Aberrant glycosylation of MUC1 and tumour-associated macrophages (TAMs)

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Prof Joy Burchell and Dr Richard Beatson from King’s College London investigated the role of aberrant glycosylation in cancer; focusing on a protein called MUC1. One very common tumour-associated glycoform of MUC1 is known as MUC1-ST. MUC1-ST binds to Siglec-9, a glycan-binding protein expressed on monocytes and macrophages. This interaction drives a tumour microenvironment that promotes cancer growth, through the differentiation of macrophages into TAMs. MUC1-ST-induced macrophages could be observed in a particular location within breast cancers and were associated with poor prognosis. Future therapies may be able to target this mechanism to improve survival outcomes for breast cancer patients.

Virtually all proteins carried on the surface of cells are decorated with sugars; these combined structures are known as glycoproteins. These sugars, or glycans, are fundamental to the function of the protein and are involved in cell:cell and cell:environment interactions. Importantly, the sugars on these glycoproteins change as cancers develop and so, in cancer, the glycoproteins can interact with a new set of proteins and cells. Mucin-type O-linked glycosylation is a type of attachment of sugars to proteins where the sugars are attached to the hydroxyl group of two amino acids, serine and or threonine, present in the protein. Changes in the O-linked sugars attached to a large glycoprotein known as MUC1 have been observed for a number of years and the role of MUC1 in tumour growth has been demonstrated in murine models of mammary cancers. However, the mechanisms involved have, until recently, been poorly understood.

Macrophages are a type of white blood cell that play important roles in anti-infection immunity, tissue homeostasis, and phagocytosis (engulfing of foreign substances). Macrophages also clear away damaging materials, such as cellular debris and tumour cells. During tumorigenesis, macrophages are recruited to sites of tumour growth due to the presence of chemokines, cytokines, and other factors secreted by tumour and host cells within the tumour microenvironment (TME). These macrophages are commonly called tumour-associated macrophages (TAMs), owing to their location.

A. Rad, Mikael Häggström, Spacebirdy, RexxS, domdomegg, CC BY-SA 4.0, via Wikimedia Commons

Macrophages are traditionally classified into two types: M1-like macrophages act against invading pathogens and tumour cells, inducing anti-pathogen and anti-tumour activity, whereas M2-like macrophages are involved in wound healing, tissue repair, and foster tumor growth and invasion. However, although a useful model, it is now clear that macrophage phenotype is not as simple as this definition. The M1 and M2 classifications represent two extreme binary states that may no longer be valid when characterising the diverse populations of TAMs present in the TME.

TAMs are implicated in tumour growth; in a recent meta-analysis of sixteen studies in breast cancer, it was found that the presence and levels of TAMs are correlated with worse disease outcome. TAMs are now seen as potential therapeutic targets to regulate tumour growth. In breast cancers, the TMEs can make up to 50% of the tumour mass, which also makes it an exciting area of study to produce potential therapeutic agents.

“These experiments elucidated MUC1-ST as a driver of monocyte differentiation into macrophages resembling TAMs, which previously has not been described.”

Glycoproteins and TAMs

Prof Joy Burchell and Dr Richard Beatson from King’s College London are interested in the role of aberrant glycosylation of MUC1 in promoting tumour-growth. After researching the differences in glycan decorations between normal breast epithelial cells and breast cancers for a number of years, in a 2016 paper published in Nature Immunology and a 2020 paper published in Communications Biology, they showed that a very common tumour-associated glycoform of MUC1 generates a particular type of TAM present in a specific location.

Interaction between MUC1-ST and Siglec-9 on monocytes and macrophages

MUC1 is a protein that is detected at high levels and aberrantly decorated with O-linked glycans in the majority of breast carcinomas. The glycans on MUC1 in breast cancer cells are shorter, and more sialylated (increased sialic acid), in contrast to the long, branched chains observed in normal epithelial cells. One particular glycoform of MUC1 studied by Prof Burchell and Dr Beatson is the short trisaccharide (Neu5Acα2,3-Galβ1,3GalNAc, where Neu5Ac is a sialic acid), known as sialylated T (MUC1-ST). 83% of human primary breast cancers showed an overexpression of MUC1-ST, and MUC1-ST was shown to increase tumour growth in experimental mouse models. These studies, plus others, show that this specific form of aberrant MUC1 glycosylation is very common and enhances tumour growth. However, the mechanisms underlying these observations have not been well-elucidated previously.

