Background
Melanoma, one of the most malignant tumors, is increasing in incidence worldwide. However, there is still no curative treatment after the disease has spread beyond the primary site, owing to the proliferative ability of the cells [
1]. Melanoma is infamous for its rapid proliferation rate [
2], but until now, the exact mechanisms for the rapid proliferation of melanoma cells has remained unknown [
3]. Because of poor vascularization and high proliferation rates, melanoma is subjected to many forms of stress. Unsurprisingly, these types of stress, including hypoxia, nutrient deprivation and altered pH, result in the accumulation of unfolded and/or misfolded proteins in the endoplasmic reticulum (ER) lumen and cause ER stress. This stress induces the activation of the unfolded protein response (UPR), which restores ER homeostasis, thus leading to cells producing more proteins for neoplastic growth, particularly secretory proteins. The UPR may assist in several aspects of tumor biology, including tumorigenesis, apoptotic evasion, metastasis, angiogenesis and chemotherapy resistance [
3‐
6].
The UPR of mammalian cells is initiated by three ER transmembrane proteins: activating transcription factor 6 (ATF6), inositol-requiring enzyme 1 (IRE1), and double-stranded RNA-activated protein kinase-like ER kinase (PERK), which act as proximal sensors of ER stress. Under normal conditions, the luminal domains of these sensors are occupied by the ER chaperone glucose-regulated protein 78 (GRP78). Under ER stress, sequestration of GRP78 by unfolded proteins activates these sensors by inducing the phosphorylation and homodimerization of IRE1 and PERK along with the relocalization of ATF6 to the Golgi, where it is cleaved by Site 1 and 2 proteases, thus leading to its activation as a transcriptional factor [
7‐
9]. After its activation, IRE1 catalyzes the non-conventional splicing of the mRNA encoding X-box-binding protein 1 (XBP1) by removing a 26 nt intron, thereby producing an active spliced form (XBP1s), thus initiating an essential UPR program [
10].
A growing number of studies report that UPR is activated in various solid tumors; e.g., GRP78 expression is elevated in some cancers, including melanoma [
11‐
13]. The spliced form of XBP1 is frequently expressed in melanoma cell lines and in fresh melanoma isolates [
13]. Recently, it has been shown that UPR is be activated during early stages of melanoma initiation by the oncogenic form of HRAS (HRASG12V) [
14]. The magnitude of the nascent protein production is higher in melanoma cell lines and results in the activation of UPR pathways, including the IRE1α-XBP1 branch [
3,
4,
6].
The activation of signal transducer and activator of transcription 3 (STAT3) is usually transient in normal cells, but STAT3 has been reported to be present in a constitutively activated state and to promote tumorigenesis by enhancing cell proliferation, survival, and angiogenesis while suppressing the anticancer immune response in many different types of cancers, including colon cancer, melanoma and myeloma [
15‐
17]. In some studies of lymphoid malignancies, interleukin-6 (IL-6) has been found to act in an autocrine/paracrine manner and to provide crucial survival signals by activating STAT3 signaling [
17].
Here, we report that the IRE1α-XBP1 branch is activated and that the spliced form of XBP1 (XBP1s) is increased in human melanoma tissues. Compared with normal melanocytes, six melanoma cell lines showed higher XBP1 splicing and enhanced IL-6 expression. Ectopic expression of IRE1α or XBP1s gave rise to IL-6 expression, which in turn promoted Mel-RMu cell proliferation, whereas these effects were blocked by IL-6 antibodies. Further experiments revealed that XBP1s directly bound to the IL-6 promoter and drove its expression. Our study reveals the crucial role of the IRE1α-XBP1 branch in promoting Mel-RMu cell proliferation by regulating IL-6/STAT3 signaling.
Methods
Patient characteristics
Clinical data, including age, sex, and the primary melanoma site, were collected retrospectively from patient records and their pathology reports. All patients were diagnosed with melanoma by the Department of Pathology, Zhongshan Hospital, Fudan University. In total, 61 patients were evaluated, and clinical and pathological data were analyzed for each patient. Of these patients, the youngest was 30 years old, and the oldest was 85 years old. The average age was 57.9 years, and the median age was 59 years. Thirty-six patients were male, and 25 patients were female. The primary sites of melanoma were grouped as head and neck, trunk and limbs, of which 75.41% were in the limbs (Table
1). All of the tumors were without regional or distant metastasis. The tissue sample collection was approved by the Ethics Committee of Zhongshan Hospital, Fudan University, and informed consent was obtained from all subjects. The tissue slides were prepared from biopsy paraffin blocks. The experiments were carried out under approved guidelines and complied with the 1975 Declaration of Helsinki.
