Synergistic actions of Alpelisib and Melatonin in breast cancer cell lines with PIK3CA gene mutation.

AIMS: Breast cancer (BC) presents high mortality rate and about 25-46 % have mutation in the PIK3CA gene. Alpelisib is a PI3K inhibitor that acts on p110α, which is a subunit of the PI3K protein. The melatonin shown important anti-neoplastic effects and may increase the effectiveness of chemotherapy. This study evaluated the synergistic action of Alpelisib and Melatonin in BC lines carrying the H1047R mutation in PIK3CA, relative to the cellular dynamics and the PI3K/AKT/mTOR pathway. MAIN METHODS: MDA-MB-468 (triple-ernegative), MDA-MB-453 (H1047R PIK3CA, HER2+) and T-47D cells (H1047R PIK3CA, ER+/PR+) were divided into four treatment groups: control; Melatonin (1 mM); Alpelisib (1 µM); and Alpelisib (1 µM) + Melatonin (1 mM). Cell viability and migration were investigated using the MTT assay and Transwell assay, respectively. Protein expression of PI3K, p-AKT, mTOR, HIF-1α, and caspase-3, was verified using immunocytochemistry. KEY FINDINGS: MTT assay revealed that MDA-MB-453 and T-47D showed reduction in cell viability in all groups, especially in the MDA-MB-453 treated with Melatonin + Alpelisib. MDA-MB-468 presents reduction in cell migration only with Melatonin, while in the lines with mutation, the treatment of Melatonin + Alpelisib caused inhibition of cell migration. PI3K, p-AKT, mTOR and HIF-1α were inhibited after treatment with Melatonin + Alpelisib in MDA-MB-453 and T-47D lines. The expression of caspase-3 increased in all groups in MDA-MB-453 and T-47D cells, being the increase more pronounced in the Melatonin + Alpelisib group. SIGNIFICANCE: These results indicate that the combined use of Melatonin and Alpelisib may be more effective in inhibiting BC in women carrying the PIK3CA gene mutation than either treatment alone.

Treatment of Triple Negative Cell Lines with Olaparib to Block DNA Repair.

BACKGROUND: The most aggressive breast cancer is the triple negative histological type, and the gold standard for its treatment is platinum salts, such as carboplatin. Due to high recurrence, there is a need to test new drugs, such as PARP inhibitors (PARPi), that induce lethality in cells with DNA damage. Olaparib is a PARPi, already used in some tumors but not tested in canine species. Thus, the aim of this study was to demonstrate the efficacy of olaparib in inhibiting DNA repair and control disease progression by decreasing the migration capacity of mammary tumor cells. METHODS: The cell lines CF41.Mg and MDA-MB-468 were cultured and MTT was performed to define the best dose of carboplatin. Next, the cells were treated with 10 µM carboplatin, olaparib, and with a combination of both for 24 hours. PARP-1 protein and gene expression were evaluated by immunofluorescence, western blotting, and qRT-PCR, respectively. The analysis of cell migration was performed in transwell chambers. RESULTS: For CF41.Mg and MDA-MB-468 cell lines, there was a decrease in PARP-1 protein and gene expression after treatment with carboplatin, olaparib, and both in combination compared to the group without treatment (control) (p<0.05). Moreover, in both lines, a reduction in invasion rate was observed after treatment with carboplatin, olaparib and when combined, compared to the control group (p<0.05). CONCLUSION: Our data suggest that carboplatin and olaparib were able to block DNA repair and control the cancer invasion, especially when used in combination. The results with olaparib in the canine line are unpublished. The olaparib should be a possible agent against human breast cancer and canine mammary tumors.

Liquid biopsy can detect BRCA2 gene variants in female dogs with mammary neoplasia.

Mammary tumours (MT) are one of the most prevalent malignancies in female dogs and women. Currently, molecular analyzes have shown that each tumour type presents its own genetic signature. In this context, liquid biopsy allows a comprehensive genetic characterisation of the tumour, enabling early diagnosis and personalised treatment of patients. In women, deleterious mutations inherited in BRCA2 gene are associated with an increased risk of breast cancer, resistance to therapies and worse prognosis. In female dogs, there are many divergent data on the involvement of BRCA2 gene with mammary carcinogenesis and what its pathogenic potential is. Therefore, the objective was to identify BRCA2 gene variants in 20 plasma DNA samples, from 10 newly diagnosed dogs with mammary cancer (RD), five control (CTR) and five mastectomized patients. Eleven single nucleotide polymorphisms (SNPs) were detected, most of them in the exon 11 and two indels (deletion/insertion) in the BRCA2 gene. However, there was no statistically significant difference in the SNPs/indels detected between the groups. In addition, only one SNP (p.T1425P) and one deletion (p.L2307del) were considered deleterious using in silico computational models. Interestingly, most common variants were present in the plasma of all groups, except for the Ile2614Thr, Ile2614Val, Thr1425Pro and p.L2307del variants. Thus, we observed that SNPs are common in the BRCA2 gene of female dogs with MT, with a similar condition identified in women with breast cancer. Liquid biopsy approach in dogs with MT is useful for genetic and therapeutic proposals.

