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Oncology 2009;77(suppl 1):69–74 DOI: 10.1159/000258498

The Dark Side of the Moon:The PI3K/PTEN/AKT Pathway inColorectal Carcinoma

Nicola Silvestris a Stefania Tommasi b Daniela Petriella b Daniele Santini c Ettore Fistola a Antonio Russo d Gianmauro Numico e Giuseppe Tonini c Evaristo Maiello f Giuseppe Colucci a

a Medical and Experimental Oncology Unit, and b Clinical and Experimental Laboratory, Cancer Institute Giovanni Paolo II, Bari , c Department of Oncology, University Campus Bio-Medico, Rome , d Genetic and Molecular Oncology Unit,School of Medicine, University of Palermo, Palermo , e Oncology Unit, Regional Hospital of the Aosta Valley, Aosta ,and f Medical Oncology Unit, Hospital Casa Sollievo della Sofferenza, San Giovanni Rotondo , Italy

Among patients with metastatic colorectal carcinoma (mCRC) wild-type KRAS, the objective response rate is limited to 17% (vs. 0% in unselected patients) with pani-tumumab monotherapy [1] , 12.8% (vs. 1.2% in unselected patients) with cetuximab monotherapy [2] , and 59 and 61% (vs. 43 and 33% in unselected patients) with cetux-imab plus either irinotecan- or oxaliplatin-based chemo-therapy [3, 4] , respectively. These data indicate that the absence of KRAS mutations does not guarantee an im-proved likelihood of response to these drugs. In other words, wild-type KRAS status is required but not suffi-cient to confer sensitivity to anti-epidermal growth factor receptor (EGFR) monoclonal antibodies (mAbs) [5] . As a consequence, the investigation of other biomarkers may be useful.

In this scenario, further predictive factors for sensi-tivity to anti-EGFR mAbs may be the presence or absence of mutations that result in constitutive activation of alter-native EGFR-mediated signaling pathways, includingthe phosphoinositide 3-kinases (PI3K)/AKT/mammali-an target of rapamycin (mTOR) [6] . Recent reports showed

Key Words

AKT � Cetuximab � Panitumumab � Phosphoinositide3-kinases � PTEN � mTOR

Abstract

Wild-type KRAS status is required but not sufficient to confer sensitivity to anti-epidermal growth factor receptor (EGFR) monoclonal antibodies (mAbs) in colorectal cancer patients. As a consequence, one of the major challenges is to identify, in non-mutant KRAS patients, other markers that can predict lack of response to this therapy. Small series have investi-gated the clinical effect of PIK3CA mutations on resistance to anti-EGFR mAbs and discrepant results have been ob-served. Furthermore, PTEN loss in metastases may be predic-tive of resistance to anti-EGFR mAbs, even if PTEN determi-nation is far from an immediate clinical application. The in-troduction of modulators of the PI3K/AKT/mTOR pathway as potential targeted anticancer drugs is encouraging, but this attractive therapy option is still at an early stage of develop-ment. Copyright © 2010 S. Karger AG, Basel

Published online: February 2, 2010 Oncology

Nicola Silvestris, MD Medical and Experimental Oncology Unit Scientific Institute for Research and Treatment of Cancer ‘Giovanni Paolo II’ Via Hahnemann 10, IT–70125 Bari (Italy) Tel. +39 080 555 5240, Fax +39 080 555 5232, E-Mail n.silvestris @ oncologico.bari.it

© 2010 S. Karger AG, Basel0030–2414/09/0777–0069$26.00/0

Accessible online at:www.karger.com/ocl

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Oncology 2009;77(suppl 1):69–7470

that breast cancers with either activating mutations in PIK3CA or with loss of the phosphatase and tensin ho-mologue deleted on chromosome TEN (PTEN) expres-sion respond poorly to treatment with the Her2/neu tar-geting antibody trastuzumab [7] . Similarly, some authors investigated the importance of the PI3K/AKT/mTOR pathway in identifying the subgroup of patients with mCRC KRAS wild type who do not benefit from anti-EGFR mAbs.

