Heat Shock Transcription Factor 4 Research Articles

Abstract PO3-24-01: Mapping inter- and intra-tumor heterogeneity in Ductal Carcinoma in situ and invasive breast cancer using integrative multi-omic profiling

Abstract Background: Molecular profiling of ductal carcinoma in situ (DCIS) has shown some prognostic utility in the clinic. However, there is still an incomplete understanding of the diversity of molecular mechanisms by which DCIS progresses to invasive breast cancer (IBC).Here, we utilize integrative multi-omic profiling of co-occurring DCIS and IBC in humans as a model for mapping the relationship between tumor mutations, global gene expression and morphological changes in DCIS and IBC. Methods: We performed targeted panel DNA sequencing (Tempus xT), whole transcriptome profiling and hyperplex immunofluorescence (Lunaphore COMET) on a cohort of 39 pairs of co-occurring DCIS and IBC lesions. We performed an integrative analysis of the above multi-modal data to uncover similar and dissimilar molecular trajectories in DCIS/IBC pairs. DAVID, KEGG and Reactome databases were utilized to analyze differential pathway activity between DCIS and IBC lesions. Results: IBC samples significantly overexpressed pathways associated with cycling cells and proliferation. For DCIS, many of the enriched pathways were related to CYP enzymes and xenobiotic metabolism. We also assessed enrichment of specific transcription factor binding sites (TFBS) with the associated differentially expressed genes. The top site enriched in IBC was a promoter regulatory element of unknown function (M120 motif KRCTCNNNNMANAGC) as well as sites regulated by heat shock transcription factor 4 (HSF4). Top TFBS enriched in DCIS included MEF2, HMEF2, AR and CEBPE. The top pathways associated with IBC using DAVID were related to extracellular matrix (ECM) organization and regulation. Interestingly, the top pathways associated with DCIS also included several pathways associated with ECM organization. On Reactome pathways diagrams for ECM formation distinct components of ECM formation and organization were enriched in IBC vs DCIS lesions, with invadopodia formation components and proteoglycans specifically enriched in IBC and integrin/laminin signaling enriched in DCIS. Among the 39 cases profiled, we observed distinct patterns of DCIS/IBC pairs with highly similar mutational profiles indicative of derivation from a shared progenitor as well as pairs with little-to-no molecular concordance that likely arose independently. Even within mutationally similar DCIS/IBC pairs there were different degrees of gene expression concordance. We subdivided mutationally similar pairs into two groups based on gene expression similarity (Euclidean Distance) and identified 115 genes that differed between these groups (q < 0.05), with the dissimilar DCIS lesions hallmarked by upregulation of a variety of pathways related to B-cell function, complement cascade and cell cycle progression. Specific mutations also were related to conserved transcriptional programs in these lesions, with MCL-1 amplified tumors showing a common gene expression signature of 357 altered genes (q < 0.05) with key differences in extracellular matrix organization and adaptive immune response pathways. Dissimilarly, tumors with CKS1B copy number alterations associated with 627 differentially expressed genes (q < 0.05) with the top targets related to endothelial function and angiogenesis. Conclusions: Integrative multi-modal analysis helps us better understand tumor progression at multiple levels of biomolecular dysregulation and shows the complex interplay among genome alterations, gene expression reprogramming and tissue morphology. Such approaches may yield future biomarker signatures that better encapsulate the diverse mechanisms by which DCIS lesions evolve. Citation Format: Henry Kaplan, Alexa Dowdell, Racheli Ben Shimoli, Angela Crabtree, Ann Berry, Fred Robinson, Christopher Carney, Carlo Bifulco, Brian Piening. Mapping inter- and intra-tumor heterogeneity in Ductal Carcinoma in situ and invasive breast cancer using integrative multi-omic profiling [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-24-01.

Plant-Derived Extracts Plus Vitamin E and/or Aloe Vera Protect Against Intrinsic/Extrinsic Stressor in Human Skin: In Vitro and Clinical Evidence.

