Barbara Bedogni

VHL loss in renal cell carcinoma leads to up-regulation of CUB domain-containing protein 1 to stimulate PKCδ-driven migration

A common genetic mutation found in clear cell renal cell carcinoma (CC-RCC) is the loss of the von Hippel-Lindau (VHL) gene, which results in stabilization of hypoxia-inducible factors (HIFs), and contributes to cancer progression and metastasis. CUB-domain-containing protein 1 (CDCP1) was shown to promote metastasis in scirrhous and lung adenocarcinomas as well as in prostate cancer. In this study, we established a molecular mechanism linking VHL loss to induction of the CDCP1 gene through the HIF-1/2 pathway in renal cancer. Also, we report that Fyn, which forms a complex with CDCP1 and mediates its signaling to PKCδ, is a HIF-1 target gene. Mechanistically, we found that CDCP1 specifically regulates phosphorylation of PKCδ, but not of focal adhesion kinase or Crk-associated substrate. Signal transduction from CDCP1 to PKCδ leads to its activation, increasing migration of CC-RCC. Furthermore, patient survival can be stratified by CDCP1 expression at the cell surface of the tumor. Taken together, our data indicates that CDCP1 protein might serve as a therapeutic target for CC-RCC.

A Notch1-neuregulin1 autocrine signaling loop contributes to melanoma growth.

The Notch pathway is an evolutionary conserved signaling cascade that has an essential role in melanoblast and melanocyte stem cell homeostasis. Notch signaling is emerging as a key player in melanoma, the most deadly form of skin cancer. In melanoma, Notch1 is inappropriately reactivated and contributes to melanoma tumorigenicity. Here, we propose a novel mechanism by which Notch1 promotes the disease. We found that Notch1 directly regulates the transcription of neuregulin1 (NRG1) by binding to its promoter region. NRG1 is the ligand for ERBB3 and 4, members of the epidermal growth factor family of receptors that are involved in the genesis and progression of a number of cancers. Notch1 and NRG1 expression are associated in melanoma and inhibition of NRG1 signaling leads to melanoma cell growth inhibition and tumor growth delay. Mechanistically, these effects are associated with the inhibition of the PI3Kinase/Akt signaling pathway and with the accumulation of p27Kip1. On the other end, addition of recombinant NRG1 can partially restore melanoma cell growth that is inhibited by Notch1 ablation. Taken together, our findings underline a new, previously undescribed autocrine signaling loop between Notch1 and NRG1 that controls melanoma growth and provide experimental evidence that the targeting of Notch and ERBB signaling may represent a novel potential therapeutic approach in melanoma.

MT1-MMP modulates melanoma cell dissemination and metastasis through activation of MMP2 and RAC1.

Metastatic melanoma remains the deadliest of all skin cancers with a survival rate at five years of less than 15%. MT1‐MMP is a membrane‐associated matrix metalloproteinase that controls pericellular proteolysis and is an important, invasion‐promoting, pro‐tumorigenic MMP in cancer. We show that deregulation of MT1‐MMP expression happens as early as the transition from nevus to primary melanoma and continues to increase during melanoma progression. Furthermore, MT1‐MMP expression is associated with poor melanoma patient outcome, underscoring a pivotal role of MT1‐MMP in melanoma pathogenesis. We demonstrate that MT1‐MMP is directly required for melanoma cells to metastasize, as cells deprived of MT1‐MMP fail to form distant metastasis in an orthotopic mouse melanoma model. We show that MT1‐MMP affects cell invasion by activating its target MMP2. Importantly, we demonstrate, for the first time, that activation of MMP2 by MT1‐MMP is required to sustain RAC1 activity and promote MT1‐MMP‐dependent cell motility. These data highlight a novel MT1‐MMP/MMP2/RAC1 signaling axis in melanoma that may represent an intriguing molecular target for the treatment of invasive melanoma.

The Apoptosis Repressor with a CARD Domain (ARC) Gene Is a Direct Hypoxia-Inducible Factor 1 Target Gene and Promotes Survival and Proliferation of VHL-Deficient Renal Cancer Cells

ABSTRACT The induction of hypoxia-inducible factors (HIFs) is essential for the adaptation of tumor cells to a low-oxygen environment. We found that the expression of the apoptosis inhibitor ARC (apoptosis repressor with a CARD domain) was induced by hypoxia in a variety of cancer cell types, and its induction is primarily HIF1 dependent. Chromatin immunoprecipitation (ChIP) and reporter assays also indicate that the ARC gene is regulated by direct binding of HIF1 to a hypoxia response element (HRE) located at bp −190 upstream of the transcription start site. HIFs play an essential role in the pathogenesis of renal cell carcinoma (RCC) under normoxic conditions, through the loss of the Von Hippel-Lindau gene (VHL). Accordingly, our results show that ARC is not expressed in normal renal tissue but is highly expressed in 65% of RCC tumors, which also express high levels of carbonic anhydrase IX (CAIX), a HIF1-dependent protein. Compared to controls, ARC-deficient RCCs exhibited decreased colony formation and increased apoptosis in vitro. In addition, loss of ARC resulted in a dramatic reduction of RCC tumor growth in SCID mice in vivo. Thus, HIF-mediated increased expression of ARC in RCC can explain how loss of VHL can promote survival early in tumor formation.