Modulation of Epithelial Mesenchymal Transition after AGTR-1 Gene Edition by Crispr/Cas9 and Losartan Treatment in Mammary Tumor Cell Line: A Comparative Study between Human and Canine Species.

Breast cancer is the most prevalent tumor type among women and female dogs. Tumor malignancy is characterized by the epithelial-to-mesenchymal transition (EMT) which leads to the metastasis formation. The inhibition of angiotensin II type I receptor (AGTR1) by an antagonist such as losartan can suppress angiogenesis, consequently contributing to the metastasis control. The aim of this study was to analyze the capacity of losartan and AGTR-1 gene edition to modulate the EMT process in triple negative/metastatic mammary tumor cells, compared to existing treatment protocols such as carboplatin. The cell lines CF41.Mg and MDA-MB-468, were cultured and treated with carboplatin, losartan, or submitted to AGTR-1 gene edition by CRISPR/Cas9. EMT markers and PARP-1 protein and gene expression were evaluated by immunofluorescence or immunocytochemistry and qRT-PCR, respectively. Cell migration capacity was also evaluated. For CF41.Mg and MDA-MB-468 cell lines, there was an increase in E-cadherin and a decrease in N-cadherin and PARP-1 protein and gene expression after treatment with carboplatin, losartan, both in combination and after AGTR-1 gene edition. There was a decrease in VEGF and PARP-1 protein and gene expression after AGTR-1 gene edition. Moreover, in both lines, reduction in invasion rate was observed after all treatments. Our data suggest that losartan and the gene edition of AGTR-1 by CRISPR/Cas9 were able to block the DNA repair and control the EMT process, such as carboplatin. The results in the canine species are unprecedented, as there are no data in the literature that demonstrate the action of losartan in this tumor type.

Liquid Biopsy as a Diagnostic and Prognostic Tool for Women and Female Dogs with Breast Cancer.

INTRODUCTION: Breast cancer (BC) is the malignant neoplasm with the highest mortality rate in women and female dogs are good models to study BC. OBJECTIVE: We investigated the efficacy of liquid biopsy to detect gene mutations in the diagnosis and follow-up of women and female dogs with BC. MATERIALS AND METHODS: In this study, 57 and 37 BC samples were collected from women and female dogs, respectively. After core biopsy and plasma samples were collected, the DNA and ctDNA of the tumor fragments and plasma were processed for next generation sequencing (NGS) assay. After preprocessing of the data, they were submitted to the Genome Analysis ToolKit (GATK). RESULTS: In women, 1788 variants were identified in tumor fragments and 221 variants in plasma; 66 variants were simultaneously detected in tumors and plasma. Conversely, in female dogs, 1430 variants were found in plasma and 695 variants in tumor fragments; 59 variants were simultaneously identified in tumors and plasma. The most frequently mutated genes in the tumor fragments of women were USH2A, ATM, and IGF2R; in female dogs, they were USH2A, BRCA2, and RRM2. Plasma of women showed the most frequent genetic variations in the MAP3K1, BRCA1, and GRB7 genes, whereas plasma from female dogs had variations in the NF1, ERBB2, and KRT17 genes. Mutations in the AKT1, PIK3CA, and BRIP genes were associated with tumor recurrence, with a highly pathogenic variant in PIK3CA being particularly prominent. We also detected a gain-of-function mutation in the GRB7, MAP3K1, and MLH1 genes. CONCLUSION: Liquid biopsy is useful to identify specific genetic variations at the beginning of BC manifestation and may be accompanied over the entire follow-up period, thereby supporting the clinicians in refining interventions.

Melatonin regulates tumor aggressiveness under acidosis condition in breast cancer cell lines.

Breast cancer progression is composed of multiple steps that are influenced by tumor cell adaptations to survive under acidic conditions in the tumor microenvironment. Regulation of this cell survival behavior is a promising strategy to avoid cancer development. Melatonin is a natural hormone produced and secreted by the pineal gland capable of modulating different biological pathways in cancer. Although the anti-cancer effects of melatonin are currently widespread, its role in the acid tumor microenvironment remains poorly understood. The aim of the present study was to investigate the effect of low pH (6.7) on human breast cancer cell lines MCF-7 and MDA-MB-231, and the effectiveness of melatonin in acute acidosis survival mechanisms. Cell viability was measured by a MTT assay and the protein expression of glucose transporter (GLUT)-1, Ki-67 and caspase-3 was evaluated by immunocytochemical (ICC) analysis following low pH media and melatonin treatment. In both cell lines the viability was decreased after melatonin treatment (1 mM) under acidosis conditions for 24 h. ICC analysis showed a significant increase in GLUT-1 and Ki-67 expression at pH 6.7, and a decrease after treatment with melatonin for 12 and 24 h. The low pH media decreased the expression of caspase-3, which was increased after melatonin treatment for 12 and 24 h. Overall, the results of the present study revealed melatonin treatment increases apoptosis, as indicated by changes in caspase-3, and decreases proliferation, indicated by changes to Ki-67, and GLUT-1 protein expression under acute acidosis conditions in breast cancer cell lines.