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The globular domains of PG-M/versican modulate the proliferation–apoptosis equilibrium and invasive capabilities of tumor cells ¹

SABRINA CATTARUZZA^*,, MONICA SCHIAPPACASSI, KOJI KIMATA, ALFONSO COLOMBATTI^, and ROBERTO PERRIS^*,,2

^* Department of Evolutionary and Function Biology, University of Parma, Parma (PR), Italy;
Division for Experimental Oncology 2, The National Cancer Institute, IRCCS-CRO, Aviano (PN), Italy;
Institute of Molecular Science of Medicine, Aichi Medical University Nagakute, Aichi, Japan; and
Department of Technical and Biomedical Sciences & Microgravity, Aging, Training and Immobility Centre of Excellence, University of Udine, Udine (UD), Italy

²Correspondence: Division for Experimental Oncology 2, The National Cancer Institute, IRCCS-CRO, Via Pedemontana Occidentale 12, 33082 Aviano (PN), Italy. E-mail: rperris{at}cro.it

SPECIFIC AIMS

Studies have suggested that both globular domains of PG-M/versicans mediate several actions of these PGs on cellular processes whereas glycosaminoglycan chains are primarily responsible for sequestering signaling molecules and selectin binding. To further clarify the role of these globular domains on the behavior of transformed cells, we selectively overexpressed either domain in human sarcoma cells and have assayed the effect of these manipulations in vitro and in vivo, focusing on the phenomena of cell adhesion, migration, ECM invasion, proliferation, and apoptosis.

PRINCIPAL FINDINGS

1. Diverse effects of G1-D and G3-D on sarcoma cell proliferation
Sarcoma SK-LMS-1 cells stably transfected with G1-D or G3-D fused to the reporter GFP displayed a higher proliferation rate than wild-type and mock-transfected ones (i.e., transfected with the GFP vector alone) that was largely independent of the substrate onto which cells were grown and the presence of serum factors. This confirmed the previously reported promitotic effect of G3-D and indicated that G1-D harbored the capacity to confer similar proliferation-promoting stimuli.

2. Overexpression of G1-D and G3-D diversely affects cell adhesion and migration
Overexpression of versican globular domains did not affect cell adhesion to representative ECM molecules when assessed using centrifugal assay for fluorescence-based cell adhesion, nor did it markedly modify the random and haptotactic motility response of G1-D⁺ and G3-D⁺ cells as examined through fluorescence-assisted transmigration invasion and migration assay. In contrast, G1-D (but not G3-D) overexpression significantly increased the invasive capability of SK-LMS-1 through Matrigel, suggesting this phenomenon could be associated with a modification of the ECM exerted by the secreted G1-D (possibly in the form of a major retention of HA around cells; see below) that favored its penetration by the cells (Fig. 1 ).

View larger version (82K): [in this window] [in a new window]   Figure 1. Proliferation rates, adhesion, migration, and invasion of G1-D or G3-D overexpressing SK-LMS-1 cells. Proliferation rate was assayed in cell cycle synchronized cells grown on plastic in the absence (A) or presence (B) of serum (analogous results were obtained when cells were grown on collagen type I, Coll I). Data are expressed as the mean of 3 experiments each performed in triplicate ± SD. C) Patterns of cell adhesion of modified and unmodified cells to a selected panel of purified ECM molecules (FN, fibronectin; Coll VI, collagen type VI; LN, laminin-1). D) Haptotactic migration in response to the same ECM molecules. E) The extent of invasion of G1-D+, G3-D+, vector-transfected, and wild-type cells seeded on Matrigel-coated membranes and allowed to migrate through this basement membrane-like matrix for 24 h in the absence or presence of medium conditioned by NIH3T3 fibroblasts (NIHCM). In all cases, data represent the means ± SD of 3 independent experiments. F–H) Representative phase contrast photographs showing the ability of G1-D+ (F) and G3-D+ (G) cells to migrate across a 3D Matrigel-based substrate compared with mock-transfected ones (H).

3. Overproduction of G1-D⁺ alters tumor growth in vivo
Subcutaneous inoculation of wild-type, mock-transfected, and G1-D⁺ cells gave rise to local and slowly growing tumor masses that became conspicuous starting 4 wk after inoculation. However, tumor lesions formed by G1-D⁺ cells grew significantly faster and reached larger sizes than those generated by mock-transfected cells. These lesions also had a higher degree of vascularization, but this did not seem to be directly dependent on G1-D overexpression. This since coculturing of G1-D⁺ cells with HUVEC did not cause an augmented tubular formation by endothelial cells.

4. Effect of HA on the growth behavior of G1-D transduced cells
To verify the possibility that enhanced G1-D secretion could lead to higher accumulation of HA around the cells and thereby affect cell proliferation, we grew G1-D⁺, G3-D⁺, and control cells in the presence of increasing concentrations of soluble high M_r HA, hyaluronidase-derived HA oligosaccharides, or chondroitin sulfates. There was a dose-dependent decrease of the proliferation rate of G1-D⁺ cells, whereas G3-D⁺ and control cells were unaffected. The relative uptake of extracellular HA by G1-D-, G3-D- and mock-transfected cells was determined by incubation with fluorescein-conjugated HA. Mock-transfected and G3-D⁺ cells largely failed to retain HA on their cell surfaces, and only a smaller number of cells were able to internalize glycosaminoglycan at physiological conditions. In contrast, a significant number of G1-D⁺ cells retained HA on their surfaces and incorporated the glycosaminoglycan intracellularly. This was not due to increased levels of CD44 expression on G1-D+ cells or to divergent levels of endogenous HA production in G3-D⁺ vs. G1-D⁺ cells. In fact, preincubation of transfected and nontransfected cells with function-blocking anti-CD44 antibodies did not affect their growth rate.

