LC3A-mediated autophagy regulates lung cancer cell plasticity.

ABBREVIATIONS: ATG14: autophagy related 14; CDH2: cadherin 2; ChIP-qPCR: chromatin immunoprecipitation quantitative polymerase chain reaction; CQ: chloroquine; ECAR: extracellular acidification rate; EMT: epithelial-mesenchymal transition; EPCAM: epithelial cell adhesion molecule; MAP1LC3A/LC3A: microtubule associated protein 1 light chain 3 alpha; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MAP1LC3C/LC3C: microtubule associated protein 1 light chain 3 gamma; NDUFV2: NADH:ubiquinone oxidoreductase core subunit V2; OCR: oxygen consumption rate; ROS: reactive oxygen species; RT-qPCR: reverse-transcriptase quantitative polymerase chain reaction; SC: scrambled control; shRNA: short hairpin RNA; SNAI2: snail family transcriptional repressor 2; SOX2: SRY-box transcription factor 2; SQSTM1/p62: sequestosome 1; TGFB/TGF-ß: transforming growth factor beta; TOMM20: translocase of outer mitochondrial membrane 20; ZEB1: zinc finger E-box binding homeobox 1.

Cross-talk between SOX2 and TGFß Signaling Regulates EGFR-TKI Tolerance and Lung Cancer Dissemination.

Regulation of the stemness factor, SOX2, by cytokine stimuli controls self-renewal and differentiation in cells. Activating mutations in EGFR are proven therapeutic targets for tyrosine kinase inhibitors (TKI) in lung adenocarcinoma, but acquired resistance to TKIs inevitably occurs. The mechanism by which stemness and differentiation signaling emerge in lung cancers to affect TKI tolerance and lung cancer dissemination has yet to be elucidated. Here, we report that cross-talk between SOX2 and TGFß signaling affects lung cancer cell plasticity and TKI tolerance. TKI treatment favored selection of lung cancer cells displaying mesenchymal morphology with deficient SOX2 expression, whereas SOX2 expression promoted TKI sensitivity and inhibited the mesenchymal phenotype. Preselection of EGFR-mutant lung cancer cells with the mesenchymal phenotype diminished SOX2 expression and TKI sensitivity, whereas SOX2 silencing induced vimentin, but suppressed BCL2L11, expression and promoted TKI tolerance. TGFß stimulation downregulated SOX2 and induced epithelial-to-mesenchymal transdifferentiation accompanied by increased TKI tolerance, which can interfere with ectopic SOX2 expression. SOX2-positive lung cancer cells exhibited a lower dissemination capacity than their SOX2-negative counterparts. Tumors expressing low SOX2 and high vimentin signature were associated with worse survival outcomes in patients with EGFR mutations. These findings provide insights into how cancer cell plasticity regulated by SOX2 and TGFß signaling affects EGFR-TKI tolerance and lung cancer dissemination. SIGNIFICANCE: These findings suggest the potential of SOX2 as a prognostic marker in EGFR-mutant lung cancer, as SOX2-mediated cell plasticity regulated by TGFß stimulation and epigenetic control affects EGFR-TKI tolerance and cancer dissemination.

Expression of Neuroendocrine Factor VGF in Lung Cancer Cells Confers Resistance to EGFR Kinase Inhibitors and Triggers Epithelial-to-Mesenchymal Transition.

Mutations in EGFR drive tumor growth but render tumor cells sensitive to treatment with EGFR tyrosine kinase inhibitors (TKI). Phenotypic alteration in epithelial-to-mesenchymal transition (EMT) has been linked to the TKI resistance in lung adenocarcinoma. However, the mechanism underlying this resistance remains unclear. Here we report that high expression of a neuroendocrine factor termed VGF induces the transcription factor TWIST1 to facilitate TKI resistance, EMT, and cancer dissemination in a subset of lung adenocarcinoma cells. VGF silencing resensitized EGFR-mutated lung adenocarcinoma cells to TKI. Conversely, overexpression of VGF in sensitive cells conferred resistance to TKIs and induced EMT, increasing migratory and invasive behaviors. Correlation analysis revealed a significant association of VGF expression with advanced tumor grade and poor survival in patients with lung adenocarcinoma. In a mouse xenograft model of lung adenocarcinoma, suppressing VGF expression was sufficient to attenuate tumor growth. Overall, our findings show how VGF can confer TKI resistance and trigger EMT, suggesting its potential utility as a biomarker and therapeutic target in lung adenocarcinoma. Cancer Res; 77(11); 3013-26. ©2017 AACR.

M2 macrophage polarization modulates epithelial-mesenchymal transition in cisplatin-induced tubulointerstitial fibrosis.

Cisplatin-induced nephrotoxicity leaded to apoptosis of tubular epithelial cells (ECs) and tubulointerstitial fibrosis through ROS stress and inflammatory cytokines. Tubulointerstitial fibrosis caused by cisplatin might be via activation of resident fibroblasts and epithelial-mesenchymal transition (EMT) of tubular ECs. Inflammatory niche was crucial for progression of fibroblast activation or EMT. It had been reported that M1/M2 macrophage polarization regulated pro-inflammation or pro-resolving phase in damage repairing. However, the role of macrophage polarization on cisplatin-induced EMT of tubular ECs had not been well elucidated. In this study, we used co-cultured cell model and condition medium to examine the interaction between tubular ECs, fibroblasts and M1/M2 macrophages. Our data showed that cisplatin alone induced incomplete EMT of tubular ECs, whereas fibroblasts co-cultured with cisplatin-treated ECs could lead to fibroblast activation by detection of α-SMA and collagen-1. Moreover, decrease of iNOS and increase of argenase-1 and CD206 expression indicated that macrophages co-cultured with cisplatin-treated ECs would turn to M2 phenotype. Finally, we found that condition medium of M2 macrophages could promote complete EMT of cisplatin-treated ECs. Taken together, cisplatin created an inflammatory niche via tubular ECs to activate fibroblasts and stimulated M2 macrophage polarization. M2 macrophages could turn back to promote EMT of cisplatin-treated ECs. These results revealed the cooperative roles of tubular ECs, fibroblast and M2 macrophages to facilitate the progression of renal fibroblasis.

