Numb was originally identified as a determinant of cell fate in Drosophila, and more recently as a tumor suppressor in different human cancers. Our lab, in particular, has contributed to the characterization of Numb as a tumor suppressor. Indeed, we showed that Numb protein expression is lost in ~30% of breast cancers (BCs) and associates with an adverse prognostic outcome. At the cellular level, Numb loss results in uncontrolled proliferation, impaired response to genotoxic treatments and emergence and amplification of the cancer stem cell pool, while at the molecular level, it leads to enhanced Notch oncogenic signaling and reduced expression of the tumor suppressor protein p53. Under physiological conditions, Numb acts to stabilize p53 protein by binding to and inhibiting the activity of Mdm2, the E3 ligase that mediates p53 ubiquitination and subsequent degradation. A detailed structural characterization of the Numb:Mdm2 binding interface, recently performed in our lab, has revealed that a short Numb region encoded by the alternatively spliced exon 3 was necessary and sufficient to bind to Mdm2 and prevent p53 degradation. This region is only present in two out of the four main Numb isoforms (Numb 1 and 2, hereafter referred to as Numb 1/2). Based on these findings, we hypothesized that the tumor suppressor role of Numb might be mediated specifically by isoforms 1 and 2, and that altered splicing could represent a novel mechanism responsible for the functional loss of Numb in tumors. To test these possibilities, we performed a series of experiments aimed at elucidating the specific roles of Numb isoforms in cancer and their contribution to tumorigenesis. In particular, we characterized Numb isoforms expression in BC cells and investigated the involvement of the expressed isoforms in the regulation of the p53 pathway. Our results point to the exclusive role of Numb 1/2 isoforms in the regulation of the Mdm2-p53 circuitry. Loss of Numb 1/2 in p53-WT BCs leads to reduced p53 levels and activity and an accumulation of DNA damage upon genotoxic stress. Accordingly, primary BC cells, displaying reduced levels of Numb 1/2, exhibited increased chemoresistance, indicating that Numb splicing patterns could influence therapy response in BC patients. Moreover, we performed a large-scale RT-qPCR-based screening of a case-cohort of 890 breast tumors to uncover the impact of the different Numb isoforms on clinical outcome. We unmasked an increased risk of distance metastasis in patients expressing low levels of Numb 1/2, which was more evident in the p53-WT BCs, in accordance with our results on the role of Numb 1/2 in p53 regulation. A similar prognostic effect was also observed in BCs displaying low levels of total Numb mRNA, revealing that, alongside the well-characterized mechanism of Numb hyperdegradation, also pre-translational mechanisms contribute to Numb loss in BC. Our analysis also revealed worse prognosis in BC patients displaying low levels of Numb isoforms 1/3 and 2/4 (with or without the exon 9 coded-region, respectively), suggesting a more complex scenario, in which the differential role of Numb isoforms in pathways other than p53, such as the Notch pathway, might contribute to tumorigenesis. Together, our results indicate that the aberrant alternative splicing of Numb towards exon 3 skipping events (leading to a reduction in Numb 1/2 levels), could increase chemoresistance and contribute to tumor progression in p53-WT BCs. Moreover, additional layers of Numb deregulation, independent of hyperdegradation, could account for loss of Numb in BCs. These findings offer new strategies for stratifying BC patients according to Numb status, and possibly new therapeutic options to re-establish the Numb-p53 physiological circuitry in breast tumors.

RELEVANCE OF NUMB ISOFORMS IN BREAST CANCER / V. D'uva ; added supervisors: S. Pece, I. Colaluca ; supervisors: P. Di Fiore. - : . DIPARTIMENTO DI ONCOLOGIA ED EMATO-ONCOLOGIA, 2019 Jan 28. ((30. ciclo, Anno Accademico 2018. [10.13130/d-uva-veronica_phd2019-01-28].

