To generate mice with inducible expression of human p110α H1047R, we injected a DNA segment consisting of seven direct repeats of the tetracycline (tet)-operator sequence, followed by hPIK3CA H1047R
cDNA and SV40 polyA
into FVB/N fertilized eggs as described previously 2,3
(Materials and Methods). Ten Tet-op-hPIK3CA
founders were identified and then crossed to CCSP-rtTA
mice (that specifically targets expression of the reverse tetracycline trans-activator protein (rtTA) in type II alveolar epithelial cells4
) to generate inducible, bitransgenic mouse cohorts harboring both the activator and the responder transgenes 4,5
. The Tet-op-hPIK3CA
copy numbers from the two most utilized founders were determined by quantitative real-time PCR (Supplementary Fig. 1a
To induce expression p110-α H1047R in mouse lung epithelial cells, we administered doxycycline (doxy) to bitransgenic mice from each of the founder lines, monitored them for labored breathing, and imaged dyspneic mice with MRI to identify abnormalities. Three founder lines #13, #121, and #3011demonstrated labored breathing and MRI images consistent with lung tumors after 12, 26, and 60 weeks respectively. These mice were sacrificed, and gross inspection revealed multiple small tumor nodules. Histological analyses revealed mixed adenocarcinomas with bronchioloalveolar features (). As founder line #13 demonstrated the shortest latency period, it was utilized for subsequent experiments.
Development of a Tet-inducible PIK3CA H1047R mouse model of lung tumorigenesis
The inducibility of the PIK3CA
mutant transgene expression in the lung was evaluated at the RNA level using RT-PCR. PIK3CA H1047R expression was readily observed after 12 weeks of doxycycline administration (Supplementary Fig. 1b
). Doxycycline withdrawal led to a loss of mutant PIK3CA expression. We observed expression of mutant p110-α protein in PI3K immunoprecipitations only from the bitransgenic mice induced with doxycycline (Supplementary Fig. 1c
). Of note, expression of the transgene did not substantially increase total p110-α protein levels. This is expected since p110-α that is not bound to p85 is unstable, and any p110-α expressed in excess of p85 is rapidly degraded 6-8
Withdrawal of doxycycline led to rapid and dramatic tumor regression thereby demonstrating that these established lung tumors require continued expression of p110-α H1047R (). After doxycycline withdrawal, histological examination showed focal pulmonary fibrosis and scarring and no evidence of cancer (). Of note, complete tumor regression was also observed in the other founder line (#121) that was examined for reversibility (Supplemental Fig. 2
). Thus, these lung tumors require continued p110-α H1047R expression for their maintenance.
To inhibit PI3K signaling in vivo
, we treated mice with NVP-BEZ235, a potent dual pan-PI3K/mTOR inhibitor currently under clinical development by Novartis Pharma Ag (Supplementary Fig. 3a
. This drug blocks the kinase activity of all four p110 isoforms and mutant p110-α H1047R with similar potencies 9
. To identify a dose that adequately blocks PI3K in lung tissue, we treated control mice with one dose ranging from 30-52.5 mg/kg, and lungs were harvested either 3 or 8 hours later. At most of the dose levels examined, NVP-BEZ235 induced suppression of PI3K signaling as indicated by decreased P-Akt levels (Supplementary Fig. 3b
). We then evaluated if this compound could inhibit PI3K signaling in the lung tumors induced by the p110-α H1047R mutant. One oral treatment of NVP-BEZ235 35 mg/kg led to substantial suppression of Akt, S6, and 4e-bp1 phosphorylation in these mouse tumors ().
NVP-BEZ235 downregulates PI3K signaling in p110-α H1047R induced lung tumors and leads to rapid tumor regression
We next evaluated the clinical efficacy of NVP-BEZ235 against p110-α H1047R induced mouse lung tumors. Tumor responses were assessed by MRI, PET-CT scans, and histological analyses. Doxycycline was administered to bitransgenic mice, and MRI screening identified mice with established tumors prior to initiating treatment. We observed that four days of treatment with NVP-BEZ235 at 35mg/kg per day led to a substantial reduction in the tumor's 18
FDG avidity as measured by PET imaging and also led to a dramatic decrease in their size as judged by CT ( and Supplemental Movie
for three dimensional reconstruction PET images). This data supports the notion that 18
FDG-PET imaging may be an important pharmacodynamic marker for efficacy of PI3K inhibitors in the clinic. Histopathological analysis after short-term treatments demonstrate decreased cellularity and increased interstitial thickening within the residual tumor nodule with no evidence of adenocarcinoma ().
