Sm Annunziata Hospital Regional Melanoma Referral Center

Firenze, Italy

Sm Annunziata Hospital Regional Melanoma Referral Center

Firenze, Italy

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Santini R.,Core Research Laboratory Instituto Toscano Tumori CRL ITT | Vinci M.C.,Core Research Laboratory Instituto Toscano Tumori CRL ITT | Pandolfi S.,Core Research Laboratory Instituto Toscano Tumori CRL ITT | Penachioni J.Y.,Core Research Laboratory Instituto Toscano Tumori CRL ITT | And 7 more authors.
Stem Cells | Year: 2012

The question of whether cancer stem/tumor-initiating cells (CSC/TIC) exist in human melanomas has arisen in the last few years. Here, we have used nonadherent spheres and the aldehyde dehydrogenase (ALDH) enzymatic activity to enrich for CSC/TIC in a collection of human melanomas obtained from a broad spectrum of sites and stages. We find that melanomaspheres display extensive in vitro self-renewal ability and sustain tumor growth in vivo, generating human melanoma xenografts that recapitulate the phenotypic composition of the parental tumor. Melanomaspheres express high levels of Hedgehog (HH) pathway components and of embryonic pluripotent stem cell factors SOX2, NANOG, OCT4, and KLF4. We show that human melanomas contain a subset of cells expressing high ALDH activity (ALDHhigh), which is endowed with higher self-renewal and tumorigenic abilities than the ALDHlow population. A good correlation between the number of ALDHhigh cells and sphere formation efficiency was observed. Notably, both pharmacological inhibition of HH signaling by the SMOOTHENED (SMO) antagonist cyclopamine and GLI antagonist GANT61 and stable expression of shRNA targeting either SMO or GLI1 result in a significant decrease in melanoma stem cell self-renewal in vitro and a reduction in the number of ALDHhigh melanoma stem cells. Finally, we show that interference with the HH-GLI pathway through lentiviral-mediated silencing of SMO and GLI1 drastically diminishes tumor initiation of ALDHhigh melanoma stem cells. In conclusion, our data indicate an essential role of the HH-GLI1 signaling in controlling self-renewal and tumor initiation of melanoma CSC/TIC. Targeting HH-GLI1 is thus predicted to reduce the melanoma stem cell compartment. © AlphaMed Press.


Pandolfi S.,Core Research Laboratory Instituto Toscano Tumori CRL ITT | Montagnani V.,Core Research Laboratory Instituto Toscano Tumori CRL ITT | Penachioni J.Y.,Core Research Laboratory Instituto Toscano Tumori CRL ITT | Vinci M.C.,Core Research Laboratory Instituto Toscano Tumori CRL ITT | And 3 more authors.
Oncogene | Year: 2013

The Hedgehog-GLI (HH-GLI) signaling plays a critical role in controlling growth and tissue patterning during embryogenesis and is implicated in a variety of human malignancies, including those of the skin. Phosphorylation events have been shown to regulate the activity of the GLI transcription factors, the final effectors of the HH-GLI signaling pathway. Here, we show that WIP1 (or PPM1D), an oncogenic phosphatase amplified/overexpressed in several types of human cancer, is a positive modulator of the HH signaling. Mechanistically, WIP1 enhances the function of GLI1 by increasing its transcriptional activity, nuclear localization and protein stability, but not of GLI2 nor GLI3. We also find that WIP1 and GLI1 are in a complex. Modulation of the transcriptional activity of GLI1 by WIP1 depends on the latter's phosphatase activity and, remarkably, does not require p53, a known WIP1 target. Functionally, we find that WIP1 is required for melanoma and breast cancer cell proliferation and self-renewal in vitro and melanoma xenograft growth induced by activation of the HH signaling. Pharmacological blockade of the HH pathway with the SMOOTHENED antagonist cyclopamine acts synergistically with inhibition of WIP1 in reducing growth of melanoma and breast cancer cells in vitro. Overall, our data uncover a role for WIP1 in modulating the activity of GLI1 and in sustaining cancer cell growth and cancer stem cell self-renewal induced by activation of the HH pathway. These findings open a novel therapeutic approach for human melanomas and, possibly, other cancer types expressing WIP1 and with activated HH pathway. © 2013 Macmillan Publishers Limited All rights reserved 0950-9232/13.


