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Lectin RCA-I specifically binds to metastasis-associated cell surface glycans in triple-negative breast cancer.

Zhou SM, Cheng L, Guo SJ, Wang Y, Czajkowsky DM, Gao H, Hu XF, Tao SC - Breast Cancer Res. (2015)

Bottom Line: Using the lectin microarray, we found that the bindings of RCA-I to TNBC cells are proportional to their metastatic capacity.Additionally, a membrane glycoprotein, POTE ankyrin domain family member F (POTEF), with different galactosylation extents in high/low metastatic TNBC cells was identified by LC-MS/MS as a binder of RCA-I.We discovered RCA-I, which bound to TNBC cells to a degree that is proportional to their metastatic capacities, and found that this binding inhibits the cell invasion, migration, and adhesion, and identified a membrane protein, POTEF, which may play a key role in mediating these effects.

View Article: PubMed Central - PubMed

ABSTRACT

Introduction: Triple-negative breast cancer (TNBC) patients often face a high risk of early relapse characterized by extensive metastasis. Previous works have shown that aberrant cell surface glycosylation is associated with cancer metastasis, suggesting that altered glycosylations might serve as diagnostic signatures of metastatic potential. To address this question, we took TNBC as an example and analyzed six TNBC cell lines, derived from a common progenitor, that differ in metastatic potential.

Methods: We used a microarray with 91 lectins to screen for altered lectin bindings to the six TNBC cell lines. Candidate lectins were then verified by lectin-based flow cytometry and immunofluorescent staining assays using both TNBC/non-TNBC cancer cells. Patient-derived tissue microarrays were then employed to analyze whether the staining of Ricinus communis agglutinin I (RCA-I), correlated with TNBC severity. We also carried out real-time cell motility assays in the presence of RCA-I. Finally, liquid chromatography-mass spectrometry/tandem spectrometry (LC-MS/MS) was employed to identify the membrane glycoproteins recognized by RCA-I.

Results: Using the lectin microarray, we found that the bindings of RCA-I to TNBC cells are proportional to their metastatic capacity. Tissue microarray experiments showed that the intensity of RCA-I staining is positively correlated with the TNM grades. The real-time cell motility assays clearly demonstrated RCA-I inhibition of adhesion, migration, and invasion of TNBC cells of high metastatic capacity. Additionally, a membrane glycoprotein, POTE ankyrin domain family member F (POTEF), with different galactosylation extents in high/low metastatic TNBC cells was identified by LC-MS/MS as a binder of RCA-I.

Conclusions: We discovered RCA-I, which bound to TNBC cells to a degree that is proportional to their metastatic capacities, and found that this binding inhibits the cell invasion, migration, and adhesion, and identified a membrane protein, POTEF, which may play a key role in mediating these effects. These results thus indicate that RCA-I-specific cell surface glycoproteins may play a critical role in TNBC metastasis and that the extent of RCA-I cell binding could be used in diagnosis to predict the likelihood of developing metastases in TNBC patients.

