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. Author manuscript; available in PMC: 2016 Oct 1.
Published in final edited form as: Cancer Prev Res (Phila). 2015 Aug 14;8(10):952–961. doi: 10.1158/1940-6207.CAPR-15-0003

Colon tumors with the simultaneous induction of driver mutations in APC, KRAS, and PIK3CA still progress through the adenoma-to-carcinoma sequence

Jamie N Hadac 1, Alyssa A Leystra 1, Terrah J Paul Olson 2, Molly E Maher 3, Susan N Payne 4, Alexander E Yueh 3, Alexander R Schwartz 5, Dawn M Albrecht 5, Linda Clipson 1, Cheri A Pasch 4, Kristina A Matkowskyj 4,6,7, Richard B Halberg 4,5, Dustin A Deming 3,4,7
PMCID: PMC4596777  NIHMSID: NIHMS712206  PMID: 26276752

Abstract

Human colorectal cancers often possess multiple mutations, including 3–6 driver mutations per tumor. The timing of when these mutations occur during tumor development and progression continues to be debated. More advanced lesions carry a greater number of driver mutations, indicating that colon tumors might progress from adenomas to carcinomas through the stepwise accumulation of mutations following tumor initiation. However, mutations that have been implicated in tumor progression have been identified in normal-appearing epithelial cells of the colon, leaving the possibility that these mutations might be present prior to the initiation of tumorigenesis. We utilized mouse models of colon cancer to investigate whether tumorigenesis still occurs through the adenoma-to-carcinoma sequence when multiple mutations are present at the time of tumor initiation. To create a model in which tumors could concomitantly possess mutations in Apc, Kras, and Pik3ca, we developed a novel minimally invasive technique to administer an adenovirus expressing Cre recombinase to a focal region of the colon. Here we demonstrate that the presence of these additional driver mutations at the time of tumor initiation results in increased tumor multiplicity and an increased rate of progression to invasive adenocarcinomas. These cancers can even metastasize to retroperitoneal lymph nodes or the liver. However, despite having as many as three concomitant driver mutations at the time of initiation, these tumors still proceed through the adenoma-to-carcinoma sequence.

Keywords: colorectal cancer, canonical pathway, APC, KRAS, PIK3CA

INTRODUCTION

Colorectal cancer (CRC) is the second-leading cause of cancer-related mortality in the United States (1). An improved understanding of the processes by which tumorigenesis occurs will allow for the rational development of chemopreventive and therapeutic agents. The canonical adenoma-to-carcinoma sequence has been proposed to describe the processes by which mutations in driver genes accumulate over time causing the progression of adenomas to invasive adenocarcinomas in the colon (2, 3).

An important early step in tumorigenesis is the acquisition of alterations in the Adenomatous Polyposis Coli (APC) tumor suppressor gene. Loss of this gatekeeper gene is thought to be the initiating event in the majority of sporadic human colorectal cancers with approximately 80–90% of human colon cancers harboring somatic mutations in APC (4–6). In the canonical sequence, tumor initiation caused by loss of APC is followed by mutations in other genes including KRAS, TP53, PIK3CA, and BRAF (7). Some studies have questioned whether the accumulation of mutations over time is necessary for colon cancer development (8–11). For example, KRAS mutations have been detected in non-neoplastic tissue of the colon (8, 12). This observation indicates that somatic mutations occurring prior to tumor initiation might subsequently influence tumor development and progression (13). In addition, it could be a potential explanation for interval cancers that form between routine screening colonoscopies.

Due to the prevalence of APC loss in human CRC, mice carrying mutations in Apc, the murine homolog, have been generated to more fully understand intestinal tumorigenesis and investigate pharmacologic strategies for chemoprevention (14–16). Here we utilized genetically engineered mouse models of colon cancer to examine the adenoma-to-carcinoma sequence in the setting of the concurrent induction of common driver mutations. We developed a minimally invasive procedure for inoculating an adenovirus expressing the Cre recombinase (Adeno-Cre) to induce multiple mutations at a desired location in the colon. This method allows for tumors to be initiated with a predetermined mutation profile at a specific time and location desired by the investigator.

