Phosphorylation of mTOR Ser2481 is a key target limiting the efficacy of rapalogs for treating hepatocellular carcinoma - PubMed (original) (raw)

Phosphorylation of mTOR Ser2481 is a key target limiting the efficacy of rapalogs for treating hepatocellular carcinoma

Kosuke Watari et al. Oncotarget. 2016.

Abstract

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. Although recent studies facilitate the identification of crucial genes and relevant regulatory pathways, therapeutic approaches against advanced HCC are insufficiently effective. Therefore, we aimed here to develop potent therapeutics to provide a reliable biomarker for the therapeutic efficacy in patients with HCC. To this end, we first compared the cytotoxic effects of various anti-cancer drugs between well differentiated (HAK-1A) and poorly differentiated (HAK-1B) cell lines established from a single HCC tumor. Of various drug screened, HAK-1B cells were more sensitive by a factor of 2,000 to the mTORC1 inhibitors (rapalogs), rapamycin and everolimus, than HAK-1A cells. Although rapalogs inhibited phosphorylation of mTOR Ser2448 in HAK-1A and HAK-1B cells, phosphorylation of mTOR Ser2481 was specifically inhibited only in HAK-1B cells. Silencing of Raptor induced apoptosis and inhibited the growth of only HAK-1B cells. Further, three other cell lines established independently from the tumors of three patients with HCC were also approximately 2,000-fold times more sensitive to rapamycin, which correlated closely with the inhibition of mTOR Ser2481 phosphorylation by rapamycin. Treatment with everolimus markedly inhibited the growth of tumors induced by poorly differentiated HAK-1B and KYN-2 cells and phosphorylation of mTOR Ser2481 in vivo. To our knowledge, this is the first study showing that the phosphorylation of mTOR Ser2481 is selectively inhibited by rapalogs in mTORC1-addicted HCC cells and may be a potential reliable biomarker for the therapeutic efficacy of rapalogs for treating HCC patients.

Keywords: Raptor; hepatocellular carcinoma; mTOR Ser2481; mTORC1; rapalogs.

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Conflict of interest statement

The authors declare that no conflicts of interest exist.

Figures

Figure 1

Figure 1. Comparison of the biological and biochemical characteristics of HAK-1A and HAK-1B cells

(A) Morphology of HCC cell lines in culture. HAK-1A shows cobblestone-like morphology, and HAK-1B shows a fibroblastic morphology when cultured in plastic dishes. A single HCC tumor showing a nodule-in-nodule appearance. The well differentiated HAK-1A and poorly differentiated HAK-1B cell lines were derived from the outer and inner nodules of the same tumor, respectively. (B, C) Comparison of cell proliferation rates in vitro (B), and tumor growth rates on days 30 and 50 in nude mice (C) engrafted with HAK-1A and HAK-1B cells (n = 3). Each bar is the average ± standard deviation (SD). (D) Comparison of colony formation under “Matrigel on top” culture conditions between HAK-1A and HAK-1B cells. Representative images of colonies of HAK-1A and HAK-1B cells incubated for 5 days (upper panel). The number of colonies > 50 μm (lower panel) (n = 3). Each bar is the average ± standard deviation (SD), *P < 0.05 (two-tailed Student t test). (original magnification ×40) (E) Comparison of invasion of Matrigel between HAK-1A and HAK-1B cells. Representative images of invaded cells incubated for 24 hr (upper panel), and the number of invaded cells (lower panel) (n = 3). Each bar is an average ± SD, *P < 0.05 (two-tailed Student t test). (original magnification ×40) (F) Comparison of expression levels of NDRG1 and growth factor receptors in HAK-1A and HAK-1B. β-actin served as loading control. (G) Comparison of the expression of downstream effectors in HAK-1A and HAK-1B cells. GAPDH served as loading control.

Figure 2

Figure 2. The effect of mTOR inhibitors on the activation of mTOR-related signaling molecules in HAK-1A and HAK-1B cells

(A) Sensitivity to mTOR inhibitors of HAK-1A and HAK-1B cells. Cells were exposed to various concentrations of the indicated drugs for 72 hr, and drug sensitivity was determined using a WST assay. Each value is the average ± SD of triplicate wells, and each percentage value was calculated by normalizing the raw data to that of the control without drugs (100%). (B) The effect of selective mTORC1 inhibitors on the expression and phosphorylation of mTOR and its related signaling molecules. Cells were treated with each inhibitor at the indicated concentrations for 6 hr, and lysates were analyzed using western blotting. The lower panel shows the quantification of expression levels of p-mTOR (Ser2448 and 2481) and p-AKT Ser473 normalized to the value in the absence of drugs. GAPDH served as loading control. (C) The effect of 6 hr treatment with AZD8055 on the expression and phosphorylation of mTOR and its related signaling molecules. The lower panel shows the relative expression levels of p-mTOR (Ser2448 and Ser2481) and p-AKT Ser473 normalized to the value in the absence of drugs. GAPDH served as loading control. (D) Kinetics of the effect of rapamycin (1 nM) on the expression and phosphorylation of mTOR and its related signaling molecules. In the lower panel, expression levels of p-mTOR (S2448 and S2481) and p-AKT Ser473 are presented as their values normalized to the value at time 0. GAPDH served as loading control. (E) Kinetics of the effect of AZD8055 (20 nM) on the expression and phosphorylation of mTOR and its related signaling molecules. In the lower panel, expression levels of p-mTOR (Ser2448 and Ser2481) and p-AKT Ser473 were normalized to the value at time 0. GAPDH served as loading control.

