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Supplementary Tables S1-S5. Table S1: Antiproliferative effects of PI3K inhibitors on SKMES-1 and PIK3CA amplified/mutated clones Tables S2-S4: densitometric analysis of Western blotting of Figure 1D Table S5: Antiproliferative effects of PI3K inhibitors on H596 and HCC2450 cells

Supplementary Figures S1-S5. Figure S1: p-AKT levels in PIK3CA transfected clones Figure S2: Induction of cell death after treatment with PI3K inhibitors Figure S3: Time course evaluation of PI3K/AKT/mTOR pathway activity after exposure to PI3K inhibitors Figure S4: Evaluation of cell migration after treatment with PI3K inhibitors Figure S5: CD133 expression in tumor spheres

SummaryPatients with cancer of unknown primary (CUP) carry the double burden of an aggressive disease and reduced access to therapies. Experimental models summing up CUP features are pivotal for CUP biology investigation and drug testing. We derived two CUP cell lines (CUP#55 and #96), and corresponding patient-derived xenografts (PDXs), from ascites tumor cells. CUP cell lines and PDXs underwent histological, immune-phenotypical, molecular, and genomic characterization confirming the features of the original tumor. Genetic testing and FISH analysis identified FGFR2 amplification as therapeutic target in tumor tissues and patient-derived models. Drug-screening assays were performed to test the activity of FGFR2 targeting drug BGJ-398 (infigratinib) and the combination treatment with the MEK inhibitor trametinib, which proved to be synergic and exceptionally active, bothin vitroandin vivo. This study brings personalized therapy closer to CUP patients and paves the way to future applications of personalized medicine for metastatic patients with adverse prognosis.

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.

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. 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. 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. 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. 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.

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.

Second- and third-generation inhibitors of EGFR possess an acrylamide group which alkylates Cys797, allowing to overcome resistance due to insurgence of T790M mutation. Less reactive warheads, yet capable to bind the target cysteine, may be useful to design newer and safer inhibitors. In the present work, we synthesized a 2-chloro-N-(4-(phenylamino)quinazolin-6-yl)acetamide (8) derivative as a prototype of EGFR inhibitor potentially able to react with Cys797 by nucleophilic substitution. We then tuned the reactivity of the acetamide fragment by replacing the chlorine leaving group with (hetero)-aromatic thiols or carboxylate esters. Among the synthesized derivatives, the 2-((1H-imidazol-2-yl)thio)acetamide 16, while showing negligible reactivity with cysteine in solution, caused long-lasting inhibition of wild-type EGFR autophosphorylation in A549 cells, resulted able to bind recombinant EGFR L858R/T790M in a time-dependent manner, and inhibited both EGFR autophosphorylation and proliferation in gefitinib-resistant H1975 lung cancer cells (expressing EGFR L858R/T790M mutant) at low micromolar concentration.

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-&gamma, 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.

Cell metabolic reprogramming is one of the main hallmarks of cancer and many oncogenic pathways that drive the cancer-promoting signals also drive the altered metabolism. This review focuses on recent data on the use of oncogene-targeting agents as potential modulators of deregulated metabolism in different solid cancers. Many drugs, originally designed to inhibit a specific target, then have turned out to have different effects involving also cell metabolism, which may contribute to the mechanisms underlying the growth inhibitory activity of these drugs. Metabolic reprogramming may also represent a way by which cancer cells escape from the selective pressure of targeted drugs and become resistant. Here we discuss how targeting metabolism could emerge as a new effective strategy to overcome such resistance. Finally, accumulating evidence indicates that cancer metabolic rewiring may have profound effects on tumor-infiltrating immune cells. Modulating cancer metabolic pathways through oncogene-targeting agents may not only restore more favorable conditions for proper lymphocytes activation, but also increase the persistence of memory T cells, thereby improving the efficacy of immune-surveillance.

The possibility to influence the physiological concentration of copper ions through the careful choice of ligands is emerging as a novel intriguing strategy in the treatment of pathologies such as cancer and Alzheimer. Thiosemicarbazones play an important role in this field, because they offer a wide variety of potential functionalizations and different kinds of coordination modes. Here we report the synthesis of some 8-hydroxyquinoline thiosemicarbazone ligands containing an ONN'S donor set and their Zn(II) and Cu(II) complexes. The metal complexes were characterized in solution and in the solid state and the X-ray structure of one of the copper(II) complex is reported. The Cu(II) complexes were characterized also by means of quantum mechanical calculations. The Cu(II) complexes displayed cytostatic activity in different cancer cell models. In particular, the most active Cu(II) complex significantly inhibited cell proliferation with an IC50 value lower than 1 μM; this effect was associated with a block of the cell cycle in the G2/M phase. This Cu(II) complex induced neither the production of reactive oxygen species (ROS) nor the accumulation of p53 protein, suggesting the lack of DNA damage.