MATERIALS AND METHODS

Mouse Husbandry

All animal studies were conducted under protocols approved by the Institutional Animal Care and Use Committee at the University of Wisconsin-Madison, following the guidelines of the American Association for the Assessment and Accreditation of Laboratory Animal Care. FC mice (FVB/N-Tg(Fabp1-Cre)1Jig/Nci; NCI Mouse Repository, strain number 01XD8), KrasG12D/+ mice (C57BL/6N.Cg-Krastm4Tyj/CjDswJ; The Jackson Laboratory, stock number 019104), Pik3cap110* mice (C57BL/6-Gt(ROSA)26Sortm7(Pik3cap110*,EGFP)Rsky/, J; The Jackson Laboratory, stock number 012343), Apcfl/fl mice (C57BL/6.Cg-Apctm2Rak/Nci; NCI Mouse Repository, strain number 01XAA), and mT/mG mice (B6.129(Cg)-Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo/J; The Jackson Laboratory, stock number 007676) were maintained and genotyped as previously described (17–21). For FC and mT/mG a 1 denotes carrier and 0 non-carrier. Pik3cap110* and KrasG12D/+ mice were also crossed to Apcfl/fl mice to generate Apcfl/fl KrasG12D/+, Apcfl/fl Pik3cap110*, and Apcfl/fl KrasG12D/+ Pik3cap110* mice on a homogeneous B6 genetic background for Adeno-Cre delivery. In addition, mT/mG1 mice were crossed to generate B6 mT/mG1 Apcfl/fl KrasG12D/+ Pik3cap110*, mice. All Pik3cap110* mice were hemizygous for this allele.

Nonsurgical exposure of the colon to Adeno-Cre

Polyethylene tubing (I.D. 1.4 mm, O.D. 1.90 mm; Becton Dickinson, Sparks, MD) was cut to sizes appropriate for mice (7–12 cm). A 1 cm window was notched into the tubing and the end of the tubing was closed with edges being rounded to avoid perforation of the bowel (Figure 1A). Marks corresponding to 1 cm intervals were made on the tubing. A longitudinal stripe was also applied corresponding to the orientation of the window. Similarly sized polyethylene tubing was cut to size without a slot to be used as a sheath for a 2.2 mm caliber soft bristle brush (DenTek Oral Care, Maryville TN).

Figure 1.

Figure 1

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A minimally invasive technique for inoculation of the colon with Adeno-Cre initiates tumorigenesis in Apcfl/fl mice. We have developed a novel technique that can be utilized to sequester Adeno-Cre within the colon without laparotomy, as was required with prior methodologies. A, in this technique a slotted tube housing GelFoam allows for the treatment of the colon with trypsin. Abrasion of the colon is then performed prior to incubation with Adeno-cre-soaked GelFoam at the desired location. B, colonic tumors are visualized by endoscopy in 3–7 weeks following treatment with Adeno-Cre and monitored over time with serial endoscopy. C, interestingly, these tumors have varying growth patterns over 14 weeks of observation, despite a similar initiation. D, histological evaluation demonstrated that the majority (83%) of these lesions were adenomas with nuclear CTNNB1 consistent with activation of the WNT pathway. The area indicated by the rectangle is shown enlarged at the right; at the far right the same area in an adjacent slide stained for CTNNB1 is shown. Size bar = 1mm.

Mice were anesthetized using 2% isoflurane. The colon was irrigated with phosphate buffered saline (PBS). A narrow ribbon of GelFoam (Pharmacia and Upjohn, NY, NY) was inserted into the window cut into the polyethylene tube. 200 µL of 0.05% trypsin (Hyclone, Logan, VT) was injected into the tubing and inserted into the mouse colon at the desired depth and radial orientation. After 10 minutes, the slotted tube was removed and the sheath with the small soft bristle brush was introduced at the same intraluminal location. The brush was then used to abrade the epithelium for up to 3 minutes. After PBS irrigation, a slotted tubing containing GelFoam was then filled with 200µL PBS containing 109 PFU of Adeno-Cre (Ad5CMVCre and Ad5CMVEmpty, University of Iowa Gene Transfer Vector Core, IA). After 30 minutes of incubation, the tubing was removed. Owing to the anatomical limitations of the mouse, only the most distal half of the colon (~4 cm) could be inoculated in this way. Mice recovered quickly after the procedure and did not exhibit overt signs of pain or distress following the procedure, as they quickly became active. Note that nonsteroidal anti-inflammatory drugs were not used for post-procedure analgesia as these agents have been shown to suppress intestinal tumorigenesis in both humans and laboratory mice.

Murine Colonoscopy/PET Imaging

Mice were anesthetized using 2% isoflurane and the colons were flushed with PBS. The Coloview System was used to monitor tumor formation and growth in the distal half of the colon as previously described (Karl Storz, Goleta, CA) (22). ImageJ analysis was utilized to measure the percent lumen occlusion as previously described (23).

