Targeting glucosylceramide synthase induces antiproliferative and proapoptotic effects in osimertinib-resistant NSCLC cell models.

The EGFR tyrosine kinase inhibitor osimertinib has been approved for the first-line treatment of EGFR-mutated Non-Small Cell Lung Cancer (NSCLC) patients. Despite its efficacy, patients develop resistance. Mechanisms of resistance are heterogeneous and not fully understood, and their characterization is essential to find new strategies to overcome resistance. Ceramides are well-known regulators of apoptosis and are converted into glucosylceramides (GlcCer) by glucosylceramide synthase (GCS). A higher content of GlcCers was observed in lung pleural effusions from NSCLC patients and their role in osimertinib-resistance has not been documented. The aim of this study was to determine the therapeutic potential of inhibiting GCS in NSCLC EGFR-mutant models resistant to osimertinib in vitro and in vivo. Lipidomic analysis showed a significant increase in the intracellular levels of glycosylceramides, including GlcCers in osimertinib resistant clones compared to sensitive cells. In resistant cells, the GCS inhibitor PDMP caused cell cycle arrest, inhibition of 2D and 3D cell proliferation, colony formation and migration capability, and apoptosis induction. The intratumoral injection of PDMP completely suppressed the growth of OR xenograft models. This study demonstrated that dysregulation of ceramide metabolism is involved in osimertinib-resistance and targeting GCS may be a promising therapeutic strategy for patients progressed to osimertinib.

Targeting metabolic adaptive responses induced by glucose starvation inhibits cell proliferation and enhances cell death in osimertinib-resistant non-small cell lung cancer (NSCLC) cell lines.

Osimertinib, a tyrosine kinase inhibitor targeting mutant EGFR, has received approval for initial treatment in patients with Non-Small Cell Lung Cancer (NSCLC). While effective in both first- and second-line treatments, patients eventually develop acquired resistance. Metabolic reprogramming represents a strategy through which cancer cells may resist and adapt to the selective pressure exerted by the drug. In the current study, we investigated the metabolic adaptations associated with osimertinib-resistance in NSCLC cells under low glucose culture conditions. We demonstrated that, unlike osimertinib-sensitive cells, osimertinib-resistant cells were able to survive under low glucose conditions by increasing the rate of glucose and glutamine uptake and by shifting towards mitochondrial metabolism. Inhibiting glucose/pyruvate contribution to mitochondrial respiration, glutamine deamination to glutamate, and oxidative phosphorylation decreased the proliferation and survival abilities of osimertinib-resistant cells to glucose starvation. Our findings underscore the remarkable adaptability of osimertinib-resistant NSCLC cells in a low glucose environment and highlight the pivotal role of mitochondrial metabolism in mediating this adaptation. Targeting the metabolic adaptive responses triggered by glucose shortage emerges as a promising strategy, effectively inhibiting cell proliferation and promoting cell death in osimertinib-resistant cells.

A Photoactive Supramolecular Complex Targeting PD-L1 Reveals a Weak Correlation between Photoactivation Efficiency and Receptor Expression Levels in Non-Small-Cell Lung Cancer Tumor Models.

Photo-immunotherapy uses antibodies conjugated to photosensitizers to produce nanostructured constructs endowed with targeting properties and photo-inactivation capabilities towards tumor cells. The superficial receptor density on cancer cells is considered a determining factor for the efficacy of the photodynamic treatment. In this work, we propose the use of a photoactive conjugate that consists of the clinical grade PD-L1-binding monoclonal antibody Atezolizumab, covalently linked to either the well-known photosensitizer eosin or the fluorescent probe Alexa647. Using single-molecule localization microscopy (direct stochastic optical reconstruction microscopy, dSTORM), and an anti-PD-L1 monoclonal antibody labelled with Alexa647, we quantified the density of PD-L1 receptors exposed on the cell surface in two human non-small-cell lung cancer lines (H322 and A549) expressing PD-L1 to a different level. We then investigated if this value correlates with the effectiveness of the photodynamic treatment. The photodynamic treatment of H322 and A549 with the photo-immunoconjugate demonstrated its potential for PDT treatments, but the efficacy did not correlate with the PD-L1 expression levels. Our results provide additional evidence that receptor density does not determine a priori the level of photo-induced cell death.

