Characterization of CDCP1 Expression and Glycosylation in Triple-Negative Breast Cancer
Poster #: 122
Session/Time: A
Author:
Paul Cheddie, BS, MS
Mentor:
LiFang Yang, MD, PhD
Research Type: Basic Science
Abstract
INTRODUCTION:
Triple-negative breast cancer (TNBC), accounting for ~15-20% of breast cancers, is a lethal subtype of breast cancer characterized by its aggressive behavior, high rates of metastatic recurrence, and limited treatment options. This disease exhibits considerable biological heterogeneity, comprising at least four recognized transcriptomic subtypes, which poses significant challenges to treating TNBC effectively. CUB-domain-containing protein 1 (CDCP1), a cell-surface type I transmembrane glycoprotein, plays an oncogenic role in many human cancers. Overexpression of CDCP1 has been reported in TNBC and is associated with adverse outcomes. Mechanistically, CDCP1 is a master regulator of tumor invasion and metastasis in TNBC by acting as an oncogenic signaling hub to control multiple intracellular biological processes. It is well known that CDCP1 harbors a larger ectodomain with heavy N-glycosylation modification, suggesting its potential function in intercellular communication. We previously reported dysregulated expression of CDCP1 in prostate cancer, and the extent of sialylation of CDCP1 was correlated with the metastatic potential of prostate cancer cells. However, the structure and role of CDCP1 glycosylation in TNBC remains unexplored. The objective of this study was to characterize CDCP1 expression and sialylation in TNBC and explore the role of CDCP1 sialylation in tumor-immune interactions.
METHODS:
Six breast cell lines-five TNBC representing basal-like (HCC1143, HCC1937) and mesenchymal-like (MDA-MB-436, MDA-MB-231, and BT549) subtypes, and one benign epithelial mammary cell line (MCF10A)-were cultured under standard conditions. CDCP1 expression at the mRNA and protein levels was assessed by RT-qPCR and Western blot. CDCP1 N-glycosylation and sialylation were examined by two approaches: (1) in vitro enzymatic deglycosylation with PNGase F and α2,3/6/8-neuraminidase A followed by Western blots; (2) affinity pulldown assays with linkage-specific lectins Sambucus nigra agglutinin (SNA) and Maackia amurensis lectin II (MALII), which recognize α2,6-linked and α2,3-linked sialic acids, respectively.
RESULTS:
Compared to non-tumorigenic breast MCF10A cells, CDCP1 is overexpressed, both at RNA and protein levels, in TNBC cells, with the most pronounced expression in mesenchymal-like TNBC cells (BT549, MDA-MB-231, and MDA-MB-436). Tumor-associated proteolytic cleavage produced heterogeneous expression of full-length (flCDCP1, ~135-140 kDa) and cleaved (cCDCP1, ~70 kDa) species in TNBC cells. PNGase F treatment yielded clear downward molecular weight shifts of both CDCP1 variants (~40 kDa for flCDCP1, ~15 kDa for cCDCP1) in breast cells, confirming extensive N-glycosylation of CDCP1. Neuraminidase treatment caused an apparent mobility shift of flCDCP1 in all tested cells, indicating substantial terminal sialylation on flCDCP1. SNA pulldown enriched flCDCP1 in MCF10A, BT549 and MDA-MB-436 cells while MALII pulldown enriched flCDCP1 in HCC1937 cells, suggesting various sialoglycan compositions of CDCP1 in breast cells.
CONCLUSION:
Our findings suggest that altered sialylation of CDCP1, along with overexpression, is associated with more aggressive TNBC subtypes. Further validation of CDCP1 sialylation characteristics and functional interrogation of their impact on TNBC progression, specifically in immune escape, is our next step.
Triple-negative breast cancer (TNBC), accounting for ~15-20% of breast cancers, is a lethal subtype of breast cancer characterized by its aggressive behavior, high rates of metastatic recurrence, and limited treatment options. This disease exhibits considerable biological heterogeneity, comprising at least four recognized transcriptomic subtypes, which poses significant challenges to treating TNBC effectively. CUB-domain-containing protein 1 (CDCP1), a cell-surface type I transmembrane glycoprotein, plays an oncogenic role in many human cancers. Overexpression of CDCP1 has been reported in TNBC and is associated with adverse outcomes. Mechanistically, CDCP1 is a master regulator of tumor invasion and metastasis in TNBC by acting as an oncogenic signaling hub to control multiple intracellular biological processes. It is well known that CDCP1 harbors a larger ectodomain with heavy N-glycosylation modification, suggesting its potential function in intercellular communication. We previously reported dysregulated expression of CDCP1 in prostate cancer, and the extent of sialylation of CDCP1 was correlated with the metastatic potential of prostate cancer cells. However, the structure and role of CDCP1 glycosylation in TNBC remains unexplored. The objective of this study was to characterize CDCP1 expression and sialylation in TNBC and explore the role of CDCP1 sialylation in tumor-immune interactions.
METHODS:
Six breast cell lines-five TNBC representing basal-like (HCC1143, HCC1937) and mesenchymal-like (MDA-MB-436, MDA-MB-231, and BT549) subtypes, and one benign epithelial mammary cell line (MCF10A)-were cultured under standard conditions. CDCP1 expression at the mRNA and protein levels was assessed by RT-qPCR and Western blot. CDCP1 N-glycosylation and sialylation were examined by two approaches: (1) in vitro enzymatic deglycosylation with PNGase F and α2,3/6/8-neuraminidase A followed by Western blots; (2) affinity pulldown assays with linkage-specific lectins Sambucus nigra agglutinin (SNA) and Maackia amurensis lectin II (MALII), which recognize α2,6-linked and α2,3-linked sialic acids, respectively.
RESULTS:
Compared to non-tumorigenic breast MCF10A cells, CDCP1 is overexpressed, both at RNA and protein levels, in TNBC cells, with the most pronounced expression in mesenchymal-like TNBC cells (BT549, MDA-MB-231, and MDA-MB-436). Tumor-associated proteolytic cleavage produced heterogeneous expression of full-length (flCDCP1, ~135-140 kDa) and cleaved (cCDCP1, ~70 kDa) species in TNBC cells. PNGase F treatment yielded clear downward molecular weight shifts of both CDCP1 variants (~40 kDa for flCDCP1, ~15 kDa for cCDCP1) in breast cells, confirming extensive N-glycosylation of CDCP1. Neuraminidase treatment caused an apparent mobility shift of flCDCP1 in all tested cells, indicating substantial terminal sialylation on flCDCP1. SNA pulldown enriched flCDCP1 in MCF10A, BT549 and MDA-MB-436 cells while MALII pulldown enriched flCDCP1 in HCC1937 cells, suggesting various sialoglycan compositions of CDCP1 in breast cells.
CONCLUSION:
Our findings suggest that altered sialylation of CDCP1, along with overexpression, is associated with more aggressive TNBC subtypes. Further validation of CDCP1 sialylation characteristics and functional interrogation of their impact on TNBC progression, specifically in immune escape, is our next step.