Heat treatment-induced autophagy promotes breast cancer cell invasion and metastasis via TGF-β2-mediated epithelial-mesenchymal transitions

Cell culture and heat treatment

The human BC cell lines MCF-7 and MDA-MB-231 (MDA-231) were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and cultured in minimal essential medium (MEM) with 10% fetal calf serum (Gibco, Thermo Fisher Scientific, Dreieich, Germany), 2 mM L-glutamine, non-essential amino acids, 1 mM sodium pyruvate, 100 units/mL of penicillin and 100 µg/mL of streptomycin. The cells were maintained in a humidified atmosphere of 5% CO₂ at 37 °C.

MCF-7 and MDA-231 cells were grown on six-well plates (5 × 10⁴ cells/well), and after 24 h of incubation, they were exposed to heat treatment. Heat treatment was performed by sealing the tops of culture flasks with parafilm and submerging the flasks in an isothermic water bath set to the target temperatures of 37 °C, 42 °C, 45 °C, 47 °C, 50 °C, 53 °C and 55 °C for 30 min. Next, the cells were seeded into 96-well culture plates (1 ×10⁴ cells per well) in 100 µL DMEM and maintained at 37 °C for 24 h for recovery. The culture medium was exchanged every 8 h for 24 h to remove dead cells and debris. One day after heat treatment, cell viability was measured using a CCK-8 assay according to the manufacturer’s instructions. We used a nonlinear regression curve fitting by Prism 6.0 (GraphPad Software; GraphPad, Inc., San Diego, CA, USA) to calculate IT₅₀, the temperature that induced a 50% reduction in cell viability relative to that in the 37 °C control cells, . The IT₅₀ data were used for subsequent experiments to simulate sublethal heat treatment conditions. Heat treatment dose–response curves of BC cells showed that IT₅₀ was 47.2 °C for MCF-7 and 46.8 °C for MDA-231. Our autophagy detection experiments showed a significant heat-induced autophagy at 47 °C for 30 min. On the basis of these data, we selected 47 °C for 30 min to simulate the effects of insufficient MWA and maintained cells at 37 °C for in vitro control experiments. After heat treatment, cells were cultured at 37 °C for recovery.

Antibodies and reagents

Chloroquine (CQ), 3-methyladenine (3-MA), rapamycin and TGF- β₂ were purchased from Sigma-Aldrich (St Louis, MO, USA). LY2109761 was from Selleck Chemicals (Houston, TX, USA). Antibodies against microtubule-associated protein 1 light chain 3-I (LC3-I), LC3-II, p62, Beclin 1, ATG7, poly (ADP-ribose) polymerase (PARP), cleaved-PARP, caspase 9, cleaved-caspase 9, Bax, and Bcl-x1 were from Abcam (Cambridge, MA, USA). Antibodies against TGF-β₂, Smad2, p-Smad2, E-cadherin, α-catenin, β-catenin, N-cadherin, fibronectin, ZO-1, Vimentin, MMP-9, Snail, Slug and β-actin were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Primary antibodies of LC3-II and E-cadherin and secondary antibodies Alexa Fluor 568 anti-mouse IgG and Alexa Flour 568 anti-rabbit antibodies were from Jackson Immuno Research (Lancaster, PA, USA).

Cell viability and clonogenic assays

Cells were seeded into 96-well plates at a density of 1 × 10⁴ cells per well and cultured overnight for attachment. Next, cells were exposed to heat treatment, and/or 3-MA (8 mM) for the indicated times (Dai et al., 2018). After incubation for 24, 48, 72, 96, and 120 h, we determined cell viabilities using a Cell Counting Kit-8 (CCK-8) assay (DoJindo, Tokyo, Japan), according to the manufacturer’s instructions as previously reported (Chen et al., 2019a; Chen et al., 2019b). The absorbance was measured at a wavelength of 450 nm with a reference wavelength of 650 nm using a microplate reader.