Intrinsic Resistance to Osimertinib in EGFR Mutated NSCLC Cell Lines Induced by Alteration in Cell-Cycle Regulators.

BACKGROUND: Cell-cycle regulators are mutated in approximately 40% of all cancer types and have already been linked to worse outcomes in non-small cell lung cancer adenocarcinomas treated with osimertinib. However, their exact role in osimertinib resistance has not been elucidated. OBJECTIVE: In this study, we aimed to evaluate how the CDK4/6-Rb axis may affect the sensitivity to osimertinib. METHODS: We genetically increased the level of CCND1 (Cyclin D1) and reduced the levels of CDKN2A (p16) in two different adenocarcinoma cell lines, PC9 and HCC827. We also retrospectively evaluated the outcome of patients with epidermal growth factor receptor-mutated advanced non-small cell lung cancer depending on their level of Cyclin D1 and p16. RESULTS: The modified clones showed higher proliferative capacity, modifications in cell-cycle phases, and higher migratory capacity than the parental cells. Cyclin D1-overexpressing clones were highly resistant to acute osimertinib treatment. CDKN2A knockdown conferred intrinsic resistance as well, although a longer time was required for adaption to the drug. In both cases, the resistant phenotype was epidermal growth factor receptor independent and associated with a higher level of Rb phosphorylation, which was unaffected by osimertinib treatment. Blocking the phosphorylation of Rb using abemaciclib, a CDK4/6 inhibitor, exerted an additive effect with osimertinib, increasing sensitivity to this drug and reverting the intrinsic resistant phenotype. In a group of 32 patients with epidermal growth factor receptor-mutated advanced non-small cell lung cancer, assessed for Cyclin D1 and p16 expression, we found that the p16-deleted group presented a lower overall response rate compared with the control group. CONCLUSIONS: We conclude that perturbation in cell-cycle regulators leads to intrinsic osimertinib resistance and worse patient outcomes.

Three-Dimensional Bioprinting of Organoid-Based Scaffolds (OBST) for Long-Term Nanoparticle Toxicology Investigation.

The toxicity of nanoparticles absorbed through contact or inhalation is one of the major concerns for public health. It is mandatory to continually evaluate the toxicity of nanomaterials. In vitro nanotoxicological studies are conventionally limited by the two dimensions. Although 3D bioprinting has been recently adopted for three-dimensional culture in the context of drug release and tissue regeneration, little is known regarding its use for nanotoxicology investigation. Therefore, aiming to simulate the exposure of lung cells to nanoparticles, we developed organoid-based scaffolds for long-term studies in immortalized cell lines. We printed the viscous cell-laden material via a customized 3D bioprinter and subsequently exposed the scaffold to either 40 nm latex-fluorescent or 11-14 nm silver nanoparticles. The number of cells significantly increased on the 14th day in the 3D environment, from 5 × 105 to 1.27 × 106, showing a 91% lipid peroxidation reduction over time and minimal cell death observed throughout 21 days. Administered fluorescent nanoparticles can diffuse throughout the 3D-printed scaffolds while this was not the case for the unprinted ones. A significant increment in cell viability from 3D vs. 2D cultures exposed to silver nanoparticles has been demonstrated. This shows toxicology responses that recapitulate in vivo experiments, such as inhaled silver nanoparticles. The results open a new perspective in 3D protocols for nanotoxicology investigation supporting 3Rs.

Interaction between Occupational and Non-Occupational Arsenic Exposure and Tobacco Smoke on Lung Cancerogenesis: A Systematic Review.

