Oncogene (2013) 32, 3231–3245 & 2013 Macmillan Publishers Limited All rights reserved 0950-9232/13 www.nature.com/onc ORIGINAL ARTICLE Bach1 is critical for the transformation of mouse embryonic fibroblasts by RasV12 and maintains ERK signaling A Nakanome1,2, A Brydun1,3, M Matsumoto1,4,KOta1, R Funayama5,6, K Nakayama5, M Ono7, K Shiga2, T Kobayashi2 and K Igarashi1,3,6 Reactive oxygen species (ROS), by-products of aerobic respiration, promote genetic instability and contribute to the malignant transformation of cells. Among the genes related to ROS metabolism, Bach1 is a repressor of the oxidative stress response, and a negative regulator of ROS-induced cellular senescence directed by p53 in higher eukaryotes. While ROS are intimately involved in carcinogenesis, it is not clear whether Bach1 is involved in this process. We found that senescent Bach1-deficient mouse embryonic fibroblasts (MEFs) underwent spontaneous immortalization the same as did the wild-type cells. When transduced with constitutively active Ras (H-RasV12), the proliferation and colony formation of these cells in vitro were markedly reduced. When transplanted into athymic nude mice, the growth and vascularization of tumors derived from Bach1-deficient cells were also decreased. Gene expression profiling of the MEFs revealed a new H-RasV12 signature, which was distinct from the previously reported signatures in epithelial tumors, and was partly dependent on Bach1. The Bach1-deficient cells showed diminished phosphorylation of MEK and ERK1/2 in response to H-RasV12, which was consistent with the alterations in the gene expression profile, including phosphatase genes. Finally, Bach1-deficient mice were less susceptible to 4-nitroquinoline-1-oxidide (4-NQO)- induced tongue carcinoma than wild-type mice. Our data provide evidence for a critical role of Bach1 in cell transformation and tumor growth induced by activated H-RasV12. Oncogene (2013) 32, 3231–3245; doi:10.1038/onc.2012.336; published online 30 July 2012 Keywords: Bach1; ROS; cancer; Ras; ERK INTRODUCTION reduced in Bach1-deficient mice compared with wild-type Reactive oxygen species (ROS) are inevitable by-products of controls,9,10 suggesting that Bach1 determines the ROS levels by aerobic respiration. ROS have conventionally been regarded as fine-tuning the expression of oxidative stress-response genes, having carcinogenic potential and profound associations with including Hmox1. tumor promotion.1 First, ROS are one of the major causes of DNA Intriguingly, Bach1 represses the oxidative stress-induced damage in vivo and increase mutations in DNA.2 Second, the cellular senescence directed by p53.11 Senescence, induced by production of ROS is induced in response to growth factors and the activation of tumor suppressors, inhibits the development of oncogene activation, and can modulate critical intracellular cancer by arresting the proliferation of damaged or stressed cells signaling molecules by affecting their redox regulation.3 Third, that are at risk for malignant transformation.12,13 Bach1 inhibits ROS affect heterotypic cell–cell interactions within tumor tissues p53-mediated cellular senescence by forming a complex with p53, and also affect angiogenesis.4 Therefore, changes in ROS histone deacetylase 1 and nuclear receptor co-repressor, thereby metabolism potentially affect both the prevention and repressing the transcriptional activity of p53.11 Although oxidative promotion of cancer. stress is one of the major causes of the senescence of mouse In higher eukaryotes, the transcriptional responses toward embryonic fibroblasts (MEFs),14 oncogenic Ras with activating oxidative stress are regulated by two major transcription factor mutations also induces cellular senescence.15 Importantly, families, the NF-kB/Rel family and the AP-1 superfamily, which although ROS are essential for Ras-induced cell transformation,16 includes Jun.5 Bach1 is a heme-regulated transcriptional repressor little is known about the involvement of the oxidative stress found in vertebrates and is a member of the AP-1 superfamily.6 response in Ras-driven tumorigeneisis. The regulation of ROS Bach1 forms heterodimers with small Maf oncoproteins (MafK, metabolism and cellular senescence by Bach1 suggest that it may MafF, MafG), binds to the Maf recognition element, which be relevant to tumorigenesis. encompasses an AP-1-binding site,7 and represses the Activating mutations in Ras family proto-oncogenes are very expression of oxidative stress-responsive genes, including heme common in human cancer, and modulators of aberrant Ras oxygenase-1 (Hmox1).8 These genes are activated by Nrf2, which signaling have an important role in tumorigenesis.17 Recently, also belongs to the AP-1 superfamily and forms heterodimers with there have