Role of MEK1 and DIAPH3 expression in colorectal adenoma-carcinoma sequence.

BACKGROUND: It is well established that most colorectal carcinomas arise from conventional adenomas through the adenoma-carcinoma sequence (ACS) model. mitogen-activated protein kinases (MAPKs) pathway has been reported as a crucial player in tumorigenesis. The MAPK signaling pathway is activated by different extracellular signals involving the "mitogen-activated/extracellular signal-regulated kinase 1 (MEK1)", and this induces the expression of genes involved in proliferation and cellular transformation. Diaphanous-related formin-3 (DIAPH3) acts as a potential metastasis regulator through inhibiting the cellular transition to amoeboid behavior in different cancer types. OBJECTIVE: The aim of the study was to investigate the pattern of immunohistochemical expression of MEK1 and DIAPH3 in colorectal adenoma (CRA) and corresponding colorectal carcinoma (CRC) specimens. METHODS: The immunohistochemical expression of DIAPH3 and MEK1 was examined in 43 cases of CRC and their associated adenomas using tissue microarray technique. RESULTS: MEK1 was overexpressed in 23 CRC cases (53.5%) and in 20 CRA cases (46.5%). DIAPH3 was overexpressed in 11 CRA cases (about 29%) which were significantly lower than CRC (22 cases; 58%) (P = 0.011). Both MEK1 and DIAPH3 overexpression were significantly correlated in CRC (P = 0.009) and CRA cases (P = 0.002). Tumors with MEK1 overexpression had a significantly higher tumor grade (P = 0.050) and perineural invasion (P = 0.017). CONCLUSIONS: Both MEK1 and DIAPH3 are overexpressed across colorectal ACS with strong correlation between them. This co- expression suggests a possible synergistic effect of MEK1 and DIAPH-3 in colorectal ACS. Further large-scale studies are required to investigate the potential functional aspects of MEK1 and DIAPH3 in ACS and their involvement in tumor initiation and the metastatic process.

Integrated analysis of transcriptome and genome variations in pediatric T cell acute lymphoblastic leukemia: data from north Indian tertiary care center.

INTRODUCTION: T-cell acute lymphoblastic leukemia (T-ALL) is a genetically heterogeneous disease with poor prognosis and inferior outcome. Although multiple studies have been perform on genomics of T-ALL, data from Indian sub-continent is scarce. METHODS: In the current study we aimed to identify the genetic variability of T-ALL in an Indian cohort of pediatric (age ≤ 12 years) T-ALL patients (n = 25) by whole transcriptome sequencing along with whole exome sequencing and correlated the findings with clinical characteristics and disease outcome. RESULTS: The median age was 7 years (range 3 -12 years). RNA sequencing revealed a definitive fusion event in 14 cases (56%) (including a novel fusions) with STIL::TAL1 in 4 (16%), followed by NUP21::ABL1, TCF7::SPI1, ETV6::HDAC8, LMO1::RIC3, DIAPH1::JAK2, SETD2::CCDC12 and RCBTB2::LPAR6 in 1 (4%) case each. Significant aberrant expression was noted in RAG1 (64%), RAG2 (80%), MYCN (52%), NKX3-1 (52%), NKX3-2 (32%), TLX3 (28%), LMO1 (20%) and MYB (16%) genes. WES data showed frequent mutations in NOTCH1 (35%) followed by WT1 (23%), FBXW7 (12%), KRAS (12%), PHF6 (12%) and JAK3 (12%). Nearly 88.2% of cases showed a deletion of CDKN2A/CDKN2B/MTAP genes. Clinically significant association of a better EFS and OS (p=0.01) was noted with RAG2 over-expression at a median follow up of 22 months, while a poor EFS (p=0.041) and high relapse rate (p=0.045) was observed with MYB over-expression. CONCLUSION: Overall, the present study demonstrates the frequencies of transcriptomic and genetic alterations from Indian cohort of pediatric T-ALL and is a salient addition to current genomics data sets available in T-ALL.

