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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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Increased Notch signaling in Numb-negative tumors. (A) Primary tumor cells from class-1(type-0) (top) and class-3 (bottom) patients were treated with MG132 (+) or mock treated (−) for 1 h and stained with anti-Notch. Note the lower basal levels of Notch expression in class-1 MG132-untreated cells and the presence of nuclear Notch in the same class upon MG132 treatment. (B) CBF1-responsive reporter gene activity was evaluated in normal and tumor cells from class-1(type-0) and class-3 patients. (C) HES-1 mRNA expression in total RNAs from normal and tumor cells from class-1(type-0) and class-3 patients. In B and C, the mean fold induction (± SD) from two independent experiments performed in triplicate is shown. In all panels, results are representative of those obtained with primary cultures from three class-1(type-0) and three class-3 patients (not depicted).
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fig4: Increased Notch signaling in Numb-negative tumors. (A) Primary tumor cells from class-1(type-0) (top) and class-3 (bottom) patients were treated with MG132 (+) or mock treated (−) for 1 h and stained with anti-Notch. Note the lower basal levels of Notch expression in class-1 MG132-untreated cells and the presence of nuclear Notch in the same class upon MG132 treatment. (B) CBF1-responsive reporter gene activity was evaluated in normal and tumor cells from class-1(type-0) and class-3 patients. (C) HES-1 mRNA expression in total RNAs from normal and tumor cells from class-1(type-0) and class-3 patients. In B and C, the mean fold induction (± SD) from two independent experiments performed in triplicate is shown. In all panels, results are representative of those obtained with primary cultures from three class-1(type-0) and three class-3 patients (not depicted).

Mentions: Therefore, we used primary tumor cells to monitor the subcellular distribution and activation state of Notch. In class-1(type-0), Notch immunostaining appeared lower than in class-3 tumors or normal cells (Fig. 4 A). We reasoned that this finding might be consistent with increased processing of Notch at the plasma membrane followed by increased nuclear translocation of the ICD into the nucleus, whereby it is promptly degraded by the nuclear proteasomal machinery. Indeed, even brief (1 h) MG132 treatment revealed nuclear accumulation of Notch (ICD) in all class-1(type-0) cells, but not in class-3 tumors or normal counterparts (Fig. 4 A). We measured Notch function by following luciferase activity driven from a Notch-dependent CBF1-responsive reporter (6x-RBP-Jk-luc) transfected into primary cells. Luciferase activity was similar among class-3 and normal cultures but clearly increased in all class-1 cultures (Fig. 4 B). Furthermore, by using quantitative RT-PCR, we found that endogenous expression of the HES-1 mRNA, a known target gene for Notch transcriptional activity (Sasai et al., 1992; Jarriault et al., 1998), was significantly higher in class-1 tumor cells than in class-3 cells or their normal counterparts (Fig. 4 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)

Increased Notch signaling in Numb-negative tumors. (A) Primary tumor cells from class-1(type-0) (top) and class-3 (bottom) patients were treated with MG132 (+) or mock treated (−) for 1 h and stained with anti-Notch. Note the lower basal levels of Notch expression in class-1 MG132-untreated cells and the presence of nuclear Notch in the same class upon MG132 treatment. (B) CBF1-responsive reporter gene activity was evaluated in normal and tumor cells from class-1(type-0) and class-3 patients. (C) HES-1 mRNA expression in total RNAs from normal and tumor cells from class-1(type-0) and class-3 patients. In B and C, the mean fold induction (± SD) from two independent experiments performed in triplicate is shown. In all panels, results are representative of those obtained with primary cultures from three class-1(type-0) and three class-3 patients (not depicted).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172557&req=5

fig4: Increased Notch signaling in Numb-negative tumors. (A) Primary tumor cells from class-1(type-0) (top) and class-3 (bottom) patients were treated with MG132 (+) or mock treated (−) for 1 h and stained with anti-Notch. Note the lower basal levels of Notch expression in class-1 MG132-untreated cells and the presence of nuclear Notch in the same class upon MG132 treatment. (B) CBF1-responsive reporter gene activity was evaluated in normal and tumor cells from class-1(type-0) and class-3 patients. (C) HES-1 mRNA expression in total RNAs from normal and tumor cells from class-1(type-0) and class-3 patients. In B and C, the mean fold induction (± SD) from two independent experiments performed in triplicate is shown. In all panels, results are representative of those obtained with primary cultures from three class-1(type-0) and three class-3 patients (not depicted).
Mentions: Therefore, we used primary tumor cells to monitor the subcellular distribution and activation state of Notch. In class-1(type-0), Notch immunostaining appeared lower than in class-3 tumors or normal cells (Fig. 4 A). We reasoned that this finding might be consistent with increased processing of Notch at the plasma membrane followed by increased nuclear translocation of the ICD into the nucleus, whereby it is promptly degraded by the nuclear proteasomal machinery. Indeed, even brief (1 h) MG132 treatment revealed nuclear accumulation of Notch (ICD) in all class-1(type-0) cells, but not in class-3 tumors or normal counterparts (Fig. 4 A). We measured Notch function by following luciferase activity driven from a Notch-dependent CBF1-responsive reporter (6x-RBP-Jk-luc) transfected into primary cells. Luciferase activity was similar among class-3 and normal cultures but clearly increased in all class-1 cultures (Fig. 4 B). Furthermore, by using quantitative RT-PCR, we found that endogenous expression of the HES-1 mRNA, a known target gene for Notch transcriptional activity (Sasai et al., 1992; Jarriault et al., 1998), was significantly higher in class-1 tumor cells than in class-3 cells or their normal counterparts (Fig. 4 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