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Aberrant HGF-MET (hepatocyte growth factor-met proto-oncogene) signaling activation via interactions with surrounding stromal cells in tumor microenvironment has significant roles in malignant tumor progression. However, extracellular proteolytic regulation of HGF activation, which is influenced by the tumor microenvironment, and its consequential effects on melanoma malignancy remain uncharacterized. In this study, we identified SPINT2 (serine peptidase inhibitor Kunitz type 2), a proteolytic inhibitor of hepatocyte growth factor activator (HGFA), which has a significant role in the suppression of the HGF-MET pathway and malignant melanoma progression. SPINT2 expression is significantly lower in metastatic melanoma tissues compared with those in early-stage primary melanomas, which also corresponded with DNA methylation levels isolated from tissue samples. Treatment with the DNA-hypomethylating agent decitabine in cultured melanoma cells induced transcriptional reactivation of SPINT2, suggesting that this gene is epigenetically silenced in malignant melanomas. Furthermore, we show that ectopically expressed SPINT2 in melanoma cells inhibits the HGF–induced MET-AKT (v-Akt murine thymoma viral oncogene) signaling pathway and decreases malignant phenotype potential such as cell motility and invasive growth of melanoma cells. These results suggest that SPINT2 is associated with tumor-suppressive functions in melanoma by inhibiting an extracellular signal regulator of HGF, which is typically activated by tumor–stromal interactions. These findings indicate that epigenetic impairment of the tightly regulated cytokine-receptor communications in tumor microenvironment may contribute to malignant tumor progression.
Hepatocyte growth factor/scatter factor (HGF/SF) and its receptor tyrosine kinase MET (met proto-oncogene) have critical roles in embryogenesis and wound healing (
). Upon ligand binding, MET activates various intracellular signaling cascades including PI3K-AKT (v-Akt murine thymoma viral oncogene), RAC1-CDC42, RAP1, and RAS-MAPK pathways for mitogenic and motogenic effects (
). Cancer cells take advantage of the HGF/SF-MET exerted signaling pathways for promoting tumor progression and metastasis by stimulating cell proliferation, dissociation, migration, invasion, angiogenesis, and survival (
). As a major mechanism of cancer progression and resistance to therapy, functional cross-talk between HGF/SF–activated MET and other receptor tyrosine kinases such as EGFR, ERBB2, and IGFR, as well as receptors of developmental signal pathways such as WNT and TGFR, has been reported (
MET kinase inhibitor SGX523 synergizes with epidermal growth factor receptor inhibitor erlotinib in a hepatocyte growth factor-dependent fashion to suppress carcinoma growth.
). These new insights of HGF/SF-MET roles in cancer progression and therapy resistance also support the validity of the HGF/SF-MET signaling pathway as potential targets in cancer therapy (
Proteolytic activation of pro-HGF/SF to the active form of ligand (HGF/SF), which is primarily mediated by three distinctive forms of serine proteinase, hepatocyte growth factor activator (HGFA), matriptase, and hepsin, is an important regulatory step (
Pericellular activation of hepatocyte growth factor by the transmembrane serine proteases matriptase and hepsin, but not by the membrane-associated protease uPA.
). In contrast to matriptase and hepsin, which are membrane-bound proteinases, HGFA is a soluble protein mainly secreted by the liver and circulates in the plasma of the human body (
) and is a potent activator of HGF/SF. HGFA converts inactive single-chain pro-HGF to the active double-chain form, which is known to bind and activate the MET signaling pathway. Proteolytic activation of HGF/SF by HGFA is tightly controlled by another layer of regulatory mechanism mediated by two known serine peptidase inhibitors Kunitz type 1 and type 2 (SPINT1 and SPINT2, respectively) (
). In cancer cells, balance between these two groups of proteases and protease inhibitors in the interface of stromal–tumor cells may determine HGF exerted malignant phenotypes; for example, decreased levels of SPINT1 and SPINT2 that were observed in aggressive cancer cells (
Tumor suppressor activity and epigenetic inactivation of hepatocyte growth factor activator inhibitor type 2/SPINT2 in papillary and clear cell renal cell carcinoma.
