Microenvironmental influences in melanoma progression

An often overlooked facet of tumor biology research is the involvement of the surrounding tumor microenvironment. Increasing evidence is being presented to support a major role for stromal components in all stages of tumorigenesis including initiation, progression, and metastasis. Melanoma serves as a model for studying cellular and stromal interactions within the tumor microenvironment due to the array of cell types localized to these lesions. Here, we discuss the both the molecular mechanisms, as well as the extracellular and contextual input that contribute to melanoma progression. Special emphasis is given to the assorted cell types and their interactions with the extracellular matrix and adjacent cells. Melanoma progression also initiates development of intralesional hypoxic regions; the relative significance of hypoxia in disease is also addressed. Lastly, a number of laboratories are currently developing innovative strategies to study melanoma within a microenvironmental platform. These promising model systems and their potential for closing current gaps in knowledge of disease are reviewed. The development of such models holds translational value that cannot be achieved with most current systems.     

IL-8 expression in malignant melanoma: implications in growth and metastasis

This review article has described briefly studies supporting the concept that IL-8 expression and its regulation by inflammatory cytokines like IL-1 may play an important role in controlling the phenotypes associated with melanoma progression and metastasis. It is clear from the experiments presented here that IL-8 is an important autocrine multifunctional cytokine that modulates melanoma/cell proliferation, migration by induction of extracellular matrix degradation enzymes and induces neovascularization, all of which are critical for melanoma growth and metastasis. In addition, their expression in melanoma tumor specimens suggests an association between IL-8 expression and tumor aggressiveness. Further, inflammatory cytokines produced by either tumor cells or stromal cells may regulate IL-8 expression, which can control melanoma growth and enhance our current knowledge regarding melanoma progression and metastasis. Understanding these events and their significance will allow us to design novel therapeutic approaches for treatment of melanoma.     

The disintegrin-like and cysteine-rich domains of ADAM-9 mediate interactions between melanoma cells and fibroblasts

A characteristic of malignant cells is their capacity to invade their surrounding and to metastasize to distant organs. During these processes, proteolytic activities of tumor and stromal cells modify the extracellular matrix to produce a microenvironment suitable for their growth and migration. In recent years the family of ADAM proteases has been ascribed important roles in these processes. ADAM-9 is expressed in human melanoma at the tumor-stroma border where direct or indirect interactions between tumor cells and fibroblasts occur. To analyze the role of ADAM-9 for the interaction between melanoma cells and stromal fibroblasts, we produced the recombinant disintegrin-like and cysteine-rich domain of ADAM-9 (DC-9). Melanoma cells and human fibroblasts adhered to immobilized DC-9 in a Mn(2+)-dependent fashion suggesting an integrin-mediated process. Inhibition studies showed that adhesion of fibroblasts was mediated by several β1 integrin receptors independent of the RGD and ECD recognition motif. Furthermore, interaction of fibroblasts and high invasive melanoma cells with soluble recombinant DC-9 resulted in enhanced expression of MMP-1 and MMP-2. Silencing of ADAM-9 in melanoma cells significantly reduced cell adhesion to fibroblasts. Ablation of ADAM-9 in fibroblasts almost completely abolished these cellular interactions and melanoma cell invasion in vitro. In summary, these results suggest that ADAM-9 expression plays an important role in mediating cell-cell contacts between fibroblasts and melanoma cells and that these interactions contribute to proteolytic activities required during invasion of melanoma cells.     

Why target the tumor stroma in melanoma?

Melanoma metastasis is fatal. Melanoma cells are often characterized by an activated extracellular signal–regulated kinase (ERK) pathway downstream of mutations in BRAF. Therapies targeting these BRAF mutations are useful for a while; however, patients ultimately develop resistance to these therapies. Recent evidence suggests that this resistance occurs when tumor cells leave their microenvironment and migrate on a stiff, activated tumor stroma; that is, this resistance is linked to the presence of an extracellular matrix reminiscent of a fibrotic micronvironment. These data suggest that agents targeting fibrosis might be used to treat melanoma. We therefore discuss what is known about the tumor stroma in melanoma. An emergent target, CCN2 (CTGF), that is required for fibrosis, may also be a good target for drug-resistant melanoma. Intriguingly, anti-CCN2 antibodies are currently under clinical development.     