Functional genetic variants of the disulfidptosis-related INF2 gene predict survival of hepatitis B virus-related hepatocellular carcinoma.

Disulfidptosis is a novel form of programmed cell death involved in migration and invasion of cancer cells, but few studies investigated the roles of genetic variants in disulfidptosis-related genes in survival of patients with hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC). We used Cox proportional hazards regression analyses, Kaplan-Meier curves and receiver operating characteristic curves to assess effects of genetic variants in 14 disulfidptosis-related genes on overall survival of 866 HBV-HCC patients. The Bayesian false discovery probability was used for multiple testing corrections. We also investigated biological mechanisms of the significant variants through expression quantitative trait loci analyses using the data from publicly available databases, luciferase reporter assays and differential expression analyses. As a result, we identified two independently functional single nucleotide polymorphisms (SNPs) (INF2 rs4072285 G > A and INF2 rs4444271 A > T) that predicted overall survival of HBV-HCC patients, with adjusted hazard ratios of 1.60 (95% CI = 1.22-2.11, P = 0.001) and 1.50 (95% CI = 1.80-1.90, P < 0.001), respectively, after multiple testing correction. Luciferase reporter assays indicated that both INF2 rs4072285 A and INF2 rs4444271 T alleles increased INF2 mRNA expression levels (P < 0.001) that were also higher in HCC tumor tissues than in adjacent normal tissues (P < 0.001); such elevated INF2 expression levels were associated with a poorer survival of HBV-HCC patients (P < 0.001) in the TCGA database. In summary, this study supported that INF2 rs4072285 and INF2 rs4444271 may be novel biomarkers for survival of HBV-HCC patients.

KIF20B and MET, hub genes of DIAPHs, predict poor prognosis and promote pancreatic cancer progression.

BACKGROUND: The DIAPHs (DIAPH1, DIAPH2, and DIAPH3) are members of the diaphanous subfamily of the formin family. KIF20B and MET, hub genes of DIAPHs, play crucial roles in cytoskeletal remodeling, cell migration, and adhesion. However, their combined prognostic and treatment value in pancreatic adenocarcinoma (PC) warrants further investigation. METHODS: Multiomics analysis tools were used to comprehensively assess the genomic expression and prognostic value of KIF20B and MET in PC. Immune cell infiltration, functional enrichment, single-cell RNA-seq (scRNA) analysis, potential therapeutic drugs, and nomograms were established and analyzed. CCK-8 levels, transwell assay, Co-IP assay, mass spectrometry, and western blotting were performed to assess the role of KIF20B and MET as modulators of ß-catenin and Lactate Dehydrogenase A (LDHA) in vitro. Xenograft tumor models were used to evaluate the anti-tumor effects in vivo. RESULTS: DIAPHs, KIF20B, and MET were overexpressed and functioned as poor prognostic markers of PC. Immunoinfiltration analysis revealed that pDC and NK cells were enriched with low expression levels of KIF20B and MET, whereas Th2 cells were enriched with high expression levels of these two genes. The copy number variations (CNVs) in KIF20B and MET were positively correlated with B cell and CD4 + T cell infiltration. Immunological checkpoints NT5E and CD44 were positively correlated with KIF20B and MET expression. Moreover, the nomogram constructed based on KIF20B and MET demonstrated predictive value for overall survival. scRNA-Seq analysis indicated that KIF20B and MET were enriched in endothelial, malignant, B, T, and CD8 + T cells, which correlated with glycolysis and the epithelial-mesenchymal transition (EMT). The interactions of KIF20B and MET with ß-catenin and LDHA were verified by Co-IP assay and mass spectrometry. Knockdown of KIF20B and MET downregulates ß-catenin and LDHA in vitro. Furthermore, dual knockdown of KIF20B and MET exhibited a synergistic suppressive effect on PC progression in vitro and in vivo. CONCLUSION: DIAPHs, KIF20B, and MET are promising candidates for the prognosis and treatment of PC. More importantly, downregulation of KIF20B and MET inhibited pancreatic cancer progression by regulating LDHA and EMT.

