Graduation Year

2023

Document Type

Dissertation

Degree

Ph.D.

Degree Name

Doctor of Philosophy (Ph.D.)

Degree Granting Department

Biology (Cell Biology, Microbiology, Molecular Biology)

Major Professor

Eric E. Lau, Ph.D.

Committee Member

Peter P. Forsyth, Ph.D.

Committee Member

Subhra S. Mohapatra, Ph.D.

Committee Member

John J. Koomen, Ph.D.

Committee Member

Andriy Marusyk, Ph.D.

Keywords

Brain metastasis, Invasion, Fucosylation, Polio virus receptor, Cancer-associated fibroblasts, Protein secretion, Hypoxia

Abstract

Breast cancer (BC) continues to be the most diagnosed cancer and the second leading cause of cancer deaths in women worldwide. High mortality, in most cancers, is due to metastasis; BC is the most common source of one of the deadliest forms of metastasis–brain metastasis (BM) in female. Patients with triple negative breast cancer (TNBC) exhibit the highest incidence of BM and poorest survival outcomes upon diagnosis with BM. The prognosis of breast cancer brain metastasis (BCBM) remains poor because the blood–brain barrier (BBB) prevents the uptake of drug substances, including chemotherapeutic agents, targeted agents, and toxins, from the brain. Therefore, local treatments including neurosurgical resection, stereotactic radiosurgery (SRS), and whole-brain radiation therapy (WBRT) are currently considered as gold standard treatments for BCBM. However, these approaches often result in impaired neurological/cognitive functions and reduced quality and expectancy of life. Hence, there is an urgent need to elucidate molecular mechanisms driving BCBM so that new treatment, prediction, and prevention strategies can be developed.

Metastatic BC cells develop aggressive characteristics based on cues from the tumor microenvironment (TME). Among tumor stromal cells, cancer-associated fibroblasts (CAFs) are one of the prominent cell types within the BC TME and play significant roles in BC metastasis. Cancer-associated fibroblasts can regulate every aspect of metastasis process, including EMT, migration, invasion, survival in circulation, colonization of disseminated tumor cells, and dormancy. Cancer-associated fibroblasts from metastatic organs have been shown to promote drug resistance and enhance the metastatic process. Targeting CAFs has represented an attractive strategy for cancer treatment, and despite significant advances, none of the therapeutic intervention efforts have effectively translated from preclinical studies to clinical trials. Thus, delineation of alternative CAF-mediated approaches is more desirable. Unlike other distal metastatic sites, the brain was presumed to be devoid of CAFs, given the lack of normal fibroblasts in the brain. However, brain metastasis-CAFs (bmCAFs) have been isolated from BCBM tissues and are emerging as crucial players in the development of BCBM. Nevertheless, our understanding of the underlying molecular mechanisms of how bmCAFs enhance the BCBM process is limited. This dissertation research elucidates the role of fucosylation (post-translational modification of proteins by the dietary sugar L-fucose) in the contribution of bmCAFs in BCBM pathogenesis.

Fucosylation, the post-translational conjugation of the dietary sugar L-fucose (L-fuc) on glycoproteins or glycolipids, plays crucial roles in immunological and organ developmental processes. The activity of key receptors, EGFR, TGF, and NOTCH, are fine-tuned by fucosylation, which can influence their ligand binding, dimerization, and signaling capacities. In BC, fucosylated serum proteins correlate with tumor presence, but studies delineating mechanisms underlying fucosylation-induced biological changes are few and correlative. Moreover, how aberrantly fucosylated proteins alter tumor:CAFs interactions to promote BCBM is not known.

