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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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Identification of metastasis-specific lectin binding to TNBC cells. (a) Schematic of the probing of fluorescently labeled cells on the lectin microarray. (b) Layout of the lectin microarray containing 91 lectins. (c) Representative lectin microarray binding patterns of the six TNBC cell lines. (d) Four lectins exhibited different extents of binding to the six different TNBC cell lines. The indicated intensities are represented as the mean values ± standard deviation (SD), n = 4. (e) Clustered heat map of the lectin cell-binding profiles. This map was generated and clustered according to the lectin-binding pattern and mean intensity. The order of the cells in this map closely matches their order of metastatic capacity. TNBC, triple-negative breast cancer.
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Fig1: Identification of metastasis-specific lectin binding to TNBC cells. (a) Schematic of the probing of fluorescently labeled cells on the lectin microarray. (b) Layout of the lectin microarray containing 91 lectins. (c) Representative lectin microarray binding patterns of the six TNBC cell lines. (d) Four lectins exhibited different extents of binding to the six different TNBC cell lines. The indicated intensities are represented as the mean values ± standard deviation (SD), n = 4. (e) Clustered heat map of the lectin cell-binding profiles. This map was generated and clustered according to the lectin-binding pattern and mean intensity. The order of the cells in this map closely matches their order of metastatic capacity. TNBC, triple-negative breast cancer.

Mentions: Six TNBC cell lines (SCP4, SCP6, MDA-MB-231, SCP28, SCP2 and 4175) that differ in metastatic capacity were chosen in this study owing to their common origin and extensive previous characterization [28,29]. To identify the cell surface glycan profiles associated with each of these cell lines, freshly harvested cells were first stained by CFDA-SE and then incubated on a lectin microarray containing 91 lectins, in which each lectin was present in triplicate [14] (Figure 1a-c). Overall, we found that these cells bound between 43 and 49 lectins (Figure S1 in Additional file 1). Of these, 41 were common among all cell lines, and, of the remaining, six were common among at least two cell lines. This high degree of overlap is probably owing to the fact that these cells were derived from a common progenitor. Among the lectins that were observed to bind to these cell lines, four (namely, RCA-I, Triticum vulgaris agglutinin (WGA), Wisteria floribunda agglutinin (WFA) and Sambucus nigra agglutinin I (SNA-I)) were found to show noticeably different extents of binding to different cell lines (Figure 1d). Of note, the binding of RCA-I (a protein found in the plant, Ricinus communis) to these cell lines was proportional to their metastatic capacity, while that of WGA was inversely proportional. Surprisingly, SNA-I, which was previously reported to show an enhanced binding to highly metastatic cancer cells [30] did not exhibit stronger binding to the TNBC cells of greater metastatic potential, indicating that the sialic acid content of the surface glycans may not correlate with the metastatic capacity of these cells. To better compare the overall lectin-binding profiles of these cells, a heat map was generated and clustered according to the lectin-binding pattern and intensity (Figure 1e). Clear differences in lectin-binding patterns, and thus of the accessible cell surface glycans, were present between the six cell lines. Interestingly, the order of the TNBC cell lines in this cluster is the same as the order of their known metastatic capacity, except for the switch in order of the two cell lines with the highest metastatic potential (4175 and SCP2). This finding was also observed if the binding patterns of RCA-I and WGA were excluded from the heat map (data not shown).Figure 1


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)

Identification of metastasis-specific lectin binding to TNBC cells. (a) Schematic of the probing of fluorescently labeled cells on the lectin microarray. (b) Layout of the lectin microarray containing 91 lectins. (c) Representative lectin microarray binding patterns of the six TNBC cell lines. (d) Four lectins exhibited different extents of binding to the six different TNBC cell lines. The indicated intensities are represented as the mean values ± standard deviation (SD), n = 4. (e) Clustered heat map of the lectin cell-binding profiles. This map was generated and clustered according to the lectin-binding pattern and mean intensity. The order of the cells in this map closely matches their order of metastatic capacity. TNBC, triple-negative breast cancer.
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Related In: Results  -  Collection

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Fig1: Identification of metastasis-specific lectin binding to TNBC cells. (a) Schematic of the probing of fluorescently labeled cells on the lectin microarray. (b) Layout of the lectin microarray containing 91 lectins. (c) Representative lectin microarray binding patterns of the six TNBC cell lines. (d) Four lectins exhibited different extents of binding to the six different TNBC cell lines. The indicated intensities are represented as the mean values ± standard deviation (SD), n = 4. (e) Clustered heat map of the lectin cell-binding profiles. This map was generated and clustered according to the lectin-binding pattern and mean intensity. The order of the cells in this map closely matches their order of metastatic capacity. TNBC, triple-negative breast cancer.
Mentions: Six TNBC cell lines (SCP4, SCP6, MDA-MB-231, SCP28, SCP2 and 4175) that differ in metastatic capacity were chosen in this study owing to their common origin and extensive previous characterization [28,29]. To identify the cell surface glycan profiles associated with each of these cell lines, freshly harvested cells were first stained by CFDA-SE and then incubated on a lectin microarray containing 91 lectins, in which each lectin was present in triplicate [14] (Figure 1a-c). Overall, we found that these cells bound between 43 and 49 lectins (Figure S1 in Additional file 1). Of these, 41 were common among all cell lines, and, of the remaining, six were common among at least two cell lines. This high degree of overlap is probably owing to the fact that these cells were derived from a common progenitor. Among the lectins that were observed to bind to these cell lines, four (namely, RCA-I, Triticum vulgaris agglutinin (WGA), Wisteria floribunda agglutinin (WFA) and Sambucus nigra agglutinin I (SNA-I)) were found to show noticeably different extents of binding to different cell lines (Figure 1d). Of note, the binding of RCA-I (a protein found in the plant, Ricinus communis) to these cell lines was proportional to their metastatic capacity, while that of WGA was inversely proportional. Surprisingly, SNA-I, which was previously reported to show an enhanced binding to highly metastatic cancer cells [30] did not exhibit stronger binding to the TNBC cells of greater metastatic potential, indicating that the sialic acid content of the surface glycans may not correlate with the metastatic capacity of these cells. To better compare the overall lectin-binding profiles of these cells, a heat map was generated and clustered according to the lectin-binding pattern and intensity (Figure 1e). Clear differences in lectin-binding patterns, and thus of the accessible cell surface glycans, were present between the six cell lines. Interestingly, the order of the TNBC cell lines in this cluster is the same as the order of their known metastatic capacity, except for the switch in order of the two cell lines with the highest metastatic potential (4175 and SCP2). This finding was also observed if the binding patterns of RCA-I and WGA were excluded from the heat map (data not shown).Figure 1

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