Although the polyphenol EGCG has various beneficial biological effects, its effect on cytoskeletal activities during cancer invasion is not well defined, and the precise molecular mechanisms are largely unknown. Here, we provide molecular evidence on the anti-invasion effect of EGCG in OSCC cells using an in vitro 3-D culture system and in vivo athymic mouse model. Briefly, EGCG exerted an inhibitory effect on the Matrigel-based Transwell invasion and migration of OSCC cells. These effects were not due to decreased cell viability or adhesion capacity to ECM. EGCG-treated OSCC cells possessed fully extended actin fibers without invadopodia, indicating a loss of ECM degradation capacity. Decreased phosphorylation of Src, CTTN, and FAK also followed EGCG treatment. Additionally, EGCG reduced activation of RhoA in dominant-negative RhoA N19 and constitutively active RhoA Q63E cells, and inhibited the invasive capability of these cells in the 3-D cell growth model. Furthermore, the administration of EGCG led to substantial inhibition of tumor growth and activation of invadopodial proteins in the tumor tissues of mice inoculated with OSCC cells. The data indicate the potential value of EGCG as an invadopodia-targeted anti-invasive agent in cancer therapy.

Cortactin (CTTN) is overexpressed in various tumors, including head and neck squamous cell carcinoma and colorectal cancer (CRC), and can serve as a biomarker of cancer metastasis. We observed that CTTN promotes cancer cell proliferation in vitro and increases CRC tumor xenograft growth in vivo. CTTN expression increases EGFR protein levels and enhances the activation of the MAPK signaling pathway. CTTN expression also inhibits the ubiquitin-mediated degradation of EGFR by suppressing the coupling of c-Cbl with EGFR. CoIP experiments indicate CTTN can interact with c-Cbl in CRC cells. These results demonstrate that CTTN promotes the proliferation of CRC cells and suppresses the degradation of EGFR.

Cortactin (CTTN) is a substrate of the Src kinase Lyn that is known to play an actin cytoskeletal regulatory role involved in cell migration and cancer progression following its phosphorylation at Y421. We recently demonstrated that Cortactin is overexpressed in patients with chronic lymphocytic leukaemia (CLL). This work was aimed at defining the functional role of Cortactin in these patients. We found that Cortactin is variably expressed in CLL patients both in the peripheral blood and lymph nodes and that its expression correlates with the release of matrix metalloproteinase 9 (MMP-9) and the motility of neoplastic cells. Cortactin knockdown, by siRNA, induced a reduction in MMP-9 release as well as a decrease of migration capability of leukaemic B cells in vitro, also after chemotactic stimulus. Furthermore, Cortactin phosphorylation was lowered by the Src kinase-inhibitor PP2 with a consequent decrease of MMP-9 release in culture medium. An impaired migration, as compared to control experiments without Cortactin knockdown, was observed following CXCL12 triggering. Reduced Cortactin expression and phosphorylation were also detected both in vivo and in vitro after treatment with Ibrutinib, a Btk inhibitor. Our results highlight the role of Cortactin in CLL as a check-point molecule between the BCR and CXCR4 signalling pathways.

OBJECTIVE

Various studies searching for biomarkers to predict tumor metastasis or prognosis in both esophageal squamous cell carcinoma (ESCC) and head and neck squamous cell carcinoma (HNSCC) are currently underway. However, few data have been reported on its association with colorectal cancer (CRC). Single nucleotide polymorphisms (SNPs) are the most common known form of human genetic variation and may contribute to an increased susceptibility to cancer including CRC. The present study aimed to investigate whether the polymorphisms in the CTTN gene are associated with susceptibility to CRC in the Korean population.

METHODS

A case-control study was performed to examine the relationship between the CTTN g.-9101C>T, g.-8748C>T, and g.72C>T polymorphisms and the risk of CRC. Polymerase chain reaction-restriction fragment length polymorphism analysis of g.-8748C>T, g.-9101C>T and Taqman analysis of g.72C>T were performed on blood samples from 218 patients with CRC and 533 control individuals. The g.-9101C>T, g.-8748C>T, and g.72C>T SNPs in CTTN and their haplotypes were analyzed.