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The RCA-I-binding tendency is constant with the invasion ability of TNBC cells rather than those of non-TNBC cells. (a) FCM analysis confirms the proportional, inversely proportional, and uncorrelated binding to the metastatic capacity of these cells with RCA-I, WGA, and WFA, respectively. (b) Incubation of biotinylated RCA-I, WFA, or WGA, followed by the addition of Cy3-conjugated streptavidin and direct inspection with microscopy further confirmed the cell-binding tendencies of these lectins. (c) The RCA-I-binding positive rates of five TNBC (red) and two non-TNBC cells (green) were analyzed by FCM, which were referred to their relative metastatic abilities. (d) The immunofluorescent staining of RCA-I binding to another two TNBC cells (SUM159 and BT549) and two non-TNBC cells (MCF7 and SKBR3). FCM, flow cytometry; RCA-I, Ricinus communis agglutinin I; TNBC, triple-negative breast cancer; WFA, Wisteria floribunda agglutinin; WGA, Triticum vulgaris agglutinin.
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Fig2: The RCA-I-binding tendency is constant with the invasion ability of TNBC cells rather than those of non-TNBC cells. (a) FCM analysis confirms the proportional, inversely proportional, and uncorrelated binding to the metastatic capacity of these cells with RCA-I, WGA, and WFA, respectively. (b) Incubation of biotinylated RCA-I, WFA, or WGA, followed by the addition of Cy3-conjugated streptavidin and direct inspection with microscopy further confirmed the cell-binding tendencies of these lectins. (c) The RCA-I-binding positive rates of five TNBC (red) and two non-TNBC cells (green) were analyzed by FCM, which were referred to their relative metastatic abilities. (d) The immunofluorescent staining of RCA-I binding to another two TNBC cells (SUM159 and BT549) and two non-TNBC cells (MCF7 and SKBR3). FCM, flow cytometry; RCA-I, Ricinus communis agglutinin I; TNBC, triple-negative breast cancer; WFA, Wisteria floribunda agglutinin; WGA, Triticum vulgaris agglutinin.

Mentions: To verify the results from the lectin microarray, representative interactions were evaluated by lectin-based flow cytometry, using biotinylated lectins and Cy3-labeled streptavidin for detection. The lectins investigated were representative of three categories: namely, those that bound proportionally (RCA-I), inversely proportionally (WGA), or whose binding was uncorrelated with metastatic capacity (WFA). As summarized in Figure 2a, and Figure S2a in Additional file 2, we found that the FCM data indeed exhibited the same binding tendencies as that of the lectin microarray. To further confirm this result in more TNBC cells and analyze whether RCA-I binding exhibited a similar tendency in non-TNBC cancer cells, an additional four breast cancer cell lines (two TNBCs and two non-TNBCs) were also evaluated with RCA-I-based FCM. As shown in Figure 2c, RCA-I indeed bound strongly to these other TNBC cells. Interestingly, RCA-I also exhibited extensive binding to the non-TNBC cancer cells.Figure 2


Lectin RCA-I specifically binds to metastasis-associated cell surface glycans in triple-negative breast cancer.

Zhou SM, Cheng L, Guo SJ, Wang Y, Czajkowsky DM, Gao H, Hu XF, Tao SC - Breast Cancer Res. (2015)

The RCA-I-binding tendency is constant with the invasion ability of TNBC cells rather than those of non-TNBC cells. (a) FCM analysis confirms the proportional, inversely proportional, and uncorrelated binding to the metastatic capacity of these cells with RCA-I, WGA, and WFA, respectively. (b) Incubation of biotinylated RCA-I, WFA, or WGA, followed by the addition of Cy3-conjugated streptavidin and direct inspection with microscopy further confirmed the cell-binding tendencies of these lectins. (c) The RCA-I-binding positive rates of five TNBC (red) and two non-TNBC cells (green) were analyzed by FCM, which were referred to their relative metastatic abilities. (d) The immunofluorescent staining of RCA-I binding to another two TNBC cells (SUM159 and BT549) and two non-TNBC cells (MCF7 and SKBR3). FCM, flow cytometry; RCA-I, Ricinus communis agglutinin I; TNBC, triple-negative breast cancer; WFA, Wisteria floribunda agglutinin; WGA, Triticum vulgaris agglutinin.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4384317&req=5

