Use of phosphoprotein profiling with high-density fluorescence protein microarray to reveal a response predictor to a multikinase inhibitor sorafenib.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. e15080-e15080
Author(s):  
Tesshi Yamada ◽  
Mari Masuda ◽  
Wei-Yu Chen ◽  
Chi-Long Chen ◽  
Kazufumi Honda

e15080 Background: The efficacy of sorafenib in patients with unresectable hepatocellular carcinoma (HCC) has been revealed in the recent phase III SHARP (Sorafenib HCC Assessment Randomized Protocol) clinical trial, but only a small proportion of patients achieved the anticipated therapeutic outcomes. It is essential to identify biomarkers that will discriminate such patients. Methods: We newly developed high-density fluorescence reverse-phase protein arrays and used them to profile the phosphorylation status of 180 signaling molecules in the 120 pathways registered on the NCI-Nature curated database in 23 HCC cell lines with different sensitivities to sorafenib. Results: Among the 180 signaling nodes, we found that the level of ribosomal protein S6 phosphorylated at serine residues 235/236 (p-rpS6 S235/236) was the most significantly correlated with the sensitivity of HCC cells to sorafenib. The high expression of p-rpS6 S235/236 was confirmed immunohistochemically in biopsy samples obtained from HCC patients who responded poorly to sorafenib. Sorafenib-resistant HCC cells showed constitutive activation of the mammalian target of rapamycin (mTOR) and/or the mitogen-activated protein kinase (MAPK) pathways downstream of c-RAF. Conclusions: The present results indicate that p-rpS6 S235/236 is a potential predictor of unresponsiveness to sorafenib. Cross-talk between the mTOR and MAPK signaling pathways in sorafenib-resistant HCC cell lines was also revealed. A combination of mTOR and MAPK inhibitors may improve the efficacy of molecular targeting therapy against HCC.

2016 ◽  
Vol 25 (139) ◽  
pp. 71-76 ◽  
Author(s):  
Emmanuelle Kempf ◽  
Benoît Rousseau ◽  
Benjamin Besse ◽  
Luis Paz-Ares

KRASmutations are the most frequent molecular abnormalities found in one out of four nonsmall cell lung cancers (NSCLC). Their incidence increases in cases of adenocarcinoma, smokers and Caucasian patients. Their negative value in terms of prognosis and responsiveness to both standard chemotherapy and targeted therapies remains under debate. Many drugs have been developed specifically forKRAS-mutated NSCLC patients. Direct inhibition ofRASactivation failed to show any clinical efficacy. Inhibition of downstream targets of the mitogen-activated protein kinase (MEK) pathway is a promising strategy: phase II combinations of MEK 1/2 kinase inhibitors with chemotherapy doubled patients’ clinical outcomes. One phase III trial in such a setting is ongoing. Double inhibition of MEK and epidermal growth factor receptor proteins is currently being assessed in early-phase trials. The association with mammalian target of rapamycin pathway inhibition leads to non-manageable toxicity. Other strategies, such as inhibition of molecular heat-shock proteins 90 or focal adhesion kinase are currently assessed. Abemaciclib, a cyclin-dependent kinase 4/6 inhibitor, showed promising results in a phase I trial, with a 54% disease control rate. Results of an ongoing phase III trial are warranted. Immunotherapy might be the next relevant step inKRAS-mutated NSCLC management due to the high burden of associated mutations and neo-antigens.


2021 ◽  
Vol 118 (3) ◽  
pp. e2012408118
Author(s):  
Corleone S. Delaveris ◽  
Shannon H. Chiu ◽  
Nicholas M. Riley ◽  
Carolyn R. Bertozzi

Inflammatory pathologies caused by phagocytes lead to numerous debilitating conditions, including chronic pain and blindness due to age-related macular degeneration. Many members of the sialic acid-binding immunoglobulin-like lectin (Siglec) family are immunoinhibitory receptors whose agonism is an attractive approach for antiinflammatory therapy. Here, we show that synthetic lipid-conjugated glycopolypeptides can insert into cell membranes and engage Siglec receptors in cis, leading to inhibitory signaling. Specifically, we construct a cis-binding agonist of Siglec-9 and show that it modulates mitogen-activated protein kinase (MAPK) signaling in reporter cell lines, immortalized macrophage and microglial cell lines, and primary human macrophages. Thus, these cis-binding agonists of Siglecs present a method for therapeutic suppression of immune cell reactivity.


