Targeting Post-Translational Modifications To Improve Combinatorial Therapies in Breast Cancer: The Role Of Fucosylation Part 2

3. Fucosylation as Druggable Target: From Pre-Clinical Studies to Clinical Translation

3.1. Specific Fucosylation Inhibitors

RNAi silencing of FUT8 has been shown to reduce core-fucosylation of cancer cells and functionally inhibit their migration and invasion in vitro [10,18], as well as tumor growth capacity in vivo [18]. To provide patients with feasible treatment protocols, genetic engineering strategies cannot be directly translated into clinics, and a handy drug is needed. 

Fucose is a very special polysaccharide compound, and its molecular structure contains a large number of sulfate groups. Core fucose is the most stable and complex structure among fucose. It has many important biological functions, the most important of which is its influence on the immune system.

The role of core fucose in the immune system is mainly achieved through the interferon (IFN) signaling pathway. Interferon is an important biological signal molecule, which can activate the innate immune system and adaptive immune system, thereby playing anti-virus, anti-cancer, and other effects. The core fucose can improve the antibacterial and antiviral capabilities of the immune system through the interferon signaling pathway, thereby enhancing human immunity.

In addition to enhancing immunity, core fucose can also improve the body's resistance through other mechanisms. For example, it can lower blood sugar, lower blood lipids, and reduce the incidence of cardiovascular disease. At the same time, core fucose also has various biological activities such as anti-inflammation and anti-oxidation, which can help the body resist the invasion of various diseases.

In daily life, you can enhance your body's immunity by eating some foods containing fucose. For example, kelp, seaweed, seaweed, etc. are all foods rich in fucose, which can help the body obtain enough fucose to improve immunity.

In short, core fucose is a very important biologically active substance, which plays an important role in improving human immunity. We should increase the body's intake of fucose through proper dietary adjustments, to help the body enhance immunity, and prevent and treat various diseases. From this point of view, we need to improve our immunity. Cistanche can significantly improve immunity because Cistanche also has anti-virus and anti-cancer effects, which can strengthen the immune system’s ability to fight and improve the body’s immunity.

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It is currently the most promising strategy to reduce fucosylation in cancer to develop orally acting fucose analogs that compete with physiological fucose in the Golgi and engulf the fucosyltransferase machinery [56]. In this context, 2-Fluoro-Fucose (2FF), a cell-permeable fluorinated fucose derivative, has been tested in both pre-clinical models as well as in human patients as a treatment for a variety of cancer types after oral, intraperitoneal (IP), or intravenous (IV) administration (Table 1).

3.2. 2FF Use in Various Cancers

To our knowledge, Okeley et al. were the first to test 2FF in vivo and demonstrated the efficacy of different compounds in enhancing ADCC activity of monoclonal antibodies (MAbs) and inducing reversible neutrophilia and also demonstrated that the drug had a direct anti-tumor effect in lymphoma and colorectal cancer models [58]. They obtained systematic data on tolerability and bioavailability for oral (drinking water or gavage), IP, and IV administration schedules, paving the way for future studies [58]. 2FF was evaluated in the context of hepatocellular carcinoma, where increased levels of core-fucosylation are already associated with worse outcomes [11]. As a result of demonstrating significant inhibition of HepG2 cell proliferation and integrin-mediated cell migration in vitro, Zhou et al. found that, after inoculating HepG2 cells pre-treated with 2FF and then injected with intra-tumoral drug injections, the tumor volume shrank consistently in subcutaneous HCC models [11].

In line with those findings, Pieri et al. examined the role of core-fucosylation in the mesenchymal subgroup of glioblastoma (GBM), the one associated with worse prognosis and chemoradiation resistance [18]. In orthotopic xenografts of human GBM, 2FF delivered intratumorally via micro-infusion pumps resulted in significantly reduced tumor volume and increased survival. Moreover, glycoproteomic profiling of patient-derived GBM cells revealed high levels of core-fucosylated proteins related to extracellular matrix adhesion and integrin-mediated signaling pathways, fundamental mediators of tumor aggressiveness, which are turned off by 2FF treatment [18].

Aside from testing 2FF as a monotherapy, combinatorial approaches with immunotherapies also appeared promising. Based on the finding that core-fucosylation is required for proper PD1 expression and ligand-receptor interaction, Okada et al. tuned the posttranslational regulatory mechanisms of PD1 to optimize the anti-tumor immune response [61]. In particular, 2FF attenuated PD1 expression in T cells and strengthened their antitumoral attack against melanoma, further supporting its use in combination with pembrolizumab [61].

