TRB3 Mediates Insulin/IGF-promoted Tumorigenesis And Progression

Type 2 Diabetes (T2D) is a serious and growing health problem worldwide and is associated with severe acute and chronic complications. Accruing epidemiological and clinical evidences have suggested that diabetes can promote carcinogenesis and tumor progression. Indeed, cancer and T2D share a number of metabolic risk factors, among which high insulin/IGF-1, hyperglycaemia, glucose deprivation, hypoxia and inflammatory factors have been considered to be potential biologic links between the two diseases. High insulin and high insulin-like growth factor are considered to be the major risk factors for cancer, but the underlying molecular mechanism remains unclear.Recently, we found that the pseudokinase tribbles homolog 3 (TRB3), a stress and metabolic sensor, plays a crucial role in transforming growth factor (TGF)-β1-mediated cancer invasion and migration by interacting with signal molecule SMAD3. Given that TRB3 senses a variety of metabolic and stress signals and that the enhanced TRB3 expression leads to insulin resistance. We seek to establish a TRB3-mediated mechanism connecting metabolic stresses to tumor promotion. We have recently demonstrated a positive correlation between TRB3 and phosphorylated insulin receptor substrate-1 (p-IRS-1) levels in several tumor tissues. Moreover, the expression levels of TRB3 and p-IRS-1 are negatively correlated with survival rates of patients with these cancers. Metabolic stresses, such as hyperglycemia, hyperinsulinemia, high 1GF-1 can all induce TRB3 expression. TRB3 mediates insulin/IGF-1-induced reactive oxygen species production, DNA damage, evasion of apoptosis and proliferation in cancer cells, whereas silencing TRB3 impedes the malignancy-promoting actions. What is more, high expression of TRB3 is responsible for the enhanced tumor proliferation and metastasis in diabetic mice. These data indicate that TRB3 mediates the tumour-promoting actions of insulin/IGF-1.Because autophagy plays an important role in cancer development and progression, we examined the effect of changed TRB3 level on autophagic signals in cancer cells. Overexpression of TRB3 in normal human bronchial epithelium cells inhibits LC3-Ⅰ/-Ⅱ conversion and increases the level of both soluble and insoluble p62. In contrast, silencing TRB3 decreases the basal and IGF-1-induced accumulation of soluble and insoluble p62 in HepG2 cells, a human liver carcinoma cell line, suggesting that TRB3 mediates the IGF-1-induced p62 accumulation and autophagy inhibition. To verify the inhibitory role of TRB3 in control of the autophagic flux, the flux rate of autophagy was measured with live-cell imaging using an mRFP-GFP-LC3 reporter construct. Transmission electron microscopy was also used to confirm that TRB3 mediates the IGF-1-suppressed autophagic flux. Our previous study revealed that TRB3 interacted with the LIR (LC3-interacting region, LIR) motif and UBA (ubiquitin-associated. UBA) domain of p62. TRB3 interacts with p62 to interfere with the binding of p62 to LC3 and ubiquitinated substrates, which causes the accumulation of p62 and the inhibition of p62-mediated selective autophagy. Indeed, the expression of TRB3 and p62 are increased in human HCC, colon and lung cancer tissues in comparison with normal liver, colon and lung tissues. These tumour tissues showed an enhanced interaction of TRB3/p62 as well as TRB3/p62 colocalization. The TRB3/p62 interaction results in the defect of p62-mediated selective autophagy and autophagy-dependent clearance of ubiquitinated proteins.We next examined how TRB3 mediated the insulin/IGF-induced p62 accumulation. TRB3 did not change p62 transcription but rather markedly enhanced the half-life of p62. Interestingly, the p62 accumulation and dysfunction of selective autophagy by TRB3 inhibit UPS (ubiquitin proteasome system)-dependent substrate degradation. Defective autophagy and UPS result in accumulation of cancer-promoting factors, including EGFR, COX-2. MMP-1/2, MT-MMP, c-Myc as well as Snail and Twist that play critical roles in regulation of tumour initiation, growth and metastasis.In previous study, we identified a TRB3-binding a-helical peptide (Pep2-A2), which could interrupt the TRB3/p62 interaction. Here, our study demonstrated that Pep2-A2 could restore the selective autophagy degradation and accelerate the ubiquitin proteasome system clearance of the tumor-promoting factors. Pep2-A2 produces potent antitumour efficacies against tumour growth and metastasis.We conclude that TRB3 acts as a bridge to connect the metabolic risk factors to tumor promotion. Many metabolic stresses including insulin/IGF-1 can increase the expression of TRB3 and enhance the interaction between TRB3 and p62. The TRB3/p62 interaction induces the blockage of autophagic flux and subsequent UPS defect. Dysfunction of both two degradation systems results in the accumulation of cancer-promoting factors, which act as enhancers of tumor development and progression. Targeting the TRB3/p62 interaction by an a-helical peptide (Pep2-A2) can produce potent antitumor efficacies. In short, our studies elucidate the molecular mechanism about high prevalence of tumor in diabetic population, and we also provide a proof-of-concept for targeting this interaction as a potential novel strategy against cancers with diabetes. These efforts provide theoretical foundation and leading compound to develop novel anticancer peptide drugs.