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1.
J Immunother Cancer ; 8(1)2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31959728

RESUMO

BACKGROUND: Immunotherapy has become an important treatment option for patients with advanced non-small cell lung cancer (NSCLC). At present, none of these existing biomarkers can effectively stratify true responders and there is an urgent need for identifying novel biomarkers. Exosomes derived from the serum of patients with cancer have been proven to be reliable markers for cancer diagnosis. Here, we explored the possibility of using plasma-derived exosomal microRNAs as potential biomarkers for optimal selection of patients with advanced EGFR / ALK negative NSCLC to immunotherapy. METHODS: From June 2017 to February 2019, 30 patients with advanced EGFR / ALK wild-type (WT) NSCLC who received PD-1/PD-L1 inhibitors were enrolled. The efficacy evaluation was conducted after every three cycles of treatment according to RECIST 1.1. Plasma samples of these patients were collected before the administration of PD-1/PD-L1 inhibitors as baseline, and after every three cycles if the patients achieved partial response (PR) or complete response. Plasma from seven healthy individuals was also collected as normal control. Exosomes were prepared by ultracentrifugation followed by total RNA extraction, and exosome-derived miRNAs were profiled using small RNA next-generation sequencing followed by differential expression analysis. RESULTS: In order to identify biomarker for better response, all five patients who achieved PR and four patients with progressive disease (PD) at efficacy evaluation were included for differential expression analysis. Based on unsupervised hierarchical clustering, exosomal miRNA expression profile was significantly altered in patients with NSCLC compared with normal controls with a total of 155 differentially expressed exosomal miRNAs. Interestingly, hsa-miR-320d, hsa-miR-320c, and hsa-miR-320b were identified significantly upregulated in the PD groups compared with the PR group at baseline before the treatment. In addition, we identified that hsa-miR-125b-5p, a T-cell suppressor, showed a trend of increased expression in the PD group at baseline and was significantly downregulated in the post-treatment plasma exosomes compared with pre-treatment samples of the PR patients. CONCLUSION: Patients with NSCLC represent unique plasma exosomal miRNA profiles. Hsa-miR-320d, hsa-miR-320c, and hsa-miR-320b were identified as potential biomarkers for predicting the efficacy of immunotherapy in advanced NSCLCs. When T-cell suppressor hsa-miR-125b-5p was downregulated during the treatment, the patients may obtain increased T-cell function and respond well to immunotherapy.

2.
Mol Genet Genomic Med ; 8(2): e1079, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31867841

RESUMO

BACKGROUND: One of the major challenges in managing invasive breast cancer (BC) is the lack of reliable biomarkers to track response. Circulating tumor DNA (ctDNA) from liquid biopsy, as a candidate biomarker, provides a valuable assessment of BC patients. In this retrospective study, we evaluated the utility of ctDNA to reflect the efficacy of treatment and to monitor resistance mechanisms. METHODS: Targeted next-generation sequencing (NGS) of 416 cancer-relevant genes was performed on 41 plasma biopsy samples of 19 HER2+ and 12 HER2- BC patients. Longitudinal ctDNA samples were analyzed in three BC patients over the treatment course for detecting acquired mutations. RESULTS: In HER2+ BC patients, ERBB2 somatic copy numbers in ctDNA samples were significantly higher in patients progressed on HER2-targeted therapy than those who were still responding to the treatment. Recurrent acquired mutations were detected in genes including ERBB2, TP53, EGFR, NF1, and SETD2, which may contribute to trastuzumab resistance. In longitudinal analyses, the observed mutation allele frequencies were tracked closely in concordance with treatment responses. A novel ERBB2 p.(Leu869Arg) mutation was acquired in one patient upon resistant to trastuzumab therapy, which was further validated as an oncogenic mutation in vitro and contributed to resistance. In HER2- BC patients with chemotherapy resistance, genetic alterations on TP53, PIK3CA, and DNA damage repair genes were frequently observed. CONCLUSIONS: In summary, ctDNA monitoring, particularly longitudinal analyses, provides valuable insights into the assessment of targeted therapy efficacy and gene alterations underlying trastuzumab resistance and chemotherapy resistance in HER2+ and HER2- BC patients, respectively.

