SARS-CoV-2 variants evolve convergent strategies to remodel the host response.

SARS-CoV-2 variants of concern (VOCs) emerged during the COVID-19 pandemic. Here, we used unbiased systems approaches to study the host-selective forces driving VOC evolution. We discovered that VOCs evolved convergent strategies to remodel the host by modulating viral RNA and protein levels, altering viral and host protein phosphorylation, and rewiring virus-host protein-protein interactions. Integrative computational analyses revealed that although Alpha, Beta, Gamma, and Delta ultimately converged to suppress interferon-stimulated genes (ISGs), Omicron BA.1 did not. ISG suppression correlated with the expression of viral innate immune antagonist proteins, including Orf6, N, and Orf9b, which we mapped to specific mutations. Later Omicron subvariants BA.4 and BA.5 more potently suppressed innate immunity than early subvariant BA.1, which correlated with Orf6 levels, although muted in BA.4 by a mutation that disrupts the Orf6-nuclear pore interaction. Our findings suggest that SARS-CoV-2 convergent evolution overcame human adaptive and innate immune barriers, laying the groundwork to tackle future pandemics.

The Global Phosphorylation Landscape of SARS-CoV-2 Infection.

The causative agent of the coronavirus disease 2019 (COVID-19) pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has infected millions and killed hundreds of thousands of people worldwide, highlighting an urgent need to develop antiviral therapies. Here we present a quantitative mass spectrometry-based phosphoproteomics survey of SARS-CoV-2 infection in Vero E6 cells, revealing dramatic rewiring of phosphorylation on host and viral proteins. SARS-CoV-2 infection promoted casein kinase II (CK2) and p38 MAPK activation, production of diverse cytokines, and shutdown of mitotic kinases, resulting in cell cycle arrest. Infection also stimulated a marked induction of CK2-containing filopodial protrusions possessing budding viral particles. Eighty-seven drugs and compounds were identified by mapping global phosphorylation profiles to dysregulated kinases and pathways. We found pharmacologic inhibition of the p38, CK2, CDK, AXL, and PIKFYVE kinases to possess antiviral efficacy, representing potential COVID-19 therapies.

Brahma safeguards canalization of cardiac mesoderm differentiation.

Differentiation proceeds along a continuum of increasingly fate-restricted intermediates, referred to as canalization1,2. Canalization is essential for stabilizing cell fate, but the mechanisms that underlie robust canalization are unclear. Here we show that the BRG1/BRM-associated factor (BAF) chromatin-remodelling complex ATPase gene Brm safeguards cell identity during directed cardiogenesis of mouse embryonic stem cells. Despite the establishment of a well-differentiated precardiac mesoderm, Brm-/- cells predominantly became neural precursors, violating germ layer assignment. Trajectory inference showed a sudden acquisition of a non-mesodermal identity in Brm-/- cells. Mechanistically, the loss of Brm prevented de novo accessibility of primed cardiac enhancers while increasing the expression of neurogenic factor POU3F1, preventing the binding of the neural suppressor REST and shifting the composition of BRG1 complexes. The identity switch caused by the Brm mutation was overcome by increasing BMP4 levels during mesoderm induction. Mathematical modelling supports these observations and demonstrates that Brm deletion affects cell fate trajectory by modifying saddle-node bifurcations2. In the mouse embryo, Brm deletion exacerbated mesoderm-deleted Brg1-mutant phenotypes, severely compromising cardiogenesis, and reveals an in vivo role for Brm. Our results show that Brm is a compensable safeguard of the fidelity of mesoderm chromatin states, and support a model in which developmental canalization is not a rigid irreversible path, but a highly plastic trajectory.

A Quantitative Genetic Interaction Map of HIV Infection.

We have developed a platform for quantitative genetic interaction mapping using viral infectivity as a functional readout and constructed a viral host-dependency epistasis map (vE-MAP) of 356 human genes linked to HIV function, comprising >63,000 pairwise genetic perturbations. The vE-MAP provides an expansive view of the genetic dependencies underlying HIV infection and can be used to identify drug targets and study viral mutations. We found that the RNA deadenylase complex, CNOT, is a central player in the vE-MAP and show that knockout of CNOT1, 10, and 11 suppressed HIV infection in primary T cells by upregulating innate immunity pathways. This phenotype was rescued by deletion of IRF7, a transcription factor regulating interferon-stimulated genes, revealing a previously unrecognized host signaling pathway involved in HIV infection. The vE-MAP represents a generic platform that can be used to study the global effects of how different pathogens hijack and rewire the host during infection.

