Neutrophil extracellular traps (NETs), in addition to their function as a host defense mechanism, play a relevant role in thrombus formation and metastatic dissemination of cancer cells. Here we screened different cancer cell lines endogenously expressing a variety of integrins for their ability to bind to NETs. To this end, we used NETs isolated from neutrophil-like cells as a substrate for adhesion assays of HT1080, U-87 MG, H1975, DU 145, PC-3 and A-431 cells. Levels of α5, αIIb, αv, β1, β3 and β5 chains were determined by western blot analysis in all cell lines and levels of whole integrins on the plasma membrane were assessed by fluorescence-activated cell sorting (FACS) analysis. We found that high levels of α5β1, αvβ3 and αvβ5 enhance cell adhesion to NETs, whereas low expression of α5β1 prevents cell attachment to NETs. Excess of cyclic RGD peptide inhibited cell adhesion to NETs by competing with fibronectin within NETs. The maximal reduction of such adhesion was similar to that obtained by DNase 1 treatment causing DNA degradation. Our findings indicate that NETs from neutrophil-like cells may be used as a substrate for large screening of the adhesion properties of cancer cells expressing a variety of RGD-binding integrins.

Since many oncogenes, including BCR-ABL, may promote the acquisition and maintenance of the glycolytic phenotype, we tested whether treatment of BCR-ABL-driven human leukemia cells with imatinib, a selective BCR-ABL inhibitor, can modulate the expression of key glycolytic enzymes and mitochondrial complex subunits thus causing alterations of glucose metabolism. BCR-ABL-driven K562 and KCL-22 cells were incubated with increasing concentrations of imatinib to preliminarily test drug sensitivity. Then untreated and treated cells were analyzed for levels of BCR-ABL signaling mediators and key proteins of glycolytic cascade and oxidative phosphorylation. Effective inhibition of BCR-ABL caused a concomitant reduction of p-ERK1/2, p-AKT, phosphorylated form of STAT3 (at Tyr705 and Ser727), c-Myc and cyclin D1 along with an increase of cleaved PARP and caspase 3 at 48 h after treatment. Furthermore, a strong reduction of the hexokinase II (HKII), phosphorylated form of PKM2 (at Tyr105 and Ser37) and lactate dehydrogenase A (LDH-A) was observed in response to imatinib along with a strong upregulation of mitochondrial complexes (OXPHOS). According to these findings, a significant reduction of glucose consumption and lactate secretion along with an increase of intracellular ATP levels was observed in response to imatinib. Our findings indicate that imatinib treatment of BCR-ABL-driven human leukemia cells reactivates mitochondrial oxidative phosphorylation thus allowing potential co-targeting of BCR-ABL and OXPHOS.

Despite the recent advances in lung cancer biology, molecular pathology, and treatment, this malignancy remains the leading cause of cancer-related death worldwide and non-small cell lung cancer (NSCLC) is the most common form found at diagnosis. Accurate staging of the disease is a fundamental prognostic factor that correctly predicts progression-free (PFS) and overall survival (OS) of NSCLC patients. However, outcome of patients within each TNM staging group can change widely highlighting the need to identify additional prognostic biomarkers to better stratify patients on the basis of risk. 18F-FDG PET/CT plays an essential role in staging, evaluation of treatment response, and tumoral target delineation in NSCLC patients. Moreover, a number of studies showed the prognostic role of imaging parameters derived from PET images, such as metabolic tumor volume (MTV) and total lesion glycolysis (TLG). These parameters represent three-dimensional PET-based measurements providing information on both tumor volume and metabolic activity and previous studies reported their ability to predict OS and PFS of NSCLC patients. This review will primarily focus on the studies that showed the prognostic and predictive role of MTV and TLG in NSCLC patients, addressing also their potential utility in the new era of immunotherapy of NSCLC.

Here, we tested whether co-targeting of glucose metabolism and oncogene drivers may enhance tumor response to tyrosine kinase inhibitors (TKIs) in NSCLC. To this end, pyruvate dehydrogenase kinase 1 (PDK1) was stably downregulated in oncogene-driven NSCLC cell lines exposed or not to TKIs. H1993 and H1975 cells were stably transfected with scrambled (shCTRL) or PDK1-targeted (shPDK1) shRNA and then treated with MET inhibitor crizotinib (1 µM), double mutant EGFR^L858R/T790M inhibitor WZ4002 (1 µM) or vehicle for 48 h. The effects of PDK1 knockdown on glucose metabolism and apoptosis were evaluated in untreated and TKI-treated cells. PDK1 knockdown alone did not cause significant changes in glycolytic cascade, ATP production and glucose consumption, but it enhanced maximal respiration in shPDK1 cells when compared to controls. When combined with TKI treatment, PDK1 downregulation caused a strong enhancement of OXPHOS and a marked reduction in key glycolytic enzymes. Furthermore, increased levels of apoptotic markers were found in shPDK1 cells as compared to shCTRL cells after treatment with TKIs. Co-immunoprecipitation studies showed that PDK1 interacts with PKM2, Bcl-2 and Bcl-xL, forming macromolecular complexes at the ER–mitochondria interface. Our findings showed that downregulation of PDK1 is able to potentiate the effects of TKIs through the disruption of macromolecular complexes involving PKM2, Bcl-2 and Bcl-xL.

Notch1 plays a key role in epithelial-mesenchymal transition (EMT) and in the maintenance of cancer stem cells. In the present study we tested whether high levels of activated Notch1 in oncogene-driven NSCLC can induce a reversible shift of driver dependence from EGFR to Notch1, and thus causing resistance to EGFR inhibitors. Adherent cells (parental) and tumor spheres (TS) from NSCLC H1975 cells and patient-derived CD133-positive cells were tested for EGFR and Notch1 signaling cascade. The Notch1-dependent modulation of EGFR, NCID, Hes1, p53, and Sp1 were then analyzed in parental cells by binding assays with a Notch1 agonist, DLL4. TS were more resistant than parental cells to EGFR inhibitors. A strong upregulation of Notch1 and a concomitant downregulation of EGFR were observed in TS compared to parental cells. Parental cell exposure to DLL4 showed a dose-dependent decrease of EGFR and a simultaneous increase of NCID, Hes1, p53, and Sp1, along with the dislocation of Sp1 from the EGFR promoter. Furthermore, an enhanced interaction between p53 and Sp1 was observed in TS. In NSCLC cells, high levels of active Notch1 can promote a reversible shift of driver dependence from EGFR to Notch1, leading to resistance to EGFR inhibitors.