3.
J Mol Cell Biol ; 2019 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-31863092

RESUMO

Faithful segregation of mitotic chromosomes requires bi-orientation of sister chromatids, which relies on the sensing of correct attachments between spindle microtubules and kinetochores. Although the mechanisms underlying PLK1 activation have been extensively studied, the regulatory mechanisms that couple PLK1 activity to accurate chromosome segregation are not well understood. In particular, PLK1 is implicated in stabilizing kinetochore-microtubule attachments, but how kinetochore PLK1 activity is regulated to avoid hyperstabilized kinetochore-microtubules in mitosis remains elusive. Here, we show that kinetochore PLK1 kinase activity is modulated by SET7/9 via lysine methylation during early mitosis. The SET7/9-elicited dimethylation occurs at the Lys191 of PLK1, which tunes down its activity by limiting ATP utilization. Overexpression of the non-methylatable PLK1 mutant or chemical inhibition of SET7/9 methyltransferase activity resulted in mitotic arrest due to destabilized kinetochore-microtubule attachments. These data suggest that kinetochore PLK1 is essential for stable kinetochore-microtubule attachments and methylation by SET7/9 promotes dynamic kinetochore-microtubule attachments for accurate error correction. Our findings define a novel homeostatic regulation at the kinetochore that integrates protein phosphorylation and methylation with accurate chromosome segregation for maintenance of genomic stability.

4.
Sci Rep ; 9(1): 13029, 2019 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-31506556

RESUMO

Prothrombin complex concentrates (PCC) are fractionated plasma protein drugs that reverse warfarin anticoagulation. PCC may control more general bleeding. We sought to identify the dominant procoagulant factor in PCC in vivo. We tested PCC or coagulation factor (F) treatment in CD1 mice made coagulopathic by exchange of whole blood for washed red cells. Anesthetized mice were transfused with murine fresh-frozen plasma (mFFP), PCC, mixtures of human vitamin K-dependent proteins (VKDP) (prothrombin, FVII, FIX, or FX), or purified single human VKDP, immediately prior to tail transection (TT), liver laceration (LL), or intravascular laser injury (ILI). Plasma donor mice were treated with vehicle or control antisense oligonucleotide (ASO-CON) or ASO specific for prothrombin (FII) (ASO-FII) to yield mFFP or ASO-CON mFFP or ASO-FII mFFP. Blood losses were determined spectrophotometrically (TT) or gravimetrically (LL). Thrombus formation was quantified by intravital microscopy of laser-injured arterioles. PCC or four factor- (4F-) VKDP or prothrombin significantly reduced bleeding from TT or LL. Omission of prothrombin from 4F-VKDP significantly reduced its ability to limit bleeding. Mice transfused with ASO-FII mFFP demonstrated inferior haemostasis versus those transfused with ASO-FII following TT, LL, or ILI. Prothrombin is the dominant procoagulant component of PCC and could limit bleeding in trauma.

5.
Future Oncol ; 15(22): 2585-2593, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31339066

RESUMO

Aim: Crizotinib has been used to counter MET amplification in different human malignancies. However, transient responses were observed in some patients with rapid acquisition of resistant mutations in MET. Materials & methods: MET mutations stably expressed Ba/F3 cell lines were used for IC50 detection. Signaling pathway analysis was done using 293T cell line. Results: Four MET mutations conferred resistance to crizotinib with sustained activation of downstream signaling pathways of MET. On the other hand, the four MET mutations displayed different response to type II tyrosine kinase inhibitors with variable deterioration of the downstream signals. Conclusion: This study suggested that patients carrying MET V1092L, D1228G or Y1230H mutations could benefit from type II tyrosine kinase inhibitor treatment, but not patients with G1163R or D1228Y/N mutations.


Assuntos
Crizotinibe/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-met/genética , Neoplasias Gástricas/tratamento farmacológico , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Crizotinibe/efeitos adversos , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Mutação , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia
7.
J Thorac Oncol ; 14(4): 732-736, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30610926

RESUMO

INTRODUCTION: Inherited genetic determinants of lung cancer risk remain relatively elusive. Germline mutations in EGFR and erb-b2 receptor tyrosine kinase 2 (ERBB2) have been previously reported in lung cancers that may be associated with genetic susceptibility to lung cancer. METHODS: We retrospectively analyzed a cohort of 12,833 Chinese lung cancer patients tested by targeted next-generation sequencing. Patients with EGFR and ERBB2 germline mutations were identified, and their clinical information and family history were summarized. Growth factor independency of EGFR germline mutations was further analyzed in vitro. RESULTS: Eight different heterozygous EGFR germline mutations from 14 adenocarcinoma patients (0.12%) were identified within or adjacent to the kinase domain, including K757R (n = 5), R831H (n = 2), D1014N (n = 2), G724S, V786M, T790M, L792F, and L844V. Only one patient harbored the ERBB2-V1128I germline mutation. Five of 15 patients had family history of cancer. Notably, the patient with EGFR-T790M germline mutation had multiple maternal family members diagnosed with lung cancers, strongly supporting its role in inherited lung cancer. Concurrent known somatic driver mutations were not detected in 5 patients at diagnosis, 1 of whom harbored the EGFR-L844V germline mutation and showed superior response to afatinib. Consistently, EGFR-K757R and L844V mutations were able to be interleukin 3 - independent in vitro and were sensitive to EGFR tyrosine kinase inhibitors. CONCLUSIONS: EGFR/ERBB2 germline mutations were found to be rare in Chinese lung cancer patients with more diversity other than the previously reported EGFR-T790M, with EGFR-K757R being the most common EGFR germline mutation. Patients with EGFR germline mutations without other known driver mutations might benefit from tyrosine kinase inhibitor treatment.

