K-RAS Mutant Pancreatic Tumors Show Higher Sensitivity to MEK than to PI3K Inhibition <em>In Vivo</em>

<div><p>Activating K-RAS mutations occur at a frequency of 90% in pancreatic cancer, and to date no therapies exist targeting this oncogene. K-RAS signals via downstream effector pathways such as the MAPK and the PI3K signaling pathways, and much effort has been focused on developing drugs targeting components of these pathways. To better understand the requirements for K-RAS and its downstream signaling pathways MAPK and PI3K in pancreatic tumor maintenance, we established an inducible K-RAS knock down system that allowed us to ablate K-RAS in established tumors. Knock down of K-RAS resulted in impaired tumor growth in all pancreatic xenograft models tested, demonstrating that K-RAS expression is indeed required for tumor maintenance of K-RAS mutant pancreatic tumors. We further examined signaling downstream of K-RAS, and detected a robust reduction of pERK levels upon K-RAS knock down. In contrast, no effect on pAKT levels could be observed due to almost undetectable basal expression levels. To investigate the requirement of the MAPK and the PI3K pathways on tumor maintenance, three selected pancreatic xenograft models were tested for their response to MEK or PI3K inhibition. Tumors of all three models regressed upon MEK inhibition, but showed less pronounced response to PI3K inhibition. The effect of MEK inhibition on pancreatic xenografts could be enhanced further by combined application of a PI3K inhibitor. These data provide further rationale for testing combinations of MEK and PI3K inhibitors in clinical trials comprising a patient population with pancreatic cancer harboring mutations in K-RAS.</p> </div

GDC0941 and AZD6244 <i>in vivo</i> treatment inhibits pAKT and pERK respectively.

<p>Indicated tumor-bearing mice were treated with a single dose of either GDC0941 100 mg/kg p.o., or AZD6244 50 mg/kg p.o., or with vehicle control. Animals were sacrificed 1 h after treatment, plasma samples were collected, analyzed and quantified by mass spectrometry for GDC0941 (A) or AZD6244 (B). Tumors were excised and analyzed by Western Blot for total AKT, pAKT (Ser473), total ERK or pERK (Thr202/Tyr204) for the model Rat1-myr-p110α (C) or the model Panc 10.05 (D).</p

K-RAS knock down impairs tumor growth of pancreatic models <i>in vivo</i>.

<p>(A) For each xenograft model indicated, tumors were grown subcutaneously in female nude mice and groups of at least 4 mice each were formed once tumors had reached a size of 200–300 mm³. The first group was given normal drinking water, whereas the second was given drinking water containing 2 mg/ml doxycycline and 10% sucrose. Mice were sacrificed after one week of treatment (after 18 days in case of the K-RAS wild type model), and tumors were analyzed by qPCR for K-RAS. K-RAS levels were normalized to ribosomal protein s18. Obtained p-values were as follows: Capan-1 shNT: p = 0.35, Capan-1 sh236: p = 0.011, Panc 10.05 sh236: p = 0.009, AsPC-1 sh236: p = 0.002, L3.3 sh236: p = 0.004, NCI-H1437 sh236: p = 0.007. (B/C) As in (A), except that mice were randomized to groups of at least 6 mice each, with the exception of the Panc 10.05 model, where the group size was n = 4. Treatment was started once tumors had reached a size of 100 mm³, tumor size was followed over time, and mice were sacrificed once tumors of the control group reached a size of 1000 mm³ at most. Statistically significant differences of tumor volumes between groups (*) as well as the area under the curve (AUC/mm³ x treatment days) are indicated. Obtained p-values for AUC at the end of the study were as follows: Capan-1 shNT: p = 0.57, Capan-1 sh236: p = 0.04, Panc 10.05 sh236: p = 0.01, AsPC-1 sh236: p = 0.01, L3.3 sh236: p = 0.0003, NCI-H1437: p = 0.22. The PANC-1 cells could not be grown <i>in vivo</i>, and for this reason this model was only examined <i>in vitro</i>.</p

Combining MEK and PI3K inhibition <i>in vivo</i> is superior to single agent treatment.

