Background
HSF1-dependent stress response is an adaptive mechanism which enhances the survival of somatic cells facing diverse arrays of environmental and physiological challenges (such as heat shock, ischemic injury, neurodegeneration, and others) [
1,
2]. Activation of HSF1 results in induced expression of a set of highly conserved proteins, known as heat shock proteins (HSPs). HSPs act as molecular chaperones by assisting protein folding during their synthesis or repair under proteotoxic conditions. Mammalian HSPs are classified according to molecular size into several families including HSPH (HSP110), HSPC (HSP90), HSPA (HSP70), HSPD (HSP60), and HSPB (small HSPs, sHSPs). Each gene family includes members that are constitutively expressed, inducibly regulated, and/or targeted to different cellular compartments [
3].
The primary role of HSF1 in cells is associated with the regulation of
HSPs expression in response to heat shock or other stress conditions. Moreover, there is some evidence indicating the importance of HSF1 in the processes associated with development, growth and fertility [
4‐
7]. Furthermore, HSF1 facilitates cell survival upon imbalanced cell signaling associated with neoplastic transformation. Convincing evidence of HSF1 involvement in carcinogenesis has emerged from data gathered from a murine tumor model. Namely, lack of HSF1 expression protected mice against tumorigenesis in a chemically-induced skin carcinogenesis model and in a genetic model driven by a clinically relevant oncogenic mutation in p53 (p53R172H) [
8]. The role of HSF1 in carcinogenesis includes protecting cancer cells from programmed cell death, overriding cell cycle checkpoints and enhancing metastasis [
9‐
11]. HSF1 also orchestrates a broad network of core cellular functions associated with proliferation, survival, protein synthesis and glucose metabolism, thus enhancing oncogenic transformation [
8,
9].
Activation of HSF1-dependent stress response, a cytoprotective mechanism, may greatly influence development of an adaptive and protective phenotype in cancer cells subjected to anticancer agents. Elevated expression of HSPs (e.g., HSP90, HSP70, HSP27) has been reported in many types of human malignancies and was linked to cancer resistance to apoptosis induced by chemotherapeutic agents [
12‐
14]. The antiapoptotic function of HSPs was shown for monoblastoid U937 cells and murine fibrosarcoma WEHI-S cells treated with actinomycin-D, camptothecin and etoposide [
15] as well as rat brain tumor cells treated with vincristine [
16]. In addition, HSP-independent mechanism may be involved in HSF1 regulated resistance of cancer cells to chemotherapeutics. HSF1-binding elements were found in
ABCB1 (
MDR1) gene promoter coding for P-glycoprotein (P-gp), an energy-dependent drug efflux pump [
17,
18].
In this study, we established mouse and human melanoma cells overexpressing hHSF1 to study the effect of HSF1 on the survival of cancer cells treated with cytotoxic agents used in chemotherapy. Here, we generated melanoma cells with different mutant forms of human HSF1, leading either to constitutive HSPs activation (transcriptionally active) or lacking the ability to activate HSPs expression (dominant-negative). We also obtained mouse melanoma B16F10 cells with a silenced HSF1 expression. We were thus able to evaluate the contribution of HSF1 and HSPs level in the development of drug resistance by melanoma cells.
Methods
Cell lines and cell culture
Melanoma cell lines, B16F10 (mouse), WM793B and 1205Lu (human), were obtained from American Type Cell Culture Collection (ATCC, Manassas, VA). Cells were routinely cultured according to ATCC protocol. Doubling time for B16F10 cells is approximately 24 h, for WM793B and 1205Lu – approximately 48 h. Heat shock was performed by placing plates with logarithmically growing cells in an incubator (Heraeus), at 42°C for 1 hour. For transcriptional studies, cells were allowed to recover at 37°C for 30 minutes or for protein studies were lysed immediately after heat shock or after 6-hour recovery.
