Histone methylation in pre-cancerous liver diseases and hepatocellular carcinoma: recent overview

Hepatocellular carcinoma (HCC) is the prevalent form of liver cancer in adults and the fourth most common cause of cancer-related death worldwide. HCC predominantly arises in the context of cirrhosis as a result of chronic liver disease, injury and inflammation. Full-blown HCC has poor prognosis because it is highly aggressive and resistant to therapy. Consequently, interventions that can prevent or restrain HCC emergence from pre-cancerous diseased liver are a desirable strategy. Histone methylation is a dynamic, reversible epigenetic modification involving the addition or removal of methyl groups from lysine, arginine or glutamine residues. Aberrant activity of histone methylation writers, erases and readers has been implicated in several cancer types, including HCC. In this review, we provide an overview of research on the role of histone methylation in pre-cancerous and cancerous HCC published over the last 5 years. In particular, we present the evidence linking environmental factors such as diet, viral infections and carcinogenic agents with dysregulation of histone methylation during liver cancer progression with the aim to highlight future therapeutic possibilities (AU)

Histone methylation in pre-cancerous liver diseases and hepatocellular carcinoma: recent overview.

Hepatocellular carcinoma (HCC) is the prevalent form of liver cancer in adults and the fourth most common cause of cancer-related death worldwide. HCC predominantly arises in the context of cirrhosis as a result of chronic liver disease, injury and inflammation. Full-blown HCC has poor prognosis because it is highly aggressive and resistant to therapy. Consequently, interventions that can prevent or restrain HCC emergence from pre-cancerous diseased liver are a desirable strategy. Histone methylation is a dynamic, reversible epigenetic modification involving the addition or removal of methyl groups from lysine, arginine or glutamine residues. Aberrant activity of histone methylation writers, erases and readers has been implicated in several cancer types, including HCC. In this review, we provide an overview of research on the role of histone methylation in pre-cancerous and cancerous HCC published over the last 5 years. In particular, we present the evidence linking environmental factors such as diet, viral infections and carcinogenic agents with dysregulation of histone methylation during liver cancer progression with the aim to highlight future therapeutic possibilities.

LincRNAs and snoRNAs in Breast Cancer Cell Metastasis: The Unknown Players.

Recent advances in research have led to earlier diagnosis and targeted therapies against breast cancer, which has resulted in reduced breast cancer-related mortality. However, the majority of breast cancer-related deaths are due to metastasis of cancer cells to other organs, a process that has not been fully elucidated. Among the factors and genes implicated in the metastatic process regulation, non-coding RNAs have emerged as crucial players. This review focuses on the role of long intergenic noncoding RNAs (lincRNAs) and small nucleolar RNAs (snoRNAs) in breast cancer cell metastasis. LincRNAs are transcribed between two protein-coding genes and are longer than 200 nucleotides, they do not code for a specific protein but function as regulatory molecules in processes such as cell proliferation, apoptosis, epithelial-to-mesenchymal transition, migration, and invasion while most of them are highly elevated in breast cancer tissues and seem to function as competing endogenous RNAs (ceRNAs) inhibiting relevant miRNAs that specifically target vital metastasis-related genes. Similarly, snoRNAs are 60-300 nucleotides long and are found in the nucleolus being responsible for the post-transcriptional modification of ribosomal and spliceosomal RNAs. Most snoRNAs are hosted inside intron sequences of protein-coding and non-protein-coding genes, and they also regulate metastasis-related genes affecting related cellular properties.

Engineered magnetoactive collagen hydrogels with tunable and predictable mechanical response.

In the present study, the synthesis of superparamagnetic collagen-based nanocomposite hydrogels with tunable swelling, mechanical and magnetic properties is reported. The fabrication strategy involved the preparation of pristine collagen type-I hydrogels followed by their immersion in highly stable aqueous solutions containing pre-formed double-layer oleic acid-coated hydrophilic magnetite nanoparticles (OA.OA.Fe3O4) at different concentrations, to interrogate nanoparticles' deposition within the 3D fibrous collagen matrix. Besides the investigation of the morphology, composition and magnetic properties of the produced materials, their mechanical properties were experimentally evaluated under confined compressive loading conditions while an exponential constitutive equation was employed to describe their mechanical response. Moreover, the deposition of the nanoparticles in the collagenous matrix was modeled mathematically with respect to the swelling of the gel and the effective stiffness of the matrix. The model recapitulated nanoparticle diffusion and deposition as well as hydrogel swelling, in terms of nanoparticles' size and concentration of OA.OA.Fe3O4 aqueous solution.