RESULTS

The genotype and allele frequencies of g.-9101C>T, g.-8748C>T, and g.72C>T did not differ between the patient group and the control group. Further, the haplotype of CTTN g.-9101C>T, g.-8748C>T, and g.72C>T did not differ between patient group and the control group. However, the genotype and allele frequencies of CTTN g.-9101C>T were significantly increased in the lymph node positive CRC group compared to the control group.

CONCLUSIONS

The CTTN g.-9101C>T polymorphism may influence lymph node positive CRC.

Bmal1 is an essential component of the molecular clockwork, which drives circadian rhythms in cell function. In Bmal1-deficient (Bmal1-/-) mice, chronodisruption is associated with cognitive deficits and progressive brain pathology including astrocytosis indicated by increased expression of glial fibrillary acidic protein (GFAP). However, relatively little is known about the impact of Bmal1-deficiency on astrocyte morphology prior to astrocytosis. Therefore, in this study we analysed astrocyte morphology in young (6-8 weeks old) adult Bmal1-/- mice. At this age, overall GFAP immunoreactivity was not increased in Bmal1-deficient mice. At the ultrastructural level, we found a decrease in the volume fraction of the fine astrocytic processes that cover the hippocampal mossy fiber synapse, suggesting an impairment of perisynaptic processes and their contribution to neurotransmission. For further analyses of actin cytoskeleton, which is essential for distal process formation, we used cultured Bmal1-/- astrocytes. Bmal1-/- astrocytes showed an impaired formation of actin stress fibers. Moreover, Bmal1-/- astrocytes showed reduced levels of the actin-binding protein cortactin (CTTN). Cttn promoter region contains an E-Box like element and chromatin immunoprecipitation revealed that Cttn is a potential Bmal1 target gene. In addition, the level of GTP-bound (active) Rho-GTPase (Rho-GTP) was reduced in Bmal1-/- astrocytes. In summary, our data demonstrate that Bmal1-deficiency affects morphology of the fine astrocyte processes prior to strong upregulation of GFAP, presumably because of impaired Cttn expression and reduced Rho-GTP activation. These morphological changes might result in altered synaptic function and, thereby, relate to cognitive deficits in chronodisruption.

Polycyclic aromatic hydrocarbons (PAHs) are widespread environmental DNA-damaging agents regarded as risk factors for human disease, including lung and breast cancer. The biotransformation of PAHs to carcinogenic metabolites is mediated by the aromatic hydrocarbon receptor (AhR), which activates transcription at xenobiotic responsive elements (XREs = 5'-GCGTG-3') found in the promoter regions of genes encoding for detoxifying enzymes, including CYP1A1 and CYP1B1. In this study, we wished to identify novel biomarkers that may be useful in monitoring critical carcinogenic events of the breast induced by PAHs. Using a GeneMAP CancerArray, we analyzed in breast cancer MCF-7 cells the temporal effects of the AhR agonist benzo[a]pyrene (B[a]P), which is a prototype PAH and known environmental carcinogen. Genes upregulated at least threefold by B[a]P and containing potential XREs within their promoter regions included CYP1A1, CYP1B1, paired box gene 3 (PAX3), cortactin (CTTN/EMS1), beta-2-microglobulin (B2M), and transferrin receptor (TfR). The stimulatory effects of B[a]P on expression of these genes were abrogated by cotreatment with the AhR antagonist flavonoid, alpha-napthoflavone (ANF). The TfR gene was selected for further analysis as its promoter region contains two potential XREs and its expression has been shown to be increased in breast cancer cells. Accumulation of TfR mRNA in B[a]P-treated cells was confirmed by quantitative real time PCR. Transient transfection studies indicated that the transcriptional activity of the TfR promoter was stimulated by B[a]P, whereas ANF counteracted this induction. These results indicate that the TfR gene may be a potential biomarker of PAH exposure.

