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.

MicroRNA­130a­3p inhibition suppresses cervical cancer cell progression.

MicroRNAs (miRNAs or miRs) play essential roles in the initiation and progression of human tumors, including cervical cancer. However, the mechanisms underlying their actions in cervical cancer remain unclear. The present study aimed to evaluate the functional role of miR­130a­3p in cervical cancer. Cervical cancer cells were transfected with a miRNA inhibitor (anti­miR­130a­3p) and a negative control. Adhesion­independent cell proliferation, migration and invasion were evaluated. The findings presented herein demonstrated that miR­130a­3p was overexpressed in HeLa, SiHa, CaSki, C­4I and HCB­514 cervical cancer cells. The inhibition of miR­130a­3p significantly reduced the proliferation, migration and invasion of cervical cancer cells. The canonical delta­like Notch1 ligand (DLL1) was identified as a possible direct target of miR­103a­3p. The DLL1 gene was further found to be significantly downregulated in cervical cancer tissues. On the whole, the present study demonstrates that miR­130a­3p contributes to the proliferation, migration and invasion of cervical cancer cells. Therefore, miR­130a­3p may be used as a biomarker to determine cervical cancer progression.

RAD50 Deficient in a Breast Cancer Model Predicts Sensitivity to PARP Inhibitors.

BACKGROUND: Breast and ovarian tumors with pathogenic variants in BRCA1 or BRCA2 genes are more sensitive to poly (ADP-ribose) polymerase inhibitors (PARPi) treatment than wildtype tumors. Pathogenic variants in non-BRCA1/2 homologous recombination repair genes (HRR) also concede sensitivity to PARPi treatment. RAD50 participates in the Mre11-RAD50-Nbn (MRN) complex of the HRR pathway and plays an important role in DNA repair. OBJECTIVE: The objective of this study is to evaluate whether RAD50 protein deficiency modulates the PARPi response in breast cancer cell lines. METHODS: T47D breast cancer cell line was modified using small interfering RNA and CRISPR/Cas9 technology, to knockout the RAD50 gene. PARPi response (niraparib, olaparib and rucaparib alone or in combination with carboplatin), in T47D and T47D-edited clones, was evaluated by cell viability, cell cycle, apoptosis and protein expression analyses. RESULTS: Treatment with niraparib and carboplatin exerted a synergistic effect on T47D-RAD50 deficient cells and an antagonistic effect on T47D cells parental. Cell cycle analysis demonstrated an increase in the G2/M population in cells treated with niraparib or rucaparib alone or in combination with carboplatin. T47D-RAD50 deficient cells treated with rucaparib and carboplatin exhibited twofold levels in late apoptosis, also showing differences in PARP activation. All T47D RAD50 deficient clones treated with niraparib or rucaparib combined with carboplatin, or rucaparib alone showed increased levels of H2AX phosphorylation. CONCLUSIONS: T47D RAD50 deficient cells treated with PARP inhibitors alone or in combination with carboplatin showed cell cycle arrest in the G2/M phase, leading to death by apoptosis. Thus, RAD50 deficiency may be a good biomarker for predicting PARPi response.

Neuroprotective Properties of Chlorogenic Acid and 4,5-Caffeoylquinic Acid from Brazilian arnica (Lychnophora ericoides) after Acute Retinal Ischemia.

Lychnophora is a genus of South American flowering plants in the daisy family, popularly known as "Brazilian arnica". It is used in traditional medicine as an anti-inflammatory and analgesic agent, whose active components are derived from chlorogenic acid (CGA) and C-flavonoids. Since the drugs currently used are ineffective to treat glaucoma, agents with antioxidant and anti-inflammatory properties may represent new alternatives in preventing cellular lesions in retinal ischemia. In this study, we report the neuroprotective effects of CGA and 4,5-di-O-[E]-caffeoylquinic (CQA) acid, isolated from Lychnophora plants, in a rodent glaucoma model. Wistar rats were administered intravitreally with 10 µg CGA or CGA, and then subjected to acute retinal ischemia (ISC) by increasing intraocular pressure (IPO) for 45 minutes followed (or not) by 15 minutes of reperfusion (I/R). Qualitative and quantitative analyses of neurodegeneration were performed using hematoxylin-eosin or Fluoro-Jade C staining protocols. All retinas submitted to ISC or I/R exhibited matrix disorganization, pyknotic nuclei, and pronounced vacuolization of the cytoplasm in the ganglion cell layer (GCL) and inner nuclear layer (INL). Pretreatment with CGA or CQA resulted in the protection of the retinal layers against matrix disorganization and a reduction in the number of vacuolized cells and pyknotic nuclei. Also, pretreatment with CGA or CQA resulted in a significant reduction in neuronal death in the GCL, the INL, and the outer nuclear layer (ONL) after ischemic insult. Our study demonstrated that CGA and CQA exhibit neuroprotective activities in retinas subjected to ISC and I/R induced by IPO in Wistar rats.