To further dissect the potential cooperative role of CD44 versican G1-D in the sarcoma cells’ response to HA, we incubated transfected cells in the concomitant presence of HA and anti-CD44 antibodies. In this case, a significant restoration of the perturbing activity of HA on G1-D-triggered cell proliferation was observed. Thus, this finding was consistent with a role for the CD44-HA interaction in mediating the negative HA effect on mitosis of sarcoma cells overexpressing G1-D and ruled out that enhanced proliferation of the G1-D⁺ was directly related to a growth-promoting activity of cell surface-associated HA.

5. Anchorage-independent modulatory role of HA in G1-D-induced cell proliferation
The negative effect of HA on G1-D-induced cell proliferation could have been restricted to substrate-bound cells (2D configuration) or exerted on cells growing in a 3D configuration, typical of neoplastic cells. To verify the latter possibility, we embedded transfected and nontransfected SK-LMS-1 cells in soft agar and determined their relative capacity to form colonies in the presence or absence of exogenous HA. G1-D⁺ cells showed a significantly greater colony formation (Table 1 ), with a relative ratio of "aggregated" (i.e., large aggregates and colonies) vs. "nonaggregated" (i.e., doublets and small aggregates) cells seen after 15 days that was equal for G1-D-transfected and nontransfected cells (see below).

Formation of colonies by G1-D⁺ cells was dose-dependently abrogated by inclusion of HA but was unaffected in G3-D⁺, wild-type, and control cells. This indicated that HA specifically impeded anchorage-independent growth of tumor cells overproducing G1-D.

6. G1-D, but not G3-D, overexpression confers apoptotic resistance
From the potentiation of anchorage-independent grow induced by G1-D, we deduced that this globular domain of versican could be implicated in altering the proliferation–apoptosis equilibrium of the cells, which could have explained the sustained cell propagation observed under serum deprivation and in vivo. We therefore challenged transfectants and nontransfected cells with the cytotoxic drugs vincristine and paclitaxel or with an apoptosis-inducing anti-Fas antibody. Mock-transfected, wild-type, and G3-D overexpressing cells were highly sensitive to these treatments, showing a rapid and marked degree of programmed cell death after drug/antibody exposure as confirmed by enhanced cell surface translocation of annexin-V, activation of caspase-3, and eventually DNA fragmentation. Conversely, G1-D⁺ cells were found to be largely resistant to these apoptosis-inducing agents. We found apparently higher expression of the Bax protein G1-D⁺ cells, indicating that the protection from programmed cell death may have implicated the mitochondrial apoptotic genes.

CONCLUSIONS

Overexpression of versican G1-D and G3-D in SK-LMS-1 leiomyosarcoma cells did not appear to perturb the transcription, translation or secretion of endogenous versican and failed to affect ECM ligand preference and the levels of cell substratum avidity of the cells. Forced overproduction of the domains did not seem to influence random cell motility in response to various ECM molecules but, in the case of G1-D, altered the capability of the cells to invade and migrate through a composite basement membrane-like ECM. G1-D⁺/G3-D⁺ cells divided more extensively than unmodified cells when firmly anchored to the substrate and when forced to grow without ECM anchorage, i.e., in soft agar. Once again, we attribute this phenomenon to a potential autocrine stimulatory loop that may integrate a fine equilibrium between the ECM immobilization of HA in proximity of the cells and binding of the glycosaminoglycan to its cell surface receptor CD44. Thus, the well-known proliferation-promoting, anti-apoptotic effect mediated by the CD44-HA binding could be dose modulated by G1-mediated mobilization of HA in proximity of the cell membrane; this event could secondarily govern the internalization rate of CD44 upon occupancy.

The incremented colony formation of G1-D⁺ cells in soft agar suggested that overgrowth of the manipulated cells could have been accounted for by a shift in the proliferation/apoptosis equilibrium. Accordingly, G1-D⁺ cells overexpressed Bax and were markedly resistant to programmed cell death induced by cytotoxic drugs and engagement of the Fas pathway. The apparent perturbation of the proliferation–apoptosis balance in G1-D⁺ cells may not have been a mere artificial phenomenon of cells grown in vitro and exposed to unnatural experimental situations: after subcutaneous implantation into nude mice, G1-D⁺ cells formed substantially larger tumor masses than the unmanipulated cells. Thus, we propose that overgrowth of G1-D overexpressing tumor cells links the potentially perturbed regulation of ECM assembly and an altered pericellular HA milieu with a dysregulation of the reciprocal sensitivity of the cells to proliferation- vs. apoptosis-inducing cues (Fig. 2 ).

View larger version (36K): [in this window] [in a new window]   Figure 2. Schematic diagram.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0660fje; doi: 10.1096/fj.03-0660fje

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