Transcriptional responses to direct and indirect TGFB1 stimulation in cancerous and noncancerous mammary epithelial cells.

BACKGROUND: Transforming growth factor beta (TGFß) is important for the morphogenesis and secretory function of the mammary gland. It is one of the main activators of the epithelial-mesenchymal transition (EMT), a process important for tissue remodeling and regeneration. It also provides cells with the plasticity to form metastases during tumor progression. Noncancerous and cancer cells respond differently to TGFß. However, knowledge of the cellular signaling cascades triggered by TGFß in various cell types is still limited. METHODS: MCF10A (noncancerous, originating from fibrotic breast tissue) and MCF7 (cancer, estrogen receptor-positive) breast epithelial cells were treated with TGFB1 directly or through conditioned media from stimulated cells. Transcriptional changes (via RNA-seq) were assessed in untreated cells and after 1-6 days of treatment. Differentially expressed genes were detected with DESeq2 and the hallmark collection was selected for gene set enrichment analysis. RESULTS: TGFB1 induces EMT in both the MCF10A and MCF7 cell lines but via slightly different mechanisms (signaling through SMAD3 is more active in MCF7 cells). Many EMT-related genes are expressed in MCF10A cells at baseline. Both cell lines respond to TGFB1 by decreasing the expression of genes involved in cell proliferation: through the repression of MYC (and the protein targets) in MCF10A cells and the activation of p63-dependent signaling in MCF7 cells (CDKN1A and CDKN2B, which are responsible for the inhibition of cyclin-dependent kinases, are upregulated). In addition, estrogen receptor signaling is inhibited and caspase-dependent cell death is induced only in MCF7 cells. Direct incubation with TGFB1 and treatment of cells with conditioned media similarly affected transcriptional profiles. However, TGFB1-induced protein secretion is more pronounced in MCF10A cells; therefore, the signaling is propagated through conditioned media (bystander effect) more effectively in MCF10A cells than in MCF7 cells. CONCLUSIONS: Estrogen receptor-positive breast cancer patients may benefit from high levels of TGFB1 expression due to the repression of estrogen receptor signaling, inhibition of proliferation, and induction of apoptosis in cancer cells. However, some TGFB1-stimulated cells may undergo EMT, which increases the risk of metastasis.

Transforming growth factor beta (TGFß) is a multifaceted cytokine that controls numerous physiological and pathological processes during development and carcinogenesis. Its best-known function is the activation of epithelial­mesenchymal transition (EMT), a process crucial for tissue remodeling and regeneration. During EMT, epithelial cells lose their connections to adjacent cells and acquire mesenchymal characteristics, such as migratory ability. Compared with cancer cells, normal (nontumorigenic) cells usually respond differently to TGFß stimulation. Typically, TGFß inhibits the proliferation of epithelial cells and may promote cell death. In cancer cells, TGFß often promotes tumor progression. TheTGFß-mediated induction of EMT increases cell mobility, which is associated with the formation of metastases and tumor invasion.In this work, we compared the transcriptional response of noncancerous (MCF10A) and cancerous (MCF7; estrogen receptor-positive) breast epithelial cells to direct stimulation by TGFB1 and its indirect effect through conditioned media. Some of TGFB1-induced changes, including inhibition of proliferation and induction of EMT, were similar in both cell lines. However, these changes were associated with different upstream signaling pathways. Other changes were more specific, such as disruption of estrogen-related signaling or induction of cell death in MCF7 cells. Direct incubation with TGFB1 and treatment of cells with conditioned media similarly affected target cells, indicating the presence of a bystander effect. Moreover, transcript profiling by RNA-seq revealed that the TGFß signaling pathway is already active in untreated MCF10A cells, which may be due to their origination from a fibrotic lesion.

The human testis-enriched HSPA2 interacts with HIF-1α in epidermal keratinocytes, yet HIF-1α stability and HIF-1-dependent gene expression rely on the HSPA (HSP70) activity.