Animals were fasted for at least 6 hours prior to injection of 18F-FDG (160 µCi; IBA Molecular, Romeoville, IL). After injection, the animals were kept under anesthesia for 60 minutes and then prepared for microPET colonography as described previously (24).

Histology and Immunohistochemistry

Mice were euthanized and the colons were excised and opened longitudinally. Tumor tissue was fixed in 10% buffered formalin. Fixed tumors were embedded in paraffin and cut into 5µm sections. Every tenth section was stained with hematoxylin and eosin (H&E) for histological review. Immunohistochemistry (IHC) was carried out as described previously (25) with the exception that tissues were blocked with Background Sniper (Biocare Medical, Concord, CA) for 10 minutes. The primary antibodies included rabbit anti-phospho-AKT (Ser473, 1:100, Cell Signaling Technology #4060), rabbit anti-phospho-S6 Ribosomal Protein (Ser235/236, 1:50, Cell Signaling Technology #4858), rabbit anti-CTNNB1 (1:100–1:200, Cell Signaling Technology #8480), rabbit anti-Ki67 (1:400, Cell Signaling Technology #12202), and rabbit anti-phospho-ERK1/2 (1:400, Cell Signaling Technology #4370). The Ki67 proliferation index was measured as the percent of nuclei staining positive for Ki67 per tumor using ImmunoRatio, an ImageJ plugin (http://jvsmicroscope.uta.fi/sites/default/files/software/immunoratio-plugin/index.html).

Recombination Testing

To validate that all alleles were recombined within tumors, neoplastic tissue was scraped from FFPE sections with a sterile size 10 surgical blade with normal epithelium left behind.. DNA was isolated from the scraped tissue using the Maxwell® 16 FFPE Tissue LEV DNA Purification Kit [Promega, Madison WI]. Samples with a sufficient amount of DNA were analyzed for Cre-mediated recombination between loxP sites.

Recombination of the loxP sites within the Apcfl allele and upstream of the KRASG12D allele was confirmed using previously described PCR primers and protocols (http://jacks-lab.mit.edu/protocols/genotyping/kras_cond) (20). Recombination of the loxP sites upstream of the Pik3cap110* allele was confirmed using the forward primer 5'- CGCGGTTGAGGACAAACTCT-3' and the reverse primer 5'- ACCATAATTCCACCACCACCA-3'. Cycling conditions were one cycle of 94°C for 3 min, followed by 35 cycles of 94°C for 20 s, 61°C for 60 s and 72°C for 60 s, with one final extension cycle at 72°C for 2 min. Recombined DNA resulted in amplification of a 561 bp product.

RESULTS

Colon tumors can be initiated using a noninvasive inoculation of the Adeno-Cre virus

KRAS and PIK3CA mutations have been identified as occurring concomitantly with the loss of APC in human adenomas and carcinomas (26, 27). Multiple investigations have examined murine models with combinations of loss of APC and activating mutations in either KRAS or PIK3CA (28–31). These studies had limitations. C57BL/6 (B6) mice carrying the Min allele of Apc (ApcMin/+) have a ubiquitous spatial and temporal expression of a mutant germline allele resulting in alterations of homeostasis along the entire intestinal tract (14). The life expectancy of these mice is limited due to their high tumor burden, especially of the small intestine, resulting in anemia or intestinal obstruction. B6 ApcMin/+ mice do not develop advanced colon cancers as a consequence of this shortened lifespan. Finally, triggering multiple mutations in the colon concurrently by expressing Cre recombinase from the FABP-1 promoter is lethal (Supplementary Fig. S1). To determine whether additional concomitant mutations would alter the progression of colon tumors through the adenoma-to-carcinoma sequence alternative methodologies were required. A model allowing for spatial control of tumor initiation that can be used to express a number of mutations was needed to further explore their effects on tumorigenesis.

While there are multiple methods of expressing Cre in the mouse, including organ-specific promoter-based systems, the delivery of an adenovirus carrying Cre recombinase allows for the greatest spatio-temporal control of initiation of colon tumors (18). To sequester the virus in the colon, an invasive laparotomy procedure has been described (23). Surgical clips were directly placed both proximally and distally around the colon after exposure of the peritoneal cavity. Adeno-Cre was then injected into the area between the clips. This surgical Adeno-Cre instillation technique allows for the development of colon tumors without the co-morbidities related to Apc mutations occurring outside of the area of interest. However, this surgical intervention is no trivial task; preparation, surgery, and recovery may all contribute detrimental effects to the overall health of the animal. Unintended consequences from surgical wounds may alter various cellular mechanisms, including inflammatory and wound healing responses, involved in tumorigenesis (32).

