Downregulation of AKT/mTOR signaling pathway for Salmonella-mediated autophagy in human anaplastic thyroid cancer

Abstract

Thyroid cancer has been known as the most common endocrine malignancy. Although majority of thyroid cancer types respond well to conventional treatment including surgery and radioactive iodine therapy, about 10% of those with differentiated thyroid cancer will present distant metastasis and will have persistent or recurrent disease. Even more serious is a rare type of thyroid cancer called anaplastic thyroid cancer (ATC), which accounts for about 1%, has been demonstrated as the most lethal and aggressive form of human malignancy. Unfortunately, these tumors are also frequently resistant to traditional therapy. Previous study have shown that Salmonella inhibits tumor growth, in part, by inducing autophagy - a cellular process that is important in the innate and adaptive immunity in response to viral or bacterial infection. In our study, we intended to investigate whether Salmonella can inhibit tumor growth by inducing autophagy, specifically in thyroid cancer and elucidate the possible molecular mechanism. In order to determine the signaling pathway involved in tumor cell autophagy, we used Salmonella to treat ATC cells line ASH-3 and KMH-2 in vitro. The autophagic markers, particularly autophagy-related gene 6 (Beclin-1), microtubule-associated protein 1A/1B-light chain 3 (LC3) and p62, were observed to be differentially expressed after infection with Salmonella indicating an activated autophagy in ATC cells. In addition, the protein expression levels of phospho-protein kinase B (P-AKT), phospho-mammalian targets of rapamycin (P-mTOR), phospho-p70 ribosomal s6 kinase (P-p70S6K) in tumor cells were decreased after Salmonella infection. In vivo, we also found that substantial cell numbers of Salmonella targeted tumor tissue, and regulated anti-tumor mechanisms. Our findings showed that Salmonella activated autophagic signaling pathway and inhibited ATC tumor growth via downregulation of AKT/mTOR pathway.

Keywords: AKT/mTOR; Autophagy; Salmonella; anaplastic thyroid cancer.

Figure 1

Cell viability of ASH-3 and KMH-2 human tumor cells infected with Salmonella. The ASH-3 (A) and KMH-2 cells (2 × 10⁴ cells/well) (B) were seeded into 96-well plates and incubated at 37 °C for 24 hours. After treatment with various MOI of Salmonella for 90 min, each well were loaded with proliferation reagent WST-1. Then, the absorbance was read and analyze to measure the cell viability (mean ± SD, n=8). (C) For monitoring cell proliferation, ATC cells were treated with various MOI of Salmonella for 90 min. Then cells were fixed and stained for BrdU immunoreactivity and nuclei were counterstained with Hoechst 33342. The cells were counted under a fluorescence microscope (mean ± SD, n=6). (D) After treated with Salmonella (100 MOI), the ASH-3 and KMH-2 cells were stained with Annexin V and the frequency of cells were examined by flow-cytometry (mean ± SD, n=6). *** p < 0.001; ** p < 0.01.

Figure 2

Effect of Salmonella on autophagic punctate formation. The ASH-3 and KMH-2 cells (10⁶ cells/well) were placed into 6-well plates and incubated at 37 °C for 24 hours. Cells were transfected with the plasmid encoding GFP-LC3 for 6 hours and then infected with Salmonella (10⁸ cfu) or PBS for 90 min. (A) The LC3 puncta were visualized by fluorescence microscopy after treatment with Salmonella. (B) Shows the quantification of autophagosome punctate percentage of cells (mean ± SD, n=11). *** p < 0.001.

Figure 3

Salmonella induced autophagic signaling pathway. The ASH-3 (A) and KMH-2 (B) cells (5 × 10⁵ cells/well) were placed into 6-well plates and incubated at 37 °C for 24 hours and then the cells were infected with various MOI of Salmonella for 90 min. The expression level of AKT/mTOR/p70S6K proteins and autophagic marker in cells were analyzed by Western blotting. β-actin was used as an internal loading control. Quantification histograms are presented beneath each Western blotting plot. Data are expressed as the mean ± SD of hexaplicate determinations. Each experiment was repeated three times with similar results.

Figure 4

Effect of Salmonella on constitutively-AKT and autophagic pathway. The ASH-3 (A) and KMH-2 (B) cells (10⁶ cells/well) were placed into 6-well plates and incubated at 37 °C for 24 hours. The cells were transfected with control or constitutively active AKT plasmids (5ug) at 37 °C for 6 hours and then infected with 0 or 100 MOI Salmonella for 90 min. The expression level of AKT/mTOR/p70S6K proteins and autophagic marker in cells were analyzed by Western blotting. β-actin was used as an internal loading control. Quantification histograms are presented beneath each Western blotting plot. Data are expressed as the mean ± SD of hexaplicate determinations. Each experiment was repeated three times with similar results.

Figure 5

Salmonella (S.C.) targeting tumor potential and induction of tumor autophagy in vivo. (A) The mice bearing ASH-3 were injected intraperitoneally with Salmonella (2×10⁶ cfu) at day 0. The mice were sacrificed at day 7, 14, 21, and 28 to calculate the amounts of Salmonella in the tumor, liver and spleen. (B) The mice bearing KMH-2 were injected intraperitoneally with Salmonella (2×10⁶ cfu) at day 0. The mice were sacrificed at day 7 and 14 to calculate the amounts of Salmonella in the tumor, liver and spleen. Each value represents mean ± SD from 3 mice. (C) Tumors were excised on day 14 and the protein expression of autophagic markers in tumor cells derived from Salmonella-treated mice or control mice was determined by Western blotting. Inserted values indicated relative protein expressions in comparison with β-actin. Tumor were excised at day 7 (D) and day 14 (E), the expression of P-AKT, P-mTOR, F4/80, Ly-6G, PCNA, and LC3-II were detected by immunohistochemistry. Scale bar = 30 µm (200X).

Figure 5

Salmonella (S.C.) targeting tumor potential and induction of tumor autophagy in vivo. (A) The mice bearing ASH-3 were injected intraperitoneally with Salmonella (2×10⁶ cfu) at day 0. The mice were sacrificed at day 7, 14, 21, and 28 to calculate the amounts of Salmonella in the tumor, liver and spleen. (B) The mice bearing KMH-2 were injected intraperitoneally with Salmonella (2×10⁶ cfu) at day 0. The mice were sacrificed at day 7 and 14 to calculate the amounts of Salmonella in the tumor, liver and spleen. Each value represents mean ± SD from 3 mice. (C) Tumors were excised on day 14 and the protein expression of autophagic markers in tumor cells derived from Salmonella-treated mice or control mice was determined by Western blotting. Inserted values indicated relative protein expressions in comparison with β-actin. Tumor were excised at day 7 (D) and day 14 (E), the expression of P-AKT, P-mTOR, F4/80, Ly-6G, PCNA, and LC3-II were detected by immunohistochemistry. Scale bar = 30 µm (200X).

Figure 6

Antitumor effects of Salmonella on mice bearing ATC. BALB/c nude mice were inoculated subcutaneously ASH-3 (10⁷) at day 0 and intraperitoneal injected Salmonella (2×10⁶ cfu) or PBS at day 7. (A) The body weight (mean ± SD, n = 10) were compared with PBS group. (B) Tumor volumes (mean ± SD, n = 10) in mice bearing ASH-3 tumors were compared with PBS group. *P < 0.05; **P < 0.01. (C) Kaplan-Meier survival curves of ASH-3 bearing mice with Salmonella or PBS are shown (p < 0.01 for mice bearing ASH-3 treated with Salmonella versus PBS).