S100A8/A9 activate key genes and pathways in colon tumor progression - PubMed (original) (raw)

S100A8/A9 activate key genes and pathways in colon tumor progression

Mie Ichikawa et al. Mol Cancer Res. 2011 Feb.

Erratum in

Abstract

The tumor microenvironment plays an important role in modulating tumor progression. Earlier, we showed that S100A8/A9 proteins secreted by myeloid-derived suppressor cells (MDSC) present within tumors and metastatic sites promote an autocrine pathway for accumulation of MDSC. In a mouse model of colitis-associated colon cancer, we also showed that S100A8/A9-positive cells accumulate in all regions of dysplasia and adenoma. Here we present evidence that S100A8/A9 interact with RAGE and carboxylated glycans on colon tumor cells and promote activation of MAPK and NF-κB signaling pathways. Comparison of gene expression profiles of S100A8/A9-activated colon tumor cells versus unactivated cells led us to identify a small cohort of genes upregulated in activated cells, including Cxcl1, Ccl5 and Ccl7, Slc39a10, Lcn2, Zc3h12a, Enpp2, and other genes, whose products promote leukocyte recruitment, angiogenesis, tumor migration, wound healing, and formation of premetastatic niches in distal metastatic organs. Consistent with this observation, in murine colon tumor models we found that chemokines were upregulated in tumors, and elevated in sera of tumor-bearing wild-type mice. Mice lacking S100A9 showed significantly reduced tumor incidence, growth and metastasis, reduced chemokine levels, and reduced infiltration of CD11b(+)Gr1(+) cells within tumors and premetastatic organs. Studies using bone marrow chimeric mice revealed that S100A8/A9 expression on myeloid cells is essential for development of colon tumors. Our results thus reveal a novel role for myeloid-derived S100A8/A9 in activating specific downstream genes associated with tumorigenesis and in promoting tumor growth and metastasis.

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Figures

Figure 1

Figure 1

A. RAGE and mAbGB3.1 glycans are expressed on colon tumor cells. MC38 colon tumor cells in culture were analyzed for surface expression of mAbGB3.1 glycans and RAGE by flow cytometry. Cells were stained with mAbGB3.1 or anti-RAGE followed by FITC-conjugated anti-mouse or anti-rabbit Ig. Unstained cells (filled) and cells stained with secondary antibody alone (dark line) served as negative control. B and C. Receptor on colon tumor cells for S100A8/A9 identified by co-immunoprecipitation. MC38 cells were incubated with purified mouse S100A8/A9, S100A8 or S100A9 and bound proteins were immunoprecipitated with anti-S100A8 and/or anti-S100A9, or an irrelevant control rabbit IgG. To confirm potential interaction of RAGE with endogenous S100 proteins, immunoprecipitation was also performed using MC38 cells in which endogenous S100A9 was silenced using target-specific siRNA. Whole cell lysates and immunoprecipitated proteins were separated on SDS-PAGE gels, transferred and immunoblotted with anti-RAGE (B) or anti-TLR4 (C).

Figure 2

Figure 2

S100A8/A9 activates MAPK signaling pathways in colon tumor cells. (A) MC38 cells were incubated with purified endotoxin-free S100A8/A9 for different periods of time, and cell lysates were analyzed by Western blotting using respective antibodies against phosphorylated MAPK. As loading controls, separate lanes with lysate proteins were incubated with rabbit polyclonal antibodies for total ERK1/ERK2, p38, SAPK/JNK or β-actin B. MC38 or Caco-2 cells were incubated with purified endotoxin-free mouse or human S100A8/A9 for 15 minutes in the presence or absence of mAbGB3.1 or RAGE, and cell lysates were analyzed by Western blot using respective antibodies against phosphorylated or total ERK1/ERK2 or β-actin. C. MC38 cells were incubated with purifed mouse S100A8 or S100A9 homodimers for different periods of time, and cell lysates were analyzed by Western blot using phosphorylated or total ERK1/ERK2 or β-actin.

