[Nature] New treatment plan for non-small cell lung cancer: targeted degradation of EGFR

Lung cancer is one of the most common malignant tumors in the world, and it has become the first cause of death from malignant tumors in the urban population of our country. Non-small cell lung cancer (NSCLC) includes squamous cell carcinoma, adenocarcinoma, and large cell carcinoma. Compared with small cell carcinoma, cancer cells grow and divide more slowly, and spread and metastasize relatively late. Non-small cell lung cancer accounts for about 80% of all lung cancers, and about 75% of patients are already in the advanced stage when they are discovered.
Epidermal growth factor receptor (EGFR) is a transmembrane protein composed of an extracellular ligand binding domain, a hydrophobic transmembrane domain, and an intracellular kinase domain. Its intracellular structure contains 1 A tyrosine kinase domain and a carboxyl terminal with multiple autophosphorylation sites belong to the receptor tyrosine kinase family. EGFR is essential for controlling the growth and survival of epithelial cells, and is usually used to treat epithelial malignancies such as non-small cell lung cancer (NSCLC).

Currently, small molecule tyrosine kinase inhibitors (TKIs) and monoclonal antibodies targeting EGFR are focused on inhibiting EGFR kinase activity or inducing antibody and complement-mediated cytotoxicity. Among them, antibodies targeting EGFR are mainly used to treat advanced colorectal cancer and head and neck tumors, but their clinical treatment effects are not good, so they are not suitable for the treatment of NSCLC. So far, three generations of EGFR tyrosine kinase inhibitors (TKIs) have been developed to reversibly (first generation) or irreversibly (second and third generations) inhibit EGFR tyrosine kinase activity, and have been widely used in NSCLC treatment .

Gefitinib and Erlotinib are the first-generation EGFR TKIs designed for wild-type EGFR, but they show strong and selective inhibition of active EGFR mutations.

The second-generation EGFR TKIs (such as afatinib, dacomitinib) were designed to overcome the acquired T790M resistance of the first-generation EGFR TKIs, but failed due to unacceptable toxicity.

The newly developed third-generation EGFR TKIs (such as osimertinib and nostenib) irreversibly bind to the cysteine-797 residue on the EGFR ATP binding site. Compared with WT-EGFR, the carrier activates The EGFR form of mutation or T790M resistance mutation showed preferential activity.

A large number of studies have shown that the spatial distribution and stability of EGFR are also key determinants of regulating lung cancer progression. Even in mutated EGFR-driven lung adenocarcinoma, EGFR degradation dysregulation further accelerates the occurrence and development of tumors. Spatial dysregulation of epidermal growth factor receptors increases the availability of plasma membrane receptors and induces continuous signal output. Depletion of sterol-C4-methyloxidase-like and NAD(P)H steroid dehydrogenase-like proteins, both of which are involved in the sterol biosynthesis pathway, inhibit the recycling of EGFR, and make A431 xenografts suitable for Cetuximab is sensitive to treatment. Golgi membrane protein 1 interacts with EGFR to promote the circulation of EGFR to the membrane, leading to prolonged EGFR activation time and progression of hepatocellular carcinoma. These findings emphasize that promoting EGFR degradation is an alternative strategy for targeting EGFR-related cancers.

A recent study determined that the increase in TRIB3 expression and the stability, recirculation, and signal activity of EGFR are related to the increase in the progression of NSCLC, revealing the potential effect of interfering with TRIB3-EGFR interaction in the treatment of NSCLC by accelerating EGFR degradation. The results of this study were discovered by a team led by researcher Hu Zhuowei and researcher Hua Fang, Institute of Materia Medica, Chinese Academy of Medical Sciences, and published in the journal Nature.

As a pressure sensor, TRIB3 can respond to various pressure sources and participate in chronic inflammation, metabolism and malignant diseases through the interaction with signal proteins and functional proteins. Studies have confirmed that TRIB3 weakens the degradation functions of autophagy and proteasome by interacting with the autophagy receptor p62, thereby promoting the occurrence and development of a variety of cancers. The loss of TRIB3 can cause a significant decrease in the expression of multiple tumor promoting factors (including EGFR) in various cancers.