The Hypoxia-Inducible Factor 1 (HIF-1) is essential for cellular adaptation to reduced oxygen levels. It also facilitates the maintenance and re-establishment of skin homeostasis. Among others, it is involved in regulating keratinocyte differentiation. The stability of the oxygen-liable HIF-1α subunit is regulated by various non-canonical oxygen-independent mechanisms, which among others involve Heat Shock Proteins of the A family (HSPA/HSP70). This group of highly homologous chaperones and proteostasis-controlling factors includes HSPA2, a unique member crucial for spermatogenesis and implicated in the regulation of keratinocyte differentiation. HIF-1 can control the HSPA2 gene expression. In this study, we revealed that HIF-1α is the first confirmed client of HSPA2 in human somatic cells. It colocalises and interacts directly with HSPA2 in the epidermis in situ and immortalised keratinocytes in vitro. Using an in vitro model based on HSPA2-overexpressing and HSPA2-deficient variants of immortalised keratinocytes we showed that changes in HSPA2 levels do not affect the levels and intracellular localisation of HIF-1α or influence the ability of HIF-1 to modulate target gene expression. However, HIF-1α stability in keratinocytes appears critically reliant on HSPAs as a group of functionally overlapping chaperones. In addition to HSPA2, HIF-1α colocalises and forms complexes with HSPA8 and HSPA1, representing housekeeping and stress-inducible HSPA family paralogs, respectively. Chemical inhibition of HSPA activity, but not paralog-specific knockdown of HSPA8 or HSPA1 expression reduced HIF-1α levels and HIF-1-dependent gene expression. These observations suggest that pharmacological targeting of HSPAs could prevent excessive HIF-1 signalling in pathological skin conditions.

An Increase in HSF1 Expression Directs Human Mammary Epithelial Cells toward a Mesenchymal Phenotype.

HSF1 is a well-known heat shock protein expression regulator in response to stress. It also regulates processes important for growth, development or tumorigenesis. We studied the HSF1 influence on the phenotype of non-tumorigenic human mammary epithelial (MCF10A and MCF12A) and several triple-negative breast cancer cell lines. MCF10A and MCF12A differ in terms of HSF1 levels, morphology, growth in Matrigel, expression of epithelial (CDH1) and mesenchymal (VIM) markers (MCF10A are epithelial cells; MCF12A resemble mesenchymal cells). HSF1 down-regulation led to a reduced proliferation rate and spheroid formation in Matrigel by MCF10A cells. However, it did not affect MCF12A proliferation but led to CDH1 up-regulation and the formation of better organized spheroids. HSF1 overexpression in MCF10A resulted in reduced CDH1 and increased VIM expression and the acquisition of elongated fibroblast-like morphology. The above-mentioned results suggest that elevated levels of HSF1 may direct mammary epithelial cells toward a mesenchymal phenotype, while a lowering of HSF1 could reverse the mesenchymal phenotype to an epithelial one. Therefore, HSF1 may be involved in the remodeling of mammary gland architecture over the female lifetime. Moreover, HSF1 levels positively correlated with the invasive phenotype of triple-negative breast cancer cells, and their growth was inhibited by the HSF1 inhibitor DTHIB.

Circulating HPV16 DNA in Blood Plasma as Prognosticator and Early Indicator of Cancer Recurrence in Radio-Chemotherapy for Anal Cancer.

BACKGROUND: Implementation of anal squamous cell carcinoma (ASCC) treatment modifications requires reliable patient risk stratification. The circulating tumor-related human papillomavirus type 16 (ctHPV16) may play a role in predicting survival or assessing treatment response. METHODS: The study included 62 ASCC patients treated with chemoradiotherapy. A threshold of 2.5 was used to determine the maximum standardized uptake value (SUVmax). The ctHPV16 viral load (VL) was quantified by qPCR. RESULTS: In the multivariate Cox analysis, lower SUVmax (p = 0.047) and ctHPV16-positive (p = 0.054) proved to be independent prognostic factors for favorable overall survival (OS). In the subgroup with the higher SUVmax, ctHPV16 and nodal (N) status were independent prognostic factors with p = 0.022 for ctHPV16 and p = 0.053 for N. The best survival rate (95%) presented ctHPV16-positive/N-negative patients. High ctHPV16 VL tended to be slightly specific for patients younger than 63 years (p = 0.152). The decrease in ctHPV16 VL to undetectable level after the end of treatment correlated with the overall clinical response. CONCLUSIONS: A prognostic stratification by SUVmax, ctHPV16 and N-positive status allows consideration of more aggressive treatment in high-risk patients (those with high SUVmax, ctHPV16-negative, and N-positive) or de-intensification of therapy in low-risk patients (those with low SUVmax, ctHPV16-positive and N-negative). However, prospective clinical trials on a large group are needed.

