Low expression of BCL6 in GC indicates more malignant clinical features and a worse prognosis
We analyzed the mRNA expression levels of BCL6 in 36 pairs of GC tissues and adjacent non-tumor tissues using qPCR and found that the mRNA level of BCL6 in 30 cancer tissues was lower than that in the matched para-carcinoma tissues (N = 36, P < 0.001, Fig. 1A). Subsequently, we examined BCL6 protein using immunohistochemistry (IHC), which revealed that BCL6 was enriched in the cytoplasm and nucleus of gastric mucosal cells, especially in the nucleus (Fig. 1B). As shown in Fig. 1C, the immunohistochemical score showed that the level of the BCL6 protein in GC tissues was significantly lower than that in the corresponding adjacent non-tumor tissues (N = 137, P < 0.001, Fig. 1C).
The patients were then divided into low and high groups according to the median histochemical BCL6 score. The clinicopathological features of the two groups are summarized in Supplementary Table S1. Univariate analysis showed that compared with the high expression group of BCL6, the low expression group of BCL6 exhibited advanced pN stage (P = 0.018), increased lymph node (LN) metastasis (9.05 ± 8.94 vs. 7.38 ± 11.73, P = 0.011; Fig. 1D), and long tumor diameter (7.04 ± 3.47 vs. 5.88 ± 3.19, P = 0.016; Fig. 1E). To determine the effect of BCL6 expression on the prognosis of patients with GC, we performed a Kaplan–Meier analysis. As shown in Fig. 1F, the 5-year survival rate of GC patients with low BCL6 expression was lower than that of GC patients with high BCL6 expression (P < 0.001). Multivariate Cox regression analysis showed that BCL6 was an independent predictor of prognosis (HR 0.582, 95% CI, 0.378–0.897; P = 0.014 ) (Table 1). We also evaluated the effects of tumor size and LN metastasis on survival outcomes by stratifying the maximum tumor diameter and pN stage. Low BCL6 expression predicted poor survival in patients with TMD < 5 cm (P = 0.015, Fig. 1G) and pN0 stage (P = 0.006, Fig. 1H).
Table 1
Log-ranktest and multivariate Cox proportional hazard models for overall survival of gastric cancer patients
Characteristics | Case | 5-YSR(%) | P-value† | Hazard ratio(95%CI) | P-value‡ |
Gender | | | 0.142 | | |
Male | 103 | 35.51 | | | |
Female | 40 | 32.45 | | | |
Age(year) | | | 0.091 | | |
<65 | 90 | 37.18 | | | |
≥ 65 | 53 | 31.09 | | | |
pT stage | | | 0.940 | | |
pT2 | 10 | 33.98 | | | |
pT3 | 9 | 34.56 | | | |
pT4 | 124 | 32.19 | | | |
pN stage | | | 0.002 | 1.371(1.097–1.682) | 0.030 |
pN0 | 34 | 50.73 | | | |
pN1 | 15 | 36.14 | | | |
pN2 | 34 | 31.82 | | | |
pN3 | 60 | 28.65 | | | |
Tumor location | | | 0.145 | | |
Upper third | 19 | 38.74 | | | |
Middle third | 14 | 40.13 | | | |
Lower third | 63 | 37.52 | | | |
More than 2/3 stoma | 28 | 35.26 | | | |
Tumor size(cm) | | | 0.065 | | |
< 5 | 49 | 38.13 | | | |
≥ 5 | 94 | 33.62 | | | |
Lauren type | | | 0.733 | | |
Intestinal | 34 | 33.05 | | | |
Diffuse | 105 | 31.78 | | | |
Mixed | 3 | 32.13 | | | |
Bormann type | | | 0.275 | | |
Ⅰ | 3 | 40.37 | | | |
Ⅱ | 24 | 38.51 | | | |
Ⅲ | 67 | 37.62 | | | |
Ⅳ | 10 | 35.89 | | | |
BCL6 expression* | | | <0.001 | 0.582(0.378–0.897) | 0.014 |
Low | 93 | 27.37 | | | |
High | 50 | 38.93 | | | |
Values in parentheses are 95 percent confidence intervals. |
*Determined by immunohistochemical staining. |
†Log-rank test. |
‡Cox proportional hazards model. |
In conclusion, these results suggest that a decrease of BCL6 expression may promote GC progression. Furthermore, low BCL6 expression in GC indicates larger tumor size, more LNs, and poor prognosis.
