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Cell Death & Disease May 2019Triple-negative breast cancer (TNBC) treatment remains a great challenge for clinical practice and novel therapeutic strategies are urgently needed. UCHL3 is a...
Triple-negative breast cancer (TNBC) treatment remains a great challenge for clinical practice and novel therapeutic strategies are urgently needed. UCHL3 is a deubiquitinase that is overexpressed in TNBC and correlates with poor prognosis. UCHL3 deubiquitinates RAD51 thereby promoting the recruitment of RAD51 to DNA damage sites and augmenting DNA repair. Therefore, UCHL3 overexpression can render cancer cells resistant to DNA damage inducing chemo and radiotherapy, and targeting UCHL3 can sensitize TNBC to radiation and chemotherapy. However, small molecule inhibitors of UCHL3 are yet to be identified. Here we report that perifosine, a previously reported Akt inhibitor, can inhibit UCHL3 in vitro and in vivo. We found low dose (50 nM) perifosine inhibited UCHL3 deubiquitination activity without affecting Akt activity. Furthermore, perifosine enhanced Olaparib-induced growth inhibition in TNBC cells. Mechanistically, perifosine induced RAD51 ubiquitination and blocked the RAD51-BRCA2 interaction, which in turn decreased ionizing radiation-induced foci (IRIF) of Rad51 and, thereby, homologous recombination (HR)-mediated DNA double strand break repair. In addition, combination of perifosine and Olaparib showed synergistic antitumor activity in vivo in TNBC xenograft model. Thus, our present study provides a novel therapeutic approach to optimize PARP inhibitor treatment efficiency.
Topics: Animals; Antineoplastic Agents; Apoptosis; BRCA2 Protein; Cell Line, Tumor; Female; Humans; Mice; Mice, Nude; Phosphorylcholine; Phthalazines; Piperazines; Poly(ADP-ribose) Polymerase Inhibitors; Rad51 Recombinase; Radiation, Ionizing; Recombinational DNA Repair; Transplantation, Heterologous; Triple Negative Breast Neoplasms; Ubiquitin Thiolesterase; Ubiquitination; RNA, Guide, CRISPR-Cas Systems
PubMed: 31113933
DOI: 10.1038/s41419-019-1628-8 -
Leukemia & Lymphoma Nov 2019Lipid rafts are ordered membrane domains, which provide an environment for the proteins participating in signal transduction. Perifosine is an alkylphospholipid (APL)...
Lipid rafts are ordered membrane domains, which provide an environment for the proteins participating in signal transduction. Perifosine is an alkylphospholipid (APL) that inhibits the AKT pathway, cytotoxic to neoplastic cells. We have shown that the lipid raft adaptor protein NTAL is a target of APLs in leukemic cells. Using human mantle cell lymphoma (MCL) Granta-519 cell line we showed here that perifosine decreased NTAL in lipid raft fractions reducing AKT phosphorylation before apoptosis. We also showed that the NTAL-knockdown by shRNA induced a state of reduced AKT activation. Experimental NTAL-knockdown in NSG mouse MCL xenografts reduced AKT activity, increased the basal apoptotic rate by 3-fold ( = 8) and decreased tumor weight by 2.7-fold ( = 5), indicating that NTAL participates in tumor growth. NTAL protein was detected by western blotting in circulating cells of 7 of 8 MCL patients in the leukemic phase, suggesting involvement in the progression of the disease.
Topics: Adaptor Proteins, Signal Transducing; Aged; Animals; Apoptosis; Biomarkers, Tumor; Cell Proliferation; Female; Gene Expression Regulation, Neoplastic; Humans; Lymphoma, Mantle-Cell; Mice; Mice, Inbred NOD; Mice, SCID; Middle Aged; Phosphorylcholine; Prognosis; Proto-Oncogene Proteins c-akt; Survival Rate; Tumor Cells, Cultured; Xenograft Model Antitumor Assays
PubMed: 31060403
DOI: 10.1080/10428194.2019.1607326 -
Experimental and Molecular Pathology Aug 2019Although tenascin-C (TNC), an extracellular matrix protein, has been shown to be widely expressed in stromal fibroblasts in various cancers, the role of its expression...
