214

ALCAM

activated leukocyte cell adhesion molecule;ALCAM;activated leukocyte cell adhesion molecule

CD166 antigen

3q13.1

protein-coding

The activated leukocyte cell adhesion molecule (ALCAM) is involved in cell migration and adhesion. Decreased levels of ALCAM expression in breast cancer tissue are known to correlate with poor prognosis. The current study specifically investigated the ALCAM expression in tumours which developed skeletal metastasis. Fresh frozen primary breast cancer tissues (n=234) and non-neoplastic mammary tissue (n=34) were used. The distribution and location of ALCAM was assessed using immunohistochemical methods and the level of ALCAM was determined using quantitative RT-PCR. The results were analysed against the clinical and pathological data. ALCAM staining was largely membranous and cytoplasmic in normal epithelial cells and is significantly stronger than in cancer cells (p=0.023) and patients who develop skeletal metastasis (p=0.048). The ALCAM transcript levels were lowest in patients with skeletal metastasis (p=0.0048) but were also significantly lower in patients who developed local recurrence (p=0.040) and in those who died from breast cancer (p=0.0075). Patients with moderate and poor prognostic indices have a lower level than those with a good index (p=0.05 and p=0.0089 respectively) and ER-positive tumours show a lower level than ER-negative (p=0.043). Ductal carcinomas, 86% of the cohort, have a similar pattern of changes with skeletal metastasis patients having significantly lower levels (p=0.015). This study has, for the first time, shown that patients who develop skeletal metastasis tend to have the lowest levels of ALCAM transcripts in their breast cancers, a finding potentially useful for clinical practice.

BACKGROUND: Activated leukocyte cell adhesion molecule (ALCAM) is implicated in the prognosis of multiple cancers with low level expression associated with metastasis and early death in breast cancer. Despite this significance, mechanisms that regulate ALCAM gene expression and ALCAM's role in adhesion of pre-metastatic circulating tumor cells have not been defined. We studied ALCAM expression in 20 tumor cell lines by real-time PCR, western blot and immunochemistry. Epigenetic alterations of the ALCAM promoter were assessed using methylation-specific PCR and bisulfite sequencing. ALCAM's role in adhesion of tumor cells to the vascular wall was studied in isolated perfused lungs. RESULTS: A common site for transcription initiation of the ALCAM gene was identified and the ALCAM promoter sequenced. The promoter contains multiple cis-active elements including a functional p65 NF-kappaB motif, and it harbors an extensive array of CpG residues highly methylated exclusively in ALCAM-negative tumor cells. These CpG residues were modestly demethylated after 5-aza-2-deoxycytidine treatment. Restoration of high-level ALCAM expression using an ALCAM cDNA increased clustering of MDA-MB-435 tumor cells perfused through the pulmonary vasculature of ventilated rat lungs. Anti-ALCAM antibodies reduced the number of intravascular tumor cell clusters. CONCLUSION: Our data suggests that loss of ALCAM expression, due in part to DNA methylation of extensive segments of the promoter, significantly impairs the ability of circulating tumor cells to adhere to each other, and may therefore promote metastasis. These findings offer insight into the mechanisms for down-regulation of ALCAM gene expression in tumor cells, and for the positive prognostic value of high-level ALCAM in breast cancer.

In the study, we enrolled 150 breast cancer cases to investigate the expression status of activated leukocyte cell adhesion molecule (ALCAM), and the relationships between ALCAM expression and clinical-pathological characteristics and prognosis of breast cancer. It was observed that ALCAM was expressed at higher levels in breast cancer tissue compared to levels observed for tumor-adjacent tissue. Compared to cancers with low membranous ALCAM expression, cancers with high membranous ALCAM expression were prone to lymph node metastasis (chi2=15.910, P=0.010) and metastasis in general (chi2=5.211, P=0.029). High cytoplasmic ALCAM expression was noticeably correlated with local recurrence (chi2=7.379, P=0.012), especially for short-term recurrence (interval<2 years) (chi2=5.562, P=0.037), while not associated to long-term local recurrence (interval>2 years). The content of ALCAM protein is closely associated with the expression of estrogen receptor (ER) (P=0.024). The disease-free survival of patients with high cytoplasmic ALCAM expression was significantly shorter compared to the cases with low cytoplasmic ALCAM expression (P=0.036). In conclusion, ALCAM expressed at high levels in breast cancer. High membranous expression of ALCAM probably resulted in weakened adherent ability and metastasis. In addition, high cytoplasmic ALCAM expression strengthened invasive ability of malignant cells and then promoted tumor development.

