In long-lasting cellular stress phases of melanoma cells, stress granules are dissolved by HSP70

Abstract

Cancer cells must adapt to harsh microenvironmental conditions such as nutrient deprivation, hypoxia and immune pressure to sustain their high proliferative potential. One adaptive mechanism involves the formation of stress granules (SGs), which promote cell survival under stress. Interestingly, melanoma cells appear to tolerate such stressors, including hypoxia and chemotherapeutic agents, with minimal dependence on SG formation. In this study, we identify heat shock protein 70 (HSP70) as a key mediator of stress resistance in melanoma cells and the participation of the kinase CK2 in this process. We demonstrate that melanoma cells express high endogenous levels of HSP70, which preserves protein homeostasis and inhibits apoptosis under both environmental and drug-induced stress. Silencing of HSP70 led to increased SG formation and sensitized melanoma cells to apoptosis, particularly in response to the BRAF inhibitor Vemurafenib. These findings suggest that HSP70 plays a central role in melanoma cell survival by compensating for the need for SGs and promoting resistance to therapeutic stress.

Keywords: Cellular stress; Melanoma; Stress granules; Vemurafenib-resistance.

Fig. 1

G3BP1 and (phospho-)EIF2S1 expression in malignant melanoma. (a) Western blot analysis of G3BP1 protein expression in normal human epidermal melanocytes (NHEM) from different donors and passages (P5–P14), and in melanoma cell lines derived from metastases (SK-Mel-28, SK-Mel-3) and primary tumors (WM1366, Mel Juso, Mel Ho, WM35). ACTINB served as a loading control. Densitometric quantification of G3BP1 level is shown. (b) Analysis of G3BP1 expression in skin cutaneous melanoma (SKCM) samples (red) compared to non-tumor samples (grey). Data obtained from GEPIA database, p-value cut-off = 0.01, |Log2FC| Cut-off = 1, retrieved on August 21, 2025. (c) Immunofluorescence staining of G3BP1 (green) in tissue sections of malignant melanoma, illustrating representative samples with positive (+) and negative (–) G3BP1 staining. DAPI (blue) was used for nuclear counterstaining. Hematoxylin and eosin (HE) staining was performed to visualize tissue morphology. Quantification of G3BP1-positive and -negative samples in tissue microarrays (nevi: n = 12; melanoma: n = 13) is shown. (d) Western blot analysis of phosphorylated (P-) and total EIF2S1 in melanoma and non-melanoma cell lines following sodium arsenite treatment (+ SA) (600 µM) or under untreated conditions. (e) Quantification of the P-EIF2S1/EIF2S1 ratio in each cell line (mean of n = 3 biological replicates). (f) Immunofluorescence staining of G3BP1 in Mel Im melanoma cells following treatment with 600 µM sodium arsenite (SA), 2,2′-Dipyridyl (DP), 2-(N-morpholino)ethanesulfonic acid (MES), or hydrogen peroxide (H₂O₂), to assess stress-induced SG formation. Magnification: 40x. Data are presented as mean ± SEM (**p < 0.01)

Fig. 2

Stress granule (SG) formation in response to hypoxic, oxidative, and osmotic stress. (a) Immunofluorescence staining of G3BP1 in melanoma cell lines Mel Juso and Mel Im following treatment with 600 µM sodium arsenite (SA) or Desferrioxamine (DFX), an iron chelator that mimics hypoxia. SG formation is indicated by punctate G3BP1-positive structures. (b) G3BP1 immunofluorescence staining of Mel Juso cells incubated under hypoxic (1% O₂) or normoxic (21% O₂) conditions for 24 h. SG formation was quantified as the percentage of G3BP1-positive cells. Data represent the mean ± SEM from three independent experiments (unpaired Student’s t-test; ns = not significant). (c) Immunofluorescence staining of G3BP1 in normal human epidermal melanocytes (NHEM) treated with or without 600 µM sodium arsenite, under control conditions or following exposure to 80 mJ/cm² UVC irradiation (image sections *, #: 40x magnification)