Systems-level effects of allosteric perturbations to a model molecular switch.

Molecular switch proteins whose cycling between states is controlled by opposing regulators1,2 are central to biological signal transduction. As switch proteins function within highly connected interaction networks3, the fundamental question arises of how functional specificity is achieved when different processes share common regulators. Here we show that functional specificity of the small GTPase switch protein Gsp1 in Saccharomyces cerevisiae (the homologue of the human protein RAN)4 is linked to differential sensitivity of biological processes to different kinetics of the Gsp1 (RAN) switch cycle. We make 55 targeted point mutations to individual protein interaction interfaces of Gsp1 (RAN) and show through quantitative genetic5 and physical interaction mapping that Gsp1 (RAN) interface perturbations have widespread cellular consequences. Contrary to expectation, the cellular effects of the interface mutations group by their biophysical effects on kinetic parameters of the GTPase switch cycle and not by the targeted interfaces. Instead, we show that interface mutations allosterically tune the GTPase cycle kinetics. These results suggest a model in which protein partner binding, or post-translational modifications at distal sites, could act as allosteric regulators of GTPase switching. Similar mechanisms may underlie regulation by other GTPases, and other biological switches. Furthermore, our integrative platform to determine the quantitative consequences of molecular perturbations may help to explain the effects of disease mutations that target central molecular switches.

Profiling the proximal proteome of the activated µ-opioid receptor.

The µ-opioid receptor (µOR) represents an important target of therapeutic and abused drugs. So far, most understanding of µOR activity has focused on a subset of known signal transducers and regulatory molecules. Yet µOR signaling is coordinated by additional proteins in the interaction network of the activated receptor, which have largely remained invisible given the lack of technologies to interrogate these networks systematically. Here we describe a proteomics and computational approach to map the proximal proteome of the activated µOR and to extract subcellular location, trafficking and functional partners of G-protein-coupled receptor (GPCR) activity. We demonstrate that distinct opioid agonists exert differences in the µOR proximal proteome mediated by endocytosis and endosomal sorting. Moreover, we identify two new µOR network components, EYA4 and KCTD12, which are recruited on the basis of receptor-triggered G-protein activation and might form a previously unrecognized buffering system for G-protein activity broadly modulating cellular GPCR signaling.

A SARS-CoV-2 protein interaction map reveals targets for drug repurposing.

A newly described coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is the causative agent of coronavirus disease 2019 (COVID-19), has infected over 2.3 million people, led to the death of more than 160,000 individuals and caused worldwide social and economic disruption1,2. There are no antiviral drugs with proven clinical efficacy for the treatment of COVID-19, nor are there any vaccines that prevent infection with SARS-CoV-2, and efforts to develop drugs and vaccines are hampered by the limited knowledge of the molecular details of how SARS-CoV-2 infects cells. Here we cloned, tagged and expressed 26 of the 29 SARS-CoV-2 proteins in human cells and identified the human proteins that physically associated with each of the SARS-CoV-2 proteins using affinity-purification mass spectrometry, identifying 332 high-confidence protein-protein interactions between SARS-CoV-2 and human proteins. Among these, we identify 66 druggable human proteins or host factors targeted by 69 compounds (of which, 29 drugs are approved by the US Food and Drug Administration, 12 are in clinical trials and 28 are preclinical compounds). We screened a subset of these in multiple viral assays and found two sets of pharmacological agents that displayed antiviral activity: inhibitors of mRNA translation and predicted regulators of the sigma-1 and sigma-2 receptors. Further studies of these host-factor-targeting agents, including their combination with drugs that directly target viral enzymes, could lead to a therapeutic regimen to treat COVID-19.

A proximity proteomics pipeline with improved reproducibility and throughput.

Proximity labeling (PL) via biotinylation coupled with mass spectrometry (MS) captures spatial proteomes in cells. Large-scale processing requires a workflow minimizing hands-on time and enhancing quantitative reproducibility. We introduced a scalable PL pipeline integrating automated enrichment of biotinylated proteins in a 96-well plate format. Combining this with optimized quantitative MS based on data-independent acquisition (DIA), we increased sample throughput and improved protein identification and quantification reproducibility. We applied this pipeline to delineate subcellular proteomes across various compartments. Using the 5HT2A serotonin receptor as a model, we studied temporal changes of proximal interaction networks induced by receptor activation. In addition, we modified the pipeline for reduced sample input to accommodate CRISPR-based gene knockout, assessing dynamics of the 5HT2A network in response to perturbation of selected interactors. This PL approach is universally applicable to PL proteomics using biotinylation-based PL enzymes, enhancing throughput and reproducibility of standard protocols.

