2858?The?Journal?of?Clinical?Investigation? ? ? http://www.jci.org? ? ? Volume 120? ? ? Number 8? ? ? August 2010
Deregulation of the PI3K and KRAS signaling
pathways in human cancer cells determines
their response to everolimus
Federica Di Nicolantonio,1,2 Sabrina Arena,1 Josep Tabernero,3 Stefano Grosso,4,5
Francesca Molinari,6 Teresa Macarulla,3 Mariangela Russo,1 Carlotta Cancelliere,1 Davide Zecchin,1
Luca Mazzucchelli,6 Takehiko Sasazuki,7 Senji Shirasawa,8 Massimo Geuna,9 Milo Frattini,6
José Baselga,3 Margherita Gallicchio,10 Stefano Biffo,4,5 and Alberto Bardelli1,2
1Laboratory of Molecular Genetics, Institute for Cancer Research and Treatment, IRCC, University of Turin Medical School, Turin, Italy.
2FIRC, Institute of Molecular Oncology, Milan, Italy. 3Medical Oncology Department, Vall d’Hebron University Hospital, Universitat Autònoma de Barcelona,
Barcelona, Spain. 4Laboratory of Molecular Histology and Cell Growth, Division of Oncology, San Raffaele Scientific Institute, Milan, Italy.
5DISAV, University of Eastern Piedmont, Alessandria, Italy. 6Laboratory of Molecular Diagnostic, Istituto Cantonale di Patologia, Locarno, Switzerland.
7International Medical Center of Japan, Tokyo, Japan. 8Department of Cell Biology, School of Medicine, Fukuoka University, Fukuoka, Japan.
9Laboratory of Immunopathology, Anatomia Patologica, Ospedale Mauriziano Umberto I, Turin, Italy.
10Department of Anatomy, Pharmacology, and Forensic Medicine, University of Turin, Turin, Italy.
The PI3K/PTEN/AKT signaling pathway is frequently deregu-
lated in human cancer (1). This pathway plays a crucial role in
cell growth, survival, and proliferation; hence, its components
have recently emerged as attractive therapeutic targets for can-
cer therapy. Accordingly, multiple drugs are being investigated
either at the preclinical or clinical level to inhibit PI3K itself or its
downstream effectors, such as AKT1, PDK1, and the mammalian
target of rapamycin (mTOR) (2, 3). Among these novel agents, the
rapamycin derivatives temsirolimus and everolimus have reached
late-phase clinical development and have gained FDA approval
for the treatment of metastatic renal cell carcinoma (4–6). These
compounds bind to FKBP12 and inhibit mTOR complex–1
(mTORC1), resulting in suppression of both tumor growth and
angiogenesis (7). Clinical trials are currently being carried out on
several tumor types, such as non-Hodgkin lymphoma, glioblas-
toma, non-small cell lung, neuroendocrine, endometrial, colorec-
tal, gastric, breast, and prostate carcinomas (6). mTOR inhibitors
elicit predominantly disease-stabilizing, cytostatic responses,
rather than tumor regression (8). Even in the best settings, such
as renal cell carcinoma or neuroendocrine tumors, response rate
as assessed by conventional Response Evaluation Criteria in Solid
Tumors (RECIST) criteria is very low; however, these drugs signif-
icantly affect progression-free and overall survival (8). Data from
early-phase studies indicate that only a subset of patients derive
significant clinical benefit from treatment with mTOR inhibitors
(9–12). The molecular basis of sensitivity and resistance to mTOR
inhibitors such as rapamycin, temsirolimus, and everolimus is
presently largely unknown. Understanding such mechanisms pro-
vides the key to select patients likely to respond, and to develop
rational drug combinations to circumvent resistance.
PIK3CA mutations sensitize non-transformed human cells to everolimus.
We have recently developed a panel of isogenic cell lines by employ-
ing homologous recombination to introduce (by knock-in [KI])
frequently mutated cancer alleles in human breast immortalized
epithelial cells (hTERT-HME1) (13). Our previous profiling of a
drug library on this panel of KI cell lines has shown that phos-
phoinositide-3-kinase, catalytic, α polypeptide (PIK3CA) H1047R
mutated cells are selectively sensitive to a group of drugs compris-
ing rapamycin and its derivative everolimus (13). We extended and
substantiated this finding by showing that cells knocked in for the
other frequently occurring PIK3CA allele, E545K, also showed an
increased response to everolimus (Figure 1A). We confirmed the
correlation between the KI of PIK3CA mutations and sensitization
to everolimus in another cell background (MCF10A), thus suggest-
ing that this effect could be cell type–independent (Figure 1B).
Authorship?note: Federica Di Nicolantonio and Sabrina Arena contributed equally
to this work.
Conflict?of?interest: José Baselga and Josep Tabernero have been members of advi-
sory boards for Novartis, the manufacturer of everolimus.
Citation?for?this?article: J Clin Invest. 2010;120(8):2858–2866. doi:10.1172/JCI37539.
Related Commentary, page 2655
?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 120 Number 8 August 2010
To better characterize the preferential effect induced by everoli-
mus in PIK3CA KI clones, we performed FACS analysis. We found
that treatment with everolimus of hTERT-HME1 cells resulted in
a cytostatic effect that was more evident in PIK3CA KI clones com-
pared with their WT counterpart (Supplemental Figure 1A; sup-
plemental material available online with this article; doi:10.1172/
JCI37539DS1). Upon treatment, all hTERT-HME1 PIK3CA KI
clones accumulated in the G0/G1 phase of the cell cycle (Supple-
mental Figure 1B), and, accordingly, the proportions of cells in the
S and G2/M phases decreased (Supplemental Table 1). Apoptosis
was almost undetectable and did not vary between vehicle only– and
drug-treated cells (Supplemental Figure 1B and data not shown).
