Identification of MYC as an antinecroptotic protein that stifles RIPK1-RIPK3 complex formation.

The underlying mechanism of necroptosis in relation to cancer is still unclear. Here, MYC, a potent oncogene, is an antinecroptotic factor that directly suppresses the formation of the RIPK1-RIPK3 complex. Gene set enrichment analyses reveal that the MYC pathway is the most prominently down-regulated signaling pathway during necroptosis. Depletion or deletion of MYC promotes the RIPK1-RIPK3 interaction, thereby stabilizing the RIPK1 and RIPK3 proteins and facilitating necroptosis. Interestingly, MYC binds to RIPK3 in the cytoplasm and inhibits the interaction between RIPK1 and RIPK3 in vitro. Furthermore, MYC-nick, a truncated form that is mainly localized in the cytoplasm, prevented TNF-induced necroptosis. Finally, down-regulation of MYC enhances necroptosis in leukemia cells and suppresses tumor growth in a xenograft model upon treatment with birinapant and emricasan. MYC-mediated suppression of necroptosis is a mechanism of necroptosis resistance in cancer, and approaches targeting MYC to induce necroptosis represent an attractive therapeutic strategy for cancer.

QKI, a miR-200 target gene, suppresses epithelial-to-mesenchymal transition and tumor growth.

The microRNA-200 (miR-200) family plays a major role in specifying epithelial phenotype by preventing expression of the transcription repressors ZEB1 and ZEB2, which are well-known regulators of the epithelial-to-mesenchymal transition (EMT) in epithelial tumors including oral squamous cell carcinoma (OSCC). Here, we elucidated whether miR-200 family members control RNA-binding protein quaking (QKI), a newly identified tumor suppressor that is regulated during EMT. We predicted that miR-200a and miR-200b could recognize QKI 3'-UTR by analyzing TargetScan and The Cancer Genome Atlas head and neck squamous cell carcinoma (HNSCC) dataset. Forced expression of miR-200b/a/429 inhibited expression of ZEB1/2 and decreased cell migration in OSCC cell lines CAL27 and HSC3. QKI expression was also suppressed by miR-200 overexpression, and the 3'-UTR of QKI mRNA was directly targeted by miR-200 in luciferase reporter assays. Interestingly, shRNA-mediated knockdown of QKI led to pronounced EMT and protumor effects in both in vitro and in vivo studies of OSCC. Furthermore, high expression of QKI protein is associated with favorable prognosis in surgically resected HNSCC and lung adenocarcinoma. In conclusion, QKI increases during EMT and is targeted by miR-200; while, it suppresses EMT and tumorigenesis. We suggest that QKI and miR-200 form a negative feedback loop to maintain homeostatic responses to EMT-inducing signals.

Structure of ADC-68, a novel carbapenem-hydrolyzing class C extended-spectrum ß-lactamase isolated from Acinetobacter baumannii.

Outbreaks of multidrug-resistant bacterial infections have become more frequent worldwide owing to the emergence of several different classes of ß-lactamases. In this study, the molecular, biochemical and structural characteristics of an Acinetobacter-derived cephalosporinase (ADC)-type class C ß-lactamase, ADC-68, isolated from the carbapenem-resistant A. baumannii D015 were investigated. The blaADC-68 gene which encodes ADC-68 was confirmed to exist on the chromosome via Southern blot analysis and draft genome sequencing. The catalytic kinetics of ß-lactams and their MICs (minimum inhibitory concentrations) for A. baumannii D015 and purified ADC-68 (a carbapenemase obtained from this strain) were assessed: the strain was resistant to penicillins, narrow-spectrum and extended-spectrum cephalosporins, and carbapenems, which were hydrolyzed by ADC-68. The crystal structure of ADC-68 was determined at a resolution of 1.8 Å. The structure of ADC-68 was compared with that of ADC-1 (a non-carbapenemase); differences were found in the central part of the Ω-loop and the C-loop constituting the edge of the R1 and R2 subsites and are close to the catalytic serine residue Ser66. The ADC-68 C-loop was stabilized in the open conformation of the upper R2 subsite and could better accommodate carbapenems with larger R2 side chains. Furthermore, a wide-open conformation of the R2-loop allowed ADC-68 to bind to and hydrolyze extended-spectrum cephalosporins. Therefore, ADC-68 had enhanced catalytic efficiency against these clinically important ß-lactams (extended-spectrum cephalosporins and carbapenems). ADC-68 is the first reported enzyme among the chromosomal class C ß-lactamases to possess class C extended-spectrum ß-lactamase and carbapenemase activities.

Oncogenic KRAS mutation confers chemoresistance by upregulating SIRT1 in non-small cell lung cancer.

