors, stratifying patients according to the clinical outcome. Future pre-clinical studies should consider options for increasing asporin expression in TNBC as a promising strategy for targeted therapy.

No MeSH data available.


Related in: MedlinePlus

Asporin is produced by breast fibroblasts in response to conditioned medium from breast cancer cells.(A) Western blot of total cell extracts (upper panel) and qRT-PCR analysis for asporin expression (lower panel) in breast cancer cell lines and NBFs incubated for 48 h with CM collected from a panel of breast cancer cells. (B) Western blot of total cell extracts (upper panel) and qRT-PCR analysis of asporin expression (lower panel) in non-cancerous epithelial breast cell line MCF-10A cells and NBFs incubated for 48 h with CM collected from MCF-10A. Fibroblasts treated with MCF-7 CM were used as the positive control for asporin expression induction. (C) Validation of NBFs and CAFs isolated from patient material. MCF-7 and MDA-MB-231 cells were used as epithelial controls. (D) Western blot analysis of asporin expression in total cell extracts of CAFs obtained from three different patients and treated with the CM of breast cancer cell lines. (A and B): The data are presented as mean ± SD. All panels: HSC70 was used as loading control; Western blots show representative data of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4556693&req=5

pmed.1001871.g002: Asporin is produced by breast fibroblasts in response to conditioned medium from breast cancer cells.(A) Western blot of total cell extracts (upper panel) and qRT-PCR analysis for asporin expression (lower panel) in breast cancer cell lines and NBFs incubated for 48 h with CM collected from a panel of breast cancer cells. (B) Western blot of total cell extracts (upper panel) and qRT-PCR analysis of asporin expression (lower panel) in non-cancerous epithelial breast cell line MCF-10A cells and NBFs incubated for 48 h with CM collected from MCF-10A. Fibroblasts treated with MCF-7 CM were used as the positive control for asporin expression induction. (C) Validation of NBFs and CAFs isolated from patient material. MCF-7 and MDA-MB-231 cells were used as epithelial controls. (D) Western blot analysis of asporin expression in total cell extracts of CAFs obtained from three different patients and treated with the CM of breast cancer cell lines. (A and B): The data are presented as mean ± SD. All panels: HSC70 was used as loading control; Western blots show representative data of three independent experiments.

Mentions: Owing to the observations made above, in which asporin was found deposited in the extracellular matrix of the tumor, we sought to investigate which cells are responsible for producing the protein. As demonstrated in Fig 2A, none of the human breast cancer cell lines tested showed detectable asporin expression levels (both protein and mRNA). NBFs isolated from the mammary tissue of healthy individuals responded to the CM of several breast cancer cell lines by expressing asporin. The results indicated that tumorigenic and highly metastatic triple-negative breast cancer (TNBC) cells of the basal-like subtype (e.g., MDA-MB-231 and MDA-MB-468) [33–35] did not induce asporin expression in NBFs. This was different in noninvasive luminal-like hormone receptor (HR) positive cell lines (e.g., T47D and MCF-7) [33–35], which activated very high asporin expression in NBFs. Both observations were confirmed at the protein and gene expression levels. Similar experiments with immortalized, non-transformed mammary epithelial cells (MCF-10A) demonstrated that such cells are unable to induce asporin expression in NBFs (Fig 2B). We next sought to examine whether CAFs would express asporin following their isolation from cancer tissue and whether they would react similarly to NBFs when exposed to the CM of breast cancer cells. We isolated CAFs from three breast cancer patients and validated these as pure CAF populations, negative for cytokeratins and overexpressing α-smooth muscle actin (Fig 2C). As shown in Fig 2D, CAFs isolated from HR+ tumors expressed high levels of asporin, which they were able to maintain in vitro (for several weeks) without the need to be in contact with cancer cells. However, CAFs challenged with the CM of TNBC cells (MDA-MB-231) responded by lowering asporin expression. The CM of HR+ cells (MCF-7) was unable to further increase asporin levels in CAFs.


Asporin Is a Fibroblast-Derived TGF-β1 Inhibitor and a Tumor Suppressor Associated with Good Prognosis in Breast Cancer.

Maris P, Blomme A, Palacios AP, Costanza B, Bellahcène A, Bianchi E, Gofflot S, Drion P, Trombino GE, Di Valentin E, Cusumano PG, Maweja S, Jerusalem G, Delvenne P, Lifrange E, Castronovo V, Turtoi A - PLoS Med. (2015)

Asporin is produced by breast fibroblasts in response to conditioned medium from breast cancer cells.(A) Western blot of total cell extracts (upper panel) and qRT-PCR analysis for asporin expression (lower panel) in breast cancer cell lines and NBFs incubated for 48 h with CM collected from a panel of breast cancer cells. (B) Western blot of total cell extracts (upper panel) and qRT-PCR analysis of asporin expression (lower panel) in non-cancerous epithelial breast cell line MCF-10A cells and NBFs incubated for 48 h with CM collected from MCF-10A. Fibroblasts treated with MCF-7 CM were used as the positive control for asporin expression induction. (C) Validation of NBFs and CAFs isolated from patient material. MCF-7 and MDA-MB-231 cells were used as epithelial controls. (D) Western blot analysis of asporin expression in total cell extracts of CAFs obtained from three different patients and treated with the CM of breast cancer cell lines. (A and B): The data are presented as mean ± SD. All panels: HSC70 was used as loading control; Western blots show representative data of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4556693&req=5

