Targeting the PI3K/Akt/mTOR pathway: Effective combinations and clinical considerations
Introduction
Signaling through the PI3K/Akt/mTOR pathway can be initiated by several mechanisms, all of which increase activation of the pathway in cancer cells. Once activated, the PI3K/Akt/mTOR pathway can be propagated to various substrates, including mTOR, a master regulator of protein translation. Initial activation of the pathway occurs at the cell membrane, where the signal for pathway activation is propagated through class IA PI3K (Fig. 1). Activation of PI3K can occur through tyrosine kinase growth factor receptors such as epidermal growth factor receptor (EGFR) and insulin-like growth factor-1 receptor (IGF-1R), cell adhesion molecules such as integrins, G-protein-coupled receptors (GPCRs), and oncogenes such as Ras. PI3K catalyzes phosphorylation of the D3 position on phosphoinositides to generate the biologically active moieties phosphatidylinositol-3,4,5-triphosphate (PI(3,4,5)P3) and phosphatidylinositol-3,4-bisphosphate (PI(3,4)P2). Upon generation, PI(3,4,5)P3 binds to the pleckstrin homology (PH) domains of PDK-1 (3′-phosphoinositide-dependent kinase 1) and the serine/threonine kinase Akt, causing both proteins to be translocated to the cell membrane where they are subsequently activated. The tumor suppressor PTEN (phosphatase and tensin homolog deleted on chromosome 10) antagonizes PI3K by dephosphorylating PI(3,4,5)P3 and (PI(3,4)P2), thereby preventing activation of Akt and PDK-1.
Akt exists as three structurally similar isoforms, Akt1, Akt2 and Akt3, which are expressed in most tissues (Zinda et al., 2001). Activation of Akt1 occurs through two crucial phosphorylation events, the first of which occurs at T308 in the catalytic domain by PDK-1 (Andjelkovic et al., 1997, Walker et al., 1998). Full activation requires a subsequent phosphorylation at S473 in the hydrophobic motif, which can be mediated by several kinases such as PDK-1 (Balendran et al., 1999), integrin-linked kinase (ILK) (Delcommenne et al., 1998, Lynch et al., 1999), Akt itself (Toker and Newton, 2000), DNA-dependent protein kinase (Feng et al., 2004, Hill et al., 2002), or mTOR (when bound to Rictor in so-called TORC2 complexes (Santos et al., 2001)). Phosphorylation of homologous residues in Akt2 and Akt3 occurs by the same mechanism. Phosphorylation of Akt at S473 is also controlled by a recently described phosphatase, PHLPP (PH domain leucine-rich repeat protein phosphatase), that has two isoforms that preferentially decrease activation of specific Akt isoforms (Brognard et al., 2007). In addition, amplification of Akt1 has been described in human gastric adenocarcinoma (Staal, 1987), and amplification of Akt2 has been described in ovarian, breast, and pancreatic carcinoma (Bellacosa et al., 1995, Cheng et al., 1996). Although mutation of Akt itself is rare, Carpten et al. recently described somatic mutations occurring in the PH domain of Akt1 in a small percentage of human breast, ovarian, and colorectal cancers (Carpten et al., 2007).
