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RAS-ERK (extracellular signal–regulated kinase) pathway signals are modulated by scaffold proteins that assemble the components of different kinase tiers into a sequential phosphorylation cascade. In the prevailing model scaffold proteins function as isolated entities, where the flux of phosphorylation events progresses downstream linearly, to achieve ERK phosphorylation. We show that different types of scaffold proteins, specifically KSR1 (kinase suppressor of Ras 1) and IQGAP1 (IQ motif-containing guanosine triphosphatase activating protein 1), can bind to each other, forming a complex whereby phosphorylation reactions occur across both species. MEK (mitogen-activated protein kinase kinase) bound to IQGAP1 can phosphorylate ERK docked at KSR1, a process that we have named “trans-phosphorylation.” We also reveal that ERK trans-phosphorylation participates in KSR1-regulated adipogenesis, and it also underlies the modest cytotoxicity exhibited by KSR-directed inhibitors. Overall, we identify interactions between scaffold proteins and trans-phosphorylation as an additional level of regulation in the ERK cascade, with broad implications in signaling and the design of scaffold protein–aimed therapeutics.

ERK 1/2 proteregulatory proteins like scaffold proteins, that link the components of MAP kinases sequential tiers to form a multienzymatic complex. This way ERK signals are fine-tune (amplitude, intensity and duration), afford signal fidelity and have spatial selectivity. Some examples of scaffold proteins are KSR1, that bind C/BRAF, MEK1/2 and ERK1/2, and IQGAP1, that binds BRAF, MEK1/2 AND ERK1/2. In both cases depletion or blockade of this scaffold functions prevents RAS-ERK pathway-driven neoplasia. We demostrated that ERK can binds constitutively to scaffold, to be cis-phosphorylated therein by scaffold-bound MEK which would enable its binding to a free, phosphorylated ERK monomer to form a dimer. We have found that KSR1 y IQGAP1 can functionally associate with each other. As a consecuence, ERK docked at KSR1 can be phosphorylated by MEK bound to IQGAP1, a process named ¿trans-phosphorylarion¿. The results identify interactions betweeen scaffold proteins as a higher-order level of regulation in the ERK cascade. ins are major actors in the transduction of signals conveyed by external stimuli to the interior of the cell, being regulators of biochemical processes such as proliferation, differentiation or survival. ERK signals are modulated by different

The transcription factor MYC regulates cell proliferation, transformation, and survival in response to growth factor signaling that is mediated in part by the kinase activity of ERK2. Because ERK2 can also bind to DNA to modify gene expression, we investigated whether it more directly regulates MYC transcription. We identified ERK2 binding sites in the MYC promoter and detected ERK2 at the promoter in various serum-stimulated cell types. Expression of nuclear-localized ERK2 constructs in serum-starved cells revealed that ERK2 in the nucleus—regardless of its kinase activity—increased MYC mRNA expression and MYC protein abundance. ERK2 bound to the promoter through its amino-terminal insert domain and to the cyclin-dependent kinase CDK9 (which activates RNA polymerase II) through its carboxyl-terminal conserved docking domain. Both interactions were essential for ERK2-induced MYC expression, and depleting ERK impaired CDK9 occupancy and RNA polymerase II progression at the MYC promoter. Artificially tethering CDK9 to the MYC promoter by fusing it to the ERK2 insert domain was sufficient to stimulate MYC expression in serum-starved cells. Our findings demonstrate a role for ERK2 at the MYC promoter acting as a kinase-independent anchor for the recruitment of CDK9 to promote MYC expression.

