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, somatic mutations are induced in epidermal cells by a single topical application of the mutagen dimethylbenzanthracene (DMBA). Tumor promotion is then achieved by repeated applications of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Early on, the overwhelming majority of the resulting skin tumors are benign papillomas. A small portion of these tumors spontaneously progress 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 (), and this difference was manifested in several ways. First, the latency period before the development of any tumors was 5 weeks longer in Hsf1-/- mice than in Hsf1+/+ mice (). Second, Hsf1-/- mice exhibited a marked reduction in tumor incidence (Hsf1+/+ 93.1% versus Hsf1-/- 60.9% at week 24, p = 0.0047, chi-square test) (). Third, they had a much lower overall tumor burden. This applied both to the number of tumors that arose () and to the size the tumors achieved (). Fourth, and most importantly, Hsf1-/- mice survived much longer than their wild-type counterparts ().
HSF1 Deficiency Suppresses Chemical Skin Carcinogenesis
To further investigate the extraordinary resistance of Hsf1-/-
mice to carcinogen-induced tumorigenesis, an independent experiment was performed in which Hsf1-/-
mice and their wild-type littermates were treated with a second mutagen, NMMG (N-methyl-N’-nitro-N-nitrosoguanidine), 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 tumors formed in the Hsf1+/+
mice, the percentage 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. Furthermore, 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 malignancies (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 development of tumors in mice carrying a germline mutation in the tumor suppressor p53
, the most frequently mutated gene in human cancers. We crossed mice heterozygous for a clinically relevant hot spot mutation (p53R172H
; Olive et al., 2004
) with Hsf1+/-
mice. Three genotypes, all heterozygous for p53 R172H,
were examined: (1) Hsf1+/+
, (2) Hsf1+/-
, and (3) Hsf1-/-
. Mice were allowed to age with no intervention and were monitored for tumor formation and overall survival. Moribund mice were sacrificed and subjected to full necropsy to detect potential tumor formation at the gross and microscopic levels in all major tissues and organs.
Tumor-free survival was dramatically prolonged in Hsf1-/- mice carrying a mutant p53 allele (). Strikingly, even Hsf1+/- mice survived longer than the Hsf1+/+ mice, indicating a dosage-dependent effect of HSF1. Median survivals were 427 days for Hsf1+/+ mice, 470 days for Hsf1+/- , and >622 days for Hsf1-/- mice. Thus, as with the skin carcinogenesis model, Hsf1-deficient mice had a surprising and profound survival advantage compared to wild-type animals.
HSF1 Deficiency Suppresses Tumorigenesis Driven by Mutant p53
Nature of p53 Mutant Tumors
Histopathological review revealed that both Hsf1+/+ and Hsf1+/- mice produced a broad spectrum of tumor types when carrying the p53R172H hot spot mutation. These included sarcomas, lymphomas, and carcinomas (). Intriguingly, in this model, compromise of Hsf1 function appeared to alter the distribution of tumor types. Hsf1+/- mice had an increase in carcinoma frequency and a decrease in sarcoma frequency compared to Hsf1+/+ mice (). Note, however, that changes in the tumor spectrum of Hsf1-/- mice could not be determined because so very few mice of this genotype developed tumors.
Hsf1 Status Does Not Alter Intrinsic Cell Growth Rates
To pursue observations made in mice at a cellular and molecular level, we examined the effect of Hsf1
status on several classical parameters of neoplastic transformation in cell culture. First, we examined freshly isolated mouse embryonic fibroblasts (MEFs). Hsf1+/+
cells had comparable growth rates in vitro (data not shown). Moreover, staining cells for DNA content followed by flow cytometry revealed similar cell-cycle profiles in both genotypes (Figure S1B
). Thus, the dramatic resistance of Hsf1-/-
mice to tumor formation was not due to an intrinsic defect in cell proliferation or cell-cycle progression. Next, we used MEFs that had been previously immortalized, but not transformed, by stable expression of the E6 and E7 proteins of human papilloma virus (HPV)(McMillan et al., 1998
) and transformed them with a variety of oncogenes.
HSF1 Enables Cellular Transformation Initiated by Oncogenic RAS
To directly investigate susceptibility to transformation by RAS, MEFs were transduced with retroviruses encoding green fluorescent protein (GFP), mouse Hsf1, or oncogenic H-RASV12D, a RAS mutation commonly found in human cancers. After several weeks in culture, cells were fixed and stained to visualize the transformed foci that had arisen as a consequence of the classic RAS-mediated loss of contact inhibition of growth.