HSF1 Can Prevent Inflammation following Heat Shock by Inhibiting the Excessive Activation of the ATF3 and JUN&FOS Genes.

Heat Shock Factor 1 (HSF1), a transcription factor frequently overexpressed in cancer, is activated by proteotoxic agents and participates in the regulation of cellular stress response. To investigate how HSF1 level affects the response to proteotoxic stress, we integrated data from functional genomics analyses performed in MCF7 breast adenocarcinoma cells. Although the general transcriptional response to heat shock was impaired due to HSF1 deficiency (mainly chaperone expression was inhibited), a set of genes was identified, including ATF3 and certain FOS and JUN family members, whose stress-induced activation was stronger and persisted longer than in cells with normal HSF1 levels. These genes were direct HSF1 targets, suggesting a dual (activatory/suppressory) role for HSF1. Moreover, we found that heat shock-induced inflammatory response could be stronger in HSF1-deficient cells. Analyses of The Cancer Genome Atlas data indicated that higher ATF3, FOS, and FOSB expression levels correlated with low HSF1 levels in estrogen receptor-positive breast cancer, reflecting higher heat shock-induced expression of these genes in HSF1-deficient MCF7 cells observed in vitro. However, differences between the analyzed cancer types were noted in the regulation of HSF1-dependent genes, indicating the presence of cell-type-specific mechanisms. Nevertheless, our data indicate the existence of the heat shock-induced network of transcription factors (associated with the activation of TNFα signaling) which includes HSF1. Independent of its chaperone-mediated cytoprotective function, HSF1 may be involved in the regulation of this network but prevents its overactivation in some cells during stress.

Heat shock factor 1 (HSF1) cooperates with estrogen receptor α (ERα) in the regulation of estrogen action in breast cancer cells.

Heat shock factor 1 (HSF1), a key regulator of transcriptional responses to proteotoxic stress, was linked to estrogen (E2) signaling through estrogen receptor α (ERα). We found that an HSF1 deficiency may decrease ERα level, attenuate the mitogenic action of E2, counteract E2-stimulated cell scattering, and reduce adhesion to collagens and cell motility in ER-positive breast cancer cells. The stimulatory effect of E2 on the transcriptome is largely weaker in HSF1-deficient cells, in part due to the higher basal expression of E2-dependent genes, which correlates with the enhanced binding of unliganded ERα to chromatin in such cells. HSF1 and ERα can cooperate directly in E2-stimulated regulation of transcription, and HSF1 potentiates the action of ERα through a mechanism involving chromatin reorganization. Furthermore, HSF1 deficiency may increase the sensitivity to hormonal therapy (4-hydroxytamoxifen) or CDK4/6 inhibitors (palbociclib). Analyses of data from The Cancer Genome Atlas database indicate that HSF1 increases the transcriptome disparity in ER-positive breast cancer and can enhance the genomic action of ERα. Moreover, only in ER-positive cancers an elevated HSF1 level is associated with metastatic disease.