To overcome limitations of the surgical approach, we developed a device to non-surgically inoculate Adeno-Cre into the colon in homozygous Apcfl/fl mice (Fig. 1A) (20). Upon expression of Cre recombinase in these mice, exon 14 of Apc is excised, resulting in the expression of a truncated, non-functional APC protein. Cre expression controls the location and time at which tumors form, in turn alleviating many of the limitations associated with models carrying germline Apc mutations. This non-surgical approach induced tumorigenesis in a physiologically relevant setting, while minimizing the systemic effects of an operative procedure.

Biocompatible materials have been used to enhance the delivery of viruses in a variety of research settings (33). One such biomaterial, GelFoam (Pharmacia and Upjohn, NY, NY), is a collagen-based sponge. It has been used to deliver viruses in gene therapy research due to its ability to localize and protect virus (34). GelFoam saturated in adenovirus solution has been implanted in mice and rats to facilitate gene expression for the study of wound healing and to elicit immune responses in tumors (35, 36). These studies have demonstrated that GelFoam can be used to improve the sustained delivery of adenovirus in vivo. Therefore, we found it an attractive alternative to surgery for use in localizing the virus in specific areas of the mouse.

A slotted tube housing GelFoam soaked with Adeno-Cre was placed 1.5 to 3.5 cm into the colon of each prepared Apcfl/fl mouse (see Materials and Methods). After the procedure, tumor formation and growth were monitored by endoscopy. Tumors were visible as early as 3 weeks post-treatment and these tumors were monitored over 14 weeks post-treatment. 58% (18/31) of treated Apcfl/fl mice developed tumors. Tumors originated at the desired location, both in radial orientation and depth in the colon (Fig. 1B). No tumors developed in mice infected with adenovirus carrying an empty vector.

Differential growth patterns occur in tumors after the simultaneous loss of both normal APC alleles

Small animal endoscopy was performed every other week over a 14-week period to follow tumor growth in homozygous Apcfl/fl mice treated with Adeno-Cre. Tumors visible one month after adenoviral treatment were relatively small, occluding less than 30% of the lumen. However, in the following weeks, tumors exhibited many different patterns of growth (Fig. 1C). Some tumors grew rapidly to eventually occlude the entire lumen, while others grew more incrementally, or even remained stable in size over this time. One small tumor ultimately regressed. These dynamic growth patterns, observed even in the earliest adenomas were similar to tumor growth patterns in other murine colon tumor models with loss of APC (25).

After 14 weeks of monitoring, necropsy was performed. None of the mice became moribund prior to this time point. Histological evaluation was completed on 6 tumors which revealed that 83% were adenomas, and 17% were invasive adenocarcinomas with invasion into the muscularis mucosa (Fig. 1D). No evidence of regional nodal disease or metastatic disease was observed.

Epithelial tumors arise secondary to inoculation of the colon with Adeno-Cre

When the Adeno-Cre virus is instilled into the colon it is possible that the virus is infecting cells other than the epithelial cells lining the intestine. To make certain that only epithelial cells were being infected with the Adeno-Cre virus, mT/mG1 Apcfl/fl KrasG12D/+ Pik3cap110* mice were treated with Adeno-Cre. mT/mG is a reporter from which red (tdTomato) fluorescent protein is ubiquitously expressed preceding Cre recombinase expression and green fluorescent protein is expressed following Cre recombinase expression. Green fluorescence was detected only within the colonic epithelium confirming the intended cell type was being infected with the Adeno-Cre (Fig. 2). Nuclear localization of CTNNB1 (β-catenin) and activation of the anticipated downstream signaling cascades were observed within the transformed epithelial cells indicating that recombination following Cre expression was occurring as anticipated (Fig. 2).

Figure 2.

