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

King JA, Tan F, Mbeunkui F, Chambers Z, Cantrell S, Chen H, Alvarez D, Shevde LA, Ofori-Acquah SF - Mol. Cancer (2010)

Bottom Line: A common site for transcription initiation of the ALCAM gene was identified and the ALCAM promoter sequenced.These CpG residues were modestly demethylated after 5-aza-2-deoxycytidine treatment.Anti-ALCAM antibodies reduced the number of intravascular tumor cell clusters.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Lung Biology, University of South Alabama, Mobile, AL 36688, USA.

ABSTRACT

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-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.

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.

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ALCAM clusters tumor cells circulating through the lung. A) Ectopic expression of ALCAM in MDA-MB-435 tumor cells. Left panel; Western blot analysis of ALCAM in MDA-MB-435 clones transfected with an empty vector (lane 1) or, an ALCAM-GFP vector (lane 2). Protein loading was verified by probing the same western blot filter for the house keeping protein EF1-α. Right panel; Monolayer of MDA-MB-435-ALCAM-GFP cells were examined by live-cell microscopy to reveal expression of ALCAM at sites of cell-cell contact. B) Medium power image show virtual absence of control MDA-MB-435 cells, except for a single cell (arrow) while the ALCAM-expressing MDA-MB-435 clones form clusters (brown stain) after 90 minute perfusion in rat lungs. C) Number of tumor cells retained in rat lungs in experiments using ALCAM-positive (n = 7), or ALCAM-negative MDA-MB-435 cells (n = 4) pre-treated with non-immune IgG or monoclonal anti-ALCAM antibody. D) Number of tumor cell clusters retained in rat lungs in experiments using ALCAM-positive and ALCAM-negative MDA-MB-435 cells pre-treated with non-immune IgG (n = 7) or monoclonal anti-ALCAM antibody (n = 7). E) Number of single tumor cells retained in rat lungs in experiments using ALCAM-positive and ALCAM-negative MDA-MB-435 cells pre-treated with non-immune IgG (n = 7) or monoclonal anti-ALCAM antibody (n = 7).
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Figure 7: ALCAM clusters tumor cells circulating through the lung. A) Ectopic expression of ALCAM in MDA-MB-435 tumor cells. Left panel; Western blot analysis of ALCAM in MDA-MB-435 clones transfected with an empty vector (lane 1) or, an ALCAM-GFP vector (lane 2). Protein loading was verified by probing the same western blot filter for the house keeping protein EF1-α. Right panel; Monolayer of MDA-MB-435-ALCAM-GFP cells were examined by live-cell microscopy to reveal expression of ALCAM at sites of cell-cell contact. B) Medium power image show virtual absence of control MDA-MB-435 cells, except for a single cell (arrow) while the ALCAM-expressing MDA-MB-435 clones form clusters (brown stain) after 90 minute perfusion in rat lungs. C) Number of tumor cells retained in rat lungs in experiments using ALCAM-positive (n = 7), or ALCAM-negative MDA-MB-435 cells (n = 4) pre-treated with non-immune IgG or monoclonal anti-ALCAM antibody. D) Number of tumor cell clusters retained in rat lungs in experiments using ALCAM-positive and ALCAM-negative MDA-MB-435 cells pre-treated with non-immune IgG (n = 7) or monoclonal anti-ALCAM antibody (n = 7). E) Number of single tumor cells retained in rat lungs in experiments using ALCAM-positive and ALCAM-negative MDA-MB-435 cells pre-treated with non-immune IgG (n = 7) or monoclonal anti-ALCAM antibody (n = 7).

Mentions: The isolated ventilated perfused rat lung system has previously been used to investigate the fate of circulating tumor cells, and was employed here to test the influence ALCAM has on these cells. Stable transfection of an ALCAM-GFP construct conferred high level ALCAM expression in MDA-MB-435 cells, and importantly localized ALCAM to sites of cell-cell contact in confluent cultures (Fig. 7A). Perfusion of this clone into rat lungs caused congestion of tumor cells in the pulmonary vasculature, while relatively few cells were retained in experiments using a control clone expressing an empty vector (Fig. 7B). Quantitative analysis revealed more than 2-fold increase in the number of the ALCAM-positive MDA-MB-435 cells retained in the rat lung (Fig. 7C), compared to the ALCAM-negative variant. Pre-treating ALCAM-positive MDA-MB-435 cells with anti-ALCAM antibodies prior to perfusion significantly reduced the number of tumor cells in the rat lung (Fig. 7C). Additional control experiments showed that anti-ALCAM antibodies did not alter the total number of ALCAM-negative MDA-MB-435 tumor cells retained in the rat lung.