DNA constructs
Human HSF1 (hHSF1) coding sequence (Accession no. NM_005526.2) was amplified by PCR using cDNA from WM793B cells as a template; the sequence recognized by
HindIII restriction enzyme was introduced into primers. HSF1 cDNA fragment was inserted downstream of the human β-actin promoter into the pHβApr-1-neo expression vector. The hHSF1ΔRD construct containing a constitutively active form of human HSF1 (aHSF1; with 221–315 amino acid deletion) driven by the human β-actin promoter in the pHβApr-1-neo expression vector, was kindly provided by Dr. A. Nakai [
6]. A plasmid containing dominant negative human HSF1 (hHSF1-DN; with deletion of amino acids 453–523; [
19]) was constructed by PCR-mediated site-directed mutagenesis consisting of two-step PCR, using two overlapping internal primers at the mutagenic site and two outer general primers each flanked by
HindIII site. The internal primers were as follows: forward 5′-GAGCCCCCCAGGCCTCCCAAGGACCCCACTGTCTTC; reverse 5′-GAAGACAGTGGGGTCCTTGGGAGGCCTGGGGGGCTG. The mutant hHSF1-DN cDNA fragment was inserted downstream of the human β-actin promoter into the pHβApr-1-neo expression vector. The hHSF1, aHSF1, hHSF1-DN sequences were also cloned into the pLNCX2 retrovirus expression vector downstream of the CMV promoter (Clontech). Nucleotide sequence of all constructs was verified by DNA sequencing. Schematic diagram of a structure of analyzed hHSF1 proteins is shown in Additional file
1: Figure S1.
Stable transfections
Mouse melanoma B16F10 cells were transfected with vectors containing hHSF1, aHSF1, and hHSF1-DN cDNA using Lipofectamine™2000 according to the manufacturer’s protocol (Life Technologies). To select clones that stably express the integrated vector, cells were cultured for 7 days with G-418 (1 mg/ml, Life Technologies). Then, cells were seeded on a 96-well plate (1 cell/well) in the presence of G-418. When colonies were formed, 7–11 individual clones were collected for each construct. Clones expressing the introduced HSF1 (as estimated by Western blotting) were pooled together for further experiments. Stably transfected human melanoma WM793B and 1205Lu cells were obtained by retroviral gene transfer of hHSF1, aHSF1, hHSF1-DN cDNA cloned in the pLNCX2 vector according to the manufacturer’s protocol (Clontech Laboratories, Inc.). Cells were infected in the presence of polybrene (8 μg/ml) and selected in the presence of G-418 (200 μg/ml - WM793B cells, and 400 μg/ml - 1205Lu cells).
Generation of HSF1-shRNA vectors
The shRNA target sequence for mouse HSF1 was selected using the RNAi Target Sequence Selector (Clontech) and according to a previous report [
8]. The target sequences were: HSF1-1 (1856–1876, NM_008296.2) - 5′ GCTGCATACCTGCTGCCTTTA; and HSF1-2 (341–359, NM_008296.2) - 5′AGCACAACAACATGGCTAG. Sense and antisense oligonucleotides were annealed and inserted into the pRNAi-Ready-Siren-RetroQ vector (Clontech) at
BamHI/
EcoRI site. Infectious retroviruses were generated by transfecting DNA into PT67 cells and virus-containing supernatant was collected. Mouse melanoma B16F10 cells were transduced with retroviruses containing HSF1 shRNAs and selected using a medium supplemented with 1 μg/ml puromycin (Life Technologies).
RNA isolation and RT-PCR
Extraction of total RNA, purification from DNA contamination, synthesis of cDNA and RT-PCR were performed as described in [
20]. For RT-PCR 1–2 μl of cDNA template was used and 25–35 cycles were applied depending on the primers set. Quantitative RT-PCR was performed using a Bio-Rad CFX 96TM Real-Time PCR Detection System. A total of 5 pmoles of forward and reverse primers, cDNA template were added to the Real-Time 2× PCR Master Mix SYBR A (A&A Biotechnology, Gdynia, Poland). Primers used in the analyses are listed in Additional file
2: Table S2.
Protein extraction and Western blotting
Whole cell extracts were prepared using RIPA buffer. Proteins (25 μg) were separated on 8-10% SDS-PAGE gels and blotted to 0.45-μm pore nitrocellulose filter (Millipore) [
21]. Primary antibodies against HSF1 (rabbit polyclonal, ADI-SPA-901, Enzo Life Sciences), HSP70 (mouse monoclonal, ADI-SPA-810, Enzo Life Sciences), HSP25 (rabbit polyclonal, ADI-SPA-801, Enzo Life Sciences), HSP105 (rabbit polyclonal, 3390–100, BioVision), or actin (mouse monoclonal, clone C4, MAB1501, Millipore) were used. The primary antibody was detected by appropriate secondary antibody conjugated with horseradish peroxidase (ThermoScientific) and visualized by ECL kit (ThermoScientific).
