PKCα promotes the mesenchymal to amoeboid transition and increases cancer cell invasiveness.

BACKGROUND: The local invasion of tumor cells into the surrounding tissue is the first and most critical step of the metastatic cascade. Cells can invade either collectively, or individually. Individual cancer cell invasion can occur in the mesenchymal or amoeboid mode, which are mutually interchangeable. This plasticity of individual cancer cell invasiveness may represent an escape mechanism for invading cancer cells from anti-metastatic treatment. METHODS: To identify new signaling proteins involved in the plasticity of cancer cell invasiveness, we performed proteomic analysis of the amoeboid to mesenchymal transition with A375m2 melanoma cells in a 3D Matrigel matrix. RESULTS: In this screen we identified PKCα as an important protein for the maintenance of amoeboid morphology. We found that the activation of PKCα resulted in the mesenchymal-amoeboid transition of mesenchymal K2 and MDA-MB-231 cell lines. Consistently, PKCα inhibition led to the amoeboid-mesenchymal transition of amoeboid A375m2 cells. Next, we showed that PKCα inhibition resulted in a considerable decrease in the invading abilities of all analyzed cancer cell lines. CONCLUSIONS: Our results suggest that PKCα is an important protein for maintenance of the amoeboid morphology of cancer cells, and that downregulation of PKCα results in the amoeboid to mesenchymal transition. Our data also suggest that PKCα is important for both mesenchymal and amoeboid invasiveness, making it an attractive target for anti-metastatic therapies.

The transcription factor EGR1 regulates metastatic potential of v-src transformed sarcoma cells.

Metastatic spreading of cancer cells is a highly complex process directed primarily by the interplay between tumor microenvironment, cell surface receptors, and actin cytoskeleton dynamics. To advance our understanding of metastatic cancer dissemination, we have developed a model system that is based on two v-src transformed chicken sarcoma cell lines-the highly metastatic parental PR9692 and a non-metastasizing but fully tumorigenic clonal derivative PR9692-E9. Oligonucleotide microarray analysis of both cell lines revealed that the gene encoding the transcription factor EGR1 was downregulated in the non-metastatic PR9692-E9 cells. Further investigation demonstrated that the introduction of exogenous EGR1 into PR9692-E9 cells restored their metastatic potential to a level indistinguishable from parental PR9692 cells. Microarray analysis of EGR1 reconstituted cells revealed the activation of genes that are crucial for actin cytoskeleton contractility (MYL9), filopodia formation (MYO10), the production of specific extracellular matrix components (HAS2, COL6A1-3) and other essential pro-metastatic abilities.

RNA-seq Characterization of Melanoma Phenotype Switch in 3D Collagen after p38 MAPK Inhibitor Treatment.

Melanoma phenotype plasticity underlies tumour dissemination and resistance to therapy, yet its regulation is incompletely understood. In vivo switching between a more differentiated, proliferative phenotype and a dedifferentiated, invasive phenotype is directed by the tumour microenvironment. We found that treatment of partially dedifferentiated, invasive A375M2 cells with two structurally unrelated p38 MAPK inhibitors, SB2021920 and BIRB796, induces a phenotype switch in 3D collagen, as documented by increased expression of melanocyte differentiation markers and a loss of invasive phenotype markers. The phenotype is accompanied by morphological change corresponding to amoeboid-mesenchymal transition. We performed RNA sequencing with an Illumina HiSeq platform to fully characterise transcriptome changes underlying the switch. Gene expression results obtained with RNA-seq were validated by comparing them with RT-qPCR. Transcriptomic data generated in the study will extend the present understanding of phenotype plasticity in melanoma and its contribution to invasion and metastasis.

Sustained Inflammatory Signalling through Stat1/Stat2/IRF9 Is Associated with Amoeboid Phenotype of Melanoma Cells.

The invasive behaviour of cancer cells underlies metastatic dissemination; however, due to the large plasticity of invasion modes, it is challenging to target. It is now widely accepted that various secreted cytokines modulate the tumour microenvironment and pro-inflammatory signalling can promote tumour progression. Here, we report that cells after mesenchymal-amoeboid transition show the increased expression of genes associated with the type I interferon response. Moreover, the sustained activation of type I interferon signalling in response to IFNß mediated by the Stat1/Stat2/IRF9 complex enhances the round amoeboid phenotype in melanoma cells, whereas its downregulation by various approaches promotes the mesenchymal invasive phenotype. Overall, we demonstrate that interferon signalling is associated with the amoeboid phenotype of cancer cells and suggest a novel role of IFNß in promoting cancer invasion plasticity, aside from its known role as a tumour suppressor.

