Stabilizing vimentin phosphorylation inhibits stem-like cell properties and metastasis of hybrid epithelial/mesenchymal carcinomas.

Epithelial-mesenchymal transition (EMT) empowers epithelial cells with mesenchymal and stem-like attributes, facilitating metastasis, a leading cause of cancer-related mortality. Hybrid epithelial-mesenchymal (E/M) cells, retaining both epithelial and mesenchymal traits, exhibit heightened metastatic potential and stemness. The mesenchymal intermediate filament, vimentin, is upregulated during EMT, enhancing the resilience and invasiveness of carcinoma cells. The phosphorylation of vimentin is critical to its structure and function. Here, we identify that stabilizing vimentin phosphorylation at serine 56 induces multinucleation, specifically in hybrid E/M cells with stemness properties but not epithelial or mesenchymal cells. Cancer stem-like cells are especially susceptible to vimentin-induced multinucleation relative to differentiated cells, leading to a reduction in self-renewal and stemness. As a result, vimentin-induced multinucleation leads to sustained inhibition of stemness properties, tumor initiation, and metastasis. These observations indicate that a single, targetable phosphorylation event in vimentin is critical for stemness and metastasis in carcinomas with hybrid E/M properties.

Proactive and reactive roles of TGF-ß in cancer.

Cancer cells adapt to varying stress conditions to survive through plasticity. Stem cells exhibit a high degree of plasticity, allowing them to generate more stem cells or differentiate them into specialized cell types to contribute to tissue development, growth, and repair. Cancer cells can also exhibit plasticity and acquire properties that enhance their survival. TGF-ß is an unrivaled growth factor exploited by cancer cells to gain plasticity. TGF-ß-mediated signaling enables carcinoma cells to alter their epithelial and mesenchymal properties through epithelial-mesenchymal plasticity (EMP). However, TGF-ß is a multifunctional cytokine; thus, the signaling by TGF-ß can be detrimental or beneficial to cancer cells depending on the cellular context. Those cells that overcome the anti-tumor effect of TGF-ß can induce epithelial-mesenchymal transition (EMT) to gain EMP benefits. EMP allows cancer cells to alter their cell properties and the tumor immune microenvironment (TIME), facilitating their survival. Due to the significant roles of TGF-ß and EMP in carcinoma progression, it is essential to understand how TGF-ß enables EMP and how cancer cells exploit this plasticity. This understanding will guide the development of effective TGF-ß-targeting therapies that eliminate cancer cell plasticity.

Mechanisms of cancer metastasis.

Metastatic cancer is almost always terminal, and more than 90% of cancer deaths result from metastatic disease. Combating cancer metastasis and post-therapeutic recurrence successfully requires understanding each step of metastatic progression. This review describes the current state of knowledge of the etiology and mechanism of cancer progression from primary tumor growth to the formation of new tumors in other parts of the body. Open questions, avenues for future research, and therapeutic approaches with the potential to prevent or inhibit metastasis through personalization to each patient's mutation and/or immune profile are also highlighted.

Synthesis and biological evaluation of novel FiVe1 derivatives as potent and selective agents for the treatment of mesenchymal cancers.

Epithelial-mesenchymal transition (EMT) endows stem cell-like properties to cancer cells. Targeting this process represents a potential therapeutic approach to overcome cancer metastasis and chemotherapy resistance. FiVe1 was identified from an EMT-based synthetic lethality screen and was found to inhibit the stem cell-like properties and proliferation of not only cancer cells undergoing EMT, but also more broadly in mesenchymal cancers that include therapeutically intractable soft tissue sarcomas. FiVe1 functions by directly binding to the type III intermediate filament protein vimentin (VIM) in a mode that induces hyperphosphorylation of Ser56, which results in selective disruption of mitosis and induced multinucleation in transformed VIM-expressing mesenchymal cancer cell types. Cell-based potency (IC50 = 1.6 µM, HT-1080 fibrosarcoma), poor solubility (<1 µM) and low oral bioavailability limits the direct application of FiVe1 as an in vivo probe or therapeutic agent. To overcome these drawbacks, we performed structure-activity relationship (SAR) studies and synthesized a set of 35 new compounds, consisting of diverse modifications of the FiVe1 scaffold. Among these compounds, 4e showed a marked improvement in potency (IC50 = 44 nM, 35-fold improvement, HT-1080) and cell type selectivity (19-fold improvement), when compared to FiVe1. Improvements in the potency of 4e, in terms of overall cytotoxicity, directly correlate with VIM Ser56 phosphorylation status and the oral bioavailability and pharmacokinetic profiles of 4e in mouse are superior to FiVe1. Successful optimization also resulted in potent and selective derivatives 11a, 11j and 11k, which exhibited superior pharmacological profiles, in terms of metabolic stability and aqueous solubility. Collectively, these optimization efforts have resulted in the development of promising FiVe1 analogs with potential applications in the treatment of mesenchymal cancers, as well as in the study of VIM-related biology.

