Spatially resolved subcellular protein-protein interactomics in drug-perturbed lung-cancer cultures and tissues.

Protein-protein interactions (PPIs) regulate signalling pathways and cell phenotypes, and the visualization of spatially resolved dynamics of PPIs would thus shed light on the activation and crosstalk of signalling networks. Here we report a method that leverages a sequential proximity ligation assay for the multiplexed profiling of PPIs with up to 47 proteins involved in multisignalling crosstalk pathways. We applied the method, followed by conventional immunofluorescence, to cell cultures and tissues of non-small-cell lung cancers with a mutated epidermal growth-factor receptor to determine the co-localization of PPIs in subcellular volumes and to reconstruct changes in the subcellular distributions of PPIs in response to perturbations by the tyrosine kinase inhibitor osimertinib.

Rictor, an essential component of mTOR complex 2, undergoes caspase-mediated cleavage during apoptosis induced by multiple stimuli.

Caspase-mediated cleavage of proteins ensures the irreversible commitment of cells to undergo apoptosis, and is thus a hallmark of apoptosis. Rapamycin-insensitive companion of mTOR (rictor) functions primarily as a core and essential component of mTOR complex 2 (mTORC2) to critically regulate cellular homeostasis. However, its role in the regulation of apoptosis is largely unknown.

Regulation of Cancer Metastasis by TRAIL/Death Receptor Signaling.

Death ligands such as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL; TNFSF10) and their corresponding death receptors (e.g., DR5) not only initiate apoptosis through activation of the extrinsic apoptotic pathway but also exert non-apoptotic biological functions such as regulation of inflammation and cancer metastasis.

RAD52 Adjusts Repair of Single-Strand Breaks via Reducing DNA-Damage-Promoted XRCC1/LIG3α Co-localization.

Radiation sensitive 52 (RAD52) is an important factor for double-strand break repair (DSBR). However, deficiency in vertebrate/mammalian Rad52 has no apparent phenotype. The underlying mechanism remains elusive.

Ionizing radiation-induced growth in soft agar is associated with miR-21 upregulation in wild-type and DNA double strand break repair deficient cells.

DNA double strand breaks (DSBs) are a severe threat to genome integrity and a potential cause of tumorigenesis, which is a multi-stage process and involves many factors including the mutation of oncogenes and tumor suppressors, some of which are transcribed microRNAs (miRNAs). Among more than 2000 known miRNAs, miR-21 is a unique onco-miRNA that is highly expressed in almost all types of human tumors and is associated with tumorigenesis through its multiple targets. However, it remains unclear whether there is any functional link between DSBs and miR-21 expression and, if so, does the link contribute to DSB-induced genomic instability/tumorigenesis.

Monocyte chemotactic protein-induced protein-1 enhances DR5 degradation and negatively regulates DR5 activation-induced apoptosis through its deubiquitinase function.

Monocyte chemotactic protein-induced protein-1 (MCPIP1; also called Regnase-1) encoded by the ZC3H12A gene critically regulates inflammatory responses and immune homeostasis primarily by RNase-dependent and -independent mechanisms. However, the relationship of MCPIP1 with apoptosis and cancer and the underlying mechanisms are largely unclear. The current study has demonstrated a previously uncovered connection between MCPIP1 and the negative regulation of death receptor 5 (DR5; also known as TRAIL-R2 or killer/DR5), a cell surface receptor for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), which is produced endogenously by various immune cells such as T cells.

The proteasome deubiquitinase inhibitor b-AP15 enhances DR5 activation-induced apoptosis through stabilizing DR5.

b-AP15 and its derivatives block proteasome deubiquitinase (DUB) activity and have been developed and tested in the clinic as potential cancer therapeutic agents. b-AP15 induces apoptosis in cancer cells, but the underlying mechanisms are largely undefined. The current study focuses on studying the modulatory effects of b-AP15 on death receptor 5 (DR5) levels and DR5 activation-induced apoptosis as well as on understanding the underlying mechanisms.

Overcoming Acquired Resistance to AZD9291, A Third-Generation EGFR Inhibitor, through Modulation of MEK/ERK-Dependent Bim and Mcl-1 Degradation.