Dysregulation of the cell cycle is a hallmark of cancer that leads to aberrant cellular proliferation. CDK4/6 are cyclin-dependent kinases activated in response to proliferative signaling, which induce RB hyper-phosphorylation and hence activation of E2F transcription factors, thus promoting cell cycle progression through the S phase. Pharmacologic inhibition of CDK4/6 by palbociclib, ribociclib, or abemaciclib has been showing promising activity in multiple cancers with the best results achieved in combination with other agents. Indeed, CDK4/6 inhibitors are currently approved in combination with endocrine therapy for the treatment of estrogen receptor-positive, human epidermal growth factor receptor 2-negative advanced or metastatic breast cancer. Moreover, a number of clinical trials are currently underway to test the efficacy of combining CDK4/6 inhibitors with different drugs not only in breast but also in other types of cancer. Beyond the inhibition of cell proliferation, CDK4/6 inhibitors have recently revealed new effects on cancer cells and on tumor microenvironment. In particular, it has been reported that these agents induce a senescent-like phenotype, impact on cell metabolism and exert both immunomodulatory and immunogenic effects. Here we describe recent data on the anti-tumor effects of CDK4/6 inhibitors as single agents or in combined therapies, focusing in particular on their metabolic and immunomodulatory activities.

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.

Patients with occult primary cancer or cancer of unknown primary (CUP) carry the double burden of an aggressive disease and reduced access to therapies, which are typically tumor-type oriented. Moreover, experimental in-vitro and in-vivo models summing up CUP features and heterogeneity are currently unavailable. We derived and expanded two CUP cell lines, and corresponding patient-derived xenografts (PDXs), from ascites tumor cells. CUP cell lines, one spontaneously growing as organoid, and PDXs were subjected to histological, immune-phenotypical, molecular and genomic (CNV and target sequencing) characterization to show their ability to recapitulate the original tumor. MicroRNA profile and genomic information were used to identify a possible primary site and patient-specific therapeutic approaches. Data on CUP most frequently altered genes were used to design a custom CUP-specific, 92-gene panel for target NGS analysis with Sure Select XS technology (Agilent Technology). Drug-screening assays were performed in vitro and in vivo on two models targeting the most promising actionable pathways. We obtained unexpected and promising results on drug combinations efficacy. This study brings personalized therapy closer to CUP patients and paves the way to future applications of personalized medicine for metastatic patients with adverse prognosis. This work was supported by grants from the Italian Association for Cancer Research (AIRC) to MF (IG 18464). Citation Format: Noemi Laprovitera, Claudia Fumarola, Elisa Porcellini, Stefano Cairo, Giulia Gallerani, Francesco Gelsomino, Irene Salamon, Maria Naddeo, Andrea Ardizzoni, Andrea Cavazzoni, Manuela Ferracin. Cancer of Unknown Primary: novel therapeutic opportunities from patient-derived cell cultures and in vivo models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3011.

Simple Summary Immune checkpoint inhibitors (ICIs) have emerged as a very promising therapeutic option for the treatment of many difficult-to-treat cancers and a number of clinical trials have explored their efficacy in malignant pleural mesothelioma patients. ICIs were initially evaluated in the salvage setting, resulting in a modest activity, not superior to chemotherapy. However, in the last year the combination of nivolumab and ipilimumab as first line treatment has proved a superior efficacy compared to chemotherapy especially in the non-epithelioid subtype, obtaining the FDA approval in October 2020. Encouraging results are also emerging from other immunological approaches that take advantage of tumor-specific antigens, such as advanced cell-based therapies with the CAR-T cells and tumor vaccines. Abstract Malignant pleural mesothelioma (MPM) is a rare and aggressive malignant disease affecting the mesothelium, commonly associated to asbestos exposure. The current therapeutic actions, based on cisplatin/pemetrexed treatment, are limited due to the late stage at which most patients are diagnosed and to the intrinsic chemo-resistance of the tumor. Another relevant point is the absence of approved therapies in the second line setting following progression of MPM after chemotherapy. Considering the poor prognosis of the disease and the fact that the incidence of this tumor is expected to increase in the next decade, novel therapeutic approaches are urgently needed. In the last few years, several studies have investigated the efficacy and safety of immune-checkpoint inhibitors (ICIs) in the treatment of unresectable advanced MPM, and a number of trials with immunotherapeutic agents are ongoing in both first line and second line settings. In this review, we describe the most promising emerging immunotherapy treatments for MPM (ICIs, engineered T cells to express chimeric antigen receptors (CARs), dendritic cells (DCs) vaccines), focusing on the biological and immunological features of this tumor as well as on the issues surrounding clinical trial design.

