TRIM69 suppressed the anoikis resistance and metastasis of gastric cancer through ubiquitin‒proteasome-mediated degradation of PRKCD

The tripartite motif (TRIM) protein family has been investigated in multiple human cancers, including gastric cancer (GC). However, the role of TRIM69 in the anoikis resistance and metastasis of GC cells remains to be elucidated. We identified the differentially expressed genes in anoikis-resistant GC cells using RNA-sequencing analysis. The interaction between TRIM69 and PRKCD was analyzed by coimmunoprecipitation and mass spectrometry. Our results have shown that TRIM69 was significantly downregulated in anoikis-resistant GC cells. TRIM69 overexpression markedly suppressed the anoikis resistance and metastasis of GC cells in vitro and in vivo. TRIM69 knockdown had the opposite effects. Mechanistically, TRIM69 interacted with PRKCD through its B-box domain and catalyzed the K48-linked polyubiquitination of PRKCD. Moreover, TRIM69 inhibited BDNF production in a PRKCD-dependent manner. Importantly, overexpression of PRKCD or BDNF blocked the effects of TRIM69 on the anoikis resistance and metastasis of GC cells. Interestingly, a TRIM69-PRKCD+BDNF+ cell subset was positively associated with metastasis in GC patients. TRIM69-mediated suppression of the anoikis resistance and metastasis of GC cells via modulation of the PRKCD/BDNF axis, with potential implications for novel therapeutic approaches for metastatic GC.


INTRODUCTION
Gastric cancer (GC) is a malignancy of the digestive system with the highest mortality rate worldwide [1].The high rate of metastasis is the major reason for the poor prognosis of patients with GC [2].Therefore, the identification of key targets and characterization of their molecular mechanisms involved in GC metastasis would contribute to the development of new treatment options.
Tumor metastasis is a multistep biological process in which cancer cells disperse from a primary site; cross the basement membrane (BM), the extracellular matrix (ECM), and vessel walls; and finally colonize distant organs through the blood and lymphatic circulation [3][4][5].Anoikis is a form of apoptosis resulting from loss of cell adhesion or adhesion-mediated signaling [6,7].It is well known that anoikis resistance (AR) is a necessary condition for the occurrence of tumor metastasis [8,9].Previous studies have shown that tumor cells can avoid anoikis by inducing epithelial-mesenchymal transition (EMT), inhibiting apoptotic signaling pathways, changing integrin expression, regulating microRNAs, activating autophagy or inducing oxidative stress [10][11][12][13][14][15].Regrettably, the underlying mechanism of anoikis resistance in GC has not been clearly elucidated.
The proteins of the tripartite motif (TRIM) family, a large group of E3 ubiquitin ligases, are characterized by the presence of a RING finger domain, a B-box domain, a coiled-coil region, and a variable C-terminal region [16].Recently, the TRIM family emerged as a potentially important regulator of various functions in cellular processes, including anoikis resistance pathways [17,18].TRIM69 is a member of the TRIM family, and it not only has been reported to function in the defense against viral infections [19,20] but also has been identified to be closely related to apoptosis [21,22].Rong et al. showed that TRIM69 overexpression attenuated ultraviolet B-induced apoptosis and ROS production by inducing p53 ubiquitination [23].Another report suggested that TRIM69 inhibited apoptosis and inflammation through ASK1 inactivation in mice with high-fat diet (HFD)-induced hippocampal injury [24].Unfortunately, no studies have reported the function of TRIM69 in anoikis resistance in GC cells.
In the present study, we aimed to investigate the effects of TRIM69 on anoikis resistance in GC cells (AR GC cells).Here, we report the discovery that TRIM69 expression was low in AR GC cells.Overexpression of TRIM69 promoted the anoikis and inhibited the metastasis of AR GC cells via the ubiquitinproteasome degradation pathway, which reduced the expression of PRKCD, in vitro and in vivo.In addition, TRIM69 downregulated BDNF expression through PRKCD-mediated signaling pathways.Importantly, we found that low expression of TRIM69 contributed to distant metastasis by analysis of clinical specimens.Thus, our results offer novel mechanistic insight into the distant metastasis of GC and provide a resource for future studies focused on GC pathogenesis or the discovery of therapeutic targets.