3.3. Focus on 2FF Use in Breast Cancer

Two genetically distinct transgenic breast cancer models have been shown to benefit from therapeutic fucosylation inhibition via 2FF-the TgMMTV-neu (HER2+ luminal B) and the C3(1)-Tag (basal-like) [63]. Compared to those isolated from untreated mice, IgG isolated from treated mice showed enhanced tumor cell lysis, suggesting enhanced ADCC function and tumor-specific reactogenicity. Moreover, 2FF treatment at two different doses in a prophylactic anti-tumor experimental setting delayed tumor formation prevented cancer development in 33% of TgMMTV-neu and 26% of C3(1)-Tag models, and enhanced splenocyte reactogenicity upon exposure to tumor-lysate. Additionally, pro-inflammatory cytokines (such as interleukin-6, IL-6; IL12-p40; and granulocyte-colony stimulating factor, G-CSF) were elevated throughout the body. Importantly, the anti-tumor effect of 2FF was greatly reduced upon CD4 T cell depletion, suggesting an active role of the immune system in mediating the anti-cancer activity upon fucosylation inhibition [63].

The role of fucosylation in modulating anti-cancer immunity and combining therapeutic approaches has also been characterized in a TNBC pre-clinical model, primarily using 4T1 cells [65]. In this work, Huang et al. first confirmed that excessive glycosylation of the immune-suppressive checkpoint B7-H3 protein, present on tumor and/or antigen-presenting cells, retains a negative prognostic value in TNBC patients. The N-glycosylation of B7-H3 at Asn-X-Ser/Thr motifs (where X is any amino acid except proline) leads to increased stabilization and membrane expression. The key enzyme involved in this glycosylation step was shown to be FUT8, which positively correlated with B7-H3 mRNA expression, but not transcription and also correlated with worse prognosis in patients with TNBC. As a result of scoring FUT8 immunohistochemical (IHC) expression by membrane intensity and percentage of positive cells, patients were almost equally divided into low and high groups, indicating that FUT8 expression in TNBC patients is supposedly heterogeneous. 

Functionally, B7-H3 core-fucosylation led to reduced immune system engagement, as evidenced by in vitro and in vivo experiments. Both B7-H3 wild-type and B7-H3-4NQ tumors grew similarly in SCID mice, but the former showed faster kinetics in syngeneic, immunocompetent BALB/c mice. B7-H3 wild-type tumors also had a reduced infiltration of T lymphocytes, both CTLs, and CD4, as well as NK cells. To further corroborate these findings, treatment of B7-H3 wild-type tumors in syngeneic mice with both the core-fucosylation inhibitor 2FF and anti-PDL1 mAb resulted in reduced tumor growth kinetics, decreased B7-H3 expression on tumor cells, and in increased infiltration of IFNγ+ NK cells as well as of IFNγ+ CD8 or CD4 T lymphocytes [65].

Overall, these studies provide evidence that core-fucosylation plays a significant role in tumor biology, invasiveness, metastatic seeding, as well as tumor-immune interactions. The research supports the use of fucosylation inhibitors, including 2FF, in various clinical situations, including breast cancer, either alone or in combination with immune-stimulating therapies.

3.4. First 2FF-Based Clinical Trial

The abovementioned pre-clinical data prompted for clinical testing of 2FF in a Firstin-Human, First-in-Class, Phase 1 clinical trial in patients with advanced solid tumors, either alone or in combination with pembrolizumab (NCT02952989) [66]. A total of 46 patients were enrolled, mostly (33/46) in part A dose-escalation monotherapy arm. A dose-proportional pharmacokinetic profile, with target inhibition of fucosylation, was demonstrated, with the identification of the Maximum Tolerated Dose (MTD) of 10 g daily. According to RECIST v1.1 criteria, 10 patients (36%) reached stable disease after 10 cycles among the 28 patients evaluated for response in part A, of whom one patient with triple-negative breast cancer showed a 51% disease reduction and a partial response (PR) based on immune-related RECIST criteria. While nausea, fatigue, and diarrhea were the most common toxicities (47%) in part A and part C, thromboembolic events (grades 2–5) were detected in 16% (5/32) and 14% (1/7) of patients, despite concurrent prophylactic anticoagulation, which led to the early termination of the study [66]. As a result of this experience, the oral 2FF fucosylation inhibitor was found to have promising anti-tumor activity, which may be exploitable as monotherapy or in combination with other therapies in the clinic in the future. However, a more refined selection of patients, alternative thromboembolic drug prophylaxis, and/or second-generation inhibitors are required.