been several reports describing that there is a large the small Maf proteins. Atherosclerosis and ischemic reperfusion group of genes/proteins that are not oncogenes, but if targeted, injury of the heart, both involving ROS-mediated damage, are can cause reduced proliferation of transformed cells.18 These 1Department of Biochemistry, Tohoku University Graduate School of Medicine, Sendai, Japan; 2Department of Otolaryngology-Head and Neck Surgery, Tohoku University Graduate School of Medicine, Sendai, Japan; 3Center for Regulatory Epigenome and Diseases, Tohoku University Graduate School of Medicine, Sendai, Japan; 4Department of Obstetrics and Gynecology, Tohoku University Graduate School of Medicine, Sendai, Japan; 5Division of Cell Proliferation, Tohoku University Graduate School of Medicine, Sendai, Japan; 6CREST, Japan Science and Technology Agency, Sendai, Japan and 7Department of Pathology, Tohoku University Graduate School of Medicine, Sendai, Japan. Correspondence: Professor K Igarashi, Department of Biochemistry, Tohoku University Graduate School of Medicine, Seiryo-machi 2–1, Sendai, Japan. E-mail: [email protected] Received 3 September 2011; revised 7 June 2012; accepted 20 June 2012; published online 30 July 2012 Bach1 as a possible target of non-oncogene addiction A Nakanome et al 3232 phenomena have been interpreted in such a way that activated deficiency did not prevent the immortalization of MEFs despite oncogenes rely on genes that are themselves not oncogenes, thus their enhanced propensity for cellular senescence. However, leading to the so-called ‘non-oncogene addiction’.19 For instance, Bach1 was nearly indispensable for the transformation of the Ras family is dependent on the heat shock response in human immortalized cells with activated Ras (H-RasV12) in vitro, and cancer cells.18 enhanced the tumor formation in vivo. Bach1 deficiency reduced In this report, we examined the roles of Bach1 in the ROS levels, and profoundly affected the transcriptional response proliferation and ROS metabolism of transformed cells. Bach1 to H-RasV12, including the negative feedback loop of extracellular Figure 1. Immortalization of Bach1-deficient MEFs. (a) Wild-type and Bach1-deficient primary cell cultures sequentially acquired morphologies typical of senescent and immortal cells (scale bar ¼ 100 mm). (b) The rates of spontaneous immortalization of 25 wild-type and 16 Bach1- deficient independent MEF cultures were monitored. (c) Top, the protein levels of Bach1 in primary and immortalized MEFs. Tubulin was used as loading control. N ¼ 3, the error bars indicate the s.d. Bottom, the result of a typical Western blot analysis is shown. (d) The expression levels of p19ARF mRNA were examined in the primary and two independently immortalized wild-type (black bars) and Bach1-deficient (gray bars) MEF cultures (N ¼ 3, the error bars indicate the s.d.). (e) The expression levels of p53 mRNA were compared as in (d). (f) The proliferation rates of immortalized Bach1-deficient and wild-type cells with high or low p19ARF expression were compared. The data represent typical results of three independent experiments. Oncogene (2013) 3231 – 3245 & 2013 Macmillan Publishers Limited Bach1 as a possible target of non-oncogene addiction A Nakanome et al 3233 signal-regulated kinase (ERK). These results suggest that Bach1 is a regulate the inhibition of proliferation induced by cell-to-cell critical factor for Ras-induced transformation and may represent a contact. Because the inactivation of p19ARF appeared to be a target gene of non-oncogene addiction by Ras. random event in immortalization, we selected only the i-MEF cultures that had lost p19ARF expression for the further analyses. RESULTS Bach1 facilitates H-RasV12-induced proliferation and Bach1-deficient MEFs bypass senescence in vitro transformation Senescent MEFs eventually resume proliferation because of the Unlike primary MEFs, i-MEFs can be transformed by single inactivation of p53 and/or p19ARF, resulting in their immortaliza- oncogenes, such as H-RasV12.21 We therefore compared the tion.20 To examine whether Bach1 deficiency would affect this transformation of wild-type and Bach1-deficient i-MEFs by process beyond the premature entry into senescence, we compared H-RasV12. When transduced with H-RasV12,theBach1-deficient the kinetics of immortalization using senescent wild-type and Bach1- i-MEFs produced smaller numbers of colonies in mono-layered deficient MEFs. While senescent cells developed typical phenotypes, culture (Figure 2a). When reconstituted with a Bach1-expressing including flattened morphology and cell cycle arrest, immortalized plasmid, Bach1-deficient i-MEFs formed more colonies (Figure 2a). cells (i-MEFs) regained their typical fibroblastic morphology and