Although tenascin-C (TNC), an extracellular matrix protein, has been shown to be widely expressed in stromal fibroblasts in various cancers, the role of its expression in esophageal squamous cell carcinoma (ESCC) cells remains unclear. Using immunohistochemistry, we investigated the expression of cancer stem-like cell (CSC) markers, epithelial-to-mesenchymal transition (EMT)-related genes, and the Akt/hypoxia-inducible factor-1α (HIF1α) signal pathway in ESCC tissue specimens from 154 patients. We further addressed the effects of TNC on the Akt/HIF1α axis and its putative association with cancer stemness in several ESCC cell lines by immunofluorescence imaging and western blot analysis. Our data suggest that TNC expression was positively correlated with the expression of the CSC marker SOX2 (p = .002), and TNC-expressing cancer cells expressed SOX2 in ESCC tissues. Moreover, TNC expression was strongly associated with EMT-related gene Snail (p = .022) and positively correlated with pAkt-Ser473 (p = .004) and HIF1α (p = .003). Furthermore, TNC-silencing down-regulated the expression of CSC marker SOX2 (p < .001) and EMT-related marker Snail (p < .001). The Akt inhibitor Perifosine inhibited the protein expression of pAkt-Ser473, Akt, HIF1α, and TNC in TE10 (an ESCC cell line) cells. Short-term exposure of TE10 cells to cobalt chloride caused an increase in protein expression of HIF1α, TNC, and SOX2 in a time-dependent manner. Taken together, these results suggest that TNC may enhance the cancer stem-like properties and promote EMT-like changes via the Akt/HIF1α axis.
Topics: Biomarkers, Tumor; Cell Line, Tumor; Disease-Free Survival; Epithelial-Mesenchymal Transition; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; Gene Expression Regulation, Neoplastic; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Immunohistochemistry; Neoplastic Stem Cells; Proto-Oncogene Proteins c-akt; RNA Interference; Signal Transduction; Tenascin
PubMed: 30904401
DOI: 10.1016/j.yexmp.2019.03.007 -
Molecular & Cellular Proteomics : MCP Jun 2019Redox stress is a well-known contributor to aging and diseases in skin. Reductants such as dithiothreitol (DTT) can trigger a stress response by disrupting disulfide...
Redox stress is a well-known contributor to aging and diseases in skin. Reductants such as dithiothreitol (DTT) can trigger a stress response by disrupting disulfide bonds. However, the quantitative response of the cellular proteome to reductants has not been explored, particularly in cells such as fibroblasts that produce extracellular matrix proteins. Here, we have used a robust, unbiased, label-free SWATH-MS proteomic approach to quantitate the response of skin fibroblast cells to DTT in the presence or absence of the growth factor PDGF. Of the 4487 proteins identified, only 42 proteins showed a statistically significant change of 2-fold or more with reductive stress. Our proteomics data show that reductive stress results in the loss of a small subset of reductant-sensitive proteins (including the collagens COL1A1/2 and COL3A1, and the myopathy-associated collagens COL6A1/2/3), and the down-regulation of targets downstream of the MAPK pathway. We show that a reducing environment alters signaling through the PDGF-associated MAPK/Akt pathways, inducing chronic dephosphorylation of ERK1/2 at Thr202/Tyr204 and phosphorylation of Akt at Ser473 in a growth factor-independent manner. Our data highlights collagens as sentinel molecules for redox stress downstream of MAPK/Akt, and identifies intervention points to modulate the redox environment to target skin diseases and conditions associated with erroneous matrix deposition.
Topics: Antioxidants; Collagen; Dermis; Dithiothreitol; Down-Regulation; Endoplasmic Reticulum Stress; Extracellular Matrix Proteins; Fibroblasts; Homeostasis; Humans; Mitogen-Activated Protein Kinases; Oxidation-Reduction; Phosphorylation; Phosphorylcholine; Platelet-Derived Growth Factor; Proto-Oncogene Proteins c-akt; Signal Transduction
PubMed: 30890563
DOI: 10.1074/mcp.RA118.001140 -
Biochemical and Biophysical Research... Apr 2019Developing novel therapeutic agents against chondrosarcoma is important. SF2523 is a PI3K-Akt-mTOR and bromodomain-containing protein 4 (BRD4) dual inhibitor. Its...
