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Fibroblastic reticular cells (FRC) are immunologically specialized myofibroblasts that control the elasticity of the lymph node, in part through their contractile properties. Swelling of tumor-draining lymph nodes is a hallmark of lymphophilic cancers such as cutaneous melanoma. Melanoma displays high intratumoral heterogeneity with the coexistence of melanoma cells with variable differentiation phenotypes from melanocytic to dedifferentiated states. Factors secreted by melanoma cells promote premetastatic lymph node reprograming and tumor spreading. Elucidating the impact of the melanoma secretome on FRC could help identify approaches to prevent metastasis. Here we show that melanocytic and dedifferentiated melanoma cells differentially impact the FRC contractile phenotype. Factors secreted by dedifferentiated cells, but not by melanocytic cells, strongly inhibited actomyosin-dependent contractile forces of FRC by decreasing the activity of the RHOA-RHO-kinase (ROCK) pathway and the mechano-responsive transcriptional coactivator Yes1 associated transcriptional regulator (YAP). Transcriptional profiling and biochemical analyses indicated that actomyosin cytoskeleton relaxation in FRC is driven by inhibition of the JAK1-STAT3 pathway. This FRC relaxation was associated with increased FRC proliferation and activation and with elevated tumor invasion in vitro. The secretome of dedifferentiated melanoma cells also modulated the biomechanical properties of distant lymph node in premetastatic mouse models. Finally, IL1 produced by dedifferentiated cells was involved in the inhibition of FRC contractility. These data highlight the role of the JAK1-STAT3 and YAP pathways in spontaneous contractility of resting FRC. They also suggest that dedifferentiated melanoma cells specifically target FRC biomechanical properties to favor tumor spreading in the premetastatic lymph node niche. Targeting this remote communication could be an effective strategy to prevent metastatic spread of the disease., Significance: Communication between dedifferentiated melanoma cells and lymph node fibroblasts reprograms the biomechanical properties of the premetastatic lymph node niche to promote tumor invasion. See related commentary by Lund, p. 1692., (©2022 American Association for Cancer Research.)

Resistance to BRAF/MEK inhibitor therapy in BRAF V600 -mutated advanced melanoma remains a major obstacle that limits patient benefit. Microenvironment components including the extracellular matrix (ECM) can support tumor cell adaptation and tolerance to targeted therapy; however, the underlying mechanisms remain poorly understood. Here, we investigated the process of matrix-mediated drug resistance (MMDR) in response to BRAF V600 pathway inhibition in melanoma. We demonstrate that physical and structural cues from fibroblast-derived ECM abrogate anti-proliferative responses to BRAF/MEK inhibition. MMDR is mediated by drug-induced linear clustering of phosphorylated DDR1 and DDR2, two tyrosine kinase collagen receptors. Depletion and pharmacological targeting of DDR1 and DDR2 overcome ECM-mediated resistance to BRAF-targeted therapy. In xenografts, targeting DDR with imatinib enhances BRAF inhibitor efficacy, counteracts drug-induced collagen remodeling, and delays tumor relapse. Mechanistically, DDR-dependent MMDR fosters a targetable pro-survival NIK/IKKα/NF-κB2 pathway. These findings reveal a novel role for a collagen-rich matrix and DDR in tumor cell adaptation and resistance. They also provide important insights into environment-mediated drug resistance and a preclinical rationale for targeting DDR signaling in combination with targeted therapy in melanoma., (© 2021 The Authors. Published under the terms of the CC BY 4.0 license.)

