Cancer cell plasticity generates heterogeneous oncogenic subpopulations in tumors. How macroautophagy/autophagy, a catabolic system required for sustaining cell homeostasis, affects cancer cell plasticity, remains elusive. In this study, we report that MAP1LC3A/LC3A (microtubule associated protein 1 light chain 3 alpha), a key molecule in autophagy, is negatively associated with histological grade and distant metastasis of lung cancer. This is achieved in part, if not all, by maintaining the mitochondria and energy homeostasis to meet the proliferation demand of lung cancer cells driven by SOX2 (SRY-box transcription factor 2) signaling. Basal autophagy is preferentially active in SOX2-positive lung cancer cells with high-proliferative and low-invasive properties. The high-proliferative cancer cells exhibit higher oxygen consumption rate (OCR), elevated reactive oxygen species (ROS), and profound fragmented mitochondrial patterns compared to their high-invasive counterparts. SOX2 expression promotes LC3A expression and enhances proliferation but attenuates invasion in lung cancer cells. LC3A silencing enriches cells harboring low-proliferative and high-invasive features, concomitant with decreased OCR and ROS levels and reduced expression of SOX2. Our findings provide novel insights into how basal autophagy cross talks with SOX2 proliferation signaling to regulate mitochondrial metabolism and determines cancer cell plasticity with an impact on lung tumor progression.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-β: transforming growth factor beta; TOMM20: translocase of outer mitochondrial membrane 20; ZEB1: zinc finger E-box binding homeobox 1
Tumor metastasis is considered as the main cause that contributes to high mortality during lung cancer progression. Autophagy is a self-cleaning process to maintain cell integrity of intracellular organelles and proteins. To date, the role of autophagy in lung tumor metastasis remains elusive. Here, we report that lung tumors display a heterogeneous expression pattern of the autophagy mediator LC3A, the high expression of which is associated with low metastasis risk. We found a group of lung cancer cells contain differential LC3A expression levels and exhibit plasticity characterized by distinct proliferative and invasive properties. Immunofluorescence staining and immunoblotting assays revealed that LC3A-mediated autophagy is more active in highly proliferative but minimally invasive lung cancer cells than their lowly proliferative but highly invasive counterpart. Clonogenic analysis and cell cycle assays showed that LC3A silencing attenuated cellular growth, causing G1/S cell cycle arrest in highly proliferative lung cancer cells. Highly proliferative lung cancer cells exhibited a higher oxygen consumption rate, accompanied with an elevated oxygen species (ROS) level, compared to their lowly proliferative counterpart. Pharmacological inhibition of autophagy with chloroquine promoted the ROS production in highly proliferative lung cancer cells but not in the highly invasive counterpart. SOX2 expression enhanced LC3A expression and promoted proliferation but inhibited invasiveness in lung cancer cells. Knockdown of LC3A expression in SOX2-high proliferative cells enriched SOX2-low cells, exhibiting decreased proliferation but increased invasiveness. Combined, our findings provide substantial evidence to suggest that LC3A cooperates with SOX2 signaling to regulate lung cancer cell plasticity.Citation Format: Chia-Cheng Miao, Wen Hwang, Yu-Ting Chou. Heterogeneous LC3A expression regulates lung cancer cell plasticity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4279.
Abstract Tumor metastasis is considered as the main cause that contributes to high mortality during lung cancer progression. Autophagy is a self-cleaning process to maintain cell integrity of intracellular organelles and proteins. To date, the role of autophagy in lung tumor metastasis remains elusive. Here, we report that lung tumors display a heterogeneous expression pattern of the autophagy mediator LC3A, the high expression of which is associated with low metastasis risk. We found a group of lung cancer cells contain differential LC3A expression levels and exhibit plasticity characterized by distinct proliferative and invasive properties. Immunofluorescence staining and immunoblotting assays revealed that LC3A-mediated autophagy is more active in highly proliferative but minimally invasive lung cancer cells than their lowly proliferative but highly invasive counterpart. Clonogenic analysis and cell cycle assays showed that LC3A silencing attenuated cellular growth, causing G1/S cell cycle arrest in highly proliferative lung cancer cells. Highly proliferative lung cancer cells exhibited a higher oxygen consumption rate, accompanied with an elevated oxygen species (ROS) level, compared to their lowly proliferative counterpart. Pharmacological inhibition of autophagy with chloroquine promoted the ROS production in highly proliferative lung cancer cells but not in the highly invasive counterpart. SOX2 expression enhanced LC3A expression and promoted proliferation but inhibited invasiveness in lung cancer cells. Knockdown of LC3A expression in SOX2-high proliferative cells enriched SOX2-low cells, exhibiting decreased proliferation but increased invasiveness. Combined, our findings provide substantial evidence to suggest that LC3A cooperates with SOX2 signaling to regulate lung cancer cell plasticity. Citation Format: Chia-Cheng Miao, Wen Hwang, Yu-Ting Chou. Heterogeneous LC3A expression regulates lung cancer cell plasticity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4279.