Cortactin is a protein encoded by the CTTN gene, localized on chromosome band 11q13. As a result of the amplification of this band, an important event in oral carcinogenesis, CTTN is also usually amplified, promoting the frequent overexpression of cortactin. Cortactin enhances cell migration in oral cancer, playing a key role in the regulation of filamentous actin and of protrusive structures (invadopodia and lamellipodia) on the cell membrane that are necessary for the acquisition of a migratory phenotype. We also analyze a series of emerging functions that cortactin may exert in oral cancer (cell proliferation, angiogenesis, regulation of exosomes, and interactions with the tumor microenvironment). We review its molecular structure, its most important interactions (with Src, Arp2/3 complex, and SH3-binding partners), the regulation of its functions, and its specific oncogenic role in oral cancer. We explore the mechanisms of its overexpression in cancer, mainly related to genetic amplification. We analyze the prognostic implications of the oncogenic activation of cortactin in potentially malignant disorders and in head and neck cancer, where it appears to be relevant in the development of lymph node metastasis. Finally, we discuss its usefulness as a therapeutic target and suggest future research lines.

Cortactin (CTTN), a major substrate of the Src tyrosine kinase, has been implicated in cell proliferation, motility and invasion in various types of cancer. However, the molecular mechanisms of CTTN-driven malignant behavior remain unclear. In the current study, we determined the expression of CTTN in colorectal cancer and investigated its underlying mechanism in the metastasis of colorectal cancer. We confirmed increased CTTN expression in lymph node-positive CRC specimens and highly invasive CRC cell lines. Further study has shown that overexpression of CTTN promoted CRC cell migration and invasion, whereas CTTN silencing inhibited CRC cell migratory and invasive capacities in vitro. Mechanistically, CTTN increases expression of dedicator of cytokinesis 1 (DOCK1) and gene silencing of DOCK1 partially abolishes the migration and invasion capacity by CTTN. Our findings indicate that CTTN promotes metastasis of CRC cells by increasing DOCK1 expression and this could offer a promising therapeutic target for colorectal cancer treatment.

OBJECTIVE

miRNAs have been confirmed to be related to cell proliferation and apoptosis. In this study, we detected the potential effect of miR-448 on glioma cell proliferation and apoptosis.

METHODS

miR-448 and CTTN expression levels were detected in glioma cell lines with quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). Cells were transfected with miR-448 mimics and inhibitor by using lipofectamine 2000 respectively. The proliferative ability of transfected cells was detected via methyl thiazolyl tetrazolium (MTT) and cell counting kit-8 (CCK8) assays. Cell apoptosis and cell-cycle were tested using flow cytometry. The regulatory correlation between miR-448 and CTTN was explored by bioinformatics analysis and luciferase reporter assay.

RESULTS

Lower expression of miR-448 and higher level of CTTN were detected in glioma cells. MiR-448 could regulate cell proliferation, cell apoptosis, and cell cycle. CTTN was negatively regulated by miR-448.

CONCLUSIONS

miR-448 downregulates CTTN to inhibit cell proliferation and promote apoptosis in glioma, which indicates a potential therapeutic target of glioma.

Cell motility and migration requires the reorganization of the cortical cytoskeleton at the leading edge of cells and extracellular Ca2+ entry is essential for this reorganization. However the molecular nature of the regulators of this pathway is unknown. This work contributes to understanding the role of STIM1 and ORAI1 in the promotion of membrane ruffling by showing that phospho-STIM1 localizes at the leading edge of cells, and that both phospho-STIM1 and ORAI1 co-localize with cortactin (CTTN), a regulator of the cytoskeleton at membrane ruffling areas. STIM1-KO and ORAI1-KO cell lines were generated by CRISPR/Cas9 genome editing in U2OS cells. In both cases, KO cells presented a notable reduction of store-operated Ca2+ entry (SOCE) that was rescued by expression of STIM1-mCherry and ORAI1-mCherry. These results demonstrated that SOCE regulates membrane ruffling at the leading edge of cells. Moreover, endogenous ORAI1 and overexpressed ORAI1-GFP co-immunoprecipitated with endogenous CTTN. This latter result, in addition to the KO cells' phenotype, the preservation of ORAI1-CTTN co-localization during ruffling, and the inhibition of membrane ruffling by the Ca2+-channel inhibitor SKF96365, further supports a functional link between SOCE and membrane ruffling.