About 70% of breast cancers rely on supplies of a hormone called estrogen ­ which is the main hormone responsible for female physical characteristics ­ to grow. Breast cancer cells that are sensitive to estrogen possess proteins known as estrogen receptors and are classified as estrogen-receptor positive. When estrogen interacts with its receptor in a cancer cell, it stimulates the cell to grow and migrate to other parts of the body. Therefore, therapies that decrease the amount of estrogen the body produces, or inhibit the receptor itself, are widely used to treat patients with estrogen receptor-positive breast cancers. When estrogen interacts with an estrogen receptor known as ERα it can also activate a protein called HSF1, which helps cells to survive under stress. In turn, HSF1 regulates several other proteins that are necessary for ERα and other estrogen receptors to work properly. Previous studies have suggested that high levels of HSF1 may worsen the outcomes for patients with estrogen receptor-positive breast cancers, but it remains unclear how HSF1 acts in breast cancer cells. Vydra, Janus, Kus et al. used genetics and bioinformatics approaches to study HSF1 in human breast cancer cells. The experiments revealed that breast cancer cells with lower levels of HSF1 also had lower levels of ERα and responded less well to estrogen than cells with higher levels of HSF1. Further experiments suggested that in the absence of estrogen, HSF1 helps to keep ERα inactive. However, when estrogen is present, HSF1 cooperates with ERα and enhances its activity to help cells grow and migrate. Vydra, Janus, Kus et al. also found that cells with higher levels of HSF1 were less sensitive to two drug therapies that are commonly used to treat estrogen receptor-positive breast cancers. These findings reveal that the effect HSF1 has on ERα activity depends on the presence of estrogen. Therefore, cancer therapies that decrease the amount of estrogen a patient produces may have a different effect on estrogen receptor-positive tumors with high HSF1 levels than tumors with low HSF1 levels.

Inhibition of the Heat Shock Protein A (HSPA) Family Potentiates the Anticancer Effects of Manumycin A.

Manumycin A (MA) is a well-tolerated natural antibiotic showing pleiotropic anticancer effects in various preclinical in vitro and in vivo models. Anticancer drugs may themselves act as stressors to induce the cellular adaptive mechanism that can minimize their cytotoxicity. Heat shock proteins (HSPs) as cytoprotective factors can counteract the deleterious effects of various stressful stimuli. In this study, we examined whether the anticancer effects of MA can be counteracted by the mechanism related to HSPs belonging to the HSPA (HSP70) family. We found that MA caused cell type-specific alterations in the levels of HSPAs. These changes included concomitant upregulation of the stress-inducible (HSPA1 and HSPA6) and downregulation of the non-stress-inducible (HSPA2) paralogs. However, neither HSPA1 nor HSPA2 were necessary to provide protection against MA in lung cancer cells. Conversely, the simultaneous repression of several HSPA paralogs using pan-HSPA inhibitors (VER-155008 or JG-98) sensitized cancer cells to MA. We also observed that genetic ablation of the heat shock factor 1 (HSF1) transcription factor, a main transactivator of HSPAs expression, sensitized MCF7 cells to MA treatment. Our study reveals that inhibition of HSF1-mediated heat shock response (HSR) can improve the anticancer effect of MA. These observations suggest that targeting the HSR- or HSPA-mediated adaptive mechanisms may be a promising strategy for further preclinical developments.

The periphilin 1-like BFAR isoform 3 is highly expressed in transcriptionally silent oocytes and involved in RNA metabolism.

The mouse 3110001I22Rik gene located in the first intron of Bfar is considered as a Bfar variant coding for the BFARv3 protein. However, it differs from other BFAR isoforms and resembles periphilin 1 (PPHLN1) due to its two (Lge1 and serine-rich) conserved domains. We identified the BFARv3/EGFP-interacting proteins by co-immunoprecipitation coupled to mass spectrometry, which revealed 40S ribosomal proteins (RPS3, RPS14, RPS19, RPS25, RPS27), histones (H1.2, H1.4, H3.3C), proteins involved in RNA processing and splicing (SFPQ, SNRPA1, HNRNPA3, NONO, KHDRBS3), calcium signaling (HPCAL1, PTK2B), as well as HSD17B4, GRB14, POSTN, and MYO10. Co-immunoprecipitation revealed that both Lge1 and Ser-rich domains of BFARv3 were necessary for binding to RNA-interacting factors NONO and SFPQ, known to be components of paraspeckles. Reciprocal co-immunoprecipitation and the proximity ligation assay confirmed that both BFARv3 and PPHLN1 could interact with NONO and SFPQ, suggesting a new function for PPHLN1 as well. BFARv3 and its Lge1 or Ser-rich-deficient mutants preferentially localize in the nucleus. We found an accumulation of BFARv3/EGFP (but not its mutated forms) in the nuclear granules, which was enhanced in response to arsenite treatment and ionizing radiation. Although Bfar v3 is expressed ubiquitously in mouse tissues, its expression is the highest in metaphase II oocytes. The BFARv3 interactome suggests its role in RNA metabolism, which is critical for the transcriptionally silent MII oocyte. Mouse BFARv3 has no ortholog in the human genome, thus it may contribute to the differences between these two species observed in oocyte maturation and early embryonic development.