TRIM69 expression was low in anoikis-resistant GC cells
To explore the mechanism of anoikis resistance in GC cells, we first established anoikis-resistant (AR) AGS and MKN45 cells.As shown in Fig. 1A, the number of surviving cells was significantly increased in the AR AGS and MKN45 cell groups compared with the control cell groups under detachment conditions.The levels of cleaved caspase-3 and cleaved PARP were markedly decreased in AR AGS and MKN45 cells in response to detachment (Fig. 1B and Supplementary Fig. 1A).The apoptosis rate of AR AGS and MKN45 cells was obviously reduced after culture under detachment conditions (Fig. 1C).The acquisition of anoikis resistance is of crucial importance for the survival of cancer cells in a stressful microenvironment [25].As shown in Supplementary Fig. 1B, the numbers of colonies of AR AGS and MKN45 cells were greater than those of WT AGS and MKN45 cells under low-serum conditions.Moreover, the migration and invasion abilities of AR AGS and MKN45 cells were significantly enhanced compared with those of control cells (Supplementary Fig. 1C).These results suggested that we successfully established anoikis-resistant GC cells.
Next, we performed RNA-seq analysis to analyze the transcriptomic differences between WT MKN45 cells and AR MKN45 cells.As shown in Fig. 1D and E, there were 457 upregulated genes and 119 downregulated genes in AR MKN45 cells compared to WT MKN45 cells.Among these, 17 DEGs meeting the logarithmic fold change criteria (logFC > 4 and logFC < −3.4) were identified (Fig. 1F).Moreover, RT-qPCR was performed to validate the results of the RNA-seq analysis, and the results showed that consistent with the RNA-seq results, WBP2NL, SERPINA1, and PIP5K1B expression levels were increased but FOXI1, TRIM69, and GABRR2 expression levels were decreased in AR AGS and MKN45 cells compared to the corresponding control cells (Fig. 1G).Among these downregulated genes, TRIM69, an E3 ubiquitin ligase, attracted our attention.Both the mRNA and protein levels of TRIM69 were lower in AR GC cells (AGS, MKN28, MKN45, and HGC27) than in WT GC cells (Fig. 1H, I).

TRIM69 suppressed the anoikis resistance and metastasis of GC cells in vitro and in vivo
To investigate the role of TRIM69 in the anoikis resistance and metastasis of GC cells, we generated GC cells stably overexpressing TRIM69 using AR GC cells (Fig. 2A).TRIM69 overexpression markedly increased caspase-3 and PARP cleavage as well as the apoptosis rate in AR AGS, MKN28, MKN45 and HGC27 cells under detachment conditions (Fig. 2A, B and Supplementary Fig. 2A).Additionally, overexpression of TRIM69 led to a decrease in the survival rate of AR GC cells (Fig. 2C).Using a colony formation assay, we observed that all TRIM69-overexpressing clones had lower growth rates than the corresponding controls under low-serum conditions (Fig. 2D).Moreover, compared with those of scrambled control cells, the migration and invasion abilities of TRIM69-overexpressing AR GC cells were obviously decreased (Fig. 2E).To further substantiate the regulatory effect of TRIM69 overexpression on tumor metastasis in vivo, we performed a lung metastasis assay in nude mice using AR MKN28 cells injected via the tail vein.The results showed that the numbers of metastatic nodules in the lungs of mice in the TRIM69 overexpression group were significantly less than those in the control group (Fig. 2F).Moreover, we observed that the expression of TRIM69 was higher in the lung metastatic nodules in the TRIM69 overexpression group than that in the control group (Supplementary Fig. 2B) Complementary loss-of-function studies were performed to further examine the effect of TRIM69 on the anoikis resistance and metastasis of GC cells.We used three nonoverlapping siRNAs targeting TRIM69 (TRIM69 siRNA-001, siRNA-002, and siRNA-003) to knock down TRIM69 expression in AGS, MKN28, MKN45 and HGC27 cells (Supplementary Fig. 3).TRIM69 siRNA-002 and siRNA-003 were selected for further studies.TRIM69 silencing resulted in decreases in the levels of apoptosis-related proteins (cleaved caspase-3 and cleaved PARP) and the apoptosis rate in AGS, MKN28, MKN45, and HGC27 cells under detachment conditions (Supplementary Fig. 4A, B).The trypan blue exclusion assay showed that knocking down TRIM69 significantly increased the survival rate of GC cells in response to detachment (Supplementary Fig. 4C).In addition, TRIM69 depletion increased the proliferation rate of AGS, MKN28, MKN45 and HGC27 cells under low-serum conditions (Supplementary Fig. 4D).Furthermore, transfection with TRIM69 siRNA-002 and siRNA-003 markedly enhanced the migration and invasion of GC cells (Supplementary Fig. 4E).These results suggest that TRIM69 is an important regulator of anoikis resistance and metastasis in GC cells.

