Late stage inhibition of hematogenous melanoma metastasis by cystatin C over-expression

Background  

Tumor metastasis is a frequent cause of treatment failure for cancer patients. A key feature of metastatic cancer cells is their invasive ability. Cysteine proteases contribute to invasive properties of many cancer cell types. To analyze the contribution of cysteine proteases to metastasis we have over-expressed in B16 melanoma cells the natural cysteine protease inhibitor, cystatin C. We measured in vitro invasion of cystatin over-expression clones with Boyden chamber type assays. Tail-vein injections of cells were used to compare lung tumor colonization. Subcutaneous tumor growth and tumor cell metastasis from primary tumors were also analyzed. Apoptosis of tumor cells was measured in lung tissues following melanoma cell injection.     

Continuous Traveling Waves for Prey-Taxis

Spatially moving predators are often considered for biological control of invasive species. The question arises as to whether introduced predators are able to stop an advancing pest or foreign population. In recent studies of reaction–diffusion models, it has been shown that the prey invasion can only be stopped if the prey dynamics observes an Allee effect. In this paper, we include prey-taxis into the model. Prey-taxis describe the active movement of predators to regions of high prey density. This effect leads to the observation that predators are drawn away from the leading edge of a prey invasion where its density is low. This leads to counterintuitive result that prey-taxis can actually reduce the likelihood of effective biocontrol.     

Exosomes Derived from the Human Primary Colorectal Cancer Cell Line SW480 Orchestrate Fibroblast‐Led Cancer Invasion

In localized tumors, basement membrane (BM) prevents invasive outgrowth of tumor cells into surrounding tissues. When carcinomas become invasive, cancer cells either degrade BM or reprogram stromal fibroblasts to breach BM barrier and lead invasion of cancer cells into surrounding tissues in a process called fibroblast‐led invasion. However, tumor‐derived factors orchestrating fibroblast‐led invasion remain poorly understood. Here it is shown that although early‐stage primary colorectal adenocarcinoma (SW480) cells are themselves unable to invade Matrigel matrix, they secrete exosomes that reprogram normal fibroblasts to acquire de novo capacity to invade matrix and lead invasion of SW480 cells. Strikingly, cancer cells follow leading fibroblasts as collective epithelial‐clusters, thereby circumventing need for epithelial to mesenchymal transition, a key event associated with invasion. Moreover, acquisition of pro‐invasive phenotype by fibroblasts treated with SW480‐derived exosomes relied on exosome‐mediated MAPK pathway activation. Mass spectrometry‐based protein profiling reveals that cancer exosomes upregulate fibroblasts proteins implicated in focal adhesion (ITGA2/A6/AV, ITGB1/B4/B5, EGFR, CRK), regulators of actin cytoskeleton (RAC1, ARF1, ARPC3, CYFIP1, NCKAP1, ICAM1, ERM complex), and signalling pathways (MAPK, Rap1, RAC1, Ras) important in pro‐invasive remodeling of extracellular matrix. Blocking tumor exosome‐mediated signaling to fibroblasts therefore represents an attractive therapeutic strategy in restraining tumors by perturbing stroma‐driven invasive outgrowth.     

Epithelial-mesenchymal transition and the invasive potential of tumors

The development of metastasis requires the movement and invasion of cancer cells from the primary tumor into the surrounding tissue. To acquire such invasive abilities, epithelial cancer cells must undergo several phenotypic changes. Some of these, including alterations in cell adhesion and migration, are reminiscent of those observed during the developmental process termed epithelial-mesenchymal transition (EMT). Several master gene regulatory programs known to promote EMT during development have recently been discovered to play key roles in cancer progression. In particular, the regulation of cell adhesion molecules and the signaling pathways linking them to mechanisms of gene regulation has emerged as an important determinant of tumor cell invasion and metastasis. A deeper understanding of these mechanisms should allow both better diagnosis and the development of specific treatments for invasive cancer.     

