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DISCUSSION AND CONCLUSIONS

[39]. However, the emerging acquired resistance together with the low survival benefit prompt further studies to improve this therapeutic approach. The necessity to overcome the resistance due to the T790M secondary mutation of EGFR led to the development of several second-generation irreversible TKIs of this target [3]. We recently synthesized a series of novel 3-aminopropanamide compounds which demonstrated to be active against H1975 NSCLC cells harboring the EGFR-T790M mutation by irreversible binding to the receptor following intra-cellular activation to a cysteine-trapping chemical species [22]. The present study demonstrates that the two most promising compounds inhibit cell proliferation and induce apoptosis in different NSCLC cell lines at significantly lower concentration than gefitinib. Since the EGFR-TKI erlotinib has been approved for the treatment of advanced PDAC in combination with gemcitabine [30], we also evaluated the new molecules in PANC-1 and MIA PaCa-2 cells.

In these cell lines both our compounds were as active as gefitinib or even more active than the first-generation EGFR-TKI. However, the very slight increase in survival obtained with the use of erlotinib [40], and the recent success of multicombinatorial approaches with conventional cytotoxic drugs reduce the impact of EGFR-TKIs in the treatment of PDAC [41].

As expected on the basis of their anti-EGFR activity, the main downstream mediators of the EGFR signal transduction pathways (p-Akt and p-p44/42) were also affected by our novel compounds. However, despite EGFR inhibition was already detectable by Western blot in the nano-molar range of concentrations, the downstream signaling molecules were slightly inhibited at 1 µM, particularly by UPR1268, and more clearly at the highest used concentration (10 µM) for both the inhibitors. Furthermore, using a commercially available 144-peptide array, we detected other tyrosine kinases affected by UPR1282. The restricted subset of the peptides spotted on the array, together with the limited specificity and the use of lysates which cannot reproduce subcellular compartmentalization and protein docking or scaffolding represent the main interdictions of this study [42]. However, several additional tyrosine kinases were identified which were significantly inhibited by UPR1282, including the fibroblast growth factor receptor 3 (FGFR3), whose upregulation has been correlated to radioresistance in squamous cell carcinoma [43]. Of note, EphB4 and the focal adhesion kinase 1 (FAK1), which are both involved in the neoplastic transformation process, were also inhibited by UPR1282 in H1975 cells [44,45]. Additionally, the treatment of the cell lysates with UPR1282 also inhibited c-Met, which amplification represents the second cause of acquired resistance to the treatment with EGFR-TKIs. In particular, the activity of the receptor was reduced of around 53% by UPR1282. Importantly, many kinases, including the previously mentioned, had relevant (although not always below the 66.6% threshold) levels of inhibition in both H1975 cell line and xenografts exposed to UPR1282.

These results potentially extend the use of UPR1282 to the treatment of NSCLCs which harbor mechanisms of resistance different than the EGFR-T790M mutation.

The invasive and metastastic capacity of tumor cells constitute two pivotal mechanisms of malignant tumor development. The poor prognosis of lung cancer is related to strong metastasis potential, which is directly correlated with EMT [33]. The transformation of epithelial cells to motile, invasive and migratory mesenchymal cells is the major feature of such process. Cell adhesion and detachment are regulated by interactions between cell–cell and cell–extracellular matrix (ECM) which coordinate the invasive mechanism [46]. Several classes of proteins participate to invasive cancer phenotype. Recent studies reported the loss of function of the cadherin adhesion complex, and in particular E-cadherin, as hallmark of EMT which leads

to increased proliferation, invasiveness and metastasis [47]. Thus, we investigated whether the exposure to UPR1282 or UPR1268 might affect E-cadherin gene expression. While treatment with gefitinib did not affect the level of mRNA coding for E-cadherin in H1975 cells, a significant increase in its gene expression was observed after exposure to our novel compounds. Furthermore, since recent studies demonstrated that EGF modifies cell–ECM adhesion [48], we tested whether our compounds might affect migration of EGFR-T790M H1975 cells. Differently from what observed after exposure to gefitinib, the novel 3-aminopropanamide compounds significantly reduced cell migration after few hours of treatment, as detected by the wound-healing assay. Such reduction in cell motility agrees with previous evidence on FAK inhibition and increased E-cadherin gene expression.

Accumulating evidence suggests that specific sub-populations of cancer cells within the bulk of tumors undergo different degree of EMT, and this process has been related with the acquisition of stem cell-like characteristics. Both EMT and stemness are implicated in the pathogenesis and chemoresistance of heterogeneous malignant tumors [21,49]. Treatment strategies for the elimination of cancer, therefore, need to consider the presence of CSCs. Sphere assay has proven to be an excellent technique to isolate CSCs and several studies demonstrated that the three-dimensional aggregates can contain different cell proliferation and metabolic gradients, such as an outer rim of viable and actively proliferating cells and an inner necrotic region [35]. Thus, after generating tumor spheres which overexpress the stem/initiating-cell marker CD133, we explored whether our novel EGFR-TKIs affected spheroids growth. Conversely to gefitinib, both the 3-aminopropanamide derivatives significantly reduced tumor spheres dimensions.

Furthermore, we demonstrated that the level of CD133 mRNA was reduced after exposure to UPR1282 and UPR1268 [37,50].

The evaluation of the activity of UPR1282 was also extended to xenograft models of EGFR-T790M NSCLC. The chronic administration of UPR1282 in athymic mice was well tolerated and produced significant inhibition of tumor growth, which might result from inhibition of both EGFR and downstream kinases activity.

Of note, while tumor shrinkage induced by canertinib treatment was accompanied by significant toxicity, UPR1282 did not induce significant loss of body weight and toxicity even after a prolonged treatment. This might be, at least in part, related to the masking of acrylamide warhead which results in reduced risk of covalent interaction with off-target molecules [22].

In conclusion, our 3-aminopropanamide irreversible EGFR-TKIs seem very promising anticancer agents by attacking different key mechanisms involved in the resistance of NSCLC cells to the first-generation EGFR-TKIs gefitinib and erlotinib, including the EMT process. These data should prompt future trials that will give the ultimate proof of the utility of these novel anticancer agents for the treatment of lung cancer.

ACKNOWLEDGEMENTS

This work was supported by: Associazione Italiana per la Ricerca sul Cancro (AIRC), Milan grant IG 8856 (Andrea Ardizzoni); the Netherlands Organization for Scientific Research (NWO, Veni grant, Elisa Giovannetti); AIRC-Marie Curie (International Fellowship, Elisa Giovannetti); FIRC grant (Fellowship for abroad; E. Galvani).

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Chapter 8

Molecular mechanisms and