Programmed death ligand 1 (PDL1) suppresses T-cell immunity by engaging programmed cell death protein 1 (PD1), and its blockade can activate T-cell responses. Although PDL1 is a transmembrane protein, its intrinsic signaling role in regulating oncogenesis remains unclear. Our study reveals lung adenocarcinomas (ADCs) exhibit deficient PDL1 expression, which correlates with poor patient prognosis. TGF-β stimulation induced PDL1 expression, while silencing PDL1 in PDL1-high lung ADC cells enhanced colony formation, and PDL1 overexpression inhibited lung cancer cell growth. Cell cycle analysis indicated that PDL1 silencing increased S-phase entry in lung ADC cells. Furthermore, PDL1 expression reduced FAK, ERK, and AKT phosphorylation, increasing cell detachment from the substrate. Gene expression profiling identified TGFBI as a downstream molecule of PDL1. TGF-β induced TGFBI expression, and knockdown of TGFBI increased the growth of lung ADC cells. Given that TGF-β regulates CITED2 and p21CIP1 to initiate cell growth arrest, we examined the PDL1-TGFBI axis's impact on these molecules. Knockdown of PDL1 or TGFBI induced CITED2 expression but decreased p21CIP1 expression in lung ADC cells. Moreover, inhibiting FAK via pharmacologic or genetic approaches decreased CITED2 but increased p21CIP1 expression in PDL1-silenced lung ADC cells. These findings suggest that intrinsic PDL1-TGFBI signaling inhibits FAK activation, affecting the CITED2 molecular switch, which induces p21CIP1, ultimately leading to cell growth arrest. Our study provides insights into intrinsic PDL1 signaling in lung ADC oncogenesis and indicates that PDL1 expression could be a biomarker for lung ADC progression.

CEACAM family proteins have been extensively studied as cell adhesion molecules, yet the biological and clinical significance of CEACAM6 remains relatively unexplored. Our research identifies a significant increase in CEACAM6 expression in lung adenocarcinoma, particularly correlating with EGFR mutation status. In EGFR-mutated lung cancer cells, CEACAM6 knockdown induced apoptosis and reduced p-ERK1/2 signaling downstream of EGFR. Treatment with EGFR-tyrosine kinase inhibitors (TKIs) decreased CEACAM6 levels, leading to TKI-resistant lung cancer cells that exhibited reduced p-ERK1/2 and increased epithelial-mesenchymal transition (EMT) characteristics. Co-immunoprecipitation assays revealed an interaction between CEACAM6 and EGFR. Although CEACAM6 expression was lost in EGFR-TKI resistant cells, its re-expression stabilized EGFR and increased sensitivity to EGFR-TKIs. TGF-β treatment, which induced EMT, also decreased CEACAM6 expression and improved EGFR-TKI resistance. Further analysis showed that EGFR-TKI resistant lung cancer cells had lower H3K27ac epigenetic modification levels at the CEACAM6 locus than EGFR-TKI sensitive cells. Treatment with HDAC1/2 inhibitors in EGFR-TKI sensitive cells reduced CEACAM6 expression, induced EMT and TGF-β-ligand/receptor gene expression, and enhanced EGFR-TKI resistance. These data highlight the crucial role of CEACAM6 in maintaining oncogenic EGFR signaling and its regulation by cytokine stimulation and epigenetic modification, influencing EGFR-TKI sensitivity. Our findings underscore CEACAM6’s potential as a valuable biomarker in EGFR-driven lung adenocarcinoma and its intricate involvement in EGFR-related pathways.

Squamous cell carcinoma (SCC) poses a significant global health challenge due to the lack of effective treatments. Boron neutron capture therapy (BNCT), a targeted particle therapy, has shown promising results in various cancers. SLC7A5, a transporter of essential amino acids and boronophenylalanine (BPA) used in BNCT, emerges as a potential therapeutic target. However, its expression across different histological subtypes and the role of SLC7A5 inhibition in developing drug resistance to BPA-BNCT remain poorly understood. Our study reveals elevated SLC7A5 expression in most SCCs, particularly in lung squamous cell carcinoma (LUSC), where it is significantly higher compared to other lung cancer subtypes. Increased SLC7A5 expression and a higher tumor-to-normal (T/N) ratio in LUSC are associated with poor patient prognosis. SLC7A5 knockdown in LUSC cells reduces colony formation and induces apoptosis. RNA-seq analysis of SLC7A5 knockout LUSC cells shows downregulated mTORC1 signaling, reduced expression of other amino acid transporters, and upregulated autophagy genes, indicating a potential cancer metabolic shift. Furthermore, SLC7A5 knockout LUSC cells demonstrate resistance to BPA-BNCT but sensitivity to the autophagy inhibitor chloroquine. Post-BPA-BNCT treatment, surviving wild-type LUSC cells exhibit reduced SLC7A5 levels and increased sensitivity to chloroquine, highlighting a vulnerability in BPA-BNCT-resistant cells. Our findings elucidate the interplay between SLC7A5, mTOR signaling, and autophagy pathways, providing insights into potential strategies to overcome drug resistance in BPA-BNCT therapy.

