Mechanisms of transcriptional regulation and prognostic significance of activated leukocyte cell adhesion molecule in cancer

Abstract

BackgroundActivated 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.ResultsA 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-κB 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.ConclusionOur 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.

Human T-Lymphotropic Virus Type 1-Induced Overexpression of Activated Leukocyte Cell Adhesion Molecule (ALCAM) Facilitates Trafficking of Infected Lymphocytes through the Blood-Brain Barrier

“…alcam upregulation by Tax was previously suggested in microarray studies (24), although the role of the NF-B pathway was not addressed. Tax directly recruits the canonical NF-B complex to the promoter: it was previously shown that the p65 NF-B subunit is able to bind to two functional B sites, located Ϫ962 and Ϫ754 nucleotides upstream of the initiation codon, on the human alcam promoter (34,35). We confirmed the functional role of the distal B site here.…”

“…Previous studies have reported the presence of two potential B sites in this region, located between Ϫ973 and Ϫ962 (B 1), and Ϫ765 and Ϫ754 (B 2) nucleotides upstream of the initiation codon, on the human alcam promoter (34,35). We investigated which site on the alcam promoter participates in Tax-induced promoter activation.…”

miR-214 Coordinates Melanoma Progression by Upregulating ALCAM through TFAP2 and miR-148b Downmodulation

“…ALCAM connections are essential for cell movement (9), for efficient conversion of pro-MMP-2 to its active form in metastatic melanoma cells (11) and for the retention of intravascular breast tumor cell clusters in the lung vessels (38). Consistently, we showed that ALCAM overexpression promotes cell migration, whereas silencing blocks it in different melanoma cell lines.…”

“…It is known that ALCAM expression is regulated by promoter CpG islands methylation (38) and by transcriptional regulation exerted by NF-kB (38) and GATA1 (39) transcription factors. Here, we propose that miR-214 regulates ALCAM indirectly via a transcriptional and a posttranscriptional control.…”

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

“…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).…”