Ras family of small gtpases in crc: New perspectives for overcoming drug resistance

Colorectal cancer remains among the cancers with the highest incidence, prevalence, and mortality worldwide. Although the development of targeted therapies against the EGFR and VEGFR membrane receptors has considerably improved survival in these patients, the appearance of resistance means that their success is still limited. Overactivation of several members of the Ras-GTPase family is one of the main actors in both tumour progression and the lack of response to cytotoxic and targeted therapies. This fact has led many resources to be devoted over the last dec-ades to the development of targeted therapies against these proteins. However, they have not been as successful as expected in their move to the clinic so far. In this review, we will analyse the role of these Ras-GTPases in the emergence and development of colorectal cancer and their relationship with resistance to targeted therapies, as well as the status and new advances in the design of targeted therapies against these proteins and their possible clinical implications.This research was funded by Spanish Health Institute Carlos III (ISCIII), grant number PI19/01231. A.R-V was funded by a contract PFIS from Spanish Health Institute Carlos III (ISCIII) (FI20/00213) associated with the project PI19/01231

Overcoming PLK1 inhibitor resistance by targeting mevalonate pathway to impair AXL-TWIST axis in colorectal cancer

© 2021 The Author(s).New therapeutic targets are revolutionizing colorectal cancer clinical management, opening new horizons in metastatic patients’ outcome. Polo Like Kinase1 (PLK1) inhibitors have high potential as antitumoral agents, however, the emergence of drug resistance is a major challenge for their use in clinical practice. Overcoming this challenge represents a hot topic in current drug discovery research. BI2536-resistant colorectal cancer cell lines HT29R, RKOR, SW837R and HCT116R, were generated in vitro and validated by IG50 assays and xenografts models by the T/C ratio. Exons 1 and 2 of PLK1 gene were sequenced by Sanger method. AXL pathway, Epithelial-to-Mesenchymal transition (EMT) and Multidrug Resistance (MDR1) were studied by qPCR and western blot in resistant cells. Simvastatin as a re-sensitizer drug was tested in vitro and the drug combination strategies were validated in vitro and in vivo. PLK1 gene mutation R136G was found for RKOR. AXL pathway trough TWIST1 transcription factor was identified as one of the mechanisms involved in HT29R, SW837R and HCT116R lines, inducing EMT and upregulation of MDR1. Simvastatin was able to impair the mechanisms activated by adaptive resistance and its combination with BI2536 re-sensitized resistant cells in vitro and in vivo. Targeting the mevalonate pathway contributes to re-sensitizing BI2536-resistant cells in vitro and in vivo, raising as a new strategy for the clinical management of PLK1 inhibitors.This study has been funded by Instituto de Salud Carlos III (ISCIII) -Fondos FEDER proyects PI16/01468 and PI19/01231

UNR/CDSE1 expression as prognosis biomarker in resectable pancreatic ductal adenocarcinoma patients: A proof-of-concept

Pancreatic ductal adenocarcinoma is an aggressive form of pancreatic cancer and the fourth leading cause of cancer-related death. When possible, curative approaches are based on surgical resection, though not every patient is a candidate for surgery. There are clinical guidelines for the management of these patients that offer different treatment options depending on the clinical and pathologic characteristics. However, the survival rates seen in this kind of patients are still low. The CDSE1 gene is located upstream of NRAS and encodes an RNA-binding protein termed UNR. The aim of this study was to analyze UNR expression and its correlation with outcome in patients with resectable pancreatic ductal adenocarcinoma (PDAC). For this, samples from resectable PDAC patients who underwent duodenopancreatectomy were used to evaluate UNR protein expression by immunohistochemistry using a tissue microarray. Here, we observed that low UNR expression was significantly associated with shorter progression-free survival after surgery (P = 0.010). Moreover, this prognostic marker remained significant after Cox proportional hazards model (P = 0.036). We further studied the role of CDSE1 expression in patient’s prognosis using data from public repositories (GEO and TGCA), confirming our results. Interestingly, CDSE1 expression correlated with that of genes characteristic of an immunogenic molecular subtype of pancreatic cancer. Based on these findings, UNR may be considered a potential prognostic biomarker for resectable PDAC and may serve to guide subsequent adjuvant treatment decisionsThis work has been carried out with the support of the RNA-Reg CONSOLIDER Network CSD2009-00080 (J.M.-U. and J.G.-F.), and Spanish Health Research Project Funds PI16/ 01468 from ªInstituto de Salud Carlos IIIº (A.C. and J.G.-F.), both of the Spanish Ministry of Economy, Industry and Competitivenes

Choline kinase inhibition induces exacerbated endoplasmic reticulum stress and triggers apoptosis via CHOP in cancer cells