Humans are exposed to physical, biological, chemical, and psychological stressor throughout their life span. In recent years many medicinal plants have been shown to induce stress adapting and protective functions. Plant-derived extracts and vitamin E exhibit stress protection or resistance by normalizing cellular homeostasis and enhancing resistance to toxic stimuli to overcome cellular damage. Here we report the evaluation of a topical preparation (product test materials; PTM) containing an ingredient blend of Rhodiola Rosea, Eleutherococcus Senticosus (Siberian Ginseng), Rhaponticum Carthamoides, Inonotus Obliqus, and Slegainella Lepidophylla as the base formula and tested the addition of Lespedeza Capitata (leaf/stem) extract plus vitamin E and/or Aloe Vera to determine the induced protective functions in human skin when challenged with intrinsic and extrinsic stressors. The base topical preparation plus Lespedeza Capitata extract plus vitamin E or the base topical preparation plus vitamin E and Aloe Vera were assayed in vitro on (a) intrinsically stressed excised abdominoplasty skin, (b) full thickness (FT) skin equivalent models post-treated with a combination of ultra-violet (UV) B light (250 mJ/cm2) and diesel particular matter (DPM) (75 µg/mL) skin, for their effect on antioxidant, inflammation, and stress biomarker geners. Additionally, the bioadaptive activity of the PTMs was confirmed in providing resilience and protection against UV-induced erythema. For example, in a clinical study, daily topical application of the PTMs on the buttocks of 20 woman (18-78 years old), average age of 51.1 years, median body mass index (BMI) of 26.5 for 8 weeks followed by 2 minimal erythema dose (MED) of UVB exposure was accessed 24 hours after irradiation. Statistical analysis was performed by t-test and ANOVA, repectively. Pretreatment with the topical PTMs on intrsinically stressed skin significantly reduced the expression of the stress gene biomarkers, p53, pro-inflammatory cytokines Interleukin-1β (IL-1β) and Tumor Necrosis Factor-α (TNFα) and the pro-apoptotic BCL2 associated X, apoptosis regulator (BAX) values compared to controls. Topical application of the PTMs on Full Thickness (FT) human skin treated with UVB light and DPM significantly enhanced the stress response by activating heat shock transcription factor 4 (HSF4) and heat shock protein family B (small) member 1 (HSPB1) gene levels belonging to the heat shock protein (HSP) family by significantly increasing the expression of heme oxygenase 1 (HMOX1). At the same time, significantly reducing IL-1β levels were observed plus protection of skin cells from toxicity ocurred by significantly increasing the expression of B-cell lymphoma 2 (BCL2) (anti-apoptotic gene). In the clinical study, daily topical applications of the PTMs for 8 weeks followed by 2MED of UVB irradiation with clinical assessment 24 hours later revealed a significantly reduced intensity of erythema when compared to the buttock region treated with UVB alone. The PTMs containing adaptogen ingredients may confer stress resistance and induce stress protective responses against intrinsic as well as extrinsic stressors as demonstrated by the obtained in vitro and clinical evidence.

Bioinformatics analysis of microarray data to explore the key genes involved in HSF4 mutation-induced cataract.

To reveal the mechanisms of heat-shock transcription factor 4 (HSF4) mutation-induced cataract. GSE22362, including 3 HSF4-null lens and 3 wild-type lens, was obtained from Gene Expression Omnibus database. After data preprocessing, the differentially expressed genes (DEGs) were identified using the limma package. Based on Database for Annotation, Visualization and Integrated Discovery (DAVID) tool, functional and pathway enrichment analyses were performed for the DEGs. Followed by protein-protein interaction (PPI) network was constructed using STRING database and Cytoscape software. Furthermore, the validated microRNA (miRNA)-DEG pairs were obtained from miRWalk2.0 database, and then miRNA-DEG regulatory network was visualized by Cytoscape software. A total of 176 DEGs were identified in HSF4-null lens compared with wild-type lens. In the PPI network, FBJ osteosarcoma oncogene (FOS), early growth response 1 (EGR1) and heme oxygenase (decycling) 1 (HMOX1) had higher degrees and could interact with each other. Besides, mmu-miR-15a-5p and mmu-miR-26a-5p were among the top 10 miRNAs in the miRNA-DEG regulatory network. Additionally, mmu-miR-26a-5p could target EGR1 in the regulatory network. FOS, EGR1, HMOX1, mmu-miR-26a-5p and mmu-miR-15a-5p might function in the pathogenesis of HSF4 mutation-induced cataract.