Of note, while vehicle only–treated cells proliferated at a compa-
rable rate, prolonged exposure to everolimus slowed cell growth in
all genotypes, with the effect being particularly evident in PIK3CA
H1047R, less pronounced in PIK3CA E545K, and only minimal in
WT cells (Supplemental Figure 1A). The subtle difference observed
between cells carrying these two mutations might suggest a differ-
ent downstream signaling of catalytic domain versus helical domain
PI3K alterations, as recently suggested by Vasudevan et al. (14).
Occurrence of PIK3CA and KRAS mutations determines response of
cancer cells to everolimus. The above pharmacogenomic analysis of
non-transformed cells carrying cancer alleles pointed to a relation-
ship between the occurrence of PIK3CA mutations and response to
everolimus. We next assessed whether and to what extent these find-
ings might be applicable to human cancer cells in which mutations
in the PI3K pathway occur naturally alongside additional genetic
alterations. To this end, we treated with everolimus a panel of cell
lines derived from glioblastoma, breast, ovarian, prostate, endome-
trial, and colorectal carcinomas, which are known to carry genetic
alterations in PIK3CA or PTEN (phosphatase and tensin homolog)
(Figure 1C and Supplemental Table 2). Interestingly, tumor cells
could be classified in two main groups based on their response
to everolimus. Drug-resistant cells (such as HT-29, HCT116, and
DLD-1) carried mutations in both PIK3CA and KRAS/BRAF. Evero-
limus-sensitive cells displayed PI3K pathway alterations but no
mutations in the KRAS/BRAF genes (Figure 1C).
Genetic ablation of the KRAS D13 mutation restores antiproliferative
response of cancer cells to everolimus. We hypothesized that genetic alter-
ations of the KRAS pathway could represent a major genetic deter-
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 muta-
tions. 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.
2860? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 120 Number 8 August 2010
minant of everolimus resistance in tumor cells carrying PIK3CA
oncogenic alleles. To formally test this hypothesis, we took advan-
tage of HCT116 cells in which the KRAS D13 mutant allele had
been genetically deleted by homologous recombination (15). Strik-
ingly, we found that HCT116-derivative cells retaining only the
KRAS WT allele (named HKh-2 and HKe-3; Supplemental Table 3)
were sensitive to everolimus, while the parental and the isogenic
cells carrying mutated KRAS were equally resistant to this com-
pound (Figure 2A). As a further control, we employed HCT116
cells in which the PIK3CA mutation H1047R had been deleted by
targeted homologous recombination (16). As expected, since all
clones retained a mutated KRAS allele, the derivative isogenic cells
were nonresponsive to everolimus (Supplemental Figure 2).
To further substantiate these results, we performed forward genetic
rescue experiments. We found that restoration of mutated KRAS D13
expression in HCT116-derivative cells that had only the KRAS WT
allele (HKe-3) prevented response to everolimus in vitro (Figure 2B).
In vivo effect of everolimus on tumors bearing PIK3CA and/or KRAS
mutations. We next evaluated the effect of everolimus in vivo by
injecting subcutaneously two independent cancer cell models into
immunocompromised mice. We found that HKe-3 cells (HCT116-
derivative cells lacking mutated KRAS) formed tumors, albeit after
a prolonged latency. Oral administration of everolimus to animals
carrying HKe-3 xenografts resulted in long-term tumor growth
arrest (Figure 2C). In contrast, the growth of tumors formed by
HKe-3 cells transduced with oncogenic KRAS was not significantly
affected by everolimus treatment (Figure 2C).
As a second cell model, we used the endometrial cancer cell line
ME-180, which carries the PIK3CA E545K mutation but is WT for
KRAS and is sensitive to everolimus. In vitro, expression of KRAS
D13 in ME-180 cells abrogated the antiproliferative effect of evero-
limus, thus confirming our hypothesis (Supplemental Figure 3A).
Parental and KRAS D13–expressing ME-180 cells formed tumors
of comparable size when injected subcutaneously in mice. While
everolimus had a prominent cytostatic effect on tumors originat-
ing from parental (PIK3CA mutated but KRAS WT) cells, it did
not significantly influence the growth of tumors formed by cells
expressing oncogenic KRAS D13 (Supplemental Figure 3B).
Next, we established whether the results obtained with ectopic
expression of oncogenic KRAS D13 could be replicated in isogenic
cells expressing physiological levels of the mutated allele. To this
end, we recapitulated the genetic background of the HCT116
colorectal cancer cells in hTERT-HME1 cells by introducing
via homologous recombination both KRAS G13D and PIK3CA
H1047R alleles in their genome. This approach generated double-
KI (hTERT-HME1 DKI) cells, in which each mutation is expressed
under the corresponding gene’s promoter. When exposed to evero-
limus, these double mutant cells displayed a delay in cell prolifera-
tion comparable to that observed in the parental WT population
(Supplemental Figure 4 and Supplemental Table 1). We found that
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.
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 120 Number 8 August 2010
these hTERT-HME1 isogenic cells were unable to grow as xenograft
in immunocompromised mice (data not shown), which prevented
us from testing the effect of everolimus on this cell model in vivo.