Simple Summary Osimertinib, a third-generation irreversible epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), has shown marked clinical benefit for non-small cell lung cancer (NSCLC) patients with EGFR activating mutations. However, resistance to osimertinib inevitably develops and heterogeneous mechanisms of acquired resistance have been documented. Therefore, new strategies to bypass resistance are urgently needed. In this study, we investigated the potential activity of abemaciclib as second-line therapeutic approach after osimertinib progression and the effect of combining abemaciclib with osimertinib on the appearance of resistance in osimertinib-sensitive models. Abstract 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.

Background Cell cycle regulators have gain attention as potential targets for anticancer therapy. Palbociclib is a selective inhibitor of the cyclin-dependent kinases 4 and 6 (CDK4/6), which coordinate the G1-S transition. Palbociclib is currently approved for the treatment of hormone receptor positive, HER2-negative advanced breast cancer (BC) in association with letrozole or fulvestrant. In contrast, its efficacy in triple negative BC (TNBC), either alone or in combined therapies, has not been fully investigated to date. Methods Here we evaluated the potential of combining palbociclib with PI3K/mTOR inhibitors in Rb-proficient TNBC cells comparing different schedules of treatment: simultaneous, sequential, or sequential combined treatment (pre-incubation with palbociclib followed by exposure to both palbociclib and PI3K/mTOR inhibitors). We assessed the effects on cell proliferation, cell death, and cell cycle distribution, and looked at the impact of such treatments on glucose metabolism. Results Palbociclib exerted cytostatic effects in Rb-positive TNBC cells, inducing a reversible blockade in G0/G1 cell cycle phase associated with down-regulation of CDK6, Rb, and c-myc expression and/or activity. Palbociclib treatment induced AKT signaling, providing a rationale for its combination with PI3K/mTOR inhibitors. The simultaneous or sequential treatment resulted in an additive inhibition of cell proliferation. On the other hand, the sequential combined treatment in which palbociclib was maintained also during exposure to PI3K/mTOR inhibitors gave rise to synergistic anti-proliferative and pro-apoptotic effects, by inhibiting both CDK4/6/Rb/myc and PI3K/mTOR signaling. Interestingly, the inhibition of the Rb/E2F/myc axis mediated by palbociclib resulted in a significant down-regulation of glucose metabolism; most importantly, these inhibitory effects were enhanced by the combination of palbociclib with BYL719 (specific inhibitor of the p110α PI3K-subunit), which promoted a stronger inhibition of GLUT-1 glucose transporter expression, glucose uptake and consumption in comparison with individual treatments, under both normoxic and hypoxic conditions. Conclusions Combination of palbociclib with PI3K/mTOR inhibitors may represent a promising therapeutic option for the treatment of Rb-proficient TNBC, with the sequential combined schedule showing a superior efficacy over the other schedules. In addition our results demonstrate that the impairment of glucose metabolism may contribute to the anti-tumor activity of such drug combinations.

Squamous cell lung carcinoma (SCC) accounts for 30% of patients with NSCLC and to date, no molecular targeted agents are approved for this type of tumor. However, recent studies have revealed several oncogenic mutations in SCC patients, including an alteration of the PI3K/AKT pathway, i.e. PI3K point mutations and amplification, AKT mutations and loss or reduced PTEN expression. Prompted by our observation of a correlation between PTEN loss and FAK phosphorylation in a cohort of patients with stage IV SCC, we evaluated the relevance of PTEN loss in cancer progression as well as the efficacy of a new combined treatment with the pan PI3K inhibitor buparlisip and the FAK inhibitor defactinib. An increase in AKT and FAK phosphorylation, associated with increased proliferation and invasiveness, paralleled by the acquisition of mesenchymal markers, and overexpression of the oncomir miR-21 were observed in SKMES-1-derived cell clones with a stable reduction of PTEN. Notably, the combined treatment induced a synergistic inhibition of cell proliferation, and a significant reduction in cell migration and invasion only in cells with reduced PTEN. The molecular mechanisms underlying these findings were unraveled using a specific RTK array that showed a reduction in phosphorylation of key kinases such as JNK, GSK-3 α/β, and AMPK-α2, due to the concomitant decrease in AKT and FAK activation. In conclusion, the combination of buparlisib and defactinib was effective against cells with reduced PTEN and warrants further studies as a novel therapeutic strategy for stage IV SCC patients with loss of PTEN expression.