Although a higher lung cancer risk has been already associated with arsenic exposure, the contribution of arsenic and its compounds to the carcinogenic effects of other agents, such as tobacco smoke, is not well characterized. This systematic review examined the relationship between occupational and non-occupational arsenic exposure and tobacco smoking on lung cancer risk using papers published from 2010 to 2022. Two databases, PUBMED and Scifinder, were used for the searches. Among the sixteen human studies included, four were about occupational exposure, and the others were about arsenic in drinking water. Furthermore, only three case-control studies and two cohort studies evaluated an additive or multiplicative interaction. The interaction between arsenic exposure and tobacco smoke seems to be negligible at low arsenic concentrations (<100 µg/L), while there is a synergistic effect at higher concentrations. Finally, it is not yet possible to assess whether a linear no-threshold (LNT) model for lung cancer risk can be applied to the co-exposure to arsenic and tobacco smoke. Although the methodological quality of the included studies is good, these findings suggest that rigorous and accurate prospective studies on this topic are highly needed.

A New ABCB1 Inhibitor Enhances the Anticancer Effect of Doxorubicin in Both In Vitro and In Vivo Models of NSCLC.

In tumors, the multi drug resistance phenomenon may occur through the efflux of chemotherapeutic drugs out of cancer cells, impeding their accumulation, and eventually reducing their toxicity. This process is mediated by transporters overexpressed in the plasma membranes of tumor cells, among which is the P-glycoprotein/multidrug resistance 1/ATP-binding cassette B1 (P-gp/MDR1/ABCB1). The aim of this study was to explore the effect of a new molecule, called AIF-1, on ABCB1 activity. In a cellular model of non-small cell lung cancer (NSCLC), AIF-1 significantly inhibited ABCB1 activity, which was evaluated by the fluorimetric measurement of the intracellular accumulation of calcein. AIF-1 also significantly increased the intracellular content of doxorubicin, which was evaluated by confocal microscopy and LC-MS/MS analysis. This effect translated to higher cytotoxicity of doxorubicin and reduced cellular proliferation. Finally, in a murine xenograft model, the tumor volume increased by 267% and 148% on average in mice treated with vehicle and doxorubicin alone, respectively. After the co-administration of doxorubicin with AIF-1, tumor volume increased by only 13.4%. In conclusion, these results suggest enhancement of the efficacy of the chemotherapeutic drug doxorubicin by AIF-1, laying the basis for the future development of new ABCB1 inhibitors for tumor treatment.

CDK4/6 Inhibition Enhances the Efficacy of Standard Chemotherapy Treatment in Malignant Pleural Mesothelioma Cells.

BACKGROUND: The loss of the CDKN2A/ARF (cyclin-dependent kinase inhibitor 2A/alternative reading frame) gene is the most common alteration in malignant pleural mesothelioma (MPM), with an incidence of about 70%, thus representing a novel target for mesothelioma treatment. In the present study, we evaluated the antitumor potential of combining the standard chemotherapy regimen used for unresectable MPM with the CDK4/6 (cyclin-dependent kinase 4 or 6) inhibitor abemaciclib. METHODS: Cell viability, cell death, senescence, and autophagy induction were evaluated in two MPM cell lines and in a primary MPM cell culture. RESULTS: The simultaneous treatment of abemaciclib with cisplatin and pemetrexed showed a greater antiproliferative effect than chemotherapy alone, both in MPM cell lines and in primary cells. This combined treatment induced cellular senescence or autophagic cell death, depending on the cell type. More in detail, the induction of cellular senescence was related to the increased expression of p21, whereas autophagy induction was due to the impairment of the AKT/mTOR signaling. Notably, the effect of the combination was irreversible and no resumption in tumor cell proliferation was observed after drug withdrawal. CONCLUSION: Our results demonstrated the therapeutic potential of CDK4/6 inhibitors in combination with chemotherapy for the treatment of MPM and are consistent with the recent positive results in the MiST2 arm in abemaciclib-treated patients.

CDK4/6 inhibitors improve the anti-tumor efficacy of lenvatinib in hepatocarcinoma cells.

