Heat shock factor 1 (HSF1) has long been recognized as the master transcription factor that regulates heat shock proteins (HSPs). More recently HSF1 has been associated with a broader role in regulating response to a variety of cellular stresses beyond heat-shock. We previously found that high HSF1 expression is associated with poor outcome in lung, breast and colon cancers. Importantly, however, the HSF1 signature correlated with poor outcome in these studies was not related to the heat shock response, which suggested that tumor outcome associated with high HSF expression may be due to processes other than stress response. Hence, we explored the question whether high HSF1 expression might be associated with the cancer stem cell (CSC) phenotype. To do so, we examined the association of HSF1 with CSC phenotype by FACS and immunofluorescence. In addition, we evaluated the effects of HSF1 over-expression and knock-down on sphere formation and CSC marker expression in breast cancer cell lines. Here, we report results demonstrating that high HSF1 not only correlates with CSC marker expression, but inducible HSF1 over-expression augments and HSF1 knock-down inhibits CSC phenotype. Furthermore, HSF1 expression confers resistance to chemotherapeutic drugs and increases CSC frequency. In conclusion, our study indicates that one of the potential HSP-independent HSF1 driven mechanisms that may contribute to poor outcome in human tumors involves regulation of the CSC phenotype. Hence, therapeutic inhibition of HSF1 may be one route to target CSCs in human tumors.
Cancer stem cells (CSCs) are subpopulation of tumor cells that are capable of self-renewal and differentiation [1–4]. CSCs have the ability to initiate tumors with extremely low seeding numbers in immunocompromised mice and differentiate asymmetrically into non-CSCs while maintaining a pool of CSCs. It is thought that CSCs may be resistant to chemotherapy and radiation due to their low rate of cell division .
While the presence of CSCs has been documented in multiple human tumors, the mechanisms that regulate CSCs remain to be fully elucidated. Several pathways including Hedgehog, NOTCH, and Wnt/β-catenin signaling have been associated with the CSC phenotype [6–8]. However, the rare nature of CSCs coupled with the methodological challenges associated with isolating, maintaining, and expanding them in vitro has slowed progress. Hence, much remains to be learned about CSC biology.
Heat shock factor 1 (HSF1) is the master transcription regulator of heat shock response (HSR). In addition, HSF1 coordinates the response of cells to diverse arrays of environmental and physiological stresses, such as ischemic injury, heavy metals, chemicals, free-radicals, inflammation, etc. .
We recently demonstrated that HSF1 is highly expressed in multiple human tumors including lung, colon, breast, and ovarian cancers. Importantly, these studies showed that HSF1 over-expression is associated with significantly worse outcome in these tumors [10, 11]. Through genome-wide ChIP-Seq analysis, we also discovered that HSF1 regulates two classes of genes in tumor cells; one set of genes included well-known stress response related targets such as HSPs; however, a second set of genes were involved with various other cellular processes, including cell cycle, signaling, metabolism, adhesion, and protein translation . Somewhat surprisingly, it was the non-heat shock HSF1 target genes that were closely associated with the more aggressive tumor phenotype, poor outcome, metastasis, and death in breast, colon, and lung tumors isolated directly from patients . High HSF1 expression has also been associated with poor outcome, advanced stage or metastasis in endometrial Carcinoma, hepatocellular carcinoma, melanoma, and squamous cell carcinoma [12–15].
In the present study, we provide evidence that the heat shock-independent activation of HSF1 augments the breast cancer stem cell phenotype. These results provide a new mechanism for the association between high HSF1 expression and poor outcome observed in human tumors.
Materials and methods
BPLER and HMLER cells were described previously  and obtained from live tumor culture core (LTCC, http://sylvester.org/shared-resources/Live-Tumor-Culture-Core). In brief, the BPLER cells are cultured in BMI-T medium obtained from LTCC, and HMLER cells are cultured in MEGM medium (Lonza, Cat. No. CC-3150). The numbers subsequent to the cell name (i.e., 2, 3, and 4) indicate cell lines established from different patient donors. SUM159 and SUM190 were kindly provided by Dr. Stephen Ethier and cultured in their recommended media (Ham’s F12 contains 5 % fetal bovine serum, 5 µg/ml insulin, and 1 µg/ml hydrocortisone). All other cell lines were obtained from the American Type Culture Collection (ATCC) and cultured in recommended conditions. All the cell lines were validated by STR analysis and tested negative for mycoplasma.
