The dual role of TRIM3 in colorectal cancer dependent on p53 status by retaining p53 in the cytoplasm to decrease its expression in the nuclei

doi:10.21203/rs.3.rs-1983354/v1

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The dual role of TRIM3 in colorectal cancer dependent on p53 status by retaining p53 in the cytoplasm to decrease its expression in the nuclei

https://doi.org/10.21203/rs.3.rs-1983354/v1

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Colorectal cancer is a very heterogeneous disease caused by the interaction of genetic and environmental factors. P53, a few exceptions, is a frequent mutation and plays a critical role in the adenoma-carcinoma transition during the tumorous pathological process. Our team screened the series of genes’ expression by high-content screening techniques to discover TRIM3 a newly tumor-associated gene in CRC. TRIM3 demonstrated both tumor-suppressive and tumorigenic features dependent on p53 wild or mutant status in cell experiments in our study. TRIM3 could directly interact with the C terminus of p53 (residues 320 to 393), a common segment of wtp53 and mutp53. Moreover, TRIM3 could decrease p53 levels in the nuclei by retaining them in the cytoplasm to exert different neoplastic biological features in a wtp53 or mutp53 dependent pathway. Chemotherapy resistance develops in nearly all patients with advanced CRC and seriously limits the therapeutic efficacies of anticancer drugs. TRIM3 could successfully reverse the chemotherapy resistance of oxaliplatin in mutp53 CRC cells by decreasing mutp53 in the nuclei to downregulate the multidrug resistance gene. Therefore, TRIM3 could be a potential therapeutic strategy to improve the survival of CRC patients with mutp53 by degradation mutp53 in the nuclei.

Colorectal cancer (CRC) is the third most common cancer and the fourth leading cause of cancer death in the world^1,2. Although early detection and perioperative treatment have improved survival, the advanced diseases remain incurable^3,4. CRC is a very heterogeneous disease caused by the interaction of genetic and environmental factors, except for a few mutations of the APC, k-Ras, and p53 genes. It is thought that p53 mutation plays a critical role in the adenoma-carcinoma transition during the tumorous pathological process^5–7. The frequency of p53 mutation in CRC is approximately 40–50%⁸. Different types of p53 mutation could determine different biologic behaviors of CRC, such as invasive depth, metastatic site, and even poor prognosis^8,9. P53 mutation can demonstrate abnormal gain-of-functions (GOF) to facilitate oncogenesis, metastasis, and chemoresistance¹⁰. Therefore, restoration wild-type function of mutant p53 (mutp53) or inactivation of mutp53 has great potential as a novel therapeutic strategy for CRC. However, the majority of molecules capable of making mutp53 loss of function or inhibition expression have only been tested in cell lines and animal models, there is no a good solution existed in clinical trials^6,11,12.

Our team designed and prepared an RNA interference library targeting a large number of genes in CRC previously. We screened a series of genes’ expression after RNA interference by high-content screening (HCS) technique¹³. We discovered that TRIM3 knockdown could significantly inhibit proliferation of RKO cells. Tripartite-motif containing 3 (TRIM3) is a member of the TRIM protein superfamily implicated in a wide range of physiologic processes such as immunity, proliferation, antiviral process, oncogenesis, and transcriptional regulation^14,15.TRIM3 is highly enriched in neurons and has been reported to be abnormally expressed in several human cancers, including glioblastoma and hepatocellular carcinoma^16–20. However, detailed expression and mechanism of TRIM3 in CRC remain unknown to date.

In the present study, we discovered that TRIM3 exhibited both pro-tumorigenic and tumor-suppressive effects dependend on p53 wild or mutant status in cell experiments. TRIM3 could directly interact with the C terminus of p53, a common segment of wtp53 and mutp53, to decrease p53 levels in the nuclei by retaining them in the cytoplasm. The reduced p53 could exert a pro-tumorigenic or tumor-suppressive role of colorectal carcinogenesis in a wtp53 or mutp53 dependent pathway. Moreover, TRIM3 could successfully reverse the sensitivity to oxaliplatin in mutp53 CRC cells by decreasing mutp53 in the nuclei to downregulate the multidrug resistance gene (MDR1). Therefore, TRIM3 could be a potential therapeutic strategy to improve the survival of CRC patients with mutp53 by degradation mutp53 in the nuclei.

Knockdown TRIM3 significantly inhibited RKO cell proliferation

Our team designed and prepared an RNA interference library targeting a series of genes associated with CRC globally by HCS technology. RKO cell, as a common of human colon cancer cell model, was used to transfect and screen their cellular proliferation abilities. The dynamic growth of cells with GFP-labeled was monitored twice a day for 3 consecutive days using HCS (Fig.1a). As illustrated in Fig.1a, the number of GFP-labeled cells in the TRIM3-shRNA group was greatly decreased compared to the control group in a time-dependent manner.

