BRAF(V600E) melanoma cells secrete factors that activate stromal fibroblasts and enhance tumourigenicity.

BACKGROUND: Melanoma, the most lethal form of skin cancer, is responsible for over 80% of all skin cancer deaths and is highly metastatic, readily spreading to the lymph nodes or metastasising to other organs. The frequent genetic mutation found in metastatic melanoma, BRAF(V600E), results in constitutive activation of the mitogen-activated protein kinase pathway. METHODS: In this study, we utilised genetically engineered melanoma cell lines and xenograft mouse models to investigate how BRAF(V600E) affected cytokine (IL-1ß, IL-6, and IL-8) and matrix metalloproteinase-1 (MMP-1) expression in tumour cells and in human dermal fibroblasts. RESULTS: We found that BRAF(V600E) melanoma cells expressed higher levels of these cytokines and of MMP-1 than wild-type counterparts. Further, conditioned medium from the BRAF(V600E) melanoma cells promoted the activation of stromal fibroblasts, inducing expression of SDF-1 and its receptor CXCR4. This increase was mitigated when the conditioned medium was taken from melanoma cells treated with the BRAF(V600E) specific inhibitor, vemurafenib. CONCLUSIONS: Our findings highlight the role of BRAF(V600E) in activating the stroma and suggest a mechanistic link between BRAF(V600E) and MMP-1 in mediating melanoma progression and in activating adjacent fibroblasts in the tumour microenvironment.

A matrix metalloproteinase-1/protease activated receptor-1 signaling axis promotes melanoma invasion and metastasis.

Hallmarks of malignant melanoma are its propensity to metastasize and its resistance to treatment, giving patients with advanced disease a poor prognosis. The transition of melanoma from non-invasive radial growth phase (RGP) to invasive and metastatically competent vertical growth phase (VGP) is a major step in tumor progression, yet the mechanisms governing this transformation are unknown. Matrix metalloproteinase-1 (MMP-1) is highly expressed by VGP melanomas, and is thought to contribute to melanoma progression by degrading type I collagen within the skin to facilitate melanoma invasion. Protease activated receptor-1 (PAR-1) is activated by MMP-1, and is also expressed by VGP melanomas. However, the effects of MMP-1 signaling through PAR-1 have not been examined in melanoma. Here, we demonstrate that an MMP-1/PAR-1 signaling axis exists in VGP melanoma, and is necessary for melanoma invasion. Introduction of MMP-1 into RGP melanoma cells induced gene expression associated with tumor progression and promoted invasion in vitro, and enhanced tumor growth and conferred metastatic capability in vivo. This study demonstrates that both the type I collagenase and PAR-1 activating functions of MMP-1 are required for melanoma progression, and suggests that MMP-1 may be a major contributor to the transformation of melanoma from non-invasive to malignant disease.

Matrix metalloproteinase-1 promotes breast cancer angiogenesis and osteolysis in a novel in vivo model.

Matrix metalloproteinase-1 (MMP-1) is critical for mediating breast cancer metastasis to bone. We investigated the role of MMP-1 in breast cancer invasion of soft tissues and bone using human MDA MB-231 breast cancer cells stably transfected with shRNAs against MMP-1 and a novel murine model of bone invasion. MMP-1 produced by breast cancer cells with control shRNA facilitated invasion of tumors into soft tissue in vivo, which correlated with enhanced blood vessel formation at the invasive edge, compared to tumors with silenced MMP-1 expression. Tumors expressing MMP-1 were also associated with osteolysis in vivo, whereas tumors with inhibited MMP-1 levels were not. Additionally, tumor-secreted MMP-1 activated bone-resorbing osteoclasts in vitro. Together, these data suggest a mechanism for MMP-1 in the activation of osteoclasts in vivo. We conclude that breast cancer-derived MMP-1 mediates invasion through soft tissues and bone via mechanisms involving matrix degradation, angiogenesis, and osteoclast activation.

Molecular targets in osteoarthritis: metalloproteinases and their inhibitors.

