The Roles And Mechanisms Of CUL4B In KRAS-driven Tumor Microenvironment Reprogramming In Lung Adenocarcinoma

Lung cancer is the most common malignant tumor with the highest incidence and mortality rate in China.Lung cancer can be divided into non-small cell lung cancer(NSCLC,about 85%)and small cell lung cancer(SCLC,about 15%).Lung adenocarcinoma(ADC)is the most common subtype of NSCLC.One-third of ADC harbored KRAS mutations.Despite the targeted therapies and immune checkpoint inhibitors having improved the prognosis of patients with advanced lung cancer,few drugs targeting KRAS have been utilized therapeutically.Cullin-4B(CUL4B)is a scaffold protein in the Cullin-Ring E3 ubiquitin ligase complex,which is involved in the regulation of various growth and development processes.Previous studies showed that the role of CUL4B in cancer is dichotomous.On the one hand,it is overexpressed in many solid tumors and is crucial for maintaining the malignant phenotype of tumor cells.On the other hand,it limits the accumulation and activation of myeloid-derived suppressive cells(MDSCs),and acts as a tumor suppressor in the tumor microenvironment.We previously constructed the ADC model driven by KRASG12D and urethane(which primarily induces tumor in the lung with a single driver mutation of KRASQ61L/R),and found that specific knockout of Cul4b in alveolar type Ⅱ epithelial cells promoted the growth of lung ADC in both KRASG12D and urethane-induced models,suggesting the tumor-suppressive role of CUL4B in Kras-driven ADCs.However,its mechanisms have not been characterized.The purpose of this study is to explore the mechanism of CUL4B suppressing tumorigenesis in Kras-driven ADC.Part Ⅰ CUL4B suppresses KRAS-driven lung adenocarcinoma growth by reprogramming the tumor microenvironmentTo explore the role of CUL4B in the tumorigenesis of KRAS-driven lung adenocarcinoma,we used the public database,KRASG12D-driven and urethane-induced lung ADC mouse models,and obtained the following results:1.Analysis of the human ADC samples from the public database showed that low expression of CUL4B predicted poor prognosis of lung ADC patients with smoking history,and the expression of CUL4B in tumors of lung cancer and lung ADC patients with KRAS mutation was significantly lower than that in patients without KRAS mutation.These observations imply that downregulation of CUL4B might cooperate with KRAS mutations to contribute to ADC development.2.To determine the effect of Cul4b loss on Kras-driven ADC,Cul4b gene in alveolar typeⅡ epithelial cells was deleted in Kras-driven lung ADC mouse model.We found that Cul4b loss in lung epithelial promoted Kras-driven ADC development.Spc-Cre;KrasG12D;Cul4f/f(SKC)mice exhibited more tumor lesions,higher tumor burden and shorter lifespan than SpcCre;KrasG12D(SK)mice.3.Immunohistochemical staining and flow cytometry assays showed that knockout of Cul4b in alveolar type Ⅱ epithelial cells reprogrammed the tumor microenvironment of KRASG12D-driven and urethane-induced lung adenocarcinoma,with significant decreased CD4+and CD8+T cells,and increased MDSCs and angiogenesis.However,knockout of Cul4b in alveolar type Ⅱ epithelial cells did not affect lung development and the microenvironment of tumor-free mice.Part Ⅱ Accelerated KRAS-driven tumor growth led by CUL4B depletion is mediated by MDSCsGiven knockout of CUL4B leads to increased MDSCs accumulation in the KRAS-driven tumor microenvironment,mouse Lewis Lung(adeno)Carcinoma(LLC,harboring KRASG12C)cell line was employed for tumor graft model,and did the following analyses:1.Cck-8 assays showed that there was no significant difference in proliferation between Cul4b knockdown cells(shCUL4B)and control(NC)LLC cells in vitro.LLC