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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

High asporin expression in human breast cancer matches with luminal-like tumor type and good patient outcome.(A) Representative IHC staining of asporin expression in human breast cancer tissues (upper panel). Box plots of asporin expression in 180 breast cancer patients with different status of HER2, ER, and PR are also shown (lower panel). The black line denotes the median expression, and the red line the mean expression. Significant differences in asporin expression were detected among all different subtypes of breast cancer. (B) Analysis of asporin mRNA expression in breast cancer tumors from different molecular subtypes (n = 1,280) and evaluation of asporin mRNA expression in breast cancer of different pathological grades (n = 1,411). (C) Representative IHC staining of asporin expression (upper panel), box plot showing the IHC score (middle panel) in breast cancer tissues from 60 patients with different outcomes, and ROC curve analysis of data obtained from 60 patients with different outcome (lower panel). Scoring and statistics were performed as outlined in the Methods section. (D) Kaplan-Meier survival curve based on asporin mRNA expression in untreated breast cancer with post-operative follow-up of 25 y (n = 375). Images in panels were taken at 100× magnification. All analyses outlined in (B) and (D) were performed using publicly deposited gene expression datasets [31,32] and according to procedures outlined in the Methods. AUC, area under the curve; HR, hazard ratio; SE, standard error.
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pmed.1001871.g007: High asporin expression in human breast cancer matches with luminal-like tumor type and good patient outcome.(A) Representative IHC staining of asporin expression in human breast cancer tissues (upper panel). Box plots of asporin expression in 180 breast cancer patients with different status of HER2, ER, and PR are also shown (lower panel). The black line denotes the median expression, and the red line the mean expression. Significant differences in asporin expression were detected among all different subtypes of breast cancer. (B) Analysis of asporin mRNA expression in breast cancer tumors from different molecular subtypes (n = 1,280) and evaluation of asporin mRNA expression in breast cancer of different pathological grades (n = 1,411). (C) Representative IHC staining of asporin expression (upper panel), box plot showing the IHC score (middle panel) in breast cancer tissues from 60 patients with different outcomes, and ROC curve analysis of data obtained from 60 patients with different outcome (lower panel). Scoring and statistics were performed as outlined in the Methods section. (D) Kaplan-Meier survival curve based on asporin mRNA expression in untreated breast cancer with post-operative follow-up of 25 y (n = 375). Images in panels were taken at 100× magnification. All analyses outlined in (B) and (D) were performed using publicly deposited gene expression datasets [31,32] and according to procedures outlined in the Methods. AUC, area under the curve; HR, hazard ratio; SE, standard error.

Mentions: Considering the in vitro and in vivo data, we expanded our observations using IHC to 180 breast cancer patients, subdivided in four categories with 45 cases each: (i) ER−/PR−/HER2− (triple-negative), (ii) ER+/PR+/HER2+ (triple-positive), (iii) ER+/PR+/HER2− (HR+), and (iv) ER−/PR−/HER2+ (HER2+) (Fig 7A; S1 Table). In our cohort, patients of all subgroups had similar age and showed similar tumor size. Triple-negative and HER2+ cases had higher tumor grade (Bloom 3 versus 2) and a stronger percentage of proliferating cells (Ki67+ cells: ~43% versus ~17%) than the other two subgroups. The frequency of metastasis was highest in TNBC patients (22%), followed by HER2+ and triple-positive breast cancer patients, who displayed similar frequencies (~9%). The IHC results showed that HR+ tumors had a high asporin expression, which was significantly elevated (up to 4-fold) in comparison to TNBC and HER2+ tumors. The latter subgroups had low, and in some cases non-detectable, asporin expression. Intrigued by these findings we aimed to examine the validity of our observations in more individuals. To do so, we used GOBO and publicly deposited mRNA expression data from breast cancer patients [31]. Analysis of asporin mRNA expression in tumors from different molecular subtypes (Fig 7B) confirmed the results obtained with IHC analysis, demonstrating that asporin expression is high in luminal-A and low in basal-like subtypes (n = 1,280). Evaluation of asporin mRNA expression in tumors of different pathological grades showed that its expression is higher in grade 1 and lower in grade 3 tumors (n = 1,411). This gradual decrease of asporin expression with the grade of the tumor suggested a relationship between asporin expression and breast cancer progression. Thus, we sought to verify how asporin expression correlates with breast cancer patient outcome. We assessed asporin protein expression retrospectively using IHC and tissues from 60 breast cancer patients with over 10-y follow-up (Fig 7C; S2 Table). The patients were divided into two groups: (i) good outcome (signified by no metastatic disease in the follow-up period and following the resection of the primary tumor) and (ii) poor outcome (patients who developed metastases). The two groups showed no major difference in age, tumor size, tumor grade, and ER, PR, HER2, and Ki67 status. The IHC results evidenced significantly higher levels of asporin in patients with good outcome than in patients with poor outcome (2-fold; p = 0.001). The suitability of asporin as a biomarker candidate for predicting metastasis in breast cancer patients was evaluated using a ROC curve. As indicated in Fig 7C, the area under the curve was 0.87 (95% CI 0.78–0.96; p = 0.0001). These data warranted further examination in a larger cohort of patients, with longer survival follow-up. We therefore examined mRNA expression in breast cancers using Kaplan-Meier Plotter and publicly deposited data [32], where the corresponding patients had a post-operative follow-up of 25 y and had no adjuvant treatment (n = 375). The Kaplan-Meier survival curve obtained from these data confirmed the IHC results and demonstrated that low asporin mRNA expression is significantly associated with decreased overall survival (hazard ratio = 0.58; 95% CI 0.37–0.91; logrank p = 0.017) (Fig 7D).