Effect of everolimus treatment on signal transduction. To gain insight
into the mechanisms of genotype-dependent proliferation upon
everolimus treatment, we performed biochemical analysis on all
hTERT-HME1 KI cells and their WT counterparts. While a wide
range of concentrations was tested in proliferation experiments, a
single clinically relevant dose of everolimus (50 nM) was employed
for biochemical and metabolic profiling. This was chosen on the
basis of plasma levels of everolimus in patients from phase I trials
(11) and additional pharmacodynamic-pharmacokinetic studies
showing that intratumor concentrations of everolimus can exceed
by 10-fold plasma levels in animal models (17).
We analyzed the short-term effect of everolimus on the downstream
effectors of RAS and PI3K (Supplemental Figure 5). Briefly, everoli-
mus resulted in the effective inhibition of the mTORC1 effector S6
kinase (S6K1) (reviewed in ref. 18) in all genotypes (Supplemental
Figure 6). Rapamycin analogs have been shown to activate indirectly
mTORC2-dependent phosphorylation of AKT at Ser473 (19, 20).
We found an increase in AKT phosphorylation upon short everoli-
mus treatment in WT cells, but not in cells carrying either PIK3CA
H1047R or KRAS G13D mutations (Supplemental Figure 6). In
addition, all PIK3CA or KRAS G13D mutated clones showed the
expected increase in basal phosphorylation of AKT (Supplemental
Figure 6), when compared with parental cells. KI of oncogenic KRAS
increased activated GTP-bound RAS expression (13) and resulted in
augmented levels of basal ERK1/2 phosphorylation (Supplemental
Figure 6). ERK1/2 is a downstream effector of active RAS, and it has
been reported to be transiently phosphorylated in response to rapa-
mycin treatment (21). However, while the levels of activated ERK1/2
increased upon treatment in parental cells, everolimus failed to
induce a transient increase in ERK1/2 phosphorylation in all tested
hTERT-HME1–derivative mutant clones (Supplemental Figure 6).
To better elucidate the molecular mechanisms responsible for
resistance to everolimus by oncogenic KRAS, we extended all bio-
chemical analyses to tumor cells employed for in vivo studies. After
transduction with KRAS D13, both HKe-3 and ME-180 cells result-
ed in increased phosphorylation of basal ERK1/2 levels, while only
ME-180 cells displayed an increase in phosphorylated AKT (Figure 3).
Everolimus administration resulted in the effective inhibition of
S6K1 in all treated cells. While everolimus did not change the levels
of activated ERK1/2, it increased phosphorylation of AKT at Ser473
in HKe-3 and ME-180 parental but not in KRAS-transduced cells.
These experiments suggested that whether short treatment with
everolimus results in increased phosphorylation of AKT or MAPK
depends on cell type rather than genotype. In addition to AKT and
ERK1/2, we also performed immunoblotting against p90RSK to
determine whether this could represent a key modulator of response.
We found that all cells with oncogenic KRAS D13 displayed elevated
phosphorylation levels of this protein, which were unaffected by
treatment with everolimus (Figure 3). p90RSK is an AGC kinase of
the RSK family that is phosphorylated and activated by ERK1/2 in
response to many growth factors, hormones, and neurotransmitters
(22). p90RSK phosphorylates a wide range of substrates, including
members of the translational machinery such as eIF4B (23) and ribo-
somal protein S6 (rpS6) at Ser235/236 (24). Consistent with this,
HKe-3 cells transduced with oncogenic KRAS D13 displayed not only
increased levels of phosphorylated p90RSK, but also increased acti-
vation of rpS6, which was not modulated by everolimus (Figure 3).
These results raised the possibility that translation could be used as
a readout of everolimus inhibition and Ras activation.
KRAS and PIK3CA oncogenic mutations affect everolimus-dependent
translation. RSK has been involved in activating the translational
machinery by promoting recruitment of rpS6 and ribosomal sub-
units to the translation preinitiation complex (22). On the other
hand, rapamycin and its derivatives have been shown to inhibit
translation (25). Our results pinpointed p90RSK as a possible medi-
ator of resistance to everolimus in cells with mutated KRAS. Accord-
ingly, we tested the hypothesis that oncogenic KRAS could activate
translation through an mTORC1-independent pathway and there-
fore could bypass everolimus-mediated mTOR inhibition.
Thus, we analyzed whether the global rate of translation of hTERT-
HME1 WT and KI cells was sensitive to everolimus. WT cells showed a
mild sensitivity to everolimus; clones in which PI3K was constitutively
active were sensitive to everolimus, and their translation rate dropped
by 30%. Intriguingly, KRAS mutant cells were not affected by everolim-
us (Figure 4A). These data provide the first evidence to our knowledge
that sensitivity of the translation rate to mTORC1 inhibition is pro-
portional to PI3K activation and is reduced by oncogenic KRAS. Of
note, the lack of sensitivity of KRAS mutants to everolimus inhibition
was not due to a depression in the basal level of translation, which was
comparable to that of PIK3CA mutants (Supplemental Figure 7).
We tried then to correlate the response of cancer cells to evero-
limus to their translation rate. Thus, we analyzed the sensitivity
of translation to everolimus in the cells that we used for tumor
xenografts, i.e., ME-180 with or without KRAS D13 and HKe-3
cells with or without KRAS D13. We observed that a short time
Effect of everolimus on RAS/PI3K signaling in PIK3CA and KRAS
mutant cells. PIK3CA mutant ME-180 and HKe-3 cells were trans-
duced 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.