Hepatocellular carcinoma (HCC) is the most frequent primary liver cancer with a poor prognosis and limited treatment options. Considering that alterations of the CDK4/6-cyclin D-Rb pathway occur frequently in HCC, we tested the efficacy of two CDK4/6 inhibitors, abemaciclib and ribociclib, in combination with lenvatinib, a multi-kinase inhibitor approved as first-line therapy for advanced HCC, in a panel of HCC Rb-expressing cell lines. The simultaneous drug combinations showed a superior anti-proliferative activity as compared with single agents or sequential schedules of treatment, either in short or in long-term experiments. In addition, the simultaneous combination of abemaciclib with lenvatinib reduced 3D cell growth, and impaired colony formation and cell migration. Mechanistically, these growth-inhibitory effects were associated with a stronger down-regulation of c-myc protein expression. Depending on the HCC cell model, reduced activation of MAPK, mTORC1/p70S6K or src/FAK signaling was also observed. Abemaciclib combined with lenvatinib arrested the cells in the G1 cell cycle phase, induced p21 accumulation, and promoted a stronger increase of cellular senescence, associated with elevation of ß-galactosidase activity and accumulation of ROS, as compared with single treatments. After drug withdrawal, the capacity of forming colonies was significantly impaired, suggesting that the anti-tumor efficacy of abemaciclib and lenvatinib combination was persistent. Our pre-clinical results demonstrate the effectiveness of the simultaneous combination of CDK4/6 inhibitors with lenvatinib in HCC cell models, suggesting that this combination may be worthy of further investigation as a therapeutic approach for the treatment of advanced HCC.

Interaction between occupational radon exposure and tobacco smoke: a systematic review.

INTRODUCTION: The risk of lung cancer from radon exposure was small compared to tobacco smoking (BEIR VI), but the relationship between these two carcinogenic agents has yet to be quantitatively estimated. The objective of this systematic review was to evaluate the last evidences on the role of radon occupational exposures and tobacco smoke on lung cancer risk. AREAS COVERED: Thirteen articles were selected using two different databases, PubMed and Scifinder, and were limited to those published from 2010 to 2021. The reference list of selected studies was reviewed to identify other relevant papers. EXPERT OPINION: Seven papers included in this systematic review did not deal with the multiplicative or the additive type of interaction between radon exposure and smoking habit. Six papers discussed the nature of this interaction with a prevalence of the sub-multiplicative model compared to the additive one. Altogether, smoking adjustment did not significantly change lung cancer risk. The included studies might constitute a starting point for updating the models for risk assessment in occupational and residential scenarios, promoting concomitantly the exposure reduction to radon and other cofactors, as recently introduced by Italian Legislative Decree number 101 of 31 July 2020, an application of Euratom Directive 59/2013.

This paper offers an updated overview on lung cancer risk due to occupational exposure to radon together with tobacco smoking habits. It evidences the quantitative role of smoking on radon exposure and discusses their interaction. The results show that smoking adjustments do not significantly modify lung cancer risk at both high and low radon concentrations.

Occupational exposure to graphene and silica nanoparticles. Part II: pilot study to identify a panel of sensitive biomarkers of genotoxic, oxidative and inflammatory effects on suitable biological matrices.

The available biomonitoring studies on workers producing/handling nanomaterials (NMs) focused on potential effects on respiratory, immune and cardio-vascular system. Aim of this study was to identify a panel of sensitive biomarkers and suitable biological matrices to evaluate particularly genotoxic and oxidative effects induced on workers unintentionally exposed to graphene or silica nanoparticles during the production process. These nanomaterials have been chosen for 'NanoKey' project, integrating the workplace exposure assessment (reported in part I) with the biomonitoring of exposed workers reported in the present work. Simultaneously to workplace exposure characterization, we monitored the workers using: Buccal Micronucleus Cytome (BMCyt) assay, fpg-comet test (lymphocytes), oxidized DNA bases 8-oxoGua, 8-oxoGuo and 8-oxodGuo measurements (urine), analysis of oxidative stress biomarkers in exhaled breath condensate (EBC), FENO measurement and cytokines release detection (serum). Since buccal cells are among the main targets of NM occupational exposure, particular attention was posed to the BMCyt assay that represents a noninvasive assay. This pilot study, performed on 12 workers vs.11 controls, demonstrates that BMCyt and fpg-comet assays are the most sensitive biomarkers of early, still reparable, genotoxic and oxidative effects. The findings suggest that these biomarkers could represent useful tools for the biomonitoring of workers exposed to nanoparticles, but they need to be confirmed on a high number of subjects. However, such biomarkers don't discriminate the effects of NM from those due to other chemicals used in the NM production process. Therefore, they could be suitable for the biomonitoring of workers exposed to complex scenario, including nanoparticles exposure.

