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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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Expression of ALCAM in MUM-2C cells does not enhance wound-gap closure speed or invasive capacity.Immunostaining of MUM-2C cells (A) reveals that ALCAM expression is virtually undetectable in these cells. DAPI-stained nuclei are shown in blue; ALCAM staining is shown in red. In the 2C-ALC cell line (B), engineered to stably overexpress ALCAM, DAPI-stained nuclei are shown in blue, and ALCAM staining is shown in red. ALCAM localizes to points of contact between cells in the 2C-ALC cell line. The expression of ALCAM in 2C-ALC is confirmed by western blot in panel (C), and is undetectable in MUM-2C. Tubulin is shown as a loading control. The expression of ALCAM in 2C-ALC did not alter closure rate in a wound-gap assay (D), nor did it enhance invasive capacity of 2C-ALC cells when compared with MUM-2C in a transwell migration assay (t-test; p>0.05; the average number of invasive MUM-2C cells per three 10× fields was 16; E). Growth and survival of MUM-2C and 2C-ALC is similar, as assayed and described in Fig. 3 (F).
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pone-0039330-g004: Expression of ALCAM in MUM-2C cells does not enhance wound-gap closure speed or invasive capacity.Immunostaining of MUM-2C cells (A) reveals that ALCAM expression is virtually undetectable in these cells. DAPI-stained nuclei are shown in blue; ALCAM staining is shown in red. In the 2C-ALC cell line (B), engineered to stably overexpress ALCAM, DAPI-stained nuclei are shown in blue, and ALCAM staining is shown in red. ALCAM localizes to points of contact between cells in the 2C-ALC cell line. The expression of ALCAM in 2C-ALC is confirmed by western blot in panel (C), and is undetectable in MUM-2C. Tubulin is shown as a loading control. The expression of ALCAM in 2C-ALC did not alter closure rate in a wound-gap assay (D), nor did it enhance invasive capacity of 2C-ALC cells when compared with MUM-2C in a transwell migration assay (t-test; p>0.05; the average number of invasive MUM-2C cells per three 10× fields was 16; E). Growth and survival of MUM-2C and 2C-ALC is similar, as assayed and described in Fig. 3 (F).

Mentions: If ALCAM expression is necessary for motility and invasiveness in the MUM-2B uveal melanoma cell line, is ALCAM expression sufficient to increase motility and confer invasiveness in the normally ALCAM-negative MUM-2C line? To test this, we created a stable cell line, termed 2C-ALC, by transducing MUM-2C with a virus encoding full-length ALCAM. Expression of the full-length ALCAM construct was confirmed by both western blot (Fig. 4C) and immunohistochemistry (Fig. 4A, B). Expression level of ALCAM in 2C-ALC was roughly comparable to that of MUM-2B. As expected, ALCAM localized to cell-cell contacts in 2C-ALC cells (Fig. 4B). Overexpression of ALCAM in the 2C-ALC cell line, however, failed to enhance the velocity of cells in the gap closure assay (Fig. 4D). 2C-ALC cells still often moved as individual cells (similar to native 2C cells; Fig. 1F), and not as a cohesive sheet like MUM-2B cells (data not shown). Overexpression of ALCAM was also not sufficient to enhance the invasive capacity of 2C-ALC cells (Fig. 4E), nor did it affect the survival or proliferation of the cell line (Fig. 4F).


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

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

Expression of ALCAM in MUM-2C cells does not enhance wound-gap closure speed or invasive capacity.Immunostaining of MUM-2C cells (A) reveals that ALCAM expression is virtually undetectable in these cells. DAPI-stained nuclei are shown in blue; ALCAM staining is shown in red. In the 2C-ALC cell line (B), engineered to stably overexpress ALCAM, DAPI-stained nuclei are shown in blue, and ALCAM staining is shown in red. ALCAM localizes to points of contact between cells in the 2C-ALC cell line. The expression of ALCAM in 2C-ALC is confirmed by western blot in panel (C), and is undetectable in MUM-2C. Tubulin is shown as a loading control. The expression of ALCAM in 2C-ALC did not alter closure rate in a wound-gap assay (D), nor did it enhance invasive capacity of 2C-ALC cells when compared with MUM-2C in a transwell migration assay (t-test; p>0.05; the average number of invasive MUM-2C cells per three 10× fields was 16; E). Growth and survival of MUM-2C and 2C-ALC is similar, as assayed and described in Fig. 3 (F).
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Related In: Results  -  Collection

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pone-0039330-g004: Expression of ALCAM in MUM-2C cells does not enhance wound-gap closure speed or invasive capacity.Immunostaining of MUM-2C cells (A) reveals that ALCAM expression is virtually undetectable in these cells. DAPI-stained nuclei are shown in blue; ALCAM staining is shown in red. In the 2C-ALC cell line (B), engineered to stably overexpress ALCAM, DAPI-stained nuclei are shown in blue, and ALCAM staining is shown in red. ALCAM localizes to points of contact between cells in the 2C-ALC cell line. The expression of ALCAM in 2C-ALC is confirmed by western blot in panel (C), and is undetectable in MUM-2C. Tubulin is shown as a loading control. The expression of ALCAM in 2C-ALC did not alter closure rate in a wound-gap assay (D), nor did it enhance invasive capacity of 2C-ALC cells when compared with MUM-2C in a transwell migration assay (t-test; p>0.05; the average number of invasive MUM-2C cells per three 10× fields was 16; E). Growth and survival of MUM-2C and 2C-ALC is similar, as assayed and described in Fig. 3 (F).
Mentions: If ALCAM expression is necessary for motility and invasiveness in the MUM-2B uveal melanoma cell line, is ALCAM expression sufficient to increase motility and confer invasiveness in the normally ALCAM-negative MUM-2C line? To test this, we created a stable cell line, termed 2C-ALC, by transducing MUM-2C with a virus encoding full-length ALCAM. Expression of the full-length ALCAM construct was confirmed by both western blot (Fig. 4C) and immunohistochemistry (Fig. 4A, B). Expression level of ALCAM in 2C-ALC was roughly comparable to that of MUM-2B. As expected, ALCAM localized to cell-cell contacts in 2C-ALC cells (Fig. 4B). Overexpression of ALCAM in the 2C-ALC cell line, however, failed to enhance the velocity of cells in the gap closure assay (Fig. 4D). 2C-ALC cells still often moved as individual cells (similar to native 2C cells; Fig. 1F), and not as a cohesive sheet like MUM-2B cells (data not shown). Overexpression of ALCAM was also not sufficient to enhance the invasive capacity of 2C-ALC cells (Fig. 4E), nor did it affect the survival or proliferation of the cell line (Fig. 4F).

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