Biologic role of activated leukocyte cell adhesion molecule overexpression in breast cancer cell lines and clinical tumor tissue

Abstract

The activated leukocyte cell adhesion molecule (ALCAM) is overexpressed in many mammary tumors, but controversial results about its role and prognostic impact in breast cancer have been reported. Therefore, we evaluated the biologic effects of ALCAM expression in two breast cancer cell lines and a larger cohort of mammary carcinomas. By stable transfections, MCF7 cells with ALCAM overexpression and MDA-MB231 cells with reduced ALCAM levels were generated and analyzed in functional assays and cDNA microarrays. In addition, an immunohistochemical study on 347 patients with breast cancer with long-term follow-up and analysis of disseminated tumor cells (DTCs) was performed. In both cell lines, high ALCAM expression was associated with reduced cell motility. In addition, ALCAM silencing in MDA-MB231 cells resulted in lower invasive potential, whereas high ALCAM expression was associated with increased apoptosis in both cell lines. Among genes which were differentially expressed in clones with altered ALCAM expression, there was an overlap of 15 genes between both cell lines, among them cathepsin D, keratin 7, gelsolin, and ets2 whose deregulation was validated by western blot analysis. In MDA-MB231 cells, we observed a correlation with VEGF expression which was validated by enzyme-linked immuno sorbent assay (ELISA). Our IHC results on primary breast carcinomas showed that ALCAM expression was associated with an estrogen receptor-positive phenotype. In addition, strong ALCAM immunostaining correlated with nodal involvement and the presence of tumor cells in bone marrow. By Kaplan-Meier analysis, strong ALCAM expression in ductal carcinomas correlated with shorter recurrence-free intervals (P=0.048) and overall survival (OAS, P=0.003). Our results indicate that the biologic role of ALCAM in breast cancer is complex, but overexpression might be relevant for outcome in ductal carcinomas.

Loss of ALCAM expression is linked to adverse phenotype and poor prognosis in breast cancer: A TMA-based immunohistochemical study on 2,197 breast cancer patients

“…For example, Piao et al (18) found high membranous ALCAM expression to be statistically correlated with a worse clinical course and high cytoplasmic staining with local recurrence. hein et al (17) found high ALCAM expression linked to shorter overall survival compared to patients with low/moderate ALCAM scores, while ALCAM-negative tumors had an intermediate position. Burkhardt et al (15) reported that strong cytoplasmic ALCAM expression was significantly associated with earlier disease progression, while there was no correlation between membranous ALCAM expression and clinicopathological data.…”

“…Burkhardt et al (15), for example, found 75% ALCAM-expressing cancers among 160 analyzed cases. Ihnen et al (27) showed that 95% of their 162 breast cancers were ALCAM-positive, while hein et al (17) demonstrated 79% ALCAM-expressing breast cancers in their cohort comprising 347 cases. Another study analyzing 84 cases found 96% of the tested breast cancers to be ALCAM-positive (28).…”

“…Ihnen et al (27) also failed to find an association of ALCAM protein or mRNA expression with histological type, grading or stage in a series of 162 breast cancers. It may be that some of these contradictory data are due to the relatively small patient cohorts analyzed, ranging from 56 to 347 (14,15,17,18,21,27). The different methods used in those studies could be an additional source explaining these conflicting results.…”

“…The different methods used in those studies could be an additional source explaining these conflicting results. For example, some studies estimated levels of ALCAM using western blotting (26,27), while others performed real-time PCR (14) or IhC (15,17,(27)(28)(29).…”

“…Some studies revealed an increased ALCAM expression to be associated with poor prognosis (15,17) or lymph node metastasis and local recurrence (18), while others found a decreased ALCAM expression to be linked to a poorer clinical course (14,19,20). One explanation for these conflicting data potentially lies in the relatively small number of breast cancer cases included in those studies, ranging from 56 to 347 (14,15,17,21). The aim of the present study was to clarify the prevalence and prognostic role of ALCAM expression in breast cancer using a pre-existing large-scale tissue microarray (TMA) including more than 2,000 breast cancer samples.…”