ILK silencing inhibits migration and invasion of more invasive glioblastoma cells by downregulating ROCK1 and Fascin-1.

Glioblastoma multiforme (GBM) is the most aggressive type of brain tumor and it is associated with poor survival. Integrin-linked kinase (ILK) is a serine/threonine protein pseudo-kinase that binds to the cytoplasmic domains of ß1 and ß3 integrins and has been previously shown to promote invasion and metastasis in many cancer types, including GBM. However, little is known regarding the exact molecular mechanism implicating ILK in GBM aggressiveness. In this study, we used two brain cell lines, the non-invasive neuroglioma H4 cells, and the highly invasive glioblastoma A172 cells, which express ILK in much higher levels than H4. We studied the effect of ILK silencing on the metastatic behavior of glioblastoma cells in vitro and elucidate the underlying molecular mechanism. We showed that siRNA-mediated silencing of ILK inhibits cell migration and invasion of the highly invasive A172 cells while it does not affect the migratory and invasive capacity of H4 cells. These data were also supported by respective changes in the expression of Rho-associated kinase 1 (ROCK1), fascin actin-bundling protein 1 (FSCN1), and matrix metalloproteinase 13 (MMP13), which are known to regulate cell migration and invasion. Our findings were further corroborated by analyzing the Cancer Genome Atlas Glioblastoma Multiforme (TCGA-GBM) dataset. We conclude that ILK promotes glioblastoma cell invasion through activation of ROCK1 and FSCN1 in vitro, providing a more exact molecular mechanism for its action.

Ras Suppressor-1 (RSU1) in Cancer Cell Metastasis: A Tale of a Tumor Suppressor.

Cancer is a multifactorial disease responsible for millions of deaths worldwide. It has a strong genetic background, as mutations in oncogenes or tumor suppressor genes contribute to the initiation of cancer development. Integrin signaling as well as the signaling pathway of Ras oncogene, have been long implicated both in carcinogenesis and disease progression. Moreover, they have been involved in the promotion of metastasis, which accounts for the majority of cancer-related deaths. Ras Suppressor-1 (RSU1) was identified as a suppressor of Ras-induced transformation and was shown to localize to cell-extracellular matrix adhesions. Recent findings indicate that its expression is elevated in various cancer types, while its role in regulating metastasis-related cellular processes remains largely unknown. Interestingly, there is no in vivo work in the field to date, and thus, all relevant knowledge stems from in vitro studies. In this review, we summarize recent studies using breast, liver and brain cancer cell lines and highlight the role of RSU1 in regulating cancer cell invasion.

Silencing of Growth Differentiation Factor-15 Promotes Breast Cancer Cell Invasion by Down-regulating Focal Adhesion Genes.

BACKGROUND/AIM: As metastasis accounts for most breast cancer (BC)-related deaths, identifying key players becomes research priority. Growth differentiation factor-15 (GDF15), a member of the transforming growth factor-ß superfamily, is affected by the actin cytoskeleton and has been associated with cancer. However, its exact role in BC cell invasiveness is vague. MATERIALS AND METHODS: GDF15 short-hairpin (shRNA)-mediated silencing was used to inhibit GDF15 expression in MCF-7 and MDA-MB-231 BC cells and gene expression of relevant focal adhesion (FA) genes, cell migration, invasion and tumor spheroid invasion were subsequently analyzed. RESULTS: GDF15 silencing promoted cell migration, cell invasion as well as tumor spheroid invasion and up-regulated urokinase plasminogen activator (uPA) and FA genes, integrin-linked kinase (ILK), LIM zinc finger domain containing 1 (LIMS1), α-parvin (PARVA), and RAS suppressor-1 (RSU1). Computational analysis of Cancer Genome Atlas BC dataset however, revealed no significant correlation between GDF15 expression and metastasis pointing towards a more complex molecular interplay between GDF15, actin cytoskeleton and FA-related genes which ultimately affects their expression pattern, in vivo. CONCLUSION: GDF15 suppresses BC cell invasion in vitro through down-regulation of FA genes but its role in BC is more complicated in vivo and warrants further investigation.

TGF-ß inhibition combined with cytotoxic nanomedicine normalizes triple negative breast cancer microenvironment towards anti-tumor immunity.