Metallothionein-3 has poorly characterized functions in neuroblastoma. Cisplatin-based chemotherapy is a major regimen to treat neuroblastoma, but its clinical efficacy is limited by chemoresistance. We investigated the impact of human metallothionein-3 (hMT3) up-regulation in neuroblastoma cells and the mechanisms underlying the cisplatin-resistance. We confirmed the cisplatin-metallothionein complex formation using mass spectrometry. Overexpression of hMT3 decreased the sensitivity of neuroblastoma UKF-NB-4 cells to cisplatin. We report, for the first time, cisplatin-sensitive human UKF-NB-4 cells remodelled into cisplatin-resistant cells via high and constitutive hMT3 expression in an in vivo model using chick chorioallantoic membrane assay. Comparative proteomic analysis demonstrated that several biological pathways related to apoptosis, transport, proteasome, and cellular stress were involved in cisplatin-resistance in hMT3 overexpressing UKF-NB-4 cells. Overall, our data confirmed that up-regulation of hMT3 positively correlated with increased cisplatin-chemoresistance in neuroblastoma, and a high level of hMT3 could be one of the causes of frequent tumour relapses.

Metastatic melanoma is a highly immunogenic tumor with very poor survival rates due to immune system escape-mechanisms. Immune checkpoint inhibitors (ICIs) targeting the cytotoxic T-lymphocyte-associated protein 4 (CTLA4) and the programmed death-1 (PD1) receptors, are being used to impede immune evasion. This immunotherapy entails an increment in the overall survival rates. However, melanoma cells respond with evasive molecular mechanisms. ERK cascade inhibitors are also used in metastatic melanoma treatment, with the RAF activity blockade being the main therapeutic approach for such purpose, and in combination with MEK inhibitors improves many parameters of clinical efficacy. Despite their efficacy in inhibiting ERK signaling, the rewiring of the melanoma cell-signaling results in disease relapse, constituting the reinstatement of ERK activation, which is a common cause of some resistance mechanisms. Recent studies revealed that the combination of RAS–ERK pathway inhibitors and ICI therapy present promising advantages for metastatic melanoma treatment. Here, we present a recompilation of the combined therapies clinically evaluated in patients.

Background: RAS-to-ERK signaling is crucial for the onset and progression of advanced thyroid carcinoma, and blocking ERK dimerization provides a therapeutic benefit in several human carcinomas. Here we analyzed the effects of DEL-22379, a relatively specific ERK dimerization inhibitor, on the activation of the RAS-to-ERK signaling cascade and on tumor-related processes in vitro and in vivo. Methods: We used a panel of four human anaplastic thyroid carcinoma (ATC) cell lines harboring BRAF or RAS mutations to analyze ERK dynamics and tumor-specific characteristics. We also assessed the impact of DEL-22379 on the transcriptional landscape of ATC cell lines using RNA-sequencing and evaluated its therapeutic efficacy in an orthotopic mouse model of ATC. Results: DEL-22379 impaired upstream ERK activation in BRAF- but not RAS-mutant cells. Cell viability and metastasis-related processes were attenuated by DEL-22379 treatment, but mostly in BRAF-mutant cells, whereas in vivo tumor growth and dissemination were strongly reduced for BRAF-mutant cells and mildly reduced for RAS-mutant cells. Transcriptomics analyses indicated that DEL-22379 modulated the transcriptional landscape of BRAF- and RAS-mutant cells in opposite directions. Conclusions: Our findings establish that BRAF- and RAS-mutant thyroid cells respond differentially to DEL-22379, which cannot be explained by the previously described mechanism of action of the inhibitor. Nonetheless, DEL-22379 demonstrated significant anti-tumor effects against BRAF-mutant cells in vivo with an apparent lack of toxicity, making it an interesting candidate for the development of combinatorial treatments. Our data underscore the differences elicited by the specific driver mutation for thyroid cancer onset and progression, which should be considered for experimental and clinical approaches.

It is known that Rho GTPases control different aspects of the biology of skin stem cells (SSCs). However, little information is available on the role of their upstream regulators under normal and tumorigenic conditions in this process. To address this issue, we have used here mouse models in which the activity of guanosine nucleotide exchange factors of the Vav subfamily has been manipulated using both gain- and loss-of-function strategies. These experiments indicate that Vav2 and Vav3 regulate the number, functional status, and responsiveness of hair follicle bulge stem cells. This is linked to gene expression programs related to the reinforcement of the identity and the quiescent state of normal SSCs. By contrast, in the case of cancer stem cells, they promote transcriptomal programs associated with the identity, activation state, and cytoskeletal remodeling. These results underscore the role of these Rho exchange factors in the regulation of normal and tumor epidermal stem cells.