Multiple roles of integrins in cell motility

Motility is essential for many important biological events, including embryonic development, inflammatory responses, wound healing, and tumor metastasis. During these events cells are in dynamic contact with the extracellular matrix through integrins. Integrins are the primary receptors for extracellular matrix proteins and consequently are required for cell motility. Cells have evolved multiple mechanisms to modulate integrin adhesive functions, which impact cell migration. In addition to providing a mechanism that allows cells to contact the extracellular matrix, integrins also promote intracellular signals that stimulate and regulate cell movement. Here we discuss the role of integrins during the multiple steps of cell migration.     

Cryptic collagen elements as signaling hubs in the regulation of tumor growth and metastasis

Structural remodeling of the extracellular matrix is a well-established process associated with tumor growth and metastasis. Tumor and stromal cells that compose the tumor mass function cooperatively to promote the malignant phenotype in part by physically interacting with intact and structurally altered matrix proteins. To this end, collagen represents the most abundant component of the extracellular matrix and is known to control the behavior of histologically distinct tumor types as well as a diversity of stromal cells. Although a significant molecular understanding has been established concerning how cellular interactions with intact collagen govern signaling pathways that control tumor progression, considerably less is known concerning how interactions with cryptic or hidden regions within remodeled collagen may selectively alter signaling cascades, or whether inhibition of these cryptic signaling pathways may represent clinically effective therapeutic strategies. Here, we review the emerging evidence concerning the possible mechanisms for the selective generation of cryptic or hidden elements within collagen and their potential cell surface receptors that may facilitate signal transduction. We discuss the concept that cellular communication links between cell surface receptors and these cryptic collagen elements may serve as functional signaling hubs that coordinate multiple signaling pathways operating within both tumor and stromal cells. Finally, we provide examples to help illustrate the possibility that direct targeting of these unique cryptic signaling hubs may lead to the development of more effective therapeutic strategies to control tumor growth and metastasis.     

Antagonistic effects of laminin and fibronectin in cell-to-cell and cell-to-matrix interactions in MCF-7 cultures

Summary During morphogenesis, tumor progression and metastasis, cell adhesion, dissociation, and migration result from a complex balance between cell-to-cell and cell-to-matrix interactions. Two different organization patterns of MCF-7 cells were induced by different extracellular matrix proteins. When plated on plastic or polymeric type I collagen gel used as a model of interstitial matrix, MCF-7 cells spread and grew in monolayer. When cultured on a solid gel of basement membrane (BM) proteins (85% laminin) used as a model of BM, cells formed clusters attached to the matrix. Matrix proteins regulated these two types of cell organization by preferentially promoting cell-to-cell or cell-support interactions. On plastic in the presence of soluble laminin or on laminin-coated dishes, cells also formed clusters. Addition of soluble fibronectin induced spreading of the cells, suggesting that laminin and fibronectin have competitive antagonistic effects on MCF-7 cell morphology. Antilaminin antibodies inhibited cluster formation and attachment, emphasizing the important role of this glycoprotein not only in promoting cluster attachment but also in cell-to-cell contact formation. Such effects of extracellular matrix proteins could play significant roles in tumor progression and metastasis. This work was supported by grants 3.4512.85 and 3.4514.85 from the Belgian Fonds de la Recherche Scientifique Médicale and the Fonds Cancérologique de la CGER.     