Tumor normalization strategies aim to improve tumor blood vessel functionality (i.e., perfusion) by reducing the hyper-permeability of tumor vessels or restoring compressed vessels. Despite progress in strategies to normalize the tumor microenvironment (TME), their combinatorial antitumor effects with nanomedicine and immunotherapy remain unexplored. Methods: Here, we re-purposed the TGF-ß inhibitor tranilast, an approved anti-fibrotic and antihistamine drug, and combined it with Doxil nanomedicine to normalize the TME, increase perfusion and oxygenation, and enhance anti-tumor immunity. Specifically, we employed two triple-negative breast cancer (TNBC) mouse models to primarily evaluate the therapeutic and normalization effects of tranilast combined with doxorubicin and Doxil. We demonstrated the optimized normalization effects of tranilast combined with Doxil and extended our analysis to investigate the effect of TME normalization to the efficacy of immune checkpoint inhibitors. Results: Combination of tranilast with Doxil caused a pronounced reduction in extracellular matrix components and an increase in the intratumoral vessel diameter and pericyte coverage, indicators of TME normalization. These modifications resulted in a significant increase in tumor perfusion and oxygenation and enhanced treatment efficacy as indicated by the notable reduction in tumor size. Tranilast further normalized the immune TME by restoring the infiltration of T cells and increasing the fraction of T cells that migrate away from immunosuppressive cancer-associated fibroblasts. Furthermore, we found that combining tranilast with Doxil nanomedicine, significantly improved immunostimulatory M1 macrophage content in the tumorigenic tissue and improved the efficacy of the immune checkpoint blocking antibodies anti-PD-1/anti-CTLA-4. Conclusion: Combinatorial treatment of tranilast with Doxil optimizes TME normalization, improves immunostimulation and enhances the efficacy of immunotherapy.

Coordinated Expression of Ras Suppressor 1 (RSU-1) and Growth Differentiation Factor 15 (GDF15) Affects Glioma Cell Invasion.

Glioblastoma multiforme (GBM) is the most aggressive type of brain tumor due to its invasive phenotype. Ras suppressor 1 (RSU-1) is a cell-extracellular matrix adhesion protein and we recently found that it promotes cell invasion in aggressive cells and inhibits it in non-invasive. Growth differentiation factor-15 (GDF15) is known to be involved in actin cytoskeleton reorganization and metastasis. In this study, we used three brain cell lines (H4, SW1088 and A172) with increasing RSU-1 expression levels and invasive capacity and decreasing GDF15 levels to investigate the interplay between RSU-1 and GDF15 with regard to cell invasion. Four experimental approaches were used: (a) GDF15 treatment, (b) Rsu-1 silencing, (c) GDF15 silencing, and (d) combined GDF15 treatment and RSU-1 silencing. We found that the differential expression of RSU-1 and GDF15 in H4 and A172 cells leading to inhibition of cell invasion in H4 cells and promotion in A172 through respective changes in PINCH1, RhoA and MMP-13 expression. Interestingly SW1088, with intermediate RSU-1 and GDF15 expression, were not affected by any treatment. We conclude that there is a strong connection between RSU-1 and GDF15 in H4, SW1088 and A172 cells and the relative expression of these two proteins is fundamental in affecting their invasive fate.

Ras suppressor-1 (RSU-1) promotes cell invasion in aggressive glioma cells and inhibits it in non-aggressive cells through STAT6 phospho-regulation.

Most gliomas are invasive tumors formed from glial cells and associated with high mortality rates. In this study, we characterized four glioma cell lines of varying degree of aggressiveness (H4, SW1088, A172 and U87-MG) in terms of morphology, cytoskeleton organization and stiffness, and evaluated their invasive potential by performing invasion, colony forming and spheroid invasion assays. Cells were divided into two distinct groups: aggressive cell lines (A172 and U87-MG) with more elongated, softer and highly invasive cells and less aggressive cells (H4 and SW088). Interestingly, we found that Ras Suppressor-1 (RSU-1), a cell-matrix adhesion protein involved in cancer cell invasion, was significantly upregulated in more aggressive glioma cells compared to less aggressive. Importantly, RSU-1 silencing had opposing effects on glioma cell invasion depending on their aggressiveness, inhibiting migration and invasion of aggressive cells and promoting those of less aggressive cells. Finally, we found that RSU-1 silencing in aggressive cells led to decreased Signal Transducer and Activator of Transcription6 (STAT6) phosphorylation and Matrix Metalloproteinase13 (MMP13) expression in contrast to less invasive cells. Our study demonstrates that RSU-1 promotes invasion of aggressive glioma cells and inhibits it in the non-aggressive cells, indicating that it could serve as a predictor of gliomas progression.

Collagen content and extracellular matrix cause cytoskeletal remodelling in pancreatic fibroblasts.

