The increased expression of Y box-binding protein 1 in melanoma stimulates proliferation and tumor invasion, antagonizes apoptosis and enhances chemoresistance

Authors


Abstract

In previous studies we identified the transcription/translation factor Y-box-binding protein (YB-1) as a gene that is upregulated in primary melanoma and melanoma metastases when compared to benign melanocytic nevi. To analyze whether YB-1 expression correlates with melanoma progression in vitro and in vivo, we performed expression analysis on melanoma cell lines representing different stages of melanoma progression and on tissues of melanocytic nevi, primary melanoma and melanoma metastases. Our data indicate that compared to benign melanocytes YB-1 expression is increased in melanoma cells in vitro and in vivo and that YB-1 is translocated into the nucleus in invasive and metastatic melanoma cells. To reveal the functional role of YB-1 in melanoma progression we achieved a stable downregulation of YB-1 using shRNA in metastatic melanoma cells. Interestingly, YB-1 downregulation resulted in a pronounced reduced rate of proliferation and an increased rate of apoptotic cell death. In addition, migration and invasion of melanoma cells in monolayer and in a three-dimensional skin reconstruct in vitro was significantly reduced. These effects were accompanied by downregulation of genes involved in proliferation, survival and migration/invasion of melanoma cells such as MMP-2, bcl-2, Cyclin D1, p53 and p16INK4A. Furthermore, melanoma cells with a reduced YB-1 expression showed a decreased resistance to the chemotherapeutic agents cisplatin and etoposide. These data suggest that YB-1 is involved in malignant transformation of melanocytes and contributes to the stimulation of proliferation, tumor invasion, survival and chemoresistance. Thus, YB-1 may be a promising molecular target in melanoma therapy. © 2007 Wiley-Liss, Inc.

Malignant melanoma, a highly malignant tumor of the pigment-producing cells in the skin, is among the human cancers whose incidence has increased most rapidly in the last few decades.1 When diagnosed early, melanoma is well curable by surgical excision. However, in patients with thick primary melanomas, or with metastasis to regional lymph nodes or distant sites, the disease is highly resistant to all current forms of therapy.2 One focus of melanoma research has therefore been to identify molecular targets for the development of novel treatment strategies. To date, a number of molecular changes that occur during melanoma progression have been described3; however, until now targeted therapy of malignant melanoma was not successful. To identify new molecular targets for melanoma therapy we used PCR-amplified subtractive hybridization of melanocytic nevi and primary melanoma tissues. We isolated the DNA-binding protein dbpB/YB-1 overexpressed in primary melanoma.4

Y-box binding protein 1 (YB-1), also known as p50 or dbpB, is a multifunctional protein regulating transcription and translation. It binds to single-stranded RNA and DNA, and double-stranded DNA.5 As a mRNA binding protein, YB-1 is part of messenger ribonucleoprotein particels (mRNPs) and controls translation in a dose-dependent manner.6, 7 YB-1 can inhibit translation by masking of mRNA and regulates mRNA stability.8, 9 As a transcription factor YB-1 binds to promoters containing the Y-box motif and either activates or represses gene expression.10, 11, 12, 13 It has also been implicated in repair, replication, recombination of DNA and alternative mRNA splicing.13, 14 YB-1 controls the expression of genes involved in tumor progression including matrix metalloproteinase 2 (MMP-2) and the multidrug resistance gene 1 (MDR 1).11, 15, 16 YB-1 might play a role in promoting cell proliferation through the transcriptional regulation of various relevant genes, including proliferating cell nuclear antigen (PCNA), epidermal growth factor receptor (EGFr), DNA topoisomerase IIa, thymidine kinase and DNA polymerase α.13, 17, 18 In summary, YB-1 plays a critical role in cell proliferation, DNA replication and drug resistance. YB-1 also appears to protect mammalian cells from the cytotoxic effects induced by DNA damage.

YB-1 is broadly expressed throughout development, and its expression level closely correlates with the cell proliferation state.19 YB-1 is induced in various cell types in response to mitogenic stimuli, such as cytokine-stimulated T cells,20 serum-activated fibroblasts21 and agonist-stimulated endothelial cells.22 It has been shown that targeted disruption of YB-1 in mice is lethal, that YB-1 is essential for early mammalian development, in the prevention of premature senescence in cultured primary cells, and is important for cellular responses to a variety of stress stimuli.19 YB-1 deficiency resulted in a premature accumulation of the G1-specific CDK- inhibitors p16INK4a and p21CIP in cells undergoing early senescence.19 Recently, it has been shown that aberrant YB-1 expression induces defective proliferation of mammary epithelial cells and the development of chromosomal instability.23 YB-1 provokes the induction of genetic instability that emerges from mitotic failure and centrosome amplification.