Akt recognizes and phosphorylates the consensus sequence RXRXX(S/T) when surrounded by hydrophobic residues. Because this sequence is present in many proteins, numerous Akt substrates have been identified and validated (Obenauer et al., 2003). These substrates control key cellular processes such as apoptosis, cell cycle progression, transcription, and translation. For instance, Akt phosphorylates the FoxO subfamily of forkhead family transcription factors, which inhibits transcription of several pro-apoptotic genes, e.g., Fas-L, IGFBP1 and Bim (Datta et al., 1997, Nicholson and Anderson, 2002). Additionally, Akt can directly regulate apoptosis by phosphorylating and inactivating pro-apoptotic proteins such as BAD, which controls release of cytochrome c from mitochondria, and ASK1 (apoptosis signal-regulating kinase-1), a mitogen-activated protein kinase kinase involved in stress- and cytokine-induced cell death (Datta et al., 1997, Del Peso et al., 1997, Zha et al., 1996). In contrast, Akt can phosphorylate IKK, which indirectly increases the activity of nuclear factor kappa B (NF-kB) and stimulates the transcription of pro-survival genes (Ozes et al., 1999, Romashkova and Makarov, 1999, Verdu et al., 1999). Cell cycle progression can also be effected by Akt through its inhibitory phosphorylation of the cyclin-dependent kinase inhibitors, p21WAF1/CIP1 and p27KIP1 (Liang et al., 2002, Shin et al., 2002, Zhou et al., 2001), and inhibition of GSK3β by Akt stimulates cell cycle progression by stabilizing cyclin D1 expression (Diehl et al., 1998). Recently, a novel pro-survival Akt substrate, PRAS40 (proline-rich Akt substrate of 40 kDa), has been described (Vander Haar et al., 2007), whereby phosphorylation of PRAS40 by Akt attenuates its ability to inhibit mTORC1 kinase activity. It has been suggested that PRAS40 may be a specific substrate of Akt3 (Madhunapantula et al., 2007). Thus, Akt inhibition might have pleiotropic effects on cancer cells that could contribute to an antitumor response.
The best-studied downstream substrate of Akt is the serine/threonine kinase mTOR (mammalian target of rapamycin). Akt can directly phosphorylate and activate mTOR, as well as cause indirect activation of mTOR by phosphorylating and inactivating TSC2 (tuberous sclerosis complex 2, also called tuberin), which normally inhibits mTOR through the GTP-binding protein Rheb (Ras homolog enriched in brain). When TSC2 is inactivated by phosphorylation, the GTPase Rheb is maintained in its GTP-bound state, allowing for increased activation of mTOR. mTOR exists in two complexes: the TORC1 complex, in which mTOR is bound to Raptor, and the TORC2 complex, in which mTOR is bound to Rictor. In the TORC1 complex, mTOR signals to its downstream effectors S6 kinase/ribosomal protein S6 and 4EBP-1/eIF4E to control protein translation. Although mTOR is generally considered a downstream substrate of Akt, mTOR can also phosphorylate Akt when bound to Rictor in TORC2 complexes, perhaps providing a level of positive feedback on the pathway (Sarbassov et al., 2005). Finally, the downstream mTOR effector S6 kinase-1 (S6K1) can also regulate the pathway by catalyzing an inhibitory phosphorylation on insulin receptor substrate (IRS) proteins. This prevents IRS proteins from activating PI3K, thereby inhibiting activation of Akt (Harrington et al., 2004, Shah et al., 2004).
In addition to preclinical studies, many clinical observations support targeting the PI3K/Akt/mTOR pathway in human cancer. First, immunohistochemical studies using antibodies that recognize Akt when phosphorylated at S473 have shown that activated Akt is detectable in cancers such as multiple myeloma (MM), lung cancer, head and neck cancer, breast cancer, brain cancer, gastric cancer, acute myelogenous leukemia, endometrial cancer, melanoma, renal cell carcinoma, colon cancer, ovarian cancer, and prostate cancer (Alkan and Izban, 2002, Choe et al., 2003, Dai et al., 2005, Ermoian et al., 2002, Gupta et al., 2002, Horiguchi et al., 2003, Hsu et al., 2001, Kanamori et al., 2001, Kreisberg et al., 2004, Kurose et al., 2001, Malik et al., 2002, Min et al., 2004, Nakayama et al., 2001, Nam et al., 2003, Perez-Tenorio and Stal, 2002, Roy et al., 2002, Schlieman et al., 2003, Sun et al., 2001, Terakawa et al., 2003, Yuan et al., 2000). Immunohistochemical analysis has also been used to demonstrate prognostic significance of Akt activation. Phosphorylation of Akt at S473 has been associated with poor prognosis in cancers of the skin (Dai et al., 2005), pancreas (Schlieman et al., 2003, Yamamoto et al., 2004), liver (Nakanishi et al., 2005), prostate (Kreisberg et al., 2004), breast (Perez-Tenorio and Stal, 2002), endometrium (Terakawa et al., 2003), stomach (Nam et al., 2003), brain (Ermoian et al., 2002), and blood (Min et al., 2004). Tsurutani et al. recently extended these studies by using antibodies against two sites of Akt phosphorylation, S473 and T308, to show that Akt activation is selective for NSCLC tumors versus normal tissue and is a better predictor of poor prognosis in NSCLC tumors than S473 alone (Tsurutani et al., 2006). In addition, amplification of Akt isoforms has been observed in some cancers, albeit at a lower frequency (Bellacosa et al., 1995, Cheng et al., 1996, Ruggeri et al., 1998, Staal, 1987).
