The present study has examined the effect of PDK1 transgene expression on mammary carcinogenesis, and how it impacts the tumor promoting effects of PPARδ activation. Although PDK1 transgenic mice did not exhibit changes in mammary gland development, function and tumorigenicity, they were markedly sensitive to GW501516 treatment, where median tumor-free survival was reduced four-fold in comparison to a two-fold reduction in GW501516-treated wild-type mice. This striking effect correlated with an increase in a specific metabolic gene signature indicative of glycolysis and fatty acid biosynthesis that was not present in either control wild-type or transgenic mice (, Supporting information Figure S3
). It is well-known that human cancers exhibit a near ubiquitous expression of metabolic genes 
that is widely regarded to support high rates of proliferation 
. This phenotype is consistent with the acceleration of mammary tumorigenesis by GW501516 
, the increase in fatty acid biosynthesis by GW501516 in muscle cells 
, and our analysis of the mammary gland metabolome in GW501516-treated animals. Some of these changes may be related to increased AKT activation in GW501516-treated animals, since it is an important regulator of glucose and lipid metabolism in both normal and malignant cells, including breast cancer 
. AKT phosphorylates and activates ATP:citrate lyase (Acly) to promote tumor growth 
, and loss of Acly counteracts AKT-driven tumorigenesis 
. In addition, the PPARδ target gene, Pdk4, 
reduces the flux of pyruvate into the tricarboxylic acid cycle, and Acss2, which increases the flux of lactate to acetylCoA, were increased in GW501516-treated animals (, ). These changes are consistent with the greater levels of fatty acid and phospholipid metabolites in GW501516-treated transgenic mice in comparison to treated wild-type mice (), suggesting their involvement in enhanced tumorigenesis. This association is also in agreement with the increase in serum lysophospholipids in women with high grade ovarian cancer 
Schematic of the regulation of glucose and fatty acid metabolism by PDK1 and PPARδ.
The lack of tumorigenicity of PDK1 transgene expression is in agreement with previous studies that found PDK1 over-expression per se
was not oncogenic unless expressed in a heterozygous PTEN background 
or together with a growth factor receptor with oncogenic potential, such as erbB2 
. The lack of change in pmTOR, p4EBP1, pS6, pPKCα () and pRSK (results not shown) in mammary tissue from PDK1 transgenic mice is also consistent with the lack of change in S6K and RSK activation following treatment of PDK1 hypomorphic mice with insulin 
. Thus, low residual levels of PDK1 appear to be sufficient for mammary gland development and function and for downstream signaling.
One seminal finding in our study was the increase in PPARδ expression in the transgenic mammary gland. PPARδ expression is induced by K-Ras via ERK activation 
, and although PDK1 has been reported to regulate MEK1/2 activation 
, ERK activation remained unchanged in MMTV-PDK1 mice (results not shown). Another possible mechanism is that increased pS9GSK3β by AKT inhibits the ability of GSK3β to phosphorylate and destabilize β-catenin by proteasomal degradation, and thus results in enhanced transcription of TCF target genes, such as PPARδ 
. However, no evidence of nuclear β-catenin accumulation was noted in GW501516-treated wild-type animals despite increased pT308AKT expression (), suggesting other signaling mechanisms. The changes in PPARδ observed in transgenic mice may also have resulted from post-translational stabilization against proteasomal degradation when ligand-bound 
. This view is supported by our results in PDK1-transduced mouse mammary epithelial cells, which expressed increased PPARδ and PPARδ-dependent reporter gene activity (). It is equally plausible that endogenous PPARδ ligands generated by fatty acid metabolism serve as PPARδ ligands 
to increase expression post-translationally. Lastly, the co-association of PDK1 and PPARδ noted in mammary tumors 
may also have enhanced resistance of the receptor to ubiquitination and degradation.
In addition to increased expression of PPARδ in MMTV-PDK1 mice, PDK1 levels were increased by PPARδ agonist GW501516 (). The mouse PDK1 locus contains a PPRE in an upstream enhancer region 
, and deletion of PPARδ resulted in a 50% reduction in PDK1 mRNA and >80% decrease in PDK1 protein expression 
. This could result in a feed forward mechanism resulting from the effect of PDK1 and PPARδ on each others expression, and could explain their synergism in tumorigenesis.
Many of the genes whose expression increased more than 3-fold in MMTV-PDK1 mice were associated with muscle architecture or motor function, e.g. myosin, nebulin, troponin 1, tropomyosin and titin. A recent study profiling gene expression in the mammary gland side population also noted a muscle-specific expression pattern 
. Although the function of these genes in non-myogenic cells is unknown, troponin 1 has been found to bind ERRα, enhance its transcriptional activity 
and regulate the metabolic switch to oxidative phosphorylation 
. Thus, the expression of these genes may be a factor in the increase in fatty acid transport and oxidation in muscle cells treated with GW501516 
In summary, PDK1 expression in the mammary gland was not oncogenic, but accelerated tumor formation in conjunction with a PPARδ agonist. GW501516-enhanced tumorigenesis was associated with a distinct gene and metabolomic signature related to glycolysis and fatty acid biosynthesis. These results suggest that PDK1 and PPARδ may drive tumorigenesis by enhancing energy metabolism.