2862?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 120 Number 8 August 2010
pulse with everolimus (30 minutes) was sufficient to inhibit trans-
lation in ME-180 and HKe-3 cells, but not in their counterpart
transduced with KRAS D13 (Figure 4, B and C). Thus, tumor cells
sensitive to everolimus treatment may be predicted by a drop in
the global rate of translation, after a short pulse with the drug.
Taken together, these data imply that oncogenic RAS activates
translation through an mTORC1-independent pathway. Activa-
tion of the RAS-ERK1/2 cascade and of RSK1 may provide an
alternative route to translational control.
Genetic alterations of the PI3K and KRAS pathways affect cancer patients’
response to everolimus. In order to determine the relevance and the appli-
cability of the above findings to human cancer therapy, we assessed
the mutational status of PIK3CA, KRAS, and BRAF in a cohort of
cancer patients who had received single-agent everolimus as part of
phase I and phase II studies at a single institution (11). Due to the
limited amount of tumor tissue available, only KRAS exon 2, BRAF
exon 15, and PIK3CA exons 9 and 20 could be amplified and subject-
ed to direct sequencing (Table 1). These exons were selected because
they include codons where the large majority of mutations occur in
these genes (Catalogue of Somatic Mutations in Cancer, http://www.
sanger.ac.uk/genetics/CGP/cosmic/). We also assessed the expression
status of PTEN by immunostaining. In the cohort, only one partial
response (PR) occurred in a patient with a heavily pretreated meta-
static colorectal cancer lasting 5.3 months (disease control during 9
months). Molecular analysis of this case revealed no mutations in the
sequenced exons of BRAF, KRAS, or PIK3CA, but lack of expression
of PTEN protein. PIK3CA mutations could only be detected in two
samples (Table 1 and Figure 5). Because of the low frequency, we were
unable to perform any meaningful statistical analysis. However, it
should be noted that one of the PIK3CA mutant tumors also had a
KRAS mutation and did not respond to therapy (progressive disease
[PD]), while the other displayed concomitant PTEN loss and resulted
in disease stabilization (stable disease [SD]). PTEN staining was nega-
tive in 18 of 35 evaluable cases. When patients with SD or PR were
considered, 8 individuals with tumors displaying PTEN loss had some
clinical benefit from treatment with everolimus. Of the remaining 10
PTEN-negative cases that progressed, 6 were found to carry a concur-
rent KRAS or BRAF mutation. The combined results suggest that can-
cer patients whose tumors carry PIK3CA kinase domain mutations or
PTEN loss of function can benefit from everolimus treatment, except
when BRAF/KRAS mutations occur in concomitance with PI3K path-
way alterations (Figure 5). Although this association reached statisti-
cal significance (P = 0.0128, 2-tailed Fisher’s exact test; Table 2), these
results should be confirmed in additional studies including a larger
number of patients. Importantly, when KRAS status was considered
in univariate analysis, the occurrence of KRAS mutations negatively
and significantly affected clinical benefit of everolimus. Eleven of the
12 patients with KRAS mutant tumors had disease progression, while
only 16 of 31 of WT cases did not benefit from treatment (P = 0.0171,
2-tailed Fisher’s exact test; Table 2). Because the distribution of KRAS
mutations is highly skewed in colorectal cancers, we restricted the
analysis to this tumor subgroup and confirmed the negative predic-
tive role of KRAS mutations (P = 0.0288, 2-tailed Fisher’s exact test;
Table 2). These results strongly support our preclinical findings that
oncogenic KRAS is able to bypass mTORC1 inhibition, thus repre-
senting what we believe to be a novel mechanism of resistance to rapa-
We present an integrated strategy to identify relationships between
tumor genotypes (biomarkers) and a molecularly targeted drug (26).
The approach involves as an initial step the use of homologous recom-
bination to introduce individual or multiple cancer mutations into
the genome of non-transformed epithelial cells (13). As a result, the
heterozygous mutated genes are expressed under their endogenous
promoters, thus closely recapitulating the lesions observed in human
tumors. In the subsequent step, mutated and WT cells were exploit-
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 vec-
tor encoding for KRAS D13 or a control vector (empty) and treated with
everolimus (50 nM) or left untreated. After 45 minutes of 35S-methio-
nine pulse, methionine incorporation was measured. All results are
expressed as percent reduction in incorporation between treated and
untreated cells (mean ± SEM).
?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 120 Number 8 August 2010
ed to identify compounds selectively targeting oncogenic PIK3CA
variants. Using this approach, we found that everolimus, a rapamy-
cin derivative, had selectivity for cells carrying PIK3CA. The strategy
subsequently involves translating the findings obtained using non-
transformed epithelial cells in a panel of human cancer cells in which
the same mutations were found to be present. We found that tumor
cells carrying oncogenic PIK3CA mutations or PTEN loss of function
were sensitive to everolimus, except when KRAS or BRAF mutations
were concomitantly present. Further analysis revealed that selective
genetic removal of the mutated KRAS allele restored sensitivity to
everolimus. These data were corroborated by forward genetic analysis
in which introduction of oncogenic KRAS abrogated the everolimus
response of PIK3CA mutated cells. As the isogenic cells were unable
to grow as xenograft in mouse (13, 15), we were unable to test the
effect of everolimus in vivo. To overcome this limitation, we trans-
duced the KRAS D13 allele in tumorigenic and everolimus-sensitive
cells carrying PIK3CA mutations. After engraftment in immunocom-
promised mice, oral administration of everolimus induced growth
arrest in PIK3CA mutant tumors, but not in their corresponding
tumors concomitantly expressing a KRAS mutation.