Developing novel therapeutic agents against chondrosarcoma is important. SF2523 is a PI3K-Akt-mTOR and bromodomain-containing protein 4 (BRD4) dual inhibitor. Its activity in human chondrosarcoma cells is tested. Our results show that SF2523 potently inhibited survival, proliferation and migration, and induced apoptosis activation in SW1353 cells and primary human chondrosarcoma cells. The dual inhibitor was yet non-cytotoxic to the primary human osteoblasts and OB-6 osteoblastic cells. SF2523 blocked Akt-mTOR activation and downregulated BRD4-regulated genes (Bcl-2 and c-Myc) in chondrosarcoma cells. It was more efficient in killing chondrosarcoma cells than other established PI3K-Akt-mTOR and BRD4 inhibitors, including JQ1, perifosine and OSI-027. In vivo, intraperitoneal injection of SF2523 (30 mg/kg) potently inhibited subcutaneous SW1353 xenograft tumor growth in severe combined immunodeficient mice. Akt-mTOR inhibition as well as Bcl-2 and c-Myc downregulation were detected in SF2523-treated SW1353 tumor tissues. In conclusion, targeting PI3K-Akt-mTOR and BRD4 by SF2523 potently inhibited chondrosarcoma cell growth in vitro and in vivo.
Topics: Adult; Animals; Antineoplastic Agents; Bone Neoplasms; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Chondrosarcoma; Humans; Male; Mice, SCID; Middle Aged; Morpholines; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Pyrans; TOR Serine-Threonine Kinases; Transcription Factors
PubMed: 30824188
DOI: 10.1016/j.bbrc.2019.02.080 -
Translational Cancer Research Dec 2018Perifosine, is a third generation alkylphospholipid analog which has promising anti-tumor efficacy in clinical trials of refractory/recurrent neuroblastoma (NB)....
BACKGROUND
Perifosine, is a third generation alkylphospholipid analog which has promising anti-tumor efficacy in clinical trials of refractory/recurrent neuroblastoma (NB). However, perifosine's mechanism of action remains unclear. Previously, we have shown that perifosine changes global proteome and acetylome profiles in NB.
METHODS
To obtain a more comprehensive understanding of the perifosine mechanism, we performed a quantitative assessment of the lysine ubiquitylome in SK-N-AS NB cells using SILAC labeling, affinity enrichment and high-resolution liquid chromatography combined with mass spectrometry analysis. To analyse the data of ubiquitylome, we performed enrichment analysis with gene ontology (GO), the Encyclopedia of Genes and Genomes (KEGG) pathway, ubiquitylated lysine motif, protein complex and protein domain. Protein-protein interaction was conducted to explore the crosstalk between ubiquitylome and previous global proteome/acetylome. Co-immunoprecipitation and western blotting were used to validate the results of the ubiquitylome analysis.
RESULTS
Altogether, 3,935 sites and 1,658 proteins were quantified. These quantified ubiquitylated proteins participated in various cellular processes such as binding, catalytic activity, biological regulation, metabolic process and signaling pathways involving non-homologous end-joining, steroid biosynthesis and Ras signaling pathway. Ubiquitylome and proteome presented negative connection. We identified 607 sites which were modified with both ubiquitination and acetylation. We selected 14 proteins carrying differentially quantified lysine ubiquitination and acetylation sites at the threshold of 1.5 folds as potential targets. These proteins were enriched in activities associated with ribosome, cell cycle and metabolism.
CONCLUSIONS
Our study extends our understanding of the spectrum of novel targets that are differentially ubiquitinated after perifosine treatment of NB tumor cells.
PubMed: 30761266
DOI: 10.21037/tcr.2018.11.30 -
Journal of Cellular Biochemistry Jul 2019To investigate the effect of substance P (SP) on human corneal epithelial cells (HCECs) that have been stressed by a high urea environment and to determine the...