BACKGROUND:Serum carcinoembryonic antigen (CEA) has potential prognostic and monitoring significance in lung adenocarcinoma (LUAD) patients undergoing different treatments, such as epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) and chemotherapy. The changes in CEA expression during relapses, influenced by resistance mechanisms involving cytokines and epigenetic factors, may impact its utility in disease prognosis, monitoring, and management. METHODS:This retrospective study analyzed advanced LUAD patients treated between 2011 and 2018, including 182 patients receiving EGFR-TKIs and 102 undergoing chemotherapies. Serum CEA levels were measured at baseline and relapse. Associations between CEA levels, treatment modalities, and survival outcomes were assessed using Cox regression and Kaplan-Meier analyses. Gene expression profiling and in vitro experiments explored the regulation of CEACAM5 expression by cytokines and epigenetic mechanisms in EGFR-TKI-resistant cells. RESULTS:Elevated baseline CEA (≥ 5 ng/mL) was associated with significantly worse overall survival (OS) in patients treated with EGFR-TKIs but showed no prognostic value in chemotherapy-treated patients. During the relapse, EGFR-TKI-treated patients were more likely to exhibit a transition to CEA-negative status (< 5 ng/mL) compared to those receiving chemotherapy. Mechanistic studies revealed that EGFR-TKI-resistant cells displayed reduced CEACAM5 expression and increased epithelial-to-mesenchymal transition (EMT) markers, driven by cytokine signaling and epigenetic modifications. CONCLUSIONS:Serum CEA is a stronger prognostic biomarker for LUAD patients treated with EGFR-TKIs while offering consistent monitoring capabilities in chemotherapy-treated patients. These findings highlight the differential clinical value of serum CEA in guiding therapeutic strategies and monitoring disease progression across treatment modalities.

AbstractMetabolic reprogramming is a pivotal characteristic of cancer, yet the intricate interplay between glycolysis and the pentose phosphate pathway (PPP) remains elusive. This study unveils the pivotal role of 6-phosphofructokinase liver type (PFKL) in glycolysis and ribose 5-phosphate isomerase A (RPIA) in PPP, orchestrating liver tumorigenesis. PFKL, the rate-limiting enzyme in glycolysis, stabilizes RPIA by impeding ubiquitination/proteasome activity. The pro-inflammatory and tumor cytokine interleukin 6 activates pSTAT3 which binds to the promoter region and activates AMPK and PFKL transcription. Furthermore, pAMPK stabilizes PFKL protein by preventing proteasome degradation in hepatoma cells. Inhibiting PFKL, AMPK, and STAT3 genetically or pharmacologically can reduce glycolysis, ATP production, resulting in reduction of hepatoma cell proliferation and migration. Intriguingly, the PFKL, AMPK, RPIA, and PKM2 are co-localized in the Glycolytic body (G-body) which starts forming at chronic hepatitis, dramatically increases during active hepatitis, and the size of G-bodies becomes bigger from cirrhosis to hepatocellular carcinoma. Furthermore, using Bimolecular fluorescence complementation (BiFC) assay, we demonstrated that PFKL and RPIA direct interacts. Targeting AMPK or STAT3 significantly reduced tumor formation and lipid accumulation in zebrafish models, suggesting the STAT3/AMPK/PFKL axis as a potential therapeutic avenue for liver cancer treatment.

