RESUMEN
Neurofibromin 1 (NF1) is a tumor suppressor that has been previously reported to regulate RASMAPK signaling. The present study investigated the possible relationship between NF1 expression and antiEGFR antibody (cetuximab) sensitivity in colorectal cancer cell lines. In addition, primary or metastatic colorectal cancer samples from patients treated with cetuximab were assessed for the association of cetuximab sensitivity. The quantities of the NF1 transcript, NF1related pathway enrichment and NF1 mutation profile were measured and investigated using RNA sequencing and targeted DNA sequencing. Based on growth inhibition and colony formation assay results, cell lines were designated to be cetuximabsensitive (NCIH508 and Caco2) or cetuximabresistant (KM12C and SM480). Western blotting revealed NF1 was highly expressed in cetuximabsensitive cell lines whilst there was little expression in their cetuximabresistant counterparts. Knocking down NF1 expression using small interfering RNA in the cetuximabsensitive cell lines enhanced the phosphorylation of MEK and ERK according to western blotting. NF1 knockdown also reduced apoptosis, as observed by the decreased number of apoptotic bodies by DAPI nuclear staining and reduced cleavage of caspase and poly(ADP ribose) polymerase. NF1 overexpression by transfection with GTPaseactivating proteinrelated domain subunit rendered the cetuximabresistant cell lines, KM12C and SW480, more susceptible to cetuximabinduced apoptosis. RNA sequencing of 111 RAS and BRAFV600 wildtype tumor samples collected from cetuximabtreated patients with metastatic colorectal cancer revealed that the pretreatment NF1 expression levels were not associated with the cetuximab response. However, tumor samples obtained after cetuximab treatment displayed slightly lower NF1 transcript levels compared with those in the pretreatment samples, suggesting that exposure to the antiEGFR antibody may be associated with reduced NF1 expression levels. Nextgeneration sequencing revealed that the frequency of inactivating mutations in NF1 were rare (1.8%) in patients with colorectal cancer and were not associated with the protein expression levels of NF1 except for in a small number of cases (0.5%), where the biallelic inactivation of NF1 was observed. To conclude, the present study showed that modification of NF1 expression can affect sensitivity to cetuximab in colorectal cancer cell lines, though a limitation exists in terms of its potential application as a biomarker for RAS and BRAFV600 wildtype tumors.
Asunto(s)
Cetuximab/administración & dosificación , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Resistencia a Antineoplásicos/genética , Neurofibromina 1/genética , Células CACO-2 , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , HumanosRESUMEN
Transcription factor EB (TFEB) is a master regulator of lysosomal function and autophagy. In addition, TFEB has various physiological roles such as nutrient sensing, cellular stress responses, and immune responses. However, the precise roles of TFEB in pancreatic cancer growth remain unclear. Here, we show that pancreatic cancer cells exhibit a significantly elevated TFEB expression compared with normal tissue samples and that the genetic inhibition of TFEB results in a significant inhibition in both glutamine and mitochondrial metabolism, which in turn suppresses the PDAC growth both in vitro and in vivo. High basal levels of autophagy are critical for pancreatic cancer growth. The TFEB knockdown had no significant effect on the autophagic flux under normal conditions but interestingly caused a profound reduction in glutaminase (GLS) transcription, leading to an inhibition of glutamine metabolism. We observed that the direct binding of TFEB to the GLS and TFEB gene promotors regulates the transcription of GLS. We also found that the glutamate supplementation leads to a significant recovery of the PDAC growth that had been reduced by a TFEB knockdown. Taken together, our current data demonstrate that TFEB supports the PDAC cell growth by regulating glutaminase-mediated glutamine metabolism.
RESUMEN
Branched-chain amino acid (BCAA) catabolism and high levels of enzymes in the BCAA metabolic pathway have recently been shown to be associated with cancer growth and survival. However, the precise roles of BCAA metabolism in cancer growth and survival remain largely unclear. Here, we found that BCAA metabolism has an important role in human pancreatic ductal adenocarcinoma (PDAC) growth by regulating lipogenesis. Compared with nontransformed human pancreatic ductal (HPDE) cells, PDAC cells exhibited significantly elevated BCAA uptake through solute carrier transporters, which were highly upregulated in pancreatic tumor tissues compared with normal tissues. Branched-chain amino-acid transaminase 2 (BCAT2) knockdown markedly impaired PDAC cell proliferation, but not HPDE cell proliferation, without significant alterations in glutamate or reactive oxygen species levels. Furthermore, PDAC cell proliferation, but not HPDE cell proliferation, was substantially inhibited upon knockdown of branched-chain α-keto acid dehydrogenase a (BCKDHA). Interestingly, BCKDHA knockdown had no significant effect on mitochondrial metabolism; that is, neither the level of tricarboxylic acid cycle intermediates nor the oxygen consumption rate was affected. However, BCKDHA knockdown significantly inhibited fatty-acid synthesis, indicating that PDAC cells may utilize BCAAs as a carbon source for fatty-acid biosynthesis. Overall, our findings show that the BCAA metabolic pathway may provide a novel therapeutic target for pancreatic cancer.
Asunto(s)
Aminoácidos de Cadena Ramificada/metabolismo , Metabolismo de los Lípidos/fisiología , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Adenosina Trifosfato/metabolismo , Animales , Carcinoma Ductal Pancreático/metabolismo , Proliferación Celular/fisiología , Femenino , Ácido Glutámico/metabolismo , Humanos , Lentivirus/genética , Metabolómica/métodos , Ratones SCID , Antígenos de Histocompatibilidad Menor/metabolismo , Consumo de Oxígeno/fisiología , Proteínas Gestacionales/metabolismo , ARN Mensajero/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Transaminasas/metabolismoRESUMEN
Matrine is a natural compound extracted from the herb Sophora flavescens Ait which is widely used in traditional Chinese medicine for treating various diseases. Recently, matrine was reported to have antitumor effects against a variety of cancers without any obvious side effects; however, the molecular mechanisms of its antiproliferative effects on cancer are unclear. Here, we report that matrine inhibits autophagy-mediated energy metabolism, which is necessary for pancreatic cancer growth. We found that matrine significantly reduces pancreatic cancer growth in vitro and in vivo by insufficiently maintaining mitochondrial metabolic function and energy level. We also found that either pyruvate or α-ketoglutarate supplementation markedly rescues pancreatic cancer cell growth following matrine treatment. Inhibition of mitochondrial energy production results from matrine-mediated autophagy inhibition by impairing the function of lysosomal protease. Matrine-mediated autophagy inhibition requires stat3 downregulation. Furthermore, we found that the antitumor effect of matrine on pancreatic cancer growth depends on the mutation of the KRAS oncogene. Together, our data suggest that matrine can suppress the growth of KRAS-mutant pancreatic cancer by inhibiting autophagy-mediated energy metabolism.