PI3K/PTEN/AKT Pathway

PI3Ks are a family of lipid kinases that phosphorylate the 3 � -hydroxyl group of phosphoinositides with conver-sion of phosphatidylinositol-4,5-biphosphate (PIP2) to phosphatidylinositol-3,4,5-trisphosphate (PIP3) [8] . PIP3 is a critical second messenger that activates AKT (also known as protein kinase B) through phosphorylation. Once activated, phospho-AKT phosphorylates up to 100 other proteins, including mTOR. mTOR complexes with raptor (regulatory associated protein of mTOR) and ric-tor (rapamycin-insensitive companion of mTOR) to form mTOR complex 1 (mTORC1) and mTORC2, respectively ( fig. 1 ).

The 8 members of the PI3K family have been classi-fied into 3 groups based on their primary sequences,in vitro substrate preferences, domain structures and modes of regulation. Only the class IA PI3Ks have been implicated in human cancer. These PI3Ks are hetero-dimers composed of a regulatory subunit (p85 � , p55 � , p50 � , p85 � , p55 � ) and a catalytic subunit (p110 � , p110 � , p110 � ). Activating mutations in PIK3CA, the gene en-coding the p110 � catalytic subunit of PI3K, were identi-fied as novel mechanisms of inducing oncogenic PI3K signaling [9] . The most frequent mutant proteins (E542K, E545K and H1047R) observed in human tumors are on-cogenic in animal models. PIK3CA is somatically mu-tated in over 25% of colorectal, gastric, breast and certain brain tumors, and is mutated at significant frequencies in other tumor types [9, 10] . From these mutation fre-quencies, PIK3CA appears to be one of the most highly mutated oncogenes yet identified in human cancers. In particular, PIK3CA mutations occurring in the hotspots located in exon 9 and exon 20 are oncogenic in CRC cel-lular models [11] and are associated with KRAS muta-tions [12] and microsatellite instability [10] . Less fre-quently occurring mutations of PIK3CA have been de-scribed in exons 1 and 2. In CRC, a double mutation of the PIK3CA gene is observed in 6–9% of mutant cases.

PIP2

PIP3

PI3K

AKT

mTOR

INSIDE

OUTSIDEEGFR

Anti-EGFRmAbs

Ligands

PTEN

Angiogenesis

Cell proliferationCell survival

Invasion

RAS

MEK

RAF

ERK

Differentiation

PerifosineGSK690693

EverolimusDeforolimus

PI103BGT226BEZ235XL765XL147

Colo

r ver

sion

ava

ilabl

e on

line

Fig. 1. Overview of the main EGFR sig-naling pathways and their effects on cell behavior. PI3K/AKT pathway (shaded el -l ipses) and RAS/RAF/MAPK pathway (shaded rectangles). PTEN prevents AKT activation. The agents targeting the signal-ing proteins are indicated in boxes.

Alternative EGFR Pathway Oncology 2009;77(suppl 1):69–74 71

Among patients with wild-type KRAS CRC, the pres-ence of PIK3CA mutations was recently correlated with a significant increase in colon cancer-specific mortality [13] . In contrast, the activation of the PI3K/AKT pathway apparently does not play an important role in tumor ag-gressiveness on a background where the KRAS oncogene is not activated.

In lung cancer patients, AKT phosphorylation has been correlated with the response to gefitinib [14] . AKT is hyperphosphorylated in about 60% of mCRC patients [15] .

PTEN is a phosphatase that antagonizes the PI3K/AKT signaling pathway by dephosphorylating PIP3 to inhibit activation of AKT with hyperactivation of PI3K signaling [16] . Indeed, recent studies demonstrated that PTEN protects the genome from instability [17] . The PTEN gene is activated in cancer cells by a combination of different molecular mechanisms (inactivating muta-tions, allelic losses, and hypermethylation of the enhanc-er region). Sporadic missense mutations frequently occur in central nervous system (20%), endometrial (39%), skin (17%) and colorectal (9%) cancers.