) may lead to the higher levels of available proteolytic HGF activators (HGFA, matriptase, and hepsin), resulting in the promotion of tumor growth and invasion (
SPINT2 (also known as HAI2, HGF activator inhibitor 2) is a transmembrane protein with two Kunitz domains within its extracellular region. SPINT2 is suggested to have an important role during embryogenesis as deletion of SPINT2 is embryonic lethal in mice (
). Although decreased expression of SPINT2, which correlates with poor prognosis, has been shown to be epigenetically silenced in cancer via DNA hypermethylation (
), physiological consequences of SPINT2 silencing in the context of malignant melanoma development have not been studied. Here, we report the malignant progression-suppressive functions of SPINT2 by inhibiting the oncogenic MET-AKT pathway in melanoma.
Results
SPINT2 is epigenetically silenced in melanoma tissue samples and cell lines
To discover novel epigenetically silenced melanoma suppressor genes, we performed a comparative analysis of genome-wide DNA methylation profiles between short-term cultured normal human primary melanocytes (HPMs) and melanoma cell lines. We identified a gene signature of differential promoter DNA methylation (Figure 1a). Furthermore, to identify genes that are epigenetically silenced and functionally associated with metastatic melanoma development, we compared the methylation signature genes with a genome-wide expression profile of primary melanoma tissues obtained from two groups of patients (patients with recurrent metastasis within 4 years after initial diagnosis vs. patients without recurrences) (
) and identified SPINT2 as the only gene in common between the two independent sets of signatures (DNA methylation and gene expression). We then validated the hypermethylation of SPINT2 gene in melanoma cell lines compared with normal HPM using methylation-specific PCR (Figure 1b and c, Supplementary Figure S1 online) and bisulfite sequencing analysis, showing that most CpG dinucleotides were hypermethylated in melanoma cell lines, whereas aberrant methylation was significantly less in HPM cells (Figure 1d). Comparative measurement of SPINT2 mRNA expression levels by semiquantitative reverse-transcriptase–PCR (RT–PCR) analysis revealed that melanoma cells express significantly lower levels of SPINT2 mRNA compared with those of HPMs (Figure 2a), suggesting that DNA hypermethylation is the primary cause of SPINT2 silencing in melanoma cells. Furthermore, treatment with a DNA-hypomethylating agent (decitabine) in a panel of melanoma cell lines showed dose-dependent increased levels of SPINT2 mRNA, whereas no significant difference was seen in primary melanocytes (Figure 2b). On the basis of these observations along with potential biochemical function of SPINT2 in inhibition of HGF/SF proteolytic activation, we hypothesized that epigenetic loss of SPINT2 may contribute to malignant melanoma progression.
Figure 1Identification of epigenetically silenced putative metastasis suppressor genes in melanoma. (a) Comparative analysis of global methylation profiles of melanoma cells and human primary melanocytes (HPMs). Heatmap shows top 30 differentially methylated genes between 10 melanoma cell lines and HPMs. (b) Schematic depiction of examined 85 CpG sites surrounding the transcription start site of serine peptidase inhibitor Kunitz type 2 (SPINT2) gene is shown. A region analyzed by methylation-specific PCR (MSP) is indicated by a black bar below the CpG sites. Black arrows indicate location of bisufite-specific PCR primers. (c) MSP analysis of SPINT2 gene CpG islands in melanoma cell lines and HPMs. (d) Bisulfite sequencing analysis of SPINT2 gene in melanoma cells and HPMs. Methylated and unmethylated CpG dinucleotides are shown by closed and open circles, respectively. Each line of circles represents analysis of a single cloned allele.
Figure 2Decreased expression of serine peptidase inhibitor Kunitz type 2 (SPINT2) gene in melanoma compared with melanocyte cells and transcriptional reactivation by a DNA-hypomethylating agent (decitabine) treatment in melanoma cells. (a) Relative transcript levels of SPINT2 were examined in melanoma cell lines compared with normal melanocytes (human primary melanocyte (HPM)1 passage no. 2 and HPM2 passage no. 10). (b) Normal melanocytes (HPM1 and HPM2) and a panel of melanoma cell lines were treated with 0, 0.25, 0.4, and 0.5 μM of 5-aza-2-deoxycytidine (5-aza-dC) (decitabine) for 72 hours. Isolated total RNAs from treated cells were used to examine SPINT2 transcript levels via reverse-transcriptase–PCR (RT–PCR). Asterisk denotes a statistically significant difference between dimethyl sulfoxide (DMSO)– and 5-aza-dC-treated samples (P<0.05).