OBJECTIVE: The MEMD gene was reported to be overexpressed in human esophageal squamous cell carcinoma (ESCC), using differential display. The aim of this study was to determine the clinical significance of MEMD/ALCAM in esophageal tumorigenesis. METHODS: Analysis of MEMD/ALCAM expression in esophageal tissues was carried out at protein and RNA level using immunohistochemistry and semiquantitative RT-PCR, respectively. RESULTS: Increased MEMD/ALCAM expression was observed in 42/65 (65%) ESCCs (p = 0.000, odds ratio, OR = 3.665) and in 17/25 (68%) dysplasias (p = 0.000, OR = 4.248) compared to paired distant histologically normal esophageal tissues. Increased MEMD mRNAlevels were observed in ESCCs and dysplasias showing overexpression of MEMD/ALCAM protein. Interestingly, increased membranous MEMD/ALCAM expression was observed in dysplasias in comparison with ESCCs (p = 0.002, OR = 3.177). MEMD/ALCAM overexpression in ESCCs was associated with late clinical stage (p = 0.002, OR = 3.619), enhanced tumor invasiveness (p = 0.002, OR = 3.619), and nodal metastasis (p = 0.000, OR = 4.206). CONCLUSION: To our knowledge, this is the first report showing MEMD expression at pre-malignant stage (dysplasia), suggesting that MEMD/ALCAM expression is an early event in the development of esophageal cancer. Furthermore, in ESCCs its correlation with late clinical stage, enhanced tumor invasiveness and nodal metastasis suggests an association with aggressive tumor behavior. Our data suggest that MEMD/ALCAM may serve as a potential marker for early diagnosis, tumor invasion and nodal metastasis in ESCCs.

BACKGROUND: Cellular adhesion molecules (CAMs), which are normally associated with leukocyte trafficking, have also been shown to play an essential role in tumor metastasis to non-CNS sites. However, the role played by CAMs in brain metastasis is largely unexplored. It is known that leukocyte recruitment to the brain is very atypical and that mechanisms of disease in peripheral tissues cannot be extrapolated to the brain. Here, we have established the spatiotemporal expression of 12 key CAMs in the initial phases of tumor seeding in 2 different models of brain metastasis. METHODS: BALB/c or SCID mice were injected intracardially (10(5) cells/100 muL phosphate-buffered saline with either 4T1-GFP or MDA231BR-GFP cells, respectively (n = 4-6/group), and expression of the CAMs was determined by immunohistochemistry and immunofluorescence colocalisation. RESULTS: Endothelial expression of E-selectin, VCAM-1, ALCAM, ICAM-1, VLA-4, and beta4 integrin was markedly increased early in tumor seeding. At the same time, the natural ligands to these adhesion molecules were highly expressed on the metastatic tumor cells both in vitro and in vivo. Two of these ligands showed particularly high tumor cell expression (ALCAM and VLA-4), and consequently their functional role in tumor seeding was determined. Antibody neutralization of either ALCAM or VLA-4 significantly reduced tumor seeding within the brain (>60% decrease in tumor number/mm(2) brain; P < .05-0.01). CONCLUSIONS: These findings suggest that ALCAM/ALCAM and VLA-4/VCAM-1 interactions play an important functional role in the early stages of metastasis seeding in the brain. Moreover, this work identifies a specific subset of ligand-receptor interactions that may yield new therapeutic and diagnostic targets for brain metastasis.