Transitional cell carcinoma (TCC) accounts for > 90% of canine malignant tumors occurring in urinary bladder, and the prognosis is poor. Our previous study, using RNA sequencing, showed that the oncogene human epidermal growth factor 2 (HER2) was the significantly activated gene pathway related to carcinogenesis in canine TCC. However, information on HER2 in canine TCC is lacking. The aims of this study were to examine the antitumor effect of lapatinib, a tyrosine kinase inhibitor of HER2, on canine TCC cell lines in vitro and in vivo and to assess HER2 protein expression in dogs with TCC.
Five canine TCC cell lines (TCCUB, Love, Sora, LTCC, and CTCC) were used. Cells were incubated with lapatinib. Expression and phosphorylation of downstream molecules in the HER2 signaling pathway were then analyzed by western blotting. Cell proliferation, cell cycle, and apoptosis were examined by methylthiazolyl tetrazolium assay, flow cytometry, and TdT-mediated dUTP nick end labeling (TUNEL) method, respectively. For the in vivo experiments, the canine TCC cells were subcutaneously injected into nude mice. Lapatinib or vehicle were administered daily via intraperitoneal administration for 14 days. At the endpoint, the mice were euthanized, and the tumors were collected. Histologically, necrotic areas in the tumor tissues were evaluated. Finally, immunohistochemistry of HER2 was performed using urinary bladder tissues of 23 dogs with TCC and 8 healthy dogs. Intensive staining localized to cell membrane was defined as HER2 protein overexpression.
HER2 protein expression was observed in all of the canine TCC cell lines. Lapatinib inhibited phosphorylation of HER2 and the downstream molecules and cell proliferation in a dose-dependent manner. Cell cycle analyses showed that lapatinib significantly increased the sub-G1 and G0/G1 phase fractions and significantly decreased the S and G2/M phase fractions in the cells. TUNEL staining showed few apoptotic cells after treatment with lapatinib. Six to fourteen days after treatment, tumor volumes of the canine TCC-engrafted mice were significantly smaller in the lapatinib group compared to the vehicle control group. Lapatinib treatment significantly increased the necrotic areas in the tumor tissues. Immunohistochemistry of HER2 showed that HER2 protein overexpression was observed in 14/23 (61%) dogs with TCC but not in the healthy dogs.
This study demonstrated that the antitumor effects of lapatinib on the canine TCC cell lines by inhibiting HER2 signaling and inducing cell cycle arrest. Importantly, lapatinib also exerted the antitumor effects on the canine TCC-engrafted mice. In addition, HER2 protein was overexpressed in more than half of the dogs with TCC. These results suggest that lapatinib has therapeutic potential for dogs with TCC.
Graduate School of Agricultural and Life Sciences, The University of Tokyo
Thursday, June 14
4:45 PM – 5:00 PM
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