High-throughput transcriptomic and proteomic profiling of mesenchymal-amoeboid transition in 3D collagen.

The plasticity of cancer cell invasion represents substantial hindrance for effective anti-metastatic therapy. To better understand the cancer cells' plasticity, we performed complex transcriptomic and proteomic profiling of HT1080 fibrosarcoma cells undergoing mesenchymal-amoeboid transition (MAT). As amoeboid migratory phenotype can fully manifest only in 3D conditions, all experiments were performed with 3D collagen-based cultures. Two previously described approaches to induce MAT were used: doxycycline-inducible constitutively active RhoA expression and dasatinib treatment. RNA sequencing was performed with ribo-depleted total RNA. Protein samples were analysed with tandem mass tag (TMT)-based mass spectrometry. The data provide unprecedented insight into transcriptome and proteome changes accompanying MAT in true 3D conditions.

Microtubule-targeting agents and their impact on cancer treatment.

Microtubule-targeting agents (MTAs) constitute a diverse group of chemical compounds that bind to microtubules and affect their properties and function. Disruption of microtubules induces various cellular responses often leading to cell cycle arrest or cell death, the most common effect of MTAs. MTAs have found a plethora of practical applications in weed control, as fungicides and antiparasitics, and particularly in cancer treatment. Here we summarize the current knowledge of MTAs, the mechanisms of action and their role in cancer treatment. We further outline the potential use of MTAs in anti-metastatic therapy based on inhibition of cancer cell migration and invasiveness. The two main problems associated with cancer therapy by MTAs are high systemic toxicity and development of resistance. Toxic side effects of MTAs can be, at least partly, eliminated by conjugation of the drugs with various carriers. Moreover, some of the novel MTAs overcome the resistance mediated by both multidrug resistance transporters as well as overexpression of specific ß-tubulin types. In anti-metastatic therapy, MTAs should be combined with other drugs to target all modes of cancer cell invasion.

Increased Level of Long Non-Coding RNA MALAT1 is a Common Feature of Amoeboid Invasion.

The ability of cancer cells to adopt various migration modes (the plasticity of cancer cell invasiveness) is a substantive obstacle in the treatment of metastasis, yet still an incompletely understood process. We performed a comparison of publicly available transcriptomic datasets from various cell types undergoing a switch between the mesenchymal and amoeboid migration modes. Strikingly, lncRNA MALAT1 (metastasis-associated lung adenocarcinoma transcript 1) was one of three genes that were found upregulated in all amoeboid cells analyzed. Accordingly, downregulation of MALAT1 in predominantly amoeboid cell lines A375m2 and A2058 resulted in decrease of active RhoA (Ras homolog family member A) and was accompanied by the amoeboid-mesenchymal transition in A375m2 cells. Moreover, MALAT1 downregulation in amoeboid cells led to increased cell proliferation. Our work is the first to address the role of MALAT1 in MAT/AMT (mesenchymal to amoeboid transition/amoeboid to mesenchymal transition) and suggests that increased MALAT1 expression is a common feature of amoeboid cells.

RNA-seq of macrophages of amoeboid or mesenchymal migratory phenotype due to specific structure of environment.

M2-polarized macrophages have been shown to adapt their 3D migration mode to physical properties of surrounding extracellular matrix. They migrate in the integrin-mediated adhesion and proteolytic activity-dependent "mesenchymal" mode in stiff matrices and in the integrin and protease-independent "amoeboid" mode in low density, porous environments. To find out what impact the switching between the migration modes has on expression of both protein-coding and non-coding genes we employed RNA sequencing of total RNA depleted of ribosomal RNA isolated from macrophages migrating in either mode in 3D collagens. Differentially expressed genes from both categories have been detected and the changes in expression of selected genes were further validated with RT-qPCR. The acquired data will facilitate better understanding of how mechanical properties of tissue microenvironment reflect in macrophage immune function and how the transitions between mesenchymal and amoeboid migratory modes are regulated at the gene expression level.

Migrastatics-Anti-metastatic and Anti-invasion Drugs: Promises and Challenges.

