Expression of the type-1 repeats of thrombospondin-1 inhibits tumor growth through activation of transforming growth factor-beta

Abstract

In the present study, the type-1 repeats of thrombospondin-1 (TSP-1) were transfected into A431 cells. Expression of all three type-1 repeats (3TSR) and expression of just the second type-1 repeat containing the transforming growth factor (TGF)-beta activating sequence KRFK (TSR2 + KRFK) significantly inhibited in vivo tumor angiogenesis and growth in nude mice. These tumors expressed increased levels of both active and total TGF-beta. A431 cells expressing the second type-1 repeat without the KRFK sequence (TSR2 - KRFK) produced tumors that were slightly larger than the 3TSR and TSR2 + KRFK tumors. These tumors expressed elevated levels of active TGF-beta but levels of total TGF-beta were not different from control tumors. Injection of the peptide, LSKL, which blocks TSP-1 activation of TGF-beta, reversed the growth inhibition observed with cells expressing TSR2 + KRFK to a level comparable to controls. Various residues in the WSHWSPW region and the VTCG sequence of both TSR2+/- KRFK were mutated. Although mutation of the VTCG sequence had no significant effect on tumor growth, mutation of the WSHWSPW sequence reduced inhibition of tumor growth. These findings suggest that the inhibition of tumor angiogenesis and growth by endogenous TSP-1 involves regulation of both active and total TGF-beta and the sequences KRFK and WSHWSPW in the second type-1 repeat.

Figure 1

A: Diagram of TSP-1 highlighting the different functional domains of the molecule. B: TSR expression level in the stably transfected A431 clones. Twenty μg of RNA isolated from one stably transfected A431 clone were probed with TSP-1 type 1 repeat and β-actin probes in a RNase protection assay.

Figure 2

A: In vivo growth of stably transfected A431 clones in nude mice. Ten tumors from five mice were measured for each clone. Average tumor volume of two clones (pSecTag) and three clones (TSR2 with and without the KRFK sequence and 3TSR) are shown ± SD. B: TSR RNA levels in the tumors. RNA was isolated from five different tumors derived from one clone for each construct. Twenty μg of RNA isolated from tumors were probed with TSR and β-actin probes in a RNase protection assay.

Figure 3

A to D: Immunofluorescence double staining showing TSR (green)- and CD31 (red)-positive blood vessels in tumor sections. E to H: Macroscopic appearance of orthotopic skin tumors after 3 weeks of in vivo growth. I to L: Immunohistochemical staining of tumor blood vessels using a CD31-specific antibody. A, E, I: PSecTag; B, F, J: TSR2 − KRFK; C, G, K: TSR2 + KRFK; D, H, L: 3TSR. Scale bars: 200 μm (A); 5 mm (E); 200 μm (I).

Figure 4

Morphometric vessel analyses. A: Vessel density; B: average vessel size; C: distribution of vessel size; D: total vessel area. CD31-stained blood vessels were counted in three different ×10 fields. For each clone, tumors from five mice were evaluated. Data are expressed as mean values ± SD.

Figure 5

Hematoxylin stain of orthotopic tumors showing extensive areas of necrosis (asterisk) in 3TSR (C) and TSR2 + KRFK (D) tumor samples that are not present in pSecTag (A) and TSR2 − KRFK (B) tumors. TUNEL immunohistochemistry staining on TSR2 − KRFK (E) and TSR2 + KRFK (F) tumor sections. Red = propidium iodide (nuclei); green = TUNEL. Scale bars: 200 μm (A); 400 μm (E).

Figure 6

A: Orthotopic tumor growth in the presence of a peptide inhibitor of TSP-1 activation of latent TGF-β, LSKL, or the control peptide, SLLK. Average tumor volume of 10 tumors from five mice ± SD. B and C: TGF-β1 immunohistochemistry staining of TSR2 + KRFK tumor treated with the SLLK peptide (B) and LSKL peptide (C). Red = propidium iodide; green = activated TGF-β1. D: CD31 staining of TSR2 + KRFK tumor treated with the SLLK peptide. Scattered areas of necrosis are marked with an asterisk. E: CD31 staining of TSR2 + KRFK tumor after LSKL peptide treatment. Large angiogenic blood vessels are marked with an arrow. Morphometric analyses on average vessel size (F) or total vessel area (G) for TSR2 + KRFK tumors treated with either LSKL or SLLK peptides. CD31-stained blood vessels were counted in three different ×10 fields. Tumors from five mice were evaluated for each clone. Data are expressed as mean values ± SD. Scale bars: 400 μm (B); 200 μm (D).

Figure 7

Summary of in vivo growth of stably transfected A431 clones in nude mice. Each data line represents the average tumor measurements from at least 10 tumors in five mice. Except for the VTCG mutation, experiments were performed at least twice using two different clones. A: Average tumor volume of pSecTag, 3TSR, TSR2 + KRFK, TSR2 + KRFK mutated WSXWSPW mutated, VTCG mutated, and both sequences mutated. B: Average tumor volume of pSecTag, TSR2 − KRFK, TSR2 − KRFK mutated WSXWSPW sequence, mutated VTCG sequence, and both sequences mutated.

Figure 8

A: Active TGF-β. B: Total TGF-β. Mink lung epithelial cells expressing a luciferase construct with a TGF-β response element were used to measure TGF-β levels in tumor cryostat sections. The experiment was done in triplicates and repeated twice using the same clone and one time using a different clone. Shown is a representational experiment with one tumor clone. Data are expressed as mean values ± SD and are normalized to pSecTag control. Asterisks indicate there is a statistical significant difference between that group and the control group (P < 0.05).

Figure 9

Schematic representation of the effect of expression of the TSR-containing proteins in A431 cell tumor microenvironment. KRFK-dependent activation of TGF-β by TSR2 + KRFK (shaded box) results in suppression of tumor cell growth. TSR2 + KRFK expression also results in an increase in the level of total TGF-β that may be derived from the tumor cells or stromal cells. The increased level of active TGF-β in the TSR2 − KRFK-expressing tumors indicates that these proteins up-regulate another mechanism for activation. This activation may take place in the extracellular matrix or on the surface of stromal cells. Published data indicate that the TSR-containing proteins inhibit angiogenesis through interaction with CD36 on endothelial cells. In this study, changes in vessel morphology are only observed with 3TSR and TSR2 + KRFK indicating that active TGF-β may also contribute to the inhibition of angiogenesis.