Proteolytic processing of PD-L1 by ADAM proteases in breast cancer cells.

Expression of programmed death ligand 1 (PD-L1) on the surface of tumor cells and its interaction with programmed cell death protein 1 (PD-1) on tumor-infiltrating lymphocytes suppress anti-tumor immunity. In breast tumors, PD-L1 expression levels are the highest in estrogen receptor-negative, progesterone receptor-negative, and human epidermal growth factor receptor 2-negative (triple-negative) cancers. In this study, we show that a portion of PD-L1 exogenously expressed in several triple-negative breast cancer cell lines, as well as endogenous PD-L1, is proteolytically cleaved by cell surface metalloproteases. The cleavage generates an ~ 37-kDa N-terminal PD-L1 fragment that is released to the media and a C-terminal PD-L1 fragment that remains associated with cells but is efficiently eliminated by lysosomal degradation. We identify ADAM10 and ADAM17, two closely related members of the ADAM family of cell surface metalloproteases, as enzymes mediating PD-L1 cleavage. Notably, treatment of cells with ionomycin, a calcium ionophore and a known activator of ADAM10, or with phorbol 12-myristate 13-acetate, an activator of ADAM17, dramatically increases the release of soluble PD-L1 to the media. We postulate that ADAM10 and/or ADAM17 may contribute to the regulation of the PD-L1/PD-1 pathway and, ultimately, to anti-tumor immunity in triple-negative breast cancer.

Metalloprotease-disintegrin ADAM12 actively promotes the stem cell-like phenotype in claudin-low breast cancer.

BACKGROUND: ADAM12 is upregulated in human breast cancers and is a predictor of chemoresistance in estrogen receptor-negative tumors. ADAM12 is induced during epithelial-to-mesenchymal transition, a feature associated with claudin-low breast tumors, which are enriched in cancer stem cell (CSC) markers. It is currently unknown whether ADAM12 plays an active role in promoting the CSC phenotype in breast cancer cells. METHODS: ADAM12 expression was downregulated in representative claudin-low breast cancer cell lines, SUM159PT and Hs578T, using siRNA transfection or inducible shRNA expression. Cell characteristics commonly associated with the CSC phenotype in vitro (cell migration, invasion, anoikis resistance, mammosphere formation, ALDH activity, and expression of the CD44 and CD24 cell surface markers) and in vivo (tumor formation in mice using limiting dilution transplantation assays) were evaluated. RNA sequencing was performed to identify global gene expression changes after ADAM12 knockdown. RESULTS: We found that sorted SUM159PT cell populations with high ADAM12 levels had elevated expression of CSC markers and an increased ability to form mammospheres. ADAM12 knockdown reduced cell migration and invasion, decreased anoikis resistance, and compromised mammosphere formation. ADAM12 knockdown also diminished ALDEFLUOR+ and CD44hi/CD24-/lo CSC-enriched populations in vitro and reduced tumorigenesis in mice in vivo. RNA sequencing identified a significant overlap between ADAM12- and Epidermal Growth Factor Receptor (EGFR)-regulated genes. Consequently, ADAM12 knockdown lowered the basal activation level of EGFR, and this effect was abolished by batimastat, a metalloproteinase inhibitor. Furthermore, incubation of cells with exogenously added EGF prevented the downregulation of CD44hi/CD24-/lo cell population by ADAM12 knockdown. CONCLUSIONS: These results indicate that ADAM12 actively supports the CSC phenotype in claudin-low breast cancer cells via modulation of the EGFR pathway.

Metalloprotease-dependent activation of EGFR modulates CD44+/CD24- populations in triple negative breast cancer cells through the MEK/ERK pathway.

PURPOSE: The CD44+/CD24- cell phenotype is enriched in triple negative breast cancers, is associated with tumor invasive properties, and serves as a cell surface marker profile of breast cancer stem-like cells. Activation of Epidermal Growth Factor Receptor (EGFR) promotes the CD44+/CD24- phenotype, but the specific signaling pathway downstream of EGFR responsible for this effect is not clear. The purpose of this study was to determine the role of the MEK/ERK pathway in the expansion of CD44+/CD24- populations in TNBC cells in response to EGFR activation. METHODS: Representative TNBC cell lines SUM159PT (claudin-low) and SUM149PT (basal) were used to evaluate cell surface expression of CD44 and CD24 by flow cytometry in response to EGFR and MEK inhibition or activation. EGFR and ERK phosphorylation levels were analyzed by Western blotting. The relationship between EGFR phosphorylation and MEK activation score in basal and claudin-low tumors from the TCGA database was examined. RESULTS: Inhibition of ERK activation with selumetinib, a MEK1/2 inhibitor, blocked EGF-induced expansion of CD44+/CD24- populations. Sustained activation of ERK by overexpression of constitutively active MEK1 was sufficient to expand CD44+/CD24- populations in cells in which EGFR activity was blocked by either erlotinib, an EGFR kinase inhibitor, or BB-94, a metalloprotease inhibitor that prevents generation of soluble EGFR ligands. In basal and claudin-low tumors from the TCGA database, there was a positive correlation between EGFR_pY1068 and MEK activation score in tumors without genomic loss of DUSP4, a negative regulator of ERK, but not in tumors harboring DUSP4 deletion. CONCLUSION: Our results demonstrate that ERK activation is a key event in EGFR-dependent regulation of CD44+/CD24- populations. Furthermore, our findings highlight the role of ligand-mediated EGFR signaling in the control of MEK/ERK pathway output in TNBC tumors without DUSP4 loss.

Protein disulfide isomerases in the endoplasmic reticulum promote anchorage-independent growth of breast cancer cells.

Metastatic breast cancer cells are exposed to stress of detachment from the extracellular matrix (ECM). Cultured breast cancer cells that survive this stress and are capable of anchorage-independent proliferation form mammospheres. The purpose of this study was to explore a link between mammosphere growth, ECM gene expression, and the protein quality control system in the endoplasmic reticulum (ER). We compared the mRNA and protein levels of ER folding factors in SUM159PT and MCF10DCIS.com breast cancer cells grown as mammospheres versus adherent conditions. Publicly available gene expression data for mammospheres formed by primary breast cancer cells and for circulating tumor cells (CTCs) were analyzed to assess the status of ECM/ER folding factor genes in clinically relevant samples. Knock-down of selected protein disulfide isomerase (PDI) family members was performed to examine their roles in SUM159PT mammosphere growth. We found that cells grown as mammospheres had elevated expression of ECM genes and ER folding quality control genes. CTC gene expression data for an index patient indicated that upregulation of ECM and ER folding factor genes occurred at the time of acquired therapy resistance and disease progression. Knock-down of PDI, ERp44, or ERp57, three members of the PDI family with elevated protein levels in mammospheres, in SUM159PT cells partially inhibited the mammosphere growth. Thus, breast cancer cell survival and growth under detachment conditions require enhanced assistance of the ER protein folding machinery. Targeting ER folding factors, in particular members of the PDI family, may improve the therapeutic outcomes in metastatic breast cancer.