RESUMEN
BRAF mutations occur early in serrated colorectal cancers, but their long-term influence on tissue homeostasis is poorly characterized. We investigated the impact of short-term (3 days) and long-term (6 months) expression of BrafV600E in the intestinal tissue of an inducible mouse model. We show that BrafV600E perturbs the homeostasis of intestinal epithelial cells, with impaired differentiation of enterocytes emerging after prolonged expression of the oncogene. Moreover, BrafV600E leads to a persistent transcriptional reprogramming with enrichment of numerous gene signatures indicative of proliferation and tumorigenesis, and signatures suggestive of metabolic rewiring. We focused on the top-ranking cholesterol biosynthesis signature and confirmed its increased expression in human serrated lesions. Functionally, the cholesterol lowering drug atorvastatin prevents the establishment of intestinal crypt hyperplasia in BrafV600E-mutant mice. Overall, our work unveils the long-term impact of BrafV600E expression in intestinal tissue and suggests that colorectal cancers with mutations in BRAF might be prevented by statins.
Asunto(s)
Neoplasias Colorrectales , Proteínas Proto-Oncogénicas B-raf , Animales , Humanos , Ratones , Colesterol , Neoplasias Colorrectales/genética , Metabolismo de los Lípidos , Proteínas Proto-Oncogénicas B-raf/genética , Activación TranscripcionalRESUMEN
Research into the metabolism of the non-essential amino acid (NEAA) proline in cancer has gained traction in recent years. The last step in the proline biosynthesis pathway is catalyzed by pyrroline-5-carboxylate reductase (PYCR) enzymes. There are three PYCR enzymes: mitochondrial PYCR1 and 2 and cytosolic PYCR3 encoded by separate genes. The expression of the PYCR1 gene is increased in numerous malignancies and correlates with poor prognosis. PYCR1 expression sustains cancer cells' proliferation and survival and several mechanisms have been implicated to explain its oncogenic role. It has been suggested that the biosynthesis of proline is key to sustain protein synthesis, support mitochondrial function and nucleotide biosynthesis. However, the links between proline metabolism and cancer remain ill-defined and are likely to be tissue specific. Here we use a combination of human dataset, human tissue and mouse models to show that the expression levels of the proline biosynthesis enzymes are significantly increased during colorectal tumorigenesis. Functionally, the expression of mitochondrial PYCRs is necessary for cancer cells' survival and proliferation. However, the phenotypic consequences of PYCRs depletion could not be rescued by external supplementation with either proline or nucleotides. Overall, our data suggest that, despite the mechanisms underlying the role of proline metabolism in colorectal tumorigenesis remain elusive, targeting the proline biosynthesis pathway is a suitable approach for the development of novel anti-cancer therapies.
Asunto(s)
Neoplasias ColorrectalesRESUMEN
Cancer is a disease of cellular evolution where single base changes in the genetic code can have significant impact on the translation of proteins and their activity. Thus, in cancer research there is significant interest in methods that can determine mutations and identify the significant binding sites (epitopes) of antibodies to proteins in order to develop novel therapies. Nano molecularly imprinted polymers (nanoMIPs) provide an alternative to antibodies as reagents capable of specifically capturing target molecules depending on their structure. In this study, we used nanoMIPs to capture KRAS, a critical oncogene, to identify mutations which when present are indicative of oncological progress. Herein, coupling nanoMIPs (capture) and liquid chromatography-mass spectrometry (detection), LC-MS has allowed us to investigate mutational assignment and epitope discovery. Specifically, we have shown epitope discovery by generating nanoMIPs to a recombinant KRAS protein and identifying three regions of the protein which have been previously assigned as epitopes using much more time-consuming protocols. The mutation status of the released tryptic peptide was identified by LC-MS following capture of the conserved region of KRAS using nanoMIPS, which were tryptically digested, thus releasing the sequence of a non-conserved (mutated) region. This approach was tested in cell lines where we showed the effective genotyping of a KRAS cell line and in the plasma of cancer patients, thus demonstrating its ability to diagnose precisely the mutational status of a patient. This work provides a clear line-of-sight for the use of nanoMIPs to its translation from research into diagnostic and clinical utility.
