The proprotein convertase PC5/6 is protective against intestinal tumorigenesis: in vivo mouse model

Tg(Vil-cre) mice (stock number 004586) [40] and Apc^Min/+mice (stock number 002020) [39] were from The Jackson Laboratory. Conditional knockout mice, in which the proximal promoter and exon 1 of Pcsk5 were flanked with loxP sites (Pcsk5^flox/flox) [15], were crossed with Tg(Vil-cre) mice that express Cre under the control of the villin promoter. After two generations,Pcsk5^flox/floxmice carrying (intestinal KO; iKO) or not (wild type; WT) one copy of the transgene were obtained and further intercrossed, yielding the F4 progeny used in this study, which exhibits a mixed background consisting of ~ 70% C57BL/6; 25% 129Sv and less than 5% SJL. When expressed, Cre leads to the recombination of the two loxP sites present in Pcsk5, resulting in the excision of ~ 3 kb of DNA including exon 1 (Δ1 alleles) and thereby gene inactivation.

Four month old mice were sacrificed by CO₂ asphyxiation, and the whole intestine was immediately removed and rinsed with ice-cold PBS. The intestine was divided into duodenum, jejunum, ileum and colon. All sections were carefully split longitudinally, fixed in 8% paraformaldehyde, stained with 8% methylene blue and the tumors were counted under a binocular microscope.

Tissue samples were dissected from PBS-rinsed intestine. Total RNA was extracted using Trizol reagent (Invitrogen), as recommended by the manufacturer. Typically, 250 ng of total RNA were used for cDNA synthesis in a total volume of 20 μL using SuperScript II reverse transcriptase, 25 μg/mL oligo(dT)_12-18, 0.5 mM 2'-deoxynucleoside 5'-triphosphates, and 40 U of RNaseOUT, all products from Life Technologies, and used according to the recommendations of the manufacturer. cDNAs of human adenocarcinomas were purchased from Origene. The quantitative PCR (QPCR) was performed as previously described [41]. Specific primers (Table 1) were used for the simultaneous amplification of the normalizing cDNA for ribosomal protein S14 (human) or S16 (mouse), and the gene of interest.

Mouse cRNA probes corresponding to the coding region for aa 20 to 348 of PC5/6 were synthesized using ³⁵S-UTP and ³⁵S-CTP (>1,000 Ci/mmol; Amersham Bioscience, Piscataway, NJ). Cryosections (8-10 μm) were fixed for 1 hour in 4% formaldehyde and hybridized overnight at 55°C as previously described [42]. For autoradiography, the sections were dipped in photographic emulsion (NTB-2, Kodak, Rochester, NY), exposed for 6-12 days, and developed in D19 solution (Kodak).

Tissues were fixed overnight in 4% paraformaldehyde at 4°C and embedded in paraffin. Proliferation cell nuclear antigen (PCNA) was visualized in sections of 6 μm thickness by incubation with a mouse antibody (1:50; Vector laboratories, Burlingame, CA) and a biotin-labeled secondary antibody (PerkinElmer, Boston, MA), and revelation with the Vectastain kit (Vector laboratories). Sections were also counterstained with hematoxylin and eosin.

Mining cancer gene expression database http://​www.​oncomine.​org revealed that PC5/6 expression was significantly reduced in 7 out of 10 tumor types (P < 0.0001); [see Additional file 1: figure S1]. Since PC5/6 expression is highest in the adult small intestine [29, 31], and as no data were available for intestinal cancers, PC5/6 mRNA levels were analyzed by QPCR in 22 human colon tumors at stages I, II, III or IV and compared to those of their match-paired normal adjacent tissue (Figure 1A). PC5/6 expression was on average ~ 7.6-fold lower in these human tumors. To assess whether PC5/6 was similarly regulated in mouse, we used the Apc^Min/+mice, which spontaneously develop numerous tumors in the small intestine due to the heterozygote mutation Min in the Apc gene. This mutation was originally discovered in patients suffering from familial adenomatous polyposis and frequently found in sporadic colorectal cancers [38, 39]. Apc^Min/+-induced tumors in the mouse small intestine constitute a good model for colonic tumorigenesis in human. We first quantified the expression levels of furin, PC5/6, PACE4 and PC7, which transit through the constitutive secretory pathway and cleave their substrates after basic residues [2]. While PACE4 and PC7 did not show any significant change, furin and PC5/6 mRNA levels were on average ~ 1.5-fold higher (P = 0.003) and lower (P = 0.0008), respectively (Figure 1B). Closer analysis of the duodenum-, jejunum- and ileum-associated tumors versus their adjacent normal tissues revealed a 1.9-, 1.2- and 1.4-fold higher furin levels, respectively, and a 2-, 1.7- and 1.1-fold lower PC5/6 expression, respectively (Figure 1C). Using specific primers, we showed that this lower level primarily affected PC5/6B transcripts [see Additional file 22: figure S2], which dominate in intestine [31]. The above data thus indicated that PC5/6 is down-regulated in many tumor types, including intestinal ones, and that in the latter furin undergoes an opposite up-regulation. Both PC5/6 and furin exhibited the greatest changes in the duodenum. These data prompted us to verify if intestinal tumorigenesis was favored in absence of PC5/6.

