The metastasis suppressor NME1 inhibits melanoma cell motility via direct transcriptional induction of the integrin beta-3 gene

Abstract

Expression of the metastasis suppressor NME1 in melanoma is associated with reduced cellular motility, invasion, and metastasis, but mechanisms underlying these activities are not completely understood. Herein we report a novel mechanism through which NME1 drives formation of large, stable focal adhesions (FAs) in melanoma cells via induction of integrin β3 (ITGβ3), and in one cell line, concomitant suppression of integrin β1 (ITGβ1) transcripts. Forced expression of NME1 resulted in a strong activation of the promoter region (-301 to +13) of the ITGB3 gene. Chromatin immunoprecipitation (ChIP) analysis revealed the transcriptional induction was associated with direct recruitment of NME1 and an increase in the epigenetic activation mark, acetylation of histone 3 on lysine 27 (H3K27Ac) to a 1 kb stretch of 5'-flanking sequence of the ITGB3 gene. Unexpectedly, NME1 did not affect the amount either ITGβ1 or ITGβ3 proteins were internalized and recycled, processes commonly associated with regulating expression of integrins at the cell surface. The ability of NME1 to suppress motile and invasive phenotypes of melanoma cells was dependent on its induction of ITGβ3. Expression of ITGβ3 mRNA was associated with increased disease-free survival time in melanoma patients of the TCGA collection, consistent with its potential role as an effector of the metastasis suppressor function of NME1. Together, these data indicate metastasis suppressor activity of NME1 in melanoma is mediated by induction of ITGB3 gene transcription, with NME1-driven enrichment of ITGβ3 protein at the cell membrane resulting in attenuated cell motility through the stabilization of large focal adhesions.

Keywords: Cell motility; ITGβ3; Melanoma; Metastasis; NME1; Transcription.

Figure 1.

NME1 promotes the formation of large, slow-cycling focal adhesions in melanoma cells. (a) Visualization of focal adhesions (FAs) in serum-starved 1205LU cells (VEC, empty vector-transfected; NME1, forced NME1 expression) after retrovirus-mediated expression of dsRed-labeled paxillin. The NME1-transfected cell line expresses 3-fold higher levels of NME1 than the control VEC line . (b) TIRF time-lapse microscopy of cells in panel (a), with paxillin-labeled structures monitored and color-coded for the indicated time points (0, 15 and 30 min). “Leading” and “trailing” edges of cells are outlined with boxes in subpanels i and iv; leading edges are magnified in subpanels ii and v, and trailing edges in iii and vi. Red arrows depict net migration of the entire cell; white arrows depict movement of focal adhesions within a cell. Results shown are representative of at least three cells monitored per cell line.

Figure 2.

NME1 promotes integrin-mediated adhesion to fibronectin (FN) via regulation of integrin beta subunit expression at the cell surface. (a) Adhesion assays were conducted as described , with minor modifications (Materials and Methods). Diagram depicts domains of the FN molecule; those associated with binding to integrins (ITG-BD) and transglutaminase 2 (TG2-BD) are indicated with brackets. Regions corresponding to FN fragments used in cell adhesion studies are identified with orange lines above the molecule (42 and 110 kDa, respectively). Bar graph summarizes adhesion of 1205Lu cells stably transfected with either empty or NME1 expression vector. Cells were incubated for 15 min on plates coated with full length (FL) fibronectin (FN) or FN fragments containing either the ITG-BD (110 kD) or TG2-BD (42 kD). *p≤0.05 by Student’s t-test. (b) Cell surface expression of the indicated alpha integrin subunits (α4, α5 and αv) was quantified in 1205Lu cells (−/+ forced NME1 expression) by flow cytometry. (c) NME1 expression induces a switch in cell surface expression from predominantly ITGβ1 to ITGβ3. Cell surface expression of beta integrins (“Surface”) was determined in the indicated M14- and 1205Lu-derived cell lines by surface biotinylation and immunoblot analysis, as described (Materials and Methods). Expression of NME1 and β-tubulin (β-tub) were measured in the “intracellular” compartment by immunoblot analysis. (d) Impact of forced NME1 expression on expression of total and activated forms of focal adhesion kinase (FAK), SRC and p130^Cas was determined in M14 cells by immunoblot analysis. (e) Cell surface and intracellular expression of ITGβ1, ITGβ3 and ITGαv was measured in M14 cells (−/+ forced NME1 expression) following a 1h incubation with either vehicle (“DMSO”) or the dynamin inhibitor dynasore (“Dyn”; 80 mM).

