Membrane-bound proteases have recently emerged as critical mediators of tumorigenesis, angiogenesis, and metastasis. However, the mechanisms by which they regulate these processes remain unknown. As the cell surface serine protease fibroblast activation protein (FAP) is selectively expressed on tumor-associated fibroblasts and pericytes in epithelial tumors, we set out to investigate the role of FAP in mouse models of epithelialderived solid tumors. In this study, we demonstrate that genetic deletion and pharmacologic inhibition of FAP inhibited tumor growth in both an endogenous mouse model of lung cancer driven by the K-ras G12D mutant and a mouse model of colon cancer, in which CT26 mouse colon cancer cells were transplanted into immune competent syngeneic mice. Interestingly, growth of only the K-ras G12D -driven lung tumors was also attenuated by inhibition of the closely related protease dipeptidyl peptidase IV (DPPIV). Our results indicate that FAP depletion inhibits tumor cell proliferation indirectly, increases accumulation of collagen, decreases myofibroblast content, and decreases blood vessel density in tumors. These data provide proof of principle that targeting stromal cell-mediated modifications of the tumor microenvironment may be an effective approach to treating epithelial-derived solid tumors.

In previous studies, we showed that acute administration of iron to intact rats or to rat hepatoma cells in culture induces synthesis of the iron-storage protein ferritin by activating translation of inactive cytoplasmic ferritin mRNAs for both the heavy (H) and the light (L) subunits. In the course of activation, these ferritin mRNAs are recruited onto polysomes. To elucidate the structural features of these mRNAs involved in the translational response to iron, a chimera was constructed from the 5' and 3' untranslated regions (UTRs) of ferritin L subunit mRNA fused to the reading frame of the mRNA of bacterial chloramphenicol acetyltransferase (CAT).This chimera and deletion constructs derived from it were introduced into a rat hepatoma cell line by retrovirus-mediated gene transfer. The complete chimera showed increased CAT activity in response to iron enrichment of the medium, whereas deletion of the rst 67 nucleotides of the 5' UTR, which contain a highly conserved sequence, caused loss of regulation by iron. Whereas cis-acting sequences located in the 5' flanking regions of many genes have been repeatedly implicated in modulating their transcriptional expression, we report here a specific regulatory translational sequence found within the 5' UTR of a eukaryotic mRNA.The iron-storage protein ferritin (Mr 450,000) consists in mammals of two types of subunits, a heavier H (M, 22,000) and a slightly lighter L (Mr 20,000) type. Iron administered to rats (1) or added to rat hepatoma cells in culture (2) rapidly increases synthesis of both subunits by mobilizing inactive ferritin L and H mRNAs onto polysomes. This report explores features of the rat ferritin L mRNA involved in this activation process. The 5' untranslated regions (UTRs) of both ferritin mRNAs are unusually long (210 and 168 nucleotides, respectively, for the L and H subunits of the rat), thus differing from most eukaryotic messages with 5' UTRs commonly of 30-60 nucleotides (3). As determined by computer-aided analysis, the first 75 nucleotides of the 5' UTR of L and H subunits in human, rat, and frog yield putative stem-loop structures with calculated free energies of formation (AG values) of -46.1 kcal/mol for rat L mRNA ( Fig. 1) and -40.3 kcal/mol for rat H mRNA (1 kcal = 4.18 kJ). Within this stem-loop structure there is a highly conserved stretch of 28 nucleotides showing almost perfect homology in both H and L mRNAs so far isolated from human (4, 5), rat (6, 7), chicken (8), and frog (9) compared with overall homology of the complete L and H subunit mRNAs of only about 50% in rat and human. To examine the molecular basis for the translational regulation of ferritin mRNA, notably involvement of the untranslated regions, a chimeric gene containing the 5' and 3' UTRs of the ferritin L subunit mRNA fused to the reading frame and 3' UTR of the bacterial chloramphenicol acetyltransferase (CAT) gene has GU C G AU

A few hours after administering iron to rats, liver ferritin synthesis increases several fold. However, Northern blot analysis with cDNA probes for ferritin light (L) and heavy (H) subunit mRNAs failed to show an increase in total population of either messenger. Cytoplasmic distribution of ferritin messages was therefore investigated in control and iron administered rats killed at 3.5 hours. The liver post-mitochondrial supernatant was fractionated on a sucrose gradient to separate polyribosomes, monosomes, ribosomal subunits and cell sap. RNA extracted from each fraction and analyzed using Northern blotting showed that 65% of the total mRNA population for each subunit was present in the cell sap of control rats, presumably as mRNP particles since ribosomal RNA was absent from this fraction. After iron administration, these reserves of free mRNA were recruited onto the polysomes, reducing the free mRNA pool to 15% of the total. We interpret this to be due to activation of blocked ferritin messages on entry of iron into the cell.

Fibroblast activation protein-α (FAP) is a cell surface, serine protease of the post-prolyl peptidase family that is expressed in human breast cancer but not in normal tissues. Previously, we showed that FAP expression increased tumor growth rates in a mouse model of human breast cancer. Here the role of the proteolytic activities of FAP in promoting tumor growth, matrix degradation and invasion was investigated. Mammary fat pads of female SCID mice were inoculated with breast cancer cells that express FAP and the mice treated with normal saline or Val-boroPro (talabostat); Glu-boroPro (PT-630); or 1-[[(3-hydroxy-1-adamantyl)amino]acetyl]-2-cyano-(S)-pyrrolidine (LAF-237) that inhibit prolyl peptidases. Other mice were injected with breast cancer cells expressing a catalytically inactive mutant of FAP and did not receive inhibitor treatment. PT-630 and LAF-237 did not slow growth of tumors produced by any of the three cell lines expressing FAP. Talabostat slightly decreased the growth rates of the FAP-expressing tumors but because PT-630 and LAF-237 did not, the growth retardation was likely not related to the inhibition of FAP or the related post-prolyl peptidase dipeptidyl peptidase IV. Breast cancer cells expressing a catalytically inactive mutant of FAP (FAP(S624A)) also produced tumors that grew rapidly. In vitro studies revealed that cells expressing wild type FAP or FAP(S624A) degrade extracellular matrix (ECM) more extensively, accumulate higher levels of matrix metalloproteinase-9 (MMP-9) in conditioned medium, are more invasive in type I collagen gels, and have altered signaling compared to control transfectants that do not express FAP and form slow growing tumors. We conclude that the proteolytic activity of FAP participates in matrix degradation, but other functions of the protein stimulate increased tumor growth.

Background A lack of biomarkers that identify patients at risk for severe osteoarthritis (OA) complicates development of disease-modifying OA drugs. Objective To determine whether infl ammatory genetic markers could stratify patients with knee OA into high and low risk for destructive disease. Methods Genotype associations with knee OA severity were assessed in two Caucasian populations. Fifteen single nucleotide polymorphisms (SNPs) in six infl ammatory genes were evaluated for association with radiographic severity and with synovial fl uid mediators in a subset of the patients. Results Interleukin 1 receptor antagonist (IL1RN) SNPs (rs419598, rs315952 and rs9005) predicted KellgrenLawrence scores independently in each population. One IL1RN haplotype was associated with lower odds of radiographic severity (OR=0.15; 95% CI 0.065 to 0.349; p<0.0001), greater joint space width and lower synovial fl uid cytokine levels. Carriage of the IL1RN haplotype infl uenced the age relationship with severity.