In the final step, we translated the findings obtained in isogenic
cell models into the clinical setting by performing genetic analysis of
tumors from patients treated with everolimus. Cancer patients whose
tumors carried PIK3CA mutations in the kinase domain or PTEN
Patients with KRAS mutated tumors are less likely to respond to everolimus
BRAF exon 15
KRAS exon 2
PIK3CA exon 9
PIK3CA exon 20
List of patients who received everolimus treatment and molecular alterations determined in their tumors. Response to everolimus is indicated according to
RECIST criteria. HNSCC, head and neck squamous cell carcinoma; NE, not evaluable; PR, partial response; SD, stable disease; PD, progressive disease.
2864?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 120 Number 8 August 2010
loss of function displayed increased clinical benefit from everolimus
treatment, except when KRAS mutations were present. In particular,
in univariate analysis, the occurrence of oncogenic KRAS was sta-
tistically correlated with lack of response in the treated cohort. To
our knowledge, this is the first time that this association has ever
been reported. Data from clinical studies have mainly focused on
immunohistochemical surrogate markers of early response in biop-
sies from treated patients (11, 27), rather than on the genetic status of
tumors. It is interesting to note that occurrence of KRAS mutations
is also a validated biomarker of clinical resistance to EGFR-targeted
monoclonal antibodies in colorectal cancer (28).
Overall, the proposed strategy led us to conclude that the onco-
genic status of KRAS plays a central role in conferring resistance
to the antiproliferative effects of everolimus in human tumors
harboring genetic alterations of the PIK3CA gene. Notably, pre-
vious studies also suggested that rapamycin was inefficacious in
engineered mouse models for ovarian or colon cancer in which
the KRAS G12D allele was expressed in a tissue-specific manner
(29, 30). Two potential mechanisms of resistance to everolimus
and other mTOR inhibitors have been previously proposed. The
first consists of a negative feedback loop between mTOR and AKT:
mTORC1 inhibition leads to AKT activation, possibly through
upregulation of either receptor tyrosine
kinases such as IGF-1R or substrates such
as IRS-1 (19, 20, 31). In accordance with
these findings, we detected increased lev-
els of phosphorylated AKT after everoli-
mus treatment in a number of cell lines.
However, this upregulation was present
in a cell-specific context and was absent in
insensitive cells carrying oncogenic KRAS.
It has been proposed that AKT activation
might negatively interfere with the effi-
cacy of mTOR inhibitors (32), but the
mechanism of this effect needs further
investigation. The second mechanism of
resistance to mTOR inhibitors consists
of a different feedback loop between
mTOR and ERK1/2: mTORC1 inhibi-
tion leads to ERK1/2 activation through
an S6K1/PI3K/RAS-dependent pathway
(21). In fact, Carracedo et al. (21) showed
that a dominant negative form of RAS
(RAS N17) abrogates rapamycin-induced
ERK1/2 activation. However, we observed a variable increase in
phosphorylated ERK1/2, with this effect being present only in
some cell types and not depending on genotype.
While the analysis of commonly known RAS/PI3K effectors such
as ERK1/2 and AKT was not conclusive, we found that the intro-
duction of oncogenic KRAS was accompanied by activation of the
ERK1/2 effector p90RSK (22). In turn, p90RSK phosphorylates a
wide range of substrates including members of the translational
machinery such as eIF4B (23) and rpS6 at Ser235/236 (24). Our
results pinpoint p90RSK as a key modulator of global translation
in cells with oncogenic KRAS. Importantly, the refractoriness of
KRAS mutant cells to everolimus was mirrored by sustained levels of
global translation observed in the same cells. In contrast, in the cor-
responding cells with PIK3CA mutations, everolimus clearly affected
translation. Recent work has showed that RSK also directly targets
the mTORC1 complex by phosphorylating Raptor and thereby pro-
moting mTORC1 kinase activity (33). We found that the expression
of a KRAS D13 allele does not elicit robust mTORC1 activation (as
demonstrated by the absence of increased S6K1 phosphorylation
compared with control cells), thereby suggesting that RSK contrib-
utes to translation through an mTORC1-independent mechanism.
It has recently been proposed that in mutant KRAS-dependent
cells, the serine/threonine kinase STK33 modulates S6K1 activity
through an mTORC1-independent mechanism (34). This raises
the possibility that suppression of STK33 expression could be
exploited to sensitize KRAS mutated cells to mTOR inhibitors.
Overall, our results have a number of general implications. We
have defined an experimental strategy (based on mutated human
cells) capable of identifying biomarkers associated with response to
anticancer drugs. We show that the pharmacological relationships
established in non-transformed cells carrying defined cancer muta-
tions can be translated to cancer cells in which the corresponding
mutations naturally occur alongside additional genetic altera-
tions. This indicates that the mutated cells together with their WT
counterparts can be successfully used to de-convolute the complex
genetic background of cancer cells. The approach defined here can
be broadly applied to study how genetic relationships affect sensi-
tivity and resistance to molecularly targeted anticancer drugs.
Oncogenic KRAS mutations are associated with clinical resistance to
everolimus. Venn diagram representation of the distribution of molecu-
lar alterations in individual cancers. Response to everolimus accord-
ing to the presence of genetic abnormalities within individual tumor
samples is also shown.
