Integrated whole-exome and bulk transcriptome sequencing delineates the dynamic evolution from preneoplasia to invasive lung adenocarcinoma featured with ground-glass nodules.

OBJECTIVE: The genomic and molecular ecology involved in the stepwise continuum progression of lung adenocarcinoma (LUAD) from adenocarcinoma in situ (AIS) to minimally invasive adenocarcinoma (MIA) and subsequent invasive adenocarcinoma (IAC) remains unclear and requires further elucidation. We aimed to characterize gene mutations and expression landscapes, and explore the association between differentially expressed genes (DEGs) and significantly mutated genes (SMGs) during the dynamic evolution from AIS to IAC. METHODS: Thirty-five patients with ground-glass nodules (GGNs) lung adenocarcinomas were enrolled. Whole-exome sequencing (WES) and transcriptome sequencing (RNA-Seq) were conducted on all patients, encompassing both tumor samples and corresponding noncancerous tissues. Data obtained from WES and RNA-Seq were subsequently analyzed. RESULTS: The findings from WES delineated that the predominant mutations were observed in EGFR (49%) and ANKRD36C (17%). SMGs, including EGFR and RBM10, were associated with the dynamic evolution from AIS to IAC. Meanwhile, DEGs, including GPR143, CCR9, ADAMTS16, and others were associated with the entire process of invasive LUAD. We found that the signaling pathways related to cell migration and invasion were upregulated, and the signaling pathways of angiogenesis were downregulated across the pathological stages. Furthermore, we found that the messenger RNA (mRNA) levels of FAM83A, MAL2, DEPTOR, and others were significantly correlated with CNVs. Gene set enrichment analysis (GSEA) showed that heme metabolism and cholesterol homeostasis pathways were significantly upregulated in patients with EGFR/RBM10 co-mutations, and these patients may have poorer overall survival than those with EGFR mutations. Based on the six calculation methods for the immune infiltration score, NK/CD8+ T cells decreased, and Treg/B cells increased with the progression of early LUAD. CONCLUSIONS: Our findings offer valuable insights into the unique genomic and molecular features of LUAD, facilitating the identification and advancement of precision medicine strategies targeting the invasive progression of LUAD from AIS to IAC.

A Novel Methylation-based Model for Prognostic Prediction in Lung Adenocarcinoma.

Objectives: Specific methylation sites have shown promise in the early diagnosis of lung adenocarcinoma (LUAD). However, their utility in predicting LUAD prognosis remains unclear. This study aimed to construct a reliable methylation-based predictor for accurately predicting the prognosis of LUAD patients. Methods: DNA methylation data and survival data from LUAD patients were obtained from the TCGA and a GEO series. A DNA methylation-based signature was developed using univariate least absolute shrinkage and selection operators and multivariate Cox regression models. Results: Eight CpG sites were identified and validated as optimal prognostic signatures for the overall survival of LUAD patients. Receiver operating characteristic analysis demonstrated the high predictive ability of the eight-site methylation signature combined with clinical factors for overall survival. Conclusion: This research successfully identified a novel eight-site methylation signature for predicting the overall survival of LUAD patients through bioinformatic integrated analysis of gene methylation markers used in the early diagnosis of lung cancer.

Ivermectin induces nonprotective autophagy by downregulating PAK1 and apoptosis in lung adenocarcinoma cells.

INTRODUCTION: LUAD (Lung adenocarcinoma), the most common subtype of lung carcinoma and one of the highest incidences and mortality cancers in the world remains still a substantial treatment challenge. Ivermectin, an avermectin derivative, has been traditionally used as an antiparasitic agent in human and veterinary medicine practice during the last few decades. Though ivermectin has been shown to be effective against a variety of cancers, however, there is few available data reporting the antitumor effects of ivermectin in LUAD. METHODS: The effect of ivermectin on cell viability and proliferative ability of LUAD cells was evaluated using CCK-8 and colony formation assay. Apoptosis rate and autophagy flux were detected using flow cytometry based on PI/Annexin V staining and confocal laser scanning microscope based on LC3-GFP/RFP puncta, respectively. Western blotting experiment was conducted to verify the results of changes in apoptosis and autophagy. LUAD-TCGA and GEO databases were used to analyse the expression and predictive value of PAK1 in LUAD patients. Xenograft model and immumohistochemical staining were used for verification of the inhibitor effect of ivermectin in vivo. RESULTS: Ivermectin treatment strikingly impeded the colony formation, and the viability of the cell, along with cell proliferation, and caused the apoptosis and enhanced autophagy flux in LUAD cells. In addition, ivermectin-induced nonprotective autophagy was confirmed by treating LUAD cells with 3-MA, an autophagy inhibitor. Mechanistically, we found that ivermectin inhibited PAK1 protein expression in LUAD cells and we confirmed that overexpression of PAK1 substantially inhibited ivermectin-induced autophagy in LUAD cells. Based on TCGA and GEO databases, PAK1 was highly expressed in LUAD tissues as compared with normal tissues. Furthermore, LUAD patients with high PAK1 level have poor overall survival. Finally, in vivo experiments revealed that ivermectin efficiently suppressed the cellular growth of LUAD among nude mice. CONCLUSION: This study not only revealed the mechanism of ivermectin inhibited the growth of LUAD but also supported an important theoretical basis for the development of ivermectin during the therapy for LUAD.