BMC Biology ◽  
2020 ◽  
Vol 18 (1) ◽  
Author(s):  
Derek Wong ◽  
Lisa Sogerer ◽  
Samantha S. Lee ◽  
Victor Wong ◽  
Amy Lum ◽  
...  

Abstract Background Aberrations in Capicua (CIC) have recently been implicated as a negative prognostic factor in a multitude of cancer types through the derepression of targets downstream of the mitogen-activated protein kinase (MAPK) signaling cascade, such as oncogenic E26 transformation-specific (ETS) transcription factors. The Ataxin-family protein ATXN1L has previously been reported to interact with CIC in both developmental and disease contexts to facilitate the repression of CIC target genes and promote the post-translational stability of CIC. However, little is known about the mechanisms at the base of ATXN1L-mediated CIC post-translational stability. Results Functional in vitro studies utilizing ATXN1LKO human cell lines revealed that loss of ATXN1L leads to the accumulation of polyubiquitinated CIC protein, promoting its degradation through the proteasome. Although transcriptomic signatures of ATXN1LKO cell lines indicated upregulation of the mitogen-activated protein kinase pathway, ERK activity was found to contribute to CIC function but not stability. Degradation of CIC protein following loss of ATXN1L was instead observed to be mediated by the E3 ubiquitin ligase TRIM25 which was further validated using glioma-derived cell lines and the TCGA breast carcinoma and liver hepatocellular carcinoma cohorts. Conclusions The post-translational regulation of CIC through ATXN1L and TRIM25 independent of ERK activity suggests that the regulation of CIC stability and function is more intricate than previously appreciated and involves several independent pathways. As CIC status has become a prognostic factor in several cancer types, further knowledge into the mechanisms which govern CIC stability and function may prove useful for future therapeutic approaches.


2018 ◽  
pp. 1-18 ◽  
Author(s):  
Matthew J. Wongchenko ◽  
Antoni Ribas ◽  
Paolo A. Ascierto ◽  
Brigitte Dréno ◽  
Anna Maria di Giacomo ◽  
...  

Purpose The treatment of advanced BRAFV600-mutated melanomas with BRAF inhibitors (BRAFi) has improved survival, but the efficacy of BRAFi varies among individuals and the development of acquired resistance to BRAFi through reactivation of mitogen-activated protein kinase (MAPK) signaling is common. We performed an exploratory, retrospective analysis to investigate the effects of BRAFV600 allelic balance, coexisting oncogene mutations, cell proliferation signaling levels, and loss of PTEN expression on progression-free survival (PFS) in patients in the phase III coBRIM study, which compared the combination of the MEK inhibitor cobimetinib with the BRAFi vemurafenib versus vemurafenib as monotherapy. Methods Baseline tumor samples from the intention-to-treat population were analyzed by targeted deep sequencing at a median coverage of 3,600× and by immunohistochemistry for cell proliferation markers, BRAFV600E, and PTEN. The association of these biomarkers with PFS was assessed by Cox proportional hazards modeling. Gene expression in relation to loss of PTEN was profiled by RNA sequencing in 205 patient samples and 42 BRAFV600-mutated melanoma cell lines. Results Neither BRAFV600 allelic balance nor coexisting mutations in the RAS/RAF/RTK pathway affected PFS in either treatment group. Increased baseline MAPK signaling and cell proliferation did not affect PFS in patients treated with cobimetinib combined with vemurafenib. PTEN loss was associated with reduced PFS in patients treated with vemurafenib alone but not in patients treated with cobimetinib combined with vemurafenib. Conclusion Deeper inhibition of the MAPK pathway through targeting of both MEK and BRAF may override the effects of tumor heterogeneity and improve PFS in all patients with advanced BRAFV600 melanoma.


Author(s):  
Meijin Wei ◽  
Chaochao Tan ◽  
Zhouqin Tang ◽  
Yingying Lian ◽  
Ying Huang ◽  
...  

