ERK implication in cell cycle regulation.

The Ras/Raf/MEK/ERK signaling cascade that integrates an extreme variety of extracellular stimuli into key biological responses controlling cell proliferation, differentiation or death is one of the most studied intracellular pathways. Here we present some evidences that have been accumulated over the last 15 years proving the requirement of ERK in the control of cell proliferation. In this review we focus (i) on the spatio-temporal control of ERK signaling, (ii) on the key cellular components linking extracellular signals to the induction and activation of cell cycle events controlling G1 to S-phase transition and (iii) on the role of ERK in the growth factor-independent G2/M phase of the cell cycle. As ERK pathway is often co-activated with the PI3 kinase signaling, we highlight some of the key points of convergence leading to a full activation of mTOR via ERK and AKT synergies. Finally, ERK and AKT targets being constitutively activated in so many human cancers, we briefly touched the cure issue of using more specific drugs in rationally selected cancer patients.

Genetic Disruption of the Multifunctional CD98/LAT1 Complex Demonstrates the Key Role of Essential Amino Acid Transport in the Control of mTORC1 and Tumor Growth.

The CD98/LAT1 complex is overexpressed in aggressive human cancers and is thereby described as a potential therapeutic target. This complex promotes tumorigenesis with CD98 (4F2hc) engaging ß-integrin signaling while LAT1 (SLC7A5) imports essential amino acids (EAA) and promotes mTORC1 activity. However, it is unclear as to which member of the heterodimer carries the most prevalent protumoral action. To answer this question, we explored the tumoral potential of each member by gene disruption of CD98, LAT1, or both and by inhibition of LAT1 with the selective inhibitor (JPH203) in six human cancer cell lines from colon, lung, and kidney. Each knockout respectively ablated 90% (CD98 KO: ) and 100% (LAT1 KO: ) of Na(+)-independent leucine transport activity. LAT1 KO: or JPH203-treated cells presented an amino acid stress response with ATF4, GCN2 activation, mTORC1 inhibition, and severe in vitro and in vivo tumor growth arrest. We show that this severe growth phenotype is independent of the level of expression of CD98 in the six tumor cell lines. Surprisingly, CD98 KO: cells with only 10% EAA transport activity displayed a normal growth phenotype, with mTORC1 activity and tumor growth rate undistinguishable from wild-type cells. However, CD98 KO: cells became extremely sensitive to inhibition or genetic disruption of LAT1 (CD98 KO: /LAT1 KO: ). This finding demonstrates that the tumoral potential of CD98 KO: cells is due to residual LAT1 transport activity. Therefore, these findings clearly establish that LAT1 transport activity is the key growth-limiting step of the heterodimer and advocate the pharmacology development of LAT1 transporter inhibitors as a very promising anticancer target. Cancer Res; 76(15); 4481-92. ©2016 AACR.

Total ERK1/2 activity regulates cell proliferation.

Regulating ERK activity is essential for normal cell proliferation to occur. In mammals and most vertebrates ERK activity is provided by ERK1 and ERK2 that are highly similar, ubiquitously expressed and share activators and substrates. By combining single and double silencings of ERK1 and ERK2 we recently demonstrated that the apparent dominant role of ERK2 to regulate cell proliferation was due to its markedly higher expression level than ERK1. The contribution of ERK1 was revealed when ERK2 activation was clamped to avoid compensating over-activation of ERK2. We found no evidences in the literature for insulated isoform-specific modules in the Ras/Raf/MEK signaling cascade that could activate specifically ERK1 or ERK2. Obviously in frogs all signal integration and fine modulation provided by three Ras and three Raf isoforms is conducted by only one MEK and one ERK isoform. In mammals, ERK1 and ERK2 display similar specific activities and are activated respectively to their expression levels. After integrating signals from Ras, Raf and MEK isoforms, ERK1 and ERK2 regulate positively cell proliferation according to their expression levels.

Single and combined silencing of ERK1 and ERK2 reveals their positive contribution to growth signaling depending on their expression levels.

The proteins ERK1 and ERK2 are highly similar, are ubiquitously expressed, and share activators and substrates; however, erk2 gene invalidation is lethal in mice, while erk1 inactivation is not. We ablated ERK1 and/or ERK2 by RNA interference and explored their relative roles in cell proliferation and immediate-early gene (IEG) expression. Reducing expression of either ERK1 or ERK2 lowered IEG induction by serum; however, silencing of only ERK2 slowed down cell proliferation. When both isoforms were silenced simultaneously, compensating activation of the residual pool of ERK1/2 masked a more deleterious effect on cell proliferation. It was only when ERK2 activation was clamped at a limiting level that we demonstrated the positive contribution of ERK1 to cell proliferation. We then established that ERK isoforms are activated indiscriminately and that their expression ratio correlated exactly with their activation ratio. Furthermore, we determined for the first time that ERK1 and ERK2 kinase activities are indistinguishable in vitro and that erk gene dosage is essential for survival of mice. We propose that the expression levels of ERK1 and ERK2 drive their apparent biological differences. Indeed, ERK1 is dispensable in some vertebrates, since it is absent from chicken and frog genomes despite being present in all mammals and fishes sequenced so far.

TRB3 inhibits the transcriptional activation of stress-regulated genes by a negative feedback on the ATF4 pathway.

The integrated stress response (ISR) is defined as a highly conserved response to several stresses that converge to the induction of the activating transcription factor 4 (ATF4). Because an uncontrolled response may have deleterious effects, cells have elaborated several negative feedback loops that attenuate the ISR. In the present study, we describe how induction of the human homolog of Drosophila tribbles (TRB3) attenuates the ISR by a negative feedback mechanism. To investigate the role of TRB3 in the control of the ISR, we used the regulation of gene expression by amino acid limitation as a model. The enhanced production of ATF4 upon amino acid starvation results in the induction of a large number of target genes like CHOP (CAAT/enhancer-binding protein-homologous protein), asparagine synthetase (ASNS), or TRB3. We demonstrate that TRB3 overexpression inhibits the transcriptional induction of CHOP and ASNS whereas TRB3 silencing induces the expression of these genes both under normal and stressed conditions. In addition, transcriptional profiling experiments show that TRB3 affects the expression of many ISR-regulated genes. Our results also suggest that TRB3 and ATF4 belong to the same protein complex bound to the sequence involved in the ATF4-dependent regulation of gene expression by amino acid limitation. Collectively, our data identify TRB3 as a negative feedback regulator of the ATF4-dependent transcription and participates to the fine regulation of the ISR.