The debilitating destruction of joint tissues seen in osteoarthritis (OA) is due, in large part, to the degradative activity of metalloproteinase (MP) enzymes that target extracellular matrix (ECM) components within articular cartilage. Although successful in suppressing the pain and inflammation associated with this disease, conventional OA therapeutics do not inhibit the underlying tissue catabolism, allowing the disease to progress into irreversible ECM loss and chronic disability. Therapeutic inhibition of metalloproteinase activity is not a new concept, however, its transfer into clinical use has been frustrating. Disappointing results from clinical trials with small molecule inhibitors of metalloproteinases have highlighted the critical importance of inhibitor specificity, and the need to identify the individual metalloproteinases responsible for joint destruction. We discuss strategies of inhibition using small molecule inhibitors and tissue inhibitors of metalloproteinases (TIMPs) engineered to increase inhibitory specificity, and present new data using of new reagents such as ribozymes and inhibitory RNAs that repress expression of specific enzymes. Recent data has implicated the disease stage-dependent involvement of matrix metalloproteinase-1, -2, -3, -9, -13, ADAM-17/TACE (tumor-necrosis factor-alpha converting enzyme), and ADAMTS-5 (a disintegrin and metalloproteinase with thrombospondin 1 motifs) as major in vivo mediators of the ECM degradation seen in OA, and as such, they represent promising therapeutic targets. We conclude that the concept of molecular polypharmacy, in which the relevant enzymes are selectively targeted with multiple directed therapies, may offer a new therapeutic strategy that prevents joint destruction and minimizes toxicities.

IL-1 induces collagenase-3 (MMP-13) promoter activity in stably transfected chondrocytic cells: requirement for Runx-2 and activation by p38 MAPK and JNK pathways.

Osteoarthritic chondrocytes secrete matrix metalloproteinase-13 (MMP-13) in response to interleukin-1 (IL-1), causing digestion of type II collagen in cartilage. Using chondrocytic cells, we previously determined that IL-1 induced a strong MMP-13 transcriptional response that requires p38 MAPK, JNK and the transcription factor NF-kappaB. Now, we have studied the tissue-specific transcriptional regulation of MMP-13. Constitutive expression of the transcription factor Runx-2 correlated with the ability of a cell type to express MMP-13 and was required for IL-1 induction; moreover, Runx-2 enhanced IL-1 induction of MMP-13 transcription by synergizing with the p38 MAPK signaling pathway. Transiently transfected MMP-13 promoters were not IL-1 inducible. However, -405 bp of stably integrated promoter was sufficient for 5- to 6-fold IL-1 induction of reporter activity and this integrated reporter required the same p38 MAPK pathway as the endogenous gene. Finally, mutation of the proximal Runx binding site and the proximal AP-1 site blunted the transcriptional response to IL-1, and double mutation synergistically decreased reporter activity. In summary, our data suggest that the transcriptional MMP-13 response to IL-1 is controlled by the p38 pathway interacting at the MMP-13 promoter through the tissue-specific transcription factor Runx-2 and the ubiquitous AP-1 transcription factor.

Early response genes induced in chondrocytes stimulated with the inflammatory cytokine interleukin-1beta.

Recent work has established that IL-1beta plays a central role in the inflammation and connective tissue destruction observed in both rheumatoid arthritis and osteoarthritis. These processes result from the ability of this inflammatory cytokine to activate expression of genes for neutral proteases, such as the matrix metalloproteinases. While IL-1beta activates matrix metalloproteinase genes within several hours, it also activates immediate early genes, which are required for the later expression of matrix metalloproteinases and other arthritis-perpetuating genes, are also activated. To identify putative immediate early genes involved in IL-1beta-mediated arthritic disease, a chondrocytic cell line (SW1353) was stimulated with this cytokine for 2 hours, total RNA was isolated, and expressed genes were identified by microarray analysis. This analysis identified alterations in the expression of multiple transcription factors, cytokines, growth factors and their receptors, adhesion molecules, proteases, and signaling intermediates that may contribute to inflammation and cartilage destruction in arthritis. Interestingly, confirmation of the expression of activating protein-1 family members by reverse transcriptase polymerase chain reaction revealed a preferential increase in junB, a known transcriptional antagonist of c-jun. The failure to observe induction of early growth response gene-1, which was detected by reverse transcriptase polymerase chain reaction to be substantially and transiently induced by 1 hour of IL-1 treatment, may be explained by the known instability of the message after early induction. However, this analysis has identified numerous IL-1beta-responsive genes that warrant further investigation as mediators of disease in arthritis.