Figure 2

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Multiple mutations were simultaneously expressed within the epithelium of the colon following a novel minimally invasive Adeno-Cre technique resulting in the development of colon tumors. To be certain that our mutations were being activated only in the epithelial cells of the colon with this technique we utilized mT/mG1 Apcfl/fl Pik3cap110* mice. Upon Cre recombination a gene encoding a red fluorescent marker in these mice is excised and a green fluorescent protein is expressed. A representative histological section is displayed demonstrating that Cre-mediated recombination has only occurred in the epithelial cells of these lesions. IHC confirmed CTNNB1 (β-catenin) localization to the nucleus and activation of the PI3K signaling cascade with intense staining for phosphorylated AKT (pAKT) and phosphorylated RPS6 (pRPS6), as expected. Activation of ERK1/2 was not observed in these tumors. The area indicated by each rectangle is shown enlarged at the right. Size bar = 1 mm.

Adeno-Cre tumor initiation rate depends upon the mutation profile

Our minimally invasive technique allows for temporal and spatial control of the initiation of colon tumors with multiple simultaneous mutations. Mice with Apcfl/fl, KrasG12D/+, and/or Pik3cap110* were generated and treated with Adeno-Cre (Fig. 3A). After 3–4 weeks, tumors were identified on endoscopy. When additional driver mutations were present in addition to the loss of APC, an increase in the rate of tumor development was observed. Tumors developed in 79% of Apcfl/fl, mice carrying KrasG12D and/or Pik3cap110* compared to 58% of Apcfl/fl mice without these additional driver mutations (Figure 3A, p (one-sided) = 0.031, Barnard’s exact test). All mice were treated according to the same Adeno-Cre protocol and on the same homogeneous genetic background. In the 14 samples that were successfully tested, recombination was confirmed in nearly all cases (12/14, Supplementary Fig. S2). Exceptions were found within 2/3 samples from a single Apcfl/fl KrasG12D/+ Pik3cap110*, mouse: only two of the three Cre dependent alleles appeared to recombine in two tumors, but all three Cre-dependent alleles recombined in the third tumor.

Figure 3.

Figure 3

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Apcfl/fl KrasG12D/+(A2K1P0), Apcfl/fl Pik3cap110* (A2K0P1), and Apcfl/fl KrasG12D/+ Pik3cap110* (A2K1P)) mice were treated with Adeno-Cre at 40–50 days of age. A, incidence of tumors increases with the addition of KrasG12D/+ and/or Pik3cap110* to the mutation profile. (Due to the relatively small sample sizes and the similar tumor incidence rates among A2K1P0, A2K0P1 and A2K1P1 mice, the tumor incidence data from these strains was pooled (31/39) for comparison to A2K0P0 (18/31); p (one-sided) = 0.031, Barnard’s exact test). B, tumors were identified by endoscopy in the colon as early as 3–4 weeks after viral inoculation. Histological sectioning was performed on tumors shortly after identification on endoscopy revealing that these lesions were small adenomas without evidence of invasion into the muscularis mucosa. An adenoma from an Apcfl/fl Pik3cap110* mouse is shown here. Nuclear localization of CTNNB1 and phosphorylation of RPS6 were observed without phosphorylation of ERK1/2 indicating that recombination was occurring as expected. The area indicated by each rectangle is shown enlarged at the right. Size bar = 500µm.

In the setting of multiple mutations Adeno-Cre induction resulted in adenoma formation

Mice with Apcfl/fl plus KrasG12D/+ and/or Pik3cap110* were generated and treated with Adeno-Cre to determine how the simultaneous induction of these mutations would alter tumor biology. Tumors were identified 3–8 weeks after viral treatment (Fig. 3B). Histological sectioning of tumors from revealed that all tumors from these mice carrying mutations in 2–3 genes at this early time point were small adenomas. An example of a small adenoma from an Apcfl/fl Pik3cap110* mouse is presented in Fig 3B. The expected nuclear localization of CTNNB1 and activation of the PI3K cascade with abundant phosphorylation of RPS6 was observed (Fig. 3B).

The addition of Kras and Pik3ca mutations does not significantly alter the rate of proliferation in colon tumors compared to loss of APC alone

To estimate the rate at which the tumors with different mutation profiles were growing, Apcfl/fl Pik3cap110* mice and Apcfl/fl KrasG12D/+ Pik3cap110* mice were monitored with serial endoscopy every two weeks following treatment with Adeno-Cre (Fig. 4A). Lumen occlusion by the colonic tumor was used as a marker of cellular proliferation within tumors. Occlusion of 75% of the colonic lumen was observed in 65% of tumors in Apcfl/fl Pik3cap110*, and Apcfl/fl KrasG12D/+ Pik3cap110*, mice. For the tumors that eventually occluded at least 75% of the lumen, the mean time required to reach 75% occlusion was 15 weeks. No statistically significant difference in the mean time to 75% lumen occlusion was observed between tumors with APC loss and the addition of 1 or 2 oncogenes (Fig 4B). Interestingly, in one instance, a tumor from an Apcfl/fl Pik3cap110* mouse regressed (Supplementary Fig. S3). Histological analysis of this regressed area revealed a predominant lymphocytic infiltrate, indicating a potential immune-mediated mechanism for the regulation of some colon tumors.

