El papel de los receptores KIR y FcyRIIIa en la respuesta ADCC mediada por IgG1

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 01-03-2019El anticuerpo monoclonal anti-IgG1 cetuximab es una terapia que bloquea al receptor EGFR de las células tumorales, lo que le convierte en una terapia eficaz debido al bloqueo de la ruta molecular de las MAPK responsable de la proliferación y supervivencia celular. Además, este fármaco posee la capacidad de activar la citotoxicidad celular mediada por anticuerpo (ADCC) desencadenada por las células NK del propio paciente. Lo interesante de activar la ADCC, es que este mecanismo es independiente de las mutaciones activadoras tanto de KRAS y BRAF, como de cualquier otra activación de la ruta de las MAPK por debajo del EGFR. Uno de los factores que van a determinar una mayor interacción con la fracción constante del anticuerpo cetuximab es el tipo de polimorfismo presente en el receptor de la fracción constante de las inmunoglobulinas presentes en la membrana de las células efectoras, en concreto FcyRIIa y FcyRIIIa. Además, sabemos que existen otros receptores en las células NK conocidos como receptores KIR. No todos los receptores KIR son activadores de la función de las células NK ya que estas moléculas son polimórficas. Por esta razón, nos planteamos estudiar que polimorfismos tanto en los receptores FcγR como en los genes KIR serían buenos marcadores predictivos de una mayor respuesta al cetuximab a través de la activación de la ADCC. Para ello, diseñamos un ensayo clínico multicéntrico en el que reclutamos 70 muestras de pacientes con cáncer colorrectal metastásico con mutación de KRAS, expresión del EGFR y con el polimorfismo favorable FcγRIIa-131H/R que fueron tratados con cetuximab. Sobre estas muestras evaluamos la expresión de los polimorfismos en el gen de FcyRIIIa y en los genes KIR. Tras evaluar los polimorfismos de FcγRIIIa (V158F, V158V y F158F) en nuestra cohorte de pacientes, no encontramos asociación con la supervivencia de los pacientes tratados con cetuximab. Sin embargo, encontramos una alta tendencia hacia la significancia con respecto a la supervivencia global asociada al gen KIR2DS4 (P=0.071). Profundizando en las variantes del gen KIR2DS4, encontramos que la variante completa (KIR2DS4-full) presentaba un peor pronóstico comparada con la variante con el receptor no funcional (KIR2DS4-RNF) (P=0.030). Posteriormente, diseñamos un modelo in vitro para validar la asociación entre los polimorfismos favorables encontrados y la capacidad de activación de la ADCC de las células NK mediante el cetuximab. Para ello, co-cultivamos la línea celular derivada de cáncer colorrectal con mutación de KRAS, LS147T, junto a células NK aisladas de donantes sanos positivas para los polimorfismos favorables descritos previamente. Interesantemente, los genotipos favorables con FcγRIIa-131(H/H ó H/R) y la variante delecionada que resulta en un receptor no funcional, KIR2DS4del, presentaron una mayor tasa de activación de ADCC mediada por el anticuerpo cetuximab.The anti-IgG1 monoclonal antibody cetuximab is a therapy that blocks the EGFR receptor of tumor cells. This treatment is an effective anti-neoplasic therapy to block the MAPK molecular pathway involved in cell proliferation and survival. In addition, this drug is able to activate antibody dependent cellular cytotoxicity (ADCC) triggered by NK cells. Interestingly, ADCC mechanism is independent of both KRAS and BRAF activating mutations, and other activating factors of the MAPK pathway downstream EGFR. One of the factors that could determine the interaction between cetuximab´s IgG1 constant fraction is the polymorphism expressed in the cell membrane of effector T-cells called gamma chain receptor of immunoglobulin constant fraction receptor of the immunoglobulins (FcγRIIa and FcγRIIIa). In fact, it is know that in NK cells there are other receptors known as KIR receptors. Not all KIR receptors are activators of NK cell function since these molecules are polymorphic. For this reason, we based our study on the polymorphisms expressed in FcγR and KIR and dissected whether they could be good predictive markers of cetuximab response through ADCC activation. For this, we designed a multicentre clinical trial and recruited 70 samples from patients with metastatic colorectal cancer with KRAS mutation, EGFR expression and the previously described FcγRIIa-131H/R polymorphism as favorable for cetuximab response. On these samples, we evaluated the expression of the polymorphisms in FcγRIIIa and in the KIR genes. After evaluating the polymorphisms of FcγRIIIa (V158F, V158V and F158F) in our patient cohort, we found no association with patient survival after cetuximab treatment. However, we found a high trend towards significance between overall survival and KIR2DS4 (P = 0.071). Further research on this gene revealed that full variant (KIR2DS4-full) showed a worse prognosis compared to the variant with the non-functional receptor (KIR2DS4-RNF) (P = 0.030). Subsequently, we designed an in vitro model to validate the association between the favorable polymorphisms previously found, and the activation of ADCC by cetuximab. Then, we cocultured a colorectal cancer derived cell line that carries KRAS mutation, LS147T, together with NK cells isolated from healthy donors tested to be positive for favorable polymorphisms. Interestingly, FcγRIIa-131 (H/H or H/R), and the deleted variant gene that translate a nonfunctional receptor, KIR2DS4del, showed a higher activation rate of ADCC mediated by cetuximab antibody