Figure 3.

NME1 activates transcription of the ITGβ3 gene via direct binding to the promoter region. (a) Expression of mRNA encoding ITGβ3 was measured by quantitative reverse transcriptase real-time polymerase chain reaction (qRT-PCR) in the indicated melanoma (M14, WM1158, and WM793; −/+ forced NME1 expression) and mouse embryo fibroblast (wild-type C57BL/6, “WT”; NME1 knockout, “NME1ko”) cell lines. Expression of ITGβ3 in the respective control conditions of each panel (vector or WT) is normalized to a value of 1. (b) Shown at top is a schematic representation of a promoter-reporter cassette containing the ITGB3 promoter (“ITGB3-P”; −301 to +13) in linkage with a firefly luciferase reporter mini-gene. Summarized below are amounts of luciferase activity obtained after transient transfection of the indicated melanoma cell lines with the ITGB3 promoter-luciferase plasmid in the absence or presence of forced NME1 expression. Activity is expressed as relative luciferase activity (“Rel. luc. activity”), with activity obtained in the absence of forced NME1 expression assigned a value of 1. *Denotes p < 0.05 by Student’s t-test. (c). Impact of forced NME1 expression on occupancy of the ITGB3 promoter by transcriptionally active chromatin and NME1 was assessed in M14 cells by chromatin immunoprecipitation assay (ChIP). ChIP reactions were conducted with antibodies directed to IgG, acetylated histone 3 (at lysine 27, or “H3K27ac”), or NME1 as indicated. Immunoprecipitated DNA was analyzed by qPCR with a series of five amplicons (150– 200 bp) spanning the ITGB3 promoter from −−958 to +15, as shown. *denotes p < 0.05 by Student’s t-test.

Figure 4.

NME1 suppresses motile and invasive potential of WM1158 melanoma cells by upregulating ITGβ3 expression. (a) Expression of NME1 and ITGβ3 proteins was measured by immunoblot analysis in cells receiving the indicated combinations of forced NME1 expression and shRNAs directed to ITGβ3. shRNA treatments consisted a non-targeting control shRNA (−) or one of two shRNAs specific for ITGβ3 (“a” or “b”). (b) Representative images of wound/scratch assays were acquired 24 h after wound induction in cells receiving the indicated combinations of forced NME1 expression and shRNA-mediated silencing of ITGβ3. Dotted boxes depict borders of original wounds. (c) Graph provides a quantitative analysis of individual cells migrating into wounds. Closed circles and error bars represent means (+/− SEM) derived from 3– 4 independent experiments. Asterisks denote means that are significantly different (*p≤0.05 by ANOVA with Holm-Sidak pairwise testing). (d) Expression of ITGβ3 mRNA was measured in cells used for measurement of single cell invasion activity in 3-dimensional sphere assays after the indicated combination of forced NME1 expression and shRNA sequences (-, non-targeting control; ITGβ3-directed shRNAs “c” and “a”). The shRNA sequence “c” was employed instead of sequence “b” used in panel a, as sequence “b” strongly disrupted spheroid formation. Cultures were co-infected with a lentiviral vector for expression of eGFP for enhanced imaging of invading cells. *, comparison between vector- and NME1-treated; †, denotes significant silencing of ITGβ3 RNA in absence of forced NME1 expression (black bars); ‡, denotes significant silencing of ITGβ3 RNA in presence of forced NME1 expression (white bars). p < 0.05. (e) Representative images of spheroids and invading cells in response to NME1 and shRNA treatments (24 and 72h post-infection). (f) Single cells detached from spheroids were counted as described in Materials and Methods. Asterisks denote means that are significantly different (*p≤0.05, **p≤0.01 by ANOVA with Holm-Sidak pairwise testing).

Figure 5.

Expression of ITGβ3 RNA is associated with prolonged survival expression in skin cutaneous melanoma (SKCM) patients of The Cancer Gene Atlas (TCGA). A total of 234 patients were grouped into subpopulations (117 patients per subpopulation) representing either “High” or “Low” expression of RNAs encoding ITGβ1 or ITGβ3, relative to the median level of expression for each. Shown are Kaplan-Meier plots of survival (overall and disease-free) for the various groups.