We assessed the cell colony formation ability using a clonogenic assay as previously reported (Chen et al., 2019a; Chen et al., 2019b). Briefly, cells were seeded into six-well dishes at a concentration of 1 × 10³ cells/well and allowed to grow in complete medium for 14 days after treatment with the indicated temperatures and/or drugs. The colonies obtained were washed with PBS and fixed in 4% paraformaldehyde for 20 min at room temperature, followed by staining with crystal violet. The colonies were counted and the numbers compared with those of untreated cells (control).

Analysis of cell apoptosis

Cells were treated with the indicated temperature in the absence or presence of 3-MA (10 µM) for 30 min, followed by culture at 37 °C for 24 h. An Annexin V-FITC/ propidium iodide (PI) apoptosis kit (Multi Sciences Biotech, Hangzhou, Zhejiang, China) was used to analyze the apoptosis rate. When the time point had been reached, cells were washed with cold PBS twice. After adding 5 µL Annexin V-FITC and 10 µL PI, cells were analyzed by fluorescence-activated cell sorting (FACS) with flow cytometer, as previously described (Dai et al., 2018). In addition, we detected apoptotic cell features using a Hoechst 33258 staining kit (Beyotime, Shanghai, China). Cells were seeded into a six-well plate (1 × 10⁵ cells/well), cultured for 24 h and, subsequently, treated with the indicated temperature and/or agents. Next, cells were stained with Hoechst 33258. Apoptotic morphological features (chromation condensation and nuclear fragmentation) were evaluated and imaged using fluorescent microscopy.

Western blotting and real-time quantitative PCR analysis

Before and after treatment with the indicated temperature and/or agents such as CQ (10 µM), 3-MA (8 mM) and LY2109761 (20 µM), or transfections with the siRNA against relevant genes, we prepared whole lysate proteins extracted from the cells and analyzed them using standard western blotting as previously described (Dai et al., 2018).

For real-time quantitative PCR analyses, total RNA was extracted from cell samples using Trizol reagents (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. RNA (1–2 µg) were reversely transcribed into cDNA using the Super Script III First-Strand Synthesis kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions; and the reactions were run on a BAI 7500 Fast real-time PCR system, and performed with the following conditions: 95 °C for 3 min; 95 °C for 10 s, and 40 amplification cycles (95 °C for 10 s and 65 °C for 30 s, and 60 for 15 s). The reaction system consisted of 20 µL, including 6.4 µL DNase-free (dd) water, 10 µL Mix SYBR (QP-010140 FOREGENE), 0.8 µL forward and reverse primers and 2 µL cDNA diluent. GAPDH was used as the internal control. Detection of PCR products was accomplished by measuring the emitting fluorescence (Rn) at the end of each reaction step. mRNA relative quantification was calculated using the Ct(2^−ΔΔCt) method (VanGuilder, Vrana & Freeman, 2008). The sequence of primers used in qrt-PCR are shown in Table S1.

Small interfering RNA transfection

All siRNA reagents used in this study were purchased from Guangzhou Ribobio (Guangzhou, China). Before treatment with the indicated temperatures, cells were transfected with Lipofectamine 2000 reagent according to the manufacturer’s instructions as previously published (Chen et al., 2019a; Chen et al., 2019b). The transfection efficacies were verified using western blotting.

Immunofluorescence and monodansylcadaverine staining

LC3-II puncta were detected via immunofluorescence as previously published (Dai et al., 2018; Xiang et al., 2020). Briefly, cells were seeded into 96-well plates and treated with the indicated temperature or/and agents before being fixed with 2% paraformaldehyde in PBS for 20 min at 37 °C. Following permeabilization for 3 min with PBS−0.2% Triton X-100, the cells were incubated overnight at 4 °C with anti-LC3 antibody diluted 1:100. Next, the cells were incubated for 1 h at room temperature with Alexa Fluor 488-conjugated secondary antibody at a 1:400 dilution. The cells were counterstained with Hoechst 33342 for 5 min for nuclear staining, and mounted onto glass slides. We visualized the cells using a Leica confocal laser scanning microscope (Leica, Wetzlar, Germany).

