MICALL2 as a substrate of ubiquitinase TRIM21 regulates tumorigenesis of colorectal cancer.

BACKGROUND: Molecule interacting with CasL-like protein 2 (MICALL2) is believed to regulate cytoskeleton dynamics, tight junction formation, and neurite outgrowth. However, its biological role and the underlying mechanism in colorectal cancer (CRC) remain largely elusive. METHODS: qRT-PCR, Western blotting and immunohistochemistry assays were used to detect the expression levels of different genes. Next, mass spectrometry, co-immunoprecipitation and immunofluorescence staining were used to detect the interactions of proteins. Furthermore, MTT assay, colony formation assay, wound-healing assays and xenograft tumor models were performed to demonstrate the functions of MICALL2 in CRC. In addition, transcriptome sequencing and Western blotting were conducted to verify the mechanism of MICALL2 in CRC. RESULTS: We found that both mRNA and protein levels of MICALL2 are up-regulated in colorectal cancer tissues compared with non-tumor tissues and that its overexpression is closely correlated with poor prognosis. Ubiquitin E3 ligase Tripartite motif-containing protein 21 (TRIM21) mediated MICALL2 ubiquitination and proteasome-dependent degradation, negatively correlated with MICALL2 levels, and reversely regulated the tumorigenic activity of MICALL2 in CRC. Functional studies confirmed that MICALL2 promoted colorectal cancer cell growth and migration via the Wnt/ß-catenin signaling pathway. CONCLUSIONS: As a substrate of ubiquitinase TRIM21, MICALL2 enhances the growth and migration of colorectal cancer cells and activates the Wnt/ß-catenin signaling pathway. Video abstract.

Analyzing gene expression profiles with preliminary validations in cardiac hypertrophy induced by pressure overload.

The aim of this study was to identify the key genes involved in the cardiac hypertrophy (CH) induced by pressure overload. mRNA microarray data sets GSE5500 and GSE18801 were downloaded from the Gene Expression Omnibus database, and differentially expressed genes (DEGs) were screened using the Limma package; then, functional and pathway enrichment analysis were performed for common DEGs using the Database for Annotation, Visualization and Integrated Discovery database. Furthermore, the top DEGs were further validated using quantitative PCR in the hypertrophic heart tissue induced by isoprenaline. A total of 113 common DEGs with absolute fold change > 0.5, including 60 significantly upregulated DEGs and 53 downregulated DEGs, were obtained. Gene ontology term enrichment analysis suggested that common upregulated DEG were mainly enriched in neutrophil chemotaxis, extracellular fibril organization, and cell proliferation; and the common downregulated genes were significantly enriched in ion transport, endoplasmic reticulum, and dendritic spine. Kyoto Encyclopedia of Genes and Genomes pathway analysis found that the common DEGs were mainly enriched in extracellular matrix receptor interaction, phagosome, and focal adhesion. Additionally, the expression of Mfap4, Ltbp2, Aspn, Serpina3n, and Cnksr1 were upregulated in the model of CH, while the expression of Anp32a was downregulated. The current study identified the key deregulated genes and pathways involved in the CH, which could shed new light to understand the mechanism of CH.

USP33 mediates Slit-Robo signaling in inhibiting colorectal cancer cell migration.

Originally discovered in neuronal guidance, the Slit-Robo pathway is emerging as an important player in human cancers. However, its involvement and mechanism in colorectal cancer (CRC) remains to be elucidated. Here, we report that Slit2 expression is reduced in CRC tissues compared with adjacent noncancerous tissues. Extensive promoter hypermethylation of the Slit2 gene has been observed in CRC cells, which provides a mechanistic explanation for the Slit2 downregulation in CRC. Functional studies showed that Slit2 inhibits CRC cell migration in a Robo-dependent manner. Robo-interacting ubiquitin-specific protease 33 (USP33) is required for the inhibitory function of Slit2 on CRC cell migration by deubiquitinating and stabilizing Robo1. USP33 expression is downregulated in CRC samples, and reduced USP33 mRNA levels are correlated with increased tumor grade, lymph node metastasis and poor patient survival. Taken together, our data reveal USP33 as a previously unknown tumor-suppressing gene for CRC by mediating the inhibitory function of Slit-Robo signaling on CRC cell migration. Our work suggests the potential value of USP33 as an independent prognostic marker of CRC.

