Highlights
- •Brain met-CAFs exhibit high level of secreted, fucosylated polio virus receptor (sfPVR)
- •Hypoxia stimulates fucosyltransferase 11-mediated fucosylation and secretion of PVR
- •sfPVR alters cell-cell junctions and actin cytoskeleton dynamics, driving BC cell invasion
- •sfPVR/FUT11 expression by bmCAFs enhances BC invasion in the brain
Summary
Brain metastasis cancer-associated fibroblasts (bmCAFs) are emerging as crucial players in the development of breast cancer brain metastasis (BCBM), but our understanding of the underlying molecular mechanisms is limited. In this study, we aim to elucidate the pathological contributions of fucosylation (the post-translational modification of proteins by the dietary sugar L-fucose) to tumor-stromal interactions that drive the development of BCBM. Here, we report that patient-derived bmCAFs secrete high levels of polio virus receptor (PVR), which enhance the invasive capacity of BC cells. Mechanistically, we find that HIF1α transcriptionally upregulates fucosyltransferase 11, which fucosylates PVR, triggering its secretion from bmCAFs. Global phosphoproteomic analysis of BC cells followed by functional verification identifies cell-cell junction and actin cytoskeletal signaling as modulated by bmCAF-secreted, -fucosylated PVR. Our findings delineate a hypoxia- and fucosylation-regulated mechanism by which bmCAFs contribute to the invasiveness of BCBM in the brain.
Introduction
Breast cancer (BC) is the second most common source of brain metastasis (BM), which occurs in 10%–30% of metastatic BC (MBC) patients.1,2,3 Patients with triple-negative breast cancer (TNBC), the most aggressive subtype of BC, exhibit the highest BM incidence rates and poorest survival outcomes upon diagnosis with BM.4,5 As brain screening for MBC patients is not recommended in the US National Comprehensive Cancer Network and European Society for Medical Oncology guidelines,6 effective early detection remains a challenge: most BC patients are diagnosed with BM after presentation of neurological symptoms (well after dissemination of cancer cells within the brain). Effective therapies are also lacking. Since the blood-brain barrier (BBB) is a major obstacle for the effective delivery of many drugs into the brain, local treatments including surgery, stereotactic radiation therapy, and whole-brain radiation therapy are currently considered as gold-standard treatments for BCBM.2,3,6,7 Unfortunately, these approaches often result in impaired neurocognitive function and significantly reduced quality of life. Thus, new early-detection/screening modalities and more effective targeted/personalized therapies with fewer comorbidities are urgently needed, particularly for high-risk BC patients.
Increasing studies have identified cancer-associated fibroblasts (CAFs) as major players within the tumor microenvironment (TME) of breast and other cancer types that promote tumor cell migration and invasion.8,9,10 Thus, targeting CAFs has been an attractive and promising strategy for cancer treatment. Significant advances have been made in CAF-targeted therapies that generally aim to (1) directly or indirectly deplete CAFs using antibodies or vaccines or (2) normalize or reprogram CAFs to a non-pathological, benign state.11,12,13,14,15 However, none of these approaches have translated from pre-clinical models to therapeutic efficacy in human trials. Thus, the delineation of alternative approaches (i.e., targeted inhibition of CAF intrinsic or paracrine signaling mechanisms that more effectively block CAF-mediated tumorigenesis) in humans is needed.
While CAFs and their protumoral effects have been demonstrated in primary breast tumors, CAFs have been presumed absent in BCBM, as fibroblasts do not reside in the healthy brain.16 However, CAFs have been isolated from BCBM tissues, suggesting that they either traffic with cancer cells from primary tumors or transdifferentiate from brain-resident stromal cells (e.g., pericytes or mesenchymal stem cells).17,18 Notably, these brain-metastasis-associated CAFs (bmCAFs) have been found to promote metastatic colonization by producing high levels of chemokines (e.g., CXCL12 and CXCL16) that enhance the migration of patient-derived cancer cells, induce BBB permeability, and provide survival, proliferative, and invasive advantages to tumor cells.19,20,21 However, definitive underlying and targetable mechanisms are unknown. More in-depth studies elucidating CAF-mediated BCBM biology are crucial and anticipated to result in new innovative diagnostic and therapeutic approaches for targeting CAF-mediated mechanisms to suppress BCBM.
