RESUMEN
BACKGROUND AND PURPOSE: Overcoming radioresistance is a critical challenge in pancreatic ductal adenocarcinoma (PDAC). Our study investigates the targeting of Cyclin-dependent kinase-1 (CDK1) through genetic and pharmaceutical inhibition to radiosensitize PDAC cells. MATERIALS AND METHODS: Mass spectrometry and phosphoproteomics were used to analyze engineered radiation-resistant PDAC cell lines (MIA PaCa-2 and PANC-1) compared to parental controls. The TCGA PDAC database was queried for clinical outcomes and patients were dichotomized based on the median CDK1 mRNA expression. We generated a microRNA-based TET-on inducible shRNA to inhibit CDK1 expression in two PDAC cell lines. We used an orthotopic model of PDAC to test the radiation sensitivity of PDAC tumors with or without doxycycline treatment. We targeted CDK1 activation with a selective CDK1 inhibitor, RO-3306, followed by in vitro experiments employing immunoblotting, immunocytochemistry, and clonogenic assays. RESULTS: Phosphoproteomics analysis revealed that phospho-CDK1 (Tyr15) was significantly elevated in the resistant clones. We found that high CDK1 expression was associated with worse OS in PDAC patients. Radiation exposure increased CDK1 phosphorylation. In MIA PaCa-2 and PANC-1 cells, CDK1 inhibition synergized with radiation therapy to delay tumor growth in vivo. CDK1 inhibition via. RO-3306 resulted in a significant shift of cells into the G2/M phase and disrupted DNA repair after radiation exposure. In vitro, pre-treatment with RO-3306 led to enhanced radiosensitivity of PDAC cells. CONCLUSION: CDK1 plays a crucial role in PDAC radioresistance. Targeting CDK1 with radiotherapy holds promise for further investigation in PDAC treatment.
RESUMEN
Oncogenic RAS and RAF signaling has been implicated in contributing to radioresistance in pancreatic and thyroid cancers. In this study, we sought to better clarify molecular mechanisms contributing to this effect. We discovered that miRNA 296-3p (miR-296-3p) is significantly correlated with radiosensitivity in a panel of pancreatic cancer cells, and miR-296-3p is highly expressed in normal cells, but low in cancer cell lines. Elevated expression of miR-296-3p increases radiosensitization while decreasing the expression of the DNA repair enzyme RAD18 in both pancreatic and thyroid cancer cells. RAD18 is overexpressed in both pancreatic and thyroid tumors compared to matched normal controls, and high expression of RAD18 in tumors is associated with poor prognostic features. Modulating the expression of mutant KRAS in pancreatic cancer cells or mutant BRAF in thyroid cancer cells demonstrates a tight regulation of RAD18 expression in both cancer types. Depletion of RAD18 results in DNA damage and radiation-induced cell death. Importantly, RAD18 depletion in combination with radiotherapy results in marked and sustained tumor regression in KRAS mutant pancreatic cancer orthotopic tumors and BRAF mutant thyroid heterotopic tumors. Overall, our findings identify a novel coordinated RAS/RAF-miR-296-3p-RAD18 signaling network in pancreatic and thyroid cancer cells, which leads to enhanced radioresistance.
Asunto(s)
Proteínas de Unión al ADN , Regulación Neoplásica de la Expresión Génica , MicroARNs , Neoplasias Pancreáticas , Proteínas Proto-Oncogénicas p21(ras) , Tolerancia a Radiación , Transducción de Señal , Neoplasias de la Tiroides , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Tolerancia a Radiación/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/radioterapia , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/metabolismo , Neoplasias de la Tiroides/genética , Neoplasias de la Tiroides/radioterapia , Neoplasias de la Tiroides/patología , Neoplasias de la Tiroides/metabolismo , Línea Celular Tumoral , Animales , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteínas Proto-Oncogénicas B-raf/genética , Ratones Desnudos , Mutación , Daño del ADN , Ensayos Antitumor por Modelo de Xenoinjerto , Proteínas ras/genética , Proteínas ras/metabolismo , TransfecciónRESUMEN
Murine norovirus (MNV), which can be used as a model system to study human noroviruses, can infect macrophages/ monocytes, neutrophils, dendritic, intestinal epithelial, T and B cells, and is highly prevalent in laboratory mice. We previously showed that MNV infection significantly reduces bone marrow B cell populations in a Stat1-dependent manner. We show here that while MNV-infected Stat1-/- mice have significant losses of bone marrow B cells, splenic B cells capable of mounting an antibody response to novel antigens retain the ability to expand. We also investigated whether increased granulopoiesis after MNV infection was causing B cell loss. We found that administration of anti-G-CSF antibody inhibits the pronounced bone marrow granulopoiesis induced by MNV infection of Stat1-/- mice, but this inhibition did not rescue bone marrow B cell losses. Therefore, MNV-infected Stat1-/- mice can still mount a robust humoral immune response despite decreased bone marrow B cells. This suggests that further investigation will be needed to identify other indirect factors or mechanisms that are responsible for the bone marrow B cell losses seen after MNV infection. In addition, this work contributes to our understanding of the potential physiologic effects of Stat1-related disruptions in research mouse colonies that may be endemically infected with MNV.