Journal: Oncogene

Article Title: c-Met-induced epithelial carcinogenesis is initiated by the serine protease matriptase

doi: 10.1038/onc.2010.586

Figure Lengend Snippet: A. Western blot of normal immortalized human oral keratinocytes (NOK, lane 1) and eight human SCC cell lines (HN6-HN31, lanes 2–9). Positions of molecular weight markers (kDa) at left and matriptase (Mat) and GAPDH loading control at right. Expression of ST14 , encoding matriptase (B), and SPINT1 , encoding the matriptase inhibitor, hepatocyte growth factor inhibitor (HAI)-1 (B') in eight gene expression array studies of human SCC of the head and neck, or skin. Data are expressed as fold change relative to corresponding normal tissue. *, P<0.05. **, P<0.01. Matriptase (C and D) and Ki67 (C' and D') immunohistochemistry of skin (C and C') and oral (D and D') SCC demonstrating matriptase expression in proliferating tumor cells at sites of invasion (examples with arrowheads). E. Expression of HGFR , encoding c-Met, in the eight array studies analyzed in B and B'. *, P<0.05; **, P<0.01. F. Anatomical location of the 72 SCC biopsies used for analyzing matriptase and c-Met expression. Numbers in the pie chart indicate number of tumors analyzed from each location. Representative immunohistochemistry for matriptase (G–J) and c-Met (G'–J') in serial sections from four arrayed HNSCCs showing co-expression of matriptase and c-Met at the invasive front (examples with arrowheads) and other locations. Stars show examples of stroma. Size bars: C–D' 50 μm, G–J' 200 μm.

Article Snippet: The protein concentration was determined by BCA assay (Pierce, Rockford, IL) and 40 μg of total protein (Pierce, Rockford, IL) was loaded on 4–12% reducing SDS-PAGE and analyzed by Western blotting using a polyclonal sheep anti-human Matriptase (R&D Systems), rabbit anti-human phospho cMet, phospho-Gab1, or rabbit anti human GAPDH primary antibodies (all Cell Signaling Technology, Beverly, MA).

Techniques: Western Blot, Molecular Weight, Control, Expressing, Gene Expression, Immunohistochemistry

Journal: Oncogene

Article Title: c-Met-induced epithelial carcinogenesis is initiated by the serine protease matriptase

doi: 10.1038/onc.2010.586

Figure Lengend Snippet: Matriptase (A–D) and Ki67 (A'–D') immunohistochemistry of normal epidermis (A and A'), hyperplasia (B and B'), dysplasia (C and C'), and squamous cell carcinoma (D and D') during murine chemical multi-stage carcinogenesis. Expression of matriptase in proliferating basal keratinocytes of hyperplastic and dysplastic lesions (examples with arrowheads in B and C) as well as in tumor cells at the invasive front (examples with arrowheads in D), but not in basal keratinocytes of normal epidermis (examples with arrowheads in A). The keratinocyte and tumor cell compartments expressing matriptase have high rates of proliferating cells as shown by expression of Ki67 (examples with arrowheads B', C', and D'). Stars in A–D' indicates the location of the dermis or the tumor stroma. E–E". Double immunofluorescence staining of squamous cell carcinoma for matriptase (E, red, examples with arrowheads) and c-Met (E', green, examples with arrowheads). Overlay in E" shows co-localization of matriptase and c-Met on the tumor cell surface (yellow, examples with arrowheads). Stars in E–E" shows location of the tumor stroma. Size bars: A–B', C, and D, 50 μm; C', D', E–E", 25 μm.

Article Snippet: The protein concentration was determined by BCA assay (Pierce, Rockford, IL) and 40 μg of total protein (Pierce, Rockford, IL) was loaded on 4–12% reducing SDS-PAGE and analyzed by Western blotting using a polyclonal sheep anti-human Matriptase (R&D Systems), rabbit anti-human phospho cMet, phospho-Gab1, or rabbit anti human GAPDH primary antibodies (all Cell Signaling Technology, Beverly, MA).

Techniques: Immunohistochemistry, Expressing, Double Immunofluorescence Staining

Journal: Oncogene

Article Title: c-Met-induced epithelial carcinogenesis is initiated by the serine protease matriptase