The formation of acidic vascular organelles (AVOs) was detected using monodansylcadaverine (MDC) staining as previously reported (Dai et al., 2018; Xiang et al., 2020). Briefly, cells were seeded onto 24-well plates and treated with the indicated temperature or/and agents. Next, the cells were incubated with 0.5 mM MDC for 2 h in serum-free medium at room temperature. We detected cellular fluorescence changes at an excitation of 380 nm and an emission of 510 nm as observed under an epi-fluorescence microscope (Olympus, Tokyo, Japan).

For the detection of the intracellular localization of proteins, cells were seeded on gelatin-coated coverslips and treated with the indicated temperature and/or agents. Next cells were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100 for 10 min and blocked with 0.5% BSA for 30 min. Target proteins in cells were visualized after incubation with the corresponding antibodies overnight, followed by incubation with FITC-conjugated secondary antibodies for 1 h. We subsequently used rhodamine-conjugated phalloidin and 4, 6-diamidino-2-phynylindol (DAPI) to localize F-actin and nuclei. Images were acquired with a confocal microscope (Leica, Wetzlar, Germany).

Transmission electron microscopy (TEM)

Autophagosomes were observed using TEM as previously reported (Dai et al., 2018). Briefly, cells (10 × 10⁶ cells/mL) were treated with the indicated temperatures and fixed with Karanovsky’s fixation for 1 h at room temperature. Dehydration was performed with graded ethanol and propylene oxide, and cells were embedded in araldite using a kit (Merck, Rahway, NJ, USA). Ultrathin sections were prepared on an LKBIII Ultratome using a diamond knife (Diatome, Switzerland), and the sections were mounted on Formvar-coated 200-mesh nicked grids. The grids were double stained for 1 h with saturated uranyl acetate in 50% methanol. The sections were examined in a JEOL-100 CX transmission electron microscope, at 80 kV.

Cell migration and invasion assays

We assessed cell migration using wound healing assays as previously reported (Xiang et al., 2020). After heat treatment, cells were cultured for 24 h and grown in medium containing 10% FBS to form nearly confluent cell monolayers. We used pipette tips to carefully scratch the surface creating a denuded zone (gap) of constant width. Subsequently, we washed the cellular debris with PBS. The wound closure was monitored and photographed at 0 and 72 h under a Levia inverted microscope (Olympus, Tokyo, Japan). To quantify the migrated cells, pictures of the initial wounded monolayers were compared with the corresponding pictures of cells at the end of the incubation period.

We assessed cell invasion using a Transwell membrane culture system coated with Matrigel as previously reported (Xiang et al., 2020). After treatment with the indicated temperature and/or agents, cells were seeded on the upper wells of a precoated Transwell plate (2 × 10⁴ cells per well). The lower wells of each Transwell plate contained the same medium with 10% FBS. After 48 h of incubation, the cells on the upper well and the membranes coated with Matrigel were swabbed with a Q-tip, fixed with methanol, and stained with 20% Giemsa solution. The cells attached to the lower surface of the polycarbonate filter were counted under an inverted microscope (we determined that the percentage of invading control cells to be 100%).

In vivo xenograft tumor assay

Female BALB/c nude mic (specific pathogen-free grade, 5–6 weeks of age) were obtained from the Animal Core Facility of Nanjing Medical University (Nanjing, China) and maintained under standard pathogen-free conditions. All animal procedures were performed in accordance with the protocols approved by the Institutional Animal Care and Use Committee of the Jiangsu University (JSDX-2022-068). MDA-231 cells were pretreated with 47 °C for 30 min or maintained at 37 °C (control). After recovery at 37 °C for 24 h, cells (5 × 6⁶ cells in 100 µl of serum-free RPMI 1640 medium) were subcutaneously injected into the mammary fat pad of 20 nude mice. Mice were divided into four group (n = 5 per group): vehicle (37 °C treatment), CQ (37 °C treatment), heat treatment (47 °C), and a combination of CQ and heat treatment. Starting on the second day, mice in the vehicle and heat treatment groups received intraperitoneal injections of 100 µl of PBS once every other day, while mice in the CQ and combination treatment groups received intraperitoneal injections of 60 mg/kg CQ in 100 µl of PBS once every other day for a total of 14 injections. Tumor size was measured once per week for 6 weeks, and tumor volumes were calculated using the formula: V =(Length × Width²)/2. By the end of the experiment, mice were euthanized, the tumors were excised and weighed, and half of the tumor samples were fixed for histological examination. The remaining tumor samples were stored at −80 °C for western blotting.