Matrix metalloprotease-1a promotes tumorigenesis and metastasis

Matrix metalloprotease-1 (MMP1), a collagenase and activator of the G protein-coupled protease activated receptor-1 (PAR1), is an emerging new target implicated in oncogenesis and metastasis in diverse cancers. However, the functional mouse homologue of MMP1 in cancer models has not yet been clearly defined. We report here that Mmp1a is a functional MMP1 homologue that promotes invasion and metastatic progression of mouse lung cancer and melanoma. LLC1 (Lewis lung carcinoma) and primary mouse melanoma cells harboring active BRAF express high levels of endogenous Mmp1a, which is required for invasion through collagen. Silencing of either Mmp1a or PAR1 suppressed invasive stellate growth of lung cancer cells in three-dimensional matrices. Conversely, ectopic expression of Mmp1a conferred an invasive phenotype in epithelial cells that do not express endogenous Mmp1a. Consistent with Mmp1a acting as a PAR1 agonist in an autocrine loop, inhibition or silencing of PAR1 resulted in a loss of the Mmp1a-driven invasive phenotype. Knockdown of Mmp1a on tumor cells resulted in significantly decreased tumorigenesis, invasion, and metastasis in xenograft models. Together, these data demonstrate that cancer cell-derived Mmp1a acts as a robust functional homologue of MMP1 by conferring protumorigenic and metastatic behavior to cells.     

Multi-step pericellular proteolysis controls the transition from individual to collective cancer cell invasion

Invasive cell migration through tissue barriers requires pericellular remodelling of extracellular matrix (ECM) executed by cell-surface proteases, particularly membrane-type-1 matrix metalloproteinase (MT1-MMP/MMP-14). Using time-resolved multimodal microscopy, we show how invasive HT-1080 fibrosarcoma and MDA-MB-231 breast cancer cells coordinate mechanotransduction and fibrillar collagen remodelling by segregating the anterior force-generating leading edge containing beta1 integrin, MT1-MMP and F-actin from a posterior proteolytic zone executing fibre breakdown. During forward movement, sterically impeding fibres are selectively realigned into microtracks of single-cell calibre. Microtracks become expanded by multiple following cells by means of the large-scale degradation of lateral ECM interfaces, ultimately prompting transition towards collective invasion similar to that in vivo. Both ECM track widening and transition to multicellular invasion are dependent on MT1-MMP-mediated collagenolysis, shown by broad-spectrum protease inhibition and RNA interference. Thus, invasive migration and proteolytic ECM remodelling are interdependent processes that control tissue micropatterning and macropatterning and, consequently, individual and collective cell migration.     

Melanoma invasion – current knowledge and future directions

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.     

Melanoma invasion - current knowledge and future directions

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.     

Smurf1 regulates tumor cell plasticity and motility through degradation of RhoA leading to localized inhibition of contractility

Rho GTPases participate in various cellular processes, including normal and tumor cell migration. It has been reported that RhoA is targeted for degradation at the leading edge of migrating cells by the E3 ubiquitin ligase Smurf1, and that this is required for the formation of protrusions. We report that Smurf1-dependent RhoA degradation in tumor cells results in the down-regulation of Rho kinase (ROCK) activity and myosin light chain 2 (MLC2) phosphorylation at the cell periphery. The localized inhibition of contractile forces is necessary for the formation of lamellipodia and for tumor cell motility in 2D tissue culture assays. In 3D invasion assays, and in in vivo tumor cell migration, the inhibition of Smurf1 induces a mesenchymal-amoeboid-like transition that is associated with a more invasive phenotype. Our results suggest that Smurf1 is a pivotal regulator of tumor cell movement through its regulation of RhoA signaling.     

Collective cell migration in morphogenesis and cancer

The movement of cells that maintain cell-cell junctions yet protrude along or within tissues is an important mechanism for cell positioning in morphogenesis, tissue repair and cancer. Collective cell migration shares similarities but also important differences to individually migrating cells. Coherent groups of cells are arranged and held together by cell-cell adhesion molecules, including cadherins, integrins, ALCAM and NCAM. Integrins of the beta 1 and beta 3 families further provide polarized interactions with the extracellular tissue environment, while matrix-degrading proteases become focalized to substrate contacts to widen tissue space for the advancing cell mass. By generating one functional unit, in contrast to individual cell migration, collective migration provides the active and passive translocation of mobile and non-mobile cells, respectively. This review highlights cellular and molecular principles of collective migration in the context of morphogenic tissue patterning and tumor cell invasion.     

