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Osimertinib induces a marked response in non–small‐cell lung cancer (NSCLC) patients harboring epidermal growth factor receptor (EGFR) gene mutations. However, acquired resistance to osimertinib remains an inevitable problem. In this study, we aimed to investigate osimertinib‐resistant mechanisms and evaluate the combination therapy of afatinib and chemotherapy. We established osimertinib‐resistant cell lines (PC‐9‐OR and H1975‐OR) from EGFR‐mutant lung adenocarcinoma cell lines PC‐9 and H1975 by high exposure and stepwise method. Combination therapy of afatinib plus carboplatin (CBDCA) and pemetrexed (PEM) was effective in both parental and osimertinib‐resistant cells. We found that expression of thrombospondin‐1 (TSP‐1) was upregulated in resistant cells using cDNA microarray analysis. We demonstrated that TSP‐1 increases the expression of matrix metalloproteinases through integrin signaling and promotes tumor invasion in both PC‐9‐OR and H1975‐OR, and that epithelial‐to‐mesenchymal transition (EMT) was involved in H1975‐OR. Afatinib plus CBDCA and PEM reversed TSP‐1‐induced invasion ability and EMT changes in resistant cells. In PC‐9‐OR xenograft mouse models (five female Balb/c‐Nude mice in each group), combination therapy strongly inhibited tumor growth compared with afatinib monotherapy (5 mg/kg, orally, five times per week) or CBDCA (75 mg/kg, intraperitoneally, one time per week) + PEM (100 mg/kg, intraperitoneally, one time per week) over a 28‐day period. These results suggest that the combination of afatinib plus CBDCA and PEM, which effectively suppresses TSP‐1 expression, may be a promising option in EGFR‐mutated NSCLC patients after the acquisition of osimertinib resistance. [ABSTRACT FROM AUTHOR]

Introduction: A neurotrophic tropomyosin receptor kinase (NTRK)‐tyrosine kinase inhibitor (TKI) has shown dramatic efficacy against malignant tumors harboring an NTRK fusion gene. However, almost all tumors eventually acquire resistance to NTRK‐TKIs. Method: To investigate the mechanism of resistance to NTRK‐TKIs, we established cells resistant to three types of NTRK‐TKIs (larotrectinib, entrectinib, and selitrectinib) using KM12 colon cancer cells with a TPM3‐NTRK1 rearrangement. Result: Overexpression of 3‐hydroxy‐3‐methylglutaryl‐CoA synthase 2 (HMGCS2) was observed in three resistant cells (KM12‐LR, KM12‐ER, and KM12‐SR) by microarray analysis. Lower expression of sterol regulatory element‐binding protein 2 (SREBP2) and peroxisome proliferator activated receptor α (PPARα) was found in two cells (KM12‐ER and KM12‐SR) in which HMGCS2 was overexpressed compared to the parental KM12 and KM12‐LR cells. In resistant cells, knockdown of HMGCS2 using small interfering RNA improved the sensitivity to NTRK‐TKI. Further treatment with mevalonolactone after HMGCS2 knockdown reintroduced the NTRK‐TKI resistance. In addition, simvastatin and silibinin had a synergistic effect with NTRK‐TKIs in resistant cells, and delayed tolerance was observed after sustained exposure to clinical concentrations of NTRK‐TKI and simvastatin in KM12 cells. In xenograft mouse models, combination treatment with entrectinib and simvastatin reduced resistant tumor growth compared with entrectinib alone. Conclusion: These results suggest that HMGCS2 overexpression induces resistance to NTRK‐TKIs via the mevalonate pathway in colon cancer cells. Statin inhibition of the mevalonate pathway may be useful for overcoming this mechanistic resistance. [ABSTRACT FROM AUTHOR]