TRIM3 expression showed no significant difference in CRC tissues

Among the 32 paired cancerous and adjacent normal mucosal samples, TRIM3 mRNA expression was elevated in 56.3% (18/32) CRC tissues, compared to matched adjacent normal mucosa (Fig.1b). Subsequent western blot analysis demonstrated that TRIM3 protein levels were higher in cancerous tissues but not obvious (Fig.1c). Moreover, immunohistochemical staining indicated that, of the 348 adjacent non-cancerous mucosa samples in the paired TMA, 29% (101/348) were TRIM3 positive expression, In contrast, among the 348 CRC specimens, 58% (202/348) were TRIM3 positive and nearly half were negative expression (Fig.1d, Table.1).TRIM3 expression was quite discrepancy even in the same differentiated tumors.

TRIM3 displayed both pro-tumorigenic and tumor-suppressive features dependent on p53 wild or mutant status in CRC cells

To investigate the role of TRIM3 in CRC progression, we explored the capacities of proliferative, invasive, and migratory in CRC cells after TRIM3 knockdown or overexpression. The selection of CRC cell lines was based on TRIM3 expression (Fig.S1) (wtp53 cells: LoVo, RKO; mutp53 cells: SW480, HT29). After TRIM3 was knocked down in LoVo cells (wtp53), the cell growth curves of TRIM3-shRNA cells at the time points of 48 and 72h were significantly reduced compared with the control group (Fig.2a). The number of cells across the membrane in the matrigel-free and matrigel chamber in TRIM3-shRNA group was significantly lower in comparison to the control group (Fig.2b1). Meanwhile, the number and size of colonies in TRIM3-shRNA displayed a dramatic decrease of 2 fold relative to the control group (Fig.2c1). However, when TRIM3 was inhibited in HT29 cells(mutp53), the abilities of cell viability, migration, invasion, and colony formation were significantly increased compared with the control group, which was opposed to the results from LoVo cells(wtp53) (Fig.2a/2b1/2c1). When TRIM3 was overexpressed in RKO cells (wtp53), the abilities of cell proliferation, colony formation, migration, and invasion were obviously increased in comparison with the empty vector-transfected control group (Fig.2a/2b2/2c2). However, after TRIM3 was overexpressed in SW480 cells(mutp53), the abilities of cell proliferation, migration, invasion, and colony formation were significantly weakened, which was opposed to the results from RKO cells(wtp53) (Fig.2a/2b2/2c2).

Moreover, TRIM3 overexpression in RKO cells (wtp53) was associated with the cell cycle. The proportions of RKO cells with TRIM3 overexpression in the G0/ G1 phase of the cell cycle were obviously decreased compared with the control group (Fig.2d1). Meanwhile, TRIM3 overexpression in SW480 cells (mutp53) was related to cell apoptosis. The rates of apoptosis in SW480 cells with TRIM3 overexpression were significantly increased than the control group (Fig.2d2).

Therefore, there seemed to be a relationship between TRIM3 neoplastic features and p53 status (wild or mutant type). TRIM3 overexpression in wtp53 cells (LoVo and RKO) seemed to promote cancer and arrest cell cycle, while TRIM3 overexpression in mutp53 cells (HT29 and SW480) seemed to suppress cancer and promote cell apoptosis.

TRIM3 association with p53 in expression and direct binding to the C terminus of p53

The phenomenon that TRIM3 pro-tumorigenic and tumor-suppressive features were closely related with p53 wild or mutant status led us to explore the relationship between TRIM3 and p53. As shown in Table 1 and 2, of the 348 paired TMA, p53 expression was obviously up-regulated in cancerous tissues compared with that in the corresponding non-cancerous mucosa (P<0.001). Of note, TRIM3 expression was significantly correlated with p53 expression in clinicopathological parameters (P<0.001). The expression trends of TRIM3 and p53 were fundamentally consistent in the identical area of the same tumor tissues, as demonstrated by immunohistochemistry (Fig.3a). Moreover, confocal microscopy was conducted to analyze the localization of TRIM3-p53 co-staining. TRIM3 was located in the cytoplasm. P53 was predominantly throughout the nuclei, partly in the cytoplasm (Fig.3b). TRIM3 and p53 displayed co-staining as distinct punctate in the cytoplasm of RKO and SW480 cells with TRIM3 overexpression (Fig.3b). In addition, with Flag-tagged TRIM3 coprecipitation with HA-tagged p53 from 293T cell lysates, TRIM3 was discovered to interact with p53 directly (Fig.3c). Furthermore, HA-tagged wtp53 and C-terminal (320-393) truncation mutp53 were constructed and transfected into 293T cells expressing v5-TRIM3. The results indicated that TRIM3 interacted directly with the C terminus of p53 (residues 320 to 393), a common segment of wild and mutant p53 (Fig.3d).

