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.

The leucine zipper–like transcriptional regulator 1 (LZTR1) protein, an adaptor for cullin 3 (CUL3) ubiquitin ligase complex, is implicated in human disease, yet its mechanism of action remains unknown. We found that Lztr1 haploinsufficiency in mice recapitulates Noonan syndrome phenotypes, whereas LZTR1 loss in Schwann cells drives dedifferentiation and proliferation. By trapping LZTR1 complexes from intact mammalian cells, we identified the guanosine triphosphatase RAS as a substrate for the LZTR1-CUL3 complex. Ubiquitome analysis showed that loss of Lztr1 abrogated Ras ubiquitination at lysine-170. LZTR1-mediated ubiquitination inhibited RAS signaling by attenuating its association with the membrane. Disease-associated LZTR1 mutations disrupted either LZTR1-CUL3 complex formation or its interaction with RAS proteins. RAS regulation by LZTR1-mediated ubiquitination provides an explanation for the role of LZTR1 in human disease.

The comprehension of the basic biology of stem cells is expected to provide a useful insight into the pathogenesis of cancer. In particular, there is evidence that hypoxia promotes stem cell renewal in vitro as well as in vivo. It therefore seems reasonable that stem cell survival and hypoxia response are strictly connected at molecular level. We here report that the 66-kDa isoform of the SHC gene (p66Shc) is induced in a breast cancer cell line by the exposure to hypoxic environment and that it controls the expression of the stem cell regulatory gene Notch-3. Then, we show that p66Shc/Notch-3 interplay modulates self-renewal (by inducing the Notch-ligand Jagged-1) and hypoxia survival (by inducing the hypoxia-survival gene carbonic anhydrase IX) in mammary gland stem/progenitor cells, expanded in vitro as multicellular spheroids (mammospheres). We conclude that mechanisms that regulate stem cell renewal and hypoxia survival are integrated at the level of the p66Shc/Notch3 interplay. Because Notch-3, Jagged-1, and carbonic anhydrase IX are dysregulated in breast cancer, and because p66Shc is an aging-regulating gene, we envision that these data may help in understanding the relationship among aging, cancer, and stem cells. STEM CELLS 2007;25:807-815

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 (ALDH high ), which is endowed with higher self-renewal and tumorigenic abilities than the ALDH low population. A good correlation between the number of ALDH high 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 ALDH high melanoma stem cells. Finally, we show that interference with the HH-GLI pathway through lentiviralmediated silencing of SMO and GLI1 drastically diminishes tumor initiation of ALDH high 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.

Malignant melanoma is among the most aggressive cancers and its incidence is increasing worldwide. Targeted therapies and immunotherapy have improved the survival of patients with metastatic melanoma in the last few years; however, available treatments are still unsatisfactory. While the role of the BRAF-MEK1/2-ERK1/2 pathway in melanoma is well established, the involvement of mitogen-activated protein kinases MEK5-ERK5 remains poorly explored. Here we investigated the function of ERK5 signaling in melanoma. We show that ERK5 is consistently expressed in human melanoma tissues and is active in melanoma cells. Genetic silencing and pharmacological inhibition of ERK5 pathway drastically reduce the growth of melanoma cells and xenografts harboring wild-type (wt) or mutated BRAF (V600E). We also found that oncogenic BRAF positively regulates expression, phosphorylation, and nuclear localization of ERK5. Importantly, ERK5 kinase and transcriptional transactivator activities are enhanced by BRAF. Nevertheless, combined pharmacological inhibition of BRAFV600E and MEK5 is required to decrease nuclear ERK5, that is critical for the regulation of cell proliferation. Accordingly, combination of MEK5 or ERK5 inhibitors with BRAFV600E inhibitor vemurafenib is more effective than single treatments in reducing colony formation and growth of BRAFV600E melanoma cells and xenografts. Overall, these data support a key role of the ERK5 pathway for melanoma growth in vitro and in vivo and suggest that targeting ERK5, alone or in combination with BRAF-MEK1/2 inhibitors, might represent a novel approach for melanoma treatment.

The HEDGEHOG-GLI (HH-GLI) signalling is a key pathway critical in embryonic development, stem cell biology and tissue homeostasis. In recent years, aberrant activation of HH-GLI signalling has been linked to several types of cancer, including those of the skin, brain, lungs, prostate, gastrointestinal tract and blood. HH-GLI signalling is initiated by binding of HH ligands to the transmembrane receptor PATCHED and is mediated by transcriptional effectors that belong to the GLI family, whose activity is finely tuned by a number of molecular interactions and post-translation modifications. Several reports suggest that the activity of the GLI proteins is regulated by several proliferative and oncogenic inputs, in addition or independent of upstream HH signalling. The identification of this complex crosstalk and the understanding of how the major oncogenic signalling pathways interact in cancer is a crucial step towards the establishment of efficient targeted combinatorial treatments. Here we review recent findings on the cooperative integration of HH-GLI signalling with the major oncogenic inputs and we discuss how these cues modulate the activity of the GLI proteins in cancer. We then summarise the latest advances on SMO and GLI inhibitors and alternative approaches to attenuate HH signalling through rational combinatorial therapies.

One undisputed milestone of traditional oncology is neoplastic progression, which consists of a progressive selection of dedifferentiated cells driven by a chance sequence of genetic mutations. Recently it has been demonstrated that the overexpression of well-defined transcription factors reprograms somatic cells to the pluripotent stem status. The demonstration raises crucial questions as to whether and to what extent this reprogramming contributes to tumorigenesis, and whether the epigenetic changes involved in it are reversible. Here, we show for the first time that a tumor produced in vivo by a chemical carcinogen is the product of the interaction between neoplastic progression and reprogramming. The experimental model employed the prototype of ascites tumors, the Yoshida AH130 hepatoma and other neoplasias, including human melanoma. AH130 hepatoma was started in the liver by the carcinogen o-aminoazotoluene. This compound binds to and abolishes the p53 protein, producing a genomic instability that promotes both the neoplastic progression and the hepatoma reprogramming. Eventually this tumor contained 100% CD133(+) elements and pO(2)-dependent percentages of the three embryonic transcription factors Nanog, Klf4 and c-Myc. Once transferred into aerobic cultures, the minor cellular fraction expressing this triad generates various types of adherent cells, which are progressively substituted by non-tumorigenic elements committed to fibromuscular, neuronal and glial differentiation. This reprogramming appears to be accomplished stepwise, with the assembly of the triad into a sophisticated transcriptional, oxygen-dependent circuitry, in which Nanog and Klf4 antagonistically regulate c-Myc, and hence, cell hypoxia survival and cell cycle activation.

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.