Fig2: The RCA-I-binding tendency is constant with the invasion ability of TNBC cells rather than those of non-TNBC cells. (a) FCM analysis confirms the proportional, inversely proportional, and uncorrelated binding to the metastatic capacity of these cells with RCA-I, WGA, and WFA, respectively. (b) Incubation of biotinylated RCA-I, WFA, or WGA, followed by the addition of Cy3-conjugated streptavidin and direct inspection with microscopy further confirmed the cell-binding tendencies of these lectins. (c) The RCA-I-binding positive rates of five TNBC (red) and two non-TNBC cells (green) were analyzed by FCM, which were referred to their relative metastatic abilities. (d) The immunofluorescent staining of RCA-I binding to another two TNBC cells (SUM159 and BT549) and two non-TNBC cells (MCF7 and SKBR3). FCM, flow cytometry; RCA-I, Ricinus communis agglutinin I; TNBC, triple-negative breast cancer; WFA, Wisteria floribunda agglutinin; WGA, Triticum vulgaris agglutinin.
Mentions: To verify the results from the lectin microarray, representative interactions were evaluated by lectin-based flow cytometry, using biotinylated lectins and Cy3-labeled streptavidin for detection. The lectins investigated were representative of three categories: namely, those that bound proportionally (RCA-I), inversely proportionally (WGA), or whose binding was uncorrelated with metastatic capacity (WFA). As summarized in Figure 2a, and Figure S2a in Additional file 2, we found that the FCM data indeed exhibited the same binding tendencies as that of the lectin microarray. To further confirm this result in more TNBC cells and analyze whether RCA-I binding exhibited a similar tendency in non-TNBC cancer cells, an additional four breast cancer cell lines (two TNBCs and two non-TNBCs) were also evaluated with RCA-I-based FCM. As shown in Figure 2c, RCA-I indeed bound strongly to these other TNBC cells. Interestingly, RCA-I also exhibited extensive binding to the non-TNBC cancer cells.Figure 2

Bottom Line: Using the lectin microarray, we found that the bindings of RCA-I to TNBC cells are proportional to their metastatic capacity.Additionally, a membrane glycoprotein, POTE ankyrin domain family member F (POTEF), with different galactosylation extents in high/low metastatic TNBC cells was identified by LC-MS/MS as a binder of RCA-I.We discovered RCA-I, which bound to TNBC cells to a degree that is proportional to their metastatic capacities, and found that this binding inhibits the cell invasion, migration, and adhesion, and identified a membrane protein, POTEF, which may play a key role in mediating these effects.

View Article: PubMed Central - PubMed

ABSTRACT

Introduction: Triple-negative breast cancer (TNBC) patients often face a high risk of early relapse characterized by extensive metastasis. Previous works have shown that aberrant cell surface glycosylation is associated with cancer metastasis, suggesting that altered glycosylations might serve as diagnostic signatures of metastatic potential. To address this question, we took TNBC as an example and analyzed six TNBC cell lines, derived from a common progenitor, that differ in metastatic potential.

Methods: We used a microarray with 91 lectins to screen for altered lectin bindings to the six TNBC cell lines. Candidate lectins were then verified by lectin-based flow cytometry and immunofluorescent staining assays using both TNBC/non-TNBC cancer cells. Patient-derived tissue microarrays were then employed to analyze whether the staining of Ricinus communis agglutinin I (RCA-I), correlated with TNBC severity. We also carried out real-time cell motility assays in the presence of RCA-I. Finally, liquid chromatography-mass spectrometry/tandem spectrometry (LC-MS/MS) was employed to identify the membrane glycoproteins recognized by RCA-I.

Results: Using the lectin microarray, we found that the bindings of RCA-I to TNBC cells are proportional to their metastatic capacity. Tissue microarray experiments showed that the intensity of RCA-I staining is positively correlated with the TNM grades. The real-time cell motility assays clearly demonstrated RCA-I inhibition of adhesion, migration, and invasion of TNBC cells of high metastatic capacity. Additionally, a membrane glycoprotein, POTE ankyrin domain family member F (POTEF), with different galactosylation extents in high/low metastatic TNBC cells was identified by LC-MS/MS as a binder of RCA-I.

Conclusions: We discovered RCA-I, which bound to TNBC cells to a degree that is proportional to their metastatic capacities, and found that this binding inhibits the cell invasion, migration, and adhesion, and identified a membrane protein, POTEF, which may play a key role in mediating these effects. These results thus indicate that RCA-I-specific cell surface glycoproteins may play a critical role in TNBC metastasis and that the extent of RCA-I cell binding could be used in diagnosis to predict the likelihood of developing metastases in TNBC patients.

Show MeSH
Related in: MedlinePlus