SPINT2 expression is significantly lower in clinically aggressive metastatic melanomas
We next examined whether tumors derived from clinically different stages of melanoma exhibit differential levels of SPINT2 gene expression correlative to disease progression. SPINT2 mRNA expression was assessed by quantitative RT–PCR from surgically removed clinical tissue samples of early-stage primary and metastatic lesions of 24 melanoma patients (12 patients for each group). Differential expression of SPINT2 mRNA levels was verified as shown in the significant decrease in SPINT2 expression in metastatic melanoma tissue samples than that of primary melanoma samples (P-value=0.014) (Figure 3a). To correlate decreased SPINT2 mRNA expression in metastatic melanoma with epigenetic silencing of the gene, specifically DNA hypermethylation, semiquantitative methylation-specific PCR of the SPINT2 gene was performed on bisulfite-treated genomic DNA isolated from available clinical tissue samples. Two of the four primary melanoma samples failed to amplify, whereas three of the four metastatic samples showed amplification (Figure 3b). The methylation-specific amplification linear fold change of each sample was normalized to the lowest amplified primary melanoma and shows a statistically higher level of SPINT2 gene methylation in metastatic tissue samples than that in primary tissue samples. These results from clinical tissue samples suggest that abrogation in SPINT2 expression by DNA hypermethylation may contribute to advancement in melanoma malignancy.
Figure 3Transcriptional serine peptidase inhibitor Kunitz type 2 (SPINT2) mRNA expression level in metastatic melanoma tissue is less than primary tumor. (a) Quantitative reverse-transcriptase–PCR (RT–PCR) was performed for SPINT2 gene expression in early-stage primary and metastatic melanoma tissue samples, and β-actin serves as a negative control. Data are represented as box plot. Asterisk denotes a statistically significant difference between primary and metastatic tissue samples (P<0.015). (b) Semiquantitative methylation-specific PCR of SPINT2 gene in early-stage primary and metastatic melanoma tissue samples; n=4 for each primary and metastatic group. Fold change in methylation was normalized to primary melanoma tissue sample 4. Asterisk denotes a statistically significant difference between primary and metastatic tissue samples (P<0.05).
SPINT2 regulates proliferation and migration of melanoma cells
The observed silencing of SPINT2 in aggressive clinical tissue samples suggests a potential metastasis-suppressive role of SPINT2 in malignant melanoma progression. To test this hypothesis, stable melanoma cells overexpressing SPINT2 were generated using a lentiviral gene delivery system. SPINT2 overexpression was confirmed by immunoblot analysis (Figure 4a). Cell proliferation was assessed over a 72-hour period after seeding in which SPINT2 overexpression resulted in decreased growth compared with empty vector controls (Figure 4c). To obtain further evidence of decreased cell growth, cell cycle profile analysis was performed (Figure 4e). In melanoma cells overexpressing SPINT2, the percentage of the cell population in the G0/G1 stage increased and the percentage in the G2/M stage decreased significantly compared with control cells, confirming the observed decrease in cell growth. SPINT2-expressing WM1552C cells (Figure 3c and d) were then chosen for lentiviral SPINT2 shRNA transduction for knockdown (Figure 4b) and showed that cell proliferation significantly increased compared with scramble control (Figure 4d). These data suggest that SPINT2 may have an important role in regulating the cell cycle and that epigenetic silencing of SPINT2 may result in increased tumor cell growth in melanomas.