BCL6 impairs malignant phenotypes of GC cells in vitro
To study the role of BCL6 in GC cells, we restored the expression of BCL6 in AGS and SGC-7901 cells, and the increased expression was confirmed using western blotting (Fig. 2A). We found that BCL6 inhibited the proliferation and viability of AGS and SGC-7901 cells, as shown by the CCK8 analysis (Fig. 2B). Similarly, BCL6 inhibited the clonogenic ability of both the GC cell lines (Fig. 2C). As mentioned earlier, low BCL6 expression in GC was positively correlated with LN metastasis. Therefore, we investigated the effects of BCL6 on the migration and invasion of AGS and SGC-7901 cells. The scratch assay showed that BCL6 significantly shortened the migration distance in the BCL6 overexpression group compared with the control group (AGS cells at 24 h, P < 0.001; SGC-7901 cells at 24 h, P < 0.001; Fig. 2D). In addition, the transwell assay showed that BCL6 inhibited the migration and invasion of GC cells (migration analysis: AGS, P < 0.001; SGC-7901, P = 0.004; invasion analysis: AGS, P = 0.001; SGC-7901, P < 0.001; Fig. 2E). Therefore, we examined the expression of related indicators of the epithelial–mesenchymal transformation (EMT) process (E-cadherin, N-cadherin, vimentin, and MMP9), which are closely related to tumor metastasis. The results showed that BCL6 could upregulate E-cadherin and downregulate N-cadherin, vimentin, and MMP9, indicating that BCL6 could inhibit the EMT process of GC cells (Fig. 2F).
In conclusion, these results suggest that BCL6 inhibits the proliferation, invasion, and migration of GC cells.
Bcl6 Inhibits The Growth And Intraperitoneal Dissemination Of Gc Cells In Vivo
We subcutaneously injected BCL6-overexpressing or control AGS and SGC-7901 GC cells into the flanks of nude mice, and tumor growth was closely observed over 22 days. The growth rate of BCL6-overexpressing SGC-7901 and AGS cells was significantly lower than that of control cells (Fig. 2G, Supplementary Figure S1). Simultaneously, the weight of the harvested tumor mass showed that BCL6 inhibited the proliferation of SGC-7901 and AGS cells in nude mice (Fig. 2G). Tumors from nude mice were sectioned and subjected to hematoxylin and eosin (HE) staining and IHC analysis. The results showed that the staining intensity of BCL6 in the tumor tissue of the BCL6-overexpressing group was significantly stronger than that in the control group (Fig. 2H). Subsequently, we constructed an intraperitoneal dissemination tumor model to explore the effect of BCL6 on the metastasis of GC cells in nude mice. The results showed that compared with the control group, the tumor metastases in the abdominal cavity of nude mice in the BCL6 overexpression group were substantially reduced, and the weight of the corresponding metastases was also significantly reduced (Fig. 2I). Tumor metastases from nude mice were sectioned and subjected to HE and IHC staining (Fig. 2J).
Bcl6 Suppresses The Activation Of Thewnt/β-catenin Pathway By Transcriptionally Repressing Fzd7 In Gc Cells
BCL6 is a transcriptional repressor. Immunohistochemical studies revealed that BCL6 is highly expressed in the nuclei of gastric mucosal cells. Thus, we speculated that this protein could regulate gene expression in GC cells. Therefore, we performed transcriptome sequencing to analyze the mRNA profiles in the control group and BCL6-overexpressing AGS cells, which indicated that the expression of 2426 genes (DEGs) changed significantly overall (FC > 1.5) (Fig. 3A). GO analysis of the sequencing results showed that the Wnt signaling pathway was enriched in BCL6 downstream genes (Fig. 3B). Meanwhile, FZD7, the key receptor for Wnt/β-catenin has attracted our attention (FC > 2) (Fig. 3C). The expression of FZD7 in patients with GC and their impact on prognosis were analyzed online using The Cancer Genome Atlas (TCGA) data. The results showed that FZD7 was highly expressed in patients with GC and predicted poor prognosis (Fig. 3D). The expression of FZD7 was further verified using qPCR in the control group and BCL6-overexpressing AGS and SGC-7901 cells (Fig. 3E). We further investigated β-catenin, the key molecule of Wnt/β-catenin pathway, after treatment with a Wnt inhibitor, the upregulation of β-catenin caused by BCL6 knockdown was completely abolished in the two GC cell lines (Supplementary Figure S2A).
