mTOR Inhibition Elicits a Dramatic Response in PI3K-Dependent Colon Cancers

The phosphatidylinositide-3-kinase (PI3K) signaling pathway is critical for multiple cellular functions including metabolism, proliferation, angiogenesis, and apoptosis, and is the most commonly altered pathway in human cancers. Recently, we developed a novel mouse model of colon cancer in which tumors are initiated by a dominant active PI3K (FC PIK3ca*). The cancers in these mice are moderately differentiated invasive mucinous adenocarcinomas of the proximal colon that develop by 50 days of age. Interestingly, these cancers form without a benign intermediary or aberrant WNT signaling, indicating a non-canonical mechanism of tumorigenesis. Since these tumors are dependent upon the PI3K pathway, we investigated the potential for tumor response by the targeting of this pathway with rapamycin, an mTOR inhibitor. A cohort of FC PIK3ca* mice were treated with rapamycin at a dose of 6 mg/kg/day or placebo for 14 days. FDG dual hybrid PET/CT imaging demonstrated a dramatic tumor response in the rapamycin arm and this was confirmed on necropsy. The tumor tissue remaining after treatment with rapamycin demonstrated increased pERK1/2 or persistent phosphorylated ribosomal protein S6 (pS6), indicating potential resistance mechanisms. This unique model will further our understanding of human disease and facilitate the development of therapeutics through pharmacologic screening and biomarker identification.     

Oncogenic Mutations of PIK3CA in Human Cancers

Phosphatidylinositol 3-kinases (PI3Ks) are important regulators of signaling pathways. To determine whether PI3Ks are genetically altered in human cancers, we recently analyzed the sequences of the PI3K gene family and discovered that one member, the PIK3CA gene encoding the p110? catalytic subunit, was frequently mutated in cancers of the colon, breast, brain and lung. The majority of mutations clustered near two positions within the PI3K helical or catalytic domains and at least one hotspot mutation appeared to increase kinase activity. PIK3CA represents one of the most highly mutated oncogenes identified in human cancers and may be a useful diagnostic and therapeutic target.     

Breast tumor cells with PI3K mutation or HER2 amplification are selectively addicted to Akt signaling

Background

Dysregulated PI3K/Akt signaling occurs commonly in breast cancers and is due to HER2 amplification, PI3K mutation or PTEN inactivation. The objective of this study was to determine the role of Akt activation in breast cancer as a function of mechanism of activation and whether inhibition of Akt signaling is a feasible approach to therapy.

Methodology/Principal Findings

A selective allosteric inhibitor of Akt kinase was used to interrogate a panel of breast cancer cell lines characterized for genetic lesions that activate PI3K/Akt signaling: HER2 amplification or PI3K or PTEN mutations in order to determine the biochemical and biologic consequences of inhibition of this pathway. A variety of molecular techniques and tissue culture and in vivo xenograft models revealed that tumors with mutational activation of Akt signaling were selectively dependent on the pathway. In sensitive cells, pathway inhibition resulted in D-cyclin loss, G1 arrest and induction of apoptosis, whereas cells without pathway activation were unaffected. Most importantly, the drug effectively inhibited Akt kinase and its downstream effectors in vivo and caused complete suppression of the growth of breast cancer xenografts with PI3K mutation or HER2 amplification, including models of the latter selected for resistance to Herceptin. Furthermore, chronic administration of the drug was well-tolerated, causing only transient hyperglycemia without gross toxicity to the host despite the pleiotropic normal functions of Akt.

Conclusions/Significance

These data demonstrate that breast cancers with PI3K mutation or HER2 amplification are selectively dependent on Akt signaling, and that effective inhibition of Akt in tumors is feasible and effective in vivo. These findings suggest that direct inhibition of Akt may represent a therapeutic strategy for breast and other cancers that are addicted to the pathway including tumors with resistant to Herceptin.     