Figure 4.

Figure 4

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The addition of Kras and Pik3ca mutations to the loss of APC has a modest effect on tumor proliferation. A, Apcfl/fl, (A2K0P0), Apcfl/fl Pik3cap110* (A2K0P1) and Apcfl/fl KrasG12D/+ Pik3cap110* (A2K1P1) mice were treated with Adeno-Cre and followed with serial endoscopy. B, change in percent lumen occlusion was used as a marker of tumor proliferation. The time required for tumors to occlude 75% of the lumen was determined. No difference was seen between groups. C, to confirm these results these tumors were sectioned and stained for Ki67. The Ki67 proliferation index was calculated for each tumor. D, no statistically significant increase in Ki67 staining was observed when additional mutations occurred concomitantly with loss of APC (p = 0.35, Kruskal-Wallis test). The area indicated by each rectangle is shown enlarged at the right. Size bar = 1mm.

To confirm that mean time to lumen occlusion as is a useful marker for cellular proliferation within tumors, histological sectioning of tumors from Apcfl/fl, Apcfl/fl Pik3cap110*, and Apcfl/fl KrasG12D/+ Pik3cap110* mice was performed and these sections were stained for Ki67, a standard marker of cell proliferation (Fig. 4C). The Ki67 proliferative index (Ki67 PI) was measured as the percent of nuclei staining positive for Ki67 per tumor. The mean Ki67 PI of Apcfl/fl tumors was 11.1% (range 0.8–24.8), Apcfl/fl Pik3cap110* tumors was 18.5% (range 7.3–25.8), and Apcfl/fl KrasG12D/+ Pik3cap110*, was 15.7% (range 11.3–20.8). A trend for increased Ki67 PI was observed in the presence of additional mutations, but this was not statistically significant (p=0.22).

Tumors with simultaneous loss of Apc and oncogenic mutations in Kras and Pik3ca progress from adenomas to adenocarcinomas to metastatic disease

Additional Apcfl/fl Pik3cap110* and Apcfl/fl KrasG12D/+ Pik3cap110* mice were monitored with serial endoscopies (Supplementary Table S1). After 14 weeks, subsets of these mice underwent necropsy. At this time-point, approximately 50% of tumors from each these genetic profiles were invasive adenocarcinomas. The remaining tumors were adenomas with many of them exhibiting high-grade dysplasia.

The remaining Apcfl/fl Pik3cap110* and Apcfl/fl KrasG12D/+ Pik3cap110* mice were aged until moribund. No difference in survival was observed between the two groups with a median survival of approximately 200 days (Fig. 5A). The tumors progressed from small polypoid lesions to invasive adenocarcinomas that filled the majority of the colonic lumen (Fig. 5B). These tumors even progressed to "apple-core" lesions often seen in advanced colon cancers in humans (Fig. 5B far right panel) (37). When these mice were moribund, only 20% of their tumors were pre-invasive lesions, all of which were associated with high-grade dysplasia. The remaining 80% of tumors in these mice were invasive adenocarcinomas, many of which had a significant proportion of the tumor extending beyond the muscularis propria to involve the serosa (Fig. 5C). Metastatic disease was identified in two of these mice, including metastases to a retroperitoneal para-aortic lymph node (Fig. 6A) and metastatic cancer within the liver (Fig. 6B). These are common locations for human colorectal cancer to metastasize. No lung metastases were identified.

Figure 5.

Figure 5

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Tumors with multiple driver mutations present at the time of initiation progressed through the adenoma to carcinoma sequence. Apcfl/fl Pik3cap110* (A2K0P1) and Apcfl/fl KrasG12D/+ Pik3cap110* (A2K1P1) mice were treated with Adeno-Cre, as described, at 40–50 days of age and aged until moribund. A, no difference in survival between these two groups was noted. B, the resulting tumors were followed with serial endoscopy; they developed initially as polypoid lesions within the colon and progressed to occlude the majority of the lumen. These tumors could be followed as long as 12 months post viral inoculation and developed into "apple-core" lesions, as seen in the far right photo, similar to advanced colorectal tumors in humans. These mice became moribund due to anemia or intestinal obstruction. At necropsy, large colon tumors approached 1 cm in diameter. C, upon histological analysis, these lesions were invasive adenocarcinomas penetrating through muscularis propria and into the serosa in many instances. H&E staining of a large invasive cancer from an Apcfl/fl KrasG12D/+ Pik3cap110* (A2K1P1) mouse is shown. Nuclear localization of CTNNB1 and activation of AKT and ERK1/2 signaling was observed. The rea indicated by each rectangle shown enlarged at the right. Scale bar = 1 mm.