Background: Osimertinib is a third‐generation epidermal growth factor receptor‐tyrosine kinase inhibitor (EGFR‐TKI) approved for the treatment of patients with EGFR‐mutant non‐small cell lung cancer (NSCLC). However, the mechanisms of acquired drug resistance to osimertinib have not as yet been clarified. Exosomes and microRNAs (miRNAs) are involved in carcinogenesis and drug resistance in human cancers. Methods: We used previously established osimertinib‐resistant HCC827 (HCC827‐OR) and PC‐9 (PC‐9‐OR) cells. We evaluated the profiles of exosomal miRNA associated with resistance to osimertinib in EGFR‐mutant NSCLC cells. Results: Epithelial–mesenchymal transition (EMT) phenomenon was observed in HCC827‐OR and PC‐9‐OR cells. Microarray and quantitative reverse transcription‐polymerase chain reaction analysis revealed that miR‐210‐3p was co‐upregulated in exosomes isolated from HCC827‐OR and PC‐9‐OR cells compared with those isolated from parental HCC827 and PC‐9 cells. HCC827‐OR cell‐derived exosomes induced EMT changes and resistance to osimertinib in HCC827 cells. Subsequently, the induction of miR‐210‐3p directly promoted the EMT phenomenon and resistance to osimertinib in HCC827 cells. Conclusions: Exosomal miR‐210‐3p may play a crucial role in resistance to osimertinib in the tumor microenvironment of EGFR‐mutant NSCLC. [ABSTRACT FROM AUTHOR]

Abstract Background Phase III trials evaluating the efficacy of gefitinib (IRESSA) in non-small cell lung cancer (NSCLC) lend support to the need for improved patient selection in terms of gefitinib use. Mutation of the epidermal growth factor receptor (EGFR) gene is reported to be associated with clinical responsiveness to gefitinib. However, gefitinib-sensitive and prolonged stable-disease-defined tumors without EGFR gene mutation have also been reported. Methods To identify other key factors involved in gefitinib sensitivity, we analyzed the protein expression of molecules within the EGFR family, PI3K-Akt and Ras/MEK/Erk pathways and examined the sensitivity to gefitinib using the MTT cell proliferation assay in 23 lung cancer cell lines. Results We identified one highly sensitive cell line (PC9), eight cell lines displaying intermediate-sensitivity, and 14 resistant cell lines. Only PC9 and PC14 (intermediate-sensitivity) displayed an EGFR gene mutation including amplification. Eight out of the nine cell lines showing sensitivity had Akt phosphorylation without ligand stimulation, while only three out of the 14 resistant lines displayed this characteristic (P = 0.0059). Furthermore, the ratio of phosphor-Akt/total Akt in sensitive cells was higher than that observed in resistant cells (P = 0.0016). Akt phosphorylation was partially inhibited by gefitinib in all sensitive cell lines. Conclusion These results suggest that Akt phosphorylation without ligand stimulation may play a key signaling role in gefitinib sensitivity, especially intermediate-sensitivity. In addition, expression analyses of the EGFR family, EGFR gene mutation, and FISH (fluorescence in situ hybridization) analyses showed that the phosphorylated state of EGFR and Akt might be a useful clinical marker of Akt activation without ligand stimulation, in addition to EGFR gene mutation and amplification, particularly in adenocarcinomas.

Abstract background The effect of current therapies in improving the survival of lung cancer patients remains far from satisfactory. It is consequently desirable to find more appropriate therapeutic opportunities based on informed insights. A molecular pharmacological analysis was undertaken to design an improved chemotherapeutic strategy for advanced lung cancer. Methods We related the cytotoxic activity of each of commonly used anti-cancer agents (docetaxel, paclitaxel, gemcitabine, vinorelbine, 5-FU, SN38, cisplatin (CDDP), and carboplatin (CBDCA)) to corresponding expression pattern in each of the cell lines using a modified NCI program. Results We performed gene expression analysis in lung cancer cell lines using cDNA filter and high-density oligonucleotide arrays. We also examined the sensitivity of these cell lines to these drugs via MTT assay. To obtain our reproducible gene-drug sensitivity correlation data, we separately analyzed two sets of lung cancer cell lines, namely 10 and 19. In our gene-drug correlation analyses, gemcitabine consistently belonged to an isolated cluster in a reproducible fashion. On the other hand, docetaxel, paclitaxel, 5-FU, SN-38, CBDCA and CDDP were gathered together into one large cluster. Conclusion These results suggest that chemotherapy regimens including gemcitabine should be evaluated in second-line chemotherapy in cases where the first-line chemotherapy did not include this drug. Gene expression-drug sensitivity correlations, as provided by the NCI program, may yield improved therapeutic options for treatment of specific tumor types.

