mTOR inhibitor everolimus reduces invasiveness of melanoma cells

Abstract The mammalian target of rapamycin (mTOR) plays a key role in several cellular processes: proliferation, survival, invasion, and angiogenesis, and therefore, controls cell behavior both in health and in disease. Dysregulation of the mTOR signaling is involved in some of the cancer hallmarks, and thus the mTOR pathway is an important target for the development of a new anticancer therapy. The object of this study is recognition of the possible role of mTOR kinase inhibitors—everolimus single and in combination with selected downstream protein kinases inhibitors: LY294002 (PI3 K), U0126 (ERK1/2), GDC-0879 (B-RAF), AS-703026 (MEK), MK-2206 (AKT), PLX-4032 (B-RRAF) in cell invasion in malignant melanoma. Treatment of melanoma cells with everolimus led to a significant decrease in the level of both phosphorylated: mTOR (Ser2448) and mTOR (Ser2481) as well as their downstream effectors. The use of protein kinase inhibitors produced a significant decrease in metalloproteinases (MMPs) activity, as well as diminished invasion, especially when used in combination. The best results in the inhibition of both MMPs and cell invasiveness were obtained for the combination of an mTOR inhibitor— everolimus with a B-RAF inhibitor—PLX-4032. Slightly less profound reduction of invasiveness was obtained for the combinations of an mTOR inhibitor—everolimus with ERK1/2 inhibitor—U126 or MEK inhibitor—AS-703026 and in the case of MMPs activity decrease for PI3 K inhibitor—LY294002 and AKT inhibitor—MK-2206. The simultaneous use of everolimus or another new generation rapalog with selected inhibitors of crucial signaling kinases seems to be a promising concept in cancer treatment.

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Receptor-mediated endocytosis of urokinase-type plasminogen activator is regulated by cAMP-dependent protein kinase

Journal of Cell Science ◽

10.1242/jcs.110.12.1395 ◽

1997 ◽

Vol 110 (12) ◽

pp. 1395-1402 ◽

Cited By ~ 1

Author(s):

L. Goretzki ◽

B.M. Mueller

Keyword(s):

Protein Kinase ◽

Plasminogen Activator ◽

Kinase Inhibitors ◽

Melanoma Cells ◽

Protein Kinase Inhibitors ◽

Dependent Protein Kinase ◽

Camp Dependent Protein Kinase ◽

Type Plasminogen Activator ◽

Urokinase Type Plasminogen Activator ◽

Dependent Protein

Internalization of the urokinase-type plasminogen activator (uPA) requires two receptors, the uPA receptor (uPAR) and the low density lipoprotein receptor-related protein (LRP)/alpha2-macroglobulin (alpha2M) receptor. Here, we address whether protein kinases are involved in the internalization of uPA by human melanoma cells. Initially, we found that the internalization of uPA was significantly inhibited by the serine/threonine protein kinase inhibitors staurosporine, K-252a and H-89, but not by the tyrosine kinase inhibitors, genistein and lavendustin A. Internalization of uPA was also inhibited by a pseudosubstrate peptide for cAMP-dependent protein kinase (PKA), but not by a pseudosubstrate peptide for protein kinase C. We confirmed a requirement for PKA-activity and implicated a specific isoform by using an antisense oligonucleotide against the regulatory subunit RI alpha of PKA which suppresses PKA-I activity. Exposure of cells to this oligonucleotide led to a specific, dose-dependent decrease in RI alpha protein and to a significant inhibition in the rate of uPA internalization. We further demonstrate that treatment of melanoma cells with either H-89 or PKA RI alpha antisense oligonucleotides also resulted in a decreased internalization of two other ligands of LRP, activated alpha2M and lactoferrin, indicating that PKA activity is associated with LRP. Finally, we demonstrate that PKA activity is also required for the internalization of transferrin, but not for the internalization of the epidermal growth factor or adenovirus 2, suggesting that in melanoma cells, PKA activity is not generally required for clathrin-mediated endocytosis, but is rather associated with specific internalization receptors.