The study investigated the anti-tumour effect of zoledronic acid (ZA) administered alone in a biological window therapy in naïve bone-only metastatic and locally advanced breast cancer (LABC) patients. 33 patients with LABC (Group 1) and 20 patients with a first diagnosis of bone metastasis only (Group 2) received 4 mg single dose of ZA, 14 days (biological window) before starting any treatment. In Group 1, Ki67, CD34, p53/bcl-2 and caspase 3 expression along with the adenosine triphosphate (ATP) levels and RNA disruption index were evaluated as markers of tumor growth in tumour specimens obtained before and after ZA administration (basal, day 14). In Group 2, the total enumeration of circulating tumour cells (CTCs), and of M30+ve CTCs along with the soluble marker of cell death (M30/M65) were carried-out as markers of tumor dissemination at baseline, at 48 h and day 14th. In Group 1, there was a significant reduction in Ki67, CD34, bcl-2 expression after 14 days ZA based-treatment (p = 0.0032; p = 0.0001, p < 0.00001 respectively). ZA showed a significant increase of RNA disruption (p < 0.0076). In Group 2, we observed a significant reduction of CTCs number after 48 h (p = 0.0012), followed by a significant rebound at 14 days (p = 0.012). The apoptotic CTCs/M30+ve and M65 levels significantly increased under treatment (p = 0.018 and p = 0.039 respectively) after drug administration when compared to the baseline. These results are the first prospective in vivo data showing the direct pure anti-tumour effect (either on the tumour cell or on CTCs) of ZA.

// Claudia Fumarola 1 , Daniele Cretella 1 , Silvia La Monica 1 , Mara A. Bonelli 1 , Roberta Alfieri 1 , Cristina Caffarra 1 , Federico Quaini 1 , Denise Madeddu 1 , Angela Falco 1 , Andrea Cavazzoni 1 , Graziana Digiacomo 1 , Giulia Mazzaschi 1 , Valentina Vivo 2 , Elisabetta Barocelli 2 , Marcello Tiseo 3 , Pier Giorgio Petronini 1, * and Andrea Ardizzoni 4, * 1 Department of Medicine and Surgery, University of Parma, Parma, Italy 2 Food and Drug Department, University of Parma, Parma, Italy 3 Medical Oncology Unit, University Hospital of Parma, Parma, Italy 4 Division of Medical Oncology, Sant’Orsola-Malpighi University Hospital, Bologna, Italy * Joint last authors Correspondence to: Claudia Fumarola, email: claudia.fumarola@unipr.it Mara A. Bonelli, email: mara.bonelli@unipr.it Keywords: FGFR1, glucose metabolism, dovitinib, NVP-BGJ398, SQCLC Received: March 03, 2017 Accepted: June 18, 2017 Published: July 17, 2017 ABSTRACT Fibroblast Growth Factor Receptor (FGFR) signaling is a complex pathway which controls several processes, including cell proliferation, survival, migration, and metabolism. FGFR1 signaling is frequently deregulated via amplification/over-expression in NSCLC of squamous histotype (SQCLC), however its inhibition has not been successfully translated in clinical setting. We determined whether targeting downstream signaling implicated in FGFR1 effects on glucose metabolism potentiates the anti-tumor activity of FGFR1 inhibition in SQCLC. In FGFR1 amplified/over-expressing SQCLC cell lines, FGF2-mediated stimulation of FGFR1 under serum-deprivation activated both MAPK and AKT/mTOR pathways and increased glucose uptake, glycolysis, and lactate production, through AKT/mTOR-dependent HIF-1α accumulation and up-regulation of GLUT-1 glucose transporter. These effects were hindered by PD173074 and NVP-BGJ398, selective FGFR inhibitors, as well as by dovitinib, a multi-kinase inhibitor. Glucose metabolism was hampered by the FGFR inhibitors also under hypoxic conditions, with consequent inhibition of cell proliferation and viability. In presence of serum, glucose metabolism was impaired only in cell models in which FGFR1 inhibition was associated with AKT/mTOR down-regulation. When the activation of the AKT/mTOR pathway persisted despite FGFR1 down-regulation, the efficacy of NVP-BGJ398 could be significantly improved by the combination with NVP-BEZ235 or other inhibitors of this signaling cascade, both in vitro and in xenotransplanted nude mice. Collectively our results indicate that inhibition of FGFR1 signaling impacts on cancer cell growth also by affecting glucose energy metabolism. In addition, this study strongly suggests that the therapeutic efficacy of FGFR1 targeting molecules in SQCLC may be implemented by combined treatments tackling on glucose metabolism.