Figure 3

Figure 3. The effect of knockdown of Raptor or Rictor on mTOR and AKT phosphrylation in HAK-1A and HAK-1B cells

(A) Association of Raptor with mTOR in the absence or presence of rapamycin. Cells were treated with rapamycin at the indicated concentrations for 6 hr, and lysates were analyzed using an immunoprecipitation assay. The immunoprecipitates were subjected to western blot analysis using the indicated antibodies against mTOR or Raptor. GAPDH served as loading control. (B) The effect of Raptor or Rictor knockdown on cell growth. Cells were incubated with siRNAs for 1 and 3 days. Each bar is an average ± SD of triplicate wells, **P < 0.01 (two-tailed Student t test). (C) The effect of Raptor knockdown on the expression of cleaved-PARP. Cells were incubated with siRNA for ≤ 3 days. GAPDH served as loading control. (D, E) The effect of Raptor (D) or Rictor (E) knockdown on the phosphorylation of mTOR, S6K, and AKT. Cells were incubated with siRNA for 1 and 3 days. In the lower panels, the expression levels of p-mTOR (Ser2481) and p-AKT (Ser473) in siRNAs-treated HAK-1A and HAK-1B cells are normalized to their values on day 0. GAPDH served as loading control.

Figure 4

Figure 4. Hypersensitivity to rapamycin and mTORC1-addicted growth of other human HCC cell lines

(A, B) The sensitivities of eight human HCC cell lines to rapamycin (A) and AZD8055 (B) were assessed using a WST assay. Each bar is the average ± SD of triplicate wells. (C) Comparison of the expression and phosphorylation of mTOR and its related signaling molecules among eight human HCC cell lines, including HAK-1A and HAK-1B cells. GAPDH served as loading control. (D) The effect of rapamycin on the phosphorylation of mTOR and S6K in six cell lines. Cells were treated with rapamycin at indicated concentration for 6 hr, and lysates were analyzed using western blotting. In the lower panel, expression levels of p-mTOR (Ser2481) are normalized to the values in the absence of drugs. GAPDH served as loading control. (E) The effect of Raptor knockdown on cell growth. Cells were incubated with a _Raptor_-siRNA for < 5 days. Each bar is the average ± SD of triplicate wells, *P < 0.05; **P < 0.01 (two-tailed Student t test).

Figure 5

Figure 5. Effect of everolimus on tumor growth and activation of mTOR-related signaling molecules in vivo

(A, B) Antitumor effects of everolimus on the growth of HAK-1B (A) and KYN-2 (B) xenografts. Mice were inoculated subcutaneously with HCC cells, and mice with tumors >100 mm3 were treated orally with everolimus (2 mg/kg/day) or a control CMC daily from day 0 until days 35 or 14 (n = 5 mice per each group). Each bar is the average ± SD, *P < 0.05; **P < 0.01 (two-tailed Student t test). (C, D) Effects of everolimus on S6 phosphorylation in tumors formed by HAK-1B (C) or KYN-2 (D) cells analyzed on days 35 or 14 using immunohistochemistry with an anti-p-S6 antibody. Three tumors of the control and treated groups are shown. (original magnification ×100) (E) Inhibitory effects of everolimus on the activation of mTOR-related signaling molecules in tumors. Western blot analysis of the expression of phosphorylated mTOR, S6K, and S6 in five tumors treated with or without everolimus in vivo. α-tubulin served as loading control.

Figure 6

Figure 6. Hypothetical model of the acquisition of hypersensitivity to rapalogs by HCC cells

(A) Normally, PI3K/AKT activation is under feedback regulation by mTORC1/S6K, such as in well differentiated HCC cells (e.g. HAK-1A cells). Rapalogs inhibit feedback suppression by mTORC1/S6K, possibly through drug-induced inhibition of the phosphorylation of mTOR Ser2448, which is accompanied by PI3K/AKT activation. Rapalogs do not affect the phosphorylation mTOR Ser2481 (mTORC1 complex), and the cells survive in the presence of drugs. (B) In contrast, PI3K/AKT is constitutively activated in poorly differentiated HCC cells (e.g. HAK-1B cells), and feedback control of PI3K/AKT by mTORC1 is dysregulated in this cell line. Rapalogs block the phosphorylation of both mTORC1 Ser2448 and Ser2481 that induces the death of HAK-1B cells through suppression of mTORC1-dependent cell growth, survival, and metabolism.

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