Biological and therapeutic implications of RKIP in Gastrointestinal Stromal Tumor (GIST): an integrated transcriptomic and proteomic analysis.

BACKGROUND: Gastrointestinal stromal tumors (GIST) represent a significant clinical challenge due to their metastatic potential and limited treatment options. Raf kinase inhibitor protein (RKIP), a suppressor of the MAPK signaling pathway, is downregulated in various cancers and acts as a metastasis suppressor. Our previous studies demonstrated low RKIP expression in GIST and its association with poor outcomes. This study aimed to expand on the previous findings and investigate the biological and therapeutic implications of RKIP loss on GIST. METHODS: To validate the RKIP prognostic significance, its expression was evaluated by immunohistochemistry in 142 bona fide GIST cases. The functional role of RKIP was evaluated in vitro, using the GIST-T1 cell line, which was knocked out for RKIP. The biological and therapeutic implications of RKIP were evaluated by invasion, migration, apoptosis, and 2D / 3D viability assays. Additionally, the transcriptome and proteome of RKIP knockout cells were determined by NanoString and mass spectrometry, respectively. RESULTS: Immunohistochemical analysis revealed the absence of RKIP in 25.3% of GIST cases, correlating with a tendency toward poor prognosis. Functional assays demonstrated that RKIP knockout increased GIST cells' invasion and migration potential by nearly 60%. Moreover, we found that RKIP knockout cells exhibited reduced responsiveness to Imatinib treatment and higher cellular viability in 2D and 3D in vitro models, as assessed by apoptosis-related protein expression. Through comprehensive genetic and proteomic profiling of RKIP knockout cells, we identified several putative RKIP-regulated proteins in GIST, such as COL3A1. CONCLUSIONS: Using a multidimensional integrative analysis, we identified, for the first time in GIST, molecules and pathways modulated by RKIP that may potentially drive metastasis and, consequently, poor prognosis in this disease.

Correction: Alves et al. WNK2 Inhibits Autophagic Flux in Human Glioblastoma Cell Line. Cells 2020, 9, 485.

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The Barretos Cancer Hospital Animal Facility: Implementation and Results of a Dedicated Platform for Preclinical Oncology Models.

The Barretos Cancer Hospital Animal Facility (BCHAF) is a unique facility in Brazil exclusively dedicated to working with animal models for cancer research. In this article, we briefly present our modern facility and the main experiments performed, focusing on mutant strains of mice (PTCH-knockout and ApcMin mice), xenograft models, and patient-derived xenografts (PDXs). Our results show the progress and challenges in establishing these models and the need for having an appropriate representation of our cancer population to better understand tumor biology and to identify cancer biomarkers, which could be putatively targeted, allowing for personalized therapy.

WNK2 Inhibits Autophagic Flux in Human Glioblastoma Cell Line.

Autophagy is a cell-survival pathway with dual role in tumorigenesis, promoting either tumor survival or tumor death. WNK2 gene, a member of the WNK (with no lysine (K)) subfamily, acts as a tumor suppressor gene in gliomas, regulating cell migration and invasion; however, its role in autophagy process is poorly explored. The WNK2-methylated human glioblastoma cell line A172 WT (wild type) was compared to transfected clones A172 EV (empty vector), and A172 WNK2 (WNK2 overexpression) for the evaluation of autophagy using an inhibitor (bafilomycin A1-baf A1) and an inducer (everolimus) of autophagic flux. Western blot and immunofluorescence approaches were used to monitor autophagic markers, LC3A/B and SQSTM1/p62. A172 WNK2 cells presented a significant decrease in LC3B and p62 protein levels, and in LC3A/B ratio when compared with control cells, after treatment with baf A1 + everolimus, suggesting that WNK2 overexpression inhibits the autophagic flux in gliomas. The mTOR pathway was also evaluated under the same conditions, and the observed results suggest that the inhibition of autophagy mediated by WNK2 occurs through a mTOR-independent pathway. In conclusion, the evaluation of the autophagic process demonstrated that WNK2 inhibits the autophagic flux in glioblastoma cell line.

Loss of 5'-Methylthioadenosine Phosphorylase (MTAP) is Frequent in High-Grade Gliomas; Nevertheless, it is Not Associated with Higher Tumor Aggressiveness.

The 5'-methylthioadenosine phosphorylase (MTAP) gene is located in the chromosomal region 9p21. MTAP deletion is a frequent event in a wide variety of human cancers; however, its biological role in tumorigenesis remains unclear. The purpose of this study was to characterize the MTAP expression profile in a series of gliomas and to associate it with patients' clinicopathological features. Moreover, we sought to evaluate, through glioma gene-edited cell lines, the biological impact of MTAP in gliomas. MTAP expression was evaluated in 507 glioma patients by immunohistochemistry (IHC), and the expression levels were associated with patients' clinicopathological features. Furthermore, an in silico study was undertaken using genomic databases totalizing 350 samples. In glioma cell lines, MTAP was edited, and following MTAP overexpression and knockout (KO), a transcriptome analysis was performed by NanoString Pan-Cancer Pathways panel. Moreover, MTAP's role in glioma cell proliferation, migration, and invasion was evaluated. Homozygous deletion of 9p21 locus was associated with a reduction of MTAP mRNA expression in the TCGA (The Cancer Genome Atlas) - glioblastoma dataset (p < 0.01). In addition, the loss of MTAP expression was markedly high in high-grade gliomas (46.6% of cases) determined by IHC and Western blotting (40% of evaluated cell lines). Reduced MTAP expression was associated with a better prognostic in the adult glioblastoma dataset (p < 0.001). Nine genes associated with five pathways were differentially expressed in MTAP-knockout (KO) cells, with six upregulated and three downregulated in MTAP. Analysis of cell proliferation, migration, and invasion did not show any significant differences between MTAP gene-edited and control cells. Our results integrating data from patients as well as in silico and in vitro models provide evidence towards the lack of strong biological importance of MTAP in gliomas. Despite the frequent loss of MTAP, it seems not to have a clinical impact in survival and does not act as a canonic tumor suppressor gene in gliomas.