Figure 3

Figure 3

S100A8/A9 activates NF-κB in colon tumor cells. A. MC38 or Caco-2 cells were incubated with purified endotoxin-free mouse or human S100A8/A9 for different periods of time, and cell lysates were analyzed by Western blot using respective antibodies against phosphorylated or total IκBα or β–actin. B. MC38 or Caco-2 cells were treated with respective purified S100A8/A9 for 6 h in the presence or absence of mAbGB3.1 or anti-RAGE and nuclear extracts were analyzed for NF-κBp65 using DNA-binding ELISA.

Figure 4

Figure 4

A. Profile of differentially expressed genes from S100A8/A9-activated MC38 cells compared to unstimulated cells obtained by global gene expression analysis. Fold variation represent the mean of replicate (n=2) analysis. B. Cellular mRNA levels of chemokines. mRNA levels of Cxcl1, Ccl7 and Ccl5 were measured by RQ-PCR using RNA samples isolated from control MC38 cells, MC38 cells starved overnight and either untreated or stimulated with S100A8/A9. The expression values were normalized relative to GAPDH. The levels of mRNA in unstimulated or stimulated cells are shown relative to control non-starved MC38 cells considered as 100%. Each value is the mean level ± SD in two different samples for each condition, each sample assayed in duplicate. C. CXCL1 is secreted into medium from activated MC38 cultures. MC38 cultures were activated with S100A8/A9 in presence or absence of mAbGB3.1 or anti-RAGE, and CXCL1 in culture supernatants harvested at different time points was measured by ELISA.

Figure 5

Figure 5

A. Representative H&E stained colon “Swiss rolls” obtained from wild type and S100A9 null mice subjected to the AOM-DSS protocol 12 weeks after initiation (10× magnification). Arrows indicate regions of dysplasia and adenoma. B. Colonic tumor incidence in S100A9 null and wild type mice 12 and 20 weeks after AOM-DSS (n=5 mice per group per time point). C. Representative sections of tumor regions and normal adjacent tissue in colons of wild type and S100A9 null mice subjected to the AOM-DSS protocol 20 weeks after initiation stained for CXCL1 or CCL7 (400×) D. CXCL1 in sera of wild type and S100A9 null mice before and 12 weeks after AOM-DSS (n=5 mice). E. Colonic tumor incidence in bone marrow chimeric mice 12 weeks after AOM-DSS (n=4 recipient mice per group).

Figure 6

Figure 6

A. Tumor volumes of ectopic MC38 tumors in wild type (n=10) and S100A9 null mice (n=12) 3 weeks after sc injection of 1×106 cells. 6 out of 12 S100A9 null mice showed significantly reduced tumor growth shown here. In addition, 2 of the remaining six S100A9 null mice completely rejected the tumors. B. CXCL1 in sera of wild type and S100A9 null mice before and 3 weeks after MC38 tumor growth. C. Quantitation of CD11b+ cells co-staining with Gr1 or GB3.1 glycans or RAGE from bone marrow of MC38 tumor-bearing wild type mice D. Tumors were examined for infiltrating macrophages and tumor endothelial cells by immunochemical staining for S100A9+ cells, CD11b+Gr1+ cells (merged images of Alexa-488 stained CD11b+ cells and Alexa-594 stained Gr1+ cells showing double positive yellow cells), and CD31+ and F4/80+ cells (200×). E. Representative sections showing CD11b+Gr1+ cells in premetastatic livers of tumor-free and tumor-bearing mice. F. Quantitation of average number of CD11b+Gr1+ cells in premetastatic livers in 3 high power fields.

Figure 7

Figure 7

S100A9 null mice exhibit reduced metastatic tumors. A. Representative livers from wild type and S100A9 null mice 2 wks after intrasplenic injection of MC38 cells. Arrows indicate visible tumors B. Histology of representative livers stained by hematoxylin and eosin (25× magnification). C. Numbers of metastatic nodules in the livers, and total tumor area represented as % of liver tissue, 2 wks after intrasplenic injection of MC38 cells (wild type (n=6) and S100A9 null mice (n=5).

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