Figure 6.

Figure 6

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Metastatic colon adenocarcinoma was identified within Adeno-Cre treated mice. An Apcfl/fl KrasG12D/+ Pik3cap110* mouse became moribund after treatment with Adeno-Cre. A, 18F-FDG PET imaging demonstrated an avid para-arotic lymph node in addition to increased avidity of the primary tumor. Enlarged adenopathy was identified at necropsy. On histological sectioning, well-differentiated adenocarcinoma consistent with the colonic primary tumor was observed. Staining for CTNNB1 demonstrated nuclear localization consistent with loss of APC. B, in addition, metastatic adenocarcinoma in the liver was identified in a moribund Adeno-Cre treated Apcfl/fl Pik3cap110* mouse. A hepatic mass was observed grossly. H&E staining of histological sections demonstrated a well-differentiated adenocarcinoma arising within the liver. Staining demonstrated nuclear localization of CTNNB1 consistent with the colonic primary tumor. The area indicated by each rectangle in photos of stained sections is shown enlarged at the right. Size bars = 500µm.

DISCUSSION

The progression from an adenoma to an invasive carcinoma is a histological determination defined by spread of tumor into the submucosa. Invasion into the submucosa has been associated with the presence of additional driver mutations beyond the loss of APC, but does not necessitate molecular progression beyond mutations that have been observed in adenomas. An important consideration, often ignored, regarding colon cancer tumorigenesis is the time necessary for the acquired mutations to alter the phenotype of the tumor. This is important when investigating the potential timing of acquired mutations.

Activation of the WNT signaling cascade following the loss of APC or mutations in CTNNB1 result in tumorigenesis in the majority of human colorectal cancers (38, 39). Although activation of the WNT pathway is important for tumor initiation, it is not sufficient for progression to an invasive cancer (40). In fact, the vast majority of polyps that form due to loss of APC will not become invasive cancers and some will actually regress (41). Additional genetic alterations including KRAS, BRAF, PIK3CA, and TP53 mutations are important for adenomas to develop high grade dysplasia and progress to invasive adenocarcinomas (42). The timing of when these additional genetic changes occur has been debated (43), though these additional genetic changes likely occur very early in tumorigenesis.

Due to the proliferative nature of the intestine, mutations are known to accumulate over time in the normal epithelium (13). In colon cancers, an average of approximately 90 mutations are seen per tumor (44). This number changes significantly depending upon the age at which the patient develops cancer. Most of these mutations are considered to be passenger mutations and are not thought to engender a selective growth advantage (45). It has been estimated that half, if not more, of these mutations occur prior to tumor initiation (13). To investigate whether driver mutations can occur prior to tumor initiation, investigations into the presence of KRAS mutations in the normal epithelium have been performed. KRAS mutations have been identified in colon cancers, adenomas, tumor-associated epithelial tissue, and completely normal appearing mucosa (46). This indicates that cells with mutations that are important for tumor progression, such as KRAS, might be present prior to tumor initiation.

If driver mutations are present within intestinal epithelial cells before or at least at the time of tumor initiation, tumor biology may be dramatically affected. Here we show that the presence of KRAS and PIK3CA mutations at the time of initiation owing to loss of APC results in increased tumor multiplicity and an increased rate of progression to invasive adenocarcinomas. The tumors in the models described here even metastasize to retroperitoneal lymph nodes and the liver at relatively short intervals, indicating the potential for an increased probability of early metastatic spread. These results could explain interval cancers that develop in patients that are routinely screened following current recommendations. Of note, the results from these experiments might not be directly translated for all mutation profiles and it is possible that certain mutations might be able to circumvent the adenoma-to-carcinoma sequence and need to be studied further.