Pro-death signaling of cytoprotective heat shock factor 1: upregulation of NOXA leading to apoptosis in heat-sensitive cells.

Heat shock can induce either cytoprotective mechanisms or cell death. We found that in certain human and mouse cells, including spermatocytes, activated heat shock factor 1 (HSF1) binds to sequences located in the intron(s) of the PMAIP1 (NOXA) gene and upregulates its expression which induces apoptosis. Such a mode of PMAIP1 activation is not dependent on p53. Therefore, HSF1 not only can activate the expression of genes encoding cytoprotective heat shock proteins, which prevents apoptosis, but it can also positively regulate the proapoptotic PMAIP1 gene, which facilitates cell death. This could be the primary cause of hyperthermia-induced elimination of heat-sensitive cells, yet other pro-death mechanisms might also be involved.

17ß-Estradiol Activates HSF1 via MAPK Signaling in ERα-Positive Breast Cancer Cells.

Heat Shock Factor 1 (HSF1) is a key regulator of gene expression during acute environmental stress that enables the cell survival, which is also involved in different cancer-related processes. A high level of HSF1 in estrogen receptor (ER)-positive breast cancer patients correlated with a worse prognosis. Here we demonstrated that 17ß-estradiol (E2), as well as xenoestrogen bisphenol A and ERα agonist propyl pyrazole triol, led to HSF1 phosphorylation on S326 in ERα positive but not in ERα-negative mammary breast cancer cells. Furthermore, we showed that MAPK signaling (via MEK1/2) but not mTOR signaling was involved in E2/ERα-dependent activation of HSF1. E2-activated HSF1 was transcriptionally potent and several genes essential for breast cancer cells growth and/or ERα action, including HSPB8, LHX4, PRKCE, WWC1, and GREB1, were activated by E2 in a HSF1-dependent manner. Our findings suggest a hypothetical positive feedback loop between E2/ERα and HSF1 signaling, which may support the growth of estrogen-dependent tumors.

Functional redundancy of HSPA1, HSPA2 and other HSPA proteins in non-small cell lung carcinoma (NSCLC); an implication for NSCLC treatment.

Heat shock proteins (HSPs) are a large group of chaperones considered critical for maintaining cellular proteostasis. Their aberrant expression in tumors can modulate the course of processes defined as hallmarks of cancer. Previously, we showed that both stress-inducible HSPA1 and testis-enriched HSPA2, highly homologous members of the HSPA (HSP70) family, are often overexpressed in non-small cell lung carcinoma (NSCLC). HSPA1 is among the best characterized cancer-related chaperones, while the significance of HSPA2 for cancer remains poorly understood. Previously we found that in primary NSCLC, HSPA1 was associated with good prognosis while HSPA2 correlated with bad prognosis, suggesting possible different roles of these proteins in cancer. Therefore, in this work we investigated the impact of HSPA1 and HSPA2 on NSCLC cell phenotype. We found that neither paralog-selective nor simultaneous knockdown of HSPA1 and HSPA2 gene expression reduced growth and chemoresistance of NSCLC cells. Only blocking of HSPA proteins using pan-HSPA inhibitors, VER-155008 or JG-98, exerted potent anticancer effect on NSCLC cells, albeit the final outcome was cell type-dependent. Pan-HSPA inhibition sensitized NSCLC cells to bortezomib, but not to platinum derivates. Our result suggests the inhibitors of proteasome and HSPAs seem an effective drug combination for pre-clinical development in highly aggressive NSCLC.

Interplay between HSF1 and p53 signaling pathways in cancer initiation and progression: non-oncogene and oncogene addiction.

