Rabbit Anti-Human FN1 (internal) Polyclonal antibody

Metabolic reprogramming is a molecular hallmark of cancer. Recently, we have reported the overexpression of glyoxalase 1 (encoded byGLO1), a glutathione-dependent enzyme involved in detoxification of the reactive glycolytic byproduct methylglyoxal, in human malignant melanoma cell culture models and clinical samples. However, the specific role ofGLO1in melanomagenesis remains largely unexplored. Here, using genetic target modulation, we report the identification ofGLO1as a novel molecular determinant of invasion and metastasis in malignant melanoma. First, A375 human malignant melanoma cells withGLO1deletion (A375-GLO1_KO) were engineered using CRISPR/Cas9, and genetic rescue clones were generated by stable transfection of KO clones employing a CMV-drivenGLO1construct (A375-GLO1_R). After confirmingGLO1target modulation at the mRNA and protein levels (RT-qPCR, immunodetection, enzymatic activity), phenotypic characterization indicated that deletion ofGLO1does not impact proliferative capacity while causing significant sensitization to methylglyoxal-, chemotherapy-, and starvation-induced cytotoxic stress. Employing differential gene expression array analysis (A375-GLO1_KO versus A375-GLO1_WT), pronounced modulation of epithelial mesenchymal transition (EMT)-related genes [upregulated:CDH1,OCLN,IL1RN, PDGFRB, SNAI3; (downregulated):BMP1, CDH2, CTNNB1, FN1, FTH1, FZD7, MELTF, MMP2, MMP9, MYC, PTGS2, SNAI2, TFRC, TWIST1, VIM, WNT5A, ZEB1,andZEB2(up to tenfold;p< 0.05)] was observed-all of which are consistent with EMT suppression as a result ofGLO1deletion. Importantly, these expression changes were largely reversed upon genetic rescue employing A375-GLO1_R cells. Differential expression ofMMP9as a function ofGLO1status was further substantiated by enzymatic activity and ELISA analysis; phenotypic assessment revealed the pronounced attenuation of morphological potential, transwell migration, and matrigel 3D-invasion capacity displayed by A375-GLO1_KO cells, reversed again in genetic rescue clones. Strikingly, in a SCID mouse metastasis model, lung tumor burden imposed by A375-GLO1_KO cells was strongly attenuated as compared to A375-GLO1_WT cells. Taken together, these prototype data provide evidence in support of a novel function ofGLO1in melanoma cell invasiveness and metastasis, and ongoing investigations explore the function and therapeutic potential ofGLO1as a novel melanoma target.

Objectives: Atypical femoral fractures (AFF) are rare, often related to long-term bisphosphonate (BPs) treatment. Their pathogenic mechanisms are not precisely known and there is no evidence to identify patients with a high risk of AFF. The aim of this work is to study the genetic bases of AFFs. Material and methods: Whole-exome sequencing was carried out on 3 sisters and 3 unrelated additional patients, all treated with BPs for more than 5 years. Low frequency, potentially pathogenic variants shared by the 3 sisters, were selected, were selected and a network of gene and protein interactions was constructed with the data found. Results: We identified 37 rare variants (in 34 genes) shared by the 3 sisters, some not previously described. The most striking variant was the p.Asp188Tyr mutation in the enzyme geranylgeranyl pyrophosphate synthase (encoded by the GGPS1 gene), from the mevalonate pathway and essential for osteoclast function. Another noteworthy finding was two mutations (one in the 3 sisters and one in an unrelated patient) in the CYP1A1 gene, involved in the metabolism of steroids. We identified other variants that could also be involved in the susceptibility to AFFs or in the underlying osteoporotic phenotype, such as those present in the SYDE2, NGEF, COG4 and FN1 genes. Conclusions: Our data are compatible with a model where the accumulation of susceptibility variants could participate in the genetic basis of AFFs.