Tail vein metastatic assays

MDA-231 cells pretreated with 47 °C and maintained at 37 °C were suspended in 100 µl PBS and injected through the tail vein into female BALB/c nude mice. Mice were divided into three group (n = 5 per group): vehicle (37 °C treatment), heat treatment (47 °C) and a combination of heat treatment and CQ. One day after tail vein injection, mice in the vehicle and heat treatment groups received intraperitoneal injections of 100 µl of PBS thrice weekly, while mice in the combination treatment group received intraperitoneal injections of 60 mg/kg CQ in 100 µl of PBS thrice weekly. Five weeks after the injection treatment, the mice were sacrificed and the lung tissues were isolated and made into serial sections for hematoxylin & eosin (HE) staining and observed under a light microscope for counting the number of pulmonary metastatic foci.

Statistical analysis

We performed all the experiments in triplicate, and the results are presented as means ± SDs. To compare the two groups, we used a two-tailed unpaired t-test. To compare multiple groups, we applied one-way ANOVA analyses followed by Tukey post hoc tests. p-values <0.05 were considered significant. All statistical data were analyzed using SPSS (version 24.0) or GraphPad Prism (7.0 version) software.

Sublethal heat treatment induces apoptosis but promotes proliferation of surviving BC cells

We first examined the viability changes of BC cells at different time points following heat treatment at different temperatures to evaluate the influence of heat treatment on the proliferation of BC cells. MCF-7 and MDA-231 cells were exposed to heat treatment of 42 °C and 47 °C, respectively, for 30 min to simulate insufficient MWA conditions. We used cells incubated at 37 °C for the control experiments. Thereafter, heat-treated cells were cultured at 37 °C for recovery. After continuous incubation at 24, 48, 72, 96, and 120 h, we determined the cells viabilities using a CCK-8 assay at the corresponding time points. The viability rates of the cells treated at 42 °C and 47 °C decreased gradually in a temperature-dependent manner during the first four days as compared to the viability rates of the control cells. However, cells treated at 47 °C showed an increase in their viability rate on the fifth day after the heat treatment (Figs. 1A and 1B). Moreover, heat treatment induced a temperature-dependent cell apoptosis (Fig. 1C), which was paralleled by an increase in cleaved-PARP, cleaved-caspase 9, and Bax, and by a decrease in Bcl-xl in a time-dependent manner (Fig. 1D). It is worth nothing that heat-treated cells displayed more colony formations than cells treated at 37 °C (Fig. 1E). The data suggest that heat treatment promoted a delayed proliferation of the BC cells, although the cell viability rates were reduced shortly after the heat treatment.

Sublethal heat treatment induces autophagy of BC cells

Sublethal heat treatment is known to induce autophagy in Hela, A549 and HCC cells (Jiang et al., 2019a; Jiang et al., 2019b; Zhao et al., 2009). We tested whether this phenomenon also occurred in BC cells by detecting autophagy-associated protein changes in surviving heat-treated BC cells. We observed increased protein expressions of LC3-II and Beclin 1, and decreased protein expression of p62 in heat-treated BC cells as compared with the same expressions in control cells (Fig. 2A). We confirmed these protein expression changes in a time-dependent manner using western blotting (Fig. 2B). We next analyzed autophagosome formation via LC3-II immunofluorescence staining. Heat treatment increased the formation of autophagosomes in a temperature-dependent manner as indicated by an increase in the number of LC3-II-positive puncta in MCF-7 and MDA-231 cells (Fig. 2C). The MDC staining of heat-treated cells showed an increase in temperature-dependent fluorescence intensity (Fig. 2D), indicative of the presence of acidic vesicular organelles (AVOs) including lysosomes and autolysosomes. Further examination using TEM revealed an increased number of autophagosomes in heat-treated BC cells (Fig. 2E). An increase of LC3-II protein levels can result from an upregulation of autophagosome formation (stimulated flux) as well as a blockage of autophagosome degradation (interrupted flux). An increase in LC3-II protein levels between conditions when flux is briefly blocked with an inhibitor of autophagosome degradation is indicative of upregulated autophagy (Klionsky et al., 2016). Hence, we treated cells with a autophagy inhibitor, CQ, which inhibits late stage autophagy through the suppression of autophagosome degradation by inhibiting lysosomal acidification (Pasquier, 2016). Our results showed that CQ treatment resulted in the further accumulation of LC3-II in the heat-treated cells (Fig. 2F), indicating that sublethal heat treatment induced the increase of autophagosome formation rate and autophagy flux in BC cells.