8.
Blood ; 132(6): 622-634, 2018 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-29794068

RESUMO

Thrombopoietin (TPO), a hematopoietic growth factor produced predominantly by the liver, is essential for thrombopoiesis. Prevailing theory posits that circulating TPO levels are maintained through its clearance by platelets and megakaryocytes via surface c-Mpl receptor internalization. Interestingly, we found a two- to threefold decrease in circulating TPO in GPIbα-/- mice compared with wild-type (WT) controls, which was consistent in GPIbα-deficient human Bernard-Soulier syndrome (BSS) patients. We showed that lower TPO levels in GPIbα-deficient conditions were not due to increased TPO clearance by GPIbα-/- platelets but rather to decreased hepatic TPO mRNA transcription and production. We found that WT, but not GPIbα-/-, platelet transfusions rescued hepatic TPO mRNA and circulating TPO levels in GPIbα-/- mice. In vitro hepatocyte cocultures with platelets or GPIbα-coupled beads further confirm the disruption of platelet-mediated hepatic TPO generation in the absence of GPIbα. Treatment of GPIbα-/- platelets with neuraminidase caused significant desialylation; however, strikingly, desialylated GPIbα-/- platelets could not rescue impaired hepatic TPO production in vivo or in vitro, suggesting that GPIbα, independent of platelet desialylation, is a prerequisite for hepatic TPO generation. Additionally, impaired hepatic TPO production was recapitulated in interleukin-4/GPIbα-transgenic mice, as well as with antibodies targeting the extracellular portion of GPIbα, demonstrating that the N terminus of GPIbα is required for platelet-mediated hepatic TPO generation. These findings reveal a novel nonredundant regulatory role for platelets in hepatic TPO homeostasis, which improves our understanding of constitutive TPO regulation and has important implications in diseases related to GPIbα, such as BSS and auto- and alloimmune-mediated thrombocytopenias.


Assuntos
Síndrome de Bernard-Soulier/sangue , Plaquetas/fisiologia , Fígado/metabolismo , Complexo Glicoproteico GPIb-IX de Plaquetas/fisiologia , Trombopoetina/biossíntese , Animais , Síndrome de Bernard-Soulier/genética , Células Cultivadas , Glicosilação , Hepatócitos/metabolismo , Homeostase , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Ácido N-Acetilneuramínico/metabolismo , Transfusão de Plaquetas , Domínios Proteicos , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/metabolismo , Trombopoetina/sangue
9.
J Mol Cell Biol ; 4(5): 331-40, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22831836

RESUMO

Histone methylation performs multiple functions such as DNA replication, transcription regulation, heterochromatin formation, and chromatin condensation. How this methylation gradient is orchestrated in the centromere during chromosome segregation is not known. Here we examine the temporal dynamics of protein methylation in the centromere by SUV39H1 methyltransferase, a key mitotic regulator, using fluorescence resonance energy transfer-based sensors in living HeLa cells and immunofluorescence of native SUV39H1 substrates. A quantitative analysis of methylation dynamics, using centromere-targeted sensors, reveals a temporal change during chromosome segregation. These dynamics result in an accurate chromosome congression to and alignment at the equator as an inhibition of methylation dynamics using SUV39H1 inhibitor perturbs chromosome congression in living HeLa cells. Surprisingly, this inhibition of methylation results in a brief increase in Aurora B kinase activity and an enrichment of microtubule depolymerase MCAK in the centromere with a concomitant kinetochore-microtubule destabilization and a reduced tension across the sister kinetochores with ultimate chromosome misalignments. We reason that SUV39H1 generates a gradient of methylation marks at the kinetochore that provides spatiotemporal information essential for accurate chromosome segregation in mitosis.


Assuntos
Centrômero/metabolismo , Segregação de Cromossomos/fisiologia , Metiltransferases/metabolismo , Mitose , Proteínas Repressoras/metabolismo , Aurora Quinase B , Aurora Quinases , Células HeLa , Humanos , Cinetocoros/metabolismo , Metilação , Metiltransferases/genética , Microtúbulos/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Repressoras/genética
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