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.

• Keywords
• amoeboid invasion, cancer, melanoma, metastasis, phenotype switch,
• MeSH
• Cell Differentiation drug effects   genetics   MeSH
• Phenotype MeSH
• Gene Ontology MeSH
• Imidazoles pharmacology   MeSH
• Protein Kinase Inhibitors pharmacology   MeSH
• Collagen metabolism   MeSH
• Humans MeSH
• Melanoma genetics   pathology   MeSH
• p38 Mitogen-Activated Protein Kinases antagonists & inhibitors   metabolism   MeSH
• Cell Line, Tumor MeSH
• Tumor Microenvironment drug effects   genetics   MeSH
• Naphthalenes pharmacology   MeSH
• Reverse Transcriptase Polymerase Chain Reaction MeSH
• Cell Proliferation drug effects   genetics   MeSH
• Pyrazoles pharmacology   MeSH
• Pyridines pharmacology   MeSH
• Gene Expression Regulation, Neoplastic drug effects   MeSH
• RNA-Seq methods   MeSH
• Gene Expression Profiling methods   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)imidazole MeSH Browser
• doramapimod MeSH Browser
• Imidazoles MeSH
• Protein Kinase Inhibitors MeSH
• Collagen MeSH
• p38 Mitogen-Activated Protein Kinases MeSH
• Naphthalenes MeSH
• Pyrazoles MeSH
• Pyridines MeSH

Observation and analysis of cancer cell behaviour in 3D environment is essential for full understanding of the mechanisms of cancer cell invasion. However, label-free imaging of live cells in 3D conditions is optically more challenging than in 2D. Quantitative phase imaging provided by coherence controlled holographic microscopy produces images with enhanced information compared to ordinary light microscopy and, due to inherent coherence gate effect, enables observation of live cancer cells' activity even in scattering milieu such as the 3D collagen matrix. Exploiting the dynamic phase differences method, we for the first time describe dynamics of differences in cell mass distribution in 3D migrating mesenchymal and amoeboid cancer cells, and also demonstrate that certain features are shared by both invasion modes. We found that amoeboid fibrosarcoma cells' membrane blebbing is enhanced upon constriction and is also occasionally present in mesenchymally invading cells around constricted nuclei. Further, we demonstrate that both leading protrusions and leading pseudopods of invading fibrosarcoma cells are defined by higher cell mass density. In addition, we directly document bundling of collagen fibres by protrusions of mesenchymal fibrosarcoma cells. Thus, such a non-invasive microscopy offers a novel insight into cellular events during 3D invasion.

• MeSH
• Cell Membrane metabolism   MeSH
• Cell Culture Techniques methods   MeSH
• Fibrosarcoma diagnostic imaging   pathology   MeSH
• Holography instrumentation   methods   MeSH
• Intravital Microscopy instrumentation   methods   MeSH
• Neoplasm Invasiveness diagnostic imaging   pathology   MeSH
• Collagen metabolism   MeSH
• Humans MeSH
• Cell Line, Tumor MeSH
• Cell Movement * MeSH
• Pseudopodia metabolism   MeSH
• Imaging, Three-Dimensional instrumentation   methods   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Collagen MeSH

Apico-basal polarity is typical of cells present in differentiated epithelium while front-rear polarity develops in motile cells. In cancer development, the transition from epithelial to migratory polarity may be seen as the hallmark of cancer progression to an invasive and metastatic disease. Despite the morphological and functional dissimilarity, both epithelial and migratory polarity are controlled by a common set of polarity complexes Par, Scribble and Crumbs, phosphoinositides, and small Rho GTPases Rac, Rho and Cdc42. In epithelial tissues, their mutual interplay ensures apico-basal and planar cell polarity. Accordingly, altered functions of these polarity determinants lead to disrupted cell-cell adhesions, cytoskeleton rearrangements and overall loss of epithelial homeostasis. Polarity proteins are further engaged in diverse interactions that promote the establishment of front-rear polarity, and they help cancer cells to adopt different invasion modes. Invading cancer cells can employ either the collective, mesenchymal or amoeboid invasion modes or actively switch between them and gain intermediate phenotypes. Elucidation of the role of polarity proteins during these invasion modes and the associated transitions is a necessary step towards understanding the complex problem of metastasis. In this review we summarize the current knowledge of the role of cell polarity signaling in the plasticity of cancer cell invasiveness.

• Keywords
• AMT, EMT, invasion, plasticity, polarity,
• MeSH
• Neoplasm Invasiveness pathology   MeSH
• Humans MeSH
• Neoplasms pathology   MeSH
• Cell Polarity physiology   MeSH
• Signal Transduction physiology   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Review MeSH

BACKGROUND: Although there is extensive evidence for the amoeboid invasiveness of cancer cells in vitro, much less is known about the role of amoeboid invasiveness in metastasis and the importance of Rho/ROCK/MLC signaling in this process. RESULTS: We analyzed the dependence of amoeboid invasiveness of rat and chicken sarcoma cells and the metastatic activity of chicken cells on individual elements of the Rho/ROCK/MLC pathway. In both animal models, inhibition of Rho, ROCK or MLC resulted in greatly decreased cell invasiveness in vitro, while inhibition of extracellular proteases using a broad spectrum inhibitor did not have a significant effect. The inhibition of both Rho activity and MLC phosphorylation by dominant negative mutants led to a decreased capability of chicken sarcoma cells to metastasize. Moreover, the overexpression of RhoA in non-metastatic chicken cells resulted in the rescue of both invasiveness and metastatic capability. Rho and ROCK, unlike MLC, appeared to be directly involved in the maintenance of the amoeboid phenotype, as their inhibition resulted in the amoeboid-mesenchymal transition in analyzed cell lines. CONCLUSION: Taken together, these results suggest that protease-independent invasion controlled by elements of the Rho/ROCK/MLC pathway can be frequently exploited by metastatic sarcoma cells.

• MeSH
• Neoplasm Invasiveness MeSH
• rho-Associated Kinases metabolism   MeSH
• Rats MeSH
• Chickens MeSH
• Myosin Light Chains metabolism   MeSH
• Cell Line, Tumor MeSH
• Cell Movement MeSH
• rho GTP-Binding Proteins metabolism   MeSH
• Sarcoma metabolism   pathology   MeSH
• Signal Transduction MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• rho-Associated Kinases MeSH
• Myosin Light Chains MeSH
• rho GTP-Binding Proteins MeSH

During malignant neoplastic progression the cells undergo genetic and epigenetic cancer-specific alterations that finally lead to a loss of tissue homeostasis and restructuring of the microenvironment. The invasion of cancer cells through connective tissue is a crucial prerequisite for metastasis formation. Although cell invasion is foremost a mechanical process, cancer research has focused largely on gene regulation and signaling that underlie uncontrolled cell growth. More recently, the genes and signals involved in the invasion and transendothelial migration of cancer cells, such as the role of adhesion molecules and matrix degrading enzymes, have become the focus of research. In this review we discuss how the structural and biomechanical properties of extracellular matrix and surrounding cells such as endothelial cells influence cancer cell motility and invasion. We conclude that the microenvironment is a critical determinant of the migration strategy and the efficiency of cancer cell invasion.

• Publication type
• Journal Article MeSH