Figure 4Serine peptidase inhibitor Kunitz type 2 (SPINT2) overexpression in metastatic melanoma cell lines inhibits cell proliferation. (a) Immunoblot image of SPINT2 from WM983B and 1205Lu total cell lysates. Wild-type (WT), empty vector control (Control-EV), and SPINT2–overexpressing cells (SPINT2). β-Actin is shown as a loading control. (b) Reverse-transcriptase–PCR (RT–PCR) of SPINT2 mRNA from WM1552C treated with scramble or SPINT2 short hairpin RNA (shRNA). Asterisk denotes a statistically significant difference between scramble control and SPINT2 shRNA samples (P<0.05). (c) The PICO green proliferation assay of SPINT2–overexpressing WM983B and 1205Lu cells 24, 48, and 72 hours after seeding. Data shown are mean±SD from triplicate wells of two separate experiments. Asterisk denotes a statistically significant difference between empty vector control and SPINT2–overexpressing samples (P<0.05). (d) The PICO green proliferation assay of SPINT2 knockdown WM1552C cells 24, 72, and 120 hours after seeding. Asterisk denotes a statistically significant difference between scramble control and SPINT2 shRNA samples (P<0.05). (e) Cell cycle analysis of SPINT2–overexpressing WM983B and 1205Lu cells. Data are shown as the percentage of total cell population at indicated cell cycle phase and from duplicate samples of three separate experiments. Asterisk denotes a statistically significant difference between empty vector control and SPINT2–overexpressing samples (P<0.05).
To determine the role of SPINT2 in regulating cell migration, a wound healing assay was performed in melanoma cells with SPINT2 expression. To establish that wound closure is dependent on cell migration and not proliferation, cell growth in serum-free media was observed for 18 hours and no significant difference was seen between control and SPINT2–expressing cells (Figure 5c), which was consistent with the proliferation time-course data (Figure 4c). Confluent monolayer of cells was scratched, and wound closure was examined 18 hours later. As determined by the percentage of wound closure, SPINT2–overexpressing cells migrated significantly less compared with control cells with or without activated pro-HGF 18 hours after wounding (Figure 5a and b). Additional scratch wound healing assay performed in 1205Lu melanoma cell lines confirmed decreased wound closure at various time points independent of cell proliferation with SPINT2 overexpression (Supplementary Figure S2 online).
Figure 5Stable serine peptidase inhibitor Kunitz type 2 (SPINT2) expression in metastatic melanoma cells suppresses metastatic phenotypes of migration and invasive growth. (a) Representative images of control and SPINT2–overexpressing WM983B cell migration 18 hours after wounding (migrating edge denoted by black line) with or without pro-hepatocyte growth factor (HGF) (50 ng ml−1) treatment. (b) Quantification of SPINT2–overexpressing WM983B cell migration with or without pro-HGF. Data shown are from three separate experiments. Asterisk denotes a statistically significant difference between empty vector control and SPINT2–overexpressing samples (P<0.05). (c) The PICO green proliferation assay of SPINT2–overexpressing WM983B cells 18 hours after seeding with or without pro-HGF (50 ng ml−1) treatment in serum-free media. Data shown are from triplicate experiment. (d) Anchorage-independent colony formation assay of malignant melanoma cell lines (1205Lu, WM983B, and WM983A). Cells are treated either with or without pro-HGF (100 ng ml−1). Data shown are colony number per field from a triplicate sample assay of two independent experiments. (e) Quantification of colony size related to respective empty vector controls of no pro-HGF treatment groups. Data shown are mean from a triplicate assay of two independent experiments. Asterisk denotes a statistically significant difference between empty vector control and SPINT2–overexpressing samples (P<0.05). (f) Representative images of colonies in soft agar colony formation assay.
The effect of SPINT2 on anchorage-independent tumor growth was also evaluated by the soft agar colony formation assay. SPINT2 re-expression did not inhibit the ability of melanoma cell lines to form colonies in anchorage-independent environments with or without pro-HGF stimulation (Figure 5d). However, stimulation with pro-HGF significantly increased colony size in control cells, whereas the colony size of melanoma cells with SPINT2 expression was significantly decreased (Figure 5e and f), suggesting that SPINT2 re-expression may suppress invasive tumor growth even in the presence of pro-HGF stimulation. Taken together, these data suggest that epigenetic loss of SPINT2 expression in metastatic melanoma cell lines results in enhanced metastatic phenotypes such as tumor cell motility and invasive growth.