Figure 1. Examples of positive MUC1-ST IHC staining in breast cancers. https://www.nature.com/articles/s42003-020-01359-5

The researchers discovered that the sialic acid binding receptor, Siglec-9 is involved in this mechanism. Siglec-9 is expressed on the cell surface of various immune cells, including neutrophils, monocytes, macrophages, and T cells. It is involved in the regulation of immune responses, with siglecs commonly being thought of as inhibitory receptors, preventing immune activation. Surprisingly, however, the binding of MUC1-ST to Siglec-9 on monocytes (precursors to macrophages) and macrophages caused cellular activation, the secretion of multiple tumour-promoting factors, and the generation of TAMs. Many of these processes and factors are associated with normal wound-healing; it appears that tumour cells are ‘using’ a normal physiological mechanism to trick the immune system to stop attacking the cancer and promote tumour growth instead. This research strongly aligns with the paradigm that cancers are ‘wounds that do not heal’.

MUC1-ST-Siglec-9 interactions promote tumour growth

How do tumour cells achieve this? Prof Burchell and Dr Beatson showed that once MUC1-ST binds to monocytes, the monocytes secrete several factors that are associated with inflammation, wound-healing and tumour progression, such as interleukin 6 (IL-6), macrophage colony-stimulating factor (M-CSF), and plasminogen-activator inhibitor (PAI-1). These activated monocytes then differentiate into macrophages expressing high levels of PD-L1 and low CD86, both causing diminished T cell activity. In turn, the macrophages present in the TME can also bind MUC1-ST and secrete factors that will induce tumour growth. Binding of Siglec-9 on macrophages to MUC1-ST also induces the remodelling of the TME through the recruitment of monocytes and neutrophils.

They also showed that in vitro, binding of MUC1-ST to Siglec-9, without any other factor, leads to differentiation of monocytes into macrophages. The binding of MUC1-ST to monocytes and their differentiation to macrophages was dependent on the sialic acid being present and could be inhibited by the presence of an anti-Siglec-9 antibody. This shows that the mechanism is dependent on both sialic acids and Siglec-9 and highlights the role macrophages play in tumour progression. Prof Burchell and Dr Beatson found that MUC1-ST binding to Siglec-9 induced cellular activation, specifically calcium flux leading to the activation of the MEK-ERK pathway. Inhibition of the MEK-ERK pathway in monocytes before addition of MUC1-ST showed inhibition of both factor secretion and the differentiation into macrophages. This revealed that MUC1-ST-Siglec-9 effects are dependent on the MEK-ERK pathway. Research on fully understanding this novel mechanism of activation is currently ongoing.

“The results can be used to build a case for the development of new
myeloid-cell-checkpoint inhibitors, such as anti-Siglec-9 immunotherapy.”

Moreover, the binding of MUC1-ST to differentiated macrophages via Siglec-9 induces a specific TAM phenotype that favours tumour growth. There was increased secretion of M-CSF, PAI-1, EGF and CHI3L1, all of which are involved the progression of tumours. There were higher levels of CD206, CD163, IDO, and PD-L1 by the macrophages, with these proteins also being associated with TAMs. Previously, it was proposed that tumours produce factors that recruit and ‘educate’ monocytes as well as repolarise ‘resident’ macrophages into TAMs. These experiments elucidated MUC1-ST as one such factor and a driver of monocyte differentiation into macrophages resembling TAMs, which previously had not been described.

Next, the researchers compared MUC1-ST-induced monocyte-derived macrophages against the ‘gold-standard’ agent of monocyte-to-macrophage differentiation, M-CSF. The MCSF-induced macrophages and MUC1-ST-induced macrophages were compared using RNA sequencing. Neutrophils have been shown to contribute to breast cancer metastasis and MUC1-ST-induced macrophages showed increased expression of genes associated with enhanced neutrophil recruitment and survival. There was also an increased expression of genes involved in extracellular matrix (ECM) disassembly, such as MMP14, suggesting that MUC1-ST-induced macrophages could degrade the basement membrane and remodel the ECM, enhancing tumour metastasis. Additionally, an increased expression of genes involved in the inhibition of T cell function was observed, such as the T cell check point inhibitor PD-L1 which, upon binding to PD1 on T cells, induces T cell anergy. Genes associated with blood clotting were also over-expressed in MUC1-ST educated cells and, finally, MUC1-ST macrophages showed a decrease in genes linked to phagocytosis. All of these ‘genetic signatures’ are associated with enhanced tumour growth and spread, and were validated in in vitro functional assays.

https://www.nature.com/articles/s42003-020-01359-5

Previous studies have shown a correlation between the increased levels of TAMs and poor disease outcome. The research team found that MUC1-ST induced macrophages predominantly reside on the edge of the tumour, with their number correlating with the amount of MUC1-ST within the tumour. Macrophages that reside in this specific area have been correlated with poor prognosis in patients previously. In addition, Gentles et al. identified immune-associated genes that are associated with poor prognosis across all cancers. Eight out of the top 10 genes were upregulated in MUC1-ST-induced macrophages, whilst a MUC1-ST educated macrophage gene expression signature was associated with a poor prognosis in breast cancers. Given that MUC1 and increased sialic acids are expressed by the majority of solid tumours, MUC1-ST-induced macrophages may also be present in other cancer types.

