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

Vitamin C activates pyruvate dehydrogenase (PDH) targeting the mitochondrial tricarboxylic acid (TCA) cycle in hypoxic KRAS mutant colon cancer

Background: In hypoxic tumors, positive feedback between oncogenic KRAS and HIF-1α involves impressive metabolic changes correlating with drug resistance and poor prognosis in colorectal cancer. Up to date, designed KRAS-targeting molecules do not show clear benefits in patient overall survival (POS) so pharmacological modulation of aberrant tricarboxylic acid (TCA) cycle in hypoxic cancer has been proposed as a metabolic vulnerability of KRAS-driven tumors. Methods: Annexin V-FITC and cell viability assays were carried out in order to verify vitamin C citotoxicity in KRAS mutant SW480 and DLD1 as well as in Immortalized Human Colonic Epithelial Cells (HCEC). HIF1a expression and activity were determined by western blot and functional analysis assays. HIF1a direct targets GLUT1 and PDK1 expression was checked using western blot and qRT-PCR. Inmunohistochemical assays were perfomed in tumors derived from murine xenografts in order to validate previous observations in vivo. Vitamin C dependent PDH expression and activity modulation were detected by western blot and colorimetric activity assays. Acetyl-Coa levels and citrate synthase activity were assessed using colorimetric/fluorometric activity assays. Mitochondrial membrane potential (Δψ) and cell ATP levels were assayed using fluorometric and luminescent test. Results: PDK-1 in KRAS mutant CRC cells and murine xenografts was downregulated using pharmacological doses of vitamin C through the proline hydroxylation (Pro402) of the Hypoxia inducible factor-1(HIF-1)α, correlating with decreased expression of the glucose transporter 1 (GLUT-1) in both models. Vitamin C induced remarkable ATP depletion, rapid mitochondrial Δψ dissipation and diminished pyruvate dehydrogenase E1-α phosphorylation at Serine 293, then boosting PDH and citrate synthase activity. Conclusion: We report a striking and previously non reported role of vitamin C in the regulation of the pyruvate dehydrogenase (PDH) activity, then modulating the TCA cycle and mitochondrial metabolism in KRAS mutant colon cancer. Potential impact of vitamin C in the clinical management of anti-EGFR chemoresistant colorectal neoplasias should be further considered.This research is supported by Ministerio de Ciencia e Innovación, Centro para el Desarrollo Tecnológico Industrial (CDTI) and Universidad Católica San Antonio (Murcia

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

Aplikasi Herbisida 2,4-d Dan Penoxsulam Pada Pertumbuhan Dan Hasil Tanaman Padi Sawah (Oryza Sativa L.)

Salah satu teknik budidaya untuk meningkatkan produksi tanaman padi sawah yaitu dengan mengurangi persaingan antara tanaman dengan gulma. Pengendalian dengan kimiawi merupakan salah satu cara mengurangi pertumbuhan gulma di pertanaman padi. Cara kimiawi merupakan cara yang praktis, efektif dan efisien untuk mengendalikan gulma. Penelitian ini bertujuan untuk mempelajari pengaruh dari aplikasi herbisida 2,4-D dan penoxsulam dalam meningkatkan pertumbuhan dan hasil padi sawah serta menentukan dosis aplikasi herbisida 2,4-D dan penoxsulam baik secara tunggal maupun campuran dalam mengendalikan gulma pada tanaman padi sawah. Penelitian telah dilaksanakan pada bulan Maret-Juli 2014 di Desa Campurasri, Ngawi. Penelitian menggunakan Rancangan Acak Kelompok sederhana, dengan menempatkan 11 perlakuan yaitu H1 : kontrol herbisida 2,4-D; H2 : 2,4-D 11,25 kg ha-1; H3 : 2,4-D 22,5 kg ha-1; H4 : 2,4-D 33,75 kg ha-1; H5 : kontrol herbisida penoxsulam; H6 : penoxsulam 200 ml ha-1; H7 : penoxsulam 400 ml ha-1; H8 : penoxsulam 600 ml ha-1; H9 : 2,4-D 11,25 kg ha-1 dan penoxsulam 200 ml ha-1; H10 : 2,4-D 22,5 kg ha-1 dan penoxsulam 400 ml ha-1; H11 : 2,4-D 33,75 kg ha-1 dan penoxsulam 600 ml ha-1. Hasil penelitian menunjukkan bahwa perlakuan herbisida 2,4-D 11,25 kg ha-1 dan penoxsulam 200 ml menghasilkan bobot kering total tanaman dengan peningkatan sebesar 34,62 % dibandingkan dengan kontrol. Pada produksi tanaman padi peningkatan terjadi sebesar 29,77 % pada perlakuan herbisida 2,4-D 33,75 kg ha-1 dan penoxsulam 600 ml dibandingkan dengan kontrol

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

Detección y seguimiento in vivo de la inflamación pulmonar aguda en respuesta al humo de tabaco: aplicación de la técnica de imagen molecular de fluorescencia en un modelo murino

