Previous studies showed that the combination of an anti-Epidermal growth factor (EGFR) and a MEK-inhibitor is able to prevent the onset of resistance to anti-EGFR monoclonal antibodies in KRAS-wild type colorectal cancer (CRC), while the same combination reverts anti-EGFR primary resistance in KRAS mutated CRC cell lines. However, rapid onset of resistance is a limit to combination therapies in KRAS mutated CRC. We generated four different KRAS mutated CRC cell lines resistant to a combination of cetuximab (an anti-EGFR antibody) and refametinib (a selective MEK-inhibitor) after continuous exposure to increasing concentration of the drugs. We characterized these resistant cell lines by evaluating the expression and activation status of a panel of receptor tyrosine kinases (RTKs) and intracellular transducers by immunoblot and qRT-PCR. Oncomine comprehensive assay and microarray analysis were carried out to investigate new acquired mutations or transcriptomic adaptation, respectively, in the resistant cell lines. Immunofluorescence assay was used to show the localization of RTKs in resistant and parental clones. We found that PI3K-AKT pathway activation acts as an escape mechanism in cell lines with acquired resistance to combined inhibition of EGFR and MEK. AKT pathway activation is coupled to the activation of multiple RTKs such as HER2, HER3 and IGF1R, though its pharmacological inhibition is not sufficient to revert the resistant phenotype. PI3K pathway activation is mediated by autocrine loops and by heterodimerization of multiple receptors. PI3K activation plays a central role in the acquired resistance to the combination of anti-EGFR and MEK-inhibitor in KRAS mutated colorectal cancer cell lines. PI3K activation is cooperatively achieved through the activation of multiple RTKs such as HER2, HER3 and IGF1R.

The toxicity of metallic nanoparticles (MNPs) has been fully ascertained, but the mechanisms underlying their cytotoxicity remain still largely unclear. Here we demonstrate that the cytotoxicity of MNPs is strictly reliant on the pathway of cellular internalization. In particular, if otherwise toxic gold, silver, and iron oxide NPs are forced through the cell membrane bypassing any form of active mechanism (e.g., endocytosis), no significant cytotoxic effect is registered. Pneumatically driven NPs across the cell membrane show a different distribution within the cytosol compared to NPs entering the cell by active endocytosis. Specifically, they exhibit free random Brownian motions within the cytosol and do not accumulate in lysosomes. Results suggest that intracellular accumulation of metallic nanoparticles into endo-lysosomal compartments is the leading cause of nanotoxicity, due to consequent nanoparticle degradation and in situ release of metal ions.

The EPHA2 tyrosine kinase receptor is implicated in tumor progression and targeted therapies resistance. We evaluated EPHA2 as a potential resistance marker to the antiepidermal growth factor receptor (EGFR) monoclonal antibody cetuximab in colorectal cancer. We studied activation of EPHA2 in a panel of human colorectal cancer cell lines sensitive or resistant to anti-EGFR drugs. The in vitro and in vivo effects of ALW-II-41-27 (an EPHA2 inhibitor) and/or cetuximab treatment were tested. Formalin-fixed paraffin-embedded tumor specimens from 82 RAS wild-type (WT) metastatic colorectal cancer patients treated with FOLFIRI + cetuximab as first-line therapy in the CAPRI-GOIM trial were assessed for EPHA2 expression by immunohistochemistry and correlated with treatment efficacy. EPHA2 was differentially activated in colorectal cancer cell lines. Combined treatment with ALW-II-41-27 plus cetuximab reverted primary and acquired resistance to cetuximab, causing cell growth inhibition, inducing apoptosis and cell-cycle G1–G2 arrest. In tumor xenograft models, upon progression to cetuximab, ALW-II-41-27 addition significantly inhibited tumor growth. EPHA2 protein expression was detected in 55 of 82 tumor samples, frequently expressed in less-differentiated and left-sided tumors. High levels of EPHA2 significantly correlated with worse progression-free survival [8.6 months; confidence interval (CI) 95%, 6.4–10.8; vs. 12.3 months; CI 95%, 10.4–14.2; P = 0.03] and with increased progression rate (29% vs. 9%, P = 0.02). A specific EPHA2 inhibitor reverts in vitro and in vivo primary and acquired resistance to anti-EGFR therapy. EPHA2 levels are significantly associated with worse outcome in patients treated with FOLFIRI + cetuximab. These results highlight EPHA2 as a potential therapeutic target in metastatic colorectal cancer.

The discovery of bioactive compounds from natural sources entails an extremely lengthy process due to the timescale and complexity of traditional methodologies. In our study, we used a rapid NMR based metabolomic approach as tool to identify secondary metabolites with anti-proliferative activity against a panel of human colorectal cancer cell lines with different mutation profiles. For this purpose, fourteen Fabaceae species of Mediterranean vegetation were investigated using a double screening method: ¹H NMR profiling enabled the identification of the main compounds present in the mixtures, whilst parallel biological assays allowed the selection of two plant extracts based on their strong anti-proliferative properties. Using high-resolution 2D NMR spectroscopy, putative active constituents were identified in the mixture and isolated by performing a bio-guided fractionation of the selected plant extracts. As a result, we found two active principles: a cycloartane glycoside and protodioscin derivative. Interestingly, these metabolites displayed a preferential anti-proliferative effect on colon cancer cell lines with an intrinsic resistance to anti-EGFR therapies. Our work provides an NMR-based metabolomic approach as a powerful and efficient tool to discover natural products with anticancer activities circumventing time-consuming procedures.