ARID3A promotes the development of colorectal cancer by upregulating AURKA.

Colorectal cancer (CRC) is one of the most common malignant tumours, and its morbidity and mortality rates are relatively high. However, the aetiology and pathogenesis of CRC have not been clearly elucidated to date. AT-rich interaction domain 3A (ARID3A) is a member of the ARID3 family and a transcription factor that can bind to specific DNA sites to regulate gene expression. It was reported that ARID3A is involved in various biological processes and may be related to carcinogenesis. In this study, by assessing the mRNA level of ARID3A in TCGA database, we found that ARID3A expression increased in CRC tissues, and proposed that ARID3A could act as a tumour-promoting factor in the development of CRC. To verify this hypothesis, we used cell proliferation, migration and invasion assays to assess the effect of ARID3A on CRC cells. We revealed that ARID3A overexpression enhanced tumour cell proliferation, migration and invasion. ARID3A could target Aurora kinase A (AURKA) to facilitate the malignant phenotype of CRC cells, and patients with a higher ratio of AURKA and ARID3A had a better overall survival. Conclusively, this study showed that ARID3A targeted AURKA to facilitate the development of CRC. The ratio of ARID3A and AURKA could be used as a potential biomarker to predict prognosis, providing a new strategy for the diagnosis and prognosis of CRC.

Alternative splicing: An important regulatory mechanism in colorectal carcinoma.

Alternative splicing (AS) is a process that produces various mRNA splicing isoforms via different splicing patterns of mRNA precursors (pre-mRNAs). AS is the primary mechanism for increasing the types and quantities of proteins to improve biodiversity and influence multiple biological processes, including chromatin modification, signal transduction, and protein expression. It has been reported that AS is involved in the tumorigenesis and development of colorectal carcinoma (CRC). In this review, we delineate the concept, types, regulatory processes, and technical advances of AS and focus on the role of AS in CRC initiation, progression, treatment, and prognosis. This summary of the current knowledge about AS will contribute to our understanding of CRC initiation and development. This study will help in the discovery of novel biomarkers and therapeutic targets for CRC prognosis and treatment.

A germline mutation in Rab43 gene identified from a cancer family predisposes to a hereditary liver-colon cancer syndrome.

BACKGROUND: Hereditary cancer syndromes have inherited germline mutations which predispose to benign and malignant tumors. Understanding of the molecular causes in hereditary cancer syndromes has advanced cancer treatment and prevention. However, the causal genes of many hereditary cancer syndromes remain unknown due to their rare frequency of mutation. METHODS: A large Chinese family with a history of hereditary liver-colon cancer syndrome was studied. The genomic DNA was extracted from the blood samples of involved family members, whole-exome sequencing was performed to identify genetic variants. Functional validation of a candidate variant was carried out using gene expression, gene knockout and immunohistochemistry. RESULTS: The whole-exome of the proband diagnosed with colon cancer was sequenced in comparison with his mother. A total of 13 SNVs and 16 InDels were identified. Among these variants, we focused on a mutation of Rab43 gene, a GTPase family member involving in protein trafficking, for further validation. Sanger DNA sequencing confirmed a mutation (c: 128810106C > T, p: A158T) occurred in one allele of Rab43 gene from the proband, that heterozygous mutation also was verified in the genome of the proband's deceased father with liver cancer, but not in his healthy mother and sister. Ectopic expression of the Rab43 A158T mutant in Huh7 cells led to more enhanced cell growth, proliferation and migration compared to the expression of wild type Rab43. Conversely, knockout of Rab43 in HepG2 cells resulted in slow cell growth and multiple nuclei formation and impaired activation of Akt. Finally, a positive correlation between the expression levels of Rab43 protein and cancer development in that family was confirmed. CONCLUSIONS: A germline mutation of Rab43 gene is identified to be associated with the onset of a familial liver-colon cancer syndrome. Our finding points to a potential role of protein trafficking in the tumorigenesis of the familial cancer syndrome, and helps the genetic counseling to the affected family members.

