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Loss of negative regulation by Numb over Notch is relevant to human breast carcinogenesis.

Pece S, Serresi M, Santolini E, Capra M, Hulleman E, Galimberti V, Zurrida S, Maisonneuve P, Viale G, Di Fiore PP - J. Cell Biol. (2004)

Bottom Line: Conversely, Numb silencing increases Notch signaling in normal breast cells and in Numb-positive breast tumors.Finally, growth suppression of Numb-negative, but not Numb-positive, breast tumors can be achieved by pharmacological inhibition of Notch.Thus, the Numb/Notch biological antagonism is relevant to the homeostasis of the normal mammary parenchyma and its subversion contributes to human mammary carcinogenesis.

View Article: PubMed Central - PubMed

Affiliation: Istituto Europeo di Oncologia, 20141 Milan, Italy.

ABSTRACT
The biological antagonism between Notch and Numb controls the proliferative/differentiative balance in development and homeostasis. Although altered Notch signaling has been linked to human diseases, including cancer, evidence for a substantial involvement of Notch in human tumors has remained elusive. Here, we show that Numb-mediated control on Notch signaling is lost in approximately 50% of human mammary carcinomas, due to specific Numb ubiquitination and proteasomal degradation. Mechanistically, Numb operates as an oncosuppressor, as its ectopic expression in Numb-negative, but not in Numb-positive, tumor cells inhibits proliferation. Increased Notch signaling is observed in Numb-negative tumors, but reverts to basal levels after enforced expression of Numb. Conversely, Numb silencing increases Notch signaling in normal breast cells and in Numb-positive breast tumors. Finally, growth suppression of Numb-negative, but not Numb-positive, breast tumors can be achieved by pharmacological inhibition of Notch. Thus, the Numb/Notch biological antagonism is relevant to the homeostasis of the normal mammary parenchyma and its subversion contributes to human mammary carcinogenesis.

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Loss of Numb expression in tumors is due to enhanced ubiquitination and proteasomal degradation. (A) Matched normal (top) and tumor (bottom) primary cells from class-1 (right) and class-3 (left) patients were treated with MG132 (+) for 12 h or mock treated (−) and stained with anti-Numb. (B) Total cellular lysates from the same cells as in A were immunoblotted with anti-Numb (top). Molecular mass is indicated in kilodaltons on the right. Typically, two Numb-specific bands (each probably corresponding to a tightly spaced doublet) are detected in human mammary cells. Equal loading was checked with anti-actin (bottom). (C) Primary tumor mammary cells were either mock treated (−) or exposed to MG132 (+) for 12 h. Lysates were immunoblotted (WB) with the indicated antibodies. (D) Tumor mammary cells from class-1 and class-3 patients were either mock treated (−) or exposed to MG132 (+) for 6 h. Lysates were immunoprecipitated (IP) with a monoclonal anti-Numb antibody and immunoblotted (WB) with the indicated antibodies. Molecular mass is indicated in kilodaltons on the right. Results in all panels are representative of three independent experiments. In addition, similar results were obtained with primary cultures from three class-1(type-0) and three class-3 patients (not depicted).
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fig2: Loss of Numb expression in tumors is due to enhanced ubiquitination and proteasomal degradation. (A) Matched normal (top) and tumor (bottom) primary cells from class-1 (right) and class-3 (left) patients were treated with MG132 (+) for 12 h or mock treated (−) and stained with anti-Numb. (B) Total cellular lysates from the same cells as in A were immunoblotted with anti-Numb (top). Molecular mass is indicated in kilodaltons on the right. Typically, two Numb-specific bands (each probably corresponding to a tightly spaced doublet) are detected in human mammary cells. Equal loading was checked with anti-actin (bottom). (C) Primary tumor mammary cells were either mock treated (−) or exposed to MG132 (+) for 12 h. Lysates were immunoblotted (WB) with the indicated antibodies. (D) Tumor mammary cells from class-1 and class-3 patients were either mock treated (−) or exposed to MG132 (+) for 6 h. Lysates were immunoprecipitated (IP) with a monoclonal anti-Numb antibody and immunoblotted (WB) with the indicated antibodies. Molecular mass is indicated in kilodaltons on the right. Results in all panels are representative of three independent experiments. In addition, similar results were obtained with primary cultures from three class-1(type-0) and three class-3 patients (not depicted).

Mentions: To gain insight into the molecular mechanisms responsible for loss of Numb expression, we established primary cultures from class-1(type-0) and class-3 mammary tumors and from normal breast tissues from the same patients, and analyzed them within the first two passages in vitro (Fig. S1 B). All primary cultures from normal breast and tumor cultures from class-3 patients displayed high levels of Numb expression, which were only marginally affected by treatment with the proteasome inhibitor MG132 (Fig. 2, A and B). In contrast, primary cultures from class-1(type-0) patients displayed little, if any, basal Numb expression. However, Numb levels were restored to high levels by treatment with MG132 (Fig. 2, A and B). Reduction of Numb levels in class-1 tumors did not appear to be the consequence of a generally increased proteasomal activity, as the basal levels of other cellular proteins also regulated by proteasomal degradation, such as β-catenin, were not affected under the same experimental conditions (Fig. 2 C).