Synergistic induction of matrix metalloproteinase 1 by interleukin-1alpha and oncostatin M in human chondrocytes involves signal transducer and activator of transcription and activator protein 1 transcription factors via a novel mechanism.

OBJECTIVE: To investigate the mechanism of interleukin-1alpha (IL-1alpha) and oncostatin M (OSM) synergistic regulation of matrix metalloproteinase 1 (MMP-1) in human chondrocytes. METHODS: Using an immortalized human chondrocyte cell line (T/C28a4), we investigated regulation of the MMP-1 gene. Northern blotting and flow cytometric analysis were used to assess changes in receptor, MMP-1, and c-fos expression. Transient transfections using MMP-1 promoter/luciferase constructs, electrophoretic mobility shift assay, and site-directed mutagenesis were used to investigate MMP-1 promoter activation. RESULTS: We found no alteration in the expression of receptors used by these cytokines after stimulation with IL-1alpha/OSM. Using MMP-1 promoter/luciferase reporter constructs, we found that the proximal (-517/+63) region of the MMP-1 promoter was sufficient to support a synergistic activation. A role for activated signal transducers and activators of transcription (STAT-3) was demonstrated, although no binding of STAT-3 to the MMP-1 promoter was found. However, constitutive binding of activator protein 1 (AP-1) was detected, and changes in c-fos expression could modulate promoter activity. CONCLUSION: Since no changes in receptor expression were observed, receptor modulation cannot account for the IL-1alpha/OSM synergy observed. Instead, the interplay of various intracellular signaling pathways is a more likely explanation. STAT activation is required, but STAT proteins do not interact directly with the MMP-1 promoter. We propose that activated STATs stimulate c-fos expression, and changes in expression of the AP-1 components regulate MMP-1 expression. We highlight a new mechanism for MMP-1 regulation in human chondrocytes that could provide potential new therapeutic targets.

A synthetic triterpenoid selectively inhibits the induction of matrix metalloproteinases 1 and 13 by inflammatory cytokines.

OBJECTIVE: To address the effects of a novel synthetic triterpenoid, 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid (CDDO), on the induction of matrix metalloproteinases 1 and 13 (MMP-1, MMP-13) by inflammatory cytokines. METHODS: Human chondrosarcoma cells stimulated with inflammatory cytokines (interleukin-1beta [IL-1beta], tumor necrosis factor alpha) were used to study the effects of CDDO on the induction of MMPs and the invasion of cells through a collagen matrix. RESULTS: CDDO selectively reduced the induction of MMP-1 and MMP-13 at the levels of messenger RNA and protein. Treatment with CDDO prior to cytokine stimulation enhanced this inhibition, and we demonstrated that CDDO functions at the level of transcription. Additionally, CDDO reduced IL-1beta-mediated invasion of cells through a collagen matrix. CONCLUSION: This study demonstrates that CDDO is a novel inhibitor of MMP-1 and MMP-13 gene expression mediated by inflammatory cytokines. Thus, CDDO may have therapeutic potential for the inhibition of joint degradation in osteoarthritis.

Manganese superoxide dismutase signals matrix metalloproteinase expression via H2O2-dependent ERK1/2 activation.

Manganese-superoxide dismutase (Sod2) removes mitochondrially derived superoxide (O(2)) at near-diffusion limiting rates and is the only antioxidant enzyme whose expression is regulated by numerous stimuli. Here it is shown that Sod2 also serves as a source of the intracellular signaling molecule H(2)O(2). Sod2-dependent increases in the steady-state levels of H(2)O(2) led to ERK1/2 activation and subsequent downstream transcriptional increases in matrix metalloproteinase-1 (MMP-1) expression, which were reversed by expression of the H(2)O(2)-detoxifying enzyme, catalase. In addition, a single nucleotide polymorphism has recently been identified (1G/2G) at base pair--1607 that creates an Ets site adjacent to an AP-1 site at base pair --1602 and has been shown to dramatically enhance transcription of the MMP-1 promoter. Luciferase promoter constructs containing either the 1G or 2G variation were 25- or 1000-fold more active when transiently transfected into Sod2-overexpressing cell lines, respectively. The levels of MMP-2, -3, and -7 were also increased in the Sod2-overexpressing cell lines, suggesting that Sod2 may function as a "global" redox regulator of MMP expression. In addition, Sod2(-/+) mouse embryonic fibroblasts failed to respond to the cytokine-mediated induction of the murine functional analog of MMP-1, MMP-13. This study provides evidence that the modulation of Sod2 activity by a wide array of pathogenic and inflammatory stimuli may be utilized by the cell as a primary signaling mechanism leading to matrix metalloproteinase expression.