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Tesis realizada en el Laboratorio de Neumología Experimental perteneciente al Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD) y al Centro de Investigación Biomédica en Red de Enfermedades Respiratorias (CIBERES). Fecha de lectura: 21 de Diciembre de 2010

Early Detection of Susceptibility to Acute Lung Inflammation by Molecular Imaging in Mice Exposed to Cigarette Smoke

Matrix metalloproteinases (MMPs) are extracellular proteolytic enzymes involved in acute lung inflammation in response to cigarette smoke exposure (CSE). We present the in vivo detection of MMP activity using a specific MMP-activatable, near-infrared, polymer-based proteolytic probe in strains of mice with different susceptibility to developing smoking-induced emphysema (susceptible mice, C57BL/6j, and resistant mice, 129S2/SvHsd) to characterize the distinctive profile of CSE-induced acute inflammation. In vivo imaging of pulmonary inflammation expressing MMPs revealed a significantly different median ratio twofold higher in smoker than in nonsmoker susceptible mice (C57BL/6j) and no significant differences between the smoker and the nonsmoker group in resistant mice (129S2/SvHsd). Ex vivo imaging of the lungs of each group of mice confirmed the same in vivo experiment results obtained for both strains of mice. In the biochemical study of lung tissue, the proteolytic signal colocalized with the endogenously expressed MMP protein levels, with MMP-9 levels that are 2.2 times higher than in the nonsmoke-exposed group in C57BL/6j mice and no significant differences in the 129S2/SvHsd mice. The MMP-activatable probe provides a useful reagent for the in vivo and ex vivo detection of MMP-selective proteolytic activity. We are able to distinguish between susceptible and resistant strains of mice in terms of the profile of MMP activity in the early stages of pulmonary disease

Role of recently migrated monocytes in cigarette smoke-induced lung inflammation in different strain of mice.

This study investigates the role of proinflammatory monocytes recruited from blood circulation and recovered in bronchoalveolar lavage (BAL) fluid in mediating the lung damage in a model of acute cigarette smoke (CS)-induced lung inflammation in two strains of mice with different susceptibility to develop emphysema (susceptible -C57BL/6J and non susceptible -129S2/SvHsd). Exposure to whole-body CS for 3 consecutive research cigarettes in one single day induced acute inflammation in the lung of mice. Analysis of BAL fluid showed more influx of recently migrated monocytes at 72 h after CS-exposition in susceptible compared to non susceptible mice. It correlated with an increase in MMP-12 and TNF-α protein levels in the lung tissue, and with an increment of NF-κB translocation to the nucleus measured by electrophoretic mobility shift assay in C57BL/6J mice. To determine the functional role of these proinflammatory monocytes in mediating CS-induced airway inflammation, alveolar macrophages and blood monocytes were transiently removed by pretreatment with intratracheal and intravenous liposome-encapsulated CL2MDP, given 2 and 4 days prior to CS exposure and their repopulation was studied. Monocytes/macrophages were maximally depleted 48 h after last liposome application and subsequently recently migrated monocytes reappeared in BAL fluid of susceptible mice at 72 h after CS exposure. Recently migrated monocytes influx to the lung correlated with an increase in the MMP-12 protein level in the lung tissue, indicating that the increase in proinflammatory monocytes is associated with a major tissue damaging. Therefore our data confirm that the recruitment of proinflammatory recently migrated monocytes from the blood are responsible for the increase in MMP-12 and has an important role in the pathogenesis of lung disease induced by acute lung inflammation. These results could contribute to understanding the different susceptibility to CS of these strains of mice

Early Detection of Susceptibility to Acute Lung Inflammation by Molecular Imaging in Mice Exposed to Cigarette Smoke

Matrix metalloproteinases (MMPs) are extracellular proteolytic enzymes involved in acute lung inflammation in response to cigarette smoke exposure (CSE). We present the in vivo detection of MMP activity using a specific MMP-activatable, near-infrared, polymer-based proteolytic probe in strains of mice with different susceptibility to developing smoking-induced emphysema (susceptible mice, C57BL/6j, and resistant mice, 129S2/SvHsd) to characterize the distinctive profile of CSE-induced acute inflammation. In vivo imaging of pulmonary inflammation expressing MMPs revealed a significantly different median ratio twofold higher in smoker than in nonsmoker susceptible mice (C57BL/6j) and no significant differences between the smoker and the nonsmoker group in resistant mice (129S2/SvHsd). Ex vivo imaging of the lungs of each group of mice confirmed the same in vivo experiment results obtained for both strains of mice. In the biochemical study of lung tissue, the proteolytic signal colocalized with the endogenously expressed MMP protein levels, with MMP-9 levels that are 2.2 times higher than in the nonsmoke-exposed group in C57BL/6j mice and no significant differences in the 129S2/SvHsd mice. The MMP-activatable probe provides a useful reagent for the in vivo and ex vivo detection of MMP-selective proteolytic activity. We are able to distinguish between susceptible and resistant strains of mice in terms of the profile of MMP activity in the early stages of pulmonary disease