All these data suggest that YB-1 plays an important role in tumor progression and drug resistance. However, the role of YB-1 in melanoma development and progression has not been investigated until now. Therefore, we analyzed in this study (i) the expression of YB-1 during melanoma progression in vitro and in vivo, (ii) the functional role of YB-1 in proliferation, migration and invasion of melanoma cells and (iii) the role in chemoresistance of melanoma cells.

Material and methods

Isolation and culture of human cells

After obtaining informed consent, human fibroblasts were isolated from human foreskin following routine circumcision. The skin samples were stored at 4°C in Hank's balanced salt solution without Ca2+ or Mg2+ (HBSS w/o Ca2+ or Mg2+) containing penicillin, gentamicin and amphotericin. The subcutaneous fat was trimmed off and the remaining cutis cut into pieces and digested in solution B containing 0.25% Trypsin as active ingredient for ∼19 h at 4°C. The action of the Trypsin was stopped with solution A, following which the epidermis was separated from the dermis. Human fibroblasts were obtained from dermal explants of human foreskin and cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS). Fibroblasts up to passage 7 were used for melanoma reconstructs. SKMEL28 melanoma cells (ATCC HTB-72) were cultured in RPMI 1640 medium with 10% FBS.

In vitro reconstruction of melanomas in organotypic skin equivalents

The in vitro reconstruction of melanoma is based on the organotypic human skin culture technique.24 SKMEL28 cells are invasive metastatic melanoma cells which grow in the dermal compartment of human skin. Therefore, dermal skin reconstructs were used for this study. A cell-free buffered collagen solution was prepared consisting of rat tail collagen type I (BD Biosciences, Bedford, MA, USA) at a final concentration of 1.35 mg/ml in Dulbecco's modified Eagle's medium with 10% FBS. 1.0 ml of the cell-free collagen solution was added to tissue culture inserts (Millicell PC, Millipore, Bedford, MA, USA) placed in 6-well tissue culture plates. While the acellular collagen layer was solidifying, a second collagen solution was prepared similar to the first with the addition of human fibroblasts and the melanoma cells SKMEL28. Human fibroblasts and human melanoma cells from subconfluent cultures were trypsinized, washed and resuspended in the second collagen solution at a density of 1.5 × 105/ml and a fibroblast to melanoma cell ratio of 1:1. 3.0 ml of the fibroblast and melanoma cell - containing collagen solution were placed over the solidified acellular collagen layer. After 5 days of incubation at 37°C, the fibroblast contraction force causes the collagen gel to contract. This structure represents the melanoma reconstruct in a dermal equivalent. For submerged culture conditions, 3 ml of melanoma cell culture medium supplemented with 10% FBS were added beneath the insert and 2 ml inside the insert to allow proliferation of the seeded cells. The culture medium was changed every 2 days. After 10–14 days of submerged culture, the melanoma reconstructs were harvested and evaluated.

Transfection of melanoma cells and generation of stable clones

The shRNA expression vector for YB-1 (YB-1-pSUPER with the target sequence GAAGGTCATCGCAACGAAG, shortly shYB-1) for generating YB-1 specific shRNAs and control shRNAs have been described previously.25 This vector does not contain a selectable marker. Therefore, for neomycin selection SKMEL28 melanoma cells were transfected with the YB-1-pSUPER vector and cotransfected with the pEGFP-vector (Clontech, Saint-Germain-en-Laye, France) using lipofectamin 2000 as described by the manufacturer (Roche, Mannheim, Germany). SKMEL28 or pEGFP-transfected SKMEL28 melanoma cells served as controls. Following transfection and selection of transfected cells using G418 (1 mg/ml), clones were picked and stable clones generated. The stable clones A3 and C6 from the shYB-1/EGFP transfection were further analyzed.

