Analysis of BCL3 expression in tamoxifen adapted cell lines (MCF-7-TamR)
In addition to earlier studies on anti-estrogen resistance and the effect of cancer-associated fibroblasts, we initially investigated the BCL3 expression in our model for acquired tamoxifen resistance. In this model, the luminal A cell line MCF-7 was adapted to 4OH-tamoxifen for at least 12 weeks [23, 24]. Here, we were particularly interested whether BCL3 localization and abundance has changed. In our cDNA array data, BCL3 mRNA was not significantly altered during tamoxifen adaption of MCF-7 . However, Western blots of three independently generated MCF7-TamR lines detected decreased amounts of BCL3 protein (Fig. 1) although with significant variation. In immunofluorescence analysis, MCF-7-TamR cells showed BCL3 mainly localized to the nucleus in a dotted appearance compared to MCF-7 (Fig. 1). The signal ratio cytosol to nucleus was determined to be 0.48 ± 0.14 and 0.37 ± 0.13 for MCF-7 and MCF-7-TamR, respectively (p = 0.014).
Distribution of BCL3 abundance by immunohistochemistry in the patient cohort
We then stained paraffin-embedded tissue of our breast cancer cohort for BCL3 by immunohistochemistry. Here, we observed a specific staining of BCL3 in tumor cells in both, the cytoplasmic and nuclear compartment but this varied between the specimens (Fig. 2). As consequence, we scored the IHC signal for nucleus and cytosol separately. A cut-off value for the immuno-reactive score (IRS) was determined separately for nuclear and cytosolic staining by optimizing the log-rank p-value in Kaplan–Meier survival analysis and using the receiver operator curve (ROC) for relapse-free survival. A cut-off value was set to IRS > 8 for both localizations (Fig. 3). The distribution of high and low abundance of BCL3 according to clinico-pathological parameters is summarized in Table 1. Overall, high cytoplasmic BCL3 was detected in 31.7% of all cases, whereas high nuclear BCL3 was found for 22.8% of the tumors. There was an intermediate correlation of cytosolic and nuclear BCL3 IRS (Spearman’s rho = 0.24, p = 0.001). Only 16% of low cytoplasmic BCL3 cases had high nuclear BCL3 and 42.1% of high cytoplasmic BCL3 tumors exhibited also high nuclear BCL3 IRS (Fisher’s exact test p < 0.001). There was a weak negative association of cytoplasmic score (p = 0.063) with ER-status. A positive association of the cytoplasmic score was found for tumor size (T > 2, p = 0.059) and Ki-67-status (p = 0.001). Most interestingly both the high cytoplasmic and high nuclear BCL3 IRS correlated strongly with the appearance of a relapse under tamoxifen therapy (p < 0.001). However, in contrast to the cytosolic BCL3-IRS, nuclear BCL3-status did not correlate with the other factors tested.
We next evaluated the significance of BCL3-IRS for survival. Both high cytoplasmic and nuclear BCL3 IRS (> 8) were significantly associated with poor relapse-free survival (RFS, Fig. 3 and Fig. 4A) but not with overall survival. In cases that were high for BCL3 in both localizations, the correlation to RFS was even more pronounced (Fig. 4A). Concerning breast cancer subgroups, both scores were significant for tamoxifen treatment, lobular histology, G2, ER + , PR + , HER2-, Ki-67-low, and cases not treated by chemotherapy. Only the nuclear score was significantly correlated with RFS in post-menopausal cases, ductal histology, larger tumors and treatment by radiotherapy and chemotherapy (Fig. 5, Table 2). Interestingly, in aromatase inhibitor–treated cases, BCL3 IRS was not significant. Notably, the Kaplan–Meier curve for cases with low BCL3-IRS for aromatase inhibitor (AI)–treated patients was above the curve for BCL3-IRS high cases, suggesting a better response to this drug. When we restricted the Kaplan–Meier analysis to ER-negative cases, similar results were found (Table 2, Fig. 4A).
In multivariate Cox regression, we adjusted the hazard ratio (HR) of cytosolic and nuclear BCL3 for the parameters ER status and lymph node metastasis (Table 3). In both cases, BCL3 IRS turned out to be independent from these factors with an associated HR of about 1.8 and 2.9, respectively. Additionally, we adjusted nuclear for cytosolic BCL3-score by Cox regression analysis and found that the nuclear score was the predominant factor for relapse-free survival (HR = 2.5; CI: 1.35–4.57; p = 0.003). When we restricted this analysis to ER-positive cases treated with tamoxifen, the significance for cytosolic and nuclear BCL3-IRS increased even further (Table 3).
Distribution of BCL3 mRNA in public BC datasets
Additionally, we were interested in the distribution of BCL3 mRNA abundance in a larger cohort of breast cancer cases. By using the GEPIA2 website , we found that BCL3 mRNA was more abundant in cancerous than in normal tissue with the exception of the basal subtype (Fig. 6). We additionally analyzed the gene expression data of the METABRIC cohort with respect to the 3-gene classifier subtypes, based upon ER- and HER2- as well as proliferation status (Fig. 6). It turned out for both datasets that HER2-over-expressing cases had the highest amounts of BCL3 mRNA, whereas ER-/HER2-cases had the lowest abundance of this RNA. ER-positive cases ranged in between these two subtypes with no significant difference between low and high proliferating cases (luminal A and B).