Cordyceps sobolifera extract ameliorates lipopolysaccharide-induced renal dysfunction in the rat.

Cordyceps Sobolifera (CS), an economic traditional Chinese herb, may ameliorate nephrotoxicity-induced renal dysfunction in the rat via antioxidant, anti-apoptosis, and anti-autophagy mechanisms. We investigated the water extract of fermented whole broth of CS on lipopolysaccharide (LPS)-induced renal cell injury in vitro and in vivo. CS effect on LPS-induced epithelial Lilly pork kidney (PK1) and Madin-Darby canine kidney epithelial (MDCK) cell death was detected with MTT assay. Two-month treatment of CS effects on renal blood flow (RBF), glomerular filtration rate (GFR), plasma blood urea nitrogen, creatinine level and leukocytes (WBC) count were determined in the LPS-treated rats. We further examined the effects of CS supplement on renal tubular oxidative stress, endoplasmic reticulum stress, apoptosis and autophagy by Western blot analysis. LPS dose-dependently induced PK1 and MDCK cell death, which can be ameliorated by CS treatment. LPS significantly decreased RBF and GFR and increased blood leukocyte counts, plasma blood urea nitrogen and creatinine level in the rat after 24 hours of injury. LPS enhanced renal tubular ER stress, autophagy and apoptosis via by increase protein expressions of GRP78, caspase 12, Beclin-1 and Bax/Bcl-2 ratio. These findings are associated with the significant staining in renal proximal and distal tubular ED-1, GRP78, Beclin-1 autophagy, and TUNEL apoptosis in the LPS-treated kidneys. Two months of CS supplement significantly improved RBF, GFR and WBC values and reduced ED-1, GRP78, Beclin-1 autophagy and TUNEL apoptosis in the LPS-treated kidneys. Long-term CS treatment reduced LPS-induced stress responses and tissue damage possibly via blocking LPS-triggered signaling pathways.

Enhanced expression of nitric oxide synthase in the early stage after increased pulmonary blood flow in rats.

OBJECTIVE: Evidence that vasodilator nitric oxide mediates normal pulmonary vascular tone has led to the hypothesis that endothelial injury induced by congenital heart disease with increased pulmonary blood flow disrupts these regulatory mechanisms and its associated altered vascular reactivity. Therefore, we hypothesized that increased pulmonary blood flow results in altered expression of endothelial nitric oxide synthase (eNOS). METHODS: We created an arteriovenous shunt in female Wistar (5-week-old) and measured the change of pulmonary blood flow and pressure immediately after and 1 month after the shunt operation. The protein levels of eNOS in the lung tissues of rats were assessed. RESULTS: The shunt immediately resulted in a significant increase in pulmonary blood flow (16.5 +/- 11.8% , pulmonary artery pressure (2.3 +/- 0.7 mm Hg), and blood O(2) saturation (16.1 +/- 11.8%) in the pulmonary artery. After 4 weeks, there was a significant increase in pulmonary blood flow (30.7 +/- 1.6%), pulmonary artery pressures (4.3 +/- 1.1 mm Hg), and blood O(2) content (43.3 +/- 17.5%). Western blot analysis demonstrated that eNOS protein was increased in the shunt lung 72 h after surgery and recovered to the control level 1 week later. CONCLUSION: This simple shunt model can induce early upregulation of eNOS expression with increased pulmonary blood flow and pulmonary artery pressure in rats.

Simple methods to elevate pulmonary arterial pressure by pre- and post-tricuspid shunts in rats.

BACKGROUND: Arteriovenous shunt in the rat is an extremely useful experimental animal model for investigating cardiac hypertrophy as well as the hemodynamics and endocrine aspects of chronic heart failure. AIMS: The present study was to develop 2 pre-tricuspid and 1 post-tricuspid models of arteriovenous shunt to induce right ventricular hypertrophy and increase pulmonary blood flow in growing rats. METHODS: In the first model, an arteriovenous shunt was created from the common iliac artery to the inferior vena cava (ICS). The second model was shunted from the common carotid artery to the external jugular vein (CJS). A post-tricuspid shunt (the third model) was made by introducing the right common carotid artery into the right ventricular outflow tract (CVS). RESULTS: Four weeks after the shunt surgery, the pulmonary artery pressure was 14.4 +/- 0.5 mmHg in the control group, 15.8 +/- 0.8 mmHg in the ICS group, 21.2 +/- 0.7 mmHg in the CJS group, and 20.2 +/- 1.1 mmHg in the CVS group. The percentage of increasing pulmonary blood flow was 33.0 +/- 1.0% in the CJS group and 26.9 +/- 1.3% in the ICS group four weeks after shunt operation. The oxygen partial pressure of pulmonary artery blood was 30.9 +/- 0.7 mmHg in the control group, 33.6 +/- 1.0 mmHg in the ICS group, 43.7 +/- 1.4 mmHg in the CJS group and 41.1 +/- 2.5 mmHg in the CVS group. The CJS and CVS groups had significant right ventricle hypertrophy. CONCLUSIONS: These three models can provide for study of the flow-pressure effect of the right heart and pulmonary circulation.