Oncogenic PI3K Research Articles

Abstract 5137: The PI3K pathway regulates the actin cytoskeleton, tumor cell migration and metastasis via the atypical tumor suppressor p27

Abstract The CDK inhibitor p27 plays a well-established role in cell cycle progression, regulating the G1-S phase transition. A growing body of evidence indicates a potential role for p27 as a mediator of tumor cell motility and invasiveness. The present data provides additional evidence for p27 as a mediator of cancer metastasis. These activities appear to require cytoplasmic mislocalization, and are independent of p27's action on CDKs and the cell cycle. p27 phosphorylation at T157 and T198 is mediated by the AGC kinases RSK, SGK, and AKT to promote cytoplasmic mislocalization and facilitate p27 binding to the RhoA cytoskeletal regulatory protein. p27-mediated inhibition of RhoA/ROCK signaling is associated with dynamic rearrangement of the actin cytoskeleton and increased cell migration and invasion. Overexpression of the PI3K effectors RSK, SGK, and mTOR all promote cytoplasmic mislocalization of p27, increased p27: RhoA binding, and all enhance directed cell migration as assayed by modified transwell assay. This increased tumor cell motility is dependent upon p27, as shRNA-mediated p27 knockdown reverts transwell invasion to parental levels. Overexpression of mTOR or these AGC kinases reduces cellular levels of RhoA-GTP and p-cofilin, both of which revert following shRNA p27 treatment. The increased p27: RhoA binding, decreased RhoA-GTP, and decreased p-cofilin correlate with dramatic loss of polymerized actin filaments as assayed by phalloidin staining, and all of these effects are rescued by shRNA p27. The in vivo relevance of these findings has been pursued in the highly metastatic human breast cancer sub-lines MDA-MB-231-1833 (bone-tropic) and MDA-MB-231-4175 (lung-tropic). Both highly metastatic variants demonstrate hyperactivation of the PI3K effectors SGK, RSK, and AKT relative to the parental MDA-MB-231 line and show markedly elevated p27 levels, with a shift in p27 protein localization to the cytoplasm, increased p27: RhoA binding, decreased p27-dependent levels of RhoA-GTP and p-cofilin, and increased p27-dependent invasiveness by modified transwell assay. The cell cycle profiles of MDA-MB-231-4175 and 1833 variants were unchanged by shRNA p27 supporting the notion that cytoskeletal effects of p27 in these lines are independent of cell cycle/Cdk regulatory activity. Of particular interest, the increase in lung tumor formation by MDA-MB-231-4175 following tail vein injection was strikingly reduced to near-parental levels after shRNA-mediated knockdown of p27. These results support a model whereby PI3K activation promotes p27 phosphorylation, mislocalization to the cytoplasm, enhanced RhoA binding and subsequent remodeling of the actin cytoskeleton. This cascade of events may be a major route whereby oncogenic PI3K activation mediates tumor cell acquisition of the invasive potential necessary for overt metastasis in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5137.

Abstract C68: Endogenous expression of an oncogenic PI3K mutation leads to epithelial-mesenchymal transition (EMT) and invasiveness

Abstract The PTEN/PI3K pathway is commonly mutated in cancer and therefore represents a therapeutic target. In order to investigate the primary phenotypes mediated by mutant PIK3CA in a controlled and patient-relevant context, we utilized human non-tumorigenic MCF10A parental and isogenic knock-in cell lines that harbor a common activating PIK3CA mutation (H1047R). We found that endogenous introduction of a mutated PIK3CA allele results in a marked epithelial-mesenchymal transition (EMT) and invasive phenotype compared to isogenic wild-type cells. Moreover, a potent and selective inhibitor of PIK3CA (GDC-0941) was highly effective in reversing this phenotype in 3D cell culture. Our results suggest that invasion assays and biomarkers should be used to reveal PIK3CA ‘oncogeneaddiction’ responses of pathway-targeted agents. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C68.

The PI3K/mTOR Inhibitor NVP-BEZ235 Is a Promising Pre-Clinical Candidate for Therapeutic Intervention in Acute Myeloid Leukemia.

