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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 expression in tumor cells. A) Quantification of ALCAM mRNA by qRT-PCR. Data shown for each cell line is the mean of three analyses each in triplicate. B) Western blot analysis of ALCAM protein in the indicated cells lines. Whole cell lysates (20 μg) were probed with anti-ALCAM antibody, and the filter stripped and re-probed for α-tubulin. C) Immunocytochemical analysis for ALCAM expression and sub-cellular localization in breast cancer cell lines HCC1806 and HCC1500. Cells were stained for ALCAM (red) and nucleus (blue) using the DAPI reagent.
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Figure 1: ALCAM expression in tumor cells. A) Quantification of ALCAM mRNA by qRT-PCR. Data shown for each cell line is the mean of three analyses each in triplicate. B) Western blot analysis of ALCAM protein in the indicated cells lines. Whole cell lysates (20 μg) were probed with anti-ALCAM antibody, and the filter stripped and re-probed for α-tubulin. C) Immunocytochemical analysis for ALCAM expression and sub-cellular localization in breast cancer cell lines HCC1806 and HCC1500. Cells were stained for ALCAM (red) and nucleus (blue) using the DAPI reagent.

Mentions: ALCAM mRNA is significantly reduced in primary breast tumors from patients with metastatic disease however the amount of ALCAM in breast cancer cells at metastatic sites remains poorly understood. In this study, ALCAM mRNA in sixteen breast cancer cell lines derived from metastatic breast cancer tumors in the brain, lymph node and the pleural cavity, and primary breast tumors in ductal epithelium were quantified by qRT-PCR. Most cell lines derived from pleural effusions (MB-157, MDA-MB-435, HCC1428, MDA-MB-453, MCF-7, MDA-MB-231 and SK-BR-3) expressed relatively low levels of ALCAM mRNA, while cells originating from the lymph node (HCC70, HCC1008 and BT549) expressed relatively high amounts of ALCAM mRNA (Fig. 1A). ALCAM mRNA was virtually not detectable in MDA-MB-435. Regarding melanoma, ALCAM mRNA was markedly elevated in most of the cell lines (LOX, C8161.9, MelJuso) in agreement with the increased expression in primary tumors (Fig. 1A). Figure 1B shows that ALCAM protein levels determined by western blot analysis showed good correlation with ALCAM mRNA in most tumor cells. Most notably we did not detect ALCAM protein in MDA-MB-435 and FEMX-I tumor cells (Fig. 1B). ALCAM was generally expressed at cell-cell contacts of confluent tumor cell cultures although cytoplasmic localization was also detected (Fig. 1C). These data indicate that ALCAM is variably expressed in breast cancer cell lines, with the lowest level of expression, predominantly in cells derived from distant metastatic sites.


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 expression in tumor cells. A) Quantification of ALCAM mRNA by qRT-PCR. Data shown for each cell line is the mean of three analyses each in triplicate. B) Western blot analysis of ALCAM protein in the indicated cells lines. Whole cell lysates (20 μg) were probed with anti-ALCAM antibody, and the filter stripped and re-probed for α-tubulin. C) Immunocytochemical analysis for ALCAM expression and sub-cellular localization in breast cancer cell lines HCC1806 and HCC1500. Cells were stained for ALCAM (red) and nucleus (blue) using the DAPI reagent.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 1: ALCAM expression in tumor cells. A) Quantification of ALCAM mRNA by qRT-PCR. Data shown for each cell line is the mean of three analyses each in triplicate. B) Western blot analysis of ALCAM protein in the indicated cells lines. Whole cell lysates (20 μg) were probed with anti-ALCAM antibody, and the filter stripped and re-probed for α-tubulin. C) Immunocytochemical analysis for ALCAM expression and sub-cellular localization in breast cancer cell lines HCC1806 and HCC1500. Cells were stained for ALCAM (red) and nucleus (blue) using the DAPI reagent.
Mentions: ALCAM mRNA is significantly reduced in primary breast tumors from patients with metastatic disease however the amount of ALCAM in breast cancer cells at metastatic sites remains poorly understood. In this study, ALCAM mRNA in sixteen breast cancer cell lines derived from metastatic breast cancer tumors in the brain, lymph node and the pleural cavity, and primary breast tumors in ductal epithelium were quantified by qRT-PCR. Most cell lines derived from pleural effusions (MB-157, MDA-MB-435, HCC1428, MDA-MB-453, MCF-7, MDA-MB-231 and SK-BR-3) expressed relatively low levels of ALCAM mRNA, while cells originating from the lymph node (HCC70, HCC1008 and BT549) expressed relatively high amounts of ALCAM mRNA (Fig. 1A). ALCAM mRNA was virtually not detectable in MDA-MB-435. Regarding melanoma, ALCAM mRNA was markedly elevated in most of the cell lines (LOX, C8161.9, MelJuso) in agreement with the increased expression in primary tumors (Fig. 1A). Figure 1B shows that ALCAM protein levels determined by western blot analysis showed good correlation with ALCAM mRNA in most tumor cells. Most notably we did not detect ALCAM protein in MDA-MB-435 and FEMX-I tumor cells (Fig. 1B). ALCAM was generally expressed at cell-cell contacts of confluent tumor cell cultures although cytoplasmic localization was also detected (Fig. 1C). These data indicate that ALCAM is variably expressed in breast cancer cell lines, with the lowest level of expression, predominantly in cells derived from distant metastatic sites.

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