Altered Cell-Surface Glycosylation Patterns in Endocrine Resistant Breast Cancer
Poster #: 138
Session/Time: A
Author:
Linsong Zhang, MS, BS
Mentor:
LiFang Yang, MD, PhD
Research Type: Basic Science
Abstract
INTRODUCTION:
Breast cancer is the most common diagnosed non-cutaneous cancer in women worldwide. Estrogen receptor (ER) is expressed over 70% of cases. Endocrine therapy is the mainstay treatment for ER+ breast cancer, and tamoxifen and fulvestrant are the most common used drugs that target ER. However, 20-30% of these patients have cancer recurrence within 10 years after initial diagnosis, highlighting the unmet need to overcome endocrine resistance for this largest population of breast cancer. Glycosylation is a post-translational modification of cell-surface and soluble proteins. Abnormal glycosylation has been implicated in tumor malignancy, progression, and therapeutic resistance in multiple cancer types. However, the specific glycosylation alterations associated with endocrine-resistant breast cancer remain largely unexplored. The objective of this study is to characterize cell-surface glycosylation patterns and underlying glycoproteins associated with endocrine resistance in ER+ breast cancer.
METHODS:
An endocrine resistance model including MCF10A (non-tumorigenic), MCF7 (endocrine-sensitive and tumorigenic), and LCC9 (derived from MCF7 with dual resistance to tamoxifen and fulvestrant) breast cell lines were employed. Cell proliferation assays, RT-qPCR, and Western blotting under different drug treatments were performed to examine their estrogen and drug response phenotypes. Cell-surface glycosylation was analyzed by flow cytometry (FC) using two orthogonal strategies: 1) lectin affinity staining for linkage-specific sialic acid and complex N-glycan epitopes; and 2) metabolic labeling and copper-free click chemistry (N-azidoacetylmannosamine ManNAz and DBCO-Biotin) for nascent sialic acid content. Correspondingly, the expression of underlying glycoprotein carriers was compared by lection blotting of cellular membrane fractions and Western blotting of metabolic labelled/clicked samples.
RESULTS:
Cell proliferation assays, RT-qPCR, and Western blotting confirmed different status in estrogen dependence, expression of ERalpha and estrogen-induced protein (pS2), and tamoxifen/fulvestrant sensitivity among three cell lines. FC quantitation of cell-surface lectins recognizing linkage-specific sialoglycans and complex N-glycans revealed significantly reduced cell-surface α(2,6)- and α(2,3)- sialylation, as well as bisecting/β1,6-branched N-glycosylation in LCC9 cells compared to MCF10A and/or MCF7 cells. The alternation in sialylation was further supported by decreased metabolic labelled signal in LCC9 cells. Moreover, these glycosylation changes are reciprocated on the cell-surface glycoproteins discerned by the combination of acetone precipitation and lectin/Western blotting, suggesting the glycoproteins are main players responsible for altered glycosylation.
CONCLUSION:
The present work shows the correlation of reduction in cell-surface sialylation and complex N-glycosylation with endocrine resistance in ER+ breast cancer. Cell-surface glycoproteins harboring these altered glycans have great potential as biomarkers for monitoring resistance and therapeutic targets for intervention. Specific glycoprotein candidates will be identified and characterized with future profiling and functional studies.
Breast cancer is the most common diagnosed non-cutaneous cancer in women worldwide. Estrogen receptor (ER) is expressed over 70% of cases. Endocrine therapy is the mainstay treatment for ER+ breast cancer, and tamoxifen and fulvestrant are the most common used drugs that target ER. However, 20-30% of these patients have cancer recurrence within 10 years after initial diagnosis, highlighting the unmet need to overcome endocrine resistance for this largest population of breast cancer. Glycosylation is a post-translational modification of cell-surface and soluble proteins. Abnormal glycosylation has been implicated in tumor malignancy, progression, and therapeutic resistance in multiple cancer types. However, the specific glycosylation alterations associated with endocrine-resistant breast cancer remain largely unexplored. The objective of this study is to characterize cell-surface glycosylation patterns and underlying glycoproteins associated with endocrine resistance in ER+ breast cancer.
METHODS:
An endocrine resistance model including MCF10A (non-tumorigenic), MCF7 (endocrine-sensitive and tumorigenic), and LCC9 (derived from MCF7 with dual resistance to tamoxifen and fulvestrant) breast cell lines were employed. Cell proliferation assays, RT-qPCR, and Western blotting under different drug treatments were performed to examine their estrogen and drug response phenotypes. Cell-surface glycosylation was analyzed by flow cytometry (FC) using two orthogonal strategies: 1) lectin affinity staining for linkage-specific sialic acid and complex N-glycan epitopes; and 2) metabolic labeling and copper-free click chemistry (N-azidoacetylmannosamine ManNAz and DBCO-Biotin) for nascent sialic acid content. Correspondingly, the expression of underlying glycoprotein carriers was compared by lection blotting of cellular membrane fractions and Western blotting of metabolic labelled/clicked samples.
RESULTS:
Cell proliferation assays, RT-qPCR, and Western blotting confirmed different status in estrogen dependence, expression of ERalpha and estrogen-induced protein (pS2), and tamoxifen/fulvestrant sensitivity among three cell lines. FC quantitation of cell-surface lectins recognizing linkage-specific sialoglycans and complex N-glycans revealed significantly reduced cell-surface α(2,6)- and α(2,3)- sialylation, as well as bisecting/β1,6-branched N-glycosylation in LCC9 cells compared to MCF10A and/or MCF7 cells. The alternation in sialylation was further supported by decreased metabolic labelled signal in LCC9 cells. Moreover, these glycosylation changes are reciprocated on the cell-surface glycoproteins discerned by the combination of acetone precipitation and lectin/Western blotting, suggesting the glycoproteins are main players responsible for altered glycosylation.
CONCLUSION:
The present work shows the correlation of reduction in cell-surface sialylation and complex N-glycosylation with endocrine resistance in ER+ breast cancer. Cell-surface glycoproteins harboring these altered glycans have great potential as biomarkers for monitoring resistance and therapeutic targets for intervention. Specific glycoprotein candidates will be identified and characterized with future profiling and functional studies.