Transduction with H-RASV12D
induced high rates of focus formation in wild-type cells, but such foci were rare in cells derived from mice carrying the germline deletion (Hsf1-/-
cells; and Figure S2A
). Cells from both the abundant Hsf1+/+
foci and the rare Hsf1-/-
foci were fully transformed as measured by soft agar cloning and tumor formation following subcutaneous injection into nude mice (data not shown). As expected, transduction with virus encoding GFP did not induce focus formation. Importantly, focus formation was also not observed with Hsf1
overexpression ( and Figure S2A
). Thus, by this criterion, Hsf1
acts as a powerful modifier of tumorigenesis rather than as an oncogene per se.
HSF1 Enables Cellular Transformation Initiated by Oncogenic RAS and PDGF-B
Reduced transformation of Hsf1-/-
cells was not due to an intrinsic growth defect, nor was it due to reduced viral transduction efficiency. Both immortalized cell types displayed comparable saturation densities (, mean ± SD, Hsf1+/+
2832 ± 267 nuclei per field versus Hsf1-/-
3411 ± 275, n = 5, p = 0.0096) and proliferation characteristics (Figure S4
). If anything, Hsf1-/-
MEFs displayed slightly greater gene transfer efficiencies as measured by flow cytometry after transduction with GFP-encoding retrovirus (mean ± SD, Hsf1+/+
, 9.0% ± 0.6% positive versus Hsf1-/-
, 12.7% ± 1.4% positive, n = 4, p = 0.003).
To control for the unlikely possibility that reduced transformation in Hsf1-/-
MEFs was due to a potent but unknown polymorphism that happened to be closely linked to the HSF1 gene, we took advantage of short hairpin RNA interference (shRNAi) technology. Independent stable Hsf1
knockdown cell lines were generated from Hsf1+/+
MEFs using five different lentiviral vectors, each encoding a distinct Hsf1
-targeted sequence. Two knockdown lines with differential gene silencing, C2 and C3, were chosen for further experiments. C3 cells, in which HSF1 levels were only partially reduced, behaved like wild-type cells in the RAS
transformation assay. Isogenic C2 cells, in which HSF1 levels were dramatically reduced, behaved like Hsf1-/-
MEFs from the germline knockout (Figures S3A-S3C
HSF1 Enables Cellular Transformation by the Proto-Oncogene PDGF-B
To determine whether transformation-permissive effects of HSF1 at the cellular level extend beyond activating mutations of H-RAS
, we tested another potent protooncogene: platelet-derived growth factor B (PDGF-B
). As with RAS, Hsf1-/-
MEFs displayed dramatic resistance to focus formation induced by PDGF-B
compared to Hsf1+/+
cells ( and Figure S2B
). These findings, too, were confirmed with shRNAi experiments (Figures S3D-S3F
). Thus, HSF1 also exerts a marked effect on cellular transformation initiated by overexpression of PDGF-B, an oncoprotein that activates multiple signaling cascades in addition to the RAS/MAPK pathway.
HSF1 Enhances Proliferation and Survival in Response to Diverse Oncogenic Stimuli
Neoplastic stimuli can increase the rates of cell proliferation, cell survival, or both. To determine which of these processes is influenced by HSF1, immortalized Hsf1+/+ and Hsf1-/- MEFs were transduced with retroviruses encoding GFP or several mechanistically distinct oncogenes. Retroviral transduction of H-RAS and PDGF-B drove a marked increase in cell number in Hsf1+/+ cells, but not in Hsf1-/- cells (). This was due to increased proliferation of Hsf1+/+ cells rather than increased death in Hsf1-/- cells ().
Unlike RAS and PDGF-B, which act as mitogenic signal transducers, c-MYC and LTA act primarily as regulators of cell-cycle progression and might not be expected to dramatically increase proliferation in these already-immortalized cell lines. Indeed, c-MYC
and SV40 Large T Antigen
) did not significantly increase cell accumulation () or induce focus formation (data not shown). LTA and c-MYC can, however, predispose cells to apoptosis (Evan et al., 1992
; Yin et al., 1997
). Indeed, in contrast with RAS
sharply increased cell death in Hsf1-/-
cultures, but not in Hsf1+/+
cultures (). Thus, depending upon the nature of the oncogenic stimuli involved, HSF1 enables oncogenic transformation in at least two ways, by permitting increased cell proliferation and/or by decreasing cell death.
HSF1 Modulates Signal Transduction
The ability to sustain dysregulated signaling is crucial to human cancers. In light of our observation that Hsf1-/- MEFs are resistant to RAS-driven transformation, we sampled downstream effectors in the RAS/MAPK signaling pathways. HSF1 deficiency caused a marked reduction in the levels of kinase suppressor of RAS 1 (KSR1) protein, both in Hsf1-/- MEFs and in shRNAi knockdown lines (). Furthermore, activation of the downstream effector, ERK, was blunted in Hsf1-/- MEFs following serum stimulation ().
