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Establishment of Canine Transitional Cell Carcinoma Cell Lines Harboring BRAF V595E Mutation as a Therapeutic Target.

ABSTRACT: Transitional cell carcinoma (TCC) is the most common malignant tumor of the canine urinary tract and tends to have a poor prognosis due to its invasive potential. Recent studies have reported that up to 80% of canine urothelial carcinoma has the BRAF V595E mutation, which is homologous to the human V600E mutation. Activating the BRAF mutation is an actionable target for developing effective therapeutic agents inhibiting the BRAF/mitogen-activated protein kinase (MAPK) pathway in canine cancer as well as human cancer. We established novel canine TCC cell lines from two tumor tissues and one metastatic lymph node of canine TCC patients harboring the BRAF V595E mutation. Tumor tissues highly expressed the BRAF mutant and phosphorylated extracellular signal-related kinases (ERK)1/2 proteins. The derived cell lines demonstrated activated MAPK pathways. We also evaluated the cell lines for sensitivity to BRAF inhibitors. Sorafenib, a multiple kinase inhibitor targeting RAF/vascular endothelial growth factor receptor (VEGFR), successfully inhibited the BRAF/MAPK pathway and induced apoptosis. The established canine TCC cell lines responded with greater sensitivity to sorafenib than to vemurafenib, which is known as a specific BRAF inhibitor in human cancer. Our results demonstrated that canine TCC cells showed different responses compared to human cancer with the BRAF V600E mutation. These cell lines would be valuable research materials to develop therapeutic strategies for canine TCC patients.

SUBMITTER: Jung H

PROVIDER: S-EPMC8430554 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The presence of BRAF variant V595E, as well as an increased cyclooxygenase-2 (COX-2) expression in canine transitional cell carcinoma (TCC) are well-described in the literature. The aim of the present study was to investigate the correlation between breed (terrier versus non-terrier dogs), histological grade, COX-2 expression, and BRAF mutation in canine TCC. Therefore, transmural TCC biopsies from 65 dogs (15 terriers, 50 non-terriers) were graded histologically into low- and high-grade. Immunohistochemical evaluation of the intensity of COX-2 expression was performed using an immunoreactive score (IRS). Exon 15 of chromosome 16 was examined for the BRAF variant c.1799T>A by TaqMan® SNP assay. TCC was low-grade in 20 cases (one terrier, 19 non-terriers) and high-grade in 45 cases (14 terriers, 31 non-terriers). Contrary to humans, histological grade was not significantly correlated to the intensity of COX-2 expression. BRAF mutation was detected in 11/15 (73%) TCC of terriers and in 18/50 (36%) TCC of non-terriers. Histological grade and BRAF mutation were not correlated significantly (p = 0.2912). Terriers had a considerably higher prevalence of high-grade tumors (p < 0.0001), as well as of BRAF mutation (p ≤ 0.05) compared to non-terriers. In non-terriers, neoplasms with BRAF mutation showed a significantly higher intensity of COX-2 expression than those without BRAF mutation (p ≤ 0.05). In conclusion, in contrast to humans, testing for BRAF mutation in canine TCC is a sensitive diagnostic method especially in terriers (73%) and may be recommended as a screening test. However, evidence of BRAF mutation in canine TCC is not a predictor for the histological grade. Moreover, a positive correlation between histological grade and the intensity of COX-2 expression was not found. Further studies are necessary to clarify the clinical and prognostic relevance of the elevated intensity of COX-2 expression of TCC with BRAF mutation detected in non-terriers.

Project description:Background: Canine prostate adenocarcinoma (PAC) and transitional cell carcinoma (TCC) are typically characterized by metastasis and chemoresistance. Cell lines are important model systems for developing new therapeutic strategies. However, as they adapt to culturing conditions and undergo clonal selection, they can diverge from the tissue from which they were originally derived. Therefore, a comprehensive characterization of cell lines and their original tissues is paramount. Methods: This study compared the transcriptomes of nine canine cell lines derived from PAC, PAC metastasis and TCC to their respective original primary tumor or metastasis tissues. Special interests were laid on cell culture-related differences, epithelial to mesenchymal transition (EMT), the prostate and bladder cancer pathways, therapeutic targets in the PI3K-AKT signaling pathway and genes correlated with chemoresistance towards doxorubicin and carboplatin. Results: Independent analyses for PAC, PAC metastasis and TCC revealed 1,743, 3,941 and 463 genes, respectively, differentially expressed in the cell lines relative to their original tissues (DEGs). While genes associated with tumor microenvironment were mostly downregulated in the cell lines, patient-specific EMT features were conserved. Furthermore, examination of the prostate and bladder cancer pathways revealed extensive concordance between cell lines and tissues. Interestingly, all cell lines preserved downstream PI3K-AKT signaling, but each featured a unique therapeutic target signature. Additionally, resistance towards doxorubicin was associated with G2/M cell cycle transition and cell membrane biosynthesis, while carboplatin resistance correlated with histone, m- and tRNA processing. Conclusions: Comparative whole-transcriptome profiling of cell lines and their original tissues identifies models with conserved therapeutic target expression. Moreover, it is useful for selecting suitable negative controls, i.e., cell lines lacking therapeutic target expression, increasing the transfer efficiency from in vitro to primary neoplasias for new therapeutic protocols. In summary, the dataset presented here constitutes a rich resource for canine prostate and bladder cancer research.

Project description:Canine prostate adenocarcinoma (PAC) and transitional cell carcinoma (TCC) of prostate and urinary bladder are highly invasive and metastatic tumors of closely neighbored organs. Cell lines are valuable tools to investigate tumor mechanisms and therapeutic approaches in vitro. PAC in dogs is infrequent, difficult to differentiate from TCC and usually characterized by poor prognosis, enhancing the value of the few available cell lines. However, as cell lines adapt to culturing conditions, a thorough characterization, ideally compared to original tissue, is indispensable. Herein, six canine PAC cell lines and three TCC cell lines were profiled by immunophenotype in comparison to respective original tumor tissues. Three of the six PAC cell lines were derived from primary tumor and metastases of the same patient. Further, two of the three TCC cell lines were derived from TCCs invading into or originating from the prostate. Cell biologic parameters as doubling times and chemoresistances to commonly used drugs in cancer treatment (doxorubicin, carboplatin and meloxicam) were assessed. All cell lines were immunohistochemically close to the respective original tissue. Compared to primary tumor cell lines, metastasis-derived cell lines were more chemoresistant to doxorubicin, but equally susceptive to carboplatin treatment. Two cell lines were multiresistant. COX-2 enzyme activity was demonstrated in all cell lines. However, meloxicam inhibited prostaglandin E2 production in only seven of nine cell lines and did neither influence metabolic activity, nor proliferation. The characterized nine cell lines represent excellent tools to investigate PAC as well as TCC in prostate and urinary bladder of the dog. Furthermore, the profiled paired cell lines from PAC primary tumor and metastasis provide the unique opportunity to investigate metastasis-associated changes PAC cells undergo in tumor progression. The combination of nine differently chemoresistant PAC and TCC cell lines resembles the heterogeneity of canine lower urinary tract cancer.

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Establishment of Canine Transitional Cell Carcinoma Cell Lines Harboring BRAF V595E Mutation as a Therapeutic Target.

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