Supplementary Figure S6. Downregulated phosphorylations after IRAK4 inhibitor treatment.

Supplementary Figure S2. Uncropped Western Blots for Figure 6A data presented in the manuscript. Also see Data Availability Statement.

Supplementary Figure S1. Uncropped Western Blots for Figure 4C data presented in the manuscript. Also see Data Availability Statement.

Supplementary Table S1. Manuscript Figure 1 data - The Excel file has data points for easy access to infer numerical values for data presented in the manuscript. Also see Data Availability Statement.

Supplementary Figure S5. Annotated MS/MS spectrum of the phosphopeptide of MYH9 modified with serine 1943 phosphorylation. Red and blue lines indicate matches between observed and expected fragment masses (red is the b series, while blue is the y series). Fragments annotated as “-Phos” indicate that the phosphorylation fragmented out of the peptide leaving a diagnostic mass of -96 Da.

Supplementary Figure S4. Immunofluorescence using CD68 Cy5 on mouse lung tissues harvested after 18 months of intratracheal treatment. Magenta stain represent Cy5 stain on CD68 antibody and blue represent nuclear counterstain DAPI. Left, representative image for intratracheal PBS administration. Right, representative image for mice administered NB (NNK and BaP).

Supplementary Table S7. Phosphoproteomics results from purified microtubules in cells treated with vehicle vs CA4948 for 72 hours.

Supplementary Figure S3. Exposure to carcinogens leads to carcinogenesis in murine lungs: Representative histological examples of mouse lung carcinogenesis observed during the study to assess the effect of smoking carcinogens.

Abstract Background Alterations in several tripartite motif-containing (TRIM) family proteins have been implicated in the pathogenesis of lung cancer. TRIM28, a member of the TRIM E3 ligase family, has been associated with tumorigenesis, cell proliferation, and inflammation. However, little is known about TRIM28 expression and its role in the immune microenvironment of non-small cell lung cancer (NSCLC). Methods We assessed the clinical significance of TRIM28 in tissue microarrays and TCGA cohorts. We investigated the function of TRIM28 in syngeneic mouse tumor models, the Kras LSL−G12D/+ ; Tp53 fl/fl (KP) mouse model, and humanized mice. Immune cell composition was analyzed using flow cytometry and immunohistochemistry. Results Our findings revealed a positive correlation between TRIM28 expression and the infiltration of suppressive myeloid-derived suppressor cells (MDSCs) in NSCLC. Moreover, silencing TRIM28 enhanced the efficacy of anti-PD-1 immunotherapy by reshaping the inflamed tumor microenvironment. Mechanistically, we demonstrated that TRIM28 could physically interact with receptor-interacting protein kinase 1 (RIPK1) and promote K63-linked ubiquitination of RIPK1, which is crucial for sustaining activation of the NF-κB pathway. Mutagenesis of the E3 ligase domain corroborated the essential role of E3 ligase activity in TRIM28-mediated NF-κB activation. Further experiments revealed that TRIM28 could upregulate the expression of CXCL1 by activating NF-κB signaling. CXCL1 could bind to CXCR2 on MDSCs and promote their migration to the tumor microenvironment. TRIM28 knockdown increased responsiveness to anti-PD-1 therapy in immunocompetent mice, characterized by increased CD8+T tumor-infiltrating lymphocytes and decreased MDSCs. Conclusion The present study identified TRIM28 as a promoter of chemokine-driven recruitment of MDSCs through RIPK1-mediated NF-κB activation, leading to the suppression of infiltrating activated CD8+T cells and the development of anti-PD-1 resistance. Understanding the regulation of MDSC recruitment and function by TRIM28 provides crucial insights into the association between TRIM28 signaling and the development of an immunosuppressive tumor microenvironment. These insights may inform the development of combination therapies to enhance the effectiveness of immune checkpoint blockade therapy in NSCLC.

Recent studies have indicated that some members of the tripartite motif (TRIM) proteins function as important regulators for non-small cell lung cancer (NSCLC), However, the regulatory mechanism underpinning aberrant expression of TRIM in NSCLC remains unclear. Here we report that TRIM15 plays important roles in NSCLC progression through modulating Keap1-Nrf2 signaling pathway. TRIM15 expression was evaluated by western blot analysis, tissue microarray-based immunohistochemistry analysis. The interactions between TRIM15 and Keap1 were analyzed by co-immunoprecipitation (Co-IP) and immunofluorescence co-localization assay. The correlation between TRIM15 and Keap1 was measured by Co-IP and ubiquitination analysis in vitro. Gain- and lost-of-function experiments were used to detect TRIM15 promotes proliferation and invasion of NSCLC cells both in vitro and vivo. Here, we revealed that TRIM15 was frequently upregulated in NSCLC samples and associated with poor prognosis. Functionally, TRIM15 knockdown resulted in decreased cancer cell proliferation and metastasis, whereas ectopic TRIM15 expression facilitated tumor cancer cell proliferation and metastasis in vitro and in vivo. Moreover, TRIM15 promoted cell proliferation and metastasis depends on its E3 ubiquitin ligase. Mechanistically, TRIM15 directly targeted Keap1 by ubiquitination and degradation, the principal regulator of Nrf2 degradation, leading to Nrf2 escaping from Keap1-mediated degradation, subsequently promoting antioxidant response and tumor progression. Therefore, our study characterizes the pivotal roles of TRIM15 promotes NSCLC progression via Nrf2 stability mediated by promoting Keap1 ubiquitination and degradation and could be a valuable prognostic biomarker and a potential therapeutic target in NSCLC.