Somatic Mutations of PIK3R1 Promote Gliomagenesis

The phosphoinositide 3-kinase (PI3K) pathway is targeted for frequent alteration in glioblastoma (GBM) and is one of the core GBM pathways defined by The Cancer Genome Atlas. Somatic mutations of PIK3R1 are observed in multiple tumor types, but the tumorigenic activity of these mutations has not been demonstrated in GBM. We show here that somatic mutations in the iSH2 domain of PIK3R1 act as oncogenic driver events. Specifically, introduction of a subset of the mutations identified in human GBM, in the nSH2 and iSH2 domains, increases signaling through the PI3K pathway and promotes tumorigenesis of primary normal human astrocytes in an orthotopic xenograft model. Furthermore, we show that cells that are dependent on mutant P85α-mediated PI3K signaling exhibit increased sensitivity to a small molecule inhibitor of AKT. Together, these results suggest that GBM patients whose tumors carry mutant PIK3R1 alleles may benefit from treatment with inhibitors of AKT.     

Phosphatidylinositol-3-OH kinase or RAS pathway mutations in human breast cancer cell lines

Constitutive activation of the phosphatidylinositol-3-OH kinase (PI3K) and RAS signaling pathways are important events in tumor formation. This is illustrated by the frequent genetic alteration of several key players from these pathways in a wide variety of human cancers. Here, we report a detailed sequence analysis of the PTEN, PIK3CA, KRAS, HRAS, NRAS, and BRAF genes in a collection of 40 human breast cancer cell lines. We identified a surprisingly large proportion of cell lines with mutations in the PI3K or RAS pathways (54% and 25%, respectively), with mutants for each of the six genes. The PIK3CA, KRAS, and BRAF mutation spectra of the breast cancer cell lines were similar to those of colorectal cancers. Unlike in colorectal cancers, however, mutational activation of the PI3K pathway was mutually exclusive with mutational activation of the RAS pathway in all but 1 of 30 mutant breast cancer cell lines (P = 0.001). These results suggest that there is a fine distinction between the signaling activators and downstream effectors of the oncogenic PI3K and RAS pathways in breast epithelium and those in other tissues.     

Glucose metabolism measured by [¹⁸F]fluorodeoxyglucose positron emission tomography is independent of PTEN/AKT status in human colon carcinoma cells

The phosphoinositide 3-kinase (PI3K) signaling pathway is one of the most altered in cancer, leading to a range of cellular responses including enhanced proliferation, survival, and metabolism, and is thus an attractive target for anticancer drug development. Stimulation of the PI3K pathway can be initiated by alterations at different levels of the signaling cascade including growth factor receptor activation, as well as mutations in PIK3CA, PTEN, and AKT genes frequently found in a broad range of cancers. Given its role in glucose metabolism, we investigated the utility of [(18)F]fluorodeoxyglucose positron emission tomography ([(18)F]FDG PET) as a pharmacodynamic biomarker of PI3K pathway-induced glucose metabolism. PTEN deletion in human colon carcinoma cells led to constitutive AKT activation but did not confer a phenotype of increased cell proliferation or glucose metabolism advantage in vivo relative to isogenic tumors derived from cells with a wild-type allele. This was not due to the activation context, that is, phosphatase activity, per se because PIK3CA activation in xenografts derived from the same lineage failed to increase glucose metabolism. Acute inhibition of PI3K activity by LY294002, and hence decreased activated AKT expression, led to a significant reduction in tumor [(18)F]FDG uptake that could be explained at least in part by decreased membrane glucose transporter 1 expression. The pharmacodynamic effect was again independent of PTEN status. In conclusion, [(18)F]FDG PET is a promising pharmacodynamic biomarker of PI3K pathway inhibition; however, its utility to detect glucose metabolism is not directly linked to the magnitude of activated AKT protein expression.     