Proteomic Profiling of Paclitaxel Treated Cells Identifies a Novel Mechanism of Drug Resistance Mediated by PDCD4.

Paclitaxel (PTX) is a widely used chemotherapeutic drug effective against numerous cancers. To elucidate cellular pathways targeted by PTX and identify novel mechanisms of PTX resistance, we used a SILAC based quantitative proteomic approach to evaluate global changes of cellular protein abundance in HeLa cells. We identified 347 proteins involved in a number of biological processes including spindle assembly, mitotic exit, and extracellular adhesion whose abundance changes upon PTX treatment. Notably, the tumor suppressor PDCD4 involved in translation suppression was down-regulated by PTX. We demonstrated that PDCD4 is a cell-cycle regulated protein and that changes in its abundance are sufficient to alter PTX sensitivity in multiple human cancer cell lines. Immunoprecipitation of PDCD4-RNA complexes and RT-PCR revealed that PDCD4 mediated PTX sensitivity acts through its interaction with mRNA of UBE2S, a ubiquitin K11 linkage conjugating enzyme critical for mitotic exit. Lastly, high levels of PDCD4 in lung cancer tissues are positively correlated with the longer overall survival time of the examined lung cancer patients with PTX involved adjuvant therapy. Therefore, our proteomic screen for paclitaxel targets not only provided novel insight into the cellular resistance to paclitaxel via the PDCD4-mitotic exit regulation axis, but also offered a predictive biomarker for paclitaxel-based personalized chemotherapy in the treatment of lung cancer.

The role of alternative splicing in lung cancer.

Aberrant alternative splicing (AS) events are frequently observed in lung cancer, which can be attributed to aberrant gene AS, alterations in splicing regulatory factors, or changes in splicing regulatory mechanisms. Consequently, the dysregulation of alternative RNA splicing is the fundamental cause of lung cancer. In this review, we have summarized the pivotal role of AS in the development, progression, invasion, metastasis, angiogenesis, and drug resistance of lung cancer. Ultimately, this review emphasizes the potential of AS as biomarkers in lung cancer prognosis and diagnosis, and introduces some applications of AS isoform in the treatment of lung cancer. The comprehension of the AS may provide a glimmer of hope for the eradication of lung cancer.

Loss of tumor suppressor inositol polyphosphate 4-phosphatase type B impairs DNA double-strand break repair by destabilization of DNA tethering protein Rad50.

Genome instability is the fundamental hallmark of malignant tumors. Tumor suppressors often play a role in maintaining genome stability. Our previous genetic screen identified inositol polyphosphate 4-phosphatase type B (INPP4B), primarily hydrolyzing phosphatidylinositol 3, 4-disphosphate, is a potential tumor suppressor in lung cancer cells. How INPP4B regulates the genome stability of lung cancer cells is unclear. Here we report knockout of INPP4B in lung adenocarcinoma A549 cells by Crispr-Cas9 gene editing leads to sensitization to ionizing radiation (IR), PARP inhibitor olaparib and impaired DNA homologous recombination repair. Re-introduction of a Crispr-Cas9 resistant INPP4B gene in the INPP4B knockout cells partially restored their resistance to IR, indicating loss of INPP4B protein is relevant to the increased IR sensitivity. Furthermore, we showed ectopic expressed INPP4B in A549 cells responds to IR irradiation by redistribution from cytoplasm to nucleus and endogenous INPP4B protein interacts with Rad50, a crucial MRN complex component for tethering DNA double-strand breaks. Loss of INPP4B protein results in decreased stability of Rad50 in vivo, suggesting an unanticipated role of tumor suppressor INPP4B in maintaining genome integrity via facilitating Rad50 mediated DNA double-strand break repair. Taken together, our findings support a dual role of INPP4B in suppression of tumorigenesis by safeguarding genome stability, as well as inhibiting of PI3K-Akt-mTOR signaling, and offer a new therapeutic strategy for personalized cancer treatment to patients with INPP4B defects or deficiency in the clinic.