Arginine methylation catalyzed by protein arginine methyltransferases (PRMTs) performs essential roles in regulating cancer initiation and progression, but its implication in pancreatic ductal adenocarcinoma (PDAC) requires further elucidation. In this study, asymmetric dimethylarginine (ADMA)-containing peptides in PDAC cell line PANC-1 were identified by label-free quantitative proteomics combined with affinity purification, using human non-cancerous pancreatic ductal epithelium cell line HPDE6c7 as the control. In total, 289 ADMA sites in 201 proteins were identified in HPDE6c7 and PANC-1 cells, including 82 sites with lower dimethylation and 37 sites with higher dimethylation in PANC-1 cells compared with HPDE6c7 cells. These ADMA-containing peptides demonstrated significant enrichment of glycine and proline residues in both cell lines. Importantly, leucine residues were significantly enriched in ADMA-containing peptides identified only in HPDE6c7 cells or showing lower dimethylation in PANC-1 cells. ADMA-containing proteins were significantly enriched in multiple biological processes and signaling cascades associated with cancer development, such as spliceosome machinery, the Wnt/β-catenin, Hedgehog, tumor growth factor beta (TGF-β), and mitogen-activated protein kinase (MAPK) signaling pathways. Moreover, PDAC cell lines with enhanced cell viability showed lower PRMT4 protein abundance and global ADMA-containing protein levels compared with HPDE6c7. PRMT4 overexpression partially recovered ADMA-containing protein levels and repressed viability in PANC-1 cells. These results revealed significantly altered ADMA-containing protein profiles in human pancreatic carcinoma cells, which provided a basis for elucidating the pathogenic roles of PRMT-mediated protein methylation in pancreatic cancer.


2020 ◽  
Vol 21 (15) ◽  
pp. 5362
Author(s):  
Ming-Shan Chen ◽  
Hsuan-Te Yeh ◽  
Yi-Zhen Li ◽  
Wen-Chun Lin ◽  
Ying-Ray Lee ◽  
...  

Autophagy is a potential target for the treatment of triple negative breast cancer (TNBC). Because of a lack of targeted therapies for TNBC, it is vital to find optimal agents that avoid chemoresistance and metastasis. Flavopereirine has anti-proliferation ability in cancer cells, but whether it regulates autophagy in breast cancer cells remains unclear. A Premo™ Tandem Autophagy Sensor Kit was used to image the stage at which flavopereirine affects autophagy by confocal microscopy. A plasmid that constitutively expresses p-AKT and siRNA targeting p38 mitogen-activated protein kinase (MAPK) was used to confirm the related signaling pathways by Western blot. We found that flavopereirine induced microtubule-associated protein 1 light chain 3 (LC3)-II accumulation in a dose- and time-dependent manner in MDA-MB-231 cells. Confocal florescent images showed that flavopereirine blocked autophagosome fusion with lysosomes. Western blotting showed that flavopereirine directly suppressed p-AKT levels and mammalian target of rapamycin (mTOR) translation. Recovery of AKT phosphorylation decreased the level of p-p38 MAPK and LC3-II, but not mTOR. Moreover, flavopereirine-induced LC3-II accumulation was partially reduced in MDA-MB-231 cells that were transfected with p38 MAPK siRNA. Overall, flavopereirine blocked autophagy via LC3-II accumulation in autophagosomes, which was mediated by the AKT/p38 MAPK signaling pathway.


Antioxidants ◽  
2019 ◽  
Vol 8 (10) ◽  
pp. 501 ◽  
Author(s):  
María José López-Grueso ◽  
Raúl González ◽  
Jordi Muntané ◽  
José Antonio Bárcena ◽  
C. Alicia Padilla

Sorafenib is the first-line recommended therapy for patients with advanced hepatocarcinoma (HCC) in de-differentiation stage (presenting epithelial–mesenchymal transition, EMT). We studied the role of the thioredoxin system (Trx1/TrxR1) in the sensitivity or resistance of HCC cells to the treatment with Sorafenib. As a model, we used a set of three established HCC cell lines with different degrees of de-differentiation as occurs in metastasis. By quantitative proteomics, we found that the expression levels of Trx1 and TrxR1 followed the same trend as canonical EMT markers in these cell lines. Treatment with Sorafenib induced thiol redox reductive changes in critical elements of oncogenic pathways in all three cell lines but induced drastic proteome reprograming only in HCC cell lines of intermediate stage. Trx1 downregulation counteracted the thiol reductive effect of Sorafenib on Signal Transducer and Activator of Transcription 3 (STAT3) but not on Mitogen-Activated Protein Kinase (MAPK) or Protein Kinase B (Akt) and transformed advanced HCC cells into Sorafenib-sensitive cells. Ten targets of the combined Sorafenib–siRNATrx1 treatment were identified that showed a gradually changing expression trend in parallel to changes in the expression of canonical EMT markers, likely as a result of the activation of Hippo signaling. These findings support the idea that a combination of Sorafenib with thioredoxin inhibitors should be taken into account in the design of therapies against advanced HCC.