Autosomal recessive congenital cataract in captive-bred vervet monkeys (Chlorocebus aethiops).

The aim of the study was to evaluate the genetic predisposition of congenital cataract in a colony of captive-bred vervet monkeys. Four congenital cataract genes: glucosaminyl (N-acetyl) transferase 2 (GCNT2), heat shock transcription factor 4 (HSF4), crystallin alpha A (CRYAA) and lens intrinsic membrane protein-2 (LIM2) were screened, sequenced and analysed for possible genetic variants in 36 monkeys. Gene expression was also evaluated in these genes. Fifteen sequence variants were identified in the coding regions of three genes (GCNT2, HSF4 and CRYAA). Of these variations, only three were missense mutations (M258V, V16I and S24N) and identified in the GCNT2 transcripts A, B and C, respectively, which resulted in a downregulated gene expression. Although the three missense mutations in GCNT2 have a benign effect, a possibility exists that the candidate genes (GCNT2, HSF4 and CRYAA) might harbour mutations that are responsible for total congenital cataract.

A novel missense mutation in HSF4 causes autosomal-dominant congenital lamellar cataract in a British family.

PurposeInherited cataract, opacification of the lens, is the most common worldwide cause of blindness in children. We aimed to identify the genetic cause of isolated autosomal-dominant lamellar cataract in a five-generation British family.MethodsWhole exome sequencing (WES) was performed on two affected individuals of the family and further validated by direct sequencing in family members.ResultsA novel missense mutation NM_001040667.2:c.190A>G;p.K64E was identified in the DNA-binding-domain of heat-shock transcription factor 4 (HSF4) and found to co-segregate with disease.ConclusionWe have identified a novel mutation in HSF4 in a large British pedigree causing dominant congenital lamellar cataract. This is the second mutation in this gene found in the British population. This mutation is likely to be dominant negative and affect the DNA-binding affinity of HSF4.

HSF4 regulates lens fiber cell differentiation by activating p53 and its downstream regulators

Cataract refers to opacities of the lens that impede the passage of light. Mutations in heat shock transcription factor 4 (HSF4) have been associated with cataract; however, the mechanisms regarding how mutations in HSF4 cause cataract are still obscure. In this study, we generated an hsf4 knockout zebrafish model using TALEN technology. The mutant zebrafish developed an early-onset cataract with multiple developmental defects in lens. The epithelial cells of the lens were overproliferated, resulting in the overabundance of lens fiber cells in hsf4null zebrafish lens. Consequently, the arrangement of the lens fiber cells became more disordered and irregular with age. More importantly, the terminal differentiation of the lens fiber cell was interrupted as the organelles cannot be cleaved in due time. In the cultured human lens epithelial cells, HSF4 could stabilize and retain p53 in the nucleus to activate its target genes such as fas cell surface death receptor (Fas) and Bcl-2-associated X apoptosis regulator (Bax). In the hsf4null fish, both p53 and activated-caspase3 were significantly decreased. Combined with the finding that the denucleation defect could be partially rescued through microinjection of p53, fas and bax mRNA into the mutant embryos, we directly proved that HSF4 promotes lens fiber cell differentiation by activating p53 and its downstream regulators. The data we presented suggest that apoptosis-related genes are involved in the lens fiber cell differentiation. Our finding that HSF4 functions in the upstream to activate these genes highlighted the new regulatory modes of HSF4 in the terminal differentiation of lens fiber cell.