Heat-induced autophagy promotes EMT in BC cells by up-regulating TGF-β2/Smad2 signaling

Autophagy has been reported to be necessary for TGF-β2-induced EMT through Smad2 signaling in HCC cells (Dash et al., 2018). Therefore, we conjectured whether the heat-induced autophagy would facilitate EMT in BC cells by up-regulating the TGF-β2/Smad2 signaling pathway. We examined the effects of autophagy induction or inhibition on TGF-β2 and EMT, and we explored the reciprocal role between autophagy and TGF-β2 during the process. We found that heat treatment activated autophagy and also up-regulated the TGF-β2 expression and phosphorylation level of Smad2. Co-treatment with 3-MA, an early phase autophagy inhibitor that can impedes autophagosome formation via its inhibitory effect on class III PI3K (Pasquier, 2016; Miller et al., 2010), suppressed both heat-induced autophagy, TGF-β2 expression and Smad2 phosphorylation (Figs. 3A and 3B). We obtained similar results with the BC cells co-treated with ATG7 knock-down and heat exposure (Fig. 3C; Figs. S1A) and S1B). To inhibit the TGF-β2 signaling pathway in BC cells, we co-treated the cells with heat exposure and LY2109761, a selective TGF-β2 receptor type I/II inhibitor (Yang et al., 2009). Our results showed that LY2109761 attenuated the heat treatment-induced TGF-β2 upregulation and inhibited Smad2 phosphorylation, while also suppressing autophagy induction (Figs. 3D and 3E). Moreover, we treated the BC cells with TGF-β2 to directly stimulate the FGF- β2/Smad2 pathway, and we observed a significant increase in Smad2 phosphorylation and autophagy levels, which was similar to the results in the cells treated with rapamycin (20 nM), an autophagy inducer (Figs. S2).

Because TGF-β2 is a well-known EMT inducer (Kim et al., 2015), we next detected EMT molecule marker expressions following heat treatment using western blots and real-time PCR. Our findings revealed that heat treatment clearly increased the protein expressions of mesenchymal and promigratory markers, such as N-cadherin, fibronectin, vimentin, snail, slug and MMP-9, and concurrently reduced the protein expressions of epithelial markers such as E-cadherin, α-catenin, β-catenin and ZO-1 (Fig. 3F; Fig. S3A). Real-time PCR analysis showed similar changes in the transcript levels of these EMT-associated markers after heat treatment (Figs. S3B-S3E). Moreover, co-treatment with 3-MA inhibited the heat treatment-induced EMT-associated marker changes in protein and mRNA levels (Fig. 3F; Figs. S3A–S3E).

Using immunofluorescence, we found LC3-II and E-cadherin colocalized in the perinuclear regions after heat treatment unless 3-MA was added (Fig. 3G), suggesting that heat-induced autophagy may entrap E-cadherin from the plasma membrane into autophagosomes for degradation by lysosomes. Taken together, our findings indicate that heat treatment-induced autophagy up-regulates TGF-β2/Smad2 signaling and thereby promotes the EMT of BC cells, while at the same time the upregulated TGF-β2 facilitates autophagy induction.