SPINT2 overexpression attenuates basal and pro-HGF/SF–stimulated MET-AKT phosphorylation
Previous studies regarding pro-HGF/SF proteolytic activation have shown SPINT2 to bind and inhibit HGFA’s proteolytic function of binding and processing pro-HGF/SF to the active form (
). However, the inhibitory function of SPINT2 in MET phosphorylation and downstream signaling cascade activation has yet to be experimentally demonstrated. Basal MET phosphorylation levels in all the tested melanoma cell lines 1205Lu, WM983B, WM902B, and A375 were minimal, and there was no discernible effect of SPINT2 on MET phosphorylation (Figure 6a). However, when pro-HGF/SF was treated, SPINT2 overexpression abrogated active HGF/SF–induced MET phosphorylation compared with control cells. Consistent with decreased MET phosphorylation, SPINT2–overexpressing cells exhibited decreased AKT phosphorylation during pro-HGF/SF stimulation. ERK phosphorylation was not affected with SPINT2 overexpression in melanoma cells, which may be attributed to the constitutional activation of BRAF (v-raf murine sarcoma viral oncogene homolog B) in melanoma cell lines that harbor the BRAFV600E mutation. However, pro-HGF/SF treatment in HeLa cells shows that SPINT2 overexpression within a BRAF–wild-type background is capable of blocking not only MET/AKT activation but also ERK signaling during pro-HGF/SF stimulation (Figure 6b). When SPINT2 expression was knocked down in WM1552C cells, pro-HGF stimulation increased MET phosphorylation in which one of the two SPINT2 knockdown cell lines exhibited increased total MET receptor protein levels, which may enhance downstream signaling (Figure 6d). These data suggest that SPINT2 abrogation of invasive growth and migration in cancer cell may be via inhibition of MET and downstream AKT activation.
Figure 6Serine peptidase inhibitor Kunitz type 2 (SPINT2) overexpression attenuates pro-hepatocyte growth factor/scatter factor (HGF/SF)–induced met proto-oncogene (MET) and downstream target phosphorylation. (a) Immunoblot images of MET/AKT/ERK phosphorylation with or without pro-HGF (50 ng ml−1) in 1205Lu, WM983B, WM902B, and A375 cells constitutively expressing SPINT2. (b) Immunoblot image of MET/AKT/ERK phosphorylation pro-HGF/SF treatment in BRAF (v-raf murine sarcoma viral oncogene homolog B)–wild-type background HeLa cells. (c) Immunoblot of SPINT2 overexpression validating constitutive expression of SPINT2 in melanoma and HeLa cell lines. (d) Immunoblot image of MET phosphorylation in the SPINT2 knockdown WM1552C cell line.
Transgenic mouse models of melanoma have shown that ectopic expression of HGF/SF and MET amplification have important roles in spontaneous melanoma tumorigenesis and acquisition of metastatic phenotype (
). These studies suggested that the HGF-MET pathway further has crucial roles in metastatic progression. SPINT2 has been characterized as one of the prominent serine protease inhibitors to block pro-HGF activation by binding and inhibiting HGFA (
). As shown in this study, epigenetic silencing of SPINT2 expression may directly contribute to tumor formation and malignancy progression by abnormal activation of the oncogenic HGF-MET pathway. In fact, ectopic expression of SPINT2 abrogates the pro-HGF–stimulated MET and attenuates its downstream target AKT and ERK activation (Figure 6). Other proteases, such as matriptase (
). However, basal expression of matriptase was minimal in melanoma cells (Supplementary Figure S3 online) and does not seem to be the prominent activator of pro-HGF in melanoma, although we cannot completely rule out a probable role of matriptase in metastatic phenotype development.