Association between response to everolimus and occurrence of genetic alterations in the
PI3K or KRAS pathway
PR + SD PD P
All tumor types
WT KRAS (31/43)
Mutant KRAS (12/43)
PTEN loss or mutant PIK3CA (12/43)
PTEN loss or mutant PIK3CA with concomitant
KRAS/BRAF alterations (7/43)
WT KRAS (13/23)
Mutant KRAS (10/23)
15 (1 PR + 14 SD)
8 (1 PR + 7 SD)
8 (1 PR + 7 SD)
The number of patients achieving some clinical benefit (PR + SD) and nonresponders (SD) is indicated
according to KRAS mutational status in all tumor types and in the subgroup of colorectal cancer. KRAS
mutations inversely correlate with response to everolimus treatment (P = 0.0171 and P = 0.0288,
2-tailed Fisher’s exact test). The number of patients achieving some clinical benefit (PR + SD) and non-
responders (PD) is indicated according to PTEN status and PIK3CA mutations on the basis of their co-
occurrence with BRAF/KRAS mutations in all tumor samples (P = 0.0128, 2-tailed Fisher’s exact test).
?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 120 Number 8 August 2010
Plasmids and viral vectors. All experimental procedures for targeting vector con-
struction, AAV production, cell infection, and screening for recombinants have
been described elsewhere (13). The list of primers employed to amplify the
AAV vectors’ homology arms for introducing the PIK3CA E545K and H1047R
alleles is detailed in Supplemental Table 4. The procedure to obtain the lentivi-
rus expressing the KRAS G13D mutation has been described elsewhere (13).
Cells and cell culture reagents. hTERT-HME1 and MCF10A cell lines were
purchased from ATCC and were cultured in growth medium containing
DMEM/F-12 (Invitrogen) supplemented with 20 ng/ml EGF, 10 μg/ml
insulin, and 100 μg/ml hydrocortisone. All other cancer cell lines (U-87
MG, Ca Ski, ME-180, MCF7, BT-474, PC-3, PANC-1, HT-29, NIH:OVCAR-3,
SK-OV-3, HCT116, and DLD-1) were also obtained from ATCC and cul-
tured according to their recommendations. All cell culture media were sup-
plemented with 5% fetal bovine serum (Sigma-Aldrich), 50 U/ml penicillin,
and 50 mg/ml streptomycin. Geneticin (G418) was purchased from Gibco
(Invitrogen). Isogenic HCT116 PIK3CA WT and mutant cells were provided
by the B. Vogelstein/V.E. Velculescu laboratories (Johns Hopkins University,
Baltimore, Maryland, USA) (16). Isogenic HCT116 KRAS WT (HKe-3, HKh-2)
and mutated (HK2-6) clones have been previously described (15).
Drug proliferation assays. Everolimus, purchased from Sigma-Aldrich (catalog
7741), was dissolved in DMSO and stored in aliquots at –80°C. The effect of
the drug on cell proliferation was determined using the CellTiter-Glo Lumines-
cent Cell Viability Assay kit (Promega), which is based on quantification of the
cellular ATP level. Cells were plated in 96-well plates at a density of 2,000–5,000
cells in triplicate. The following day, cells were treated with a range of drug con-
centrations prepared by serial dilution. After 72–96 hours of treatment, 100 μl
of luciferin luciferase–containing reagent was added to each well, and lumines-
cence measurements were recorded by the DTX 880-Multimode plate reader
(Beckman Coulter). To account for clonal variability, at least two independent
clones carrying each of the mutations were generated and analyzed.
Flow cytometric analysis. For time-course experiments, on the initial day
hTERT-HME1 cells were labeled with 3 μM CFSE (Invitrogen, C1157) in
PBS in the dark for 30 minutes. After washing and recording of baseline
fluorescence, cells were plated in media containing 1% FBS and 2 ng/ml
EGF and treatment with everolimus was initiated, with the drug replen-
ished on a daily basis. For cell cycle analysis, trypsinized cells were washed
with PBS, and cell nuclei DNA were stained with propidium iodide (PI) for
at least 120 minutes using a commercial kit (DNA con 3, Consul T.S.).
All fluorescence levels were detected by flow cytometry on a FACSCali-
bur (BD) and analyzed using CellQuest software. The number of events
collected for each sample varied between 15,000 and 50,000. After doublet
exclusion, an extended analysis of the DNA content and calculations of
the percentage of cells in each phase of the cell cycle were performed on
ModFit LT software (Verity Software House).
Protein analysis. Prior to biochemical analysis, all cells were grown in their
specific media supplemented with 5% FBS, unless otherwise indicated.
SDS-PAGE Western blotting was performed as previously described (13). The
following antibodies were used for Western blotting (from Cell Signaling Tech-
nology, except where indicated): anti-AKT (BK9272); anti–phospho-AKT S473
(BK9271S); anti–phospho-S6K1 (Thr389; catalog 9205S), and anti–total S6K1
(catalog 9202); anti–phospho-p44/42 ERK (Thr202/Tyr204) (BK9101S); anti-
p44/42 ERK (BK9102); phospho-p90RSK(Ser380) (catalog 9341); anti-RSK1/
RSK2/RSK3 (catalog 9347); phospho-rpS6 (Ser 235–236) (catalog 2211S);
anti-rpS6 (5G10) (2217); anti-actin and anti-vinculin (Sigma-Aldrich).
