Elevated S100A8 protein expression in breast cancer cells and breast tumor stroma is prognostic of poor disease outcome.

PURPOSE: Elevated S100A8 expression has been observed in cancers of the bladder, esophagus, colon, ovary, and breast. S100A8 is expressed by breast cancer cells as well as by infiltrating immune and myeloid cells. Here we investigate the association of elevated S100A8 protein expression in breast cancer cells and in breast tumor stroma with survival outcomes in a cohort of breast cancer patients. PATIENTS AND METHODS: Tissue microarrays (TMA) were constructed from breast cancer specimens from 417 patients with stage I-III breast cancer treated at the University of Michigan Comprehensive Cancer Center between 2004 and 2006. Representative regions of non-necrotic tumor and distant normal tissue from each patient were used to construct the TMA. Automated quantitative immunofluorescence (AQUA) was used to measure S100A8 protein expression, and samples were scored for breast cancer cell and stromal S100A8 expression. S100A8 staining intensity was assessed as a continuous value and by exploratory dichotomous cutoffs. Associations between breast cancer cell and stromal S100A8 expression with disease-free survival and overall survival were determined using the Kaplan-Meier method and Cox proportional hazard models. RESULTS: High breast cancer cell S100A8 protein expression (as indicated by AQUA scores), as a continuous measure, was a significant prognostic factor for OS [univariable hazard ratio (HR) 1.24, 95% confidence interval (CI) 1.00-1.55, p = 0.05] in this patient cohort. Exploratory analyses identified optimal S100A8 AQUA score cutoffs within the breast cancer cell and stromal compartments that significantly separated survival curves for the complete cohort. Elevated breast cancer cell and stromal S100A8 expression, indicated by higher S100A8 AQUA scores, significantly associates with poorer breast cancer outcomes, regardless of estrogen receptor status. CONCLUSIONS: Elevated breast cancer cell and stromal S1008 protein expression are significant indicators of poorer outcomes in early stage breast cancer patients. Evaluation of S100A8 protein expression may provide additional prognostic information beyond traditional breast cancer prognostic biomarkers.

Targeting of RAGE-ligand signaling impairs breast cancer cell invasion and metastasis.

The receptor for advanced glycation end products (RAGE) is highly expressed in various cancers and is correlated with poorer outcome in breast and other cancers. Here we tested the role of targeting RAGE by multiple approaches in the tumor and tumor microenvironment, to inhibit the metastatic process. We first tested how RAGE impacts tumor cell-intrinsic mechanisms using either RAGE overexpression or knockdown with short hairpin RNAs (shRNAs). RAGE ectopic overexpression in breast cancer cells increased MEK-EMT (MEK-epithelial-to-mesenchymal transition) signaling, transwell invasion and soft agar colony formation, and in vivo promoted lung metastasis independent of tumor growth. RAGE knockdown with multiple independent shRNAs in breast cancer cells led to decreased transwell invasion and soft agar colony formation, without affecting proliferation. In vivo, targeting RAGE shRNA knockdown in human and mouse breast cancer cells, decreased orthotopic tumor growth, reduced tumor angiogenesis and recruitment of inflammatory cells, and markedly decreased metastasis to the lung and liver in multiple xenograft and syngeneic mouse models. To test the non-tumor cell microenvironment role of RAGE, we performed syngeneic studies with orthotopically injected breast cancer cells in wild-type and RAGE-knockout C57BL6 mice. RAGE-knockout mice displayed striking impairment of tumor cell growth compared with wild-type mice, along with decreased mitogen-activated protein kinase signaling, tumor angiogenesis and inflammatory cell recruitment. To test the combined inhibition of RAGE in both tumor cell-intrinsic and non-tumor cells of the microenvironment, we performed in vivo treatment of xenografted tumors with FPS-ZM1 (1 mg/kg, two times per week). Compared with vehicle, FPS-ZM1 inhibited primary tumor growth, inhibited tumor angiogenesis and inflammatory cell recruitment and, most importantly, prevented metastasis to the lung and liver. These data demonstrate that RAGE drives tumor progression and metastasis through distinct tumor cell-intrinsic and -extrinsic mechanisms, and may represent a novel and therapeutically viable approach for treating metastatic cancers.