Loss of p53 promotes RhoA-ROCK-dependent cell migration and invasion in 3D matrices

In addition to its role in controlling cell cycle progression, the tumor suppressor protein p53 can also affect other cellular functions such as cell migration. In this study, we show that p53 deficiency in mouse embryonic fibroblasts cultured in three-dimensional matrices induces a switch from an elongated spindle morphology to a markedly spherical and flexible one associated with highly dynamic membrane blebs. These rounded, motile cells exhibit amoeboid-like movement and have considerably increased invasive properties. The morphological transition requires the RhoA-ROCK (Rho-associated coil-containing protein kinase) pathway and is prevented by RhoE. A similar p53-mediated transition is observed in melanoma A375P cancer cells. Our data suggest that genetic alterations of p53 in tumors are sufficient to promote motility and invasion, thereby contributing to metastasis.     

Peritumoral monocytes induce cancer cell autophagy to facilitate the progression of human hepatocellular carcinoma

Macroautophagy/autophagy is an important catabolic process mediating cellular homeostasis and plays critical roles in cancer development. Whereas autophagy has been widely studied in various pathological models, little is known about the distribution, clinical significance and regulatory mechanism of this process in human hepatocellular carcinoma (HCC). In the present study, we found that tumor tissues exhibited significantly increased levels of autophagy compared with non-tumor tissues, and cancer cells with higher levels of autophagy were predominantly enriched in the invading edge regions of human HCC. Increased MAP1LC3B/LC3B expression in the invading edge regions was significantly correlated with a higher density of closely located monocytes, and TNF and IL1B derived from tumor-activated monocytes synergistically induced cancer cell autophagy in the invading edge regions of HCC. Monocyte-elicited autophagy induced the epithelial-mesenchymal transition (EMT) of cancer cells and promoted tumor metastasis by activating the NFKB-SNAI1 signaling pathway. Moreover, the increase of LC3B⁺ cancer cells in the invading edge areas was associated with high mortality and reduced survival of patients with HCC. These findings indicated that cancer cell autophagy is regulated by a collaborative interaction between tumor and immune cell components in distinct HCC microenvironments, thus allowing the inflammatory monocytes to be rerouted in a tumor-promoting direction.     

Invasiveness-triggered state transition in malignant melanoma cells

Cancer cells are considered to have high morphological heterogeneity in human melanoma tissue. Here, we report that epithelial cancer cells are dominant in different development stages of human melanoma tissues. The cellular and molecular mechanisms that maintain melanoma cells in the epithelial state are further investigated in the A2058 cell line. We find that micropore (8 µm) transwell invasion, but not superficial migration in the scratch assay, can induce remarkable morphological changes between epithelial and mesenchymal melanoma cells within 4 days. The morphological switch is associated with dynamic changes of epithelial–mesenchymal transition (EMT) hallmarks E-cadherin and vimentin. Further immunoflurencent staining and co-immunoprecipitation assay showed the uncoupling of the M3 muscarinic acetylcholine receptor (mAChR) and the p75 neurotrophin receptor (p75NTR) in epithelial melanoma cells. Specific knockdown of M3 mAChR by small interfering RNA (siRNA) significantly abrogates the transition of spindle-shaped mesenchymal cells to epithelial cells. Collectively, we report a cellular model of invasiveness-triggered state transition (ITST) in which melanoma cell invasion can induce morphological changes between epithelial and mesenchymal cells. ITST is one of the biological basis for maintaining metastatic melanoma cells in the epithelial state. Furthermore, M3 mAChR receptor-mediated ITST provides a novel therapeutic strategy to inhibit the development of malignant melanoma.     

Tissue inhibitor of matrix metalloproteinase-2 regulates matrix metalloproteinase-2 activation by modulation of membrane-type 1 matrix metalloproteinase activity in high and low invasive melanoma cell lines.