Aberrant extracellular matrix (ECM) deposition and stiffening is a physical hallmark of several solid cancers and is associated with therapy failure. BRAF-mutant melanomas treated with BRAF and MEK inhibitors almost invariably develop resistance that is frequently associated with transcriptional reprogramming and a de-differentiated cell state. Melanoma cells secrete their own ECM proteins, an event that is promoted by oncogenic BRAF inhibition. Yet, the contribution of cancer cell-derived ECM and tumor mechanics to drug adaptation and therapy resistance remains poorly understood. Here, we show that melanoma cells can adapt to targeted therapies through a mechanosignaling loop involving the autocrine remodeling of a drug-protective ECM. Analyses revealed that therapy-resistant cells associated with a mesenchymal dedifferentiated state displayed elevated responsiveness to collagen stiffening and force-mediated ECM remodeling through activation of actin-dependent mechanosensors Yes-associated protein (YAP) and myocardin-related transcription factor (MRTF). Short-term inhibition of MAPK pathway also induced mechanosignaling associated with deposition and remodeling of an aligned fibrillar matrix. This provided a favored ECM reorganization that promoted tolerance to BRAF inhibition in a YAP- and MRTF-dependent manner. Matrix remodeling and tumor stiffening were also observed in vivo upon exposure of BRAF-mutant melanoma cell lines or patient-derived xenograft models to MAPK pathway inhibition. Importantly, pharmacologic targeting of YAP reversed treatment-induced excessive collagen deposition, leading to enhancement of BRAF inhibitor efficacy. We conclude that MAPK pathway targeting therapies mechanically reprogram melanoma cells to confer a drug-protective matrix environment. Preventing melanoma cell mechanical reprogramming might be a promising therapeutic strategy for patients on targeted therapies. SIGNIFICANCE: These findings reveal a biomechanical adaptation of melanoma cells to oncogenic BRAF pathway inhibition, which fuels a YAP/MRTF-dependent feed-forward loop associated with tumor stiffening, mechanosensing, and therapy resistance. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/10/1927/F1.large.jpg., (©2020 American Association for Cancer Research.)

Rounded-amoeboid cancer cells use actomyosin contractility driven by Rho-ROCK and JAK-STAT3 to migrate efficiently. It has been suggested that rounded-amoeboid cancer cells do not require matrix metalloproteinases (MMPs) to invade. Here we compare MMP levels in rounded-amoeboid and elongated-mesenchymal melanoma cells. Surprisingly, we find that rounded-amoeboid melanoma cells secrete higher levels of several MMPs, including collagenase MMP-13 and gelatinase MMP-9. As a result, rounded-amoeboid melanoma cells degrade collagen I more efficiently than elongated-mesenchymal cells. Furthermore, using a non-catalytic mechanism, MMP-9 promotes rounded-amoeboid 3D migration through regulation of actomyosin contractility via CD44 receptor. MMP-9 is upregulated in a panel of rounded-amoeboid compared with elongated-mesenchymal melanoma cell lines and its levels are controlled by ROCK-JAK-STAT3 signalling. MMP-9 expression increases during melanoma progression and it is particularly prominent in the invasive fronts of lesions, correlating with cell roundness. Therefore, rounded-amoeboid cells use both catalytic and non-catalytic activities of MMPs for invasion.

Signaling crosstalk between tumor cells and fibroblasts confers proinvasive properties to the tumor microenvironment. Here, we identify leukemia inhibitory factor (LIF) as a tumor promoter that mediates proinvasive activation of stromal fibroblasts independent of alpha-smooth muscle actin (α-SMA) expression. We demonstrate that a pulse of transforming growth factor β (TGF-β) establishes stable proinvasive fibroblast activation by inducing LIF production in both fibroblasts and tumor cells. In fibroblasts, LIF mediates TGF-β-dependent actomyosin contractility and extracellular matrix remodeling, which results in collective carcinoma cell invasion in vitro and in vivo. Accordingly, carcinomas from multiple origins and melanomas display strong LIF upregulation, which correlates with dense collagen fiber organization, cancer cell collective invasion, and poor clinical outcome. Blockade of JAK activity by Ruxolitinib (JAK inhibitor) counteracts fibroblast-dependent carcinoma cell invasion in vitro and in vivo. These findings establish LIF as a proinvasive fibroblast producer independent of α-SMA and may open novel therapeutic perspectives for patients with aggressive primary tumors., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

The acquisition of invasive behaviour is the key transition in the progression of benign melanocyte hyperplasia to life threatening melanoma. Understanding this transition and the mechanisms of invasion are the key to understanding why malignant melanoma is such a devastating disease and will aid treatment strategies. Underlying the invasive behaviour is increased cell motility caused by changes in cytoskeletal organization and altered contacts with the extra-cellular matrix (ECM). In addition, changes in the interactions of melanoma cells with keratinocytes and fibroblasts enable them to survive and proliferate outside their normal epidermal location. Proteomic and genomic initiatives are greatly increasing our knowledge of which gene products are deregulated in invasive and metastatic melanoma; however, the next challenge is to understand how these genes promote the invasion of melanoma cells. In recent years new models have been developed that more closely recapitulate the conditions of melanoma invasion in vivo. It is hoped that these models will give us a better understanding of how the genes implicated in melanoma progression affect the motility of melanoma cells and their interactions with the ECM, stromal cells and blood vessels. This review will summarise our current understanding of melanoma invasion and focus on the new model systems that can be used to study melanoma.

