Deregulation of the PI3K and KRAS signaling pathways in human cancer cells determines their response to everolimus

Abstract

Personalized cancer medicine is based on the concept that targeted therapies are effective on subsets of patients whose tumors carry specific molecular alterations. Several mammalian target of rapamycin (mTOR) inhibitors are in preclinical or clinical trials for cancers, but the molecular basis of sensitivity or resistance to these inhibitors among patients is largely unknown. Here we have identified oncogenic variants of phosphoinositide-3-kinase, catalytic, alpha polypeptide (PIK3CA) and KRAS as determinants of response to the mTOR inhibitor everolimus. Human cancer cells carrying alterations in the PI3K pathway were responsive to everolimus, both in vitro and in vivo, except when KRAS mutations occurred concomitantly or were exogenously introduced. In human cancer cells with mutations in both PIK3CA and KRAS, genetic ablation of mutant KRAS reinstated response to the drug. Consistent with these data, PIK3CA mutant cells, but not KRAS mutant cells, displayed everolimus-sensitive translation. Importantly, in a cohort of metastatic cancer patients, the presence of oncogenic KRAS mutations was associated with lack of benefit after everolimus therapy. Thus, our results demonstrate that alterations in the KRAS and PIK3CA genes may represent biomarkers to optimize treatment of patients with mTOR inhibitors.

Figure 1. Genetic alterations in the PI3K and KRAS pathways are determinants of cells’ response to everolimus.

(A and B) The effect of everolimus treatment on cellular proliferation was assessed for hTERT-HME1 (A) and MCF10A (B) cells and their isogenic clones carrying the indicated PIK3CA mutations. The average cell number was measured by determining ATP content in 3 replicate wells. Results are normalized to growth of cells treated with DMSO and are represented as mean ± SD of at least 3 independent experiments. (C) Antiproliferative effects of everolimus on a panel of cancer cell lines. Cells carrying mutant or amplified PIK3CA are depicted in red or pink, respectively; cells lacking PTEN expression are represented in brown; cells carrying mutant KRAS/BRAF with or without concomitant PIK3CA mutations are depicted in blue or black, respectively. Details on specific mutations of KRAS, BRAF, and PIK3CA and the functional status of PTEN are provided in Supplemental Table 2. Results are expressed as percent viability compared with cells treated with DMSO only (control) and represent mean ± SD of at least 3 independent observations.

Figure 2. Oncogenic KRAS D13 confers resistance to everolimus.

(A) Two independent clones of HCT116 colorectal cancer cells — in which the KRAS D13 allele was genetically deleted by homologous recombination (HKh-2 and HKe-3, depicted in red) — were more sensitive to everolimus than either their parental cells (black) or a clone in which the KRAS WT allele was knocked out but the mutated allele was retained (HK2-6, green). Data are mean ± SD of at least 3 independent observations. (B) Effect of everolimus (96 hours) on proliferation of HKe-3 (HCT116-derivative KRAS WT clone) cells infected with control or KRAS D13 lentivirus. Results are expressed as percent viability compared with cells treated with DMSO only (control) and represent mean ± SD of at least 3 independent observations. (C) NOD/SCID mice were inoculated with HKe-3 cells (5 × 10⁶) transduced with empty or KRAS D13 lentiviral vectors; once tumors were established, animals were administered everolimus at 7.5 mg/kg every other day. The arrows indicate the time point at which drug treatment was started. Results are shown as mean ± SEM.

Figure 3. Effect of everolimus on RAS/PI3K signaling in PIK3CA and KRAS mutant cells.

PIK3CA mutant ME-180 and HKe-3 cells were transduced with either empty or KRAS D13–expressing lentiviral vectors. All cells were treated for 30 minutes with everolimus (50 nM), and the corresponding lysates were blotted with total RSK1/RSK2/RSK3, phospho–p90RSK (Ser380), total S6K1, phospho-S6K1 (Thr389), total AKT, phospho-AKT (Ser473), total ERK1/2 and phospho-ERK1/2 antibodies, phospho-rpS6 (Ser235–236), and total rpS6. Vinculin was included as a loading control.

Figure 4. Oncogenic KRAS and PIK3CA mutations affect translation.

(A) hTERT-HME1 cells of the indicated genotypes were treated with everolimus (500 nM) or left untreated. After 45 minutes of ³⁵S-methionine pulse, methionine incorporation was measured in newly translated proteins. (B) ME-180 and (C) HKe-3 cells were transduced with a lentiviral vector encoding for KRAS D13 or a control vector (empty) and treated with everolimus (50 nM) or left untreated. After 45 minutes of ³⁵S-methionine pulse, methionine incorporation was measured. All results are expressed as percent reduction in incorporation between treated and untreated cells (mean ± SEM).

Figure 5. Oncogenic KRAS mutations are associated with clinical resistance to everolimus.

Venn diagram representation of the distribution of molecular alterations in individual cancers. Response to everolimus according to the presence of genetic abnormalities within individual tumor samples is also shown.