Enrichment of Cancer Stem Cells in a Tumorsphere Assay.

Cancer stem cells (CSCs) are a small subpopulation of self-renewing cancer cells that are present within tumors. CSCs possess tumor initiation potential as well as the ability to resist toxic compounds and chemotherapeutic agents through the upregulation of drug efflux transporters, DNA repair pathways, and survival cascades. Accumulating evidence suggests that CSCs are responsible for tumor relapse and resistance to chemotherapeutic agents and that targeting CSCs is critical to inhibition of cancer progression. Therefore, isolation and characterization of CSCs is important in studying tumor initiation and progression. In this chapter, we provide a detailed method for the identification and isolation of CSCs.

In Vitro Quantification of Cancer Stem Cells Using a Mammosphere Formation Assay.

Cancer stem cells (CSCs) are a small subpopulation of self-renewing cancer cells that are present within tumors. In this chapter, we provide a detailed method for the quantification of CSCs in vitro through mammosphere formation.

Limiting Dilution Tumor Initiation Assay: An In Vivo Approach for the Study of Cancer Stem Cells.

Cancer stem cells (CSCs) are a small subpopulation of self-renewing cancer cells that are present within tumors. Calculating the frequency of tumor-initiating cells is important in the assessment of the number of CSCs present in a cell population. In this chapter, we present a protocol developed for quantification of CSCs from breast cancer tumors that can be adapted to CSCs from other types of tumors.

Vimentin and cytokeratin: Good alone, bad together.

The cytoskeleton plays an integral role in maintaining the integrity of epithelial cells. Epithelial cells primarily employ cytokeratin in their cytoskeleton, whereas mesenchymal cells use vimentin. During the epithelial-mesenchymal transition (EMT), cytokeratin-positive epithelial cells begin to express vimentin. EMT induces stem cell properties and drives metastasis, chemoresistance, and tumor relapse. Most studies of the functions of cytokeratin and vimentin have relied on the use of either epithelial or mesenchymal cell types. However, it is important to understand how these two cytoskeleton intermediate filaments function when co-expressed in cells undergoing EMT. Here, we discuss the individual and shared functions of cytokeratin and vimentin that coalesce during EMT and how alterations in intermediate filament expression influence carcinoma progression.

Mitogen-activated protein kinase regulation of the phosphodiesterase RegA in early Dictyostelium development.

Mitogen-activated protein kinase (MAPK) regulation of cAMP-specific phosphodiesterase function has been demonstrated in mammalian cells and suspected to occur in other eukaryotes. Epistasis analysis in the soil amoeba Dictyostelium discoideum suggests the atypical MAPK Erk2 downregulates the function of the cAMP-specific phosphodiesterase RegA to regulate progression of the developmental life cycle. A putative MAPK docking motif located near a predicted MAPK phosphorylation site was characterized for contributions to RegA function and binding to Erk2 because a similar docking motif has been previously characterized in the mammalian PDE4D phosphodiesterase. The overexpression of RegA with alterations to this docking motif (RegAD-) restored RegA function to regA- cells based on developmental phenotypes, but low-level expression of RegAD- from the endogenous regA promoter failed to rescue wild-type morphogenesis. Co-immunoprecipitation analysis indicated that Erk2 associates with both RegA and RegAD-, suggesting the docking motif is not required for this association. Epistasis analysis between regA and the only other Dictyostelium MAPK, erk1, suggests Erk1 and RegA can function in different pathways but that some erk1- phenotypes may require cAMP signalling. These results imply that MAPK downregulation of RegA in Dictyostelium is accomplished through a different mechanism than MAPK regulation of cAMP-specific phosphodiesterases in mammalian cells and that the regulation in Dictyostelium does not require a proximal MAPK docking motif.

