Effects of H2O2 Treatment Combined With PI3K Inhibitor and MEK Inhibitor in AGS Cells: Oxidative Stress Outcomes in a Model of Gastric Cancer.

Gastric cancer is worldwide the fifth and third cancer for incidence and mortality, respectively. Stomach wall is daily exposed to oxidative stress and BER system has a key role in the defense from oxidation-induced DNA damage, whilst ErbB receptors have important roles in the pathogenesis of cancer. We used AGS cells as an aggressive gastric carcinoma cell model, treated with H2O2 alone or combined with ErbB signaling pathway inhibitors, to evaluate the effects of oxidative stress in gastric cancer, focusing on the modulation of ErbB signaling pathways and their eventual cross-talk with BER system. We showed that treatment with H2O2 combined with PI3K/AKT and MEK inhibitors influenced cell morphology and resulted in a reduction of cancer cell viability. Migration ability was reduced after H2O2 treatment alone or combined with MEK inhibitor and after PI3K/AKT inhibitor alone. Western blotting analysis showed that oxidative stress stimulated EGFR pathway favoring the MAPKs activation at the expense of PI3K/AKT pathway. Gene expression analysis by RT-qPCR showed ErbB2 and OGG1 increase under oxidative stress conditions. Therefore, we suggest that in AGS cells a pro-oxidant treatment can reduce gastric cancer cell growth and migration via a different modulation of PI3K and MAPKs pathways. Moreover, the observed ErbB2 and OGG1 induction is a cellular response to protect the cells from H2O2-induced cell death. In conclusion, to tailor specific combinations of therapies and to decide which strategy to use, administration of a chemotherapy that increases intracellular ROS to toxic levels, might not only be dependent on the tumor type, but also on the molecular targeting therapy used.

Emerging Role of Oxidative Stress on EGFR and OGG1-BER Cross-Regulation: Implications in Thyroid Physiopathology.

Thyroid diseases have a complex and multifactorial aetiology. Despite the numerous studies on the signals referable to the malignant transition, the molecular mechanisms concerning the role of oxidative stress remain elusive. Based on its strong oxidative power, H2O2 could be responsible for the high level of oxidative DNA damage observed in cancerous thyroid tissue and hyperactivation of mitogen-activated protein kinase (MAPK) and PI3K/Akt, which mediate ErbB signaling. Increased levels of 8-oxoG DNA adducts have been detected in the early stages of thyroid cancer. These DNA lesions are efficiently recognized and removed by the base excision repair (BER) pathway initiated by 8-oxoG glycosylase1 (OGG1). This study investigated the relationships between the EGFR and OGG1-BER pathways and their mutual regulation following oxidative stress stimulus by H2O2 in human thyrocytes. We clarified the modulation of ErbB receptors and their downstream pathways (PI3K/Akt and MAPK/ERK) under oxidative stress (from H2O2) at the level of gene and protein expression, according to the mechanism defined in a human non-pathological cell system, Nthy-ori 3-1. Later, on the basis of the results obtained by gene expression cluster analysis in normal cells, we assessed the dysregulation of the relationships in a model of papillary thyroid cancer with RET/PTC rearrangement (TPC-1). Our observations demonstrated that a H2O2 stress may induce a physiological cross-regulation between ErbB and OGG1-BER pathways in normal thyroid cells (while this is dysregulated in the TPC-1 cells). Gene expression data also delineated that MUTYH gene could play a physiological role in crosstalk between ErbB and BER pathways and this function is instead lost in cancer cells. Overall, our data on OGG1 protein expression suggest that it was physiologically regulated in response to oxidative modulation of ErbB, and that these might be dysregulated in the signaling pathway involving AKT in the progression of thyroid malignancies with RET/PTC rearrangements.

Oxidative Distress Induces Wnt/ß-Catenin Pathway Modulation in Colorectal Cancer Cells: Perspectives on APC Retained Functions.

Colorectal cancer (CRC) is a multistep process that arises in the colic tissue microenvironment. Oxidative stress plays a role in mediating CRC cell survival and progression, as well as promoting resistance to therapies. CRC progression is associated with Wnt/ß-Catenin signaling dysregulation and loss of proper APC functions. Cancer recurrence/relapse has been attributed to altered ROS levels, produced in a cancerous microenvironment. The effect of oxidative distress on Wnt/ß-Catenin signaling in the light of APC functions is unclear. This study evaluated the effect of H2O2-induced short-term oxidative stress in HCT116, SW480 and SW620 cells with different phenotypes of APC and ß-Catenin. The modulation and relationship of APC with characteristic molecules of Wnt/ß-Catenin were assessed in gene and protein expression. Results indicated that CRC cells, even when deprived of growth factors, under acute oxidative distress conditions by H2O2 promote ß-Catenin expression and modulate cytoplasmic APC protein. Furthermore, H2O2 induces differential gene expression depending on the cellular phenotype and leading to favor both Wnt/Catenin-dependent and -independent signaling. The exact mechanism by which oxidative distress can affect Wnt signaling functions will require further investigation to reveal new scenarios for the development of therapeutic approaches for CRC, in the light of the conserved functions of APC.

The bromodomain and extra-terminal domain degrader MZ1 exhibits preclinical anti-tumoral activity in diffuse large B-cell lymphoma of the activated B cell-like type.

