Tamoxifen-resistant breast cancer cells exhibit reactivity with Wisteria floribunda agglutinin

Abstract

Glycosylation is one of the most important post-translational modifications of cell surface proteins involved in the proliferation, metastasis and treatment resistance of cancer cells. However, little is known about the role of glycosylation as the mechanism of breast cancer cell resistance to endocrine therapy. Herein, we aimed to identify the glycan profiles of tamoxifen-resistant human breast cancer cells, and their potential as predictive biomarkers for endocrine therapy. We established tamoxifen-resistant cells from estrogen receptor-positive human breast cancer cell lines, and their membrane-associated proteins were subjected to lectin microarray analysis. To confirm differential lectin binding to cellular glycoproteins, we performed lectin blotting analyses after electrophoretic separation of the glycoproteins. Mass spectrometry of the tryptic peptides of the lectin-bound glycoproteins was further conducted to identify glycoproteins binding to the above lectins. Finally, expression of the glycans that were recognized by a lectin was investigated using clinical samples from patients who received tamoxifen treatment after curative surgery. Lectin microarray analysis revealed that the membrane fractions of tamoxifen-resistant breast cancer cells showed increased binding to Wisteria floribunda agglutinin (WFA) compared to tamoxifen-sensitive cells. Glycoproteins seemed to be responsible for the differential WFA binding and the results of mass spectrometry revealed several membrane glycoproteins, such as CD166 and integrin beta-1, as candidates contributing to increased WFA binding. In clinical samples, strong WFA staining was more frequently observed in patients who had developed distant metastasis during tamoxifen treatment compared with non-relapsed patients. Therefore, glycans recognized by WFA are potentially useful as predictive markers to identify the tamoxifen-resistant and relapse-prone subset of estrogen receptor-positive breast cancer patients.

Fig 1. Lectin microarray analysis of T47D and ZR75-1 cells and their TAM-resistant derivative T47D-TAM^R and ZR75-1-TAM^R cells.

The relative intensities of WFA and SNA in (A) T47D (sensitive) and T47D-TAM^R (TAM-resistant) cells and (B) ZR75-1 (sensitive) and ZR75-1-TAM^R (TAM-resistant) cells, based on normalized average data. Error bars indicate standard deviations. t tests were employed for statistical comparisons.

Fig 2. Lectin blotting analysis of T47D and ZR75-1 cells and their corresponding TAM-resistant cells.

The hydrophobic fractions from sensitive and resistant cells were electrophoretically separated and the binding of WFA and SNA were examined. (A) The results of lectin blotting in T47D and T47D-TAM^R cells. (B) The results of lectin blotting in ZR75-1 and ZR75-1-TAM^R cells.

Fig 3. Precipitation of WFA binding glycoproteins in T47D-TAM^R and ZR75-1-TAM^R cells.

(A) Blotting of WFA binding fractions with WFA. (B) Silver staining of WFA binding fractions. Red rectangles indicate bands around 100 kDa, which were cut out and used for mass spectrometry.

Fig 4. Histochemical WFA staining of sections from primary breast cancer specimens.

Primary tumors from patients who developed distant metastasis during adjuvant TAM treatment (n = 20) and those who did not develop recurrent disease (n = 20) were analyzed. Strong positive staining (dark grey) was significantly more frequent in relapsed patients (13 patients, 65%) than in the control group (4 patients, 20%; p = 0.011).