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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

Asporin reduces primary breast cancer tumor growth and lung metastasis formation in vivo.(A) Western blot analysis of asporin expression in the CM of MDA-MB-468 stable clones expressing asporin, used for subcutaneous injection in mice. Ponceau red is shown as loading control. (B) Bioluminescence imaging of control and asporin-expressing xenografts at day 28 after tumor engraftment (left panel). The color scale indicates the fluorescent intensity. The mean (± SEM) volume (in cubic millimeters) of primary tumors measured weekly (from day 14 onwards) for the time-matched cohort is also shown (n = 20 for each group) (right panel). Statistical significance was calculated using Student’s t-test (**0.01 < p < 0.001; ***0.001 < p < 0.0001). (C) Representative hematoxylin and eosin (H&E), asporin, and Ki67 IHC staining in MDA-MB-468 xenografts collected 7 wk post-engraftment. Control xenografts consistently displayed an invasion in the muscle layer (M). An extended necrotic (N) area was present in the peri-tumoral zone of MDA-MB-468-aspn mice tumors. (D) Quantification of the stem cell population in xenografted tumors expressing asporin using ALDH+ and CD44high/CD24low stemness markers (7 wk post-engraftment). (E) Post-operative follow-up of mice that had primary tumors removed at the same time (time-matched). (F) Mean (± SEM) volume (in cubic millimeters) of primary tumors measured weekly for the size-matched cohort (n = 20 for each group). (G) Post-operative follow-up of mice that had primary tumors removed at the same volume (size-matched). (E and G): IHC evaluation of vimentin in lung necropsies and quantification of metastatic deposits. All images of representative fields were taken at 40×, 100×, and 400× magnification. (D, E, and G): The data are presented as mean ± SD. Statistical significance was calculated using Student’s t-test.
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pmed.1001871.g006: Asporin reduces primary breast cancer tumor growth and lung metastasis formation in vivo.(A) Western blot analysis of asporin expression in the CM of MDA-MB-468 stable clones expressing asporin, used for subcutaneous injection in mice. Ponceau red is shown as loading control. (B) Bioluminescence imaging of control and asporin-expressing xenografts at day 28 after tumor engraftment (left panel). The color scale indicates the fluorescent intensity. The mean (± SEM) volume (in cubic millimeters) of primary tumors measured weekly (from day 14 onwards) for the time-matched cohort is also shown (n = 20 for each group) (right panel). Statistical significance was calculated using Student’s t-test (**0.01 < p < 0.001; ***0.001 < p < 0.0001). (C) Representative hematoxylin and eosin (H&E), asporin, and Ki67 IHC staining in MDA-MB-468 xenografts collected 7 wk post-engraftment. Control xenografts consistently displayed an invasion in the muscle layer (M). An extended necrotic (N) area was present in the peri-tumoral zone of MDA-MB-468-aspn mice tumors. (D) Quantification of the stem cell population in xenografted tumors expressing asporin using ALDH+ and CD44high/CD24low stemness markers (7 wk post-engraftment). (E) Post-operative follow-up of mice that had primary tumors removed at the same time (time-matched). (F) Mean (± SEM) volume (in cubic millimeters) of primary tumors measured weekly for the size-matched cohort (n = 20 for each group). (G) Post-operative follow-up of mice that had primary tumors removed at the same volume (size-matched). (E and G): IHC evaluation of vimentin in lung necropsies and quantification of metastatic deposits. All images of representative fields were taken at 40×, 100×, and 400× magnification. (D, E, and G): The data are presented as mean ± SD. Statistical significance was calculated using Student’s t-test.

Mentions: Therefore, we next sought to engraft asporin-overexpressing cancer cells that would maintain constant asporin expression in the tumor. This was performed with stably transduced asporin-expressing MDA-MB-468 cells (Fig 6A). The control and asporin-expressing MDA-MB-468 cells were implanted subcutaneously in NOD-SCID mice. Primary tumor growth was monitored weekly. The results indicated that asporin-expressing tumors were significantly smaller, reaching up to 2-fold lower volumes at 7 wk post-engraftment (day 49 post-engraftment: 124.1 mm3 smaller than control; 95% CI 75.2–180.4; p = 0.001) (Fig 6B). Histological evaluation demonstrated invasive control tumors developing towards the muscle layers, whereas this was not observed in asporin-expressing counterparts (Fig 6C). Further analysis of asporin-expressing tumors evidenced extensive zones of tumor necrosis in the central areas (Fig 6C), as well as numerous cells with condensed chromatin. In the control conditions necrosis was less pronounced, whereas transparent chromatin staining and the presence of nucleoli further characterized tumor cells. The latter suggested a higher proliferation rate in control tumors. The assessment of tumor proliferation based on Ki67 staining showed stronger and more frequent nuclear positivity in the control tumors in comparison to the asporin-expressing counterparts (Fig 6C). IHC evaluation of asporin expression in the experimental tumors evidenced the expected asporin overexpression.


