| Abstract |
This Ph.D. thesis studies how small leucine-rich proteoglycans (SLRPs) interact with the molecular mechanisms that regulate mesenchymal origin tumors' biological functions. Sarcomas are tumors that develop from differentiated cells of the human body's connective tissues. They can arise from tissues, like bone, cartilage, blood, or vessels, and they are classified into two categories depending on the tissue and the cell type of their origin. Thus, there are bone sarcomas, like osteosarcoma, chondrosarcoma, and sarcomas of soft tissues, like fibrosarcoma. In this study, we aimed to identify the interaction of lumican and biglycan with the signaling pathways that participate in bone sarcomas progression in four human cell lines, HTB94 human chondrosarcoma cell line and MG63, U-2OS and Saos-2 human osteosarcoma cells.
Extracellular matrix (ECM) is a complex system of proteoglycans (PGs), fibrous proteins, and hyaluronic acid, which supports cells in tissues but participates in the modulation of the cellular signaling cascades. The ECM components create and transfer different signals between cells/tissues, affecting their characteristics and functions. ECM also acts as a reservoir for growth factors like insulin-like growth factor I (IGF-I) and other biologically significant macromolecules able to change cellular behavior. Malignant bone cells produce an aberrant non-mineralized or partly mineralized ECM compared to healthy cells, whose components participate in signaling pathways connected to specific bone pathogenic phenotypes. The deregulated expression of different ECM molecules affects sarcomas’ biological functions.
PGs are molecules that consist of glycosaminoglycan chains (GAGs) covalently bound into a protein core. These proteins undergo various post-translational changes and can affect cellular function and cancer progression through a plethora of signaling pathways. Many studies have shown that the secreted to the ECM PGs, SLRPs, directly interact with the tumor microenvironment, transmit cues from the ECM to the cells, and finally act as signaling molecules that control cellular functions, like proliferation migration, differentiation, and also modulate the ECM composition. It is also shown that SLRPs interact with tyrosine kinase receptors, which affects their downstream signaling and cellular behavior. More specifically, some SLRPs, like biglycan and lumican, participate in bone formation and homeostasis regulation and have specific roles in different phases of these processes. Therefore, PGs act as regulators of signaling molecules and pathways and could be used as target molecules either for therapeutic purposes or as diagnostic molecules for pathological conditions.
IGF-I is a growth hormone with proven oncogenic properties. Most of its functions are performed through two main signaling pathways, PI3K/Akt (phosphoinositide 3-kinase/ serine/threonine kinase Akt) and MAPK (mitogen-activated protein kinase). We found that endogenous lumican is a novel regulator of HTB94 cell growth. More specifically, it was shown that lumican is the main expressed and secreted SLRP in HTB94 human chondrosarcoma cells, while decorin and biglycan are poorly expressed. Lumican positively affects the proliferation of HTB94 cells as lumican deficiency significantly inhibits basal and IGF-I-induced HTB94 cell growth. The oncogenic action of IGF-I is mediated by its receptor, IGF-IR, whose phosphorylation levels are strongly attenuated in lumican-deficient cells. Furthermore, lumican expression levels affect ERK1/2 phosphorylation, which seems crucial to IGF-I-dependent HTB94 cell growth. Moreover, lumican deficiency led to increased mRNA levels of the oncosuppressive gene, p53 suggesting that lumican facilitates chondrosarcoma cell growth through an IGF-IR/ ERK1/2/p53 signaling cascade.