Aim: Bromodomain and extra-terminal domain (BET) proteins are epigenetic readers that play a fundamental role in transcription regulation. Preclinical and early clinical evidence sustain BET targeting as an anti-cancer approach. BET degraders are chimeric compounds comprising of a BET inhibitor, which allows the binding to BET bromodomains, linked to a small molecule, binder for an E3 ubiquitin ligase complex, triggering BET proteins degradation via the proteasome. These degraders, called proteolysis-targeting chimeras (PROTACs), can exhibit greater target specificity compared to BET inhibitors and overcome some of their limitations, such as the upregulation of the BET proteins themselves. Here are presented data on the anti-tumor activity and the mechanism of action of the BET degrader MZ1 in diffuse large B cell lymphoma (DLBCL) of the activated B-cell like (ABC, ABC DLBCL), using a BET inhibitor as a comparison. Methods: Established lymphoma cell lines were exposed for 72 h to increasing doses of the compounds. Cell proliferation was evaluated by using an 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazoliumbromide (MTT) assay. Fluorescent-Activated Cell Sorter (FACS) analysis was performed to measure apoptotic activation and RNA sequencing (RNA-Seq) to study the transcriptional changes induced by the compounds. Results: MZ1, and not its negative control epimer cisMZ1, was very active with a median half maximal inhibitory concentration (IC50) of 49 nmol/L. MZ1 was more in vitro active than the BET inhibitor birabresib (OTX015). Importantly, MZ1 induced cell death in all the ABC DLBCL cell lines, while the BET inhibitor was cytotoxic only in a fraction of them. BET degrader and inhibitor shared partially similar changes at transcriptome level but the MZ1 effect was stronger and overlapped with that caused cyclin-dependent kinase 9 (CDK9) inhibition. Conclusions: The BET degrader MZ1 had strong cytotoxic activity in all the ABC DLBCL cell lines that were tested, and, at least in vitro, it elicited more profound effects than BET inhibitors, and encourages further investigations.

Relationship between MUTYH, OGG1 and BRCA1 mutations and mRNA expression in breast and ovarian cancer predisposition.

The aetiology of breast and ovarian cancer (BC/OC) is multi-factorial. At present, the involvement of base excision repair (BER) glycosylases (MUTYH and OGG1) in BC/OC predisposition is controversial. The present study investigated whether germline mutation status and mRNA expression of two BER genes, MUTHY and OGG1, were correlated with BRCA1 in 59 patients with BC/OC and 50 matched population controls. In addition, to evaluate the relationship between MUTYH, OGG1 and BRCA1, their possible mutual modulation and correlation among mutational spectrum, gene expression and demographic characteristics were evaluated. The results identified 18 MUTYH and OGG1 variants, of which 4 were novel (2 MUTYH and 2 OGG1) in 44 of the 59 patients. In addition, two pathogenic mutations were identified: OGG1 p.Arg46Gln, detected in a patient with BC and a family history of cancer, and MUTYH p.Val234Gly in a patient with OC, also with a family history of cancer. A significant reduced transcript expression in MUTYH was observed (P=0.033) in cases, and in association with the presence of rare variants in the same gene (P=0.030). A significant correlation in the expression of the two BER genes was observed in cases (P=0.004), whereas OGG1 and BRCA1 was significantly correlated in cases (P=0.001) compared with controls (P=0.010). The results of the present study indicated that the relationship among mutational spectrum, gene expression and demographic characteristics may improve the genetic diagnosis and primary prevention of at-risk individuals belonging to families with reduced mRNA expression, regardless of mutation presence.

Germline TP53 mutation spectrum in Sudanese premenopausal breast cancer patients: correlations with reproductive factors.

PURPOSE: The role of non-genetic factors as modifiers of TP53-related hereditary breast cancer (BC) risk is debated. In this regard, little is known about the impact of germline TP53 mutations on BC in sub-Saharan Africa, where the disease often presents in non-contraceptive multiparous premenopausal women with extended history of breastfeeding. Herein, we report the germline TP53 mutations found in a series of 92 Sudanese premenopausal BC patients characterized for reproductive history. METHODS: The entire TP53 coding sequence, including intron-exon boundaries and UTRs, was analyzed via DHPLC and direct sequencing, and the association of TP53 genotypes with BC risk and with individual lifetime exposures to reproductive factors was investigated with statistical tools. RESULTS: The germline TP53 mutation spectrum comprised 20 variants, 15 in the non-coding and 5 in the coding region. The latter included a deleterious missense mutation, c.817C>T (p.Arg273Cys), in a unique patient, and the common and functionally relevant coding polymorphism at amino acid 72 [Pro72Arg (rs1042522)]. The non-coding mutations included c.919+1G>A, a known deleterious splice site mutation, also in a unique patient. Notably, the 2 carriers of deleterious TP53 mutations clustered in the subset of cases with stronger reproductive history relative to childbearing age. When analyzed in comparison to population controls, the codon 72 polymorphism did not reveal associations with BC. CONCLUSIONS: Our study suggests that the codon 72 Arg>Pro polymorphism is not implicated in premenopausal BC susceptibility, whereas multiparity and breastfeeding might be BC risk factors for carriers of deleterious TP53 mutations.