The Hedgehog-GLI (HH-GLI) pathway is a highly conserved signaling that plays a critical role in controlling cell specification, cell-cell interaction and tissue patterning during embryonic ...development. Canonical activation of HH-GLI signaling occurs through binding of HH ligands to the twelve-pass transmembrane receptor Patched 1 (PTCH1), which derepresses the seven-pass transmembrane G protein-coupled receptor Smoothened (SMO). Thus, active SMO initiates a complex intracellular cascade that leads to the activation of the three GLI transcription factors, the final effectors of the HH-GLI pathway. Aberrant activation of this signaling has been implicated in a wide variety of tumors, such as those of the brain, skin, breast, gastrointestinal, lung, pancreas, prostate and ovary. In several of these cases, activation of HH-GLI signaling is mediated by overproduction of HH ligands (e.g., prostate cancer), loss-of-function mutations in
or gain-of-function mutations in
, which occur in the majority of basal cell carcinoma (BCC), SHH-subtype medulloblastoma and rhabdomyosarcoma. Besides the classical canonical ligand-PTCH1-SMO route, mounting evidence points toward additional, non-canonical ways of GLI activation in cancer. By non-canonical we refer to all those mechanisms of activation of the GLI transcription factors occurring independently of SMO. Often, in a given cancer type canonical and non-canonical activation of HH-GLI signaling co-exist, and in some cancer types, more than one mechanism of non-canonical activation may occur. Tumors harboring non-canonical HH-GLI signaling are less sensitive to SMO inhibition, posing a threat for therapeutic efficacy of these antagonists. Here we will review the most recent findings on the involvement of alternative signaling pathways in inducing GLI activity in cancer and stem cells. We will also discuss the rationale of targeting these oncogenic pathways in combination with HH-GLI inhibitors as a promising anti-cancer therapies.
Despite the development of new targeted and immune therapies, the prognosis of metastatic melanoma remains bleak. Therefore, it is critical to better understand the mechanisms controlling advanced ...melanoma to develop more effective treatment regimens. Hedgehog/GLI (HH/GLI) signaling inhibitors targeting the central pathway transducer Smoothened (SMO) have shown to be clinical efficacious in skin cancer; however, several mechanisms of non-canonical HH/GLI pathway activation limit their efficacy. Here, we identify a novel SOX2-BRD4 transcriptional complex driving the expression of GLI1, the final effector of the HH/GLI pathway, providing a novel mechanism of non-canonical SMO-independent activation of HH/GLI signaling in melanoma. Consistently, we find a positive correlation between the expression of GLI1 and SOX2 in human melanoma samples and cell lines. Further, we show that combined targeting of canonical HH/GLI pathway with the SMO inhibitor MRT-92 and of the SOX2-BRD4 complex using a potent Proteolysis Targeted Chimeras (PROTACs)-derived BRD4 degrader (MZ1), yields a synergistic anti-proliferative effect in melanoma cells independently of their BRAF, NRAS, and NF1 mutational status, with complete abrogation of GLI1 expression. Combination of MRT-92 and MZ1 strongly potentiates the antitumor effect of either drug as single agents in an orthotopic melanoma model. Together, our data provide evidence of a novel mechanism of non-canonical activation of GLI1 by the SOX2-BRD4 transcriptional complex, and describe the efficacy of a new combinatorial treatment for a subset of melanomas with an active SOX2-BRD4-GLI1 axis.
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.
Malignant melanoma is the deadliest skin cancer, with a poor prognosis in advanced stages. We recently showed that the extracellular signal-regulated kinase 5 (ERK5), encoded by the
gene, plays a ...pivotal role in melanoma by regulating cell functions necessary for tumour development, such as proliferation. Hedgehog-GLI signalling is constitutively active in melanoma and is required for proliferation. However, no data are available in literature about a possible interplay between Hedgehog-GLI and ERK5 pathways. Here, we show that hyperactivation of the Hedgehog-GLI pathway by genetic inhibition of the negative regulator Patched 1 increases the amount of ERK5 mRNA and protein. Chromatin immunoprecipitation showed that GLI1, the major downstream effector of Hedgehog-GLI signalling, binds to a functional non-canonical GLI consensus sequence at the
promoter. Furthermore, we found that ERK5 is required for Hedgehog-GLI-dependent melanoma cell proliferation, and that the combination of GLI and ERK5 inhibitors is more effective than single treatments in reducing cell viability and colony formation ability in melanoma cells. Together, these findings led to the identification of a novel Hedgehog-GLI-ERK5 axis that regulates melanoma cell growth, and shed light on new functions of ERK5, paving the way for new therapeutic options in melanoma and other neoplasms with active Hedgehog-GLI and ERK5 pathways.
Among the mechanisms of mitochondrial quality control (MQC), generation of mitochondria-derived vesicles (MDVs) is a process to avoid complete failure of mitochondria determining lysosomal ...degradation of mitochondrial damaged proteins. In this context, RAB7, a late endocytic small GTPase, controls delivery of MDVs to late endosomes for subsequent lysosomal degradation. We previously demonstrated that RAB7 has a pivotal role in response to cisplatin (CDDP) regulating resistance to the drug by extracellular vesicle (EVs) secretion.