Loss of heterozygosity on chromosome 11q22-23 in melanoma is associated with retention of the insertion polymorphism in the matrix metalloproteinase-1 promoter.

Matrix metalloproteinase-1 (MMP-1, collagenase-1), which degrades interstitial collagen, is expressed at high levels by some tumor cells and is thought to enhance their invasiveness and metastatic potential. We recently described a common single nucleotide insertion polymorphism (2G allele) at -1,607 bp in the promoter of the MMP-1 gene that creates a binding site for the ETS family of transcription factors, and that is associated with enhanced transcription of this gene and increased enzyme activity. Allelic loss at the MMP-1 locus on chromosome 11 occurs in many tumors including melanoma, an invasive and aggressive cancer. We hypothesized that although loss of either the 1G or 2G allele from 1G/2G heterozygotes is random, retention of the transcriptionally more active 2G allele would favor tumor invasion and metastasis. As a result, a higher proportion of metastases would contain the 2G genotype than the 1G genotype. We report here the development of quantitative methods for assessing allelic loss at the MMP-1 locus, and demonstrate that 83% of the metastatic melanomas with loss of heterozygosity at this locus retained the 2G allele. This supports the hypothesis that retention of the 2G allele favors tumor invasion and metastasis in melanoma.

Confocal assay for invasion: use of propidium iodide fluorescence and laser reflectance to quantify the rate of migration of cells through a matrix.

BACKGROUND: Most assays used to measure invasion are based on manual counting of the number of cells that have migrated completely through commercial coated filters. We describe here a confocal fluorescence-imaging method that can assess the relative rates of invasion of cells into a matrix. METHODS: After being seeded on the matrix and a period of incubation, the cells are fixed and treated with RNase. Propidium iodide is then added to stain the double-stranded DNA. A confocal microscope system is used to obtain high-resolution images of the red propidium iodide fluorescence and laser reflectance from optical sections at increasing depths in the matrix. The section with high laser reflectance marks the top of the matrix. RESULTS: Data were calculated as the total area of red fluorescence above background in each section and were plotted as a percentage of the summed fluorescent areas in all sections. CONCLUSIONS: Because the distance into the matrix of the nuclei can be calculated by measuring from the reflective upper surface of the matrix, the method is useful for assessing the rate of cell migration and for comparing the ability of different cells to invade through different matrices under varying conditions.

Interleukin-1 induction of collagenase 3 (matrix metalloproteinase 13) gene expression in chondrocytes requires p38, c-Jun N-terminal kinase, and nuclear factor kappaB: differential regulation of collagenase 1 and collagenase 3.

OBJECTIVE: To examine the mechanism of interleukin-1 (IL-1)-induced collagenase 3 (matrix metalloproteinase 13 [MMP-13]) gene expression in cultured chondrocytes for the purpose of better understanding how the gene is induced in these cells, and how it contributes to cartilage degradation in osteoarthritis. METHODS: The transcriptional and posttranscriptional responses of the MMP-13 gene to IL-1 were assessed first. Then, direct inhibitors of mitogen-activated protein kinase (MAPK) signaling pathways and a constitutive repressor of nuclear factor kappaB (NF-kappaB) were used to assess the role of each pathway in IL-1-mediated induction of MMP-13. RESULTS: We found that IL-1 induction of MMP-13 requires p38 activity, c-Jun N-terminal kinase (JNK) activity and NF-kappaB translocation. These results suggest that both NF-kappaB and activator protein 1 transcription factors are necessary for IL-1 induction of MMP-13. We also compared the signaling pathways necessary for IL-1 to stimulate collagenase 1 (MMP-1) in articular chondrocytes and chondrosarcoma cells and found that IL-1 induction of MMP-1 requires different pathways from those required by MMP-13. In chondrosarcoma cells, MMP-1 induction depends on p38 and MEK (an MAPK kinase of the extracellular signal-regulated kinase pathway) and does not require JNK or NF-kappaB. In articular chondrocytes, inhibition of MEK had no effect, while inhibition of p38 gave variable results. CONCLUSION: These studies demonstrate, for the first time, that p38, JNK, and NF-kappaB are required for IL-1 induction of MMP-13. The results also highlight the differential requirements for signaling pathways in the induction of MMP-1 and MMP-13. Additionally, they demonstrate that induction of MMP-1 by IL-1 in chondrocytic cells depends on unique combinations of signaling pathways that are cell type-specific.

