Exosome biogenesis: machinery, regulation, and therapeutic implications in cancer.

Exosomes are well-known key mediators of intercellular communication and contribute to various physiological and pathological processes. Their biogenesis involves four key steps, including cargo sorting, MVB formation and maturation, transport of MVBs, and MVB fusion with the plasma membrane. Each process is modulated through the competition or coordination of multiple mechanisms, whereby diverse repertoires of molecular cargos are sorted into distinct subpopulations of exosomes, resulting in the high heterogeneity of exosomes. Intriguingly, cancer cells exploit various strategies, such as aberrant gene expression, posttranslational modifications, and altered signaling pathways, to regulate the biogenesis, composition, and eventually functions of exosomes to promote cancer progression. Therefore, exosome biogenesis-targeted therapy is being actively explored. In this review, we systematically summarize recent progress in understanding the machinery of exosome biogenesis and how it is regulated in the context of cancer. In particular, we highlight pharmacological targeting of exosome biogenesis as a promising cancer therapeutic strategy.

Reprogramming of m6A epitranscriptome is crucial for shaping of transcriptome and proteome in response to hypoxia.

Hypoxia causes a series of responses supporting cells to survive in harsh environments. Substantial post-transcriptional and translational regulation during hypoxia has been observed. However, detailed regulatory mechanism in response to hypoxia is still far from complete. RNA m6A modification has been proven to govern the life cycle of RNAs. Here, we reported that total m6A level of mRNAs was decreased during hypoxia, which might be mediated by the induction of m6A eraser, ALKBH5. Meanwhile, expression levels of most YTH family members of m6A readers were systematically down-regulated. Transcriptome-wide analysis of m6A revealed a drastic reprogramming of m6A epitranscriptome during cellular hypoxia. Integration of m6A epitranscriptome with either RNA-seq based transcriptome analysis or mass spectrometry (LC-MS/MS) based proteome analysis of cells upon hypoxic stress revealed that reprogramming of m6A epitranscriptome reshaped the transcriptome and proteome, thereby supporting efficient generation of energy for adaption to hypoxia. Moreover, ATP production was blocked when silencing an m6A eraser, ALKBH5, under hypoxic condition, demonstrating that m6A pathway is an important regulator during hypoxic response. Collectively, our studies indicate that crosstalk between m6A and HIF1 pathway is essential for cellular response to hypoxia, providing insights into the underlying molecular mechanisms during hypoxia.

CDC20 overexpression predicts a poor prognosis for patients with colorectal cancer.

BACKGROUND: The cell division cycle 20 homolog (CDC20) is an essential cofactor of the anaphase-promoting complex (APC/C). CDC20 overexpression has been detected in many types of human cancers; however, its clinical role in colorectal cancer remains unknown. METHODS: Western blotting and immunohistochemistry were used to compare CDC20 expression in adjacent non-cancerous, cancerous and liver metastatic tissues as well as in colon cancer cell lines and normal colon epithelial cell lines. Additionally, the correlation of CDC20 expression with patient clinical parameters and its diagnostic value were statistically analyzed. RESULTS: CDC20 was overexpressed in colon cancer cell lines/primary cancer tissues compared with normal colon epithelial cell lines/adjacent noncancerous tissue samples. Interestingly, CDC20 expression was further increased in metastatic liver tissues. CDC20 protein expression was significantly correlated with clinical stage (P = 0.008), N classification (P = 0.020), M classification (P = 0.013) and pathologic differentiation (P = 0.008). Patients with higher CDC20 expression had a shorter overall survival than those with lower CDC20 expression. Univariate and multivariate analyses indicated that CDC20 expression was an independent prognostic factor (P < 0.001). CONCLUSION: CDC20 may serve as a potential prognostic biomarker of human colorectal cancer.

Prognostic relevance of BRD7 expression in colorectal carcinoma.

BACKGROUND: BRD7 is a member of bromodomain-containing protein and was found to be a cofactor of P53. Down-regulation of BRD7 has been shown in nasopharyngeal carcinoma cell lines and tissues. However, the clinical role of BRD7 in colorectal cancer remains unknown. MATERIALS AND METHODS: Real-time PCR, Western blotting analysis and immunohistochemistry were employed to examine BRD7 expression in CRC cell lines/tissues compared with normal epithelia cells/adjacent non-tumorous tissues. In addition, statistical analyses were applied to evaluate the diagnostic value and associations of BRD7 expression with clinical parameters of patient samples. RESULTS: BRD7 was down-regulated in colorectal cancer cell lines and cancerous tissues compared with that in normal colon epithelial cells and adjacent noncancerous tissue samples. BRD7 protein expression was positively correlated with clinical stage (P  <  0·001), T classification (P  =  0·001), N classification (P  <  0·001), M classification (P <  0·001) and pathologic differentiation (P =  0·008). Patients with low/none BRD7 expression had shorter overall survival time than those with higher BRD7 expression. Univariate and multivariate analyses indicated BRD7 expression was an independent prognostic factor (P < 0·001). CONCLUSION: BRD7 may serve as a potential prognostic biomarker of human colorectal cancer.

Molecular cloning and expression of orange-spotted grouper (Epinephelus coioides) CD8α and CD8ß genes.