Mouse xenografts. All animal procedures were approved by the Ethical Com-
mission of the University of Turin and by the Italian Ministry of Health.
Lentiviral vector–transduced HKe-3 and ME-180 cells (5 × 106 cells/mouse)
were injected subcutaneously into the right posterior flanks of 7-week-old
immunodeficient NOD/SCID male mice (6 mice/group; Charles River).
Tumor formation was monitored twice a week, and tumor volume based
on caliper measurements was calculated by the modified ellipsoid formula
(tumor volume = 1/2[length × width2]). When tumors reached a volume of
approximately 0.3 cm3, mice were randomized to receive either vehicle (1%
methylcellulose in sterile water) or everolimus. Everolimus was adminis-
tered by oral gavage every other day at a dosage of 7.5 mg/kg/treatment.
Metabolic assay. Subconfluent hTERT-HME1 cell clones were plated in
6-well plates. Cells were incubated at 37°C with 80% methionine-free medi-
um (Sigma-Aldrich), 20% DMEM for 45 minutes. Preincubation with evero-
limus was performed 30 minutes before methionine pulse. Cells were pulsed
with 33 μCi per well of 35S-labeled methionine (PerkinElmer) for 45 minutes.
Cells were lysed by scraping in 50 μl RIPA buffer without SDS (10 mM Tris-
HCl pH 7.5, 1% Na-deoxycholate, 1% Triton X-100, 150 mM NaCl, 1 mM
EDTA, proteases inhibitor cocktail). Ten-microliter extracts were TCA pre-
cipitated and counted as previously described (35). Obtained values were
normalized to sample protein content, quantified by bicinchoninic acid
(BCA) protein assay (Pierce). Each sample was assayed at least in triplicate.
Patient population. We retrospectively analyzed primary or metastatic tissue
samples from 43 patients with advanced solid tumors treated with single-
agent everolimus at the Medical Oncology Department, Vall d’Hebron Uni-
versity Hospital. These patients were treated in the context of a phase I and
a phase II study, and the population included patients with different tumor
types refractory to standard therapies, the most frequent tumor type being
colorectal cancer (11). Tumor types are summarized in Table 2. Everolimus
was administered either as a single weekly oral dose (20, 50, and 70 mg) or as
a continuous daily oral dose (5 and 10 mg). Treatment was continued until
PD or toxicity occurred, according to the standard criteria. The Ethics Com-
mittee of Vall d’Hebron University Hospital approved the studies, which fol-
lowed the Declaration of Helsinki. The molecular analysis of these samples
was done at Istituto Cantonale di Patologia, Locarno, or at the Laboratory
of Molecular Genetics, Institute for Cancer Research and Treatment (Turin,
Italy). All patients provided informed consent for their tissues to be used for
the molecular analyses that were performed in this study.
Clinical evaluation and tumor response criteria. Objective tumor measure-
ments were evaluated in accordance with RECIST criteria (36) after 8 weeks
of treatment, and thereafter every 8 weeks. Objective tumor responses were
classified as PR, SD, and PD. Patients with either SD or PR at the first
workup evaluation were considered to have clinical benefit for the purpose
of this molecular study.
Molecular analyses. Formalin-fixed, paraffin-embedded tumor blocks
were reviewed for quality and tumor content. Macrodissection was per-
formed on a single representative block from the primary tumor or from
metastatic lesions, in order to have at least 70% malignant cells. Genomic
DNA was extracted using the QIAamp Mini kit (QIAGEN) according to the
Mutational analysis of KRAS, BRAF, and PIK3CA. We searched for KRAS
(exon 2), for BRAF (exon 15) and for PIK3CA (exons 9 and 20) mutations
by direct sequencing. KRAS exon 2 includes codons 12 and 13; BRAF exon
15 includes codon 600; PIK3CA exon 9 includes codons 542 and 545; and
PIK3CA exon 20 includes codon 1,047, where the large majority of muta-
tions occur. The list of primers used for mutational analyses has been pre-
viously reported (37). All samples were subjected to automated sequencing
by ABI PRISM 3730 (Applied Biosystems). All mutated cases were con-
firmed at least twice starting from independent PCR reactions. Mutational
analysis was concomitantly performed by the laboratories of Molecular
Genetics, Institute for Cancer Research and Treatment (Turin, Italy) and
Istituto Cantonale di Patologia, Locarno, without knowledge of clinical
data or results of molecular experiments.
PTEN expression. PTEN protein expression status by IHC on 3-μm tissue sec-
tions was performed and evaluated according to the literature (38). Anti-PTEN
2866? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 120 Number 8 August 2010
Ab-2 (Neomarkers) was applied at a 1:50 dilution. PTEN protein expression
was detected mainly at the cytoplasmic level, although occasional nuclear posi-
tivity was present. Tumors were considered negative, i.e., with loss of PTEN
expression, when absence or reduction of immunostaining was seen in more
than 50% of neoplastic cells as compared with internal controls (i.e., vascular
endothelial cells and nerves). Normal endometrium tissue was used as external
positive control. The evaluations were performed by two independent patholo-
gists without knowledge of clinical data or results of molecular analyses.
Statistics. Statistical analyses were performed by the 2-tailed t test with
Bonferroni’s multiple comparisons correction and the Fisher’s exact test
using the Instat program (GraphPad Software). For all tests, the level of
statistical significance was set at P < 0.05. Error bars throughout the fig-
ures indicate SD or SEM, as detailed in each legend.