Quantitative real-time RT-PCR was performed using SuperScript® III First-Strand Synthesis kit and SsoFast ™ EvaGreen® Supermix according to the manufacturers’ instructions and analyzed with BioRad CFX Manager Software. GAPDH mRNA was used as an internal control to normalize RNA inputs, and expression levels were calculated according to the relative ΔCt method. Primers sequences are annotated in Supplemental Table 1.
Flow cytometry and cell sorting
Cells were harvested at 80 % confluence with non-enzymatic dissociation buffer (Life Technologies, Cat. No. 13151-014) and cell pellets were washed, resuspended in Hank’s Balanced Salt Solution (HBSS) buffer containing 0.2 % BSA, and stained with fluorophore-conjugated monoclonal antibodies per manufacture’s instruction. Antibodies used for cell sorting included anti-CD44, APC conjugated (BD Pharmingen, 559942) and CD166, PE conjugated (BD Pharmingen, 559263). Flow sorting was performed using BD FACS Aria II and purity of sorted cells was confirmed to be between 90-95 %. Sorted cells were washed once with HBSS before protein extraction.
Cells were plated on 8-chamber slides, fixed with 4 % PFA for 15 min at room temperature, and then permeabilized for 10 min with 0.2 % Tween-20. After blocking for 1 h in 10 % Goat serum, slides were incubated overnight with primary antibodies at 4 °C, followed by 45 min incubation with Alexa Fluorophore-labeled secondary antibodies. Fluoro-Gel II containing DAPI (Electron Microscopy Sciences, Cat. No. 17985-50) was used to mount slides, and images were taken on an EVOS fluorescence microscope (AMG & Life Tech). Primary antibodies and their dilutions used in our experiments are listed in Supplemental Table 2.
Doxycycline inducible HSF1 over-expression and knockdown
For inducible HSF1 over-expression, a complete HSF1 ORF was subcloned into pENTR1A gateway entry vector (ENTR) and transferred into pLenti CMV/TO Hygro DEST plasmid (Addgene, 17291) provided by Dr. Eric Campeau . pLenti CMV TetR Blast was used for Tet-repressor expression (Addgene 17492). For inducible HSF1 knockdown, specific HSF1 shRNA sequences were obtained from Dr. Susan Lindquist  and cloned into pLKO-Tet-On-Neo plasmid provided by Dr. Dmitri Wiederschain from Addgene (Cat. No. 21916) . Non-target shRNA (NT shRNA) was cloned into the same plasmid to serve as a control. Lentiviral packaging was achieved using the second generation packaging plasmid pCMV-dR8.2 dvpr and pCMV-VSV-G (Addgene Cat. No. 8455 and 8454). Lentivirus-infected cells were selected with antibiotic treatment. Doxycycline (DOX) was added into cell culture to induce HSF1 over-expression or knockdown before experiments. To ensure that the experiments were carried out below toxic DOX doses, first serial dilutions of DOX were tested and 1 ng/ml was determined to be optimal for use in HSF1 over-expression or knockdown.
Tumorsphere formation assays were performed following Clarke et al. with slight modification . In brief, 3000 to 5000 cells were plated on 6-well plates pretreated with polyhema and maintained in 4-ml tumorsphere media. The tumorsphere medium is composed of a 1:1 ratio of DMEM/F12 containing 2 % B27 (Invitrogen), 20 ng/ml EGF, 20 ng/ml bFGF (BD Biosciences), 4 µg/ml heparin (Sigma), and 0.5 % methyl cellulose. For inducible HSF1 over-expression or knockdown groups, cells were pretreated with DOX for 48 h before sphere assay and sphere medium was supplemented with DOX to maintain the induction. After 10–14 days of incubation, spheres were consolidated into a 96-well plate and imaged on an EVOS fluorescence microscope (AMG & Life Tech) after DAPI staining and they were also counted using GelCount™ (Oxford Optronix) following iodonitratetrazolium chloride staining. Each group included triplicate samples and at least 3 independent experiments were carried out.
