Exo70 is a direct interacts wit LAMTOR1
To investigate how LAMTOR1 exocytosis,co-expression in cells was carried out(Figure.1a).Candidate LAMTOR1 interacting proteins co-precipitated with Sec13, Rab11 and Exo70.Therefore,we investigated the role of exocytosis regulated by LAMTOR1, we investigated the role of LAMTOR1 protein-protein interaction, which are critically involved in endogenous LAMTOR1-Sec13 and LAMTOR1-Rab11 interaction(Figure.1b,c).We sought to determine whether LAMTOR1 interacts with Exo70 to promote endocytosis,which a subunit of the exocyst complex.Using endogenous proteins in different cell lines by IP assays, we show Exo70 can interact directly with LAMTOR1(Fig.1d), and then with tagged proteins exogenously expressed in cell lines(Fig.1e,f).Co-localization of LAMTOR1 with Exo70 critical for membrane trafficking was observed by immunofluorescence microscopy in HCC patients(Fig.1g).
LAMTOR1 phosphorylate Exo70 at Serine 133
To determine whether Exo70 is due to LAMTOR1 directly interaction, we constructed GST-Exo70, suggesting that LAMTOR1 activity is required for LAMTOR1 interaction(Fig.2a).For more detailed mapping of the region of Exo70, three domains were expressed(Fig.2b).Binding of these constructs to LAMTOR1 was assessed using in vitro GST pull down.Deletion of part of construct 2 or 3 domain abrogated LAMTOR1 binding(Fig.2c).We next investigated whether LAMTOR1 phosphorylates Exo70.Using an in vitro kinase assay, we found that Exo70 is a sub-strate for LAMTOR1 phosphorylation(Fig.2d).To determine whether LAMTOR1 directly phosphorylates Exo70, we performed in vitro assays using both recombinant LAMTOR1 and a purified Exo70(Fig.2e).Together,these data indicating that Exo70(1-140 amino acid) is a alternative phosphorylation sites of LAMTOR1.Through in vitro assays, we found that LAMTOR1 is a substrate for Exo70 phosphorylation, and complete loss of phosphorylation in the S133A mutant resulted in inability to bind to LAMTOR1, suggesting a phosphorylation site leading to exocytosis(Fig.2f). Additionally, using recombinant LAMTOR1 and purified Exo70, suggesting that Ser133 was indeed crucial for LAMTOR1 to phosphorylate Exo70(Fig.2g), suggesting that LAMTOR1 activity is required for Exo70 interaction and directly phosphorylate Exo70 to regulate exocytosis.
Exo70 phosphorylation promotes its interaction with Rab11
How does LAMTOR1 phosphorylation of Exo70 affect exocytosis? Since the assembly of the exocyst complex is regulated from Golgi processing and linked to the plasma membrane via associated vesicle trafficking,we investigated whether phosphorylation of Exo70 by LAMTOR1 regulates the interaction of Sec13 as well as Rab11 to mediate vesicle fusion. Flag-tagged Exo70 or Exo70(S133A) were transduced in 293T cells, Exo70 was immediately immunoprecipitated with anti-Flag antibodies against Sec13 and Rab11, which have previously been indicated to interact directly in the exocyst complex.To further identify the impact on binding, we performed mutual immunoprecipitation experiments. Flag-tagged Rab11 were co-transfected with Myc-tagged Exo70 or Exo70(S133A), Rab11-Flag was immunoprecipitated with an anti-Flag antibody, and Myc-Exo70 and Sec13 were detected by western blotting. As shown in Figure.3a,b, Exo70 binding to Sec13 and Rab11.The binding of the Exo70(S113A) mutant to Sec13 and Rab11 was reduced.
To simulate the interaction between Exo70 and Rab11 at the endogenous level in cells under stress, we treated performed immunoprecipitation experiments using a monoclonal antibody against endogenous Exo70 treat with HBSS in HCC.The amount of Rab11 that co-immunoprecipitated with Exo70 increased upon HBSS stimulation(Figure 3c).As shown in results, HCC tissues expressed a punctate cytoplasmic distribution of Rab11 that co-localized with LAMTOR1(Figure.3d) and Exo70(Figure.3e).Taken together, these data indicate that Exo70 enhances its binding to Sec3 and Rab11.