HSF4 promotes G1/S arrest in human lens epithelial cells by stabilizing p53

The differentiation from constantly dividing epithelial cells into secondary fiber cells is a key step during lens development. Failure in this process, which requires cell proliferation inhibition and cell cycle exit, causes cataract formation. HSF4 (Heat Shock Transcription Factor 4) gene mutations may lead to both congenital and senile cataract. However, how HSF4 mutations induce cataract formation remains obscure. In this study, we demonstrate that HSF4 can suppress the proliferation of human lens epithelial cells (HLECs) by promoting G1/S arrest in a p53-dependent manner. In contrast, HSF4 with cataract causative mutations fail to cause cell cycle arrest and have no obvious effect on cell proliferation. We further identify that HSF4 recruits p53 in the nucleus and promotes its transcriptional activity, leading to the expression of its target gene p21 in HLECs. HSF4, but not its cataract-causing mutants, stabilizes p53 protein and inhibits its ubiquitin degradation. Our data reveal that HSF4 may work as a switch between lens epithelial cell proliferation and secondary fiber cell differentiation, a process which mainly depends on p53. Through demonstration of this novel downstream pathway of HSF4, our results help uncover the pathogenic mechanisms caused by HSF4 mutations.

Expression of the HSF4 DNA binding domain-EGFP hybrid gene recreates early childhood lamellar cataract in transgenic mice.

The clinical management of cataracts in infancy involves surgical removal of the lens to ensure transmission of light to the retina, which is essential for normal neural development of the infant. This surgery, however, entails a lifelong follow-up and impaired vision. To our knowledge, no animal models recapitulate human lamellar opacities, the most prevalent form of early childhood cataracts. We present data on the recreation of the human lamellar cataract phenotype in transgenic mice. Mutations in the DNA binding domain (DBD) of the heat shock transcription factor 4 (HSF4) are known to be associated with early childhood autosomal dominant lamellar cataract. We used bacterial artificial chromosome (BAC) transgenesis to express a hybrid gene: Hsf4 (DBD)-enhanced green fluorescent protein (EGFP), by recombineering EGFP sequences into the DBD of the Hsf4 gene, to interfere with the DNA binding properties of Hsf4. We recapitulated the human lamellar cataract, in its temporal as well as spatial presentation, within the transgenic mouse lens. This phenotype was reproduced faithfully using four different BACs, indicating that EGFP can be used to target transcription factor function in transgenic mice. Molecular and cell biological examination of early postnatal transgenic lens reveals impairment of secondary fiber cell differentiation. Recreation of the human lamellar cataract phenotype in mice allows investigation of this human pathology at a level not possible previously and points to the relevance of fiber cell heterogeneity dictated by fiber cell-specific gene activity in the biogenesis of the lamellar cataract.

HSF4 mutation p.Arg116His found in age-related cataracts and in normal populations produces childhood lamellar cataract in transgenic mice.

The p.Arg116His mutation in the heat shock transcription factor-4 (HSF4) has been associated with age-related cataracts, but it is also seen in 2% of the normal population, indicating either reduced penetrance or that the normal subjects were not old enough to express the phenotype. Based on the proximity of p.Arg116His to two known mutations in the DNA-binding domain of HSF4, namely, p.Leu114Pro and p.Arg119Cys, which segregate with childhood lamellar cataract, we tested the possibility that this phenotype may have been missed by the ophthalmologist and/or that it did not spread to the visual axis so as to affect vision significantly. Here, we demonstrate via BAC (bacterial artificial chromosome) transgenesis that p.Arg116His recreates the childhood lamellar cataract in mice suggesting that incomplete penetrance associated with early cataracts may not be an absence but a limitation of the detection of the phenotype.

A Novel HSF4 Gene Mutation Causes Autosomal-Dominant Cataracts in a Chinese Family

Congenital cataracts are a significant cause of visual impairment or blindness in children. One-third of cases estimated to have a genetic cause. We carried out gene analysis and bioinformatics analysis to map the locus and to identify the underlying genetic defect in a 12-member, four-generation Chinese family affected with bilateral congenital cataracts. We screened individuals of the family and discovered a distinct missense mutation in HSF4 (a gene at this locus that encodes teat-shock transcription factor 4). Bioinformatics analysis was used to determine possible changes in the protein structure that could affect the phenotype. Sequencing of the candidate genes showed a heterozygous c.69 G→T change in the heat shock transcription factor 4 (HSF4) gene, which resulted in the substitution of a lysine with an asparagine (p. K23N). This mutation cosegregated with all affected individuals and was not observed in unaffected family members. Bioinformatics analysis indicated that the p. K23N mutation was predicted to be disease causing. This is the first report of the novel missense mutation, c.69 G→T (p. K23N), in exon 3 of the HSF4 locus on 16q21-q22 associated with bilateral congenital cataracts in a Chinese family. This novel mutation could enable propergenetic diagnostics and counseling in affected families and could lead to a better understanding of the structure and function of HSF4 in health and disease.