Transforming growth factor beta inhibitory element in the rabbit matrix metalloproteinase-1 (collagenase-1) gene functions as a repressor of constitutive transcription.

Transforming growth factor beta (TGF-beta) is a potent modulator of the extracellular matrix, enhancing collagen synthesis and regulating expression of several genes that encode the matrix metalloproteinases (MMPs), enzymes that degrade the extracellular matrix. In this study, we explored the mechanisms whereby TGF-beta inhibits expression of the MMP-1 (collagenase 1) gene. We used transient transfection and gel mobility shift assays to characterize a TGF beta inhibitory element (TIE) at -249 bp in the rabbit MMP-1 promoter, which is also conserved at -246 bp in the human gene. This sequence shares homology to a previously identified TIE in the rat stromelysin-1 (MMP-3) promoter, where it is located at -709 bp. Mutational analyses and transient transfections indicate that MMP-1 TIE functions both as a constitutive repressor of MMP-1 gene expression and, in the presence of TGF-beta, as an antagonist of transcriptional induction by phorbol esters. c-Fos binds to the TIE in the rabbit MMP-1 promoter, along with other nuclear proteins, even in the absence of treatment with TGF-beta. However, the pattern of proteins binding to the TIE is altered in the presence of nuclear extracts from TGF-beta-treated cells, suggesting that TGF-beta leads to an alteration in protein/DNA interaction, with subsequent modulation of MMP-1 gene expression. We conclude that in the rabbit MMP-1 promoter, the TIE has dual functions as a repressor of basal transcription and as a mediator of the biologic effects of TGF-beta. Furthermore, these dual functions provide additional and subtle mechanisms for regulating MMP-1 gene expression under a variety of biological and pathological conditions.

Interstitial collagenases as markers of tumor progression.

Degradation of the extracellular matrix is the sine qua non of tumor invasion and metastasis. Most of this degradation is mediated by matrix metalloproteinases (MMPs), a family of enzymes that, collectively, degrades the extracellular matrix. Although the basement membrane-degrading enzymes, MMP-2 and MMP-9, have been given considerable attention for their roles in invasion and metastasis, the interstitial collagenases, a subfamily of MMPs that cleaves the stromal collagens types I and III, have received relatively little recognition for their part in these processes. This subfamily is comprised of collagenase 1 (MMP-1), collagenase 3 (MMP-13), and the MT-MMPs, membrane-bound MMPs, and numerous reports over the last several years document the expression of these MMPs in a wide variety of advancing tumors. Of particular interest is a single nucleotide polymorphism in the MMP-1 promoter that increases the transcription of this gene and that is associated with melanoma and with ovarian and endometrial cancers. The collagenases can mediate tumor invasion through several mechanisms, which include constitutive production of enzyme by the tumor cells, induction of collagenase production in the neighboring stromal cells, and interactions between tumor/ stromal cells to induce collagenase production by one or both cell types. Thus, evidence indicates that elevated expression of the interstitial collagenases is associated with a poor prognosis in a variety of cancers, and therefore, these MMPs can serve as a marker of tumor progression.

Human breast cancer cells activate procollagenase-1 and invade type I collagen: invasion is inhibited by all-trans retinoic acid.