Preclinical Data

In a preclinical study 22 colon cancer cell lines were screened for cetuximab response and sensitive and resis-tant cell lines were identified [18] . Cell lines with activat-ing PIK3CA mutations or loss of PTEN expression (PTEN null) were more resistant to cetuximab than PIK3CA wild-type/PTEN-expressing cell lines. Indeed, cell lines that were PIK3CA mutant/PTEN null and KRAS/BRAF mutant were highly resistant to cetuximab compared with those without dual mutations/PTEN loss, suggest-ing that activating mutations of the KRAS and PIK3CA pathways confer maximal resistance to this mAbs.

Clinical Studies

Frattini et al. [19] showed for the first time that PTEN loss of expression was associated with non-responsive-ness to cetuximab in 27 mCRC patients. In particular, the PTEN protein was normally expressed in 16 patients, and 10 of them achieved a partial response. On the other hand, no benefit was documented in 11 patients with loss of PTEN activity (p ! 0.001). The 6 subjects with intact PTEN expression who did not benefit from cetuximab had KRAS mutation in 4 cases.

The results of the study by Perrone et al. [20] con-firmed, in a cohort of 32 cetuximab-treated mCRC pa-tients, that PIK3CA/PTEN gene deregulation (due to di-rect PIK3CA mutations or as an indirect result of the loss of PTEN gene function/expression due to gene mutations or allele loss) significantly correlated with an impaired response to cetuximab (p = 0.02). Cumulatively, all muta-tions of the PIK3CA gene (13% of samples) were foundin non-responder patients. Furthermore, all the patients with a decreased PTEN gene copy number (13% of cases) or with PTEN mutations (10% of cases) were non-re-sponders to cetuximab therapy.

In a larger cohort of 110 mCRC patients, Sartore-Bianchi et al. [21] evaluated if mutations activatingPIK3CA could preclude responsiveness to EGFR-target-ed mAbs through a mechanism similar to the constitu-tive activation of KRAS. The authors observed thatPIK3CA mutations were significantly associated with lack of response to panitumumab or cetuximab, with none of the mutated patients achieving objective tumor response (p = 0.038). When only KRAS wild-type tu-mors were analyzed, the statistical correlation was stron-ger (p = 0.016). Indeed, PIK3CA mutations explained lack of OR in 17% of wild-type KRAS patients. Further-more, patients with PIK3CA mutated tumors had a worse clinical outcome in terms of progression-free survival, compared with wild-type tumors. In addition, loss of PTEN was associated with either lack of tumor response or worse overall survival. Overall, these data indicated that the concomitant evaluation of the molecular status of the PIK3CA/PTEN and KRAS pathways is able to identify up to 70% of mCRC patients unlikely to respond to anti-EGFR mAbs.

Collectively, these studies provided clinical evidence that the mutational status of the PI3K signaling pathway should be considered before treatment with anti-EGFR mAbs. Nevertheless, the above results were not con-firmed by Prenen et al. [22] in a much larger group (n = 200) of mCRC patients treated with cetuximab in mono-therapy or in combination with irinotecan. In fact, they found no correlation between the presence of a PIK3CA mutation and impaired response to cetuximab. PIK3CA mutations were detected in 5/39 (13%) of the responders and 18/60 (11%) of the non-responders (p = 0.781). In pa-tients with wild-type KRAS, the objective response rate was 32/108 (29.6%) in PIK3CA wild type versus 5/14 (35.7%) in PIK3CA mutants (p = 0.75). PIK3CA and KRAS mutant status was confirmed to correlate with worse progression-free survival and overall survival. In conclusion, these findings did not provide any evidence

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for a strong role of PIK3CA mutations as a marker in de-termining response to cetuximab. The authors suggested several arguments that could explain these unexpected results: the lack of definition of the functional aspect of some mutations, the increased frequency of PIK3CA mu-tations in metastases, the evaluation of only primary tu-mors in their study, and the negative prognostic role of PIK3CA mutations which could be reversed by treating these patients with cetuximab.