Multiple phosphorylation sites on the RegA phosphodiesterase regulate Dictyostelium development.

In Dictyostelium, the intracellular cAMP-specific phosphodiesterase RegA is a negative regulator of cAMP-dependent protein kinase (PKA), a key determinant in the timing of developmental morphogenesis and spore formation. To assess the role of protein kinases in the regulation of RegA function, this study identified phosphorylation sites on RegA and characterized the role of these modifications through the analysis of phospho-mimetic and phospho-ablative mutations. Mutations affecting residue T676 of RegA, a presumed target of the atypical MAP kinase Erk2, altered the rate of development and impacted cell distribution in chimeric organisms suggesting that phosphorylation of this residue reduces RegA function and regulates cell localization during multicellular development. Mutations affecting the residue S142 of RegA also impacted the rate developmental morphogenesis but in a manner opposite of changes at T676 suggesting the phosphorylation of the S142 residue increases RegA function. Mutations affecting residue S413 residue altered aggregate sizes and delayed developmental progression suggesting that PKA operates in a negative feedback mechanism to increase RegA function. These results suggest that the phosphorylation of different residues on RegA can lead to increased or decreased RegA function and therefore in turn regulate developmental processes such as aggregate formation, cell distribution, and the kinetics of developmental morphogenesis.

Dictyostelium Erk2 is an atypical MAPK required for chemotaxis.

The Dictyostelium genome encodes only two MAPKs, Erk1 and Erk2, and both are expressed during growth and development. Reduced levels of Erk2 expression have been shown previously to restrict cAMP production during development but still allow for chemotactic movement. In this study the erk2 gene was disrupted to eliminate Erk2 function. The absence of Erk2 resulted in a complete loss of folate and cAMP chemotaxis suggesting that this MAPK plays an integral role in the signaling mechanisms involved with this cellular response. However, folate stimulation of early chemotactic responses, such as Ras and PI3K activation and rapid actin filament formation, were not affected by the loss of Erk2 function. The erk2- cells had a severe defect in growth on bacterial lawns but assays of bacterial cell engulfment displayed only subtle changes in the rate of bacterial engulfment. Only cells with no MAPK function, erk1-erk2- double mutants, displayed a severe proliferation defect in axenic medium. Loss of Erk2 impaired the phosphorylation of Erk1 in secondary responses to folate stimulation indicating that Erk2 has a role in the regulation of Erk1 activation during chemotaxis. Loss of the only known Dictyostelium MAPK kinase, MekA, prevented the phosphorylation of Erk1 but not Erk2 in response to folate and cAMP confirming that Erk2 is not regulated by a conventional MAP2K. This lack of MAP2K phosphorylation of Erk2 and the sequence similarity of Erk2 to mammalian MAPK15 (Erk8) suggest that the Dictyostelium Erk2 belongs to a group of atypical MAPKs. MAPK activation has been observed in chemotactic responses in a wide range of organisms but this study demonstrates an essential role for MAPK function in chemotactic movement. This study also confirms that MAPKs provide critical contributions to cell proliferation.

Acanthamoeba and Dictyostelium Use Different Foraging Strategies.

Amoeba often use cell movement as a mechanism to find food, such as bacteria, in their environment. The chemotactic movement of the soil amoeba Dictyostelium to folate or other pterin compounds released by bacteria is a well-documented foraging mechanism. Acanthamoeba can also feed on bacteria but relatively little is known about the mechanism(s) by which this amoeba locates bacteria. Acanthamoeba movement in the presence of folate or bacteria was analyzed in above agar assays and compared to that observed for Dictyostelium. The overall mobility of Acanthamoeba was robust like that of Dictyostelium but Acanthamoeba did not display a chemotactic response to folate. In the presence of bacteria, Acanthamoeba only showed a marginal bias in directed movement whereas Dictyostelium displayed a strong chemotactic response. A comparison of genomes revealed that Acanthamoeba and Dictyostelium share some similarities in G protein signaling components but that specific G proteins used in Dictyostelium chemotactic responses were not present in current Acanthamoeba genome sequence data. The results of this study suggest that Acanthamoeba does not use chemotaxis as the primary mechanism to find bacterial food sources and that the chemotactic responses of Dictyostelium to bacteria may have co-evolved with chemotactic responses that facilitate multicellular development.