Fig. 3

Melanoma cells exhibit high levels of HSP70 and p-G3BP1.(a) Western blot analysis of phosphorylated G3BP1 (P-G3BP1, Ser149) in normal human epidermal melanocytes (NHEM; passages P5 and P6) and melanoma cell lines. Additional tumor cell lines including Panc-1 (pancreatic carcinoma), Hep3B and HepG2 (hepatocellular carcinoma), and HT29 and SW480 (colorectal carcinoma) were included for comparison. ACTINB served as a loading control. (b) Densitometric quantification of P-G3BP1/G3BP1 protein ratio in NHEM, SK-Mel-28, WM1366, Mel Ho, Mel Juso, Sk-Mel-3 and WM35 cells. NHEM protein expression was set to 1. Normalized to ACTINB. (c) RNA sequencing analysis of HSP70 paralogues (HSPA1A, HSPA1B) in melanoma cell lines and NHEM. Read counts were normalized to library size. (d) Quantitative real-time PCR of HSPA1A and HSPA1B mRNA levels in melanoma cell lines and NHEM. (e) Western blot analysis of total HSP70 protein expression in NHEM (passages P5 and P8) and melanoma cell lines. ACTINB served as a loading control. (f) Western blot analysis of p-G3BP1 expression following treatment of Mel Juso and Mel Im cells with hypoxia-mimetic agents Desferrioxamine (DFX) and Dimethyloxalylglycine (DMOG). ACTINB was used as a loading control. (g) Western blot analysis of P-G3BP1 and G3BP1 expression in Mel Juso cells exposed to hypoxic conditions (1% O₂). ACTINB served as loading controls. (h) Western blot analysis of HSP70 expression in Mel Juso and Mel Im cells treated with DFX and DMOG. ACTINB served as a loading control. (i) Western blot analysis of HSP70 expression in Mel Juso cells under hypoxic conditions (1% O₂). ACTINB was used as a loading control

Fig. 4

HSP70 suppresses stress granule (SG) formation and protects melanoma cells from stress-induced apoptosis. (a) Quantitative real-time PCR analysis of HSP70 (HSPA1A/B) mRNA levels in Mel Juso cells 24 h after transfection with an HSP70 siRNA pool (siHSP70). (b) Western blot analysis of HSP70 protein levels in Mel Juso cells following siHSP70 transfection for 24 h. ACTINB served as a loading control. (c) Western blot analysis of HSP70, phosphorylated EIF2S1 (P-EIF2S1), and total EIF2S1 in Mel Juso cells transfected with siHSP70 for 24 h and subsequently treated with 600 µM sodium arsenite (SA) to induce stress. EIF2S1 served as the internal control. (d) Immunofluorescence staining of G3BP1 (red) in Mel Juso cells transfected with either siHSP70 or non-targeting control siRNA (sictrl), followed by SA treatment. Arrows indicate SG formation. Quantification of G3BP1-positive cells is shown; data represent the mean of three independent experiments. (e) Western blot analysis of total G3BP1 and phosphorylated G3BP1 (P-G3BP1) in Mel Juso cells after transfection with siHSP70 or sictrl and SA treatment. ACTINB was used as a loading control. Densitometric quantification from three independent experiments is shown. (f) Western blot analysis of PARP/cleaved PARP and CASPASE9/cleaved CASPASE9 in Mel Juso cells transfected with siHSP70 or sictrl, followed by SA treatment. Full-length PARP and CASPASE9 served as loading controls. Densitometric analysis from three independent experiments is provided. (g) Flow cytometric analysis of apoptotic Mel Juso cells after transfection with siHSP70 or sictrl and subsequent SA treatment. Data are presented as mean ± SEM (*p < 0.05, **p < 0.01 and ***p < 0.001)