Abstract 1024Poster Board I-46The treatment of acute myeloid leukemia (AML) still represents a challenge. As the growth and survival of AML cells are enhanced by the deregulation of signaling pathways such as PI3K, ERK/MAPK or mTOR, major efforts are made to develop targeted molecules against these activated pathways. The mTOR serine/threonine kinase belongs to two separate complexes, mTORC1 and mTORC2. The mTORC1 complex, defined by the interaction between mTOR and raptor, is inhibited by rapamycin in most models. It controls mRNA translation through the phosphorylation of the physiological translation repressor 4E-BP1. A constitutive activation of mTORC1 is usually detected in primary AML cells, indicating this pathway as a major target for AML therapy. However, inhibition of mTORC1 with rapamycin has only modest effects in this disease (Recher, Beyne-Rauzy et al., Blood 2005; Xu, Thompson et al.,Blood 2005). Accordingly, we recently showed that the translation process is deregulated and rapamycin resistant in AML, although specifically targeting translation with the 4E-BP1 mimetic 4EGI-1 markedly impairs AML cells survival (Tamburini, Green et al., Blood 2009). The mTORC2 complex is defined by the interaction between rictor and mTOR and is generally reported as resistant to rapamycin. An emerging role for mTORC2 has recently been rose in cancer biology, including in AML, in part through its ability to activate the oncogenic kinase Akt by phosphorylation on S473 (Zeng, Sarbassov dos et al., Blood 2007). In AML, the PI3K and mTOR activities are closely connected: inhibition of mTORC1 activates an IGF-1/IGF-1R autocrine loop that overactivates PI3K but mTORC1 activity is PI3K-independent (Tamburini, Chapuis et al., Blood 2008). These results strongly suggest a rational for simultaneous inhibition of PI3K and mTOR to enhance the killing of AML cells (Park, Chapuis et al., Leukemia 2008; Tamburini, Chapuis et al., Blood 2008). The NVP-BEZ235 compound (Novartis®) is an ATP-competitive inhibitor specific of PI3K and mTOR kinases, actually in phase I/II trial in oncology. We tested for the first time NVP-BEZ235 in 3 human AML cell lines (MV4-11, MOLM-14 and OCI-AML3) and in primary AML samples (n=10). This compound inhibited PI3K and mTOR signaling without off-target effects against other kinases usually activated in AML cells (ERK1/2 and STAT5). In contrast to rapamycin, NVP-BEZ235 dose-dependently inhibited the catalytic activity of mTOR as attested by inhibition of the mTOR autophosphorylation site on S2481. Consequently, NVP-BEZ235 fully inhibited the rapamycin-resistant phosphorylation of the translation repressor 4E-BP1 on T37/46, T70 and S65 residues. This resulted in a marked inhibition of mRNA translation in AML cells, attested by (i) an inhibition of the assembly of the translation initiating complex eIF4F, (ii) a shift from large to small polysomes in NVP-BEZ235-treated AML cells and (iii) a decreased expression of the oncogenic cap-dependant proteins Bcl-XL, c-myc and cyclinD1. Moreover, to study the effects of NVP-BEZ235 on mTORC2 activity independently of Akt, we investigated the regulation of the mTORC2 substrate paxillin (Jacinto, Loewith et al., Nat Cell biol 2004). We first confirmed that paxillin Y118 residue was a substrate for mTORC2 activity in primary AML cells as the siRNA-mediated rictor knockdown dramatically inhibited this phosphorylation event. In primary AML samples, NVP-BEZ235 inhibited paxillin Y118 phosphorylation in a dose-dependent way and this closely correlated to the inhibition of Akt S473 phosphorylation. Targeting the catalytic activity of mTOR represses therefore both mTORC1 and mTORC2 activity. Accordingly, the NVP-BEZ235 compound had marked anti-leukemic activity in AML. Indeed, it strongly reduced the proliferation of AML cells and blocked the cell-cycle as attested by a decrease of AML cells in S-phase and an accumulation in G1-phase. Finally, NVP-BEZ235 induced an important apoptotic response in AML blast cells but its effects on normal hematopoiesis were limited ex vivo. Overall, NVP-BEZ235 strongly inhibited protein synthesis and induced the killing of AML cells through the catalytic inhibition of the oncogenic PI3K and mTOR kinases. Given these results, NVP-BEZ235 may be tested urgently in clinical trials in AML. Disclosures:No relevant conflicts of interest to declare.

