Cadherin-5: a biomarker for metastatic breast cancer with optimum efficacy in oestrogen receptor-positive breast cancers with vascular invasion.

BACKGROUND: A glycoproteomic study has previously shown cadherin-5 (CDH5) to be a serological marker of metastatic breast cancer when both protein levels and glycosylation status were assessed. In this study we aimed to further validate the utility of CDH5 as a biomarker for breast cancer progression. METHODS: A nested case-control study of serum samples from breast cancer patients, of which n=52 had developed a distant metastatic recurrence within 5 years post-diagnosis and n=60 had remained recurrence-free. ELISAs were used to quantify patient serum CDH5 levels and assess glycosylation by Helix pomatia agglutinin (HPA) binding. Clinicopathological, treatment and lifestyle factors associated with metastasis and elevated biomarker levels were identified. RESULTS: Elevated CDH5 levels (P=0.028) and ratios of CDH5:HPA binding (P=0.007) distinguished patients with metastatic disease from those that remained metastasis-free. Multivariate analysis showed that the association between CDH5:HPA ratio and the formation of distant metastases was driven by patients with oestrogen receptor (ER+) positive cancer with vascular invasion (VI+). CONCLUSIONS: CDH5 levels and the CDH5 glycosylation represent biomarker tests that distinguish patients with metastatic breast cancer from those that remain metastasis-free. The test reached optimal sensitivity and specificity in ER-positive cancers with vascular invasion.

Identification of O-Linked Glycoproteins Binding to the Lectin Helix pomatia Agglutinin as Markers of Metastatic Colorectal Cancer.

BACKGROUND: Protein glycosylation is an important post-translational modification shown to be altered in all tumour types studied to date. Mucin glycoproteins have been established as important carriers of O-linked glycans but other glycoproteins exhibiting altered glycosylation repertoires have yet to be identified but offer potential as biomarkers for metastatic cancer. METHODOLOGY: In this study a glycoproteomic approach was used to identify glycoproteins exhibiting alterations in glycosylation in colorectal cancer and to evaluate the changes in O-linked glycosylation in the context of the p53 and KRAS (codon 12/13) mutation status. Affinity purification with the carbohydrate binding protein from Helix pomatia agglutinin (HPA) was coupled to 2-dimensional gel electrophoresis with mass spectrometry to enable the identification of low abundance O-linked glycoproteins from human colorectal cancer specimens. RESULTS: Aberrant O-linked glycosylation was observed to be an early event that occurred irrespective of the p53 and KRAS status and correlating with metastatic colorectal cancer. Affinity purification using the lectin HPA followed by proteomic analysis revealed annexin 4, annexin 5 and CLCA1 to be increased in the metastatic colorectal cancer specimens. The results were validated using a further independent set of specimens and this showed a significant association between the staining score for annexin 4 and HPA and the time to metastasis; independently (annexin A4: Chi square 11.45, P = 0.0007; HPA: Chi square 9.065, P = 0.0026) and in combination (annexin 4 and HPA combined: Chi square 13.47; P = 0.0002). CONCLUSION: Glycoproteins showing changes in O-linked glycosylation in metastatic colorectal cancer have been identified. The glycosylation changes were independent of p53 and KRAS status. These proteins offer potential for further exploration as biomarkers and potential targets for metastatic colorectal cancer.

A targeted glycoproteomic approach identifies cadherin-5 as a novel biomarker of metastatic breast cancer.

Aberrant glycosylation has long been recognised as a hallmark of cancer, and is increasingly being exploited in biomarker discovery studies. Helix pomatia agglutinin (HPA) is known to bind aberrant glycans associated with metastatic breast cancer, and was used here to isolate glycoproteins from pooled breast cancer serum samples of (i) patients with recurrent breast cancer and (ii) patients with no sign of recurrence 5years after diagnosis of their primary tumour. Pregnancy zone protein, the polymeric immunoglobulin receptor and cadherin-5 emerged as potential markers of metastasis following proteomic identification of HPA binding glycoproteins. ELISAs were developed to verify these findings, and to assess protein glycosylation, in individual patient sera. The cadherin-5 ELISA discriminated serum samples of patients with recurrent breast cancer from those with no sign of recurrence, and analysis of cadherin-5 glycosylation by HPA also showed a significant difference between the two sample groups. The targeted glycoproteomic and validatory approach developed here has shown that when taking into account both the protein levels and HPA binding, serum cadherin-5 discriminated patients with recurrent breast cancer from those with no sign of recurrence with 90% specificity.