Fig. 5

Caseinkinase 2a regulates Stress Granule Formation by modulating G3BP1 phosphorylation (a) Analysis of CSNK2A1 expression in skin cutaneous melanoma (SKCM; T) samples (red) compared to non-tumor skin (N) samples (grey). Data were obtained from the GEPIA database with a p-value cut-off of 0.01 and |Log₂FC| cut-off of 1 (retrieved August 21, 2025). (b) Representative western blot analysis of casein kinase 2α (CK2α) expression in normal human epidermal melanocytes (NHEMs) from different donors and melanoma cell lines (SK-MEL-28, WM1366, SK-MEL-3, Mel Juso, Mel Ho, and WM35). ACTINB was used as a loading control. (c) Schematic representation of the experimental timeline for TBCA treatment. (d) Immunofluorescence staining of G3BP1 (red) in Mel Juso cells treated with TBCA or DMSO for 45 min (stress phase) or after 24 h (recovery phase). Nuclei were counterstained with DAPI (blue). Quantification of stress granule (SG)-positive cells per field of view is shown as the mean of three independent experiments (lower panels). (e) Representative western blot analysis of HSP70, phosphorylated G3BP1 (P-G3BP1), and CK2α in cells treated with TBCA or DMSO, followed by sodium arsenite (SA) treatment for 45 min (stress phase) or 24 h (recovery phase). ACTINB served as a loading control

Fig. 6

Phosphorylated EIF2S1 and stress granule (SG) dynamics in BRAF-mutant melanoma cell lines. (a) Representative western blot analysis of phosphorylated (p-) and total EIF2S1 in BRAF-mutant melanoma cell lines SK-Mel-28 and 451Lu, comparing non-resistant (NR) and vemurafenib-resistant (R) cells. Treatments included no treatment (NT), sodium arsenite (SA), vemurafenib (V), or a combination of both (SA/V). ACTINB was used as a loading control. Bar graph depicts the ratio of p-EIF2S1 to total EIF2S1 based on densitometry from three independent experiments. (b) Immunofluorescence staining of G3BP1 (red) and EIF2S1 (green) in SK-Mel-28 and 451Lu NR and R cells after treatment with SA or SA/V. Nuclei were counterstained with DAPI (blue). Quantification of SG-positive cells per field of view is shown as the mean of three independent experiments. Data are presented as mean ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001; ns = not significant)

Fig. 7

Role of HSP70 in vemurafenib-resistant melanoma cells. (a) Representative western blot analysis of HSP70 and CK2α protein expression in SK-Mel-28 non-resistant (NR) and vemurafenib-resistant (R) cells. ACTINB was used as a loading control. (b) Western blot analysis of HSP70 and phosphorylated EIF2S1 (p-EIF2S1) expression in SK-Mel-28-R cells transfected with HSP70 siRNA (siHSP70) or non-targeting control siRNA (sictrl) for 24 h. Total EIF2S1 and ACTINB were used as loading controls. Densitometric quantification from three independent experiments is shown. (c) Immunofluorescence staining of G3BP1 (red) in SK-Mel-28-R cells following sodium arsenite (SA) treatment (600 µM) for 24 h. Nuclei were counterstained with DAPI (blue). Quantification of SG-positive cells per field of view is shown as the mean of three independent experiments. (d) Western blot analysis of phosphorylated G3BP1 (p-G3BP1) in SK-Mel-28-R cells transfected with siHSP70 or sictrl, followed by SA treatment for 24 h. Total G3BP1 and ACTINB served as loading controls. Quantification from three independent experiments is included. (e) Western blot analysis of full-length and cleaved PARP and CASPASE9 in SK-Mel-28-R cells after transfection with siHSP70 or sictrl, with or without additional SA treatment. ACTINB was used as a loading control. (f) Flow cytometric analysis of apoptosis in SK-Mel-28-R cells after siHSP70 or sictrl transfection (24 h), followed by SA treatment. Data reflect one representative experiment of three independent replicates. Data are presented as mean ± SEM (*p < 0.05 and ****p < 0.0001; ns = not significant)