BACKGROUND: The p53 and HSF1 transcription factors are key players in cellular responses to stress. They activate important signaling pathways triggering adaptive mechanisms that maintain cellular homeostasis. HSF1 is mainly activated by proteotoxic stress, and its induction leads to the synthesis of chaperones that provide proteome integrity. The p53 protein, which is primarily activated in response to DNA damage, causes cell cycle arrest allowing for DNA repair or directs cells to apoptosis, thereby maintaining genome integrity. Both signaling pathways are also involved in neoplastic transformation and tumor progression. Loss of tumor suppressor abilities of the wild-type p53 protein results in oncogenesis, whereas proper HSF1 action, though non-oncogenic itself, actively supports this process. CONCLUSIONS: Here, we describe in detail the interplay between the p53 and HSF1 signaling pathways, with particular emphasis on the molecular mechanisms involved, as well as their importance for normal cellular behavior, cancer development, the effectiveness of anti-cancer therapies and their toxicity. Detailed knowledge of the complex interplay between HSF1 and p53 may form a basis for the design of new protocols for cancer treatment.

PHLDA1 Does Not Contribute Directly to Heat Shock-Induced Apoptosis of Spermatocytes.

Spermatocytes are among the most heat-sensitive cells and the exposure of testes to heat shock results in their Heat Shock Factor 1 (HSF1)-mediated apoptosis. Several lines of evidence suggest that pleckstrin-homology-like domain family A, member 1 (PHLDA1) plays a role in promoting heat shock-induced cell death in spermatogenic cells, yet its precise physiological role is not well understood. Aiming to elucidate the hypothetical role of PHLDA1 in HSF1-mediated apoptosis of spermatogenic cells we characterized its expression in mouse testes during normal development and after heat shock. We stated that transcription of Phlda1 is upregulated by heat shock in many adult mouse organs including the testes. Analyzes of the Phlda1 expression during postnatal development indicate that it is expressed in pre-meiotic or somatic cells of the testis. It starts to be transcribed much earlier than spermatocytes are fully developed and its transcripts and protein products do not accumulate further in the later stages. Moreover, neither heat shock nor expression of constitutively active HSF1 results in the accumulation of PHLDA1 protein in meiotic and post-meiotic cells although both conditions induce massive apoptosis of spermatocytes. Furthermore, the overexpression of PHLDA1 in NIH3T3 cells leads to cell detachment, yet classical apoptosis is not observed. Therefore, our findings indicate that PHLDA1 cannot directly contribute to the heat-induced apoptosis of spermatocytes. Instead, PHLDA1 could hypothetically participate in death of spermatocytes indirectly via activation of changes in the somatic or pre-meiotic cells present in the testes.

SPEN protein expression and interactions with chromatin in mouse testicular cells.

SPEN (spen family transcription repressor) is a nucleic acid-binding protein putatively involved in repression of gene expression. We hypothesized that SPEN could be involved in general downregulation of the transcription during the heat shock response in mouse spermatogenic cells through its interactions with chromatin. We documented predominant nuclear localization of the SPEN protein in spermatocytes and round spermatids, which was retained after heat shock. Moreover, the protein was excluded from the highly condensed chromatin. Chromatin immunoprecipitation experiments clearly indicated interactions of SPEN with chromatin in vivo However, ChIP-Seq analyses did not reveal any strong specific peaks both in untreated and heat shocked cells, which might suggest dispersed localization of SPEN and/or its indirect binding to DNA. Using in situ proximity ligation assay we found close in vivo associations of SPEN with MTA1 (metastasis-associated 1), a member of the nucleosome remodeling complex with histone deacetylase activity, which might contribute to interactions of SPEN with chromatin.

The Role of Heat Shock Factors in Mammalian Spermatogenesis.

Heat shock transcription factors (HSFs), as regulators of heat shock proteins (HSPs) expression, are well known for their cytoprotective functions during cellular stress. They also play important yet less recognized roles in gametogenesis. All HSF family members are expressed during mammalian spermatogenesis, mainly in spermatocytes and round spermatids which are characterized by extensive chromatin remodeling. Different HSFs could cooperate to maintain proper spermatogenesis. Cooperation of HSF1 and HSF2 is especially well established since their double knockout results in meiosis arrest, spermatocyte apoptosis, and male infertility. Both factors are also involved in the repackaging of the DNA during spermatid differentiation. They can form heterotrimers regulating the basal level of transcription of target genes. Moreover, HSF1/HSF2 interactions are lost in elevated temperatures which can impair the transcription of genes essential for spermatogenesis. In most mammals, spermatogenesis occurs a few degrees below the body temperature and spermatogenic cells are extremely heat-sensitive. Pro-survival pathways are not induced by heat stress (e.g., cryptorchidism) in meiotic and postmeiotic cells. Instead, male germ cells are actively eliminated by apoptosis, which prevents transition of the potentially damaged genetic material to the next generation. Such a response depends on the transcriptional activity of HSF1 which in contrary to most somatic cells, acts as a proapoptotic factor in spermatogenic cells. HSF1 activation could be the main trigger of impaired spermatogenesis related not only to elevated temperature but also to other stress conditions; therefore, HSF1 has been proposed to be the quality control factor in male germ cells.