MICAL-L2, as an estrogen-responsive gene, is involved in ER-positive breast cancer cell progression and tamoxifen sensitivity via the AKT/mTOR pathway.

Endocrine treatment, particularly tamoxifen, has shown significant improvement in the prognosis of patients with estrogen receptor-positive (ER-positive) breast cancer. However, the clinical utility of this treatment is often hindered by the development of endocrine resistance. Therefore, a comprehensive understanding of the underlying mechanisms driving ER-positive breast cancer carcinogenesis and endocrine resistance is crucial to overcome this clinical challenge. In this study, we investigated the expression of MICAL-L2 in ER-positive breast cancer and its impact on patient prognosis. We observed a significant upregulation of MICAL-L2 expression in ER-positive breast cancer, which correlated with a poorer prognosis in these patients. Furthermore, we found that estrogen-ERß signaling promoted the expression of MICAL-L2. Functionally, our study demonstrated that MICAL-L2 not only played an oncogenic role in ER-positive breast cancer tumorigenesis but also influenced the sensitivity of ER-positive breast cancer cells to tamoxifen. Mechanistically, as an estrogen-responsive gene, MICAL-L2 facilitated the activation of the AKT/mTOR signaling pathway in ER-positive breast cancer cells. Collectively, our findings suggest that MICAL-L2 could serve as a potential prognostic marker for ER-positive breast cancer and represent a promising molecular target for improving endocrine treatment and developing therapeutic approaches for this subtype of breast cancer.

Alternative Pre-mRNA Splicing, Cell Death, and Cancer.

Alternative splicing is one of the most powerful mechanisms for generating functionally distinct products from a single genetic loci and for fine-tuning gene activities at the post-transcriptional level. Alternative splicing plays important roles in regulating genes critical for cell death. These cell death genes encode death ligands, cell surface death receptors, intracellular death regulators, signal transduction molecules, and death executor enzymes such as caspases and nucleases. Alternative splicing of these genes often leads to the formation of functionally different products, some of which have antagonistic effects that are either cell death-promoting or cell death-preventing. Differential alternative splicing can affect expression, subcellular distribution, and functional activities of the gene products. Molecular defects in splicing regulation of cell death genes have been associated with cancer development and resistance to treatment. Studies using molecular, biochemical, and systems-based approaches have begun to reveal mechanisms underlying the regulation of alternative splicing of cell death genes. Systematic studies have begun to uncover the multi-level interconnected networks that regulate alternative splicing. A global picture of the complex mechanisms that regulate cell death genes at the pre-mRNA splicing level has thus begun to emerge.

MICAL1 inhibits colorectal cancer cell migration and proliferation by regulating the EGR1/ß-catenin signaling pathway.

MICAL1 has been reported to be involved in the malignant processes of several types of cancer cells, however, the roles of MICAL1 in colorectal cancer (CRC) have not been well-characterized. This study aims to investigate the cellular functions and molecular mechanisms of MICAL1 in CRC cells. Here, we found that both mRNA and protein levels of MICAL1 were down-regulated in colorectal cancer tissues compared with matched adjacent non-tumor tissues, and the expression level of MICAL1 was correlated with the metastatic status of colorectal cancer. Importantly, overexpression of MICAL1 significantly inhibited colorectal cancer cell migration and growth, and increased the level of E-cadherin and Occludin, and suppressed the expression level of Vimentin and N-cadherin; while silencing of MICAL1 promoted CRC cell migration and enhanced EMT. In addition, MICAL1 overexpression significantly inhibited the proliferation and growth of CRC in vitro and in vivo. Moreover, RNA sequencing and bioinformatics analysis identified that MICAL1 was closely correlated with "cell migration", "cell cycle" and "ß-catenin signaling" genesets. Mechanistically, overexpression of MICAL1 downregulated the mRNA level of EGR1 and ß-catenin, decreased the protein level and nuclear translocation of ß-catenin, and inhibited the transcriptions of ß-catenin downstream targets, c-myc and cyclin D1. The ectopic expression of EGR1 or ß-catenin can significantly block the MICAL1-mediated inhibitory effects. Collectively, MICAL1 is down-regulated in CRC, and plays an inhibitory role in the migration and growth of CRC cells by suppressing the ERG1/ß-catenin signaling pathway.