The mechanisms accounting for anticancer activity of AZD9291 (osimertinib or TAGRISSO), an approved third-generation EGFR inhibitor, in EGFR-mutant non-small cell lung cancer (NSCLC) cells and particularly for the subsequent development of acquired resistance are unclear and thus are the focus of this study. AZD9219-resistant cell lines were established by exposing sensitive cell lines to AZD9291. Protein alterations were detected with Western blotting.

DR5 suppression induces sphingosine-1-phosphate-dependent TRAF2 polyubiquitination, leading to activation of JNK/AP-1 and promotion of cancer cell invasion.

Background: Death receptor (DR5), a well-characterized death domain-containing cell surface pro-apoptotic protein, has been suggested to suppress cancer cell invasion and metastasis. However, the underlying mechanisms have not been fully elucidated. Our recent work demonstrates that DR5 suppression promotes cancer cell invasion and metastasis through caspase-8/TRAF2-mediated activation of ERK and JNK signaling and MMP1 elevation.

Expression of Death Receptor 4 Is Positively Regulated by MEK/ERK/AP-1 Signaling and Suppressed upon MEK Inhibition.

Death receptor 4 (DR4) is a cell surface receptor for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and triggers apoptosis upon ligation with TRAIL or aggregation. MEK/ERK signaling is a well known and the best-studied effector pathway downstream of Ras and Raf. This study focuses on determining the impact of pharmacological MEK inhibition on DR4 expression and elucidating the underlying mechanism.

Met gene amplification and protein hyperactivation is a mechanism of resistance to both first and third generation EGFR inhibitors in lung cancer treatment.

The 3rd generation epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs; e.g., AZD9291), which selectively and irreversibly inhibit EGFR activating and T790M mutants, represent very promising therapeutic options for patients with non-small cell lung cancer (NSCLC) that has become resistant to 1st generation EGFR-TKIs due to T790M mutation.

Paradoxical activation of MEK/ERK signaling induced by B-Raf inhibition enhances DR5 expression and DR5 activation-induced apoptosis in Ras-mutant cancer cells.

B-Raf inhibitors have been used for the treatment of some B-Raf-mutated cancers. They effectively inhibit B-Raf/MEK/ERK signaling in cancers harboring mutant B-Raf, but paradoxically activates MEK/ERK in Ras-mutated cancers. Death receptor 5 (DR5), a cell surface pro-apoptotic protein, triggers apoptosis upon ligation with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or aggregation.

Suppression of death receptor 5 enhances cancer cell invasion and metastasis through activation of caspase-8/TRAF2-mediated signaling.

The role of death receptor 5 (DR5), a well-known cell surface pro-apoptotic protein, in the negative regulation of invasion and metastasis of human cancer cells and the underlying mechanisms are largely unknown and were hence the focus of this study. In this report, we have demonstrated that DR5 functions to suppress invasion and metastasis of human cancer cells, as evidenced by enhanced cancer cell invasion and metastasis upon genetic suppression of DR5 either by gene knockdown or knockout. When DR5 is suppressed, FADD and caspase-8 may recruit and stabilize TRAF2 to form a metastasis and invasion signaling complex, resulting in activation of ERK and JNK/AP-1 signaling that mediate the elevation and activation of matrix metalloproteinase-1 (MMP1) and eventual promotion of cancer invasion and metastasis.

The novel proteasome inhibitor carfilzomib activates and enhances extrinsic apoptosis involving stabilization of death receptor 5.

Carfilzomib (CFZ) is a second generation proteasome inhibitor approved for the treatment of patients with multiple myeloma. It induces apoptosis in human cancer cells; but the underlying mechanisms remain undefined. In the present study, we show that CFZ decreases the survival of several human cancer cell lines and induces apoptosis.

Oncogenic Ras and B-Raf proteins positively regulate death receptor 5 expression through co-activation of ERK and JNK signaling.

Oncogenic mutations of ras and B-raf frequently occur in many cancer types and are critical for cell transformation and tumorigenesis. Death receptor 5 (DR5) is a cell surface pro-apoptotic death receptor for tumor necrosis factor-related apoptosis-inducing ligand and has been targeted in cancer therapy. The current study has demonstrated induction of DR5 expression by the oncogenic proteins Ras and B-Raf and revealed the underlying mechanisms.

Establishment of novel cell lines latently infected with human immunodeficiency virus 1.

