NFE2L2 and STAT3 converge on common targets to promote survival of primary lymphoma cells

NFE2L2 and STAT3 are key pro-survival molecules, and thus, their targeting may represent a promising anti-cancer strategy. In this study, we found that a positive feedback loop occurred between them and provided evidence that their concomitant inhibition efficiently impaired the survival of PEL cells, a rare, aggressive B cell lymphoma associated with the gammaherpesvirus KSHV and often also EBV. At the molecular level, we found that NFE2L2 and STAT3 converged in the regulation of several pro-survival molecules and in the activation of processes essential for the adaption of lymphoma cells to stress. Among those, STAT3 and NFE2L2 promoted the activation of pathways such as MAPK3/1 and MTOR that positively regulate protein synthesis, sustained the antioxidant response, expression of molecules such as MYC, BIRC5, CCND1, and HSP, and allowed DDR execution. The findings of this study suggest that the concomitant inhibition of NFE2L2 and STAT3 may be considered a therapeutic option for the treatment of this lymphoma that poorly responds to chemotherapies

Genomic, Pathway Network, and Immunologic Features Distinguishing Squamous Carcinomas

This integrated, multiplatform PanCancer Atlas study co-mapped and identified distinguishing molecular features of squamous cell carcinomas (SCCs) from five sites associated with smokin

5-AZA upregulates SOCS3 and PTPN6/SHP1, inhibiting STAT3 and potentiating the effects of AG490 against primary effusion lymphoma cells

Epigenetic modifications, including aberrant DNA methylation occurring at the promoters of oncogenes and oncosuppressor genes and histone modifications, can contribute to carcinogenesis. Aberrant methylation mediated by histone methylatransferases, alongside histones, can affect methylation of proteins involved in the regulation of pro-survival pathways such as JAK/STAT and contribute to their activation. In this study, we used DNA or histone demethylating agents, 5-Azacytidine (5-AZA) or DS-3201 (valemetostat), respectively, to treat primary effusion lymphoma (PEL) cells, alone or in combination with AG490, a Signal transducer and activator of transcription 3 (STAT3) inhibitor. Cell viability was investigated by trypan blue assay and FACS analysis. The molecular changes induced by 5-AZA and/or AG490 treatments were investigated by Western blot analysis, while cytokine release by PEL cells treated by these drugs was evaluated by Luminex. Statistical analyses were performed with Graphpad Prism (R) software (version 9) and analyzed by Student's t test or a nonparametric one-way ANOVA test. The results obtained in this study suggest that 5-AZA upregulated molecules that inhibit STAT3 tyrosine phosphorylation, namely Suppressor of Cytokine Signaling 3 (SOCS3) and tyrosine-protein phosphatase non-receptor type (PTPN) 6/Src homology region 2 domain-containing phosphatase-1 (SHP-1), reducing STAT3 activation and downregulating several STAT3 pro-survival targets in PEL cells. As this lymphoma is highly dependent on the constitutive activation of STAT3, 5-AZA impaired PEL cell survival, and when used in combination with AG490 JAK2/STAT3 inhibitor, it potentiated its cytotoxic effect. Differently from 5-AZA, the inhibition of the EZH1/2 histone methyltransferase by DS-3201, reported to contribute to STAT3 activation in other cancers, slightly affected STAT3 phosphorylation or survival in PEL cells, either alone or in combination with AG490. This study suggests that 5-AZA, by upregulating the expression level of SOCS3 and PTPN6/SHP1, reduced STAT3 activation and improved the outcome of treatment targeting this transcription factor in PEL cells

ATF4 suppresses hepatocarcinogenesis by inducing SLC7A11 (xCT) to block stress-related ferroptosis

