Brigatinib, a newly discovered AXL inhibitor, suppresses AXL-mediated acquired resistance to osimertinib in EGFR-mutated non-small cell lung cancer.

In addition to the classical resistance mechanisms, receptor tyrosine-protein kinase AXL is a main mechanism of resistance to third-generation epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) osimertinib in EGFR-mutated non-small cell lung cancer (NSCLC). Developing an effective AXL inhibitor is important to sensitize osimertinib in clinical application. In this study we assessed the efficacy of brigatinib, a second-generation of anaplastic lymphoma kinase (ALK)-TKI, as a novel AXL inhibitor, in overcoming acquired resistance to osimertinib induced by AXL activation. We established an AXL-overexpression NSCLC cell line and conducted high-throughput screening of a small molecule chemical library containing 510 anti-tumor drugs. We found that brigatinib potently inhibited AXL expression, and that brigatinib (0.5 µM) significantly enhanced the anti-tumor efficacy of osimertinib (1 µM) in AXL-mediated osimertinib-resistant NSCLC cell lines in vitro. We demonstrated that brigatinib had a potential ability to bind AXL kinase protein and further inhibit its downstream pathways in NSCLC cell lines. Furthermore, we revealed that brigatinib might decrease AXL expression through increasing K48-linked ubiquitination of AXL and promoting AXL degradation in HCC827OR cells and PC-9OR cells. In AXL-high expression osimertinib-resistant PC-9OR and HCC827OR cells derived xenograft mouse models, administration of osimertinib (10 mg·kg-1·d-1) alone for 3 weeks had no effect, and administration of brigatinib (25 mg·kg-1·d-1) alone caused a minor inhibition on the tumor growth; whereas combination of osimertinib and brigatinib caused marked tumor shrinkages. We concluded that brigatinib may be a promising clinical strategy for enhancing osimertinib efficacy in AXL-mediated osimertinib-resistant NSCLC patients.

Electrotaxis of alveolar epithelial cells in direct-current electric fields.

PURPOSE: This study aims to elucidate the electrotaxis response of alveolar epithelial cells (AECs) in direct-current electric fields (EFs), explore the impact of EFs on the cell fate of AECs, and lay the foundation for future exploitation of EFs for the treatment of acute lung injury. METHODS: AECs were extracted from rat lung tissues using magnetic-activated cell sorting. To elucidate the electrotaxis responses of AECs, different voltages of EFs (0, 50, 100, and 200 mV/mm) were applied to two types of AECs, respectively. Cell migrations were recorded and trajectories were pooled to better demonstrate cellular activities through graphs. Cell directionality was calculated as the cosine value of the angle formed by the EF vector and cell migration. To further demonstrate the impact of EFs on the pulmonary tissue, the human bronchial epithelial cells transformed with Ad12-SV40 2B (BEAS-2B cells) were obtained and experimented under the same conditions as AECs. To determine the influence on cell fate, cells underwent electric stimulation were collected to perform Western blot analysis. RESULTS: The successful separation and culturing of AECs were confirmed through immunofluorescence staining. Compared with the control, AECs in EFs demonstrated a significant directionality in a voltage-dependent way. In general, type Ⅰ alveolar epithelial cells migrated faster than type Ⅱ alveolar epithelial cells, and under EFs, these two types of cells exhibited different response threshold. For type Ⅱ alveolar epithelial cells, only EFs at 200 mV/mm resulted a significant difference to the velocity, whereas for, EFs at both 100 mV/mm and 200 mV/mm gave rise to a significant difference. Western blotting suggested that EFs led to an increased expression of a AKT and myeloid leukemia 1 and a decreased expression of Bcl-2-associated X protein and Bcl-2-like protein 11. CONCLUSION: EFs could guide and accelerate the directional migration of AECs and exert antiapoptotic effects, which indicated that EFs are important biophysical signals in the re-epithelialization of alveolar epithelium in lung injury.

Metformin synergistically enhances the antitumor activity of the third-generation EGFR-TKI CO-1686 in lung cancer cells through suppressing NF-κB signaling.