Pharmaceutics ◽  
2021 ◽  
Vol 13 (1) ◽  
pp. 112
Author(s):  
Jisoo Song ◽  
Jiyeon Ham ◽  
Taeyeon Hong ◽  
Gwonhwa Song ◽  
Whasun Lim

Fraxetin is a coumarin scaffold compound extracted from Fraxinus rhynchophylla. It has antioxidant, anti-inflammatory, hepatoprotective, and antifibrotic effects. Furthermore, fraxetin has anticancer effects in breast and lung cancer. We aimed to evaluate whether fraxetin has anticancer activity in hepatocellular carcinoma (HCC) cells and its underlying mechanism. We demonstrated the anticancer effects of fraxetin in the HCC cell lines Huh7 and Hep3B. We confirmed that fraxetin inhibited cell proliferation (42% ± 10% Huh7; 52% ± 7% Hep3B) by arresting the cell cycle and inducing apoptosis in both cell lines. Moreover, fraxetin increased reactive oxygen species production (221% ± 55% Huh7; 460% ± 73% Hep3B), depolarized the mitochondrial membranes (ΔΨm) (345% ± 160% Huh7; 462% ± 140% Hep3B), and disrupted calcium homeostasis in both HCC cell lines. Chelating calcium ions with BAPTA-AM restored proliferation in fraxetin-treated Huh7 cells but not in Hep3B cells. Fraxetin did not affect the phosphorylation of extracellular-signal-regulated kinase 1/2, whereas it decreased JNK and phosphoinositide 3-kinase signaling. Furthermore, fraxetin and mitogen-activated protein kinase pharmacological inhibitors had synergistic antiproliferative effects on HCC cells. Although our study was limited to in vitro data that require validation, we suggest that fraxetin is a potential therapeutic agent against HCC progression.


2021 ◽  
Vol 19 (1) ◽  
pp. 1233-1243
Author(s):  
Yanning Ma ◽  
Dongheng Huang ◽  
Xingtong Li ◽  
Wanqin Cheng ◽  
Xiaomin Huang ◽  
...  

Abstract This study aimed to investigate the key role and mechanism of GADD45B in the radiation resistance of nasopharyngeal carcinoma (NPC) cell lines. Radiotherapy-resistant HONE1 (HONE1-R) cells with stable genetic radioresistance were cultured under continuous radiation stimulation. CCK-8 and clone formation assays were used to verify the radioresistance of the cell line. Transcriptome sequencing was used to identify the most important differential signaling pathway in the cell line. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot analysis were used to verify the sequencing results. GADD45B-siRNA was used to knock down the key gene so as to verify the downstream gene expression and analyze its mechanism. The transcriptome analysis showed that 702 genes were upregulated and 772 genes were downregulated in the HONE1-R cell lines. The core differential signaling pathway was mitogen-activated protein kinase (MAPK) signaling pathway, and the core differential gene was GADD45B. After GADD45B was knocked down, the cell viability and proliferation ability of HONE1-R cell lines significantly decreased under radiation, and the expression of cyclin B1 and p-CDK1 decreased significantly. MAPK is the core signaling pathway in radioresistance of NPC. GADD45B plays an important role by affecting cell viability and proliferation in NPC radioresistance. GADD45B is a potential target of radioresistance in NPC.


2012 ◽  
Vol 03 (04) ◽  
pp. 200-200
Author(s):  
Birgit Pohlmann

Eine potenzielle neue Standardtherapie für Patienten mit metastasiertem bzw. inoperablem GIST nach Imatinib-und Sunitinib-Versagen sieht Prof. Carsten Bokemeyer, Hamburg, in dem oralen Multikinase-Inhibitor Regorafenib. In der randomisierten Phase-III-Studie GRID wurde Regorafenib als Drittlinientherapie nach Imatinib- und Sunitinib-Versagen gegen best-supportiv-care verglichen und erreichte eine statistisch hoch signifikante Verlängerung der PFS, dem primären Studienendpunkt (4,8 vs. 0,9 Monaten; HR 0,27; p˂0,0001).


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