TRIM3 retaining p53 in the cytoplasm to decrease p53 levels in the nuclei to exert a dual neoplastic feature

To explore the mechanisms of how TRIM3 bound with p53 to exert a dual neoplastic feature, we used TRIM3 and p53 as seeds to construct their related interaction network by Ingenuity Pathway Analysis (IPA) software. The predicted target genes and downstream pathway of TRIM3 and p53 were showed in Fig.4a. TRIM3 could retain p53 in the cytoplasm to decrease p53 levels in the nuclei by direct binding with each other to influence cell cycle and apoptosis-related proteins.

To verify the accuracy of prediction, we detected p53 expression in the nucleus and cytoplasm after TRIM3 overexpression. The results showed that TRIM3 overexpression in either RKO cells (wtp53) or SW480 cells (mutp53) could obviously increase p53 expression in the cytoplasm and decrease its expression in the nucleus but basically make no change in total protein levels (Fig.4b). Furthermore, to validate whether TRIM3 exerted a dual neoplastic feature in a wtp53 or mutp53 dependent pathway as predicted, HCT116^+/+ (p53 wild type), HCT116^-/- (p53-null) and SW480 (p53 mutant) were selected for further experiments.

As expected, western blot showed that TRIM3 overexpression in HCT116^+/+ (wtp53+) decreased the expression of P21 but had no obvious effect on apoptosis-related proteins such as Bcl-2, Bax, caspase 3/8. Moreover, in SW480 (p53 mutant) cells with TRIM3 overexpression, Bcl-2 was down-regulated, and Bax and caspase 3/8 were up-regulated, whereas there was no obvious change on P21. However, there was no significant effect on either P21 or apoptosis-related proteins in HCT116-/-(p53-null) cells at the absence of p53 (Fig.4c).

The patients of TRIM3 overexpression with chemotherapy had better prognosis

Cox univariate analysis suggested that decreased overall survival (OS) and disease-free survival (DFS) were associated with age, pT stage, pN stage, pM stage, and AJCC stage, without TRIM3. Multivariate analysis confirmed that only age and AJCC stage remained as independent prognostic factors (Table.3). TRIM3 was not an independent prognostic marker for CRC. Moreover, Kaplan-Meier curves showed that there was no significant difference in OS and DFS between TRIM3 positive patients and TRIM3 negative ones (Fig.4d). Further stratification analysis showed that it induced better outcomes in patients with chemotherapy than without chemotherapy in the TRIM3 positive group, especially with FOLFOX and CapeOX, whereas there was no significant difference in survival between the groups with chemotherapy and without chemotherapy in the TRIM3 negative group of CRC patients (Fig.4d). Next, we sought to explore the underlying molecular mechanisms of how CRC patients with TRIM3 positive expression could gain benefit from chemotherapy.

We compared the growth curves of colon cells with TRIM3 overexpression treated with 5-Fu and oxaliplatin (regimens of FOLFOX and CapeOX). It was shown that the half-maximal inhibitory concentration (IC50) valve of RKO(wtp53) and SW480(mutp53) cells with TRIM3 overexpression had no obvious change after treatment with 5-Fu, whereas IC50 had changed after treatment with oxaliplatin. The IC50 of oxaliplatin in RKO cells (wtp53) with TRIM3 overexpression was increased compared with the control group, but no statistical significance. However, the IC50 of oxaliplatin in SW480 cells (mutp53) with TRIM3 overexpression was significantly decreased (supplementary Fig.2a). TRIM3 overexpression could greatly increase the sensitivity of oxaliplatin in mutp53 CRC cells.

The decreased p53 in the nucleus could alter chemosensitivity to oxaliplatin by affecting MDR1

As shown in the IPA analysis of TRIM3 and p53 downstream pathway (Fig.4a), TRIM3 could decrease the levels of p53 in the nucleus by retaining p53 in the cytoplasm to alter ABCB1 (ATP-binding cassette subfamily B member 1, MDR1). The decreased wtp53 could arrest the cell cycle and activate MDR1. However, the decreased mutp53 in the nucleus could inhibit apoptosis and repress MDR1 to reverse chemotherapy resistance.

To explore the chemosensitivity of CRC cells with TRIM3 overexpression as predicted, we compared the downstream pathway of TRIM3 after treatment with oxaliplatin by western blot. The results showed that the levels of p53 in either RKO (wtp53) or SW480 cells(mutp53) were obviously increased treated with oxaliplatin (Fig.4e). However, compared with the control, the expression of p53 after TRIM3 overexpression was relatively decreased. Moreover, we analyzed differential expressions of p53 proteins between the nuclei and cytoplasm. We discovered that the levels of p53 in the cytoplasm were slightly increased whereas its expressions in the nuclei were significantly increased after treatment with oxaliplatin. Nevertheless, the expression level of p53 in the nuclei after TRIM3 overexpression was still reduced as compared with the control group, which was consistent with expression of total p53 (supplementary Fig 2b). Furthermore, the expression of p21 was reduced whereas MDR1 protein was increased in RKO cells (wtp53) with TRIM3 overexpression compared with the control group. However, in SW480 cells (mutp53), caspase 3/8 proteins were elevated, while MDR1 expression was decreased in response to TRIM3 overexpression, especially after treatment with oxaliplatin (Fig.4e).

