Tyrosine Kinase Inhibitors Are Promising Therapeutic Tools for Cats with HER2-Positive Mammary Carcinoma

Abstract

Feline mammary carcinoma (FMC) is a common neoplasia in cat, being HER2-positive the most prevalent subtype. In woman's breast cancer, tyrosine kinase inhibitors (TKi) are used as a therapeutic option, by blocking the phosphorylation of the HER2 tyrosine kinase domain. Moreover, clinical trials demonstrated that TKi produce synergistic antiproliferative effects in combination with mTOR inhibitors, overcoming resistance to therapy. Thus, to uncover new chemotherapeutic strategies for cats, the antiproliferative effects of two TKi (lapatinib and neratinib), and their combination with a mTOR inhibitor (rapamycin), were evaluated in FMC cell lines (CAT-M, FMCp and FMCm) and compared with a human breast cancer cell line (SkBR-3). Results revealed that both TKi induced antiproliferative effects in all feline cell lines, by blocking the phosphorylation of EGFR members and its downstream effectors. Furthermore, combined treatments with rapamycin presented synergetic antiproliferative effects. Additionally, the DNA sequence of the her2 TK domain (exons 18 to 20) was determined in 40 FMC tissue samples, and despite several mutations were found none of them were described as inducing resistance to therapy. Altogether, our results demonstrated that TKi and combined protocols may be useful in the treatment of cats with mammary carcinomas, and that TKi-resistant FMC are rare.

Keywords: HER2; feline her2 TK mutations; feline mammary carcinoma; targeted therapies; tyrosine kinase inhibitors.

Figure 1

Lapatinib and neratinib showed strong cytotoxic effects in feline mammary carcinoma cell lines. (A) CAT-M cell line presented higher cytotoxic effects when incubated with lapatinib (IC₅₀ = 3930 nM ± 49) than with neratinib. (B) FMCp cell line was susceptible to both lapatinib and neratinib (100% and 74.9% of cytotoxicity, respectively; IC₅₀ = 4870 nM ± 100 for lapatinib). (C) FMCm cell line also showed high cytotoxic effects in the presence of lapatinib (IC₅₀ = 17,470 nM ± 100), contrasting with the neratinib. (D) SkBR-3 cells showed a maximum cytotoxic effect of 91.1% (IC₅₀ = 16,220 nM ± 1040) after exposure to lapatinib and 66.0% when exposed to neratinib. For graphical convenience, lapatinib was represented in a µM range, while neratinib was defined in a nM range. The experiments were performed in triplicates, in three independent assays.

Figure 2

Feline cell lines presented low HER2 expression, showing altered phosphorylation levels of HER2 and its downstream effectors after TKi exposure. (A) CAT-M and FMCm cell lines presented low HER2 expression levels, with no expression detected in the FMCp cell line. (B) After exposure to lapatinib, the CAT-M cell line showed decreased phosphorylation levels of the HER1 (pY1173), HER2 (pY1221 + Y1222) and of its downstream effectors, AKT1 (pS473) and ERK1/2 (pT202/pY204 + pT185/pY187), coupled in an increased of PTEN phosphorylation at Thr366. Results were less perceptible when cells were exposed to neratinib. (C) TKi exposure, particularly, lapatinib, increased HER2 levels not only in CAT-M cells, but also in (D) FMCp cells. β-actin was used as loading control.

Figure 3

Rapamycin showed small cytotoxicity in all tested tumor cell lines, although it lead to a decrease in the phosphorylation levels of mTOR at S2448. (A) The most susceptible cell line to rapamycin exposure was FMCp (43.4% of cytotoxicity) followed by the FMCm (41.3% of cytotoxicity) and the CAT-M cell lines (38.7% of cytotoxicity). The human SkBR-3 cell line was used as control, presenting a maximum of 29.6% of cytotoxicity. The experiments were performed in triplicates and repeated three times. (B) Rapamycin reduces the phosphorylation levels of the mTOR at S2448. β-actin was used as loading control.

Figure 4

Combinations of TKi and mTOR inhibitor (lapatinib plus rapamycin and neratinib plus rapamycin) showed synergistic antiproliferative effects in feline mammary tumor cell lines. (A) CAT-M cells presented a significantly higher antiproliferative response when exposed to lapatinib at 3125 nM plus rapamycin at 6.25 nM or incubated with neratinib at 12.5 nM plus rapamycin at 6.25 nM (57.5% of cytotoxicity, * p < 0.05; 49.9% of cytotoxicity, ** p < 0.01, respectively). (B) FMCp cells showed significant synergistic effects of conjugations between lapatinib at 780 nM plus rapamycin at 0.196 nM or at 6.25 nM (45.5% of cytotoxicity, ** p < 0.01; 54.4% of cytotoxicity, *** p < 0.001, respectively), and when neratinib at 3.125 nM plus rapamycin at 0.196 nM or at 6.25 nM were used (32.1% of cytotoxicity, * p < 0.05; 44.5% of cytotoxicity, ** p < 0.01, respectively). (C) FMCm cells presented an increased cytotoxic response to the conjugations of lapatinib at 22,650 nM plus rapamycin at 0.196 nM or at 6.25 nM (42.6% of cytotoxicity, ** p < 0.01; 95.2% of cytotoxicity, *** p < 0.001, respectively) and neratinib at 25 nM plus rapamycin at 0.196 nM or at 6.25 nM (67.7% of cytotoxicity, ** p < 0.01; 76.3% of cytotoxicity, *** p < 0.001, respectively). (D) SkBR-3 cells used as control, displayed similar antiproliferative responses to feline cell lines. All the assays were performed in triplicate and repeated a total of three separated times.

Figure 5

Mutations identified in feline her2 TK domain are not reported to induce therapeutic resistance in woman breast cancer. Most of the detected mutations were localized at intronic regions, although some of them were identified in exons, as a very frequent mutation in the exon 22 (c.20940 T > G; 85%; 34/40). Regarding the exon 20 that encodes for the protein region recognized by TKi, none of the seven identified mutations were reported as being related to therapeutic resistance in woman breast cancer.

Figure 6

A significant positive correlation was found between the tumor size and the presence of c.19573A > T mutation in exon 18 (* p = 0.045). The mutation c.19573A > T was associated to larger tumor sizes (4.13 cm ± 1.44 vs. 2.64 cm ± 1.13).