mDia formins form hetero-oligomers and cooperatively maintain murine hematopoiesis.

mDia formin proteins regulate the dynamics and organization of the cytoskeleton through their linear actin nucleation and polymerization activities. We previously showed that mDia1 deficiency leads to aberrant innate immune activation and induces myelodysplasia in a mouse model, and mDia2 regulates enucleation and cytokinesis of erythroblasts and the engraftment of hematopoietic stem and progenitor cells (HSPCs). However, whether and how mDia formins interplay and regulate hematopoiesis under physiological and stress conditions remains unknown. Here, we found that both mDia1 and mDia2 are required for HSPC regeneration under stress, such as serial plating, aging, and reconstitution after myeloid ablation. We showed that mDia1 and mDia2 form hetero-oligomers through the interactions between mDia1 GBD-DID and mDia2 DAD domains. Double knockout of mDia1 and mDia2 in hematopoietic cells synergistically impaired the filamentous actin network and serum response factor-involved transcriptional signaling, which led to declined HSPCs, severe anemia, and significant mortality in neonates and newborn mice. Our data demonstrate the potential roles of mDia hetero-oligomerization and their non-rodent functions in the regulation of HSPCs activity and orchestration of hematopoiesis.

Sorafenib induces cardiotoxicity through RBM20-mediated alternative splicing of sarcomeric and mitochondrial genes.

Sorafenib, a multi-targeted tyrosine kinase inhibitor, is a first-line treatment for advanced solid tumors, but it induces many adverse cardiovascular events, including myocardial infarction and heart failure. These cardiac defects can be mediated by alternative splicing of genes critical for heart function. Whether alternative splicing plays a role in sorafenib-induced cardiotoxicity remains unclear. Transcriptome of rat hearts or human cardiomyocytes treated with sorafenib was analyzed and validated to define alternatively spliced genes and their impact on cardiotoxicity. In rats, sorafenib caused severe cardiotoxicity with decreased left ventricular systolic pressure, elongated sarcomere, enlarged mitochondria and decreased ATP. This was associated with alternative splicing of hundreds of genes in the hearts, many of which were targets of a cardiac specific splicing factor, RBM20. Sorafenib inhibited RBM20 expression in both rat hearts and human cardiomyocytes. The splicing of RBM20's targets, SLC25A3 and FHOD3, was altered into fetal isoforms with decreased function. Upregulation of RBM20 during sorafenib treatment reversed the pathogenic splicing of SLC25A3 and FHOD3, and enhanced the phosphate transport into mitochondria by SLC25A3, ATP synthesis and cell survival.We envision this regulation may happen in many drug-induced cardiotoxicity, and represent a potential druggable pathway for mitigating sorafenib-induced cardiotoxicity.

Super Enhancer-Regulated LncRNA LINC01089 Induces Alternative Splicing of DIAPH3 to Drive Hepatocellular Carcinoma Metastasis.