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

Tractament del càncer; Expressió gènica; Anàlisi de supervivènciaTratamiento del cáncer; Expresión génica; Análisis de supervivenciaCancer treatment; Gene expression; Survival analysisPancreatic 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 decisions.This 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 “Instituto de Salud Carlos III” (A.C. and J.G.-F.), both of the Spanish Ministry of Economy, Industry and Competitiveness

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

Nuclear DICKKOPF-1 as a biomarker of chemoresistance and poor clinical outcome in colorectal cancer

Sporadic colorectal cancer (CRC) insurgence and progression depend on the activation of Wnt/β-catenin signaling. Dickkopf (DKK)-1 is an extracellular inhibitor of Wnt/β-catenin signaling that also has undefined β-catenin-independent actions. Here we report for the first time that a proportion of DKK-1 locates within the nucleus of healthy small intestine and colon mucosa, and of CRC cells at specific chromatin sites of active transcription. Moreover, we show that DKK-1 regulates several cancer-related genes including the cancer stem cell marker aldehyde dehydrogenase 1A1 (ALDH1A1) and Ral-binding protein 1-associated Eps domain-containing 2 (REPS2), which are involved in detoxification of chemotherapeutic agents. Nuclear DKK-1 expression is lost along CRC progression; however, it remains high in a subset (15%) of CRC patients (n = 699) and associates with decreased progression-free survival (PFS) after chemotherapy administration and overall survival (OS) [adjusted HR, 1.65; 95% confidence interval (CI), 1.23-2.21; P = 0.002)]. Overexpression of ALDH1A1 and REPS2 associates with nuclear DKK-1 expression in tumors and correlates with decreased OS (P = 0.001 and 0.014) and PFS. In summary, our findings demonstrate a novel location of DKK-1 within the cell nucleus and support a role of nuclear DKK-1 as a predictive biomarker of chemoresistance in colorectal cancer

UNR/<i>CSDE1</i> Expression Is Critical to Maintain Invasive Phenotype of Colorectal Cancer through Regulation of c-MYC and Epithelial-to-Mesenchymal Transition

CSDE1 (cold shock domain containing E1) gene is located upstream of the N-RAS locus, and codes for an RNA-binding protein named Upstream of N-Ras (UNR). In cancer, CSDE1 has been shown to regulate c-Fos, c-Myc, Pten, Rac1, or Vimentin. UNR/CSDE1 has been studied in breast, melanoma, pancreatic and prostate cancer. Then, the aim of this study is to evaluate the role of CSDE1/UNR in colorectal cancer progression and maintenance of aggressive phenotype. We firstly evaluated UNR/CSDE1 expression in human colon cancer derived cell lines and patient samples. Subsequently, we performed functional experiments by UNR/CSDE1 downregulation. We also evaluated UNR/CSDE1 prognostic relevance in two independent sets of patients. Not only was UNR/CSDE1 expression higher in tumor samples compared to untransformed samples, but also in colonospheres and metastatic origin cell lines than their parental and primary cell lines, respectively. Downregulation of UNR/CSDE1 reduced cell viability and migration throughout a restrain of epithelial-to-mesenchymal transition and increases sensitivity to apoptosis. Interestingly, high UNR/CSDE1 expression was associated with poor prognosis and correlated positively with c-MYC expression in colorectal cancer samples and cell lines. Here, we show for the first time compelling data reporting the oncogenic role of UNR/CSDE1 in human colorectal cancer

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 decisions.This 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 “Instituto de Salud Carlos III” (A.C. and J.G.-F.), both of the Spanish Ministry of Economy, Industry and Competitiveness

Additional file 1: Table S1. of Predictive value of vrk 1 and 2 for rectal adenocarcinoma response to neoadjuvant chemoradiation therapy: a retrospective observational cohort study

Relationship between clinicopathological characteristics and biomarkers. (DOCX 21 kb