Molecular mechanism of congenital cataract induced by heat shock transcription factor 4 gene mutation in lop11 mouse

Background Mutation of the heat shock transcription factor 4 （HSF4） gene causes autosomal recessive hereditary cataract in lens opacity locus 11 （lopl I ） mouse, but the molecular mechanism of the pathogenesis has not been determined. Objective This study was to investigate the molecular mechanism of congenital cataract induced by HSF4 gene mutation in lopll mouse. Methods Twenty-four lopll mice and 24 wild type C57BL/6 mice were used in this study. The animals were sacrificed and the lenses were obtained on postnatal days 1,7 and 12. Regular pathological examination was carried out to evaluate the morphological changes of the lens and count the number of lens epithelial ceils （LECs） in the mice. The ctB-Crystallin （CRYAB） content in the lenses was detected in postnatal day 1 mice by Western blot. The expression of the fibroblast growth factor （FGF） mRNA in the lenses was assayed by quantitative PCR （q-PCR）. The data were compared between the lopl 1 mice and wild type C57BL/6 mice with independent sample t test. The use and care of the experimental animals complied with the ARVO statement. Results The morphology and array of LECs were uniform and regular in the wild type C57BL/6 mice, while proliferation of LECs and disorder of lens fibers were seen in the lopll mice on postnatal day 1. On postnatal day 7,vacuolar degeneration of the lens appeared in 7-day-old lopll mice. The numbers of LECs were （417±19）, （467±16） and （489 ±21 ） in lopll mice on the postnatal day 1,7 and 12, respectively, and those of wild type C57BL/6 mice were （ 378 ± 13 ） , （ 391 ±9 ） and （ 395 ±7 ） , respectively, showing statistically significant differences between them （1 day：t=6.696,P=0. O00;7 days：t=6.578,P=0.000;14 days：t=7.240,P=0.000）. The expression of CRYAB in the lenses was evidently weaker in the 1-day-old lopll mice compared with wild type C57BL/6 mice. The relative folds of expression of FGF-1, FGF-4, FGF-7 mRNA in the lenses were significantly higher in the lopll mice than those in the wild type C57BL/6 mice （2.04±0. 13 vs. 1. 037±0.06;2.0±0.08 vs. 0.97± 0.08;4.59±0.12 vs.1.0±0.04） （FGF-1 mRNA：t=14.000,P〈0.001;FGF-4 mRNA：t=15.510,P〈0.01;FGF-7 mRNA：t = 29.41 ,P〈0.01 ）. Conclusions HSF4 mutation leads to the abnormal development of the lens in lopl 1 mice by arresting the expression of cLB-Crystallin protein and increasing the expression of the FGF gene. Key words: Gene mutation; Heat shock transcription factor; Cataract/congenital; Crystallin; Fibroblast growth factor

SKAP2, a novel target of HSF4b, associates with NCK2/F-actin at membrane ruffles and regulates actin reorganization in lens cell

In addition to roles in stress response, heat shock factors (HSFs) play crucial roles in differentiation and development. Heat shock transcription factor 4 (HSF4) deficiency leads to defect in lens epithelial cell (LEC) differentiation and cataract formation. However, the mechanism remains obscure. Here, we identified Src kinase-associated phosphoprotein 2 (SKAP2) as a downstream target of HSF4b and it was highly expressed at the anterior tip of lens elongating fibre cells in vivo. The HSF4-deficient lenses showed reduced SKAP2 expression and defects in actin reorganization. The disassembly of stress fibres and formation of cortical actin fibres are critical for the initiation of LEC differentiation. SKAP2 localized at actin-rich ruffles in human LECs (SRA01/04 cells) and knockdown SKAP2 using RNA interference impaired the disassembly of cellular stress fibres in response to fibroblast growth factor (FGF)-b. Overexpression of SKAP2, but not the N-terminal deletion mutant of SKAP2, induced the actin remodelling. We further found that SKAP2 interacted with the SH2 domain of non-catalytic region of tyrosine kinase adaptor protein 2 (NCK2) via its N-terminus. The complex of SKAP2-NCK2-F-actin accumulated at the leading edge of the lamellipodium, where FGF receptors and focal adhesion were also recruited. These results revealed an essential role for HSF4-mediated SKAP2 expression in the regulation of actin reorganization during lens differentiation, likely through a mechanism that SKAP2 anchors the complex of NCK2/focal adhesion to FGF receptors at the lamellipodium in lens epithelial cells.