GPR56 and TG2: Possible Roles in Suppression of Tumor Growth by the Microenvironment

Metastasis is a complex process that involves multiple levels of cell-cell interaction. Among these interactions, tumor-stroma interactions are being actively investigated. Metastatic cells are hypothesized to show gene expression changes that contribute to their survival and growth at the distant site. Such chances could contribute either to enhancement of growth or to evasion of growth inhibition by the normal tissue environment thus allowing growth as metastases. Our recent report that tumors from highly metastatic melanoma derivatives express low levels of a suppressor of tumor progression, GPR56, is consistent with such a model. GPR56 associates in a complex with Gαq and the tetraspanin CD81. We further identified a ligand that interacts with GPR56 in the extracellular matrix (ECM) as TG2, a major crosslinking enzyme in the matrix. TG2 also binds to fibronectin and integrins and affects their cell adhesion functions. TG2 itself has been implicated in suppression of tumor progression; therefore TG2 might serve as a host defense against the invading metastatic cells. The highly metastatic cells may escape from this inhibition by down-regulation of GPR56. Much future work will be needed to test this hypothesis and further our understanding of metastasis in general.     

Liposomes modified with a synthetic Arg-Gly-Asp mimetic inhibit lung metastasis of B16BL6 melanoma cells

Administration of large amounts of synthetic peptides based on the Arg-Gly-Asp (RGD) sequence has been shown to suppress tumor metastasis. To overcome the rapid degradation of peptides in the circulation, an RGD mimetic, L-arginyl-6-aminohexanoic acid (NOK), was synthesized and conjugated with phosphatidylethanolamine (PE) (NOK-PE) for liposomalization. Cell adhesion assays revealed that B16BL6 murine melanoma cells adhered to immobilized NOK-PE. This adhesion was inhibited by addition of either soluble RGDS or NOK at similar concentration in a dose-dependent manner. Administration of NOK-PE liposomes (equivalent to ca. 500 microg RGD peptides) via the tail vein completely inhibited lung colonization of B 16BL6 cells. The same dose of soluble NOK was not effective in inhibition of the tumor metastasis. In addition, injection of NOK-PE liposomes via the tail vein inhibited spontaneous lung metastasis of B16BL6 cells from the primary tumor site in the hind footpad. These results suggest that NOK, a structural mimetic of RGD, is capable of suppressing metastasis by blockade of the binding of the integrins present on tumor cells to the RGD-containing extracellular matrix.     

Tamoxifen inhibits tumor cell invasion and metastasis in mouse melanoma through suppression of PKC/MEK/ERK and PKC/PI3K/Akt pathways

In melanoma, several signaling pathways are constitutively activated. Among these, the protein kinase C (PKC) signaling pathways are activated through multiple signal transduction molecules and appear to play major roles in melanoma progression. Recently, it has been reported that tamoxifen, an anti-estrogen reagent, inhibits PKC signaling in estrogen-negative and estrogen-independent cancer cell lines. Thus, we investigated whether tamoxifen inhibited tumor cell invasion and metastasis in mouse melanoma cell line B16BL6. Tamoxifen significantly inhibited lung metastasis, cell migration, and invasion at concentrations that did not show anti-proliferative effects on B16BL6 cells. Tamoxifen also inhibited the mRNA expressions and protein activities of matrix metalloproteinases (MMPs). Furthermore, tamoxifen suppressed phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt through the inhibition of PKCα and PKCδ phosphorylation. However, other signal transduction factor, such as p38 mitogen-activated protein kinase (p38MAPK) was unaffected. The results indicate that tamoxifen suppresses the PKC/mitogen-activated protein kinase kinase (MEK)/ERK and PKC/phosphatidylinositol-3 kinase (PI3K)/Akt pathways, thereby inhibiting B16BL6 cell migration, invasion, and metastasis. Moreover, tamoxifen markedly inhibited not only developing but also clinically evident metastasis. These findings suggest that tamoxifen has potential clinical applications for the treatment of tumor cell metastasis.     

