PIK3CA mutations can initiate pancreatic tumorigenesis and are targetable with PI3K inhibitors.

Aberrations in the phosphoinositide 3-kinase (PI3K) signaling pathway have a key role in the pathogenesis of numerous cancers by altering cell growth, metabolism, proliferation and apoptosis. Interest in targeting the PI3K signaling cascade continues, as new agents are being clinically evaluated. PIK3CA mutations result in a constitutively active PI3K and are present in a subset of pancreatic cancers. Here we examine mutant PIK3CA-mediated pancreatic tumorigenesis and the response of PIK3CA mutant pancreatic cancers to dual PI3K/mammalian target of rapamycin (mTOR) inhibition. Two murine models were generated expressing a constitutively active PI3K within the pancreas. An increase in acinar-to-ductal metaplasia and pancreatic intraepithelial neoplasms (PanINs) was identified. In one model these lesions were detected as early as 10 days of age. Invasive pancreatic ductal adenocarcinoma developed in these mice as early as 20 days of age. These cancers were highly sensitive to treatment with dual PI3K/mTOR inhibition. In the second model, PanINs and invasive cancer develop with a greater latency owing to a lesser degree of PI3K pathway activation in this murine model. In addition to PI3K pathway activation, increased ERK1/2 signaling is common in human pancreatic cancers. Phosphorylation of ERK1/2 was also investigated in these models. Phosphorylation of ERK1/2 is demonstrated in the pre-neoplastic lesions and invasive cancers. This activation of ERK1/2 is diminished with dual PI3K/mTOR inhibition. In summary, PIK3CA mutations can initiate pancreatic tumorigenesis and these cancers are particularly sensitive to dual PI3K/mTOR inhibition. Future studies of PI3K pathway inhibitors for patients with PIK3CA mutant pancreatic cancers are warranted.

PIK3CA and APC mutations are synergistic in the development of intestinal cancers.

Human colorectal cancers are known to possess multiple mutations, though how these mutations interact in tumor development and progression has not been fully investigated. We have previously described the FCPIK3ca* murine colon cancer model, which expresses a constitutively activated phosphoinositide-3 kinase (PI3K) in the intestinal epithelium. The expression of this dominantly active form of PI3K results in hyperplasia and invasive mucinous adenocarcinomas. These cancers form via a non-canonical mechanism of tumor initiation that is mediated through activation of PI3K and not through aberrations in WNT signaling. Since the Adenomatous Polyposis Coli (APC) gene is mutated in the majority of human colon cancers and often occurs simultaneously with PIK3CA mutations, we sought to better understand the interaction between APC and PIK3CA mutations in the mammalian intestine. In this study, we have generated mice in which the expression of a constitutively active PI3K and the loss of APC occur simultaneously in the distal small intestine and colon. Here, we demonstrate that expression of a dominant active PI3K synergizes with loss of APC activity resulting in a dramatic change in tumor multiplicity, size, morphology and invasiveness. Activation of the PI3K pathway is not able to directly activate WNT signaling through the nuclear localization of CTNNB1 (ß-catenin) in the absence of aberrant WNT signaling. Alterations at the transcriptional level, including increased CCND1, may be the etiology of synergy between these activated pathways.

A resistant genetic background leading to incomplete penetrance of intestinal neoplasia and reduced loss of heterozygosity in ApcMin/+ mice.

Previous studies of Min/+ (multiple intestinal neoplasia) mice on a sensitive genetic background, C57BL/6 (B6), showed that adenomas have lost heterozygosity for the germ-line ApcMin mutation in the Apc (adenomatous polyposis coli) gene. We now report that on a strongly resistant genetic background, AKR/J (AKR), Min-induced adenoma multiplicity is reduced by about two orders of magnitude compared with that observed on the B6 background. Somatic treatment with a strong mutagen increases tumor number in AKR Min/+ mice in an age-dependent manner, similar to results previously reported for B6 Min/+ mice. Immunohistochemical analyses indicate that Apc expression is suppressed in all intestinal tumors from both untreated and treated AKR Min/+ mice. However, the mechanism of Apc inactivation in AKR Min/+ mice often differs from that observed for B6 Min/+ mice. Although loss of heterozygosity is observed in some tumors, a significant percentage of tumors showed neither loss of heterozygosity nor Apc truncation mutations. These results extend our understanding of the effects of genetic background on Min-induced tumorigenesis in several ways. First, the AKR strain carries modifiers of Min in addition to Mom1. This combination of AKR modifiers can almost completely suppress spontaneous intestinal tumorigenesis associated with the Min mutation. Second, even on such a highly resistant genetic background, tumor formation continues to involve an absence of Apc function. The means by which Apc function is inactivated is affected by genetic background. Possible scenarios are discussed.

Intestinal neoplasia in the ApcMin mouse: independence from the microbial and natural killer (beige locus) status.

We have tested the hypothesis that enteric bacteria are necessary for formation of intestinal adenomas in C57BL/6-ApcMin/+ mouse. Germ-free mice developed 2-fold fewer adenomas than conventional controls in the medial small intestine (7.3 versus 14.9; P < 0.003), but there were no significant differences in the rest of the intestinal tract. We conclude that microbial status does not strongly alter the adenoma phenotype in this mouse model of familial adenomatous polyposis. In parallel, we have found that C57BL/6-ApcMin/+ mice mutated at the beige locus, which controls natural killer activity, are also unaltered in adenoma multiplicity.

Homozygosity for the Min allele of Apc results in disruption of mouse development prior to gastrulation.

Mutation of the APC (adenomatous polyposis coli) gene is an early event in colon tumor development in humans. Mice carrying Min (multiple intestinal neoplasia), a mutant allele of Apc, develop intestinal and mammary tumors as adults. To study the role of the Apc gene in development, we have investigated the phenotype of embryos homozygous for ApcMin (Min). Development of the primitive ectoderm fails prior to gastrulation in homozygous Min embryos. By midgestation, the presumed homozygotes consist of a mass of trophoblast giant cells with an additional cluster of much smaller embryonic cells. These results indicate that functional Apc is required for normal growth of inner cell mass derivatives.

Teratocarcinoma transplantation antigens are encoded in the H-2 region.

Evidence is presented for the existence of teratocarcinoma transplantation antigens (Gt) encoded within the H-2 complex and present also on adult tissues. It has not been possible to separate these Gt loci from H-2 by recombination, and Gt factors map to each end of the H-2 complex. Previous reports indicating separation of all Gt loci from H-2 are reinterpreted. One class of such apparent recombinants has been shown to result from the outgrowth of tumor variants in mice of resistant genotype.

Strong teratocarcinoma transplantation loci, Gt-1 and Gt-2, flank H-2.

Evidence is presented for the existence of two strong murine teratocarcinoma transplantation antigens (Gt) on the cell line PCC3. It is shown that the loci governing expression of these antigens are linked to the H-2 complex. These loci have been further mapped with respect to the brachyury marker (T) and H-2: Gt-1 lies 5 +/- 2 crossover units proximal to H-2 and 12 +/- 2 crossover units distal to T; Gt-2 lies 21 +/- 4 crossover units distal to H-2. It is possible that these strong transplantation antigens provide an embryonic analogue to the adult major histocompatibility system.