Activation of pro-matrix metalloproteinase (MMP)-2 on the surface of malignant cells by membrane-bound MT1-MMP is believed to play a critical role during tumor progression and metastasis. In this study we present evidence that MT1-MMP plays a key role for the in vitro invasiveness of malignant melanoma. Melanoma cell lines secreted latent MMP-2 when cultured on plastic. However, when cells were grown in floating type I collagen lattices, only high invasive melanoma cells activated proMMP-2. Activation could be inhibited by antibodies against MT1-MMP, by addition of recombinant tissue inhibitor of metalloproteinases (TIMP)-2 and by inhibition of MT1-MMP cleavage. MT1-MMP protein was detected as an inactive protein in all cell lines cultured as monolayers, whereas in collagen gels, active MT1-MMP protein was detected in the membranes of both high and low invasive melanoma cells. Production of TIMP-2 was about 10-fold higher in low invasive cells as compared with high invasive melanoma cells and was further increased in the low invasive cells upon contact to collagen. Thus, in melanoma cells TIMP-2 expression levels might regulate MT1-MMP-mediated activation of proMMP-2. High invasive melanoma cells displayed increased in vitro invasiveness, which was inhibited by TIMP-2. These data indicate the importance of these enzymes for the invasion processes and support a role for MT1-MMP as an activator of proMMP-2 in malignant melanoma.     

Heparanase degrades syndecan-1 and perlecan heparan sulfate: functional implications for tumor cell invasion

Heparanase (HPSE-1) is involved in the degradation of both cell-surface and extracellular matrix (ECM) heparan sulfate (HS) in normal and neoplastic tissues. Degradation of heparan sulfate proteoglycans (HSPG) in mammalian cells is dependent upon the enzymatic activity of HPSE-1, an endo-beta-d-glucuronidase, which cleaves HS using a specific endoglycosidic hydrolysis rather than an eliminase type of action. Elevated HPSE-1 levels are associated with metastatic cancers, directly implicating HPSE-1 in tumor progression. The mechanism of HPSE-1 action to promote tumor progression may involve multiple substrates because HS is present on both cell-surface and ECM proteoglycans. However, the specific targets of HPSE-1 action are not known. Of particular interest is the relationship between HPSE-1 and HSPG, known for their involvement in tumor progression. Syndecan-1, an HSPG, is ubiquitously expressed at the cell surface, and its role in cancer progression may depend upon its degradation. Conversely, another HSPG, perlecan, is an important component of basement membranes and ECM, which can promote invasive behavior. Down-regulation of perlecan expression suppresses the invasive behavior of neoplastic cells in vitro and inhibits tumor growth and angiogenesis in vivo. In this work we demonstrate the following. 1) HPSE-1 cleaves HS present on the cell surface of metastatic melanoma cells. 2) HPSE-1 specifically degrades HS chains of purified syndecan-1 or perlecan HS. 3) Syndecan-1 does not directly inhibit HPSE-1 enzymatic activity. 4) The presence of exogenous syndecan-1 inhibits HPSE-1-mediated invasive behavior of melanoma cells by in vitro chemoinvasion assays. 5) Inhibition of HPSE-1-induced invasion requires syndecan-1 HS chains. These results demonstrate that cell-surface syndecan-1 and ECM perlecan are degradative targets of HPSE-1, and syndecan-1 regulates HPSE-1 biological activity. This suggest that expression of syndecan-1 on the melanoma cell surface and its degradation by HPSE-1 are important determinants in the control of tumor cell invasion and metastasis.     

FHOD1 formin is upregulated in melanomas and modifies proliferation and tumor growth

The functional properties of actin-regulating formin proteins are diverse and in many cases cell-type specific. FHOD1, a formin expressed predominantly in cells of mesenchymal lineage, bundles actin filaments and participates in maintenance of cell shape, migration and cellular protrusions. FHOD1 participates in cancer-associated epithelial to mesenchymal transition (EMT) in oral squamous cell carcinoma and breast cancer. The role of FHOD1 in melanomas has not been characterized. Here, we show that FHOD1 expression is typically strong in cutaneous melanomas and cultured melanoma cells while the expression is low or absent in benign nevi. By using shRNA to knockdown FHOD1 in melanoma cells, we discovered that FHOD1 depleted cells are larger, rounder and have smaller focal adhesions and inferior migratory capacity as compared to control cells. Importantly, we found FHOD1 depleted cells to have reduced colony-forming capacity and attenuated tumor growth in vivo, a finding best explained by the reduced proliferation rate caused by cell cycle arrest. Unexpectedly, FHOD1 depletion did not prevent invasive growth at the tumor margins. These results suggest that FHOD1 participates in key cellular processes that are dysregulated in malignancy, but may not be essential for melanoma cell invasion.     