tumor graft assays in athymic nude mice showed that knockdown of CUL4B did not affect tumor growth.Correspondingly,there was no significant difference in the percentage of Ki67 positive cells between these two groups.However,the number of Gr-1 positive cells was significantly elevated in shCUL4B group,compared with NC group.2.Tumor graft assay in immunocompetent C57 mice showed that the tumors formed by shCUL4B LLC cells were significantly larger than those formed by NC LLC cells.Flow cytometry analysis showed that the percentage of CD4+and CD8+T cells significantly reduced while the percentage of MDSCs significantly increased in shCUL4B tumor grafts,compared with NC tumor grafts.3.Rescue experiment by Gr-1 neutralizing antibody and gemcitabine revealed that depletion of MDSCs effectively blocked the accelerated tumor grafts growth caused by Cul4b knockdown in C57 mice,and significantly reduced the percentage of MDSCs and increased the percentage of CD4+and CD8+T cells in shCUL4B tumor grafts.Part Ⅲ CRL4B/HDAC complexes epigenetically repress Cxcl2 transcription and restrict the recruitment of MDSCsTo clarify the mechanism that Cul4b depletion in tumor cells promoted MDSCs infiltration,we did the following analyses:1.To test the ability of tumor tissues to recruit MDSCs,PKH26-labeled bone marrow cells were injected into KrasG12D mice through tail vein.Flow cytometry assays detected more PKH26-MDSCs in the lung of Cul4b-deficient KrasG12D mice(SKC),compared with control KrasG12D mice(SK).Chemotaxis assays in vitro revealed that the conditioned medium from Cul4b knockdown LLC cells recruited more MDSCs than that from control LLC cells.2.RNA-seq analysis showed that Cul4b-deletion in KrasG12D mouse lung epithelial cells caused upregulated expression of cytokine/chemokines,such as Il1β,Ccl3,Cxcl1,Cxcl2,Cxcl3,Cxcl5,Ccl4,Csf3,Ccl2 as well as Il6,which were confirmed in Cul4b knockdown LLC cells.3.Protein microarray assays revealed enhanced CXCL2 expression in the conditioned medium from LLC shCUL4B cells.ELISA and Western blot assays confirmed increased CXCL2 expression in Cul4b-deficient LLC cells and KrasG12D lung epithelial cells,as well as the bronchoalveolar lavage fluid(BALF)of SKC mice.However,no significantly increased CXCL2 expression was detected in BALF from Spc-Cre;Cul4bf/f mice without tumor.4.To determine whether increased MDSCs accumulation was mediated by increased CXCL2 expression,we did rescue experiment with CXCR2 antagonist SB-265610 and found that inhibition of CXCR2 could effectively block accelerated tumor growth caused by Cul4b knockdown.Flow cytometry analysis showed that SB-265610 treatment significantly reduced the percentage of MDSCs and increase the percentage of CD4+and CD8+T cells in tumor grafts formed by Cul4b knockdown LLC cells.5.ChIP assays showed that CUL4B could directly bind to the region at-485 to-295 bp upstream of the Cxcl2 transcription start site.Further analysis indicated that CRL4B complex,coordinated with HD AC complexes,promoted H2AK119 monoubiquitination and histone H3 deacetylation,and thus repressed Cxcl2 transcription.Conclusions:1.CUL4B-deficient tumor cells reprogramed the tumor microenvironment in KRASdriven lung adenocarcinoma,presenting as increased MDSCs,decreased CD4+and CD8+T cells,and increased angiogenesis.2.Accelerated KRAS-driven tumor growth led by lack of CUL4B was mediated by enhanced MDSCs infiltration.3.CRL4B complex coordinated with HDAC complexes,and promoted H2AK119 monoubiquitination and histone deacetylation,thus repressing the transcription of Cxcl2.Cul4b depletion in Kras-driven lung adenocarcinoma led to enhanced CXCL2 production and secretion and thus increased accumulation of MDSCs and tumor growth.