Identification of DHX36 as a tumour suppressor through modulating the activities of the stress-associated proteins and cyclin-dependent kinases in breast cancer.

The nucleic acid guanine-quadruplex structures (G4s) are involved in many aspects of cancer progression. The DEAH-box polypeptide 36 (DHX36) has been identified as a dominant nucleic acid helicase which targets and disrupts DNA and RNA G4s in an ATP-dependent manner. However, the actual role of DHX36 in breast cancer remains unknown. In this study, we observed that the gene expression of DHX36 was positively associated with patient survival in breast cancer. The abundance of DHX36 is also linked with pathologic conditions and the stage of breast cancer. By using the xenograft mouse model, we demonstrated that the stable knockdown of DHX36 via lentivirus in breast cancer cells significantly promoted tumour growth. We also found that, after the DHX36 knockdown (KD), the invasion of triple-negative breast cancer cells was enhanced. In addition, we found a significant increase in the number of cells in the S-phase and a reduction of apoptosis with the response to cisplatin. DHX36 KD also desensitized the cytotoxic cellular response to paclitaxel and cisplatin. Transcriptomic profiling analysis by RNA sequencing indicated that DHX36 altered gene expression profile through the upstream activation of TNF, IFNγ, NFκb and TGFß1. High throughput signalling analysis showed that one cluster of stress-associated kinase proteins including p53, ROCK1 and JNK were suppressed, while the mitotic checkpoint protein-serine kinases CDK1 and CDK2 were activated, as a consequence of the DHX36 knockdown. Our study reveals that DHX36 functions as a tumour suppressor and may be considered as a potential therapeutic target in breast cancer.

Distinct mechanisms by which two forms of miR-140 suppress the malignant properties of lung cancer cells.

In this study we attempted to determine the molecular mechanisms underlying the two mature products of pre-miR-140 (3p and 5p) in malignant properties of lung cancer cells. The differential expression of the two forms of miR-140 in both NSCLC tissues and cell lines was determined by quantitative real-time PCR (qRT-PCR). The effects of the miR-140 mimics on the malignant properties of lung cancer cells were evaluated using invasion assay, adhesion assay, tubule formation assay and metabolite profiling. Biotin-miRNA pulldown and transcriptome profiling by RNA-seq were utilized to distinguish their mRNA targets of the miR-140 strands. Their downstream signalling pathways were unveiled using a high-throughput antibody array. Although both strands of the miR-140 are downregulated in the NSCLC, miR-140-3p is more predominant compared to miR-140-5p in lung cancer cell lines. Both miR-140 mimics suppress the invasion of lung cancer cells and the inhibitory effect of the miR-140 on adhesion is cell-dependent. Tumor conditioned media from A549 cells after treatment with miR-140-3p mimic reduce the tubule formation ability of the endothelial cells. Metabolite profiling indicates the alteration of glycine in both lung cancer cells following treatment with miR-140 mimics. The data from the RNA-sequencing and antibody array indicate that two miR-140 strands present different targeting and signalling profiles despite the existence of mutual targets such as IGF1R and FOS. In conclusion, two forms of miR-140 both suppress the malignant properties of lung cancer cells but through distinct and multiple mechanisms.

The Satellite Cell Niche Regulates the Balance between Myoblast Differentiation and Self-Renewal via p53.

Satellite cells are adult muscle stem cells residing in a specialized niche that regulates their homeostasis. How niche-generated signals integrate to regulate gene expression in satellite cell-derived myoblasts is poorly understood. We undertook an unbiased approach to study the effect of the satellite cell niche on satellite cell-derived myoblast transcriptional regulation and identified the tumor suppressor p53 as a key player in the regulation of myoblast quiescence. After activation and proliferation, a subpopulation of myoblasts cultured in the presence of the niche upregulates p53 and fails to differentiate. When satellite cell self-renewal is modeled ex vivo in a reserve cell assay, myoblasts treated with Nutlin-3, which increases p53 levels in the cell, fail to differentiate and instead become quiescent. Since both these Nutlin-3 effects are rescued by small interfering RNA-mediated p53 knockdown, we conclude that a tight control of p53 levels in myoblasts regulates the balance between differentiation and return to quiescence.