Accurate nodal staging is pivotal for treatment planning in early (stage I-II) oral cancer. Unfortunately, current imaging modalities lack sensitivity to detect occult nodal metastases. Chromosomal region 11q13, including genes CCND1, Fas-associated death domain (FADD), and CTTN, is often amplified in oral cancer with nodal metastases. However, evidence in predicting occult nodal metastases is limited.

In 158 patients with early tongue and floor of mouth (FOM) squamous cell carcinomas, both CCND1 amplification and cyclin D1, FADD, and cortactin protein expression were correlated with occult nodal metastases.

CCND1 amplification and cyclin D1 expression correlated with occult nodal metastases. Cyclin D1 expression was validated in an independent multicenter cohort, confirming the correlation with occult nodal metastases in early FOM cancers.

Cyclin D1 is a predictive biomarker for occult nodal metastases in early FOM cancers. Prospective research on biopsy material should confirm these results before implementing its use in routine clinical practice. © 2016 Wiley Periodicals, Inc. Head Neck 39: 326-333, 2017.

A dynamic, properly organised actin cytoskeleton is critical for the production and haemostatic function of platelets. The Wiskott Aldrich Syndrome protein (WASp) and Actin-Related Proteins 2 & 3 Complex (Arp2/3 complex) are critical mediators of actin polymerisation and organisation in many cell types. In platelets and megakaryocytes, these proteins have been shown to be important for proper platelet production and function. The cortactin family of proteins (Cttn & HS1) are known to regulate WASp-Arp2/3-mediated actin polymerisation in other cell types and so here we address the role of these proteins in platelets using knockout mouse models. We generated mice lacking Cttn and HS1 in the megakaryocyte/platelet lineage. These mice had normal platelet production, with platelet number, size and surface receptor profile comparable to controls. Platelet function was also unaffected by loss of Cttn/HS1 with no differences observed in a range of platelet function assays including aggregation, secretion, spreading, clot retraction or tyrosine phosphorylation. No effect on tail bleeding time or in thrombosis models was observed. In addition, platelet actin nodules, and megakaryocyte podosomes, actin-based structures known to be dependent on WASp and the Arp2/3 complex, formed normally. We conclude that despite the importance of WASp and the Arp2/3 complex in regulating F-actin dynamics in many cells types, the role of cortactin in their regulation appears to be fulfilled by other proteins in platelets.

Decabromodiphenyl ether (decaBDE) is a brominated flame retardant used in many commercial products such as televisions, computers, and textiles. Recent reports indicate that decaBDE adversely affects male reproductive organs in mice, but the underlying molecular mechanisms remain unknown. We hypothesized that decaBDE affects mouse testes by altering the expression and phosphorylation level of cortactin (CTTN), an F-actin-binding protein that is similar to flutamide, and we performed western blot analyses on testicular samples from mice subcutaneously injected with decaBDE (0.025, 0.25, and 2.5 mg/kg body weight/day) on postnatal days 1 to 5. Mice treated with low-dose decaBDE (0.025 mg/kg) showed reduced testicular weight, sperm count, elongated spermatid and Sertoli cell numbers, as well as induced Tyr phosphorylation of CTTN and reduced the expression level of p60 Src tyrosine kinase (SRC). Further, 0.25 and 2.5 mg/kg decaBDE-exposed groups produced an decrease the expression level of CTTN. High-dose decaBDE (2.5 mg/kg) showed increased abnormal germ cells, as well as induced Ser phosphorylation of CTTN and activated extracellular signal-regulated kinase (ERK1/2); however, high-dose decaBDE did not affect testicular weight and sperm count. These findings suggest that postnatal exposure to low-dose decaBDE inhibits mouse testicular development by increasing Tyr phosphorylation of CTTN, although different mechanisms may be involved depending on the dose of decaBDE.

