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ALCAM regulates motility, invasiveness, and adherens junction formation in uveal melanoma cells.

Jannie KM, Stipp CS, Weiner JA - PLoS ONE (2012)

Bottom Line: Previous studies addressing ALCAM's role in cancer have, however, yielded conflicting results.Additionally, we stably overexpressed ALCAM in a low-ALCAM cell line (MUM-2C); intriguingly, these cells did not exhibit any increase in motility or invasiveness, indicating that ALCAM is necessary but not sufficient to promote metastasis-associated cell behaviors.In these ALCAM-overexpressing cells, however, recruitment of ß-catenin and N-cadherin to adherens junctions was enhanced.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, The University of Iowa, Iowa City, Iowa, United States of America.

ABSTRACT
ALCAM, a member of the immunoglobulin superfamily, has been implicated in numerous developmental events and has been repeatedly identified as a marker for cancer metastasis. Previous studies addressing ALCAM's role in cancer have, however, yielded conflicting results. Depending on the tumor cell type, ALCAM expression has been reported to be both positively and negatively correlated with cancer progression and metastasis in the literature. To better understand how ALCAM might regulate cancer cell behavior, we utilized a panel of defined uveal melanoma cell lines with high or low ALCAM levels, and directly tested the effects of manipulating these levels on cell motility, invasiveness, and adhesion using multiple assays. ALCAM expression was stably silenced by shRNA knockdown in a high-ALCAM cell line (MUM-2B); the resulting cells displayed reduced motility in gap-closure assays and a reduction in invasiveness as measured by a transwell migration assay. Immunostaining revealed that the silenced cells were defective in the formation of adherens junctions, at which ALCAM colocalizes with N-cadherin and ß-catenin in native cells. Additionally, we stably overexpressed ALCAM in a low-ALCAM cell line (MUM-2C); intriguingly, these cells did not exhibit any increase in motility or invasiveness, indicating that ALCAM is necessary but not sufficient to promote metastasis-associated cell behaviors. In these ALCAM-overexpressing cells, however, recruitment of ß-catenin and N-cadherin to adherens junctions was enhanced. These data confirm a previously suggested role for ALCAM in the regulation of adherens junctions, and also suggest a mechanism by which ALCAM might differentially enhance or decrease invasiveness, depending on the type of cadherin adhesion complexes present in tissues surrounding the primary tumor, and on the cadherin status of the tumor cells themselves.

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ALCAM-silenced cells display reduced MMP-2 activity.Levels of pro-MMP-2 were assayed in media conditioned by each cell line by gelatin zymography (A). Clear bands indicating pro-MMP-2 activity (which is activated by SDS; a faint “active” cleaved MMP2 band was present in some gels but often too weak for robust quantification) are present in MUM-2B, sh6, and the sh5rxd cell lines, but are reduced in the ALCAM-silenced sh5 cell line. MUM-2C and 2C-ALC display pro-MMP-2 levels that are lower than MUM-2B, and comparable to sh5 cells; the overexpression of ALCAM in 2C-ALC fails to increase pro-MMP-2 activity beyond that of MUM-2C. A minimum of three independent gelatin zymography trials (except 2C-ALC, 2 trials) are quantified in (B). (C) Western blots of cell lysates shows that the activation of pro-MMP-2 in the sh5 cell line is reduced (higher molecular weight band is pro-MMP-2; lower molecular weight band is active MMP-2) compared to MUM-2B, sh6, and the sh5rxd rescue cell line.
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pone-0039330-g005: ALCAM-silenced cells display reduced MMP-2 activity.Levels of pro-MMP-2 were assayed in media conditioned by each cell line by gelatin zymography (A). Clear bands indicating pro-MMP-2 activity (which is activated by SDS; a faint “active” cleaved MMP2 band was present in some gels but often too weak for robust quantification) are present in MUM-2B, sh6, and the sh5rxd cell lines, but are reduced in the ALCAM-silenced sh5 cell line. MUM-2C and 2C-ALC display pro-MMP-2 levels that are lower than MUM-2B, and comparable to sh5 cells; the overexpression of ALCAM in 2C-ALC fails to increase pro-MMP-2 activity beyond that of MUM-2C. A minimum of three independent gelatin zymography trials (except 2C-ALC, 2 trials) are quantified in (B). (C) Western blots of cell lysates shows that the activation of pro-MMP-2 in the sh5 cell line is reduced (higher molecular weight band is pro-MMP-2; lower molecular weight band is active MMP-2) compared to MUM-2B, sh6, and the sh5rxd rescue cell line.