Smoothened transduces Hedgehog signals via activity-dependent sequestration of PKA catalytic subunits.

The Hedgehog (Hh) pathway is essential for organ development, homeostasis, and regeneration. Dysfunction of this cascade drives several cancers. To control expression of pathway target genes, the G protein-coupled receptor (GPCR) Smoothened (SMO) activates glioma-associated (GLI) transcription factors via an unknown mechanism. Here, we show that, rather than conforming to traditional GPCR signaling paradigms, SMO activates GLI by binding and sequestering protein kinase A (PKA) catalytic subunits at the membrane. This sequestration, triggered by GPCR kinase (GRK)-mediated phosphorylation of SMO intracellular domains, prevents PKA from phosphorylating soluble substrates, releasing GLI from PKA-mediated inhibition. Our work provides a mechanism directly linking Hh signal transduction at the membrane to GLI transcription in the nucleus. This process is more fundamentally similar between species than prevailing hypotheses suggest. The mechanism described here may apply broadly to other GPCR- and PKA-containing cascades in diverse areas of biology.

Mucin 3A's Promotion of the Proliferation and Migration of Gastric Cancer Cells Through Activation of the mTOR Pathway.

Context: Gastric cancer (GC) is a common and life-threatening gastrointestinal malignancy. Although mucin 3A (MUC3A) is an essential oncogenic factor in several cancers, limited information is available on its expression in GC tissues and its impact on prognosis. Objective: The study aimed to characterize MUC3A in GC and to explore its potential involvement in regulating GC cells' behavior through the mammalian target of rapamycin (mTOR) signaling pathway. Design: The research team conducted a retrospective genetic analysis. Setting: The study took place as Huzhou Central Hospital, an Affiliated Central Hospital of Huzhou University in Huzhou, Zhejiang, China. Participants: Participants were 47 patients with GC who had received treatment at the department of general surgery at the hospital and who gave consent for the use of their tissue samples for the genetic analysis. Outcome Measures: The research team: (1) performed a differential analysis of MUC3A using GC and normal tissue samples purchased from the American Type Culture Collection; (2) investigated the exposure of cancer tissues to MUC3A and its effects in the tumor, node, metastasis (TNM) stages of GC, using the real-time quantitative polymerase chain reaction (rt-qPCR) method; (3) performed clone formation and conducted transwell assays by knocking down or overexpressing MUC3A to analyze the effects on the behavior of GC cells; and (4) assessed the content of related marker proteins and the phosphoinositide 3-kinase (PI3K)/ protein kinase B (Akt)/ mammalian target of rapamycin (mTOR) pathway proteins, using a Western blot analysis. Results: A high level of MUC3A existed in GC tissues, and it was associated with TNM staging. Silencing of the MUC3A inhibited GC-cell migration and proliferation, and MUC3A overexpression had the opposite effect. The addition of agonist M05856 restored the inhibitory effect of silencing MUC3A on GC cell proliferation and migration, suggesting that MUC3A regulates GC cells' behavior through the PI3K/Akt/mTOR pathway. Conclusions: MUC3A plays an oncogenic role in GC and may regulate GC cell behavior through the PI3K/Akt/mTOR pathway.

CDBN-YGXZ, a Novel Small-Molecule Drug, Shows Efficacy against Clostridioides difficile Infection and Recurrence in Mouse and Hamster Infection Models.

