The complexity of the phosphatase, PP2A, is being unravelled and current research is increasingly providing information on the association of deregulated PP2A function with cancer initiation and progression. It has been reported that decreased activity of PP2A is a recurrent observation in many types of cancer, including colorectal and breast cancer (Baldacchino et al. EPMA J. 5:3, 2014; Cristobal et al. Mol Cancer Ther. 13:938-947, 2014). Since deregulation of PP2A and its regulatory subunits is a common event in cancer, PP2A is a potential target for therapy (Baldacchino et al. EPMA J. 5:3, 2014). In this review, the structural components of the PP2A complex are described, giving an in depth overview of the diversity of regulatory subunits. Regulation of the active PP2A trimeric complex, through phosphorylation and methylation, can be targeted using known compounds, to reactivate the complex. The endogenous inhibitors of the PP2A complex are highly deregulated in cancer, representing cases that are eligible to PP2A-activating drugs. Pharmacological opportunities to target low PP2A activity are available and preclinical data support the efficacy of these drugs, but clinical trials are lacking. We highlight the importance of PP2A deregulation in cancer and the current trends in targeting the phosphatase.
BackgroundThe most commonly used biomarkers to predict the response of breast cancer patients to therapy are the oestrogen receptor (ER), progesterone receptor (PgR), and human epidermal growth factor receptor 2 (HER2). Patients positive for these biomarkers are eligible for specific therapies such as endocrine treatment in the event of ER and PgR positivity, and the monoclonal antibody, trastuzumab, in the case of HER2-positive patients. Patients who are negative for these three biomarkers, the so-called triple negatives, however, derive little benefit from such therapies and are associated with a worse prognosis. Deregulation of the protein serine/threonine phosphatase type 2A (PP2A) and its regulatory subunits is a common event in breast cancer, providing a possible target for therapy.MethodsThe data portal, cBioPortal for Cancer Genomics was used to investigate the incidence of conditions that are associated with low phosphatase activity. Four (4) adherent human breast cancer cell lines, MDA-MB-468, MDA-MB-436, Hs578T and BT-20 were cultured to assess their viability when exposed to various dosages of rapamycin or FTY720. In addition, RNA was extracted and cDNA was synthesised to amplify the coding sequence of PPP2CA. Amplification was followed by high-resolution melting to identify variations.Results and conclusionThe sequence of PPP2CA was found to be conserved across a diverse panel of solid tumour and haematological cell lines, suggesting that low expression of PPP2CA and differential binding of inhibitory PPP2CA regulators are the main mechanisms of PP2A deregulation. Interestingly, the cBioPortal for Cancer Genomics shows that PP2A is deregulated in 59.6% of basal breast tumours. Viability assays performed to determine the sensitivity of a panel of breast cancer cell lines to FTY720, a PP2A activator, indicated that cell lines associated with ER loss are sensitive to lower doses of FTY720. The subset of patients with suppressed PP2A activity is potentially eligible for treatment using therapies which target the PI3K/AKT/mTOR pathway, such as phosphatase activators.
Introduction: At a global level breast cancer is the second most common cancer, and the most frequent in women. Statins are 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase) inhibitors. HMG-CoA reductase is a rate-limiting enzyme in the mevalonate pathway, making it important in the pathology of cancer. The serine/threonine kinase, mammalian target of rapamycin (mTOR) regulates cell survival, proliferation and growth via translational initiation control, and this pathway is deregulated in many types of cancer which are associated with phosphatidylinositol-3-kinase (PI3K)/Akt signaling activation. HMG-CoA reductase expression is dependent on eukaryotic translation initiation factor 4E (eIF4E), and requires high translation initiation efficiency for polysome recruitment. eIF4E availability is reduced by mTOR inhibitors such as rapamycin and metformin, leading to a reduction in HMG-CoA reductase expression. Objectives: This research aims to study the proposed combinatory treatments of rapamycin or metformin with HMG-CoA reductase inhibitors, in order to identify breast cancer subtypes sensitive to the proposed drug combinations. The specific mechanisms of action will also be studied. Methodology: The breast cancer cell lines of Hs587T, MCF-7 and MDA-MB-468 were seeded in 96-well plates. Following 24 hours, triplicate wells of each cell line were treated with 0, 2, 4, 6, 8, 10, 12, and 14mM metformin. MTT assays were carried out 24, 48, and 72 hours post treatment. Successively, similar procedures for each cell line were carried out using 0, 10, 25, 35, 50, 65, 75, 100ng/mL rapamycin. For every cell line the time point