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CD133 Is Associated with Increased Melanoma Cell Survival after Multikinase Inhibition

Journal of Oncology ◽

10.1155/2019/6486173 ◽

2019 ◽

Vol 2019 ◽

pp. 1-19 ◽

Cited By ~ 3

Author(s):

Cynthia M. Simbulan-Rosenthal ◽

Anirudh Gaur ◽

Hengbo Zhou ◽

Maryam AbdusSamad ◽

Qing Qin ◽

...

Keyword(s):

Drug Resistance ◽

Kinase Inhibitors ◽

Braf Inhibitor ◽

Mek Inhibitor ◽

Melanoma Cells ◽

Stem Cell Marker ◽

High Dose ◽

Recurrence Rates ◽

Mapk Inhibitors

FDA-approved kinase inhibitors are now used for melanoma, including combinations of the MEK inhibitor trametinib, and BRAF inhibitor dabrafenib for BRAFV600 mutations. NRAS-mutated cell lines are also sensitive to MEK inhibitionin vitro, and NRAS-mutated tumors have also shown partial response to MEK inhibitors. However, melanoma still has high recurrence rates due to subpopulations, sometimes described as “melanoma initiating cells,” resistant to treatment. Since CD133 is a putative cancer stem cell marker for different cancers, associated with decreased survival, we examined resistance of patient-derived CD133(+) and CD133(-) melanoma cells to MAPK inhibitors. Human melanoma cells were exposed to increasing concentrations of trametinib and/or dabrafenib, either before or after separation into CD133(+) and CD133(-) subpopulations. In parental CD133-mixed lines, the percentages of CD133(+) cells increased significantly (p<0.05) after high-dose drug treatment. Presorted CD133(+) cells also exhibited significantly greater (p<0.05) IC50s for single and combination MAPKI treatment. siRNA knockdown revealed a causal relationship between CD133 and drug resistance. Microarray and qRT-PCR analyses revealed that ten of 18 ABC transporter genes were significantly (P<0.05) upregulated in the CD133(+) subpopulation, while inhibition of ABC activity increased sensitivity, suggesting a mechanism for increased drug resistance of CD133(+) cells.

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Dual Inhibition of AKT and MEK Pathways Potentiates the Anti-Cancer Effect of Gefitinib in Triple-Negative Breast Cancer Cells

Cancers ◽

10.3390/cancers13061205 ◽

2021 ◽

Vol 13 (6) ◽

pp. 1205

Author(s):

Kyu Sic You ◽

Yong Weon Yi ◽

Jeonghee Cho ◽

Yeon-Sun Seong

Keyword(s):

Breast Cancer ◽

Triple Negative Breast Cancer ◽

Kinase Inhibitors ◽

Triple Negative ◽

Protein Kinase Inhibitors ◽

Akt Inhibitor ◽

Term Survival ◽

Viral Oncogene ◽

Dual Inhibition ◽

Viral Oncogene Homolog

There is an unmet medical need for the development of new targeted therapeutic strategies for triple-negative breast cancer (TNBC). With drug combination screenings, we found that the triple combination of the protein kinase inhibitors (PKIs) of the epidermal growth factor receptor (EGFR), v-akt murine thymoma viral oncogene homolog (AKT), and MAPK/ERK kinase (MEK) is effective in inducing apoptosis in TNBC cells. A set of PKIs were first screened in combination with gefitinib in the TNBC cell line, MDA-MB-231. The AKT inhibitor, AT7867, was identified and further analyzed in two mesenchymal stem-like (MSL) subtype TNBC cells, MDA-MB-231 and HS578T. A combination of gefitinib and AT7867 reduced the proliferation and long-term survival of MSL TNBC cells. However, gefitinib and AT7867 induced the activation of the rat sarcoma (RAS)/ v-raf-1 murine leukemia viral oncogene homolog (RAF)/MEK/ extracellular signal-regulated kinase (ERK) pathway. To inhibit this pathway, MEK/ERK inhibitors were further screened in MDA-MB-231 cells in the presence of gefitinib and AT7867. As a result, we identified that the MEK inhibitor, PD-0325901, further enhanced the anti-proliferative and anti-clonogenic effects of gefitinib and AT7867 by inducing apoptosis. Our results suggest that the dual inhibition of the AKT and MEK pathways is a novel potential therapeutic strategy for targeting EGFR in TNBC cells.

