Additional file 2: Figure S1. of Iroquois homeobox 2 suppresses cellular motility and chemokine expression in breast cancer cells

Gene reporter assay with reporter plasmid containing proximal CCL5 promoter fragment. The reporter plasmid was transfected into BT-549 cells that over express IRX2 and into the control cell line.  For normalization, a co-transfection with the pGL4.74 plasmid containing the Renilla luciferase was performed. Each experiment was performed in triplicate. Error bars represent the standard deviation of the mean. (PDF 18 kb

Clinical Relevance of Loss of 11p15 in Primary and Metastatic Breast Cancer: Association with Loss of PRKCDBP Expression in Brain Metastases

<div><p>The occurrence of brain metastases among breast cancer patients is currently rising with approximately 20–25% incidence rates, underlining the importance of the identification of new therapeutic and prognostic markers. We have previously screened for new markers for brain metastasis by array CGH. We found that loss of 11p15 is common among these patients. In this study, we investigated the clinical significance of loss of 11p15 in primary breast cancer (BC) and breast cancer brain metastases (BCBM). 11p15 aberration patterns were assessed by allelic imbalance (AI) analysis in primary BC (n = 78), BCBM (n = 21) and metastases from other distant sites (n = 6) using six different markers. AI at 11p15 was significantly associated with BCBM (p = 0.002). Interestingly, a subgroup of primary BC with a later relapse to the brain had almost equally high AI rates as the BCBM cases. In primary BC, AI was statistically significantly associated with high grade, negative hormone receptor status, and triple-negative (TNBC) tumors. Gene expression profiling identified <em>PRKCDBP</em> in the 11p15 region to be significantly downregulated in both BCBM and primary BC with brain relapse compared to primary tumors without relapse or bone metastasis (fdr<0.05). qRT-PCR confirmed these results and methylation was shown to be a common way to silence this gene. In conclusion, we found loss at 11p15 to be a marker for TNBC primary tumors and BCBM and <em>PRKCDBP</em> to be a potential target gene in this locus.</p> </div

Quantitative real-time RT-PCR results for PRKCDBP expression in BCBM and primary BC patients.

<p>Relative PRKCDBP transcript levels were determined by normalization to the reference gene RPLP0 and universal human reference (UHR) using the ΔΔCt method.</p

11p allelic imbalances and association to clinical factors in primary tumors.

<p>HR: hormone receptor.</p><p>TNBC: triple-negative tumors (HR negative/HER2 negative tumors).</p

Microsatellite analyses for AI on 11p in primary breast cancers and metastases.

<p>Base pair position and the markers used are indicated on the top line. The result for each marker is shown as follows: AI: black; non-informative: light gray; unavailable measurement: dark gray; and informative without changes: white box.</p

Differentially expressed genes at 11p between BCBM and primary BC without relapse, bone, or lung relapse.

*<p>fold change down regulated in brain metastases samples compared to primary breast tumors.</p>**<p>significantly down regulated genes among primary tumors with brain relapse compared to primary tumors with bone relapse.</p>***<p>significantly down regulated genes among primary tumors with brain relapse compared to primary tumors with lung relapse.</p

Frequencies and p-values for AI at chromosome 11p in BCBM, primary tumors and metastases.

*<p>FUP missing for 6 patients.</p><p>n.s.: not significant.</p

Estimated probability of disease free survival.

<p>A: Copy number loss at position 4q21.23 (RP11-570L13) revealed a trend towards shorter median disease free survival (DFS) in univariate analysis (12.5 months versus 28.0 months, <i>P = </i>0.056). B: Copy number loss at position 4q21.23 (RP11-570L13) in the squamous cell carcinoma (SqCC) subgroup showed strong correlations with shorter DFS (11.2 months versus 39.1 months) <i>P = </i>0.031. C: Copy number loss at position 4q22.1 (RP11-1053C2) showed significant correlations with shorter DFS (12.5 months versus 32.7 months), <i>P = </i>0.010. D: Copy number loss at position 4q22.1 (RP11-1053C2) showed significant correlations with shorter DFS in SqCC subgroup (11.7 months versus 35.5 months), <i>P = </i>0.027. Survival plots for gain aberrations were faded out for the sake of clarity.</p

Estimated probability of overall survival.

<p>A: Copy number loss at position 4q21.23 (RP11-570L13) revealed strong correlations with shorter median overall survival (OS) in univariate analysis (23.9 months versus 42.6 months), <i>P = </i>0.033. B: Copy number loss at position 4q21.23 (RP11-570L13) in the squamous cell carcinoma (SqCC) subgroup showed strong correlations with shorter OS (12.5 months versus 41 months), <i>P = </i>0.031 C: Copy number loss at position 4q22.1 (RP11-1053C2) showed significant correlations with shorter DFS (25.8 months versus 40.3 months), <i>P = </i>0.012. D: Copy number loss at position 4q22.1 (RP11-1053C2) showed significant correlations with shorter DFS in adenocarcinoma subgroup (10.0 months versus 43.1 months), <i>P = </i>0.035. Survival plots for gain aberrations were faded out for the sake of clarity.</p

Multivariate analysis RP11-1053C2.

<p>Cox regression hazard model was used for multivariate analysis to assess the prognostic value of aberrations.</p><p>HR, hazard ratio; CI, confidence interval; UICC, Union for International Cancer Control.</p><p>Multivariate analysis RP11-1053C2.</p