The challenge of clonal heterogeneity of CRC and the consequent possible absence of concordance of EGFR downstream proteins between primaries and metastases was considered by Loupakis et al. [23] in a retrospective study investigating the role of PTEN loss, AKT phos-phorylation, and KRAS mutations on the activity of ce-tuximab plus irinotecan in 102 mCRC patients. Forty-five pairs of primary tumors and related metastases were assessable for PTEN immunohistochemistry (IHC). Re-sults were concordant in 27 (60%) instances. Seven (16%) positive and 11 (24%) negative primary tumors were as-sociated with negative and positive correspondent metas-tases, respectively. Among 85 assessable primary tumors, 11 (22%) of the 49 PTEN-positive and 4 (11%) of the 36 PTEN-negative patients were responders (p = 0.178), sug-gesting a lack of a predictive role for PTEN status tested on primary tumors. None of the patients with KRAS wild-type status who bore PTEN-negative metastases ob-tained a response, suggesting that these patients may not be candidates for treatment with anti-EGFR mAbs. Con-versely, when PTEN-IHC was performed on metastatic samples, responders were 12/33 (26%) and 1/22 (5%) of PTEN-positive and PTEN-negative patients, respectively (p = 0.007). Indeed, the median progression-free survival of patients with PTEN-positive metastases was 4.7 months compared with 3.3 months for those with PTEN-negative metastases (p ! 0.005). In this study, AKT phosphoryla-tion seemed not to be a predictive/prognostic marker, probably because of its involvement in many different pathways. The relatively small, retrospective cohort and the exploratory nature of PTEN-IHC make these data only hypothesis-generating.

Therapeutic Approaches Targeting the

PI3K/AKT/mTOR Pathway

Given the important role of this pathway in tumor pathogenesis, its inhibition seems an obvious means to induce anti-tumor activity. In fact, a number of the intra-cellular components of this pathway have been targeted

during anticancer drug discovery activities, leading to the current panoply of clinical trials of inhibitors ofPI3K, AKT and mTOR [24] .

Currently, there is limited clinical experience withinhibitors of PI3K. The molecules that are undergoing phase I evaluation include PI-103, BGT-226, BEZ-235, XL-765 and XL-147 [25] .

Given AKT’s key role in this pathway, AKT inhibition produces theoretically more severe side effects than mTOR inhibition. So far, the development of this class of agents has been disappointing. Perifosine, a lipid-based derivative of miltefosine, is perhaps the best character-ized AKT inhibitor in human testing so far [26] . Never-theless, despite encouraging evidence in preclinical stud-ies, this drug failed to demonstrate significant single-agent anticancer activity in several malignancies during phase II testing.

mTORC1 has been probably the most studied target of this pathway, with 4 drugs explored for clinical use: rapa-mycin, temsirolimus, everolimus and deforolimus [27] . mTORC1 inhibitors failed to inhibit tumor progression in a variety of cancers, with the exception of kidney can-cer [28, 29] .

Conclusion

In CRC, as KRAS mutations are clearly associated with resistance to anti-EGFR mAbs, one of the major challenges is to identify, in non-mutant KRAS patients, other markers that can predict lack of response to this therapy. Concerning PIK3CA mutations, small series have investigated their clinical effect on resistance to anti-EGFR mAbs, and discrepant results have been ob-served. In particular, a recent study does not provide any evidence for a strong role of PIK3CA mutations as a sin-gle marker in determining response to cetuximab in che-morefractory mCRC. As PTEN negatively regulates the PI3K/AKT pathway, it is easy to speculate that PTEN in-activation downstream of the EGFR could lead to a resis-tance to the EGFR inhibitors. In particular, PTEN loss in metastases may be predictive of resistance to anti-EGFR mAbs. Nevertheless, PTEN determination is far from an immediate clinical application.

The introduction of modulators of the PI3K/AKT/mTOR pathway as potential targeted anticancer drug is attractive, but is still in early stage. In particular, although this pathway is often described in a linear-vertical fash-ion, its regulation is rather complex, with internal feed-back loops signaling axes, including the MAPK pathway.

Alternative EGFR Pathway Oncology 2009;77(suppl 1):69–74 73

These relationships highlight the requirement of patient stratification and the use of drug combinations or of the so-called ‘dirty drugs’ which can target multiple mole-cules or multiple kinases at once.

In this scenario, the battle against cancer looks like playing chess with a very skilled player against whom, until now, we have succeeded in making the game last longer but who continues to win, each year killing mil-lions of people worldwide [30] .

Disclosure Statement

The authors declare that they have no financial conflict of interest.

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