Low ERK Activity Research Articles

KRAS(G12D) drives lepidic adenocarcinoma through stem-cell reprogramming.

Many cancers originate from stem or progenitor cells hijacked by somatic mutations that drive replication, exemplified by adenomatous transformation of pulmonary alveolarepithelial type II (AT2) cells1. Here we demonstrate a different scenario: expression of KRAS(G12D) in differentiated AT1 cells reprograms them slowly and asynchronously back into AT2 stem cells that go on to generate indolent tumours. Like human lepidic adenocarcinoma, the tumour cells slowly spread along alveolar walls in a non-destructive manner and have low ERK activity. We find that AT1 and AT2 cells act as distinct cells of origin and manifest divergent responses to concomitant WNT activation and KRAS(G12D) induction, which accelerates AT2-derived but inhibits AT1-derived adenoma proliferation. Augmentation of ERK activity in KRAS(G12D)-induced AT1 cells increases transformation efficiency, proliferation and progression from lepidic to mixed tumour histology. Overall, we have identified a new cell of origin for lung adenocarcinoma, the AT1 cell, which recapitulates features of human lepidic cancer. In so doing, we also uncover a capacity for oncogenic KRAS to reprogram a differentiated and quiescent cell back into its parent stem cell en route to adenomatous transformation. Our work further reveals that irrespective of a given cancer's current molecular profile and driver oncogene, the cell of origin exerts a pervasive and perduring influence on its subsequent behaviour.

Inherent low Erk and p38 activity reduce Fas Ligand expression and degranulation in T helper 17 cells leading to activation induced cell death resistance.

Activation Induced Cell Death of T helper cells is central to maintaining immune homeostasis and a perturbation often manifests in aberrant T helper cells that is associated with immunopathologies. Significant presence of T cells positive for IL-17A (Th17) and dual positive for IFN-γ/IL-17A (Th1/Th17) in both effector (CD45RA+RO+) and memory (CD45RA−RO+) compartments with differential FasL protein in RA peripheral blood suggested their differential TCR AICD sensitivity. Lowered active caspase-3 in Th17 and Th1/Th17 over Th1 cells confirmed their capability to resist AICD and pointed to early upstream events. Differential MAPK activities, FasL protein and downstream caspase-3 activities in murine Th1 and Th17 cells established distinct TCR mediated signaling pathways and suggested low Erk and p38 activity as pivotal for AICD sensitivity. We extrapolated our mouse and human data and report that Fas-FasL is the preferred death pathway for both Th1 and Th17 and that inherently low Erk2 activity protected Th17 cells from TCR AICD. The presence of significantly higher numbers of aberrant T helper cells in RA also suggest an inflammatory cytokine milieu and AICD insensitive T cell link to sustained inflammation. Re sensitization to apoptosis by targeting MAPK activity especially Erk2 in RA might be of therapeutic value.

Isoproterenol acts as a biased agonist of the alpha-1A-adrenoceptor that selectively activates the MAPK/ERK pathway.

The α1A-AR is thought to couple predominantly to the Gαq/PLC pathway and lead to phosphoinositide hydrolysis and calcium mobilization, although certain agonists acting at this receptor have been reported to trigger activation of arachidonic acid formation and MAPK pathways. For several G protein-coupled receptors (GPCRs) agonists can manifest a bias for activation of particular effector signaling output, i.e. not all agonists of a given GPCR generate responses through utilization of the same signaling cascade(s). Previous work with Gαq coupling-defective variants of α1A-AR, as well as a combination of Ca2+ channel blockers, uncovered cross-talk between α1A-AR and β2-AR that leads to potentiation of a Gαq-independent signaling cascade in response to α1A-AR activation. We hypothesized that molecules exist that act as biased agonists to selectively activate this pathway. In this report, isoproterenol (Iso), typically viewed as β-AR-selective agonist, was examined with respect to activation of α1A-AR. α1A-AR selective antagonists were used to specifically block Iso evoked signaling in different cellular backgrounds and confirm its action at α1A-AR. Iso induced signaling at α1A-AR was further interrogated by probing steps along the Gαq /PLC, Gαs and MAPK/ERK pathways. In HEK-293/EBNA cells transiently transduced with α1A-AR, and CHO_α1A-AR stable cells, Iso evoked low potency ERK activity as well as Ca2+ mobilization that could be blocked by α1A-AR selective antagonists. The kinetics of Iso induced Ca2+ transients differed from typical Gαq- mediated Ca2+ mobilization, lacking both the fast IP3R mediated response and the sustained phase of Ca2+ re-entry. Moreover, no inositol phosphate (IP) accumulation could be detected in either cell line after stimulation with Iso, but activation was accompanied by receptor internalization. Data are presented that indicate that Iso represents a novel type of α1A-AR partial agonist with signaling bias toward MAPK/ERK signaling cascade that is likely independent of coupling to Gαq.

