Endometriosis is a well-known risk factor for ovarian cancer. The genetic changes that characterise endometriosis are poorly understood; however, the mechanistic target of rapamycin (mTOR) pathway is involved. In this study, we investigated the expression of key mTOR components in endometriosis and the effects of rapalogues using an endometrioid ovarian carcinoma cell line (MDAH 2774) as an in vitro model. Gene expression of mTOR, DEPTOR, Rictor and Raptor was assessed by qPCR in 24 endometriosis patients and in silico in ovarian cancer patients. Furthermore, the effects of Rapamycin, Everolimus, Deforolimus, Temsirolimus, Resveratrol, and BEZ235 (Dactolisib, a dual kinase inhibitor) on mTOR signalling components was assessed. mTOR showed a significant increase in the expression in endometriosis and ovarian endometrioid adenocarcinoma patients compared to non-affected controls. DEPTOR, an inhibitor of mTOR, was downregulated in the advanced stages of ovarian cancer (III and IV) compared to earlier stages (I and II). Treatment of MDAH-2774 cells with the mTOR inhibitors resulted in the significant upregulation of DEPTOR mRNA, whereas treatment with rapamycin and BEZ-235 (100 nM) resulted in downregulation of the mTOR protein expression after 48 h of treatment. None of the treatments resulted in translocation of mTOR from cytoplasm to nucleus. Upregulation of DEPTOR is a positive prognostic marker in ovarian cancer and is increased in response to mTOR pathway inhibition suggesting that it functions as a tumour suppressor gene in endometrioid ovarian carcinoma. Collectively, our data suggest the mTOR pathway as a potential connection between endometriosis and ovarian cancer and may be a potential target in the treatment of both conditions.

The mammalian or mechanistic target of rapamycin (mTOR) is a Ser/Thr protein kinase that, in response to nutrient stimulation, regulates cellular growth, proliferation, survival, protein synthesis and gene transcription. It has also been implicated in Alzheimer’s disease (AD) with neuronal cells and hippocampal slices of AD transgenic mice experiencing dysregulated mTOR and synaptic plasticity in response to treatment with the toxic amyloid β (Aβ1–42) peptide, which has been implicated in AD. DEP domain-containing mTOR-interacting protein (DEPTOR) is a protein which can bind to mTOR and cause its inhibition, and functions as a regulatory protein of mTOR to control its activity. The inhibition of mTOR has been shown to have a neuroprotective effect; in an animal model, it was shown to protect against Aβ-induced neurotoxicity. In the present study, to investigate to role of DEPTOR in a model of AD, we neuronally differentiated the SH-SY5Y cell line and examined the effects of treatment with an Aβ42 peptide, thus mimicking plaque formation. This resulted in a significant increase in mTOR and a significant decrease in DEPTOR expression compared to the unstimulated controls. Moreover, to the best of our knowledge, we demonstrate for the first time a reduction in the protein level of DEPTOR in the precentral gyrus, postcentral gyrus and occipital lobe of a brain with AD compared to a normal control, as well as a significant reduction in DEPTOR expression in samples from late-onset AD (LOAD) compared to early-onset familial AD (EOFAD). The reduction in DEPTOR expression in cases of AD compared to healthy controls can lead to an augmentation of mTOR signalling, leading to Aβ accumulation, which in turn leads to a further reduction in DEPTOR expression. This results in the accumulation of amyloid plaque, shifting the balance from neuroprotection to neurodegeneration.

Signaling via the B-cell receptor (BCR) is a key driver and therapeutic target in chronic lymphocytic leukemia (CLL). BCR stimulation of CLL cells induces expression of eIF4A, an initiation factor important for translation of multiple oncoproteins, and reduces expression of PDCD4, a natural inhibitor of eIF4A, suggesting that eIF4A may be a critical nexus controlling protein expression downstream of the BCR in these cells. We, therefore, investigated the effect of eIF4A inhibitors (eIF4Ai) on BCR-induced responses. We demonstrated that eIF4Ai (silvestrol and rocaglamide A) reduced anti-IgM-induced global mRNA translation in CLL cells and also inhibited accumulation of MYC and MCL1, key drivers of proliferation and survival, respectively, without effects on upstream signaling responses (ERK1/2 and AKT phosphorylation). Analysis of normal naïve and non-switched memory B cells, likely counterparts of the two main subsets of CLL, demonstrated that basal RNA translation was higher in memory B cells, but was similarly increased and susceptible to eIF4Ai-mediated inhibition in both. We probed the fate of MYC mRNA in eIF4Ai-treated CLL cells and found that eIF4Ai caused a profound accumulation of MYC mRNA in anti-IgM treated cells. This was mediated by MYC mRNA stabilization and was not observed for MCL1 mRNA. Following drug wash-out, MYC mRNA levels declined but without substantial MYC protein accumulation, indicating that stabilized MYC mRNA remained blocked from translation. In conclusion, BCR-induced regulation of eIF4A may be a critical signal-dependent nexus for therapeutic attack in CLL and other B-cell malignancies, especially those dependent on MYC and/or MCL1.

Quantitative polymerase chain reaction (qPCR) is a method of amplifying and detecting small samples of genetic material in real time and is in routine use across many laboratories. Speed and thermal uniformity, two important factors in a qPCR test, are in direct conflict with one another in conventional peltier-driven thermal cyclers. To overcome this, companies are developing novel thermal systems for qPCR testing. More recently, qPCR technology has developed to enable its use in point-of-care testing (POCT), where the test is administered and results are obtained in a single visit to a health provider, particularly in developing countries. For a system to be suitable for POCT it must be rapid and reliable. In the present study, the speed and thermal uniformity of four qPCR thermal cyclers currently available were compared, two of which use the conventional peltier/block heating method and two of which use novel heating and cooling methods. The time required to complete 40 cycles varied between 12 and 58 min, and the Ct values were comparable, ranging between 13.6 and 16.8. Therefore, the novel technologies investigated in the present study for qPCR instrumentation performed equally well compared with conventional qPCR instruments, in terms of amplification efficiency and thermal uniformity.

Ovarian cancer is the second most common gynaecological malignancy and was diagnosed in over 7,000 women in 2011 in the UK. There are currently no reliable biomarkers available for use in a regular screening assay for ovarian cancer and due to characteristic late presentation (78% in stages III and IV) ovarian cancer has a low survival rate (35% after 10 years). The mTOR pathway is a central regulator of growth, proliferation, apoptosis and angiogenesis; providing balance between available resources such as amino acids and growth factors, and stresses such as hypoxia, to control cellular behaviour accordingly. Emerging data links mTOR with the aetiopathogenesis of ovarian cancer. We hypothesised that mTOR inhibitors could play a therapeutic role in ovarian cancer treatment. In this study we began by validating the expression of four main mTOR pathway components, mTOR, DEPTOR, rictor and raptor, at gene and protein level in in vitro models of endometrioid (MDAH-2774) and clear cell (SKOV3) ovarian cancer using qPCR and ImageStream technology. Using a wound healing assay we show that inhibition of the mTOR pathway using rapamycin, rapalogues, resveratrol and NVP BEZ-235 induces a cytostatic and not cytotoxic response up to 18 h in these cell lines. We extended these findings up to 72 h with a proliferation assay and show that the effects of inhibition of the mTOR pathway are primarily mediated by the dephosphorylation of p70S6 kinase. We show that mTOR inhibition does not involve alteration of mTOR pathway components or induce caspase 9 cleavage. Preclinical studies including ovarian tissue of ovarian cancer patients, unaffected controls and patients with unrelated gynaecological conditions show that DEPTOR is reliably upregulated in ovarian cancer.

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