Santini R.,Core Research Laboratory Instituto Toscano Tumori CRL ITT | Pietrobono S.,Core Research Laboratory Instituto Toscano Tumori CRL ITT | Pandolfi S.,Core Research Laboratory Instituto Toscano Tumori CRL ITT | Montagnani V.,Core Research Laboratory Instituto Toscano Tumori CRL ITT | And 5 more authors.
Oncogene | Year: 2014

Melanoma is one of the most aggressive types of human cancer, characterized by enhanced heterogeneity and resistance to conventional therapy at advanced stages. We and others have previously shown that HEDGEHOG-GLI (HH-GLI) signaling is required for melanoma growth and for survival and expansion of melanoma-initiating cells (MICs). Recent reports indicate that HH-GLI signaling regulates a set of genes typically expressed in embryonic stem cells, including SOX2 (sex-determining region Y (SRY)-Box2). Here we address the function of SOX2 in human melanomas and MICs and its interaction with HH-GLI signaling. We find that SOX2 is highly expressed in melanoma stem cells. Knockdown of SOX2 sharply decreases self-renewal in melanoma spheres and in putative melanoma stem cells with high aldehyde dehydrogenase activity (ALDH(high)). Conversely, ectopic expression of SOX2 in melanoma cells enhances their self-renewal in vitro. SOX2 silencing also inhibits cell growth and induces apoptosis in melanoma cells. In addition, depletion of SOX2 progressively abrogates tumor growth and leads to a significant decrease in tumor-initiating capability of ALDH(high) MICs upon xenotransplantation, suggesting that SOX2 is required for tumor initiation and for continuous tumor growth. We show that SOX2 is regulated by HH signaling and that the transcription factors GLI1 and GLI2, the downstream effectors of HH-GLI signaling, bind to the proximal promoter region of SOX2 in primary melanoma cells. In functional studies, we find that SOX2 function is required for HH-induced melanoma cell growth and MIC self-renewal in vitro. Thus SOX2 is a critical factor for self-renewal and tumorigenicity of MICs and an important mediator of HH-GLI signaling in melanoma. These findings could provide the basis for novel therapeutic strategies based on the inhibition of SOX2 for the treatment of a subset of human melanomas.


Santini R.,Core Research Laboratory Instituto Toscano Tumori CRL ITT | Pietrobono S.,Core Research Laboratory Instituto Toscano Tumori CRL ITT | Pandolfi S.,Core Research Laboratory Instituto Toscano Tumori CRL ITT | Montagnani V.,Core Research Laboratory Instituto Toscano Tumori CRL ITT | And 5 more authors.
Oncogene | Year: 2014

Melanoma is one of the most aggressive types of human cancer, characterized by enhanced heterogeneity and resistance to conventional therapy at advanced stages. We and others have previously shown that HEDGEHOG-GLI (HH-GLI) signaling is required for melanoma growth and for survival and expansion of melanoma-initiating cells (MICs). Recent reports indicate that HH-GLI signaling regulates a set of genes typically expressed in embryonic stem cells, including SOX2 (sex-determining region Y (SRY)-Box2). Here we address the function of SOX2 in human melanomas and MICs and its interaction with HH-GLI signaling. We find that SOX2 is highly expressed in melanoma stem cells. Knockdown of SOX2 sharply decreases self-renewal in melanoma spheres and in putative melanoma stem cells with high aldehyde dehydrogenase activity (ALDHhigh). Conversely, ectopic expression of SOX2 in melanoma cells enhances their self-renewal in vitro. SOX2 silencing also inhibits cell growth and induces apoptosis in melanoma cells. In addition, depletion of SOX2 progressively abrogates tumor growth and leads to a significant decrease in tumor-initiating capability of ALDHhigh MICs upon xenotransplantation, suggesting that SOX2 is required for tumor initiation and for continuous tumor growth. We show that SOX2 is regulated by HH signaling and that the transcription factors GLI1 and GLI2, the downstream effectors of HH-GLI signaling, bind to the proximal promoter region of SOX2 in primary melanoma cells. In functional studies, we find that SOX2 function is required for HH-induced melanoma cell growth and MIC self-renewal in vitro. Thus SOX2 is a critical factor for self-renewal and tumorigenicity of MICs and an important mediator of HH-GLI signaling in melanoma. These findings could provide the basis for novel therapeutic strategies based on the inhibition of SOX2 for the treatment of a subset of human melanomas.Oncogene advance online publication, 31 March 2014; doi:10.1038/onc.2014.71.

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