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Therapeutic care in metastatic renal cell carcinoma during the follow-up phase of the RECORD-1 phase III trial

Journal of Clinical Oncology ◽

10.1200/jco.2009.27.15_suppl.e17531 ◽

2009 ◽

Vol 27 (15_suppl) ◽

pp. e17531-e17531

Author(s):

D. Wiederkehr ◽

R. Casciano ◽

L. Stern ◽

J. Zheng ◽

J. Baladi

Keyword(s):

Clinical Trial ◽

Mtor Inhibitor ◽

Kinase Inhibitors ◽

Study Drug ◽

Protein Kinase Inhibitors ◽

Phase Iii ◽

Drug Discontinuation ◽

Study Drug Discontinuation ◽

Trial Setting

e17531 Background: Following drug discontinuation for progression or adverse event in a clinical trial for relapsed or stage IV kidney cancer, supportive care including surgery, palliative radiotherapy, or bisphosphonates continue to be recommended by National Comprehensive Cancer Network (NCCN). However, published data on active therapeutic agents given to patients following study drug discontinuation in recent clinical trials is limited. Methods: World Health Organization Anatomical Therapeutic Chemical codes or therapeutic names, captured from the follow-up phase in a phase III clinical trial (RECORD-1) of patients with metastatic renal cell carcinoma (mRCC) patients, were used to describe antineoplastic therapies following discontinuation of study drug. Prior to trial, patients had progressed on at least one VEGFr-TKI therapy. Results: Of the 130 patients with follow-up after discontinuation of study drug, 78.5% received at least one of the following: corticosteroids, radiotherapy, protein kinase inhibitors, mTOR inhibitor, pyrimidine analogues, monoclonal antibodies, interferons, and investigational drugs. Among patients who received an active agent, nearly three-quarters (73.5%) utilized targeted therapy (protein kinase inhibitors, mTOR inhibitor, monoclonal antibodies). Conclusions: In a clinical trial setting with mRCC patients who have received several classes of systemic therapy, care delivered following study drug discontinuation often includes an active antineoplastic agent, despite the limited supportive evidence in this setting. While the placebo control with supportive care in a double-blind phase is acceptable to evaluate the efficacy and safety of a therapy for regulatory approval purposes, decision makers must also consider how these data may inform comparisons with the usual alternatives available to and used by physicians and patients in the non-trial setting. [Table: see text]

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Signaling pathways regulating FSH- and amphiregulin-induced meiotic resumption and cumulus cell expansion in the pig

Reproduction ◽

10.1530/rep-12-0191 ◽

2012 ◽

Vol 144 (5) ◽

pp. 535-546 ◽

Cited By ~ 43

Author(s):

R Prochazka ◽

M Blaha ◽

L Nemcova

Keyword(s):

Protein Kinase ◽

Signaling Pathways ◽

Kinase Inhibitors ◽

Egf Receptor ◽

Cumulus Cell ◽

Cumulus Cells ◽

Protein Kinase Inhibitors ◽

Akt Inhibitor ◽

Cumulus Expansion

To define signaling pathways that drive FSH- and epidermal growth factor (EGF)-like peptide-induced cumulus expansion and oocyte meiotic resumption, in vitro cultured pig cumulus–oocyte complexes were treated with specific protein kinase inhibitors. We found that FSH-induced maturation of oocytes was blocked in germinal vesicle (GV) stage by protein kinase A (PKA), MAPK14, MAPK3/1, and EGF receptor (EGFR) tyrosine kinase inhibitors (H89, SB203580, U0126, and AG1478 respectively) whereas phosphoinositide-3-kinase/v-akt murine thymoma viral oncogene homolog (PI3K/AKT) inhibitor (LY294002) blocked maturation of oocytes in metaphase I (MI). Amphiregulin (AREG)-induced maturation of oocytes was efficiently blocked in GV by U0126, AG1478, and low concentrations of LY294002; H89, SB203580, and high concentrations of LY294002 allowed the oocytes to undergo breakdown of GV and blocked maturation in MI. Both FSH- and AREG-induced cumulus expansion was incompletely inhibited by H89 and completely inhibited by SB203580, U0126, AG1478, and LY294002. The inhibitors partially or completely inhibited expression of expansion-related genes (HAS2, PTGS2, and TNFAIP6) with two exceptions: H89 inhibited only TNFAIP6 expression and LY294002 increased expression of PTGS2. The results of this study are consistent with the idea that PKA and MAPK14 pathways are essential for FSH-induced transactivation of the EGFR, and synthesis of EGF-like peptides in cumulus cells and MAPK3/1 is involved in regulation of transcriptional and posttranscriptional events in cumulus cells required for meiotic resumption and cumulus expansion. PI3K/AKT signaling is important for regulation of cumulus expansion, AREG-induced meiotic resumption, and oocyte MI/MII transition. The present data also indicate the existence of an FSH-activated and PKA-independent pathway involved in regulation of HAS2 and PTGS2 expression in cumulus cells.