Efficacy of the CDK4/6 Dual Inhibitor Abemaciclib in EGFR-Mutated NSCLC Cell Lines with Different Resistance Mechanisms to Osimertinib.

Abemaciclib is an inhibitor of cyclin-dependent kinases (CDK) 4 and 6 that inhibits the transition from the G1 to the S phase of the cell cycle by blocking downstream CDK4/6-mediated phosphorylation of Rb. The effects of abemaciclib alone or combined with the third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) osimertinib were examined in a panel of PC9 and HCC827 osimertinib-resistant non-small cell lung cancer (NSCLC) cell lines carrying EGFR-dependent or -independent mechanisms of intrinsic or acquired resistance. Differently from sensitive cells, all the resistant cell lines analyzed maintained p-Rb, which may be considered as a biomarker of osimertinib resistance and a potential target for therapeutic intervention. In these models, abemaciclib inhibited cell growth, spheroid formation, colony formation, and induced senescence, and its efficacy was not enhanced in the presence of osimertinib. Interestingly, in osimertinib sensitive PC9, PC9T790M, and H1975 cells the combination of abemaciclib with osimertinib significantly inhibited the onset of resistance in long-term experiments. Our findings provide a preclinical support for using abemaciclib to treat resistance in EGFR mutated NSCLC patients progressed to osimertinib either as single treatment or combined with osimertinib, and suggest the combination of osimertinib with abemaciclib as a potential approach to prevent or delay osimertinib resistance in first-line treatment.

Simultaneous Combination of the CDK4/6 Inhibitor Palbociclib With Regorafenib Induces Enhanced Anti-tumor Effects in Hepatocarcinoma Cell Lines.

Advanced hepatocarcinoma (HCC) is an aggressive malignancy with poor prognosis and limited treatment options. Alterations of the cyclin D-CDK4/6-Rb pathway occur frequently in HCC, providing the rationale for its targeting at least in a molecular subset of HCC. In a panel of HCC cell lines, we investigated whether the CDK4/6 inhibitor palbociclib might improve the efficacy of regorafenib, a powerful multi-kinase inhibitor approved as second-line treatment for advanced HCC after sorafenib failure and currently under clinical investigation as first-line therapy in combination with immunotherapy. In Rb-proficient cells, the simultaneous drug combination, but not the sequential schedules, inhibited cell proliferation, either in short or in long-term experiments, and induced cell death more strongly than individual treatments. Moreover, the combination significantly reduced spheroid cell growth and inhibited cell migration/invasion. The superior efficacy of palbociclib plus regorafenib emerged also under hypoxia and was associated with a significant down-regulation of CDK4/6-Rb-myc and mTORC1/p70S6K signaling. Moreover, regorafenib suppressed palbociclib-induced expression of cyclin D1 contributing to the cytotoxic effects of the combination. Besides these inhibitory effects on cell viability/proliferation, palbociclib and regorafenib reduced glucose uptake, although this effect was dependent on the cell model and on the oxygen availability (normoxia or hypoxia). Palbociclib and regorafenib combination impaired glucose uptake and utilization, down-regulating basal and hypoxia-induced expression of HIF-1α, HIF-2α, GLUT-1, and MCT4 proteins as well as the activity/expression of glycolytic enzymes (HK2, PFKP, aldolase A, PKM2). In addition, regorafenib alone reduced mitochondrial respiration. The combined treatment impaired glucose metabolism and respiration without enhancing the effects of the single agents. Our findings provide pre-clinical evidence for the effectiveness of palbociclib and regorafenib combination in HCC cell models.

Dual Inhibition of CDK4/6 and PI3K/AKT/mTOR Signaling Impairs Energy Metabolism in MPM Cancer Cells.