Therapeutic Role of MiR-140-5p for the Treatment of Non-small Cell Lung Cancer.

BACKGROUND/AIM: Lung cancer is the second most common cancer in both men and women, after prostate and breast cancer, respectively. There are two main types of primary lung cancer, non-small cell lung cancer (NSCLC), which accounts for approximately 85-90% of all lung cancer cases, and small cell lung cancer (SCLC), which accounts for the other 10-15% of lung cancers. MiRNAs are small molecules that post-transcriptionally regulate many genes and contribute to many disease aetiologies, including tumours. In lung cancer, the down-regulation of miR-140-5p leads to disease progression. MATERIALS AND METHODS: In this study a miR-140-5p-only treatment and miR-140-5p combined with other chemotherapeutics have been studied in vitro. RESULTS: When transfected into NSCLC, the overexpression of miR-140-5p reduced the migration and invasion properties of malignant cells and, also improved their adhesion onto the artificial extracellular matrix. When miRNA-140-5p replacement treatment was combined with other drugs commonly used in clinical practice, such as gefinitib, DMH1 and cisplatin, it enhanced their efficacy by reducing the migration and invasion ability of cancer cells, thus suggesting that it acts synergistically with known compounds for the treatment of NSCLC. Additionally, some endothelial mesenchymal transition (EMT) markers appeared to be regulated by miR-140-5p. CONCLUSION: Novel direct targets of miR-140-5p have not been investigated in this study, but our results indicate the involvement of miR-140-5p in lung cancer invasion. The preliminary data from this study imply that when miR-140-5p levels are restored; maybe synergistically support current therapies for NSCLC though further validation, especially in vivo is required.

The Axis of CXCR4/SDF-1 Plays a Role in Colon Cancer Cell Adhesion Through Regulation of the AKT and IGF1R Signalling Pathways.

BACKGROUND/AIM: Colorectal cancer (CRC) is the third most common cancer in the world. The high mortality of this tumor is mainly due to its invasive properties, as it forms metastases in multiple organs, preferentially in the liver. There has evidence showing that C-X-C chemokine receptor type 4 (CXCR-4) and its ligand, stromal cell-derived factor-1 (SDF-1), plays an important role in cancer progression and metastasis. However, the molecular mechanism underling the CRCR4-mediated CRC metastasis has not been well characterized. In this study, we aimed to investigate the roles of CXCR4 in colorectal cancer using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-based genomic editing technique. MATERIALS AND METHODS: We knocked-down CXCR4 using specific guide-RNA linked CRISPR/Cas9 in HT115 and COLO201 colon cancer cell lines which exhibited high levels of endogenous CXCR4 gene expression. Stable HT115 cells with CXCR4 knock-down were established by CRISPR plasmid transfection and validation was confirmed using T7 endonuclease 1 (T7EN1), flow cytometry (FACS) and western blotting assays. RESULTS: Knock-down of CXCR4 did not decrease proliferation of HT115 cells, but decreased the adhesion potential of cells to the human umbilical vein endothelial cells (HUVEC) and extracellular matrix. We further demonstrated that the AKT and type 1 insulin-like growth factor receptor (IGF1R) signalling pathways may be involved in the alteration of adhesion in CRC cells when CXCR4 is knocked down. CONCLUSION: Our data suggest that CXCR4 plays a key role in colorectal cancer progression via the mediation of tumor cell adhesion.

Knockdown of EPHA1 Using CRISPR/CAS9 Suppresses Aggressive Properties of Ovarian Cancer Cells.