Xenograft tumor growth and chemosensitivity of TRIM3 in vivo

Except in vitro results described above, we proceeded to investigate the dual neoplastic features and chemosensitivity of TRIM3 in vivo. As shown in Fig.5a1/2, the growth index of the tumors in the RKO of TRIM3 overexpression group (wtp53) was significantly higher than that of tumors in the control group. In contrast, the tumor volumes were obviously decreased in the SW480 of TRIM3 overexpression group (mutp53), compared with the control group (Fig.5b1/2). Meanwhile, we conducted RT-PCR for the specimen of tumor xenograft to analyze expression of p53 target genes. The results were consistent with those in vitro. It was showed that p21 mRNA levels were obviously increased while there was no obvious change of p53 and Bax expression in the tumor xenograft of RKO withTRIM3 overexpression (wtp53). Meanwhile, Bax mRNA expression was obviously increased whereas there was no obvious change of p53 and p21 in the tumor xenograft of SW480 with TRIM3 overexpression (mutp53).

Furthermore, we compared chemosensitivity to oxaliplatin of tumor xenograft with TRIM3 overexpression in RKO and SW480 cells (Fig.5c1). We discovered that the tumor volume of RKO with TRIM3 overexpression (wtp53) had no significant difference after treatment with oxaliplatin (Fig.5c2). However, the tumor volumes of TRIM3 overexpression in SW480 cells (mutp53) were obviously decreased after treatment with oxaliplatin (Fig.5c3).

P53, as a transcription factor, regulates a large number of diverse downstream genes to exert regulative function in multiple signaling processes. It is thought that p53 mutations play a critical role in the CRC adenoma-carcinoma transition process²¹.

P53 mutations in CRC not only impair wild-type p53 function (loss-of-function) but also provide neo-morphic (gain-of-function) activities such as promoting cell proliferation, invasion, metastasis, and chemoresistance, thereby promoting cancer progression⁹⁸. Therefore, making mutp53 loss of function or expression inhibition appears a straightforward and logical therapy in CRC. Recently, different approaches to target mutant p53 have been exploited including restoration of wild-type like activities to mutant p53, selective degradation of mutant p53, inhibition of novel protein–protein interactions involved in mediating gain-of-functions of mutant p53, exploitation of synthetic lethality vulnerabilities of mutant p53, inhibition of downstream survival pathways augmented by mutant p53, and immunotherapy based on the recognition of mutant p53 neoantigens²². However, current strategies most remain in the theoretical stage, except compound some small molecule inhibitors of p53^23,24.

TRIM3 is a member of the TRIM protein superfamily, containing an N-terminal zinc-finger and coiled-coil domains and a C-terminal NHL domain with E3 ubiquitin ligase activity²⁵. TRIM3 was identified as the candidate tumor suppressor gene in glioblastomas¹⁹, cervical cancer²⁶, esophageal squamous cell carcinoma²⁷, and hepatocellular carcinoma²⁸. In human gliomas, TRIM3 is a direct transcriptional target of p53 that in turn mediates degradative ubiquitination of the p53 response gene product CDKN1a/p21^17,29. TRIM3 could mediate p53 activity by ubiquitination in a double-edged way. However, the detailed expression and mechanism of TRIM3 in CRC remained unknown up to date.

It was reported that TRIM3 could function as a tumor suppressor in CRC progression through regulation of p53 protein in CRC cells³⁰. However, the expression of TRIM3 from TCGA or Oncomine database displayed the character between tumor suppressor and promoting tumor, preferred toward tumor suppressor. In our study, the proportion of TRIM3 negative expression was in agreement with the common rate of p53 mutation in CRC. Moreover, it was interesting to find that TRIM3 demonstrated a dual neoplastic feature related to p53 status (wild-type/mutant) in cell experiments. TRIM3 could act as a pro-tumorigenic gene in wtp53 cells (LoVo, RKO), while TRIM3 could serve as a tumor-suppressive gene in mutp53 cells (SW480, HT29). Moreover, TRIM3 was discovered to directly interact with p53 at the C terminus of p53 (residues 320 to 393), the common segment of wtp53 and mutp53. TRIM3 overexpression in either wtp53 or mutp53 cells could retain p53 in the cytoplasm and decrease its expression in the nucleus but make no change in total protein levels. Furthermore, TRIM3 exerted a dual role of neoplastic features in a p53-dependent manner confirmed by using three p53-specific cell lines. TRIM3 could decrease the levels of p53 in the nucleus to influence the cell cycle or apoptosis by retaining p53 in the cytoplasm dependent on p53 wild or mutant status (Fig. 5d). The decreased wtp53 could arrest the cell cycle to exert promoting cancer, while the reduced mutp53 could inhibit apoptosis to play the role of suppressing cancer (Fig. 5d). That was the reason why TRIM3 demonstrated a dual neoplastic feature of pro-tumorigenic gene in wtp53 cells and tumor-suppressive gene in mutp53 cells.