pmed.1001871.g002: Asporin is produced by breast fibroblasts in response to conditioned medium from breast cancer cells.(A) Western blot of total cell extracts (upper panel) and qRT-PCR analysis for asporin expression (lower panel) in breast cancer cell lines and NBFs incubated for 48 h with CM collected from a panel of breast cancer cells. (B) Western blot of total cell extracts (upper panel) and qRT-PCR analysis of asporin expression (lower panel) in non-cancerous epithelial breast cell line MCF-10A cells and NBFs incubated for 48 h with CM collected from MCF-10A. Fibroblasts treated with MCF-7 CM were used as the positive control for asporin expression induction. (C) Validation of NBFs and CAFs isolated from patient material. MCF-7 and MDA-MB-231 cells were used as epithelial controls. (D) Western blot analysis of asporin expression in total cell extracts of CAFs obtained from three different patients and treated with the CM of breast cancer cell lines. (A and B): The data are presented as mean ± SD. All panels: HSC70 was used as loading control; Western blots show representative data of three independent experiments.
Mentions: Owing to the observations made above, in which asporin was found deposited in the extracellular matrix of the tumor, we sought to investigate which cells are responsible for producing the protein. As demonstrated in Fig 2A, none of the human breast cancer cell lines tested showed detectable asporin expression levels (both protein and mRNA). NBFs isolated from the mammary tissue of healthy individuals responded to the CM of several breast cancer cell lines by expressing asporin. The results indicated that tumorigenic and highly metastatic triple-negative breast cancer (TNBC) cells of the basal-like subtype (e.g., MDA-MB-231 and MDA-MB-468) [33–35] did not induce asporin expression in NBFs. This was different in noninvasive luminal-like hormone receptor (HR) positive cell lines (e.g., T47D and MCF-7) [33–35], which activated very high asporin expression in NBFs. Both observations were confirmed at the protein and gene expression levels. Similar experiments with immortalized, non-transformed mammary epithelial cells (MCF-10A) demonstrated that such cells are unable to induce asporin expression in NBFs (Fig 2B). We next sought to examine whether CAFs would express asporin following their isolation from cancer tissue and whether they would react similarly to NBFs when exposed to the CM of breast cancer cells. We isolated CAFs from three breast cancer patients and validated these as pure CAF populations, negative for cytokeratins and overexpressing α-smooth muscle actin (Fig 2C). As shown in Fig 2D, CAFs isolated from HR+ tumors expressed high levels of asporin, which they were able to maintain in vitro (for several weeks) without the need to be in contact with cancer cells. However, CAFs challenged with the CM of TNBC cells (MDA-MB-231) responded by lowering asporin expression. The CM of HR+ cells (MCF-7) was unable to further increase asporin levels in CAFs.

Bottom Line: In two in vivo murine models of TNBC, we observed that tumors expressing asporin exhibit significantly reduced growth (2-fold; p = 0.01) and metastatic properties (3-fold; p = 0.045).Survival analysis, based on gene expression (n = 375; 25-y follow-up), confirmed that low asporin levels are associated with a reduced likelihood of survival (hazard ratio = 0.58; 95% CI 0.37-0.91; p = 0.017).High asporin expression is significantly associated with less aggressive tumors, stratifying patients according to the clinical outcome.

View Article: PubMed Central - PubMed

Affiliation: Metastasis Research Laboratory, GIGA-Cancer, University of Liège, Liège, Belgium.

ABSTRACT

Background: Breast cancer is a leading malignancy affecting the female population worldwide. Most morbidity is caused by metastases that remain incurable to date. TGF-β1 has been identified as a key driving force behind metastatic breast cancer, with promising therapeutic implications.

Methods and findings: Employing immunohistochemistry (IHC) analysis, we report, to our knowledge for the first time, that asporin is overexpressed in the stroma of most human breast cancers and is not expressed in normal breast tissue. In vitro, asporin is secreted by breast fibroblasts upon exposure to conditioned medium from some but not all human breast cancer cells. While hormone receptor (HR) positive cells cause strong asporin expression, triple-negative breast cancer (TNBC) cells suppress it. Further, our findings show that soluble IL-1β, secreted by TNBC cells, is responsible for inhibiting asporin in normal and cancer-associated fibroblasts. Using recombinant protein, as well as a synthetic peptide fragment, we demonstrate the ability of asporin to inhibit TGF-β1-mediated SMAD2 phosphorylation, epithelial to mesenchymal transition, and stemness in breast cancer cells. In two in vivo murine models of TNBC, we observed that tumors expressing asporin exhibit significantly reduced growth (2-fold; p = 0.01) and metastatic properties (3-fold; p = 0.045). A retrospective IHC study performed on human breast carcinoma (n = 180) demonstrates that asporin expression is lowest in TNBC and HER2+ tumors, while HR+ tumors have significantly higher asporin expression (4-fold; p = 0.001). Assessment of asporin expression and patient outcome (n = 60; 10-y follow-up) shows that low protein levels in the primary breast lesion significantly delineate patients with bad outcome regardless of the tumor HR status (area under the curve = 0.87; 95% CI 0.78-0.96; p = 0.0001). Survival analysis, based on gene expression (n = 375; 25-y follow-up), confirmed that low asporin levels are associated with a reduced likelihood of survival (hazard ratio = 0.58; 95% CI 0.37-0.91; p = 0.017). Although these data highlight the potential of asporin to serve as a prognostic marker, confirmation of the clinical value would require a prospective study on a much larger patient cohort.

Conclusions: Our data show that asporin is a stroma-derived inhibitor of TGF-β1 and a tumor suppressor in breast cancer. High asporin expression is significantly associated with less aggressive tumors, stratifying patients according to the clinical outcome. Future pre-clinical studies should consider options for increasing asporin expression in TNBC as a promising strategy for targeted therapy.

No MeSH data available.


Related in: MedlinePlus