Kirsten rat sarcoma viral oncogene homologue (KRAS) is a frequent oncogenic driver of solid tumors, including non-small cell lung cancer (NSCLC). The treatment and outcomes of KRAS-mutant cancers have not been dramatically revolutionized by direct KRAS-targeted therapies because of the lack of deep binding pockets for specific small molecule inhibitors. Here, we demonstrated that the mRNA and protein levels of the class III histone deacetylase SIRT1 were upregulated by the KRASMut-Raf-MEK-c-Myc axis in KRASMut lung cancer cells and in lung tumors of a mouse model with spontaneous KrasG12D expression. KRASMut-induced SIRT1 bound to KRASMut and stably deacetylated KRASMut at lysine 104, which increased KRASMut activity. SIRT1 knockdown (K/D) or the SIRT1H363Y mutation increased KRASMut acetylation, which decreased KRASMut activity and sensitized tumors to the anticancer effects of cisplatin and erlotinib. Furthermore, in KrasG12D/+;Sirt1co/co mice, treatment with cisplatin and erlotinib robustly reduced the tumor burden and increased survival rates compared with those in spontaneous LSL-KrasG12D/+;Sirt1+/+ mice and mice in each single-drug treatment group. Then, we identified p300 as a KRASMut acetyltransferase that reinforced KRASMut lysine 104 acetylation and robustly decreased KRASMut activity. KRASMut lysine 104 acetylation by p300 and deacetylation by SIRT1 were confirmed by LC‒MS/MS. Consistent with this finding, the SIRT1 inhibitor EX527 suppressed KRASMut activity, which synergistically abolished cell proliferation and colony formation, as well as the tumor burden in KRASMut mice, when combined with cisplatin or erlotinib. Our data reveal a novel pathway critical for the regulation of KRASMut lung cancer progression and provide important evidence for the potential application of SIRT1 inhibitors and p300 activators for the combination treatment of KRASMut lung cancer patients.

Midkine Is a Potential Therapeutic Target of Tumorigenesis, Angiogenesis, and Metastasis in Non-Small Cell Lung Cancer.

Hypoxia-inducible factors (HIFs) induced by reduced O2 availability activate the transcription of target genes encoding proteins that play important roles in communication between cancer and stromal cells. Cancer cells were incubated under hypoxic conditions: H1299, A549 (NSCLC); Hep3B, HepG2 (HCC); HCT116, CT26 (Colon cancer); MCF-7, MDAMB231 (Breast cancer); MKN1, MKN5 (Gastric cancer); U87MG, SHSY5Y (Brain cancer); and SKOV3, SNU840 (Ovary cancer). All cells expressed HIF-1α and HIF-2α mRNA and proteins. However, cell proliferation of NSCLC, breast, gastric, and brain cancer cells under hypoxia was more dependent on HIF-1α except for HCC cells where it was more dependent on HIF-2α. Among HIF-1α dependent cells H1299 was the most affected in terms of cell proliferation by HIF-1α knockdown. To examine which cytokines are secreted in NSCLC cells by HIF-1α to communicate with stromal cells, we performed a cytokine-profiling array with H1299. We screened the top 14 cytokines which were dependent on the HIF-1α expression pattern. Among them, midkine (MDK) expression was affected the most in response to HIF-1α. MDK is a heparin-binding growth factor that promotes angiogenesis and carcinogenesis. Indeed, MDK significantly increased HUVEV endothelial cell migration and neo- vascularization in chick chorioallantoic membrane assay (CAM) assay via paracrine signaling. In addition, MDK secreted from NSCLC cells interacted with Notch2 which activated the Notch signaling pathway and induced EMT, upregulated NF-κB, and increased cancer promotion. However, in response to MDK knock down, siRNA or the MDK inhibitor, iMDK treatment not only decreased MDK-induced migration and angiogenesis of endothelial cells but also abrogated the progression and metastasis of NSCLC cells in in vitro and in vivo orthotopic and spontaneous lung metastasis models. Consequently, iMDK treatment significantly increased mice survival rates compared with the control or MDK expression group. MDK plays a very important role in the progression and metastasis of NSCLC cells. Moreover, the MDK targeting strategy provides a potential therapeutic target for the treatment of MDK-expressing lung cancers.

Dual Targeting of ERBB2/ERBB3 for the Treatment of SLC3A2-NRG1-Mediated Lung Cancer.

We characterized the SLC3A2-NRG1 fusion gene in non-small cell lung cancer (NSCLC) and established an effective therapy for patients with SLC3A2-NRG1 fusion-positive cancer. The SLC3A2-NRG1 fusion product was composed of the SLC3A2 transmembrane domain and the EGF-like domain of the neuregulin 1 (NRG1) protein. The NRG1 family is classified as a ligand of the ERBB family. We identified ERBB3 and ERBB4 in the ERBB family as binding partners of the SLC3A2-NRG1 fusion protein via ligand and receptor binding assays. We confirmed that SLC3A2-NRG1 increased formation of a heterocomplex of ERBB3 with ERBB2. Activation of the ERBB2-ERBB3 heterocomplex by SLC3A2-NRG1 increased colony formation and tumor growth through PI3K-AKT and MAP kinase. The specific siRNAs for ERBB2 and ERBB3, pertuzumab, lumretuzumab, and afatinib all decreased ERBB2-ERBB3 heterocomplex formation, phosphorylation of each protein, and their downstream signaling. In addition, single treatment with pertuzumab, lumretuzumab, or afatinib decreased tumor volume and weight, whereas combination treatment with these drugs and taxol enhanced generation of cleaved caspase 3, PARP, and TUNEL-positive cells compared with each single treatment. Thus, the SLC3A2-NRG1 fusion gene plays an important role in lung cancer cell proliferation and tumor growth by promoting generation of the ERBB2-ERBB3 heterocomplex, its phosphorylation, and activation of the PI3K/ERK/mTOR signaling pathway. Inhibition of either ERBB2 or ERBB3 alone did not completely shut down downstream signaling of ERBB2 and ERBB3; however, inhibition of both ERBB2 and ERBB3 blocked downstream signaling activated by SLC3A2-NRG1 fusion. ERBB2 and ERBB3 might be promising targets for treatment of SLC3A2-NRG1-positive tumors. Mol Cancer Ther; 17(9); 2024-33. ©2018 AACR.