1A top, primary HFKs stably expressing GFP; middle and lower panel, 1205Lu-Vc and 1205Lu-Smad7 melanoma cell lines stably expressing DsRed, respectively. Each figure contains 20X phase-contrast insets. 1B, Day 12 Grafts. Left-to-right: grayscale images, DsRed-expressing 1205Lu cells, GFP-expressing primary HFKs and overlaid images far right (animal autofluorescence is depicted in pink). Small palpable tumors present in 1205Lu-Vc animals were detectable by day 12, while 1205Lu-Smad7 expansion appeared minimal. 1C, Day 25 Grafts. 1205Lu-Vc animals had a large tumor mass depicted via DsRed, while 1205Lu-Smad7 growth remained comparable to day 12. In addition, 1205Lu-Vc animals frequently contained localized necrosis (upper panel, arrowhead). Four animals were used per condition.

3A, 20X epifluorescence microscopy. 1205Lu-Smad7 graft showed Smad7-expressing cells reside just below the dermal-epidermal junction (arrow), while 1205Lu-Vc grafts depict an invading melanoma. Overlaid images are shown far right. Epidermis and dermis are depicted in the right-hand margin. 3B, 40X confocal microscopy, via DsRed, showed the predominant localization of 1205Lu-Smad7 cells (upper panel) seen in most grafts. Occasionally, 1205Lu-Smad7 cells were found residing in the upper-epidermal layer (lower panel). Overlaid images are shown right and depict: GFP-keratinocytes (green); DsRed-Smad7 cells (red) and DAPI-stained nuclei (blue). White arrowheads identify the positioning of 1205Lu-Smad7 cells within the grafts.

5A, from top to bottom: 30μg of total cell lysate was probed for β-catenin, α-catenin, E-cadherin, full-length (FL) N-cadherin and vimentin. HFK total cell lysate was used as a positive control. Total β-catenin appeared increased in Smad7 cells (p<0.05). Full-length N-cadherin (~130kDa) was also significantly enhanced in 1205Lu-Smad7 cells when compared to 1205Lu-Vc and represented a 4.5 fold increase over vector control (p< 0.001). Full-length N-cadherin was detected using monoclonal N-cadherin clone-32 antibody (BD Biosciences). Vimentin, an intermediate filament commonly used to diagnose malignant melanoma, was significantly reduced in cells expressing Smad7 when compared to control (p<0.001). Figure 5C, D densitometry for each of the cell adhesion proteins assayed: β-catenin, N-cadherin and vimentin each reached statistical significance. 5C, Endogenous β-catenin immunoprecipitation was performed and followed by immunoblot with: anti-N-cadherin, anti-β-catenin, and anti-Smad7. β-catenin immunoprecipitated Smad7; Smad7 is shown on left panel below the IgG heavy chain (IgG_HC). β-catenin also immunoprecipitated a proportional amount of N-cadherin in Smad7 cells. Molecular weights are indicated. 15% Inputs are shown on right in each figure. 5D, anti-Smad7-FLAG immunoprecipitated Smad7 bound to endogenous β-catenin, confirmed by probing with both anti-β-catenin and anti-Smad7. 5E, 120nM of Smad7-siRNA reduced both β-catenin and N-cadherin, indicating the effect was Smad7-specific. 5F and G, 50μg protein was probed for phosphorylated-β-catenin T⁴¹/S⁴⁵. Smad7 expression reduced the amount of β-catenin targeted for degradation (p<0.001). Experiments were performed three independent times. Results are the means ± SDs of three biological replicates of a representative experiment.

1) E-cadherin (shown in black) directs cell-cell adhesion between normal melanocytes and normal keratinocytes. 2) In melanoma, E-cadherin expression is often reduced resulting in a loss of attachment to keratinocytes. 3) A concomitant increase in N-cadherin (shown in light blue) establishes linkage to dermal fibroblasts. 4) During invasion, reduction of N-cadherin can permit transformed cells to migrate through the dermis. This results in loss of a stable interaction between melanoma and fibroblasts thereby contributing to disease progression. Smad7 expression can limit invasion by stabilizing the cell-adhesion related proteins β-catenin and subsequently N-cadherin, while 1205Lu cells lacking Smad7 continue to invade and show reduced N-cadherin expression at their surface, although N-cadherin may be re-upregulated at distant metastatic sites.