Absence of AKT1 Mutations in Glioblastoma

Background

Oncogenic activation of the PI3K signalling pathway plays a pivotal role in the development of glioblastoma multiforme (GBM). A central node in PI3K downstream signalling is controlled by the serine-threonine kinase AKT1. A somatic mutation affecting residue E17 of the AKT1 gene has recently been identified in breast and colon cancer. The E17K change results in constitutive AKT1 activation, induces leukaemia in mice, and accordingly, may be therapeutically exploited to target the PI3K pathway. Assessing whether AKT1 is activated by somatic mutations in GBM is relevant to establish its role in this aggressive disease.

Methodology/Principal Findings

We performed a systematic mutational analysis of the complete coding sequence of the AKT1 gene in a panel of 109 tumor GBM samples and nine high grade astrocytoma cell lines. However, no somatic mutations were detected in the coding region of AKT1.

Conclusions/Significance

Our data indicate that in GBM oncogenic deregulation of the PI3K pathway does not involve somatic mutations in the coding region of AKT1.     

Integrative genomic approaches identify IKBKE as a breast cancer oncogene

The karyotypic chaos exhibited by human epithelial cancers complicates efforts to identify mutations critical for malignant transformation. Here we integrate complementary genomic approaches to identify human oncogenes. We show that activation of the ERK and phosphatidylinositol 3-kinase (PI3K) signaling pathways cooperate to transform human cells. Using a library of activated kinases, we identify several kinases that replace PI3K signaling and render cells tumorigenic. Whole genome structural analyses reveal that one of these kinases, IKBKE (IKKepsilon), is amplified and overexpressed in breast cancer cell lines and patient-derived tumors. Suppression of IKKepsilon expression in breast cancer cell lines that harbor IKBKE amplifications induces cell death. IKKepsilon activates the nuclear factor-kappaB (NF-kappaB) pathway in both cell lines and breast cancers. These observations suggest a mechanism for NF-kappaB activation in breast cancer, implicate the NF-kappaB pathway as a downstream mediator of PI3K, and provide a framework for integrated genomic approaches in oncogene discovery.     

Genotype Directed Therapy in Murine Mismatch Repair Deficient Tumors

The PI3K/AKT/mTOR pathway has frequently been found activated in human tumors. We show that in addition to Wnt signaling dysfunction, the PI3K/AKT/mTOR pathway is often upregulated in mouse Msh2^−/− initiated intestinal tumors. NVP-BEZ235 is a dual PI3K/mTOR inhibitor toxic to many cancer cell lines and currently involved in clinical trials. We have treated two mouse models involving Msh2 that develop small intestinal and/or colonic tumors with NVP-BEZ235, and a subset of animals with NVP-BEZ235 and MEK inhibitor ADZ4266. The disease phenotype has been followed with pathology, ¹⁸F FDG PET imaging, and endoscopy. Intestinal adenocarcinomas are significantly decreased in multiplicity by both drug regimens. The majority of tumors treated with combined therapy regress significantly, while a small number of highly progressed tumors persist. We have examined PTEN, AKT, MEK 1&2, MAPK, S6K, mTOR, PDPK1, and Cyclin D1 and find variable alterations that include downregulation of PTEN, upregulation of AKT and changes in its phosphorylated forms, upregulation of pMEK 1&2, p42p44MAPK, pS6K, and Cyclin D1. Apoptosis has been found intact in some tumors and not in others. Our data indicate that NVP-BEZ235 alone and in combination with ADZ4266 are effective in treating a proportion of colorectal cancers, but that highly progressed resistant tumors grow in the presence of the drugs. Pathways upregulated in some resistant tumors also include PDPK1, suggesting that metabolic inhibitors may also be useful in treating these tumors.     