Confocal laser microscopy

Melanoma tissues from benign melanocytic nevi, primary melanoma or melanoma skin metastases were fixed with 4% formaldehyde for 8–9 h, dehydrated and embedded in paraffin. From paraffin sections of melanoma tissues immunofluorescence for YB-1 was performed. Paraffin sections were blocked for 30 min with donkey serum (1:20 diluted in PBS/0.1% Tween) and then incubated overnight with an affinity-purified polyclonal YB-1 antiserum generated in rabbits by immunization with a peptide corresponding (with modifications) to amino acids 299–313 of the YB-1 protein (DGKETKAADPPAENS). Sections were washed with PBS and incubated for 1 hr with the secondary antibody, a Cy3-coupled donkey-anti-rabbit antibody (Dianova, Hamburg, Germany). Nuclei were detected with YOPRO (Molecular Probes, Leiden Netherlands). Melanocytes and melanoma cells were identified by incubating the sections with an antibody against MelanA (Dako, Hamburg, Germany) and a donkey anti-mouse secondary antibody coupled to Cy5.

Proliferation and chemosensitivity assay

Cells were seeded as triplicates in 96 well plates at a density of 1,500 cells per well in 150 μl medium (1 × 104 cells/ml). For the measurement of proliferation, the assay was stopped at 24, 48, 72 and 96 hr after plating. Medium was discarded and each well was washed twice with PBS (without Ca2+ and Mg2+) and 100 μl of a solution containing 100 μg MUH (4-methylumbelliferyl-hepanoat)/ml PBS was added. Plates were incubated at 37°C for 1 hr and measured in a Fluoroskan II (Labsystems, Helsinki), with an λem of 355 nm and an λex of 460 nm. The intensity of fluorescence indicates the number of vital cells in the wells.26, 27 For the analysis of chemosensitivity, cells were incubated 24 hr after plating with cisplatin, etoposide or doxorubicin at the indicated concentrations and 72 hr later the assay using MUH as a substrate was performed as described above. The percentage of living cells was calculated compared to the respective nontreated control cells.

Scratch assay

Melanoma cells were seeded in a petri dish at a density of 1 × 106 cells/ml and grown in their respective medium until the cells were nearly confluent. The medium was removed and 2 lines were drawn with a pipette tip. The cells were washed twice with HBSS and the respective medium was added to the cells. After 0, 1, 24, 48 and 72 hr photographs were made with a Zeiss IM microscope at an 40-fold magnification.

Migration and matrigel invasion assay

Invasion assays were performed in Boyden chambers containing polycarbonate filters with a pore size of 8 μM coated with a Matrigel basement membrane matrix (BD Biocoat Matrigel invasion chambers, BD Biosciences, Heidelberg, Germany). For the analysis of cell migration polycarbonate filters with the same pore size were used without matrigel (Control inserts, BD Biosciences). Chambers were incubated with RPMI medium for 2 hr at 37°C to rehydrate them. SKMEL28 melanoma cells were plated at a cell density of 8 × 104 cells in the upper compartment containing fibroblast-conditioned medium with 0.1% FCS. The lower compartment was filled with fibroblast-conditioned medium containing 10% FCS as a chemoattractant. After incubation for 24 hr at 37°C noninvading cells remaining on the upper surface of the chamber were removed by scrubbing with a cotton-tipped swab and the invaded cells adhering to the bottom surface of the chamber membrane were fixed and counted after cell staining with hematoxilin–eosin. The assays were performed in triplicates, at least 6 fields were counted per filter and mean cell numbers and standard deviations were calculated.

Apoptosis assays using flow cytometry

For measurement of apoptosis induction the flow cytometric detection of cells with fragmented DNA was used by measuring the percentage of cells with sub- G1 DNA profiles.28 Melanoma cell apoptosis was measured 72 hr after the start of chemotherapeutic treatment. Both floating and adherent cells were collected, washed in PBS and fixed in 75% ethanol. Cells were incubated with propidium iodide (50 μg/ml) and RNAse A (100 μg/ml), incubated on ice for 30 min and analyzed by flow cytometry on a FACScalibur and the CellQuest software (BD Biosciences, Heidelberg). Cell debris was excluded from the analysis by appropriate forward and light scatter gating. After clusters and fragments were excluded, cell cycles profiles were analyzed and cells with sub-G1 DNA profiles were identified.

Northern blot analysis

10 μg total RNA isolated from several melanoma cell lines were fractionated on a 1% formaldehyde agarose gel and transferred overnight to a positively charged nylon membrane (Roche, Mannheim, Germany). The blot was hybridized with DIG-labeled YB-1 cDNA or 28S rRNA oligonucleotide. Hybridization was performed overnight at 50°C in DIG Easy Hyb (Roche, Mannheim, Germany). The filters were washed twice in 1× SSC/0.1% SDS at room temperature for 5 min and twice in 0.5× SSC/0.1% SDS at 50°C for 15 min, and subjected to chemiluminescent Digoxigenin detection as described by the manufacturer. Hybridization signals were quantified by scanning the film and volume integration using the Scion Image program.

