| Abstract |
Fibrosarcoma is a rare malignant tumor originating from fibroblasts. Different
cell lines of fibroblastic origin have been shown to have an abundant ECM with a
high content and turnover of hyaluronan (HA) and proteoglycans. HA modulates key
cancer cell functions through interaction with its CD44 and receptor for hyaluronic
acid-mediated motility (RHAMM) receptors. HA was recently found to regulate the
migration of fibrosarcoma cells in a manner specifically dependent on its size. It is
observed in neonates, in middle-aged and elderly and represents a 10-20% of tumors
that occur in childhood. More than half of the patients die within 5 years. The
treatment of choice is the excision of the tumor and chemotherapy. The causes of of
this cancer type development remain unknown and its diagnosis is usually made by
exclusion.
The extracellular matrices (ECMs) are complicated structures that surround
and support cells within tissues. The ECM acts as a physical scaffold to which tumor
cells attach and migrate and thus is crucial for the regulation of cell motility,
proliferation, invasion, and metastasis. HA, a glycosaminoglycan, is a ubiquitous
component of the extracellular matrix that provides tissue homeostasis and is known
to have a fundamental role in maintaining the ECM architecture.
High levels of HA reported in tumor cells and peri-tumor stroma are suggested
to be strong independent prognostic indicators of poor outcome in breast, ovarian,
gastric, and colorectal cancers. In tumor cell microenvironment systems, HA is able to
transmit signals originating from the ECM into the cell, and changes in its metabolism
have been linked to the promotion of cell motility, adhesion, migration, and
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metastasis. Considerable evidence indicates that HA mediates these biological
processes mostly via specific interactions with its receptors CD44 και RHAMM.
CD44 is the best characterized HA receptor and is indicated to be the principal
mediator of HA signaling. RHAMM receptor (receptor for hyaluronic acid-mediated
motility) is unique among the hyaladherins due to its variable distribution on the cell
surface, within the cytoplasm, in the nucleus, or secreted to the ECM.
The process of cell adhesion including the molecules that are involved in it
play a fundamental role in carcinogenesis. It has been reported that the reduction of
adhesion forces between cells and between cell-extracellular matrix allows some
cancer cells to separate from each other and detach from an initial tumor site to and to
migrate into another (metastasis). An important molecule that is located in adhesion
sites, focal adhesion kinase (FAK), induces the adhesive ability of the cells by
activating intracellular signaling pathways. The activity of FAK is triggered by
various molecules such as Src and ERK kinases that induce its phosphorylation.
Moreover, carcinogenesis is a process characterized by deregulated cell
proliferation with a simultaneous suppression of apoptosis leading to tumor formation
and loss of tissue organization. The canonical Wnt signaling pathway regulates the
expression of several fundamental genes that participate in crucial cellular functions
such as proliferation, cell differentiation, and survival. The dysfunction of the Wnt
cascade is associated with cancer pathogenesis in various tissues. An intracellular
protein, β-catenin, is a crucial downstream mediator of the Wnt signaling cascade
which can enhance carcinogenic events under pathological conditions. The β-catenin
protein has the ability to induce cell proliferation by translocating to the nucleus
where it upregulates the transcriptional activity of T-Cell Factor (TCF)/Lymphoid
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Enhancer Factor (LEF) and the expression of specific target genes, such as cyclin-D1,
c-Jun and c-Myc.
The aim of this study was to investigate HA/RHAMM interactions in the
regulation of cell adhesion and proliferation of HT1080 fibrosarcoma cells. Moreover,
we explored potential extracellular and intracellular signaling pathways that may
regulate these specific cellular functions. Our ultimate goal was to identify new
diagnostic markers for fibrosarcoma as well as target molecules suitable for the
treatment of the disease.
