Thrombospondin-1 in a Murine Model of Colorectal Carcinogenesis

Colorectal Cancer (CRC) is one of the late complications observed in patients suffering from inflammatory bowel diseases (IBD). Carcinogenesis is promoted by persistent chronic inflammation occurring in IBD. Understanding the mechanisms involved is essential in order to ameliorate inflammation and prevent CRC. Thrombospondin 1 (TSP-1) is a multidomain glycoprotein with important roles in angiogenesis. The effects of TSP-1 in colonic tumor formation and growth were analyzed in a model of inflammation-induced carcinogenesis. WT and TSP-1 deficient mice (TSP-1-/-) of the C57BL/6 strain received a single injection of azoxymethane (AOM) and multiple cycles of dextran sodium sulfate (DSS) to induce chronic inflammation-related cancers. Proliferation and angiogenesis were histologically analyzed in tumors. The intestinal transcriptome was also analyzed using a gene microarray approach. When the area containing tumors was compared with the entire colonic area of each mouse, the tumor burden was decreased in AOM/DSS-treated TSP-1-/- versus wild type (WT) mice. However, these lesions displayed more angiogenesis and proliferation rates when compared with the WT tumors. AOM-DSS treatment of TSP-1-/- mice resulted in significant deregulation of genes involved in transcription, canonical Wnt signaling, transport, defense response, regulation of epithelial cell proliferation and metabolism. Microarray analyses of these tumors showed down-regulation of 18 microRNAs in TSP-1-/- tumors. These results contribute new insights on the controversial role of TSP-1 in cancer and offer a better understanding of the genetics and pathogenesis of CRC

The highly deregulated genes, up-regulated (left column) and down-regulated (right column), in the TSP-1 deficient mice after AOM-DSS treatment are categorized according to molecular function/ontology, below. Numbers in brackets represent fold-change: first value refers to fold-change in the comparison between treated TSP-1^-/- vs water-TSP-1^-/-; second value refers to fold-change comparing treated TSP-1^-/- vs treated WT, p<0.05).

<p>The highly deregulated genes, up-regulated (left column) and down-regulated (right column), in the TSP-1 deficient mice after AOM-DSS treatment are categorized according to molecular function/ontology, below. Numbers in brackets represent fold-change: first value refers to fold-change in the comparison between treated TSP-1^-/- vs water-TSP-1^-/-; second value refers to fold-change comparing treated TSP-1^-/- vs treated WT, p<0.05).</p

Validation of transcripts involved in inflammation and cancer by IHC.

<p>The expression of some transcripts involved in epithelial proliferation and the immune response was examined by IHC. PLA2g2 immunostaining of these tumors showed a focal localization in glands of WT tumors (A). This same protein was diffusely expressed in the glands and stroma of TSP-1^-/- tumors (B). SSTR2 was intensely expressed in WT tumors (C) while the staining in TSP-1^-/- lesions was rather light or even negative (D). WIF1 was focally located in the lamina propria of WT cancers (E). Cytoplasmic staining was detected in isolated foci of cells in malignant glands in TSP-1^-/- tumors (F). Neurotensin (NTS) was predominantly localized in the malignant epithelial cells. While WT tumors showed intense and diffuse staining (G), some of the tumors developed in TSP-1^-/- showed focal areas lacking NTS staining (H).</p

Analysis of microvascular density (MVD) and proliferation in TSP-1^-/- and WT tumors.

<p>Blood vessels were blindly evaluated in each tumor section stained with MECA and CD31 antibodies in TSP-1^-/- (A) and WT colons (B). Tumors lacking TSP-1 showed significantly higher MVD as compared to the MVD in the WT tumors (p = 0.0159), (C). IHC for PCNA was performed and positive nuclei were counted in the tumor sections of TSP-1 deficient mice (D) and WT mice (E). PCNA rates were significantly higher in tumors of TSP-1^-/- mice compared to WT tumors (p<.0001), (F).</p

Gross and microscopic features of AOM/DSS tumors originated in WT and TSP-1^-/- colons.

<p>Tumors in TSP-1^-/- colons (A, asterisks) consisted of well-delimited polypoid lesions separated by wide areas of normal mucosa. WT tumors (B, asterisks) were grossly characterized by multiple coalescing tumors delimited by little intervening normal mucosa When the area containing tumors was compared with the entire colonic area of each mouse, the tumor burden was decreased in AOM/DSS-treated TSP-1^-/- versus WT mice (p = 0.0060), (C). All the microscopic dysplastic foci were analyzed in TSP-1^-/- (D) and WT colonic sections (E). No significant differences were observed in the number of dysplasic lesions detected by microscopic field (p = 0.14) (F).</p