Background & aimsHepatocellular carcinoma (HCC), a leading cause of cancer-related death, is associated with viral hepatitis, non-alcoholic steatohepatitis (NASH), and alcohol-related steatohepatitis, all of which trigger endoplasmic reticulum (ER) stress, hepatocyte death, inflammation, and compensatory proliferation. Using ER stress-prone MUP-uPA mice, we established that ER stress and hypernutrition cooperate to cause NASH and HCC, but the contribution of individual stress effectors, such as activating transcription factor 4 (ATF4), to HCC and their underlying mechanisms of action remained unknown.MethodsHepatocyte-specific ATF4-deficient MUP-uPA mice (MUP-uPA/Atf4Δhep) and control MUP-uPA/Atf4F/F mice were fed a high-fat diet to induce NASH-related HCC, and Atf4F/F and Atf4Δhep mice were injected with diethylnitrosamine to model carcinogen-induced HCC. Histological, biochemical, and RNA-sequencing analyses were performed to identify and define the role of ATF4-induced solute carrier family 7a member 11 (SLC7A11) expression in hepatocarcinogenesis. Reconstitution of SLC7A11 in ATF4-deficient primary hepatocytes and mouse livers was used to study its effects on ferroptosis and HCC development.ResultsHepatocyte ATF4 ablation inhibited hepatic steatosis, but increased susceptibility to ferroptosis, resulting in accelerated HCC development. Although ATF4 activates numerous genes, ferroptosis susceptibility and hepatocarcinogenesis were reversed by ectopic expression of a single ATF4 target, Slc7a11, coding for a subunit of the cystine/glutamate antiporter xCT, which is needed for glutathione synthesis. A ferroptosis inhibitor also reduced liver damage and inflammation. ATF4 and SLC7A11 amounts were positively correlated in human HCC and livers of patients with NASH.ConclusionsDespite ATF4 being upregulated in established HCC, it serves an important protective function in normal hepatocytes. By maintaining glutathione production, ATF4 inhibits ferroptosis-dependent inflammatory cell death, which is known to promote compensatory proliferation and hepatocarcinogenesis. Ferroptosis inhibitors or ATF4 activators may also blunt HCC onset.Impact and implicationsLiver cancer or hepatocellular carcinoma (HCC) is associated with multiple aetiologies. Most HCC aetiologies cause hepatocyte stress and death, as well as subsequent inflammation, and compensatory proliferation, thereby accelerating HCCdevelopment. The contribution of individual stress effectors to HCC and their underlying mechanisms of action were heretofore unknown. This study shows that the stress-responsive transcription factor ATF4 blunts liver damage and cancer development by suppressing iron-dependent cell death (ferroptosis). Although ATF4 ablation prevents hepatic steatosis, it also increases susceptibility to ferroptosis, due to decreased expression of the cystine/glutamate antiporter SLC7A11, whose expression in human HCC and NASH correlates with ATF4. These findings reinforce the notion that benign steatosis may be protective and does not increase cancer risk unless accompanied by stress-induced liver damage. These results have important implications for prevention of liver damage and cancer

Genomic, Pathway Network, and Immunologic Features Distinguishing Squamous Carcinomas

This integrated, multiplatform PanCancer Atlas study co-mapped and identified distinguishing molecular features of squamous cell carcinomas (SCCs) from five sites associated with smoking and/or human papillomavirus (HPV). SCCs harbor 3q, 5p, and other recurrent chromosomal copy-number alterations (CNAs), DNA mutations, and/or aberrant methylation of genes and microRNAs, which are correlated with the expression of multi-gene programs linked to squamous cell stemness, epithelial-to-mesenchymal differentiation, growth, genomic integrity, oxidative damage, death, and inflammation. Low-CNA SCCs tended to be HPV(+) and display hypermethylation with repression of TET1 demethylase and FANCF, previously linked to predisposition to SCC, or harbor mutations affecting CASP8, RAS-MAPK pathways, chromatin modifiers, and immunoregulatory molecules. We uncovered hypomethylation of the alternative promoter that drives expression of the ΔNp63 oncogene and embedded miR944. Co-expression of immune checkpoint, T-regulatory, and Myeloid suppressor cells signatures may explain reduced efficacy of immune therapy. These findings support possibilities for molecular classification and therapeutic approaches

