(A) Representative sequence chromatograms of mutations identified in ADAMTS18. In each case, the lower sequence chromatogram was from melanoma tumor. The top sequence chromatograms were from normal tissue of the relevant patient. Arrows indicate the location of missense mutations. The nucleotide and amino acid alterations are shown at the bottom of each chromatogram. (B) Protein schematic of ADAMTS18 is presented with conserved functional domains indicated as colored blocks. Black arrow heads indicate positions of nonsynonymous somatic mutations identified in 79 melanoma specimens. Mutations found in the validation melanoma or colorectal specimens are labeled as red or blue arrows, respectively. The mutations analyzed in this study are indicated with an asterisk. Propep: Reprolysin family propeptide domain; Repro: Reprolysin family zinc metalloprotease domain; ACR: ADAM cysteine-rich domain; TS: Thrombospondin type 1 domain; Spacer: ADAMTS Spacer 1 domain; PLAC: Protease and lacunin domain.

(A) Focus formation assay of A375 (i) or Mel-STR (ii) pooled clones expressing the indicated constructs was performed. Graph indicates number of colonies observed after two weeks of growth. (B) Cellular proliferation of A375 (i and iii) or Mel-STR (ii and iv) pooled clones transduced with an empty vector, wild-type ADAMTS18 or the indicated ADAMTS18 mutants was assessed in plastic culture plates in the presence of 10% serum (i and ii), or reduced serum (iii and iv) for 12 days. Average cell number at each time point was measured by determining DNA content in eight replicate wells using SYBR Green I.

(A) Adhesion assay of A375 clones expressing the indicated constructs was performed. Laminin-I (i) or fibronectin (ii) coated plates were assessed for adhesion after 1hr incubation by crystal violet staining. Plates were analyzed by reading the absorbance at 610nm. (B) Localization of ADAMTS18 proteins were assessed using immunoprecipitation and immunofluorescent staining. (i) Cellular condition media and lysates were immunoprecipitated using anti-FLAG (M2) beads and analyzed by western blot analysis. Cell lysates were blotted with anti-alpha-tubulin as a loading control. (ii) A375 pooled ADAMTS-18 clones (WT, G312E, C638S, P1035S or empty vector) were plated and fixed on slides for immunofluorescent staining with anti-FLAG and DAPI for nuclear localization. (C) A375 clones expressing the indicated constructs were grown in Boyden chambers and assessed for their ability to migrate. (i) Graph indicates the number of cells that migrated 18 hr after seeding (ii) Representative pictures of migrated cells. (D) Melanoma cell lines expressing wild-type ADAMTS18 (i and ii) or mutant ADAMTS18 (iii and iv) were infected with either control shRNAs or two different shRNA constructs targeted against ADAMTS18 (#4 and #5). Migration ability of the cells was assessed and plotted. Representative images of migrated cells are shown in v and vi.

Mel-STR clones expressing empty vector, wild-type or mutant ADAMTS18 (G312E, C638S, Q904X, P1035S) were subcutaneously injected into NOD/SCID mice. 22 days after injection, the mice were evaluated for lung metastasis formation by examining hematoxylin/eosin (H&E) stained sections of paraffin embedded lungs. (A) Graph shows the number of mice with ulcerations where 0.5 was used to score minor tumor ulcerations (< 2mm) and 1 was used to score major ulcerative tumors (~5–30mm). (B) Graph indicates the number of mice that had lung metastases. (C) Representative images of lung sections showing patterns of metastatic spread of the different mutants and the vector control are presented. (i) Mutant G312E mice, a group of neoplastic cells have impacted into a blood vessel. The malignant cells are beginning to grow into the lung parenchyma (arrow). The inset at higher magnification shows the characteristic atypia and an apoptotic cell (arrow). (ii) Mutant C638S mice, the metastatic tumor is actively growing and has invaded the surrounding lung tissues. Two bronchi are trapped within the tumor (arrows). Several mitoses are shown in the inset. (iii) Mutant C638S mice, the invading neoplastic tissue grows surrounding a bronchial structure (arrow). The atypia of the malignant cells is evident in the inset. (iv) Mutant Q904X mice, a web-like network of malignant cell is expanding into the lung parenchyma, spilling into alveolar spaces (black arrow). Disruption of the alveolar walls due to altered mechanical properties of the lung tissue is evident in the area surrounding the metastasis (white arrow). (v) Mutant P1035S mice, a group of neoplastic cells have impacted into a blood vessel, and tumor expansions are already growing into the lung (arrow). The cancer cells are shown at a higher magnification in the inset. (vi) Vector mice, a subpleural (black arrow, pleura) metastatic growth (white arrow) is growing into the lung parenchyma. The atypia of the neoplastic cells is shown in the inset.