TRIM69 directly degrades PRKCD via the ubiquitinproteasome pathway
To explore the potential mechanism by which TRIM69 suppresses the anoikis resistance and metastasis of GC cells, Co-IP and LC-MS/MS were conducted to identify potential substrates of TRIM69 related to apoptosis (Fig. 3A).PRKCD, a member of the novel PKC subfamily, has been demonstrated to be closely associated with the apoptotic process [26,27] (Fig. 3B).Co-IP assays showed that TRIM69 was able to interact with PRKCD in GC cells (Fig. 3C).Similarly, endogenous PRKCD coimmunoprecipitated with exogenous TRIM69 in GC cells (Fig. 3D).Moreover, the interaction between exogenous TRIM69 and PRKCD was demonstrated in HEK293T cells (Fig. 3E).To further explore the functional domains involved in the TRIM69-PRKCD interaction, we constructed several truncations of TRIM69 and PRKCD.As shown in Fig. 3F, four TRIM69 truncation mutants lacking the RING domain (ΔRING), B-box domain (ΔB-box), Coiled-coil domain (ΔCoiled-coil), or B30.2 domain (ΔB30.2) were constructed [21].The results of the co-IP assay showed that the B-box domain of TRIM69 is required for interaction with PRKCD (Fig. 3G).In addition, we generated three PRKCD truncation mutants lacking the C2 domain (ΔC2), C1 domain (ΔC1), or protein kinase domain (ΔPKD) (Fig. 3H).As shown in Fig. 3I, we found that the C1 domain of PRKCD is required for interaction with TRIM69.
Considering that TRIM69 is an E3 ligase, we hypothesized that TRIM69 can regulate the protein stability of PRKCD through ubiquitination [21].The western blot results showed that TRIM69 overexpression markedly decreased the PRKCD protein level (Fig. 4A and Supplementary Fig. 5A).In the presence of the protein synthesis inhibitor cycloheximide (CHX), the half-life of the PRKCD protein in TRIM69-overexpressing cells was much shorter than that in control cells (Fig. 4B).Then, we determined which degradation system predominantly regulates the TRIM69mediated degradation of PRKCD.Treatment with the ubiquitin-proteasome system inhibitor MG132 rescued TRIM69 overexpression-mediated PRKCD degradation in AGS, MKN28, MKN45, and HGC27 cells (Fig. 4C and Supplementary Fig. 5B).Moreover, TRIM69 overexpression led to accumulation of ubiquitinated PRKCD in the presence of MG132 (Fig. 4D).To determine which lysine (K)-linked polyubiquitin chains are conjugated to PRKCD, we overexpressed several ubiquitin mutants, in which the K48 ubiquitin construct lacked all lysine residues except K48, while the K63 construct lacked all lysine residues except K63.The results of co-IP showed that K48-and K63-linked ubiquitination were the primary types of ubiquitination of PRKCD mediated by TRIM69 Fig. 1 TRIM69 expression was low in anoikis-resistant GC cells.A Trypan blue exclusion assay was used to measure the viability of anoikisresistant AGS and MKN45 cells in suspension culture.B Western blotting was used to measure the levels of cleaved caspase-3 and cleaved PARP in WT and AR AGS and MKN45 cells.β-Actin served as the loading control.C Flow cytometric analysis of the apoptosis rate of WT and AR AGS and MKN45 cells in suspension culture.D, E Heatmap and volcano plots showing the differential gene expression of WT MKN45 and anoikis-resistant (AR) MKN45 cells in RNA-seq data.F DEGs meeting the logarithmic fold change criteria (logFC > 4 and logFC < −3.4) were identified.G The expression of WBP2NL, SERPINA1, PIP5K1B, FOXI1, TRIM69, and GABRR2 was analyzed by RT-qPCR in WT and AR AGS and MKN45 cells.H The TRIM69 mRNA levels in WT and AR AGS, MKN28, MKN45, and HGC27 cells were measured by RT-qPCR.I The TRIM69 protein levels in WT and AR AGS, MKN28, MKN45, and HGC27 cells were measured by western blotting.GAPDH served as a loading control.Each experiment was performed in triplicate.The data are presented as the means ± SD and were analyzed by Student's t-test.(*p < 0.05, **p < 0.01, ***p < 0.001).(Fig. 4E, F).Altogether, these data demonstrated that TRIM69 acted as a negative regulator of PRKCD protein expression and destabilized PRKCD by enhancing its ubiquitination and degradation.
PRKCD is required for TRIM69 to suppress the anoikis resistance and metastasis of GC cells To investigate whether TRIM69 inhibits GC anoikis resistance and metastasis through PRKCD, we cotransfected the TRIM69 and PRKCD expression plasmids into AR AGS and MKN28 cells (Fig. 5A).
PRKCD overexpression significantly decreased the cleavage of caspase-3 and PARP in AR GC cells, which was increased in AR GC cells after transfection with the TRIM69 expression plasmid (Fig. 5A and Supplementary Fig. 6A).Moreover, PRKCD overexpression partially reversed the effects of TRIM69 overexpression on the apoptosis, survival, and growth rates (Fig. 5B-D).Additionally, overexpression of TRIM69 markedly decreased the migration and invasion of AR GC cells, and this decrease was reversed by PRKCD overexpression (Fig. 5E, F).Importantly, the in vivo lung metastasis assay showed that PRKCD overexpression abolished the inhibitory effect of TRIM69 on the lung metastatic ability of AR GC cells (Fig. 5G and Supplementary Fig. 6B, C).In conclusion, these results demonstrated that TRIM69 negatively regulated GC anoikis resistance and metastasis through PRKCD.