In solid cancers, invasion and metastasis account for more than 90% of mortality. However, in the current armory of anticancer therapies, a specific category of anti-invasion and antimetastatic drugs is missing. Here, we coin the term 'migrastatics' for drugs interfering with all modes of cancer cell invasion and metastasis, to distinguish this class from conventional cytostatic drugs, which are mainly directed against cell proliferation. We define actin polymerization and contractility as target mechanisms for migrastatics, and review candidate migrastatic drugs. Critical assessment of these antimetastatic agents is warranted, because they may define new options for the treatment of solid cancers.

The role of focal adhesion anchoring domains of CAS in mechanotransduction.

CAS is a docking protein, which was shown to act as a mechanosensor in focal adhesions. The unique assembly of structural domains in CAS is important for its function as a mechanosensor. The tension within focal adhesions is transmitted to a stretchable substrate domain of CAS by focal adhesion-targeting of SH3 and CCH domain of CAS, which anchor the CAS protein in focal adhesions. Mechanistic models of the stretching biosensor propose equal roles for both anchoring domains. Using deletion mutants and domain replacements, we have analyzed the relative importance of the focal adhesion anchoring domains on CAS localization and dynamics in focal adhesions as well as on CAS-mediated mechanotransduction. We confirmed the predicted prerequisite of the focal adhesion targeting for CAS-dependent mechanosensing and unraveled the critical importance of CAS SH3 domain in mechanosensing. We further show that CAS localizes to the force transduction layer of focal adhesions and that mechanical stress stabilizes CAS in focal adhesions.

The oncoprotein v-Myb activates transcription of Gremlin 2 during in vitro differentiation of the chicken neural crest to melanoblasts.

The neural crest (NC) is a transient dynamic structure of ectodermal origin, found in early vertebrate embryos. The multipotential NC cells migrate along well defined routes, differentiate to various cell types including melanocytes and participate in the formation of various permanent tissues. As there is only limited information about the molecular mechanisms controlling early events in melanocyte specification and development, we exploited the AMV v-Myb transcriptional regulator, which directs differentiation of in vitro chicken NC cells to the melanocyte lineage. This activity is strictly dependent on v-Myb specifically binding to the Myb recognition DNA element (MRE). The two tamoxifen-inducible v-Myb alleles were constructed one which recognizes the MRE and one which does not. These were activated in ex ovo NC cells, and the expression profiles of resulting cells were analyzed using Affymetrix microarrays and RT-PCR. These approaches revealed up-regulation of the BMP antagonist Gremlin 2 mRNA, and down-regulation of mRNAs encoding several epithelial genes including KRT19 as very early events following the activation of melanocyte differentiation by v-Myb. The enforced v-Myb expression in neural tubes of chicken embryos resulted in detectable presence of Gremlin 2 mRNA. However, expression of Gremlin 2 in NC cells did not promote formation of melanocytes suggesting that Gremlin 2 is not the master regulator of melanocytic differentiation.

Molecular analysis of the TGF-beta controlled gene expression program in chicken embryo dermal myofibroblasts.

The myofibroblast is a mesenchymal cell characterized by synthesis of the extracellular matrix, plus contractile and secretory activities. Myofibroblasts participate in physiological tissue repair, but can also cause devastating fibrosis. They are present in the tumor stroma of carcinomas and contribute to tumor growth and spreading. As myofibroblasts derive from various cell types and appear in a variety of tissues, there is marked variability in their phenotype. As regulatory mechanisms of wound healing are likely conserved among vertebrates, detailed knowledge of these mechanisms in more distant species will help to distinguish general from specific phenomena. To provide this as yet missing comparison, we analyzed the impact of the chemical inhibition of TGF-beta signaling on gene expression in chicken embryo dermal myofibroblasts. We revealed genes previously reported in mammalian systems (e.g. SPON2, ASPN, COMP, LUM, HAS2, IL6, CXCL12, VEGFA) as well as novel TGF-beta dependent genes, among them PGF, VEGFC, PTN, FAM180A, FIBIN, ZIC1, ADCY2, RET, HHIP and DNER. Inhibition of TGF-beta signaling also induced multiple genes, including NPR3, AGTR2, MTUS1, SOD3 and NOV. We also analyzed the effects of long term inhibition, and found that it is not able to induce myofibroblast dedifferentiation.

Effective myofibroblast dedifferentiation by concomitant inhibition of TGF-ß signaling and perturbation of MAPK signaling.