Abstract Background Fourteen-membered ring macrolides have been effective in reducing chronic airway inflammation and also preventing lung injury and fibrosis in bleomycin-challenged mice via anti-inflammatory effects. EM703 is a new derivative of erythromycin (EM) without the bactericidal effects. We investigated the anti-inflammatory and antifibrotic effects of EM703 in an experimental model of bleomycin-induced lung injury and subsequent fibrosis in mice. Methods Seven-week-old male ICR mice were used. All experiments used eight mice/group, unless otherwise noted in the figure legends. Bleomycin was administered intravenously to the mice on day 0. EM703 was orally administered daily to mice. All groups were examined for cell populations in the bronchoalveolar lavage (BAL) fluid and for induction of messenger RNA (mRNA) of Smad3 and Smad4 in the lung tissues by reverse transcriptase (RT)-polymerase chainreaction (PCR) on day 7. Fibroblastic foci were assessed histologically, and the hydroxyproline content was chemically determined in the lung tissues on day 28. We performed assay of proliferation and soluble collagen production, and examined the induction of mRNA of Smad3 and Smad4 by RT-PCR in murine lung fibroblast cell line MLg2908. We also examined Smad3, Smad4 and phosphorylated Smad2/3 (p-Smad2/3) protein assay by western blotting in MLg2908. Results Bleomycin-induced lung fibrosis, and the infiltration of macrophages and neutrophils into the airspace were inhibited by EM703. The expression of Smad3 and Smad4 mRNA was clearly attenuated by bleomycin, but was recovered by EM703. EM703 also inhibited fibroblast proliferation and the collagen production in lung fibroblasts induced by Transforming growth factor-beta (TGF-β). The expression of Smad3 and Smad4 mRNA in murine lung fibroblasts disappeared due to TGF-β, but was recovered by EM703. EM703 inhibited the expression of p-Smad2/3 and Smad4 protein in murine lung fibroblasts induced by TGF-β. Conclusion These findings suggest that EM703 improves bleomycin-induced pulmonary fibrosis in mice by actions of anti-inflammation and regulation of TGF-β signaling in lung fibroblasts.

It was previously revealed that Wnt signaling is activated in mesothelioma cells. Although epidermal growth factor receptor (EGFR) is expressed in mesothelioma cells, EGFR-tyrosine kinase inhibitors (TKIs) are not effective for mesothelioma treatment. However, in non-small cell lung cancer, the blocking of Wnt signaling has been identified to enhance the anticancer effect of EGFR-TKIs. To confirm the anticancer effect of blocking Wnt signaling in combination with EGFR-TKI treatment in mesothelioma, the present study evaluated the effect of simultaneous suppression of human dishevelled-3 (Dvl-3) expression with Dvl-3 small interfering RNA (siRNA) and of EGFR inhibition with gefitinib on mesothelioma cell viability. Mesothelioma cell lines with and without β-catenin gene expression were transfected with Dvl-3 siRNA and were cultured with gefitinib, and cell viability, colony formation and cell cycle analyses were performed. Dvl-3 siRNA downregulated the expression of Dvl-3 in mesothelioma cells. The combination of Dvl-3 siRNA with gefitinib acted synergistically to induce concomitant suppression of cell viability and colony formation, suggesting that inhibition of Wnt signaling by downregulating Dvl-3 with siRNA and inhibiting EGFR with gefitinib leads to significant antitumor effects. [ABSTRACT FROM AUTHOR]