Germline PIK3CA and AKT1 Mutations in Cowden and Cowden-like Syndromes

Cowden syndrome (CS) is a difficult-to-recognize multiple hamartoma syndrome with high risks of breast, thyroid, and other cancers. Germline mutations in PTEN on 10q23 were found to cause 85% of CS when accrued from tertiary academic centers, but prospective accrual from the community over the last 12 years has revealed a 25% PTEN mutation frequency. PTEN is the phosphatase that has been implicated in a heritable cancer syndrome and subsequently in multiple sporadic cancers and developmental processes. PTEN antagonizes the AKT1/PI3K signaling pathway and has roles in cell cycle, migration, cell polarity, and apoptosis. We report that 8 of 91 (8.8%) unrelated CS individuals without germline PTEN mutations carried 10 germline PIK3CA mutations (7 missense, 1 nonsense, and 2 indels) and 2 (2.2%) AKT1 mutations. These mutations result in significantly increased P-Thr308-AKT and increased cellular PIP3. Our observations suggest that PIK3CA and AKT1 are CS susceptibility genes.     

Phosphoinositide 3-kinase signaling pathway mediated by p110α regulates invadopodia formation

Invadopodia are extracellular matrix-degrading protrusions formed by invasive cancer cells that are thought to function in cancer invasion. Although many invadopodia components have been identified, signaling pathways that link extracellular stimuli to invadopodia formation remain largely unknown. We investigate the role of phosphoinositide 3-kinase (PI3K) signaling during invadopodia formation. We find that in human breast cancer cells, both invadopodia formation and degradation of a gelatin matrix were blocked by treatment with PI3K inhibitors or sequestration of D-3 phosphoinositides. Functional analyses revealed that among the PI3K family proteins, the class I PI3K catalytic subunit p110α, a frequently mutated gene product in human cancers, was selectively involved in invadopodia formation. The expression of p110α with cancerous mutations promoted invadopodia-mediated invasive activity. Furthermore, knockdown or inhibition of PDK1 and Akt, downstream effectors of PI3K signaling, suppressed invadopodia formation induced by p110α mutants. These data suggest that PI3K signaling via p110α regulates invadopodia-mediated invasion of breast cancer cells.     

The Unique Non-Catalytic C-Terminus of P37delta-PI3K Adds Proliferative Properties In Vitro and In Vivo

The PI3K/Akt pathway is central for numerous cellular functions and is frequently deregulated in human cancers. The catalytic subunits of PI3K, p110, are thought to have a potential oncogenic function, and the regulatory subunit p85 exerts tumor suppressor properties. The fruit fly, Drosophila melanogaster, is a highly suitable system to investigate PI3K signaling, expressing one catalytic, Dp110, and one regulatory subunit, Dp60, and both show strong homology with the human PI3K proteins p110 and p85. We recently showed that p37δ, an alternatively spliced product of human PI3K p110δ, displayed strong proliferation-promoting properties despite lacking the catalytic domain completely. Here we functionally evaluate the different domains of human p37δ in Drosophila. The N-terminal region of Dp110 alone promotes cell proliferation, and we show that the unique C-terminal region of human p37δ further enhances these proliferative properties, both when expressed in Drosophila, and in human HEK-293 cells. Surprisingly, although the N-terminal region of Dp110 and the C-terminal region of p37δ both display proliferative effects, over-expression of full length Dp110 or the N-terminal part of Dp110 decreases survival in Drosophila, whereas the unique C-terminal region of p37δ prevents this effect. Furthermore, we found that the N-terminal region of the catalytic subunit of PI3K p110, including only the Dp60 (p85)-binding domain and a minor part of the Ras binding domain, rescues phenotypes with severely impaired development caused by Dp60 over-expression in Drosophila, possibly by regulating the levels of Dp60, and also by increasing the levels of phosphorylated Akt. Our results indicate a novel kinase-independent function of the PI3K catalytic subunit.     

