Fig. 1. GREMLIN 1 mRNA is elevated in BCC tumor-derived stromal cultures ex vivo and in BCC tumors in vivo. (A and B) Gene expression in stromal cells from BCC tumor and nontumor skin. Each row in the heat map represents a gene, and each column represents a sample; cultures derived from BCC and nontumor skin are indicated by red and black branches, respectively. (A) Hierarchical clustering of samples based on expression of 403 array elements selected for differential expression by two-class Significance Analysis of Microarrays analysis at a false discovery rate of 15%. The level of expression of each gene in each stromal cell sample is relative to the mean level of expression of that gene across all samples and is represented by using a red-green color scale. (B) Hierarchical clustering of samples according to their expression of a more stringently selected set of genes (false discovery rate of 5%). (C) RT-PCR analysis of whole-tissue samples of BCC tumor or adjacent nontumor skin from eight patients. The relative level of GREMLIN 1 RNA in each sample was normalized to GAPDH for that sample.
Fig. 2. Expression of GREMLIN 1 and BMP 2 and 4 in BCC tumor tissues. (A and B) ISH for GREMLIN 1 RNA in normal scalp (A) and BCC tumor skin (B). GREMLIN 1 is expressed by stromal cells surrounding the tumor (indicated by arrows) but is undetectable in normal scalp. Positive signal appears as dark purple dots. (C and D) IHC for gremlin 1 protein in normal scalp (C) and BCC tumor skin (D). Positive signal appears as diffuse brown staining. (E and F) IHC with antibodies against BMP 2 (E) and 4 (F) in large sections of human BCC. Signal is represented by brown color. (G–N) serial sections of a BCC tumor showing that GREMLIN 1-expressing cells have properties of fibroblasts. (G and H) RNA ISH for GREMLIN 1 RNA in nontumor (G) and tumor (H) skin. GREMLIN 1 expression is indicated by dark purple dots. (I–N) IHC for cell lineage markers vimentin (I), CD45 (J), CD31 (K), desmin (L), pancytokeratin (M), and GFAP (N). Signal is represented by brown color.
Fig. 3. Effects of BMP and gremlin 1 on BCC cell differentiation and expansion in vitro. (A) Cells were cultured from a human BCC tumor and then treated for 7 days in culture with recombinant human BMP 4 (833 ng/ml), recombinant mouse gremlin 1 (2,105 ng/ml), or both. Populations were then compared by using quantitative RT-PCR to detect the levels of SPRR1A, SPRR1B, SPRR3, and SPRR4 transcripts. (B) Cells were cultured in vitro from human BCCs then treated with varying concentrations of gremlin 1 or BMP 4 protein for 7 days. Cells were counted by using a hemacytometer, with triplicate counts taken for each measurement; each measurement is the average of duplicate experiments.
Fig. 4. GREMLIN 1 RNA is widely expressed in cancer. (A) Representative images for ISH in other carcinomas. Tissue microarrays representing 774 human cancers of diverse tissue types were analyzed for expression of GREMLIN 1 RNA. Positive signal is denoted by dark brown staining. (B) Tabulation of results for all tumor types examined for which n was >10.
Fig. 5. A possible parallel for the role of BMPs and BMP antagonists in the tumor stem cell niche and normal stem cell niche. BMPs can induce differentiation and block expansion of normal stem and progenitor cells. (Upper) BMP antagonists can reverse this effect and favor expansion. Signals that are critical for stem cell self renewal, such as BMP antagonists, derive from the stem cell niche. BMPs may often come from epithelial or stem cells. (Lower) In tumor biology, BMPs can also inhibit expansion of tumor cells. BMP antagonists secreted by the tumor cell niche may reverse this inhibition, allowing tumor cell expansion to continue.