Deregulation of the phosphatase, PP2A is a common event in breast cancer, predicting sensitivity to FTY720

We would like to thank Professor Andrew Hanby, Professor Valerie Speirs and Dr Thomas A Hughes for the breast cancer cell lines used in this study. This research was supported by the Faculty of Medicine and Surgery, University of Malta.Background: The 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. Methods: The 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 conclusion: The 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.peer-reviewe

Targeting mTOR and prenylation

Protein prenylation is a post-translational addition of a lipophilic farnesyl or geranylgeranyl moiety derived from the pyrophosphate substrates, intermediates of the mevalonate pathway. Prenylation of Ras-related small GTP-binding proteins and heterotrimeric G proteins constitute protein activation events associated with cellular proliferation. The monoterpenes limonene and perillyl alcohol inhibit protein isoprenylation resulting in cell cycle arrest and induction of apoptosis. Clinical trials of d-limonene and perillyl alcohol resulted in dose limiting toxicity. Reducing the dosage of isoprenoids, while maintaining the antiproliferative effect is a challenge. Interestingly, the limiting factor of the mevalonate pathway, HMG CoA reductase, is controlled at translation level and hence sensitive to mTOR activity. The purpose of this study was to investigate the dose response using a combinatory treatment of isoprenoids and rapamycin (mTOR inhibitor), on various solid tumour cell line models. 3 solid tumour cell lines, namely PC3 (prostate) C32 (melanoma) and A549 (lung), were chosen on the basis of sensitivity to the mTOR inhibitor, rapamycin. Cytotoxicity assays using XTT were performed on the cell lines treated with Limonene, Perillyl alcohol and α-Pinene. In this study XTT assays were used to quantify viable cells after treatment with Isoprenoids and Rapamycin alone or in combination. Dosages at IC50s were used to measure apoptosis by Annexin V staining and flow cytometry. The C32 and PC3 cell lines were sensitive to rapamycin treatment, resulting in a decrease in cell viability by more than 20%, but retaining a constant growth curve thereafter. A549 was not sensitive to rapamycin at all concentrations tested. For all combinatory treatments, 50ng/ml rapamycin was selected. The viability of PC3 and C32 cell lines decreased significantly by the combinatory effect of 50ng/ml rapamycin and isoprenoids. Although cell death was enhanced after sensitisation with rapamycin, other mechanisms of loss of cellular viability, other than apoptosis, have a major role. Our results show a statistically significant reduction in IC50 of various isoprenoids, following pre-sensitization with 50ng/ml of rapamycin in the prostate cell line (PC3) and the melanoma cell line (C32). Hence this novel combination of drugs targeting two mechanisms that converge on a common target, provide a higher efficacy compared to using either drug on its own. This merits further investigation to characterise the mechanism/s of viability suppression in the solid tumour cellular models.peer-reviewe

Acute Effects on the Human Peripheral Blood Transcriptome of Decompression Sickness Secondary to Scuba Diving

Decompression sickness (DCS) develops due to inert gas bubble formation in bodily tissues and in the circulation, leading to a wide range of potentially serious clinical manifestations. Its pathophysiology remains incompletely understood. In this study, we aim to explore changes in the human leukocyte transcriptome in divers with DCS compared to closely matched unaffected controls after uneventful diving. Cases (n = 7) were divers developing the typical cutis marmorata rash after diving with a confirmed clinical diagnosis of DCS. Controls (n = 6) were healthy divers who surfaced from a ≥25 msw dive without decompression violation or evidence of DCS. Blood was sampled at two separate time points—within 8 h of dive completion and 40–44 h later. Transcriptome analysis by RNA-Sequencing followed by bioinformatic analysis was carried out to identify differentially expressed genes and relate their function to biological pathways. In DCS cases, we identified enrichment of transcripts involved in acute inflammation, activation of innate immunity and free radical scavenging pathways, with specific upregulation of transcripts related to neutrophil function and degranulation. DCS-induced transcriptomic events were reversed at the second time point following exposure to hyperbaric oxygen. The observed changes are consistent with findings from animal models of DCS and highlight a continuum between the responses elicited by uneventful diving and diving complicated by DCS. This study sheds light on the inflammatory pathophysiology of DCS and the associated immune response. Such data may potentially be valuable in the search for novel treatments targeting this disease

Differential expression of breast cancer signature genes following rapamycin treatment

Introduction: Breast cancer is classified into intrinsic molecular subtypes, each relating to predictive prognostic and clinical outcomes. Rapamycin inhibits the mammalian target of rapamycin (mTOR) pathway, which is often deregulated in various types of cancer. mTOR inhibitors are associated with antiproliferation and apoptosis. Aim: Investigating the differential expression of breast cancer signature genes following rapamycin treatment in various breast cancer subtypes.peer-reviewe

Molecular classifiers of breast cancer patients using multiplex assays

Introduction: Breast cancer patients can be classified using receptor status or based on expression of specific signature genes. Classification of patients provides the basis to select specific targeted therapy and to identify new molecular subytpes with potential therapeutic options. Data from the cBioPortal for cancer genomics demonstrate that PP2A function is likely to be reduced in up to 60% of basal breast tumours. Tumours exhibit either homozygous deletion or underexpression of PP2A, but also overexpression of PP2A inhibitors. In this study we classify molecularly breast cancer cell lines and a cohort of Maltese patients. In addition, we assessed the effect of PP2A activity restoration on the cellular models.peer-reviewe

Acute effects on the human peripheral blood transcriptome of decompression sickness secondary to scuba diving

Decompression sickness (DCS) develops due to inert gas bubble formation in bodily tissues and in the circulation, leading to a wide range of potentially serious clinical manifestations. Its pathophysiology remains incompletely understood. In this study, we aim to explore changes in the human leukocyte transcriptome in divers with DCS compared to closely matched unaffected controls after uneventful diving. Cases (n = 7) were divers developing the typical cutis marmorata rash after diving with a confirmed clinical diagnosis of DCS. Controls (n = 6) were healthy divers who surfaced from a ≥25 msw dive without decompression violation or evidence of DCS. Blood was sampled at two separate time points—within 8 h of dive completion and 40–44 h later. Transcriptome analysis by RNA-Sequencing followed by bioinformatic analysis was carried out to identify differentially expressed genes and relate their function to biological pathways. In DCS cases, we identified enrichment of transcripts involved in acute inflammation, activation of innate immunity and free radical scavenging pathways, with specific upregulation of transcripts related to neutrophil function and degranulation. DCS-induced transcriptomic events were reversed at the second time point following exposure to hyperbaric oxygen. The observed changes are consistent with findings from animal models of DCS and highlight a continuum between the responses elicited by uneventful diving and diving complicated by DCS. This study sheds light on the inflammatory pathophysiology of DCS and the associated immune response. Such data may potentially be valuable in the search for novel treatments targeting this disease