The Rho Guanine Nucleotide Exchange Factor DRhoGEF2 Is a Genetic Modifier of the PI3K Pathway in Drosophila

The insulin/IGF-1 signaling pathway mediates various physiological processes associated with human health. Components of this pathway are highly conserved throughout eukaryotic evolution. In Drosophila, the PTEN ortholog and its mammalian counterpart downregulate insulin/IGF signaling by antagonizing the PI3-kinase function. From a dominant loss-of-function genetic screen, we discovered that mutations of a Dbl-family member, the guanine nucleotide exchange factor DRhoGEF2 (DRhoGEF2^2(l)04291), suppressed the PTEN-overexpression eye phenotype. dAkt/dPKB phosphorylation, a measure of PI3K signaling pathway activation, increased in the eye discs from the heterozygous DRhoGEF2 wandering third instar larvae. Overexpression of DRhoGEF2, and it’s functional mammalian ortholog PDZ-RhoGEF (ArhGEF11), at various stages of eye development, resulted in both dPKB/Akt-dependent and -independent phenotypes, reflecting the complexity in the crosstalk between PI3K and Rho signaling in Drosophila.     

Hot-spot mutations in p110α of phosphatidylinositol 3-kinase (PI3K): Differential interactions with the regulatory subunit p85 and with RAS

The phosphatidylinositol 3-kinase (PI3K) signaling pathway is frequently upregulated in cancer. PIK3CA, the gene coding for the catalytic subunit p110α of PI3K, is mutated in about 12% of all human cancers. Most of these mutants are single amino acid substitutions that map to three positions (hot spots) in the helical or kinase domains of the enzyme. The mutant proteins show gain of enzymatic function, constitutively activate AKT signaling and induce oncogenic transformation in vitro and in animal model systems. We have shown previously that hot-spot mutations in the helical domain and kinase domain of the avian p110α have different requirements for interaction with the regulatory subunit p85 and with RAS-GTP. Here, we have carried out a genetic and biochemical analysis of these "hot-spot" mutations in human p110α. The present studies add support to the proposal that helical and kinase domain mutations in p110α trigger a gain of function by different molecular mechanisms. The gain of function induced by helical domain mutations requires interaction with RAS-GTP. In contrast, the kinase domain mutation is active in the absence of RAS-GTP binding, but depends on the interaction with p85.     

Design,synthesis and biological evaluation of novel series of 2H-benzo[b][1,4]oxazin-3(4H)-one and 2H-benzo[b][1,4]oxazine scaffold derivatives as PI3Kα inhibitors

The abnormal activation of PI3K signaling pathway leads to the occurrence of various cancers. The PI3Kα is frequently mutated and overexpressed in many human cancers. Therefore, the PI3Kα was considered as a promising target in therapeutic treatment of cancer. In this study, two series of compounds containing 2H-benzo[b][1,4]oxazin-3(4H)-one and 2H-benzo[b][1,4]oxazine scaffold were synthesized and evaluated antiproliferative activities against three cancer cell lines, including HCT-116, MDA-MB-231 and SNU638. Compound 7f with the most potent antiproliferative activity was selected for further evaluation on normal cells and PI3K kinase. Studies indicated that compound 7f could decrease the phospho-Akt (T308) in a dose-dependent manner. Four key hydrogen bonding interactions were found in the docking of 7f with PI3K enzyme. All the results suggested that 7f was a potent PI3Kα inhibitor.     

PI3K: a potential therapeutic target for cancer

Phosphatidylinositol 3-kinase (PI3K), one member of lipid kinase family, has been demonstrated to play a key role in regulating cell proliferation, adhesion, survival, and motility. Recent studies indicate that PI3K related signaling pathway is one of the most commonly activated pathways in human cancers. Accordingly, pharmacological inhibition of key nodes in this signaling cascade has been a focus in developmental therapeutics. To date, Inhibitors targeting PI3K or nodes in this pathway, AKT and mTOR, are best studied and have reached clinical trials. In this review, we will focus on recent progress on understanding of PI3Ks signaling pathway and the development of PI3K inhibitors.