To investigate the effect of substance P (SP) on human corneal epithelial cells (HCECs) that have been stressed by a high urea environment and to determine the relationship between SP and the protein kinase B (AKT)/glycogen synthase kinase-3β (GSK-3β) signaling pathway. An in vitro model of chronic renal failure (CRF)-related dry eye was used to study HCECs that were treated with high urea concentrations. Cell proliferation was assayed using a cell counting kit-8 test. Besides, cell apoptosis was evaluated by flow cytometry. Furthermore, the effects of SP and the AKT inhibitor perifosine on the urea-treated HCECs were examined using immunofluorescence, quantitative real time polymerase chain reaction (qRT-PCR), and Western blot analysis. SP markedly reduced the number of apoptotic HCECs and decreased the cleaved caspase-3 expression levels while contributing to increased cellular proliferation (P < 0.05). The Western blot analysis and qRT-PCR experiments revealed that SP significantly increased the expression of p-AKT and p-GSK-3β (P < 0.05); additionally, these increases were attenuated after the perifosine inhibition of the AKT signaling pathway (P < 0.05). These in vitro experiments demonstrated that SP may protect against the apoptotic damage of HCECs caused by the high urea condition. The underlying mechanism may be related to the activation of the AKT/GSK-3β signaling pathway.
PubMed: 30724378
DOI: 10.1002/jcb.28410 -
Frontiers in Aging Neuroscience 2018Neuroinflammation and autophagy dysfunction are closely related to the development of neurodegeneration such as Parkinson's disease (PD). However, the role of autophagy...
Neuroinflammation and autophagy dysfunction are closely related to the development of neurodegeneration such as Parkinson's disease (PD). However, the role of autophagy in microglia polarization and neuroinflammation is poorly understood. TNF-α, which is highly toxic to dopaminergic neurons, is implicated as a major mediator of neuroinflammation in PD. In this study, we found that TNF-α resulted in an impairment of autophagic flux in microglia. Concomitantly, an increase of M1 marker (iNOS/NO, IL-1β, and IL-6) expression and reduction of M2 marker (Arginase1, Ym1/2, and IL-10) were observed in TNF-α challenged microglia. Upregulation of autophagy via serum deprivation or pharmacologic activators (rapamycin and resveratrol) promoted microglia polarization toward M2 phenotype, as evidenced by suppressed M1 and elevated M2 gene expression, while inhibition of autophagy with 3-MA or Atg5 siRNA consistently aggravated the M1 polarization induced by TNF-α. Moreover, Atg5 knockdown alone was sufficient to trigger microglia activation toward M1 status. More important, TNF-α stimulated microglia conditioned medium caused neurotoxicity when added to neuronal cells. The neurotoxicity was further aggravated when Atg5 knockdown in BV2 cells but alleviated when microglia pretreatment with rapamycin. Activation of AKT/mTOR signaling may contribute to the changes of autophagy and inflammation as the AKT specific inhibitor perifosine prevented the increase of LC3II (an autophagic marker) in TNF-α stimulated microglia. Taking together, our results demonstrate that TNF-α inhibits autophagy in microglia through AKT/mTOR signaling pathway, and autophagy enhancement can promote microglia polarization toward M2 phenotype and inflammation resolution.
PubMed: 30515090
DOI: 10.3389/fnagi.2018.00378 -
Molecular Cancer Therapeutics Feb 2019Hyperactivated AKT kinase due to loss of its negative regulator PTEN influences many aspects of cancer biology, including chromatin. AKT primarily regulates acetyl-CoA...