HSF1 Modulates Signal Transduction
We also asked whether Hsf1
affects the G protein-coupled receptor (GPCR) pathway, which increases cAMP levels, drives PKA activation, and is implicated in many human cancers (Bossis et al., 2004
). A marked reduction in the phosphorylation of endogenous PKA substrates was observed in cells with germline Hsf1
deletion (). shRNAi-mediated Hsf1
knockdown in wild-type cells produced a similar effect. (In this case, dosage sensitivity was apparent.) These differences in PKA activity were verified by directly measuring the phosphorylation of a standard peptide substrate. Lysates from Hsf1-/-
cells demonstrated less than half the PKA activity of lysates from Hsf1+/+
cells (mean ± SD, Hsf1+/+
2452 ± 451 versus Hsf1-/-
917 ± 100, n = 2, p < 0.05) (). Having found that HSF1 modulates at least two classical oncogenic signaling pathways, we asked if it affects other crucial, but more recently recognized, cancer-related processes: ribosomal biogenesis and translation control.
HSF1 Modulates the Translation Machinery
The dysregulated growth of cancer cells requires growth factor independence in the control of ribosome biogenesis and protein translation. To reveal a potential role for HSF1 in this process, we cultured MEFs under growth factor-depleted conditions—that is, serum starvation. HSF1 status caused no difference in the levels of eIF4E, an mRNA capbinding protein, β-actin, or glyceraldehyde-3-phosphate dehydrogenase (GAPDH). However, HSF1-deficient MEFs consistently had reduced levels of the three ribo-somal subunits tested, L26RP, L28RP, and S6RP, whether they were deficient from germline knockout or from an shRNAi (). Furthermore, much lower levels of phosphorylated ribosomal protein S6 kinase (p70 S6K), a potent regulator of translational activity, were observed in Hsf1-deficient cells compared to wild-type cells (). Importantly, in both germline-deleted and shRNAi knockdown cells, comparable levels of these ribosomal proteins were restored when the cells were returned to culture in serum-replete medium (). Thus, HSF1 deficiency enforces tighter growth factor dependency on the translational machinery.
HSF1 Modulates Translation Machinery
Inhibition of mTOR function by rapamycin impairs protein translation and reduces cell size. Eliminating its downstream effector, p70 S6K, recapitulates this phenotype (Fingar et al., 2002
). The lower levels of p70 S6K phosphorylation in Hsf1
-deficient cells, together with the smaller size of Hsf1-/-
mice (Xiao et al., 1999
; and data not shown) led us to ask if individual cells derived from Hsf1-/-
mice are smaller than Hsf1+/+
cells. Indeed, the mean cell volume (pL) of Hsf1-/-
MEFs was 20% less than that of Hsf1+/+
MEFs (1925 ± 49 versus 2401 ± 184, mean ± SD, n = 3, p < 0.05). This suggested that the mTOR pathway might be affected by Hsf1
status. Indeed, Hsf1-/-
cells were significantly more sensitive than Hsf1+/+
cells to rapamycin-induced growth inhibition ( and Figure S5A
). The hypersensitivity of Hsf1-/-
cells was not due to increased cell death (data not shown). Instead, rapamycin caused the same type of cell-cycle arrest in G1
typical of mTOR inhibition, but it was more profound in Hsf1-/-
than in Hsf1+/+
cells (). Notably, rapamycin neither induced a heat shock response nor impaired it (Figure S5B
). Thus, independently of a classic proteotoxic stress response, HSF1 maintains the activity of the translation machinery and permits continued cell-cycle progression in immortalized but nontransformed cells under growth factor-reduced conditions in a manner that likely involves the role of mTOR in regulating protein translation.
HSF1 Modulates Glucose Metabolism
Unlike normal cells, virtually all cancer cells preferentially catabolize glucose by glycolysis, even under normoxic conditions, and thereby produce high levels of lactic acid (Bissell et al., 1976
; Gatenby and Gillies, 2004
). Recent evidence indicates that increased glycolysis is a consequence of oncogenic transformation and is advantageous to tumor growth and survival (Fantin et al., 2006
; Matoba et al., 2006
). To determine whether Hsf1
status alters glucose metabolism, we first examined glucose uptake, which is almost universally increased in cancers.