To explore the in vivo role of PC5/6 in intestinal tumor formation, we specifically inactivated its gene in enterocytes using a loxP/Cre system. Pcsk5^flox/floxmice were bred to Tg(Vil-cre) mice that expressed the Cre recombinase under the direction of the villin promoter, specifically expressed in enterocytes [40]. Pcsk5^flox/floxmice carrying one copy of the transgene (iKO; Tg^+/0) or none (WT; Tg^0/0) were generated. To verify that the presence of the transgene resulted in an efficient inactivation of Pcsk5 in enterocytes, we analyzed PC5/6 mRNA levels using QPCR and in situ hybridization in 3 mice of each genotype. Duodenum, jejunum, ileum and colon sections were dissected for further RNA extraction and tissue sectioning. Cre expression under the villin promoter in iKO mice was highest in duodenum and progressively diminished along the intestinal tract to reach ~ 25% of the duodenum level in the distal colon (Figure 2A). In WT mice, PC5/6 expression is elevated in the small intestine, especially in the duodenum, as compared to colon (Figure 2B). Indicative of the Cre efficiency all along the intestine, the absolute numbers of PC5/6 mRNA remaining in all sections of iKO intestine were very similar: 1.6 to 3.1 PC5/6 mRNA/1000 S16 mRNA. Furthermore, in situ hybridization with a PC5/6 cRNA probe confirmed that PC5/6 transcripts were strongly reduced in iKO intestinal enterocytes (Figure 3). The low residual expression observed by QPCR (Figure 2B) and in situ hybridization labeling suggest that in the small intestine PC5/6 is mainly expressed in enterocytes, but to a much less extent expressed in other cell types all along the intestine. Finally, the morphology and proliferation of enterocytes was assessed by immunohistochemistry. No gross malformation was observed and labeling with PCNA, a marker for proliferation, was not significantly different between the two genotypes [see Additional file 3: figure S3].

Intercrossing of [Pcsk5^flox/floxTg(Vil-cre)^+/0] with [Pcsk5^flox/floxApc^Min/+] generates 25% mice that carry only the Min mutation (WT^Min), and exhibit normal levels of PC5/6 in intestine. Another 25% of these mice carry both the Min mutation and the Cre transgene (iKO^Min), and lack PC5/6 expression in enterocytes. Duodenum, jejunum and ileum from 11 WT^Min mice and 17 iKO^Min mice were dissected out, opened longitudinally and stained with methylene blue (Figure 4A). All the tumors, including those exceeding 2 mm in diameter, were counted along the entire section of each tissue. The average tumor density (tumors/cm) in the duodenum of iKO^Min mice was significantly higher than that in WT^Min mice (P = 0.01; Figure 4B). In iKO mice, the duodenum is the tissue in which the PC5/6 drop was the most drastic (Figure 2). However, although this trend was observed in other intestinal sections, it did not reach statistical significance, and the total number of tumors in iKO^Min mice, 58 versus 46 in WT mice, was not significantly higher (Figure 4C). In addition, the numbers of large tumors (>2 mm; Figure 4C) were very similar in both cases. Overall, this analysis indicates that only in duodenum does the loss of PC5/6 significantly enhance intestinal tumorigenesis.

Apc ^Min/+mice having a pure C57BL/6 background were reported to die by 120 days of age [38, 39], likely due to severe chronic anemia [38]. In this study, WT^Min mice exhibited a longer half-life of 180 days, possibly due to their mixed background (see Methods). However, in the absence of intestinal PC5/6, this half-life was significantly shortened to 140 days (P = 0.03; Figure 5), suggesting that PC5/6 exerts a protective effect on these mice. Apc^Min/+mice develop anemia with a severity that seems to depend on the density of intestinal adenomas [38]. Considering that iKO^Min mice had a trend for higher numbers of tumors, especially in the duodenum, premature death of iKO^Min mice could be the result of more severe chronic anemia [38], which could be exacerbated by multiple hemorrhages, as observed in the liver and subcutaneously in PC5/6 knockout mice [15]. In the future, it may be valuable to examine whether PC5/6 levels correlate with the survival rate, or intestinal bleeding/anemia of patients that suffer from colorectal carcinomas.