and the dose at which a suppression of cell viability (not reaching IC50) occurred for 3 consecutive doses, were defined as the sensitization time point (Ts) and the sensitization dose (Ds) respectively. Ongoing research: The same cell lines are currently being pre-treated with Ds of metformin and rapamycin separately, and at time Ts simvastatin is added at a range of µM concentrations. Following these conditions MTT assays are being carried out, and compared to control experiments using simvastatin alone. Annexin V FITC / propidium iodide flow cytometry based apoptosis assays, as well as Western Blot analysis for the quantification of HDJ2, HMG-CoA reductase, and eukaryotic translation initiation factor 4E binding protein 1 proteins are being planned to be carried out at a later stage. Results: Time Ts and dose Ds for MCF-7 cells treated with rapamycin were found to be 72 hours and 12.5ng/mL respectively. MCF-7 cells were found to exhibit sensitivity to metformin that was too high to be considered for further combinatory treatment with simvastatin, since IC50 was reached post 48 hours of treatment with 10mM. Substantial sensitivity of all breast cancer cell lines studied was also observed for rapamycin or metformin treatment alone. In addition, previous work published by our group shows that the breast cancer cell lines MDA-MB-436 and BT-20 are sensitive to rapamycin. Conclusion: Cell viability assays have shown that response to mTOR modulators is time, cell type and dose dependent. This project will determine optimum conditions for the combinatory treatment described above. The targeting of these two specific mechanisms converging onto a common target is novel, and expected to result in a higher efficacy, as well as decreased unwanted effects in an in vivo situation due to the lower individual doses used. Drug combinations would thus be superior to using either drug alone. Citation Format: Vanessa Petroni, Marie Therese Camilleri Podesta, Anthony George Fenech, Godfrey Grech. Identification of novel drug combinations to target molecular pathways involved in breast cancer. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr B22.
IntroductionInterferon-gamma (IFN-γ) release assay is used to identify patients with latent tuberculosis prior to biological treatment initiation. A lack of stimulation capacity of lymphocytes by the test control results in an indeterminate result. This is not uncommon in inflammatory bowel disease (IBD) patients receiving immunosuppressive agents.1,2,3 The aim of this study is to compare the clinical and laboratory characteristics of patients with indeterminate results to those with determinate results.MethodsThe national database of IBD patients receiving biological agents was accessed. IFN-γ release assay (QuantiFERON-TB Gold) results were noted. Parameters at the time of testing were collected, namely age, diagnosis, disease activity, blood results and drug treatment. Comparisons between the indeterminate group and determinate group were made using IBM® SPSS® Statistics Version 20. The study was approved by the University and Research Ethics Committee of Malta.ResultsData from 136 subjects was analysed. 8.8% (n = 12) had an indeterminate IFN-γ release assay result, 2.2% (n = 3) had a positive result, while 89% (n = 121) had a negative result. The indeterminate group had a significantly lower lymphocyte count (p = 0.041), globulin level (p = 0.042), immunoglobulin A (IgA) (p = 0.031) and haemoglobin (p = 0.016). C-reactive protein was higher in the indeterminate group (p = 0.024). No significant differences were recorded for age, gender, diagnosis, disease activity, white cell count, neutrophils, platelets, renal/liver function, albumin levels, IgM, IgG, or drug treatment. Critically, only 25% of indeterminate result subjects had a repeat test, all reported as negative.ConclusionIndeterminate results are common in IBD patients, affecting nearly 1 in 10 patients. Patients with low lymphocyte counts, low globulin, low haemoglobin, low IgA and high CRP levels are more likely to have an indeterminate result when tested. The potential of a false negative result is likely to be higher in these patients. One must exert caution when requesting IFN-γ release assays in such IBD patients since the probability of a determinate result may be low. We advise test repetition in all patients with indeterminate results and screening with chest x-ray.References1 Helwig U, Müller M, Hedderich J, et al, Corticosteroids and immunosuppressive therapy influence the result of QuantiFERON TB Gold testing in inflammatory bowel disease patients. J Crohns and Colitis 2012;6:419–424.2 Jeong SJ, Han SH, Kim CO, et al, Predictive factors for indeterminate result on the QuantiFERON test in an intermediate tuberculosis-burden country. J Infect. 2011;62:347–354.3 Kobashi Y, Sugiu T, Mouri Y, et al, Indeterminate results of QuantiFERON TB-2G test performed in routine clinical practice. Eur Respir J 2009;33:812–815.Disclosure of InterestNone Declared
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