Chemical Oral Cancerogenesis Is Impaired in PI3Kγ Knockout and Kinase-Dead Mice

Abstract

We investigated the role of PI3Kγ in oral carcinogenesis by using a murine model of oral squamous carcinoma generated by exposure to 4-nitroquinoline 1-oxide (4NQO) and the continuous human cancer cell line HSC-2 and Cal-27. PI3Kγ knockout (not expressing PI3Kγ), PI3Kγ kinase-dead (carrying a mutation in the PI3Kγ gene causing loss of kinase activity) and wild-type (WT) C57Bl/6 mice were administered 4NQO via drinking water to induce oral carcinomas. At sacrifice, lesions were histologically examined and stained for prognostic tumoral markers (EGFR, Neu, cKit, Ki67) and inflammatory infiltrate (CD3, CD4, CD8, CD19 and CD68). Prevalence and incidence of preneoplastic and exophytic lesions were significantly and similarly delayed in both transgenic mice versus the control. The expression of prognostic markers, as well as CD19⁺ and CD68⁺ cells, was higher in WT, while T lymphocytes were more abundant in tongues isolated from transgenic mice. HSC-2 and Cal-27 cells were cultured in the presence of the specific PI3Kγ-inhibitor (IPI-549) which significantly impaired cell vitality in a dose-dependent manner, as shown by the MTT test. Here, we highlighted two different mechanisms, namely the modulation of the tumor-infiltrating cells and the direct inhibition of cancer-cell proliferation, which might impair oral cancerogenesis in the absence/inhibition of PI3Kγ.

Keywords: 4NQO; PI3Kγ; chemical carcinogenesis; oral squamous cell carcinoma; transgenic mice.

Figure 1

(A) Total lesion prevalence, (B) total lesion multiplicity, (C) OPL prevalence, (D) OPL multiplicity, (E) ExL prevalence, and (F) ExL multiplicity of WT, PI3Kγ^KD/KD and PI3Kγ^−/− mice treated with 4NQO. Values represent mean ± SEM. PI3Kγ^KD/KD/PI3Kγ^−/− vs. WT: * p < 0.05, ** p < 0.01 and *** p < 0.001.

Figure 2

Mean pathological score (as the sum of the score of every single lesion per animal) of WT, PI3Kγ^KD/KD and PI3Kγ^−/− mice receiving 4NQO treatment are shown. PI3Kγ^KD/KD/PI3Kγ^−/− vs. WT: * p < 0.05, # p < 0.01 and § p < 0.001.

Figure 3

(A) Histological analysis: global overview of WT, PI3Kγ^KD/KD and PI3Kγ^−/− mice lesions at sacrifice. (B–D) Representative H&E staining of tongue sections in (B) WT, (C) PI3Kγ^KD/KD and (D) PI3Kγ^−/− mice at the end of 4NQO treatment (22 weeks). Values represent mean ± SEM. PI3Kγ^KD/KD/PI3Kγ^−/− vs. WT: # p < 0.01. Scale bars, 100 μm.

Figure 4

(A) Western blot analysis of PI3Kγ expression in HeLa, 293T, SG, HSC-2 and Cal-27 whole cell lysates. HeLa and 293T represent, respectively, the positive and the negative control for PI3Kγ expression. (B) Residual cell vitality in presence of the PI3Kγ specific inhibitor IPI-549 (0–160 µM) as detected by MTT assay. Values, expressed as a percentage of controls, represent mean ± standard error (SEM), p are detailed in (C).