[Expression, purification of recombinant cationic peptide AIK in Escherichia coli and its antitumor activity].

AIK is a novel cationic peptide with potential antitumor activity. In order to construct the AIK expression vector by Gateway technology, and establish an optimal expression and purification method for recombinant AIK, a set of primers containing AttB sites were designed and used to create the AttB-TEV-FLAG-AIR fusion gene by overlapping PCR. The resulting fusion gene was cloned into the donor vector pDONR223 by attB and attP mediated recombination (BP reaction), then, transferred into the destination vector pDESTl 5 by attL and attR mediated recombination (LR reaction). All the cloning was verified by both colony PCR and DNA sequencing. The BL21 F. coli transformed by the GST-AIR expression plasmid was used to express the GST-AIK fusion protein with IPTG induction and the induction conditions were optimized. GST-AIR fusion protein was purified by glutathione magnetic beads, followed by rTEV cleavage to remove GST tag and MTS assay to test the growth inhibition activity of the recombinant AIR on human leukemia HL-60 cells. We found that a high level of soluble expression of GST-AIK protein (more than 30% out of the total bacterial proteins) was achieved upon 0.1 mmol/L ITPG induction for 4 h at 37 °C in the transformed BL21 F. coli with starting OD600 at 1.0. Through GST affinity purification and rTEV cleavage, the purity of the resulting recombinant AIK was greater than 95%. And the MTS assays on HL-60 cells confirmed that the recombinant AIK retains an antitumor activity at a level similar to the chemically synthesized AIK. Taken together, we have established a method for expression and purification of recombinant AIK with a potent activity against tumor cells, which will be beneficial for the large-scale production and application of recombinant AIK in the future.

An ShRNA Based Genetic Screen Identified Sesn2 as a Potential Tumor Suppressor in Lung Cancer via Suppression of Akt-mTOR-p70S6K Signaling.

BACKGROUND: Lung cancer is emerging rapidly as the leading death cause in Chinese cancer patients. The causal factors for Chinese lung cancer development remain largely unclear. Here we employed an shRNA library-based loss-of-function screen in a genome-wide and unbiased manner to interrogate potential tumor suppressor candidates in the immortalized human lung epithelial cell line BEAS-2B. METHODS/RESULTS: Soft agar assays were conducted for screening BEAS-2B cells infected with the retroviral shRNA library with the acquired feature of anchorage-independent growth, large (>0.5mm in diameter) and well-separated colonies were isolated for proliferation. PCRs were performed to amplify the integrated shRNA fragment from individual genomic DNA extracted from each colony, and each PCR product is submitted for DNA sequencing to reveal the integrated shRNA and its target gene. A total of 6 candidate transformation suppressors including INPP4B, Sesn2, TIAR, ACRC, Nup210, LMTK3 were identified. We validated Sesn2 as the candidate of lung cancer tumor suppressor. Knockdown of Sesn2 by an shRNA targeting 3' UTR of Sesn2 transcript potently stimulated the proliferation and malignant transformation of lung bronchial epithelial cell BEAS-2B via activation of Akt-mTOR-p70S6K signaling, whereas ectopic expression of Sens2 re-suppressed the malignant transformation elicited by the Sesn2 shRNA. Moreover, knockdown of Sesn2 in BEAS-2B cells promoted the BEAS-2B cell-transplanted xenograft tumor growth in nude mice. Lastly, DNA sequencing indicated mutations of Sesn2 gene are rare, the protein levels of Sesn2 of 77 Chinese lung cancer patients varies greatly compared to their adjacent normal tissues, and the low expression level of Sesn2 associates with the poor survival in these examined patients by Kaplan Meier analysis. CONCLUSIONS: Our shRNA-based screen has demonstrated Sesn2 is a potential tumor suppressor in lung epithelial cells. The expression level of Sesn2 may serve as a prognostic marker for Chinese lung cancer patients in the clinic.