Hyperactivated AKT kinase due to loss of its negative regulator PTEN influences many aspects of cancer biology, including chromatin. AKT primarily regulates acetyl-CoA production and phosphorylates many histone-modulating enzymes, resulting in their activation or inhibition. Therefore, understanding the therapeutic impact of AKT inhibition on chromatin-related events is essential. Here, we report that AKT inhibition in prostate-specific PTEN knockout mice significantly induces di- and trimethylation of H3K4 with concomitant reduction in H3K9 acetylation. Mechanistically, we observed that AKT inhibition reduces expression of the H3K4 methylation-specific histone demethylases KDM5 family, especially KDM5B expression at transcriptional levels. Furthermore, we observed that AKT negatively regulates miR-137 levels, which transcriptionally represses KDM5B expression. Overexpression of miR-137 significantly reduced KDM5B and increased H3K4 methylation levels but failed to change AKT phosphorylation. Overall, we observed that AKT transcriptionally regulates KDM5B mainly via repression of miR-137. Our data identify a mechanism by which AKT kinase modulates the prostate cancer epigenome through regulating H3K4 methylation. Additional studies on AKT inhibition-mediated induction of H3K4 methylation will help in designing strategies to enhance the therapeutic efficacy of PI3K/AKT inhibitors.
Topics: Acetylation; Animals; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Gene Knockout Techniques; Humans; Jumonji Domain-Containing Histone Demethylases; Male; Methylation; Mice; MicroRNAs; Nuclear Proteins; PTEN Phosphohydrolase; Phosphorylcholine; Prostatic Neoplasms; Proto-Oncogene Proteins c-akt; Repressor Proteins; Xenograft Model Antitumor Assays
PubMed: 30446585
DOI: 10.1158/1535-7163.MCT-18-0141 -
Experimental Cell Research Dec 2018The role of Hedgehog (HH)/ glioma-associated oncogene homolog 1 (GLI1) pathway has been implicated in a variety of cancer entities, and the targeted pathway inhibition...
The role of Hedgehog (HH)/ glioma-associated oncogene homolog 1 (GLI1) pathway has been implicated in a variety of cancer entities, and the targeted pathway inhibition mediated by GLI1 is of therapeutic relevance. However, its oncogenicity and cross-talks with other cancer pathways including PI3K/Akt/NFκB, which modulates the HH/GLI1 signal strength, have rarely been explored in colorectal adenocarcinoma. We assessed the expression of GLI1 and its relationship with other cancer stemness genes, cell cycle markers, epithelial-mesenchymal transition (EMT), PI3K/Akt/NFκB signaling pathway genes, and HIF1α in 100 paraffin-embedded colorectal adenocarcinoma tissue samples using immunohistochemistry. We further addressed the effect of GLI1 on EMT, cell cycle, and its putative interaction with the PI3K/Akt/NFκB cascade in colorectal adenocarcinoma cell lines. The expression of GLI1 in colorectal adenocarcinoma tissues was found to correlate with the clinical stages, and distant metastasis. Moreover, GLI1 was found to be an independent predictor of poor overall survival and disease-free survival in colorectal adenocarcinoma. GLI1-expressing cancer cells also expressed their representative cancer stem-like cell (CSC) markers (SOX9 and CD133), as well as HIF1α. GLI1 expression was also strongly linked to EMT-related and PI3K/Akt/NFκB signaling genes. Downregulation of GLI1 by inhibitor treatment in colorectal adenocarcinoma cell lines resulted in reduced expression of CSC markers, cell clonogenicity, S-phase subpopulations, as well as the migration and invasion ability. Importantly, Akt inhibitor Perifosine significantly inhibited the expression of pAkt and GLI1 in colorectal adenocarcinoma cells. Combination of GLI1 inhibitor GANT61 and NFκB p65 inhibitor QZN exhibited much higher inhibition compared to using any of them individually on colorectal adenocarcinoma cells. We suggested that GLI1 may be a novel stem cell marker, and cancer stemness was activated via PI3K/Akt/NFκB pathway. In addition, co-targeting GLI1 and PI3K/Akt/NFκB signaling simultaneously might provide an alternative therapeutic strategy for colorectal adenocarcinoma patients.
Topics: Adenocarcinoma; Biomarkers, Tumor; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Epithelial-Mesenchymal Transition; Female; Humans; Male; Middle Aged; NF-kappa B; Neoplastic Stem Cells; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Signal Transduction; Zinc Finger Protein GLI1
PubMed: 30321514
DOI: 10.1016/j.yexcr.2018.10.006