Emerging studies have highlighted mechanistic roles for fucosylation in the pathogenesis of multiple cancer types, including those within the brain. Fucosylation is the post-translational conjugation of the dietary sugar L-fucose (L-fuc) onto N- or O-linked oligosaccharide chains on glycoproteins or onto glycolipids. Fucosylation begins with cytoplasmic synthesis of GDP-L-fucose (GDP-L-fuc) that is transported into the ER/Golgi by the transporters SLC35C1/2, where it is conjugated onto proteoglycans by fucosyltransferases (FUTs).22,23 Thirteen FUTs catalyze structurally distinct fucose linkages in humans,24 and the aberrant up- and downregulation of individual FUTs is associated with tumorigenesis. The activity of key receptors (e.g., integrins, epidermal growth factor receptor, transforming growth factor receptor β, and Notch) are fine-tuned by fucosylation in terms of ligand binding, dimerization, and signaling capacities.25,26,27,28,29 Profiling of cancer patient sera for altered glycosylation-fucosylation states/levels of secreted proteins is a promising new diagnostic approach; increased serum levels of L-fuc and fucosylated proteins correlate with BC progression.30,31,32 Notably, altered abundance of L-fuc and N– and O-fucosylated glycans have been reported in brain cancers including glioblastoma (GBM),33,34,35 consistent with pathological contributions. However, how aberrantly fucosylated proteins alter tumor-stromal interactions (e.g., tumor:CAF interactions) to promote progression of tumors in the brain is not known.
Here, we report that compared with normal and primary tumor fibroblasts, patient-derived bmCAFs uniquely secrete fucosylated proteins that induce migration/invasion of TNBC cells. We identified polio virus receptor (PVR/CD155) as a highly bmCAF-fucosylated/secreted protein that potently drives BC cell migration/invasion. Consistent with the hypoxic environment in the brain parenchyma, our studies delineate that fucosylation of PVR is facilitated by FUT11 that is directly transcriptionally upregulated by hypoxia-inducible factor 1α (HIF1α). Intracranial mouse modeling of BCBM using a brain-homing BC cell line reveals the crucial roles played by secreted fucosylated PVR and FUT11 in CAF-mediated invasion and expansion of BCBM in the brain. Analysis of BCBM patient tissues reveals that fucosylated PVR is enhanced in bmCAF compared to BC populations. Together, our findings highlight a fucosylation- and hypoxia-regulated mechanism underlying bmCAF-mediated BCBM pathogenesis.
Results
BC CAFs exhibit high fucosylation levels that correlate with metastasis
The importance of fucosylation in cellular signaling and tumor progression continues to be defined in emerging studies. However, our understanding of the roles of fucosylation in key cell type(s) within the TME and their contributions to tumorigenesis remains limited. To understand the dynamics of fucosylation within the TME of breast tumors, we performed immunofluorescent (IF) staining of MDA-MB-231 tumor xenograft sections using three different types of lectins that recognize different fucosylation structural linkages: AAL: α(1,2), α(1,3), α(1,4), and α(1,6); UEA-1: α(1,2); and LTL: α(1,3) and α(1,4).24
AAL exhibited the highest signals within the tumors compared to UEA-1 and LTL (Figure S1A). More detailed inspection of the intratumoral IF staining revealed that SMA+ cells exhibited ∼20-fold higher levels of AAL-recognized fucosylation compared to pan-cytokeratin (Pan-CK)+ BC cells (Figures S1A and S1B). To confirm the specificity of AAL for immunostaining of fucosylated proteins, we performed a control L-fuc wash during AAL staining of the tumors, which abolished all AAL IF signals (Figure S1C). We subsequently assessed the correlation between AAL-recognized fucosylation and CAFs in human BC by multiplexed IF staining and single-cell segmented imaging analysis of a 50-patient breast tumor microarray (TMA), which contained one primary and one metastatic core biopsy pair per patient (Figures 1A and S1D). Total AAL-recognized fucosylation levels were increased in metastatic compared to patient-matched primary tumors (Figure 1B). Intriguingly, we discovered that relative AAL-recognized fucosylation levels in CAFs were significantly higher than in BC cells (Figure 1C), and metastatic CAFs (mCAFs) exhibited significantly higher fucosylation levels compared to matched tumor CAFs (tCAFs) (Figure 1D). Notably, Pearson analysis revealed a correlation (r = 0.3732) between the number of CAFs and their level of fucosylation (Figure 1E). Moreover, intratumoral CAF populations (absolute and percentage of total tumor cells) correlated with increased clinical staging and metastasis (Figure S1E), consistent with previous reports,36,37,38 suggesting that increased intratumoral fucosylation in breast tumors is attributed to CAF-specific fucosylation that increases with intratumoral CAF density during progression. These observations suggest that CAF-specific fucosylation plays an essential role in BC progression. However, why CAFs exhibit such high fucosylation levels and whether and how CAF fucosylated proteins might impact BC biology was unclear.