The TRIM69/PRKCD axis inhibits anoikis resistance and metastasis via BDNF in GC cells
Cytokines are key proteins in signaling in the tumor microenvironment (TME) that have pleiotropic effects and are associated with cancer progression, invasion, and metastasis fore, we explored whether TRIM69 modulates anoikis resistance and metastasis via one or more cytokines.A Human Cytokine Array G5 was employed to identify differentially expressed cytokines between OE-NC and OE-TRIM69 AR MKN45 cells.The results showed that 12 cytokines were downregulated and 29 were upregulated (Fig. 6A, B). Figure 6C shows the top 3 downregulated (MCP-1, BDNF, and NAP-2) and upregulated (TNFRSF11B, IGFBP-3, and IL-16) cytokines.Previous studies have shown that BDNF/TrkB is related to anoikis resistance, tumor progression, and poor prognosis in many types of malignancies [31][32][33].Therefore, we inferred that BDNF might be involved in the TRIM69-mediated regulation of anoikis resistance and metastasis.The ELISA data showed that the level of BDNF was lower in the OE-TRIM69 group than in the OE-NC group (Fig. 6D).Similar results were also found by RT-qPCR and western blot analysis (Fig. 6E, F Supplementary Fig. 7A).Furthermore, PRKCD overexpression reversed the effects of TRIM69 overexpression on the mRNA and protein expression of BDNF in AR AGS and MKN28 cells (Fig. 6G-I and Supplementary Fig. 7B).These results suggested that TRIM69 inhibited BDNF expression in GC cells in a PRKCD-dependent manner.
We next verified whether BDNF is involved in the TRIM69mediated anoikis resistance and metastasis of GC cells.As shown in Fig. 6J, supplementation with human recombinant BDNF (hr BDNF) reduced the levels of cleaved caspase-3 and cleaved PARP in AR GC cells, which were increased after TRIM69 overexpression (Supplementary Fig. 7C).Moreover, addition of hr BDNF reversed the effects of TRIM69 on the apoptosis and survival rates of AR GC cells (Fig. 6K, L).Additionally, treatment with hr BDNF abolished the TRIM69 overexpression-induced decrease in cell growth under low-serum conditions as well as the decreases in the migration and invasion abilities of AR GC cells (Fig. 6M, N).Altogether, these findings indicate that the roles of the TRIM69/PRKCD axis in preventing anoikis resistance and metastasis are dependent on BDNF.

The proportion of TRIM69 -PRKCD + BDNF + cells was positively associated with metastasis in GC patients
To analyze the associations between TRIM69, PRKCD and BDNF expression and metastasis in 162 GC patients, an mIHC assay was performed.As shown in Fig. 7A and B, compared with that in nonmetastatic (M0) tissues, the expression of TRIM69 in metastatic GC (M1) tissues was significantly decreased in the epithelial region.Importantly, low TRIM69 expression (TRIM69 -) was significantly positively correlated with pathologic differentiation (P = 0.047), more advanced clinical stage (P = 0.044), and distant metastasis (P = 0.007), as shown in (Supplementary Table 1).Moreover, we examined PRKCD and BDNF protein expression in tissue samples from patients with GC (Fig. 7A).As shown in Fig. 7B, the expression of PRKCD was markedly increased in M1 tissues compared with M0 tissues.However, there was no difference in BDNF expression between M0 and M1 tissues (Fig. 7B).Interestingly, we observed that tumors in patients with high proportions of TRIM69 -PRKCD + , TRIM69 -BDNF + , PRKCD + BDNF + and TRIM69 -PRKCD + BDNF + tumor cell subsets showed higher metastatic potential (Fig. 7C, D).As shown in Fig. 7E, patients with high proportions of TRIM69 -PRKCD + had poorer overall survival (OS).

