To investigate the role of Ras in the pathogenesis of thyroid cancer, we have generated a mouse strain in which an oncogenic Kras mutant allele, G12D, is conditionally expressed in the thyroid epithelium through Cre-mediated deletion of a floxed STOP cassette preventing KrasG12D
expression. This allele is targeted to its endogenous genomic locus, thus yielding physiological expression levels of the mutant transcript (9
). Tissue specificity is dictated by the human thyroid peroxidase promoter (TPO), driving Cre expression in the follicular cells starting at day 14.5 of embryonic development (10
TPO-Cre efficiently excised the STOP cassette (Suppl. Fig. 1A
), leading to the expression of the oncogenic allele at endogenous levels. Kras
mutant mice were born at the expected Mendelian frequencies, and throughout their lives were indistinguishable from their wild-type littermates. Surprisingly, constitutive activation of Kras did not lead to morphological () or functional () alterations of the thyroid gland, at least up to one year of age. This result is in striking contrast with the ability of the same allele to induce pancreatic intraepithelial neoplasia by two weeks of age (13
), colonic hyperplasia two weeks after activation (14
), and lung hyperplasia two weeks after activation (9
Figure 1 Simultaneous PI3K and Kras activation transforms thyrocytes. A, histopathology of thyroids from 12-week old mice. Note the normal follicles in KrasG12D mice, compared to the hyperplasia in Pten-/- mutants and the solid lesion with mitotic figures (inset) (more ...)
Figure 2 Hormonal and molecular characterization of mutant strains. A, TSH and T4 levels in 6 to12-week old mice (n=8-15), shown as box plots. The ends of the box are the 25th and 75th quantiles. The line across the box identifies the median value. Whiskers extend (more ...)
Ras activation, in certain cell types, leads to a negative feedback that down-regulates MAPK signaling and may induce senescence (15
). On the other hand, genetic (7
) and epigenetic (18
) alterations causing the activation of the PI3K pathway have recently been identified in most thyroid cancers. Thus, in thyroid follicular cells, Kras
activating mutations might need the simultaneous activation of PI3K signaling to fully realize their oncogenic potential.
To test this hypothesis, we crossed the KrasG12D
;TPO-Cre mice with PtenL/L
;TPO-Cre mice, which have hyperplastic thyroids as a result of constitutively active PI3K signaling (, Suppl. Fig. 1B
), and progress to develop adenomas by 10 months of age (19
). Strikingly, all the double mutant (DM) mice rapidly developed thyroid follicular carcinomas (). 50% of the mice died within seven weeks from birth, and none survived over four months of age (). These mice developed thyroids 200 to 500-fold larger than control glands (data not shown). PCR analysis showed that both the Pten
and the Kras
locus had undergone appropriate Cre-mediated recombination (). Accordingly, follicular cells in both Pten-/-
glands showed dramatically increased pAKT levels (Suppl. Fig. 1B
). Histopathology of the DM mice revealed that 30 to 90% of the thyroid glands was replaced by microfollicular to solid areas (Suppl. Fig. 2
) presenting many hallmarks of thyroid follicular cancer, including capsular, muscle, and vascular invasion. In addition, all the mice surviving at least 12 weeks had developed thyroglobulin-positive lung metastases (, Suppl. Fig. 3
To assess the thyroid functional status in the four strains, and to determine whether the tumors depend on TSH signaling, we measured TSH and T4 in the serum of control and mutants. While single gene mutation did not alter the levels of either hormone, TSH was drastically reduced and T4 increased in the DM mice, suggesting that simultaneous PI3K and Kras activation may confer a certain degree of thyroid autonomy (). Although we still do not know the basis for this TSH-independent thyroid hormone synthesis, TSH suppression strongly suggests that tumor development, in this model, does not rely on TSH-mediated proliferative signals.
In several mouse models, endogenous Kras activation seems to be unable to fully activate MAPK signaling (4
). We analyzed the phosphorylation of MEK and its direct target ERK in the thyroids of the four strains. PI3K activation in Pten-/-
thyroids did not alter the phosphorylation of these kinases. Surprisingly, while oncogenic Kras, alone, substantially increased MEK phosphorylation, this had no significant effect on ERK activation (). Conversely, simultaneous activation of Kras and PI3K increased MEK phosphorylation at levels similar to oncogenic Kras alone, but, in this case, also drastically increased ERK phosphorylation, strongly suggesting that, at least in thyroid cells, PI3K signaling removes or overcomes a negative feedback uncoupling MEK and ERK activation. In addition, PI3K signaling increased MEK protein levels (), and specifically MEK2 mRNA (), providing an additional possible mechanism for ERK activation in the DM mice.
We used cell lines generated from DM tumors and pharmacological inhibitors of PI3K (LY294002) and MEK1 (PD98059) to further investigate the cooperation between PI3K- and Kras-dependent signaling. While MEK inhibition could decrease only to a certain degree the growth rate of the DM cells, targeting PI3K was much more effective, and the two compounds cooperated to completely inhibit the tumor cell growth ().
Figure 3 Modulation of PI3K signaling reduces cell proliferation in vitro and in vivo. A, growth curve of tumor cells upon treatment with LY294002 and PD98059. The graph represents one of at least four independent experiments, on three different cell lines. B (more ...)
Strikingly, we found that protracted PI3K inhibition in tumor-derived cells reduced ERK phosphorylation to the same extent as direct MEK inhibition (), and led to the establishment of senescence (), strongly suggesting that activation of Kras alone is not transforming because of a PI3K-sensitive negative feedback that uncouples MEK and ERK, terminates the MAPK cascade, and establishes senescence.
To test in vivo
the permissive role of PI3K in Kras-dependent transformation, we administered LY294002 twice a week to DM mice at a low dose of 25mg/kg (21
), starting at three weeks of age. PI3K inhibition significantly prolonged the survival of LY-treated mice, compared to untreated mice (). These data are consistent with the results obtained on the tumor-derived cell lines, and support the notion that continuous PI3K signaling is necessary to permit the transforming activity of oncogenic Kras mutations.
We then used a qRT-PCR array to measure the expression of 84 genes associated with MAPK signaling in thyroid RNA from the four strains, trying to identify relevant targets of the cooperation between PI3K and Kras. Analysis of the expression of these genes identified a subgroup of transcripts specifically up-regulated in the DM mice, compared to wild-type thyroids (). We have initially focused on these genes as prime candidates to mediate the combined transforming activity of PI3K and Kras, and in particular on cyclin D1.
Figure 4 Cyclin D1 is a target of PI3K and Kras cooperation. A, heat map showing the expression of significantly deregulated genes in the thyroids of mutant mice, relative to controls. The group of genes cooperatively up-regulated by PI3K and Kras activation is (more ...)
Cyclin D1 transcript levels were up-regulated over four-fold in the compound mutants, compared to both wild-type and single mutant mice (). In contrast, the expression levels of the other D-type cyclins, D2 and D3, were not significantly altered in any of the mutant strains. Accordingly, when we inhibited PI3K and/or MAPK signaling in tumor-derived cell lines, cyclin D1 transcript levels (but not D2 or D3) were down-regulated (again, about four-fold) by simultaneous LY294002 and PD98059 treatment (), thus validating our data obtained in vivo. Finally, we utilized Western blot analysis to prove that cyclin D1 protein expression followed the same pattern observed at the mRNA level. DM thyroids expressed much higher cyclin D1 protein than any of the single mutant strains, and simultaneous PI3K and MAPK inhibition dramatically reduced cyclin D1 protein levels in tumor-derived cell lines ().