Abstract P5-05-04: Vitamin K dependent gamma-carboxylation in breast cancer
Authors: Welsh J , Beaudin S , Tenniswood M 
Published: March 2017

Abstract
The secreted protein periostin (PN) is strongly linked to breast cancer aggressiveness and serum PN is a promising marker of disease activity. However, the regulation and function of PN in relation to the cancer phenotype has yet to be clearly defined. PN is one of the rare proteins (&lt;15 in mammalian systems) that is subject to the post-translational addition of γ-carboxylated glutamatic acid (GLA) residues. However, no studies have addressed whether γ-carboxylation of PN has functional relevance in breast cancer, despite evidence that the proteins that catalyze γ-carboxylation are expressed in normal mammary gland. Using publically available datasets we found that GGCX, VKORC1 and VKORC1L1 (genes that mediate γ-carboxylation) are overexpressed in 24% of breast cancers. Furthermore, survival of patients whose tumors overexpress these genes is significantly worse than that of patients whose tumors do not overexpress these genes. Follow-up studies utilized the Human Protein Atlas to examine expression of GGCX, VKORC1 and VKORC1L1 proteins in normal breast and invasive ductal carcinomas (IDC). Normal ductal epithelial cells showed intense staining for GGCX, patchy moderate staining for VKORC1 and diffuse low signal for VKORC1L1. All cases of IDC exhibited strong staining for GGCX, 80% had moderate-intense staining for VKORC1 and 77% had diffuse but low staining for VKORC1L1. Staining for all 3 proteins was localized only in tumor cells indicating that stromal cells are unlikely to contribute to protein γ-carboxylation. Next we explored expression of the γ-carboxylation pathway genes using in vitro models of epithelial-mesenchymal transition (EMT) and tumor progression. The EMT model consists of human mammary epithelial cells immortalized with telomerase and SV40 (HMLE cells) that constitutively express GFP (control), TWIST, SNAIL or TGFβ. Relative to control cells, GGCX, VKORC1 and VKORC1L1 were up-regulated in HMLE-TWIST cells, GGCX and VKORC1 were up-regulated in HMLE-SNAIL cells and VKORC1 was up-regulated in HMLE-TGFβ cells. These data suggest that γ-carboxylation may be triggered early in breast cancer oncogenesis. In the tumor progression model, we found up-regulation of GGCX and VKORC1 in HMLE+RAS cells relative to HMLE cells. Introduction of SV40 alone did not alter expression of these genes. In HMLE-TWIST and HMLE-RAS cells with the highest expression of GGCX, VKORC1 and VKORC1L1, PN was up-regulated 200-300-fold, supporting the concept that PN may be a relevant target for γ-carboxylation during EMT and tumor progression. In summary, available genomic and proteomic data suggest that the vitamin K dependent pathway genes GGCX, VKORC1 and VKORC1L1 are present in normal mammary gland but up-regulated in a subset of invasive breast cancers that are characterized by poor overall survival. Genes in this pathway are also up-regulated in mammary epithelial cells expressing triggers of EMT (particularly TWIST) or oncogenes such as RAS. Since the only known function of these genes is in γ-carboxylation, studies to confirm PN as a GGCX substrate and to identify additional γ-carboxylated proteins and their functions in breast cancer are clearly warranted. Our limited understanding of this ancient, conserved pathway may be masking an important therapeutic opportunity.
Citation Format: Welsh J, Beaudin S, Tenniswood M. Vitamin K dependent gamma-carboxylation in breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P5-05-04.