Influences of the tumor microenvironment in regulating metastasis, changes in tumor metabolism, and therapy resistance have been established (
). One example of this stromal–tumor interaction is the changes in tumor cell behaviors induced by the high level of HGF/SF secreted from cancer-associated fibroblasts (CAFs) into the tumor microenvironment, which binds to MET receptor of the neighboring carcinoma cells. HGF/SF also has been shown to promote endothelial cell growth and migration as an angiogenesis factor (
), thus enabling cancer cells to establish a habitable tumor microenvironment. This paracrine communication of HGF/SF has a critical role in invasive and metastatic characteristics of tumors (
), tumor cells express HGF/SF along with elevated levels of MET expression. In addition, HGFA is mainly secreted by the liver and circulates in plasma throughout the human body (
). Minimal levels of HGFA protein were expressed in melanoma cells compared with primary human liver cells (THLE-2) (Supplementary Figure S4 online), suggesting that circulating HGFA from plasma is the primary source of HGFA in the tumor microenvironment of melanomas. These observations indicate that disruption of tightly regulated HGF/SF-MET signaling occurs at multiple levels and is an important factor for melanoma progression. Interestingly, recent studies characterizing intrinsic resistance to BRAF targeted therapy have suggested that HGF/SF secreted from CAFs causes innate resistance (
). The plethora of data indicating HGF as a prominent mediator of cancer metastasis and therapy resistance suggest that pro-HGF regulator SPINT2 may have an important role maintaining homeostasis within the tumor microenvironment.
Although a significant number of genes associated with cancer metastasis has been reported, causality of epigenetic mechanism in metastasis development, particularly silencing potential metastasis suppressor gene by hypermethylation of CpG islands, has not been well established (
suggests that DNA methylation alterations have the potential for producing a selectable driver event in cancer progression. This finding indicates that there is a strong association of epigenetic silencing of critical genetic loci by DNA hypermethylation with metastatic phenotype development in cancer. Identification of SPINT2 as an epigenetically silenced potential metastasis suppressor as shown in our current study supports the notion of the causal effect of epigenetic alterations in melanoma development and progression. Loss of SPINT2 expression and its association with poor disease prognosis in various cancer types was also reported previously (
Tumor suppressor activity and epigenetic inactivation of hepatocyte growth factor activator inhibitor type 2/SPINT2 in papillary and clear cell renal cell carcinoma.
), suggesting that the suppressive function of SPINT2 in malignant cancer progression is not limited to melanoma and epigenetic alteration is a pervasive mechanism of tumor development. Further studies should be directed to elucidate whether this epigenetic trait can be used as a biomarker for malignant progression and also as a therapeutic and prophylactic target for melanoma treatment.
Materials and methods
Cells culture
Six melanoma cell lines (WM35, SBCl2, WM1552C, WM852, WM983B, and 1205Lu) were obtained from Dr Meenard Herlyn (The Wistar Institute, Philadelphia, PA). A375, HeLa, and THLE-2 cells were obtained from ATCC (Manassas, VA). These cell lines were maintained in Dulbecco's modified eagle medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum, L-glutamine (2 mM), and 1% penicillin/streptomycin. HPMs were purchased from Life Technologies (Grand Island, NY) and maintained in Medium 254 with human melanocyte growth supplements.
Patients and tissue samples
This study was approved by the Institutional Review Board of Boston University School of Medicine. Clinical pathologic diagnosis of the patient biopsies was described in the previous study (
WM1552C cells were treated with SPINT2 shRNA or scramble containing lentiviral particles (Invitrogen). Cells were selected with puromycin 48 hours after transduction to create stable cell lines. SPINT2 knockdown was determined by quantitative RT–PCR.
Quantitative RT–PCR
Quantitative RT–PCR analysis of SPINT2 expression in clinical tissue specimens and SPINT2 knockdown cells was performed using the Taqman real-time assay method as described previously (
). Gene expression analysis of melanoma cell lines was performed using the SYBR Green qRT–PCR method. Detailed descriptions are shown in Supplementary Information online.
Genome-wide DNA methylation profiling
DNA (1 μg) from melanoma cells and normal melanocytes was processed in the Microarray Core Facility at the Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins according to Illumina’s protocols. Illumina Infinium Human Methylation27 Bead Chip was used to simultaneously interrogate promoter DNA methylation at 14,495 RefSeq genes. After initial data extraction and normalization, comparative analysis was performed using Significant Analysis of Microarrays to identify differentially methylated genes between normal human HPMs and melanoma cells as described previously (
). The genome-wide methylation profile data set is accessible (GSE53516) in a data repository site (http://www.ncbi.nlm.nih.gov/geo).