Interestingly, we demonstrated that despite having both Kras and Pik3ca mutations at the time of tumor initiation from loss of APC, these cancers still develop through the adenoma-to-carcinoma sequence. Following inoculation of the colon with Adeno-Cre, a visible tumor can be identified in just a few weeks. These tumors are pre-malignant adenomas. The formation of the early polyp is most likely related to WNT signaling (47). The loss of APC leads to the nuclear accumulation of CTNNB1, which results in cellular proliferation and polyp formation. Without additional genetic events, the polyps tend to stabilize in size and do not progress to cancers and may actually regress (48). With time the vast majority of the tumors possessing mutations in KRAS, PIK3CA, or both will develop into invasive adenocarcinomas. The selective advantage conferred upon a cell carrying an additional driver mutation has been estimated to increase the probability of proliferation by 0.4% in silico (49). Since driver mutations are being identified in tumors containing less than 109 cells (~1cm in diameter), clones carrying these mutations are likely developing very early. This possibility might explain why a statistically significant change in the proliferation rate could not be detected in the described experiments when additional driver mutations were added. Other factors including angiogenesis and stromal cell signaling might also limit the proliferation rate in vivo. Interestingly, since driver mutations are identified in tumors containing less than 109 cells (~1cm in diameter), clones carrying these mutations are likely developing very early (50). For these clones to outcompete others in the same tumor to a detectable extent, these additional driver mutations are likely occurring when the tumor consists of 104 cells or less. The small probability of proliferation causes dramatic changes as the number of cells in these tumors enlarges. These driver mutations in vivo augment tumor progression by more than just increasing cell proliferation, but also by suppressing apoptosis, stimulating angiogenesis, recruiting stroma, and evading immune surveillance, among others (50).

The presence of KRAS and PIK3CA mutations at the time of tumor initiation in the setting of loss of APC does not eliminate the formation of the pre-malignant intermediary. This is important because there is still the possibility to prevent invasion of these tumors if the polyps can be resected at colonoscopy. In addition the malignant potential of these lesions might not be able to be predicted purely on tumor growth rate and morphology as it appears based on the experiments presented here that loss of APC may be the key mediator of early tumor growth. The increased rate of tumor progression to invasive disease in the setting of concomitant driver mutations indicates that it is likely beneficial to understand the biology of the polyps that are removed from patients. Prognostic stratification of premalignant lesions might be beneficial for determining the likelihood of these tumors harboring foci of invasive disease and potentially the risk of future lesions developing. Further investigation is needed, but it might be necessary to screen patients more frequently if they develop adenomas with high-risk molecular features.

The combinatorial accumulation of driver mutations, opposed to the timing of potential sequential mutations, has the greatest impact on tumor development and progression (49). When these mutations occur is very important, however, as this will impact the amount of time necessary for these lesions to become invasive cancers. In many instances, the canonical mechanism of tumorigenesis with mutations slowly accumulating over time does likely occur. This study and others demonstrate, though, that there are likely some instances when the canonical pathway is not followed. This could explain the development of interval cancers between colonoscopies. We have characterized an effective mouse model for further investigations into the mechanisms of tumorigenesis that can ultimately be applied to the better understanding of the molecular progression of colon tumors. These studies will allow for improved risk stratification to dictate screening intervals and the development of better chemopreventive strategies.

Supplementary Material

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ACKNOWLEDGMENT

This work was partially funded by Funk Out Cancer, a memorial event dedicated to Kate Gates Falaschi.

Financial support

This project was supported by the Conquer Cancer Foundation of the American Society of Clinical Oncology through A Young Investigator Award (D.A.Deming); T32 CA09017 (T.J. Paul Olson), T32 CA009135 (J.N. Hadac. and A.A. Leystra), R21 CA170876 (R.B. Halberg) and R01 CA123438 (R.B.Halberg) from the National Institutes of Health; 2012 AACR Career Development Award for Colorectal Cancer Research, Grant Number 12-20-01-HALB (R.B. Halberg); start-up funds (R.B. Halberg) from the UW Division of Gastroenterology and Hepatology, the UW Department of Medicine, and the UW School of Medicine and Public Health; and start-up funds (D.A. Deming) from the UW Carbone Cancer Center, UW Department of Medicine, UW School of Medicine and Public Health, and the UW Graduate School through the Wisconsin Alumni Research Foundation; P30 CA014520 (Core Grant, University of Wisconsin Carbone Cancer Center); UW Carbone Cancer Center Gastrointestinal Disease Oriented Working Group (R.B. Halberg and D.A. Deming; Funk Out Cancer (D.A. Deming).

Footnotes

No conflicts of interest.

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