Utility of whole exome sequencing analysis in differentiating intrapulmonary metastatic multiple ground-glass nodules (GGNs) from multiple primary GGNs.

PURPOSE: Clinical evidence of metastasis with ground-glass nodules (GGNs) has been reported, including pulmonary metastasis and distant metastasis. However, the clonal relationships of multiple GGNs at the genetic level remain unclear. EXPERIMENTAL DESIGN: Sixty tissue specimens were obtained from 19 patients with multiple GGN lung cancer who underwent surgery in 2019. Whole exome sequencing (WES) was performed on tissue samples, and genomic profiling and clone evolution analysis were conducted to investigate the genetic characteristics and clonality of multiple GGNs. RESULTS: A total of 15,435 nonsynonymous mutations were identified by WES, and GGNs with shared nonsynonymous mutations were observed in seven patients. Copy number variant (CNV) analysis showed that GGNs in ten patients had at least one shared arm-level CNV. Mutational spectrum analysis showed that GGNs in three patients had similar six substitution profiles and GGNs in fou patients had similar 96 substitution profiles. According to the clone evolution analysis, we found that GGNs in five patients had shared clonal driver gene mutations. Taken together, we identified that 5 patients may have multiple primary GGNs without any similar genetic features, 2 patients may have intrapulmonary metastatic GGNs with ≥ 3 similar genetic features, and the other 12 patients cannot be determined due to insufficient evidences in our cohort. CONCLUSIONS: Our findings suggest that the intrapulmonary metastasis exist in multiple GGNs, but the number of GGNs was not associated with the probability of metastasis. Application of genomic profiling may prove to be important to precise management of patients with multiple GGNs.

Plasmodium Circumsporozoite Protein Enhances the Efficacy of Gefitinib in Lung Adenocarcinoma Cells by Inhibiting Autophagy via Proteasomal Degradation of LC3B.

Background: Almost all lung adenocarcinoma (LUAD) patients with EGFR mutant will develop resistance to EGFR-TKIs, which limit the long-term clinical application of these agents. Accumulating evidence shows one of the main reasons for resistance to EGFR-TKIs is induction of autophagy in tumor cells. Our previous study found that circumsporozoite protein (CSP) in Plasmodium can suppress autophagy in host hepatocytes. However, it is unknown whether CSP-mediated inhibition of autophagy could improve the anti-tumor effect of EGFR-TKIs. Methods: We constructed A549 and H1975 cell lines with stable overexpression of CSP (OE-CSP cells). CCK-8, Lactate Dehydrogenase (LDH), flow cytometry, and colony analysis were performed to observe the effect of CSP overexpression on cell viability, apoptosis rate, and colony formation ratio. The sensitizing effect of CSP on gefitinib was evaluated in vivo using a subcutaneous tumor model in nude mice and immunohistochemical assay. The role of CSP in regulation of autophagy was investigated by laser confocal microscopy assay and western blotting. A transcriptome sequencing assay and real-time polymerase chain reaction were used to determine the levels of mRNA for autophagy-related proteins. Cycloheximide (CHX), MG132, TAK-243, and immunoprecipitation assays were used to detect and confirm proteasomal degradation of LC3B. Results: OE-CSP A549 and H1975 cells were more sensitive to gefitinib, demonstrating significant amounts of apoptosis and decreased viability. In the OE-CSP group, autophagy was significantly inhibited, and there was a decrease in LC3B protein after exposure to gefitinib. Cell viability and colony formed ability were recovered when OE-CSP cells were exposed to rapamycin. In nude mice with xenografts of LUAD cells, inhibition of autophagy by CSP resulted in suppression of cell growth, and more marked apoptosis during exposure to gefitinib. CSP promoted ubiquitin-proteasome degradation of LC3B, leading to inhibition of autophagy in LUAD cells after treatment with gefitinib. When LUAD cells were treated with ubiquitin activating enzyme inhibitor TAK-243, cell viability, apoptosis, and growth were comparable between the OE-CSP group and a control group both in vivo and in vitro. Conclusion: CSP can inhibit gefitinib-induced autophagy via proteasomal degradation of LC3B, which suggests that CSP could be used as an autophagy inhibitor to sensitize EGFR-TKIs.