doi: 10.1038/onc.2010.586

Figure Lengend Snippet: A. Efficient activation of recombinant proHGF/SF by the activated matriptase serine protease domain in solution. proHGF/SF (40 nM) was incubated with 4 (lane 2), 2 (lane 3), 1 (lane 4), and 0.5 (lane 5) nM matriptase or vehicle (lanes 1 and 6) for 1 h at 37 °C. Lanes 1 and 7 are single-chain proHGF/SF and two-chain HGF standards, respectively. Positions of single-chain proHGF/SF, heavy (hcHGF) and light (lcHGF) chains of two-chain HGF/SF are indicated right. Molecular weight markers (kDa) are indicated left. B. Elevated matriptase expression in cultured primary K5-matriptase transgenic keratinocytes. Cell lysates (lanes 1 and 2) and conditioned medium (lanes 3 and 4) from newborn wildtype (lanes 1 and 3) and littermate K5-matriptase +/0 (lanes 2 and 4) keratinocyte cultures and matriptase expression was analyzed by Western blot. Positions of full-length (Mat L) and SEA domain-processed (Mat S) forms of matriptase are indicated with arrowheads. Positions of molecular weight markers (kDa) are indicated at left. C. Elevated matriptase expression increases c-Met and Gab1 phosphorylation in response to single-chain proHGF/SF, but not to active two-chain HGF/SF. Primary keratinocytes from newborn wildtype (panels 1, 3 and 5 from top) and littermate K5-matriptase +/0 (panels 2, 4, and 6 from top) were treated with either 2.5 nM proHGF/SF (lanes 1–7) or active HGF/SF (lanes 8 and 9) for 0 (lane 1), 5 (lanes 8 and 9), 10 (lane 2), 20 (lane 3), 30 (lanes 4 and 7), 45 (lane 5), and 60 (lane 6) min in the absence (lanes 1–6, and 8) or presence (lanes 7 and 9) of the serine protease inhibitor, aprotinin. Phosphorylated c-Met (panels 1 and 2 from top), phosphorylated Gab1 (panels 3 and 4 from top), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (bottom two panels) were detected by Western blotting. D–J. Elevated matriptase expression amplifies the migratory response of primary keratinocytes to single-chain proHGF/SF, but not to active two-chain HGF/SF. Scrape wounds were generated in confluent monolayers of primary keratinocytes from newborn wildtype (WT) (D–F') and littermate K5-matriptase +/0 (K5) (G–I') mice, and the monolayers were treated with vehicle (D, D', G, and G'), 2.5 nM proHGF/SF (E, E', H, and H') or proHGF/SF with 2 μM aprotinin (Ap) (F, F', I, and I') for 24 h. Dashed yellow lines indicate the margins of denuded area at 0 h. Examples of keratinocytes that have migrated into the denuded area after 24 h are shown in E' and H'. Size bars: 100 μm. J. Quantitation of average migration distance of wildtype (blue bars) and littermate K5-matriptase +/0 keratinocytes (green bars) in response to vehicle, proHGF/SF, proHGF/SF and aprotinin (Ap), active HGF/SF and active HGF/SF with aprotinin. Results are shown as mean migration distance ± standard deviation of the mean.

Article Snippet: The protein concentration was determined by BCA assay (Pierce, Rockford, IL) and 40 μg of total protein (Pierce, Rockford, IL) was loaded on 4–12% reducing SDS-PAGE and analyzed by Western blotting using a polyclonal sheep anti-human Matriptase (R&D Systems), rabbit anti-human phospho cMet, phospho-Gab1, or rabbit anti human GAPDH primary antibodies (all Cell Signaling Technology, Beverly, MA).

Techniques: Activation Assay, Recombinant, Incubation, Molecular Weight, Expressing, Cell Culture, Transgenic Assay, Western Blot, Phospho-proteomics, Protease Inhibitor, Generated, Quantitation Assay, Migration, Standard Deviation

Journal: Oncogene

Article Title: c-Met-induced epithelial carcinogenesis is initiated by the serine protease matriptase

doi: 10.1038/onc.2010.586

Figure Lengend Snippet: A–C'. Matriptase-induced follicular metaplasia depends on keratinocyte c-Met. Outwards appearance of untreated four (A–C) and 12 (A'–C') months-old control (A and A'), c-Met-sufficient K5-Mat (K5Mat;cMet+) (B and B'), and c-Met-deficient K5-Mat (K5Mat;cMet-) (C an C') mice. D–G. Loss of keratinocyte c-Met blocks matriptase-induced epidermal hyperplasia, dysplasia, and expression of stress keratin. Hematoxylin and eosin (D), BrdU (E), and keratin-6 (K6) (F) immunohistochemistry of epidermis from control (top panels), keratinocyte c-Met-deficient (second panels from top), c-Met sufficient K5-Mat (third panels from top), and c-Met-deficient K5-Mat mice (bottom panels) at 12 months of age. Arrowheads in D and F show location of the basal layer of the epidermis and stars in D and F show location of dermis. Arrowheads in E are examples of BrdU incorporating keratinocytes. G. Enumeration of BrdU incorporation in basal keratinocytes in the four groups of mice. P values by Student's t-test, two tailed. H–K. Matriptase-induced dermal fibrosis and hypercellularity is prevented by loss of keratinocyte c-Met. Masson's trichrome staining of skin of control (H), c-Met-deficient (I), c-Met sufficient K5-Mat (J), and c-Met-deficient K5-Mat mice (K) at 12 months of age. Size bars: 50 μm.