Melanomas are malignant tumors of melanocytes that, if not detected early, are highly aggressive and poorly treatable. Activation of extracellular signal-regulated (ERK)/mitogen-activated protein (MAP) kinase signaling is commonly found in melanomas mainly through oncogenic mutations of B-Raf. We previously reported that activation of ERK/MAP kinase stimulates synthesis of fibronectin by upregulating the transcription factor early growth response-1 (Egr-1). To further analyze the link between ERK/MAP kinase pathway and fibronectin in melanoma, we have studied the regulation and role of fibronectin produced by melanoma cells bearing oncogenic B-Raf mutation. We show that fibronectin is expressed in situ during tumor progression and that high fibronectin and Egr-1 levels are found in cells expressing this mutation. Expression of active mutants of B-Raf induces fibronectin, whereas endogenous fibronectin is inhibited by small interfering RNA (siRNA)-mediated depletion of B-Raf or Egr-1. In contrast, stimulation of ERK pathway is insufficient to promote fibronectin upregulation in normal melanocytes. Finally, we show that suppression of fibronectin by siRNA leads to decreased melanoma cell invasiveness in vitro. These results reveal a tumor-specific regulation of fibronectin by constitutive ERK/MAP kinase signaling and indicate that self-production of fibronectin may play a role in melanoma tumorigenesis, by promoting tumor cell invasion.

During progression of melanoma, loss of the cell-cell adhesion molecule E-cadherin contributes to uncontrolled growth and invasive behavior of transformed melanocytes. Secreted protein acidic and rich in cysteine (SPARC) is a nonstructural matricellular protein that regulates cell-matrix interactions leading to alterations in cell adhesion and proliferation. Overexpression of SPARC has been associated with progression of various cancers, including melanoma; however, its role in primary tumor development is not well defined. We show that normal human melanocytes overexpressing SPARC adopt a fibroblast-like morphology, concomitant with loss of E-cadherin and P-cadherin expression, and increased expression of mesenchymal markers. Concurrent with these changes, SPARC expression stimulates melanocyte motility and melanoma cell invasion. Expression of SPARC results in transcriptional down-regulation of E-cadherin that correlates with induction of Snail, a repressor of E-cadherin. Conversely, SPARC depletion leads to up-regulation of E-cadherin and reduces Snail levels, and SPARC-null cells exhibit a marked change in their mesenchymal phenotype. Finally, analysis of SPARC, Snail, and E-cadherin levels in melanocytes and malignant melanoma cell lines further supports the functional relationship among these proteins during melanoma progression. Our findings provide evidence for the role of SPARC in early transformation of melanocytes and identify a novel mechanism, whereby tumor-derived SPARC promotes tumorigenesis by mediating Snail induction and E-cadherin suppression.

Microphthalmia-associated transcription factor (MITF) M-form is a melanocyte-specific transcription factor that plays a key role in melanocyte development, survival, and differentiation. Here, we identified MITF as a new substrate of caspases and we characterized the cleavage site after Asp 345 in the C-terminal domain. We show that expression of a noncleavable form of MITF renders melanoma cells resistant to apoptotic stimuli, and we found that the C-terminal fragment generated upon caspase cleavage is endowed with a proapoptotic activity that sensitizes melanoma cells to death signals. The proapoptotic function gained by MITF following its processing by caspases provides a tissue-restricted means to modulate death in melanocyte and melanoma cells.