Tumor metastasis is one of death causes for patients of prostate carcinoma. PIWI-interacting RNAs (piRNAs) are a subtype of noncoding protein RNAs that are involved in tumorigenesis, but the effect of piRNAs in prostate carcinoma (PCa) remains unclear. This article showed the identification of piRNAs was performed using a piRNA microarray screen in PCa tissues and several piRNAs were identified as dysregulated. The two up-regulated piRNAs (piR-19004 and piR-2878) and one down-regulated piR-19166 have been validated in the tissues and cell lines of PCa using quantitative reverse transcription polymerase chain reaction (qRT-PCR). Further studies showed that piR-19166 is transfected into PCa cells to suppress its migration and metastasis. Mechanistically, cortactin (CTTN) 3' untranslated region (UTR) was complementary combined with piR-19166 by bioinformatic prediction and identified as a direct target of piR-19166 through dual-luciferase reporter assay. Over-expression and knockdown of CTTN could respectively rescue and simulate the effects induced by piR-19166. Finally, piR-19166 suppresses migration and metastasis by the CTTN/matrix metalloproteinases (MMPs) pathway in PCa cells. Thus, these findings suggested that piR-19166 targets the CTTN of prostate cancer cells to inhibit migration and distant metastasis, and may represent a new marker of diagnosis and treatment for PCa patients in early stages.

Keywords: CTTN; metastasis; piR-19166; piRNAs; prostate carcinoma.

BACKGROUND

This study was designed to identify the pathogenic mutation in a Chinese family with arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D) using whole genome sequencing (WGS).

RESULTS

Probands II:1 and II:2 underwent routine examinations for diagnosis. Genomic DNA was extracted from the peripheral blood of family members and analyzed using WGS. A total of 60,285 single-nucleotide polymorphisms (SNP) and 13,918 insertions/deletions (InDel) occurring in the exonic regions of genes and predisposing to cardiomyopathies and arrhythmias were identified. When filtered using the 1000 Genomes Project (2014 version), NHLBI ESP6500, and ExAC databases, 12 missense SNP and 2 InDel in exonic regions remained, the allele frequencies of which were <0.01 or unknown. The potentially pathogenic mutations that occurred in the genes DSG2, PKP4, PRKAG2, FOXD4, CTTN, and DMD, which were identified by SIFT or PolyPhen-2 software as "damaging," were validated using Sanger sequencing. Probands II:1 and II:2 shared an extremely rare homozygous mutation in the DSG2 (p.F531C) gene, which was also demonstrated using intersection analysis of WGS data from probands II:1 and II:2. Electron microscopy and histological staining of myocardial biopsies showed widened and destroyed intercalated discs, and interrupted, atrophic, and disarranged myocardial fibers, and hyperplastic interstitial fibers, collagen fibers, and adipocytes were infiltrated and invaded.

CONCLUSIONS

A homozygous mutation of DSG2 p.F531C was identified as the pathogenic mutation in patients with ARVC/D involving both ventricles, as a result of widened and impaired intercalated discs, interrupted myocardial fibers, and abnormally hyperplastic interstitial fibers, collagen fibers, and adipocytes.