Malignant pleural mesothelioma (MPM) is a rare and aggressive malignant disease affecting the mesothelium, commonly associated to asbestos exposure. Therapeutic actions are limited due to the late stage at which most patients are diagnosed and the intrinsic chemo-resistance of the tumor. The recommended systemic therapy for MPM is cisplatin/pemetrexed regimen with a mean overall survival of about 12months and a median progression free survival of less than 6months. Considering that the incidence of this tumor is expected to increase in the next decade and that its prognosis is poor, novel therapeutic approaches are urgently needed. For some tumors, such as lung cancer and breast cancer, druggable oncogenic alterations have been identified and targeted therapy is an important option for these patients. For MPM, clinical guidelines do not recommend biological targeted therapy, mainly because of poor target definition or inappropriate trial design. Further studies are required for a full comprehension of the molecular pathogenesis of MPM and for the development of new target agents. This review updates pre-clinical and clinical data on the efficacy of targeted therapy and immune checkpoint inhibition in the treatment of mesothelioma. Finally, future perspectives in this deadly disease are also discussed.

Silk fibroin fibres from two different sources, Bombyx mori pure-breed silkworms and polyhybrid cross-bred silkworm cocoons, were treated with formic acid under planar stirring conditions to prepare non-woven nets. The treatment partially dissolved the fibres, which bound together and formed a non-woven micrometric net with fibres coated by a thin layer of low molecular weight fibroin matrix. The starting fibres, net materials and fibroin coating layer were characterized in terms of amino acid composition, molecular weight and calorimetric properties. In vitro cell culture tests with rat fibroblasts were performed to investigate cell proliferation, morphology and spreading. Moreover, host-rat fibroblasts were preseeded on the afore-mentioned nets and implanted in the thorax of rats for histological analysis. In spite of the chemical differences between the two starting fibroins, the response of the said materials in vitro and in vivo were very similar. These results suggest that the outcome is likely correlated with the modification of the processing technique; that during the formation of the net, a thin gel layer of similar amino acid composition was formed on the fibroin fibres. Copyright © 2012 John Wiley & Sons, Ltd.

Gefitinib, an inhibitor of epidermal growth factor receptor tyrosine kinase, has been developed and approved for treatment of advanced non-small cell lung cancer (NSCLC). In this study, we investigated the uptake of gefitinib in gefitinib-sensitive and -resistant NSCLC cell lines. The transport system was temperature-dependent, indicative of an active process and sodium- and potential-independent. Moreover, high cell densities and low extracellular pH significantly reduced the uptake of gefitinib. Inhibitors of the human organic cation transporter 1 (hOCT1) significantly decreased gefitinib uptake; however, gefitinib was not a substrate for hOCT1 or hOCT2 in overexpressing HEK293 cells. Interestingly, gefitinib significantly reduced uptake of the hOCT prototypical substrate MPP suggesting that gefitinib may exert an inhibitory effect on the intracellular accumulation of drugs transported by hOCT1 and hOCT2. After 15min of treatment at 1microM (the maximum plasma concentration of gefitinib obtained at the clinically relevant dose) gefitinib accumulated within the cell in resistant-cell lines at concentrations similar or even higher than in gefitinib-sensitive cells tending to rule out an alteration in drug uptake as a mechanism of resistance to gefitinib treatment. Moreover, our results suggest that the extrusion of lactate by crowded cells may contribute in decreasing the pH, which in turn can influence the uptake of gefinitib and as a result the inhibition of EGFR autophosphorylation.

The epidermal growth factor receptor(EGFR) is a validated target for therapy in non-small cell lung cancer (NSCLC). Most patients, however, either do not benefit or develop resistance to specific inhibitors of the EGFR tyrosine kinase activity, such as gefitinib or erlotinib. The mammalian target of rapamycin (mTOR) is a key intracellular kinase integrating proliferation and survival pathways and has been associated with resistance to EGFR tyrosine kinase inhibitors. In this study, we assessed the effects of combining the mTOR inhibitor everolimus (RAD001) with gefitinib on a panel of NSCLC cell lines characterized by gefitinib resistance and able to maintain S6K phosphorylation after gefitinib treatment. Everolimus plus gefitinib induced a significant decrease in the activation of MAPK and mTOR signaling pathways downstream of EGFR and resulted in a growth-inhibitory effect rather than in an enhancement of cell death. A synergistic effect was observed in those cell lines characterized by high proliferative index and low doubling time. These data suggest that treatment with everolimus and gefitinib might be of value in the treatment of selected NSCLC patients that exhibit high tumor proliferative activity. (C) 2009 Elsevier Inc. All rights reserved. RI goldoni, matteo/E-9153-2011; Mutti, Antonio/C-1095-2011

