KNK437, abrogates hypoxia-induced radioresistance by dual targeting of the AKT and HIF-1α survival pathways

Abstract

KNK437 is a benzylidene lactam compound known to inhibit stress-induced synthesis of heat shock proteins (HSPs). HSPs promote radioresistance and play a major role in stabilizing hypoxia inducible factor-1α (HIF-1α). HIF-1α is widely responsible for tumor resistance to radiation under hypoxic conditions. We hypothesized that KNK437 sensitizes cancer cells to radiation and overrides hypoxia-induced radioresistance via destabilizing HIF-1α. Treatment of human cancer cells MDA-MB-231 and T98G with KNK437 sensitized them to ionizing radiation (IR). Surprisingly, IR did not induce HSPs in these cell lines. As hypothesized, KNK437 abrogated the accumulation of HIF-1α in hypoxic cells. However, there was no induction of HSPs under hypoxic conditions. Moreover, the proteosome inhibitor MG132 did not restore HIF-1α levels in KNK437-treated cells. This suggested that the absence of HIF-1α in hypoxic cells was not due to the enhanced protein degradation. HIF-1α is mainly regulated at the level of post-transcription and AKT is known to modulate the translation of HIF-1α mRNA. Interestingly, pre-treatment of cells with KNK437 inhibited AKT signaling. Furthermore, down regulation of AKT by siRNA abrogated HIF-1α levels under hypoxia. Interestingly, KNK437 reduced cell survival in hypoxic conditions and inhibited hypoxia-induced resistance to radiation. Taken together, these data suggest that KNK437 is an effective radiosensitizer that targets multiple pro-survival stress response pathways.

Introduction

Stress ranging from heat stress to pathophysiological conditions induce HSPs [1]. These proteins function as molecular chaperones, highly conserved and cytoprotective [2]. Enhanced expression of HSPs in response to stress is mediated by the heat shock transcription factor 1 (HSF1) [2]. HSPs enhance DNA repair and inhibit cell death to activate an adaptive response to IR [3], [4], [5], [6]. Although, a number of HSPs have been implicated in cellular resistance to IR, only HSP90 has been effectively targeted. The most widely used HSP90 inhibitors such as geldenamycin and its clinically relevant analog 17AAG, sensitize cancer cells to radiation [7], [8]. However, these inhibitors activate HSF1 and subsequent induction of HSPs [9], [10]. Induction of HSPs by HSP90 inhibitors in turn limits their efficacy as radiosensitizers.

Hypoxia induces genetic instability and tumor invasion, resulting in a more malignant phenotype [11] and is a major limiting factor in the efficacy of IR against solid tumors. HIF-1 is an oxygen sensitive heterodimeric transcription factor responsible for cellular adaptation to hypoxic conditions. HIF-1 comprises α and β components where the α component is regulated by oxygen dependent protein degradation [12]. HIF-1 contributes to radioresistance by modulating the expression of genes involving angiogenesis and cell proliferation [13], [14]. Inhibition of HIF-1α by either genetic or pharmacological means sensitizes cancer cells to radiation [15], [16]. HIF-1α is mostly regulated at post-transcriptional levels of translation and protein stability [17], [18]. Various studies have reported that hypoxia induces HSPs which in turn stabilizes HIF-1α [19], [20], [21]. AKT signaling is an established upstream modulator of HIF-1α [18], [21], [22], [23]. Selective inhibition of AKT abrogates the accumulation of HIF-1α under hypoxia [18], [23].

KNK437 is a novel benzylidene lactam compound, isolated from an organic source library (Kaneka Corp., Osaka, Japan). KNK437 is characterized by its ability to inhibit stress induced synthesis of HSPs mediated by HSF1 [24]. The efficacy of KNK437 as a sensitizer to hyperthermia mediated cell killing has been validated in vivo [25]. When used along with the HSP90 inhibitor, 17-AAG, KNK437 abrogated the induction of HSP70 and exhibited synergy to reduce the survival of HL-60 cells [10]. In the present study, we examined whether KNK437 sensitizes cancer cells to IR and overrides hypoxia-induced radioresistance.