RAGE and arthritis: the G82S polymorphism amplifies the inflammatory response.

The receptor for advanced glycation end products (RAGE) and its proinflammatory S100/calgranulin ligands are enriched in joints of subjects with rheumatoid arthritis (RA) and amplify the immune/inflammatory response. In a model of inflammatory arthritis, blockade of RAGE in mice immunized and challenged with bovine type II collagen suppressed clinical and histologic evidence of arthritis, in parallel with diminished levels of TNF-alpha, IL-6, and matrix metalloproteinases (MMP) 3, 9 and 13 in affected tissues. Allelic variation within key domains of RAGE may influence these proinflammatory mechanisms, thereby predisposing individuals to heightened inflammatory responses. A polymorphism of the RAGE gene within the ligand-binding domain of the receptor has been identified, consisting of a glycine to serine change at position 82. Cells bearing the RAGE 82S allele displayed enhanced binding and cytokine/MMP generation following ligation by a prototypic S100/calgranulin compared with cells expressing the RAGE 82G allele. In human subjects, a case-control study demonstrated an increased prevalence of the 82S allele in patients with RA compared with control subjects. These data suggest that RAGE 82S upregulates the inflammatory response upon engagement of S100/calgranulins, and, thereby, may contribute to enhanced proinflammatory mechanisms in immune/inflammatory diseases.

Characterization of allelic and nucleotide variation between the RAGE gene on chromosome 6 and a homologous pseudogene sequence to its 5' regulatory region on chromosome 3: implications for polymorphic studies in diabetes.

Activation of the receptor for advanced glycation end products (RAGE) appears to be a key mechanism in the pathogenesis of diabetic vascular disease, making RAGE a candidate gene for investigation. RAGE is located in the major histocompatibility complex locus on chromosome 6, which contains a multitude of overlapping and duplicated genes involved predominantly in inflammatory and immune responses. The RAGE 5' flanking region from -505 in a 5' direction overlaps with PBX2, a gene that has a pseudogene copy on chromosome 3, making any studies of polymorphisms in this duplicated region potentially fraught with error. In this study we have addressed these issues by confirming RAGE as a predominantly single-copy gene and PBX2 to have two gene copies in the haploid human genome. We have characterized the gene:pseudogene differences between RAGE/PBX2 on chromosome 6 and PsiPBX2 on chromosome 3, which include a change from C to A at position -1139 RAGE/+2298 PBX2, previously reported as a polymorphism. Single chromosome-specific DNA amplification of the duplicated region has clarified five polymorphisms to be on chromosome 3 and one (at -1202 RAGE/+2234 PBX2) to be on chromosome 6. In conclusion, this study provides essential data for the study of RAGE and its genetics.

Effects of novel polymorphisms in the RAGE gene on transcriptional regulation and their association with diabetic retinopathy.

Interactions between advanced glycation end products (AGEs) and the receptor for AGE (RAGE) are implicated in the vascular complications in diabetes. We have identified eight novel polymorphisms, of which the -1420 (GGT)n, -1393 G/T, -1390 G/T, and -1202 G/A were in the overlapping PBX2 3' untranslated region (UTR), and the -429 T/C (66.5% TT, 33.5% TC/CC), -407 to -345 deletion (99% I, 1% I/D, 0% D), -374 T/A (66.4% TT, 33.6% TA/AA), and +20 T/A were in the RAGE promoter. To evaluate the effects on transcriptional activity, we measured chloramphenicol acetyl transferase (CAT) reporter gene expression, driven by variants of the -738 to +49 RAGE gene fragment containing the four polymorphisms identified close to the transcriptional start site. The -429 C, -374 A, and 63-bp deletion alleles resulted in a mean increase of CAT expression of twofold (P < 0.0001), threefold (P < 0.001), and fourfold (P < 0.05), respectively, with the -374 T and A alleles yielding highly differential binding of nuclear protein extract from both monocyte- and hepatocyte-derived cell lines. The prevalence of the functional polymorphisms were investigated in subjects with type 2 diabetes (106 with and 109 without retinopathy), with the -429 C allele showing an increase in the retinopathy group (P < 0.05). These data suggest that the polymorphisms involved in differences in RAGE gene regulation may influence the pathogenesis of diabetic vascular complications.