As the most energy- and metabolite -consuming process, protein synthesis is under the control of several intrinsic and extrinsic factors that determine its fine-tuning to the cellular microenvironment. Consequently, variations in protein synthesis rates occur under various physiological and pathological conditions, enabling an adaptive response by the cell. For example, global protein synthesis increases upon mitogenic factors to support biomass generation and cell proliferation, while exposure to low concentrations of oxygen or nutrients require translational repression and reprogramming to avoid energy depletion and cell death. To assess fluctuations in protein synthesis rates, radioactive isotopes or radiolabeled amino acids are often used. Although highly sensitive, these techniques involve the use of potentially toxic radioactive compounds and require specific materials and processes for the use and disposal of these molecules. The development of alternative, non -radioactive methods that can be easily and safely implemented in laboratories has therefore been encouraged to avoid handling radioactivity. In this context, the SUrface SEnsing of Translation (SUnSET) method, based on the classical western blot technique, was developed by Schmidt et al. in 2009. The SUnSET is nowadays recognized as a simple alternative to radioactive methods assessing protein synthesis rates.

MTOR inhibitors activate PERK signaling and favor viability of gastrointestinal neuroendocrine cell lines mTOR inhibitors activate PERK signaling and favor viability of gastrointestinal neuroendocrine cell lines

Genetic alterations in non-small cell lung cancers (NSCLC) stimulate the generation of energy and biomass to promote tumor development. However, the efficacy of the translation process is finely regulated by stress sensors, themselves often controlled by nutrient availability and chemotoxic agents. Yet, the crosstalk between therapeutic treatment and glucose availability on cell mass generation remains understudied. Herein, we investigated the impact of pemetrexed (PEM) treatment, a first-line agent for NSCLC, on protein synthesis, depending on high or low glucose availability. PEM treatment drastically repressed cell mass and translation when glucose was abundant. Surprisingly, inhibition of protein synthesis caused by low glucose levels was partially dampened upon co-treatment with PEM. Moreover, PEM counteracted the elevation of the endoplasmic reticulum stress (ERS) signal produced upon low glucose availability, providing a molecular explanation for the differential impact of the drug on translation according to glucose levels. Collectively, these data indicate that the ERS constitutes a molecular crosstalk between microenvironmental stressors, contributing to translation reprogramming and proteostasis plasticity.

Endoplasmic reticulum (ENR) stress perturbs cell homeostasis and induces the unfolded protein response (UPR). In breast cancer, this process is activated by oestrogen deprivation and is associated with tamoxifen resistance. We present evidence that the transcription factor SOX2 and the long noncoding RNA SOX2 overlapping transcript (SOX2OT) are upregulated in oestrogen receptor-positive (ER+) breast cancer and in response to oestrogen deprivation. We examined the effect of the UPR on SOX2 and SOX2OT expression and the effect of SOX2OT on UPR pathways in breast cancer cell lines. The induction of the UPR by thapsigargin or glucose deprivation upregulates SOX2OT expression. This upregulation is also shown with the anti-oestrogen 4OH-tamoxifen and mTOR inhibitor everolimus in ER + breast cancer cells that are sensitive to oestrogen deprivation or everolimus treatment. SOX2OT overexpression decreased BiP and PERK expression. This effect of SOX2OT overexpression was confirmed on BiP and PERK pathway by q-PCR. Our results show that a long noncoding RNA regulates the UPR and evince a new function of SOX2OT as a participant of ENR stress reprogramming of breast cancer cells.

E23056 Background: A number of RAS mutations confer resistance to anti-EGFR therapies routinely used in the treatment of colon cancer. The objective of this study was to evaluate the pertinence of analyzing circulating-free plasma DNA (cfDNA) as an indicator of the mutational status of a tumor, in order to use liquid biopsies instead of invasive and painful tumor biopsies during tumor progression. Methods: A cohort of 24 lung and 25 colon cancer patients was constituted in the Hospices Civils of Lyon. Liquid biopsy plasma samples were collected at diagnosis (colon cancer) and during tumor progression (lung cancer) for the purpose of the current study. KRAS and NRAS somatic alterations were quantified using three different technologies: the Droplet Digital polymerase chain reaction (ddPCR) from BioRad, the BEAMing Digital PCR from Sysmex Inostics, and the NGS NextSeq 500 by Illumina with the Accel-Amplicon 56G Oncology Panel from SWIFT BIOSCIENCES. Results: We observed a high level of sensitivity and specificity with the BEAMing technology, which provided us with excellent matches, around 96% and 73%, between solid and liquid biopsies taken at diagnosis (colon cancer) or during tumor progression (lung cancer), respectively. Indeed, when examining cfDNA from patients displaying one of the KRAS or NRAS mutations, 11 of the 13 mutations were confirmed using this technology, whereas only 5-6 matched the initial NGS status, using the two other technologies. The detection threshold was estimated at 1% for samples containing at least 0.8 ngctDNA/µL for the multiplex screening ddPCR from BioRad and for the 56G Oncology Panel from SWIFT BIOSCIENCES. The threshold was lower, at 0.03%, in samples containing only 0.25 ng ctDNA/µL for the BEAMing technology, which includes a PCR pre-amplification step. Conclusions: The advantage of the Illumina NGS technology is the larger coverage of longer gene regions, and thus the detection of more genetic mutations. Finally, the BEAMing technology enabled us to follow the appearance and disappearance of somatic alterations, with a very high level of sensitivity.