TRIM37 was overexpression in chemo-resistance ovarian cancer tissues.
It has been reported that tripartite motif (TRIM) family proteins have various functions in cellular processes including cell stress responses, signal transduction, immune system diseases and carcinogenesis[15, 24, 25]. However, the biological function of TRIM family proteins in ovarian cancer chemo-resistance remain unclear. The real-time PCR assay showed that among 64 genes of TRIM family proteins, TRIM37 were markedly overexpressed in chemo-resistance ovarian cancer tissues compared to chemo-sensitive ovarian cancer tissues (Fig. 1A). Western blotting analyses revealed that the protein level of TRIM37 was markedly overexpressed in four chemo-resistance ovarian cancer tissues, compared with four chemo-sensitive ovarian cancer tissues (Fig. 1B). Furthermore, IHC assay showed that TRIM37 expression was markedly overexpressed in 145 paraffin-embedded, archived ovarian cancer tissues which received standardized platinum-based chemotherapy (Fig. 1C). Collectively, these results indicate that TRIM37 expression is upregulation in chemotherapy resistant ovarian cancer.
TRIM37 was correlates with progression and poor prognosis in human ovarian cancer.
To determine the clinical relevance of TRIM37 in ovarian cancer, TRIM37 expression was examined and analysed in 145 paraffin-embedded, archived ovarian cancer tissues. As showed in Supplementary Table 1–2, TRIM37 levels were correlated with the FIGO stage (P < 0.001), and chemo-resistance (P = 0.008) in patients with ovarian cancer. Importantly, statistical analysis showed that ovarian cancer patients with high TRIM37 expression had significantly worse overall and disease-free survival than those with low TRIM37 expression (Fig. 2A-B). Consistent with our results, we found that TRIM37 overexpression was correlated with overall survival and progression-free survival of ovarian cancer patients with platin-resistance in multiple public GEO datasets (Fig. 2C-D). These results suggest that TRIM37 has potential clinical value as a predictive biomarker for ovarian cancer chemo-resistant.
Upregulation of TRIM37 contributes cytotoxicity of ovarian cancer cells in vitro.
GSEA analysis revealed that TRIM37 overexpression was strongly correlated with gene signatures associated with CDDP-based chemotherapy signatures, suggesting that TRIM37 overexpression may contribute to CDDP-resistance in ovarian cancer (Fig. 3A). To investigate the chemo-resistance role of TRIM37 in ovarian cancer, A2780 that stably expressed TRIM37 and A2780/cis that silencing TRIM37 cell lines were established (Fig. 3B). IC50 assay shown that A2780 that stably expressed TRIM37 enhanced cisplatin resistance compared with the vector control (IC50 values were 5.24 µM and 21.3 µM, respectively) (Fig. 3C). However, A2780/cis that depleted of TRIM37 were less resistant to cisplatin than control (IC50 values were 1.67 µM, 2.01 µM, and 16.85 µM, respectively) (Fig. 3C).
TRIM37 confers CDDP resistance in ovarian cancer in vitro.
Furthermore, the Annexin V and Tunel staining assay show that the percentage of apoptotic cells in A2780/TRIM37 ovarian cancer cells treated with CDDP was much lower compared than that in control cells, but much higher in A2780/cis TRIM37-shRNA cancer cells (Fig. 4A-B). Furthermore, the protein level of cleaved caspase 3, an apoptosis relative gene, was significantly decrease in A2780/TRIM37 overexpression ovarian cancer cells but increase in A2780/cis TRIM37-shRNA cancer cells (Fig. 4C). Interestingly, we also found that overexpressing TRIM37 or silencing TRIM37 only resulted in slightly change of apoptotic rate of ovarian cancer cells without any treatment (Fig. 4D). The above results indicating that deregulation of TRIM37 is involved in CDDP resistance of ovarian cancer cells.
Upregulation of TRIM37 activates the Wnt/β-catenin signalling pathway in ovarian cancer.
In order to better understand the mechanism of TRIM37 induced chemo-resistance, mRNA microarray (GEO-GSE8057) was performed. Gene ontology (GO) enrichment analysis showed that Wnt/β-catenin signalling pathway was enriched in TRIM37 up-regulated genes (Fig. 5A). GSEA analysis show that TRIM37 mRNA expression levels was positively correlated with Wnt/β-catenin signalling gene signatures (Fig. 5B). These results suggest that TRIM37 may play crucial roles in Wnt/β-catenin signalling regulation. As expected, overexpression of TRIM37 significantly enhanced, whereas silencing of TRIM37 reduced, the activity of TOP/FOP luciferase reporter activity in ovarian cancer cells (Fig. 5C). Moreover, western blotting revealed that the levels of nuclear β-catenin were dramatically upregulated in TRIM37-overexpressing cells but were downregulated in TRIM37-silenced cells (Fig. 5D). Furthermore, the expression levels of numerous well-characterized Wnt/β-catenin signalling downstream genes were showed to be increased in TRIM37 overexpressing cells, but were lower in TRIM37-silenced cells (Fig. 5E). These results suggesting that TRIM37 plays an important role in activating the Wnt/β-catenin signalling pathway in ovarian cancer.
Wnt/β-catenin signalling pathway is required for TRIM37 induced chemo-resistance.
Next, we investigated whether TRIM37 mediated ovarian cancer chemo-resistance through Wnt/β-catenin activation. The chemo-resistant effect of TRIM37 on ovarian cancer through Wnt/β-catenin activation was determined by Annexin V and Tunel staining assay. Strikingly, we found that blockade of the Wnt/β-catenin pathway by β-catenin siRNA significantly abrogates the effect of TRIM37 on ovarian cancer aggressiveness in both in vitro (Fig. 6A-B). Similar to the effect of β-catenin silencing in TRIM37 overexpression cells, treatment with ICG-001, a specific inhibitor of β-catenin signalling via blockage of β-catenin/CBP interaction also significantly decreased the effect of TRIM37 on ovarian cancer chemo-resistance (Fig. 6A-B). Taken together, these results indicate that activation of the Wnt/β-catenin signalling pathway exerted functional effects of TRIM37 on ovarian cancer chemo-resistance.