Left, representative H&E of day 12 1205Lu-Vc (top panel) and 1205Lu-Smad7 (middle panel) grafts. Day 12 (left panel) shows 1205Lu-Smad7 grafts formed a mature epidermal layer; while 1205Lu-Vc showed a dense, proliferating melanoma largely devoid of epithelia. Day 25 (right panel) shows representative H&E of day 25 1205Lu-Vc grafts showed a dense invading melanoma, while 1205Lu-Smad7 formed normal epithelia and lacked tumor formation. Left, bottom image, primary melanocytes correctly positioned themselves within the lower epidermis and were identifiable via brightfield microscopy (arrow). In order to identify the epidermis, HFK-GFP cells are shown as an overlay in primary melanocyte graft; 40X inset depicts melanocyte localization. As expected, primary melanocytes did not result in tumor formation and therefore serve as control for the graft itself as well as melanocyte localization. Four animals were used per condition.

4A, HFF monolayers were overlaid with either 1205Lu-Vc or 1205Lu-Smad7 cells. 1205Lu cells stained with calcein-AM were either left untreated or treated with EGTA; neutralizing MAb GC4 N-cadherin antibody; or matched IgG isotype control. 4B, same as above, though HFK’s were used in place of HFF’s. 1205Lu-Smad7 cells did not form adhesions with primary HFK; instead distinct adhesions with primary dermal fibroblasts were formed. No adhesions were present using 1205Lu-Vc cells with either HFF or HFK. Percent adherent cells are plotted, 1205Lu-Vc are depicted in black bars; Smad7 are shown in gray. Representative images of HFF cells overlaid with either 1205Lu-Vc or 1205Lu-Smad7 cells are shown below. *Note a small portion of labeled 1205Lu-Vc cells can be seen at top left of image, while in 1205Lu-Smad7 the cells appear attached throughout. This experiment was performed three independent times. Results are the means ± SDs of three biological replicates of a representative experiment.

6A, upper panel, 40X confocal immunofluorescence shows cells co-stained for β-catenin and N-cadherin (H-63 SantaCruz Biotechnology Santa Cruz, CA). Neither β-catenin nor N-cadherin was appreciably expressed at the cell surface in 1205Lu-Vc cells, demonstrating the inability of 1205Lu-Vc cells to engage in cell-cell adhesion. Lower panel, 1205Lu-Smad7 cells showed membranous co-localization of both β-catenin and N-cadherin, indicating both proteins are present at the cell surface. 6B, Cells were subjected to nuclear and cytoplasmic fractionation; 50μg of each fraction was analyzed. 1205Lu-Vc cells showed an increased proportion of β-catenin in the nucleus while cells expressing Smad7 showed cytoplasmic accumulation. 6C, representative tissue collected from day 9 grafts was stained for N-cadherin (DAPI in blue; DsRed-1205Lu in red; GFP-HFK in green; N-cadherin in light blue). Left panel, 1205Lu-Vc cells lacked N-cadherin expression (panel iii) when overlaid, confirming, in vivo, 1205Lu-Vc cells do not engage in cell-cell adhesion via N-cadherin. Lowest left panel showed fibroblasts outside the tumor border expressed N-cadherin. Right panel, N-cadherin expression was observed at the cell periphery of both 1205Lu-Smad7 cells and fibroblasts. 6D, (DAPI is shown in blue; DsRed-1205Lu in red; and N-cadherin in green) 100X confocal immunofluorescence shows 1205Lu-Vc cells lacked N-cadherin expression in vivo. Lower image, both 1205Lu-Smad7 cells and dermal fibroblasts show N-cadherin expression. White boxes isolate shared 1205Lu-Smad7/fibroblast border and arrowheads identify peripheral N-cadherin localization.