Clostridioides difficile infection (CDI) causes severe diarrhea and colitis, leading to significant morbidity, mortality, and high medical costs worldwide. Oral vancomycin, a first-line treatment for CDI, is associated with a high risk of recurrence, necessitating novel therapies for primary and recurrent CDI. A novel small-molecule compound, CDBN-YGXZ, was synthesized by modifying the benzene ring of nitazoxanide with lauric acid. The mechanism of action of CDBN-YGXZ was validated using a pyruvate:ferredoxin/flavodoxin oxidoreductase (PFOR) inhibition assay. The efficacy of CDBN-YGXZ was evaluated using the MIC test and CDI infection model in mice and hamsters. Furthermore, metagenomics was used to reveal the underlying reasons for the effective reduction or prevention of CDI after CDBN-YGXZ treatment. The inhibitory activity against PFOR induced by CDBN-YGXZ. MIC tests showed that the in vitro activity of CDBN-YGXZ against C. difficile ranging from 0.1 to 1.5 µg/mL. In the mouse and hamster CDI models, CDBN-YGXZ provided protection during both treatment and relapse, while vancomycin treatment resulted in severe relapse and significant clinical scores. Compared with global effects on the indigenous gut microbiota induced by vancomycin, CDBN-YGXZ treatment had a mild influence on gut microbes, thus resulting in the disappearance or reduction of CDI recurrence. CDBN-YGXZ displayed potent activity against C. difficile in vitro and in vivo, reducing or preventing relapse in infected animals, which could merit further development as a potential drug candidate for treating CDI.

Hierarchical modularity and the evolution of genetic interactomes across species.

To date, cross-species comparisons of genetic interactomes have been restricted to small or functionally related gene sets, limiting our ability to infer evolutionary trends. To facilitate a more comprehensive analysis, we constructed a genome-scale epistasis map (E-MAP) for the fission yeast Schizosaccharomyces pombe, providing phenotypic signatures for ~60% of the nonessential genome. Using these signatures, we generated a catalog of 297 functional modules, and we assigned function to 144 previously uncharacterized genes, including mRNA splicing and DNA damage checkpoint factors. Comparison with an integrated genetic interactome from the budding yeast Saccharomyces cerevisiae revealed a hierarchical model for the evolution of genetic interactions, with conservation highest within protein complexes, lower within biological processes, and lowest between distinct biological processes. Despite the large evolutionary distance and extensive rewiring of individual interactions, both networks retain conserved features and display similar levels of functional crosstalk between biological processes, suggesting general design principles of genetic interactomes.

Genetic interaction mapping reveals a role for the SWI/SNF nucleosome remodeler in spliceosome activation in fission yeast.

Although numerous regulatory connections between pre-mRNA splicing and chromatin have been demonstrated, the precise mechanisms by which chromatin factors influence spliceosome assembly and/or catalysis remain unclear. To probe the genetic network of pre-mRNA splicing in the fission yeast Schizosaccharomyces pombe, we constructed an epistatic mini-array profile (E-MAP) and discovered many new connections between chromatin and splicing. Notably, the nucleosome remodeler SWI/SNF had strong genetic interactions with components of the U2 snRNP SF3 complex. Overexpression of SF3 components in ΔSWI/SNF cells led to inefficient splicing of many fission yeast introns, predominantly those with non-consensus splice sites. Deletion of SWI/SNF decreased recruitment of the splicing ATPase Prp2, suggesting that SWI/SNF promotes co-transcriptional spliceosome assembly prior to first step catalysis. Importantly, defects in SWI/SNF as well as SF3 overexpression each altered nucleosome occupancy along intron-containing genes, illustrating that the chromatin landscape both affects--and is affected by--co-transcriptional splicing.

Mapping Differential Protein-Protein Interaction Networks using Affinity Purification Mass Spectrometry.

Proteins congregate into complexes to perform fundamental cellular functions. Phenotypic outcomes, in health and disease, are often mechanistically driven by the remodeling of protein complexes by protein-coding mutations or cellular signaling changes in response to molecular cues. Here, we present an affinity purification-mass spectrometry (APMS) proteomics protocol to quantify and visualize global changes in protein-protein interaction (PPI) networks between pairwise conditions. We describe steps for expressing affinity-tagged "bait" proteins in mammalian cells, identifying purified protein complexes, quantifying differential PPIs, and visualizing differential PPI networks. Specifically, this protocol details steps for designing affinity-tagged "bait" gene constructs, transfection, affinity purification, mass spectrometry sample preparation, data acquisition, database search, data quality control, PPI confidence scoring, cross-run normalization, statistical data analysis, and differential PPI visualization. Our protocol discusses caveats and limitations with applicability across cell types and biological areas. For complete details on the use and execution of this protocol, please refer to Bouhaddou et al. 20231.

A foundational atlas of autism protein interactions reveals molecular convergence.