Hsf1+/+ and Hsf1-/- MEFs were cultured overnight in the presence of a fluorescent, noncleavable glucose analog, 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyglucose (2-NBDG). Hsf1-/- MEFs accumulated much less 2-NBDG than did Hsf1+/+ cells (). Hsf1 knockdown MEFs exhibited the same trend, although greater variability was observed, as expected in the heterogeneous population of knockdown cells ().
HSF1 Modulates Glucose Metabolism
Tumor cells, which utilize glucose at a much higher rate than normal cells, are generally more sensitive to glucose deprivation. We asked if Hsf1-/-
cells are less addicted to glucose and less sensitive to its deprivation. Culturing Hsf1+/+
MEFs for 4 days in glucose-reduced medium led to a drastic decrease in total cell number and cell viability relative to cells cultured in standard high-glucose medium (). Hsf1-/-
MEFs tolerated low glucose conditions much better. Thus, HSF1 deficiency leads to reduced dependence on glucose to support cellular energy needs and/or more efficient use of the sugar. Two other findings support this hypothesis. First, the nonmetabolizable glucose analog 2-deoxy-glucose (2-DG), which competes with glucose for the key glycolytic enzyme phosphohexose isomerase, killed Hsf1+/+
cells more efficiently than Hsf1-/-
cells (Figure S6A
). Second, under glucose-replete conditions, Hsf1-/-
and shRNAi C2 cells generated significantly less lactate and possessed lower lactate dehydrogenase (LDH) activity than Hsf1+/+
cells (Figures S6B and S6C
). Thus, the resistance of Hsf1
-deficient cells to malignant transformation that we have demonstrated is associated with a pattern of basal glucose metabolism that could make it more difficult for them to undergo the glycolytic shift characteristic of cancers.
HSF1 Maintains the Transformed Phenotype
Having established in whole mice and cultured mouse cells that HSF1 is required in multifaceted ways for the initiation of transformation, we asked if it was also required for the maintenance of transformed phenotypes in established oncogenic cell lines and, at the same time, whether it was relevant to human cancers. We chose human lines of varying malignant potential, lines created in the laboratory with known oncogenes and lines derived from naturally occurring tumors with a diversity of histopathological origins and a broad spectrum of molecular genetic abnormalities. To modulate HSF1 expression, we evaluated three independent human HSF1
-targeted shRNAi constructs, hA8, hA9, and hA6, and two control constructs (a GFP-targeted shRNAi and an shRNAi with no known homology within the human genome), all of the same infectious titers (Figure S8
). Only the three HSF1-targeted vectors lowered HSF1 levels (); the most efficacious, hA6 and hA9, were selected for further analysis.
HSF1 Is Required for Maintenance of the Transformed Phenotype
We first examined three breast cell lines representing progressively more oncogenic states (): (1) primary human mammary epithelial (PHME) cells; (2) human mammary epithelial (HME) cells made immortal, but nontumorigenic, by expression of hTERT
(telomerase); and (3) HME cells rendered fully transformed and tumorigenic by introduction of LTA
in addition to hTERT
(HMLER) (Elenbaas et al., 2001
). PHME cells were little affected by transduction with HSF1
-targeted constructs. Tumorigenic HMLER cells were strongly affected. Immortalized, but nontumorigenic, cells (HME) were intermediate in sensitivity, suggesting a correlation between oncogenic state and HSF1 dependence ().
Next we examined a diverse collection of breast cell lines derived from spontaneous human tumors (). The lines varied with regard to p53 status, carrying wild-type (MCF-7) or various mutant alleles (BT-474, MDA-MB-231, and T47D) (International Agency for Research on Cancer TP53 Database) and with regard to HER2 overexpression, estrogen sensitivity, and metastatic potential (MD Anderson Breast Cancer Cell Line Database). All were strongly affected by both of the HSF1-inhibitory hairpins; none was affected by the control hairpins.
Finally, we examined malignant cells of diverse histological origins either derived from human tumors (HeLa [cervix], PC-3 [prostate], and S462 and 90-8 [peripheral nerve sheath]) or derived by in vitro transformation (293T [kidney]). All were strongly inhibited by HSF1 knockdown (). Where a difference in sensitivity to the two targeting hairpins was observed, it was always the hairpin that inhibited HSF1 expression the most severely (hA6; ) that had the stronger effect (). This strong correlation between the extent of HSF1 inhibition and phenotypic effects, together with the fact that similar results were obtained with independent human and mouse targeting sequences and with a mouse germline knockout, argue strongly against “off-target” factors being responsible for these effects. Note also that the hairpins had no effect on normal diploid human fibroblasts (WI-38; ). Therefore, we conclude that, in addition to its enabling role in tumor initiation, HSF1 function helps to maintain the growth and survival of human cancer cells with diverse underlying malignant defects.