PO-035 LKB1 deficiency renders non-small-cell lung cancer cells sensitive to ERK inhibitor

Introduction Lung Cancer is the first cause of cancer-related death in the world. The alterations in KRAS oncogene are very frequent (25%), but, unfortunately, this protein is at present undruggable. KRAS mutations determine over-activation of important pathways of growth and proliferation (PI3K-AKT-mTOR and MAPK). KRAS-mutated tumours are also frequently co-mutated in LKB1 (50%), an important regulator of metabolic homeostasis and oxidative stress in the cells. LKB1 modulates catabolic processes through AMPK-mediated mTOR inhibition. Thereafter, the inactivation of LKB1 causes in KRAS mutated-tumours further activation of PI3K-AKT-mTOR and MAPK pathways, making them particularly aggressive. The possibility to specifically target tumours with both KRAS and LKB1 alterations represent an important medical need. Material and methods We generated from the NSCLC cell line NCI-H1299 clones over-expressing KRAS WT or KRAS G12C forms. These clones have been subsequently modified through CRISPR-CAS9 system to obtain deletions in LKB1 gene. We successfully generated isogenic cells differing only for the status of KRAS and LKB1 (KRASwt/LKB1wt, KRASmut/LKB1wt, KRASwt/LKB1mut, KRASmut/LKB1mut). These clones were treated with a panel of inhibitors of MAPK and PI3K pathways. Viability was evaluated with MTS assay. Molecular characterizations were performed by western blot analysis. In vivo antitumor activity was determined after subcutaneous injection of NSCLC cells in immunodeficient mice. Results and discussions Using the isogenic system generated we tested the activity of several inhibitors of MAPK and PI3K pathways. The results highlighted a strong response of the clones with deletion in LKB1 to ERK inhibitor, independently from the KRAS status. These results were confirmed 'in vivo', where tumours with LKB1 deletion showed a significant sensitivity to ERK inhibitor, compared to LKB1 WT tumours. At molecular level we tested the activation of proteins related to MAPK and PI3K pathway such as p70, S6, 4-EBP1, ERK. The results showed that the response to ERK inhibitor was mainly due to mTOR signalling inhibition. Conclusion The results obtained highlight a possible strategy to target NSCLC with KRAS-LKB1 co-mutations, that, at moment are those with a worse prognosis. The sensitivity to ERK inhibitor is remarkable, also in presence of KRAS WT, therefore this strategy could be applied to all LKB1-mutated lung tumours, that represent 30% of all NSCLC. These studies are being confirmed in other NSCLC backgrounds and mouse models

Co-targeting triple-negative breast cancer cells and endothelial cells by metronomic chemotherapy inhibits cell regrowth and migration via downregulation of the FAK/VEGFR2/VEGF axis and autophagy/apoptosis activation

High-dose standard-of-care chemotherapy is the only option for triple-negative breast cancer (TNBC) patients, which eventually die due to metastatic tumors. Recently, metronomic chemotherapy (mCHT) showed advantages in treating TNBCs leading us to investigate the anti-metastatic and anti-angiogenic potential of metronomic 5-Fluorouracil plus Vinorelbine (5-FU+VNR) on endothelial cells (ECs) and TNBCs in comparison to standard treatment (STD). We found that 10-fold lower doses of 5-FU+VNR given mCHT vs. STD inhibits cell proliferation and survival of ECs and TNBC cells. Both schedules strongly affect ECs migration and invasion, but in TNBC cells mCHT is significantly more effective than STD in impairing cell migration and invasion. The two treatments disrupt FAK/VEGFR/VEGF signaling in both ECs and TNBC cells. mCHT, and to a much lesser extent STD treatment, induces apoptosis in ECs, whereas it switches the route of cell death from apoptosis (as induced by STD) to autophagy in TNBC cells. mCHT-treated TNBCs-derived conditioned medium also strongly affects ECs' migration, modulates different angiogenesis-associated proteins, and hampers angiogenesis in matrix sponge in vivo. In conclusion, mCHT administration of 5-FU+VNR is more effective than STD schedule in controlling cell proliferation/survival and migration/invasion of both ECs and TNBC cells and has a strong anti-angiogenic effect. Our data suggest that the stabilization of tumor growth observed in TNBC patients treated with mCHT therapy schedule is likely due not only to direct cytotoxic effects but also to anti-metastatic and anti-angiogenic effects