Generally, p53 mutant proteins get gain-of-function (GOF) properties via protein–protein interactions with other transcription factors and co-factors, or by binding directly to novel target genes²². While wild-type p53 mediates cellular responses by transactivating MDM2 for proteasomal degradation and ubiquitination in a negative feedback loop. Our team discovered that TRIM3 mediated p53 by direct interaction with p53 at the C terminus of p53 (residues 320 to 393) in CRC. Therefore, our strategy that TRIM3 made mutp53 proteins express suppression in the nuclei by retaining mutp53 in the cytoplasm belonged to a kind of selective degradation of mutant p53.

Chemotherapy is the basis of CRC treatment, except for surgery and radiotherapy³¹. However, chemotherapy therapy provides only a limited increase in overall survival due to chemotherapy resistance after treatment, especially for these advanced CRC patients. Moreover, chemotherapy resistance in CRC was often associated with overexpression of MDR1 gene, the most frequently expressed drug resistance genes in human tumors³². MDR1 overexpression has rendered many currently available chemotherapeutic agents ineffective. Studies have shown that inhibition of MDR1 reverses drug resistance in CRC³³. Our results showed that the patients of TRIM3 overexpression with chemotherapy had a better prognosis than those without chemotherapy. The chemosensitivity of oxaliplatin was significantly increased in mutp53 CRC cells after TRIM3 overexpression by decreasing the levels of wtp53 in the nucleus to inactivate MDR1 by western blot. In actuality, there were substantial evidences that p53 was involved in the regulation of MDR1. The mutp53 specifically stimulated MDR1 promoter activity whereas wtp53 exerted specific repression³⁴. Thus, making mutp53 losing function or inhibition expression could reduce or inhibit MDR1 and reverse the chemotherapy resistance of oxaliplatin³⁵.

In a word, our study was the first to demonstrate that TRIM3 could display a dual neoplastic feature of pro-tumorigenic gene in wtp53 cells and tumor-suppressive gene in mutp53 cells. TRIM3 could exert a dual role of colorectal carcinogenesis and alter chemosensitivity by retaining p53 in the cytoplasm to decrease p53 levels in the nuclei through direct binding with each other. Moreover, TRIM3 could successfully reverse the chemotherapy resistance of oxaliplatin in mutp53 cells by downregulation MDR1. Therefore, TRIM3 was a potential therapeutic strategy to improve the survival of CRC patients with mutp53 by degradation of mutp53 in the nuclei.

Patients and Tissue Specimens

A total of 32 couples of fresh tissue specimens (including tumor tissues and adjacent non-cancerous mucosa) were collected from CRC patients who underwent radical surgery at Shanghai General Hospital and Fudan University Shanghai Cancer Center and for qRT-PCR and western blot. Moreover, the total 348 pairs of preserved paraffin-embedded human CRC tissues and adjacent non-cancerous mucosa specimens were retrieved from the tumor tissue bank. The group comprised 169 males and 179 females. The number of patients with stage I, II, III, IV was 60, 155, 109 and 24 cases, respectively. These 348 pair samples were constructed into a tissue microarray to simultaneously detect protein expression in various tissues³⁶. None of the patients underwent preoperative chemotherapy or radiation therapy. 75.9% (264/348) of the patients received postoperative adjuvant chemotherapy. The detailed adjuvant chemotherapy characteristics of these 264 patients were presented in Supplementary Table 1. The four common chemotherapy regimens in our cohort were 5-Fu/LV (12 patients), Capecitabine (29 patients), FOLFOX (141 patients) and CapeOX (76 patients). Pathological diagnoses were confirmed by two pathologists according to the American Joint Committee on Cancer (AJCC).

Cell Culture and Transfection.

Human colorectal cancer cell lines (LoVo, HT29, RKO, SW480 and HCT116) were purchased from Shanghai Institute Cell Bank, Chinese Academy of Science (original source: American Type Culture Collection, ATCC, Manassas, VA, USA). HCT116^p53-/- was kindly provided by Professor Chuan-gui Wang (Translational Medicine Research Institute, Shanghai General Hospital, China). Cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 mg/ml streptomycin, and maintained at 37°C and 5% CO2 in a humidified incubator.