Abstract Changes in expression patterns of serum carcinoembryonic antigen at initial diagnosis (CEA In ) and disease progression (CEA Pd ) in lung cancer patients under EGFR-tyrosine kinase inhibitors (TKI) treatment may reflect different tumor progression profiles. Of the 1736 lung cancer patients identified from the cancer registry group between 2011 to 2016, we selected 517 patients with advanced stage adenocarcinoma, data on EGFR mutation status and CEA In , among whom were 288 patients with data on CEA Pd , eligible for inclusion in the correlation analysis of clinical characteristics and survival. Multivariable analysis revealed that CEA In expression was associated with poor progression-free survival in patients harboring mutant EGFR. Moreover, CEA In and CEA Pd were associated with the good and poor post-progression survival, respectively, in the EGFR-mutant group. Cell line experiments revealed that CEA expression and cancer dissemination can be affected by EGFR-TKI selection. EGFR-mutant patients, exhibiting high CEA In (≥5 ng/mL) and low CEA Pd (<5 ng/mL), showed a potential toward displaying new metastasis. Taken together, these findings support the conclusion that EGFR mutation status is a critical factor in determining prognostic potential of CEA In and CEA Pd in patients under EGFR-TKI treatment, and CEA In and CEA Pd are associated with distinct cancer progression profiles.
Abstract Inhibitors of DNA binding and cell differentiation (ID) proteins regulate cellular differentiation and tumor progression. Whether ID family proteins serve as a linkage between pathological differentiation and cancer stemness in colorectal cancer is largely unknown. Here, the expression of ID4, but not other ID family proteins, was enriched in LGR5-high colon cancer stem cells. Its high expression was associated with poor pathological differentiation of colorectal tumors and shorter survival in patients. Knockdown of ID4 inhibited the growth and dissemination of colon cancer cells, while enhancing chemosensitivity. Through gene expression profiling analysis, brain-derived neurotrophic factor (BDNF) was identified as a downstream target of ID4 expression in colorectal cancer. BDNF knockdown decreased the growth and migration of colon cancer cells, and its expression enhanced dissemination, anoikis resistance and chemoresistance. ID4 silencing attenuated the epithelial-to-mesenchymal transition pattern in colon cancer cells. Gene cluster analysis revealed that ID4 and BDNF expression was clustered with mesenchymal markers and distant from epithelial genes. BDNF silencing decreased the expression of mesenchymal markers Vimentin, CDH2 and SNAI1. These findings demonstrated that ID4–BDNF signaling regulates colorectal cancer survival, with the potential to serve as a prognostic marker in colorectal cancer.
Abstract High‐grade neuroendocrine tumors (NETs) of the lung consist of small‐cell lung cancer (SCLC) and large‐cell neuroendocrine carcinoma (LCNEC). Both exhibit aggressive malignancy with poor prognosis. The transformation of lung adenocarcinoma (ADC) to SCLC or LCNEC also contributes to acquired resistance to epidermal growth factor receptor (EGFR)‐tyrosine kinase inhibitors (TKIs). Despite initially being responsive to chemotherapy, high‐grade NET patients inevitably develop drug resistance; thus, novel therapeutic targets are urgently needed for these patients. Our study reported that VGF (nerve growth factor inducible), a factor mainly expressed in neurons during neural development, is highly expressed in SCLC and LCNEC as well as in a subset of ADCs, whereas targeting VGF attenuates cancer cell growth and tumor formation. High VGF expression was associated with advanced stage SCLC and predicted poor prognosis in lung ADC. In addition, EGFR‐TKI selection enriched VGF expression in TKI‐resistant ADC under epigenetic control. The VGF locus possessed the HDAC1 binding site, and treatment of ADC cells with the HDAC1 inhibitor induced VGF expression. High VGF expression was associated with chemoresistance, and silencing VGF induced BMF and BCL2L11 expression and rendered lung cancer cells sensitive to chemotherapy drugs. These findings suggested the potential of VGF as a prognostic factor and therapeutic target in lung cancers with neuroendocrine feature.
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