Western blot analysis

Melanoma cells were washed twice with PBS and incubated for 30 min at 4°C with shaking in 100–400 μl lysis buffer containing PBS with 5 mM EDTA, 0.5% Triton X-100, 20 mM sodium fluoride, 1 mM orthovanadate, 1 mM pyrophosphate and protease-inhibitors (0.1 mM PMSF, 10 μM pepstatin A, 10 μM leupeptin, 25 μg/ml aprotinin). Cell lysates were clarified by centrifugation, 30 μg protein subjected to SDS-PAGE and transferred to polyvinylidene difluoride (PVDF)-membranes. After blocking in PBS/0.1% Tween-20/5–10% dry milk, the membranes were probed with the following antibodies: anti-AKT (1:1,000), anti phospho-AKT (Ser473; 1:1,000), anti-ERK (1:1,000), antiphospho-ERK (Thr202/Tyr204; 1:1,000), anti-bcl-2 (1:1,000), anti-Cyclin D1 (1:2,000), anti-c-myc (1:1,000), anti-p16INK4A (1:1,000), anti-p21 WAF1/Cip1 (1:2,000), anti-p53 (1:1,000), anti-beta-catenin (1:1,000) (all these antibodies from Cell Signaling Technology, Beverly, MA), anti-MMP2 (1:200; Calbiochem, Darmstadt, Germany), anti-beta-actin (1:500) (Santa Cruz Biotechnology, Heidelberg, Germany), polyclonal YB-1 antiserum generated in rabbits by immunization with a peptide corresponding to the first N-terminal amino acids of the YB-1 protein (MSSEAETQQPPAAPPC). Blots were developed with biotinylated secondary antibodies (1:1,500; DAKO, Hamburg, Germany) and streptavidine-alkaline phosphatase and developed using CDP-Star (Roche, Mannheim, Germany) and by exposure using chemiluminescence.

Results

YB-1 expression is upregulated in melanoma cells and the protein translocated to the nucleus in invasive and metastatic melanoma cells in vivo

To investigate whether YB-1 expression correlates with melanoma progression in vitro we performed Northern Blot analysis with a YB-1 specific probe from RNA of several human melanoma cell lines, which represent the melanoma progression stages radial growth phase (RGP), vertical growth phase (VGP) and metastatic melanoma (MM). All cell lines expressed YB-1 RNA, however at different levels. Semiquantification with 28S rRNA revealed that compared to normal human melanocytes (NHM) YB-1 RNA expression is increased in melanoma cells on the average 9-fold (Fig. 1a). RGP-melanoma cell lines show on the average a 7.6-fold (p < 0.036), VGP-melanoma cell lies a 3.6-fold (p < 0.0037) and metastatic melanoma cells an 11.2-fold (p < 0.012) increased YB-1 expression compared to normal human melanocytes. Furthermore, there is a significant increase in YB-1 expression from the transition of primary melanoma cell lines (RGP and VGP) to metastatic melanoma cells (p < 0.015). In addition, most melanoma cell lines expressed at least twice more YB-1 than the mamma carcinoma cell line MCF-7 described to express high levels of YB-1.29 These data indicate that YB-1 expression is increased from the transition of melanocytes to melanoma cells.

Figure 1.

YB-1 expression is upregulated and the protein translocated to the nucleus during melanoma progression. (a) Expression analysis of normal human melanocytes (NHM) and melanoma cell lines representing different stages of melanoma progression—radial growth phase (RGP), vertical growth phase (VGP) and metastatic melanoma (MM)—using Northern blot analysis with a YB-1 specific probe. As a control, a probe specific for 28S RNA was used. The change in YB-1 expression was calculated by the ratio of YB-1/28S RNA expression and the YB1/28S ratio derived from melanocytes was set as 1. As can be seen all melanoma cell lines have increased RNA expression of YB-1 when compared to normal human melanocytes (NHM) and the mamma carcinoma cell line MCF-7. (b) Immunofluorescence analysis of YB-1 protein expression in melanoma tissues in vivo. Shown is a confocal laser microscopy analysis of 3 of each benign melanocytic nevi, primary melanoma (superficial spreading melanoma) and cutaneous melanoma metastases using an affinity-purified antiserum to YB-1 (red), an antibody to MelanA/MART1 to localize melanocytes and melanoma cells (blue) and YOPRO (green) as a nuclear stain. During transition from primary melanoma to melanoma metastases nuclear translocation of YB-1 was observed as visualized by the predominant yellow color (red + green) in skin metastases.