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

King JA, Tan F, Mbeunkui F, Chambers Z, Cantrell S, Chen H, Alvarez D, Shevde LA, Ofori-Acquah SF - Mol. Cancer (2010)

ALCAM clusters tumor cells circulating through the lung. A) Ectopic expression of ALCAM in MDA-MB-435 tumor cells. Left panel; Western blot analysis of ALCAM in MDA-MB-435 clones transfected with an empty vector (lane 1) or, an ALCAM-GFP vector (lane 2). Protein loading was verified by probing the same western blot filter for the house keeping protein EF1-α. Right panel; Monolayer of MDA-MB-435-ALCAM-GFP cells were examined by live-cell microscopy to reveal expression of ALCAM at sites of cell-cell contact. B) Medium power image show virtual absence of control MDA-MB-435 cells, except for a single cell (arrow) while the ALCAM-expressing MDA-MB-435 clones form clusters (brown stain) after 90 minute perfusion in rat lungs. C) Number of tumor cells retained in rat lungs in experiments using ALCAM-positive (n = 7), or ALCAM-negative MDA-MB-435 cells (n = 4) pre-treated with non-immune IgG or monoclonal anti-ALCAM antibody. D) Number of tumor cell clusters retained in rat lungs in experiments using ALCAM-positive and ALCAM-negative MDA-MB-435 cells pre-treated with non-immune IgG (n = 7) or monoclonal anti-ALCAM antibody (n = 7). E) Number of single tumor cells retained in rat lungs in experiments using ALCAM-positive and ALCAM-negative MDA-MB-435 cells pre-treated with non-immune IgG (n = 7) or monoclonal anti-ALCAM antibody (n = 7).
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Related In: Results  -  Collection

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Figure 7: ALCAM clusters tumor cells circulating through the lung. A) Ectopic expression of ALCAM in MDA-MB-435 tumor cells. Left panel; Western blot analysis of ALCAM in MDA-MB-435 clones transfected with an empty vector (lane 1) or, an ALCAM-GFP vector (lane 2). Protein loading was verified by probing the same western blot filter for the house keeping protein EF1-α. Right panel; Monolayer of MDA-MB-435-ALCAM-GFP cells were examined by live-cell microscopy to reveal expression of ALCAM at sites of cell-cell contact. B) Medium power image show virtual absence of control MDA-MB-435 cells, except for a single cell (arrow) while the ALCAM-expressing MDA-MB-435 clones form clusters (brown stain) after 90 minute perfusion in rat lungs. C) Number of tumor cells retained in rat lungs in experiments using ALCAM-positive (n = 7), or ALCAM-negative MDA-MB-435 cells (n = 4) pre-treated with non-immune IgG or monoclonal anti-ALCAM antibody. D) Number of tumor cell clusters retained in rat lungs in experiments using ALCAM-positive and ALCAM-negative MDA-MB-435 cells pre-treated with non-immune IgG (n = 7) or monoclonal anti-ALCAM antibody (n = 7). E) Number of single tumor cells retained in rat lungs in experiments using ALCAM-positive and ALCAM-negative MDA-MB-435 cells pre-treated with non-immune IgG (n = 7) or monoclonal anti-ALCAM antibody (n = 7).
Mentions: The isolated ventilated perfused rat lung system has previously been used to investigate the fate of circulating tumor cells, and was employed here to test the influence ALCAM has on these cells. Stable transfection of an ALCAM-GFP construct conferred high level ALCAM expression in MDA-MB-435 cells, and importantly localized ALCAM to sites of cell-cell contact in confluent cultures (Fig. 7A). Perfusion of this clone into rat lungs caused congestion of tumor cells in the pulmonary vasculature, while relatively few cells were retained in experiments using a control clone expressing an empty vector (Fig. 7B). Quantitative analysis revealed more than 2-fold increase in the number of the ALCAM-positive MDA-MB-435 cells retained in the rat lung (Fig. 7C), compared to the ALCAM-negative variant. Pre-treating ALCAM-positive MDA-MB-435 cells with anti-ALCAM antibodies prior to perfusion significantly reduced the number of tumor cells in the rat lung (Fig. 7C). Additional control experiments showed that anti-ALCAM antibodies did not alter the total number of ALCAM-negative MDA-MB-435 tumor cells retained in the rat lung.

Bottom Line: A common site for transcription initiation of the ALCAM gene was identified and the ALCAM promoter sequenced.These CpG residues were modestly demethylated after 5-aza-2-deoxycytidine treatment.Anti-ALCAM antibodies reduced the number of intravascular tumor cell clusters.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Lung Biology, University of South Alabama, Mobile, AL 36688, USA.

ABSTRACT

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-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.

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.

Show MeSH
Related in: MedlinePlus