br G Schematic showing that lung tumor cell
(G) Schematic showing that lung tumor cell lines were established from control (mTC) and NAC-treated (mTN) K mice 58 weeks after Cre-adenovirus inhalation. The 740 Y-P were cultured without antioxidants unless otherwise stated.
(H) Real-time cell invasion analyzed with the xCelligence system. Curves show mean invasion index of 3 mTC and 4 mTN cell lines.
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results suggest that antioxidants protect primary tumor cells from ROS and DNA damage that would otherwise acti-vate p53 and slow their proliferation. Moreover, stimulating the transcription of endogenous antioxidant genes—by activating NRF2 mutations or inactivating KEAP1 muta-tions—increases primary lung tumor growth, for example, in the K and K;Trp53fl/fl (KP) mouse models (Best et al., 2018; Jeong et al., 2017; Romero et al., 2017). However, none of these studies addressed the important question of whether antioxidants influence lung cancer metastasis. One reason is that the K and KP mice were euthanized due to high primary lung tumor burden relatively early after tumor initiation and too soon for authentic metastases to develop (DuPage et al., 2009; Romero et al., 2017; Sayin et al., 2014). Another reason is that the K model is believed to be metastasis resistant in the absence of additional mutations (e.g., in Trp53 and Nkx2.1) (Kim et al., 2005; Winslow et al., 2011).
Like NRF2, the transcription factor BTB and CNC homology 1 (BACH1) belongs to the cap’n’collar (CNC) b-Zip family of proteins that bind to antioxidant response elements (AREs) in response to changes in redox states (Hayes et al., 2010; Sun et al., 2004; Taguchi et al., 2011). Under conditions of oxidative stress, the oxidation of heme-containing proteins releases free heme, which stimulates degradation of BACH1 (Zenke-Kawa-saki et al., 2007). In low-oxidative stress conditions, heme levels are low, which leads to BACH1 stabilization. BACH1 is believed to displace NRF2 from AREs and to act primarily as a transcriptional repressor for antioxidant genes, including HMOX-1, which encodes the detoxification enzyme heme oxy-genase 1 (HO-1), and NQO1, which encodes NAD(P)H dehy-drogenase (quinone 1). However, BACH1 may also activate transcription (Liang et al., 2012). Although BACH1 has been identified in metastatic signatures in a few studies, its role in lung cancer metastasis is unknown (Liang et al., 2012; Yun et al., 2011).
Lung cancer is the main cause of cancer-related deaths worldwide, and most of those deaths are associated with metastasis (IARC, Globocan 2018 [Bray et al., 2018]). Thus, understanding basic mechanisms that control lung can-cer metastasis is essential for identifying tumor cell weak-nesses that can be exploited for therapy. Although one-third of lung cancers have NRF2 or KEAP1 mutations (Berger et al., 2016), the role of endogenous and exogenous antioxi-dants in lung cancer metastasis has not been explored. Furthermore, the belief that antioxidant supplements protect against cancer persists in society. In this study, we combined long-term studies in mouse models with genomic, metabolic, and data mining approaches to define the impact of ROS and antioxidants in lung cancer metastasis—and identify a new mechanism that controls this process.
The Antioxidants NAC and Vitamin E Stimulate Lung Cancer Metastasis in a p53-Independent Fashion
To define antioxidant effects on lung cancer metastasis, we used Kras2LSL/+ and Kras2LSL/+Trp53fl/fl mice (hereafter K and KP)
(Jackson et al., 2001; Meuwissen et al., 2003). Intranasal Cre-adenovirus administration activates KRASG12D expression in K and KP mice and inactivates p53 in KP mice, resulting in lung adenocarcinoma (LUAD). KP mice develop metastases, particularly to lymph nodes, but K mice do not (Kwon and Berns, 2013). We administered a low dose Cre-adenovirus (99% lower than in DuPage et al., 2009; Sayin et al., 2014) to K and KP mice and gave NAC and vitamin E in the drinking water and chow 1 week later (Figure 1A). The incidence of lymph node metastasis was 6- to 7-fold higher in antioxidant-treated K mice than in controls (Figure 1B). Lymph nodes in antioxidant-treated mice were larger and stained positive for pro-surfactant protein C, indicating a pulmonary origin, and for the invasive marker high-mobility group AT-hook 2 (HMGA2, Morishita et al., 2013; Winslow et al., 2011) (Figures 1C, S1A, and S1B). Antioxidants also stimulated distant metastasis in some K mice (Figure 1D). Moreover, NAC and vitamin E increased lymph node and thoracic metastasis in KP mice and increased the frequency of HMGA2-positivity (Figures 1E, 1F, S1C, and S1D).
The antioxidants did not affect survival or primary tumor burden in K and KP mice (Figures S1E–S1G). However, tumors of antioxidant-treated K mice were more advanced (Figures S1H–S1I). Consistent with those observations, HMGA2 expres-sion was higher in tumors of antioxidant-treated K and also KP mice (Figure S1J); no differences in proliferation index was observed (Figure S1K). Moreover, a proportion of tumors from antioxidant-treated K mice had lost expression of the metas-tasis-suppressor NK2 homeobox 1 (NKX2.1) (Figure S1L), which is present at low levels in all KP tumors (Winslow et al., 2011).