RELEVANCE OF NUMB ISOFORMS IN BREAST CANCER

V. D'Uva
2019-01-28

Abstract

Numb was originally identified as a determinant of cell fate in Drosophila, and more recently as a tumor suppressor in different human cancers. Our lab, in particular, has contributed to the characterization of Numb as a tumor suppressor. Indeed, we showed that Numb protein expression is lost in ~30% of breast cancers (BCs) and associates with an adverse prognostic outcome. At the cellular level, Numb loss results in uncontrolled proliferation, impaired response to genotoxic treatments and emergence and amplification of the cancer stem cell pool, while at the molecular level, it leads to enhanced Notch oncogenic signaling and reduced expression of the tumor suppressor protein p53. Under physiological conditions, Numb acts to stabilize p53 protein by binding to and inhibiting the activity of Mdm2, the E3 ligase that mediates p53 ubiquitination and subsequent degradation. A detailed structural characterization of the Numb:Mdm2 binding interface, recently performed in our lab, has revealed that a short Numb region encoded by the alternatively spliced exon 3 was necessary and sufficient to bind to Mdm2 and prevent p53 degradation. This region is only present in two out of the four main Numb isoforms (Numb 1 and 2, hereafter referred to as Numb 1/2). Based on these findings, we hypothesized that the tumor suppressor role of Numb might be mediated specifically by isoforms 1 and 2, and that altered splicing could represent a novel mechanism responsible for the functional loss of Numb in tumors. To test these possibilities, we performed a series of experiments aimed at elucidating the specific roles of Numb isoforms in cancer and their contribution to tumorigenesis. In particular, we characterized Numb isoforms expression in BC cells and investigated the involvement of the expressed isoforms in the regulation of the p53 pathway. Our results point to the exclusive role of Numb 1/2 isoforms in the regulation of the Mdm2-p53 circuitry. Loss of Numb 1/2 in p53-WT BCs leads to reduced p53 levels and activity and an accumulation of DNA damage upon genotoxic stress. Accordingly, primary BC cells, displaying reduced levels of Numb 1/2, exhibited increased chemoresistance, indicating that Numb splicing patterns could influence therapy response in BC patients. Moreover, we performed a large-scale RT-qPCR-based screening of a case-cohort of 890 breast tumors to uncover the impact of the different Numb isoforms on clinical outcome. We unmasked an increased risk of distance metastasis in patients expressing low levels of Numb 1/2, which was more evident in the p53-WT BCs, in accordance with our results on the role of Numb 1/2 in p53 regulation. A similar prognostic effect was also observed in BCs displaying low levels of total Numb mRNA, revealing that, alongside the well-characterized mechanism of Numb hyperdegradation, also pre-translational mechanisms contribute to Numb loss in BC. Our analysis also revealed worse prognosis in BC patients displaying low levels of Numb isoforms 1/3 and 2/4 (with or without the exon 9 coded-region, respectively), suggesting a more complex scenario, in which the differential role of Numb isoforms in pathways other than p53, such as the Notch pathway, might contribute to tumorigenesis. Together, our results indicate that the aberrant alternative splicing of Numb towards exon 3 skipping events (leading to a reduction in Numb 1/2 levels), could increase chemoresistance and contribute to tumor progression in p53-WT BCs. Moreover, additional layers of Numb deregulation, independent of hyperdegradation, could account for loss of Numb in BCs. These findings offer new strategies for stratifying BC patients according to Numb status, and possibly new therapeutic options to re-establish the Numb-p53 physiological circuitry in breast tumors.
DI FIORE, PIER PAOLO
PECE, SALVATORE
Numb; Numb isoforms; p53; mdm2; chemoresistance; DNA damage response
Settore BIO/10 - Biochimica
RELEVANCE OF NUMB ISOFORMS IN BREAST CANCER / V. D'uva ; added supervisors: S. Pece, I. Colaluca ; supervisors: P. Di Fiore. - : . DIPARTIMENTO DI ONCOLOGIA ED EMATO-ONCOLOGIA, 2019 Jan 28. ((30. ciclo, Anno Accademico 2018. [10.13130/d-uva-veronica_phd2019-01-28].
Doctoral Thesis
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2434/609521
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