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.

Semi-Synthetic Ingenol Derivative from Euphorbia tirucalli Inhibits Protein Kinase C Isotypes and Promotes Autophagy and S-phase Arrest on Glioma Cell Lines.

The identification of signaling pathways that are involved in gliomagenesis is crucial for targeted therapy design. In this study we assessed the biological and therapeutic effect of ingenol-3-dodecanoate (IngC) on glioma. IngC exhibited dose-time-dependent cytotoxic effects on large panel of glioma cell lines (adult, pediatric cancer cells, and primary cultures), as well as, effectively reduced colonies formation. Nevertheless, it was not been able to attenuate cell migration, invasion, and promote apoptotic effects when administered alone. IngC exposure promoted S-phase arrest associated with p21CIP/WAF1 overexpression and regulated a broad range of signaling effectors related to survival and cell cycle regulation. Moreover, IngC led glioma cells to autophagy by LC3B-II accumulation and exhibited increased cytotoxic sensitivity when combined to a specific autophagic inhibitor, bafilomycin A1. In comparison with temozolomide, IngC showed a mean increase of 106-fold in efficacy, with no synergistic effect when they were both combined. When compared with a known compound of the same class, namely ingenol-3-angelate (I3A, Picato®), IngC showed a mean 9.46-fold higher efficacy. Furthermore, IngC acted as a potent inhibitor of protein kinase C (PKC) activity, an emerging therapeutic target in glioma cells, showing differential actions against various PKC isotypes. These findings identify IngC as a promising lead compound for the development of new cancer therapy and they may guide the search for additional PKC inhibitors.

Establishment, molecular and biological characterization of HCB-514: a novel human cervical cancer cell line.

Cervical cancer is the fourth most common cancer in women. Although cure rates are high for early stage disease, clinical outcomes for advanced, metastatic, or recurrent disease remain poor. To change this panorama, a deeper understanding of cervical cancer biology and novel study models are needed. Immortalized human cancer cell lines such as HeLa constitute crucial scientific tools, but there are few other cervical cancer cell lines available, limiting our understanding of a disease known for its molecular heterogeneity. This study aimed to establish novel cervical cancer cell lines derived from Brazilian patients. We successfully established one (HCB-514) out of 35 cervical tumors biopsied. We confirmed the phenotype of HCB-514 by verifying its' epithelial and tumor origin through cytokeratins, EpCAM and p16 staining. It was also HPV-16 positive. Whole-exome sequencing (WES) showed relevant somatic mutations in several genes including BRCA2, TGFBR1 and IRX2. A copy number variation (CNV) analysis by nanostring and WES revealed amplification of genes mainly related to kinases proteins involved in proliferation, migration and cell differentiation, such as EGFR, PIK3CA, and MAPK7. Overexpression of EGFR was confirmed by phospho RTK-array and validated by western blot analysis. Furthermore, the HCB-514 cell line was sensitive to cisplatin. In summary, this novel Brazilian cervical cancer cell line exhibits relevant key molecular features and constitutes a new biological model for pre-clinical studies.

In vitro screening of cytotoxic activity of euphol from Euphorbia tirucalli on a large panel of human cancer-derived cell lines.

A large number of classic antineoplastic agents are derived from plants. Euphorbia tirucalli L. (Euphorbiaceae) is a subtropical and tropical plant, used in Brazilian folk medicine against many diseases, including cancer, yet little is known about its true anticancer properties. The present study evaluated the antitumor effect of the tetracyclic triterpene alcohol, euphol, the main constituent of E. tirucalli in a panel of 73 human cancer lines from 15 tumor types. The biological effect of euphol in pancreatic cells was also assessed. The combination index was further used to explore euphol interactions with standard drugs. Euphol showed a cytotoxicity effect against several cancer cell lines (IC50 range, 1.41-38.89 µM), particularly in esophageal squamous cell (11.08 µM) and pancreatic carcinoma cells (6.84 µM), followed by prostate, melanoma, and colon cancer. Cytotoxicity effects were seen in all cancer cell lines, with more than half deemed highly sensitive. Euphol inhibited proliferation, motility and colony formation in pancreatic cancer cells. Importantly, euphol exhibited synergistic interactions with gemcitabine and paclitaxel in pancreatic and esophageal cell lines, respectively. To the best of our knowledge, this study constitutes the largest in vitro screening of euphol efficacy on cancer cell lines and revealed its in vitro anti-cancer properties, particularly in pancreatic and esophageal cell lines, suggesting that euphol, either as a single agent or in combination with conventional chemotherapy, is a potential anti-cancer drug.

