Microfibril associated protein 4 (MFAP4) is a carrier of the tumor associated carbohydrate sialyl-Lewis x (sLe^x) in pancreatic adenocarcinoma

Highlights

• MFAP4 is a carrier of sLe^x in pancreatic adenocarcinoma (PDA).

• MFAP4 is not produced by pancreatic cancer cells but it is found in the PDA stroma.

• MFAP4-sLe^x glycoform is only found in PDA and not in pancreatic control samples.

• MFAP4-sLe^x glycoform may have a role interacting with the PDA dense fibrotic stroma and ECM proteins

Abstract

Late diagnosis of pancreatic ductal adenocarcinoma (PDA) is one of the reasons of its low 5-year survival rate and it is due to its unspecific symptoms during the first stages of the disease and the lack of reliable serological markers. Since PDA shows an altered glycan expression, here we have focused on finding novel potential biomarkers, namely glycoproteins that express the tumor associated carbohydrate structure sialyl-Lewis x (sLe^x), which is described in PDA. Through a glycoproteomic approach, we have analyzed target proteins containing sLe^x from PDA tissues by 2DE and immunodetection techniques, and have identified by mass spectrometry the protein MFAP4 as a carrier of sLe^x in PDA. MFAP4 showed a higher expression in PDA tissues compared with pancreatic control tissues. In addition, the colocalization of sLe^x over MFAP4 was found only in PDA and not in control pancreatic tissues. The analysis of MFAP4 expression in PDA cell lines and their secretome, in combination with immunohistochemistry of pancreatic tissues, revealed that MFAP4 was not produced by PDA cells, but it was found in the pancreatic extracellular matrix. The specificity of MFAP4 glycoform containing sLe^x in PDA tissues shows its relevance as a potential PDA biomarker.

Significance

Despite advances in the field of cancer research, pancreatic ductal adenocarcinoma (PDA) lacks of a specific and sensitive biomarker for its early detection, when curative resection is still possible before metastases arise. Thus, efforts to discover new PDA biomarkers represent the first line in the fight against the increase of its incidence reported in recent years. Glycan alterations on glycoconjugates, such as glycoproteins have emerged as a rich source for the identification of novel cancer markers. In the present work, we aimed to shed light on novel biomarkers based on altered glycosylation in PDA, in particular those glycoproteins of PDA tissues carrying the tumor carbohydrate antigen sialyl-Lewis x (sLe^x). Through a glycoproteomic approach, we have shown that the glycoprotein MFAP4 carries sLe^x in PDA tissues and not in control pancreatic tissues. MFAP4 is found in the extracellular matrix in PDA and although its role in cancer progression is unclear, its sLe^x glycoform could be a potential biomarker in pancreatic ductal adenocarcinoma.

Introduction

Pancreatic ductal adenocarcinoma (PDA), the most frequent pancreatic tumor, presents the lowest five-year survival rate of all solid tumors [1]. In addition to its aggressiveness and high resistance to available therapies, one of the causes of PDA dismal outcome is the absence of tumor markers for its early detection. Late diagnosis due to the lack of accurate diagnostic markers and non-specific symptoms favors the poor outcome of the patients [2], which are usually diagnosed at advanced stages, once metastases have occurred, thus reducing the possibility of curative resection and the chances of survival [3,4]. The carbohydrate antigen sialyl-Lewis a (sLe^a), also known as CA 19–9, is the only serum biomarker approved for PDA. This marker provides valuable prognostic and recurrence information, and it is useful to predict resectability and response assessment in neoadjuvant therapy [[5], [6], [7]]. However, CA 19–9 is not useful for PDA diagnosis because of its low positive predictive value (0.5–0.9%) and its sensitivity and specificity in symptomatic patients (79–81% and 82–90% respectively) [8]. Moreover, it is poorly expressed in early PDA stages and its levels can be also elevated in other pathologies such as obstructive jaundice, liver cirrhosis and inflammatory processes like pancreatitis [9,10]. In addition, CA 19–9 is not universally expressed in PDA because a percentage of the Caucasian population (7%–10% of general population) lacks a functional FUT3 allele (termed Lewis genotype negative Le a-b-), being the synthesis of CA 19–9 from the precursor carbohydrate antigen DUPAN-2 not possible in those patients and this consequently generates false-negative results [[11], [12], [13], [14], [15]]. Overall, to improve PDA patients' outcomes, there is an urgent need to find novel tumor markers that could overcome the limitations of the CA 19–9, allowing for the detection of PDA in the time-window between the initial mutations and the onset of cancer and distal metastasis.

Altered glycosylation occurs during tumor progression, and some of the tumor glycoconjugates, including glycoproteins and mucins, with altered glycosylation can reach to the bloodstream and become cancer biomarkers [16]. A high percentage of tumor biomarkers available nowadays are glycoproteins or mucins such as CA 125, CA 15–3, CA 19–9, CA 72–4, or PSA [17,18]. In this regard, there is a growing tendency to study changes in the glycosylation of protein tumor markers to improve their sensitivity and specificity [19,20]. The study of altered glycosylation of proteins from tumors represents a powerful new tool to discover novel candidates, by using high-throughput glycan structural analysis [21] or lectins and antibodies against carbohydrate antigens [[22], [23], [24]]. In this work we have focused on finding novel PDA glycoproteins carrying an aberrant carbohydrate structure associated to PDA such as sialyl-Lewis x (sLe^x). SLe^x is de novo expressed in human PDA cells and potentiates cancer features such as cell migration and invasion [[25], [26], [27], [28], [29]].

In this work, a glycoproteomic approach to identify glycoproteins containing sLe^x from human PDA tissues was performed. PDA tissues were chosen as a source of tumor proteins that can carry such glycan structure. 2D electrophoresis of protein cell lysates from tissues followed by Western Blot (WB) with antibodies against sLe^x indicated the spots of the glycoproteins that were sLe^x carriers. Mass Spectrometry (MS) analysis of these target proteins allowed us to find that microfibril associated protein 4 (MFAP4) was one of the carriers of the carbohydrate structure sLe^x in PDA tissues. Furthermore, to gain more insight on the role of MFAP4 in PDA, we analyzed its expression in different stages of PDA and its cellular location by immunohistochemistry, which showed its presence in the stroma surrounding neoplastic cells. Overall, this work reveals the presence of microfibril associated protein 4 in pancreatic cancer patients' tissue lysates and it explores for the first time the changes in MFAP4 glycan content under tumorigenesis, introducing the MFAP4-sLe^x glycoform as a novel potential PDA biomarker.