In melanocytes and melanoma cells alpha-melanocyte stimulating hormone (alpha-MSH), via the cAMP pathway, elicits a large array of biological responses that control melanocyte differentiation and influence melanoma development or susceptibility. In this work, we show that cAMP transcriptionally activates Hif1a gene in a melanocyte cell-specific manner and increases the expression of a functional hypoxia-inducible factor 1alpha (HIF1alpha) protein resulting in a stimulation of Vegf expression. Interestingly, we report that the melanocyte-specific transcription factor, microphthalmia-associated transcription factor (MITF), binds to the Hif1a promoter and strongly stimulates its transcriptional activity. Further, MITF "silencing" abrogates the cAMP effect on Hif1a expression, and overexpression of MITF in human melanoma cells is sufficient to stimulate HIF1A mRNA. Our data demonstrate that Hif1a is a new MITF target gene and that MITF mediates the cAMP stimulation of Hif1a in melanocytes and melanoma cells. Importantly, we provide results demonstrating that HIF1 plays a pro-survival role in this cell system. We therefore conclude that the alpha-MSH/cAMP pathway, using MITF as a signal transducer and HIF1alpha as a target, might contribute to melanoma progression.

The matrix fibronectin protein is a multifunctional adhesive molecule that promotes migration and invasiveness of many tumors including melanomas. Increased fibronectin synthesis has been associated with the metastatic potential of melanoma cells; however, the molecular mechanisms underlying fibronectin overexpression during melanoma development are poorly understood. We report that hepatocyte growth factor/scatter factor (HGF) induces fibronectin expression and its extracellular assembly on the surface of melanoma cells through activation of mitogen-activated protein (MAP) kinase pathway, and induction and transcriptional activation of Early growth response-1 (Egr-1). Inhibition of B-RAF/MAP kinase pathway by dominant-negative mutants and by U0126-abrogated HGF-induced Egr-1, and chromatin immunoprecipitation showed that Egr-1 is bound to the fibronectin promoter in response to HGF. Exogenously expressed Egr-1 increased fibronectin levels, while blockage of Egr-1 activation by expression of the Egr-1 corepressor NAB2 interfered with the upregulation of fibronectin synthesis induced by HGF, indicating that Egr-1 exerts a significant role in fibronectin expression in response to HGF. Finally, analysis of the expression pattern of fibronectin in melanoma cells demonstrated that fibronectin levels are correlated with constitutive MAP kinase signaling. Our data define a novel mechanism that might have important implications in regulation of melanoma progression by autocrine HGF signaling or by constitutive activation of MAP kinase pathway.

The Hepatocyte growth factor/scatter factor (HGF/SF), a fibroblast-derived cytokine that elicits mitogenic, motogenic and morphogenic responses in epithelial cells, is a mitogen for human melanocytes and has recently been implicated as a critical factor for melanoma progression and metastasis. In a search for transcription factors induced by HGF/SF in melanoma cells, we analyzed by multi-well plate real-time PCR analysis immediate early gene activation in mouse B16 melanoma cells. Here we investigate the expression of the zinc finger transcription factor Egr-1 (early growth-response factor-1) by HGF/SF in melanocytes and melanoma cells. Egr-1 has a role in many cellular processes through the transcription regulation of target genes. We observe that HGF rapidly and transiently induces Egr-1 expression in B16 melanoma cells. Likewise, Egr-1 expression is increased in several human melanoma cells treated with HGF. However, in contrast to melanoma cells, Egr-1 expression is not induced in normal human melanocytes treated by HGF/SF. Using Egr-1 promoter-luciferase constructs, we localized elements responsible for HGF/SF responsiveness to a region containing five juxtaposed SREs (Serum responsive elements) and a cluster of Ets motifs. In addition, HGF stimulates Elk-1 activation, a factor that binds and regulates SRE-dependent transcription. This suggests that HGF stimulate transcription of Egr-1 primarily through activation and binding of Elk-1 at multiple sites on the promoter. Consistent with this, inhibition of MEK1/2 by the UO126 and expression of dominant negative mutants of the Ras/RAF pathway markedly inhibited Egr-1 expression induced by HGF/SF. We conclude that Egr-1 is a target of HGF/SF action through the Ras/RAF/MEK/ERK-mediated pathway in melanoma cells. We propose that Egr-1 might be an important part of the HGF/SF-initiated changes in gene transcription associated with tumor progression. We are currently investigating the function of Egr-1 in HGF/SF-induced signalling pathway in melanoma cells. The identification of genes regulated by Egr-1 will help us to better define the role of HGF/SF through Egr-1 in melanoma progression. [ABSTRACT FROM AUTHOR]