Abstract A09: Downregulation of PI3K signaling by high ERK activity prevents transformation by oncogenic RAS

Abstract In this study, we investigated the contribution of strong ERK signals to tumor suppression. Oncogenic forms of RAS are found in up to 30% of all human cancers and are established drivers of tumor initiation and maintenance. However, strong expression of these oncogenes in normal cells induces cellular senescence, a putative tumor-suppressive barrier. RAS activates several signaling pathways, such as the PI3K/AKT pathway, the RAL pathway and the classical RAF/MEK/ERK MAP Kinase pathway. We previously found that RASV12-induced senescence of primary cells is prevented by attenuation of ERK signaling. Mechanistically, strong ERK signaling promotes senescence by inducing selective proteasome-dependent protein degradation. This “Senescence-Associated Protein Degradation” (SAPD) targets proteins required for cell cycle progression, mitochondrial functions, cell migration and cell signaling. Here we show that in addition to abrogating RAS-induced senescence, low ERK activity allows transformation of primary human cells stably expressing RasV12 and hTERT as well as transformation of RasV12-expressing rodent cells. Furthermore, in a Kras-driven mouse model of multistage pancreatic progression, decreased p-ERK levels correlate with tumor initiation. Taken together, these observations suggest that overactivated ERK may serve as a barrier for oncogenic transformation of cells acquiring activating RAS mutations. We found that the antitumor effects of aberrant ERK signaling include the inhibition of the phosphoinositide-3-kinase/AKT (PI3K/AKT) signaling pathway. Consequently, transformed RasV12-expressing cells with reduced ERK signaling show hyperactivation of the PI3K/AKT pathway, GSK3b inhibition, c-MYC stabilization and expression of stem cell-associated genes. In these cells, the hyperactivation of the PI3K/AKT pathway is essential in order to maintain their transformed phenotype since pharmacological inhibition of the pathway abrogates their tumorigenicity. Collectively, these results demonstrate a novel anti-tumor effect of strong ERK signaling and suggest that processes attenuating ERK levels and/or activity may contribute to malignant transformation of oncogenic RAS-expressing cells. Therefore, we propose a model where a moderated level of activated ERK (p-ERK) in RAS-expressing cells promotes transformation whereas higher levels limit cancer initiation by activating tumor-supressive mechanisms. This model suggests caution in clinical use of inhibitors of the ERK-MAPK pathway. Although complete pathway shut-down may counter tumorigenesis, partial reductions in ERK signaling may be deleterious by allowing a bypass of tumor-suppressive mechanisms in other premalignant lesions or in cells with strong ERK activity. Citation Format: Xavier Deschênes-Simard, Filippos Kottakis, Frédéric Lessard, Nabeel Bardeesy, Gerardo Ferbeyre. Downregulation of PI3K signaling by high ERK activity prevents transformation by oncogenic RAS. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr A09.

Abstract A32: In vivo imaging of ERK activity of mouse breast cancer cells reveals heterogeneity in response to anti-cancer drugs and tumor forming capacity

Abstract The Ras-Raf-MEK-ERK pathway is critical for normal physiological development and for carcinogenesis. However, the activity of this signaling cascade has never been examined in living cancer tissues due to technical difficulty. We have recently developed transgenic mice expressing a Förster resonance energy transfer (FRET) biosensor for ERK, which allows us to monitor cellular ERK activity in living tissues under two-photon (2P) microscopes. To study the role of ERK in mammary tumors, we crossed MMTV-Neu mice with the mice expressing the ERK FRET biosensor and observed in vivo under 2P. In the primary mammary tumors, the ERK activity exhibited significant divergence among the tumor cells. Such heterogeneity was significantly diminished in the secondary tumor cells. A MEK inhibitor PD0325901 and an EGFR/HER2 inhibitor Lapatinib suppressed ERK in both the primary mammary tumor cells and the secondary mammary tumor cells. Interestingly, we found that a small number of cells with low ERK activity in primary tumors that did not respond to Lapatinib. We sorted cells depending on ERK activity and obtained ERKlow and ERKhigh population.The ERKlow population exhibited higher tumorsphere formation in nonadherent culture condition and tumor formation in syngeneic mice. Notably, the tumors developed from ERKlow cells exhibited heterogeneous ERK activity similar to primary tumors. Taken together, our results suggest that the heterogeneous ERK activity might be related to cancer stem cell in tumors. Citation Format: Yuka Kumagai, Michiyuki Matsuda. In vivo imaging of ERK activity of mouse breast cancer cells reveals heterogeneity in response to anti-cancer drugs and tumor forming capacity. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr A32.