Fibrotic diseases are a group of pathologies with high incidence and mortality. Despite extensive research efforts, effective therapies are still not available. Understanding the molecular mechanisms driving the onset, progression and possible resolution of fibrosis is a prerequisite to the development of successful therapies. The central role of the TGF-ß pathway and myofibroblasts in the pathogenesis of fibrosis is now generally accepted. The possible mechanisms of myofibroblast elimination or dedifferentiation, on the other hand, are still almost uncharted territory. Here we show that sustained expression of some components of MAPK signaling pathway (PDGFB, Ha-Ras(G12V) or the transcription factor EGR4) in primary chicken embryo dermal myofibroblasts results in a loss of autocrine TGF-ß signaling and suppression of the myofibroblastic phenotype, characterized by the loss of alpha smooth muscle actin fibers and a substantial reduction in the production of extracellular matrix. Detailed analysis of the possible molecular mechanisms employed by EGR4 revealed FOXG1, BAMBI, NAB1, NAB2 and DUSP5 genes forming an EGR4 regulated network counteracting autocrine TGF-ß signaling. We have also found that a combination of chemical inhibition of TGF-ß signaling and perturbation of MAPK signaling with phorbol ester mimics the anti-fibrotic effects of PDGFB, Ha-Ras(G12V) and EGR4.

c-Myb inhibits myoblast fusion.

Satellite cells represent a heterogeneous population of stem and progenitor cells responsible for muscle growth, repair and regeneration. We investigated whether c-Myb could play a role in satellite cell biology because our previous results using satellite cell-derived mouse myoblast cell line C2C12 showed that c-Myb was expressed in growing cells and downregulated during differentiation. We detected c-Myb expression in activated satellite cells of regenerating muscle. c-Myb was also discovered in activated satellite cells associated with isolated viable myofiber and in descendants of activated satellite cells, proliferating myoblasts. However, no c-Myb expression was detected in multinucleated myotubes originated from fusing myoblasts. The constitutive expression of c-Myb lacking the 3' untranslated region (3' UTR) strongly inhibited the ability of myoblasts to fuse. The inhibition was dependent on intact c-Myb transactivation domain as myoblasts expressing mutated c-Myb in transactivation domain were able to fuse. The absence of 3' UTR of c-Myb was also important because the expression of c-Myb coding region with its 3' UTR did not inhibit myoblast fusion. The same results were repeated in C2C12 cells as well. Moreover, it was documented that 3' UTR of c-Myb was responsible for downregulation of c-Myb protein levels in differentiating C2C12 cells. DNA microarray analysis of C2C12 cells revealed that the expression of several muscle-specific genes was downregulated during differentiation of c-Myb-expressing cells, namely: ACTN2, MYH8, TNNC2, MYOG, CKM and LRRN1. A detailed qRT-PCR analysis of MYOG, TNNC2 and LRRN1 is presented. Our findings thus indicate that c-Myb is involved in regulating the differentiation program of myogenic progenitor cells as its expression blocks myoblast fusion.

Downregulation of HOPX controls metastatic behavior in sarcoma cells and identifies genes associated with metastasis.

UNLABELLED: Comparing the gene expression profiles of metastatic and nonmetastatic cells has the power to reveal candidate metastasis-associated genes, whose involvement in metastasis can be experimentally tested. In this study, differentially expressed genes were explored in the v-src-transformed metastatic cell line PR9692 and its nonmetastatic subclone PR9692-E9. First, the contribution of homeodomain only protein X (HOPX) in metastasis formation and development was assessed. HOPX-specific knockdown decreased HOPX expression in the nonmetastatic subclone and displayed reduced cell motility in vitro. Critically, HOPX knockdown decreased the in vivo metastatic capacity in a syngeneic animal model system. Genomic analyses identified a cadre of genes affected by HOPX knockdown that intersected significantly with genes previously found to be differentially expressed in metastatic versus nonmetastatic cells. Furthermore, 232 genes were found in both screens with at least a two-fold change in gene expression, and a number of high-confidence targets were validated for differential expression. Importantly, significant changes were demonstrated in the protein expression level of three metastatic-associated genes (NCAM, FOXG1, and ITGA4), and knockdown of one of the identified HOPX-regulated metastatic genes, ITGA4, showed marked inhibition of cell motility and metastasis formation. These data demonstrate that HOPX is a metastasis-associated gene and that its knockdown decreases the metastatic activity of v-src-transformed cells through altered gene expression patterns. IMPLICATIONS: This study provides new mechanistic insight into a HOPX-regulated metastatic dissemination signature.