To analyze YB-1 expression in vivo, immunofluorescence of each 3 melanocytic nevi, primary melanoma and metastatic melanoma was performed with an affinity-purified antiserum against YB-1. Fig. 1 shows that YB-1 is higher expressed in melanoma metastases than in benign melanocytic nevi and primary melanomas and that YB-1 is translocated to the nucleus in melanoma metastases (Fig. 1b). This indicates that YB-1 expression and nuclear translocation correlates with melanoma progression in vivo.

YB-1 blockade inhibits proliferation, migration and invasion of melanoma cells

To reveal the function of YB-1 in melanoma cells, we downregulated YB-1 in the metastatic melanoma cell line SKMEL28 using specific shRNA and selected 2 stable clones which express different levels of YB-1. Fig. 2a demonstrates that a stable downregulation of YB-1 can be achieved. In clone A3 YB-1 was 33-fold and in clone C6 125-fold downregulated compared to control SKMEL28 or EGFP-transfected control cells. Using these clones we studied the effects of YB-1 downregulation on cell proliferation, migration and invasion of melanoma cells compared to control SKMEL28 cells and EGFP-transfected control cells. YB-1 downregulation resulted in a 3- to 4-fold reduction in cell proliferation in both clones (Fig. 2a).

Figure 2.

Downregulation of YB-1 expression affects proliferation and migration of melanoma cells. (a) Proliferation assay of melanoma cells in which YB-1 was stably downregulated using shRNA. SKMEL28 melanoma cells were transfected with a plasmid containing siRNA against YB-1 and cotransfected with pEGFP as a selectable marker. The clones A3 (dotted line) and C6 (broken line) were selected, which showed a stable downregulation of YB-1 (see inserted western blot analysis with an antibody against YB-1 (upper lane) or beta-actin (lower lane). Either untransfected SKMEL28 or EGFP-control transfected SKMEL28 melanoma cells served as control cells. Proliferation was measured in triplicates over 96 hr using MUH as a substrate and the mean fluorescence with standard deviation was plotted, indicating the relative number of living cells in the indicated time period. Both melanoma cell clones in which YB-1 was downregulated show a significantly reduced rate of proliferation (p < 0.001). (b) Scratch or wound healing assay of melanoma cells in which YB-1 was downregulated. A wound was created by a scratch (day 0) in a nearly confluent culture of SKMEL28 melanoma cells in which YB-1 was downregulated (clones A3 and C6) and in the control SKMEL28 cells and EGFP-transfected control cells. Wound closure was observed over 3 days by light microscopy and photographed. After 3 days the wound was nearly closed in control SKMEL28 melanoma cells, whereas in the clones in which YB-1 was downregulated the wound was still not closed indicating that YB-1 downregulation resulted in a reduced migration of melanoma cells. Magnification: 40-fold.

Furthermore, we evaluated the effect of YB-1 downregulation on migration and invasion of SKMEL28 melanoma cells. Using a scratch assay in which an artificial wound was set, it was seen that SKMEL28 melanoma cells in which YB-1 was downregulated migrated much slower than the nontransfected or EGFP-transfected control cells over a 3 day period (Fig. 2b). Since this effect could be caused by the slower proliferation rate of the clones in which YB-1 was downregulated we performed a Boyden chamber assay, a method which enables us to quantify the number of migrating melanoma cells. In melanoma cells with downregulated YB-1 migration was reduced at least two-fold in clone A3 and nearly completely inhibited in clone C6. This indicates a dose-dependent effect of YB-1 since YB-1 expression was more suppressed in clone C6 than in clone A3 (Fig. 3a). Similarly, invasive capability of these cells was at least 5-fold inhibited in clone A3 and nearly completely inhibited in clone C6 seen in a matrigel invasion assay (Fig. 3b). Using a more physiological skin environment, we performed invasion assays using a human skin reconstruct with integrated melanoma cells. According to the data achieved in the matrigel-assay we saw a dramatic reduction in the invasive capability of SKMEL28 melanoma cells in which YB-1 was downregulated (Fig. 3c). Most cells were apoptotic as seen after staining with an antibody against active caspase 3. These data suggest that YB-1 has a major effect on proliferation, survival, migration and invasion of human melanoma cells.