Translating high-confidence (hc) autism spectrum disorder (ASD) genes into viable treatment targets remains elusive. We constructed a foundational protein-protein interaction (PPI) network in HEK293T cells involving 100 hcASD risk genes, revealing over 1,800 PPIs (87% novel). Interactors, expressed in the human brain and enriched for ASD but not schizophrenia genetic risk, converged on protein complexes involved in neurogenesis, tubulin biology, transcriptional regulation, and chromatin modification. A PPI map of 54 patient-derived missense variants identified differential physical interactions, and we leveraged AlphaFold-Multimer predictions to prioritize direct PPIs and specific variants for interrogation in Xenopus tropicalis and human forebrain organoids. A mutation in the transcription factor FOXP1 led to reconfiguration of DNA binding sites and altered development of deep cortical layer neurons in forebrain organoids. This work offers new insights into molecular mechanisms underlying ASD and describes a powerful platform to develop and test therapeutic strategies for many genetically-defined conditions.

[Expression of pGSK-3α/ß Tyr279/216 and XIAP proteins in cholangiocarcinoma and their clinical significance].

OBJECTIVE: To investigate the expressions of the active form of glycogen synthase kinase-3(GSK-3)-pGSK-3α/ß (Tyr279/216) and its downstream moleculor X-linked inhibitor of apoptosis protein (XIAP) in cholangiocarcinoma and to analyze their correlation with clinicopathological and survival significance. METHODS: Immunohistoehemistry was used to detect the expressions of the active form of GSK-3- pGSK-3α/ß (Tyr279/216) and its downstream moleculor XIAP proteins in 50 cholangiocarcinoma tissues and 20 normal bile duct tissues. RESULTS: The positive rates of pGSK-3α/ß (Tyr279/216) and XIAP were 62.0% and 68.0% in cholangiocarcinoma, and 10.0% and 25.0% in normal bile duct tissues, respectively. The intensity of pGSK-3α/ß (Tyr279/216) and XIAP expressions in cholangiocarcinoma were significantly higher than that in the normal bile duct tissues (P < 0.001), and there was a significant correlation between pGSK-3α/ß (Tyr279/216) and XIAP expressions (r = 0.544, P < 0.001). The expression of pGSK-3α/ß(Tyr279/216) protein in cholangiocarcinoma was associated with TNM stage (P = 0.042), histological grade (P = 0.031), whereas the expression of XIAP protein in cholangiocarcinoma was correlated with CEA level (P = 0.006). Patients with positive expression of pGSK-3α/ß (Tyr279/216) and XIAP demonstrate a significantly worse prognosis than that of patients with negative expression of pGSK-3α/ß (Tyr279/216) and XIAP for overall survival (P = 0.002, P = 0.018). Multivariate survival analysis revealed that positive pGSK-3α/ß (Tyr279/216) expression provided significant independent prognostic value for overall survival (P = 0.002). CONCLUSIONS: The expressions of pGSK-3α/ß(Tyr279/216) and XIAP proteins were significantly associated with the development and progression of cholangiocarcinoma. pGSK-3α/ß(Tyr279/216) may be an important prognostic factor for survival of patients with cholangiocarcinoma.

LncRNA-SNHG1 promotes paclitaxel resistance of gastric cancer cells through modulating the miR-216b-5p-hexokianse 2 axis.

Gastric cancer (GC) is one of the most malignant tumors with high incidence and poor prognosis. Currently, the combination of surgery with chemo- or radiotherapy is widely applied therapeutic strategy against GC. However, development of drug resistance severely limited the clinical application of chemotherapy. Small nucleolar RNA host gene 1 (SNHG1) has been reported to be frequently overexpressed in diverse human tumors. Yet, the biological roles and mechanisms of SNHG1 in chemoresistant GC remain unclear. Expressions of lncRNA and miRNA were detected by qRT-PCR. Responses of GC cells to Taxol treatments were evaluated by cell viability assay and apoptosis assay. Glucose metabolism rate was examined by glucose uptake and extracellular acidification rate (ECAR). The lncRNA-miRNA interaction was validated by RNA pull-down assay and luciferase assays. This study reports that expressions of SNHG1 were significantly elevated in patients with GC and gastric cancer cell lines. Silencing SNHG1 effectively suppressed GC cells migration and increased the Taxol sensitivity of GC cells. Moreover, we detected remarkedly upregulated SNHG1 expression and increased glucose metabolism in Taxol resistant cell line, MKN-45 TXR. Low glucose supply rendered Taxol resistant cells more susceptible to Taxol treatment compared with that from MKN-45 parental cells. Bioinformatical analysis, RNA pull-down and luciferase assays verified that SNHG1 functioned as a ceRNA of miR-216b-5p in GC cells. Consistently, we detected miR-216b-5p was significantly downregulated in GC tumor specimens and Taxol resistant GC cells. The hexokinase 2 (HK2), a glucose metabolism key enzyme, was predicted and validated as a direct target of miR-216b-5p in GC cells. Finally, restoration of miR-216b-5p in SNHG1-overexpressing MKN-45 TXR cells successfully overrode the SNHG1-promoted Taxol resistance through targeting the HK2-glycolysis axis. This study uncovered new biological roles and molecular mechanisms of the lncRNA-SNHG1-mediated Taxol resistance of gastric cancer, suggesting targeting the SNHG1-miR-216b-5p-HK2 axis could be a potentially therapeutic approach against chemoresistant gastric cancer.