DISCUSSION
Anoikis resistance is essential for tumor metastasis, allows tumor cells to survive during circulation in the bloodstream, and facilitates their metastasis to distant organs [34,35].However, the underlying molecular mechanism of anoikis resistance in GC remains unclear.In the present study, we established a model of anoikis resistance in GC cells in vitro and found that TRIM69 expression was significantly downregulated in anoikis-resistant GC cells.TRIM69 is a RING finger domain-containing E3 ligase that belongs to the tripartite motif (TRIM) family.It was initially discovered in a human testis cDNA library and can mediate ubiquitination in vivo, playing a very important role in the process of spermatogenesis and participating in apoptosis [21,23].However, the molecular function of TRIM69 has not been extensively studied in various types of cancer cells.In this study, we confirmed that TRIM69 overexpression inhibited but TRIM69 knockdown promoted the anoikis resistance, migration, and invasion of GC cells in vitro.Moreover, TRIM69 overexpression impaired distant metastasis in vivo.In addition, TRIM69 was expressed at low levels in metastatic GC tissues, and TRIM69 expression was negatively associated with distant metastasis.Although the mechanism by which TRIM69 is downregulated in anoikis-resistant GC cells remains unknown and needs further investigation, our data demonstrated a crucial role for TRIM69 in regulating anoikis resistance and metastasis in GC.
The results of RNA-seq analysis indicated that FOXI1, which belongs to the forkhead family of transcription factors, exhibited the most decrease in expression in AR MKN45 cells compared to WT MKN45 cells.Previous studies have shown that FOXI1 can inhibit the proliferation and promote apoptosis of GC cells [36,37].Although the relationship between FOXI1 and anoikis resistance in GC has not been reported so far, the expression of FOXI1 was significantly downregulated in AR MKN45 cells, suggesting that FOXI1 might exert key roles in anoikis resistance and metastasis in GC.Therefore, further study on the effect of FOXI1 on anoikis resistance in GC will be helpful to further understand the mechanism of anoikis resistance in GC.
PRKCD is a member of the protein kinase C family of serine-and threonine-specific protein kinases [38,39] and is involved in the progression of GC [40,41].For example, PRKCD overexpression enhances the metastasis, chemoresistance, and stem cell-like characteristics of GC cells [40].Yoshihiko Iioka et al. noted that PRKCD, in cooperation with p53, modulates cisplatin-induced caspase-3-mediated cell death in GC [41].In the current study, the results of co-IP and LC-MS/MS analysis showed that TRIM69 might interact directly with the PRKCD protein in GC cells.Importantly, PRKCD expression was regulated by TRIM69-mediated ubiquitination.Moreover, the results of the mutagenesis and co-IP assays showed that the ubiquitination of PRKCD induced by TRIM69 was both K48-and K63-linked, which are commonly observed types of ubiquitination.These results were consistent with a previous study reported by Han et al. [21].K48-linked polyubiquitination of target proteins leads to proteasome-mediated degradation, while K63linked ubiquitination has been implicated in the activation of several signaling pathways.Furthermore, K63-linked polyubiquitination has been shown to promote protein degradation through the autophagy pathway [42,43].We cannot explain the K63-linked polyubiquitination-mediated degradation of PRKCD in GC cells under autophagic conditions observed in the present study.Additionally, K6, K11, K27, K29, or K33 -linked unconventional ubiquitination has been reported to be associated with protein degradation [44,45].The possibility of TRIM69-mediated PRKCD degradation through K6, K11, K27, K29, or K33 -linked ubiquitination cannot be ruled out.Further investigations are required to explain these phenomenons.More importantly, the rescue experiment showed that PRKCD overexpression abolished the Fig. 5 PRKCD is required for TRIM69 to suppress the anoikis resistance and metastasis of GC cells.A Western blotting was used to measure the levels of cleaved caspase-3, cleaved PARP, PRKCD, and TRIM69 in OE-NC and OE-TRIM69 AR AGS and MKN28 cells treated with the PRKCD plasmid.GAPDH served as the loading control.B Flow cytometric analysis of the apoptosis rate of OE-NC and OE-TRIM69 AR AGS and MKN28 cells treated with the PRKCD plasmid in suspension culture.C A Trypan blue exclusion assay was used to measure the viability of OE-NC and OE-TRIM69 AR AGS and MKN28 cells treated with the PRKCD plasmid.D A colony formation assay was used to determine the proliferative capacity of OE-NC and OE-TRIM69 AR AGS and MKN28 cells treated with the PRKCD plasmid in low-serum medium (1% FBS).E, F Transwell migration and invasion assays (E) and the corresponding statistical results (F) in OE-NC and OE-TRIM69 AR AGS and MKN28 cells treated with the PRKCD plasmid.Scale bar, 50 μm.G Treatment with the PRKCD expression plasmid reversed the effects of TRIM69 overexpression on the metastasis of AR MKN28 cells in vivo.Lungs were observed for metastatic nodules on the surface and stained with H&E for histological analysis; the arrows indicate metastatic nodules.Representative photographs and H&E staining images are shown (n = 5 mice per group).Scale bar, 100 μm.Each experiment was performed in triplicate.The data are presented as the means ± SD and were analyzed by Student's t-test or one-way ANOVA test.(*p < 0.05, **p < 0.01, ***p < 0.001).effect of TRIM69 on GC anoikis resistance and metastasis in vitro and in vivo.Overall, we arrived at the novel conclusion that TRIM69 destabilizes PRKCD by enhancing its ubiquitination and degradation and suppresses the anoikis resistance and metastasis of GC cells through this effect on PRKCD.
BDNF is a member of the nerve growth factor family of proteins and participates in cellular differentiation, proliferation, and survival processes in the nervous system [46][47][48].A number of studies have noted that BDNF plays crucial roles in the progression of various cancers, including GC [49][50][51].For instance, lactate/BDNF/TrkB signaling is involved in the crosstalk between tumor cells and activated fibroblasts and enhances anlotinib resistance in GC [52].Ding et al. reported that BDNF expression was upregulated in GC tissues and that BDNF knockdown inhibited the proliferation, migration and invasion of GC cells [53].In this study, BDNF was significantly downregulated in TRIM69-overexpressing GC cells.
PRKCD overexpression reversed the effect of TRIM69 overexpression on BDNF expression.A previous study showed that PRKCD activation upregulated BDNF expression in retinal ganglion cells [39].These results indicate that TRIM69 negatively regulates BDNF expression in GC cells via PRKCD.However, the precise mechanism by which TRIM69/PRKCD signaling regulates BDNF expression is still unclear.Hence, we will further explore the mechanism by which TRIM69/PRKCD signaling regulates BDNF expression in our next study.Importantly, hr BDNF abolished the effects of TRIM69 on the anoikis resistance and metastasis of GC cells.Collectively, our findings indicated that the TRIM69/PRKCD axis modulated the anoikis resistance and metastasis of GC cells through BDNF.Importantly, we also identified a TRIM69 -PRKCD + BDNF + tumor cell subset, whose presence was positively correlated with distant metastasis in GC patients.
Previous studies showed that p53 played key roles in regulating anoikis of cancer cells [54,55].It has also been shown that TRIM69 could modulate p53 expression and function in cataractogenesis and zebrafish development [23,56].As shown in Supplementary Fig 8, overexpression of TRIM69 decreased, while knockdown of TRIM69 increased, the protein expression of p53 in MKN28 and HGC27 cells, which contain mutant p53.But both TRIM69 overexpression and knockdown did not influence the p53 expression in AGS and MKN45 cells, which contain wild-type p53.Therefore, we speculated that TRIM69 may inhibit anoikis resistance in GC cells via negatively regulating mutant p53 expression.It would be interesting to further elaborate on the functions of TRIM69 in modulating mutant p53 expression in AR GC cells.