To establish cell lines with stable knockdown and overexpression, TRIM3 shRNA and overexpression lentiviral vectors were commercially designed and constructed by Shanghai Genechem Company (Shanghai, China).

RNA Extraction, Reverse Transcription and qRT-PCR.

RNA extraction and reverse transcription methods were performed as described previously ³⁷.The primers used in this study were listed in Supplementary Table 2A.

Western Blot.

Total proteins were extracted and western blot was performed as described previously³⁷. Proteins (40mg) were separated for western blot analysis. The detailed antibodies and their concentration were described in Supplementary Table 2B.

Immunohistochemistry.

Immunohistochemistry was performed as previously described³⁷. The detailed antibodies and their concentration were described in Supplementary Table 2B. The evaluation was based on the staining intensity and extension as described elsewhere³⁷.

Ingenuity Pathway Analysis.

To predict the relationship between TRIM3 and p53 and their downstream pathway, we performed pathway in-silico analysis with Ingenuity Pathway Analysis (IPA) software (Ingenuity Inc., CA, USA). TRIM3 and p53 were used as seeds to construct their related interaction network available in the Ingenuity database.

Cell Counting Kit-8 Assay.

The proliferation of CRC cells was assessed using a CCK-8 assay (DOJINDO, Kumamoto, Japan), in accordance to manufacturer’ instruction. The detailed concentrations of the four types of chemotherapy in our study were described in Supplementary Table 2C.

Colony Formation Assay.

The colony formation assay was performed as previously described³⁷.

Migration and Invasion Assay.

The assays were performed using standard techniques as described previously³⁷.

Flow cytometry analysis of the cell apoptosis and cycle.

Cell apoptosis was analyzed using the Annexin V-PE /7AAD Apoptosis Detection kit (BD, CA, USA).The cells were collected and resuspended in 500μl of 1×binding buffer. The cells were then stained for 15 min in the dark at room temperature using the apoptosis kit in accordance with the manufacturer’s instructions. The apoptotic cells were analyzed by flow cytometry (BD Accuri C6, USA). For the cell cycle, cells were harvested and fixed with 5 mL of prechilled 70% ethanol overnight at 4°C. The cells were then gently centrifuged and stained for 15 min by the Cell Cycle kit (BD, CA, USA). The cells were analyzed by flow cytometry (BD Accuri C6, USA).

Immunofluorescence

Cells plated on Nunc Glass Bottom Dish (ThermoFisher Scientific, MA, USA) were allowed to grow for 48h, fixed with 4% paraformaldehyde (Sangon Biotech, Shanghai, China), permeabilized with 0.1% Triton X-100 (Sangon Biotech, Shanghai, China). Dishes were incubated overnight at 4°C with anti-TRIM3 rabbit monoclonalantibody at 1:200 (abcam, CA, USA) and anti-p53 mouse monoclonal antibody at 1:250 (CST, CA, USA). Secondary antibody staining was performed with anti-rabbit and anti-mouse (CST, CA, USA), mounted with DAPI (CST, CA, USA) and imaged with confocal microscope.

Co-immunoprecipitation.

293T cells with overexpression TRIM3 or/and p53 were lysed in ice cold HEPES buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM MgCl2, 1 mM EGTA) containing protease inhibitors. After complete lysis by sonication, the lysates were centrifuged at 14000rpm to collect the supernatants. The extracts were incubated with antibodies against Flag and HA overnight at 4°C, followed by the addition of Protein A plus-Sepharose and incubation for 4h at 4°C. After 5 times wash with cell-lysis buffer, the beads were boiled with SDS loading buffer and then analyzed by SDS-PAGE and western blotting. Moreover, to analyze the interaction between TRIM3 and p53, we constructed HA-tagged wtp53 and C-terminal(320-393) truncation mutp53³⁸. The subsequent CoIP followed the same steps mentioned above.

Nude mice xenograft models.

To clarify the effect of TRIM3 in vivo, four-week-old male BALB/C nude mice purchased from the Institute of Zoology, at the Chinese Academy of Sciences of Shanghai were used to establish CRC xenografts. CRC cells (5×10⁶) with TRIM3 overexpression were suspended in 100 μL of PBS and subcutaneously injected into flanks of the mice. To verify the chemosensitivity to oxaliplatin of TRIM3 overexpression in vivo, a single dose of 5mg/kg oxaliplatin was injected via tail intravenous after 8 days of establishment xenograft models of TRIM3 overexpression. Tumor volume (mm³) was estimated weekly by using the following formula: tumor volume (mm³) = length×width²×0.5³⁹. All the mice were sacrificed after 4 weeks. All animal studies were performed in accordance with the animal care guidelines. All efforts were made to minimize animal suffering.

Statistical Analysis.