4. Fucosylation Interplay with the Immune System and Hormonal Pathways

4.1. Macrophages

Macrophages play key roles in multiple immunological and cancer-related processes, such as antigen uptake and presentation, angiogenesis, metastatic seeding, and chemotherapy resistance [67,68]. They modulate the microenvironment by integrating multiple signals, and their modulation ranges from the inflammatory, M1-like, to the immunomodulatory, M2-like polarization [69].

Rheumatoid arthritis (RA) patients’ synovial cells express terminal-fucosylation but not core-fucosylation and this expression correlates positively with tumor necrosis factor-alpha (TNFα). In vitro, terminal-fucosylation inhibition by 2-deoxy-D-galactose (2-Dgal), blocking FUT1/2 enzymes, resulted in suppression of M1 differentiation and M1 to M2 polarization. In vivo, 2-D-gal dramatically reduced the onset of collagen IIinduced arthritis [70]. Fut8 KO macrophages also showed altered CD14 and Toll-like Receptor (TLR) 2 and 4 axis expression in an experimental model of lipopolysaccharide (LPS) stimulation. As a result, mice transplanted with Fut8 KO hematopoietic bone marrow displayed enhanced resistance to inflammation [71]. In addition, macrophages represent one the largest leukocyte population in various tumor microenvironments and have been described to display unusual glycosylation patterns, which have, in turn, been proposed as potential therapeutic targets [72–75].

4.2. T & B Lymphocytes

Glycosylation is a recognized modulator of lymphocytes’ functions, from autoimmunity to cell activation and homeostasis [76]. O-fucosylation regulates T cell development as well as lymphoid/myeloid fate specification in hematopoietic progenitors through Notch signaling [77]. Fx KO mice show an expansion of myelopoiesis and a contraction of lymphopoiesis [78]. The O-fucosylation of Notch1/2 by Protein O-Fucosyltransferase 1 (PO-FUT1) promotes B-cell and thymocyte development while reversing the myeloproliferative burst in Pofut1 knockout mice [79].

Moreover, ex vivo fucosylation of Cytotoxic T Lymphocytes (CTLs), while not affecting target specificity, has been shown to enhance homing and tumor cell killing [80]. Interestingly, while broad fucosylation inhibition via 2-FluoroFucose (2FF) has been proposed to positively impact T Cell Receptor (TCR) engagement and regulation [81], core-fucosylation of the heavy chain of the B Cell Receptor (BCR) is needed for proper B cell development and transition from the pro-B stage. Moreover, Fut8 KO mice display reduced immunoglobulin (Ig) production (IgG, IgA, IgM) [82,83]. In addition, IgG production is improved upon fucose recognition by DC-SIGN on dendritic cells (DCs), facilitating T follicular helper (TFH) cells’ differentiation [84]

Lastly, core-fucosylation influences immunologically relevant co-receptors, thereby influencing the cancer immunity cycle. The programmed death 1 (PD-1) receptor is indeed regulated by post-translational modifications, such as core-fucosylation, and blockade of it in a pre-clinical model of melanoma using 2FF has been shown to enhance T cell-driven antitumoral immunity by reducing PD-1 membrane expression [61].

4.3. Antibodies

Even though antibodies exhibit highly conserved structures, with variable heavy and light chains conferring specificity, post-translational modifications have been shown to significantly impact their effector functions, with fucosylation being the most studied [85–87]. Afucosylated Fc glycans exhibit a high affinity for FcgRIIIa glycans, increasing antibody-dependent cell cytotoxicity (ADCC) [88,89], and this property is already being exploited in drug engineering, as with Amivantamab, a bispecific antibody [90,91]. In vivo, 2FF exposure has been linked with fucosylated IgG production and displayed an anti-tumor effect in both syngeneic and xenograft models [58]. Individuals from different geographical regions have different Ig glycosylation profiles [92]; in contrast, viral-vectored vaccines can produce similar antigen-specific IgG glycosylation profiles that are influenced by inflammatory stimuli after B cell priming [93].

There is also evidence indicating that Ig fucosylation plays a role in infectious diseases, with reduced levels found in HIV elite controllers [94] and a link between dengue and COVID-19 severity [95,96]. Interestingly, fucosylated anti-SARS-CoV2 Ig increased inflammation by activating macrophages and produced prothrombotic conditions [97,98].

4.4. Hormonal Pathways

Both men and women have shown that estrogen regulates IgG glycomic composition. Indeed, post-menopausal women display increased pro-inflammatory IgG glycoforms lacking terminal galactose. Such galactosylated IgGs display enhanced complement fixation via lectin pathways and ADCC. Interestingly, testosterone aromatization has also been shown to cause such estradiol-related events in men [99].