CONCLUSIONS
In summary, we investigated the novel role of TRIM69 in anoikis resistance and metastasis in GC and revealed a new mechanism for the regulation of PRKCD expression by TRIM69-mediated ubiquitination and degradation.Furthermore, TRIM69/PRKCD signaling was found to suppress anoikis resistance and metastasis by inhibiting BDNF expression (Fig. 8).Hence, it is of great value to develop therapeutic approaches targeting TRIM69 or blocking PRKCD/BDNF as potential GC interventions.

MATERIALS AND METHODS Patients and specimens
GC tissue microarray (TMA) chips, which contained 162 pairs of GC tissue samples and adjacent normal tissues, as well as follow-up data, were obtained from Shanghai Outdo Biotech Co., Ltd.(Shanghai, China).The relevant clinical data for each patient were collected and are provided in (Supplementary Table 2).Moreover, among these GC patients, 91 patients with the OS data were used to evaluate the prognostic value.Additionally, we obtained approval for this study from the Ethics Review Board of the First Affiliated Hospital of Soochow University (Approval number: 2022-117).Written informed consent was obtained from each patient.

Construct anoikis-resistant cells
The 1 × 10 6 gastric cancer cells were seeded onto a Poly-HEMA-coated 6-well plate and allowed to grow in suspension.Following a 24 h culture period, the suspension cells were collected and centrifugally resuspended.The cells were then cultured for an additional 24 h in an attached state, during which dead cells were removed.The suspension culture was then continued for another 24 h, and the suspension/ attachment cycle was repeated.Anoikis resistance was observed after the fourth cycle.The cells obtained through this process are referred to as anoikis-resistant GC cells.

RNA-seq assay
MKN45 GC cells (1 × 10 6 ) were seeded in poly-HEMA-coated 6-well plates.After culture for 24 h, detached MKN45 cells were collected and returned to adherent culture for 24 h.This detachment/attachment cycle was repeated until the emergence of anoikis resistance for 4 cycles.The cells obtained by the above method were called anoikis-resistant cells (AR MKN45 cells).Total RNA from WT MKN45 and AR MKN45 cells was isolated with TRIzol (Vazyme, Nanjing, China) and analyzed by RiboBio Life Science Co., Ltd.(Guangzhou, China).