PURPOSE: Third-generation irreversible epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), rociletinib (CO-1686), is great efficacy against EGFR-mutated patients bearing the T790M resistance mutation. However, acquired resistance may emerge. There is a need to characterize acquired resistance mechanism(s) and to devise ways to overcome CO-1686 resistance. EXPERIMENTAL DESIGN: MTT assay, ki67 incorporation assay, transwell assay and TUNEL assay were employed to analyze the effects of metformin to reverse CO-1686 resistance in vitro. The NF-κB activity was measured by the antibody of p50, p65, p-IKBɑ, and p-IKKɑ/ß. Western blotting was used to analyze the proteins in cells. RESULTS: We have established CO-1686-resistant cell lines of PC-9GRCOR and H1975COR from two parental cell lines of PC-9GR and H1975 by long-term exposure to increasing doses of CO-1686. Compared with the parental cells, PC-9GRCOR cells and H1975COR cells showed 90-folds and 20-folds higher resistance to CO-1686, respectively. Critically, we showed that the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling molecular proteins subunits of p50, p65 and its inhibitor proteins of IKBɑ, IKKɑ/ß in phosphorylation levels in resistant cells were higher than parental cells. Accordingly, inhibition of NF-κB activity used TPCA-1 effective in decreasing viability and inducing apoptosis of resistant cells. Moreover, metformin overcame the acquired resistance to CO-1686 by reducing cell proliferation and invasion. Metformin combined with CO-1686 synergistically inhibited the p-IKBɑ, p-IKKɑ/ß, p50, and p65. CONCLUSIONS: NF-κB signaling activation induced acquired resistance to CO-1686. Metformin sensitized resistant cells to CO-1686 via inhibiting NF-κB signaling.

Combined treatment with metformin and gefitinib overcomes primary resistance to EGFR-TKIs with EGFR mutation via targeting IGF-1R signaling pathway.

AIM: Although EGFR tyrosine kinase inhibitors (TKIs) have shown dramatic effects against sensitizing EGFR mutations in non-small cell lung cancer (NSCLC), ~20%-30% of NSCLC patients with EGFR-sensitive mutation exhibit intrinsic resistance to EGFR-TKIs. The purpose of the current study was to investigate the enhanced antitumor effect of metformin (Met), a biguanide drug, in combination with gefitinib (Gef) in primary resistant human lung cancer cells and the associated molecular mechanism. EXPERIMENTAL DESIGN: H1975 cell line was treated with Met and/or Gef to examine the inhibition of cell growth and potential mechanism of action by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Ki67 incorporation assay, flow cytometry analysis, small interfering RNA technology, Western blot analysis and xenograft implantation. RESULTS: Insulin-like growth factor-1 receptor (IGF-1R) signaling pathway was markedly activated in EGFR-TKI primary resistant H1975 cells as compared to EGFR-TKI acquired resistance cells (PC-9GR, H1650-M3) and EGFR-TKI sensitivity cells (PC-9, HCC827). Inhibition of IGF-1R activity by AG-1024 (a small molecule of IGF-1R inhibitor), as well as downregulation of IGF-1R by siRNA, significantly enhanced the ability of Gef to suppress proliferation and induce apoptosis in H1975 cells via the inhibition of AKT activation and subsequent upregulation of Bcl-2-interacting mediator of cell death (BIM). Interestingly, the observation showed that Met combined with Gef treatment had similar tumor growth suppression effects in comparison with the addition of AG-1024 to therapy with Gef. A clear synergistic antiproliferative interaction between Met and Gef was observed with a combination index (CI) value of 0.65. Notably, IGF-1R silencing mediated by RNA interference (RNAi) attenuated anticancer effects of Met without obviously resensitizing H1975 cells to Gef. Finally, Met-based combinatorial therapy effectively blocked tumor growth in the xenograft with TKI primary resistant lung cancer cells. CONCLUSION: Our findings demonstrated that Met combined with Gef would be a promising strategy to overcome EGFR-TKI primary resistance via suppressing IGF-1R signaling pathway in NSCLC.

Fabrication and characterization of stable ultrathin film micropatterns containing DNA and photosensitive polymer diazoresin.

Stable, ultrathin DNA micropatterns were fabricated from photosensitive polymer diazoresin (DR) through a self-assembly technique. The micropatterns were achieved on LBL ultrathin film after UV exposure through a photomask. The patterns were characterized systematically with scanning electron microscopy, atomic force microscopy, X-ray photoelectron spectroscopy and fluorescence microscopy. All of the results indicate that the combined LBL self-assembly and photolithography technique is a promising method for constructing stable, well-defined micropatterns with a nanoscale thickness.