Initially, we investigated the effect of HA/RHAMM signaling on the ability of
HT1080 fibrosarcoma cells to adhere onto fibronectin. Low molecular weight HA
(LMWHA) significantly increased (p ≤ 0,01) the adhesion capacity of HT1080 cells,
which high molecular weight HA inhibited. The ability of HT1080 RHAMMdeficient
cells, but not of CD44-deficient ones, to adhere was significantly decreased
(p ≤ 0,001) as compared to control cells. Importantly, the effect of LMWHA on
HT1080 cell adhesion was completely attenuated in RHAMM-deficient cells. In
contrast, adhesion of RHAMM-deficient cells was not sensitive to high molecular
weight HA treatment, which identifies RHAMM as a specific conduit of the LMWHA
effect. Western blot and real time-PCR analyses indicated that LMWHA significantly
increased RHAMM transcript (p ≤ 0,05) and protein isoform levels (53%, 95 kDa;
37%, 73 kDa) in fibrosarcoma cells. Moreover, Western blot analyses showed that
LMWHA in a RHAMM-dependent manner enhanced basal and adhesion-dependent
ERK1/2 and focal adhesion kinase (FAK) phosphorylation in HT1080 cells.
Utilization of a specific ERK1/2 inhibitor completely inhibited (p ≤ 0,001) LMWHAdependent
adhesion, suggesting that ERK1/2 is a downstream effector of
LMWHA/RHAMM signaling. Likewise, the utilization of the specific ERK1 inhibitor
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resulted in a strong down-regulation of FAK activation in HT1080 cells, which
identifies ERK1/2 as a FAK upstream activator.
Moreover, we investigated the effect of HA/RHAMM signaling on HT1080
fibrosarcoma cells proliferation. An in vitro proliferation assay showed that both low
molecular weight ΗΑ and high molecular weight ΗΑ (HMWHA) significantly
increased (p ≤ 0,01 and p ≤ 0,05 respectively) ΗΤ1080 cell proliferation. In order to
investigate the direct role of RHAMM on HT1080 cell growth, we utilized siRNA
specific for the RHAMM gene. The demonstrated increase of HT1080 cells’
proliferation (p ≤ 0,01) due to LMWHA was completely inhibited in siRHAMMtreated
cells as compared with the siRHAMM controls. In an attempt to locate
candidate molecules participating in HA/RHAMM signaling pathway, we examined
factors regulating the expression of RHAMM. Specifically, with the use of a specific
ERK1/2 inhibitor, both the basal and the LMWHA induced proliferation levels were
reduced. These results show that ERK1/2 is a mediator of LMWHA/RHAMM
signaling. Furthermore, the downregulation of RHAMM led to a significant reduction
of β-catenin expression both in mRNA (p ≤ 0,05) and protein levels (p ≤ 0,01). Then,
we examined the effect of β-catenin in fibrosarcoma cell proliferation. The
downregulation of β-catenin caused a reduction in the basal levels as well as in the
LMWHA induced fibrosarcoma cell proliferation. LMWHA altered significantly the
β-catenin expression in mRNA (p ≤ 0,01) and protein levels (p ≤ 0,05). The addition
of LMWHA resulted in an increase of β-catenin protein expression in the nucleus.
Moreover, the downregulation of both β-catenin and RHAMM receptor caused a
reduction of c-myc (p ≤ 0,001; p ≤ 0,01) and c-jun (p ≤ 0,001; p = statistically non
significant) respectively, which are important modulators of the cell cycle.
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Our results suggest that RHAMM/HA interaction regulates fibrosarcoma cell
adhesion via the activation of FAK and ERK1/2 signaling pathways. The present
study also suggests that RHAMM is a novel β-catenin intracellular binding partner,
protecting β-catenin from degradation and supporting the nuclear translocation of this
key cellular mediator, which results in c-myc activation and enhanced fibrosarcoma
cell growth. From the above, therefore, it can be inferred that the RHAMM receptor
and LMWHA may pose an investigation field for obtaining useful conclusions which
aim at promoting the receptor to a diagnostic marker and both LMWHA and
RHAMM as potential target molecules for treating fibrosarcoma.
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Μελέτη της δράσης των εξωκυττάριων πρωτεογλυκανών και γλυκοζαμινογλυκανών στις λειτουργίες των κυττάρων του ινοσαρκώματος και διερεύνηση του ρόλου των αυξητικών παραγόντων
