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

Caracterización molecular de los mecanismos de resistencia a inhibidores de Polo-like kinasa 1 en cáncer colorrectal. Papel de simvastatina como modulador de la resistencia

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de Lectura: 05-04-2022Los inhibidores de Polo-like Kinasa 1 (PLK1) tienen un alto potencial como agentes antitumorales ya que van dirigidos al proceso de mitosis celular, aunque en ensayos clínicos la aparición de resistencias contra estos fármacos ha supuesto un reto para su aplicación en la práctica clínica. La caracterización de estas vías de resistencia adaptativas es el objetivo de esta investigación así como el descubrimiento de nuevas terapias combinadas que consigan revertir esta resistencia. Se emplearon 4 líneas de cáncer colorrectal (HT29, RKO, SW837 y HCT116) para generar la resistencia al inhibidor de PLK1 BI2536 mediante la exposición continuada al fármaco. Las líneas resistentes HT29R, RKOR, SW837R y HCT116R fueron validadas in vitro a través de ensayos de concentración inhibitoria del 50% del crecimiento (IG50), e in vivo mediante la evaluación del ratio T/C. Para evaluar el papel de las mutaciones como mecanismo de adquisición de resistencia, se secuenciaron los exones 1 y 2 del gen PLK1 en las cuatro líneas celulares resistentes, encontrando la presencia de la mutación R136G en la línea RKOR. En el resto de líneas (HT29R, SW837R y HCT116R) la vía de señalización de AXL a través del factor de transcripción TWIST1 fue identificada como uno de los mecanismos involucrados en estas tres líneas resistentes, induciendo la transición epitelio-mesénquima (TEM) y el aumento de expresión de MDR1. Las estatinas han demostrado un gran potencial en la quimio-sensibilización en muchos tipos de tumores así como la modulación de AXL, por ello evaluamos el impacto de simvastatina como agente re-sensibilizante in vitro, obteniendo como resultado el bloqueo del mecanismo de resistencia común activado en las tres líneas. El tratamiento combinado de BI2536 y simvastatina fue testado tanto en ensayos in vitro obteniendo una re-sensibilización al inhibidor de PLK1 en las tres líneas resistentes, como in vivo, observando una reducción del ratio T/C por debajo del punto de corte establecido. Los resultados de esta investigación demuestran que la inhibición de la vía del mevalonato contribuyó a la re-sensibilización de líneas de cáncer colorrectal resistentes a BI2536 in vitro e in vivo, planteándose como una nueva estrategia terapéutica para el manejo clínico de los inhibidores de PLK

Tecnecio 99m: radiofármaco de diagnóstico utilizado en la actualidad.

El tecnecio es un elemento químico cuyo radioisótopo 99mTc se utiliza para el diagnóstico de enfermedades en Medicina Nuclear. Se obtiene a partir de un generador de Molibdeno 99Mo99mTc, que es su radionucleido padre. El resultado es una elución salina de Pertecnetato de sodio estéril y apirógena. Es utilizado en forma de Pertecnetato de sodio directamente inyectado para el diagnóstico de alteraciones en glándulas como tiroides o glándulas salivares, para detectar defectos en la permeabilidad de los vasos, y para diagnosticar patologías más específicas mediante el marcaje de moléculas con 99mTc como alteraciones cardiacas, renales, hepáticas, óseas, alteraciones del movimiento y digestivas (radiofármacos de primera, segunda y tercera generación)

Molecular evidence of field cancerization initiated by diabetes in colon cancer patients

The potential involvement of type 2 diabetes mellitus (T2DM) as a risk factor for colon cancer (CC) has been previously reported. While several clinical studies show a higher incidence of CC and a lower survival rate in diabetics, others report no association. Our own experience indicates that diabetes does not seem to worsen the prognosis once the tumor is present. Despite this controversy, there are no wide‐spectrum molecular studies that delve into the impact of T2DM‐related mechanisms in colon carcinogenesis. Here, we present a transcriptomic and proteomic profiling of paired tumor and normal colon mucosa samples in a cohort of 42 CC patients, 23 of which have T2DM. We used gene set enrichment and network approaches to extract relevant pathways in diabetics, referenced them to current knowledge, and tested them using in vitro techniques. Through our transcriptomics approach, we identified an unexpected overlap of pathways overrepresented in diabetics compared to nondiabetics, in both tumor and normal mucosa, including diabetes‐related metabolic and signaling processes. Proteomic approaches highlighted several cancer‐related signaling routes in diabetics found only in normal mucosa, not in tumors. An integration of the transcriptome and proteome analyses suggested the deregulation of key pathways related to colon carcinogenesis which converged on tumor initiation axis TEAD/YAP‐TAZ as a potential initiator of the process. In vitro studies confirmed upregulation of this pathway in nontumor colon cells under high‐glucose conditions. In conclusion, T2DM associates with deregulation of cancer‐related processes in normal colon mucosa adjacent to tissue which has undergone a malignant transformation. These data support that in diabetic patients, the local microenvironment in normal colon mucosa may be a factor driving field cancerization promoting carcinogenesis. Our results set a new framework to study links between diabetes and colon cancer, including a new role of the TEAD/YAP‐TAZ complex as a potential driver