Evaluation of canine heat‐shock transcription factor 4 as a candidate for primary cataracts in English Cocker Spaniels and wire‐haired Kromfohrlanders

Canine heat-shock transcription factor 4 (HSF4) was evaluated as a candidate for primary cataracts in English Cocker Spaniels (ECS) and wire-haired Kromfohrlanders (KFL). We sequenced exon 9 and its flanking regions of the HSF4 gene in ECS and KFL but were not able to detect a previously reported insertion mutation associated with primary cataracts in Staffordshire Bull Terriers and Boston Terriers. Another single nucleotide polymorphism (LOC489766:g.3243A>G) in intron 9 was identified and included in linkage and association tests together with two flanking microsatellites. Linkage and association tests with primary cataracts in ECS and KFL were not significant. Therefore, we could not verify that the mutation in exon 9 of canine HSF4 gene is causative for primary cataracts in ECS and wire-haired KFL.

Locus heterogeneity in autosomal recessive congenital cataracts: linkage to 9q and germline HSF4 mutations

Isolated (non-syndromic) congenital cataract may be inherited as an autosomal dominant, autosomal recessive, or X-linked recessive trait. Considerable progress has been made in identifying genes and loci for dominantly inherited cataract, but the molecular basis for autosomal recessive disease is less well defined. Hence we undertook genetic linkage studies in four consanguineous Pakistani families with non-syndromic autosomal recessive congenital cataracts. In two families linkage to a 38 cM region 9q13-q22 was detected. Although a locus for recessive congenital cataracts had not been mapped previously to this region, the target interval encompasses the candidate region autosomal recessive adult-onset pulverulent cataracts (CAAR). The CAAR was mapped previously to 9q13-q22, and may therefore be allelic to non-syndromic autosomal recessive congenital cataracts. The other two families did not demonstrate linkage to 9q, but both had a region of homozygosity at 16q22 containing the heat shock transcription factor 4 (HSF4) gene. The HSF4 mutations have been reported in four families with autosomal dominant cataracts and, recently, in a single kindred with autosomal recessive congenital cataract. Mutation analysis of HSF4 revealed homozygous mutations (p.Arg175Pro and c.595_599delGGGCC, respectively) in the two families. These findings confirm that mutations in HSF4 may result in both autosomal dominant and autosomal recessive congenital cataract, and highlight the locus heterogeneity in autosomal recessive congenital cataract.

Unique contribution of heat shock transcription factor 4 in ocular lens development and fiber cell differentiation

Mammalian ocular lens development results via a differentiation program that is highly regulated by tissue-specific transcription factors. Central to this is the terminal differentiation of fiber cells, which develop from epithelial cells on the anterior surface of the lens, accompanied by a change in cell shape and expression of structural proteins (such as membrane proteins MP19, MIP26, connexin 43, 46, and 50, cytoskeletal proteins CP49, CP115, and alpha, beta, and gamma crystallins), creating a transparent, refractive index gradient in the lens. Mutations in genes controlling eye development and in lens structural protein genes are associated with multiple ocular developmental disorders, including cataracts and other opacities of the lens. Here we show that heat shock transcription factor 4 (HSF4) expression in the developing lens is required for correct lens development and that inactivation of hsf4 leads to early postnatal cataract formation with primary effects specific to terminal fiber cell differentiation. These data suggest that HSF4 acts as a critical transcription factor for lens-specific target gene expression, in particular regulating the small 25 kDa heat shock protein that acts as a modifier for lens opacity and cataract development. Thus, HSF4 fulfills a central role in controlling spatial and temporal expression of genes critical for correct development and function of the lens.