Heat Shock Factor 1 Is a Powerful Multifaceted Modifier of Carcinogenesis Chengkai Dai,1 Luke Whitesell,1 Arlin B. Rogers,3 and Susan Lindquist1,2,* 1Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA 2Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA 3Divison of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, MA 02139, USA *Correspondence: lindquist_admin@wi.mit.edu DOI 10.1016/j.cell.2007.07.020 SUMMARY Heat shock factor 1 (HSF1) is the master regula- tor of the heat shock response in eukaryotes, a very highly conserved protective mechanism. HSF1 function increases survival under a great many pathophysiological conditions. How it might be involved in malignancy remains largely unexplored. We report that eliminating HSF1 protects mice from tumors induced by muta- tions of the RAS oncogene or a hot spot muta- tion in the tumor suppressor p53. In cell culture, HSF1 supports malignant transformation by or- chestrating a network of core cellular functions including proliferation, survival, protein syn- thesis, and glucose metabolism. The striking effects of HSF1 on oncogenic transformation are not limited to mouse systems or tumor initi- ation human cancer lines of diverse origins show much greater dependence on HSF1 func- tion to maintain proliferation and survival than their nontransformed counterparts. While it en- hances organismal survival and longevity under most circumstances, HSF1 has the opposite effect in supporting the lethal phenomenon of cancer. INTRODUCTION The heat shock response is one of the most ancient and evolutionarily conserved protective mechanisms found in nature. Environmental insults provoke a variety of adap- tive physiological responses to help organisms cope with specific stressors. The dramatic induction of heat shock proteins (HSPs) is an important unifying component of most of these responses, and this induction has proven to be essential for survival under stressful conditions. Work over the last three decades has revealed that the major HSPs are molecular chaperones that guard against ������illicit or promiscuous interactions������ between other pro- teins. Their basal expression facilitates normal protein folding and guards the proteome from the dangers of misfolding and aggregation. In the face of proteotoxic stressors including heat, hypoxia/ischemia, free radicals, ATP depletion, and acidosis, the importance of HSPs in preventing the aggregation and promoting the refolding of other proteins becomes acute. When misfolding ex- ceeds a certain threshold, other HSPs disaggregate pro- teins and refold them or divert them to the proteasome for destruction (Whitesell and Lindquist, 2005). Regulation of HSP expression is intricate, with multiple layers of redundancy and feedback control, but a small family of transcription factors called heat shock factors (HSFs) are the primary regulators of stress-inducible ex- pression in eukaryotic cells. The structure and function of HSFs have been conserved for more than a billion years. They bind consensus heat shock elements (HSEs) within the promoter regions of HSP genes (Westerheide and Morimoto, 2005), and this binding is critical to HSP in- duction. Several HSFs are present in mammalian cells, but HSF1 is clearly the dominant factor controlling cellular re- sponses to stress. Deletion of Hsf1 in mammalian cells allows normal basal expression of HSPs but completely abrogates induction in response to heat shock and a vari- ety of other stresses (Xiao et al., 1999). In mice and Drosophila, HSF1 is dispensable for growth and survival under controlled laboratory conditions but essential for survival following stresses such as high temperature and endotoxin challenge (Jedlicka et al., 1997 Xiao et al., 1999). Hsf1-deficient mouse embryos suffer from defects in placental development and are recovered from crosses in lower numbers than expected by Mendelian segrega- tion. Other than being 20% smaller than wild-type mice, however, they display no overt organ system ab- normalities and, in the absence of acute stress, live to late adulthood (Xiao et al., 1999 A. Steele and S.L., unpublished data). Although less well understood, the activities of HSF1 extend far beyond the classical induction of HSPs. In yeast, HSF1 has now been shown to regulate up to 3% of the genome and impact genes ranging in function from energy production to signal transduction, from small molecule transport to carbohydrate metabolism, and from cytoskeletal organization to vesicular transport (Hahn et al., 2004). Immunolocalization and chromatin Cell 130, 1005���1018, September 21, 2007 ��2007 Elsevier Inc. 1005