Figure 3.

Downregulation of YB-1 expression abolishes invasive tumor growth of melanoma cells in monolayer and in organotypic skin culture. (a) Boyden chamber assay of melanoma cells in which YB-1 was downregulated (clones A3 and C6) and of control cells (untransfected and EGFP-transfected SKMEL28 cells). Analyses were performed in triplicates and the mean number of cells/field was calculated, indicating the number of migrating cells. YB-1 downregulation resulted in a reduced ability of melanoma cells to migrate. (b) Invasion assay using a Boyden chamber assay with matrigel as a substrate. The assay was performed as described already. YB-1 downregulation resulted in a markedly reduced invasive capability of melanoma cells to penetrate matrigel, which resembles the basement membrane in human skin. (c) Untransfected SKMEL28 control melanoma cells as well as SKMEL28 melanoma cells in which YB-1 was downregulated (clones A3 and C6) were incorporated into human dermal reconstructs and stained with hematoxylin (HE), an antibody directed against melanoma cells (HMB45) or an antibody against active caspase 3 to detect apoptotic cells. Control metastatic melanoma cells exhibited aggressive growth of numerous tumor cell nests and tumor cell clusters throughout the entire dermis. In melanoma cells with downregulated YB-1 number and size of melanoma cell nests were reduced with small melanoma cell nests or single melanoma cells scattered throughout the dermis. YB-1 downregulation completely suppressed invasive melanoma growth with very few rounded melanoma cells left in the dermis. Most of these cells stained positive for caspase 3, which indicates that YB-1 downregulation induces apoptotis in melanoma cells.

YB-1 regulates expression of genes involved in signal transduction, proliferation and survival of melanoma cells

To analyze the effect of YB-1 downregulation on the expression of YB-1 target genes we performed western blot analysis of critical molecules involved in tumor progression. First, we evaluated the effect of YB-1 downregulation on important members of signalling pathways activated in melanoma. Interestingly, we observed a significant decrease of total AKT- and phosphorylated AKT protein levels in SKMEL28 melanoma cells in which YB-1 was downregulated (Figs. 4a and 4b) indicating that YB-1 controls the expression of AKT. Similarly, β-catenin was also expressed at lower amounts. In contrast, ERK- and phosphorylated ERK-protein levels were only slightly downregulated, but this did not reach statistical significance (p > 0.05). In a second step we analyzed the effects of YB-1 downregulation on the expression of YB-1 target genes involved in cell cycle progression, survival and invasion. As can be seen in Figures 4a and 4b c-myc expression was only affected to a low degree. However, whereas MMP-2, bcl-2, Cyclin D1, p53 and p16INK4A expression was downregulated up to 30-fold in melanoma cells in which YB-1 was downregulated, p21CIP1 protein levels were dramatically upregulated (Figs. 4a and 4b). These effects were more pronounced in clone C6 which shows a lower expression of YB-1 than clone A3. These data indicate that YB-1 controls the expression of genes involved in melanoma proliferation, survival and invasion.

Figure 4.

YB-1 controls the expression of genes involved in melanoma proliferation, apoptosis and invasion. (a) Western Blot analysis of cell lysates from untransfected SKMEL28 or EGFP-transfected control melanoma cells as well as SKMEL28 melanoma cells in which YB-1 was downregulated (clones A3 and C6). Shown are representative blots of 3 western blots per protein detection performed. (b) Analysis of protein expression by western blot analysis comparing SKMEL28 melanoma cells to the cell clones A3 (in grey) and C6 (in black) in which YB-1 was downregulated. Shown are mean values with standard deviations of 3 western blots per protein performed. The data are represented as x-fold change of the quotient of the respective gene and the house-keeping gene beta-actin when compared to SKMEL28 control cells set as 1 (marked by a line). Significant changes in gene expression (p < 0.05) were marked with an asteriks.

YB-1 regulates chemosensitivity of melanoma cells

To evaluate whether YB-1 is involved in chemosensitivity of melanoma cells, we evaluated the effect of YB-1 downregulation on chemosensitivity of SKMEL28 melanoma cells using an assay determining the number of living cells before and after treatment. YB-1 downregulation resulted in a pronounced increased sensitivity to the chemotherapeutic agents cisplatin and etoposide (up to 2-fold), whereas the sensitivity to doxorubicin was not significantly affected (Figs. 5a5c). Next, we investigated whether YB-1 downregulation resulted in an increased apoptotic rate of melanoma cells after chemotherapy. Interestingly, YB-1 downregulation alone resulted in a 6-fold higher number of apoptotic cells compared to the control cells. As seen in Figure 5d cisplatin (p < 0.01) and etoposide (p < 0.01) resulted in an ∼2-fold increased apoptotic rate in melanoma cells in which YB-1 was downregulated and in a nonsignificant increase after doxorubicin-treatment. These data suggest that YB-1 is involved in chemoresistance of melanoma cells to cisplatin and etoposide.