Background: Malignant pleural mesothelioma (MPM) is an aggressive malignancy associated to asbestos exposure. One of the most frequent genetic alteration in MPM patients is CDKN2A/ARF loss, leading to aberrant activation of the Rb pathway. In MPM cells, we previously demonstrated the therapeutic efficacy of targeting this signaling with the CDK4/6 inhibitor palbociclib in combination with PI3K/mTOR inhibitors. Here, we investigated whether such combination may have an impact on cell energy metabolism. Methods: The study was performed in MPM cells of different histotypes; metabolic analyses were conducted by measuring GLUT-1 expression and glucose uptake/consumption, and by SeaHorse technologies. Results: MPM cell models differed for their ability to adapt to metabolic stress conditions, such as glucose starvation and hypoxia. Independently of these differences, combined treatments with palbociclib and PI3K/mTOR inhibitors inhibited cell proliferation more efficaciously than single agents. The drugs alone reduced glucose uptake/consumption as well as glycolysis, and their combination further enhanced these effects under both normoxic and hypoxic conditions. Moreover, the drug combinations significantly impaired mitochondrial respiration as compared with individual treatments. These metabolic effects were mediated by the concomitant inhibition of Rb/E2F/c-myc and PI3K/AKT/mTOR signaling. Conclusions: Dual blockade of glycolysis and respiration contributes to the anti-tumor efficacy of palbociclib-PI3K/mTOR inhibitors combination.

Pemetrexed Enhances Membrane PD-L1 Expression and Potentiates T Cell-Mediated Cytotoxicity by Anti-PD-L1 Antibody Therapy in Non-Small-Cell Lung Cancer.

Immunotherapy has significantly changed the treatment landscape for advanced non-small-cell lung cancer (NSCLC) with the introduction of drugs targeting programmed cell death protein-1 (PD-1) and programmed cell death ligand-1 (PD-L1). In particular, the addition of the anti-PD-1 antibody pembrolizumab to platinum-pemetrexed chemotherapy resulted in a significantly improved overall survival in patients with non-squamous NSCLC, regardless of PD-L1 expression. In this preclinical study, we investigated whether chemotherapy can modulate PD-L1 expression in non-squamous NSCLC cell lines, thus potentially affecting immunotherapy efficacy. Among different chemotherapeutic agents tested, only pemetrexed increased PD-L1 levels by activating both mTOR/P70S6K and STAT3 pathways. Moreover, it also induced the secretion of cytokines, such as IFN-γ and IL-2, by activated peripheral blood mononuclear cells PBMCs that further stimulated the expression of PD-L1 on tumor cells, as demonstrated in a co-culture system. The anti-PD-1/PD-L1 therapy enhanced T cell-mediated cytotoxicity of NSCLC cells treated with pemetrexed and expressing high levels of PD-L1 in comparison with untreated cells. These data may explain the positive results obtained with pemetrexed-based chemotherapy combined with pembrolizumab in PD-L1-negative NSCLC and can support pemetrexed as one of the preferable chemotherapy partners for immunochemotherapy combination regimens.

Neutrophil Extracellular Traps Profiles in Patients with Incident Systemic Lupus Erythematosus and Lupus Nephritis.

OBJECTIVE: Neutrophil extracellular traps (NET) expose modified antigens for autoantibodies in vasculitis. Little is known about levels and removal pathways of NET in systemic lupus erythematosus (SLE), especially in lupus nephritis (LN). We determined circulating levels and defined NET removal in large subsets of patients with incident SLE (iSLE), some of whom had new-onset nephritis. METHODS: Serum levels of NET (ELISA), DNase1/DNase1L3 (ELISA), and DNase activity (functional assay) were determined in 216 patients with iSLE [103 had incident LN (iLN)], in 50 patients with other primary glomerulonephritis, and in healthy controls. Ex vivo NET production by neutrophils purified from a random selection of patients was quantified as elastase/DNA release and by immunofluorescence techniques. RESULTS: Serum NET levels were very high in iSLE/iLN compared to all groups of controls and correlated with anti-dsDNA, C3-C4, and proteinuria; iLN had the highest levels. DNase activity was decreased in iLN compared to SLE (20% had one-half DNase activity) despite similar serum levels of DNase1/DNase1L3. In these cases, pretreatment of serum with protein A restored DNase efficiency; 1 patient was homozygous for a c.289_290delAC variant of DNASE1L3. Ex vivo NET production by neutrophils purified from LN, SLE, and normal controls was similar in all cases. CONCLUSION: Patients with iLN have increased circulating NET and reduced DNase activity, the latter being explained by the presence of inhibitory substances in circulation and/or by rare DNase1L3 mutations. Accumulation of NET derives from a multifactorial mechanism, and is associated and may contribute to disease severity in SLE, in particular to renal lesions. (Clinical trial registration: The Zeus study was registered at ClinicalTrials.gov, study number NCT02403115).