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)

High asporin expression in human breast cancer matches with luminal-like tumor type and good patient outcome.(A) Representative IHC staining of asporin expression in human breast cancer tissues (upper panel). Box plots of asporin expression in 180 breast cancer patients with different status of HER2, ER, and PR are also shown (lower panel). The black line denotes the median expression, and the red line the mean expression. Significant differences in asporin expression were detected among all different subtypes of breast cancer. (B) Analysis of asporin mRNA expression in breast cancer tumors from different molecular subtypes (n = 1,280) and evaluation of asporin mRNA expression in breast cancer of different pathological grades (n = 1,411). (C) Representative IHC staining of asporin expression (upper panel), box plot showing the IHC score (middle panel) in breast cancer tissues from 60 patients with different outcomes, and ROC curve analysis of data obtained from 60 patients with different outcome (lower panel). Scoring and statistics were performed as outlined in the Methods section. (D) Kaplan-Meier survival curve based on asporin mRNA expression in untreated breast cancer with post-operative follow-up of 25 y (n = 375). Images in panels were taken at 100× magnification. All analyses outlined in (B) and (D) were performed using publicly deposited gene expression datasets [31,32] and according to procedures outlined in the Methods. AUC, area under the curve; HR, hazard ratio; SE, standard error.
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Related In: Results  -  Collection

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pmed.1001871.g007: High asporin expression in human breast cancer matches with luminal-like tumor type and good patient outcome.(A) Representative IHC staining of asporin expression in human breast cancer tissues (upper panel). Box plots of asporin expression in 180 breast cancer patients with different status of HER2, ER, and PR are also shown (lower panel). The black line denotes the median expression, and the red line the mean expression. Significant differences in asporin expression were detected among all different subtypes of breast cancer. (B) Analysis of asporin mRNA expression in breast cancer tumors from different molecular subtypes (n = 1,280) and evaluation of asporin mRNA expression in breast cancer of different pathological grades (n = 1,411). (C) Representative IHC staining of asporin expression (upper panel), box plot showing the IHC score (middle panel) in breast cancer tissues from 60 patients with different outcomes, and ROC curve analysis of data obtained from 60 patients with different outcome (lower panel). Scoring and statistics were performed as outlined in the Methods section. (D) Kaplan-Meier survival curve based on asporin mRNA expression in untreated breast cancer with post-operative follow-up of 25 y (n = 375). Images in panels were taken at 100× magnification. All analyses outlined in (B) and (D) were performed using publicly deposited gene expression datasets [31,32] and according to procedures outlined in the Methods. AUC, area under the curve; HR, hazard ratio; SE, standard error.
Mentions: Considering the in vitro and in vivo data, we expanded our observations using IHC to 180 breast cancer patients, subdivided in four categories with 45 cases each: (i) ER−/PR−/HER2− (triple-negative), (ii) ER+/PR+/HER2+ (triple-positive), (iii) ER+/PR+/HER2− (HR+), and (iv) ER−/PR−/HER2+ (HER2+) (Fig 7A; S1 Table). In our cohort, patients of all subgroups had similar age and showed similar tumor size. Triple-negative and HER2+ cases had higher tumor grade (Bloom 3 versus 2) and a stronger percentage of proliferating cells (Ki67+ cells: ~43% versus ~17%) than the other two subgroups. The frequency of metastasis was highest in TNBC patients (22%), followed by HER2+ and triple-positive breast cancer patients, who displayed similar frequencies (~9%). The IHC results showed that HR+ tumors had a high asporin expression, which was significantly elevated (up to 4-fold) in comparison to TNBC and HER2+ tumors. The latter subgroups had low, and in some cases non-detectable, asporin expression. Intrigued by these findings we aimed to examine the validity of our observations in more individuals. To do so, we used GOBO and publicly deposited mRNA expression data from breast cancer patients [31]. Analysis of asporin mRNA expression in tumors from different molecular subtypes (Fig 7B) confirmed the results obtained with IHC analysis, demonstrating that asporin expression is high in luminal-A and low in basal-like subtypes (n = 1,280). Evaluation of asporin mRNA expression in tumors of different pathological grades showed that its expression is higher in grade 1 and lower in grade 3 tumors (n = 1,411). This gradual decrease of asporin expression with the grade of the tumor suggested a relationship between asporin expression and breast cancer progression. Thus, we sought to verify how asporin expression correlates with breast cancer patient outcome. We assessed asporin protein expression retrospectively using IHC and tissues from 60 breast cancer patients with over 10-y follow-up (Fig 7C; S2 Table). The patients were divided into two groups: (i) good outcome (signified by no metastatic disease in the follow-up period and following the resection of the primary tumor) and (ii) poor outcome (patients who developed metastases). The two groups showed no major difference in age, tumor size, tumor grade, and ER, PR, HER2, and Ki67 status. The IHC results evidenced significantly higher levels of asporin in patients with good outcome than in patients with poor outcome (2-fold; p = 0.001). The suitability of asporin as a biomarker candidate for predicting metastasis in breast cancer patients was evaluated using a ROC curve. As indicated in Fig 7C, the area under the curve was 0.87 (95% CI 0.78–0.96; p = 0.0001). These data warranted further examination in a larger cohort of patients, with longer survival follow-up. We therefore examined mRNA expression in breast cancers using Kaplan-Meier Plotter and publicly deposited data [32], where the corresponding patients had a post-operative follow-up of 25 y and had no adjuvant treatment (n = 375). The Kaplan-Meier survival curve obtained from these data confirmed the IHC results and demonstrated that low asporin mRNA expression is significantly associated with decreased overall survival (hazard ratio = 0.58; 95% CI 0.37–0.91; logrank p = 0.017) (Fig 7D).

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