Background: Cortactin (CTTN) and the focal adhesion kinase (FAK) are two major candidate genes to, respectively, drive 11q13- and 8q24-associated aggressive behavior in various cancers. Recent evidence uncovered their clinical relevance in early stages of tumorigenesis as promising biomarkers for cancer risk assessment.Methods: Using a multicenter validation study, CTTN and FAK expression was evaluated by immunohistochemistry (IHC) in a cohort of 109 patients with laryngeal precancerous lesions, and correlated with clinicopathologic parameters and laryngeal cancer risk. The pathophysiologic role of CTTN and FAK was further investigated using functional studies in cellular models.Results: Positive CTTN and FAK expression (scores 2 and 3) was detected in 49 (41%) and 35 (32%) laryngeal dysplasias, respectively. Univariate Cox analysis showed that CTTN and FAK expression but not histologic grading was significantly associated with both recurrence risk and laryngeal cancer risk. Patients carrying strong CTTN- or FAK-expressing lesions (score 3) experienced the highest laryngeal cancer incidence (log-rank P < 0.001). In multivariate stepwise analysis, FAK expression [HR = 13.91; 95% CI, 4.82-40.15; P < 0.001] and alcohol consumption (HR = 2.22; 95% confidence interval, 1.17-4.20; P = 0.014) were significant independent predictors of laryngeal cancer development. Targeting FAK by either RNAi or pharmacologic inhibitors effectively blocked cell growth, colony formation, and invasion into 3D collagen matrices.Conclusions: CTTN and FAK emerge as powerful predictors of laryngeal cancer risk and recurrence risk beyond histologic grading.Impact: Our work supports the applicability of IHC CTTN and FAK as complementary markers for risk stratification in patients with laryngeal precancerous lesions. Cancer Epidemiol Biomarkers Prev; 27(7); 805-13. ©2018 AACR.

Beyond their role in bone and lung homeostasis, mesenchymal stem cells (MSCs) are becoming popular in cell therapy. Various insults may disrupt the repair mechanisms involving MSCs. One such insult is smoking, which is a major risk factor for osteoporosis and respiratory diseases. Upon cigarette smoke-induced damage, a series of reparatory mechanisms ensue; one such mechanism involves Glycosaminoglycans (GAG). One of these GAGs, namely hyaluronic acid (HA), serves as a potential therapeutic target in lung injury. However, much of its mechanisms of action through its major receptor CD44 remains unexplored. Our previous studies have identified and functionally validated that both cortactin (CTTN: marker of motility) and Survivin (BIRC5: required for cell survival) act as novel HA/CD44-downstream transcriptional targets underpinning cell motility. Here, human MSCs were treated with "Water-pipe" smoke to investigate the effects of cigarette smoke condensate (CSC) on these HA-CD44 novel signaling pathways. Our results show that CSC decreased the expression of both CD44 and its downstream targets CTTN and BIRC5 in MSCs, and that HA reversed these effects. Interestingly, CSC inhibited migration and invasion of MSCs upon CD44-targeted RNAi treatment. This shows the importance of CD44-HA/CTTN and CD44-HA/BIRC5 signaling pathways in MSC motility, and further suggests that these signaling pathways may provide a novel mechanism implicated in migration of MSCs during repair of lung tissue injury. These findings suggest that one should use caution before utilizing MSC from donors with history of smoking, and further pave the way towards the development of targeted therapeutic approaches against CD44-associated diseases.

Background: IgA nephropathy (IgAN) is the most frequent type of primary glomerulonephritis globally and the leading cause of end-stage renal disease in young adults. Its pathogenesis is not fully known, but is largely attributed to genetic factors. This study was aimed to explore the prognostic values of key genes in IgAN.

Methods: The gene expression profile GSE93798 of 20 IgAN samples and 22 normal samples using glomeruli from kidney biopsy was adopted. Totally 447 upregulated and 719 downregulated differentially expressed genes were found in IgAN patients on the R software. The Gene Ontology enrichment and the Kyoto Encyclopedia of Gene and Genomes pathway were investigated on DAVID, and the protein-protein interaction network and the top 13 hub genes of the differentially expressed genes were built via the plug-in molecular complex detection and cytoHubba of Cytoscape.

Results: From the protein-protein interaction network, of the top 13 hub genes, FOS, EGFR, SIRT1, ALB, TFRC, JUN, IGF1, HIF1A, and SOCS3 were upregulated, while CTTN, ACTR2, CREB1, and CTNNB1 were downregulated. The upregulated genes took part in the HIF-1 signaling pathway, Choline metabolism in cancer, Pathways in cancer, Amphetamine addiction, Estrogen, TNF, and FoxO signaling pathways, and Osteoclast differentiation, while the downregulated genes were involved in Pathogenic Escherichia coli infection, Bacterial invasion of epithelial cells, prostate cancer, and melanogenesis.