11 p.-6 fig.Endoplasmic reticulum (ER) is a central organelle in eukaryotic cells that regulates protein synthesis and maturation.Perturbation of ER functions leads to ER stress, which has been previously associated with a broad variety of diseases. ER stress is generally regarded as compensatory, but prolonged ER stress has been involved in apoptosis induced by several cytotoxic agents. Choline kinase a (ChoKa), the first enzyme in the Kennedy pathway, is responsible for the generation of phosphorylcholine (PCho) that ultimately renders phosphatidylcholine. ChoKa overexpression and high PCho levels have been detected in several cancer types. Inhibition of ChoKa has demonstrated antiproliferative and antitumor properties; however, the mechanisms underlying these activities remain poorly understood. Here, we demonstrate that ChoKa inhibitors (ChoKIs), MN58b and RSM932A, induce cell death in cancer cells (T47D,MCF7, MDA-MB231, SW620 and H460), through the prolonged activation of ER stress response. Evidence of ChoKIs-induced ER stress includes enhanced production of glucose-regulated protein, 78 kDa (GRP78), protein disulfide isomerase, IRE1a, CHOP, CCAAT/enhancer-binding protein beta (C/EBPb) and TRB3. Although partial reduction of ChoKa levels by small interfering RNA was not sufficient to increase the production of ER stress proteins, silencing of ChoKa levels also show a decrease in CHOP overproduction induced by ChoKIs, which suggests that ER stress induction is due to a change in ChoKa protein folding after binding to ChoKIs. Silencing of CHOP expression leads to a reduction in C/EBPb, ATF3 and GRP78 protein levels and abrogates apoptosis in tumor cells after treatment with ChoKIs, suggesting that CHOP maintains ER stress responses and triggers the pro-apoptotic signal. Consistent with the differential effect of ChoKIs in cancer and primary cells previously described, ChoKIs only promoted a transient and moderated ER stress response in the non-tumorogenic cells MCF10A. In conclusion, pharmacological inhibition of ChoKa induces cancer cell death through a mechanism that involves the activation of exaggerated and persistent ER stress supported by CHOP overproduction.This work has been funded by the following grants:Comunidad de Madrid (S2010/BMD-2326), Ministerio de Economía y Competitividad (SAF2011-29699, RD06-0020-0016 and RD12/0036/0019) and EU no.259737. ESL is a Sara Borell fellow under the program ISCIII/MICINN.Peer reviewe

Choline kinase α inhibitors MN58b and RSM932A enhances the antitumor response to cisplatin in lung tumor cells

Lung cancer is one of the main causes of death in developed countries, and non-small cell lung cancer (NSCLC) is the most frequent type (80% of patients). In advanced NSCLC, platinum-based chemotherapy is the frontline palliative treatment, but less than 5% of patients achieve prolonged survival. Immunotherapy has recently been proposed as the standard of care (SoC) as either monotherapy or in combination with chemotherapy for advanced NSCLC. The levels of expression of PD-L1 are the only predictive biomarkers for patient assessment. Although around 30% of patients receiving immunotherapy achieve 5-year survival, a significant number does not benefit from this novel therapeutic approach. Therefore, there is a need for novel strategies to improve clinical outcomes. The expression level of choline kinase α (ChoKα) is increased in a large number of human tumors, including NSCLC tumors, and constitutes an independent prognostic factor for early-stage NSCLC patients. Thus, ChoKα has been postulated as a new target drug in cancer therapy. The combination of cisplatin with novel targeted drugs such as choline kinase inhibitors may improve both the survival rates and the quality of life of NSCLC patients and may serve as the basis for the development of new therapeutic approaches. To that aim, we developed several in vitro and in vivo approaches to assess the antitumor activity of a novel combination regimen using cisplatin and ChoKα inhibitors. Our results suggest that a proper combination of specific inhibitors of the NSCLC prognostic factor ChoKα and platinum-based conventional chemotherapy might constitute a new, efficient treatment approach for NSCLC patients. This novel approach may help reduce the toxicity profile associated with cisplatin since, despite the advances in NSCLC management in recent years, the overall 5-year survival rate is still poor.This research was funded by MCIN/AEI/10.13039/501100011033, Grant PID2020-116165RBC21 and Fondo de Investigaciones Sanitarias, Instituto de Salud Carlos III, Spain, Grant PI20-00335 supported by FEDER funds

Approaches for the study of cancer: towards the integration of genomics, proteomics and metabolomics

Recent technological advances, combined with the development of bioinformatic tools, allow us to better address biological questions combining -omic approaches (i.e., genomics, metabolomics and proteomics). This novel comprehensive perspective addresses the identification, characterisation and quantitation of the whole repertoire of genes, proteins and metabolites occurring in living organisms. Here we provide an overview of recent significant advances and technologies used in genomics, metabolomics and proteomics. We also underline the importance and limits of mass accuracy in mass spectrometry-based -omics and briefly describe emerging types of fragmentation used in mass spectrometry. The range of instruments and techniques used to address the study of each -omic approach, which provide vast amounts of information (usually termed >high-throughput> technologies in the literature) is briefly discussed, including names, links and descriptions of the main databases, data repositories and resources used. Integration of multiple -omic results and procedures seems necessary. Therefore, an emerging challenge is the integration of the huge amount of data generated and the standardisation of the procedures and methods used. Functional data integration will lead to answers to unsolved questions, hopefully, applicable to clinical practice and management of patients.This work has been funded by the Ministerio de Ciencia e Innovación (SAF2008-03750 and RD06-0020-0016).Peer Reviewe