Figure 5.

Downregulation of YB-1 increases chemosensitivity of melanoma cells. (ac) Survival of control melanoma cells (SKMEL28 in black and EGFP-transfected control cells in grey) and SKMEL28 melanoma cells in which YB-1 was stably downregulated using shRNA (clone A3 dotted line and clone C6 dashed line) 72 hr after treatment with increasing concentrations of cisplatin (a), etoposide (b) or doxorubicin (c). The respective untreated cell population was set as 100% to correct for the lower proliferation rate of clones A3 and C6. Shown is the percentage of living cells after treatment when compared to the respective cell population before treatment. Downregulation of YB-1 in melanoma cells resulted in a significantly increased chemosensitivity to cisplatin (p < 0.02) and etoposide (p < 0.004), but not to doxorubicin (p > 0.05). (d) Measurement of apoptosis induction by flow cytometric detection of cells with fragmented DNA. Shown is the percentage of apoptotic cells as measured by cells with sub-G1 DNA profiles. Melanoma cell apoptosis was measured 72 hr after the start of chemotherapeutic treatment. There was a significantly higher number of apoptotic cells in melanoma cells in which YB-1 was downregulated (clone A3 dotted bar and clone C6 dashed bar) after treatment with 1 μg/ml cisplatin (p < 0.01) and 1 μg/ml etoposide (p < 0.01), but a nonsignificant higher number of apoptotic cells after treatment with 0.1 μg/ml doxorubicin (p > 0.05). These results were confirmed by annexin/PI staining (data not shown).

Discussion

YB-1 is a multifunctional protein acting as a regulator of transcription and translation. YB-1, also known as p50 or DNA-binding protein B (dbpb), regulates transcription through binding to promoters containing the Y box motif and controls translation in a dose-dependent manner by binding to mRNA.13 It was shown that a low YB-1/mRNA ratio promotes translation at the stage of initiation,8, 30 whereas an increase of this ratio strongly inhibits mRNA translation in vitro and in vivo.7 The inhibition of protein synthesis seen with elevated concentrations of YB-1 may reflect a masking of mRNA that prevents access of the translational machinery.

In this study we show for the first time that YB-1 expression is upregulated and the protein translocated to the nucleus during melanoma progression, and that YB-1 is a critical factor in proliferation, survival, migration, invasion and chemosensitivity of melanoma cells. In other tumors like ovarian, breast, colon and lung cancers it has been described that YB-1 is frequently overexpressed and shows increased nuclear localization.29, 31, 32, 33, 34 Nuclear localization of YB-1 correlates with tumor progression and a poor prognosis in tumor patients.31, 32, 34 Furthermore, translocation of YB-1 to the nucleus can be achieved by UV irradiation, anticancer agents, hyperthermia, phosphorylation, or association with p53 or splicing factor SRp30c.13, 35, 36 The nuclear translocation of YB-1 seen in tumor cells might indicate that the function as a transcriptional regulator preponderates the function as a cytoplasmic mRNA-binding protein.

As a transcription factor YB-1 binds to promoters containing the Y box motif and either activates or represses gene expression.10, 11, 12, 37, 38 We could show that in melanoma cells YB-1 downregulation resulted in a decreased expression of genes involved in cell proliferation, survival, migration and signaling such as Cyclin D1, p53, p16INK4A, bcl-2, MMP-2 and AKT, whereas the expression of p21CIP1 was upregulated. Interestingly, YB-1 downregulation resulted not only in a reduced proliferation rate of melanoma cells, but also in a higher number of apoptotic cells. The reduced viability of melanoma cells after downregulation of YB-1 might therefore be the combined effect of apoptotic cell death and growth retardation due to downregulation of genes involved in cell cycle progression such as Cyclin D1 or those involved in cell survival such as bcl-2. YB-1 might play a role in promoting cell proliferation through the transcriptional regulation of various other relevant genes, including proliferating cell nuclear antigen (PCNA), epidermal growth factor receptor (EGFr), DNA topoisomerase IIa, thymidine kinase, and DNA polymerase α.13, 17, 18 YB-1 has also been implicated in repair, replication, recombination of DNA and alternative mRNA splicing.13, 14, 18 YB-1 positively controls the expression of matrix metalloproteinase 2 (MMP-2), which promotes tumor invasion39, 40 and the multidrug resistance gene 1 (MDR 1).15, 16