In this study, we examined the mechanism of action of the novel epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor 5-benzylidene-hydantoin UPR1024, whose structure was designed to interact at the ATP-binding site of EGFR. The compound had antiproliferative and proapoptotic effects when tested on the non–small cell lung cancer cell line A549. The growth inhibitory effect was associated with an accumulation of the cells in the S phase of the cell cycle. Moreover, UPR1024 induced significant level of DNA strand breaks associated with increased expression of p53 and p21WAF1 proteins, suggesting an additive mechanism of action. The presence of wild-type p53 improved the drug efficacy, although the effect was also detectable in p53 null cells. We also noted apoptotic cell death after treatment with UPR1024 at concentrations above 10 μmol/L for >24 h, with involvement of both the extrinsic and intrinsic pathways. The present data show that UPR1024 may be considered a combi-molecule capable of both blocking EGFR tyrosine kinase activity and inducing genomic DNA damage. UPR1024 or its derivatives might serve as a basis for development of drugs for the treatment of lung cancer in patients resistant to classic tyrosine kinase inhibitors. [Mol Cancer Ther 2008;7(2):361–70]

Loss of FHIT expression and p53 mutations are critical events in the early stages of lung carcinogenesis. The restoration of Fhit function in FHIT-negative cancer cells has been reported to cause tumour suppression by inhibition of cell proliferation and/or activation of apoptotic pathways. However, the studies designed to elucidate the biological role of Fhit and its potential interaction with p53 have produced conflicting results. We investigated here the effects of the simultaneous restoration of FHIT and p53 in Calu-1 cells by using a hormone-inducible gene expression system. We demonstrate that the restoration of FHIT expression reinforces the anti-proliferative effect associated with the simultaneous replacement of p53. Indeed, a more pronounced inhibition of cell proliferation associated with an earlier and higher induction of p21(waf1) mRNA and protein expression was observed in Fhit/p53-expressing cells compared with cells expressing p53 alone. This effect was not due to Fhit-mediated up-regulation of p53 expression; in fact p53 protein was expressed at the same level in both FHIT-positive and FHIT-negative cell clones. Consistent with this result, Fhit did not affect the expression of MDM2, a protein known to interact directly with p53 and target p53 for proteolytic degradation, thus down-regulating its activity.

Introduction Development of resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors is a clinical issue in patients with epidermal growth factor receptor gene ( EGFR ) - mutated non–small cell lung cancer (NSCLC). The aim of this study was to investigate the potential of combining gefitinib and pemetrexed in preventing the acquisition of resistance to EGFR tyrosine kinase inhibitors in NSCLC cell lines harboring EGFR exon 19 deletion. Methods The effect of different combinatorial schedules of gefitinib and pemetrexed on cell proliferation, cell cycle, apoptosis, and acquisition of gefitinib resistance in PC9 and HCC827 NSCLC cell lines and in PC9 xenograft models was investigated. Results Simultaneous treatment with gefitinib and pemetrexed enhanced cell growth inhibition and cell death and prevented the appearance of gefitinib resistance mediated by T790M mutation or epithelial-to-mesenchymal transition (EMT) in PC9 and HCC827 cells, respectively. In PC9 cells and in PC9 xenografts the combination of gefitinib and pemetrexed, with different schedules, prevented gefitinib resistance only when pemetrexed was the first treatment, given alone or together with gefitinib. Conversely, when gefitinib alone was administered first and pemetrexed sequentially alternated, a negative interaction was observed and no prevention of gefitinib resistance was documented. The mechanisms of resistance that developed in vivo included T790M mutation and EMT. The induction of EMT was a feature of tumors treated with gefitinib when given before pemetrexed, whereas T790M was recorded only in tumors treated with gefitinib alone. Conclusions The combination of gefitinib and pemetrexed is effective in preventing gefitinib resistance; the application of intermittent treatments requires that gefitinib not be administered before pemetrexed.

Exposure of C2C12 muscle cells to hypertonic stress induced an increase in cell content of creatine transporter mRNA and of creatine transport activity, which peaked after about 24 h incubation at 0.45 osmol (kg H2O)−1. This induction of transport activity was prevented by addition of either cycloheximide, to inhibit protein synthesis, or of actinomycin D, to inhibit RNA synthesis. Creatine uptake by these cells is largely Na+ dependent and kinetic analysis revealed that its increase under hypertonic conditions resulted from an increase in Vmax of the Na+-dependent component, with no significant change in the Km value of about 75 μmol l−1. Quantitative real-time PCR revealed a more than threefold increase in the expression of creatine transporter mRNA in cells exposed to hypertonicity. Creatine supplementation significantly enhanced survival of C2C12 cells incubated under hypertonic conditions and its effect was similar to that obtained with the well known compatible osmolytes, betaine, taurine and myo-inositol. This effect seemed not to be linked to the energy status of the C2C12 cells because hypertonic incubation caused a decrease in their ATP content, with or without the addition of creatine at 20 mmol l−1 to the medium. This induction of creatine transport activity by hypertonicity is not confined to muscle cells: a similar induction was shown in porcine endothelial cells.