The results described in these Nature Immunology and Communications Biology papers can be used to build a case for the development of new myeloid-cell-checkpoint inhibitors, such as anti-Siglec-9 immunotherapy, or the use of specific inhibitors, such as those that target the MEK/ERK pathway.

Conclusion

In conclusion, Prof Burchell and Dr Beatson provided strong arguments to explain the common expression of MUC1-ST on cancer cells, namely its role in the re-education of TME through the conversion of monocytes and macrophages into tumour-supporting TAMs, via binding to Siglec-9. Research into the glycosylation and cancer is still underrepresented, even though aberrant cancer-associated glycans are near-ubiquitous, are not typically associated with mutation, and are shared across multiple cancers. Targeting the interaction between MUC1-ST and Siglec-9 on macrophages can potentially inhibit the re-education of the TME and improve the survival outcome for breast cancer, and potentially many other cancer patients.


What combination therapy, along with Siglec-9 inhibition, do you propose for breast cancer patients? Are immune checkpoint inhibitors a good candidate due to the inhibition of PD-L1 in MUC1-ST-induced macrophages?

As more Siglec interactions other than Siglec-9 binding to MUC1-ST have been implicated in tumour growth, the combination of inhibiting sialic acid Siglec interaction with checkpoint inhibitors is certainly worthy of investigation. Inhibition of the interaction of sialic acid on tumour cells with Siglecs on immune cells could be via antibody blocking or the targeted removal of sialic acid from the cancer cells as investigated by others.

It may be beneficial to combine any of these therapies with other biologics; however, one of the benefits of targeting innate immune checkpoints is that you would, in theory, remove much of the PD-L1 within the TME simply by inhibiting its expression on TAMs. It may well prove that the best therapeutic combination would be an innate checkpoint inhibitor plus an adoptive cellular immunotherapy, such as CAR-T, where, once the TME has been rendered ‘inert’ by the innate checkpoint inhibitor, the T cells can successfully target and destroy the cancer.

 

References

  • Beatson, R., Tajadura-Ortega, V., Achkova, D., Picco, G., et al. (2016). The mucin MUC1 modulates the tumour immunological microenvironment through engagement of the lectin Siglec-9. Nature Immunology, 17(11), 1273-1281.
  • Beatson, R., Graham, R., Freile, F. G., Cozzetto, D. et al. (2020). Cancer-associated hypersialylated MUC1 drives the differentiation of human monocytes into macrophages with a pathogenic phenotype. Communications Biology, 3, 644.
  • Gentles, A. J., Newman, A. M., Liu, C. L., Bratman, S. V. et al. (2015). The prognostic landscape of genes and infiltrating immune cells across human cancers. Nature Medicine, 21, 938-945.
  • Lin, Y., Xu, J. & Lan, H. (2019). Tumor-associated macrophages in tumor metastasis: biological roles and clinical therapeutic applications. Journal of Haematology & Oncology, 12, 76.
  • Mungul, A. et al. (2004). Sialylated core-1-based O-linked glycans enhance the growth rate of mammary carcinoma cells in MUC1 transgenic mice. Int. J. Oncol., 25, 937-943.
  • Picco, G. et al. (2010). Over-expression of ST3Gal-I promotes mammary tumorigenesis. Glycobiology, 20, 1241-1250.
DOI
10.26904/RF-135-1217229982

Research Objectives

The researchers examine the role and impact of MUC1-ST in the Siglec-9 dependent generation of tumour-associated macrophages (TAMs) in breast cancers.

Funding

  • Medical Research Council
  • Breast Cancer Now
  • Cancer Research UK

Collaborators

  • Rosalind Graham
  • Fabio Grundland Freile
  • Domenico Cozzette
  • Shichina Kannambath
  • Ulla Mandel
  • Sarah Pinder
  • Thomas Noll
  • Ihssane Bouybayoune
  • Joyce Taylor-Papadimitriou

Bio

Joy Burchell, PhD is Emeritus Professor of glyco-oncology at King’s College London and Head of the Breast Cancer Biology Group. She joined King’s College London in 2007 from Cancer Research UK where she was a Senior Scientist.

Richard Beatson , PhD is a Research Fellow at King’s College London. Richard has a background in medicine (Glasgow), immunology (UCL) and glyco-oncology (KCL).

Contact
Breast Cancer Biology Group
King’s College London
Innovation Hub
Guy’s Cancer Centre
Guy’s Hospital
London SE1 9RT, UK

Joy Burchell
E: joy.burchell@kcl.ac.uk
T: +44 7710 576967
W: kclpure.kcl.ac.uk/portal/en/persons/joy-burchell(417e87d7-be97-4fd3-8547-9c9759c39035).html
Twitter: @SweetBiology

Richard Beatson
E: Richard.1.Beatson@kcl.ac.uk
T:+44 7717 414473
W: https://kclpure.kcl.ac.uk/portal/richard.1.beatson.html

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