Coimmunoprecipitation (Co-IP) assay
To detect the interaction of endogenous TRIM69 with PRKCD, AGS, MKN28 or MKN45 cell lysates were obtained by using IP lysis buffer (Beyotime, Shanghai, China) and incubated with 2 µg of an anti-PRKCD antibody or anti-rabbit IgG (Beyotime, Shanghai, China) overnight at 4 °C.Protein A + G magnetic beads (Beyotime, Shanghai, China) were added to the cell mixtures and incubated for 4 h at 4 °C.For the interaction between exogenous TRIM69 and PRKCD, AGS, MKN28, MKN45 or HEK293T cells were transfected with TRIM69 or PRKCD plasmids.After culture for 48 h, the cells were homogenized in lysis buffer for immunoprecipitation (Beyotime, Shanghai, China).Then, the cell lysates were incubated with 20 µl of anti-IgG magnetic beads (Beyotime, Shanghai, China) overnight at 4 °C.The next day, the mixtures were incubated with anti-Flag, anti-HA or anti-Myc magnetic beads (Beyotime, Shanghai, China) for 4 h at 4 °C.After the beads were washed four times with lysis buffer for IP, the precipitates were collected, and proteins were eluted with 1×SDS sample buffer.The samples were analyzed using western blotting.

Tumor metastasis in vivo
Six-to eight-week-old female BALB/c athymic nude mice were purchased from the Shanghai Laboratory Animal Center.Sample size estimate was based on our recent experience of similar lung metastasis studies.All animal experiments were approved by the Institutional Animal Care and Use Committee of Soochow University (Suzhou, China) (Ethical number, 202206A0710).The mice were randomized into control or experimental groups (n = 5 mice per group) before cancer cell inoculation.For the lung metastasis model, single-cell suspensions of 3 × 10 6 cells were injected into the tail veins of nude mice.The mice were killed 8 weeks after injection, the lungs were resected and fixed with paraformaldehyde, and the visible tumor nodules were counted.The fixed lungs were then embedded in paraffin, sectioned, and stained with hematoxylin and eosin (HE), and microscopic lung metastases were counted by light microscopy.And IHC assay was used to measure the TRIM69 expression in lung metastatic tissues.Sections were processed and stained with an anti-TRIM69 antibody (Proteintech, Wuhan, China, 1:100).
Fig. 6 The TRIM69/PRKCD axis inhibited anoikis resistance and metastasis via BDNF in GC cells.A A Human Cytokine Array G5 was used to identify the differentially expressed cytokines in the supernatants of OE-NC and OE-TRIM69 AR MKN45 cells.B Volcano plots showing the differentially expressed cytokines in the supernatants of OE-NC and OE-TRIM69 AR MKN45 cells.C Up-and downregulated cytokines are shown.D ELISA was used to measure BDNF expression in OE-NC and OE-TRIM69 AR AGS and MKN28 cell supernatants.E The BDNF mRNA level was measured by RT-qPCR in OE-NC and OE-TRIM69 AR AGS and MKN28 cells.F The BDNF protein level was measured by western blotting in OE-NC and OE-TRIM69 AR AGS and MKN28 cells.GAPDH served as the loading control.G ELISA was used to measure BDNF expression in the supernatants of OE-NC and OE-TRIM69 AR AGS and MKN28 cells after treatment with the PRKCD plasmid.H, I BDNF mRNA (H) and protein (I) levels in OE-NC and OE-TRIM69 AR AGS and MKN28 cells after treatment with the PRKCD plasmid.β-actin served as the loading control.J Western blotting was used to measure the levels of cleaved caspase-3, cleaved PARP, and TRIM69 in OE-NC and OE-TRIM69 AR AGS and MKN28 cells treated with hr BDNF.β-actin served as the loading control.K Flow cytometric analysis of the apoptosis rate of OE-NC and OE-TRIM69 AR AGS and MKN28 cells treated with hr BDNF in suspension culture.L A Trypan blue exclusion assay was used to measure the viability of OE-NC and OE-TRIM69 AR AGS and MKN28 cells treated with hr BDNF in suspension culture.M Colony formation assay of the proliferative capacity of OE-NC and OE-TRIM69 AR AGS and MKN28 cells treated with hr BDNF in low-serum medium (1% FBS).N Transwell migration and invasion assays of OE-NC and OE-TRIM69 AR AGS and MKN28 cells treated with hr BDNF.Scale bar, 50 μm.Each experiment was performed in triplicate.The data are presented as the means ± SD and were analyzed by Student's t-test or one-way ANOVA test.(*p < 0.05, **p < 0.01, ***p < 0.001).
treatment was used to remove the antibody-TSA complexes after every staining cycle, staining with the next marker was then performed until all four markers were labeled, and finally, spectral 4′,6-diamidino-2-phenylindole (DAPI) (PerkinElmer, USA) was added.The slides were mounted with VECTASHIELD® HardSet Antifade Mounting Medium (Vector Labs).Panoramic image acquisition was performed using a TissueFAXS SPECTRA multispectral panoramic tissue scanning quantitative analysis system.Spectral channel resolution was performed using TissueFAXS analysis software.DAPI channels were used to locate and identify nuclei and to identify and quantitatively analyze cells with positive cytoplasmic staining.