Asunto(s)
Impresión Molecular , Nanopartículas , Humanos , Espectrometría de Masas , Mutación , Proteínas Proto-Oncogénicas p21(ras)/genéticaRESUMEN
BACKGROUND: The dietary polyphenol resveratrol prevents various malignancies in preclinical models, including prostate cancer. Despite attempts to translate findings to humans, gaps remain in understanding pharmacokinetic-pharmacodynamic relations and how tissue concentrations affect efficacy. Such information is necessary for dose selection and is particularly important given the low bioavailability of resveratrol. OBJECTIVES: This study aimed to determine concentrations of resveratrol in prostate tissue of men after a dietary-achievable (5 mg) or pharmacological (1 g) dose. We then examined whether clinically relevant concentrations of resveratrol/its metabolites had direct anticancer activity in prostate cell lines. METHODS: A window trial was performed in which patients were allocated to 5 mg or 1 g resveratrol daily, or no intervention, before prostate biopsy. Patients (10/group) ingested resveratrol capsules for 7-14 d before biopsy, with the last dose [14C]-labeled, allowing detection of resveratrol species in prostate tissue using accelerator MS. Cellular uptake and antiproliferative properties of resveratrol/metabolites were assessed in cancer and nonmalignant cell cultures using HPLC with UV detection and cell counting, respectively. RESULTS: [14C]-Resveratrol species were detectable in prostate tissue of all patients analyzed, with mean ± SD concentrations of 0.08 ± 0.04 compared with 22.1 ± 8.2 pmol/mg tissue for the 5 mg and the 1 g dose, respectively. However, total [14C]-resveratrol equivalents in prostate were lower than we previously reported in plasma and colorectum after identical doses. Furthermore, resveratrol was undetectable in prostate tissue; instead, sulfate and glucuronide metabolites dominated. Although resveratrol reduced prostate cell numbers in vitro over 7 d, the concentrations required (≥10 µM) exceeded the plasma maximum concentration. Resveratrol mono-sulfates and glucuronides failed to consistently inhibit cell growth, partly due to poor cellular uptake. CONCLUSIONS: Low tissue concentrations of resveratrol species, coupled with weak antiproliferative activity of its conjugates, suggest daily doses of ≤1 g may not have direct effects on human prostate.This trial was registered at clinicaltrialsregister.eu as EudraCT 2007-002131-91.
Asunto(s)
Próstata/metabolismo , Resveratrol/metabolismo , Resveratrol/farmacocinética , Administración Oral , Antioxidantes/administración & dosificación , Antioxidantes/metabolismo , Antioxidantes/farmacocinética , Antioxidantes/uso terapéutico , Radioisótopos de Carbono , Línea Celular Tumoral , Dieta , Relación Dosis-Respuesta a Droga , Vías de Administración de Medicamentos , Humanos , Marcaje Isotópico , Masculino , Neoplasias de la Próstata/prevención & control , Resveratrol/administración & dosificación , Resveratrol/uso terapéuticoRESUMEN
Tamoxifen is contraindicated during pregnancy but many births have been reported in breast cancer patients taking this drug and numbers might be expected to increase with FDA approval of tamoxifen for risk reduction in women at high, risk of breast cancer. The neonatal mouse, exquisitely sensitive to xenobiotic estrogens, has been used to investigate the effects of short-term oral dosing with tamoxifen (1 mg/kg on days 2-5 after birth) on long-term changes in uterine pathology and gene expression. Increased adenomyosis incidence and severity was evident in the tamoxifen-treated mice with increasing age. Uterine weights in treated mice remained lower than the corresponding controls up until 9 months, after which they became greater but during life-time studies (up to 36 months), there was no development of uterine tumours. Pathological examination of uterine tissues showed there to be extensive down-growth of endometrial glands and stroma into thickened, abnormal myometrium that had disorganised fascicles of smooth muscle and increased interstitial collagen deposition. In advanced cases, the endometrial epithelium showed mild degrees of focal hyperplasia and squamous metaplasia but no atypical cytology suggestive of premalignant change. Microarray analysis of uterine RNA taken at 1.5, 3, 6, 9 and 12 months showed from 4500 ESTs, only 12 genes were continuously over-expressed by tamoxifen treatment over this time, while none was continuously down-regulated. Up-regulated genes include those for nerve growth factor (Ngfa), cathepsin B (Ctsb), transforming growth factor beta induced (Tqfbi) and collagens (Colla1, Colla2). Results provide a basis for understanding the mechanism for tamoxifen induced tissue remodelling and the development of adenomyosis.