The membrane tethered matrix metalloproteinase MT1-MMP at the forefront of melanoma cell invasion and metastasis

The Extracellular Matrix (ECM) plays an important role in normal physiological development and functioning of cells, tissues and organs [1]. Under normal physiological conditions degradation of the ECM is a finely regulated process, and altered homeostasis of ECM degradation (excessive or insufficient) is associated with many diseases [2-5] such as cancer, fibrosis, arthritis, nephritis, encephalomyelitis and chronic ulcers. The remodeling of the ECM is carried out by a family of enzymes known as matrix metalloproteinases (MMP). MMPs constitute a large group of multidomain, zinc dependent endopeptidases capable of hydrolyzing all protein components of the ECM [6]. Additional functions of MMPs have also been identified. MMPs, and in particular MT1-MMP, the prototypic membrane-tethered matrix metalloproteinase, are no longer only ECM remodeling enzymes but rather regulators of several cellular functions including growth, migration, invasion and gene expression. Here we will focus on the role of the membrane bound MT1-MMP in melanoma growth, invasion and metastasis. MT1-MMP has in fact emerged as a multifaceted protease capable of influencing melanoma metastasis by canonical means, i.e. ECM degradation, but also via regulation of genes involved in several pro-tumorigenic functions including tumor cell growth and motility.

An ERBB3/ERBB2 oncogenic unit plays a key role in NRG1 signaling and melanoma cell growth and survival

We recently identified neuregulin‐1 (NRG1) as a novel target of Notch1 required in Notch‐dependent melanoma growth. ERBB3 and ERBB4, tyrosine kinase receptors specifically activated by NRG1, have been shown to be either elevated in melanoma cell lines and tumors or to be mutated in 20% of melanomas, respectively. While these data support key roles of NRG1 and its receptors in the pathogenesis of melanoma, whether ERBB3 and ERBB4 display redundant or exclusive functions is not known. Here, we show that ERBB3 and ERBB4 inhibition results in distinct outcomes. ERBB3 inhibition ablates the cellular responses to NRG1, results in AKT inactivation and leads to cell growth arrest and apoptotic cell death. In contrast, ERBB4 knockdown mildly affects cell growth, has no effects on cell survival and, importantly, does not alter the responses to NRG1. Finally, we identified ERBB2 as a key coreceptor in NRG1‐dependent ERBB3 signaling. ERBB2 forms a complex with ERBB3, and its inhibition recapitulates the phenotypes observed upon ERBB3 ablation. We propose that an NRG1‐ERBB3‐ERBB2 signaling unit operates in melanoma cells where it promotes growth and survival.

Noncanonical Activation of Notch1 Protein by Membrane Type 1 Matrix Metalloproteinase (MT1-MMP) Controls Melanoma Cell Proliferation

Background: Notch1 is highly activated in melanoma where it plays protumorigenic functions, yet no Notch1-activating mutations have been identified in melanoma. Results: MT1-MMP operates as a novel protease that promotes Notch1 activation in melanoma cells. Conclusion: An MT1-MMP/Notch1 signaling pathway supports melanoma cell growth. Significance: Notch1 emerges as a new MT1-MMP substrate that plays important biological roles in melanoma. Notch1 is an evolutionarily conserved signaling molecule required for stem cell maintenance that is inappropriately reactivated in several cancers. We have previously shown that melanomas reactivate Notch1 and require its function for growth and survival. However, no Notch1-activating mutations have been observed in melanoma, suggesting the involvement of other activating mechanisms. Notch1 activation requires two cleavage steps: first by a protease and then by γ-secretase, which releases the active intracellular domain (Notch1NIC). Interestingly, although ADAM10 and -17 are generally accepted as the proteases responsible of Notch1 cleavage, here we show that MT1-MMP, a membrane-tethered matrix metalloproteinase involved in the pathogenesis of a number of tumors, is a novel protease required for the cleavage of Notch1 in melanoma cells. We find that active Notch1 and MT1-MMP expression correlate significantly in over 70% of melanoma tumors and 80% of melanoma cell lines, whereas such correlation does not exist between Notch1NIC and ADAM10 or -17. Modulation of MT1-MMP expression in melanoma cells affects Notch1 cleavage, whereas MT1-MMP expression in ADAM10/17 double knock-out fibroblasts restores the processing of Notch1, indicating that MT1-MMP is sufficient to promote Notch1 activation independently of the canonical proteases. Importantly, we find that MT1-MMP interacts with Notch1 at the cell membrane, supporting a potential direct cleavage mechanism of MT1-MMP on Notch1, and that MT1-MMP-dependent activation of Notch1 sustains melanoma cell growth. Together, the data highlight a novel mechanism of activation of Notch1 in melanoma cells and identify Notch1 as a new MT1-MMP substrate that plays important biological roles in melanoma.