Serum osteopontin, an enhancer of tumor metastasis to bone, promotes B16 melanoma cell migration

Tumor malignancy is associated with several features such as proliferation ability and frequency of metastasis. Since tumor metastasis shortens patients' lifetime, establishment of therapy for anti-metastasis is very important. Osteopontin (OPN), which abundantly expressed in bone matrix, is involved in cell adhesion, migration, extracellular matrix (ECM) invasion and cell proliferation via interaction with its receptor, that is, alphavbeta3 integrin. OPN is believed to be a positive regulator of tumor metastasis in vivo. However, how OPN regulates metastasis is largely unknown. Here, we explore the role of OPN in cell migration. Serum from wild-type mice induced cell migration of B16 melanoma cells, while serum from OPN-deficient mouse suppressed this event. The presence of recombinant OPN significantly enhanced cell migration compared to albumin containing medium. OPN-induced cell migration was suppressed by inhibiting the ERK/MAPK pathway indicating that OPN-induced cell migration depends on this pathway. Overexpression of OPN in these cancer cells per se promoted cell proliferation and tended to increase B16 cell migration suggesting that OPN promotes bone metastasis by playing dual roles both in host microenvironment and in tumor cell itself. In conclusion, the elevated OPN expression in host tissue and tumor cell itself promotes tumor cell migration reading to tumor metastasis, suggesting that neutralization of OPN-induced signal might be effective in suppression of tumor metastasis.     

Impaired desensitization of a human polymorphic α2B-adrenergic receptor variant enhances its sympatho-inhibitory activity in chromaffin cells

Recent advances in tumor biology have revealed that a detailed analysis of the complex interactions of tumor cells with their adjacent microenvironment (tumor stroma) is mandatory in order to understand the various mechanisms involved in tumor growth and the development of metastasis. The mutual interactions between tumor cells and cellular and non-cellular components (extracellular matrix = ECM) of the tumor microenvironment will eventually lead to a loss of tissue homeostasis and promote tumor development and progression. Thus, interactions of genetically altered tumor cells and the ECM on the one hand and reactive non-neoplastic cells on the other hand essentially control most aspects of tumorigenesis such as epithelial-mesenchymal-transition (EMT), migration, invasion (i.e. migration through connective tissue), metastasis formation, neovascularisation, apoptosis and chemotherapeutic drug resistance. In this mini-review we will focus on these issues that were recently raised by two review articles in CCS.     

Regulation of invadopodia by the tumor microenvironment

The tumor microenvironment consists of stromal cells, extracellular matrix (ECM), and signaling molecules that communicate with cancer cells. As tumors grow and develop, the tumor microenvironment changes. In addition, the tumor microenvironment is not only influenced by signals from tumor cells, but also stromal components contribute to tumor progression and metastasis by affecting cancer cell function. One of the mechanisms that cancer cells use to invade and metastasize is mediated by actin-rich, proteolytic structures called invadopodia. Here, we discuss how signals from the tumor environment, including growth factors, hypoxia, pH, metabolism, and stromal cell interactions, affect the formation and function of invadopodia to regulate cancer cell invasion and metastasis. Understanding how the tumor microenvironment affects invadopodia biology could aid in the development of effective therapeutics to target cancer cell invasion and metastasis.     

Regulation of invadopodia by the tumor microenvironment

The tumor microenvironment consists of stromal cells, extracellular matrix (ECM), and signaling molecules that communicate with cancer cells. As tumors grow and develop, the tumor microenvironment changes. In addition, the tumor microenvironment is not only influenced by signals from tumor cells, but also stromal components contribute to tumor progression and metastasis by affecting cancer cell function. One of the mechanisms that cancer cells use to invade and metastasize is mediated by actin-rich, proteolytic structures called invadopodia. Here, we discuss how signals from the tumor environment, including growth factors, hypoxia, pH, metabolism, and stromal cell interactions, affect the formation and function of invadopodia to regulate cancer cell invasion and metastasis. Understanding how the tumor microenvironment affects invadopodia biology could aid in the development of effective therapeutics to target cancer cell invasion and metastasis.     

