Autophagy induction contributes to GDC-0349 resistance in head and neck squamous cell carcinoma (HNSCC) cells

https://doi.org/10.1016/j.bbrc.2016.06.039Get rights and content

Highlights

  • GDC-0349 inhibits proliferation of HNSCC cells bearing high-level of p-AKT/p-S6K.

  • GDC-0349 activates caspase-dependent apoptosis in HNSCC cells.

  • Simultaneous blockage of mTORC1/2 by GDC-0349 induces autophagy activation.

  • Autophagy inhibitor or ATG-7 siRNA potentiates GDC-0349’s cytotoxicity.

  • C14 ceramide downregulates ATG-7 and sensitizes HNSCC cells to GDC-0349.

Abstract

Dysregulation of mammalian target of rapamycin (mTOR) signaling contributes to head and neck squamous cell carcinoma (HNSCC) tumorigenesis and progression. In the current study, we tested the anti-HNSCC cell activity by GDC-0349, a selective ATP-competitive inhibitor of mTOR. We showed that GDC-0349 inhibited proliferation of established and primary human HNSCC cells bearing high-level of p-AKT/p-S6K. Further, it induced caspase-dependent apoptosis in the HNSCC cells. GDC-0349 blocked mTORC1 and mTORC2 activation, yet it simultaneously induced autophagy activation in HNSCC cells. The latter was evidenced by induction of LC3B-II, Beclin-1 and Autophagy-related (ATG)-7, as well as downregulation of p62. Autophagy inhibitors (3-methyladenine and bafilomycin A1) or ATG-7 siRNA dramatically potentiated GDC-0349’s cytotoxicity against HNSCC cells. Intriguingly, we showed that ceramide (C14), a pro-apoptotic sphingolipid, also induced ATG-7 degradation, and sensitized HNSCC cells to GDC-0349. Collectively, the preclinical study provided evidences to support GDC-0349 as a promising anti-HNSCC agent. GDC-0349 sensitization may be achieved via autophagy inhibition.

Introduction

Our research focus [1] has been on identifying novel oncotargets of head and neck squamous cell carcinoma (HNSCC), and to develop strategies against the targets [1], [2], [3], [4]. Dysregulated mammalian target of rapamycin (mTOR) signaling is observed in many HNSCCs [5], [6], [7], which contributes to tumorigenesis and regulates cancer cell transformation, proliferation and metastasis [5], [6], [7], [8]. At least two distinct multiple-protein mTOR complexes have been characterized thus far, including the rapamycin-sensitive mTOR complex 1 (mTORC1) and later-discovered mTOR complex 2 (mTORC2) [9]. mTORC1 is assembled with mTOR, Raptor, mLST8, PRAS40 and DEPTOR, which phosphorylates p70S6K1 (S6K1) and 4E-binding protein 1 (4E-BP1) [9]. On the other hand, mTORC2 is composed of mTOR, Rictor, Sin1, as well as Protor and DEPTOR, which phosphorylates AKT at Ser-473 [9].

Conventional mTORC1 inhibitors (i.e. rapamycin and RAD001) only partially inhibit mTORC1 activity, and are ineffective to mTORC2 [10]. Therefore, the anti-tumor activity of these inhibitors is relatively weak [11]. Meanwhile, mTORC1 inhibition will lead to feedback activation of several key oncogenic pathways, such as PI3K-AKT and ERK-MAPK [11], [12]. Due to these limitations, recent studies have developed mTOR kinase inhibitors, or the second generation mTOR inhibitors [13]. One of the compounds, GDC-0349, was recently characterized as a potent and selective ATP-competitive inhibitor of mTOR [14]. Its potential activity in HNSCC cells has not been tested.

Autophagy is a survival response to many stresses, which attenuates the anti-cancer cell activity by a number of chemo-agents [15], [16], [17]. Autophagy is initiated by formation of autophagosomes, where a portion of cytoplasm is enclosed into a double-membrane [15], [16], [17]. Afterwards, autophagosomes will be fused with lysosomes, and the enclosed contents are digested to provide nutrients for cell survival [15], [16], [17]. Autophagy-related (ATG)-7 and other ATG proteins are required for autophagic vacuole formation [15], [16], [17]. It has been proposed that autophagy inhibition may sensitize cancer cells to mTOR inhibitors [15], [16], [17]. In the present study, we also studied the potential role of autophagy in GDC-0349-induced anti-cancer activity in HNSCC cells.

Section snippets

Chemicals and reagents

GDC-0349 was provided by Selleck (Beijing, China). All the antibodies were obtained from Cell Signaling Technology (Shanghai, China). C14 ceramide was purchased from Avanti Polar Lipids (Alabaster, AL). The general caspase inhibitor Ac-VAD-CHO, the autophagy inhibitor 3-methyladenine (3-MA), chloroquine (CQ) and bafilomycin A1 (Baf A1) were purchased from Sigma-Aldrich Chemicals (St Louis, MO). Cell culture reagents were purchased from Hyclone (Suzhou, China).

Culture of HNSCC cell lines and oral epithelial cells

Human HNSCC cell lines, including

GDC-0349 inhibits proliferation of HNSCC cells bearing high-level of p-AKT/p-S6K

First, the structure and molecular weight of GDC-0349 were presented in Fig. 1A. HNSCC cells of different background were treated with increasing concentrations of GDC-0349 (1–1000 nM). Results of Fig. 1B showed that GDC-0349 potently inhibited proliferation of PTEN-low UMSCC47 cells and EGFR-mutant SQ20B cells [25]. Note that in UMSCC47 cells and SQ20B cells, basal activation of AKT-mTOR was high due the mutations [25]. On the other hand, GDC0349’s cytotoxicity was weak in SCC90 cells, which

Discussions

AKT-mTOR-initiated signaling pathway is one of the most frequently altered signaling in HNSCC, which contributes to HNSCC tumorigenesis and progression [5], [6], [7]. Thus it represents a potential and valuable clinical target. Our previous study demonstrated that PP121 blocked AKT-mTOR signaling and potently inhibited esophageal cancer cells in vitro and in vivo [1]. Here, we showed that GDC-0349, a selective ATP-competitive inhibitor of mTOR, inhibited proliferation of HNSCC cells with

Conflict of interests

No conflict of interests were stated by authors.

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