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Background There is a likely genetic component to transsexualism, and genes involved in sex steroidogenesis are good candidates. We explored the specific hypothesis that male-to-female transsexualism is associated with gene variants responsible for undermasculinization and/or feminization. Specifically, we assessed the role of disease-associated repeat length polymorphisms in the androgen receptor (AR), estrogen receptor β (ERβ), and aromatase (CYP19) genes. Methods Subject-control analysis included 112 male-to-female transsexuals and 258 non-transsexual males. Associations and interactions were investigated between CAG repeat length in the AR gene, CA repeat length in the ERβ gene, and TTTA repeat length in the CYP19 gene and male-to-female transsexualism. Results A significant association was identified between transsexualism and the AR allele, with transsexuals having longer AR repeat lengths than non-transsexual male control subjects (p = .04). No associations for transsexualism were evident in repeat lengths for CYP19 or ERβ genes. Individuals were then classified as short or long for each gene polymorphism on the basis of control median polymorphism lengths in order to further elucidate possible combined effects. No interaction associations between the three genes and transsexualism were identified. Conclusions This study provides evidence that male gender identity might be partly mediated through the androgen receptor.

PIK3CA, the gene coding for the p110α subunit of phosphoinositide 3-kinase, is frequently mutated in a variety of human tumors including breast cancers. To better understand the role of mutant PIK3CA in the initiation and/or progression of breast cancer, we have generated mice with a conditional knock-in of the common activating mutation, Pik3caH1047R, into one allele of the endogenous gene in the mammary gland. These mice developed a ductal anaplasia and hyperplasia by 6 weeks of age characterized by multi-layering of the epithelial lining of the mammary ducts and expansion of the luminal progenitor (Lin−; CD29lo; CD24+; CD61+) cell population. The Pik3caH1047R expressing mice eventually develop mammary tumors with 100% penetrance but with a long latency (>12 months). This is significantly longer than has been reported for transgenic models where expression of the mutant Pik3ca is driven by an exogenous promoter. Histological analysis of the tumors formed revealed predominantly ERα-positive fibroadenomas, carcinosarcomas and sarcomas. In vitro induction of Pik3caH1047R in immortalized mammary epithelial cells also resulted in tumor formation when injected into the mammary fat pad of immunodeficient recipient mice. This novel model, which reproduces the scenario of a heterozygous somatic mutation occurring in the endogenous PIK3CA gene, will thus be a valuable tool for investigating the role of Pik3caH1047R mutation in mammary tumorigenesis both in vivo and in vitro.

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The phosphoinositide 3-kinase (PI3K)/AKT signalling pathway regulates many cellular functions including proliferation, migration, survival and protein synthesis. Somatic mutations in PIK3CA, the gene encoding the p110α catalytic subunit of PI3K enzyme, are commonly associated with many human cancers as well as recently being implicated in human overgrowth syndromes. However, it is not clear if such mutations can be inherited through the germline. We have used a novel mouse model with Cre recombinase (Cre)-conditional knock-in of the common H1047R mutation into the endogenous Pik3ca gene. Heterozygous expression of the Pik3ca(H1047R) mutation in the developing mouse embryo resulted in failed 'turning' of the embryo and disrupted vascular remodelling within the embryonic and extraembryonic tissues, leading to lethality prior to E10. As vascular endothelial growth factor A (VEGF-A) signalling was disrupted in these embryos, we used Cre under the control of the Tie2 promoter to target the Pik3ca(H1047R) mutation specifically to endothelial cells. In these embryos turning occurred normally but the vascular remodelling defects and embryonic lethality remained, likely as a result of endothelial hyperproliferation. Our results confirm the lethality associated with heterozygous expression of the Pik3ca(H1047R) mutation during development and likely explain the lack of inherited germline PIK3CA mutations in humans.