Loss of negative regulation by Numb over Notch is relevant to human breast carcinogenesis.

Pece S, Serresi M, Santolini E, Capra M, Hulleman E, Galimberti V, Zurrida S, Maisonneuve P, Viale G, Di Fiore PP - J. Cell Biol. (2004)

Loss of Numb expression in tumors is due to enhanced ubiquitination and proteasomal degradation. (A) Matched normal (top) and tumor (bottom) primary cells from class-1 (right) and class-3 (left) patients were treated with MG132 (+) for 12 h or mock treated (−) and stained with anti-Numb. (B) Total cellular lysates from the same cells as in A were immunoblotted with anti-Numb (top). Molecular mass is indicated in kilodaltons on the right. Typically, two Numb-specific bands (each probably corresponding to a tightly spaced doublet) are detected in human mammary cells. Equal loading was checked with anti-actin (bottom). (C) Primary tumor mammary cells were either mock treated (−) or exposed to MG132 (+) for 12 h. Lysates were immunoblotted (WB) with the indicated antibodies. (D) Tumor mammary cells from class-1 and class-3 patients were either mock treated (−) or exposed to MG132 (+) for 6 h. Lysates were immunoprecipitated (IP) with a monoclonal anti-Numb antibody and immunoblotted (WB) with the indicated antibodies. Molecular mass is indicated in kilodaltons on the right. Results in all panels are representative of three independent experiments. In addition, similar results were obtained with primary cultures from three class-1(type-0) and three class-3 patients (not depicted).
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Related In: Results  -  Collection

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fig2: Loss of Numb expression in tumors is due to enhanced ubiquitination and proteasomal degradation. (A) Matched normal (top) and tumor (bottom) primary cells from class-1 (right) and class-3 (left) patients were treated with MG132 (+) for 12 h or mock treated (−) and stained with anti-Numb. (B) Total cellular lysates from the same cells as in A were immunoblotted with anti-Numb (top). Molecular mass is indicated in kilodaltons on the right. Typically, two Numb-specific bands (each probably corresponding to a tightly spaced doublet) are detected in human mammary cells. Equal loading was checked with anti-actin (bottom). (C) Primary tumor mammary cells were either mock treated (−) or exposed to MG132 (+) for 12 h. Lysates were immunoblotted (WB) with the indicated antibodies. (D) Tumor mammary cells from class-1 and class-3 patients were either mock treated (−) or exposed to MG132 (+) for 6 h. Lysates were immunoprecipitated (IP) with a monoclonal anti-Numb antibody and immunoblotted (WB) with the indicated antibodies. Molecular mass is indicated in kilodaltons on the right. Results in all panels are representative of three independent experiments. In addition, similar results were obtained with primary cultures from three class-1(type-0) and three class-3 patients (not depicted).
Mentions: To gain insight into the molecular mechanisms responsible for loss of Numb expression, we established primary cultures from class-1(type-0) and class-3 mammary tumors and from normal breast tissues from the same patients, and analyzed them within the first two passages in vitro (Fig. S1 B). All primary cultures from normal breast and tumor cultures from class-3 patients displayed high levels of Numb expression, which were only marginally affected by treatment with the proteasome inhibitor MG132 (Fig. 2, A and B). In contrast, primary cultures from class-1(type-0) patients displayed little, if any, basal Numb expression. However, Numb levels were restored to high levels by treatment with MG132 (Fig. 2, A and B). Reduction of Numb levels in class-1 tumors did not appear to be the consequence of a generally increased proteasomal activity, as the basal levels of other cellular proteins also regulated by proteasomal degradation, such as β-catenin, were not affected under the same experimental conditions (Fig. 2 C).

Bottom Line: Conversely, Numb silencing increases Notch signaling in normal breast cells and in Numb-positive breast tumors.Finally, growth suppression of Numb-negative, but not Numb-positive, breast tumors can be achieved by pharmacological inhibition of Notch.Thus, the Numb/Notch biological antagonism is relevant to the homeostasis of the normal mammary parenchyma and its subversion contributes to human mammary carcinogenesis.

View Article: PubMed Central - PubMed

Affiliation: Istituto Europeo di Oncologia, 20141 Milan, Italy.

ABSTRACT
The biological antagonism between Notch and Numb controls the proliferative/differentiative balance in development and homeostasis. Although altered Notch signaling has been linked to human diseases, including cancer, evidence for a substantial involvement of Notch in human tumors has remained elusive. Here, we show that Numb-mediated control on Notch signaling is lost in approximately 50% of human mammary carcinomas, due to specific Numb ubiquitination and proteasomal degradation. Mechanistically, Numb operates as an oncosuppressor, as its ectopic expression in Numb-negative, but not in Numb-positive, tumor cells inhibits proliferation. Increased Notch signaling is observed in Numb-negative tumors, but reverts to basal levels after enforced expression of Numb. Conversely, Numb silencing increases Notch signaling in normal breast cells and in Numb-positive breast tumors. Finally, growth suppression of Numb-negative, but not Numb-positive, breast tumors can be achieved by pharmacological inhibition of Notch. Thus, the Numb/Notch biological antagonism is relevant to the homeostasis of the normal mammary parenchyma and its subversion contributes to human mammary carcinogenesis.

Show MeSH
Related in: MedlinePlus