Mutation of the Conserved Threonine 8 within the Human ARF Tumour Suppressor Protein Regulates Autophagy

Background: The ARF tumour suppressor plays a well-established role as a tumour suppressor, halting cell growth by both p53-dependent and independent pathways in several cellular stress response circuits. However, data collected in recent years challenged the traditional role of this protein as a tumour suppressor. Cancer cells expressing high ARF levels showed that its expression, far from being dispensable, is required to guarantee tumour cell survival. In particular, ARF can promote autophagy, a self-digestion pathway that helps cells cope with stressful growth conditions arising during both physiological and pathological processes. Methods: We previously showed that ARF is regulated through the activation of the protein kinase C (PKC)-dependent pathway and that an ARF phospho-mimetic mutant on the threonine residue 8, ARF-T8D, sustains cell proliferation in HeLa cells. We now explored the role of ARF phosphorylation in both basal and starvation-induced autophagy by analysing autophagic flux in cells transfected with either WT and ARF phosphorylation mutants by immunoblot and immunofluorescence. Results: Here, we show that endogenous ARF expression in HeLa cells is required for starvation-induced autophagy. Further, we provide evidence that the hyper-expression of ARF-T8D appears to inhibit autophagy in both HeLa and lung cancer cells H1299. This effect is due to the cells’ inability to elicit autophagosomes formation upon T8D expression. Conclusions: Our results lead to the hypothesis that ARF phosphorylation could be a mechanism through which the protein promotes or counteracts autophagy. Several observations underline how autophagy could serve a dual role in cancer progression, either protecting healthy cells from damage or aiding cancerous cells to survive. Our results indicate that ARF phosphorylation controls protein’s ability to promote or counteract autophagy, providing evidence of the dual role played by ARF in cancer progression.

Hypermethylation involved in DNA profiles of lung cancer specific tumour suppressor genes and epigenetic modification caused by an ultra-highly diluted homeopathic drug, Condurango 30C, in vitro and in vivo

Background and objectives: DNA hyper-methylation is an important aspect involved in carcinogenesis and cancer progression, which affects mainly CpG islands of DNA and causes inactivation of tumour suppressor genes. Therefore DNA hypermethylation status of the genomic DNA in both the transformed cancerous cell lines and in carcinogen-induced lung cancer was ascertained by analysis of expressions of certain major lung cancer specific tumour suppressor genes. The other objective was to examine if ultra highly diluted homeopathic drug, Condurango 30C, had ability to modulate DNA methylation. Methods: DNA methylation activity, if any, has been ascertained in H460-NSCLC cells in vitro and in BaP-induced lung cancer of rats in vivo, in respect of tumour suppressor genes like p15, p16, p18 and p53 by using PCR-SSCP analyses. The ability of modulation of DNA methylation, if any, by Condurango 30C was also verified against placebo control in a blinded manner. Results: Condurango 30C-treated DNA showed significant decrease in band-intensity of p15 and p53 genes especially in methylated condition, in vitro, at the IC50 dose (2.43µl/100µl). SSCP analysis of p15 and p53 genes in Condurango 30C-treated DNA also supported ability of Condurango 30C to modulate methylation state, in vitro. Inhibition of p15 hypermethylation was observed after post cancer treatment of rat with Condurango 30C. SSCP results gave a better indication of differences in band-position and single strand separation of p15 and p53 in Condurango 30C treated samples. Conclusion: Condurango 30C could trigger epigenetic modification in lung cancer via modulation of DNA hypermethylation but placebos could not.

Influence of dysregulated expression of circular RNA on the diagnosis and prognosis of breast cancer in Asia: a meta-analysis study

ObjectiveRecent studies have reported a correlation between non-coding RNAs such as circular RNAs (circRNAs) and clinical value of various cancers. However, the diagnostic and prognostic role of circRNA in breast cancer remains controversial.DesignSystematic review and meta-analysis.MethodsDiagnostic efficacy was estimated by sensitivity, specificity and area under the curve (AUC). Pooled HRs with 95% CIs estimated overall survival (OS), and ORs with 95% CIs investigated clinical features.ResultsBy searching PubMed, Embase, Web of Science, CNKI and Cochrane Library, we obtained a total of 29 studies with 4405 patients. A shorter survival time was associated with high expression levels of tumour-promoter circRNAs (OS: HR=2.43, 95% CI 2.20 to 2.92, p<0.001), and tumour‐suppressor circRNAs were related to a favourable prognosis (OS: HR=0.32, 95% CI 0.23 to 0.44, p<0.001). Furthermore, high expression levels of oncogenic circRNAs were associated with poor clinical outcomes; tumour-suppressor circRNAs showed the opposite result. As for the diagnostic role, the outcome indicated an AUC of 0.82 (95% CI 0.78 to 0.85), with 85% sensitivity and 86% specificity to distinguish patients with breast cancer from healthy controls.ConclusionDysregulated expression of circRNA was related to diagnosis and prognosis in breast cancer, which indicated it might be a novel biomarker and a target of therapy for breast cancer.PROSPERO registration numberCRD42020207912.