In our study we show that during melanoma progression YB-1 is translocated to the nucleus in vivo. Furthermore, downregulation of YB-1 increased the sensitivity to cisplatin and etoposide, indicating that YB-1 plays an important role in chemosensitivity of melanoma cells. Several studies indicated that the level of nuclear expression of YB-1 predicts drug resistance and patient outcome in breast tumors, ovarian cancers and synovial sarcomas.29, 33, 34, 41 Predominant nuclear localization of YB-1 is characteristic of transformed cells and is also associated with development of multiple drug resistance of cancer cells and the expression of the MDR-1 gene.29, 33, 34, 42 Targeted disruption of 1 allele of YB-1 in mouse embryonic stem cells increased sensitivity to cisplatin and mitomycin C, but not to etoposide, X-ray or UV irradiation.43 Transfection of human epidermoid cancer cells with a YB-1 antisense construct resulted in increased sensitivity to cisplatin, mitomycin and UV radiation, suggesting a protective effect of YB-1 against cytotoxic effects of agents that induce crosslinking of DNA.44 Moreover, YB-1 specifically binds to cisplatin-modified DNA and apurinic DNA and interacts with PCNA and p53, indicating that YB-1 is involved in DNA repair and DNA damage response.13, 16

YB-1 is broadly expressed throughout development, and its expression level closely correlates with the cell proliferation state.19 YB-1 is induced in various cell types in response to mitogenic stimuli, such as cytokine-stimulated T cells,20 serum-activated fibroblasts21 and agonist-stimulated endothelial cells.22 Furthermore, YB-1 may directly activate cell proliferation by controlling Cyclin A and B1 gene expression. Although YB-1 is localized predominantly in the cytoplasm throughout the cell cycle, it moves into the nucleus during the G1 to S phase transition in a pattern similar to Cyclin E.45 Furthermore, targeted disruption of 1 allele of YB-1 in a chicken pre-B lymphocyte cell line results in major defects in cell cycle with a combined phenotype of apoptotic cell death and growth retardation, implicating a crucial role of YB-1 in cell growth.46 By contrast, the targeted disruption of 1 allele of the YB-1 gene in mouse ES-1 cells had no effect on the growth rate.43 However, homozygous YB-1 deficient embryos exhibit severe growth retardation and premature senescence, revealing a nonredundant role of YB-1 in late embryonic development.19 Furthermore, fibroblasts from YB-1−/− embryos demonstrated a reduced ability to respond to oxidative, genotoxic and oncogene-induced stress. YB-1−/− cells under oxidative stress expressed high levels of the G1-specific CDK-inhibitors p16INK4A and p21CIP1 and senesced prematurely. These data suggest that YB-1 represses the transcription of the CDK inhibitors p16 and p21.19

We could show that YB-1 controls the expression of AKT in melanoma cells. The PI3K/AKT signaling pathway is known to be constitutively active in melanoma cells and is currently a molecular target in melanoma therapy.47 Therefore, it is interesting that overexpression of YB-1 leads to resistance to oncogenic transformation induced by PI3K or AKT.37, 38, 48 YB-1 interferes with PI3K-induced transformation by a specific inhibition of translation through its RNA-binding domain and by inhibition of protein synthesis.37 Activated AKT binds to and phosphorylates YB-1, which in turn activates translation. Therefore, association of YB-1 with mRNAs is regulated via phosphorylation by the serine/threonine kinase AKT, which relieves translational repression of the YB-1 bound mRNAs.30 In primary breast cancer, activated AKT positively correlated with the protein expression of YB-1. Disruption of the AKT phosphorylation site on YB-1 suppressed tumor cell growth in soft agar and in monolayer. This correlated with an inhibition of nuclear translocation by the YB-1 mutant.49 Whereas in these cells YB-1 is a target of the AKT signaling pathway, and we could show that YB-1 controls the expression of AKT in melanoma cells.

In summary, our data highlight the critical role of YB-1 in melanoma proliferation, tumor invasion, survival and chemoresistance and indicate that YB-1 may be a rewarding molecular target in melanoma therapy.

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