<p>(A). Indicated tumor-bearing mice were treated either with GDC0941 100 mg/kg p.o. once a day, or with AZD6244 5 mg/kg p.o. once a day, or with the combination of both, or with vehicle control, with 8 mice per group. Tumor volumes were measured twice a week, for the indicated period of time, and antitumor activity was plotted and quantified. (B). Indicated tumor-bearing mice were treated with a single dose of either GDC0941 100 mg/kg p.o. or of AZD6244 5 mg/kg p.o., with the combination of both or with vehicle control. Animals were sacrificed 3 h after treatment, tumors were excised and analyzed by Western Blot for total AKT, pAKT (Ser473), total ERK or pERK (Thr202/Tyr204).</p

K-RAS mutant pancreatic models show stronger response to MEK than to PI3K inhibition <i>in vivo</i>.

<p>(A/B). Indicated tumor-bearing mice were treated either with GDC0941 100 mg/kg p.o. once a day, or with AZD6244 50 mg/kg p.o. twice a day, or with vehicle control, with at least 5 mice per group. Tumor volumes were measured twice a week for the indicated period of time, and antitumor activity was plotted and quantified.</p

K-RAS knock down results in decreased pERK levels <i>in vivo</i>.

<p>For each xenograft model indicated, tumors were grown subcutaneously in female nude mice and groups of at least 4 mice each were formed once tumors had reached a size of 200–300 mm³. The first group was given normal drinking water (-dox), whereas the second was given drinking water containing 2 mg/ml doxycycline and 10% sucrose (+dox). After one week of treatment, mice were sacrificed and the tumors were removed and processed for immunohistochemistry for either pERK (Thr202/Tyr204) (A), or pAKT (Ser473) (B). The T47D model was used as an AKT dependent control model with physiological pAKT levels.</p

K-RAS knock down impairs proliferation in pancreatic lines <i>in vitro</i>.

<p>(A) Indicated cell lines (NT: non-targeting shRNA; 236 and 562: shRNAs targeting K-RAS) were either treated for 7 days with 200 ng/ml doxycycline (dox) or left untreated (no dox), followed by preparation of cell lysates. Corresponding cell extracts were then analyzed for K-RAS, total AKT, pAKT (Ser473), total ERK or pERK (Thr202/Tyr204) levels by Western Blot. (B) As in (A), except that cells were fixed on day 1 and day 7, followed by determination of relative cell number. Each cell line was tested in at least two independent experiments, and untreated samples were set to 100% of growth. Statistically significant differences (p<0.05) are indicated (*). Obtained p-values were as follows: Capan-1 shNT: p = 1, Capan-1 sh236: p = 0.002, Capan-1 sh562: p = 0.029; Panc 10.05 shNT: p = 0.33, Panc 10.05 sh236: p<0.001, Panc 10.05 sh562: p = 0.029; AsPc1 shNT: p = 0.33, AsPc1 sh236: p = 0.002, AsPc1 sh562: p = 0.029; L3.3 shNT: p = 0.187, L3.3 sh236: p<0.001, L3.3 sh562: p = 0.333; PANC-1 shNT: p = 1, PANC-1 sh236: p<0.001, PANC-1 sh562: p = 0.002.</p

K-RAS mutant pancreatic lines are independent of AKT <i>in vitro</i>.

<p>(A). Indicated cell lines were treated for 72 h with the AKT inhibitor MK2206, and effects on proliferation were determined by calculation of respective GI₅₀ values. (B/C). As in (A), except that indicated cell lines were treated for 62 h with either the PI3K inhibitor GDC0941 (B) or with the MEK inhibitor AZD6244 (C), and effects on proliferation were determined by calculation of respective GI₅₀ values. MCF7 cells were used as control for cells sensitive to GDC0941 and insensitive to AZD6244 and A375 cells were used as control for cells sensitive to AZD6244 and insensitive to GDC0941.</p

YAP-PTPN14 binding is mediated through the WW domain-PPxY motif interaction.