Protein homeostasis regulation in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a disorder that affects motor neurons in the motor cortex, brainstem and spinal cord. The lack of an effective treatment indicates the need for a deeper understanding of the pathogenesis underlying this disease. ALS, as well as the majority of neurodegenerative diseases such as Alzheimer’s disease (AD) and frontotemporal lobar degeneration (FTLD) are characterised by dysfunctions in protein homeostasis (proteostasis). The endoplasmic reticulum (ER) plays an important role in proteostasis through the unfolded protein response (UPR). We therefore started this thesis by characterising the UPR signalling pathways in human post-mortem spinal cord from sporadic ALS (SALS) and in frontal and temporal cortex from FTLD and AD cases and compared with healthy controls. In ALS, UPR activation was confirmed by a substantial expression increase of both known and novel target genes involved particularly in ER-associated degradation (ERAD), while in AD a distinct pattern emerged, with a predominant involvement of protein folding genes, such as Protein Disulphide Isomerases (PDIs). Similarly, in human motor cortex of SALS cases we found an increased expression of PDIs and other specific UPR target genes which correlated with oligodendrocyte markers. Moreover, we found that the heat shock response (HSR), a major proteostasis regulatory pathway, and ERAD genes were activated predominately in the spinal cord and strongly correlated with the motor neuron marker VAPB. Finally, we performed a meta-analysis of publicly available RNA-Seq studies derived from the spinal cord of healthy and ALS cases. We identified cholesterol metabolism, cell adhesion and regulation of vesicle-mediated transport as top disease-associated processes and 21 hub genes as central nodes in these networks. We conclude that proteostasis is strongly and selectively activated specific cell types in SALS motor cortex and spinal cord. Hence, these results provide novel insights into the pathophysiology of ALS and other neurodegenerative disorders.Open Acces

Unfolded Protein Response manipulation in cancer treatment

Cancer cells are exposed to intrinsic and extrinsic factors that alter the homeostasis of the endoplasmic reticulum (ER), resulting in constitutive ER stress. To adapt to this stress, cancer cells activate the unfolded protein response (UPR), which usually sustains their survival. However, the activation of UPR can be considered a "double-edged sword", as it may also trigger cell death, i.e. in conditions of too long or intense stress. Approaches based on the manipulation of UPR are emerging as promising strategies for the treatment of cancer, either alone or in combination with existing drugs. In this study, we shed more light on the molecular mechanisms underlying the effects of UPR targeting by using natural compounds, such as Resveratrol, Curcumin, and DPE, or drugs that specifically alter ER homeostasis, such as Thapsigargin and Tunicamycin. By pharmacologic and genetic strategies, we observed that the UPR sensors, IRE1α, PERK, and ATF6, have different, and sometimes opposite, roles in balancing death/survival in stressed cells, also depending on the cellular context and the genetic background. For example, while the inhibition of ATF6 was the most effective in decreasing the survival of colon cancer cells undergoing ER stress, the inhibition of IRE1α was the most promising strategy, both in vitro and in vivo, for the treatment of B-cell lymphomas. Furthermore, in agreement with previous studies, we evidenced the close relationship between the UPR and other adaptive responses, such as autophagy and DNA damage response (DDR), influencing the resistance to anti-tumor treatments. In this regard, we described for the first time the role of ATF6 in protecting cells from cell death and DNA damage by sustaining the expression of BRCA1 in TG- or DPE-treated cells. Moreover, we evidenced a new role of ATF6 in supporting lysosomal function, thus directly linking UPR to autophagy. The latter process, together with chaperone mediated autophagy (CMA) were activated by ER stress and contributed to the degradation of mutp53 protein, with important implications for anti-cancer therapy. From these studies, it emerges that UPR and its dynamic interplay with other adaptive responses may be manipulated with important consequences in term of death/survival of stressed cancer cells and that the outcome may be dependent on the cellular context

Investigating the genetic basis of cisplatin-induced ototoxicity in adult South African patients

