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TrkA is amplified in malignant melanoma patients and induces an anti-proliferative response in cell lines.

ABSTRACT: The nerve growth factor (NGF) receptor tyrosine-kinase TrkA is a well-known determinant of the melanocytic lineage, through modulation of the MAPK and AKT cascades. While TrkA gene is frequently rearranged in cancers, its involvement in malignant melanoma (MM) development is still unclear.We analyzed a dataset of primary cutaneous MM (n = 31) by array comparative genomic hybridization (aCGH), to identify genomic amplifications associated with tumor progression. The analysis was validated by genomic quantitative PCR (qPCR) on an extended set of cases (n = 64) and the results were correlated with the clinical outcome. To investigate TrkA molecular pathways and cellular function, we generated inducible activation of the NGF-TrkA signaling in human MM cell lines.We identified amplification of 1q23.1, where the TrkA locus resides, as a candidate hotspot implicated in the progression of MM. Across 40 amplicons detected, segmental amplification of 1q23.1 showed the strongest association with tumor thickness. By validation of the analysis, TrkA gene amplification emerged as a frequent event in primary melanomas (50 % of patients), and correlated with worse clinical outcome. However, experiments in cell lines revealed that induction of the NGF-TrkA signaling produced a phenotype of dramatic suppression of cell proliferation through inhibition of cell division and pronounced intracellular vacuolization, in a way straightly dependent on NGF activation of TrkA. These events were triggered via MAPK activity but not via AKT, and involved p21(cip1) protein increase, compatibly with a mechanism of oncogene-induced growth arrest.Taken together, our findings point to TrkA as a candidate oncogene in MM and support a model in which the NGF-TrkA-MAPK pathway may mediate a trade-off between neoplastic transformation and adaptive anti-proliferative response.

SUBMITTER: Pasini L

PROVIDER: S-EPMC4619539 | biostudies-other | 2015

REPOSITORIES: biostudies-other

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TrkA is amplified in malignant melanoma patients and induces an anti-proliferative response in cell lines.

Pasini Luigi L Re Angela A Tebaldi Toma T Ricci Gianluca G Boi Sebastiana S Adami Valentina V Barbareschi Mattia M Quattrone Alessandro A

BMC cancer 20151024

<h4>Background</h4>The nerve growth factor (NGF) receptor tyrosine-kinase TrkA is a well-known determinant of the melanocytic lineage, through modulation of the MAPK and AKT cascades. While TrkA gene is frequently rearranged in cancers, its involvement in malignant melanoma (MM) development is still unclear.<h4>Methods</h4>We analyzed a dataset of primary cutaneous MM (n = 31) by array comparative genomic hybridization (aCGH), to identify genomic amplifications associated with tumor progression. ...[more]

PMID: 26496938

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Project description:BackgroundFarnesyltransferase inhibitors (FTIs) are anticancer agents with a spectrum of activity in Ras-dependent and independent tumor cellular and xenograph models. How inhibition of protein farnesylation by FTIs results in reduced cancer cell proliferation is poorly understood due to the multiplicity of potential FTase targets. The low toxicity and oral availability of FTIs led to their introduction into clinical trials for the treatment of breast cancer, hematopoietic malignancy, advanced solid tumor and pancreatic cancer treatment, and Hutchinson-Gilford Progeria Syndrome. Although their efficacy in combinatorial therapies with conventional anticancer treatment for myeloid malignancy and solid tumors is promising, the overall results of clinical tests are far below expectations. Further exploitation of FTIs in the clinic will strongly rely on understanding how these drugs affect global cellular activity.MethodsUsing FTase inhibitor I and genome-wide chemical profiling of the yeast barcoded deletion strain collection, we identified genes whose inactivation increases the antiproliferative action of this FTI peptidomimetic. The main findings were validated in a panel of cancer cell lines using FTI-277 in proliferation and biochemical assays paralleled by multiparametric image-based analyses.ResultsABC transporter Pdr10 or p-21 activated kinase (PAK) gene deletion increases the antiproliferative action of FTase inhibitor I in yeast cells. Consistent with this, enhanced inhibition of cell proliferation by combining group I PAK inhibition, using IPA3, with FTI-277 was observed in melanoma (A375MM), lung (A549) and colon (HT29), but not in epithelial (HeLa) or breast (MCF7), cancer cell lines. Both HeLa and A375MM cells show changes in the nuclear localization of group 1 PAKs in response to FTI-277, but up-regulation of PAK protein levels is observed only in HeLa cells.ConclusionsOur data support the view that group I PAKs are part of a pro-survival pathway activated by FTI treatment, and group I PAK inactivation potentiates the anti-proliferative action of FTIs in yeast as well as in cancer cells. These findings open new perspectives for the use of FTIs in combinatorial strategies with PAK inhibitors in melanoma, lung and colon malignancy.