In many solid tumours a desmoplastic reaction takes place, which results in tumour tissue stiffening due to the extensive production of extracellular matrix (ECM) proteins, such as collagen, by stromal cells, mainly fibroblasts (FBs) and cancer-associated fibroblasts (CAFs). In this study, we investigated the effect of collagen stiffness on pancreatic FBs and CAFs, particularly on specific cytoskeleton properties and gene expression involved in tumour invasion. We found that cells become stiffer when they are cultured on stiff substrates and express higher levels of alpha-smooth muscle actin (α-SMA). Also, it was confirmed that on stiff substrates, CAFs are softer than FBs, while on soft substrates they have comparable Young's moduli. Furthermore, the number of spread FBs and CAFs was higher in stiffer substrates, which was also confirmed by Ras-related C3 botulinum toxin substrate 1 ( RAC1) mRNA expression, which mediates cell spreading. Although stress fibres in FBs become more oriented on stiff substrates, CAFs have oriented stress fibres regardless of substrate stiffness. Subsequently, we demonstrated that cells' invasion has a differential response to stiffness, which was associated with regulation of Ras homologue family member ( RhoA) and Rho-associated, coiled-coil containing protein kinase 1 ( ROCK-1) mRNA expression. Overall, our results demonstrate that collagen stiffness modulates FBs and CAFs cytoskeleton remodelling and alters their invasion properties.

Inhibition of Breast Cancer Cell Invasion by Ras Suppressor-1 (RSU-1) Silencing Is Reversed by Growth Differentiation Factor-15 (GDF-15).

Extracellular matrix (ECM)-related adhesion proteins are important in metastasis. Ras suppressor-1 (RSU-1), a suppressor of Ras-transformation, is localized to cell⁻ECM adhesions where it interacts with the Particularly Interesting New Cysteine-Histidine rich protein (PINCH-1), being connected to Integrin Linked Kinase (ILK) and alpha-parvin (PARVA), a direct actin-binding protein. RSU-1 was also found upregulated in metastatic breast cancer (BC) samples and was recently demonstrated to have metastasis-promoting properties. In the present study, we transiently silenced RSU-1 in BC cells, MCF-7 and MDA-MB-231. We found that RSU-1 silencing leads to downregulation of Growth Differentiation Factor-15 (GDF-15), which has been associated with both actin cytoskeleton reorganization and metastasis. RSU-1 silencing also reduced the mRNA expression of PINCH-1 and cell division control protein-42 (Cdc42), while increasing that of ILK and Rac regardless of the presence of GDF-15. However, the downregulation of actin-modulating genes PARVA, RhoA, Rho associated kinase-1 (ROCK-1), and Fascin-1 following RSU-1 depletion was completely reversed by GDF-15 treatment in both cell lines. Moreover, complete rescue of the inhibitory effect of RSU-1 silencing on cell invasion was achieved by GDF-15 treatment, which also correlated with matrix metalloproteinase-2 expression. Finally, using a graph clustering approach, we corroborated our findings. This is the first study providing evidence of a functional association between RSU-1 and GDF-15 with regard to cancer cell invasion.

Identification of Ras suppressor-1 (RSU-1) as a potential breast cancer metastasis biomarker using a three-dimensional in vitro approach.

Breast cancer (BC) is the most common malignant disease in women, with most patients dying from metastasis to distant organs, making discovery of novel metastasis biomarkers and therapeutic targets imperative. Extracellular matrix (ECM)-related adhesion proteins as well as tumor matrix stiffness are important determinants for metastasis. As traditional two-dimensional culture does not take into account ECM stiffness, we employed 3-dimensional collagen I gels of increasing concentration and stiffness to embed BC cells of different invasiveness (MCF-7, MDA-MB-231 and MDA-MB-231-LM2) or tumor spheroids. We tested the expression of cell-ECM adhesion proteins and found that Ras Suppressor-1 (RSU-1) is significantly upregulated in increased stiffness conditions. Interestingly, RSU-1 siRNA-mediated silencing inhibited Urokinase Plasminogen Activator, and metalloproteinase-13, whereas tumor spheroids formed from RSU-1-depleted cells lost their invasive capacity in all cell lines and stiffness conditions. Kaplan-Meier survival plot analysis corroborated our findings showing that high RSU-1 expression is associated with poor prognosis for distant metastasis-free and remission-free survival in BC patients. Taken together, our results indicate the important role of RSU-1 in BC metastasis and set the foundations for its validation as potential BC metastasis marker.