Article Snippet: The protein concentration was determined by BCA assay (Pierce, Rockford, IL) and 40 μg of total protein (Pierce, Rockford, IL) was loaded on 4–12% reducing SDS-PAGE and analyzed by Western blotting using a polyclonal sheep anti-human Matriptase (R&D Systems), rabbit anti-human phospho cMet, phospho-Gab1, or rabbit anti human GAPDH primary antibodies (all Cell Signaling Technology, Beverly, MA).

Techniques: Control, Expressing, Immunohistochemistry, BrdU Incorporation Assay, Two Tailed Test, Staining

Journal: Oncogene

Article Title: c-Met-induced epithelial carcinogenesis is initiated by the serine protease matriptase

doi: 10.1038/onc.2010.586

Figure Lengend Snippet: A–D. Immunohistochemistry for phospho-S6 ribosomal protein (pS6) during DMBA and matriptase-induced multi-stage squamous cell carcinogenesis. mTor-dependent phosphorylation of S6 protein is confined to suprabasal keratinocytes in normal and hyperplastic epidermis, whereas S6 protein is phosphorylated in all keratinocyte layers of the dysplastic epidermis, and all tumor cells of squamous cell carcinomas. E and F. Representative examples of the outward appearance of K5-Mat mice treated with vehicle (E) or rapamycin (F) for 33 weeks after DMBA treatment. G and H. Histological appearance of H&E stained epidermis of vehicle-(top panel, G) or rapamycin-treated (middle panel, G) control mice, and of rapamycin-treated (bottom panel, G) and vehicle-treated (H) K5Mat mice. High magnification (I and J) of epidermis of vehicle- (I) and rapamycin- (J) treated mice. Examples of basal keratinocytes and tumor cells at the invasive front are indicated with arrowheads in G–J. Stars in I and J indicate tumor stroma and dermis, respectively. Size bars: A–G, 50 μm; H, 100 μm; I–J, 25 μm. K. Kaplan-Meier analysis of DMBA-induced epidermal tumor formation in rapamycin-treated K5-Mat (black lines, N=15), rapamycin-treated wildtype (green lines, N=15), vehicle-treated wildtype (blue lines, N=15), and vehicle-treated K5-Mat (red lines, N=15) littermate mice. P values were determined by the log-rank test, two-tailed. L. Enumeration of BrdU incorporation in basal keratinocytes of K5-Mat and wildtype littermate mice treated with vehicle or rapamycin. P values were determined by Student's t-test, two tailed.

Article Snippet: The protein concentration was determined by BCA assay (Pierce, Rockford, IL) and 40 μg of total protein (Pierce, Rockford, IL) was loaded on 4–12% reducing SDS-PAGE and analyzed by Western blotting using a polyclonal sheep anti-human Matriptase (R&D Systems), rabbit anti-human phospho cMet, phospho-Gab1, or rabbit anti human GAPDH primary antibodies (all Cell Signaling Technology, Beverly, MA).

Techniques: Immunohistochemistry, Phospho-proteomics, Staining, Control, Two Tailed Test, BrdU Incorporation Assay

Journal: Oncogene

Article Title: c-Met-induced epithelial carcinogenesis is initiated by the serine protease matriptase

doi: 10.1038/onc.2010.586

Figure Lengend Snippet: 1. Mesenchymal cells located in close proximity to c-Met- and matriptase-expressing basal keratinocytes with high tumorigenic potential secrete single-chain proHGF/SF into the pericellular microenvironment. 2. ProHGF/SF binds c-Met with high affinity on the keratinocyte cell surface. 3. Matriptase cleaves and converts single-chain proHGF/SF to signaling-competent two-chain HGF/SF. 4. Matriptase-cleaved two-chain HGF/SF undergoes a conformational change that enables c-Met activation by autophosphorylation. 5. Activation of c-Met leads to recruitment of Gab1 and other effectors of c-Met signaling. 6. Gab1 recruitment initiates a pro-tumorgenic PI3K-Akt-mTor signaling pathway. 7. Activation of additional unidentified signaling pathway(s) located downstream from c-Met (hatched arrows) induces constitutive keratinocyte proliferation. Matriptase-induced mTor activation and mitogenic signaling, in combination with other epigenetic and genetic changes ( ras -dependent and ras -independent), causes malignant transformation. The model is synthesized on the basis of data obtained in (7), and the current study.

Article Snippet: The protein concentration was determined by BCA assay (Pierce, Rockford, IL) and 40 μg of total protein (Pierce, Rockford, IL) was loaded on 4–12% reducing SDS-PAGE and analyzed by Western blotting using a polyclonal sheep anti-human Matriptase (R&D Systems), rabbit anti-human phospho cMet, phospho-Gab1, or rabbit anti human GAPDH primary antibodies (all Cell Signaling Technology, Beverly, MA).

Techniques: Expressing, Activation Assay, Transformation Assay, Synthesized