Project description:Fibroblast Growth Factor Receptors (FGFR1-4) have a critical role in the progression of several human cancers, including Squamous Non-Small-Cell Lung Cancer (SQCLC). Both non-selective and selective reversible FGFR inhibitors are under clinical investigation for the treatment of patients with tumors harboring FGFR alterations. Despite their potential efficacy, the clinical development of these drugs has encountered several challenges, including toxicity, and the appearance of drug resistance. Recent efforts have been directed at development of irreversible FGFR inhibitors, which have the potential to exert superior anti-proliferative activity in tumors carrying FGFR alterations. With this in mind, we synthetized, and investigated a set of novel inhibitors possessing a warhead potentially able to covalently bind a cysteine in the P-loop of FGFR. Among them, the chloroacetamide UPR1376 resulted able to irreversible inhibit FGFR1 phosphorylation in FGFR1 over-expressing cells generated from SQCLC SKMES-1 cells. In addition, this compound inhibited cell proliferation in FGFR1-amplified H1581 cells with a potency higher than the reversible inhibitor BGJ398 (infigratinib), while sparing FGFR1 low-expressing cells. The anti-proliferative effects of UPR1376 were demonstrated in both 2D and 3D systems and were associated with the inhibition of MAPK and AKT/mTOR signaling pathways. UPR1376 inhibited cell proliferation also in two BGJ398-resistant cell clones generated from H1581 by chronic exposure to BGJ398, although at concentrations higher than those effective in the parental cells, likely due to the persistent activation of the MAPK pathway associated to NRAS amplification. Combined blockade of FGFR1 and MAPK signaling, by UPR1376 and trametinib respectively, significantly enhanced the efficacy of UPR1376, providing a means of circumventing resistance to FGFR1 inhibition. Our findings suggest that the insertion of a chloroacetamide warhead on a suitable scaffold, as exemplified by UPR1376, is a valuable strategy to develop a novel generation of FGFR inhibitors for the treatment of SQCLC patients with FGFR alterations.

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Project description:Malignant melanomas (MMs) are the abnormal proliferation of melanocytes and are one of the lethal skin cancers in humans, equines, and canines. Accordingly, MMs in companion animals can serve as naturally occurring animal models, completing conventional cancer models. The common constitutive activation of the MAPK and PI3K pathways in MMs has been described in all three species. Targeting the related pathways is considered a potential option in comparative oncologic approaches. Herein, we present a cross-species comparative analysis exposing a set of ten melanoma cell lines (one human, three equine, and six canine) derived from primary tumors or metastasis to a pan-RAF and RAF dimer inhibitor (LY3009120). Cellular response (proliferation, biomass, metabolism, early and late apoptosis/necrosis, and morphology) and the presence of pathogenic single-nucleotide variants (SNVs) within the mutational hotspot genes BRAF exon 11 and 15, NRAS exon 2 and 3, KRAS exon 2, and KIT exon 11 were analyzed. This study showed that equine malignant melanoma (EMM) cells (MelDuWi) harbor the KRAS p.Q61H mutation, while canine malignant melanoma (CMM) cells (cRGO1 and cRGO1.2) carry NRAS p.G13R. Except for EMM metastasis cells eRGO6 (wild type of the above-mentioned hotspot genes), all melanoma cell lines exhibited a decrease in dose dependence after 48 and 72 h of exposure to LY3009120, independent of the mutation hotspot landscape. Furthermore, LY3009120 caused significant early apoptosis and late apoptosis/necrosis in all melanoma cell lines except for eRGO6. The anti-tumor effects of LY3009120 were observed in nine melanoma cell lines, indicating the potential feasibility of experimental trials with LY3009120. The present study reveals that the irradiation-resistant canine metastasis cells (cRGO1.2) harboring the NRAS p.G13R mutation are significantly LY3009120-sensitive, while the equine metastases-derived eRGO6 cells show significant resistance to LY3009120, which make them both valuable tools for studying resistance mechanisms in comparative oncology.

Dataset Information

TrkA is amplified in malignant melanoma patients and induces an anti-proliferative response in cell lines.

Publications

TrkA is amplified in malignant melanoma patients and induces an anti-proliferative response in cell lines.

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