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Discovery of Novel Gq-Biased LPA1 Negative Allosteric Modulators

SLAS DISCOVERY Advancing Life Sciences ◽

10.1177/2472555217691719 ◽

2017 ◽

Vol 22 (7) ◽

pp. 859-866

Author(s):

Yuji Shimizu ◽

Masaharu Nakayama

Keyword(s):

G Protein ◽

High Throughput Screening ◽

Kinase Inhibitors ◽

Protein Kinase Inhibitors ◽

Allosteric Modulators ◽

Label Free ◽

Cellular Processes ◽

Recombinant Cells ◽

And Migration ◽

Cellular Phenotypes

Lysophosphatidic acid (LPA) activates the G-protein-coupled receptor LPA1, which regulates various cellular processes, including cell proliferation and migration. Although LPA1 transduces cellular responses via Gq, Gi, and G12/13, associations between these signaling molecules and cellular phenotypes remain poorly characterized due to the lack of signal-specific pharmacological tools. Here, we characterized novel signal-biased modulators using multiple assays, including label-free impedance assays. LPA caused dramatic changes in cellular impedance in LPA1-expressing recombinant cells, which were susceptible to G-protein and protein kinase inhibitors. Subsequently, Gq-biased LPA1 negative allosteric modulators (NAMs) were identified using high-throughput screening, and a nonbiased antagonist differently affected the LPA-induced cellular impedance. These NAMs provide pharmacological tools for further investigations of the biology of LPA1.

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Perturbation biology nominates upstream–downstream drug combinations in RAF inhibitor resistant melanoma cells

eLife ◽

10.7554/elife.04640 ◽

2015 ◽

Vol 4 ◽

Cited By ~ 58

Author(s):

Anil Korkut ◽

Weiqing Wang ◽

Emek Demir ◽

Bülent Arman Aksoy ◽

Xiaohong Jing ◽

...

Keyword(s):

Kinase Inhibitors ◽

Network Models ◽

Clinical Problem ◽

Melanoma Cells ◽

Drug Combinations ◽

Protein Kinase Inhibitors ◽

Cancer Therapies ◽

Anti Cancer ◽

Important Clinical Problem ◽

Targeted Cancer Therapies

Resistance to targeted cancer therapies is an important clinical problem. The discovery of anti-resistance drug combinations is challenging as resistance can arise by diverse escape mechanisms. To address this challenge, we improved and applied the experimental-computational perturbation biology method. Using statistical inference, we build network models from high-throughput measurements of molecular and phenotypic responses to combinatorial targeted perturbations. The models are computationally executed to predict the effects of thousands of untested perturbations. In RAF-inhibitor resistant melanoma cells, we measured 143 proteomic/phenotypic entities under 89 perturbation conditions and predicted c-Myc as an effective therapeutic co-target with BRAF or MEK. Experiments using the BET bromodomain inhibitor JQ1 affecting the level of c-Myc protein and protein kinase inhibitors targeting the ERK pathway confirmed the prediction. In conclusion, we propose an anti-cancer strategy of co-targeting a specific upstream alteration and a general downstream point of vulnerability to prevent or overcome resistance to targeted drugs.