A Region in Urokinase Plasminogen Receptor Domain III Controlling a Functional Association with α5β1 Integrin and Tumor Growth

Highly expressed urokinase plasminogen activator receptor (uPAR) can interact with alpha5beta1 integrin leading to persistent ERK activation and tumorigenicity. Disrupting this interaction reduces ERK activity, forcing cancer cells into dormancy. We identified a site in uPAR domain III that is indispensable for these effects. A 9-mer peptide derived from a sequence in domain III (residues 240-248) binds purified alpha5beta1 integrin. Substituting a single amino acid (S245A) in this peptide, or in full-length soluble uPAR, impairs binding of the purified integrin. In the recently solved crystal structure of uPAR the Ser-245 is confined to the large external surface of the receptor, a location that is well separated from the central urokinase plasminogen binding cavity. The impact of this site on alpha5beta1 integrin-dependent cell functions was examined by comparing cells induced to express uPAR(wt) or the uPAR(S245A) mutant. Transfecting uPAR(wt) into cells with low endogenous levels of uPAR, inactive integrin, low ERK activity, and a dormant phenotype in vivo restores these functions and reinstates growth in vivo. In contrast, transfection of the same cells with uPAR(S245A) elicits only very small changes. Incubation of highly malignant cells with the wild-type, but not the S245A mutant peptide, disrupts the uPAR integrin interaction leading to down-regulation of ERK activity. The relevance of this binding site, and of the lateral uPAR-alpha5beta1 integrin interaction, to ERK pathway activation and tumor growth implicates it as a possible specific target for cancer therapy.

Functional Characterization of Human RSK4, a New 90-kDa Ribosomal S6 Kinase, Reveals Constitutive Activation in Most Cell Types

The 90-kDa ribosomal S6 kinases (RSK1-3) are important mediators of growth factor stimulation of cellular proliferation, survival, and differentiation and are activated via coordinated phosphorylation by ERK and 3-phosphoinositide-dependent protein kinase-1 (PDK1). Here we performed the functional characterization of a predicted new human RSK homologue, RSK4. We showed that RSK4 is a predominantly cytosolic protein with very low expression and several characteristics of the RSK family kinases, including the presence of two functional kinase domains and a C-terminal docking site for ERK. Surprisingly, however, in all cell types analyzed, endogenous RSK4 was maximally (constitutively) activated under serum-starved conditions where other RSKs are inactive due to their requirement for growth factor stimulation. Constitutive activation appeared to result from constitutive phosphorylation of Ser232, Ser372, and Ser389, and the low basal ERK activity in serum-starved cells appeared to be sufficient for induction of approximately 50% of the constitutive RSK4 activity. Finally experiments in mouse embryonic stem cells with targeted deletion of the PDK1 gene suggested that PDK1 was not required for phosphorylation of Ser232, a key regulatory site in the activation loop of the N-terminal kinase domain, that in other RSKs is phosphorylated by PDK1. The unusual regulation and growth factor-independent kinase activity indicate that RSK4 is functionally distinct from other RSKs and may help explain recent findings suggesting that RSK4 can participate in non-growth factor signaling as for instance p53-induced growth arrest.

The Association of CRKII with C3G Can be Regulated by Integrins and Defines a Novel Means to Regulate the Mitogen-activated Protein Kinases

In studies to define mechanisms of ERK activation in Chinese hamster ovary cells, we have observed an inverse correlation between CRKII-C3G complex formation and ERK activity. That is, we were able to coprecipitate the guanine nucleotide exchange factor C3G with the adaptor protein CRKII in lysates from suspended cells that had low ERK activity, but we could not do so or could do so less efficiently in lysates of adherent cells with increased ERK activity. Consistent with the presence of a functional CRKII-C3G complex, we detected more GTP-loaded RAP1 in suspension than adherent lysates. Overexpression of cDNAs encoding B-RAF, CRKII W109L, and PTP1B C215S activated ERK in suspension cells, the latter two constructs also disrupting CRKII-C3G complex formation. Finally, we have also observed that certain integrin alpha subunit cytoplasmic splice variants differentially regulate ERK1/2 but also in a manner that correlated with levels of a CRKII-C3G complex. Thus, these data suggest the involvement of integrins in an ERK suppression pathway mediated by CRKII-C3G complex formation and downstream signaling from activated RAP1.

The Sos1 and Sos2 Ras-specific exchange factors: differences in placental expression and signaling properties.

Targeted disruption of both alleles of mouse sos1, which encodes a Ras-specific exchange factor, conferred mid-gestational embryonic lethality that was secondary to impaired placental development and was associated with very low placental ERK activity. The trophoblastic layers of sos1(-/-) embryos were poorly developed, correlating with high sos1 expression in wild-type trophoblasts. A sos1(-/-) cell line, which expressed readily detectable levels of the closely related Sos2 protein, formed complexes between Sos2, epidermal growth factor receptor (EGFR) and Shc efficiently, gave normal Ras.GTP and ERK responses when treated with EGF for < or =10 min and was transformed readily by activated Ras. However, the sos1(-/-) cells were resistant to transformation by v-Src or by overexpressed EGFR and continuous EGF treatment, unlike sos1(+/-) or wild-type cells. This correlated with Sos2 binding less efficiently than Sos1 to EGFR and Shc in cells treated with EGF for > or =90 min or to v-Src and Shc in v-Src-expressing cells, and with less ERK activity. We conclude that Sos1 participates in both short- and long-term signaling, while Sos2-dependent signals are predominantly short-term.