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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. 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. 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. 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.

Lung cancer is the leading cause of cancer deaths worldwide. Most of lung cancer cases are classified as non-small cell lung cancers (NSCLC). EGFR has become an important therapeutic target for the treatment of NSCLC patients, and inhibitors targeting the kinase domain of EGFR are currently used in clinical settings. Recently, an increasing interest has emerged toward understanding the mechanisms and biological consequences associated with lipid reprogramming in cancer. Increased uptake, synthesis, oxidation, or storage of lipids has been demonstrated to contribute to the growth of many types of cancer, including lung cancer. In this review, we provide an overview of metabolism in cancer and then explore in more detail the role of lipid metabolic reprogramming in lung cancer development and progression and in resistance to therapies, emphasizing its connection with EGFR signaling. In addition, we summarize the potential therapeutic approaches targeting lipid metabolism for lung cancer treatment.

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.

Females have a lower probability to develop somatic cancers and a better response to chemotherapy than males. However, the reasons for these differences are still not well understood. The X-linked gene TSPY-Like 2 (TSPYL2) encodes for a putative tumor suppressor protein involved in cell cycle regulation and DNA damage response (DDR) pathways. Here, we demonstrate that in unstressed conditions TSPYL2 is maintained at low levels by MDM2-dependent ubiquitination and proteasome degradation. Upon genotoxic stress, E2F1 promotes TSPYL2 expression and protein accumulation in non-transformed cell lines. Conversely, in cancer cells, TSPYL2 accumulates only in females or in those male cancer cells that lost the Y-chromosome during the oncogenic process. Hence, we demonstrate that while TSPYL2 mRNA is induced in all the tested tumor cell lines after DNA damage, TSPYL2 protein stability is increased only in female cancer cells. Indeed, we found that TSPYL2 accumulation, in male cancer cells, is prevented by the Y-encoded protein SRY, which modulates MDM2 protein levels. In addition, we demonstrated that TSPYL2 accumulation is required to sustain cell growth arrest after DNA damage, possibly contributing to protect normal and female cancer cells from tumor progression. Accordingly, TSPYL2 has been found more frequently mutated in female-specific cancers. These findings demonstrate for the first time a sex-specific regulation of TSPYL2 in the DDR of cancer cells and confirm the existence of sexual dimorphism in DNA surveillance pathways.

Abstract 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.

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.

Background Osimertinib is a third-generation EGFR-TKI with a high selective potency against T790M-mutant NSCLC patients. Considering that osimertinib can lead to enhanced HER-2 expression on cell surface and HER-2 overexpression is a mechanism of resistance to osimertinib, this study was addressed to investigate the potential of combining osimertinib with trastuzumab emtansine (T-DM1) in order to improve the efficacy of osimertinib and delay or overcome resistance in NSCLC cell lines with EGFR activating mutation and with T790M mutation or HER-2 amplification. Methods The effects of osimertinib combined with T-DM1 on cell proliferation, cell cycle, cell death, antibody-dependent cell-mediated cytotoxicity (ADCC), and acquisition of osimertinib resistance was investigated in PC9, PC9-T790M and H1975 cell lines. The potential of overcoming osimertinib resistance with T-DM1 was tested in a PC9/HER2c1 xenograft model. Results T-DM1 exerted an additive effect when combined with osimertinib in terms of inhibition of cell proliferation, cell death and ADCC induction in PC9, PC9-T790M and H1975 cell lines. Combining osimertinib and T-DM1 using different schedules in long-term growth experiments revealed that the appearance of osimertinib-resistance was prevented in PC9-T790M and delayed in H1975 cells when the two drugs were given together. By contrast, when osimertinib was followed by T-DM1 an antagonistic effect was observed on cell proliferation, cell death and resistance acquisition. In xenograft models, we demonstrated that HER-2 amplification was associated with osimertinib-resistance and that T-DM1 co-administration is a potential strategy to overcome this resistance. Conclusions Our data suggest that concomitant treatment with osimertinib and T-DM1 may be a promising therapeutic strategy for EGFR-mutant NSCLC.

Despite the initial response, all patients with epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer (NSCLC) eventually develop acquired resistance to EGFR tyrosine kinase inhibitors (TKIs). The EGFR-T790M secondary mutation is responsible for half of acquired resistance cases, while MET amplification has been associated with acquired resistance in about 5-15% of NSCLCs. Clinical findings indicate the retained addiction of resistant tumors on EGFR signaling. Therefore, we evaluated the molecular mechanisms supporting the therapeutic potential of gefitinib maintenance in the HCC827 GR5 NSCLC cell line harbouring MET amplification as acquired resistance mechanism. We demonstrated that resistant cells can proliferate and survive regardless of the presence of gefitinib, whereas the absence of the drug significantly enhanced cell migration and invasion. Moreover, the continuous exposure to gefitinib prevented the epithelial-mesenchymal transition (EMT) with increased E-cadherin expression and down-regulation of vimentin and N-cadherin. Importantly, the inhibition of cellular migration was correlated with the suppression of EGFR-dependent Src, STAT5 and p38 signaling as assessed by a specific kinase array, western blot analysis and silencing functional studies. On the contrary, the lack of effect of gefitinib on EGFR phosphorylation in the H1975 cells (EGFR-T790M) correlated with the absence of effects on cell migration and invasion. In conclusion, our findings suggest that certain EGFR-mutated patients may still benefit from a second-line therapy including gefitinib based on the specific mechanism underlying tumor cell resistance.

In this study, we investigated the effects and the underlying molecular mechanisms of the multi-kinase inhibitor sorafenib in a panel of breast cancer cell lines. Sorafenib inhibited cell proliferation and induced apoptosis through the mitochondrial pathway. These effects were neither correlated with modulation of MAPK and AKT pathways nor dependent on the ERα status. Sorafenib promoted an early perturbation of mitochondrial function, inducing a deep depolarization of mitochondrial membrane, associated with drop of intracellular ATP levels and increase of ROS generation. As a response to this stress condition, the energy sensor AMPK was rapidly activated in all the cell lines analyzed. In MCF-7 and SKBR3 cells, AMPK enhanced glucose uptake by up-regulating the expression of GLUT-1 glucose transporter, as also demonstrated by AMPKα1 RNA interference, and stimulated aerobic glycolysis thus increasing lactate production. Moreover, the GLUT-1 inhibitor fasentin blocked sorafenib-induced glucose uptake and potentiated its cytotoxic activity in SKBR3 cells. Persistent activation of AMPK by sorafenib finally led to the impairment of glucose metabolism both in MCF-7 and SKBR3 cells as well as in the highly glycolytic MDA-MB-231 cells, resulting in cell death. This previously unrecognized long-term effect of sorafenib was mediated by AMPK-dependent inhibition of the mTORC1 pathway. Suppression of mTORC1 activity was sufficient for sorafenib to hinder glucose utilization in breast cancer cells, as demonstrated by the observation that the mTORC1 inhibitor rapamycin induced a comparable down-regulation of GLUT-1 expression and glucose uptake. The key role of AMPK-dependent inhibition of mTORC1 in sorafenib mechanisms of action was confirmed by AMPKα1 silencing, which restored mTORC1 activity conferring a significant protection from cell death. This study provides insights into the molecular mechanisms driving sorafenib anti-tumoral activity in breast cancer, and supports the need for going on with clinical trials aimed at proving the efficacy of sorafenib for breast cancer treatment.

Thanks to an unusual reversible cause of reflex syncope, a young physician avoided pacemaker implantation. We present the treatment of a bizarre case of gastro-cardiac syndrome, an often-overlooked clinical entity.