Esophageal, gastric and colorectal cancers: Looking beyond classical serological biomarkers towards glycoproteomics-assisted precision oncology

Abstract

Esophageal (OC), gastric (GC) and colorectal (CRC) cancers are amongst the digestive track tumors with higher incidence and mortality due to significant molecular heterogeneity. This constitutes a major challenge for patients' management at different levels, including non-invasive detection of the disease, prognostication, therapy selection, patient's follow-up and the introduction of improved and safer therapeutics. Nevertheless, important milestones have been accomplished pursuing the goal of molecular-based precision oncology. Over the past five years, high-throughput technologies have been used to interrogate tumors of distinct clinicopathological natures, generating large-scale biological datasets (e.g. genomics, transcriptomics, and proteomics). As a result, GC and CRC molecular subtypes have been established to assist patient stratification in the clinical settings. However, such molecular panels still require refinement and are yet to provide targetable biomarkers. In parallel, outstanding advances have been made regarding targeted therapeutics and immunotherapy, paving the way for improved patient care; nevertheless, important milestones towards treatment personalization and reduced off-target effects are also to be accomplished. Exploiting the cancer glycoproteome for unique molecular fingerprints generated by dramatic alterations in protein glycosylation may provide the necessary molecular rationale towards this end. Therefore, this review presents functional and clinical evidences supporting a reinvestigation of classical serological glycan biomarkers such as sialyl-Tn (STn) and sialyl-Lewis A (SLe^A) antigens from a tumor glycoproteomics perspective. We anticipate that these glycobiomarkers that have so far been employed in non-invasive cancer prognostication may hold unexplored value for patients' management in precision oncology settings.

Keywords: digestive tract cancer; glycobiomarkers; glycomics; glycoproteomics; glycosylation; precision oncology.

Figure 1

Gastroesophageal and colorectal cancers main clinical challenges (inner circle), current clinical approaches (outer circle) and opportunities provided by neoantigen discovery. Current clinical challenges include: i) Lack of robust early diagnosis tools, requiring population screening and timely medical check-ups, especially for population subsets presenting known risk factors (poor diet; H. pylori infection; family history, age, gender, pre-neoplastic lesions; etc). The absence of molecular biomarkers with the necessary specificity and sensitivity to assist in this matter remains a tremendous obstacle for early cancer detection; ii) Need for patient stratification, mostly achieved based on the clinicopathological classification of the lesions and, most recently, moving towards the incorporation of molecular biomarkers; iii) Therapy selection and efficacy, currently based on clinicopathological features but rapidly evolving towards molecular-assisted settings capable of aiding therapy personalization and early definition of responders. Therapeutic management has been based on surgery, chemo and/or radiotherapy, encompassing severe toxicity and limited efficacy, particularly for advanced disease stages. However, this paradigm has started to change with the introduction of antibody-based targeted therapeutics against key oncogenic cell surface receptors and immune-check point proteins such as PD-1, PD-L1 and CTLA4. CAR-T immunotherapy is also amongst future promising approaches; iv) Non-invasive detection, necessary for real-time monitoring of disease status and evolution throughout the course of disease. The field of liquid biopsies has tremendously evolved with the evaluation of circulating tumor DNA/miRNAs, proteins, micro and nanovesicles (exosomes and others) and, more recently, the study of circulating tumor cells (CTCs). The evaluation of these biomarkers in bodily fluids has improved prognostications and helped refining therapeutic selection, evaluating responses, establishing the risk of metastasis development and the detection of radiologically occult micrometastasis; v) Molecular heterogeneity is also a critical clinical challenge. This aspect has been a major obstacle towards effective molecular-assisted oncology and the introduction of targeted therapeutics. Nevertheless, the field has experienced significant advances with next generation sequencing, which generated a significant amount of genomics and transcriptomics data that has been used to propose gastric and colorectal cancer molecular subtypes. Cancer proteomics characterization has also contributed to the identification of relevant biomarkers; however, with yet limited clinical translation; vi) Cancer neoantigens discovery also represents a critical objective and a daunting challenge. It will be crucial for the identification of cancer-specific fingerprints capable of guiding therapeutic decision and designing effective targeted therapies and immunotherapy with very limited off-targeted effects. The comprehensive integration of genomics, transcriptomics and proteomics as well as information on post-translational modifications, with emphasis on glycosylation, will be of key importance for the identification of relevant protein functional nodes and targetable biomarkers at the cell-surface.