Transcriptomics integrated with metabolomics reveals the effect of Lycium barbarum polysaccharide on apoptosis in Nile tilapia (Oreochromis niloticus).

Lycium barbarum polysaccharide (LBP) is one of the main active ingredients in the fruit of L. barbarum L. It has been used as herbal medicine for thousands of years in China. In this study, Nile tilapia (Oreochromis niloticus) was taken as the research object. After feeding tilapia with 5 different doses of LBP (0 mg/kg, 500 mg/kg, 1000 mg/kg, 1500 mg/kg, 2000 mg/kg) for 55 d, it was found that LBP could promote the growth of tilapia, and this effect was the strongest at Group 1500 mg/kg. Apoptosis analysis in the liver and spleen showed that dietary supplementation with 1000 mg/kg LBP had the best protective effect on the spleen and liver in tilapia. Combined transcriptomics and metabolomics of the spleen in tilapia at Group 0 mg/kg and 1000 mg/kg showed that the differentially expressed genes (DEGs) such as NT5C2L1, pmm1, FasL and the differentially metabolites such as xanthine, dGMP, guanine and glutamate were mainly concentrated in signaling pathways such as Purine metabolism and FoxO signaling pathway. In conclusion, LBP regulates the metabolic waste levels of tilapia mainly through Purine metabolism and the FoxO signaling pathway, thereby inhibiting cell apoptosis, improving the utilization of nutrients, and promoting the growth of tilapia. This study not only provides a theoretical basis for the application of LBP in aquatic animals but also provides useful information for the healthy development of the aquaculture.

Significance of Parkinson Family Genes in the Prognosis and Treatment Outcome Prediction for Lung Adenocarcinoma.

Epidemiological investigations have shown that patients with Parkinson's disease (PD) have a lower probability of developing lung cancer. Subsequent research revealed that PD and lung cancer share specific genetic alterations. Therefore, the utilisation of PD biomarkers and therapeutic targets may improve lung adenocarcinoma (LUAD) diagnosis and treatment. We aimed to identify a gene-based signature from 25 Parkinson family genes for LUAD prognosis and treatment choice. We analysed Parkinson family gene expression and protein levels in LUAD, utilising multiple databases. Least absolute shrinkage and selection operator (LASSO) regression was used to construct a prognostic model based on the TCGA-LUAD cohort. We validated the model in external GEO cohorts. Immune cell infiltration was compared between risk groups, and GEO data was used to explore the model's predictive ability for LUAD treatment response. Nearly all Parkinson family genes exhibited significant differential expression between LUAD and normal tissues. LASSO regression confirmed that our seven Parkinson family gene-based signature had excellent prognostic performance for LUAD, as validated in three GEO cohorts. The high-risk group was clearly associated with low tumour immune cell infiltration, suggesting that immunotherapy may not be an optimal treatment choice. This is the first Parkinson family gene-based model for the prediction of LUAD prognosis and treatment outcome. The association of these genes with poor prognosis and low immune infiltration requires further investigation.

A Noninvasive Multianalytical Approach for Lung Cancer Diagnosis of Patients with Pulmonary Nodules.

Addressing the high false-positive rate of conventional low-dose computed tomography (LDCT) for lung cancer diagnosis, the efficacy of incorporating blood-based noninvasive testing for assisting practicing clinician's decision making in diagnosis of pulmonary nodules (PNs) is investigated. In this prospective observative study, next generation sequencing- (NGS-) based cell-free DNA (cfDNA) mutation profiling, NGS-based cfDNA methylation profiling, and blood-based protein cancer biomarker testing are performed for patients with PNs, who are diagnosed as high-risk patients through LDCT and subsequently undergo surgical resections, with tissue sections pathologically examined and classified. Using pathological classification as the gold standard, statistical and machine learning methods are used to select molecular markers associated with tissue's malignant classification based on a 98-patient discovery cohort (28 benign and 70 malignant), and to construct an integrative multianalytical model for tissue malignancy prediction. Predictive models based on individual testing platforms have shown varying levels of performance, while their final integrative model produces an area under the receiver operating characteristic curve (AUC) of 0.85. The model's performance is further confirmed on a 29-patient independent validation cohort (14 benign and 15 malignant, with power > 0.90), reproducing AUC of 0.86, which translates to an overall sensitivity of 80% and specificity of 85.7%.

Emergence in protein derived nanomedicine as anticancer therapeutics: More than a tour de force.