Matrix metalloproteinases (MMPs) play an important role in tumor cell invasion and metastasis. These processes require the dissolution of the basement membrane and invasion of the stromal matrix (ECM), and are mediated by MMPs. Consequently, MMP inhibitors may be attractive as new anticancer agents. To examine the potential contribution of collagenase-1 (MMP-1) in invasion of stromal matrix, we used the highly invasive and metastatic breast cancer cell line MDA-MB-231 as a model system. These cells express procollagenase-1 constitutively and this expression can be repressed by all-trans retinoic acid. Invasion of these cells into a collagen type I matrix was assessed by scanning electron microscopy (SEM), and was quantitated with a computer program and confocal laser scanning microscopy (CLSM). We found that MDA-MB-231 cells freely invaded the collagen type I matrix, suggesting that these cells possess a mechanism for activating the latent collagenase-1. In contrast, down-regulation of collagenase-1 expression by all-trans retinoic acid caused these cells to become less invasive. To confirm a role for collagenase-1 in mediating collagen type I invasion, assays were carried out in the presence of FN-439, an inhibitor of collagenase-1 enzyme activity. We found that in the presence of the proteinase inhibitor, invasion of type I collagen by MDA-MB-231 cells was also reduced. These results indicate that collagenase-1 produced by the breast tumor cells may enhance stromal matrix degradation by enabling the tumor cells to modulate their own invasive behavior, and suggest that decreasing collagenase-1 levels may be effective in breast cancer therapy.

A novel host/tumor cell interaction activates matrix metalloproteinase 1 and mediates invasion through type I collagen.

Along with degradation of type IV collagen in basement membrane, destruction of the stromal collagens, types I and III, is an essential step in the invasive/metastatic behavior of tumor cells, and it is mediated, at least in part, by interstitial collagenase 1 (matrix metalloproteinase 1 (MMP-1)). Because A2058 melanoma cells produce substantial quantities of MMP-1, we used these cells as models for studying invasion of type I collagen. With a sensitive and quantitative in vitro invasion assay, we monitored the ability of these cells to invade a matrix of type I collagen and the ability of a serine proteinase inhibitor and all-trans-retinoic acid to block invasion. Although these cells produce copious amounts of MMP-1, they do not invade collagen unless they are co-cultured with fibroblasts or with conditioned medium derived from fibroblasts. Our studies indicate that a proteolytic cascade that depends on stromal/tumor cell interactions facilitates the ability of A2058 melanoma cells to invade a matrix of type I collagen. This cascade activates latent MMP-1 and involves both serine proteinases and MMPs, particularly stromelysin 1 (MMP-3). All-trans-retinoic acid (10(-6) M) suppresses the invasion of tumor cells by several mechanisms that include suppression of MMP synthesis and an increase in levels of tissue inhibitor of metalloproteinases 1 and 2. We conclude that invasion of stromal collagen by A2058 melanoma cells is mediated by a novel host/tumor cell interaction in which a proteolytic cascade culminates in the activation of pro-MMP-1 and tumor cell invasion.

Retinoid-mediated suppression of tumor invasion and matrix metalloproteinase synthesis.

Cancer mortality usually results from the tumor invading the local environment and metastasizing to vital organs, e.g. liver, lung, and brain. Degradation of the extracellular matrix is, therefore, the sine qua non of tumor cell invasion. this degradation is mediated mainly by MMPs, and thus, inhibition of MMP synthesis is a target for anticancer agents. Tumor cells must traverse both the basement membrane (type IV collagen) and the interstitial stroma (type I collagen). Therefore, we used scanning electron microscopy to examine the invasive behavior of several aggressive tumor cell lines, A2058 melanoma cells, and SCC and FaDu squamous cell carcinomas through these matrices; and we monitored the ability of all-trans retinoic acid and several RAR-specific ligands to block invasion. We demonstrate that several retinoids, which are specific RAR alpha, beta, or gamma agonists/antagonists, selectively inhibited MMP synthesis in the three tumor cell lines. However, there was not a common pattern of MMP inhibition by a particular retinoid. For instance, a RAR alpha antagonist suppressed MMP-1 and MMP-2 synthesis in the melanoma cell line, but not in the FaDu or SCC-25 cells. On the other hand, synthesis of MMP-1 and MMP-9 by the FaDu cells was affected hardly at all, while a RAR gamma antagonist reduced the levels of MMP-2. Only all-trans retinoic acid reduced MMP-1 synthesis in these cells. We postulate that the differences may be related to a differential pattern of RAR expression in each of these cells, and that the RARs expressed by each cell line may not be targets of these RAR specific compounds. All-trans retinoic acid is a pan ligand, binding to all three RARs and, therefore, may modulate gene expression more generally. We conclude that the power of these new ligands lies in their specificity, which can be directed towards modulating expression of certain RARs and, thus, of certain MMPs. By blocking MMP synthesis, retinoids may be effective in cancer therapy by decreasing tumor invasiveness.