Correlations of differentially expressed gap junction connexins cx26, cx30, cx32, cx43 and cx46 with breast cancer progression and prognosis.

BACKGROUND AND AIMS: Connexins and their cell membrane channels contribute to the control of cell proliferation and compartmental functions in breast glands and their deregulation is linked to breast carcinogenesis. Our aim was to correlate connexin expression with tumor progression and prognosis in primary breast cancers. MATERIALS AND METHODS: Meta-analysis of connexin isotype expression data of 1809 and 1899 breast cancers from the Affymetrix and Illumina array platforms, respectively, was performed. Expressed connexins were also monitored at the protein level in tissue microarrays of 127 patients equally representing all tumor grades, using immunofluorescence and multilayer, multichannel digital microscopy. Prognostic correlations were plotted in Kaplan-Meier curves and tested using the log-rank test and cox-regression analysis in univariate and multivariate models. RESULTS: The expression of GJA1/Cx43, GJA3/Cx46 and GJB2/Cx26 and, for the first time, GJA6/Cx30 and GJB1/Cx32 was revealed both in normal human mammary glands and breast carcinomas. Within their subfamilies these connexins can form homo- and heterocellular epithelial channels. In cancer, the array datasets cross-validated each other's prognostic results. In line with the significant correlations found at mRNA level, elevated Cx43 protein levels were linked with significantly improved breast cancer outcome, offering Cx43 protein detection as an independent prognostic marker stronger than vascular invasion or necrosis. As a contrary, elevated Cx30 mRNA and protein levels were associated with a reduced disease outcome offering Cx30 protein detection as an independent prognostic marker outperforming mitotic index and necrosis. Elevated versus low Cx43 protein levels allowed the stratification of grade 2 tumors into good and poor relapse free survival subgroups, respectively. Also, elevated versus low Cx30 levels stratified grade 3 patients into poor and good overall survival subgroups, respectively. CONCLUSION: Differential expression of Cx43 and Cx30 may serve as potential positive and negative prognostic markers, respectively, for a clinically relevant stratification of breast cancers

Kaplan-Meier plots of significant prognostic correlations of elevated (>median) Cx43 (a-d and g–i) and Cx46 (e–f) mRNA expression in 1809 breast cancers based on <i>in silico</i> analysis of Affymetrix array data.

<p>Cx43 expression was associated with improved relapse/disease free survival (RFS) in a tumor group similar to SEER prevalence (a), in ER and lymph node positive tumors (b) and in ER positive endocrine treated tumors (c), but reduced RFS in ER negative tumors (d). Elevated Cx46 levels were predictive for better RFS in the whole patient cohort (e) and in ER and lymph node positive grade 3 patients (f). Elevated Cx43 mRNA levels were also correlated with significantly better distant metastasis-free survival (DMFS) in ER positive endocrine treated patients (g) and in grade 2 cancers (h). Cx43 expression in ER negative patients was linked with reduced overall survival (OS) (i). Significance (at p<0.05) was calculated using the log-rank test. HR: hazard ratio (at 95% Confidence Interval).</p

Prognostic value of connexin protein isotype expression using multivariate Cox regression analysis.

<p>HR: hazard ratio; CI: confidence interval;</p><p>0123: score categories where _ represents thresholds.</p><p>Prognostic value of connexin protein isotype expression using multivariate Cox regression analysis.</p

Detection of connexin protein isotypes (Alexa-564, red) and the proliferation marker Ki67 protein (Alexa-518, green) in normal pre-menopausal breast tissue.

<p>Punctuate Cx43 reaction was localized to the myoepithelial cell layer of normal mammary glands and to adjacent stromal cells (arrow) (a). Both Cx32 (b and c) and Cx26 (d) proteins were found dominantly in the luminal epithelial cells. Cx46 protein was found both in the myoepithelial and luminal cells and less in stromal inflammatory cells (arrow) (e and f). High power views show Cx32 mainly linked to the luminal cells (c) and Cx46 mainly localized to the basal cells (f) involving both their cytoplasm and intercellular borders. Cx30 was revealed along myoepithelial cells and at the apex of luminal epithelium and less in the rest of luminal cells, and along vascular endothelial cells (arrow) (g). Co-localization of Cx26 (red) and Cx30 (green) in epithelial cells (yellow), involving the intercellular borders (h). Double immunofluorescence, cell nuclei are stained blue using Hoescht. Summary drawing of our results shows potential homo- or heterocellular/typic interactions of Cx26 (red), Cx32 (blue), Cx46 (violet), Cx43 (green) and Cx30 (yellow) gap junctions in a normal mammary gland (i).</p

Kaplan-Meier plots of significant correlations between overall survival (OS) and elevated (>median) Cx30 (a–c), Cx43 (d–f) and Cx46 (g–i) mRNA expression in 1988 breast cancers based on <i>in silico</i> analysis of Illumina array data.

<p>Elevated Cx30 levels were linked with reduced OS in patients with luminal B tumors (a) and in ER positive endocrine therapy treated patients (b), but with a strong tendency for improved OS in ER negative cases (c). Cx43 expression was associated with better OS in the whole cohort (d) and in ER positive endocrine treated patients (e) but showed a strong trend for reduced OS in ER negative patients (f). Cx46 expression was associated with improved OS in the whole cohort (g), in ER negative patients (h) and in HER2 positive patients (i). Significance (at p<0.05) was calculated using the log-rank test. HR: hazard ratio (at 95% Confidence Interval).</p

Significant prognostic correlations of connexin mRNA expression data resulting from the <i>in silico</i> analysis of 1809 (Affymetrix) and 1988 (Illumina) breast cancers.

<p>A: Affymetrix platform using relapse-free survival (RFS) data if not indicated, distant metastasis-free survival (DMFS) data indicated as M or overall survival data (OS).</p><p>I: Illumina platform using OS data only (not indicated).</p><p>Significant prognostic correlations of connexin mRNA expression data resulting from the <i>in silico</i> analysis of 1809 (Affymetrix) and 1988 (Illumina) breast cancers.</p