Since NVP-BEZ235 is dual PI3K/mTOR inhibitor, we determined if the effects of this compound were due to its inhibition of TORC1 (mTOR/Raptor). Therefore, we treated mice with established PIK3CA
mutated tumors with rapamycin. Treatment with rapamycin effectively blocked TORC1 in these tumors as evidenced by a loss of S6 phosphorylation (Supplementary Fig. 4
). However, unlike NVP-BEZ235, rapamycin did not shrink these tumors (). Thus, it appears that the activity of NVP-BEZ235 is not due solely to TORC1 inhibition.
Recently, a study by Downward and colleagues revealed that p110-α is required for lung tumorigenesis in the LA2 K-Ras G12
mouse model 10
. In that study, mice were generated in which the endogenous Pik3ca
gene was mutated in the Ras binding domain. This mutation abrogated the ability of K-Ras G12D to induce lung tumors. Using a different genetic approach, we also observed that loss of PI3K signaling hindered K-Ras induced lung tumorigenesis. We crossed the LSL K-Ras mice to those with genetic deletion of the p85 PI3K regulatory subunits (encoded by Pik3r1
). We previously utilized p85 knockouts to genetically ablate PI3K signaling in different tumor models 11
. The experiments were performed on a Pik3r2 -/-
background, and the Pik3r1
allele was flanked by flox sites. Inhaled adenoviral Cre leads to both its deletion and activation of K-Ras G12D. We observed that loss of both Pik3r1
alleles significantly inhibited tumorigenesis (). Of note, these genetic experiments evaluate the role of PI3K in mutant K-Ras induced tumor development
. To more closely mirror clinical treatment of patients with cancer, we evaluated if PI3K signaling was necessary for the maintenance of established K-Ras driven tumors. We utilized both the tet-inducible K-Ras G12D
transgenic model 4
as well as the LSL K-Ras
mouse model to induce lung tumors. After tumors developed, the mice were treated with the NVP-BEZ235. In contrast to the lung cancers induced by p110-α H1047R, those derived from K-Ras G12D did not shrink in response to single-agent BEZ235 as indicated by PET-CT or MRI images ( and S5a
). However, NVP-BEZ235 decreased Akt phosphorylation in these lungs as determined by western blot analysis () and immunohistochemical analysis (see below). These results suggest that PI3K may be required for K-Ras induced tumorigenesis, but may be less critical for tumor maintenance. Although both K-Ras tumor model mouse strains and the p110-α H1047R tumor model mouse strain are on similar mixed genetic backgrounds, it remains conceivable that subtle differences in genetic background may have affected responsiveness to PI3K inhibitors. However, the data with the K-Ras tumor models clearly underscores the notion that blocking tumorigenesis is not equivalent to treating a cancer that has already been established.
PI3K signaling is needed for K-Ras induced tumorigenesis but not tumor maintenance
Recently, it has become evident that cancers respond dramatically to therapies targeting receptor tyrosine kinases (RTKs) when inhibition of the RTK leads to loss of both PI3K and ERK signaling 12-15
. To recapitulate this effect in the K-Ras mutant lung tumors, we treated the mice with a combination of a PI3K and a MEK inhibitors. Whereas treatment of the K-Ras mutant mice with the MEK inhibitor, ARRY-142886 16
, led to only modest tumor regression, the combination led to marked synergistic tumor regression (), and pathological analyses at the completion of treatment revealed only scant remnants of tumor nodules (Supplementary Fig. 5b
). After two days of the combination treatment, there was marked downregulation of PI3K, Erk and downstream signaling as indicated by western blot analyses and IHC (). Of note, we invariably detected low level P-Akt staining in the K-Ras G12D nodules that was impressively lost upon treatment of the mouse with NVP-BEZ235 ().
Combined PI3K and MEK inhibition dramatically shrinks K-Ras G12D induced lung tumors
K-RAS mutated lung cancers remain a huge cancer problem as they comprise 20-30% of non-small cell lung cancers. Currently, there are no effective targeted therapies for this subset of cancers. In fact, the presence of K-RAS mutations only serves to identify those cancers that are likely not to respond to targeted therapies. The data in this study clearly demonstrate that the combination of PI3K and MEK inhibitors may be a very potent combination for these cancers. We hope this study spurs efforts to combine these classes of inhibitors for K-RAS mutated cancers.
There is not yet sufficient clinical data to determine if PI3K inhibitors will be powerful therapeutic agents as single-agent cancer therapies for patients or whether they will be effective only when combined with other targeted therapies. However, it is tempting to speculate that cancers that harbor activating mutations in PIK3CA
or loss of PTEN may be particularly sensitive to PI3K inhibitors. Although this study suggest that cancers with PIK3CA
mutations may respond to PI3K inhibitors, human cancers with PIK3CA
mutations often harbor other known oncogenic mutations such as K-RAS
(colon cancers) and HER2
amplification (breast cancer) 17-19
. These concordant oncogenic genetic changes may impact their responsiveness to PI3K inhibitors and possibly may necessitate combinations as was the case for effective treatment of the K-Ras
mutated mouse lung cancers.