Heat-induced autophagy enhances the migration and invasion capabilities of BC cells through TGF-β2-mediated EMT

EMT is considered a major driver of cancer exacerbations from initiation to metastasis and invasion (Saitoh, 2018). To further verify the occurrence of EMT, we evaluated the migration and invasion of BC cells using wound healing and Transwell assays. Our results indicate that the cell migration and invasion capabilities were markedly enhanced in heat-treated BC cells compared with those in control BC cells (Figs. 4A–4D). Adding 3-MA significantly depressed the cell migration and invasion caused by the heat treatment (Figs. 4A and 4B). Likewise, co-treatment with LY2109761 dramatically restrained the migration and invasion caused by the heat treatment in BC cells (Fig. 4C and 4D). Collectively, these results signify that autophagy induction and TGF-β2 upregulation, as well as the interaction between the two signaling pathway, promoted EMT and enhanced the migration and invasion capabilities of heat-treated BC cells.

Suppressing heat-induced autophagy facilitates apoptosis and decreases proliferation of BC cells

Accumulating evidence indicates that autophagy serves a cytoprotective role particularly during cancer treatment. In addition, autophagy inhibition can enhance therapy-induced cell death or apoptosis (Amaravadi et al., 2007; Ryter, Cloonan & Choi, 2013). We used the autophagy inhibitor 3-MA to suppress autophagosome formation and block heat-induced autophagy to determine the biological role of autophagy in heat-induced apoptosis. BC cells were treated with 3-MA, heat exposure, a combination of both. As shown in Figs. 5A–5C, co-treatment with 3-MA further reduced the cell viability rates 5 days after heat treatment and further raised the heat treatment-induced cell apoptotic rates. Cells co-treated with 3-MA exhibited more significant apoptotic morphological features such as chromatin condensation and nuclear fragmentation under enhanced bright blue fluorescence (Fig. 5D). In concurrence with these observations, the heat treatment-induced expressions of cleaved-PARP and -caspase 9 were further augmented in the presence of 3-MA (Fig. 5E). The heat treatment-increased Bax expression and -reduced Bcl-xl expression were intensified after 3-MA addition (Fig. 5E). In addition, co-treatment with 3-MA decreased the heat treatment-induced potentiation of cell proliferation (Fig. 5F). All these results demonstrate that autophagy inhibits heat treatment-induced apoptosis, thereby promoting cell proliferation.

Autophagy inhibitor potentiates the antitumor effect of heat treatment and suppresses heat treatment-promoted metastasis in BC cells

To evaluate the effect of sublethal heat treatment on tumor growth and determine whether autophagy inhibitor can enhance the antitumor effect of heat treatment in vivo, we established a xenograft tumor model of MDA-231 cells and examined the effect of vehicle, CQ, heat treatment alone, and a combination of heat treatment and CQ on tumor growth in nude mice. By the end of in vivo experiment, no significant difference in tumor volumes and weights was found between mice treated with vehicle and mice treated with CQ alone. Tumor growth was moderately inhibited by heat treatment alone. However, significant growth inhibition was observed under the combination treatment (Figs. 6A–6C). Western blotting of tumor samples showed that the combination treatment with CQ increased heat treatment induced the accumulation of LC3-II and cleaved-caspase 9, and retarded heat treatment induced the up-regulation of TGF-β2, Smad2 phosphorylation and N-cadherin and down-regulation of E-cadherin (Fig. 6D). These data were consistent with the in vitro results and further confirmed that autophagy inhibitors can enhance the antitumor effect of heat treatment.

To determine the effect of heat treatment and CQ on the in vivo metastasis of MDA-231 cells, a tail vein metastasis assay was applied. As shown in Fig. 6E, the number of metastatic foci in the lung significantly increased in mice receiving heat treated-MDA-231 cells compared with those receiving 37 °C treated-MDA-231 cells, and co-treatment with CQ markedly decreased the number of metastatic foci in lung. The results indicate that sublethal heat treatment promoted the distant metastasis of BC cells, which can be inhibited by suppressing autophagy.