All data and survival analyses were calculated by using SPSS version 19.0 statistical software (SPSS, Chicago, USA). All experiments were conducted in triplicate to verify the reproducibility of the findings. The significance of the covariate differences was determined using a two-tailed χ2 or Fisher’s exact tests where appropriate. The survival rates were calculated by the Kaplan-Meier method and the differences between the survival curves were examined by the log-rank test. A Cox proportional hazards model was used to investigate the multivariate analysis and independent prognostic factors. The in vitro and in vivo data were expressed as the mean ± SD and were determined by the two-tailed Student’s t-test. All P-values <0.05 were considered to be statistically significant.

Acknowledgements

We thank Professor Chuan-gui Wang (Translational Medicine Research Institute, Shanghai General Hospital, China) for providing us with HCT116 ^p53-/- cells.

Conflict of interest

The authors declare that they have no conflict of interest.

Author Contributions

Y.H. and H.C. performed study concept and design; C.L.S. and Y.X. performed acquisition of data; C.L.S. and Y.X. performed analysis and interpretation of data; Y.H. and S.L. made statistical analysis; Y.H., S.L. and M.Z. wrote draft of the manuscript; H.C. and M.Z. revised the manuscript:. All authors read and approved the final paper.

Ethics Approval and Consent to Participate

The study was performed in accordance with the Declaration of Helsinki. All the procedures carried out in the research involving human participants are in accordance with the ethical standards of the Research Ethics Committee of the Shanghai General Hospital of Shanghai Jiao Tong University and Fudan University Shanghai Cancer Center (Project 2018KY127). All subjects provided written informed consent. Animal research has been approved and carried out in strict accordance with the institutional ethical guidelines of the Committee on the Use of Live Animals of Shanghai General Hospital.

Data Availability Statement

The data and material that were used or analyzed during the current study are available from the corresponding author on reasonable request.

Funding

This study was funded by the grants from National Natural Science Foundation of China (No. 81802439 and No.81602055)

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Table 1

Expression of TRIM3 and P53 in adjacent non-cancerous mucosa and cancer tissues

		TRIM3 expression			P53 expression
	N	Negative(%)	Positive(%)	P-value	Negative(%)	Positive(%)	P-value
Adjacent mucosa	348	247(71%)	101(29%)	<0.001*	303(87.1%)	45(12.9%)	<0.001*
Cancer tissues	348	146(42%)	202(58%)	<0.001*	136(39.1%)	212(60.9%)	<0.001*

P-value are based on chi-squared.

*Significant difference.

Table 2

Correlation Between clinicopathological features and TRIM3 expression (N=348)

		TRIM3 expression
	N	Negative(146)	Positive(202)	P
Age				0.533
<50 years	20	6(30.0%)	14(70.0%)
50-75 years	207	90(43.5%)	117(56.5%)
>75 years	121	50(41.3%)	71(58.7%)
Sex				0.232
Male	169	65(38.5%)	104(61.5%)
Female	179	81(45.3%)	98(54.7%)
Tumor size				0.604
<4 cm	115	52(45.2%)	63(54.8%)
4-6 cm	175	69(39.4%)	106(60.6%)
>6 cm	58	25(43.1%)	33(56.9%)
Tumor location				0.403
Ascending	85	34(40.0%)	51(60.0%)
Transverse	16	6(37.5%)	10(62.5%)
Descending	30	17(56.7%)	13(43.3%)
Sigmoid	91	41(45.1%)	50(54.9%)
Rectum	126	48(38.1%)	78(61.9%)
T stage				0.172
T1	19	9(47.4%)	10(52.6%)
T2	51	28(54.9%)	23(45.1%)
T3	198	80(40.4%)	118(59.6%)
T4	80	29(36.3%)	51(63.8%)
N stage				0.920
N0	224	96(42.9%)	128(57.1%)
N1	84	34(40.5%)	50(59.5%)
N2	40	16(40.0%)	24(60.0%)
M stage				0.010*
M0	324	142(43.8%)	182(56.2%)
M1	24	4(16.7%)	20(83.3%)
AJCC stage				0.026*
I	60	31(51.7%)	29(48.3%)
II	155	63(40.6%)	92(59.4%)
III	109	48(44.0%)	61(56.0%)
IV	24	4(16.7%)	20(83.3%)
Differentiation				0.619
Well	124	50(40.3%)	74(59.7%)
Moderate	206	90(43.7%)	116(56.3%)
Poorly	18	6(33.3%)	12(66.7%)
P53				<0.001*
Negative	136	79(58.1%)	57(41.9%)
Positive	212	67(31.6%)	145(68.4%)

P-value are based on chi-squared or Fisher's exact test

*Significant difference

Table 3

Univariate and Multivariate Analysis of Overall Survival and Disease‒free Survival After Surgery (N=348)