Inhibition of murine melanoma cell-matrix adhesion and experimental metastasis by albolabrin, an RGD-containing peptide isolated from the venom of Trimeresurus albolabris

Albolabrin, a 7.5-kDa cysteine-rich protein isolated from the venom of Trimeresurus albolabris, contains the arginine-glycine-aspartic acid (RGD) cell recognition sequence found in many cell adhesion-promoting extracellular matrix proteins, such as fibronectin and laminin. Albolabrin belongs to a family of RGD-containing peptides, termed disintegrins, recently isolated from the venom of various vipers and discovered to be potent inhibitors of both platelet aggregation and cell-substratum adhesion. Here we report that albolabrin inhibited the attachment of B16-F10 mouse melanoma cells to either fibronectin or laminin absorbed on plastic. When immobilized on plastic, albolabrin promoted B16-F10 melanoma cell attachment; this was inhibited by either RGD-serine (RGDS) or antibodies to integrins, suggesting that albolabrin binds via its RGD amino sequence to integrin receptors expressed on the melanoma cell surface. In an in vivo experimental metastasis system, albolabrin at a concentration of 300-600 nM inhibited C57BL/6 mouse lung colonization by tail vein-injected mouse melanoma cells and was at least 2000 times more active than RGDS in this assay. We propose that albolabrin inhibits tumor cell metastasis by inhibiting integrin-mediated attachment of melanoma cells to RGD-containing components of the extracellular matrix in the mouse lung.     

Emergent behaviors from a cellular automaton model for invasive tumor growth in heterogeneous microenvironments

Understanding tumor invasion and metastasis is of crucial importance for both fundamental cancer research and clinical practice. In vitro experiments have established that the invasive growth of malignant tumors is characterized by the dendritic invasive branches composed of chains of tumor cells emanating from the primary tumor mass. The preponderance of previous tumor simulations focused on non-invasive (or proliferative) growth. The formation of the invasive cell chains and their interactions with the primary tumor mass and host microenvironment are not well understood. Here, we present a novel cellular automaton (CA) model that enables one to efficiently simulate invasive tumor growth in a heterogeneous host microenvironment. By taking into account a variety of microscopic-scale tumor-host interactions, including the short-range mechanical interactions between tumor cells and tumor stroma, degradation of the extracellular matrix by the invasive cells and oxygen/nutrient gradient driven cell motions, our CA model predicts a rich spectrum of growth dynamics and emergent behaviors of invasive tumors. Besides robustly reproducing the salient features of dendritic invasive growth, such as least-resistance paths of cells and intrabranch homotype attraction, we also predict nontrivial coupling between the growth dynamics of the primary tumor mass and the invasive cells. In addition, we show that the properties of the host microenvironment can significantly affect tumor morphology and growth dynamics, emphasizing the importance of understanding the tumor-host interaction. The capability of our CA model suggests that sophisticated in silico tools could eventually be utilized in clinical situations to predict neoplastic progression and propose individualized optimal treatment strategies.     

The sweet and bitter sides of galectins in melanoma progression

Melanoma is the leading cause of skin cancer-related deaths, which is due in large part to its aggressive behavior, resistance to therapy, and ability to metastasize to multiple organs such as the lymph nodes, lung, and brain. Melanoma progresses in a stepwise manner from the benign nevus, to radial spreading through the dermis, to a vertical invasive phase, and finally to metastasis. The carbohydrate-binding family of galectins has a strong influence on each phase of melanoma progression through their effects on immune surveillance, angiogenesis, cell migration, tumor cell adhesion, and the cellular response to chemotherapy. Galectins share significant homology in their carbohydrate recognition domain (CRD), which mediates binding to an array of N-glycosylated proteins located on the surface of tumor cells, endothelial cells, T-cells, and to similarly glycosylated extracellular matrix proteins. Galectins are also present within tumor cells where they perform anti-apoptotic functions and enhance intracellular signaling that results in deregulated expression of genes involved in tumor progression. The most extensively studied galectins, galectin-1 and galectin-3, have been shown to have profound effects on melanoma growth and metastasis by influencing many of these biological processes.     

