INPP4B-mediated tumor resistance is associated with modulation of glucose metabolism via hexokinase 2 regulation in laryngeal cancer cells

https://doi.org/10.1016/j.bbrc.2013.09.041Get rights and content

Highlights

  • HIF-1α-regulated INPP4B enhances glycolysis.

  • INPP4B regulates aerobic glycolysis by inducing HK2 via Akt-mTOR pathway.

  • Blockage of INPP4B and HK2 sensitizes radioresistant laryngeal cancer cells to radiation and anticancer drug.

  • INPP4B is associated with HK2 in human laryngeal cancer tissues.

Abstract

Inositol polyphosphate 4-phosphatase type II (INPP4B) was recently identified as a tumor resistance factor in laryngeal cancer cells. Herein, we show that INPP4B-mediated resistance is associated with increased glycolytic phenotype. INPP4B expression was induced by hypoxia and irradiation. Intriguingly, overexpression of INPP4B enhanced aerobic glycolysis. Of the glycolysis-regulatory genes, hexokinase 2 (HK2) was mainly regulated by INPP4B and this regulation was mediated through the Akt-mTOR pathway. Notably, codepletion of INPP4B and HK2 markedly sensitized radioresistant laryngeal cancer cells to irradiation or anticancer drug. Moreover, INPP4B was significantly associated with HK2 in human laryngeal cancer tissues. Therefore, these results suggest that INPP4B modulates aerobic glycolysis via HK2 regulation in radioresistant laryngeal cancer cells.

Introduction

Radiotherapy in combination with surgery and/or chemotherapy is a major treatment option for eradicating tumor cells in laryngeal cancer, of which the survival rates are relatively high following radiotherapy [1], [2]. Despite the effective therapeutic modality of radiotherapy for laryngeal cancer, the existence of radioresistant tumor cells contributes to locally recurrent and poor prognosis after radiotherapy. Several biological factors of the tumors such as the extent of hypoxia have been shown to contribute to resistance of these cells to radiotherapy [3]. It is well known that hypoxia as occurs in most tumors reduces the therapeutic effect by activating hypoxia-inducible factor-1α (HIF-1α) in several human cancers [3], [4]. Thus, understanding these mechanisms is important for the development of strategies to overcome resistance of tumor cells to radiotherapy.

Most cancer cells prefer to metabolize glucose by glycolysis even in the presence of sufficient oxygen, known as aerobic glycolysis or “Warburg effect” [5]. Up-regulation of glycolysis in malignant tumor cells is considered as adaption to hypoxia in pre-malignant lesions. Hypoxia and genetic alteration of oncogenes or tumor suppressor genes lead to the activation of transcription factor HIF-1α and thereby reprogram the malignant tumor cells to the increased glycolytic phenotype by inducing glycolysis-regulatory genes such as glucose transporters (e.g., GLUT1 and 2) and hexokinases (e.g., HK1 and 2) [5], [6]. The increased glycolysis promotes acidosis of the tumor microenvironment by producing lactic acid, end product of glycolysis [5]. Several reports have shown that enhanced aerobic glycolysis and acidosis are often linked to the resistant phenotype of cancer cells to radio- or chemotherapy. For example, overexpression of GLUT1 and HK2 in several cancers predicts poor prognosis after radio- or chemotherapy [7], [8], [9], [10]. Thus, increased aerobic glycolysis is not only one of the essential component of the malignant phenotype but also for resistance of tumor cells to radio- or chemotherapy [5]. However, the precise mechanisms underlying modulation of aerobic glycolysis in resistant tumor cells are still unclear.

Inositol polyphosphate 4-phosphatase type II (INPP4B) is a regulatory enzyme that selectively removes the phosphate at the fourth position of the inositol ring from phosphatidylinositol(3,4)-bisphosphate, which is involved in the phosphatidylinositol signaling pathway [11], [12], [13]. The rapid production and/or degradation of phosphoinositides participate in a wide range of cellular processes, including cell growth, differentiation, apoptosis, protein trafficking, and movement [14], [15]. Recently, we identified INPP4B as a novel tumor resistance gene by systematically analyzing Unigene libraries of the human laryngeal cancer [16]. Induction of INPP4B by radiation or anticancer drugs contributes to radioresistance and chemoresistance of tumors via regulation of extracellular signal-regulated kinase (ERK)-Akt pathway [16].

In this study, we found that INPP4B expression is regulated by HIF-1α under stress conditions. Further, INPP4B was able to modulate aerobic glycolysis by inducing HK2, which consequently contributed to the resistance of tumor cells to radiation or anticancer drug. Furthermore, we provide evidence of the clinical relevance of this regulation in human laryngeal cancer tissues.

Section snippets

Cell lines and treatment

A549 lung cancer cells, MCF7 breast cancer cells, and HEp-2 laryngeal cancer cells were obtained from the American Type Culture Collection. Cells were grown in DMEM (MCF7 and HEp-2) or RPMI (A549) supplemented with 10% fetal bovine serum (HyClone, South Logan, UT) and penicillin/streptomycin at 37 °C in a humidified 5% CO2 incubator. Radioresistant-HEp-2 (RR-HEp-2) and HEp-2 cells overexpressing INPP4B were established as previously described [16], [17]. Hypoxia was stimulated in a chamber with

HIF-1α-regulated INPP4B enhances glycolysis

As the functional hypoxia response elements which bound to HIF-1α and HIF-1β [19], were found on the promoter region of INPP4B by using VISTA analysis (Supplementary Fig. 1), we initially hypothesized that INPP4B expression might be regulated by hypoxia. Interestingly, the levels of INPP4B was induced under hypoxic condition, concomitant with increase of HIF-1α protein levels, in laryngeal cancer HEp-2 cells as assessed by Western blotting (Fig. 1A). In addition, this hypoxia-induced INPP4B

Discussion

In this study, we demonstrate that INPP4B modulates glycolytic phenotype of laryngeal cancer cells and this regulation may contribute to radioresistance and chemoresistance in tumor cells. As depicted in Fig. 4C, our data suggests that exposure of tumor cells to various stress stimuli such as hypoxia and irradiation can trigger the induction of INPP4B and is dependent on HIF-1α in radioresistant laryngeal cancer cells. Increased INPP4B has the capacity to promote glucose metabolism by

Acknowledgments

This work was supported by Basic Science Research Program (Grant No. 2012R1A1A2002955) and the Nuclear Research and Development Program through a National Research Foundation of Korea (NRF) grant funded by the Korean government (Ministry of Education, Science and Technology).

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