Table 1
Clinical characteristics of patients with melanoma
Age |
≤40 | 9 (14.75) |
40–60 | 24 (39.34) |
≥60 | 28 (45.91) |
Sex |
Male | 36 (59.02) |
Female | 25 (40.98) |
Primary sites of melanoma |
Head and face | 3 (4.92) |
Trunk | 12 (19.67) |
Limbs | 46 (75.41) |
Immunohistochemical analysis
Immunohistochemistry was conducted by using anti-human antibodies against XBP1s (1:100, BioLegend, San Diego, CA, USA). The TMA slides stained with XBP1s were evaluated by light microscopy at 200× magnification by two investigators blinded to the clinicopathologic data of the patients. To access the expression intensity of XBP1s, the integrated absorbance in the area of a 1-mm-diameter cylinder was measured by using Image-Pro Plus version 6.0 (Media Cybernetics, Inc., Rockville, MD USA). The mean XBP1s density was calculated as the product of the integrated absorbance to total area. The tissue slides were prepared from biopsy paraffin blocks. The methods were carried out under the approved guidelines and complied with the 1975 Declaration of Helsinki.
Cell culture
Melanocyte cell lines (HEMn-MP and HEMn-DP) and melanoma cell lines (Mel-RMu, MM200, Mel-CV, IgR3, A2058, and SkMel-28) were obtained from the Cell Bank of Shanghai, Chinese Academy of Sciences (Shanghai, China). All of the cell lines were cultured in high-glucose DMEM supplemented with 10% fetal bovine serum (Gibco, Thermo Fisher Scientific, Waltham, MA, USA).
Quantitative real-time PCR
Real-time PCR analyses were performed as previously described [
18,
19]. Briefly, total RNA of the cells was extracted using TRIzol reagent (Invitrogen, Waltham, MA, USA) according to the manufacturer’s protocol. Then, the RNA was reverse transcribed with an M-MLV first-strand cDNA synthesis kit (Invitrogen). Indicated mRNA levels were determined by qPCR using SYBR Premix Ex Taq (Roche, Basel, Switzerland), and human
GAPDH was used as an internal control.
Western blotting
Western blotting analysis was performed as previously described [
20‐
22]. In brief, cells were harvested and lysed in RIPA lysis buffer. Then, proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes. The membranes were washed in TBST, blocked in 10% milk, and then incubated with primary antibodies against human IRE1α (1:1000, Cell Signaling Technology, Boston, USA), XBP1s (1:500, BioLegend, San Diego, CA, USA), pSTAT3 (1:1000, Cell Signaling Technology), STAT3 (1:1000, Cell Signaling Technology) or GAPDH (1:5000, Abcam, Cambridge, UK) overnight at 4 °C, and this was followed by incubation with horseradish peroxidase-conjugated secondary antibodies. Proteins were detected with enhanced chemiluminescence assay (Thermo Fisher Scientific).
CCK8 and BrdU assays
CCK8 assays were used to detect the effect of XBP1s on cell proliferation. Briefly, 1 × 103 cells were seeded in 96-well culture plates, and these cells were then incubated with a CCK8 reagent for 2 h at 37 °C at the 24, 48, 72, 96 and 120 h time points. The staining intensity in the medium was measured by determining the absorbance at 450 nm.
BrdU assays were conducted by using a BrdU Cell Proliferation Assay Kit (#6813, Cell Signaling Technology, USA) according to the manufacturer’s instructions.
Luciferase reporter assay
The pGL3 basic plasmid containing the promoter of the human interleukin-6 gene, which corresponds to the region from −2000 to +100 nt on the putative transcription start site (denoted nucleotide +1), was constructed. The deletion of the ACGT core from the IL-6 promoter was performed by using a PCR-based strategy. HEK293T cells were co-transfected with the designed plasmids. Luciferase activity was measured using a Dual-luciferase Assay Kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Renilla luciferase activity was used as an internal control for normalization.
Chromatin immunoprecipitation (ChIP)
ChIP assays were performed with an Agarose ChIP Kit (Pierce, Cat# 26156, Thermo Fisher Scientific), according to the manufacturer’s instructions. In brief, 293T cells were subjected to cross-linking with 1% formaldehyde, and glycine solution was then added to stop the cross-linking process. Nuclear extracts were prepared. Chromatin-XBP1s complexes were immunoprecipitated with anti-Flag (Sigma, Cat# F3165; diluted 1: 500, St. Louis, MO, USA) or anti-XBP1s (BioLegend, Cat# 647501; diluted 1:100) antibodies by incubation at 4 °C overnight, and this was followed by incubation with beads from the Agarose ChIP Kit (Pierce) or Protein G-Sepharose beads (GE Health, Chicago, IL, USA) at 4 °C for 1 h with gentle rocking. After the beads were washed 5 times with wash buffer, the complexes were eluted from the beads with elution buffer and subjected to PCR analysis.