Active heat shock transcription factor 1 supports migration of the melanoma cells via vinculin down-regulation.

Heat shock transcription factor 1 (HSF1), the major regulator of stress response, is frequently activated in cancer and has an apparent role in malignant transformation. Here we analyzed the influence of the over-expression of a constitutively active transcriptionally-competent HSF1 mutant form on phenotypes of mouse and human melanoma cells. We observed that the expression of active HSF1 supported anchorage-independent growth in vitro, and metastatic spread in the animal model in vivo, although the proliferation rate of cancer cells was not affected. Furthermore, active HSF1 enhanced cell motility, reduced the adherence of cells to a fibronectin-coated surface, and affected the actin cytoskeleton. We found that although the expression of active HSF1 did not affect levels of epithelial-to-mesenchymal transition markers, it caused transcriptional down-regulation of vinculin, protein involved in cell motility, and adherence. Functional HSF1-binding sites were found in mouse and human Vcl/VCL genes, indicating a direct role of HSF1 in the regulation of this gene. An apparent association between HSF1-induced down-regulation of vinculin, increased motility, and a reduced adherence of cells suggests a possible mechanism of HSF1-mediated enhancement of the metastatic potential of cancer cells.

Crosstalk between HSF1 and HSF2 during the heat shock response in mouse testes.

Heat Shock Factor 1 (HSF1) is the primary transcription factor responsible for the response to cellular stress, while HSF2 becomes activated during development and differentiation, including spermatogenesis. Although both factors are indispensable for proper spermatogenesis, activation of HSF1 by heat shock initiates apoptosis of spermatogenic cells leading to infertility of males. To characterize mechanisms assisting such heat induced apoptosis we studied how HSF1 and HSF2 cooperate during the heat shock response. For this purpose we used chromatin immunoprecipitation and the proximity ligation approaches. We looked for co-occupation of binding sites by HSF1 and HSF2 in untreated (32 °C) or heat shocked (at 38 °C or 43 °C) spermatocytes, which are cells the most sensitive to hyperthermia. At the physiological temperature or after mild hyperthermia at 38 °C, the sharing of binding sites for both HSFs was observed mainly in promoters of Hsp genes and other stress-related genes. Strong hyperthermia at 43 °C resulted in an increased binding of HSF1 and releasing of HSF2, hence co-occupation of promoter regions was not detected any more. The close proximity of HSF1 and HSF2 (and/or existence of HSF1/HSF2 complexes) was frequent at the physiological temperature. Temperature elevation resulted in a decreased number of such complexes and they were barely detected after strong hyperthermia at 43 °C. We have concluded that at the physiological temperature HSF1 and HSF2 cooperate in spermatogenic cells. However, temperature elevation causes remodeling of chromatin binding and interactions between HSFs are disrupted. This potentially affects the regulation of stress response and contributes to the heat sensitivity of these cells.

Overlapping dose responses of spermatogenic and extragonadal testosterone actions jeopardize the principle of hormonal male contraception.