Furthermore, it was found that perimenopause is associated with decreased galactosylated glycans and increased IgG core-fucosylation by studying IgG glycome changes. In addition to promoting low-grade inflammation due to a loss of galactosylation, increased core-fucosylation has also been linked to less efficient Ig effectors [100].

Overall, these data show the vast interplay between the immune/hormonal systems and protein fucosylation, highlighting a relevant role of such post-translational modifications on relevant physiologic processes in healthy and diseased conditions and suggesting possible novel biomarkers to be investigated as well as therapeutic vulnerabilities to be addressed.

5. Discussion

Solid tumors utilize multiple complex mechanisms to facilitate cellular growth, adapt to hostile environments, evade immune recognition, and develop resistance to various therapeutic approaches. Many of these characteristics are shared among different malignancies and have been thoroughly characterized, ultimately leading to the identification of novel, tailored therapeutic approaches [101]. Solid tumors often develop a hypoxic, largely immune-suppressive tumor microenvironment (TME), which ultimately poses an insurmountable obstacle to anti-cancer therapies, including cell therapies [102,103]. In addition, malignant tumors have been shown to hijack post-translational modifications, such as glycosylation and fucosylation, to block cell-to-cell communications [104,105].

There is a growing body of evidence that fucosylation plays a role in regulating the immune and hormonal systems under physiological conditions, but many questions remain, especially when it comes to large, prospective population studies. In addition, fucosylation has also been convincingly identified as a cancer-related characteristic enabling tumor invasiveness, aggressiveness, angiogenesis, and immune evasion in several solid malignancies [6]. Clinical samples from breast cancer patients have also revealed exaggerated core-fucose PTMs upon disease progression and metastatic spread. Further, pharmacologic inhibition of fucosylation in different breast cancer pre-clinical models has shown significant anti-tumor activity, also linked to immune responses [10,18,56,58]. In line with this, synergism between fucosylation inhibitor 2FF with the anti-PD1 immune checkpoint blockers has also been documented, instructing for hypothetical combinatorial treatment strategies [61].

To date, fucosylated proteins have been largely unexplored as a prognostic or predictive biomarker. While in patients with hepatocellular carcinoma, the level of fucosylated, rather than total, alpha-fetoprotein was shown to be more specifically associated with cancer progression [21], no such specific biomarker currently exists for breast cancer. Considering this, the study of peculiar fucosylated biomarkers would be of great clinical value, especially in areas of unmet clinical need, such as in adjuvant clinical decision-making.

It has been suggested that fucosylation and, in particular, core-fucosylation is a newer hallmark of cancer that can influence cell-to-cell communications, foster the development of derailed TMEs, and ultimately influence resistance to chemotherapy. Even though most of the molecular regulators of cancer-related core-fucosylation are still largely unknown, as well as most of their functional implications, more sustained pre-clinical research is urgently needed in the coming years to guide further refined clinical testing in the future.

Author Contributions:

Conceptualization: G.A. and V.P.; Methodology: G.A., V.P., and C.C.; Supervision: C.C., N.F., G.F., F.M.P., and G.C.; Writing: G.A. and V.P.; Images & Table: G.A. and V.P.; Critical Review & Editing: all authors. All authors have read and agreed to the published version of the manuscript.

Funding:

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Institutional Review Board Statement:

Not applicable.

Informed Consent Statement:

Not applicable.

Data Availability Statement:

No new data were created or analyzed in this study. Data sharing does not apply to this article.

Conflicts of Interest:

G.C. reports the following honoraria for speaker’s engagement: Roche, Seattle Genetics, Novartis, Lilly, Pfizer, Foundation Medicine, NanoString, Samsung, Celltrion, BMS, MSD; Honoraria for providing consultancy: Roche, Seattle Genetics, NanoString; Honoraria for participating in Advisory Board: Roche, Lilly, Pfizer, Foundation Medicine, Samsung, Celltrion, Mylan; Honoraria for writing engagement: Novartis, BMS; Honoraria for participation in Ellipsis Scientific Affairs Group; Institutional research funding for conducting phase I and II clinical trials: Pfizer, Roche, Novartis, Sanofi, Celgene, Servier, Orion, AstraZeneca, Seattle Genetics, AbbVie, Tesaro, BMS, Merck Serono, Merck Sharp Dome, Janssen-Cilag, Philogen, Bayer, Medivation, MedImmune. All the competing interests were outside the submitted work. C.C. reports personal fees for consulting, advisory roles, and speakers’ bureau from Lilly, Roche, Novartis, MSD, Seagen, Gilead, and Pfizer.

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