Cancer has thwarted as a major health problem affecting the global population. With an alarming increase in the patient population suffering from diverse varieties of cancers, the global demographic data predicts sharp escalation in the number of cancer patients. This can be expected to reach 420 million cases by 2025. Among the diverse types of cancers, the most frequently diagnosed cancers are the breast, colorectal, prostate and lung cancer. From years, conventional treatment approaches like surgery, chemotherapy and radiation therapy have been practiced. In the past few years, increasing research on molecular level diagnosis and treatment of cancers have significantly changed the realm of cancer treatment. Lately, uses of advanced chemotherapy and immunotherapy like treatments have gained significant progress in the cancer therapy, but these approaches have several limitations on their safety and toxicity. This has generated lot of momentum for the evolution of new drug delivery approaches for the effective delivery of anticancer therapeutics, which may improve the pharmacokinetic and pharmacodynamic effect of the drugs along with significant reduction in the side effects. In this regard, the protein-based nano-medicines have gained wider attention in the management of cancer. Proteins are organic macromolecules essential, for life and have quite well explored in developing the nano-carriers. Furthermore, it provides passive or active tumour cell targeted delivery, by using protein based nanovesicles or virus like structures, antibody drug conjugates, viral particles, etc. Moreover, by utilizing various formulation strategies, both the animal and plant derived proteins can be converted to produce self-assembled virus like nano-metric structures with high efficiency in targeting the metastatic cancer cells. Therefore, the present review extensively discusses the applications of protein-based nano-medicine with special emphasis on intracellular delivery/drug targeting ability for anticancer drugs.

CWHM-1008 Induces Apoptosis and Protective Autophagy through the Akt/mTOR Axis in LUAD Cells.

Recent studies have revealed that antiparasitic agents showed promising inhibitory effects on tumors, raising a possibility that repositioning this class of drugs may shed new light on clinical therapy against tumors. CWHM-1008 is a novel class of antimalarial drug; however, the inhibitory impact of CWHM-1008 on lung adenocarcinoma (LUAD) cells remains unclear. This study aimed to explore the anticancer effect and underlying mechanisms of CWHM-1008 on LUAD cells in vitro and in vivo. Human LUAD cells, H358 and A549, were treated with varying concentrations of CWHM-1008 at different lengths of time. Cell viability, colony formation, cell count, flow cytometry findings, microtubule-associated protein-1 light chain 3-green- (LC3-) GFP/RFP adenovirus infection status, and the expression of apoptosis and autophagy-related proteins were examined. Potential effects of an autophagy inhibitor (LY294002) and constitutively active Akt plasmid (CA-Akt) on CWHM-1008-induced apoptosis were also examined. Our results showed that CWHM-1008 significantly inhibited proliferation, induced apoptosis, and enhanced autophagy flux by blocking the RAC-alpha serine/threonine-protein kinase/the mammalian target of rapamycin (Akt/mTOR) axis in two LUAD cells. In addition, autophagy inhibited by LY294002 or CA-Akt transfection accelerated CWHM-1008-induced apoptosis in those LUAD cells. Moreover, CWHM-1008 significantly inhibited the growth and induced apoptosis of A549 cell in nude mice in vivo. The present findings provide new insights into anticancer properties of CWHM-1008, suggesting that it may be an adjuvant treatment for LUAD treatment, warranting further study.

PINK1 Overexpression Promotes Cell Migration and Proliferation via Regulation of Autophagy and Predicts a Poor Prognosis in Lung Cancer Cases.

BACKGROUND: Lung cancer remains the leading cause of cancer-related death worldwide. The human PINK1 gene (PTEN induced kinase 1, Park6), an important gene for Parkinson's disease, was found to be associated with tumor development although the molecular mechanisms underlying this relationship remain largely unknown. OBJECTIVE: To analyze the clinical value and molecular mechanism of PINK1 in non-small cell lung cancer (NSCLC). MATERIALS AND METHODS: Western blot, qRT-PCR and Immunohistochemistry were employed to determine the levels of PINK1 in 87 paired NSCLC tissues, Oncomine and TCGA databases were also used for the evaluation of expression and prognosis of PINK1. The mitophagy, proliferation, migration, invasion, and apoptosis abilities of A549 and H1975 cells were detected, and the autophagy-related proteins in the cells were also determined. RESULTS: Immunohistochemical staining revealed higher PINK1 expression in tumor tissues, which was strongly linked to the tumor-node-metastasis classification. Survival analysis of 1085 NSCLC patients also revealed that low PINK1 expression levels were associated with significantly longer overall survival. Univariate and multivariate analyses indicated that PINK1 expression was an independent predictor of overall survival among patients with NSCLC. We also evaluated the influence of PINK1 deficiency in NSCLC cell lines (A549 and H1975), which revealed significant suppression of migration capability and cell viability, as well as a significantly elevated apoptosis ratio. In cells with stable interference of PINK1 expression, dysfunctional mitochondria accumulated while autophagy was inhibited, which indicated that cell activity suppression was mediated by the accumulation of dysfunctional mitochondria. The suppression of migration and autophagy was reversed in cells that overexpressed PINK1. CONCLUSION: Our results suggest that PINK1 may be a potential therapeutic target and prognostic biomarker in NSCLC.