Hepatocellular carcinoma (HCC) is one of the most lethal neoplasms and has a 5-year survival rate of only 18% in patients with metastatic diseases. Epigenetic modifiers and alterations, including histone modifications, long noncoding RNAs (lncRNA), RNA alternative splicing, and N6-methyladenosine (m6A) modification, are key regulators of HCC development, highlighting the importance of understanding the cross-talk between these biological processes. In the current study, we identified LINC01089 as a super enhancer (SE)-driven lncRNA that promotes epithelial-mesenchymal transition (EMT), migration, invasion, and metastasis of HCC cells in vivo and in vitro. The transcription factor E2F1 bound to a LINC01089 SE, promoting LINC01089 transcription and overexpression. LINC01089 interacted with heterogeneous nuclear ribonucleoprotein M (hnRNPM) and led to hnRNPM-mediated skipping of DIAPH3 exon 3. Knockdown of LINC01089 increased the inclusion of DIAPH3 exon 3, which contains an important m6A-modification site that is recognized by IGF2BP3 to increase DIAPH3 mRNA stability. Thus, LINC01089 loss increased DIAPH3 protein levels, which suppressed the ERK/Elk1/Snail axis and inhibited EMT of HCC cells. In conclusion, this study revealed cross-talk between different epigenetics modifiers and alterations that drives HCC progression and identified LINC01089 as a potential prognostic marker and therapeutic target for HCC. SIGNIFICANCE: LINC01089 is a super enhancer-driven long noncoding RNA that induces ERK signaling and epithelial-mesenchymal transition by regulating DIAPH3 alternative splicing that blocks N6-methyladenosine-mediated mRNA stabilization, establishing an epigenetic network that promotes hepatocellular carcinoma metastasis.

Formin-related protein 1 facilitates proliferation and aggressive phenotype of clear cell renal cell carcinoma through MAPK/MMP2 pathway.

BACKGROUND: Formin-related protein-1(FRL1) has reportedly been overexpressed in a variety of malignancies, such as clear cell renal cell carcinoma (ccRCC). However, the clinical value and molecular mechanisms underlying ccRCC tumorigenesis and progression in association with FRL1 remain poorly understood. METHODS: Immunohistochemical analysis was performed on 119 paraffin-embedded RCC tissue samples to detect FRL1 expression and analyze its prognostic value. Colony formation, the CCK-8 assay, flow cytometry, and in vivo nude mice subcutaneous experiments were used to identify the effects of FRL1 on growth and proliferation. In vitro tests for wound healing, migration, and invasion were used to assess the involvement of FRL1 in invasion and metastatic potential. The process of epithelial-mesenchymal transition process (EMT) and the MMP2 expression were detected in stably transfected RCC cells via western blotting, as well as in tumor tissue paraffin sections from xenograft model. RESULTS: Both FRL1 mRNA and protein levels were noticeably elevated in ccRCC cell lines and samples. Aberrant overexpression of FRL1 was associated with unfavorable clinicopathological features of ccRCC and indicated poor prognosis. Ectopic overexpression of FRL1 increased the growth-promoting traits of ccRCC cells as well as the migratory and invasive capacity of RCC cells, whereas FRL1-silencing caused the opposite results. In addition, FRL1 promoted epithelial-mesenchymal transition (EMT) and upregulated the expression of matrix metalloproteinase 2 (MMP2). Finally, overexpression of FRL1 upregulated phosphorylation level of ERK1/2 with no effect on total level of ERK1/2 in the RCC cells. MAPK/ERK inhibitor reversed the promotional effects of FRL1. CONCLUSION: FRL1 was overexpressed in ccRCC tissues and predicted poor prognosis. FRL1 contributes to invasion and aggressive phenotype of ccRCC by facilitating EMT through MAPK/MMP2 axis.

Aggregation tests identify new gene associations with breast cancer in populations with diverse ancestry.

BACKGROUND: Low-frequency variants play an important role in breast cancer (BC) susceptibility. Gene-based methods can increase power by combining multiple variants in the same gene and help identify target genes. METHODS: We evaluated the potential of gene-based aggregation in the Breast Cancer Association Consortium cohorts including 83,471 cases and 59,199 controls. Low-frequency variants were aggregated for individual genes' coding and regulatory regions. Association results in European ancestry samples were compared to single-marker association results in the same cohort. Gene-based associations were also combined in meta-analysis across individuals with European, Asian, African, and Latin American and Hispanic ancestry. RESULTS: In European ancestry samples, 14 genes were significantly associated (q < 0.05) with BC. Of those, two genes, FMNL3 (P = 6.11 × 10-6) and AC058822.1 (P = 1.47 × 10-4), represent new associations. High FMNL3 expression has previously been linked to poor prognosis in several other cancers. Meta-analysis of samples with diverse ancestry discovered further associations including established candidate genes ESR1 and CBLB. Furthermore, literature review and database query found further support for a biologically plausible link with cancer for genes CBLB, FMNL3, FGFR2, LSP1, MAP3K1, and SRGAP2C. CONCLUSIONS: Using extended gene-based aggregation tests including coding and regulatory variation, we report identification of plausible target genes for previously identified single-marker associations with BC as well as the discovery of novel genes implicated in BC development. Including multi ancestral cohorts in this study enabled the identification of otherwise missed disease associations as ESR1 (P = 1.31 × 10-5), demonstrating the importance of diversifying study cohorts.