Fig. 2
Fig. 2 TRIM69 suppressed the anoikis resistance and metastasis of GC cells in vitro and in vivo.A The protein levels of cleaved caspase-3, cleaved PARP, and TRIM69 in TRIM69-overexpressing (OE-TRIM69) AR AGS, MKN28, MKN45, and HGC27 cells.β-Actin served as a loading control.B Flow cytometry was used to measure the apoptosis rate of negative control-overexpressing (OE-NC) and OE-TRIM69 AR GC cells in suspension culture.C Cell viability was determined by a trypan blue exclusion assay in OE-NC and OE-TRIM69 AR GC cells in suspension culture.D A colony formation assay was used to confirm the effects of OE-TRIM69 on the proliferative capacity of AR GC cells in low-serum medium (1% FBS).E Transwell assays were performed to determine the effects of TRIM69 overexpression on the migration and invasion abilities of AR GC cells.Scale bar, 50 μm.F The effects of TRIM69 overexpression on the lung metastasis of AR MKN28 cells in vivo.Lungs were observed for metastatic nodules on the surface and stained with H&E for histological analysis; the arrows indicate metastatic nodules.Representative photographs and H&E staining images are shown (n = 5 mice per group).Each experiment was performed in triplicate.The data are presented as the means ± SD and were analyzed by Student's t-test.(*p < 0.05, **p < 0.01, ***p < 0.001).

Fig. 3
Fig. 3 TRIM69 directly interacted with PRKCD.A Interacting proteins that coimmunoprecipitated with TRIM69 in Flag-TRIM69 MKN45 cells were identified by mass spectrometry.B Six putative substrate proteins targeted by TRIM69 were predicted by the UbiBrowser 2.0 website and mass spectrometry results.C Immunoprecipitation and western blot analysis showed the endogenous interaction between TRIM69 and PRKCD in AGS, MKN28, and MKN45 cells.D HA-tagged TRIM69 interacted with endogenous PRKCD in AGS, MKN28, and MKN45 cells.E The interaction of TRIM69 and PRKCD in HEK293T cells transfected with HA-TRIM69 and Myc-PRKCD.F Schematic of the full-length and truncated TRIM69 sequences.H Schematic of the full-length and truncated PRKCD sequences.G, I Co-IP assay results showing the binding regions between TRIM69 (G) and PRKCD (I).Each experiment was performed in triplicate.

Fig. 4
Fig.4TRIM69 directly degraded PRKCD via the ubiquitin-proteasome pathway.A PRKCD protein levels were measured by western blotting in OE-NC and OE-TRIM69 GC cells.GAPDH expression was used for normalization.B Western blot analysis showed the stability of the PRKCD protein in OE-NC and OE-TRIM69 GC cells in the presence of cycloheximide (CHX, 40 μM) for the indicated times.GAPDH expression was used for normalization.C Western blot analysis showed the expression level of the PRKCD protein in OE-NC and OE-TRIM69 GC cells treated with the proteasome inhibitor MG132.Cells were treated with 20 μM MG132 for 6 h.GAPDH expression was used for normalization.D TRIM69-overexpressing AGS cells were cotreated with the HA-Ubiquitin plasmid, Myc-PRKCD plasmid, and MG132.PRKCD ubiquitination was detected by immunoprecipitation with anti-Myc beads and analyzed by immunoblotting with the indicated antibodies.E, F TRIM69overexpressing AGS cells were cotransfected with Myc-PRKCD and different linkage types of HA-Ubiquitin (WT, K48, K63), and were then treated with MG132 (E); HEK293T cells were cotransfected with Myc-PRKCD, different linkage types of HA-Ubiquitin (WT, K48, K63) and Flag-TRIM69 and were then treated with MG132 (F).Cell lysates were immunoprecipitated with anti-Myc beads and analyzed by immunoblotting with the indicated antibodies.Each experiment was performed in triplicate.

Fig. 8
Fig.8The mechanisms by which TRIM69 suppresses GC anoikis resistance and metastasis.Schematic diagram showing the regulatory mechanisms of the TRIM69/PRKCD/BDNF axis in anoikis resistance and metastasis in GC.