Metallothionein-3 has poorly characterized functions in neuroblastoma. Cisplatin-based chemotherapy is a major regimen to treat neuroblastoma, but its clinical efficacy is limited by chemoresistance. We investigated the impact of human metallothionein-3 (hMT3) up-regulation in neuroblastoma cells and the mechanisms underlying the cisplatin-resistance. We confirmed the cisplatin-metallothionein complex formation using mass spectrometry. Overexpression of hMT3 decreased the sensitivity of neuroblastoma UKF-NB-4 cells to cisplatin. We report, for the first time, cisplatin-sensitive human UKF-NB-4 cells remodelled into cisplatin-resistant cells via high and constitutive hMT3 expression in an in vivo model using chick chorioallantoic membrane assay. Comparative proteomic analysis demonstrated that several biological pathways related to apoptosis, transport, proteasome, and cellular stress were involved in cisplatin-resistance in hMT3 overexpressing UKF-NB-4 cells. Overall, our data confirmed that up-regulation of hMT3 positively correlated with increased cisplatin-chemoresistance in neuroblastoma, and a high level of hMT3 could be one of the causes of frequent tumour relapses.

[EN]: Nearly 50% of human malignancies have dysregulated RAS-ERK signalling; inhibiting it is a valid strategy for antineoplastic intervention. ERK dimerization is essential for extranuclear ERK signalling, but not for nuclear signalling. Here we describe the role of ERK dimers in tumour progression in response to different stimuli. ERK dimerization-mediated cell spreading involves actin remodelling and ERK scaffold proteins as potential targets driven during tumorigenesis. In this study, we performed chemotaxis assays and employed 2D migration, 3D organoid formation and chick embryo metastasis models. We have observed the tumorigenic role mediated by ERK dimerization at different stages of tumour progression, monitored with the ERK dimer inhibitor DEL-22379., [ES]: Casi el 50% de las neoplasias humanas presentan una señalización RAS-ERK no regulada; inhibirla es una estrategia válida para la intervención antineoplásica. La dimerización de ERK es esencial para la señalización extranuclear de las ERK, pero no para la nuclear. Aquí describimos el papel de los dímeros de ERK en la progresión tumoral en respuesta a diferentes estímulos. La diseminación celular mediada por la dimerización de ERK implica la remodelación de la actina, y las proteínas andamio de ERK como objetivos potenciales impulsados durante la tumorogénesis. En este estudio se realizan ensayos de quimiotaxis y se emplean modelos de migración 2D, de formación de organoides 3D y de metástasis de embrión de pollo. Hemos observado el papel tumorogénico mediado por la dimerización de ERK en los distintos estados de la progresión tumoral, monitorizado con el inhibidor de dímeros de ERK, DEL-22379.

Signals conveyed through the RAS-ERK pathway constitute a pivotal regulatory element in cancer-related cellular processes. Recently, RAS dimerization has been proposed as a key step in the relay of RAS signals, critically contributing to RAF activation. RAS clustering at plasma membrane microdomains and endomembranes facilitates RAS dimerization in response to stimulation, promoting RAF dimerization and subsequent activation. Remarkably, inhibiting RAS dimerization forestalls tumorigenesis in cellular and animal models. Thus, the pharmacological disruption of RAS dimers has emerged as an additional target for cancer researchers in the quest for a means to curtail aberrant RAS activity.