T-cell surface glycoprotein CD8 consists of two distinguished chains, termed α and ß chains, and functions as a co-receptor for the T-cell receptor by binding to MHC class I proteins. In this study we report the cloning and identification of both CD8α and CD8ß genes from orange-spotted grouper (Epinephelus coioides). The predicted grouper CD8α and CD8ß proteins were structurally similar to other fish especially to those of Pleuronectiformes. Real-time RT-PCR revealed that the CD8 mRNA was much higher in the thymus than in other immune organs, and the expression level were very low in stomach, liver, and brain. During embryonic development of the grouper, the highest CD8 transcripts were detected in the multi-cell stage, followed by muscle burl stage, which suggested that the multi-cell stage may be critical in CD8 transcript synthesis. Moreover, CD8 mRNA levels were examined in lymphocytes at different time treated with lipopolysaccharide (LPS), polyriboinosinic polyribocytidylic acid (PolyI:C), phytohemagglutinin (PHA), and concanavalin A (ConA). The result showed that the CD8 mRNA levels were significantly affected in time-dependent manner by PolyI:C, PHA, and ConA, but not by LPS.

LncRNA DSCAM-AS1 interacts with YBX1 to promote cancer progression by forming a positive feedback loop that activates FOXA1 transcription network.

Rationale: The forkhead box A1 (FOXA1) is a crucial transcription factor in initiation and development of breast, lung and prostate cancer. Previous studies about the FOXA1 transcriptional network were mainly focused on protein-coding genes. Its regulatory network of long non-coding RNAs (lncRNAs) and their role in FOXA1 oncogenic activity remains unknown. Methods: The Cancer Genome Atlas (TCGA) data, RNA-seq and ChIP-seq data were used to analyze FOXA1 regulated lncRNAs. RT-qPCR was used to detect the expression of DSCAM-AS1, RT-qPCR and Western blotting were used to determine the expression of FOXA1, estrogen receptor α (ERα) and Y box binding protein 1 (YBX1). RNA pull-down and RIP-qPCR were employed to investigate the interaction between DSCAM-AS1 and YBX1. The effect of DSCAM-AS1 on malignant phenotypes was examined through in vitro and in vivo assays. Results: In this study, we conducted a global analysis of FOXA1 regulated lncRNAs. For detailed analysis, we chose lncRNA DSCAM-AS1, which is specifically expressed in lung adenocarcinoma, breast and prostate cancer. The expression level of DSCAM-AS1 is regulated by two super-enhancers (SEs) driven by FOXA1. High expression levels of DSCAM-AS1 was associated with poor prognosis. Knockout experiments showed DSCAM-AS1 was essential for the growth of xenograft tumors. Moreover, we demonstrated DSCAM-AS1 can regulate the expression of the master transcriptional factor FOXA1. In breast cancer, DSCAM-AS1 was also found to regulate ERα. Mechanistically, DSCAM-AS1 interacts with YBX1 and influences the recruitment of YBX1 in the promoter regions of FOXA1 and ERα. Conclusion: Our study demonstrated that lncRNA DSCAM-AS1 was transcriptionally activated by super-enhancers driven by FOXA1 and exhibited lineage-specific expression pattern. DSCAM-AS1 can promote cancer progression by interacting with YBX1 and regulating expression of FOXA1 and ERα.

Genome-wide study of ER-regulated lncRNAs shows AP000439.3 may function as a key regulator of cell cycle in breast cancer.

Estrogen receptor (ER) plays important roles in cell growth, development and tumorigenesis. Although ER-regulated genes have been extensively investigated, little is known about roles of ER-regulated lncRNAs in breast cancer. Here, we conducted genome-wide study of ER-regulated lncRNAs by using RNA-seq, ChIP-seq and TCGA data. A total of identified 114 ER-regulated lncRNAs were identified, many of them were overexpressed in ER+ breast cancer and co-expressed with some key regulators. Silencing one of most prominent lncRNA, AP000439.3, resulted in inhibition of cell cycle progression and proliferation. Further study revealed AP000439.3 can regulate expression of CCND1 through enhancing estrogen receptor induction of CCND1. This finding revealed lncRNAs may serve as important effectors of ER in regulation of gene expression and cell phenotype in breast cancer.

ß-phenylethyl isothiocyanate reverses platinum resistance by a GSH-dependent mechanism in cancer cells with epithelial-mesenchymal transition phenotype.

Platinum (Pt)-based chemotherapy is an important regimen in the clinical treatment of cancer, but development of drug resistance presents a major challenge. One key mechanism involved in resistance to Pt drugs is the decrease of intracellular Pt due to the drug efflux through the glutathione (GSH)-mediated export, and this is particularly significant in cancer cells with stem-cell like properties. In the present study, we showed that two Pt-resistant human cancer cell lines exhibited stem-cell like EMT properties, had high cellular GSH and accumulated significantly less cellular Pt compared to their parental cells, and failed to undergo apoptosis when exposed to Pt at the drug concentrations toxic to the parental cells. Importantly, we found that the natural compound ß-phenylethyl isothiocyanate (PEITC) was able to effectively abolish this drug resistant mechanism by effective depletion of cellular GSH, leading to a significant increase in cellular Pt as well as DNA-bound Pt. A combination of PEITC and Pt showed a striking synergistic anticancer activity both in vitro and in vivo, as evidenced by an increase in drug-induced apoptosis, a loss of colony formation capacity, and significant suppression of tumor growth in mice. Taken together, our study shows a promising therapeutic strategy to overcome drug resistance to platinum-based chemotherapy and may potentially have broad implications in clinical treatment of cancer.