Systematic Characterization of Expression Patterns and Immunocorrelations of Formin-Like Genes in Breast Cancer.

Background: Members of the formin-like gene (FMNL) family are required for cytoskeleton-related processes, and their expressions are implicated to the progression of a multitude of malignancies. However, there are insufficient studies on transcription factors and promising prognosis benefit of FMNLs during the genesis of breast cancer (BrCa). Methods: The transcriptional levels of FMNL family members in primary BrCa tissues and their association with intrinsic subclasses were analyzed using the UALCAN database. Then, the prognostic values of FMNLs in BrCa patients were investigated via the Kaplan-Meier plotter. Moreover, the correlations between FMNL expression levels and immune infiltrations were analyzed using the TIMER database. In addition, the expression patterns of FMNLs in BrCa were investigated by single-cell RNA-sequencing (scRNA-seq) analysis and were validated by immunohistochemistry (IHC) staining. Results: The transcriptional level of FMNL1 was shown to be considerably increased in BrCa. It is surprising that the transcriptional quantities of FMNL2 and FMNL3 were substantially reduced. In addition, during the comparison of several BrCa subclasses, FMNL1 and FMNL2 mRNA levels of patients with HER2-positive and triple-negative BrCa subclasses increased, while FMNL3 mRNA levels reduced. With the processions of experimentation, high FMNL1 expression was hopefully linked to well clinical outcome, while high FMNL2 expression predicted poor prognosis. Moreover, FMNL1 was highly expressed in tumor-infiltrating immune cells (TIICs) in tumor tissues. Last but not least, FMNL1 was highly expressed in TIICs and served as a gene marker for TIICs. Conclusions: The fact and result which we analyzed demonstrate FMNL1 as a diagnostic marker for TIICs by comprehensively elucidating the expression patterns and changeable prognostic implications of FMNLs in BrCa clinical applications.

Formin-Like 2 Is a Potential Biomarker of Poor Prognosis in Nasopharyngeal Carcinoma.

Introduction: Nasopharyngeal carcinoma (NPC) is the most common malignant tumor in southern China and Southeast Asia. Although substantial research on NPC has been conducted, the resulting improvement in clinical outcomes remains very disappointing. NPC treatment typically involves radiation therapy and chemotherapy, but the high incidence of metastasis and recurrence in NPC patients result in poor survival. Therefore, identifying potential biomarkers and discovering therapeutic targets are necessary to design tailored treatments based on the genetic profiles of NPC patients. Methods: Correlations of protein immunostaining with clinicopathological features were analyzed by Pearson's χ2 test. The Kaplan-Meier method with a log-rank test was used to generate survival curves. Multivariate analysis was performed using the Cox proportional hazards model. Results: In this study, we comparatively analyzed cytoskeletal organization and biogenesis (GO:0007010) and tumorigenesis in the NPC transcriptome (GSE12452) and found that formin-like 2 (FMNL2) expression was significantly upregulated in NPC tumor tissues. Moreover, high FMNL2 expression was significantly correlated with primary tumor stage (p = 0.001), lymph node status (p = 0.004), cancer stage (p = 0.006), and histological grade (p = 0.040). More importantly, high FMNL2 expression was significantly correlated with poor survival in NPC patients according to univariate analysis and multivariate analysis. Conclusion: This study reveals that FMNL2 may be an important potential biomarker for evaluating the prognosis of NPC patients.

