落合 和彦

Mammary tumors are the most prevalent type of tumors in female dogs. Breast cancer 2, early onset (BRCA2) malignant mutations are associated with tumorigenesis in humans and dogs. BRCA2 plays a pivotal role in homologous recombination repair by recruiting RAD51 recombinase to DNA damage sites to maintain genome stability. To recruit RAD51, BRCA2 must interact with RAD51 via BRC repeats, but the regulation of this interaction has been unclear. In this study, we focused on a highly conserved region (HCR) near BRC repeats. Using co-immunoprecipitation and mammalian two-hybrid assay, we found that HCR suppressed the RAD51-interaction activity of BRC repeats and that substitutions of HCR phosphorylation sites affected it. In canine tumor samples, we found ten mutations, including a novel HCR mutation (I1110M) from canine tumor samples. The effect of four HCR mutations, including I1110M, on the RAD51-interaction activity of BRC repeats was tested. One of the HCR mutations found in canine mammary tumors increased the interaction, but the two mutations found in human breast cancers decreased it. This study suggested that the HCR regulated the RAD51-interacting activity of BRC repeats through HCR phosphorylation and that mutations in HCR may be related to tumorigenesis in both dogs and humans.

Canine prostate cancer (cPCa) is a malignant neoplasm with no effective therapy. The BRAF V595E mutation, corresponding to the human BRAF V600E mutation, is found frequently in cPCa. Activating BRAF mutations are recognized as oncogenic drivers, and blockade of MAPK/ERK phosphorylation may be an effective therapeutic target against BRAF-mutated tumors. The aim of this study was to establish a novel cPCa cell line and to clarify the antitumor effects of MEK inhibitors on cPCa in vitro and in vivo. We established the novel CHP-2 cPCa cell line that was derived from the prostatic tissue of a cPCa patient. Sequencing of the canine BRAF gene in two cPCa cell lines revealed the presence of the BRAF V595E mutation. MEK inhibitors (trametinib, cobimetinib, and mirdametinib) strongly suppressed cell proliferation in vitro, and trametinib showed the highest efficacy against cPCa cells with minimal cytotoxicity to non-cancer COPK cells. Furthermore, we orally administered 0.3 or 1.0 mg/kg trametinib to CHP-2 xenografted mice and examined its antitumor effects in vivo. Trametinib reduced tumor volume, decreased phosphorylated ERK levels, and lowered Ki-67 expression in xenografts in a dose-dependent manner. Although no clear adverse events were observed with administration, trametinib-treated xenografts showed osteogenesis that was independent of dosage. Our results indicate that trametinib induces cell cycle arrest by inhibiting ERK activation, resulting in cPCa tumor regression in a dose-dependent manner. MEK inhibitors, in addition to BRAF inhibitors, may be a targeted agent option for cPCa with the BRAF V595E mutation. This article is protected by copyright. All rights reserved.

Mammary adenocarcinoma, the most common cancer in female dogs, often exhibits the lymph node and lung metastases and has a higher mortality rate. However, mammary adenocarcinoma has no established treatment, except early surgical excision. Canine mammary carcinoma has many common features with human mammary carcinoma, including clinical characteristics, heterogeneity, and genetic aberrations, making it an excellent spontaneous tumor model for human breast cancer. Diverse cancers comprised heterogeneous cell populations originating from cancer stem cells (CSCs) with self-renewal ability. Therefore, in addition to conventional therapy, therapeutic strategies targeting CSCs are essential for cancer eradication. The present study aimed to extract inhibitors of canine mammary CSCs that suppress their self-renewal ability. Sphere-formation assay, which evaluates self-renewal ability, was performed for the canine mammary cancer cell lines CTBp and CNMp. The spheres formed in this assay were used in inhibitor library screening, which identified various signaling pathways such as proteosome, stress inducer, and mammalian target of rapamycin (mTOR). The present study focused on the mTOR signaling pathway. Western blotting showed higher levels of phosphorylated mTOR in sphere-forming CTBp and CNMp cells than in adherent cells. Drug sensitivity examination using the mTOR inhibitors everolimus and temsirolimus revealed dose-dependent reductions in viability among both sphere-forming cells and adherent cells. Expression of phosphorylated mTOR in adherent and sphere-forming cells decreased by everolimus and temsirolimus treatment. In mice transplanted with CTBp-derived spheres, everolimus treatment significantly decreased tumor volume compared to control. These results reveal that the mTOR signaling pathway may be a potential to be a therapeutic target in both cancer cells and CSCs. Novel therapeutic strategies for canine mammary carcinoma are expected to benefit to human breast carcinoma as well.

Due to the high incidence of mammary tumors in dogs, it is important to elucidate the pathogenesis of these tumors in veterinary medicine. Radiation therapy is often used to treat mammary tumors that target DNA lesions. RAD51 is a key molecule that repairs DNA damage via homologous recombination. We examined the relationship between RAD51 expression and radiosensitivity in mammary tumor cell lines. CHMp and CHMm from the same individual were selected based on the differences in RAD51 expression. The radiosensitivity of both cell lines was examined using MTT and scratch assays; CHMm, which has high RAD51 expression, showed higher sensitivity to radiation than CHMp. However, the nuclear focus of RAD51 during DNA repair was formed normally in CHMp, but not in most of CHMm. Since irradiation resulted in the suppression of cell cycle progression in CHMp, the expression of p21, a cell cycle regulatory factor, was detected in CHMp after 15 Gy irradiation but not in CHMm. These results indicate that functional expression is more important than the quantitative expression of RAD51 in canine mammary tumor cells in response to DNA damage.