Cytotoxicity of allitinib, an irreversible anti-EGFR agent, in a large panel of human cancer-derived cell lines: KRAS mutation status as a predictive biomarker.

BACKGROUND: The epidermal growth factor receptor (EGFR) is a member of the HER family of growth factors that activates several intracellular signaling pathways promoting proliferation and survival. EGFR over-expression is frequently associated with gene mutation or amplification, thereby constituting a major target for molecular therapies. Recently, a new generation of EGFR inhibitors has been developed with pan-HER properties and irreversible actions. Allitinib® (AST1306) is an orally active, highly selective irreversible inhibitor of the HER family of receptor tyrosine kinases with promising efficacies. In the present study we aimed to investigate the cytotoxicity of allitinib in a large panel of human cancer-derived cell lines and to correlate its efficacy to the mutational status of the EGFR, KRAS, BRAF, PI3KCA and PTEN genes. In addition, we aimed to evaluate the functional role of KRAS mutations in the response to this new inhibitor. RESULTS: In total 76 different cancer-derived cell lines, representing 11 distinct histological types, were analyzed and classified into three groups: highly sensitive (HS), moderately sensitive (MS) and resistant (R). We found that 28 (36.8 %) cancer-derived cell lines exhibited a HS phenotype, 19 (25.0 %) a MS phenotype and 29 (38.1 %) a R phenotype. Allitinib showed a stronger cytotoxicity in head and neck, esophageal, melanoma and lung cancer-derived cell lines. We found that KRAS mutations were significantly associated with the R phenotype. To substantiate these results, an allitinib-sensitive lung cancer-derived cell line (H292) was transfected with plasmids carrying the two most common activating KRAS mutations (p.G12D and p.G12S). We found that both mutations reverted the allitinib-sensitive phenotype in these cells. CONCLUSIONS: The current study represents the largest in vitro assessment of allitinib cytotoxicity performed to date. Through this study, we identified cancer types that could potentially benefit from this drug. Additionally, our findings suggest that prevalent KRAS mutations constitute potential predictive biomarkers for allitinib response.

C-Phycocyanin protects SH-SY5Y cells from oxidative injury, rat retina from transient ischemia and rat brain mitochondria from Ca2+/phosphate-induced impairment.

Oxidative stress and mitochondrial impairment are essential in the ischemic stroke cascade and eventually lead to tissue injury. C-Phycocyanin (C-PC) has previously been shown to have strong antioxidant and neuroprotective actions. In the present study, we assessed the effects of C-PC on oxidative injury induced by tert-butylhydroperoxide (t-BOOH) in SH-SY5Y neuronal cells, on transient ischemia in rat retinas, and in the calcium/phosphate-induced impairment of isolated rat brain mitochondria (RBM). In SH-SY5Y cells, t-BOOH induced a significant reduction of cell viability as assessed by an MTT assay, and the reduction was effectively prevented by treatment with C-PC in the low micromolar concentration range. Transient ischemia in rat retinas was induced by increasing the intraocular pressure to 120mmHg for 45min, which was followed by 15min of reperfusion. This event resulted in a cell density reduction to lower than 50% in the inner nuclear layer (INL), which was significantly prevented by the intraocular pre-treatment with C-PC for 15min. In the RBM exposed to 3mM phosphate and/or 100µM Ca(2+), C-PC prevented in the low micromolar concentration range, the mitochondrial permeability transition as assessed by mitochondrial swelling, the membrane potential dissipation, the increase of reactive oxygen species levels and the release of the pro-apoptotic cytochrome c. In addition, C-PC displayed a strong inhibitory effect against an electrochemically-generated Fenton reaction. Therefore, C-PC is a potential neuroprotective agent against ischemic stroke, resulting in reduced neuronal oxidative injury and the protection of mitochondria from impairment.