The Integrated Stress Response Effector atF4: Characterization of a Small Molecule Modulator of its Expression and Investigation of the Role of atF4 in Metastasis

The Integrated Stress Response (ISR) and the Unfolded Protein Response (UPR) are signaling programs that enable cellular adaptation to stressful conditions like hypoxia and nutrient deprivation in the tumor microenvironment. An important effector of these pathways is ATF4, a transcription factor that regulates genes involved in redox homeostasis, amino acid metabolism and transport, autophagy, and angiogenesis. However, prolonged activation of the UPR/ISR and resultant ATF4 expression can be cytotoxic under certain conditions. In these studies, we characterized a small molecule compound, E235, which activated the ISR and dose-dependently increased levels of ATF4 in transformed cells. A dose-dependent decrease in viability was observed in several mouse and human tumor cell lines, and knockdown of ATF4 significantly increased the anti-proliferative effects of E235. Interestingly, low μM doses of E235 induced senescence in many cell types. E235-mediated induction of senescence was not dependent on p21 or p53; however, p21 conferred protection against the growth inhibitory effects of E235. Treatment with E235 resulted in an increase in cells arrested at the G2/M phase. E235 also activated DNA damage response signaling, although E235 did not appear to cause physical DNA damage. Induction of γ-H2AX was abrogated in ATF4 knockdown cells. Together, these results suggest that modulation of the ISR pathway with the small molecule E235 could be a promising anti-tumor strategy. We also investigated the potential role of ATF4 in tumor metastasis. Constitutive knockdown of ATF4 expression in HT1080 fibrosarcoma cells completely prevented the lung colonization of these cells when injected intravenously in mice. This phenomenon may be tumor type specific however, as ATF4 ablation in mouse mammary carcinoma cells had no effect on the ability of these cells to colonize various organs of the mice. In addition, we generated HT1080 cells with an inducible knockdown of ATF4 and demonstrated an increased tumor growth rate upon downregulation of ATF4 expression. However, ATF4 knockdown did not affect the migration of HT1080 cells. This, together with results from a colleague showing that ATF4 protects cells from anoikis, led us to propose that ATF4 may be facilitating metastasis by protecting tumor cells during detachment and circulation

Transcriptional profiling of canine osteosarcoma identifies prognostic gene expression signatures with translational value for humans

Abstract Canine osteosarcoma is increasingly recognized as an informative model for human osteosarcoma. Here we show in one of the largest clinically annotated canine osteosarcoma transcriptional datasets that two previously reported, as well as de novo gene signatures devised through single sample Gene Set Enrichment Analysis (ssGSEA), have prognostic utility in both human and canine patients. Shared molecular pathway alterations are seen in immune cell signaling and activation including TH1 and TH2 signaling, interferon signaling, and inflammatory responses. Virtual cell sorting to estimate immune cell populations within canine and human tumors showed similar trends, predominantly for macrophages and CD8+ T cells. Immunohistochemical staining verified the increased presence of immune cells in tumors exhibiting immune gene enrichment. Collectively these findings further validate naturally occurring osteosarcoma of the pet dog as a translationally relevant patient model for humans and improve our understanding of the immunologic and genomic landscape of the disease in both species

Age and frailty are independently associated with increased COVID-19 mortality and increased care needs in survivors: results of an international multi-centre study

Introduction: Increased mortality has been demonstrated in older adults with coronavirus disease 2019 (COVID-19), but the effect of frailty has been unclear. Methods: This multi-centre cohort study involved patients aged 18 years and older hospitalised with COVID-19, using routinely collected data. We used Cox regression analysis to assess the impact of age, frailty and delirium on the risk of inpatient mortality, adjusting for sex, illness severity, inflammation and co-morbidities. We used ordinal logistic regression analysis to assess the impact of age, Clinical Frailty Scale (CFS) and delirium on risk of increased care requirements on discharge, adjusting for the same variables. Results: Data from 5,711 patients from 55 hospitals in 12 countries were included (median age 74, interquartile range [IQR] 54–83; 55.2% male). The risk of death increased independently with increasing age (>80 versus 18–49: hazard ratio [HR] 3.57, confidence interval [CI] 2.54–5.02), frailty (CFS 8 versus 1–3: HR 3.03, CI 2.29–4.00) inflammation, renal disease, cardiovascular disease and cancer, but not delirium. Age, frailty (CFS 7 versus 1–3: odds ratio 7.00, CI 5.27–9.32), delirium, dementia and mental health diagnoses were all associated with increased risk of higher care needs on discharge. The likelihood of adverse outcomes increased across all grades of CFS from 4 to 9. Conclusion: Age and frailty are independently associated with adverse outcomes in COVID-19. Risk of increased care needs was also increased in survivors of COVID-19 with frailty or older age.</p