An automated proximity proteomics pipeline for subcellular proteome and protein interaction mapping.

Proximity labeling (PL) coupled with mass spectrometry has emerged as a powerful technique to map proximal protein interactions in living cells. Large-scale sample processing for proximity proteomics necessitates a high-throughput workflow to reduce hands-on time and increase quantitative reproducibility. To address this issue, we developed a scalable and automated PL pipeline, including generation and characterization of monoclonal cell lines, automated enrichment of biotinylated proteins in a 96-well format, and optimization of the quantitative mass spectrometry (MS) acquisition method. Combined with data-independent acquisition (DIA) MS, our pipeline outperforms manual enrichment and data-dependent acquisition (DDA) MS regarding reproducibility of protein identification and quantification. We apply the pipeline to map subcellular proteomes for endosomes, late endosomes/lysosomes, the Golgi apparatus, and the plasma membrane. Moreover, using serotonin receptor (5HT2A) as a model, we investigated agonist-induced dynamics in protein-protein interactions. Importantly, the approach presented here is universally applicable for PL proteomics using all biotinylation-based PL enzymes, increasing both throughput and reproducibility of standard protocols.

Long noncoding RNA GAS5 inhibits proliferation and metastasis in papillary thyroid carcinoma through the IFN/STAT1 signaling pathway.

Dysregulation of the long noncoding RNA GAS5 in human cancer has been identified in recent studies. In this study, we confirmed a negative correlation between the GAS5 expression level and papillary thyroid carcinoma clinicopathologic characteristics, such as the tumor size, lymph node metastasis, the TNM stage and BRAFV600E mutation. The viability and metastasis of papillary thyroid carcinoma cells were detected by CCK-8 and transwell assays, respectively. The results showed that upregulation of GAS5 significantly inhibited papillary thyroid cancer cell growth, migration and invasion in vitro. RNA transcriptome sequencing was performed to explore the underlying targets of GAS5. Through qRT-PCR and Western blot analysis, we found that ectopic expression of GAS5 significantly increased IFI44 and STAT1 levels. Taken together, these findings suggest that GAS5 is a tumor suppressor in papillary thyroid carcinoma, and the action of GAS5 may be mediated through the IFNγ/STAT1 signaling pathway.

An Ovarian Large-Cell Neuroendocrine Carcinoma Accompanied by Clear Cell Carcinoma with Specific High Level of AFP: Case Report and Review of the Literature.

Large cell neuroendocrine carcinoma (LCNEC) is a rare histological subtype of ovarian cancer. A few cases have been reported in the literature with extreme invasiveness and a poor prognosis. However, there still have not been accepted criteria for diagnosis and treatment of LCNEC. Here we report an unmarried 37 year-old woman who was diagnosed with LCNEC associated with clear cell carcinoma and the tumor index was manifested with a specific increase of AFP. The case received six courses of etoposide and carboplatin chemotherapy as an adjuvant therapy after primary curative surgery. However, she relapsed within 6 months after surgery and metastasized rapidly to distant organs despite combined chemotherapy of paclitaxel, cisplatin, and bevacizumab, and died 18 months after primary surgery. This is the first reported case of LCNEC manifested with a specific increase of AFP and characteristically metastasized to the spine as recurrence. Reviewing our case as well as previously reported cases, LCNEC present with aggressive malignancy and vulnerable to distant metastasis through a hematogenous approach, we conjectured that adding Bevacizumab in primary chemotherapy may be beneficial to extend disease-free survival. But so far there is no recommendation of this regimen for treatment of LCNEC in current guidelines. Further research is needed to confirm this view so as to find the best treatment of LCNEC and improve the prognosis of these patients.