Many human immunodeficiency virus 1 (HIV-1) researchers focus on the developing new anti-reservoir therapy to eradicate HIV-1 provirus from the HIV-1-infected patients. HIV-1 provirus is the major obstacle for effective HIV-1 treatment because it integrates into the host genome and can produce a virus progeny after stopping highly active antiretroviral therapy (HAART). We established two novel cell lines latently infected with HIV-1 by limiting dilution cloning of A3.

ERK/ribosomal S6 kinase (RSK) signaling positively regulates death receptor 5 expression through co-activation of CHOP and Elk1.

Death receptor 5 (DR5) is a death domain-containing transmembrane receptor that triggers apoptosis upon binding to its ligand or when overexpressed. Its expression is induced by certain small molecule drugs, including celecoxib, through mechanisms that have not been fully elucidated. The current study has revealed a novel ERK/ribosomal S6 kinase (RSK)-dependent mechanism that regulates DR5 expression primarily using celecoxib as a DR5 inducer.

Novel histone deacetylase inhibitors CG05 and CG06 effectively reactivate latently infected HIV-1.

Histone deacetylase plays an important role in HIV latency. Novel histone deacetylase inhibitors, CG05 and CG06, were evaluated for their roles in HIV latency using ACH2 cells. Both inhibitors were highly efficient in reactivation of provirus and exerted lesser toxicity compared with other known histone deacetylase inhibitors.

Regulation of cyclin-dependent kinase inhibitor p21WAF1/CIP1 by protein kinase Cdelta-mediated phosphorylation.

Cyclin-dependent kinase (CDK) inhibitor p21(WAF1/CIP1(-/-))-null mice have an increased incidence of tumor formation. Here, we demonstrate that p21(WAF1/CIP1) is unstable in HeLa cells treated with siRNA duplexes that target PKCdelta. PKCdelta phosphorylates p21(WAF1/CIP1 )at a serine residue ((146)Ser) located in its C-terminal domain.

Cyclin-dependent protein kinase 2 activity is required for mitochondrial translocation of Bax and disruption of mitochondrial transmembrane potential during etoposide-induced apoptosis.

Previous studies have suggested that upregulation of Cyclin A-dependent protein kinase 2 (Cdk2) activity is an essential event in apoptotic progression and the mitochondrial permeability transition in human cancer cells. Here, we show that upregulated Cyclin A/Cdk2 activity precedes the proteolytic cleavage of PARP and is correlated with the mitochondrial translocation of Bax and the loss of mitochondrial transmembrane potential (Deltapsim) during etoposide-induced apoptosis in human cervical adenocarcinoma (HeLa) cells. Etoposide-induced apoptotic cell death is efficiently prevented in cells that overexpress a dominant negative mutant of Cdk2 (Cdk2-dn) or p21(WAF1/CIP1), a specific Cdk inhibitor.

PKCdelta modulates p21WAF1/CIP1 ability to bind to Cdk2 during TNFalpha-induced apoptosis.

Cyclin-dependent kinase 2 (Cdk2) activity is thought to be involved in cell death-associated chromatin condensation and other manifestations of apoptotic death. Here we show that during TNFalpha-induced apoptosis, PKCdelta is activated in a caspase-3-dependent manner and phosphorylates p21(WAF1/CIP1), a specific cyclin-dependent kinase inhibitor, on (146)Ser. This residue is located near a cyclin-binding motif (Cy2) that plays an important role in the interaction between p21(WAF1/CIP1) and Cdk2, and its phosphorylation modulates the ability of p21(WAF1/CIP1) to associate with Cdk2.

Caspase-3-dependent protein kinase C delta activity is required for the progression of Ginsenoside-Rh2-induced apoptosis in SK-HEP-1 cells.

Ginsenoside-Rh2 (G-Rh2) has been shown to induce apoptosis in a variety of cell types. In this study, we show that G-Rh2-induced apoptosis is accompanied by the mitochondrial release of cytochrome c and activation of caspase-3 in the human hepatoma cell line, SK-HEP-1. Furthermore, protein kinase C delta (PKCdelta) activity was markedly up-regulated in a lipid activator-independent manner with kinetics similar to those of PKCdelta and PARP cleavages during the apoptotic progression.