Differential Enhancement of Breast Cancer Cell Motility and Metastasis by Helical and Kinase Domain Mutations of Class IA Phosphoinositide 3-Kinase

Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Cancer Research (Impact Factor: 9.33). 11/2009; 69(23):8868-76. DOI: 10.1158/0008-5472.CAN-09-1968
Source: PubMed


Class IA (p85/p110) phosphoinositide 3-kinases play a major role in regulating cell growth, survival, and motility. Activating mutations in the p110alpha isoform of the class IA catalytic subunit (PIK3CA) are commonly found in human cancers. These mutations lead to increased proliferation and transformation in cultured cells, but their effects on cell motility and tumor metastasis have not been evaluated. We used lentiviral-mediated gene transfer and knockdown to produce stable MDA-MB-231 cells in which the endogenous human p110alpha is replaced with either wild-type bovine p110alpha or the two most common activating p110alpha mutants, the helical domain mutant E545K and the kinase domain mutant H1047R. The phosphoinositide 3-kinase/Akt pathway was hyperactivated in cells expressing physiologic levels of helical or kinase domain mutants. Cells expressing either mutant showed increased motility in vitro, but only cells expressing the helical domain mutant showed increased directionality in a chemotaxis assay. In severe combined immunodeficient mice, xenograft tumors expressing either mutant showed increased rates of tumor growth compared with tumors expressing wild-type p110alpha. However, tumors expressing the p110alpha helical domain mutant showed a marked increase in both tumor cell intravasation into the blood and tumor cell extravasation into the lung after tail vein injection compared with tumors expressing wild-type p110alpha or the kinase domain mutant. Our observations suggest that, when compared with kinase domain mutations in a genetically identical background, expression of helical domain mutants of p110alpha produce a more severe metastatic phenotype.

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Available from: Sumanta Goswami, Feb 21, 2014
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    • "Consistently, they further demonstrated that the p110a helical domain mutants require the RBD for their transformation activities, whereas the oncogenic activity of the H1047R mutant depends on the ABD domain. (2) Pang et al. (2009) FIGURE 3.—Structures of PI3Ks. PI3Ks are classified into three groups: class I, class II, and class III. "

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    • "One million C33A cells were plated in a 35 mm tissue culture dish and grown to confluence. Two parallel scratches were made with a 200 µL pipette tip per dish and the scratch width was measured to be the baseline [12]. The wound width was measured at a minimum of six different points for each wound. "
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    ABSTRACT: PI3K/AKT pathway alterations are associated with incomplete response to chemoradiation in human cervical cancer. This study was performed to test for mutations in the PI3K pathway and to evaluate the effects of AKT inhibitors on glucose uptake and cell viability. Mutational analysis of DNA from 140 pretreatment tumor biopsies and 8 human cervical cancer cell lines was performed. C33A cells (PIK3CAR88Q and PTENR233*) were treated with increasing concentrations of two allosteric AKT inhibitors (SC-66 and MK-2206) with or without the glucose analogue 2-deoxyglucose (2-DG). Cell viability and activation status of the AKT/mTOR pathway were determined in response to the treatment. Glucose uptake was evaluated by incubation with 18F-fluorodeoxyglucose (FDG). Cell migration was assessed by scratch assay. Activating PIK3CA (E545K, E542K) and inactivating PTEN (R233*) mutations were identified in human cervical cancer. SC-66 effectively inhibited AKT, mTOR and mTOR substrates in C33A cells. SC-66 inhibited glucose uptake via reduced delivery of Glut1 and Glut4 to the cell membrane. SC-66 (1 µg/ml-56%) and MK-2206 (30 µM-49%) treatment decreased cell viability through a non-apoptotic mechanism. Decreases in cell viability were enhanced when AKT inhibitors were combined with 2-DG. The scratch assay showed a substantial reduction in cell migration upon SC-66 treatment. The mutational spectrum of the PI3K/AKT pathway in cervical cancer is complex. AKT inhibitors effectively block mTORC1/2, decrease glucose uptake, glycolysis, and decrease cell viability in vitro. These results suggest that AKT inhibitors may improve response to chemoradiation in cervical cancer.
    Full-text · Article · Apr 2014 · PLoS ONE
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    • "The helical domain mutants require RAS binding for transformation and are independent of p85, whereas the H1047R mutant depends on p85 binding [27,28]. In another study, helical domain mutants produced a more aggressive phenotype than kinase domain mutants with regard to cellular motility and enhanced extravasation [29]. This study, however, used the MDA-MB-231 breast cancer cell line, which is known to be RAS mutant and ER-negative, so it is conceivable that the helical domain mutant could have synergized with these features. "
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    ABSTRACT: PIK3CA mutations represent one of the most common genetic aberrations in breast cancer. They have been reported to be present in over one-third of cases, with enrichment in the luminal and in human epidermal growth factor receptor 2-positive subtypes. Substantial preclinical data on the oncogenic properties of these mutations have been reported. However, whilst the preclinical data have clearly shown an association with robust activation of the pathway and resistance to common therapies used in breast cancer, the clinical data reported up to now do not support that the PIK3CA mutated genotype is associated with high levels of pathway activation or with a poor prognosis. We speculate that this may be due to the minimal use of transgenic mice models thus far. In this review, we discuss both the preclinical and clinical data associated with PIK3CA mutations and their potential implications. Prospective clinical trials stratifying by PIK3CA genotype will be necessary to determine if the mutation also predicts for increased sensitivity to agents targeting the phosphoinositide 3-kinase pathway.
    Full-text · Article · Jan 2014 · Breast Cancer Research
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