The positive impact of TRIP-Br1 on IR expression results in a higher IR/IGF1R ratio
The effects of TRIP-Br1 expression on the IR/IGF1R ratio were initially evaluated in normal (MCF10A) and breast cancer cell lines (Fig. 1A-B). While normal cells showed similar IR and IGF1R expression levels, the majority of the breast cancer cell lines showed significantly higher levels of IR expression than IGF1R expression, resulting in a high IR/IGF1R ratio (Fig. 1A-B). In particular, four cancer cell lines (MDA-MB-453, MDA-MB-468, BT20, and BT549) with very high levels of TRIP-Br1 showed a much higher IR/IGF1R ratio than the other cancer cell lines (Fig. 1A-B). In our previous and unpublished studies, we found that TRIP-Br1 gene expression was always very high regardless of stressful stimuli in these four cell lines, while TRIP-Br1 expression was greatly increased in MCF7 and MDA-MB-231 cell lines in response to various cell death-inducing stressful conditions . Therefore, the MCF7 and MDA-MB-231 cell lines were selected for further study.
First, the impact of TRIP-Br1 on IR expression was tested in MEFs isolated from TRIP-Br1 wild-type (MEFWT-TRIP-Br1) and knockout (MEFKO-TRIP-Br1) mice. Interestingly, a higher level of IR expression was found in MEFWT-TRIP-Br1 compared to MEFKO-TRIP-Br1 cells (Fig. 1C-D). The knockout of TRIP-Br1 was confirmed by genotyping, as shown in Supplementary Fig. S1A and S1B. Similar results were obtained in the confocal immunofluorescence experiment (Fig. 1E-F). Furthermore, TRIP-Br1 wild-type mice also showed approximately 2-4 fold higher IR expression levels in adipocyte and heart tissue samples compared with TRIP-Br1 knockout mice (Fig. 1G-H). MCF7 cells with wild-type TRIP-Br1 (MCF7WT-TRIP-Br1) also showed a much higher IR/IGF1R ratio than TRIP-Br1 knockdown stable MCF7 cells (MCF7KD-TRIP-Br1) (Fig. 1I-J). MCF7WT-TRIP-Br1 cells showed higher IR but lower IGF1R expression levels than MCF7KD-TRIP-Br1 cells (Fig. 1J-K). We also analyzed the total amount of IR and IGF1R using a co-antibody that recognizes both IGF1R and IR. No significant difference was detected between MCF7WT-TRIP-Br1 and MCF7KD-TRIP-Br1 cells, implying an inverse or compensatory relationship between them (Fig. 1J-K). Interestingly, we found an inverse relationship between IR and IGF1R expression. IR silencing significantly increased the IGF1R protein levels in MCF7 and MDA-MB-231 cells (Supplementary S1C-D). In a further study, much higher levels of IR ubiquitination were detected in MCF7KD-TRIP-Br1 cells than in MCF7WT-TRIP-Br1 cells after treatment with a proteasome inhibitor (MG132), but not with an autophagy inhibitor (CQ), suggesting that TRIP-Br1 suppresses proteasome-mediated degradation of IR (Fig. 1L-M).
These data clearly indicate that TRIP-Br1 positively regulates the expression of IR at the protein level, thereby enhancing the IR/IGF1R ratio.
TRIP-Br1-mediated IGF1R downregulation results in a higher IR/IGF1R ratio
TRIP-Br1 was found to contribute to a relatively high IR/IGF1R ratio by positively affecting IR expression. In addition, we examined the effects of TRIP-Br1 on IGF1R expression. First, the effect of TRIP-Br1 on IGF1R expression was tested in MCF7 and MDA-MB-231 cells. While TRIP-Br1 overexpression significantly decreased IGF1R expression (Fig. 2A-B), TRIP-Br1 silencing greatly increased IGF1R expression (Fig. 2C-D). In addition, MCF7KD-TRIP-Br1 cells also showed much higher IGF1R expression than MCF7WT-TRIP-Br1 cells (Fig. 2E-F). Furthermore, MEFKO-TRIP-Br1 cells also showed a significant increase in IGF1R expression in comparison with MEFWT-TRIP-Br1 (Fig. 2G-H). Lastly, TRIP-Br1 knockout mice showed elevated IGF1R in adipocytes (~ 20-fold) and the heart (~ 2-fold) compared to control mice (Fig. 2I-J).