Another frequent genetic event that occurs in human cancer is loss of tumor suppressor PTEN function. PTEN normally suppresses activation of the PI3K/Akt/mTOR pathway by functioning as a lipid phosphatase. Loss of PTEN function in cancer can occur through mutation, deletion, or epigenetic silencing. Multiple studies have demonstrated a high frequency of PTEN mutations or deletions in a variety of human cancers, including brain, bladder, breast, prostate, and endometrial cancers (Ali et al., 1999, Aveyard et al., 1999, Dahia, 2000, Dreher et al., 2004, Li et al., 1997, Rasheed et al., 1997), making PTEN the second most frequently mutated tumor suppressor gene (Stokoe, 2001). In tumor types where PTEN mutations are rare, such as lung cancer, epigenetic silencing may occur (Forgacs et al., 1998, Kohno et al., 1998, Yokomizo et al., 1998). Several studies have also demonstrated the prognostic significance of PTEN loss in multiple human cancers, where mutation, deletion, or epigenetic silencing of PTEN correlates with poor prognosis and reduced survival (Bertram et al., 2006, Perez-Tenorio et al., 2007, Saal et al., 2007, Smith et al., 2001, Yoshimoto et al., 2007). Collectively, these studies have established that the loss of PTEN is a common mechanism for activation of the PI3K/Akt/mTOR pathway and poor prognostic factor in human cancer.
Finally, activation of PI3K has been described in human cancers. It can result from amplification, overexpression or from mutations in the p110 catalytic or p85 regulatory subunits. Amplification of the 3q26 chromosomal region, which contains the gene PIK3CA that encodes the p110α catalytic subunit of PI3K, occurs in 40% of ovarian (Shayesteh et al., 1999) and 50% of cervical carcinomas (Ma et al., 2000). Somatic mutations of this gene have also been detected in several cancer types and result in increased kinase activity of the mutant PI3K relative to wild-type PI3K. Mutations in the regulatory p85 subunit have also been detected (Jimenez et al., 1998, Philp et al., 2001). Because any of these alterations in individual components would result in activation of the pathway, these studies suggest that pathway activation is one of the most frequent molecular alterations in cancer.
The rationale for targeting the PI3K/Akt/mTOR pathway in combination therapy comes from data describing constitutive or residual pathway activation in cells that have developed resistance to conventional chemotherapy and radiation (West et al., 2002), as well as to other targeted therapies such as EGFR antagonism. In these cases, combining chemotherapy or radiation with a pathway inhibitor can overcome acquired resistance to EGFR tyrosine kinase inhibitors (TKIs). Some standard chemotherapeutic agents appear to directly inhibit Akt in vitro, and the cytotoxicity may be a direct consequence of inhibition of Akt signaling (Asselin et al., 2001, Hayakawa et al., 2002). Because Akt is integrally involved in cellular survival, many groups have investigated the effects of combining chemotherapy with pathway inhibitors. Preclinical studies that have investigated this concept will be discussed below.