The good and the bad of chemokines/chemokine receptors in melanoma

Chemokine ligand/receptor interactions affect melanoma cell growth, stimulate or inhibit angiogenesis, recruit leukocytes, promote metastasis, and alter the gene expression profile of the melanoma associated fibroblasts. Chemokine/chemokine receptor interactions can protect against tumor development/growth or can stimulate melanoma tumor progression, tumor growth and metastasis. Metastatic melanoma cells express chemokine receptors that play a major role in the specifying the organ site for metastasis, based upon receptor detection of the chemokine gradient elaborated by a specific organ/tissue. A therapeutic approach that utilizes the protective benefit of chemokines involves delivery of angiostatic chemokines or chemokines that stimulate the infiltration of cytotoxic T cells and natural killer T cells into the tumor microenvironment. An alternative approach that tackles the tumorigenic property of chemokines uses chemokine antibodies or chemokine receptor antagonists to target the growth and metastatic properties of these interactions. Based upon our current understanding of the role of chemokine‐mediated inflammation in cancer, it is important that we learn to appropriately regulate the chemokine contribution to the tumorigenic ‘cytokine/chemokine storm’, and to metastasis.     

Study of motogenic signal in human melanoma cells

The components of the extracellular matrix (ECM) are more than just adhesion sites for migrating tumor cells: following enzymatic degradation of the ECM, the release of sequestrated growth factors increases, thus they become available for tumor cells. In a number of cancers dysfunction of epidermal growth factor receptor (EGFR) or hepatocyte growth factor receptor (c-Met) contribute to the malignant transformation that directly regulates cell proliferation, survival and motility. Furthermore, intracellular calcium level plays an important role in the regulation of the tyrosine kinase pathway. In our preclinical experiments, by administering heparin-derived oligosaccharides we influenced the interaction between human melanoma cells and ECM. In vitro cell migration was inhibited by heparin fragments. Moreover, two of the effective oligosaccharides reduced the number of lung colonies formed in SCID mice. In human melanoma cells an important element of Ca2+ homeostasis, the purinergic Ca2+ channel P2X7 proved to be an anti-apoptotic protein. EGFR and c-Met showed constitutive activity in human melanoma cells, and their inhibition in vitro caused decreased proliferation, migration and elevated apoptosis. Administration of a selective c-Met-TKI significantly decreased primary tumor growth in vivo as well as the capacity for liver colony formation in SCID mice. Selective EGFR-TKI had less inhibitory effect on metastasis formation, and had no effect on the primary tumor. Our results suggest the necessity of a rational dual-specific drug design for the purpose in the therapy of malignant melanoma.     

CSE1L,a Novel Microvesicle Membrane Protein,Mediates Ras-Triggered Microvesicle Generation and Metastasis of Tumor Cells

Tumor-derived microvesicles are rich in metastasis-related proteases and play a role in the interactions between tumor cells and tumor microenvironment in tumor metastasis. Because shed microvesicles may remain in the extracellular environment around tumor cells, the microvesicle membrane protein may be the potential target for cancer therapy. Here we report that chromosome segregation 1–like (CSE1L) protein is a microvesicle membrane protein and is a potential target for cancer therapy. v-H-Ras expression induced extracellular signal–regulated kinase (ERK)-dependent CSE1L phosphorylation and microvesicle biogenesis in various cancer cells. CSE1L overexpression also triggered microvesicle generation, and CSE1L knockdown diminished v-H-Ras–induced microvesicle generation, matrix metalloproteinase (MMP)-2 and MMP-9 secretion and metastasis of B16F10 melanoma cells. CSE1L was preferentially accumulated in microvesicles and was located in the microvesicle membrane. Furthermore, anti-CSE1L antibody–conjugated quantum dots could target tumors in animal models. Our findings highlight a novel role of Ras-ERK signaling in tumor progression and suggest that CSE1L may be involved in the “early” and “late” metastasis of tumor cells in tumorigenesis. Furthermore, the novel microvesicle membrane protein, CSE1L, may have clinical utility in cancer diagnosis and targeted cancer therapy.