Western blot assays were performed as described before . Protein lysates were harvested with RIPA buffer and run on SDS-PAGE gels. Blots were probed with antibodies listed in Table S2. Blots were developed using SuperSignal West regular or Dura ECL (ThermoScientific).
The effect of paclitaxel (Taxol) on cell proliferation was tested both in 2D and 3D cultures. For 2D culture, cells were treated with Taxol at 2.5 nM with HSF1 over-expression or knockdown for 4 days and counted with Trypan blue exclusion method using a Nexcelom Auto T4 Cellometer. For 3D culture, 5 nM taxol was added into pre-induced HSF1 over-expression or knockdown cells and maintained for 4 days. Cells were allowed to recover for 3 days before trypsinization and subjected to tumorsphere assay. Percentage inhibition on 2D or 3D cell culture was compared to control groups which only had vehicle addition.
For protein translation inhibition, 3 pairs of HMLER and BPLER cells were plated at 8000 cell/well on 96-well plate one day before adding protein translation inhibitors including anisomycin and cycloheximide. Protein translation inhibitor drugs were removed after 8 h incubation and cells were maintained in fresh drug-free media for additional four days before being subjected to CellTiter-Blue® (Promega, Cat. No. G8081) assay. Inhibition of cell proliferation inhibition was compared to the vehicle control group.
Inhibition of cell proliferation and sphere formation was analyzed using student’s t test or one-way ANOVA with significance at p < 0.05.
HSF1 is highly expressed in CSCs
We initially explored the potential role of HSF1 in CSCs using CSC-like BPLER and non-CSC-like HMLER cell lines. Both BPLER and HMLER lines do form mouse xenograft tumors that are histologically similar to human primary triple-negative breast carcinoma (TNBC), and their global mRNA profile mimics human TNBC . We previously demonstrated that only the BPLER cells possessed CSC-like features, even though the matched HMLER cell lines are isolated from the same patient and transformed with identical oncogenes [16, 20]. In vivo tumor formation studies showed that as low as ten unsorted BPLER cells could form tumors in mice. In contrast, >104 HMLER cells were required for tumor formation. Consistent with these observations, we now show that BPLER cells express high levels of CSC-associated markers such as CD326 (EpCAM), CD44v, and CD166 and form fivefold more tumor spheres compared to HMLER (Fig. 1a) .
Taking the advantage of this cell model system, we compared HSF1 expression between BPLER and HMLER cells using real-time PCR and Western blotting, and found that HSF1 is over-expressed in all three CSC-like BPLER cell lines compared to non-CSC-like HMLERs not only at mRNA level (Supplemental Figure 1), but also at protein level (Fig. 1a), which was the first indication that high HSF1 levels may be associated with the CSC phenotype.
Next, we confirmed these observations in other cancer cell lines that represent all three subtypes of breast cancers, including T47D (ER+), MCF7 (ER+), BT20 (TNBC), BT474 (Her2+), and HCC1954 (Her2+). Fluorescence-activated cell sorting (FACS) has been used to isolate CSC and non-CSC subpopulations based on cell surface marker expression [23–28]. In our work, we used CD44/CD166 double staining and found that CD44high/CD166high subpopulation has significantly greater sphere-forming capacity compared to non-CSC (CD44low/CD166low) counterparts (data not shown). By Western blot, we found that HSF1 expression is much higher in these FACS-enriched CSC subpopulations compared to the non-CSCs in all three subtypes of breast cancer cell lines, further supporting a potential correlation between HSF1 expression and CSCs phenotype (Fig. 1b).