Methylation-specific PCR and bisulfite sequencing analysis of the SPINT2 gene
Genomic DNA was isolated from cells using DNeasy column purification and processed according to the manufacturer’s protocol using the EpiTect Bisulfite Kit (Qiagen, Valencia, CA). Thirty-two CpG sites from SPINT2 promoter region were amplified from bisulfite-modified DNA. Bisulfite DNA conversion PCR conditions are as follows: 99 °C for 5 minutes, 60 °C for 25 minutes, 99 °C for 5 minutes, 60 °C for 85 minutes, 99 °C for 5 minutes, 60 °C for 175 minutes, and 20 °C for indefinitely. Methylation-specific PCR and bisulfite sequencing primers and conditions were as described previously (
SPINT2 gene inserted lentiviral vector was created with pENTR221-SPINT2 and pDEST-FG12-CMV via Gateway Technology (Invitrogen). Lentiviral particles were produced in HEK293T cells according to the manufacturer’s instructions (Invitrogen) and stored at -80 °C after 0.22 μm filtration. Cancer cells were transduced with lentiviral particles overnight. Stably transduced cells were sorted by GFP expression via FACSAria III cell sorter (BD Biosciences, Franklin Lakes, NJ) at the Boston University Medical Center Flow Cytometry Core Facility.
Immunoblotting
Immunoblotting was performed as described in the previous study (
). SPINT2 and matriptase protein was detected by mouse monoclonal antibody (R&D Systems, Minneapolis, MN). Total and phospho-MET, Akt, and ERK were detected using rabbit polyclonol antibodies (Cell Signaling, Boston, MA).
Cell cycle and proliferation analysis
Melanoma cells (5 × 105) were seeded in 6-well plates in 1% serum media. Twenty-four hours after seeding cells were trypsinized and centrifuged (1,500 r.c.f., 15 minutes at 4 °C). Pellet was washed with cold phosphate-buffered saline and centrifuged. Pellet was fixed with ice-cold 70% ethanol overnight at 4 °C. After additional phosphate-buffered saline wash, cells were stained with propidium iodide solution (50 μg ml−1 propidium iodide, 100 μg ml−1 RNAse A in phosphate-buffered saline) for 30 minutes in the dark at room temperature. Cell cycle profile was obtained with the FACScan System and analyzed via CellQuest Pro software (BD Biosciences) at Boston University Medical Center Flow Cytometry Core Facility. For cell proliferation analysis, the PICO Green assay was performed at indicated time points after seeding 2 × 103 cells in 96-well plates (Life Technologies, Carlsbad, CA).
Cell migration and colony formation
Confluent monolayer was wounded, and images were taken at indicated time points after wounding at × 100 original magnification (Nikon Eclipse TS100, Tokyo, Japan). WM983B monolayers were wounded, and cell migration in serum-free media was analyzed 18 hours after wounding. For the 1205Lu cell line, cell migration in full-growth media was quantified by calculating the difference between the wounded edges at each time point from time 0 hours and normalizing to control at 24 hours set at 100%. For the soft agar colony formation assay, 1.5 × 103 cells were seeded in 0.3% Noble agar (BD Biosciences) overlaid on top of 0.6% agar layer. Cells were treated with 200 μl of full-growth media with or without 200 ng ml−1 pro-HGF every 5 days. Colony number and size were analyzed with ImageJ (NIH, Bethesda, MD) from bright field images acquired at × 100 original magnification 20 days after seeding. Five independent image fields were taken per sample for analysis.
Statistical analysis
The values are presented as mean±SD. The statistical analysis was conducted using the Student’s t-test (*P<0.05; **P<0.01; ***P<0.005). The number of independent replicates for each experiment was indicated in the figure legends.
ACKNOWLEDGMENTS
This work was supported in part by NIH K01 CA113779 for Dr Ryu.
Tumor suppressor activity and epigenetic inactivation of hepatocyte growth factor activator inhibitor type 2/SPINT2 in papillary and clear cell renal cell carcinoma.
Pericellular activation of hepatocyte growth factor by the transmembrane serine proteases matriptase and hepsin, but not by the membrane-associated protease uPA.
MET kinase inhibitor SGX523 synergizes with epidermal growth factor receptor inhibitor erlotinib in a hepatocyte growth factor-dependent fashion to suppress carcinoma growth.
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