Conclusion: This study based on the Gene Expression Omnibus database updates the knowledge about the mechanism of IgAN and may offer new treatment targets.

Integrated analyses of multiple genomic datatypes are now common in cancer profiling studies. Such data present opportunities for numerous computational experiments, yet analytic pipelines are limited. Tools such as the cBioPortal and Regulome Explorer, although useful, are not easy to access programmatically or to implement locally. Here, we introduce the MVisAGe R package, which allows users to quantify gene-level associations between two genomic datatypes to investigate the effect of genomic alterations (e.g., DNA copy number changes on gene expression). Visualizing Pearson/Spearman correlation coefficients according to the genomic positions of the underlying genes provides a powerful yet novel tool for conducting exploratory analyses. We demonstrate its utility by analyzing three publicly available cancer datasets. Our approach highlights canonical oncogenes in chr11q13 that displayed the strongest associations between expression and copy number, including CCND1 and CTTN, genes not identified by copy number analysis in the primary reports. We demonstrate highly concordant usage of shared oncogenes on chr3q, yet strikingly diverse oncogene usage on chr11q as a function of HPV infection status. Regions of chr19 that display remarkable associations between methylation and gene expression were identified, as were previously unreported miRNA-gene expression associations that may contribute to the epithelial-to-mesenchymal transition.Significance: This study presents an important bioinformatics tool that will enable integrated analyses of multiple genomic datatypes. Cancer Res; 78(12); 3375-85. ©2018 AACR.

Background: Hepatocellular carcinoma (HCC) includes cryptogenic hepatocellular carcinomas (CR-HCC) that lack a defined cause. Specific DNA methylation patterns and comparisons of the aberrant alterations in DNA methylation between CR-HCC and adjacent peritumor tissues (APTs) have not yet been reported.

Methods: The SureSelectXT Methyl-Seq Target Enrichment System was used to sequence targeted DNA methylation in three paired CR-HCC tissues and APTs. Gene Ontology (GO) enrichment and KEGG pathway analysis were performed to investigate the DNA methylation mechanism of CR-HCC. The mRNA expression levels of HOXB-AS3, HOXB6, HOXB3, USP18, MAP3K6, TIRAP, TNNI2, SHC3, CTTN, and TFAP2A, selected from the identified signaling pathways, were evaluated by quantitative real-time PCR (qPCR).

Results: A total of 1728 differentially methylated regions (DMRs) were identified in tumor tissues compared with non-tumor tissues, of which 868 DMRs were hypermethylated and 860 were hypomethylated. The DMRs were mapped within 2091 DMR-associated genes (DMGs). The mRNA expression of HOXB-AS3, HOXB3, and MAP3K6 was downregulated in CR-HCC tissues compared to the APTs. However, the mRNA expression of TIRAP, SHC3, and CTTN was upregulated in the CR-HCC tissues. Differences between the mRNA expression of HOXB6, USP18, TNNI2, and TFAP2A in the CR-HCC and APTS tissues were not statistically significant. GO analysis showed that the molecular functions of "binding", "protein binding", and "cytoskeletal protein binding" were the main categories for the hypermethylated DMGs. The hypomethylated DMGs were mostly enriched in the molecular functions "binding", "protein binding", "calcium ion binding", among others. KEGG pathway analysis showed that the hypermethylated DMGs were enriched in several pathways such as "estrogen signaling pathway", while hypomethylated DMGs were enriched in several pathways such as "proteoglycans in cancer", suggesting that epigenetic modifications play important roles in the cryptogenic hepatocarcinogenesis.

Conclusion: These results provide useful information for future work to characterize the functions of epigenetic mechanisms on CR-HCC.

Keywords: DMR; DNA methylation; cryptogenic hepatocellular carcinomas.