Lectin array-based strategies for identifying metastasis-associated changes in glycosylation.

Since 2005, lectin microarray technology has emerged as a relatively simple yet powerful technique for the comprehensive analysis of glycoprotein glycosylation. Lectin microarrays represent a new analytical method that can be used to explore the human glycome, a unique source of markers of diseases including cancer. The lectin microarray technology is a sensitive tool with the potential to allow high-throughput analysis of cancer-associated changes in glycosylation. This chapter describes the generation of a lectin-binding signature associated with metastatic primary breast tumours that have been resected, fixed, and embedded in paraffin. Procedures concerning sample and lectin microarray preparation are explained, alongside experimental considerations and approaches to data analysis.

Lectin microarray profiling of metastatic breast cancers.

Altered protein glycosylation compared with the disease-free state is a universal feature of cancer cells. It has long been established that distinct glycan structures are associated with specific forms of cancer, but far less is known about the complete array of glycans associated with certain tumors. The cancer glycome has great potential as a source of biomarkers, but progress in this field has been hindered by a lack of available techniques for the elucidation of disease-associated glycosylation. In the present study, lectin microarrays consisting of 45 lectins with different binding preferences covering N- and O-linked glycans were coupled with evanescent-field activated fluorescent detection in the glycomic analysis of primary breast tumors and the serum and urine of patients with metastatic breast cancer. A single 50 µm section of a primary breast tumor or <1 µL of breast cancer patient serum or urine was sufficient to detect glycosylation alterations associated with metastatic breast cancer, as inferred from lectin-binding patterns. The high-throughput, sensitive and relatively simple nature of the simultaneous analysis of N- and O-linked glycosylation following minimal sample preparation and without the need for protein deglycosylation makes the lectin microarray analysis described a valuable tool for discovery phase glycomic profiling.

Cancer-associated glycoforms of gelatinase B exhibit a decreased level of binding to galectin-3.

Gelatinase B (MMP-9) and galectin-3 are widely known to participate in tumor cell invasion and metastasis. Glycans derived from MMP-9 expressed in MCF-7 breast cancer and THP-1 myeloid leukemia cells were compared with those from MMP-9 expressed in natural neutrophils. The many O-linked glycans of neutrophil gelatinase B presented a cluster of mainly galactosylated core II structures, 46% of which were ligands for galectin-3; 11% contained two to three N-acetyllactosamine repeating units that are high-affinity ligands for the lectin. The glycan epitopes thus provide MMP-9 with both high-affinity and (presumably) high-avidity interactions with galectin-3. In contrast, the O-glycans released from MMP-9 expressed in MCF-7 and THP-1 cells were predominantly sialylated core I structures. Only 10% of MCF-7 and THP-1 gelatinase B O-glycans were ligands for galectin-3 and contained only a maximum single N-acetyllactosamine repeat. Consistent with the glycan analysis, surface plasmon resonance binding assays indicated that the cancer-associated glycoforms of MMP-9 bound galectin-3 with an affinity and avidity significantly reduced compared with those of the natural neutrophil MMP-9. Galectin-3 exists as a multimer that also binds laminin, providing a means of localizing neutrophil MMP-9 in the extracellular matrix (ECM). The analytical data presented here suggest that MMP-9 glycoforms secreted by tumor cells are unlikely to be tethered at the site of secretion, thus promoting more extensive cleavage of the ECM and providing a rationale for the contribution that gelatinase B makes to cancer cell metastasis.

The hemopexin and O-glycosylated domains tune gelatinase B/MMP-9 bioavailability via inhibition and binding to cargo receptors.

Gelatinase B/matrix metalloproteinase-9 (MMP-9), a key regulator and effector of immunity, contains a C-terminal hemopexin domain preceded by a unique linker sequence of approximately 64 amino acid residues. This linker sequence is demonstrated to be an extensively O-glycosylated (OG) domain with a compact three-dimensional structure. The OG and hemopexin domains have no influence on the cleavage efficiency of MMP-9 substrates. In contrast, the hemopexin domain contains a binding site for the cargo receptor low density lipoprotein receptor-related protein-1 (LRP-1). Furthermore, megalin/LRP-2 is identified as a new functional receptor for the hemopexin domain of MMP-9, able to mediate the endocytosis and catabolism of the enzyme. The OG domain is required to correctly orient the hemopexin domain for inhibition by TIMP-1 and internalization by LRP-1 and megalin. Therefore, the OG and hemopexin domains down-regulate the bioavailability of active MMP-9 and the interactions with the cargo receptors are proposed to be the original function of hemopexin domains in MMPs.