Statistical analysis
All experiments presented in this paper were repeated more than three times. The data are presented as the mean ± standard error of mean (s.e.m.). Statistical analysis (SPSS 18.0 software, SPSS Inc., Armonk, NY, USA) was performed with two-tailed independent Student’s t tests after a demonstration of homogeneity of variance with the F test or one-way ANOVA for more than two groups. Scheffe tests were used for post hoc analysis. The threshold for statistical significance was set at P < 0.05.
Discussion
UPR pathways, including the IRE1α-XBP1 branch, have been shown to have critical functions in the development of melanoma, but the exact mechanisms have been unclear. In our present work, we found that XBP1s levels were dramatically elevated in human melanoma tissues and melanoma cell lines compared with normal tissues or melanocytes. Ectopic expression of IRE1α and XBP1s in melanocytes and melanoma cells increased IL-6 levels and then activated intracellular STAT3 signaling, which was diminished by the addition of IL-6 antibodies. We also demonstrated that XBP1s bound to the IL-6 promoter and activated IL-6 transcription directly. Furthermore, increased expression of IRE1α and XBP1s promoted Mel-RMu cell proliferation, which was dependent on secreted extracellular IL-6.
XBP1s, the potent transcription activator and product of the RNase activity of IRE1α, mediates the expression of a large group of genes, thus relieving ER stress and reestablishing ER homeostasis [
7‐
9,
24]. In addition to its critical role in ER stress, growing evidence has demonstrated that the IRE1α-XBP1 pathway controls the expression of many genes that are involved in the regulation of various biological processes, such as peroxisome proliferator-activated receptor alpha (PPARα) [
18], protein disulfide isomerase (PDI) [
25], fatty acid synthase (Fasn), and UDP-galactose-4-epimerase [
26]. The IRE1α-XBP1 pathway also exerts non-transcriptional actions such as promoting the degradation of the forkhead box O1 (FoxO1) protein [
27]. XBP1s was also found to activate
IL-
6 transcription by binding to its promoter in murine macrophages involved in innate immunity [
28]. Moreover, Toosi et al. have reported that vitiligo-induced activation of UPR pathways upregulates the expression of IL-6 and IL-8 in melanoma cells, but the molecular mechanism is not clearly defined [
29]. Here, we demonstrated that XBP1s activated
IL-
6 expression by binding to its promoter in human melanoma cells, thus indicating the conservation of XBP1 behavior in controlling IL-6 expression. Inhibition of the RNase activity of IRE1α by 4μ8C impaired IL-6 expression induced by the activation of the IRE1α-XBP1 pathway. Because IL-6 is considered to be a critical player in promoting cell proliferation [
30] and progression and even a prognostic biomarker [
30,
31] of melanoma, the RNase activity of IRE1α may be a promising therapeutic target.
Recently, Liu et al. have reported that IRE1α promotes hepatocyte proliferation and liver regeneration by regulating the STAT3 signaling pathway [
32]. Although IRE1α has been implicated in cell proliferation in pancreatic islet cells [
33] and certain cancer cell lines [
34], it remains unclear whether the IRE1α-XBP1 branch is linked to melanoma cell growth. Our work is the first to demonstrate the critical role of IRE1α-XBP1s in promoting cell proliferation of melanoma and its detailed molecular mechanism.
Constitutive activation of STAT3 signaling has been observed in aggressive forms of cancer and is crucial in regulating tumor cell proliferation and survival in diverse cancer types [
15,
35,
36]. Previous studies have revealed that STAT3 is constitutively activated in approximately 50–90% of melanomas [
37,
38], but the exact details were unknown. Our results suggested that in melanoma cells, the constitutive activation of STAT3 is activated by secreted extracellular IL-6 working in an autocrine/paracrine manner, which can be abolished by adding IL-6 antibodies to the medium, thus neutralizing IL-6. Above all, IL-6 neutralization attenuated the enhanced proliferation of melanoma cells induced by the activation of the IRE1α-XBP1 branch, thus suggesting that anti-IL-6 antibody is a promising candidate for the clinical treatment of melanoma.
Conclusion
In summary, our results reveal a novel role of the IRE1α-XBP1 branch of UPR pathways in regulating Mel-RMu cell proliferation and progression via controlling IL-6 expression and STAT3 signaling. However, in vivo studies are needed to clarify the high rate of melanoma cell proliferation caused by the constitutive activation of STAT3 signaling resulting from XBP1s-driven IL-6 expression. Our study provides a new promising therapeutic target for melanoma treatment and drug discovery.
Authors’ contributions
CC performed experiments, wrote the manuscript and prepared all the figures. XZ designed the study and analyzed the data. Both authors read and approved the final manuscript.