Testosterone (T), alone or in combination with progestin, provides a promising approach to hormonal male contraception. Its principle relies on enhanced negative feedback of exogenous T to suppress gonadotropins, thereby blocking the testicular T production needed for spermatogenesis, while simultaneously maintaining the extragonadal androgen actions, such as potency and libido, to avoid hypogonadism. A serious drawback of the treatment is that a significant proportion of men do not reach azoospermia or severe oligozoospermia, commensurate with contraceptive efficacy. We tested here, using hypogonadal luteinizing hormone/choriongonadotropin receptor (LHCGR) knockout (LHR(-/-)) mice, the basic principle of the T-based male contraceptive method, that a specific T dose could maintain extragonadal androgen actions without simultaneously activating spermatogenesis. LHR(-/-) mice were treated with increasing T doses, and the responses of their spermatogenesis and extragonadal androgen actions (including gonadotropin suppression and sexual behavior) were assessed. Conspicuously, all dose responses to T were practically superimposable, and no dose of T could be defined that would maintain sexual function and suppress gonadotropins without simultaneously activating spermatogenesis. This finding, never addressed in clinical contraceptive trials, is not unexpected in light of the same androgen receptor mediating androgen actions in all organs. When extrapolated to humans, our findings may jeopardize the current approach to hormonal male contraception and call for more effective means of inhibiting intratesticular T production or action, to achieve consistent spermatogenic suppression.

Pleiotropic role of HSF1 in neoplastic transformation.

HSF1 (Heat Shock transcription Factor 1) is the main transcription factor activated in response to proteotoxic stress. Once activated, it induces an expression of heat shock proteins (HSPs) which enables cells to survive in suboptimal conditions. HSF1 could be also activated by altered kinase signaling characteristic for cancer cells, which is a probable reason for its high activity found in a broad range of tumors. There is rapidly growing evidence that HSF1 supports tumor initiation and growth, as well as metastasis and angiogenesis. It also modulates the sensitivity of cancer cells to therapy. Functions of HSF1 in cancer are connected with HSPs' activity, which generally protects cells from apoptosis, but also are independent of its classical targets. HSF1-dependent regulation of non-HSPs genes plays a role in cell cycle progression, glucose metabolism, autophagy and drug efflux. HSF1 affects the key cell-survival and regulatory pathways, including p53, RAS/MAPK, cAMP/PKA, mTOR and insulin signaling. Although the exact mechanism of HSF1 action is still somewhat obscure, HSF1 is becoming an attractive target in anticancer therapies, whose inhibition could enhance the effects of other treatments.

Overexpression of Heat Shock Transcription Factor 1 enhances the resistance of melanoma cells to doxorubicin and paclitaxel.

BACKGROUND: Heat Shock Transcription Factor 1 (HSF1) is activated under stress conditions. In turn, it induces expression of Heat Shock Proteins (HSPs), which are well-known regulators of protein homeostasis. Elevated levels of HSF1 and HSPs were observed in many types of tumors. The aim of the present study was to determine whether HSF1 could have an effect on the survival of cancer cells treated with chemotherapeutic cytotoxic agents. METHODS: We constructed mouse (B16F10) and human (1205Lu, WM793B) melanoma cells overexpressing full or mutant form of human HSF1: a constitutively active one with a deletion in regulatory domain or a dominant negative one with a deletion in the activation domain. The impact of different forms of HSF1 on the expression of HSP and ABC genes was studied by RT-PCR and Western blotting. Cell cultures were treated with increasing amounts of doxorubicin, paclitaxel, cisplatin, vinblastine or bortezomib. Cell viability was determined by MTT, and IC50 was calculated. Cellular accumulation of fluorescent dyes and side population cells were studied using flow cytometry. RESULTS: Cells overexpressing HSF1 and characterized by increased HSPs accumulation were more resistant to doxorubicin or paclitaxel, but not to cisplatin, vinblastine or bortezomib. This resistance correlated with the enhanced efflux of fluorescent dyes and the increased number of side population cells. The expression of constitutively active mutant HSF1, also resulting in HSPs overproduction, did not reduce the sensitivity of melanoma cells to drugs, unlike in the case of dominant negative form expression. Cells overexpressing a full or dominant negative form of HSF1, but not a constitutively active one, had higher transcription levels of ABC genes when compared to control cells. CONCLUSIONS: HSF1 overexpression facilitates the survival of melanoma cells treated with doxorubicin or paclitaxel. However, HSF1-mediated chemoresistance is not dependent on HSPs accumulation but on an increased potential for drug efflux by ABC transporters. Direct transcriptional activity of HSF1 is not necessary for increased expression of ABC genes, which is probably mediated by HSF1 regulatory domain.