Cisplatin, a potent chemotherapeutic agent, is widely used in the treatment of numerous soft-tissue cancers. Although high cure rates can be achieved when cisplatin is incorporated in chemotherapy regimens, the therapeutic utility of the drug may be limited by the development of dose-limiting adverse reactions in patients. A prevalent reaction to cisplatin is ototoxicity, or drug-induced hearing loss, which occurs when the drug accumulates in and damages cells of the inner ear, leading to permanent and progressive hearing impairment. In this investigation, two approaches were employed to explore the role of genetics in cisplatin response amongst South African cancer patients (n = 214). Using a candidate gene approach, which investigated variants in six genes which are involved in drug transport and processing, potential modifiers in the genes nuclear factor, erythroid 2-like 2 (NFE2L2) and solute carrier family 22, member 2 (SLC22A2) were identified. SLC22A2 encodes a known transporter of cisplatin, and the variant rs316019 conferred potentially protective effects against Chang- and TUNE-graded ototoxicity through a reduced transport of the drug (p = 0.039 and p = 0.031, respectively). Similarly, the variant NFE2L2 rs6721961 was possibly protective, as it occurred more frequently in patients who did not develop hearing impairment according to four different ototoxicity grading scales during high-dose (≥ 200 mg/m2) cisplatin treatment (ASHA, p = 0.001; Chang, p = 0.022; CTCAE, p = 0.001; TUNE, p = 0.028). When supplementing the prospective cohort with retrospective patient data, an increased susceptibility of indigenous African patients to Chang grade > 0 ototoxicity was observed (p = 0.001). For this reason, whole-exome sequencing was conducted on a subset of the patient cohort (n = 11), focussing on individuals of African origin who represented the phenotype extremes. Potential genetic modifiers were identified in genes involved in various biological processes, including transmembrane transport, development, hearing, the response to DNA damage, immune reactions and signalling pathways, implicating many previously unreported genes in the cellular response to cisplatin as well as its ototoxicity. The results reported in this study indicate that genetic information can improve predictive models of cisplatin response, although there are many novel genes which should be explored in the South African population. Identifying these genetic modifiers, such as those in SLC22A2 and NFE2L2, has the potential to further our understanding of this adverse drug reaction, and may assist in the future personalisation of treatment plans in the management of cancer

The HBP1 tumor suppressor is a negative epigenetic regulator of MYCN driven neuroblastoma through interaction with the PRC2 complex

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Targeting Ferroptosis in Tuberous Sclerosis Cell Line Models

Ferroptosis, an iron-dependent cell death mechanism characterized by oxidative damage to phospholipids and subsequent membrane damage, presents a promising target for cancer therapy. The TSC1-TSC2 complex is crucial in cellular signalling, regulating cell growth, proliferation, and metabolism. Mutations or loss of TSC2 lead to hyperactivation of mTORC1, implicated in various cancers. This research aimed to elucidate the role of TSC2 loss in ferroptosis and its contribution to drug resistance in TSC2-cell line models, potentially guiding the development of new therapeutic strategies. Cytotoxicity testing within this study revealed that Tsc2-deficient cells have greater resistance to ferroptosis induction compared to Tsc2-positive cells. Notably, inhibition of mTORC1 did not reverse this resistance, whereas NRF2 antioxidant pathway inhibition and AMPK activation did, suggesting that resistance operates through mTORC1-independent pathways. Biochemical analysis identified altered ferroptosis markers in Tsc2-deficient cells, such as ROS-mediated lipid peroxidation, GPX4, GSH, and labile iron pools as key factors in this resistance. Further investigations into NRF2 revealed significantly elevated nuclear translocation upon ferroptosis induction in Tsc2-deficient cells during ferroptosis induction, identifying the NRF2 pathway as a potential mediator of resistance. qPCR and RNAseq analyses confirmed significant dysregulation of NRF2 and its target genes between TSC2-deficient and TSC2-expressing tumours. Additionally, inhibition of ferroptosis suppressor protein 1 (FSP1) also counteracted the cell death resistance in Tsc2-deficient cells. These cells displayed a fourfold increase in mRNA levels of FSP1, which significantly enhanced their resistance to ferroptosis. Overall, this thesis establishes that the loss of TSC2 confers resistance to ferroptosis through mechanisms that are independent of mTORC1 overactivation but dependent on NRF2 activation. This study also provides a deeper understanding of ferroptosis and additional cellular signalling pathways, such as those involving ROS regulation, lipid peroxidation, and iron metabolism, within the context of TSC2 loss. These insights will guide the development of future therapeutic strategies targeting ferroptosis in TSC2-deficient cancers