'Figure S1. Lack of T790M and amplification of MET in HCC827GR cells; Figure S2. VGF-expressing cells are resistant to rociletinib; Figure S3. VGF expression in neuroendocrine carcinoma and EGFR-TKI resistant cells; Figure S4. VGF is expressed in the independently selected EGFR-TKI resistant HCC827 pool; Figure S5. Correlation of VGF levels with EGFR-TKI resistance and EMT in EGFR-TKI selected single cell clones; Figure S6. Effect of VGF expression on cell survival; Figure S7. VGF is highly expressed in small cell lung cancer; Figure S8. VGF is highly expressed in large cell neuroendocrine carcinoma; Figure S9. Correlation of VGF, CEACAM6, SYP, and CHGB expression levels with IC50 values of gefitinib in EGFR-mutated lung cancer cell lines; Figure S10. EGFR-TKI resistance in H1975 cells expressing differential VGF levels; Figure S11. VGF-TWIST1 signaling encourages EGFR-TKI resistance; Figure S12. Effect of conditioned medium from VGF expressing cells on low serum-mediated growth; Figure S13. Effect of doxycycline alone on tumor cell growth in vitro and in vivo; Figure S14. Correlation of VGF with EMT markers in lung adenocarcinoma; Figure S15. Correlation of neuroendocrine markers or TWIST1 with survival in lung adenocarcinoma; Figure S16. Correlation of neuroendocrine markers or VGF-TWIST1 signature with survival in EGFR-mutated lung adenocarcinoma.'

Supplementary Table 4 from EGFR Promotes Lung Tumorigenesis by Activating miR-7 through a Ras/ERK/Myc Pathway That Targets the Ets2 Transcriptional Repressor ERF

This file contains technical details of experimental methods used in this study: 1. Colony formation assay; 2. Matrigel invasion assay; 3. Live cell migration assay; 4. Flow cytometry analysis; 5. Immunostaining; 6. ChIP-qPCR; 7. Cell proliferation assay; 8. Animal experiment.

Supplementary Materials and Methods

'Table S1. Q-PCR primer and probe sequences for quantifying gene expression; Table S2. Q-PCR primer and probe sequences for quantifying gene amplification; Table S3. Antibodies for Western blot; Table S4. Antibodies for immunofluorescence; Table S5. Gene expression profiling data from the public domain used in this study.'

Supplementary Methods, Figures 1-6, Tables 1-3 from EGFR Promotes Lung Tumorigenesis by Activating miR-7 through a Ras/ERK/Myc Pathway That Targets the Ets2 Transcriptional Repressor ERF

Supplementary Figures 1-4, Tables 1-4, Methods from Coexpression of Oct4 and Nanog Enhances Malignancy in Lung Adenocarcinoma by Inducing Cancer Stem Cell–Like Properties and Epithelial–Mesenchymal Transdifferentiation

This file contains figure legends.

This file contains following figures and tables: Fig. S1. Differential effects of cisplatin on SOX2- and SOX9-positive lung cancer cells (related to Fig. 1); Fig. S2. STR profiling in the derived stable clones (related to Fig. 2); Fig. S3. Expression switching between SOX2 and SOX9 (related to Fig. 2 and 3); Fig. S4. Ectopic expression of SOX2 attenuates SOX9 expression (related to Fig. 3); Fig. S5. Differential effects of SOX2 and SOX9 on cell proliferation (related to Fig. 3 and 4); Fig. S6. CCNA2-silencing attenuates cell growth in CL1-0 cells (related to Fig. 3); Fig. S7. SOX9-silencing attenuates lung invasion (related to Fig. 5); Fig. S8. Switching enrichment of H3K9me2 between CL1-1 and CL1-2 cells (related to Fig. 6); Fig. S9. Correlation analysis of SOX2 with SOX9, CCNA2 and HDAC1 in primary lung adenocarcinoma (related to Fig. 7); Fig. S10. Model for cancer cell plasticity (related to Fig. 1 to 7); Table S1. shRNA clones used in this study; Table S2. Primary antibodies used in this study; Table S3. Primers and probes used in this study; Table S4. Gene expression profiling data from the public domain used in this study.

'Establishment of gefitinib-resistant cell lines; RNA extraction and quantitative real-time polymerase chain reaction (Q-PCR); Genomic DNA extraction and DNA sequencing; AlamarBlue® viability assay; Xenograft tumorigenicity assay; RNA In Situ Hybridization; Immunomagnetic reduction (IMR) assay.'