immunoprecipitation indicate that HSF1 binds to a simi- larly broad array of non-HSP genes in Drosophila (West- wood et al., 1991 Birch-Machin et al., 2005) and human erythroleukemia cells (Trinklein et al., 2004). The HSF1-mediated stress response and the activity of specific HSPs have both been implicated in protecting or- ganisms from a broad range of pathophysiological condi- tions including thermal injury, ischemia/reperfusion, and age-related neurodegeneration (Christians et al., 2002 Westerheide and Morimoto, 2005). Intriguingly, in nema- todes, HSF1 promotes longevity under stable laboratory conditions (Hsu et al., 2003 Morley and Morimoto, 2004). Much less is known about the role of HSF1 in can- cer. It has long been noted that HSP levels increase in a wide range of tumor types (Jolly and Morimoto, 2000). Many of the signaling pathways and transcription factors that are frequently deranged in cancers display a striking dependence on the chaperone machinery, especially HSP90 (Whitesell and Lindquist, 2005). Moreover, HSF1 expression is elevated in human prostate carcinoma cell lines (Tang et al., 2005). But whether the multifaceted HSF1-mediated stress response plays a causal, sup- portive, or inhibitory role in mammalian oncogenesis is unknown. On the one hand, given its prominent role in helping cells cope with stressful insults, HSF1 might promote oncogen- esis by facilitating cellular adaptation to the malignant lifestyle. On the other hand, given its general role in enhancing longevity, HSF1 might assist organisms in combating malignancy. To investigate these possibilities, we used both whole-animal and cell-culture models in which HSF1 expression could be disrupted by genetic techniques. We find that HSF1 is a remarkably potent modifier of tumor-free survival in whole animals. Further, it modulates oncogenesis by coordinating a diverse array of core cellular functions and supports the aberrant prolif- eration and survival of human tumor cell lines carrying a wide range of molecular genetic defects. As a very an- cient adaptive mechanism, the HSF1-dependent stress response has evolved to enhance survival in the face of environmental challenges from without and disease processes within such as ischemic injury and neurode- generation. These broadly recognized beneficial effects, however, contrast sharply with its lethal role in the phe- nomenon of cancer that we now report. RESULTS HSF1 Deficiency Suppresses Chemical Skin Carcinogenesis in Mice To begin investigating the role of Hsf1 as a modifier of tumorigenesis, we used a classical multistep chemical skin carcinogenesis protocol. In this mouse model, so- matic mutations are induced in epidermal cells by a single topical application of the mutagen dimethylbenzanthra- cene (DMBA). Tumor promotion is then achieved by re- peated applications of the phorbol ester 12-O-tetradeca- noylphorbol-13-acetate (TPA). Early on, the overwhelming majority of the resulting skin tumors are benign papillo- mas. A small portion of these tumors spontaneously prog- ress to become malignant squamous cell carcinomas, which are invasive and sometimes metastatic (Yuspa, 1994). When Hsf1 wild-type mice (Hsf1+/+) and their Hsf1 null littermates (Hsf1 / ) were treated with DMBA and TPA, no obvious skin damage or irritation was noticed in either genotype after topical application of the chemicals. There was, however, a striking difference in carcinogen- induced tumorigenesis. Hsf1 / mice were far more resistant to tumor formation than Hsf1+/+ mice (Figure 1A), and this difference was manifested in several ways. First, the latency period be- fore the development of any tumors was 5 weeks longer in Hsf1 / mice than in Hsf1+/+ mice (Figure 1B). Second, Hsf1 / mice exhibited a marked reduction in tumor inci- dence (Hsf1+/+ 93.1% versus Hsf1 / 60.9% at week 24, p = 0.0047, chi-square test) (Figure 1B). Third, they had a much lower overall tumor burden. This applied both to the number of tumors that arose (Figure 1C) and to the size the tumors achieved (Figure 1D). Fourth, and most im- portantly, Hsf1 / mice survived much longer than their wild-type counterparts (Figure 1E). To further investigate the extraordinary resistance of Hsf1 / mice to carcinogen-induced tumorigenesis, an in- dependent experiment was performed in which Hsf1 / mice and their wild-type littermates were treated with a second mutagen, NMMG (N-methyl-N0-nitro-N-nitroso- guanidine), at week 25 to promote tumor progression. Once again, Hsf1 / mice developed many fewer tumors. They also had a very strong survival advantage over Hsf1+/+ mice (see Figure S1A in the Supplemental Data available with this article online). Thus, in sharp contrast to the many circumstances under which Hsf1-deficient organisms are at a survival disadvantage relative to wild- type organisms, in survival after chemically induced skin carcinogenesis they have a profound advantage. Nature of Carcinogen-Induced Tumors Although there was a large difference in the number of tu- mors formed in the Hsf1+/+ and Hsf1 / mice, the percent- age of benign versus malignant tumors (papilloma versus squamous cell carcinoma) was comparable (Figures S1C and S1D). Next we asked if the tumors harbored mutations in the H-Ras proto-oncogene. This gene is almost always activated during chemical skin carcinogenesis. Further- more, activating mutations of RAS occur in approximately 30% of all human cancers including skin cancers (Balmain et al., 1984 Sebti and Adjei, 2004). Fourteen skin lesions were randomly sampled in both genotypes. All harbored activating mutations in H-Ras. Strikingly, these occurred at positions that are known hot spots in human malignan- cies (Table S1). HSF1 Deficiency Suppresses Tumorigenesis Driven by Mutant p53 To test the generality of the detrimental effects of HSF1 on tumor-free survival, we examined its impact on the 1006 Cell 130, 1005���1018, September 21, 2007 ��2007 Elsevier Inc.