AKT1E17K in human solid tumours

The serine-threonine kinase AKT1 is a central player in the oncogenic pathway controlled by PI3K. Recently, a somatic mutation in AKT1 (E17K) has been detected in breast, colorectal, lung and ovarian cancers. The E17K change results in constitutive AKT1 activation and induces leukaemia in mice. We determined the occurrence of the E17K variant in a panel of 764 tumour samples. These included breast, lung, ovarian, colorectal and pancreatic carcinomas as well as melanomas and glioblastomas. Despite the fact that these tumours are known to bear alterations in genes involved in the PI3K signalling pathway, AKT1(E17K) was detected only in breast (16/273), colorectal (1/88) and lung (1/155) cancers. Within the neoplasms of breast origin, the AKT1(E17K) variant was mutually exclusive with respect to the PIK3CA(E454K or H1047R) alleles and was present only in ductal and lobular histotypes. Our results, showing that AKT1 mutations seem to occur in a tissue-specific fashion have basic and clinical implications. First, the activity of mutated AKT1 in oncogenic PI3K signalling could be strictly dependent on the cell and tissue milieu. Second, therapeutic efforts aimed at selective targeting the AKT1(E17K) variant could be effective mainly in specific cancer types.

Pharmacodynamic (PD) endpoints and cellular proliferation effects of NVP-BEZ235, a dual PI3K/mTOR inhibitor, in breast cancer cells and xenografts with wild-type (WT) and activating PI3K mutations

14508 Background: PI3K mutations are frequent in human cancer and play a role in the malignant phenotype and therapeutic resistance. NVP-BEZ235 is a small molecular mass compound in phase I clinical studies. NVP-BEZ235 reversibly blocks the ATP binding pocket on both the p110 catalytic subunit of PI3Ks and mTOR. Our aims were to study the activity and PD markers of NVP-BEZ235 in breast cancer cell lines with activating PI3K mutations. Methods: A panel of breast cancer cell lines with either WT, endogenous PI3K activating mutations or over- expressing oncogenic p110α mutants were treated with NVP-BEZ235. A xenograft breast cancer model was used to determine in vivo effects. Results: Target inhibition by NVP-BEZ235 was achieved as evidenced by a reduction in the level of the phosphorylated form of Akt, S6 and 4EBP-1, plus the nuclear re-localisation of FoxO3a. Cellular proliferation in all tested cancer cell lines was strongly inhibited in the nanomolar range as determined by the growth inhibitory 50 concentration values. NVP-BEZ235 induced G1 cell cycle arrest and apoptosis in the breast cancer cell lines tested as determined by fluorescence-activated cell sorting and the presence of cleaved PARP. There was a strong correlation between the concentrations that inhibited signalling and those that induced a growth inhibitory effect. Importantly, NVP-BEZ235 was significantly active in cell cultures harbouring oncogenic PI3K mutants and demonstrated activity in models of trastuzumab resistance. Statistically significant antitumor activity was observed upon oral administration of NVP-BEZ235 to mice bearing breast cancer xenografts at tolerated doses. PD endpoints (p-Akt, p-4EBP1 and p-S6) in tumour tissue were reduced after compound treatment. Conclusions: NVP- BEZ235 shows a rapid and profound inhibition of the PI3K pathway and results in tumor cell growth inhibition and apoptosis. Disease stasis and inhibition of PD biomarkers was observed in breast cancer xenograft models. The observed correlation between the in vivo antitumor activity and the selected PD endpoints suggests that these markers may be of assistance in the dose-finding studies in patients. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Novartis Pharma AG Novartis Pharma AG

Oncogenic PI3K and its role in cancer

The purpose of this review is to examine the contribution of the PI3K signaling pathway to the development of human tumors and to propose further studies to elucidate how to develop therapeutics for patients with mutations in this pathway. More than 30% of various solid tumor types were recently found to contain mutations in PIK3CA, the catalytic subunit of PI3K. Further analysis of key genes in this pathway identified an additional eight genes altered in tumors. These were generally found to be mutated in a mutually exclusive manner, thus increasing the mutation frequency of the pathway to 40% in colorectal cancers and emphasizing the importance of the PI3K pathway in tumorigenesis. Functional analyses of PIK3CA mutations revealed that they increase its enzymatic activity, stimulate AKT signaling, allow growth factor-independent growth as well as increasing cell invasion and metastasis. The PI3K signaling pathway is dysregulated by a variety of mechanisms in a large fraction of human tumors. Both mutational and functional analyses have shown that PIK3CA is an oncogene that plays an important role in tumor progression. Mutant members of the PI3K pathway, including PIK3CA, are good targets for therapeutic intervention because most of them are kinases, making them attractive for drug development. Gaining further insights into PIK3CA oncogenic mechanisms may produce new biomarkers and help the development of targeted therapeutics.