Lower Respiratory Tract Microbiome-oriented Metabolites Modulate The Aryl Hydrocarbon Receptor De-SUMOylation Modification To Inhibit The Development Of Non-small Cell Lung Cancer

BackgroundThe relationships between microorganisms and host cells,especially their roles in regulating tumor biological behaviors,have always been hot topics in the field of cancer research.In a healthy state,the microbial community and host form a complex"organism",in which microorganisms interact with host cells in this symbiotic relationship.When the regulatory mechanism of the host controlling the steady-state of the microbiota is disrupted,or the composition of the microbiota changes due to environmental factors,it may break this symbiotic relationship and promote the occurrence and development of tumors.The lungs are in an open state and undergo dynamic gas exchange with the external environment,corresponding to changes in their local microbial community.Due to close contact with lung tissue,imbalanced colonization of the lower respiratory tract microbiota may be one of the driving factors for the development of NSCLC,and adjusting the composition of the lower respiratory tract microbiota may be an effective tumor prevention measure.Research has shown that there are colonized bacteria in the lower respiratory tract,and the imbalance and metabolic differences of the bacterial community in this area are inherently related to the development of lung cancer.Although research has confirmed the potential role of gut microbiota and their metabolites in regulating the occurrence,development,and efficacy of lung cancer through the lung gut axis and other means,there is still room for exploration in the biological functions of lower respiratory tract colonizing bacteria and their metabolites in leading to lung cancer.Short chain fatty acids（SCFAs）,also known as volatile fatty acids,are organic fatty acids with a carbon chain length of less than 6 carbon atoms produced by anaerobic bacteria during the decomposition of energy substances.It is generally believed that the gut microbiota is the main source of SCFAs in the blood,and the produced SCFAs can participate in disease regulation and exert anti-tumor effects through various organ target axes through blood transport.SCFAs have a strong ability to inhibit tumor growth,but there are few literature reports on the inhibitory effect of SCFAs produced by lower respiratory tract microbiota on local tumor progression.Due to being small molecule organic acids,SCFAs often act as exogenous ligands and enter the cell through cell membrane transporters to participate in various protein post-translational modification processes,including acetylation and SUMOylation.SUMOylation,as an important type of protein post-translational modification,is widely involved in various intracellular signal communication such as protein stability and spatial signal transduction.However,there is currently no research to confirm the role and specific mechanism of SCFAs produced by these microorganisms in regulating host cell SUMOylation modification.Aryl hydrocarbon receptor is a ligand-dependent intracellular receptor,which has been regarded as an important transcription factor in initiating downstream DNA transcription and activating metabolic enzymes in previous studies.In recent years,literature has also confirmed that it can still act as a protease to regulate various biological processes in cells under ligand free activation conditions,especially as a central receptor to maintain the homeostasis of the host microecological environment.Microbial metabolites,as potential regulators of Ah R in host cells,participate in homeostasis regulation.However,there is still vast exploration space for the key roles and potential mechanisms of these microorganisms and their metabolites in activating or inhibiting Ah R signaling in host cell biological functions.Aims1)Describe the characteristics of lower respiratory tract microbiota and short chain fatty acids in lung cancer patients,distinguish the differences in bacterial composition and short chain fatty acid content between normal lung segments and tumor loaded lung segments;2)Screen key differential bacterial genera and short chain fatty acids,and identify key bacterial genera closely related to the production of SCFAs;3)Explore the function of key bacterial genera releasing protective metabolites such as SCFAs to inhibit the development of non-small cell lung cancer;4)From the perspective of post translational modifications of host cell proteins mediated by microbial metabolites,this study preliminarily explores the potential mechanism of SCFAs mediated SUMOylation of Ah R to inhibit tumor cell proliferation,providing a theoretical basis for tumor prevention interventions targeting the lower respiratory tract microbiota and its metabolites.Methods1)Continuously enrolled patients with advanced NSCLC lung cancer in our center,and collected alveolar lavage fluid from normal lung segments and tumor loaded lung segments of the enrolled patients through electronic bronchoscopy examination;Analyze the characteristics of microorganisms and metabolites in the two groups of samples through metagenomic sequencing and targeted metabolomics sequencing;Using multi omics combined analysis and machine learning,identify the differentially expressed bacterial genera and SCFA closely related to the development of lung cancer,and establish an intrinsic relationship between the both.2)Establish correlation analysis based on differential bacterial genera and metabolites,and preliminarily identify differential bacterial genera closely related to key SCFA;Cultivate the key bacterial genera mentioned above in vitro to determine their survival status and suitable conditions in the human environment;The supernatant of the above-mentioned bacterial genera was cultured in vitro with human lung cancer cell lines,and the inhibitory effect of SCFA produced by the aforementioned bacterial genera on regulating tumor cell proliferation was clarified through plate cloning experiments.3)Apply differential SCFA to treat lung cancer cell lines and detect cell proliferation ability and SUMOylation modification;Construct Ah R upregulation and downregulation expression vectors to clarify the potential impact of differential SCFA on Ah R signaling;Preliminary exploration of the mechanisms of SUMOylation and de-SUMOylation of Ah R proteins using molecular biology methods,and detection of the regulatory effect of SCFA on the SUMO modification of Ah R