In Jurkat cells, the decreased cell growth rate associated with a long-lasting deactivation of the mammalian target of rapamycin (mTOR)/p70 ribosomal S6 kinase (S6K)-signaling pathway generates a cell population of progressively reduced cellular mass and size. When promoted by rapamycin as prototype inhibitor, the mTOR deactivation-dependent cell size reduction was associated with slowed, but not suppressed, proliferation. Small-size cells were significantly protected from apoptosis induced by Fas/Apo-1 death-receptor activation (as shown by reduced procaspase cleavage and decreased catalytic activity of relevant caspases) or by stress signals-dependent mitochondrial perturbation (as shown by reduced cleavage of caspase-2, lower dissipation of mitochondrial membrane potential and decreased release of cytochorome c and apoptosis-inducing factor from mitochondria). Protection faded when reactivation of the mTOR/S6K pathway promoted the cell recovery to normal size. These results suggest that cells induced to reduce their mass by the mTOR deactivation-dependent inhibition of cell growth become more resilient to lethal assaults by curbing the cell's suicidal response.

Mammalian target of rapamycin (mTOR) mediates a signaling pathway that couples amino acid availability to S6 kinase (S6K) activation, translational initiation and cell growth rate, participating to a versatile checkpoint that inspects the energy status of the cell. The pathway is activated by branched-chain amino acids (BCAA), leucine being the most effective, whereas amino acid dearth and ATP shortage lead to its deactivation. Glutamine- or amino acid-deprivation and hyperosmotic stress induce a fast cell shrinkage (with marked decrease of the intracellular water volume) associated to mTOR-dependent S6K1 dephosphorylation. Using cultured Jurkat cells, we have measured the changes of cell content and intracellular concentration of ATP, of relevant amino acids (BCAA) and of ninhydrin-positive substances (NPS, as measure of NH(2)-bearing organic osmolytes) under conditions that deactivate (leucine-deprivation, glutamine-deprivation, amino acid withdrawal, sorbitol-induced hyperosmotic stress) or reactivate a previously deactivated, mTOR-S6K1 pathway. We have also assessed the mitochondrial function by measurements of mitochondrial transmembrane potential in cells subjected to hypertonic stress. Our results indicate that diverse control signals converge on the mTOR-S6K1 signaling pathway. In the presence of adequate energy resources, the pathway senses the amino acid availability as inward transport of effective amino acids (as BCAA and especially leucine), but its activation occurs only in the presence of an extracellular amino acid complement, with glutamine as obligatory component, and does not tolerate decrements of cell water volume incapable of maintaining adequate intracellular physicochemical conditions.

Abnormalities in the expression of the tumour suppressor fragile histidine triad (FHIT) gene have been reported in a variety of human tumours, including lung cancer and restoration of its expression in cancer cell lines resulted in the inhibition of proliferation and apoptosis induction. Most of the studies that have assigned a proapoptotic role to the FHIT gene were performed in adenoviral-FHIT-transduced cancer cells expressing high levels of the Fhit protein. The present work was the first study designed to investigate the effects of FHIT gene replacement in a human FHIT-negative non-small-cell lung cancer (NSCLC) cell line (Calu-1) by using a hormone-inducible expression system that allows tight modulation of the transgene expression. Through this approach, we demonstrated that a prolonged induction was required to accumulate the Fhit protein at levels adequate to promote a significant decrease of cell proliferation. Analysis of cell-cycle phase distribution showed an accumulation of cells in the G0/G1 phase and a concomitant decrease in the S phase. Moreover, an upregulation of p21waf1 transcript was found, which could account for the alteration of the cycling properties of the cells. The growth-inhibitory effects observed were not associated with apoptosis appearance, and although in these conditions the Fhit protein content was higher than in normal bronchial human epithelial cells (NHBE), it was still significantly lower than the level capable of inducing apoptosis in Calu-1 cells after adenoviral-mediated FHIT gene transfer. These results indicate that the tumour suppressor properties of Fhit are strictly related to its expression level and show that the Fhit protein has a dose-dependent antiproliferative effect on the Fhit-negative Calu-1 lung cancer cell line.