Combined 5-FU and ChoKα inhibitors as a new alternative therapy of colorectal cancer: Evidence in human tumor-derived cell lines and mouse xenografts

This is an open-access article distributed under the terms of the Creative Commons Attribution License.[Background]: Colorectal cancer (CRC) is the third major cause of cancer related deaths in the world. 5-fluorouracil (5-FU) is widely used for the treatment of colorectal cancer but as a single-agent renders low response rates. Choline kinase alpha (ChoKα), an enzyme that plays a role in cell proliferation and transformation, has been reported overexpressed in many different tumors, including colorectal tumors. ChoKα inhibitors have recently entered clinical trials as a novel antitumor strategy. [Methodology/Principal Findings]: ChoKα specific inhibitors, MN58b and TCD-717, have demonstrated a potent antitumoral activity both in vitro and in vivo against several tumor-derived cell line xenografts including CRC-derived cell lines. The effect of ChoKα inhibitors in combination with 5-FU as a new alternative for the treatment of colon tumors has been investigated both in vitro in CRC-tumour derived cell lines, and in vivo in mouse xenografts models. The effects on thymidilate synthase (TS) and thymidine kinase (TK1) levels, two enzymes known to play an essential role in the mechanism of action of 5-FU, were analyzed by western blotting and quantitative PCR analysis. The combination of 5-FU with ChoKα inhibitors resulted in a synergistic effect in vitro in three different human colon cancer cell lines, and in vivo against human colon xenografts in nude mice. ChoKα inhibitors modulate the expression levels of TS and TK1 through inhibition of E2F production, providing a rational for its mechanism of action. [Conclusion/Significance ]: Our data suggest that both drugs in combination display a synergistic antitumoral effect due to ChoKα inhibitors-driven modulation of the metabolization of 5-FU. The clinical relevance of these findings is strongly supported since TCD-717 has recently entered Phase I clinical trials against solid tumors. © 2013 de la Cueva et al.This work has been funded by the following grants: Comunidad de Madrid (S-BIO/0280/2006 and S2010/BMD-2326), Ministerio de Ciencia e Innovación (SAF2008-03750, SAF2011-29699, RD06-0020-0016 and RD12/0036/0019) and EU #259737.Peer Reviewe

Human urine proteomics: Building a list of human urine cancer biomarkers

In the last decade, several reports have focused on the identification and characterization of proteins present in urine. In an effort to build a list of proteins of interest as biomarkers, we reviewed the largest urine proteomes and built two updated lists of proteins of interest (available as supplementary tables). The first table includes a consensus list of 443 proteins found in urine by independent laboratories and reported on the top three largest urine proteomes currently published. This consensus list of proteins could serve as biomarkers to diagnose, monitor and manage a number of diseases. Here, we focus on a reduced list of 35 proteins with potential interest as cancer biomarkers in urine following two criteria: first, proteins previously detected in urine using bottom-up proteomic experiments, and second, those suggested as cancer protein biomarkers in human plasma. In an effort to standardize the information presented and its use in future studies, here we include the updated International Protein Index (v. 3.80) and primary Swiss-Prot accession numbers, official gene symbols and recommended full names. The main variables that influence urine proteomic experiments are also discussed. © 2011 Expert Reviews Ltd.Peer Reviewe

Lights and shadows of proteomic technologies for the study of protein species including isoforms, splicing variants and protein post-translational modifications

Recent reviews pinpointed the enormous diversity of proteins found in living organisms, especially in higher eukaryotes. Protein diversity is driven through three main processes: first, at deoxyribonucleic acid (DNA) level (i.e. gene polymorphisms), second, at precursor messenger ribonucleic acid (pre-mRNA) or messenger ribonucleic acid (mRNA) level (i.e. alternative splicing, also termed as differential splicing) and, finally, at the protein level (i.e. PTM). Current proteomic technologies allow the identification, characterization and quantitation of up to several thousands of proteins in a single experiment. Nevertheless, the identification and characterization of protein species using these technologies are still hampered. Here, we review the use of the terms >protein species> and >protein isoform.> We evidence that the appropriate selection of the database used for searches can impede or facilitate the identification of protein species. We also describe examples where protein identification search engines systematically fail in the attribution of protein species. We briefly review the characterization of protein species using proteomic technologies including gel-based, gel-free, bottom-up and top-down analysis and discuss their limitations. As an example, we discuss the theoretical characterization of the two human choline kinase species, α-1 and α-2, sharing the same catalytic activity but generated by alternative splicing on CHKA gene. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.J. C. V., A. C. and T. GdP are supported by Red Temática de Investigación Cooperativa de Cancer (RTICC, RD06-002-0016). J. C. V. and R. B. M. acknowledge funding from the National Institute for Proteomics (PROTEORED) and the Spanish Proteomics Society (SEPROT). This work has also been supported by Ministerio de Ciencia e Innovación (SAF2008-03750) and from Comunidad de Madrid (GR-SAL-0821-2004). R. B. M. acknowledges financial support from MICINN-FEDER (BIO2008-2941) and Genoma España.Peer Reviewe