Figure 2

Protein-associated glycan biosynthetic pathways. N-glycan biosynthesis begins at the endoplasmic reticulum (ER) membrane with the Glc3Man9GlcNAc2-P-P-Dol precursor transfer to Asn in Asn-X-Ser/Thr sequons of proteins by oligosaccharyltransferase (OST). Of note, “X” represents any amino acid except Pro. Subsequent N-glycan processing reactions take place in the ER from where glycoproteins exit to the Golgi apparatus carrying N-glycans with either eight or nine Man residues, termed oligomannose N-glycans. Further, biosynthesis of hybrid and complex N-glycans occurs in the medial-Golgi. Subsequent sugar additions diversify the repertoire of hybrid and complex N-glycans by elongation of branching GlcNAc residues, capping of elongated branches and N-glycan core sugar addition. Common terminal structures include sialyl lewis antigens, such as sialyl lewis A (SLe^A) through the concerted action of beta-galactoside-alpha-2,3-sialyltransferase-III and IV (ST3Gal III and IV) as well as alpha-4-fucosyltransferases (α4-FucT). Mucin type O-glycan biosynthesis is initiated in the late ER or in Golgi compartments by the Polypeptide GalNAcT- mediated transfer of N-acetylgalactosamine (GalNAc) to Ser or Thr residues of proteins in a tissue- and cell-type-specific manner. This first biosynthetic steps yield the Tn antigen (GalNAcα1-Ser/Thr), the simplest form of mucin-type O-glycosylation. Tn antigen can be sialylated into the sialyl Tn (STn) antigen by N-acetylgalactosaminide alpha-2,6-sialyltransferase I (ST6GalNAc I), abrogating further chain extension, or it can be extended into the core 1 antigen (T antigen) by N-acetylgalactosamine 3-beta-galactosyltransferase 1 (C1Gal-T1). In turn, the core 1 structure can be capped with sialic acid residues through the action of N-acetylgalactosaminide alpha-2,6-sialyltransferase 2 (ST6GalNAc II) or ST3Gal I, giving rise to sialyl T and disialyl T antigens, again preventing further extension. On the other hand, T antigen can be extended into the core 2 glycan by core 2 beta-1,6-N-acetylglucosaminyltransferase-I or III (C2GnT-I, III). Extended O-glycan structures beyond the core 2 antigen can also display terminal structures similar to N-glycan such as SLe^A.

Figure 3

Functional impact of STn and SLe^A expression in gastrointestinal tumors. Both STn and SLe^A overexpression influence tumor initiating processes as constitutive activation of several oncogenic signalling mediate by EGFR and ErbB2. Moreover, SLe^A also facilitates H. pylori adhesion to the gastric epithelium, contributing to persistent infection and potentially cancer development. STn also drives tumor progression by negatively impacting cell-cell and cell-extracellular matrix adhesion as well as galectin-3-mediated chemoresistance. Moreover, sialylated lewis antigens facilitate hematogenous metastasis of tumor cells through E-selectin interactions, while protecting tumor cells from sheer stress in circulation and hampering immune recognition.

Figure 4

STn and SLe^A clinical applications (non-invasive serological methods and molecular-assisted decisions using cancer tissues) in gastroesophageal and colorectal cancers. A) Non-invasive serological methods. The STn antigen is detected in the serum using the CA72-4 test; whereas the SLe^A antigen can be detected by the CA19-9 test. These antigens are used for prognostication, cancer screening and response to therapy monitoring. However, both lack the sensitivity and specificity for early diagnosis; B) Molecular-assisted oncology using cancer tissues. The right side of B panel highlights some of the most explored monoclonal antibodies detecting STn (orange) and SLe^A (blue), while the left side summarizes known clinical associations.