Treatment of cells with cytotoxic drugs and MTT assay
Mouse melanoma cells (1.5 × 10
3/well) or human melanoma cells (4 × 10
3/well) were seeded in 96-well plates and allowed to attach overnight. Cytotoxic agents: doxorubicin (5, 10, 20, 40, 80 ng/ml), paclitaxel (5, 10, 20, 40, 80 nM), vinblastin (1, 2, 4, 8, 16 nM), cisplatin (2, 4, 8, 16 μM) and bortezomib (2.5, 5, 10, 20 nM) were applied for 48 hours (B16F10 cells) or for 72 h (WM793B and 1205Lu cells). Cell viability was determined by MTT assay, as described in [
22]. The absorbance (λ = 570 nm) was read using Synergy 2 microplate reader (Biotek). Relative survival was determined using the formula: viability (%) = (cytotoxic agent treated-blank)/(untreated-blank)*100. All experiments were performed at least in triplicate.
Assay for the fluorescent dyes efflux
Cells suspended in phenol-free medium supplemented with 0.5% FBS (PAA) in polystyrene tubes were incubated for 30 minutes in a 37°C incubator with (i) doxorubicin (1 μg/ml; 5 × 105 cells) or (ii) eFluxx-ID™ Green Detection Reagent (Enzo Life Sciences) (2.5 × 105 cells). Next, cells were washed, resuspended in PBS, and analyzed using a FACSCanto cytometer (Becton Dickinson). Dye concentration and treatment exposure times were established experimentally to obtain the best signal-to-noise ratio.
Side population analysis
Cells were stained according to Goodell’s protocol [
23]. Briefly, cells at 1 × 10
6/ml were suspended in prewarmed phenol-free DMEM (Sigma-Aldrich) with 2% FBS. Hoechst 33342 (Sigma-Aldrich) was added to the final concentration of 5 μg/ml in the presence or absence of verapamil (50 μg/ml; Sigma-Aldrich). Cells were incubated at 37°C for 90 min with intermittent shaking. At the end of incubation, cells were washed with phenol-free DMEM, centrifuged at 4°C, and resuspended in ice-cold PBS. Propidium iodide (Sigma-Aldrich) was added to cells to gate viable cells. Analyses were performed using FACSAria III apparatus (Becton Dickinson). The Hoechst 33342 dye was excited at 357 nm and its fluorescence was dual-wavelength analyzed (blue, 402–446 nm; red, 650–670 nm).
Statistical analysis
The data were analyzed by Student’s t-test. A p-value of <0.05 was considered statistically significant.
Discussion
High levels of HSF1 and HSPs expression were observed in a broad range of human tumors [
27‐
30]. Moreover, it has been shown that increased HSF1 expression is associated with reduced survival of cancer patients. It is not surprising as HSF1 modulates an entire network of cellular functions that enable neoplastic transformation [
8,
31]. However, the impact of HSF1 overexpression on cell susceptibility to chemotherapy has not been studied so far. Chemotherapy, a major modality of cancer treatment, is effective initially in controlling the growth of many sensitive tumors, but later it often fails due to the development of resistance to the received drugs. Diverse mechanisms are involved in the acquisition of drug resistance by cancer cells. Understanding them is the key to identify new possible treatments.
In the presented work, we screened the sensitivity of mouse (B16F10) and human (WM793B and 1205Lu) melanoma cells overexpressing HSF1 to different anticancer drugs. We found that HSF1 overexpression had no effect on the survival of cells treated with cisplatin, vinblastine or bortezomib, while the survival of cells treated with doxorubicin or paclitaxel was significantly enhanced when compared to their parental wild-type cells (or control cells containing the empty vector). Surprisingly, we revealed that such selective resistance of melanoma cells was not dependent on direct transcriptional activity of HSF1 (and linked HSPs expression and accumulation). Melanoma cells expressing transcriptionally competent and constitutively active HSF1 mutant characterized by an enhanced expression of HSPs did not acquire resistance. On the other hand, HSF1 mutant form with a deletion in the transcriptional activation domain was found to be as effective as overexpression of wild-type HSF1.