We are grateful to Chris Torrance, Miriam Martini, Emily Crow-
ley, and Luca Cardone for critically reading the manuscript.
We thank Elena Casanova for technical assistance with FACS
acquisition data; Sebastjian Hobor, Alessia Bottos, and Laura
Tarditi for help with mouse handling; and Irene Marimon and
José Jimenez for technical help in the collection of tumor sam-
ples. Work in the laboratories of the authors is supported by the
Italian Association for Cancer Research (AIRC; A. Bardelli and
S. Biffo); Italian Ministry of Health (A. Bardelli and S. Biffo);
Regione Piemonte (F. Di Nicolantonio, S. Arena, S. Grosso,
and A. Bardelli); Italian Ministry of University and Research (A.
Bardelli); CRT Progetto Alfieri (A. Bardelli); European Union
FP7 Marie Curie Programme (A. Bardelli); Association for Inter-
national Cancer Research UK (A. Bardelli and S. Biffo); Europe-
an Union FP6, MCSCs contract 037297 (A. Bardelli); Oncosuisse
grants OCS-01921-08-2006 and OCS-02301-08-2008 (M. Frat-
tini); and Fondazione Ticinese per la Ricerca sul Cancro (Tessin
Foundation for Cancer Research; M. Frattini).
Received for publication October 6, 2009, and accepted in revised
form May 19, 2010.
Address correspondence to: Alberto Bardelli, Institute for Cancer
Research and Treatment, IRCC, Laboratory of Molecular Genetics,
University of Turin Medical School, SP 142, km 3.95, I-10060 Can-
diolo (Turin), Italy. Phone: 39.011.993.3235; Fax: 39.011.993.3225;
Mariangela Russo’s present address is: Horizon Discovery Ltd.
Gene Engineering Laboratory, Molecular Biotechnology Center,
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Di Nicolantonio et al., Supplemental Data File
Supplemental Table 1. Everolimus affects cell cycle in a genotype dependent
hTERT-HME1 cells of the indicated genotype were incubated for 48 h with
everolimus (0.5 µM), and the effect on cell cycle was analyzed by FACS.
Upon treatment, only hTERT-HME1 PIK3CA KI clones significantly
accumulated in the G0/G1-phase of the cell cycle. Accordingly, the
proportions of cells in the S- and G2/M-phases decreased. Means of at least 4
independent experiments are shown. Significance by paired t test was taken
KI PIK3CA E545K
KI PIK3CA H1047R
KI KRAS G13D
DKI (KRAS G13D + PIK3CA H1047R)
Everolimus 0.5 µM
Di Nicolantonio et al., Supplemental Data File
Supplemental Table 2.
Genetic alterations of PIK3CA, KRAS and BRAF in cancer cell lines displayed
in Figure 1C. The status of PTEN expression is also shown.
Di Nicolantonio et al., Supplemental Data File
Supplemental Table 3.
Genetic mutations of PIK3CA and KRAS in the isogenic cell lines employed in
CELL LINES MUTATIONS
hTERT-HME1 WT WT WT
KI PIK3CA H1047R WT
KI PIK3CA E545K WT
KI KRAS WT G13D
DKI H1047R G13D
MCF10A WT WT WT
KI PIK3CA H1047R WT
WT H1047R WT G13D
WT + + + +
HK2-6 + + - +
HKe-3 + + + -
HKh-2 + + + -
-/H1047R - + + +
WT/- + - + +
Di Nicolantonio et al., Supplemental Data File
Supplemental Table 4.
Primers used for the amplification of the homology arms of the PIK3CA targeting vectors. The position of the mutated residues and
the source of genomic DNA used for the PCR amplification are also indicated.
Homology gDNA source
Primers (F= forward; R=reverse) Restriction sites
Eco RV, Not I
Eco RI, NotI
R tgtccaTCTAGAataacttcgtataatgtatgctatacgaagttatGTGACTGCTTCCAAAACTGC Xba I, loxP
Di Nicolantonio et al., Supplemental Data File
Supplemental Figure 1.
Everolimus affects cell cycle in
a genotype-dependent fashion.
(A) CFSE-labeled cells were
analyzed by flow cytometry at
the indicated time-points. The
intensity for all samples was
recorded at day 0 (depicted in
proportional to the number of
cell divisions. hTERT-HME1
WT, PIK3CA E545K and
H1047R KI cells showed a
similar pattern of cell doublings
Exposure to everolimus 500
nM for 7 days resulted in
rate in all genotypes, with the
effect being particularly evident
in PIK3CA H1047R,
pronounced in PIK3CA E545K
and only minimal in WT cells.
(B) Cells of the indicated
genotype were incubated with
everolimus 500 nM for 48 h,
after which cell cycle was analyzed by flow cytometry. No increase of the subG1 apoptotic fraction of cells was observed upon
treatment. Representative data from 3 independent experiments are shown.
Di Nicolantonio et al., Supplemental Data File
Supplemental Figure 2.
Effect of everolimus on HCT 116 cells and their PIK3CA WT or mutant derivative
After 96 hours’ treatment with everolimus, HCT 116-derived cells that are knock-out
for the mutated 1047R allele of PIK3CA (WT/-, depicted in red) displayed similar
response as either their parental cells (WT/H1047R, in black) or a clone retaining only
the PIK3CA mutated allele (-/H1047R, indicated in green).
% of control
HCT 116 PIK3CA WT/H1047R
HCT 116 PIK3CA -/H1047R
HCT 116 PIK3CA WT/-