In order to exclude artifacts associated with FACS enrichment of CSCs, we used dual-immunofluorescence (IF) staining to examine co-expression of HSF1 and CSC markers in situ. These IF stains demonstrate that HSF1 is highly expressed in 13–35 % of the cells (HSF1high) in all of the breast cancer cell lines we examined (Fig. 1c, Supplemental Table 3). Importantly, this HSF1high subpopulation co-express higher levels of CSC markers (CD44/CD326) (Fig. 1c). In MDA-MB231 (TNBC) cell line, ~35 % cells express high levels of HSF1 and 90 % of these HSF1high cells co-express CD44. In MCF7 (ER+) cell line, 57 % of the HSF1high cells are CD44 high. In T47D (ER+) and BT474 (Her2+), approximately 56–68 % of the HSF1high cells are also CD326 high (Fig. 1c, Supplemental Table 3).
HSF1 is necessary to maintain CSC phenotype in breast cancer cell lines
Given the promising correlation between high HSF1 expression and CSC marker expression, we sought to determine whether HSF1 plays a functional role in CSC regulation. After examining endogenous HSF1 expression levels in twenty-two commonly used breast cancer cell lines, we selected T47D, MCF7, and BT474 cell lines that have high endogenous HSF1 expression levels to examine the changes in CSC phenotype after HSF1 knockdown (Supplemental Fig. 2).
We used the Tet All-in-One inducible lentiviral shRNA knockdown system (Tet-on shHSF1) with non-Target shRNA (NT-shRNA) as control for HSF1 knockdown. The induction efficiency was tested using various doses of DOX and as low as 1 ng/ml was able to achieve HSF1 knockdown (Supplemental Fig. 3). HSF1 expression was reduced after induction of HSF1-shRNA with 1 ng/ml DOX for 48 h (Fig. 2a), which is associated with a decrease in CSC markers CD133/CD326 in MCF7 and BT474, and CD133/CD166/Nanog in T47D  (Fig. 2a). Moreover, we found that knockdown of HSF1 significantly decreases 3D tumorsphere formation (Fig. 2b). In control experiments, there was no change in the HSF1 levels, CSC markers, or sphere formation with NT-shRNA cells after DOX stimulation, ensuring the results are specific to HSF1 knockdown (Supplemental Figure 4). There was also no effects on cell viability or proliferation rate in 2D cultures after HSF1 knockdown (data not shown), suggesting that the decrease in sphere formation is not due to a generic suppression of cell proliferation.
In order to examine the CSC phenotype after HSF1 over-expression, we selected SUM159 and BT20 because of relatively lower expression levels in these lines (Supplemental Fig. 2). The HSF1 over-expression in these two cell lines was achieved using lentiviral tetracycline inducible expression system (TetR-HSF1). The sphere formation assays revealed that HSF1 over-expression significantly increases the CSC markers ALDH1 and SOX2 in both cell lines compared to control group (Ctrl) (Fig. 2c), and induced the number of tumorspheres by >twofold (p < 0.05) (Fig. 2d). This effect was not due to a generic increase in cell proliferation since we did not observe any changes in cell proliferation in routine 2D culture after HSF1 over-expression (data not shown). Taken together, these data suggest that HSF1 is both sufficient and necessary to augment the CSC phenotype.
HSF1 expression confers drug resistance
Another feature of CSCs is their relative resistance to chemotherapy . To test whether HSF1 is involved with chemo-resistance, we treated cells with HSF1 over-expression or knockdown with paclitaxel (taxol) and found that HSF1 overexpressing cells are more resistant to taxol treatment compared to control both in BT20 and SUM159 cell lines (Fig. 3a). Conversely, HSF1 knockdown markedly increased cell death after taxol treatment in all three lines tested (BT474, T47D, and MCF7) (Fig. 3b).
In order to test whether changes in chemotherapy resistance after HSF1 over-expression and knockdown is related to the CSC phenotype, we carried out 3D tumorsphere assays and found that HSF1 over-expression increases sphere formation in SUM159 after taxol treatment (Taxol + HSF1) compared to the taxol alone group (Taxol+) (Fig. 3c). Conversely, in BT474 HSF1 knockdown cells treated with taxol (Taxol + HSF1 KD), there was a small yet statistically significant decrease in number of tumorspheres compared to treatment alone group (Taxol+) (Fig. 3d, insets) [9, 30]. Consistent with our results, Vydra et al. also showed that HSF1 over-expression causes chemo-resistance by increasing side-population (SP) cells in human melanoma .