BACKGROUND:Exosomes are emerging as important mediators of the cross-talk between tumor cells and the microenvironment. The communication between tumor-derived exosomes and macrophages has a critical role in facilitating tumor progression. However, the mechanisms by which exosomes modulate tumor development in lung cancer are not fully understood.METHODS:Short hairpin RNA mediated knockdown or exogenous expression of TRIM59 combined with in vitro and in vivo assays were performed to prove the functional significance of TRIM59. Western blotting, real-time PCR, co-immunoprecipitation, immunofluorescence (IF) staining assays, proximity ligation assay (PLA), ubiquitination assays, lactate secretion and lipid droplets content measurement, and rescue experiments were used to evaluate the mechanism. Lewis lung carcinoma (LLC) cells were injected via subcutaneously or tail vein into C57BL/6 wild-type (WT) and transgenic mice to assess the role of TRIM59 in vivo.RESULTS:We demonstrated that tripartite motif-containing 59 (TRIM59) was expressed in lung cancer cells-derived exosomes, and can be transferred to macrophages through the exosomes. Activated macrophages by TRIM59 promote lung cancer progression in vitro and in vivo. Mechanistic investigations revealed that TRIM59 physically interacts with abhydrolase domain containing 5 (ABHD5) and directly induced the ubiquitination of ABHD5 and led to its proteasome-dependent degradation. ABHD5, an lipolytic co-activator, deficiency induced metabolic reprogramming and enabled NLRP3 inflammasome activation in macrophages. Further studies showed that the exacerbation of NLRP3 inflammasome activation by ABHD5 deficiency, provides a positive feedback loop to promote cancer progression by preferentially secrete the proinflammatory cytokine IL-1β.CONCLUSIONS:Collectively, these data indicate that tumor-derived exosomal TRIM59 converts macrophages to tumor-promoting functions of macrophages via regulating ABHD5 proteasomal degradation, to activate NLRP3 inflammasome signaling pathway to promote lung cancer progression by IL-1β secretion. Our findings also indicate that tumor-derived exosomal TRIM59 has an important role in intercellular communication for fostering an inflammatory microenvironment and promoting lung metastasis.

Objective: To investigate the relationship between single nucleotide polymorphisms of pulmonary surfactant protein B gene and respiratory distress syndrome in premature infants. Methods: Ninety-two preterm infants with neonatal respiratory distress syndrome (NRDS) were selected as the NRDS group. Another 92 cases without respiratory distress syndrome were selected as the non-NRDS group. The single nucleotide polymorphisms of SPB-18 A/C locus and SPB-1580 C/T locus were detected in the groups, and the genotype distributions of the SPB-18 A/C locus and the SPB-1580 C/T locus were compared between the two groups. The relative risk of both sites and the onset of respiratory distress syndrome were analyzed using an chi(2) test. Results: In the SPB-18 A/C locus genotype test, AA, CC, and AC in the NRDS group were 20.65%, 35.87%, and 43.47%, respectively, which were not significantly different from 19.57%, 32.61%, and 47.83% in the non-NRDS group (P = 0.854, P = 0.641 and P = 0.554). In the SPB-1580 C/T locus genotype test, CC in the NRDS group accounted for 66.30%, which was higher than the corresponding value in the non-NRDS group (40.22%, P = 0.000). T in the NRDS group accounted for 5.43%, and CT accounted for 28.26%, which were lower than the corresponding values in the non-NRDS group (14.13%, P = 0.047; 45.65%, P = 0.015). SPB-18 A/C was not associated with NRDS in preterm infants (P = 0.833), whereas the SPB-1580 C/T site was associated with respiratory distress syndrome in preterm infants (P = 0.000). Respiratory distress syndrome in preterm delivery of C allele with SPB-1580 locus was 2.410 times higher than it was in preterm infants with a confidence interval of (1.504, 3.862). Conclusion: The SPB-1580 A/C polymorphism is associated with respiratory distress syndrome in preterm infants. Carrying the C allele of SPB-1580 may be a risk factor for respiratory distress syndrome in preterm infants.

Lung cancer ( LC ) is a devastating malignancy with no effective treatments, due to its complex genomic profile. Using bioinformatics analysis and immunohistochemical of lung carcinoma tissues, we show that TRIM 59 as a critical oncoprotein relating to LC proliferation and metastasis. In this study, high TRIM 59 expression was significantly correlated with lymph node metastasis, distant metastasis, and tumour stage. Furthermore, up‐regulation of TRIM 59 expression correlated with poorer outcomes in LC patients. Mechanistically, TRIM 59 play a key role in promoting LC growth and metastasis through regulation of extracellular‐signal regulated protein kinase (ERK) signalling pathway and epithelial‐to‐mesenchymal transition ( EMT )‐markers, as validated by loss‐of‐function studies. In‐depth bioinformatics analysis showed that there is preliminary evidence of co‐expression of TRIM 59 and cyclin dependent kinase 6 ( CDK 6) in LC . Notably, CDK 6 expression significantly decreased when TRIM 59 was knocked down in the LC cells. In contrast, exogenous up‐regulation of TRIM 59 expression also induced significant increases in the expression of CDK 6. Moreover, the expression of CDK 6 was also inhibited by the ERK signalling inhibitor, U0126. The results of both loss‐ and gain‐of‐function studies showed that TRIM 59 could regulate the expression of CDK 6. Collectively, these data provide evidence that TRIM 59 is involved in lung carcinoma growth and progression possibly through the induction of CDK 6 expression and EMT process by activation of ERK pathway.