Overall, these data strongly suggest that TRIP-Br1 negatively affects IGF1R expression, eventually increasing the IR/IGF1R ratio in breast cancer cells.
IGF1R protein levels decreased by TRIP-Br1 as an adopter protein and NEDD4-1 E3 ligase results in a higher IR/IGF1R ratio
Next, we investigated how TRIP-Br1 downregulates the IGF1R protein levels. TRIP-Br1 directly binds to two E3 ubiquitin ligases, NEDD4-1 and XIAP, as an adaptor protein [38-39, 41]. In addition, multiple lines of evidence have indicated that NEDD4-1 is an E3 ubiquitin ligase responsible for IGF1R degradation [42-46]. For example, oxidative stress-mediated NEDD4-1 upregulation degrades IGF1R during neurodegeneration . However, no direct interaction between the IGF1R and NEDD4-1 has been reported, implying a possible constraint, such as the need for an adaptor protein. Therefore, we investigated whether TRIP-Br1 is responsible for the ubiquitination and degradation of IGF1R by interacting with NEDD4-1 or XIAP. Interestingly, the IGF1R expression levels were greatly increased in TRIP-Br1 and/or NEDD4-1 silenced MCF7 and MDA-MB-231 cell lines (Fig. 3A-B). However, little change was observed in TRIP-Br1/XIAP double knockdown cells (Supplementary Fig. S2). The effect of NEDD4-1 on IGF1R degradation was also assessed. The IGF1R protein levels increased considerably after NEDD4-1 silencing in the presence of cycloheximide (CHX), a protein synthesis blocker (Fig. 3C-D). Co-immunoprecipitation experiments showed a direct interaction between TRIP-Br1 and IGF1R, as well as between NEDD4-1 (Fig. 3E-F). However, no direct interaction was observed between TRIP-Br1 and IR (Fig. 3E). Co-immunofluorescence experiments also revealed a higher co-localization of endogenous NEDD4-1 and IGF1R in MCF7WT-TRIP-Br1 cells than in MCF7KD-TRIP-Br1 cells, implying that TRIP-Br1 appears to serve as an adaptor protein to bring NEDD4 close enough to IGF1R (Fig. 3G-H).
Taken together, our data strongly suggest that TRIP-Br1 functions as an adapter protein and plays a vital role in NEDD4-1-mediated IGF1R downregulation.
TRIP-Br1/NEDD4-1 mediated IGF1R degradation through a proteasome/ubiquitination
The degradation of many ligand-induced receptors is mediated through the ubiquitination of the receptors, followed by proteasome- or lysosome-dependent degradation. The binding of IGF1 to IGF1R leads to the polyubiquitination of IGF1R .Our previous and unpublished data showed that TRIP-Br1 plays an important role in both pathways [39, 41]. Thus, we evaluated which pathway is responsible for TRIP-Br1/NEDD4-1 mediated IGF1R degradation. To test this hypothesis, siNEDD4-1 was transfected into MCF7WT-TRIP-Br1 and MCF7KD-TRIP-Br1 cells in the absence or presence of MG132 or CQ. NEDD4-1 silencing significantly increased the IGF1R protein levels in the presence of MG132 (Fig. 4A-B) but only slightly increased after CQ treatment in MCF7WT-TRIP-Br1 cells (Fig. 4C-D). Similar results were obtained for the IGF1R expression levels after treatment with MG132 (Fig. 4E-F) but not with CQ (data not shown). These findings suggest that TRIP-Br1/NEDD4-1-mediated IGF1R degradation occurs mainly through the proteasome/ubiquitination pathway rather than through a lysosomal pathway.