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A Nonradioactive Fluorimetric SPE-Based Ceramide Kinase Assay Using NBD-C6-Ceramide

Journal of Lipids ◽

10.1155/2012/404513 ◽

2012 ◽

Vol 2012 ◽

pp. 1-9 ◽

Cited By ~ 4

Author(s):

Helena Van Overloop ◽

Gerd Van der Hoeven ◽

Paul P. Van Veldhoven

Keyword(s):

High Throughput Screening ◽

Kinase Inhibitors ◽

Solid Phase ◽

Protein Kinase Inhibitors ◽

Kinase Assay ◽

Cellular Processes ◽

Ceramide Kinase ◽

C6 Ceramide ◽

Ceramide 1

Ceramide kinase (CERK) has been implicated in important cellular processes such as inflammation and apoptosis. Its activity is usually measured using radiolabeled ceramide or [γ-32P]-ATP, followed by extraction, thin-layer chromatography, and detection of the formed labeled ceramide-1-phosphate. To eliminate the use of radioactivity, we developed similarly but independently from the approach by Don and Rosen (2008), a fluorescence-based ceramide kinase assay, using N-[7-(4-nitrobenz-2-oxa-1,3-diazole)]-6-aminohexanoyl-sphingenine (NBD-C6-ceramide) as substrate. ItsKmvalue (4 μM) was comparable to that of N-hexanoyl-sphingenine (C6-ceramide). The produced fluorescent NBD-C6-ceramide-1-phosphate was captured by means of solid-phase extraction on an aminopropyl phase, resulting in a fast and sensitive CERK measurement. By performing this assay in a 96-well format, it is also suitable for high-throughput screening (HTS) to search for CERK modulators. A limited screen revealed that some protein kinase inhibitors (e.g., U-0126;IC504 μM) and ceramide analogues (e.g., fenretinide, AMG-9810;IC501.1 μM) affect CERKin vitro.

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Dual Inhibition of Mitogen-Activated Protein Kinase Kinase and Mammalian Target of Rapamycin in Differentiated and Anaplastic Thyroid Cancer

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/jc.2009-0662 ◽

2009 ◽

Vol 94 (10) ◽

pp. 4107-4112 ◽

Cited By ~ 55

Author(s):

Ning Jin ◽

Tianyun Jiang ◽

D. Marc Rosen ◽

Barry D. Nelkin ◽

Douglas W. Ball

Keyword(s):

Thyroid Cancer ◽

Growth Inhibition ◽

Differentiated Thyroid Cancer ◽

Mtor Inhibitor ◽

Mammalian Target Of Rapamycin ◽

Xenograft Model ◽

Anaplastic Thyroid Cancer ◽

Mek Inhibitor ◽

Mtor Pathway ◽

Pathway Inhibition

Context: Differentiated thyroid cancer and anaplastic thyroid cancer tumors frequently have activation of the ras/raf /MAPK kinase (MEK)/ERK and phosphatidylinositol 3-kinase (PI-3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathways. Objective: The objective of the study was to investigate the efficacy of MEK and mTOR inhibitors in preclinical thyroid cancer treatment models with defined mutation status. Experimental Design: The MEK inhibitor AZD6244 (ARRY-142886) and mTOR inhibitor rapamycin were tested separately and in combination in 10 differentiated thyroid cancer and anaplastic thyroid cancer cell lines and in a xenograft model for evidence of pathway inhibition, growth inhibition, apoptosis, and long-range adaptation and resistance. Results: Seven of 10 tested lines had evidence of significant basal activity of the PI-3K/AKT/mTOR pathway, with elevated phosphorylated AKT and phosphorylated p70 S6 kinase. Activation of ras/RAF/MEK/ERK was equally common in this panel. All 10 lines exhibited better than 60% growth inhibition with combined MEK and mTOR inhibition, including lines with BRAF, Ret-PTC, ras, and PTEN mutations. Rapamycin or AZD6244 alone achieved this threshold in six and two lines, respectively. Dual-pathway inhibition in the Ret-PTC mutant cell line TPC1 caused an intense G1 arrest in cell culture and reversible cytostatic inhibition in a xenograft model. We did not observe significant feedback up-regulation of AKT activation in either acute or prolonged exposures. Conclusion: These preclinical results support the inclusion of thyroid cancer patients in early-phase clinical trials combining ras/RAF/MEK/ERK and PI-3K/AKT/mTOR pathway inhibition. Combined treatment with a MEK inhibitor (AZD6244/ARRY-142886) plus an mTOR inhibitor (Rapamycin) inhibited growth of thyroid cancer cells in vitro, and in a xenograft model, more potently than either agent alone.

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