Section snippets
PI3 kinase inhibitors: LY294002 and wortmannin
Targeting PI3 kinase, the most proximal pathway component, has advantages over targeting more distal components such as Akt and mTOR. Inhibitors of PI3K diminish signaling to Rac as well as Akt, providing a broader inhibition of downstream signaling than distal inhibition. The pharmacologic agents LY294002 and wortmannin both target the p110 catalytic subunit of PI3K. Although these commercially available inhibitors effectively inhibit PI3K, poor solubility and high toxicity have limited their
Clinical trials with PI3K/Akt/mTOR pathway inhibitors as single agents and in combination with other therapies
Although combinations of pathway inhibitors with various types of chemotherapy have been investigated extensively in preclinical studies, only a few clinical trials with Akt inhibitors and mTOR inhibitors have been reported up to now, while no clinical trials using PI3K inhibitors have been published. These data will be discussed below (Table 1).
Some clinical considerations for the combination of pathway inhibitors with other chemotherapies
There is substantial preclinical evidence that PI3K/Akt/mTOR pathway inhibitors can be effectively combined with chemotherapy, radiotherapy and targeted agents to enhance efficacy and overcome mechanisms of resistance. The early clinical trials suggest that pathway inhibitors may be beneficial when added to other anticancer therapies, particularly other targeted therapies such as EGFR TKIs, imatinib, and bevacizumab, although there remains a paucity of phase II and phase III data to corroborate
References (233)
- et al.
Immunohistochemical localization of phosphorylated AKT in multiple myeloma
Blood
(2002) - et al.
Role of translocation in the activation and function of protein kinase B
J. Biol. Chem.
(1997) - et al.
PDK1 acquires PDK2 activity in the presence of a synthetic peptide derived from the carboxyl terminus of PRK2
Curr. Biol.
(1999) - et al.
The mTOR inhibitor RAD001 sensitizes tumor cells to DNA-damaged induced apoptosis through inhibition of p21 translation
Cell
(2005) - et al.
PHLPP and a second isoform, PHLPP2, differentially attenuate the amplitude of Akt signaling by regulating distinct Akt isoforms
Mol. Cell
(2007) - et al.
Alkyl phospholipid perifosine induces myeloid hyperplasia in a murine myeloma model
Exp. Hematol.
(2007) - et al.
Upstream signaling inhibition enhances rapamycin effect on growth of kidney cancer cells
Urology
(2007) - et al.
Phase I and pharmacological study of daily oral administration of perifosine (D-21266) in patients with advanced solid tumours
Eur. J. Cancer
(2002) - et al.
Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery
Cell
(1997) - et al.
Inhibition of phosphatidylinositol 3-kinase/Akt and histone deacetylase activity induces apoptosis in non-small cell lung cancer in vitro and in vivo
J. Thorac. Cardiovasc. Surg.
(2005)
New antitumor substances of natural origin
Cancer Treat. Rev.
A dual PI3 kinase/mTOR inhibitor reveals emergent efficacy in glioma
Cancer Cell
Identification of a PKB/Akt hydrophobic motif Ser-473 kinase as DNA-dependent protein kinase
J. Biol. Chem.
Phosphatidylinositol ether lipid analogues that inhibit AKT also independently activate the stress kinase, p38alpha, through MKK3/6-independent and -dependent mechanisms
J. Biol. Chem.
Radiation sensitization of human cancer cells in vivo by inhibiting the activity of PI3K using LY294002
Int. J. Radiat. Oncol. Biol. Phys.
Perifosine, an oral bioactive novel alkylphospholipid, inhibits Akt and induces in vitro and in vivo cytotoxicity in human multiple myeloma cells
Blood
Identification of a plasma membrane raft-associated PKB Ser473 kinase activity that is distinct from ILK and PDK1
Curr. Biol.
Elevated Akt activation and its impact on clinicopathological features of renal cell carcinoma
J. Urol.
Human brain tumor xenografts in nude mice as a chemotherapy model
Eur. J. Cancer Clin. Oncol.
The AKT kinase is activated in multiple myeloma tumor cells
Blood
Autophagy for cancer therapy through inhibition of pro-apoptotic proteins and mammalian target of rapamycin signaling
J. Biol. Chem.
Frequent loss of PTEN expression is linked to elevated phosphorylated Akt levels, but not associated with p27 and cyclin D1 expression, in primary epithelial ovarian carcinomas
Am. J. Pathol.