WTAP-mediated m6A modification of lncRNA DIAPH1-AS1 enhances its stability to facilitate nasopharyngeal carcinoma growth and metastasis.

As the most predominant RNA epigenetic regulation in eukaryotic cells, N6-methyladenosine (m6A) plays a critical role in human tumorigenesis and cancer progression. However, the biological function and molecular mechanism of m6A regulation in naso-pharyngeal carcinoma (NPC) remain elusive. Here, we showed that Wilms' tumor 1-associating protein (WTAP) expression was apparently upregulated in NPC, and increased WTAP was associated with poor prognosis. WTAP upregulated in NPC was fine-tuned by KAT3A-mediated H3K27 acetylation. Functionally, WTAP was required for the growth and metastasis of NPC. Mechanistically, lncRNA DIAPH1-AS1 was identified as a bona fide m6A target of WTAP. WTAP-mediated m6A modification of DIAPH1-AS1 enhanced its stability relying on the m6A reader IGF2BP2-dependent pathway. Furthermore, DIAPH1-AS1 acted as a molecular adaptor that promoted MTDH-LASP1 complex formation and upregulated LASP1 expression, ultimately facilitating NPC growth and metastasis. Thus, WTAP-mediated DIAPH1-AS1 m6A methylation is required for NPC tumorigenesis and metastasis.

Role of formin INF2 in human diseases.

Formin proteins catalyze actin nucleation and microfilament polymerization. Inverted formin 2 (INF2) is an atypical diaphanous-related formin characterized by polymerization and depolymerization of actin. Accumulating evidence showed that INF2 is associated with kidney disease focal segmental glomerulosclerosis and cancers, such as colorectal and thyroid cancer where it functions as a tumor suppressor, glioblastoma, breast, prostate, and gastric cancer, via its oncogenic function. However, studies on the underlying molecular mechanisms of the different roles of INF2 in diverse cancers are limited. This review comprehensively describes the structure, biochemical features, and primary pathogenic mutations of INF2.

Circ_0044556 Promotes the Progression of Colorectal Cancer via the miR-665-Dependent Expression Regulation of Diaphanous Homolog 1.

BACKGROUND: Cancer progression can be regulated by noncoding circular RNAs. A recent study has indicated that circ_0044556 facilitated the progression of colorectal cancer. AIM: This research was performed to explore the regulatory mechanism of circ_0044556 in CRC. METHODS: Circ_0044556, miR-665 and Diaphanous Homolog 1 levels were detected by the quantitative real-time polymerase chain reaction. Cell proliferation analysis was performed by cell counting kit-8 assay and Edu assay. Cell cycle progression was assessed using flow cytometry. The protein examination was conducted using western blot. Transwell assay was used to analyze cell migration and invasion. Dual-luciferase reporter assay was performed to validate the interaction between targets. In vivo research was implemented by xenograft tumor assay. RESULTS: Circ_0044556 was upregulated in colorectal cancer samples and cells. Silencing circ_0044556 inhibited cell proliferation, cell cycle progression, migration, invasion, and epithelial-mesenchymal transition in CRC cells. Circ_0044556 could directly target miR-665 and the function of circ_0044556 was associated with the regulation of miR-665. In addition, Diaphanous Homolog 1 was a target gene for miR-665 and the anti-tumor role of miR-665 in colorectal cancer was dependent on the downregulation of Diaphanous Homolog 1. Diaphanous Homolog 1 level was regulated by circ_0044556 via sponging miR-665 in CRC cells. In vivo assay suggested that circ_0044556 promoted CRC tumor growth by regulating the miR-665 and Diaphanous Homolog 1 levels. CONCLUSION: Our findings manifested that circ_0044556 functioned as an oncogenic circRNA in colorectal cancer by mediating the miR-665/Diaphanous Homolog 1 axis, elucidating the molecular mechanism of circ_0044556 in CRC progression.

