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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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Effects of perturbation of Numb or Notch activity in mammary tumor primary cells. (A) Primary normal (left) or tumor (right) cells were transfected with siRNA oligos for Numb or control (ctr) oligos for 72 h and assayed for HES-1 mRNA levels (histograms) or immunoblotted with the indicated antibody (WB). (B) Primary tumor cells from class-1(type-0) and class-3 patients were transduced with GFP or Numb-GFP and assayed for HES-1 mRNA levels 72 h after infection (protein expression controls are as from Fig. 3 B, not depicted here). Normal primary cells from the same patients behaved as class-3 tumors (not depicted). In A and B, the mean fold induction (± SD) from two independent experiments performed in triplicate is shown. (C) Primary tumor cells from class-1(type-0) (left) and class-3 (right) patients were treated with DFP-AA for 10 d or mock treated (ctr), followed by staining (bottom) to count colonies. The histograms show the average colonies (colony-forming units ± SD) in triplicate plates. Results are representative of three independent experiments. (D) HES-1 mRNA expression from cells treated as in 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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fig5: Effects of perturbation of Numb or Notch activity in mammary tumor primary cells. (A) Primary normal (left) or tumor (right) cells were transfected with siRNA oligos for Numb or control (ctr) oligos for 72 h and assayed for HES-1 mRNA levels (histograms) or immunoblotted with the indicated antibody (WB). (B) Primary tumor cells from class-1(type-0) and class-3 patients were transduced with GFP or Numb-GFP and assayed for HES-1 mRNA levels 72 h after infection (protein expression controls are as from Fig. 3 B, not depicted here). Normal primary cells from the same patients behaved as class-3 tumors (not depicted). In A and B, the mean fold induction (± SD) from two independent experiments performed in triplicate is shown. (C) Primary tumor cells from class-1(type-0) (left) and class-3 (right) patients were treated with DFP-AA for 10 d or mock treated (ctr), followed by staining (bottom) to count colonies. The histograms show the average colonies (colony-forming units ± SD) in triplicate plates. Results are representative of three independent experiments. (D) HES-1 mRNA expression from cells treated as in 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: These results prompted us to assess directly a possible functional link between Numb levels and Notch activity in tumor cells. RNAi-mediated silencing of Numb in primary normal breast cells resulted in a significant increase in HES-1 mRNA transcripts in comparison to cells transfected with a control siRNA (Fig. 5 A). As expected, a similar increase in Notch-dependent transcriptional activity was observed in class-3 tumor cells, but not in class-1 tumor cells (Fig. 5 A). Accordingly, retrovirally mediated overexpression of Numb caused a significant decrease in basal Notch activity in class-1, but not in class-3, tumor cells (Fig. 5 B) or in normal cells from the same patients (not depicted). As overexpression of Numb in class-1 tumor cells also caused a significant growth-suppression effect (Fig. 3), we directly tested the possibility that deregulated Notch activity arising from loss of Numb might be responsible for uncontrolled cell proliferation in class-1 tumors. We took advantage of the small molecule peptidomimetic presenilin inhibitor DFP-AA, which blocks Notch signaling and effectively suppresses the growth of Notch1-transformed lymphoid cell lines in vitro (Weng et al., 2003). DFP-AA treatment of class-1 tumor cells was sufficient to cause a dramatic suppression of their growth potential (Fig. 5 C), which was accompanied by a marked decrease in Notch activity, as assessed by HES-1 mRNA levels (Fig. 5 D). In contrast, no significant effect was observed in class-3 tumor cells (Fig. 5, C and D).


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)

Effects of perturbation of Numb or Notch activity in mammary tumor primary cells. (A) Primary normal (left) or tumor (right) cells were transfected with siRNA oligos for Numb or control (ctr) oligos for 72 h and assayed for HES-1 mRNA levels (histograms) or immunoblotted with the indicated antibody (WB). (B) Primary tumor cells from class-1(type-0) and class-3 patients were transduced with GFP or Numb-GFP and assayed for HES-1 mRNA levels 72 h after infection (protein expression controls are as from Fig. 3 B, not depicted here). Normal primary cells from the same patients behaved as class-3 tumors (not depicted). In A and B, the mean fold induction (± SD) from two independent experiments performed in triplicate is shown. (C) Primary tumor cells from class-1(type-0) (left) and class-3 (right) patients were treated with DFP-AA for 10 d or mock treated (ctr), followed by staining (bottom) to count colonies. The histograms show the average colonies (colony-forming units ± SD) in triplicate plates. Results are representative of three independent experiments. (D) HES-1 mRNA expression from cells treated as in 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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fig5: Effects of perturbation of Numb or Notch activity in mammary tumor primary cells. (A) Primary normal (left) or tumor (right) cells were transfected with siRNA oligos for Numb or control (ctr) oligos for 72 h and assayed for HES-1 mRNA levels (histograms) or immunoblotted with the indicated antibody (WB). (B) Primary tumor cells from class-1(type-0) and class-3 patients were transduced with GFP or Numb-GFP and assayed for HES-1 mRNA levels 72 h after infection (protein expression controls are as from Fig. 3 B, not depicted here). Normal primary cells from the same patients behaved as class-3 tumors (not depicted). In A and B, the mean fold induction (± SD) from two independent experiments performed in triplicate is shown. (C) Primary tumor cells from class-1(type-0) (left) and class-3 (right) patients were treated with DFP-AA for 10 d or mock treated (ctr), followed by staining (bottom) to count colonies. The histograms show the average colonies (colony-forming units ± SD) in triplicate plates. Results are representative of three independent experiments. (D) HES-1 mRNA expression from cells treated as in 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: These results prompted us to assess directly a possible functional link between Numb levels and Notch activity in tumor cells. RNAi-mediated silencing of Numb in primary normal breast cells resulted in a significant increase in HES-1 mRNA transcripts in comparison to cells transfected with a control siRNA (Fig. 5 A). As expected, a similar increase in Notch-dependent transcriptional activity was observed in class-3 tumor cells, but not in class-1 tumor cells (Fig. 5 A). Accordingly, retrovirally mediated overexpression of Numb caused a significant decrease in basal Notch activity in class-1, but not in class-3, tumor cells (Fig. 5 B) or in normal cells from the same patients (not depicted). As overexpression of Numb in class-1 tumor cells also caused a significant growth-suppression effect (Fig. 3), we directly tested the possibility that deregulated Notch activity arising from loss of Numb might be responsible for uncontrolled cell proliferation in class-1 tumors. We took advantage of the small molecule peptidomimetic presenilin inhibitor DFP-AA, which blocks Notch signaling and effectively suppresses the growth of Notch1-transformed lymphoid cell lines in vitro (Weng et al., 2003). DFP-AA treatment of class-1 tumor cells was sufficient to cause a dramatic suppression of their growth potential (Fig. 5 C), which was accompanied by a marked decrease in Notch activity, as assessed by HES-1 mRNA levels (Fig. 5 D). In contrast, no significant effect was observed in class-3 tumor cells (Fig. 5, C and D).

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