In this study we report a previously unrecognized role of NAC1 in regulating autophagy, and provide a possible mechanistic role for how NAC1 up-regulation, as observed in several types of human cancer, contributes to tumor progression and early recurrence in patients after chemotherapy as previously reported. We show that treatment with the chemotherapeutic drug cisplatin activates autophagy in ovarian cancer cells (), and inactivation and gene silencing of NAC1 blocks the activation of autophagy by cisplatin (). We further demonstrate that regulation of autophagy by NAC1 is mediated through its effect on HMGB1, as the functional status of NAC1 affects the expression, translocation, and release of this critical autophagy regulator (), which is known to induce autophagy by disrupting the interaction between beclin 1 and bcl-2 (Tang D et al 2010).
Because tumor recurrence as a result of resistance to chemotherapeutic drugs remains a formidable problem in managing cancer patients, the results from this study should illuminate fundamental properties of chemo-resistance and provide new therapeutic targets to treat advanced recurrent neoplastic diseases. The role of NAC1 in activation of autophagy, a cellular process of self-digestion in response to various stresses, may account, at least in part, for the prosurvival function of this cancer-associated protein. Indeed, autophagy has been appreciated as an important mechanism for conferring resistance to chemotherapy. For instance, it has been shown that induction of autophagy promotes resistance of leukemic cells to adriamycin and vincristine (Liu et al.
). Similarly, we demonstrate here that cisplatin-induced autophagy protects ovarian cancer cells from the cytotoxic effects of cisplatin (). It has been known that cisplatin induces autophagy in various types of cells (Fanzani et al.
; Kaushal et al., 2008
; Ren et al.
), but how autophagy contributes to cellular sensitivity to this drug remains to be clarified. Several studies have shown that activation of autophagy in the cisplatin-treated cells inhibited apoptosis and protected cells from cisplatin toxicity (Harhaji-Trajkovic et al., 2009
; Liu et al., 2009
; Periyasamy-Thandavan et al., 2008
; Ren et al.
). In contrast, there are also studies reporting that forced expression of Beclin 1, a key autophagy gene, activates both autophagy and apoptosis, and potentiates the cytotoxic effect of cisplatin on tumor cells (Sun et al.
). We demonstrate here that autophagy induced by cisplatin is cyto-protective, as suppression of autophagy by inhibiting NAC1 or by using small molecule inhibitors of autophagy augments the cytotoxic effect of cisplatin ( and ). Therefore, it is likely that the functional consequences of activating autophagy are context-dependent, and factors such as cell type and treatment regimen could all determine the fates of autophagic cells for their survival or death. Additionally, we observed a similar effect of NAC1 on autophagy that was induced by other stimuli (nutrient depletion, hypoxia or rapamycin treatment) in ovarian cancer cells (Supplementary Information
, Fig. S2
) and in other types of cells treated with cisplatin (Supplementary Information
, Fig. S3
, Fig. S4
and Fig. S5
), suggesting that NAC1 may act as an autophagy regulator in various types of tumor cells responding to multiple forms of stress.
Regulation of autophagy by NAC1 appears to be mediated through HMGB1, a highly conserved chromatin-associated nuclear protein and an extracellular signaling molecule that is now appreciated as a critical regulator of autophagy and apoptosis by acting as an inducer of autophagy and a suppressor of apoptosis (Ellerman et al., 2007
; Tang et al.
). The expression or function of HMGB1 has been reported to be closely linked to cancer development and progression (Ellerman et al., 2007
). Although the expression of HMGB1 is activated in cisplatin-resistant cancer cells (Nagatani et al., 2001
), the actual effects of this protein on cellular sensitivity to cisplatin vary in different cell types (He et al., 2000
; Sharma et al., 2009
; Wei et al., 2003
). We show that the expression, translocation, and release of HMGB1 are altered by the functional status of NAC1 (), which is also demonstrated to modulate the sensitivity of ovarian cancer cells to cisplatin via autophagy ( and ). Thus, it is likely that the effect of HMGB1 on cisplatin sensitivity could be dependent on the functional status of NAC1 in an individual cell type. At present, the precise mechanism by which NAC1 controls the expression, translocation and release of HMGB1 is unknown; however, considering that NAC1 predominantly localizes in the nuclei (Supplementary Information
, Fig. S6
) and its role as a transcription factor, it is likely that NAC1 activates the transcription of HMGB1, leading to increased expression, translocation and release of HMGB1. The possibility that NAC1 assists HMGB1 in nucleo-cytoplasmic shuttling is also worth investigating. Note worthily, the release of HMGB1 can also be regulated by autophagy, as reported by Thorburn et al. (Thorburn et al., 2009a
; Thorburn et al., 2009b
). It is conceivable that the increased amount of cytoplasmic HMGB1 activates autophagy, since HMGB1 is known to modulate autophagy by disrupting the interaction between beclin 1 and bcl-2 in the cytoplasm (Tang D et al 2010). In addition, consistent with the role of HMGB1 in balancing autophagy and apoptosis, our results show that silencing of HMGB1 not only inhibits autophagy but also activates apoptosis in ovarian cancer cells subjected to cisplatin treatment (). As reduced apoptosis is believed to be one of the mechanisms of cisplatin resistance, the converse relationship between autophagy and apoptosis in tumor cells treated with cisplatin, as shown in this study, might also help explain why apoptosis is suppressed in cisplatin-resistant cells.
Development of drug resistance is a complex process involving multiple genes and the pathways they control. It is most likely that NAC1 participates in chemo-resistance through several other mechanisms besides mediating autophagy. For example, NAC1 has been reported to inactivate the Gadd45 tumor suppressor pathway that is activated by chemotherapeutic compounds (Jinawath et al., 2009
; Nakayama et al., 2007
). Thus, NAC1-expressing tumor cells should have a survival advantage in the presence of cytotoxic drugs. Moreover, NAC1 has been found to play a role in maintaining pluripotency in embryonic stem cells by transcriptionally regulating gene expression of other pluripotency-associated factors including Nanog, Oct4, Sox2, Klf4, Sall1 and Sall4 (Kim et al., 2008
; Muller et al., 2008
; Wang et al., 2006
). As exportation of foreign compounds such as cytotoxic drugs is an inherent feature of stem cells, it is possible that cancer cells with NAC1 up-regulation may adopt a similar strategy as embryonic stem cells to survive during chemotherapy.
In summary, our study identified NAC1 as a novel regulator of autophagy and its involvement in modulation of cisplatin sensitivity, and demonstrated that inhibition of NAC1 can enhance sensitivity of ovarian cancer cells to this chemotherapeutic drug. These findings may expand current knowledge of the autophagic protein networks and provide new insight into how autophagy is regulated in tumor cells. Thus, NAC1 might be explored as a novel therapeutic target for prevention of tumor cells from becoming resistance to chemotherapy and for reinforcement of the efficacy of cisplatin against malignant tumors.