Pre-treatment with the CDK4/6 inhibitor palbociclib improves the efficacy of paclitaxel in TNBC cells.

Triple Negative Breast Cancer (TNBC) is a challenging disease due to the lack of druggable targets; therefore, chemotherapy remains the standard of care and the identification of new targets is a high clinical priority. Alterations in the components of the cell cycle machinery have been frequently reported in cancer; given the success obtained with the CDK4/6 inhibitor palbocicib in ER-positive BC, we explored the potential of combining this drug with chemotherapy in Rb-positive TNBC cell models. The simultaneous combination of palbociclib with paclitaxel exerted an antagonistic effect; by contrast, the sequential treatment inhibited cell proliferation and increased cell death more efficaciously than single treatments. By down-regulating the E2F target c-myc, palbociclib reduced HIF-1α and GLUT-1 expression, and hence glucose uptake and consumption both under normoxic and hypoxic conditions. Importantly, these inhibitory effects on glucose metabolism were enhanced by palbociclib/paclitaxel sequential combination; the superior efficacy of such combination was ascribed to the ability of paclitaxel to inhibit palbociclib-mediated induction of AKT and to further down-regulate the Rb/E2F/c-myc signaling. Our results suggest that the efficacy of standard chemotherapy can be significantly improved by a pre-treatment with palbociclib, thus offering a better therapeutic option for Rb-proficient TNBC.

Acquired BRAF G469A Mutation as a Resistance Mechanism to First-Line Osimertinib Treatment in NSCLC Cell Lines Harboring an EGFR Exon 19 Deletion.

BACKGROUND: Osimertinib is a new third-generation, epidermal growth factor receptor-tyrosine kinase inhibitor highly selective for the epidermal growth factor receptor with both activating and T790M mutations. A recent phase III trial showed a statistically significant progression-free survival benefit with osimertinib vs. gefitinib or erlotinib as first-line treatment for EGFR-mutated non-small cell lung cancer, and preliminary data are available on resistance mechanisms to first-line osimertinib therapy. OBJECTIVE: The objective of this study was to examine potential in vitro mechanisms of acquired resistance to osimertinib in a cell model carrying an EGFR exon 19 deletion. METHODS: PC9 cells were cultured in the presence of increasing concentrations of osimertinib (ranging from 10 to 500 nM) to generate resistant cells. Three clones resistant to osimertinib (half maximal inhibitory concentration > 1 µM) were isolated, genotyped by next-generation sequencing and tested for drug sensitivity. Cell proliferation and migration, cell death, and signaling transduction pathways were analyzed. RESULTS: Our study revealed that all the three resistant clones developed acquired resistance via the BRAF G469A mutation maintaining a constitutive activation of the ERK pathway. Stable transfection of PC9 and HCC827 cells with a plasmid containing BRAF G469A rendered the cells resistant to osimertinib. Treatment with selumetinib and trametinib, but not dabrafenib, restored the sensitivity to osimertinib and enhanced cell death in the resistant clones with the BRAF G469A mutation. CONCLUSIONS: Our in vitro studies revealed the BRAF G469A-activating mutation as a potential mechanism of acquired resistance to first-line osimertinib treatment, and provide a strategy of intervention to overcome this mechanism of resistance.

Subchronic exposure to titanium dioxide nanoparticles modifies cardiac structure and performance in spontaneously hypertensive rats.