<p>A) 293A cells were transfected with WT GST-PTPN14 and the indicated V5-YAP constructs (WT and mutants). A V5 IP was carried out and blotted for GST to study the interaction of the various YAP mutants with WT PTPN14. Input lanes were loaded with 10% of the amount of lysate used for each IP and used to compare the expression levels of each construct. B–C) 293A cells were transfected with WT V5-YAP and the indicated GST-PTPN14 constructs (WT and mutants). A V5 IP was carried out and blotted for GST to study the interaction of the various PTPN14 mutants with WT YAP. Input lanes were loaded with 10% of the amount of lysate used for each IP and used to compare the expression levels of each construct. All lanes in (C) are from a single blot and exposure. D) An SF268 cell line stably expressing the YAP-responsive MCAT_Luc reporter was transduced with lentivirus encoding for the indicated dox-inducible PTPN14 expression. Luciferase expression of each cell line was analysed 72 hours post dox induction (left panel). A Resazurin assay was carried out in parallel for each sample and used to normalize the luciferase readings. PTPN14 expression levels achieved for each construct were analysed by Western blot (right panel; arrows indicate the WT PTPN14 protein and the truncated ΔPTP PTPN14 which migrates faster; all lanes from a single blot and exposure). Tubulin serves as loading control. Luciferase results are shown as the average of at least 3 independent experiments ± STDEV. Statistical analysis was carried out with a 2-tailed paired t-test; * p<0.05. E) The mRNA levels of the indicated YAP target genes were assessed in cells from (D) 72 hours post dox induction. Statistical analysis was carried out with a 2-tailed paired t-test; * p<0.001; **p<0.05, F) 293A cells were transduced with lentivirus encoding for the indicated dox-inducible PTPN14 expression. The nuclear/cytoplasmic YAP ratio was quantified at low density after 72 hours of dox induction using a Cellomics automated imager with a conventional microscope, and expressed relative to control (left panel). Results are shown as the average of three experiments ± STDEV. Statistical analysis was carried out with a 2-tailed paired t-test; * p<0.05. For each experiment, the average ratio was calculated from three wells per sample (10 images per well). PTPN14 expression levels were analysed by Western blot (right panel; arrows indicate the WT PTPN14 protein and the truncated ΔPTP PTPN14 which migrates faster; all lanes from a single blot and exposure). Tubulin serves as a loading control G) Confocal microscopy images of 293A cells from (F) generated 72 hours post dox induction. H) The mRNA levels of the indicated YAP target genes were assessed in cells from (F) 72 hours post dox induction. Statistical analysis was carried out with a 2-tailed paired t-test; * p<0.05.</p

FGFR expression levels of the MRT cell lines A204, G401 and G402.

<p>(A) Scatter plot showing expression and copy number levels for <i>FGFR1</i> (left panel) and <i>FGFR2</i> (right panel) within the CCLE. MRT lines A204, G401 and G402 are indicated in red. (B) Quantitative RT-PCR (qRT-PCR) analysis of <i>FGFR1</i> and <i>FGFR2</i> mRNA expression in MRT cell lines and soft tissue cancer lines SKLMS1 and SKUT1. Expression values are given as average with standard errors of the mean (SEM) (n≥3) with respect to <i>GAPDH</i> mRNA levels (arbitrarily set as 100). (C) qRT-PCR and immunoblot analysis of SNF5-deficiency in MRT lines. SKLMS1 and SKUT1 cells were used as positive controls for SNF5 expression. <i>SNF5</i> mRNA expression is given as average with SEM (n≥3) with respect to <i>GAPDH</i> mRNA levels (arbitrarily set as 100). β-Tubulin expression was used to monitor equal loading.</p