Suppression of tumor growth in mice by rationally designed pseudopeptide inhibitors of fibroblast activation protein and prolyl oligopeptidase

Abstract

Tumor microenvironments (TMEs) are composed of cancer cells, fibroblasts, extracellular matrix, microvessels, and endothelial cells. Two prolyl endopeptidases, fibroblast activation protein (FAP) and prolyl oligopeptidase (POP), are commonly overexpressed by epithelial-derived malignancies, with the specificity of FAP expression by cancer stromal fibroblasts suggesting FAP as a possible therapeutic target. Despite overexpression in most cancers and having a role in angiogenesis, inhibition of POP activity has received little attention as an approach to quench tumor growth. We developed two specific and highly effective pseudopeptide inhibitors, M83, which inhibits FAP and POP proteinase activities, and J94, which inhibits only POP. Both suppressed human colon cancer xenograft growth >90% in mice. By immunohistochemical stains, M83- and J94-treated tumors had fewer microvessels, and apoptotic areas were apparent in both. In response to M83, but not J94, disordered collagen accumulations were observed. Neither M83- nor J94-treated mice manifested changes in behavior, weight, or gastrointestinal function. Tumor growth suppression was more extensive than noted with recently reported efforts by others to inhibit FAP proteinase function or reduce FAP expression. Diminished angiogenesis and the accompanying profound reduction in tumor growth suggest that inhibition of either FAP or POP may offer new therapeutic approaches that directly target TMEs.

Figure 1

FAP and POP inhibitors. (A) M83, FAP, and POP inhibitor structure: acetyl-arginyl-8-amino-3,6-dioxaoctanoyl-d-alanyl-l-proline boronic acid (MW = 529.4). (B) J94, POP-specific inhibitor structure: acetyl-lysyl-leucyl-arginyl proline boronic acid (MW = 554.5).

Figure 2

Growth of human xenografts in Foxn1nu mice. Treatment was initiated as i.p. drug injections when tumors reached ~ 0.060 cm³ in size. Tumor volumes were measured over the course of 28 days of treatment. (A) Human H441 lung cancer cell xenograft growth. Treatment consisted of 50-μl saline injections (●) or M83, 26.5 μg in 50 μl of saline (○) per day. *The M83-treated group is statistically different than the saline treatment for days 10 to 28 (P < .001). (B) Human colon cancer HCT116 xenograft growth. Mice were treated with 50 μl of saline (●) or M83 (▲), or J94 (■), 50 μg in 50 μl of saline per day. **Both the M83 and J94 treatment groups are statistically different than the saline-treated group for days 10 to 28 (P < .001). (C) Human colon cancer HCT116 xenograft growth. Mice were treated with 50 μl of saline (●), M83, 50 μg in 50 μl of saline (▲), or M83, 100 μg in 50 μl of saline (■). ***Both M83 treatment groups, 50 μg and 100 μg, are statistically different than the saline group from days 18 to 28 (P < .02 and P < .003).

Figure 3

Human HCT116 colon cancer xenograft growth in Foxn1nu mice. The growth of individual tumors was plotted over the course of the 28-day treatment period. Treatment was initiated as i.p. drug injections when tumors reached ~ 0.060 cm³ in size. (A) Tumor growth in control mice treated with daily 50-μl saline injection. (B) Tumor growth curves for mice treated with M83, 50 μg in 50 μl of saline daily.

Figure 4

Images of left hindquarters of Foxn1nu mice with HCT116 colon cancer xenografts after 28 days of treatment with daily i.p. injections of (A) saline, (B) 50 μg of M83, or (C) 100 μg of M83. The excised tumors are shown below panels A and B. The saline-treated mouse had a tumor weight of 1.95 g, while the mouse treated with 50 μg of M83 had a tumor weight of 0.26 g. Of the six tumors in the 100 μg M83 treatment group, one tumor was completely suppressed (C), a second tumor was barely detectable, and the remaining four tumors were significantly reduced in size.

Figure 5

FAP and POP identified from HCT116 mouse xenograft tissue homogenates by excision of protein bands following gel electrophoresis, which were then digested with trypsin, analyzed by HPLC–MS/MS, and identified by MASCOT database search. The complete amino acid sequence of each protein is shown beginning with the amino terminus. (A) Peptides that represented 28% of the amino acid sequence deduced from the DNA sequence were identified. Amino acids in blue are peptides unique to mouse FAP as identified in the tumor tissue, while those in red are peptides that are common to both mouse and human FAPs. (B) Peptides representing 73% of the human POP sequence were observed. Amino acids in green represent peptides unique to human POP that were identified in the tumor tissue, while those in red are peptides that confirm the identification of POP but are common to both mouse and human forms. (C) Pulverized tumors from three control mice and three mice treated with M83 at 100 μg/day were suspended in 2 × SDS sample buffer at equivalent mg/ml concentrations. Equal volumes of each were subjected to SDS-PAGE, transferred to nitrocellulose, and then Western blotted with either rabbit anti-FAP or (D) goat anti-POP. The human FAP reference protein for panel C was 5 ng of the native soluble version termed APCE (94.5 kDa), and the reference for panel D was 1.5 ng of human POP (73 kDa).

Figure 6

IHC staining of excised HCT116 xenografts. Tissues were double-stained for FAP and CD31 in row one, POP and CD31 in row three, or ssDNA and CD31 in row four. In addition, tissues were stained with picrosirius red for detection of collagen, as shown in row two. The two leftmost columns represent stained images of untreated control tumors, the two center columns show stained tumors from M83-treated mice, and the two rightmost columns show the stained tumor tissue from mice treated with J94. All images were at × 20 magnification.

Figure 7

Average remaining tumor volumes after different tumor growth suppression methods. Reported effects of four different treatment approaches are compared with our results for M83 on tumor growth inhibition. Growth inhibition by M83 treatment exceeded that given by the less specific inhibitor, PT630 , by the inhibitor, PT100 , and by shRNA knockdown of FAP expression  and surpassed tumor growth reduction observed in FAP knockout mice .