Candidate genes and pathways associated with brain metastasis from lung cancer compared with lymph node metastasis.

Brain metastasis from lung cancer (BMLC) is one of the common types of metastasis associated with poor prognosis. The aim of the present study was to elucidate the underlying molecular mechanisms of BMLC. The mRNA microarray dataset GSE18549 was downloaded from the Gene Expression Omnibus database. The Limma package of R was used to screen the differentially expressed genes (DEGs). Based on the DAVID database, functional and pathway enrichment analyses of DEGs were performed. Furthermore, the protein-protein interaction (PPI) network was predicted using the STRING database and visualized with Cytoscape software. In addition, hub genes and significant modules were selected based on the network. A total of 190 DEGs with log2|(fold change)|>1, including 129 significantly downregulated DEGs and 61 upregulated DEGs, were obtained. Gene Ontology functional enrichment analysis indicated that downregulated DEGs were mainly associated with 'immune response', 'cell activation' and 'leukocyte activation', while the upregulated DEGs were involved in 'DNA repair' and 'viral process'. Kyoto Encyclopedia of Genes and Genomes pathway analysis indicated that the downregulated DEGs were mainly enriched in 'chemokine signaling pathway', whereas the upregulated DEGs were associated with 'oocyte meiosis'. Based on the PPI network, 9 hub genes were selected, namely tumor necrosis factor, C-C motif chemokine ligand (CCL) 2, CD34, vascular cell adhesion molecule 1, CD48, CD27, CCL19, C-X-C motif chemokine receptor 6 and C-C motif chemokine receptor 2. The present study sheds light on the molecular mechanisms of BMLC and may provide molecular targets and diagnostic biomarkers for BMLC.

Identification of candidate biomarkers and pathways associated with SCLC by bioinformatics analysis.

Small cell lung cancer (SCLC) is one of the highly malignant tumors and a serious threat to human health. The aim of the present study was to explore the underlying molecular mechanisms of SCLC. mRNA microarray datasets GSE6044 and GSE11969 were downloaded from Gene Expression Omnibus database, and the differentially expressed genes (DEGs) between normal lung and SCLC samples were screened using GEO2R tool. Functional and pathway enrichment analyses were performed for common DEGs using the DAVID database, and the protein­protein interaction (PPI) network of common DEGs was constructed by the STRING database and visualized with Cytoscape software. In addition, the hub genes in the network and module analysis of the PPI network were performed using CentiScaPe and plugin Molecular Complex Detection. Finally, the mRNA expression levels of hub genes were validated in the Oncomine database. A total of 150 common DEGs with absolute fold­change >0.5, including 66 significantly downregulated DEGs and 84 upregulated DEGs were obtained. The Gene Ontology term enrichment analysis suggested that common upregulated DEGs were primarily enriched in biological processes (BPs), including 'cell cycle', 'cell cycle phase', 'M phase', 'cell cycle process' and 'DNA metabolic process'. The common downregulated genes were significantly enriched in BPs, including 'response to wounding', 'positive regulation of immune system process', 'immune response', 'acute inflammatory response' and 'inflammatory response'. Kyoto Encyclopedia of Genes and Genomes pathway analysis identified that the common downregulated DEGs were primarily enriched in the 'complement and coagulation cascades' signaling pathway; the common upregulated DEGs were mainly enriched in 'cell cycle', 'DNA replication', 'oocyte meiosis' and the 'mismatch repair' signaling pathways. From the PPI network, the top 10 hub genes in SCLC were selected, including topoisomerase IIα, proliferating cell nuclear antigen, replication factor C subunit 4, checkpoint kinase 1, thymidylate synthase, minichromosome maintenance protein (MCM) 2, cell division cycle (CDC) 20, cyclin dependent kinase inhibitor 3, MCM3 and CDC6, the mRNA levels of which are upregulated in Oncomine SCLC datasets with the exception of MCM2. Furthermore, the genes in the significant module were enriched in 'cell cycle', 'DNA replication' and 'oocyte meiosis' signaling pathways. Therefore, the present study can shed new light on the understanding of molecular mechanisms of SCLC and may provide molecular targets and diagnostic biomarkers for the treatment and early diagnosis of SCLC.