As the receptor of Wnt, FZD7 is responsible for transmitting extracellular Wnt signals intracellularly, thus affecting the expression level of β-catenin [22]. Therefore, we speculated that BCL6 may inhibit the intracellular transmission of Wnt by inhibiting FZD7. The correlation between BCL6 and FZD7 expression in GC tissues was further analyzed using human GC tissue microarrays. The results indicated that FZD7 was negatively correlated with BCL6 in GC tissues (Fig. 3F, Supplementary Figure S2B). IHC also showed that the staining intensity of FZD7 in the tumor tissue of BCL6-overexpressing nude mice was significantly weaker than that in the control group (Supplementary Figure S2C). BCL6 is a classic transcriptional suppressor; therefore, we speculate that BCL6 is a direct transcriptional repressor of FZD7, which was verified through ChIP and luciferase reporter gene assays. Several putative BCL6-binding sites in the promoter of FZD7 were predicted by the JASPAR database (Fig. 3G). Chromatin immunoprecipitation (ChIP) analysis showed that BCL6 is recruited to promoter regions containing binding sites 2 and 3 (Fig. 3H). Truncated FZD7 promoters or site-directed mutagenesis combined with luciferase reporter assays indicated that these 2 binding sites in the FZD7 promoter mediate the repression of promoter activity induced by BCL6 (Fig. 3I). Compared with GC cells infected only with the BCL6 overexpression lentivirus group, the β-catenin protein level in the BCL6 overexpression combined with the FZD7 overexpression group was significantly restored. However, the β-catenin protein level decreased significantly after the Wnt inhibitor IWP-2 was incubated based on the overexpression (Fig. 3J).
Bcl6 Undermines Viability And Motility Of Gc Cell By Suppressing Fzd7
Functional rescue experiments showed that overexpression of FZD7 significantly reverse the inhibitory effect of BCL6 on proliferation, invasion, and migration of GC cells (Fig. 4A, B, C, and D). In addition, immunofluorescence staining was performed to determine the β-catenin levels in AGS and SGC-7901 cells. The results showed that nuclear β-catenin levels were reduced after BCL6 overexpression and were restored when FZD7 was simultaneously overexpressed(Fig. 4E). WB analysis of cytoplasmic (C) and nuclear (N) extracts was performed. These results also verify this conclusion (Supplementary Figure. S2D). Therefore, BCL6 could directly repress the transcription of FZD7 to inhibit the Wnt/β-catenin signaling pathway and act as a tumor suppressor in GC.
Bcl6 Facilitates Ferroptosis Of Gc Cells By Suppressing Fzd7/β-catenin/tp63/gpx4 Pathway
Recent studies have shown that FZD7 can promote ferroptosis through β-catenin-TP63-GPX4 pathway in ovarian cancer [23]. We have confirmed that FZD7 is the direct target of BCL6 in GC. Therefore, we tried to explore whether BCL6 can affect the occurrence of ferroptosis in GC cells. First of all, we found that overexpressed BCL6 increased ferroptosis inducer RSL3- or erastin-induced cell death, indicating that BCL6 could promote the occurrence of ferroptosis in GC cells (Fig. 5A, B). then, we detected the level of lipid peroxidation (lipid-ROS) and lipid oxidation MDA, which are the key substance leading to ferroptosis [24]. The results show that BCL6 can promote the up-regulation of lipid-ROS and MDA level in GC cells (Fig. 5C, D).
In order to further clarify whether BCL6 promotes ferroptosis in GC cells through FZD7, we overexpressed BCL6 and FZD7 in GC cells at the same time, and then determined cell death. We found that FZD7 can reverse the facilitation of BCL6 on erastin or RSL3- induced cell death (Fig. 5E, F). In addition, we detected lipid-ROS and MDA level, and found that FZD7 can reverse the promoting effect of BCL6 on lipid-ROS accumulation and MDA level (Fig. 5G, H). In short, FZD7 could reverse the occurrence of ferroptosis induced by BCL6. From a mechanistic standpoint, we detected the expression of TP63 and ferroptosis marker GPX4. The results showed that BCL6 can significantly repress the expression of TP63 and GPX4 in GC cells (Fig. 6A, B). Moreover, β-catenin, TP63 and GPX4 levels could be restored by FZD7 in BCL6 overexpressed GC cells(Fig. 6C, D). Then, we explored the role of TP63 in this process, and found that depletion of TP63 could reverse the promoting effect of FZD7 on GPX4, while overexpression of TP63 could restore the inhibitory effect of BCL6 on GPX4. In addition, we also found that TP63 depletion can in turn reduce the expression of FZD7 (Supplementary Figure S3A, B). In general, we found that BCL6 promotes ferroptosis in GC cells on the FZD7-β-catenin-TP63-GPX4 pathway.