Regulation of CSC phenotype by HSF1
To examine the possible factors that may mediate the actions of HSF1 that effect CSC phenotype, we first analyzed the expression of heat shock proteins in HSF1high CSC-like BPLERs and HSFlow non-CSC-like HMLERs. Compared to the significant differential expression of HSF1 between BPLERs and HMLERs, we only observed very minor changes in the expression of HSP70 and HSP90, two of the main heat shock proteins (Fig. 4a). Next we examined whether HSF1 over-expression increases markers of epithelial-to-mesenchymal transition (EMT), which has been implicated in inducing the CSC phenotype . However, we did not observe any change in EMT markers such as E-cadherin, Vimentin, or ZEB1 with HSF1 over-expression or knockdown (Fig. 4b, c). Likewise, there was no significant change in HSP70 or HSP90 expression after HSF1 knockdown or over-expression in breast cancer cells (Fig. 4c).
It has been reported that malignant cells upregulate their protein translation to manage the high metabolic stress associated with the malignant phenotype . Santagata et al. found that the increased protein translation in cancer cells may be mediated by HSF1 and it is essential for cancer cell survival . Consistent with this, we found that both knockdown of HSF1 and inhibition of protein translation using Cycloheximide or Anisomycin cause inhibition of HSF1high-CSC-like BPLER proliferation compared to HSF1low non-CSC-like HMLER cell lines (Fig. 4d, Supplemental Figure 5).
Here we report that HSF1 plays a role in the regulation of cancer stem cell phenotype in breast cancer cell lines. Previously, we demonstrated that high HSF1 expression is associated with poor prognosis and increased mortality in more than 1800 clinical breast cancer patients’ samples . In this study, we discovered that HSF1 is highly expressed in breast CSC subpopulations. Furthermore, we found that CSC phenotype is augmented by HSF1 over-expression and inhibited by HSF1 knockdown in breast cancer cells lines. Hence, cumulatively, our results suggest that the correlation between high HSF1 expression and poor patient outcome might be partially explained by the actions of HSF1 on CSCs in breast tumor [29, 30, 33, 34].
Together these data suggest that HSF1-mediated augmentation of CSC phenotype involves mechanisms in addition to HSPs and EMT, including HSF1-mediated protein translation that may be feature of CSC survival. Interestingly, a recent genome-wide siRNA screen identified protein degradation and proteasome addiction as a vulnerability of HSF1high CSC-like BPLER cells, which provides independent corroboration of these results . The inhibition of the proteasome reduced growth of established breast cancers in mice and blocked tumor initiation by CSCs and metastasis . It is intriguing that the hypothesis-based studies converge with unbiased high-throughput screens over the protein turn-over machinery.
While the role of HSF1 has been traditionally explored in terms of its role in regulating HSPs and stress response, there is rapidly growing evidence that HSF1 regulates many non-HSPs genes that regulate many cellular processes including protein translation, cell cycle, glucose anabolic metabolism, autophagy, apoptosis, p53, Ras, MAPK, cAMP, PKA, and mTOR pathways [11, 35–37]. Our results suggest that some of these non-HSP targets of HSF1 may be involved in the regulation of CSC phenotype as well, which makes HSF1 as a potential target to develop CSC-specific therapies [32, 38–40].
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We thank Dr. Stephen Ethier for providing the SUM159 and SUM190 cell lines. This study was funded by grants from the Florida Bankhead-Coley Cancer Research Program (Grant No: 4CC12), Breast Cancer Research Foundation, NY, 1R01ES024991 and R01CA146445 (to T.A.I.).
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The authors declare no conflict of interest.
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Wang, B., Lee, CW., Witt, A. et al. Heat shock factor 1 induces cancer stem cell phenotype in breast cancer cell lines. Breast Cancer Res Treat 153, 57–66 (2015). https://doi.org/10.1007/s10549-015-3521-1