In the current Research Topic (RT), we provide insights on the small GTPase biology, from protein synthesis to disease, by presenting a Research Topic of original and review articles describing distinct GTPases, with special attention to RAS proteins. The RAS superfamily comprises a large family of small GTPases, where the members of the RAS, RHO, and RAB families are the most well-characterized. These small proteins cycle between a GDP-bound inactive and a GTP-bound active state, thus mediating several cellular processes such as intracellular trafficking, cytoskeletal organization, cell migration, proliferation, differentiation, and gene expression (Bos 2018; Gray et al., 2020). RAS is considered a central protooncogene in cancer. There are different RAS proteins (K-RAS, N-RAS, and H-RAS) and gain-of-function missense mutations in RAS genes are frequently found in a variety of tumors (Hobbs et al., 2016). Although there are different RAS isoforms, most research to date have concentrated their efforts in studying K-RAS, since it is the most mutated isoform in cancer. In addition, the K-RAS gene undergoes alternative splicing generating the splicing variants K-RAS4A and K-RAS4B. In this RT, Aran discusses the roles of both splicing variants in cancer and since K-RAS4A, the under-studied splicing variant, shares the same oncogenic point mutations with K-RAS4B and has strong transforming capability, the author emphasizes the importance of investigating K-RAS4A in RAS-driven cancer and developing anti-RAS therapies.

Nowadays, college biology textbooks teach us that most biomolecules, acting on no matter which biochemical process, generally function not as single entities, but rather, as constituents of macromolecular assemblies of variable complexity, whereby biochemical activities are promoted, regulated and fine-tuned in order to yield precise biological outcomes. In this respect, the past decades have witnessed a broad development of novel experimental approaches that, accompanied by an unprecedented surge of computational capabilities, have enabled extensive and multifaceted analyses on a large plethora of biomolecular interactions. These have substantially fostered our understanding of essential mechanistic and functional aspects underlying the activity of biomolecules that participate in key cellular processes. And in some cases, have provided the clue for deciphering the molecular essence of pathologies arising when such biomolecules go awry.

The survival rate in lung cancer remains stubbornly low and there is an urgent need for the identification of new therapeutic targets. In the last decade, several members of the SWI/SNF chromatin remodeling complexes have been described altered in different tumor types. Nevertheless, the precise mechanisms of their impact on cancer progression, as well as the application of this knowledge to cancer patient management are largely unknown. In this study, we performed targeted sequencing of a cohort of lung cancer patients on genes involved in chromatin structure. In addition, we studied at the protein level the expression of these genes in cancer samples and performed functional experiments to identify the molecular mechanisms linking alterations of chromatin remodeling genes and tumor development. Remarkably, we found that 20% of lung cancer patients show ARID2 protein loss, partially explained by the presence of ARID2 mutations. In addition, we showed that ARID2 deficiency provokes profound chromatin structural changes altering cell transcriptional programs, which bolsters the proliferative and metastatic potential of the cells both in vitro and in vivo. Moreover, we demonstrated that ARID2 deficiency impairs DNA repair, enhancing the sensitivity of the cells to DNA-damaging agents. Our findings support that ARID2 is a bona fide tumor suppressor gene in lung cancer that may be exploited therapeutically.

La presente invención se refiere a un método in vitro que permite predecir la respuesta terapéutica al tratamiento con fármacos inhibidores de BRAF, tales como vemurafenib, en pacientes de cáncer asociado a mutaciones oncogénicas en dicha kinasa, tal como meianoma BRAF positivo. Dicho método se basa en ¡a medición de los niveles de ERK1/2 citoplasmática, a través de la detección y cuantifi catión de ERK1/2 fosforiladas en serina 301/284 respectivamente, en una muestra biológica aislada del paciente. La invención también proporciona un anticuerpo específico frente a ERK1/2 fosforiladas en serina 301/284 y un kit que lo comprende, ios cuales son de utilidad en el método descrito. [ES], The present invention relates to an in vitro method which enables the prediction of therapeutic response to treatment with BRAF inhibitor drugs, such as vemurafenib, in cancer patients associated with oncogenic mutations in said kinase, such as BRAF positive melanoma. Said method is based on the measurement of cytoplasmic ERK1/2 levels through the detection and quantification of ERK1/2 phosphorylated in serine 301/284, respectively, in an isolated biological sample of the patient. The invention also provides a specific antibody against ERK1/2 phosphorylated in serine 301/284 and a kit comprising it, which are of use in the described method. [EN]