Notch1 Autoactivation via Transcriptional Regulation of Furin, Which Sustains Notch1 Signaling by Processing Notch1-Activating Proteases ADAM10 and Membrane Type 1 Matrix Metalloproteinase.

ABSTRACT Notch1 is an evolutionarily conserved transmembrane receptor involved in melanoma growth. Notch1 is first cleaved by furin in the Golgi apparatus to produce the biologically active heterodimer. Following ligand binding, Notch1 is cleaved at the cell membrane by proteases such as ADAM10 and -17 and membrane type 1 matrix metalloproteinase (MT1-MMP), the latter of which we recently identified as a novel protease involved in Notch1 processing. The final cleavage is γ-secretase dependent and releases the active Notch intracellular domain (NIC). We now demonstrate that Notch1 directly regulates furin expression. Aside from activating Notch1, furin cleaves and activates several proteases, including MT1-MMP, ADAM10, and ADAM17. By chromatin immunoprecipitation and a reporter assay, we demonstrate that Notch1 binds at position −1236 of the furin promoter and drives furin expression. The Notch1-dependent enhancement of furin expression increases the activities of MT1-MMP and ADAM10 but not that of ADAM17, as demonstrated by short hairpin RNA (shRNA) knockdown of furin, and promotes the cleavage of Notch1 itself. These data highlight a novel positive-feedback loop whereby Notch1-dependent furin expression can induce Notch1 signaling by increasing Notch1 processing and by potentiating the activity of the proteases responsible for Notch1 activation. This leads to Notch1 signal amplification, which can promote melanoma tumor growth and progression, as demonstrated by the inhibition of cell migration and invasion upon furin inhibition downstream of Notch1. Disruption of such feedback signaling might represent an avenue for the treatment of melanoma.

Synchronized Targeting of Notch and ERBB Signaling Suppresses Melanoma Tumor Growth through Inhibition of Notch1 and ERBB3.

Despite significant advances in melanoma therapy, melanoma remains the deadliest form of skin cancer, with a five-year survival of only 15%. Novel treatments are therefore required to address this disease. Notch and ERBB are evolutionarily conserved signaling cascades required for the maintenance of melanocyte precursors. We show that active Notch1 (Notch1NIC) and active (phosphorylated) ERBB3 and ERBB2 correlate significantly and are similarly expressed in both mutated and wild type BRAF melanomas, suggesting these receptors are co-reactivated in melanoma to promote survival. Indeed, while blocking either pathway triggers modest effects, combining a γ-secretase inhibitor to block Notch activation, and a tyrosine kinase inhibitor to inhibit ERBB3/2 elicits synergistic effects, reducing cell viability by 90% and by hampering melanoma tumor growth. Specific inhibition of Notch1 and ERBB3 mimics these results, suggesting these are the critical factors triggering melanoma tumor expansion. Notch and ERBB inhibition blunts AKT and NFκB signaling; Constitutive expression of NFκB partially rescues cell death. Finally, blockade of both Notch and ERBB signaling inhibits the slow cycling JARID1B positive cell population, which is critical for long-term maintenance of melanoma growth. We propose that blocking these pathways is an effective approach to treat melanoma patients regardless of whether they carry mutated or wild type BRAF.

The thiirane-based selective MT1-MMP/MMP2 inhibitor ND-322 reduces melanoma tumor growth and delays metastatic dissemination

MT1-MMP and MMP2 have been implicated as pro-tumorigenic and pro-metastatic factors in a wide variety of cancers including melanoma. We have previously demonstrated that MT1-MMP is highly expressed in melanoma where it promotes melanoma cell invasion and metastasis in part through the activation of its target MMP2. Given the accessibility of MMPs, as they are either secreted (e.g. MMP2) or membrane-tethered (e.g. MT1-MMP), they represent ideal targets for specific inhibition via small molecules. Here we show that the novel small-molecule inhibitor ND-322 with high selectivity for MT1-MMP and MMP2, effectively inhibits MT1-MMP and MMP2 activity resulting in reduced in vitro melanoma cell growth, migration and invasion. Importantly, these inhibitory effects lead to significant reduction of melanoma tumor growth and metastasis. We further show that while cell migration and invasion could be similarly hampered by specific inhibition of either MT1-MMP or MMP2 via shRNAs, the growth inhibitory activity of ND-322 could only be mirrored by specific inhibition of MT1-MMP. These data support ND-322 as a novel effective inhibitor capable of counteracting both MT1-MMP and MMP2, two key proteases involved in melanoma growth and metastasis. ND-322 may therefore represent a new inhibitor in the repertoire of treatments against melanoma.