The role of collagen and transforming growth factor-beta in mesothelioma growth
Malignant Mesothelioma (MM) is an aggressive tumour of the lung pleura with very poor patient prognosis. MM is unresponsive to all treatment regimes and over the next 30 years an estimated 100,000 fatalities will occur from this disease in Western Europe alone. MM is an extremely fibrous tumour containing abundant amounts of extracellular matrix, including collagen. Cell-matrix interactions are important for tumour progression and MM cells synthesise collagen as well as transforming growth factor-beta (TGF-beta), a key regulator of collagen production. Thiaproline, a proline analogue, was used to inhibit collagen production in MM cells in vitro and in vivo to test the hypothesis that collagen is important for MM growth. Murine MM cells (AC29) were incubated with increasing concentrations of thiaproline with and without TGF-beta1 (1ng/ml) and incorporation of tritiated thymidine and hydroxyproline levels measured as an index of cell proliferation and collagen production (nM hydroxyproline/106 cells) respectively. The effect of thiaproline on tumour growth (median weight, mg [range]) was determined in syngeneic mice subcutaneously injected in the flank with 106 MM cells. In vitro, 10mM thiaproline significantly reduced cell proliferation by over 65% (control 27400 3200; thiaproline 8950 1000 dpm/well, p 0.001) and basal and TGF-beta1-induced collagen production by over 50% (control 1.6 0.1; thiaproline 0.7 0.1, p 0.005) and 60% (TGF-beta1 3.8 0.2; TGF-beta1+thiaproline 1.3 0.1, p 0.005) respectively. At 10 days after injection of cells, 100mg/kg/day thiaproline reduced median tumour weight by over 80% (control 58 [30-105]; thiaproline 10.5 [5-12], p 0.01) but no significant difference was seen at 18 days. To summarise, thiaproline inhibited MM cell proliferation, collagen production and delayed tumour growth, suggesting an important role for collagen in MM growth. MM also secretes TGF-beta, which regulates cell growth and collagen production. MM cells produce 30 - 70 times more TGF-beta than untransformed normal mesothelial cells. To investigate the role of specific TGF-beta isoforms in MM cell proliferation and collagen production and to determine the relative importance in tumour growth, selective neutralising antibodies to TGF-beta1 and TGF-beta2 were used in conjunction with a pan-specific TGF-beta antibody which neutralised all TGF-beta isoforms. Through the use of the neutralizing antibodies it was determined that the predominant isoform produced by AC29 cells into conditioned medium was TGF-beta2. TGF-beta and control antibodies had no effect on AC29 cell proliferation or collagen production in vitro. TGF-beta and control antibodies (5mg/kg) were injected intraperitoneally into the murine flank model of MM tumour growth at three-day intervals until the end of the experiment, when the tumours were resected and weighed. TGF-beta2 antibody administered in vivo significantly decreased median tumour weight compared with PBS and control antibody (PBS control 67.5 [27-114], control antibody 57 [35-193], TGF-beta2 antibody 27 [5-51], p 0.001 for both controls). Pan-specific antibody reduced median tumour weight compared to PBS control (PBS control 46.5 [25-109], control antibody 36 [19-107], pan-specific TGF-beta antibody 30 [14-66], p 0.05 vs. PBS control). Antibodies against TGF-beta1 had no effect on tumour growth. Collagen analysis of the tumours revealed a significantly lower concentration of collagen in the pan-specific antibody treated tumours (PBS control 9.11 0.76, pan-specific TGF-beta antibody 4.03 0.68 nM hydroxyproline / mg tumour, p 0.0001). Collectively, this thesis has clearly demonstrated that TGF-beta-induced collagen production may be an important aspect of MM tumour growth, and inhibition of either collagen production or TGF-beta activity can delay MM tumour growth with a resultant decrease in tumour collagen content. A dual approach targeting both collagen and TGF-beta production may be of benefit in the treatment of this disease in which the current therapies are inadequate.