Mutations in PIK3CA, the gene encoding the p110a catalytic subunit of PI3K, are among the most common mutations found in human cancer and have also recently been implicated in a range of overgrowth syndromes in humans. We have used a novel inducible "exonswitch" approach to knock in the constitutively active Pik3ca H1047R mutation into the endogenous Pik3ca gene of the mouse. Ubiquitous expression of the Pik3ca H1047R mutation throughout the body resulted in a dramatic increase in body weight within 3 weeks of induction (mutant 150 6 5%; wild-type 117 6 3%, mean 6 SEM), which was associated with increased organ size rather than adiposity. Severe metabolic effects, including a reduction in blood glucose levels to 59 6 4% of baseline (11 days postinduction) and undetectable insulin levels, were also observed. Pik3ca H1047R mutant mice died earlier (median survival 46.5 d post-mutation induction) than wild-type control mice (100% survival > 250 days). Although deletion of Akt2 increased median survival by 44%, neither organ overgrowth, nor hypoglycemia were rescued, indicating that both the growth and metabolic functions of constitutive PI3K activity can be Akt2 independent. This mouse model demonstrates the critical role of PI3K in the regulation of both organ size and glucose metabolism at the whole animal level.-Kinross, K. M., Montgomery, K. G., Mangiafico, S. P., Hare, L. M., Kleinschmidt, M., Bywater, M. J., Poulton, I. J., Vrahnas, C., Henneicke, H., Malaterre, J., Waring, P. M., Cullinane, C., Sims, N. A., McArthur, G. A., Andrikopoulos, S., Phillips, W. A. Ubiquitous expression of the Pik3ca H1047R mutation promotes hypoglycemia, hypoinsulinemia, and organomegaly. FASEB J. 29, 1426-1434 (2015). www.fasebj.org Key Words: PI3K • p110a • mouse model • overgrowth syndrome • glucose metabolism CLASS I PI3KS ARE A UBIQUITOUS family of lipid kinases that play a key role in regulating a wide range of important cellular processes including proliferation, growth, survival, angiogenesis, metabolism, and migration (1). PI3Ks are comprised of a unique catalytic subunit (p110a, b, or d) along with one of a number of shared regulatory subunits (p85a, p85b, and p55g). PI3Ks are activated by growth factor stimulation of receptor tyrosine kinases, and catalyze the phosphorylation of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P 2 ) to form PI(3,4,5)P 3 . The second messenger PI(3,4,5)P 3 then activates a series of downstream signaling pathways, including the AKT/mammalian target of rapamycin pathway. The activity of PI3K can be opposed by the phosphatase PTEN (phosphatase and tensin homolog), which dephosphorylates PI(3,4,5)P 3 returning it back to PI(4,5)P 2 (1, 2).

PIK3CA, the gene encoding the p110α catalytic subunit of PI3K (phosphoinositide 3-kinase), is mutated in approximately 20% of sporadic CRCs (colorectal cancers), but the role of these mutations in the pathogenesis of CRC remains unclear. In the present study we used a novel mouse model to investigate the role of the Pik3caH1047R mutation, the most common PIK3CA mutation in CRC, during the development and progression of intestinal cancer. Our results demonstrate that Pik3caH1047R, when expressed at physiological levels, is insufficient to initiate intestinal tumorigenesis; however, in the context of Apc (adenomatous polyposis coli) loss, which is observed in 80% of CRCs and by itself results in benign intestinal adenomas, the Pik3caH1047R mutation promotes the development of highly aggressive and invasive adenocarcinomas in both the small and large intestines. The results of the present study show that an activating Pik3ca mutation can act in tandem with Apc loss to drive the progression of gastrointestinal cancer and thus this disease may be susceptible to therapeutic targeting using PI3K pathway inhibitors.

The PI3K/Akt signalling pathway regulates many important cellular functions including proliferation, migration, survival and angiogenesis. The PIK3CA gene, encoding the p110α catalytic subunit of the PI3K enzyme, is somatically mutated in a range of human cancers and overgrowth syndromes. We have previously described a novel mouse model with a Cre-recombinase (Cre)-mediated knock-in of the common cancer-associated Pik3ca(H1047R) mutation that can be targeted to selected tissues using various Cre-expressing mouse strains. As the mutation is knocked in to the endogenous gene, the mutant protein is expressed at normal physiological levels. We have used the Cre-deleter mouse to induce expression of the Pik3ca(H1047R) mutation ubiquitously from the 2-cell stage of development. This results in embryonic lethality by E10.5 with embryos at E9.5 demonstrating stunted growth, failed ‘turning’ and abnormal cardiovascular development in both the embryo proper and extraembryonic yolk sac. These tissues also showed increased expression of vascular endothelial growth factor (VEGF) family proteins. We then selectively targeted the mutation to the endothelial cells of the developing embryo using the Tie2-Cre mouse strain. No Tie2-Cre:Pik3ca(H1047R) mutant embryos survived to E11.5 and at E10.5, while successfully turned, embryos were severely underdeveloped and no longer viable. At E9.5, vascular remodelling was disorganised and truncated in the embryo proper and yolk sacs. Development of the heart was also affected with clear disruption to anterior cardinal vein and dorsal aorta development. Our results demonstrate the importance of PI3K/Akt signalling in embryonic development and cardiovascular formation in the mouse. The observed cell autonomous role in vessel development was further confirmed upon targeted expression to endothelial cells while the increased expression of various VEGF family proteins suggests probable crosstalk between the PI3K/Akt and VEGF signalling pathways during cardiovascular development. The lethality associated with expression of Pik3ca(H1047R) mutation during development likely explains the lack of inherited germline PIK3CA mutations in humans. Citation Format: Lauren M. Hare, Quenten Schwarz, Rajendra Gurung, Karen G. Montgomery, Christina Mitchell, Wayne A. Phillips. Heterozygous expression of an oncogenic Pik3ca mutation during murine development results in fatal embryonic and extra-embryonic defects. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 86. doi:10.1158/1538-7445.AM2014-86