Silencing of PHLPP1 promotes neuronal apoptosis and inhibits functional recovery after spinal cord injury in mice.

AIM: Spinal cord injury (SCI) causes increased apoptosis of neurons, leading to irreversible dysfunction of the spinal cord. In this study, we investigated the effects of the progression of SCI and potential regulation of apoptosis after the Pleckstrin homology (PH) domain and leucine rich repeat protein phosphatase 1 (PHLPP1) gene was silenced. MAIN METHODS: Spinal cord injection, and neuronal transfection with a recombinant adenovirus vector encoding small interfering RNA (siRNA) against PHLPP1 (AdsiPHLPP1) successfully silenced PHLPP1. These created in vivo and in vitro PHLPP1-silenced models, respectively, resulting in stable expression of the transgene in neurons. KEY FINDINGS: The results showed that silencing of PHLPP1 evidently reduced levels of the nuclear factor erythroid 2-related factor 2 (Nrf2) after SCI. Western blot analysis revealed that the mice injected with AdsiPHLPP1 showed increased the expression of pro-apoptotic factors (Bax and cleaved-caspase 3), and reduced levels of neurotrophic (BDNF) and anti-apoptotic (Bcl-2) factors, both in vivo and in vitro. The motor function of AdsiPHLPP1-injected mice was restored more slowly than that of wild type (WT) mice. In addition, the number of motor neurons surviving in the anterior horn of the spinal cord was also reduced after SCI. SIGNIFICANCE: Our results confirm that silencing of PHLPP1 promotes neuronal apoptosis and inhibits functional recovery after injury in vivo and in vitro. Consequently, PHLPP1 represents a potential therapeutic target gene for the clinical treatment of SCI.

TDP-43 regulates cancer-associated microRNAs.

Aberrant regulation of miRNA genes contributes to pathogenesis of a wide range of human diseases, including cancer. The TAR DNA binding protein 43 (TDP-43), a RNA/DNA binding protein associated with neurodegeneration, is involved in miRNA biogenesis. Here, we systematically examined miRNAs regulated by TDP-43 using RNA-Seq coupled with an siRNA-mediated knockdown approach. TDP-43 knockdown affected the expression of a number of miRNAs. In addition, TDP-43 down-regulation led to alterations in the patterns of different isoforms of miRNAs (isomiRs) and miRNA arm selection, suggesting a previously unknown role of TDP-43 in miRNA processing. A number of TDP-43 associated miRNAs, and their candidate target genes, are associated with human cancers. Our data reveal highly complex roles of TDP-43 in regulating different miRNAs and their target genes. Our results suggest that TDP-43 may promote migration of lung cancer cells by regulating miR-423-3p. In contrast, TDP-43 increases miR-500a-3p expression and binds to the mature miR-500a-3p sequence. Reduced expression of miR-500a-3p is associated with poor survival of lung cancer patients, suggesting that TDP-43 may have a suppressive role in cancer by regulating miR-500a-3p. Cancer-associated genes LIF and PAPPA are possible targets of miR-500a-3p. Our work suggests that TDP-43-regulated miRNAs may play multifaceted roles in the pathogenesis of cancer.