Intercellular invasion and the organizational stability of tissues: a role for fibronectin

Intracellular invasion is the movement of cells of one type into the fabric of other, contiguous tissues. Invasion is a signature behavior of the malignant tumor and also is found as part of the normal behavior of inflammatory blood cells and tissues engaged in the morphogenetic movements of normal embryogenesis and in a number of instances of normal and pathological tissue remodeling in the adult. Informed by the view that the underlying mechanisms of invasion will be similar for tumor cells and invasive blood and embryonic cells, this review adopts a comparative approach to the analysis of invasion. Invasion results in the development of a diffuse interface between contiguous tissues. Its alternative is the maintenance of stable, planar tissue boundaries. This is the more usual condition for contiguous tissues in the animal. This review will focus on the processes that, on the one hand, stabilize planar contact interfaces between tissues, and, on the other, promote the destabilization of tissue integrity by fostering intercellular invasion. Particular attention is devoted to a role for adhesive interactions mediated by the matrix adhesion molecule, fibronectin. In certain instances, fibronectin in the matrix promotes invasion whereas in others, the presence of fibronectin prevents invasion. The distinction appears to depend on whether the invasive tissue is migrating into an acellular extracellular matrix or whether invasion involves densely cellular tissues. In the first instance, fibronectin promotes invasion, whereas in the second, it stabilizes the interface of the contacting tissues and prevents invasion.     

Novel Functions of CD147 in the Mitochondria Exacerbates Melanoma Metastasis

Melanoma is an aggressive form of skin cancer characterized by rapid invasion and metastasis. CD147 is known to be functioning in cell invasion. In this study, we showed that CD147 was translocated from the cell membrane to the mitochondria in advanced melanoma. Melanoma patients with CD147 localized to the mitochondria confer a worse prognosis. The mitochondrial CD147 levels are correlated with the invasion potential of various melanoma cell lines as well as mitochondrial energy metabolism. Depletion of CD147 decreased the activity of mitochondrial complex V. STRING analysis for protein-protein interaction networks (PPIN) in CD147-depleted melanoma cells showed that mitochondrial proteins HSP60 and ATP5B, a subunit of mitochondrial complex V, were node proteins. HSP60 upregulation was correlated with a worse prognosis of melanoma patients. Co-immunoprecipitation (Co-IP) assay indicates that CD147 interacts with HSP60. These data suggested that mitochondrial CD147 may prompt HSP60 to activate ATP5B, thereby promoting the mitochondrial aerobic oxidation and the invasive abilities of melanoma cells. Correlation analysis of the data acquired from patients was helpful to draw a 5-year survival curve for patients who screened positive and negative for mitochondrial CD147. This study unravels the function of CD147 in tumor invasion and highlights it as a potential tumor therapeutic target.     

The tandem PDZ domains of syntenin promote cell invasion

Syntenin is a tandem PDZ protein that has recently been shown to be overexpressed in several cancer cells and tissues, and that might play an active role in tumor cell invasion and metastasis. Here we show that overexpression of the tandem PDZ domains of syntenin in non-invasive cells is necessary and sufficient to stimulate these cells to invade a collagen I matrix, and this effect can be regulated by ligand binding to the PDZ domains. Furthermore, we show that syntenin-induced invasion requires signaling through ras, rho and PI3K/MAPK signaling pathways and involves changes in cell-cell adhesion. Inversely, when we used RNA interference to inhibit syntenin expression in different invasive cancer cell lines, we observed a drastically decreased ability of these cells to migrate and invade into collagen type I or Matrigel. RNAi-treated cells also show increased cell aggregation, indicating that syntenin is important for cell-cell adhesion in epithelial cells. Together, these results suggest that downregulation of syntenin by RNA interference could provide a means of inhibiting tumor invasion and possibly metastasis in different cancers, and point to syntenin as a potential cancer biomarker and drug target.