Altered glycosylation in pancreatic cancer: development of new tumor markers and therapeutic strategies
- Autores: Pedro Enrique Guerrero
- Directores de la Tesis: Rosa Peracaula Miró (dir. tes.), Esther Llop Escorihuela (codir. tes.)
- Lectura: En la Universitat de Girona ( España ) en 2020
- Idioma: español
- Tribunal Calificador de la Tesis: Cristina Fillat Fonts (presid.), Santiago Ruiz Martínez (secret.), Cristina Esteva Font (voc.)
- Programa de doctorado: Programa de Doctorado en Biología Molecular, Biomedicina y Salud por la Universidad de Girona
- Materias:
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- Resumen
Pancreatic Ductal Adenocarcinoma (PDA) represents more than 90% of pancreatic cancers. PDA is an extremely aggressive solid tumor, with the direst prognosis among all carcinomas. This type of pancreatic cancer is characterized by the presence of a microenvironment formed by an abundant desmoplastic reaction. The fibrotic stroma of the PDA favors the survival of the tumor and contributes to its wide resistance against current antitumor therapies. For PDA patients, tumor resection surgery is currently one of the best curative options. However, this option remains only possible for a small group of patients, since it is usually detected on late and locally advanced stages of the disease, where patients already have metastases or vascular infiltration. Currently, only about 9% of patients survive 5-years after diagnosis, and recurrence of the disease is a common feature. This poor prognosis is also due to the lack of tumor markers with enough specificity and sensitivity for the diagnosis of this tumor before metastasis occurs.
During carcinogenesis, several alterations have been observed both in the genome and the cellular physiology. One of these transformations commonly found in oncogenic transformation is aberrant glycosylation that affects the carbohydrates from the cell surface and from secreted glycoconjugates. This phenomenon confers multiple advantages on tumor cells, ranging from evasion of the immune response to the promotion of migration and cell invasion events. One of the most common alterations is the expression of new carbohydrate antigens and the synthesis of truncated glycan structures, in which sialic acid residues usually play a key role. This work has been focused specially on the role of the carbohydrate antigen Sialyl Lewis X (SLeX); a glycan structure that is only present in pancreatic cells during the development of pancreatic cancer. Previous studies of the group have shown that the overexpression of this carbohydrate antigen in PDA cells favors the aggressiveness of the tumor in vitro and in vivo, playing an important role in the metastasis process.
Under these premises, in this work we have proposed the identification of glycoproteins containing SLeX in PDA tissues for their usefulness as biomarkers for this tumor. To accomplish this goal, glycoproteins carrying SLeX present in tissues from PDA patients were separated by two-dimensional electrophoresis. The use of this technique allowed us to isolate SLeX-positive bands and subsequently detect those glycoproteins of interest by mass spectrometry. This way,the microfibril-4-associated protein (MFAP4) was identified as one of the SLeX carrier glycoproteins in tissues from PDA patients. MFAP4 expression was subsequently analyzed in different pancreatic cancer cell lines, as well as in pancreatic tissues (tumor and non-tumor) using immunohistochemical and western blot techniques. MFAP4 levels in PDA tissues were higher than in non-tumor samples from PDA patients and healthy controls. Furthermore, the expression of MFAP4 in tissues was localized in their extracellular matrix and not in the pancreatic cells. No expression of MFAP4 was detected in the panel of PDA cell lines or in their corresponding conditioned media. Co-localization of SLeX over MFAP4 in PDA tissue samples was confirmed in a panel of eight PDA tissues where all of them showed this sialylated glycoform, which was not detected in pancreatic tissue samples from healthy patients. Thus, the MFAP4-SLeX glycoform is characteristic of PDA tissues and may be found in serum, opening a window of possibilities for its subsequent study as a possible PDA biomarker.
In this work, the effect of the inhibition of the expression of the main genes that code for the enzymes responsible for the final steps of SLeX biosynthesis has been studied. The main objective has been to analyze whether their inhibition can reverse the invasive phenotype of PDA. This work has focused on the α2,3-sialyltransferases (α2,3-ST) ST3Gal III and ST3Gal IV, which add sialic acid with an α2,3 linkage to galactose of type-1 or 2 (Galβ1,3GlcNAc or Galβ1,4GlcNAc) glycan structures. To do this, firstly, the sialome of a panel from seven human PDA cell lines was characterized at the level of their cell surface glycoconjugates and their secreted glycoproteins. The level of expression of α2,3-STs and fucosyltransferases involved in the biosynthesis of antigens of the sialyl Lewis (SLe) family was also analyzed. All PDA cells showed sialic acid expression with different levels of sialylated determinants such as SLeX or SLeA as well as different degrees of α2,3-ST expression, reflecting the phenotypic heterogeneity of PDA. Capan-1 and BxPC3 cell lines that expressed both ST3GAL3 and ST3GAL4 as well as moderate-high levels of sialyl Lewis antigens were chosen as candidates for inhibition of ST3GAL3 and ST3GAL4 genes by shRNAs. Silencing of ST3GAL3 and ST3GAL4 in both cell lines led to a significant reduction in SLeX and in most of them also led to a reduction in SLeA levels, with slight increases in the α2,6-sialic acid content. To assess the effect of inhibition at the phenotypic level, different in vitro tests were carried out with transwells and microfluidic devices. These showed a significant decrease in migratory (reduction between 42 and 57%) and invasive capacities (33-67%) of the cells silenced for each of both genes. We also proved that this reduction was associated, at least in part, with the decrease in SLeX expression levels. The silenced cells of both cell lines also showed a significant decrease in adhesion and rolling to E- selectin, key protein in the extravasation process that takes place during metastasis. Taken together, all of these results indicate that the ST3GAL3 and ST3GAL4 genes play an important role in these migratory, invasive and E-selectin binding processes and therefore could be considered potential therapeutic targets against PDA.
In conclusion, the results obtained from this work highlight the importance of the sialyl Lewis X antigen and the sialyltransferases involved in its biosynthesis in PDA. On one hand, this work has shown the potential of identifying glycoproteins with sialyl Lewis X as possible biomarkers of PDA. On the other hand, it has pointed to α2,3-STs as potential targets to reduce PDA migration and invasion in future in vivo tests.