Tumor suppressor p53 plays a central role in preventing tumorigenesis. Here, we unravel how p53 modulates mitochondrial dynamics to restrain the metastatic properties of cancer cells. p53 inhibits the mammalian target of rapamycin complex 1 (mTORC1) signaling to attenuate the protein level of mitochondrial fission process 1 (MTFP1), which fosters the pro-fission dynamin-related protein 1 (Drp1) phosphorylation. This regulatory mechanism allows p53 to restrict cell migration and invasion governed by Drp1-mediated mitochondrial fission. Downregulating p53 expression or elevating the molecular signature of mitochondrial fission correlates with aggressive tumor phenotypes and poor prognosis in cancer patients. Upon p53 loss, exaggerated mitochondrial fragmentation stimulates the activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling resulting in epithelial-to-mesenchymal transition (EMT)-like changes in cell morphology, accompanied by accelerated matrix metalloproteinase-9 (MMP9) expression and invasive cell migration. Notably, blocking the activation of mTORC1/MTFP1/Drp1/ERK1/2 axis completely abolishes the p53 deficiency-driven cellular morphological switch, MMP9 expression, and cancer cell dissemination. Our findings unveil a hitherto unrecognized mitochondria-dependent molecular mechanism underlying the metastatic phenotypes of p53-compromised cancers.

AbstractBackground and PurposeAlthough cytotoxic platinum‐based adjuvant chemotherapy (pACT) has been recommended for patients with completely resected early‐stage (ES) non–small‐cell lung cancer (ES‐NSCLC), therapeutic regimens for NSCLC have evolved in the past two decades. The study was aimed to examine the effectiveness of postoperative pACT for resected ES‐NSCLC patients with squamous cell carcinoma (SCC) or adenocarcinoma (ADC) according to real‐world data.Methods and PatientsInverse probability treatment weighting (IPTW) was used to adjust baseline characteristics between the group receiving pACT and those not receiving any treatment (observation, OBS) within 3 months after curative surgery. Cox regression models were used to compare overall survival (OS) and treatment failure‐free survival (TFS) between the groups.ResultsOf 31,208 patients with ES‐NSCLC, 4700 undergoing complete tumor resection were eligible, with a mean follow‐up period of 4.5 years. The pACT (n = 2347) and OBS (n = 2353) groups were well‐balanced after IPTW. The median OS differed between the pACT and OBS groups (77.2 vs. 75.5 months, adjusted hazard ratio [aHR] = 0.87, 95% confidence interval [CI] = 0.79–0.95, p = 0.003), and the 5‐year survival rates were 58.2% and 55.3%, respectively (p < 0.001). In the SCC group, pACT was superior to OBS in OS (75.0 vs. 57.4 months, aHR = 0.74, 95% CI = 0.62–0.88, p = 0.001) and TFS (32.7 vs. 21.8 months, aHR = 0.74, 95% CI = 0.63–0.86, p < 0.001). Both OS and TFS did not differ between two groups in those with ADC.ConclusionReal‐world data indicated that pACT confers a survival benefit for resected ES‐NSCLC patients with SCC but not ADC, which needs to be verified by a large sample of randomized controlled studies.

Ribose-5-phosphate isomerase A (RPIA) regulates tumorigenesis in liver and colorectal cancer. However, the role of RPIA in lung cancer remains obscure. Here we report that the suppression of RPIA diminishes cellular proliferation and activates autophagy, apoptosis, and cellular senescence in lung cancer cells. First, we detected that RPIA protein was increased in the human lung cancer versus adjust normal tissue via tissue array. Next, the knockdown of RPIA in lung cancer cells displayed autophagic vacuoles, enhanced acridine orange staining, GFP-LC3 punctae, accumulated autophagosomes, and showed elevated levels of LC3-II and reduced levels of p62, together suggesting that the suppression of RPIA stimulates autophagy in lung cancer cells. In addition, decreased RPIA expression induced apoptosis by increasing levels of Bax, cleaved PARP and caspase-3 and apoptotic cells. Moreover, RPIA knockdown triggered cellular senescence and increased p53 and p21 levels in lung cancer cells. Importantly, RPIA knockdown elevated reactive oxygen species (ROS) levels. Treatment of ROS scavenger N-acetyl-L-cysteine (NAC) reverts the activation of autophagy, apoptosis and cellular senescence by RPIA knockdown in lung cancer cells. In conclusion, RPIA knockdown induces ROS levels to activate autophagy, apoptosis, and cellular senescence in lung cancer cells. Our study sheds new light on RPIA suppression in lung cancer therapy.

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.

ABSTRACT 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