Figure 5

Glycan-based therapeutics. This includes glycan-based vaccines such as Theratope, exploiting the STn-antigen linked to a KLH protein carrier to elicited immune responses against STn-expressing cancer cells through antibody mediated killing and cytotoxic T cells effects. Other emerging immunotherapy is based on CAR-T cells engineered to target cancer cells expressing abnormal glycosylation. There are also several monoclonal antibodies capable of targeting abnormally glycosylated cells promoting antibody-dependent cellular cytotoxicity (ADCC) or blocking relevant oncogenic receptors at the cell-surface. Glycomimetics able to interfere with glycan biosynthesis or blocking glycan-receptor interactions relevant in cancer have also been developed. Finally, antibodies targeting glycans have been used to guide nanoparticles to tumor sites; thereby improving therapeutic outcomes.

Figure 6

Glycoproteomics objectives and opportunities facing clinical applications. The right top panel summarizes the objectives of glycoproteomics. Glycoproteomics provides an opportunity for identifying cancer unique molecular fingerprints at the cell-surface (glyco”neo”antigens) that are not reflected by healthy cells and non-malignant conditions, paving the way for precise cancer targeting. Alterations in glycan composition, glycosites density and distribution associated to peptide domains in clinically relevant glycoproteins may significantly contribute to this end. The bottom panel highlights the main findings achieved by glycoproteomics in gastroesophageal and colorectal tumors to this date. It mostly includes serological studies, with emphasis on the identification of alterations in the glycosylation of plasminogen and haptoglobulin showing potential for non-invasive gastric and esophageal cancer detection and early diagnosis. Targeting specific CEA and CA125 glycoforms may also improve the predictive value of existing clinical tests. In addition, glycopeptide arrays bearing different types of protein glycoforms immobilized in solid supports have shown potential to improve early cancer detection and prognosis based on the identification of autoantibodies. It may also be a relevant tool for identifying potentially immunogenic protein glycoforms as well as a decisive device for functional assays. Contrastingly, cancer tissues glycoproteomics is yet to be initiated, which will be critical foreseeing true clinical applications.

Figure 7

The quest for molecular-assisted precision oncology can be found in the crossroad between interdependent omics (genomics, transcriptomics and proteomics with PTM analysis) backed by comprehensive bioinformatics settings. This panel shows the possible connections between different omics, attempting to emphasize the importance of bringing together genomics, transcriptomics and proteomics allied to PTM (phosphorylation, methylation, acetylation and glycosylation, amongst others) analysis. It also aims to highlight the decisive role played by bioinformatics and current omics databases, which paved the way for tailored oncoproteogenomics. Namely, the comprehensive integration of genomics intel in customised databases can now greatly expand the coverage of protein annotations envisaging cancer neoantigens. This comprehensive strategy would be of key importance for accurate tumor stratification as well as identification of functional protein nodes and neoantigens for precise cancer detection and therapeutic design.

Figure 8

Main cancer challenges tackled by targeted glycoproteomics and glycan-based opportunities facing clinical translation. Glycans, particularly the STn and SLeA antigens, have been explored in the context of non-invasive cancer diagnosis, patient stratification, response to chemotherapy, disease monitoring, understanding and addressing immune response to cancer cells and design of safer targeted therapeutics. These approaches have been challenged by the lack of tumor specificity of these glycans; nevertheless, these glycobiomarkers specificity is being refined based on the integration of multiomics approaches. Fulfilling this objective will pave the way for improvements in liquid biopsies, targeted therapeutics, non-invasive cancer detection tools, patient stratification and prognostication models, novel targeted therapeutics, new immune check-point inhibitors, cancer glyconeoantigens and ultimately an improved understanding on the role of glycans in health and disease.