Formins in Human Disease.

Almost 25 years have passed since a mutation of a formin gene, DIAPH1, was identified as being responsible for a human inherited disorder: a form of sensorineural hearing loss. Since then, our knowledge of the links between formins and disease has deepened considerably. Mutations of DIAPH1 and six other formin genes (DAAM2, DIAPH2, DIAPH3, FMN2, INF2 and FHOD3) have been identified as the genetic cause of a variety of inherited human disorders, including intellectual disability, renal disease, peripheral neuropathy, thrombocytopenia, primary ovarian insufficiency, hearing loss and cardiomyopathy. In addition, alterations in formin genes have been associated with a variety of pathological conditions, including developmental defects affecting the heart, nervous system and kidney, aging-related diseases, and cancer. This review summarizes the most recent discoveries about the involvement of formin alterations in monogenic disorders and other human pathological conditions, especially cancer, with which they have been associated. In vitro results and experiments in modified animal models are discussed. Finally, we outline the directions for future research in this field.

Identification of hub genes in colorectal cancer based on weighted gene co-expression network analysis and clinical data from The Cancer Genome Atlas.

Colorectal cancer (CRC) is one of the most common tumors worldwide and is associated with high mortality. Here we performed bioinformatics analysis, which we validated using immunohistochemistry in order to search for hub genes that might serve as biomarkers or therapeutic targets in CRC. Based on data from The Cancer Genome Atlas (TCGA), we identified 4832 genes differentially expressed between CRC and normal samples (1562 up-regulated and 3270 down-regulated in CRC). Gene ontology (GO) analysis showed that up-regulated genes were enriched mainly in organelle fission, cell cycle regulation, and DNA replication; down-regulated genes were enriched primarily in the regulation of ion transmembrane transport and ion homeostasis. Weighted gene co-expression network analysis (WGCNA) identified eight gene modules that were associated with clinical characteristics of CRC patients, including brown and blue modules that were associated with cancer onset. Analysis of the latter two hub modules revealed the following six hub genes: adhesion G protein-coupled receptor B3 (BAI3, also known as ADGRB3), cyclin F (CCNF), cytoskeleton-associated protein 2 like (CKAP2L), diaphanous-related formin 3 (DIAPH3), oxysterol binding protein-like 3 (OSBPL3), and RERG-like protein (RERGL). Expression levels of these hub genes were associated with prognosis, based on Kaplan-Meier survival analysis of data from the Gene Expression Profiling Interactive Analysis database. Immunohistochemistry of CRC tumor tissues confirmed that OSBPL3 is up-regulated in CRC. Our findings suggest that CCNF, DIAPH3, OSBPL3, and RERGL may be useful as therapeutic targets against CRC. BAI3 and CKAP2L may be novel biomarkers of the disease.

Characterization of a L136P mutation in Formin-like 2 (FMNL2) from a patient with chronic inflammatory bowel disease.

Diaphanous related formins are highly conserved proteins regulated by Rho-GTPases that act as actin nucleation and assembly factors. Here we report the functional characterization of a non-inherited heterozygous FMNL2 p.L136P mutation carried by a patient who presented with severe very early onset inflammatory bowel disease (IBD). We found that the FMNL2 L136P protein displayed subcellular mislocalization and deregulated protein autoinhibition indicating gain-of-function mechanism. Expression of FMNL2 L136P impaired cell spreading as well as filopodia formation. THP-1 macrophages expressing FMNL2 L136P revealed dysregulated podosome formation and a defect in matrix degradation. Our data indicate that the L136P mutation affects cellular actin dynamics in fibroblasts and immune cells such as macrophages.

Inhibition of polar actin assembly by astral microtubules is required for cytokinesis.