LAMTOR1 enhances release the TGFB1 by interaction with Exo70
Whether exocyst influenced the cytokine effect of driving immune cells,we used Cytokine Array combined to ELISA assay to test Ctrl vs LAMTOR1 Knockdown.We identified TGFB1 and CCL5 as the major cytokine secreted in Control as compared to LAMTOR1 Knockdown(Fig.4a).These results show that LAMTOR1 expression is associated with a robust anti-inflammatory environment.We next used RNA-seq to analyze differential gene expression in HCC infiltrated by LAMTOR1 positive or negative.Biological pathway analysis performed in “Enrich R” revealed several regulated inflammatory immune response clusters and cytokine activation(Fig.4b,c).Using flow cytometry,we found that the liver cancer cell lines produced TGFB1 and CCL5(Fig.4d).It is important that cell supernatant of TGFB1 produced by tumor cells correlated with the tumor expression of LAMTOR1(Fig.4e).In HCC,we found LAMTOR1, EXO70, RAB11A and TGFB1 was a trend toward higher co-expression in the tumor nests(Fig.4f).
Next,we examined whether the effect of immunity activity on Exo70 phosphorylation by LAMTOR1.Stable Hepa1-6 cell lines expressing either Exo70 or the Exo70(S133A) mutant were generated.Treatment with the Exo70(S133A) mutant showed decreased the co-localization TGFB1 with RAB11, Exo70 in mouse HCC(Fig.4g).We also examined whether the secretion of TGFB1 in tumor cells was affected by the Exo70(S133A) mutant. As shown in Figure 4H, knockdown of Exo70 significantly reduced the level of TGFB1, while cells expressing Exo70(S133A) similarly showed reduced levels of TGFB1 secretion. These data suggest that tumor cells secrete TGFB1 via Exo70.
LAMTOR1 promotes TAMs polarization by the exocytosis release of TGFB1
Given the immune-suppressive profile of HCC, we next assessed their capability to suppress immune effector cells.Multiplex immunofluorescent staining and multispectral imaging was to identify CD4+T cells, CD8+T cells, NK cells, and TAMs.Patients with high infiltration of CD4+T cells, CD8+T cells, NK cells in adjacent and significantly higher infiltration of TAMs cells in the tumor(Fig.5a).To investigate whether TGFB1 could be a potential cytokine for the TAMs polarization,we stained tissues expressing with antibodies against CD68 and CD206.We observed that TAMs polarization accumulated on the tumor(Fig.5b).Likewise,co-staining of CD206 with antibodies targeting TGFB1 showed co-localization of CD206 with TGFB1(Fig.5c).LAMTOR1 knockdown cells block the macrophages induction of CD206 expression(Fig.5d).In in vitro experiments we found that TGFB1 significantly reduced the expression of CD163, and CD86, and simultaneously increased the expression of CD206 (Figure 5e). These data suggest that liver tumor cells produce TGFB1 to drive macrophage polarization through the expression of LAMTOR1.
LAMTOR1 co-culture macrophages inhibit T-cell activation and tumor killing
Next,we used RT-PCR assay to test tumor-conditioned macrophages(TCM) treat with Ctrl and LAMTOR1 knockdown HCC cells(Fig.6a).And then,Purified T cells from peripheral blood were co-cultured with TCM and assessed for their capacity to produce IFNgamma and proliferate.We found that TCM cytokines(IL-10 and IFNgamma) suppressed T cell activation.Also, the suppressed T-cell activation correlated with polarization of TCM(Fig.6b,c).Similarly, proliferation and IFNgamma production were suppressed in T cells co-cultured with TCM(Fig.6d,e).This shows that LAMTOR1 expression can be used as a measure of the suppressive capacity of macrophages.We also found that the T cells that were cultured with TCM were less efficient in killing(Fig.6f).Thus,LAMTOR1 expressing macrophages actively block T-cell cytotoxicity and inhibit both cytokine production and proliferation.
LAMTOR1 enhanced release of TGFB1, IL-10 in the tumor microenvironment and prevented the infiltration of CD4+ and CD8+ effector cells
Our results described above strongly argue that therapeutic strategies preventing LAMTOR1 would improve cancer immunotherapies by inducing the infiltration of immune effectors.We first showed that AAV-shLAMTOR1 treatment of HCC cells,under these experimental conditions in vitro, we observe changes in the expression of LAMTOR1,EXCO70,RAB11,TGFB1 and CD206(Fig.7a),treatment of tumor bearing mice with AAV-sh-LAMTOR1 significantly increased the infiltration of CD4+T and CD8+T cells associated with a decrease in the infiltration of TAMs(CD206) cells as well as Treg cells(Fig.7b,c).Interestingly, AAV combination therapy(AAV-sh-LAMTOR1+anti-TGFB1+anti-IL-10) completely inhibited tumor growth compared to either the combination therapy or control(Fig.7d),indicating that LAMTOR1 can improve cancer immunotherapy approaches based on immune cytokines blockade and peptide-based vaccination strategies.Our data are in line with several studies highlighted the relevance of targeting LAMTOR1 associated pathways in tumors to enhance immunotherapy efficacy(Fig.8).