Generation and characterization of MEK and ERK inhibitors- resistant non-small-cells-lung-cancer (NSCLC) cells

Abstract Background The RAS/RAF/MEK/ERK pathway is one of the most downregulated pathway in cancer. Inhibitors of RAF and MEK have established clinical use while ERK inhibitors recently faced the clinic. We aimed to generate resistant cell lines which could be helpful for defining new combinations able to overcome resistance. Methods the human NSCLC cell line NCI-H727, sensitive to both MEK and ERK inhibitors, was treated with increasing concentrations of MEK162 (as MEK inhibitor) or SCH772984 as ERK inhibitor. Results we successfully obtained a MEK resistant subline (H727/MEK, after 40 passages) as well as an ERK resistant subline (H727/SCH, after 18 passages). The two resistant sublines H727/MEK and H727/SCH were cross-resistant to ERK and MEK inhibitors, respectively, but not to RAF inhibitors. The sublines maintained the responsiveness to inhibitors of the parallel PI3K/akt/mTOR pathway as well as to agents with different mechanism of action. Mechanistically, treatment of sensitive and resistant cells with MEK or ERK inhibitors was able to induce a similar inhibition of ERK phosphorylation, while only in parental cells the drugs were able to induce a downregulation of S6 and RSK phosphorylation. Conclusions these resistant cells represent an important tool for further studies on the mechanisms of resistance and ways to overcome it

High miR-100 expression is associated with aggressive features and modulates TORC1 complex activation in lung carcinoids

Mammalian target of rapamycin (mTOR) is a promising therapeutic target in advanced lung carcinoid patients. However, the mechanisms of mTOR modulation and of responsiveness to mTOR inhibitors are largely unclear. Our aim was to analyze the expression and functional role of specific miRNAs in lung carcinoids as an alternative mechanism targeting mTOR pathway

Metronomic combination of Vinorelbine and 5Fluorouracil is able to inhibit triple-negative breast cancer cells. Results from the proof-of-concept VICTOR-0 study

Triple Negative Breast Cancer (TNBC) is an aggressive neoplasia with median Overall Survival (OS) less than two years. Despite the availability of new drugs, the chance of survival of these patients did not increase. The combination of low doses of drugs in a metronomic schedule showed efficacy in clinical trials, exhibiting an anti-proliferative and anti-tumour activity. In Victor-2 study we recently evaluated a new metronomic combination (mCHT) of Capecitabine (CAPE) and Vinorelbine (VNR) in breast cancer patients showing a disease control rate with a median Progression- Free Survival (PFS) of 4.7 months in 28 TNBC patients. Here in Victor-0 study, we examined the effect of mCHT vs standard (STD) schedule of administration of different combinations of 5-Fluorouracil (5FU), the active metabolite of CAPE, and VNR in TNBC cell lines MDA-MB-231 and BT-549. A significant antiproliferative activity was observed in cells treated with metronomic vs STD administration of 5FU or VNR alone. Combination of the two drugs showed an additive inhibitor effect on cell growth in both cell lines . Moreover, after exposure of cells to 5FU and VNR under mCHT or conventional schedule of administration we also observed a downregulation of chemoresistance factor Bcl-2, changes in pro-apoptotic protein Bax and in cleaved effector caspase-3 and increased expression of LC3A/B autophagy protein. Our results therefore suggest that molecular mechanisms implicated in apoptosis and autophagy as well as the cross-talk between these two forms of cell death in MDA-MB-231 and BT-549 cells treated with 5FU and VNR is dose- and scheduledependent and provide some insights about the roles of autophagy and senescence in 5FU/VNR-induced cell deat