Método para predecir la respuesta terapéutica a fármacos inhibidores de la serina-treonina kinasa BRAF. La presente invención se refiere a un método in vitro que permite predecir la respuesta terapéutica al tratamiento con fármacos inhibidores de BRAF, tales como vemurafenib, en pacientes de cáncer asociado a mutaciones oncogénicas en dicha kinasa, tal como melanoma BRAF positivo. Dicho método se basa en la medición de los niveles de ERK1/2 citoplasmática, a través de la detección y cuantificación de ERK1/2 fosforiladas en serina 301/284 respectivamente, en una muestra biológica aislada del paciente. La invención también proporciona un anticuerpo específico frente a ERK1/2 fosforiladas en serina 301/284 y un kit que lo comprende, los cuales son de utilidad en el método descrito.

RAS mutations are the second most common genetic alteration in thyroid tumors. However, the extent to which they are associated with the most aggressive phenotypes is still controversial. Regarding their malignancy, the majority of RAS mutant tumors are classified as undetermined, which complicates their clinical management and can lead to undesired under- or overtreatment. Using the chick embryo spontaneous metastasis model, we herein demonstrate that the aggressiveness of HRAS-transformed thyroid cells, as determined by the ability to extravasate and metastasize at distant organs, is orchestrated by HRAS subcellular localization. Remarkably, aggressiveness inversely correlates with tumor size. In this respect, we also show that RAS site-specific capacity to regulate tumor growth and dissemination is dependent on VEGF-B secretion. Furthermore, we have identified the acyl protein thioesterase APT-1 as a determinant of thyroid tumor growth versus dissemination. We show that alterations in APT-1 expression levels can dramatically affect the behavior of thyroid tumors, based on its role as a regulator of HRAS sublocalization at distinct plasma membrane microdomains. In agreement, APT-1 emerges in thyroid cancer clinical samples as a prognostic factor. As such, APT-1 levels could serve as a biomarker that could help in the stratification of HRAS mutant thyroid tumors based on their aggressiveness.

Ras GTPases convey signals from different types of membranes. At these locations, different Ras isoforms, interactors and regulators generate different biochemical signals and biological outputs. The study of Ras localisation-specific signal transduction networks has been hampered by our inability to specifically activate each of these Ras pools. Here, we describe a new set of site-specific tethered exchange factors, engineered by fusing the RasGRF1 CDC25 domain to sub-localisation-defining cues, whereby Ras pools at specific locations can be precisely activated. We show that the CDC25 domain has a high specificity for activating HRas but not NRas and KRas. This unexpected finding means that our constructs mainly activate endogenous HRas. Hence, their use enabled us to identify distinct pathways regulated by HRas in endomembranes and plasma membrane microdomains. Importantly, these new constructs unveil different patterns of HRas activity specified by their subcellular localisation. Overall, the targeted GEFs described herein constitute ideal tools for dissecting spatially-defined HRas biochemical and biological functions.

RHO GTPases are key regulators of the cytoskeletal architecture, which impact a broad range of biological processes in malignant cells including motility, invasion, and metastasis, thereby affecting tumor progression. One of the constraints during cell migration is the diameter of the pores through which cells pass. In this respect, the size and shape of the nucleus pose a major limitation. Therefore, enhanced nuclear plasticity can promote cell migration. Nuclear morphology is determined in part through the cytoskeleton, which connects to the nucleoskeleton through the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex. Here, we unravel the role of RAC1 as an orchestrator of nuclear morphology in melanoma cells. We demonstrate that activated RAC1 promotes nuclear alterations through its effector PAK1 and the tubulin cytoskeleton, thereby enhancing migration and intravasation of melanoma cells. Disruption of the LINC complex prevented RAC1-induced nuclear alterations and the invasive properties of melanoma cells. Thus, RAC1 induces nuclear morphology alterations through microtubules and the LINC complex to promote an invasive phenotype in melanoma cells.