The primary role of HSF1 is traditionally referred to the regulation of
HSP genes expression. It is generally accepted that HSPs are the fundamental component of cytoprotective reaction that enables somatic cells to survive exposure to harmful conditions. HSPs prevent protein denaturation and/or processing of denatured proteins, which limits accumulation of misfolded species [
32,
33]. Other mechanism of HSP-dependent cytoprotection involves inhibition of apoptosis. Direct physical interactions with apoptotic molecules were demonstrated for HSPA1, HSPB1 and HSP90 [
34,
35]. Regardless of well-known cytoprotective function of HSPs, its role in the effectiveness of chemotherapy is not obvious. There are several reports showing that up-regulation of HSP90, HSPA1 or HSPB1 is associated with cell resistance to cisplatin or doxorubicin [
36‐
39]. Furthermore, the damage induced by doxorubicin is more efficiently repaired following heat shock, which correlates with nuclear translocation of HSPB1 and HSPA1 [
40]. Also, it was reported that heat-induced carboplatin resistance of p53-dependent hepatoma cells is mediated by HSPA1 [
41]. Nevertheless, there are several reports demonstrating that activation of HSPs expression does not enhance cancer cell survival in various types of neoplasia upon cisplatin, colchicine, 5-fluorouracil, actinomycin D or methotrexate treatments [
42‐
46]. Moreover, diminished HSPs expression resulting from HSF1 silencing did not abrogate resistance of cervix carcinoma HeLa cells to cisplatin [
47]. Thus, it seems plausible that susceptibility of cells to chemotherapeutics does not solely depend on HSPs expression. The presence of HSPs could be just a secondary effect of HSF1 activity, while mechanisms of HSF1-dependent resistance of cancer cells to drugs could be connected to its interactions with other proteins and/or its impact (direct or indirect) on expression of non-
HSPs genes. If fact, it was already reported that HSF1 interacts with p53 and enhances p53-mediated transcription [
48] or regulates expression of ATG7 (autophagy-related protein 7) [
49]. Recent studies have shown that although HSPs expression is important for the tumor initiation [
50], a network of genes regulated by HSF1 in malignant cells is distinct from the transcriptional program induced by heat shock [
51].
In this report we show that enhanced resistance to doxorubicin and paclitaxel is associated with enhanced drug efflux. Most markedly, the ABC transporter substrate eFluxx-ID Green Reagent was more effectively removed from cells overexpressing HSF1. We found that transcription of several ABC transporters was increased not only in cells overexpressing HSF1 but also its dominant negative form, while not the constitutively active form. This finding suggests that enhanced expression of
ABC genes is not coupled directly to transcriptional activity of HSF1. The expression of
Abcb1b/
ABCB1 gene was mostly dependent on HSF1 in all three tested melanoma cell lines. It has been previously demonstrated that multidrug resistance of osteosarcoma U2-OS cells and hepatoma HepG2 cells was mediated by HSF1-dependent expression of the
ABCB1 gene, but not by HSPs expression [
52]. Additionally, the transcriptional activity of HSF1 has been required for enhanced expression of
ABCB1 gene in HeLa cells [
18]. Although HSE (heat shock element) sequences are present in
ABCB1 gene promoter [
17], it was revealed that the mere binding of HSF1 was not sufficient to transactivate the
ABCB1 expression, as it was in the case of
HSP genes [
18,
19]. Hence, a plausible posttranscriptional mechanism of
ABCB1 up-regulation in HSF1 overexpressing cells has been proposed [
52].
Different mechanisms explaining HSF1 influence on
ABC mRNAs up-regulation may be proposed. Our data indicate that the HSF1 regulatory domain, which confers repression at control temperature and heat inducibility of HSF1 is required for this effect. It could be hypothesized that HSF1 mediates,
via its regulatory domain, the activity of other transcription factors or that it affects mRNA maturation or stability. Although a role for HSF1 in RNA processing has not been fully documented, HSF1 incorporation into nuclear stress bodies, where RNA splicing could take place, was reported [
53]. Recently, it was shown that HSF1 is involved in the regulation of mRNA-binding protein ELAVL1 (HuR) which, in turn, controls mRNA stability and/or translation of many proteins involved in cancer [
54]. In spite of HSF1-dependent accumulation of
Abc/
ABC transcripts we did not confirm the corresponding accumulation of ABC proteins. However, our data confirm an enhanced drug efflux, which is considered to be the most relevant indicator of both expression of ABC transporters and its molecular catalytic activity [
55,
56].
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
NV carried out most of the molecular biology experiments, designed the study and drafted the manuscript. AT participated in the construction and characteristization of cell lines. MG-K performed analysis of fluorescent dye accumulation by flow cytometry. AG-P participated in the analysis of ABC transporters’ expression. WW designed and wrote the manuscript. All authors read and approved the final manuscript.