Mentions: In gelatin zymography, active MMP-2 appears as a clear, Coomassie-negative band of ∼64 kDa upon staining of the gel; the “pro-MMP2” band of 72 kDa is also active in this assay, in the presence of SDS [40]. Because the pro-MMP2 band was much more prominent in our conditioned media samples, we quantified this band as a measure of MMP2 levels secreted by cells; in many gels, we could see a faint ∼64 kDa active band as well, which tracked levels of the clearer pro-MMP2 band (data not shown). Gelatin-clearing MMP-2 activity was strong in MUM-2B (Fig. 5A), C918, and M619 cells (data not shown), all of which highly express ALCAM (Fig. 1C), but not in the ALCAM-negative OCM-1A (data not shown) or MUM-2C (Fig. 5A). Next, we quantified MMP-2 activity in the stable cell lines, sh5, sh6, and 2C-ALC (Fig. 5A, B). We found that MMP-2 activation was reduced in sh5 by nearly 80% compared to parental MUM-2B, control sh6 cells, and sh5rxd rescued cells (Fig. 5B). As expected from our previous results with the 2C-ALC cell line (Fig. 4) MMP-2 activity was not increased in 2C-ALC compared to parental MUM-2C (Fig. 5A, B); again, this suggests that ALCAM is necessary, but not sufficient, for an invasive cell phenotype in uveal melanoma. Pro-MMP-2 was detectable in MUM-2B, sh5, sh6, and sh5rxd cell lysates by western blot, indicating that even sh5 expressed this enzyme (Fig. 5C). Consistent with the decreased invasive capacity in sh5, the active form of MMP-2 was just barely detectable in sh5, yet was clearly present in MUM-2B, sh6, and sh5rxd (Fig. 5C). It is possible that sh5 ALCAM-silenced cells exhibit defects in both MMP2 secretion and MMP2 activation, based on our combined results.


ALCAM regulates motility, invasiveness, and adherens junction formation in uveal melanoma cells.

Jannie KM, Stipp CS, Weiner JA - PLoS ONE (2012)

ALCAM-silenced cells display reduced MMP-2 activity.Levels of pro-MMP-2 were assayed in media conditioned by each cell line by gelatin zymography (A). Clear bands indicating pro-MMP-2 activity (which is activated by SDS; a faint “active” cleaved MMP2 band was present in some gels but often too weak for robust quantification) are present in MUM-2B, sh6, and the sh5rxd cell lines, but are reduced in the ALCAM-silenced sh5 cell line. MUM-2C and 2C-ALC display pro-MMP-2 levels that are lower than MUM-2B, and comparable to sh5 cells; the overexpression of ALCAM in 2C-ALC fails to increase pro-MMP-2 activity beyond that of MUM-2C. A minimum of three independent gelatin zymography trials (except 2C-ALC, 2 trials) are quantified in (B). (C) Western blots of cell lysates shows that the activation of pro-MMP-2 in the sh5 cell line is reduced (higher molecular weight band is pro-MMP-2; lower molecular weight band is active MMP-2) compared to MUM-2B, sh6, and the sh5rxd rescue cell line.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3383762&req=5

