In our colony, we find that the penetrance of colitis is nearly 100% by 7 weeks of age. The original description likened the colitis to that seen in UC, noting the exclusively colonic localization of inflammation, and the worst colitis being present in the distal colon.12
We find that the histopathologic features are somewhat more CD-like, given the patchy distribution of inflammation that is often the most severe in the cecum, in addition to transmural disease and serositis. However, there are important features of CD that are not seen in Gia2−/− mice, which lack mucosal granulomas, prominent fissuring, and ileitis.
The most striking feature of our analysis is the incidence, location, histology, and multifocality of the colon cancers that arise in Gi2-alpha−/− mice. Of the 32 mice older than 6 months at the time of sacrifice, nearly 80% of the animals had colon cancer. More than 75% of the cancers were located in the cecum or ascending colon and were multifocal. Many of these cancers arose out of areas of nonpolypoid dysplasia, although some invasive lesions are overlaid by inflamed but nondysplastic epithelium. These tumors displayed mucinous differentiation nearly 30% of the time. Together these features are very reminiscent of those seen in human patients with IBD-associated colon cancer.22
Choi and Zelig3
reviewed the features of IBD-associated cancers in 80 patients (28 CD and 52 UC)—50% of the CD and 36% of the UC cancers were located in the right colon; 22% of the CD patients and 15% of the UC patients had mucinous cancers, and 12% of all patients had multiple synchronous tumors.
There are several animal models of colitis that also develop colon cancer. IL-10 KO mice, like Gi2-alpha−/− mice, develop transmural disease with a high incidence of colon cancer (60% at 6 months, n
although the distribution within the colon was not described further. The mucinous differentiation and relatively low-grade features seen in some Gi2-alpha−/− tumors are also seen in IL-10−/− mice. In the initial description of colon cancers in IL-10−/− mice, lesions with these features were not counted as adenocarcinomas, but rather thought to represent colitis cystica profunda, an uncommon nonneoplastic lesion that can mimic colon cancer.26
It is unlikely that all of the mucinous tumors in Gi2-alpha−/− mice represent this benign entity, as several of these mucinous tumors are invasive into adjacent structures such as the pancreas. It should also be noted that the majority of adenocarcinomas that arise are in fact non-mucinous and are composed of irregular, poorly organized glands invading into an inflamed, desmoplastic stroma.
In all segments of the colon, 100% of Smad3-KO mice develop multifocal adenocarcinomas in a Helicobacter
Review of the histopathologic spectrum of tumors observed in Smad3 mutant mice28
reveals striking similarity to those seen in Gi2-alpha−/− mice. We have performed Helicobacter
species-specific PCR on stool samples from our colony and found products specific for H. bilis, H. hepaticus
, and H. rodentium
(data not shown). We have also previously identified resistance to TGFβ signaling in T-cells from Gi2-alpha−/− mice,29
although there are no differences in Smad3 expression between WT and Gi2-alpha littermates. Because TGFβ is a negative regulator of colonic epithelial proliferation,28
it will be interesting to see whether there is decreased responsiveness to TGFβ in cells overex-pressing dominant-negative Gi2-alpha.
The features of colon cancer in Gi2-alpha−/− mice are also strongly reminiscent of those seen in patients with hereditary nonpolyposis colon cancer (HNPCC), who develop multifocal right-sided mucinous or medullary-type adenocarcinomas arising out of nonpolypoid (villous) adenomatous mucosa.30
All these features (lymphocytic infiltration, proximal location, mucinous differentiation) have been shown to be independent predictors of a tumor being MSI-H (defined as having microsatellite instability at ≥30% of the tested microsatellite markers).31
Although no direct link between Gi2-alpha signaling and mismatch repair has been identified, there is emerging evidence that microsatellite instability is commonly found not only in IBD-associated cancers14
but in the surrounding nondysplastic mucosa as well.32
If the tumors in Gi2-alpha−/− mice are found to exhibit MSI and whether MSI is a particular feature of this model or is a common feature in animal models of chronic colitis, remains to be seen.
A widely held principle in colitis-associated carcinogenesis is that carcinomas arise in areas of inflammation. Indeed, this is true in Gi2-alpha−/− mice. However, the rapid onset of dysplasia and cancer (as early as 12 weeks of age) so soon after the development of colitis prompted us to ask whether the loss of Gi2-alpha has a direct effect on epithelial proliferation and/or apoptosis, in the absence of an inflammatory milieu. We found that the Gi2-alpha−/− epithelium is hyperproliferative in the absence of colitis (). One limitation of this result is that there is evidence of widespread immune activation in Gi2-alpha−/− mice before the onset of histologically identifiable colitis,33
so inflammatory mediators might disrupt epithelial growth regulation in “precolitic” mice. We therefore evaluated proliferative and apoptotic indices in dominant-negative Gi2-alpha-expressing colon cancer cells in vitro and found comparable responses to those seen in vivo (, ). These data support the contention that Gi2-alpha signaling is a direct negative regulator of colonic epithelial cell growth.
The Gi2-alpha−/− model of colitis-associated colon cancer poses a paradox. The loss of Gi2-alpha results in decreased epithelial () and stromal11
arachidonic acid release, leading to decreased mucosal PGE2
levels, yet the epithelium is hyperproliferative. How might decreased ara-chidonate be related to the inhibition of apoptosis? Arachidonate has been shown to enhance the activity of intestinal sphingomyelinases that produce proapoptotic ceramides,34
and inhibition of AA release has clear effects on apoptosis and carcinogenesis in vivo. Animals deficient in cPLA2
develop a 5.5-fold greater tumor burden in the azoxymethane model of induced colon carcinogenesis due to significantly decreased colonic epithelial cell apoptosis seen by TUNEL assay, despite lower tissue PGE2
We are currently testing whether cell lines deficient in Gi2-alpha harbor decreased sphingomyelinase activity and ceramide generation which may underlie the observed antiapoptotic phenotype.