Exploring the Role of GlycoRNAs in Breast Cancer Endocrine Resistance
Poster #: 130
Session/Time: A
Author:
Joanna Marie Camacho, BS
Mentor:
LiFang Yang, MD, PhD
Research Type: Basic Science
Abstract
INTRODUCTION:
Breast cancer affects 1 in 8 women in the United States , with 70-80% of the cases diagnosed with the estrogen receptor positive (ER+) subtype. Despite the current availability of therapies against these subtypes, such as tamoxifen and fulvestrant, a significant portion of patients will experience recurrence due to de novo or acquired endocrine resistance, underscoring the need for continued research to understand and treat this major hurdle. Glycosylation of biomolecules such as proteins and lipids are crucial for cellular functions such as trafficking, recognition, and signaling. In breast cancer as well as many other malignancies, alteration of glycosylation patterns have been observed and are known to contribute to disease progression, epithelial to mesenchymal transition, and tumor metastasis. Recent insights in glycan modifications have revealed the presence of glycosylated RNAs, termed glycoRNAs, on the cellular surface. Further research has demonstrated potential roles of glycoRNAs in cell biology, including neutrophil recruitment and malignant transformation, however their role in breast cancer and drug resistance remains in its infancy. Herein, the aim of this lab is to determine if glycosylation alterations are associated with endocrine resistance in breast cancer specifically regarding the role of glycoRNAs in cellular communication.
METHODS:
An endocrine resistance cell model including MCF10A cells (a non-tumorigenic breast epithelial cell line), MCF7 (an endocrine-sensitive ER+ breast cancer line), and LCC9 (derived from MCF7 with dual resistance to tamoxifen and fulvestrant) was used. Sialoglycans were metabolically labelled with the chemical azide group (AC4ManNAz) and further tagged by biotin via the copper-free click reaction in total cellular RNA lysates or on intact cell surface. Then RNA was strictly purified and cleaned up using TRIzol and silica columns followed by DNA and proteins decontamination. RNA quality and quantity was measured by NanoDrop and TapeStation. Northern Blot analysis with SYBR Gold and streptavidin dyes was employed to detect total and cell-surface glycosylated signal on RNA.
DISCUSSION/results RNA with high purity (A260/280 ≥ 2.0; RIN > 9.0) was exacted from above breast cells. GlycoRNAs were detected in cells treated with AC4ManNAz and click chemistry-based RNA blots. MCF10A cells displayed higher glycoRNA abundance compared to MCF7 cells while LCC9 cells completely lost glycoRNA signal. The expression of cell-surface glycoRNAs in these cells is consistent with the expression of cellular glycoRNAs. Treatment with RNA with protease K did not affect the glycoRNA signal, whereas treatment with an RNase cocktail completely digested the total RNA and glycoRNA signal.
CONCLUSION:
GlycoRNAs are displayed on the cell surface of breast cells, and their expression levels are inversely associated with tumor aggressiveness and endocrine sensitivity. Future studies will characterize both glycan structures and underlying RNA species, as well as investigate potential functions of these glycoRNAs in endocrine resistance of breast cancer.
Breast cancer affects 1 in 8 women in the United States , with 70-80% of the cases diagnosed with the estrogen receptor positive (ER+) subtype. Despite the current availability of therapies against these subtypes, such as tamoxifen and fulvestrant, a significant portion of patients will experience recurrence due to de novo or acquired endocrine resistance, underscoring the need for continued research to understand and treat this major hurdle. Glycosylation of biomolecules such as proteins and lipids are crucial for cellular functions such as trafficking, recognition, and signaling. In breast cancer as well as many other malignancies, alteration of glycosylation patterns have been observed and are known to contribute to disease progression, epithelial to mesenchymal transition, and tumor metastasis. Recent insights in glycan modifications have revealed the presence of glycosylated RNAs, termed glycoRNAs, on the cellular surface. Further research has demonstrated potential roles of glycoRNAs in cell biology, including neutrophil recruitment and malignant transformation, however their role in breast cancer and drug resistance remains in its infancy. Herein, the aim of this lab is to determine if glycosylation alterations are associated with endocrine resistance in breast cancer specifically regarding the role of glycoRNAs in cellular communication.
METHODS:
An endocrine resistance cell model including MCF10A cells (a non-tumorigenic breast epithelial cell line), MCF7 (an endocrine-sensitive ER+ breast cancer line), and LCC9 (derived from MCF7 with dual resistance to tamoxifen and fulvestrant) was used. Sialoglycans were metabolically labelled with the chemical azide group (AC4ManNAz) and further tagged by biotin via the copper-free click reaction in total cellular RNA lysates or on intact cell surface. Then RNA was strictly purified and cleaned up using TRIzol and silica columns followed by DNA and proteins decontamination. RNA quality and quantity was measured by NanoDrop and TapeStation. Northern Blot analysis with SYBR Gold and streptavidin dyes was employed to detect total and cell-surface glycosylated signal on RNA.
DISCUSSION/results RNA with high purity (A260/280 ≥ 2.0; RIN > 9.0) was exacted from above breast cells. GlycoRNAs were detected in cells treated with AC4ManNAz and click chemistry-based RNA blots. MCF10A cells displayed higher glycoRNA abundance compared to MCF7 cells while LCC9 cells completely lost glycoRNA signal. The expression of cell-surface glycoRNAs in these cells is consistent with the expression of cellular glycoRNAs. Treatment with RNA with protease K did not affect the glycoRNA signal, whereas treatment with an RNase cocktail completely digested the total RNA and glycoRNA signal.
CONCLUSION:
GlycoRNAs are displayed on the cell surface of breast cells, and their expression levels are inversely associated with tumor aggressiveness and endocrine sensitivity. Future studies will characterize both glycan structures and underlying RNA species, as well as investigate potential functions of these glycoRNAs in endocrine resistance of breast cancer.