Inhibition of Heat Shock Factor 1 Signaling Decreases Hepatoblastoma Growth via Induction of Apoptosis

Abstract

Although rare compared with adult liver cancers, hepatoblastoma (HB) is the most common pediatric liver malignancy, and its incidence is increasing. Currently, the treatment includes surgical resection with or without chemotherapy, and in severe cases, liver transplantation in children. The effort to develop more targeted, HB-specific therapies has been stymied by the lack of fundamental knowledge about HB biology. Heat shock factor 1 (HSF1), a transcription factor, is a canonical inducer of heat shock proteins, which act as chaperone proteins to prevent or undo protein misfolding. Recent work has shown a role for HSF1 in cancer beyond the canonical heat shock response. The current study found increased HSF1 signaling in HB versus normal liver. It showed that less differentiated, more embryonic tumors had higher levels of HSF1 than more differentiated, more fetal-appearing tumors. Most strikingly, HSF1 expression levels correlated with mortality. This study used a mouse model of HB to test the effect of inhibiting HSF1 early in tumor development on cancer growth. HSF1 inhibition resulted in fewer and smaller tumors, suggesting HSF1 is needed for aggressive tumor growth. Moreover, HSF1 inhibition also increased apoptosis in tumor foci. These data suggest that HSF1 may be a viable pharmacologic target for HB treatment.

Figure 1

Evaluation of mouse hepatoblastoma model for heat shock factor 1 (HSF1) and activation of downstream genes. Representative images of immunohistochemistry for HSF1 in mouse livers transfected with β-catenin and yes-associated protein 1 (β-Y) plasmids, and wild-type controls showing staining in tumor foci and no staining in wild-type livers. Original magnification, ×10.

Figure 2

Expression of heat shock factor 1 (HSF1) in overall tumors and in more fetal- or embryonic-appearing tumors. A: Expression levels of HSF1 in tumors versus control livers in six transcriptomic data sets: GSE131329 (https://www.sciencerepository.org/gene-expression-profiling-in-hepatoblastoma-cases-of-the-japanese-study-group-for-pediatric-liver-tumors-2-jplt-2-trial_EJMC-2018-1-103), GSE75271 (https://aasldpubs.onlinelibrary.wiley.com/doi/10.1002/hep.28888), E-MEXP-1851 (https://www.ebi.ac.uk/arrayexpress/experiments/E-MEXP-1851), GSE81928 (https://www.nature.com/articles/s42003-018-0077-8), GSE104766 (https://aasldpubs.onlinelibrary.wiley.com/doi/10.1002/hep.29672), and GSE89775 (https://www.nature.com/articles/srep38347). Levels were compared using unpaired t-test for each of the six sets. A meta P value was then calculated across all sets based on Chang et al (meta P = 3 × 10⁻⁵). B: Expression levels of HSF1 in tumors differentiated by degree of differentiation, as based on Cairo et al. C1 tumors are more fetal appearing, whereas C2 tumors are more embryonic appearing. Expression levels were compared using unpaired t-test. Meta P value was calculated across all sets (meta P = 2 × 10⁻⁸). ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.

Figure 3

Correlation between heat shock factor 1 (HSF1) expression levels and mortality. Three of the examined transcriptomic data sets featured mortality data: GSE131329 (https://www.sciencerepository.org/gene-expression-profiling-in-hepatoblastoma-cases-of-the-japanese-study-group-for-pediatric-liver-tumors-2-jplt-2-trial_EJMC-2018-1-103), GSE75271 (https://aasldpubs.onlinelibrary.wiley.com/doi/10.1002/hep.28888), and E-MEXP-1851 (https://www.ebi.ac.uk/arrayexpress/experiments/E-MEXP-1851). Expression levels of HSF1 in tumors were ordered and divided into quartiles (Qs). The mortality rate in the quartile with the highest HSF1 expression was compared with the lowest three quartiles using the Fisher exact test for each data set. Mortality was significantly elevated in the highest quartile in one set (GSE131329). A meta P value was then calculated across all sets (meta P = 0.0027). ∗∗P < 0.01.

Figure 4

Analysis of livers in mice transfected with a dominant negative for heat shock factor 1 (HSF1) along with constitutively active β-catenin and yes-associated protein 1 (YAP1) plasmids (dnHSF1-β-Y mice) and mice transfect with just β-catenin and YAP1 (β-Y mice) as control. A: Representative gross images of mice transfected with β-catenin and YAP1 plasmids (left panel), dominant negative HSF1 (dnHSF1), β-catenin and YAP1 plasmid (middle panel), and wild-type control (right panel). B: Top panel: Average body weights of dnHSF1-β, β-Y, and wild-type mice analyzed by one-way analysis of variance. Bottom panel: Liver/body weight ratio was calculated and then compared by one-way analysis of variance. C: Representative images for hematoxylin and eosin (H&E) staining for β-Y mice and dnHSF1-β-Y mice (right panels) and immunohistochemistry for Myc-tag, an artificial tag on the β-catenin plasmid (left panels). ∗∗∗P < 0.001. Original magnification, ×10 (C).

Figure 5

Verification of transfection at 2 weeks after injection. Livers from both constitutively active β-catenin and yes-associated protein 1 (YAP1) plasmids (dnHSF1-β-Y) and β-catenin and YAP1 (β-Y) mice were harvested 2 weeks after injection with the plasmids. Livers from both sets of mice were stained with hematoxylin and eosin (H&E; left panels), and immunohistochemistry was performed for Myc-tag (middle panels) and V5-tag (right panels). V5-tag is an artificial tag on the dnHSF1 plasmid. Original magnification, ×20.

Figure 6

A: Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay and immunohistochemistry for cleaved caspase 3 were performed on β-catenin and yes-associated protein 1 (YAP1; β-Y) and constitutively active β-catenin and YAP1 plasmid (dnHSF-β-Y) livers from mice harvested 2 weeks after transfection. B: TUNEL and cleaved caspase 3 staining was quantified by counting the number of positively stained cells in at least 20 high-powered fields per condition. The average number of positive cells in β-Y and dnHSF-β-Y livers was compared using unpaired t-test. ∗P < 0.05. Original magnification, ×10 (A).

Supplemental Figure S1

Representative images of immunohistochemistry for glutamine synthetase (GS) and phosphorylated mammalian target of rapamycin (Phos-mTOR) in β-catenin and yes-associated protein 1 (YAP1; β-Y) and constitutively active β-catenin and YAP1 plasmid (dnHSF-β-Y) livers. Original magnification, ×10.

Supplemental Figure S2

Assessing how the dominant negative heat shock factor 1 (dnHSF1) plasmid impacts expression of heat shock protein (HSP) 70, an HSF1 target gene. HepG2 cells were transfected with the dnHSF1 plasmid and green fluorescent protein (GFP) as control. Cells were assessed both under standard incubator conditions and after heat shock (42°C for 60 minutes, followed by 30 minutes of recovery). Expression of HSP70 was measured via quantitative PCR and normalized to glyceraldehyde-3-phosphate dehydrogenase and 18S expression. ∗P < 0.05.