The combination of WNT974 and ART exhibited synergistic effect in reducing CRC cell viability (Fig. 1A) as indicated by the combination index (CI) < 1. Interestingly, the combination of WNT974 and ART significantly reduced KRAS protein levels (Fig. 1B) in the CRC cells when compared to WNT974 or ART mono-treatments. The treatment did not affect the KRAS mRNA levels (Fig. 1C). We also examined the KRAS activity in these cells by using Raf1 RBD agarose beads to selectively pull-down the active form of KRAS from the protein samples. Figure 1B showed that the combination treatment significantly reduced KRAS activity in the CRC cells when compared to control or the monotreatments. These results imply that the reduced KRAS protein expression and activity may be associated with the synergistic effect of the combination treatment in reducing CRC growth.
The combination of WNT974 and ART induces KRAS protein degradation in CRC cells
Post-translational modifications of KRAS protein such as ubiquitylation and degradation may reduce the protein expression . The ubiquitin proteasome pathway consists of concerted actions of enzymes that link chains of the polypeptide co-factor, ubiquitin, onto proteins to mark them for degradation . We next examined whether the combination treatment affected KRAS ubiquitination. As shown in Fig. 2A, the combination treatment markedly enhanced KARS ubiquitination in the CRC cells when compared to the mono-treatments. To further examine whether the treatment affected KRAS protein degradation, we treated the cells with WNT974, ART or the combination of both in presence or absence of MG132. MG132 is a specific, potent, reversible cell-permeable proteasome inhibitor . As shown in Fig. 2B and 2C, MG132 abolished the combination treatment-reduced KRAS protein expression, suggesting the combination of WNT974 and ART induces KRAS degradation via the ubiquitination proteasome pathway.
The combination of WNT974 and ART induces KRAS protein degradation by increasing anaphase promoting complex subunit 2 (ANAPC2) expression
Next, we examined how the combination treatment induced KRAS degradation in CRC cells. Firstly, we used PCR array for the human ubiquitination pathway (genes layout in Table 1) to examine whether the treatments affected the expressions of the genes that are involved in the ubiquitination pathway. As shown in Fig. 3A, the heat map indicated that the treatments affected the gene expressions, the numbers of up- or down-regulated genes were shown in Fig. 3B. The results showed that, compared to control, ART treatment upregulated 15 genes and downregulated 34 genes; WNT974 treatment upregulated 13 genes and downregulated 20 genes; and the combination treatment upregulated 18 genes and downregulated 27 genes. Compared to ART treatment, the combination treatment upregulated 12 genes and downregulated 9 genes. Compared to WNT974 treatment, the combination treatment upregulated 12 genes and downregulated 10 genes. More importantly, we found that 5 genes were upregulated by more than 2-fold under the combination treatment when compared to the mono-treatments. These ubiquitination related genes were CUL7 (cullin), ANAPC2, UBE2M (ubiquitin conjugating enzyme E2M), SYVN1 (synoviolin 1) and RNF123 (RING finger protein123) (Fig. 3C),
To examine whether these gene candidates were involved in the combination treatment reduced KRAS protein expression, we used siRNA to mediate the knockdown of the gene candidates before the treatments. The upper panels in Figs. 3D to 3H showed the siRNA-mediated knockdown of the candidates (CUL7, ANAPC2, UBE2M, SYVN1, RNF123) in the CRC cells. We found that the combination treatment could significantly reduce KRAS protein level in the CUL7-, UBE2M-, SYVN1-, RNF123-knockdown cells (Fig. 3D, 3F to 3H). However, the combination treatment failed to reduce KRAS protein level in the ANAPC2-knockout cells (Fig. 3E), implying ANAPC2 was involved in the combination treatment-reduced KRAS protein expression.
We further validated the role of ANAPC2 in KRAS protein degradation. We found that ANAPC2 overexpression (Fig. 3I) significantly reduced KRAS protein levels (Fig. 3J), which was reversed in the presence of MG132 (Fig. 3J). Furthermore, we also found that the combination treatment significantly increased ANAPC2 protein expression when compared to the mono-treatments (Fig. 3K). Our data strongly suggest that the combination treatment increases ANAPC2 expression and hence increases KRAS protein degradation in the CRC cells.
The combination of WNT974 and ART induces KRAS protein degradation by increasing β-TrCP and GSK-3β expressions
We further explored whether other gene candidates would affect KRAS expressions and degradation under the combination treatment. Other studies have reported that elF5A and PEAK increased KRAS protein synthesis; NEDD4 and β-TrCP which are E3 ligase will promote KRAS degradation; SMURF2 and UBCH5 as a critical E3:E2 complex maintaining KRAS protein stability [34–36]. In our study, we found that combination treatment did not significantly affect the mRNA expressions of elF5A, NEDD4, PEAK, SMURF2, UBCH5 (Supplementary Figure S1A to S1J), but significant increased β-TrCP mRNA level (Fig. 4A and 4B) and protein level (Fig. 4C) in the CRC cells. β-TrCP is an F-box ubiquitin ligase, it has been implicated in RAS (including KRAS) ubiquitination and degradation [35, 37, 38]. β-TrCP degrades KRAS protein and in this degradation process, glycogen synthase kinase-3 beta (GSK-3β) mediates the phosphorylation of KRAS for the priming of β-TrCP to the KRAS protein for degradation . Interestingly, the combination treatment not only increased β-TrCP expression but also GSK-3β expression (Fig. 4D).
We then examined whether β-TrCP would affect the KRAS protein expression in the CRC cells.
We used siRNA to mediate the knockdown of β-TrCP in the CRC cells (Fig. 4E to 4H). We found that in the β-TrCP-knockdown cells, KRAS protein expression was increased (Fig. 4I and 4J). More importantly, in these cells, in the presence of GSK-3β inhibitor, the combination treatment failed to induce KRAS ubiquitination (Fig. 4K) and reduce KRAS protein expression (Fig. 4L). Taken together, the data strongly suggest that the combination treatment increases β-TrCP and GSK-3β expressions that lead to the KRAS degradation in CRC.
The combination of WNT974 and ART exhibits a potent anti-CRC effect in vivo
Next, we examined the anti-CRC effect of the treatments with CRC-bearing xenograft mouse model. After 12-days of treatment, the combination treatment significantly reduces the tumor size (Fig. 5A and 5B) and the percentage increase in tumor size (Fig. 5C) when compared to the control and monotreatments. The combination treatment did not significantly affect the body weight of the mice (Fig. 5D), suggesting the treatments do not have apparent toxicity to the mice. In parallel with the in vitro data, the combination treatment significantly increased β-TrCP and GSK-3β expressions and reduced KRAS expression levels in the tumor tissues of these mice (Fig. 5E and 5F).