Western blot and immunofluorescence analysis were used to analyze structure and function of endosomes and lysosomes in CDDP chemosensitive and chemoresistant ovarian cancer cell lines. EVs were purified from chemosensitive and chemoresistant cells by ultracentrifugation or immunoisolation to analyze their mitochondrial DNA and protein content. Treatment with cyanide m-chlorophenylhydrazone (CCCP) and RAB7 modulation were used, respectively, to understand the role of mitochondrial and late endosomal/lysosomal alterations on MDV secretion. Using conditioned media from chemoresistant cells the effect of MDVs on the viability after CDDP treatment was determined. Seahorse assays and immunofluorescence analysis were used to study the biochemical role of MDVs and the uptake and intracellular localization of MDVs, respectively.
We observed that CDDP-chemoresistant cells are characterized by increased MDV secretion, impairment of late endocytic traffic, RAB7 downregulation, an increase of RAB7 in EVs, compared to chemosensitive cells, and downregulation of the TFEB-mTOR pathway overseeing lysosomal and mitochondrial biogenesis and turnover. We established that MDVs can be secreted rather than delivered to lysosomes and are able to deliver CDDP outside the cells. We showed increased secretion of MDVs by chemoresistant cells ultimately caused by the extrusion of RAB7 in EVs, resulting in a dramatic drop in its intracellular content, as a novel mechanism to regulate RAB7 levels. We demonstrated that MDVs purified from chemoresistant cells induce chemoresistance in RAB7-modulated process, and, after uptake from recipient cells, MDVs localize to mitochondria and slow down mitochondrial activity.
Dysfunctional MQC in chemoresistant cells determines a block in lysosomal degradation of MDVs and their consequent secretion, suggesting that MQC is not able to eliminate damaged mitochondria whose components are secreted becoming effectors and potential markers of chemoresistance.
Melanoma is the deadliest skin cancer with a very poor prognosis in advanced stages. Although targeted and immune therapies have improved survival, not all patients benefit from these treatments. The ...mitogen-activated protein kinase ERK5 supports the growth of melanoma cells
and
. However, ERK5 inhibition results in cell-cycle arrest rather than appreciable apoptosis. To clarify the role of ERK5 in melanoma growth, we performed transcriptomic analyses following ERK5 knockdown in melanoma cells expressing BRAFV600E and found that cellular senescence was among the most affected processes. In melanoma cells expressing either wild-type or mutant (V600E) BRAF, both genetic and pharmacologic inhibition of ERK5 elicited cellular senescence, as observed by a marked increase in senescence-associated β-galactosidase activity and p21 expression. In addition, depletion of ERK5 from melanoma cells resulted in increased levels of CXCL1, CXCL8, and CCL20, proteins typically involved in the senescence-associated secretory phenotype. Knockdown of p21 suppressed the induction of cellular senescence by ERK5 blockade, pointing to p21 as a key mediator of this process.
, ERK5 knockdown or inhibition with XMD8-92 in melanoma xenografts promoted cellular senescence. Based on these results, small-molecule compounds targeting ERK5 constitute a rational series of prosenescence drugs that may be exploited for melanoma treatment. SIGNIFICANCE: This study shows that targeting ERK5 induces p21-mediated cellular senescence in melanoma, identifying a prosenescence effect of ERK5 inhibitors that may be exploited for melanoma treatment.
Human melanomas contain a population of tumor-initiating cells that are able to maintain the growth of the tumor. We previously showed that the embryonic transcription factor SOX2 is essential for ...self-renewal and tumorigenicity of human melanoma-initiating cells. However, targeting a transcription factor is still challenging. Gentian violet (GV) is a cationic triphenylmethane dye with potent antifungal and antibacterial activity. Recently, a combination therapy of imiquimod and GV has shown an inhibitory effect against melanoma metastases. Whether and how GV affects melanoma cells remains unknown. Here we show that GV represses melanoma stem cell self-renewal through inhibition of SOX2. Mechanistically, GV hinders EGFR activation and inhibits the signal transducer and activator of transcription-3 (STAT3)/SOX2 axis. Importantly, we show that GV treatment decreases STAT3 phosphorylation at residue tyrosine 705, thus preventing the translocation of STAT3 into the nucleus and its binding to SOX2 promoter. In addition, GV affects melanoma cell growth by promoting mitochondrial apoptosis and G2 cell cycle arrest. This study shows that in melanoma, GV affects both the stem cell and the tumor bulk compartments, suggesting the potential use of GV in treating human melanoma alone or in combination with targeted therapy and/or immunotherapy.
Aberrant activation of the Hedgehog (HH) signaling is a critical driver in tumorigenesis. The Smoothened (SMO) receptor is one of the major upstream transducers of the HH pathway and a target for the ...development of anticancer agents. The SMO inhibitor Vismodegib (GDC-0449/Erivedge) has been approved for treatment of basal cell carcinoma. However, the emergence of resistance during Vismodegib treatment and the occurrence of numerous side effects limit its use. Our group has recently discovered and developed novel and potent SMO inhibitors based on acylguanidine or acylthiourea scaffolds. Here, we show that the two acylguanidine analogs, compound (1) and its novel fluoride derivative (2), strongly reduce growth and self-renewal of melanoma cells, inhibiting the level of the HH signaling target GLI1 in a dose-dependent manner. Both compounds induce apoptosis and DNA damage through the ATR/CHK1 axis. Mechanistically, they prevent G2 to M cell cycle transition, and induce signs of mitotic aberrations ultimately leading to mitotic catastrophe. In a melanoma xenograft mouse model, systemic treatment with 1 produced a remarkable inhibition of tumor growth without body weight loss in mice. Our data highlight a novel route for cell death induction by SMO inhibitors and support their use in therapeutic approaches for melanoma and, possibly, other types of cancer with active HH signaling.
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