Biglycan, a class I SLRP, has an active role in bone formation, development, and homeostasis. This study reveals novel mechanisms that biglycan regulates to enhance osteosarcoma growth and cell chemoresistant phenotype. Biglycan expression correlates to aggressive osteosarcoma phenotype and resistance to chemotherapy. A constitutive input of signaling of IGF-IR signaling in sarcoma progression has been established, which is connected with chemoresistant phenotypes. Here, we showed that biglycan, by forming a complex with the IGF-IR, activates the receptor signaling pathway to promote biglycan-secreting MG63 osteosarcoma cell growth. Computational models of IGF-IR and biglycan docking suggest that biglycan binds IGF-IR dimer via its concave surface. Our binding free energy calculations indicate the formation of a stable complex. Biglycan binding results in prolonged IGF-IR activation leading to protracted IGF-IR-dependent cell growth response of the poorly-differentiated MG63 cells. Moreover, biglycan facilitates the internalization and sumoylation-enhanced nuclear translocation of IGF-IR and its DNA binding in MG63 cells. The tyrosine kinase activity of the receptor mediates this mechanism. Furthermore, biglycan increases the expression of endothelial–mesenchymal transition (EMT) and aggressiveness markers vimentin and fibronectin in MG63 cells. Interestingly, this mechanism is invalid in moderately and well-differentiated, biglycan non-expressing U-2OS and Saos-2 OS cells. In addition, we have shown that biglycan downregulates the expression of the tumor-suppressor gene, Phosphatase, and tensin homolog (PTEN). In contrast, biglycan deficiency increases cells’ response to the chemotherapeutic drug doxorubicin.
Autophagy is a well-studied catabolic mechanism that provides cells energy and macromolecular precursors. However, it is deregulated in many pathological conditions, including cancer, and its role in tumor biology is dual and context-dependent. Rapamycin inhibits the mammalian target of the rapamycin (mTOR) signaling cascade, activating the autophagic flux. In osteosarcoma, autophagy is deregulated, affecting response to chemotherapy, while the inactivation of tumor suppressors, including PTEN, seems to regulate autophagy activation and the autophagic flux. Biglycan led to PTEN downregulation, so, next, we tested its role on autophagy activation in our model. We found that biglycan inhibits autophagy in two osteosarcoma cell lines, while rapamycin-induced autophagy decreases biglycan expression in MG63 osteosarcoma cells and abrogates the biglycan-induced cell growth increase.
Wnt/ β-catenin signaling cascade is another well-established pathway activated in osteosarcoma. Crosstalk between autophagy and Wnt/β-catenin has previously been detected at several levels in cancer models. Interestingly, in a previous study, we identified crosstalk between β-catenin and IGF-IR, where IGF-IR is activated through non-canonical β-catenin signaling, resulting in increased biglycan expression. Here, we proved that rapamycin inhibits β-catenin translocation to the nucleus, inhibiting the Wnt pathway and reducing biglycan’s colocalization with the Wnt co-receptor low-density lipoprotein receptor-related protein 6 (LRP6). The increased deposition of β-catenin inhibits autophagy and protects MG63 cells against apoptosis through a biglycan-dependent mechanism. Furthermore, biglycan exhibits protective effects against doxorubicin in MG63 osteosarcoma cells through an autophagy-dependent manner. Co-treatment of these cells with rapamycin and doxorubicin enhances cells’ response to doxorubicin by decreasing biglycan and β-catenin expression. Biglycan deficiency leads to increased caspase-3 activation, suggesting increased apoptosis of biglycan-deficient cells treated with doxorubicin.
Computational models of LRP6 and biglycan complexes suggest that biglycan changes the receptor’s ability to interact with other signaling molecules by affecting the interdomain bending angles in the receptor structure. Biglycan binding to LRP6 activates the Wnt pathway and β-catenin nuclear translocation by disrupting β-catenin degradation complex formation. Interestingly, this mechanism is not followed in moderately differentiated, biglycan non-expressing U-2OS osteosarcoma cells.
To sum up, biglycan exhibits protective effects against the doxorubicin in MG63 osteosarcoma cells by promoting IGF-IR prolonged activation and nuclear translocation, activating the Wnt signaling pathway, and inhibiting autophagy. These data reveal new aspects of biglycan’s interaction network and its role in receptor signaling. They indicate that this SLRP is an essential regulator of osteosarcoma progression and response to chemotherapy.
In conclusion, the above-described mechanisms reveal SLRP’s involvement in mesenchymal origin tumor progression. Furthermore, their interaction with different signaling pathways indicates them as potential diagnostic tools and therapeutic targets in sarcomas biology.
|
Collections