Targeting the Akt/mammalian target of rapamycin pathway for radiosensitization of breast cancer
Mol. Cancer Ther.
Mutational spectra of PTEN/MMAC1 gene: a tumor suppressor with lipid phosphatase activity
J. Natl. Cancer Inst.
A phase II trial of perifosine, an oral alkylphospholipid, in recurrent or metastatic head and neck cancer
Cancer Biol. Ther.
XIAP regulates Akt activity and caspase-3-dependent cleavage during cisplatin-induced apoptosis in human ovarian epithelial cancer cells
Cancer Res.
Randomized phase II study of multiple dose levels of CCI-779, a novel mammalian target of rapamycin kinase inhibitor, in patients with advanced refractory renal cell carcinoma
J. Clin. Oncol.
Somatic mutation of PTEN in bladder carcinoma
Br. J. Cancer
Phase II study of daily oral perifosine in patients with advanced soft tissue sarcoma
Cancer
Molecular alterations of the AKT2 oncogene in ovarian and breast carcinomas
Int. J. Cancer
Loss of PTEN is associated with progression to androgen independence
Prostate
Loss of PTEN/MMAC1/TEP in EGF receptor-expressing tumor cells counteracts the antitumor action of EGFR tyrosine kinase inhibitors
Oncogene
Signaling interactions of rapamycin combined with erlotinib in cervical carcinoma xenografts
Mol. Cancer Ther.
Dual inhibition of mTOR and estrogen receptor signaling in vitro induces cell death in models of breast cancer
Clin. Cancer Res.
LY294002 and rapamycin co-operate to inhibit T-cell proliferation
Br. J. Pharmacol.
Rapamycin synergizes with the epidermal growth factor receptor inhibitor erlotinib in non-small-cell lung, pancreatic, colon, and breast tumors
Mol. Cancer Ther.
Inhibition of mammalian target of rapamycin or apoptotic pathway induces autophagy and radiosensitizes PTEN null prostate cancer cells
Cancer Res.
Activated forms of H-RAS and K-RAS differentially regulate membrane association of PI3K, PDK-1, and AKT and the effect of therapeutic kinase inhibitors on cell survival
Mol. Cancer Ther.
Randomized 3-arm, phase 2 study of temsirolimus (CCI-779) in combination with letrozole in postmenopausal women with locally advanced or metastatic breast cancer
J. Clin. Oncol. ASCO Annu. Meet. Proc.
A transforming mutation in the pleckstrin homology domain of AKT1 in cancer
Nature
Online Collaborative Oncology Group, perifosine (P) can be combined with docetaxel (T) without dose reduction of either drug.
J. Clin. Oncol. ASCO Annu. Meet. Proc. (Part I)
Differential sensitivities of trastuzumab (herceptin)-resistant human breast cancer cells to phosphoinositide-3 kinase (PI-3K) and epidermal growth factor receptor (EGFR) kinase inhibitors
Breast Cancer Res. Treat.
Phase II study of temsirolimus (CCI-779), a novel inhibitor of mTOR, in heavily pretreated patients with locally advanced or metastatic breast cancer
J. Clin. Oncol.
Phase II study of CCI-779 in patients with recurrent glioblastoma multiforme
Invest. New Drugs
Amplification of AKT2 in human pancreatic cells and inhibition of AKT2 expression and tumorigenicity by antisense RNA
Proc. Natl. Acad. Sci. (USA)
Sensitizing HER2-overexpressing cancer cells to luteolin-induced apoptosis through suppressing p21(WAF1/CIP1) expression with rapamycin
Mol. Cancer Ther.
Analysis of the phosphatidylinositol 3′-kinase signaling pathway in glioblastoma patients in vivo
Cancer Res.
Gefitinib-induced killing of NSCLC cell lines expressing mutant EGFR requires BIM and can be enhanced by BH3 mimetics
PLoS Med.
PTEN, a unique tumor suppressor gene
Endocr. Relat. Cancer
Prognostic significance of activated Akt expression in melanoma: a clinicopathologic study of 292 cases
J. Clin. Oncol.
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