Enhanced tumor formation is associated with a higher IR/IGF1R ratio resulting from TRIP-Br1 expression
Next, we examined the IR/IGF1R ratio in TRIP-Br1-mediated tumor formation and growth using a xenograft model. MCF7WT-TRIP-Br1 and MCF7KD-TRIP-Br1 cells were subcutaneously injected into nude mice and tumor size was measured on the indicated days (Fig. 5A). Our results revealed a significant reduction in the tumor volume in MCF7KD-TRIP-Br1 pretreated mice (Fig. 5A-B). A marked decrease (>90%) in tumor weight was observed in tumors collected from null mice injected with MCF7KD-TRIP-Br1 compared with MCF7WT-TRIP-Br, suggesting that TRIP-Br1 is effective in strengthening in vivo tumor formation and growth (Fig. 5C). In agreement with the in vitro observations, an approximately 10-fold higher IR/IGF1R ratio, due to the higher IR but lower IGF1R, was detected in MCF7WT-TRIP-Br1 cells grown in null mice. This suggests that a higher IR/IGF1R ratio could enhance the growth and proliferation of breast cancer cells (Fig. 5D-F). This result is consistent with those of previous studies. Although the inhibition of IGF1R was previously reported to not affect tumor growth in preclinical trials, it was found to enhance the IR signaling pathway, enhancing multistage tumors as a result [29-30]. These results suggest that IR, rather than IGF1R, may be responsible for the better growth and survival of cancer cells. This hypothesis was tested by examining the effect of IR on the survival of MCF7 cells in response to three different anticancer drugs (doxorubicin, staurosporine, and paclitaxel), namely, resistance to anticancer-mediated cell death. The MCF7 cell line is well known for its high resistance to programmed cell death against various anticancer drugs. Cell viability was found to be lower in IR-silenced MCF7 cells than in control cells after treatment with anticancer drugs (Fig. 5G).
Overall, these results indicate that TRIP-Br1 provides breast cancer cells with a better capacity for proliferation and survival by increasing the IR/IGF1R ratio even after treatment with anticancer drugs.
TRIP-Br1 induces a higher IR/IGF1R ratio in insulin-deficient mice mimicking diabetes
The effect of TRIP-Br1 on the IR/IGF1R ratio and the inverse relationship between IR and IGF1R expression were further tested in insulin-deficient mice mimicking diabetes patients, in which decreased insulin and increased glucagon levels were used as controls (Fig. 6A-B). As in our previous studies, in which a deficiency of insulin or IGF1 was found to trigger TRIP-Br1 upregulation in breast cancer cells , a similar pattern was observed in our animal model (Fig. 6A-B). Insulin-deficient mice showed significantly elevated TRIP-Br1 protein levels, which were accompanied by increased IR but decreased IGF1R in all three examined tissue samples (heart, liver, and adipocytes), confirming the positive effect of TRIP-Br1 on the higher IR/IGF1R ratio (Fig. 6A-B). Representative images of immunohistochemical staining for TRIP-Br1, IR, and IGF1R in insulin-deficient mice and the corresponding normal mouse tissues are shown in Fig. 6C. Again, significantly higher TRIP-Br1 and IR, but lower IGF1R expression levels, were observed in insulin-deficient mice compared to normal mouse tissues, resulting in a higher IR/IGF1R ratio in insulin-deficient mice (Fig. 6D). These findings strongly suggest that TRIP-Br1 positively regulates IR but negatively regulates IGF1R expression, resulting in a higher IR/IGF1R ratio, even in insulin-deficient mice.
Taken together, these results imply that TRIP-Br1 is also at least partly responsible for the induction of a higher IR/IGF1R ratio in patients with diabetes and breast cancer.