High LIFr expression stimulates melanoma cell migration and is associated with unfavorable prognosis in melanoma

Abstract

Increased or decreased expression of LIF receptor (LIFr) has been reported in several human cancers, including skin cancer, but its role in melanoma is unknown. In this study, we investigated the expression pattern of LIFr in melanoma and assessed its prognostic value. Using tissue microarrays consisting of 441 melanomas and 96 nevi, we found that no normal nevi showed high LIFr expression. LIFr staining was significantly increased in primary melanoma compared to dysplastic nevi (P = 0.0003) and further increased in metastatic melanoma (P = 0.0000). Kaplan-Meier survival curve and univariate Cox regression analyses showed that increased expression of LIFr was correlated with poorer 5-year patient survival (overall survival, P = 0.0000; disease-specific survival, P = 0.0000). Multivariate Cox regression analyses indicated that increased LIFr expression was an independent prognostic marker for primary melanoma (P = 0.036). LIFr knockdown inhibited melanoma cell migration in wound healing assays and reduced stress fiber formation. LIFr knockdown correlated with STAT3 suppression, but not YAP, suggesting that LIFr activation might stimulate melanoma cell migration through the STAT3 pathway. Our data indicate that strong LIFr expression identifies potentially highly malignant melanocytic lesions at an early stage and LIFr may be a potential target for the development of early intervention therapeutics.

Keywords: LIFr; biomarker; cell migration; melanoma.

Figure 1. LIFr expression is increased in human advanced melanoma

Representative images of LIFr immunohistochemical staining in nevi, primary melanoma and metastatic melanoma TMA. a, d. Normal vevi with negative staining. b, e. primary melanoma with moderate positive staining. c, f. Metastatic melanoma with strong positive staining. LIFr expression was significantly increased from nevi to melanoma. (a-c) Bar = 40 μm; (d-f) bar = 10 μm. g. Increased LIFr expression correlates with melanoma progression. LIFr expression was enhanced in primary melanoma compared with dysplastic nevi (P = 0.0003, χ2 test) and further increased in metastatic melanoma compared with primary melanoma (P = 0.0000, χ2 test). **P < 0.01. DN, dysplastic nevi; LIFr, leukemia inhibitory factor receptor; MM, metastatic melanoma; NN, normal nevi; PM, primary melanoma.

Figure 2. LIFr expression is associated with patient age, tumor thickness and ulceration in primary melanoma patients and gender and AJCC in all melanoma patients

a. The high LIFr expression was significantly more frequent in primary melanoma patients aged over 60 years old (P = 0.009; χ2 test); b. patients with tumor thickness greater than 2.0 mm (P = 1e-8; χ2 test); c. melanoma with ulceration (P = 8.8e-7; χ2 test). d. In all melanoma patients, the high LIFr expression was significantly more frequent in males than females (P = 0.001; χ2 test); e. In all melanoma the percentage of cases with high LIFr expression was significantly increased in AJCC stages III and IV (P = 0.0000, χ2 test). *P < 0.05, **P < 0.01.

Figure 3. LIFr expression is significantly correlated with 5-year survival of all melanoma patients and primary melanoma patients

Patients with strong LIFr expression have a significantly worse overall a, c. and disease specific 5-year survival b, d. than those with weak staining in all melanoma patients (including primary and metastatic melanoma) and primary melanoma patients (P = 0.000 and 0.000 respectively, log-rank test). However LIFr expression did not show any correlation to the survival rate of metastatic melanoma patients e, f. (P = 0.140 and 0.102 respectively, log-rank test).

Figure 4. LIFr protein and mRNA expression are enhanced in melanoma cell lines compared with melanocytes

Whole cell extracts were obtained from normal human melanocytes and melanoma cell lines for Western blot a. and real-time reverse transcription quantitative PCR analysis b. Fold change in melanoma cell lines is relative to melanocytes as the baseline comparator (set to value of 1 fold). Bars equal to means ± SD. c. Immunofluorescence LIFr staining of melanoma cell line MMRU and melanocytes (MC).

Figure 5. Knockdown of LIFr inhibits melanoma cell migration

a. Representative images of the effects of LIFr knockdown on melanoma cell migration. b. The effects of LIFr expression inhibition on melanoma cell migration was quantified by counting the migrated cells in five random fields of each well; Bars equal to means ± SD. The data were obtained from three independent experiments. **P < 0.01. c. Western blot analysis of LIFr expression; knockdown of LIFr inhibited phosphorylated STAT3 (p-STAT3) expression.

Figure 6. Knockdown of LIFr decreases STAT3 mRNA levels and phosphorylated STAT3 expression

Forty-eight hours after transfection, cells were plated for migration assays, and the remaining cells were harvested for LIFr and STAT3 mRNA expression detection and Western blot analysis. a. Representative images of LIFr, phosphorylated STAT3 (p-STAT3) and b-actin expression in Western blotting. Analyses of band density are presented as the relative ratio of LIFr and STAT3 to actin for MMRU cells b. and PMWK cells c, d. LIFr mRNA expression in MMRU and PMWK cell lines after LIFr knockdown relative to controls (set to value of 1 fold). e. STAT3 mRNA expression in MMRU and PMWK cell lines after LIFr knockdown relative to controls (set to value of 1 fold). The data was analyzed by Student's t-test, Bars equal to mean ± SD. All experiments were carried out in triplicate. *P < 0.05, **P < 0.01.

Figure 7. Knockdown of LIFr reduces MMP2 activation

Forty-eight hours after transfection with siRNAs, then serum-free medium starved 24 hours, the proteins in the conditioned medium and MMRU cell lysates were concentrated and extracted; zymography assay and Western blot analysis were performed. The active MMP2 extracted from MMRU cell lysates showed little change, but the active MMP2 from MMRU cell conditioned medium was reduced. Western-blot showed that there were no significant changes in Pro-MMP2 protein expression.

Figure 8. Knockdown of LIFr inhibits actin stress fiber induction

a. Representative images of stress fiber formation in LIFr knockdown and control melanoma cells. Forty-eight hours after transfection with siRNAs, cells were seeded on cover slips at a density of 2 × 10⁴ cells per well in six-well plates for 24 h. Cells seeded on coverslips were serum starved overnight followed by serum stimulation with 10% fetal bovine serum for 1 h, then the cells were stained with rhodamine-conjugated phalloidin. Serum-induced stress fibers were thinner, poorly oriented and less spread in LIFr knockdown MMRU cells, whereas there were thicker, well-aligned stress fibers running across the control MMRU cells. Data were obtained from triplicate experiments. b. Quantification of stress fiber staining intensity. There was a significant reduction of stress fiber formation in LIFr knockdown MMRU cells compared with control MMRU cells.