Tumor invasion requires efficient cell migration, which is achieved by the generation of persistent and polarized lamellipodia. The generation of lamellipodia is supported by actin dynamics at the leading edge where a complex of proteins known as the WAVE regulatory complex (WRC) promotes the required assembly of actin filaments to push the front of the cell ahead. By using an U2OS osteosarcoma cell line with high metastatic potential, proven by a xenotransplant in zebrafish larvae, we have studied the role of the plasma membrane Ca²⁺ channel ORAI1 in this process. We have found that epidermal growth factor (EGF) triggered an enrichment of ORAI1 at the leading edge, where colocalized with cortactin (CTTN) and other members of the WRC, such as CYFIP1 and ARP2/3. ORAI1-CTTN co-precipitation was sensitive to the inhibition of the small GTPase RAC1, an upstream activator of the WRC. RAC1 potentiated ORAI1 translocation to the leading edge, increasing the availability of surface ORAI1 and increasing the plasma membrane ruffling. The role of ORAI1 at the leading edge was studied in genetically engineered U2OS cells lacking ORAI1 expression that helped us to prove the key role of this Ca²⁺ channel on lamellipodia formation, lamellipodial persistence, and cell directness, which are required for tumor cell invasiveness in vivo.

There is an acceptance that plasmid-based delivery of interfering RNA always generates the intended targeting sequences in cells, making it as specific as its synthetic counterpart. However, recent studies have reported on cellular inefficiencies of the former, especially in light of emerging gene discordance at inter-screen level and across formats. Focusing primarily on the TRC plasmid-based shRNA hairpins, we reasoned that alleged specificities were perhaps compromised due to altered processing; resulting in a multitude of random interfering sequences. For this purpose, we opted to study the processing of hairpin TRCN#40273 targeting CTTN; which showed activity in a miRNA-21 gain-of-function shRNA screen, but inactive when used as an siRNA duplex. Using a previously described walk-through method, we identified 36 theoretical cleavage variants resulting in 78 potential siRNA duplexes targeting 53 genes. We synthesized and tested all of them. Surprisingly, six duplexes targeting ASH1L, DROSHA, GNG7, PRKCH, THEM4, and WDR92 scored as active. QRT-PCR analysis on hairpin transduced reporter cells confirmed knockdown of all six genes, besides CTTN; revealing a surprising 7 gene-signature perturbation by this one single hairpin. We expanded our qRT-PCR studies to 26 additional cell lines and observed unique knockdown profiles associated with each cell line tested; even for those lacking functional DICER1 gene suggesting no obvious dependence on dicer for shRNA hairpin processing; contrary to published models. Taken together, we report on a novel dicer independent, cell-type dependent mechanism for non-specific RNAi gene silencing we coin Alternate Targeting Sequence Generator (ATSG). In summary, ATSG adds another dimension to the already complex interpretation of RNAi screening data, and provides for the first time strong evidence in support of arrayed screening, and questions the scientific merits of performing pooled RNAi screens, where deconvolution of up to genome-scale pools is indispensable for target identification.

Thrombocytopenia is a major side effect of a new class of anticancer agents that target histone deacetylase (HDAC). Their mechanism is poorly understood. Here, we show that HDAC6 inhibition and genetic knockdown lead to a strong decrease in human proplatelet formation (PPF). Unexpectedly, HDAC6 inhibition-induced tubulin hyperacetylation has no effect on PPF. The PPF decrease induced by HDAC6 inhibition is related to cortactin (CTTN) hyperacetylation associated with actin disorganization inducing important changes in the distribution of megakaryocyte (MK) organelles. CTTN silencing in human MKs phenocopies HDAC6 inactivation and knockdown leads to a strong PPF defect. This is rescued by forced expression of a deacetylated CTTN mimetic. Unexpectedly, unlike human-derived MKs, HDAC6 and CTTN are shown to be dispensable for mouse PPF in vitro and platelet production in vivo. Our results highlight an unexpected function of HDAC6-CTTN axis as a positive regulator of human but not mouse MK maturation.