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 reduces primary breast cancer tumor growth and lung metastasis formation in vivo.(A) Western blot analysis of asporin expression in the CM of MDA-MB-468 stable clones expressing asporin, used for subcutaneous injection in mice. Ponceau red is shown as loading control. (B) Bioluminescence imaging of control and asporin-expressing xenografts at day 28 after tumor engraftment (left panel). The color scale indicates the fluorescent intensity. The mean (± SEM) volume (in cubic millimeters) of primary tumors measured weekly (from day 14 onwards) for the time-matched cohort is also shown (n = 20 for each group) (right panel). Statistical significance was calculated using Student’s t-test (**0.01 < p < 0.001; ***0.001 < p < 0.0001). (C) Representative hematoxylin and eosin (H&E), asporin, and Ki67 IHC staining in MDA-MB-468 xenografts collected 7 wk post-engraftment. Control xenografts consistently displayed an invasion in the muscle layer (M). An extended necrotic (N) area was present in the peri-tumoral zone of MDA-MB-468-aspn mice tumors. (D) Quantification of the stem cell population in xenografted tumors expressing asporin using ALDH+ and CD44high/CD24low stemness markers (7 wk post-engraftment). (E) Post-operative follow-up of mice that had primary tumors removed at the same time (time-matched). (F) Mean (± SEM) volume (in cubic millimeters) of primary tumors measured weekly for the size-matched cohort (n = 20 for each group). (G) Post-operative follow-up of mice that had primary tumors removed at the same volume (size-matched). (E and G): IHC evaluation of vimentin in lung necropsies and quantification of metastatic deposits. All images of representative fields were taken at 40×, 100×, and 400× magnification. (D, E, and G): The data are presented as mean ± SD. Statistical significance was calculated using Student’s t-test.
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Related In: Results  -  Collection

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pmed.1001871.g006: Asporin reduces primary breast cancer tumor growth and lung metastasis formation in vivo.(A) Western blot analysis of asporin expression in the CM of MDA-MB-468 stable clones expressing asporin, used for subcutaneous injection in mice. Ponceau red is shown as loading control. (B) Bioluminescence imaging of control and asporin-expressing xenografts at day 28 after tumor engraftment (left panel). The color scale indicates the fluorescent intensity. The mean (± SEM) volume (in cubic millimeters) of primary tumors measured weekly (from day 14 onwards) for the time-matched cohort is also shown (n = 20 for each group) (right panel). Statistical significance was calculated using Student’s t-test (**0.01 < p < 0.001; ***0.001 < p < 0.0001). (C) Representative hematoxylin and eosin (H&E), asporin, and Ki67 IHC staining in MDA-MB-468 xenografts collected 7 wk post-engraftment. Control xenografts consistently displayed an invasion in the muscle layer (M). An extended necrotic (N) area was present in the peri-tumoral zone of MDA-MB-468-aspn mice tumors. (D) Quantification of the stem cell population in xenografted tumors expressing asporin using ALDH+ and CD44high/CD24low stemness markers (7 wk post-engraftment). (E) Post-operative follow-up of mice that had primary tumors removed at the same time (time-matched). (F) Mean (± SEM) volume (in cubic millimeters) of primary tumors measured weekly for the size-matched cohort (n = 20 for each group). (G) Post-operative follow-up of mice that had primary tumors removed at the same volume (size-matched). (E and G): IHC evaluation of vimentin in lung necropsies and quantification of metastatic deposits. All images of representative fields were taken at 40×, 100×, and 400× magnification. (D, E, and G): The data are presented as mean ± SD. Statistical significance was calculated using Student’s t-test.
Mentions: Therefore, we next sought to engraft asporin-overexpressing cancer cells that would maintain constant asporin expression in the tumor. This was performed with stably transduced asporin-expressing MDA-MB-468 cells (Fig 6A). The control and asporin-expressing MDA-MB-468 cells were implanted subcutaneously in NOD-SCID mice. Primary tumor growth was monitored weekly. The results indicated that asporin-expressing tumors were significantly smaller, reaching up to 2-fold lower volumes at 7 wk post-engraftment (day 49 post-engraftment: 124.1 mm3 smaller than control; 95% CI 75.2–180.4; p = 0.001) (Fig 6B). Histological evaluation demonstrated invasive control tumors developing towards the muscle layers, whereas this was not observed in asporin-expressing counterparts (Fig 6C). Further analysis of asporin-expressing tumors evidenced extensive zones of tumor necrosis in the central areas (Fig 6C), as well as numerous cells with condensed chromatin. In the control conditions necrosis was less pronounced, whereas transparent chromatin staining and the presence of nucleoli further characterized tumor cells. The latter suggested a higher proliferation rate in control tumors. The assessment of tumor proliferation based on Ki67 staining showed stronger and more frequent nuclear positivity in the control tumors in comparison to the asporin-expressing counterparts (Fig 6C). IHC evaluation of asporin expression in the experimental tumors evidenced the expected asporin overexpression.

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