A prominent role in the pathogenesis of squamous cell carcinoma of the lung (SQCLC) has been attributed to the aberrant activation of the PI3K signaling pathway, due to amplification or mutations of the p110α subunit of class I phosphatidylinositol 3-kinase (PIK3CA) gene. The aim of our study was to determine whether different genetic alterations of PIK3CA affect the biologic properties of SQCLC and to evaluate the response to specific targeting agents in vitro and in vivo. The effects of NVP-BEZ235, NVP-BKM120, and NVP-BYL719 on two-dimensional/three-dimensional (2D/3D) cellular growth, epithelial-to-mesenchymal transition, and invasiveness were evaluated in E545K or H1047R PIK3CA–mutated SQCLC cells and in newly generated clones carrying PIK3CA alterations, as well as in a xenograft model. PIK3CA mutated/amplified cells showed increased growth rate and enhanced migration and invasiveness, associated with an increased activity of RhoA family proteins and the acquisition of a mesenchymal phenotype. PI3K inhibitors reverted this aggressive phenotype by reducing metalloproteinase production, RhoA activity, and the expression of mesenchymal markers, with the specific PI3K inhibitors NVP-BKM120 and NVP-BYL719 being more effective than the dual PI3K/mTOR inhibitor NVP-BEZ235. A xenograft model of SQCLC confirmed that PIK3CA mutation promotes the acquisition of a mesenchymal phenotype in vivo and proved the efficacy of its specific targeting drug NVP-BYL719 in reducing the growth and the expression of mesenchymal markers in xenotransplanted tumors. These data indicate that PIK3CA mutation/amplification may represent a good predictive feature for the clinical application of specific PI3K inhibitors in SQCLC patients. Mol Cancer Ther; 14(8); 1916–27. ©2015 AACR.

Background BCRP/ABCG2 emerged as an important multidrug resistance protein, because it confers resistance to several classes of cancer chemotherapeutic agents and to a number of novel molecularly-targeted therapeutics such as tyrosine kinase inhibitors. Gefitinib is an orally active, selective EGFR tyrosine kinase inhibitor used in the treatment of patients with advanced non small cell lung cancer (NSCLC) carrying activating EGFR mutations. Membrane transporters may affect the distribution and accumulation of gefitinib in tumour cells; in particular a reduced intracellular level of the drug may result from poor uptake, enhanced efflux or increased metabolism. Aim The present study, performed in a panel of NSCLC cell lines expressing different ABCG2 plasma membrane levels, was designed to investigate the effect of the efflux transporter ABCG2 on intracellular gefitinib accumulation, by dissecting the contribution of uptake and efflux processes. Methods and Results Our findings indicate that gefitinib, in lung cancer cells, inhibits ABCG2 activity, as previously reported. In addition, we suggest that ABCG2 silencing or overexpression affects intracellular gefitinib content by modulating the uptake rather than the efflux. Similarly, overexpression of ABCG2 affected the expression of a number of drug transporters, altering the functional activities of nutrient and drug transport systems, in particular inhibiting MPP, glucose and glutamine uptake. Conclusions Therefore, we conclude that gefitinib is an inhibitor but not a substrate for ABCG2 and that ABCG2 overexpression may modulate the expression and activity of other transporters involved in the uptake of different substrates into the cells.

While PI3K/AKT/mTOR pathway is altered in a variety of cancers including non small cell lung cancer, abnormalities in this pathway are more common in squamous cell lung carcinoma than in adenocarcinoma of the lung. Moreover, aberrant activation of PI3K/AKT/mTOR pathway is one of the mechanisms of acquired resistance to EGFR-TK inhibitors in patients with adenocarcinoma carrying EGFR activating mutations. Several inhibitors of the PI3K pathway are undergoing evaluation in preclinical and clinical studies. These include pan and selective inhibitors of PI3K, AKT inhibitors, rapamycin and rapalogs for mTOR inhibition, dual mTORC1-mTORC2 inhibitors and dual PI3K-mTOR inhibitors. This review focuses on recent preclinical and clinical data on the efficacy of PI3K pathway inhibitors in NSCLC either as monotherapy approach or in combination with chemotherapy or with drugs that target other signaling transduction pathways.

Cell shrinkage and loss of cell viability by apoptosis have been examined in cultured CD95(Fas/Apo-1)-expressing leukemia-derived CEM and HL-60 cells subjected to acute deprivation of glutamine, a major compatible osmolyte engaged in cell volume control. Glutamine deprivation-mediated cell shrinkage promoted a ligand-independent activation of the CD95-mediated apoptotic pathway. Cell transfection with plasmids expressing FADD-DN or v-Flip viral proteins pointed to a functional clustering of CD95 receptors at the cell surface with activation of the 'extrinsic pathway' caspase cascade. Accordingly, cell shrinkage did not induce apoptosis in CD95 receptor-negative lymphoma L1210 cells. Replacement of glutamine with surrogate compatible osmolytes counteracted cell volume decrement and protected the CD95-expressing cells from apoptosis. A glutamine deprivation-dependent cell shrinkage with activation of the CD95-mediated pathway was also observed when asparaginase was added to the medium. Asparagine depletion had no role in this process. The cell-size shrinkage-dependent apoptosis induced by glutamine restriction in CD95-expressing leukemic cells may therefore be of clinical relevance in amidohydrolase enzyme therapies.