BACKGROUND: Non-communicable diseases, intended as the results of a combination of inherited, environmental and biological factors, kill 40 million people each year, equivalent to roughly 70% of all premature deaths globally. The possibility that manufactured nanoparticles (NPs) may affect cardiac performance, has led to recognize NPs-exposure not only as a major Public Health concern, but also as an occupational hazard. In volunteers, NPs-exposure is problematic to quantify. We recently found that inhaled titanium dioxide NPs, one of the most produced engineered nanomaterials, acutely increased cardiac excitability and promoted arrhythmogenesis in normotensive rats by a direct interaction with cardiac cells. We hypothesized that such scenario can be exacerbated by latent cardiovascular disorders such as hypertension. RESULTS: We monitored cardiac electromechanical performance in spontaneously hypertensive rats (SHRs) exposed to titanium dioxide NPs for 6 weeks using a combination of cardiac functional measurements associated with toxicological, immunological, physical and genetic assays. Longitudinal radio-telemetry ECG recordings and multiple-lead epicardial potential mapping revealed that atrial activation times significantly increased as well as proneness to arrhythmia. At the third week of nanoparticles administration, the lung and cardiac tissue encountered a maladaptive irreversible structural remodelling starting with increased pro-inflammatory cytokines levels and lipid peroxidation, resulting in upregulation of the main pro-fibrotic cardiac genes. At the end of the exposure, the majority of spontaneous arrhythmic events terminated, while cardiac hemodynamic deteriorated and a significant accumulation of fibrotic tissue occurred as compared to control untreated SHRs. Titanium dioxide nanoparticles were quantified in the heart tissue although without definite accumulation as revealed by particle-induced X-ray emission and ultrastructural analysis. CONCLUSIONS: The co-morbidity of hypertension and inhaled nanoparticles induces irreversible hemodynamic impairment associated with cardiac structural damage potentially leading to heart failure. The time-dependence of exposure indicates a non-return point that needs to be taken into account in hypertensive subjects daily exposed to nanoparticles.

Third generation EGFR inhibitor osimertinib combined with pemetrexed or cisplatin exerts long-lasting anti-tumor effect in EGFR-mutated pre-clinical models of NSCLC.

BACKGROUND: The third generation Epidermal Growth Factor Receptor (EGFR) Tyrosine Kinase Inhibitor (TKI) osimertinib has been initially approved for T790M positive Non-Small Cell Lung Cancer (NSCLC) and more recently for first-line treatment of EGFR-mutant T790M negative NSCLC patients. Similarly to previous generation TKIs, despite the high response rate, disease progression eventually occurs and current clinical research is focused on novel strategies to delay the emergence of osimertinib resistance. In this study we investigated the combination of osimertinib with pemetrexed or cisplatin in EGFR-mutated NSCLC cell lines and xenografts. METHODS: Tumor growth was evaluated in a PC9T790M xenograft model and tissue composition was morphometrically determined. PC9, PC9T790M and HCC827 cell lines were employed to test the efficacy of osimertinib and chemotherapy combination in vitro. Cell viability and cell death were evaluated by MTT assay and fluorescence microscopy. Protein expression and gene status were analysed by Western blotting, fluorescence in situ hybridization analysis, next-generation sequencing and digital droplet PCR. RESULTS: In xenograft models, osimertinib significantly inhibited tumor growth, however, as expected, in 50% of mice drug-resistance developed. A combination of osimertinib with pemetrexed or cisplatin prevented or at least delayed the onset of resistance. Interestingly, such combinations increased the fraction of fibrotic tissue and exerted a long-lasting activity after stopping therapy. In vitro studies demonstrated the stronger efficacy of the combination over the single treatments in inhibiting cell proliferation and inducing cell death in PC9T790M cells as well as in T790M negative PC9 and HCC827 cell lines, suggesting the potential role of this strategy also as first-line treatment. Finally, we demonstrated that osimertinib resistant clones, either derived from resistant tumors or generated in vitro, were less sensitive to pemetrexed prompting to use a chemotherapy regimen non-containing pemetrexed in patients after progression to osimertinib treatment. CONCLUSIONS: Our results identify a combination between osimertinib and pemetrexed or cisplatin potentially useful in the treatment of EGFR-mutated NSCLC patients, which might delay the appearance of osimertinib resistance with long-lasting effects.