molecules under different treatment conditions.Results1)Analyzing paired alveolar lavage fluid samples from 28 advanced NSCLC patients,we found that there was no significant statistical difference in the relative abundance between both groups.But in diversity analysis,it was found that the microorganisms in the normal lung segmentαdiversity(^**P<0.001);Compared to the normal lung segment,the microbiota in the tumor loaded lung segment ofβdiversity is relatively concentrated(Stress=0.1311;ADONIS,^**P=0.001;ANOSIM,^**P<0.001);2)The tumor loaded lung segment is mainly enriched with genera such as Campylobacter,Enterobacterium,Deinococcus,and Clostridium,while the normal lung segment is mainly enriched with genera such as Bacillus;Within the lung segment of tumor burden,C.jejuni is positively correlated with other differential bacterial genera,while negatively correlated with Bacillus genus;3)After detecting SCFAs in the above samples,it was found that the content of short chain fatty acids in both groups of samples remained at a relatively low level,with only slight differences in acetic acid between the two groups of samples;Using random forest and LASSO analysis,it was found that after constructing a joint prediction model for three types of SCFAs:CA,VA,and IBA,the diagnostic efficiency can reach over 90%,and the AUC under the ROC curve reaches 0.993;4)Bray-Curtis analysis on KEGG pathway abundance suggests that the microbiome in normal lung segments and tumor loaded lung segments may lead to different cellular functional states(ANOSIM,^**P<0.01),including pathways such as signal transduction,membrane transport proteins,vitamin metabolism,and carbohydrate metabolism;5)In the correlation analysis between the different bacterial genera in the two samples and acetic acid,it was found that there was a significant difference in the linear correlation between Paenibacillus odorifer and acetic acid（r=0.303,^*P=0.02598）;P.Odorifer,as a colonizing bacterium,is widely present in various parts of the human body and is at a low load level.Under disease conditions,the biological load of this bacterium genus decreases;In the lung cancer dataset,the relative abundance of P.Odorifer significantly decreased compared to the normal lung content;6)In vitro,model strain was found to form spindle shaped spores with swollen and protruding ends,deepening staining,and under optimal cultivation conditions;30℃as the optimal growth temperature can maintain the strain’s long-term stable activation state;At37℃,the strain showed a growth peak after 24h and was gradually inhibited during the subsequent cultivation time;7)The strain showed a peak content in AA at 24h in the 30℃group,and a peak in AA content at 48 hours in the 37℃group;As the AA content increases,cell proliferation and plate clone formation ability were gradually inhibited,while changes in AA content have no significant effect on normal bronchial epithelial cells;8)Analysis of NSCLC tissues using 4D SUMOylation modified proteomics according to the relative abundance of P.Odorifer,we found that tissues with lower relative abundance of this strain differ from the other,and these differences in SUMOylation substrates are mainly related to proteins on proliferation pathways;9)In the H460 and H1975 cell lines with high expression of Ah R,significant endogenous interactions between Ah R and SUMO1 molecules were found through immunoprecipitation experiments;The highly selective SUMO E2 inhibitor TAK-981 can significantly inhibit SUMO modification activity;Under the involvement of exogenous ligands of Ah R,the SUMOylation modification level of Ah R is inhibited by TAK-981;MG132 can effectively improve the SUMO modification level of Ah R;10)After constructing UBC9 and SENP1 downregulated expression vectors,we found that sh UBC9 cells could effectively block the SUMO1 modification of Ah R,but the SUMO modification of Ah R was preserved in sh SENP1 cells;Nuclear plasma separation and indirect immunofluorescence experiments indicate that ligand activated Ah R mainly undergoes SUMO1 modification and is mainly concentrated in the nucleus;11)The three highest levels of SCFAs（acetic acid,propionic acid,and butyric acid）can all affect the SUMO modification level of host cells;Acetic acid mediated SUMO modification has a non-linear dose-dependent effect,exhibiting SUMO inhibition at moderate concentrations;In cells with stable downregulation of UBC9 and SENP1,it was found that inhibition of SUMO modification weakened the ability of host plate clone formation after acetic acid gradient treatment;12)SUMO modification omics and structural prediction both revealed the presence of SUMO modification in the 510th lysine position of the Ah R amino acid sequence;The overall SUMO1 modification level of Ah R significantly decreased after K510 mutation.Meanwhile,as the UBC9 gradient increased,the K510 site did not affect the SUMO1modification level of Ah R;After the mutation at the Ah R K510,the protein stability of this molecule was significantly improved;Under the intervention of proteasome inhibitors MG132 and CHX,the stability of the K510 mutation was improved.Conclusion1)There is a significant difference in the microbial composition between the normal lung segment and the tumor loaded lung segment of the same patient;The acetic acid content is highest and enriched in normal lung segments.There is no significant difference in the overall SCFA content between the two groups of samples,and joint analysis has tumor predictive value;There is a significant correlation between P.odorifer and acetic acid,and the former may be an important microbial source of acetic acid in the lower respiratory tract.2)P.odorifer is widely distributed in the human body and mainly colonizes the lower respiratory tract of normal individuals,serving as an important biological source of acetic acid;Forming spindle shaped spores at suitable human temperatures and maintaining biological activity,and continuously and stably existing at low levels within normal lung segments to maintain local microecological homeostasis;3)Active P.Odorifer inhibits the proliferation of lung cancer cell lines in vitro and mediates changes in the level of Ah R SUMO modification,which is mainly regulated by SENP1 mediated de SUMO modification;The Ah R K510 site may be an important SUMO modification site and affect the nuclear transcriptional regulation and stability of Ah R;The acetic acid produced by P.odorifer enhances the SUMOylation modification of Ah R and inhibits its tumor suppressive effect.