CD146 acts as a novel receptor for netrin-1 in promoting angiogenesis and vascular development.

Angiogenesis, a process that newly-formed blood vessels sprout from pre-existing ones, is vital for vertebrate development and adult homeostasis. Previous studies have demonstrated that the neuronal guidance molecule netrin-1 participates in angiogenesis and morphogenesis of the vascular system. Netrin-1 exhibits dual activities in angiogenesis: either promoting or inhibiting angiogenesis. The anti-angiogenic activity of netrin-1 is mediated by UNC5B receptor. However, how netrin-1 promotes angiogenesis remained unclear. Here we report that CD146, an endothelial transmembrane protein of the immunoglobulin superfamily, is a receptor for netrin-1. Netrin-1 binds to CD146 with high affinity, inducing endothelial cell activation and downstream signaling in a CD146-dependent manner. Conditional knockout of the cd146 gene in the murine endothelium or disruption of netrin-CD146 interaction by a specific anti-CD146 antibody blocks or reduces netrin-1-induced angiogenesis. In zebrafish embryos, downregulating either netrin-1a or CD146 results in vascular defects with striking similarity. Moreover, knocking down CD146 blocks ectopic vascular sprouting induced by netrin-1 overexpression. Together, our data uncover CD146 as a previously unknown receptor for netrin-1 and also reveal a functional ligand for CD146 in angiogenesis, demonstrating the involvement of netrin-CD146 signaling in angiogenesis during vertebrate development.

Myo9b is a key player in SLIT/ROBO-mediated lung tumor suppression.

Emerging evidence indicates that the neuronal guidance molecule SLIT plays a role in tumor suppression, as SLIT-encoding genes are inactivated in several types of cancer, including lung cancer; however, it is not clear how SLIT functions in lung cancer. Here, our data show that SLIT inhibits cancer cell migration by activating RhoA and that myosin 9b (Myo9b) is a ROBO-interacting protein that suppresses RhoA activity in lung cancer cells. Structural analyses revealed that the RhoGAP domain of Myo9b contains a unique patch that specifically recognizes RhoA. We also determined that the ROBO intracellular domain interacts with the Myo9b RhoGAP domain and inhibits its activity; therefore, SLIT-dependent activation of RhoA is mediated by ROBO inhibition of Myo9b. In a murine model, compared with control lung cancer cells, SLIT-expressing cells had a decreased capacity for tumor formation and lung metastasis. Evaluation of human lung cancer and adjacent nontumor tissues revealed that Myo9b is upregulated in the cancer tissue. Moreover, elevated Myo9b expression was associated with lung cancer progression and poor prognosis. Together, our data identify Myo9b as a key player in lung cancer and as a ROBO-interacting protein in what is, to the best of our knowledge, a newly defined SLIT/ROBO/Myo9b/RhoA signaling pathway that restricts lung cancer progression and metastasis. Additionally, our work suggests that targeting the SLIT/ROBO/Myo9b/RhoA pathway has potential as a diagnostic and therapeutic strategy for lung cancer.

USP33, a new player in lung cancer, mediates Slit-Robo signaling.

Ubiquitin specific protease 33 (USP33) is a multifunctional protein regulating diverse cellular processes. The expression and role of USP33 in lung cancer remain unexplored. In this study, we show that USP33 is down-regulated in multiple cohorts of lung cancer patients and that low expression of USP33 is associated with poor prognosis. USP33 mediates Slit-Robo signaling in lung cancer cell migration. Downregulation of USP33 reduces the protein stability of Robo1 in lung cancer cells, providing a previously unknown mechanism for USP33 function in mediating Slit activity in lung cancer cells. Taken together, USP33 is a new player in lung cancer that regulates Slit-Robo signaling. Our data suggest that USP33 may be a candidate tumor suppressor for lung cancer with potential as a prognostic marker.