BACKGROUND/AIM: Overexpression of erythropoietin-producing hepatocellular A1 (EPHA1), a member of the EPH super family, is frequently observed in various cancer types. The dysregulated interaction of EPHA1 with its ligand Ephrin A1 has been linked to the progression of ovarian cancer (OC). However, the contribution of EPHA1 in the regulation of the aggressive properties of OC cells remains unknown. MATERIALS AND METHODS: In this study we investigated the differential expression of EPHA1 in human OC cells. The EPHA1 gene was knocked-down using the CRISPR/Cas9 technique to evaluate its effect on the progressive properties of OC cells. RESULTS: After EPHA1 was knocked-down using a CRISPR/CAS9 genomic editing system in OC cells (SKOV3 and COV504), we observed cell-cycle arrest at the G0/G1 phases in both OC cell lines. Knockdown of EPHA1 in the two OC cells inhibited their aggressive traits, including proliferation, invasion and migration, as well as improving their attachment to extracellular matrix. EPHA1 may play a role in OC through its regulation of multiple signaling pathways, such as matrix metalloproteinase-2 (MMP2), extracellular signal-regulated kinase 2 (ERK2) and proto-oncogene c-MYC. CONCLUSION: EPHA1 may promote the aggression of some OC cells and, thus, be considered a potential therapeutic target for the treatment of malignant OC.

MicroRNA-7 suppresses the homing and migration potential of human endothelial cells to highly metastatic human breast cancer cells.

BACKGROUND: MicroRNA-7 (miR-7) has been observed as a potent tumour suppressor in multiple cancer types including breast cancer. The aim of this study was to investigate the response sensitivities of metastatic breast cancer cells to miR-7 and the roles of miR-7 in the interaction of endothelial cells and metastatic cancer cells. METHODS: Expression profile of miRNAs in a breast cancer specimen cohort and breast cancer cells were determined using real-time quantitative miRNA assays. Effect of the altering expression of miR-7 on migration, invasion, proliferation, interaction and underlying molecular mechanism of breast cancer cells and endothelial cells was investigated after treatment with the synthesised mimic of miR-7. Luciferase activity analysis was performed to validate Wave-3 as a novel target of miR-7. RESULTS: miR-7 expression was negatively correlated with the stage, grade and survival of the breast cancer patients. There was also differential expression of miRNAs including miR-7 in the breast cancer cells. The synthesised mimic of miR-7 inhibits the motility and wound healing potential of breast cancer cells. The highly metastatic MDA-MB-231 cells are more sensitive to the miR-7 treatment than the poorly invasive MCF-7 cells. Treatment with miR-7 downregulated the expression of EGFR, IGF1R and Wave3 in MDA-MB-231 cells but not in MCF-7 cells. In addition, we further demonstrated that miR-7 inhibited the proliferation, migration and invasion of endothelial cells. And more importantly, miR-7 suppressed the homing and migration of endothelial cells to more aggressive tumour cell conditions. CONCLUSIONS: Given the dual inhibitory effect of miR-7 on metastatic breast cancer cells alone and the interaction of endothelial cells with the tumour-conditioned microenvironment, we suggest miR-7 may be a new therapeutic candidate for its capacity not only to prevent breast cancer cell spreading but also to inhibit tumour-associated angiogenesis in the metastatic breast cancer.

Significance and therapeutic implications of endothelial progenitor cells in angiogenic-mediated tumour metastasis.

Cancer conveys profound social and economic consequences throughout the world. Metastasis is responsible for approximately 90% of cancer-associated mortality and, when it occurs, cancer becomes almost incurable. During metastatic dissemination, cancer cells pass through a series of complex steps including the establishment of tumour-associated angiogenesis. The human endothelial progenitor cells (hEPCs) are a cell population derived from the bone marrow which are required for endothelial tubulogenesis and neovascularization. They also express abundant inflammatory cytokines and paracrine angiogenic factors. Clinically hEPCs are highly correlated with relapse, disease progression, metastasis and treatment response in malignancies such as breast cancer, ovarian cancer and non-small-cell lung carcinoma. It has become evident that the hEPCs are involved in the angiogenesis-required progression and metastasis of tumours. However, it is not clear in what way the signalling pathways, controlling the normal cellular function of human BM-derived EPCs, are hijacked by aggressive tumour cells to facilitate tumour metastasis. In addition, the actual roles of hEPCs in tumour angiogenesis-mediated metastasis are not well characterised. In this paper we reviewed the clinical relevance of the hEPCs with cancer diagnosis, progression and prognosis. We further summarised the effects of tumour microenvironment on the hEPCs and underlying mechanisms. We also hypothesized the roles of altered hEPCs in tumour angiogenesis and metastasis. We hope this review may enhance our understanding of the interaction between hEPCs and tumour cells thus aiding the development of cellular-targeted anti-tumour therapies.