To understand the cellular dynamics of fucosylation within the BC TME, we performed an immunofluorescent (IF) staining of MDA-MB-231 tumor xenografts. IF staining pattern reveal that CAF population exhibited higher fucosylation levels compared to BC cells within the TME. Multiplexed IF staining and single-cell segmented imaging analysis of BC TMA, revealed that CAFs have higher fucosylation levels, which increase with the clinical staging and metastasis. This result highlights a potential role of CAFs-driven fucosylated proteins in BC progression. Since CAFs are known to secrete tumorigenic growth factors, chemokines, and cytokines in the TME, we sought to investigate if patients’ CAFs secrete fucosylated proteins that may impact BC cell biology. We assessed proliferation and motility of BC cells treated with NBF-, tCAF-, or bmCAFs-derived conditioned media (CM) that was depleted or not of fucosylated proteins. We discovered that compared with normal and primary tumor fibroblasts, patient-derived bmCAFs uniquely secrete fucosylated proteins that induce migration of TNBC cells. To identify specific bmCAF-secreted fucosylated (sf) proteins that may stimulate BC cells motility, we performed comparative mass spectrometry (MS) profiling of fucosylated proteins secreted by bmCAFs and NBFs. In assessing the top 5 hits from the fucoproteomic profiling by lectin pulldown and IB analysis, we validated Polio Virus Receptor (PVR) as one of the highly sf-proteins from bmCAFs. We confirmed the secreted fucosylated PVR (sfPVR), in bmCAFs by two independent approaches: an enzymatic assay that liberates N-glycans from proteins, and Lectin-mediated proximity ligation assay (L-PLA) technique that can immunofluorescently visualize fucosylated Proteins. By performing lectin pulldown and IB analysis on fucosylation deficient vs. control bmCAFs, we established that fucosylation is required for secretion of PVR. To further substantiate direct fucosylation of sPVR, we overexpressed the sPVR in bmCAFs and observe that the overexpressed sPVR is also fucosylated and secreted. Together, these data demonstrated that PVR is highly fucosylated protein secreted from bmCAFs and fucosylation is required for secretion. Next, to determine how PVR fucosylation is regulated, we profiled fucosyltransferases (FUTs) in bmCAFs. Since we have already established that PVR is N-glycosylated/fucosylated, we focused on 11 FUTs that can catalyze N-linked fucosylation. We compared the expression of these FUTs; RNA-Seq data revealed that FUT11, FUT10, FUT8, and FUT4 are highly expressed in bmCAFs compared to other FUTs. Quantitative reverse transcription-PCR (qRT-PCR) confirmed that FUT11 is highly expressed in bmCAFs among the 4 potential FUTs. FUT11 is known as a hypoxia-related gene, a direct target of Hypoxia Inducible Factor 1a (HIF1[U+F061]) in multiple cancers including breast. By chromatin immunoprecipitation-quantitative reverse transcription (ChIP-qRT-PCR), we confirmed a direct binding of HIF1[U+F061] to FUT11 promoter and its transcriptional induction. At the protein level, we observed a 2-fold induction of sfPVR under hypoxia and FUT11 knockdown in bmCAFs significantly reduced fuco-PVR. These data demonstrate that FUT11 is directly transcriptionally upregulated by HIF1[U+F061] during hypoxia in bmCAFs, which drives the fucosylation and secretion of PVR.

To investigate the functional and mechanistic roles of sfPVR in promoting BCBM biology, we performed global phosphoproteomic analysis of BC cells followed by functional verification. These approaches identified adherens junction, tight junction, and actin cytoskeletal signaling as modulated by sfPVR to drive BC migration and invasion. When BC cells were treated with CM from control (shNT) or PVR-knocked down (shPVR) bmCAFs, we observed that shNT CM significantly induced migration and invasion of BC cells whereas shPVR CM did not. At molecular level, we further established that sfPVR drives BC cells migration/invasion by enhancing the localization of phosphorylated focal adhesion kinase (pFAK) at the leading edge of the cell, disrupting the tight junction, and rearranging actin cytoskeleton. These in vitro observations validated our phosphoproteomic findings. Intracranial mouse modeling of BCBM confirmed our in vitro observations, where we observed that sfPVR from bmCAF enhance the invasion and spread of BC cells into the brain. Analysis of BCBM patient tissue samples reveal that fucosylated PVR is enhanced in bmCAFs compared to BC populations.

Together, our findings highlight a novel fucosylation- and hypoxia-regulated mechanism underlying bmCAF-mediated BCBM pathogenesis. Our data support the targeting of sfPVR by neutralizing antibody, assessment of its utility as a cerebrospinal fluid biopsy biomarker, and/or targeting the cell-cell adhesion interactions (e.g., tight junction protein 1) in BC cells that are modulated by sfPVR.

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