The helicase HAGE expressed by malignant melanoma-initiating cells is required for tumor cell proliferation in vivo

Abstract

Malignant melanoma-initiating cells (MMIC) are a subpopulation of cells responsible for melanoma tumor growth and progression. They are defined by the expression of the ATP-binding cassette (ABC) subfamily B member 5 (ABCB5). Here, we identified a critical role for the DEAD-box helicase antigen (HAGE) in ABCB5+ MMIC-dependent tumorigenesis and show that HAGE-specific inactivation inhibits melanoma tumor growth mediated by this tumor-initiating population. Knockdown of HAGE led to a significant decrease in RAS protein expression with a concomitant decrease in activation of the AKT and ERK signaling pathways implicated to play an important role in melanoma progression. To confirm that the reduction in NRAS (Neuroblastoma RAS) expression was dependent on the HAGE helicase activity, we showed that NRAS, effectively silenced by siRNA, could be rescued by reintroduction of HAGE in cells lacking HAGE. Furthermore, we provide a mechanism by which HAGE promotes NRAS unwinding in vitro. We also observed using tumor transplantation in Non-obese diabetic/severe combined immunodeficiency mice that the HAGE knockdown in a ABCB5+ melanoma cell line displayed a significant decrease in tumor growth and compared with the control. Our results suggest that the helicase HAGE is required for ABCB5+ MMIC-dependent tumor growth through promoting RAS protein expression and that cancer therapies targeting HAGE helicase may have broad applications for treating malignant melanoma and potentially other cancer types.

FIGURE 1.

Expression of HAGE protein in malignant melanoma. Representative image of an immunohistochemistry with antibody to HAGE (red) on malignant melanoma and normal skin sections. The negative control was an IgG isotype from mice. Scale bars = 100 μm.

FIGURE 2.

HAGE silencing results in a decrease of proliferation in vitro and in vivo. A, immunofluorescence staining showing HAGE expression in the FM82 control and FM82 shRNA1 melanoma cell lines. HAGE aggregates are shown in the cytoplasm by arrows. Scale bars = 20 μm. Also shown is immunoblotting using an antibody to HAGE in FM82 control and FM82 shRNA1 extracts. β-actin was measured as a loading control. B, immunofluorescence staining showing HAGE expression in FM82 shRNA2 and FM82 shRNA3. Scale bars = 20 μm. Also shown is immunoblotting using an antibody to HAGE in FM82 shRNA2 and FM82 shRNA3 extracts. β-actin was measured as a loading control. C, standard ³H incorporation assays of stable knockdown and control FM82 cells carried out one day and three days post-plating. Mann-Whitney U test: **, p = < 0.01; ***, p = < 0.001. D, measurement of tumor growth in mice injected with either FM82 control cells (n = 12) or FM82 shRNA1 cells (n = 12). Student's t test: **, p = < 0.01; ***, p = < 0.001. E, immunostaining of FM82 control and FM82 shRNA1 tumors using antibody to HAGE (green) and the proliferative marker PCNA (red). Scale bars = 20 μm. The graph illustrates the percentage of HAGE- and PCNA-positive cells from 10 random counting microscopic fields. Student's t test: *, p = < 0.05; ***, p = < 0.001.

FIGURE 3.

HAGE knockdown leads to a decrease in NRAS protein expression and in phosphorylation of its downstream targets. A, comparison of protein lysates extracted from FM82 control and FM82 shRNA1 using Proteome Profiler arrays specific for the PI3K and MAPK pathways. Mann-Whitney U test: *, p = < 0.05; **, p = < 0.01. Positive controls were not specified by the manufacturer. Negative controls were IgG from goat, mouse, and rabbit. B, schematic representation of upstream regulators of RAS proteins and their downstream targets. C, immunoblotting using antibodies against AKT, p-AKT, ERK, p-ERK, GSK3β, p-GSK3β, p-FOXO, and p21^CIP1 in shRNA1 cell lysates compared with control cell lysates. β-actin was measured as a loading control. D, immunoblotting of FM82 control and FM82 shRNA1 extracts using antibodies to SRC, SOS1, GRB2, and NRAS. β-actin was measured as a loading control. E, immunoblotting using antibodies against HAGE, NRAS, p-AKT, and p-ERK in FM82 control, FM82 shRNA2, and FM82 shRNA3. β-actin was measured as a loading control. F, representative immunostaining of tumor sections using antibody against NRAS (red). Scale bars = 20 μm. The graph demonstrates the percentage of NRAS+ cells in FM82 control and FM82 shRNA1 derived from counting 10 random microscopic fields. Student's t test: **, p = < 0.01.

FIGURE 4.

HAGE promotes NRAS protein expression. A, real-time quantitative PCR of HAGE and NRAS mRNAs isolated from FM82 control and FM82 shRNA1. Mann-Whitney U test: ***, p = < 0.001. B, transient silencing of NRAS in HAGE stable knockdown and control FM82 cells followed by rescue of HAGE expression using a HAGE cDNA expression vector. β-actin was measured as a loading control. C, immunofluorescence on FM82 control and FM82 shRNA1 cells using antibody to HAGE and streptavidin Alexa Fluor 488-conjugated antibody to biotinylated NRAS mRNA. Partial colocalizations are shown by arrows. Scale bar = 20 μm. D, unwinding of biotinylated NRAS N-terminal complimentary RNA sequence-NRAS RNA duplexes in the presence of increasing HAGE protein concentration (lane 1, 0 μg; lane 2, 0.6 μg; lane 3, 1.2 μg). A biotinylated NRAS N-terminal complimentary RNA sequence was used as a loading control in lane 4.

FIGURE 5.

Expression of HAGE protein in ABCB5-positive malignant melanoma-initiating cells. A, representative image of immunohistochemistry with antibodies to ABCB5 (green) and HAGE (red) in malignant melanoma. The negative control was an IgG isotype from mice. Scale bars = 20 μm. B, immunofluorescence with antibodies to ABCB5 (green) and HAGE (red) in the FM82 control and FM82 shRNA1 melanoma cell line. Scale bars = 20 μm. Also shown is immunoblotting using an antibody to ABCB5 in FM82 control and FM82 shRNA1 extracts. β-actin was measured as a loading control. C, immunostaining of sections from FM82 control and FM82 shRNA1 tumors using antibody against ABCB5 (green) and HAGE (red). Scale bars = 20 μm.

FIGURE 6.

Sphere-forming assay with HAGE stable knockdown and control FM82 cells. The spheres were stained by immunofluorescence with antibodies to ABCB5 (green) and HAGE (red). The negative controls were IgG isotypes from both mouse and rabbit. Scale bars = 50 μm.