pone-0039330-g005: ALCAM-silenced cells display reduced MMP-2 activity.Levels of pro-MMP-2 were assayed in media conditioned by each cell line by gelatin zymography (A). Clear bands indicating pro-MMP-2 activity (which is activated by SDS; a faint “active” cleaved MMP2 band was present in some gels but often too weak for robust quantification) are present in MUM-2B, sh6, and the sh5rxd cell lines, but are reduced in the ALCAM-silenced sh5 cell line. MUM-2C and 2C-ALC display pro-MMP-2 levels that are lower than MUM-2B, and comparable to sh5 cells; the overexpression of ALCAM in 2C-ALC fails to increase pro-MMP-2 activity beyond that of MUM-2C. A minimum of three independent gelatin zymography trials (except 2C-ALC, 2 trials) are quantified in (B). (C) Western blots of cell lysates shows that the activation of pro-MMP-2 in the sh5 cell line is reduced (higher molecular weight band is pro-MMP-2; lower molecular weight band is active MMP-2) compared to MUM-2B, sh6, and the sh5rxd rescue cell line.
Mentions: In gelatin zymography, active MMP-2 appears as a clear, Coomassie-negative band of ∼64 kDa upon staining of the gel; the “pro-MMP2” band of 72 kDa is also active in this assay, in the presence of SDS [40]. Because the pro-MMP2 band was much more prominent in our conditioned media samples, we quantified this band as a measure of MMP2 levels secreted by cells; in many gels, we could see a faint ∼64 kDa active band as well, which tracked levels of the clearer pro-MMP2 band (data not shown). Gelatin-clearing MMP-2 activity was strong in MUM-2B (Fig. 5A), C918, and M619 cells (data not shown), all of which highly express ALCAM (Fig. 1C), but not in the ALCAM-negative OCM-1A (data not shown) or MUM-2C (Fig. 5A). Next, we quantified MMP-2 activity in the stable cell lines, sh5, sh6, and 2C-ALC (Fig. 5A, B). We found that MMP-2 activation was reduced in sh5 by nearly 80% compared to parental MUM-2B, control sh6 cells, and sh5rxd rescued cells (Fig. 5B). As expected from our previous results with the 2C-ALC cell line (Fig. 4) MMP-2 activity was not increased in 2C-ALC compared to parental MUM-2C (Fig. 5A, B); again, this suggests that ALCAM is necessary, but not sufficient, for an invasive cell phenotype in uveal melanoma. Pro-MMP-2 was detectable in MUM-2B, sh5, sh6, and sh5rxd cell lysates by western blot, indicating that even sh5 expressed this enzyme (Fig. 5C). Consistent with the decreased invasive capacity in sh5, the active form of MMP-2 was just barely detectable in sh5, yet was clearly present in MUM-2B, sh6, and sh5rxd (Fig. 5C). It is possible that sh5 ALCAM-silenced cells exhibit defects in both MMP2 secretion and MMP2 activation, based on our combined results.

Bottom Line: Previous studies addressing ALCAM's role in cancer have, however, yielded conflicting results.Additionally, we stably overexpressed ALCAM in a low-ALCAM cell line (MUM-2C); intriguingly, these cells did not exhibit any increase in motility or invasiveness, indicating that ALCAM is necessary but not sufficient to promote metastasis-associated cell behaviors.In these ALCAM-overexpressing cells, however, recruitment of ß-catenin and N-cadherin to adherens junctions was enhanced.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, The University of Iowa, Iowa City, Iowa, United States of America.

ABSTRACT
ALCAM, a member of the immunoglobulin superfamily, has been implicated in numerous developmental events and has been repeatedly identified as a marker for cancer metastasis. Previous studies addressing ALCAM's role in cancer have, however, yielded conflicting results. Depending on the tumor cell type, ALCAM expression has been reported to be both positively and negatively correlated with cancer progression and metastasis in the literature. To better understand how ALCAM might regulate cancer cell behavior, we utilized a panel of defined uveal melanoma cell lines with high or low ALCAM levels, and directly tested the effects of manipulating these levels on cell motility, invasiveness, and adhesion using multiple assays. ALCAM expression was stably silenced by shRNA knockdown in a high-ALCAM cell line (MUM-2B); the resulting cells displayed reduced motility in gap-closure assays and a reduction in invasiveness as measured by a transwell migration assay. Immunostaining revealed that the silenced cells were defective in the formation of adherens junctions, at which ALCAM colocalizes with N-cadherin and ß-catenin in native cells. Additionally, we stably overexpressed ALCAM in a low-ALCAM cell line (MUM-2C); intriguingly, these cells did not exhibit any increase in motility or invasiveness, indicating that ALCAM is necessary but not sufficient to promote metastasis-associated cell behaviors. In these ALCAM-overexpressing cells, however, recruitment of ß-catenin and N-cadherin to adherens junctions was enhanced. These data confirm a previously suggested role for ALCAM in the regulation of adherens junctions, and also suggest a mechanism by which ALCAM might differentially enhance or decrease invasiveness, depending on the type of cadherin adhesion complexes present in tissues surrounding the primary tumor, and on the cadherin status of the tumor cells themselves.

Show MeSH
Related in: MedlinePlus