The Expression And Role Of Bcl6 In Gc Cells Are Strengthened By The Rnf180/rhoc Pathway
Our previous studies showed that RNF180, a tumor suppressor in GC, inhibits the proliferation and movement of GC cells [25]. Gene Expression Profiling Interactive Analysis (GEPIA) database analysis showed that there was a strong positive correlation between BCL6 and RNF180 expression (Fig. 7A). And our IHC results also demonstrated that BCL6 expression was positively correlated with RNF180 in the GC tissue microarrays (Fig. 7B, Supplementary Figure. S4A). Therefore, we investigated whether RNF180 regulated the expression of BCL6. RNF180 was overexpressed in AGS and SGC-7901 GC cells to verify whether it affected the expression level of BCL6. The results showed that RNF180 increased the transcription and protein levels of BCL6 in GC cells (Fig. 7C, 7D).
RNF180 is an E3 ubiquitin ligase that directly promotes ubiquitination and subsequent degradation of its target gene at the protein level but cannot directly promote the expression of its downstream genes at the transcriptional level [26]. Therefore, RNF180 indirectly regulates BCL6 by forming a regulatory axis with a specific molecule. Our previous studies showed that RNF180 could promote proteasome-pathway-dependent degradation of the oncogene RhoC in GC cells [21]. Therefore, we investigated whether RNF180 promotes the expression of BCL6 through RhoC. First, RhoC was knocked down in AGS and SGC-7901 GC cells, and the expression of BCL6 was determined using western blotting and qPCR. The results showed that the mRNA and protein levels of BCL6 were significantly increased (Fig. 7E). IHC results also suggested a negative correlation between RhoC and BCL6 levels in GC tissues (Supplementary Figure S4B,C). Therefore, we speculated that RNF180 might reverse the decreased expression of BCL6 in GC cells by promoting the degradation of RhoC through the proteasome pathway.
Then, Cycloheximide (CHX) was incubated in control and RNF180-overexpressed AGS and SGC-7901 GC cells, and the protein levels of RhoC were examined at fixed time intervals. The results showed that RNF180 promoted the degradation of RhoC (Fig. 7F). MG132 (10 µM) was added to both cell lines. The results revealed that with the inhibition of the proteasome, RNF180 was unable to promote the proteasome-dependent degradation of RhoC; thus, the levels of RNF180 and RhoC protein were accumulated (Fig. 7G). To further determine the dependence of RhoC on the increase in BCL6 caused by RNF180, we upregulated RhoC in AGS and SGC-7901 cells overexpressing RNF180. The results showed that overexpression of RhoC reversed the upregulation of BCL6 and downregulation of its downstream genes induced by RNF180 at the mRNA and protein levels (Fig. 7H, Supplementary Figure S4D). In addition, immunofluorescence staining was performed to determine the BCL6 levels in AGS and SGC-7901 cells. We found that nuclear BCL6 levels were upregulated after RNF180 overexpression and were reversed when RhoC was simultaneously overexpressed(Fig. 7I). WB analysis of cytoplasmic (C) and nuclear (N) extracts was performed. These results equally verify this conclusion (Supplementary Figure S4E). Similarly, BCL6 depletion restored the downregulation of FZD7 and its downstream genes induced by RNF180 overexpression or RhoC depletion (Supplementary Figure S5A, B).
The results of the colony formation and transwell assays revealed that depletion of BCL6 could reverse the inhibitory effect of RhoC depletion or RNF180 overexpression on the proliferation, migration and invasion capacities, respectively, of the two GC cell lines (Fig. 8A, B, Supplementary Figure S6A, B). Meanwhile, we found that BCL6 depletion could reverse the promotion of RhoC depletion or RNF180 overexpression on erastin or RSL3- induced cell death (Fig. 8C, D, Supplementary Figure S6C, D). In short, the malignancy inhibition and ferroptosis facilitation of BCL6 mediated FZD7 repression could be strengthened by RNF180/RhoC pathway in GC cells.