During cytokinesis, the actin cytoskeleton is partitioned into two spatially distinct actin isoform specific networks: a ß-actin network that generates the equatorial contractile ring, and a γ-actin network that localizes to the cell cortex. Here we demonstrate that the opposing regulation of the ß- and γ-actin networks is required for successful cytokinesis. While activation of the formin DIAPH3 at the cytokinetic furrow underlies ß-actin filament production, we show that the γ-actin network is specifically depleted at the cell poles through the localized deactivation of the formin DIAPH1. During anaphase, CLIP170 is delivered by astral microtubules and displaces IQGAP1 from DIAPH1, leading to formin autoinhibition, a decrease in cortical stiffness and localized membrane blebbing. The contemporaneous production of a ß-actin contractile ring at the cell equator and loss of γ-actin from the poles is required to generate a stable cytokinetic furrow and for the completion of cell division.

DIAPH3 deficiency links microtubules to mitotic errors, defective neurogenesis, and brain dysfunction.

Diaphanous (DIAPH) three (DIAPH3) is a member of the formin proteins that have the capacity to nucleate and elongate actin filaments and, therefore, to remodel the cytoskeleton. DIAPH3 is essential for cytokinesis as its dysfunction impairs the contractile ring and produces multinucleated cells. Here, we report that DIAPH3 localizes at the centrosome during mitosis and regulates the assembly and bipolarity of the mitotic spindle. DIAPH3-deficient cells display disorganized cytoskeleton and multipolar spindles. DIAPH3 deficiency disrupts the expression and/or stability of several proteins including the kinetochore-associated protein SPAG5. DIAPH3 and SPAG5 have similar expression patterns in the developing brain and overlapping subcellular localization during mitosis. Knockdown of SPAG5 phenocopies DIAPH3 deficiency, whereas its overexpression rescues the DIAHP3 knockdown phenotype. Conditional inactivation of Diaph3 in mouse cerebral cortex profoundly disrupts neurogenesis, depleting cortical progenitors and neurons, leading to cortical malformation and autistic-like behavior. Our data uncover the uncharacterized functions of DIAPH3 and provide evidence that this protein belongs to a molecular toolbox that links microtubule dynamics during mitosis to aneuploidy, cell death, fate determination defects, and cortical malformation.

Novel role of LINC01013/miR-6795-5p/FMNL3 axis in the regulation of hepatocellular carcinoma stem cell features.

Cancer stem cells (CSCs) are major contributors to tumor initiation, recurrence, and metastasis of hepatocellular carcinoma (HCC). Some long non-coding RNAs have been reported as modulators of stem-like properties in cancer cells. However, the role of LINC01013 in liver CSCs has not yet been clarified. In this study, we aimed to elucidate the expression pattern and functions of LINC01013 in HCC. HCC tissues and normal controls were collected, and the expression pattern of LINC01013 and miR-6795-5p was identified by quick real-time polymerase chain reaction. Cell counting kit-8 assay, colony formation, and spheroid formation were performed to measure cell viability, proliferation, and self-renewal of HCC cell lines. The expression of stem markers was detected by western blot analysis. The effect of LINC01013 on viability, proliferation, and stem-like properties was detected through gain-of-function and loss-of-function experiments. The direct interaction among LINC01013, miR-6795-5p, and FMNL3 was testified by dual-luciferase reporter gene assay. Tumor-bearing mice were constructed to ascertain the functions of LINC01013 in vivo. HCC tissues showed increased LINC01013 and FMNL3 expression, while it showed a decreased miR-6795-5p expression as compared to the relative controls. Moreover, the high level of LINC01013 was closely related to the poor prognosis of HCC patients. LINC01013 directly binds to miR-6795-5p and subsequently relieves FMNL3. Silencing LINC01013, FMNL3, or overexpression of miR-6795-5p could suppress spheroid and colony formation, proliferation, as well as expression of stemness markers in HepG2 and SNU-182 cells. LINC01013 knockdown suppressed growth and stem-like traits of HCC cells in vivo by reducing FMNL3 expression. LINC01013/miR-6795-5p/FMNL3 axis may be a novel therapeutic target for HCC.