	Overall survival				Disease‒free survival
	Univariate analysis		Multivariate analysis		Univariate analysis		Multivariate analysis
	HR(95%CI)	P	HR(95%CI)	P	HR(95%CI)	P	HR(95%CI)	P
Age
﹤50yr	-	0.006*	-	0.002*	-	0.008*	-	0.004*
50-75yr	2.204(0.534-9.102)	0.275	2.282(0.551-9.448)	0.255	2.109(0.511-8.710)	0.302	2.152(0.520-8.913)	0.291
﹥75yr	4.082(0.987-16.882)	0.052	4.474(1.080-18.530)	0.039*	3.860(0.933-15.964)	0.062	4.158(1.004-17.220)	0.049*
Gender
Male
Female	0.993(0.651-1.516)	0.975			0.956(0.626-1.459)	0.835
Tumor size
﹤4 cm	-	0.352			-	0.405
4-6 cm	1.331(0.811-2.185)	0.258			1.308(0.797-2.147)	0.288
﹥6 cm	1.538(0.826-2.863)	0.175			1.489(0.800-2.772)	0.209
Tumor location
Ascending	-	0.142			-	0.131
Transverse	0.715(0.215-2.381)	0.584			0.685(0.206-2.282)	0.538
Descending	1.333(0.634-2.803)	0.448			1.346(0.641-2.829)	0.433
Sigmoid	0.533(0.274-1.035)	0.063			0.532(0.274-1.034)	0.063
Rectum	1.098(0.650-1.854)	0.726			1.104(0.654-1.864)	0.711
T stage
T1	-	0.046*			-	0.043*
T2	2.154(0.477-9.723)	0.318			2.123(0.471-9.582)	0.327
T3	2.271(0.551-9.366)	0.256			2.251(0.546-9.281)	0.262
T4	3.966(0.944-16.660)	0.060			3.965(0.944-16.650)	0.060
N stage
N0	-	<0.001*			-	<0.001*
N1	2.338(1.458-3.749)	<0.001*			2.294(1.431-3.677)	<0.001*
N2	2.769(1.547-4.958)	0.001*			2.717(1.518-4.864)	0.001*
M stage
M0
M1	3.426(1.896-6.192)	<0.001*			3.307(1.831-5.974)	<0.001*
AJCC stage
I	-	<0.001*	-	<0.001*	-	<0.001*	-	<0.001*
II	1.074(0.501-2.300)	0.855	1.117(0.521-2.395)	0.775	1.083(0.505-2.320)	0.838	1.114(0.520-2.388)	0.781
III	2.706(1.310-5.588)	0.007*	2.938(1.420-6.080)	0.004*	2.662(1.289-5.496)	0.008*	2.874(1.389-5.947)	0.004*
IV	5.376(2.294-12.596)	<0.001*	5.632(2.401-13.211)	<0.001*	5.178(2.211-12.126)	<0.001*	5.296(2.259-12.415)	<0.001*
Differentiation
Well	-	0.186			-	0.232
Moderate	1.557(0.966-2.509)	0.069			1.509(0.937-2.432)	0.091
Poorly	1.521(0.580-3.987)	0.394			1.503(0.573-3.940)	0.408
TRIM3
Negative
Positive	0.911(0.595-1.394)	0.667			0.904(0.590-1.384)	0.643
P53
Negative
Positive	1.489(0.944-2.349)	0.087			1.485(0.941-2.341)	0.089

HR hazard ratio;CI confidence interval

*P<0.05 indicated that the 95% CI of HR was not including 1

(Not answered)

SupplementaryFigure1.tif
Supplementary Figure 1 The TRIM3 mRNA expression in different CRC cell lines.
SupplmentaryFigure2.tif
Supplementary Figure 2 The change of IC50 values of 5-Fu and oxaliplatin and the expression of p53 in the nuclei and cytoplasm after TRIM3 overexpression treated with different concentrations of oxaliplatin. a1 The inhibition rate of RKO and SW480 cells treated with different concentrations of 5-Fu; a2 The inhibition rate of RKO and SW480 cells treated with different concentrations of oxaliplatin; a3 The change of IC50 values of 5-Fu and oxaliplatin; b The expression of p53 in the nuclei and cytoplasm after TRIM3 overexpression treated with different concentrations of oxaliplatin. All data were obtained from three independent experiments, and expressed as mean ± standard deviation. *, ** P< 0.05 in comparison with control groups.
SupplementaryTable1.docx
Supplementary Table 1 Adjuvant chemotherapy for CRC patients in our cohort (N=348)
SupplementaryTable2revised.doc
Supplementary Table 2A Primer sequences used for qPCR in this study Supplementary Table 2B Antibodies applied in this study Supplementary Table 2C The Concentrations of four kinds of chemotherapy in our study

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The dual role of TRIM3 in colorectal cancer dependent on p53 status by retaining p53 in the cytoplasm to decrease its expression in the nuclei

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