TRICL3 expression is positively correlated with EGFR in NSCLC

To determine the relationship between TRIB3 and EGFR levels in lung cancer, the researchers tested the expression of these two proteins in several human lung cancer cell lines. In most human NSCLC cell lines, high expression of TRIB3 correlates with increased expression of EGFR (a). TRIB3 depletion not only reduced EGFR expression in these cell lines and primary NSCLC cells (b), but also inhibited EGFR-responsive genes in A549 cells (c).
The researchers used the online kmplot tool to query the TCGA database to evaluate 1,416 NSCLC patients and determined that high TRIB3 mRNA levels were only associated with poor survival of lung adenocarcinoma (a), but not lung squamous cell carcinoma (b). However, it was found that high TRIB3 protein was positively correlated with poor survival rates in lung adenocarcinoma (c, d) and squamous cell carcinoma. Consistent with the expression of TRIB3 protein, the level of EGFR protein observed in human NSCLC tissue samples was higher than that of adjacent non-tumor tissue samples (d, e). In NSCLC tissues, there is a positive correlation between TRIB3 and EGFR protein levels (f). It is worth noting that the survival rate of patients with simultaneous expression of EGFR and TRIB3 was significantly lower than that of patients with single expression of EGFR or TRIB3 and simultaneous low expression (g).

TRIB3 promotes PKCα-mediated phosphorylation of EGFR Thr654

TRIB3 induces a variety of cellular functions through protein interactions. The researchers conducted high-throughput protein array screening and identified PKCa as the binding partner of TRIB3 (a).
Co-immunoprecipitation (CO-IP) analysis showed that TRIB3, EGFR and PKCα were co-precipitated by each antibody (b).
In addition, TRIB3, EGFR and PKCa co-localized in the cytoplasm after EGF stimulation (c).
It is understood that Thr654 in EGFR is the main phosphorylation site of PKCα. Under EGF treatment, a large number of EGFR and PKCa co-localized in control A549 cells (d left);
In TRIB3-deficient cells, colocalization was less than that of control cells (12±2%), and the total amount of EGFR and PKCα was reduced (d right).
Even when PKCa is ectopic expressed, TRIB3 depletion will reduce EGF-induced phosphorylation of EGFR T654 (e).
In order to map the interaction region of TRIB3 that interacts with EGFR and PKCα, a deletion mutant of TRIB3 with an HA tag was constructed and CO-IP assay was performed. It was identified that the C-terminus of the TRIB3 kinase death region interacted with EGFR (f ).
The C-terminal tail of TRIB3 is responsible for the binding between TRIB3 and PKCa (g).
In addition, the interaction between the intracellular membrane region of EGFR and TRIB3 was identified (h, i).
Restoring the expression of TRIB3 without restoring the KDC deletion mutant (M5) can reverse the inhibitory effect of TRIB3 depletion on the EGFR cycle (j).

These data all indicate that the interaction between TRIB3 and EGFR is essential for the EGFR cycle.

Targeted degradation of EGFR inhibits the development of lung cancer

Researchers found that SAH-JGZ4 can promote EGFR degradation and inhibit EGFR signaling activity, so in vitro and in vivo models were used to evaluate its anti-tumor effects. SAH-JGZ4 can not only resist the expression of core pluripotency factors induced by EGF (g), but also inhibit tumor proliferation, invasion and internal tumor formation in A549 cells (h-j).

SAH-JGZ4 treatment inhibited tumor growth in a subcutaneous xenograft model with A549 cells in a dose-dependent manner, and compared with gefitinib, a dose of 2 mg kg -1 administered twice a week had better anti-tumor effects. Tumor effect (a). SAH-JGZ4 induced a dose-dependent reduction in liver metastases, which was superior to the reduction induced by gefitinib (b). It is worth noting that SAH-JGZ4 also inhibited tumor growth and metastasis in mice inoculated with NCI-H1975 cells. NCI-H1975 cells are gefitinib-resistant lung cancer cells with T790M mutation (c, d); SAH- The anti-tumor efficacy of JGZ4 is better than gefitinib and comparable to AZD9291 (Figure c, d). In terms of mechanism, SAH-JGZ4 interferes with the in vivo interaction of EGFR-TRIB3 and EGFR-PKCα, and inhibits the expression of EGFR and PKCα in the inoculated tumor tissue (Figure e). In addition, the phosphorylation of STAT3/5 and EGFR and the expression of total EGFR, PKCa, TRIB3 and core pluripotency factor in tumor tissue samples inoculated with NCI-H1975 mice treated with SAH-JGZ4 were reduced (d, E). Using the lung orthotopic transplantation model, we found that SAH-JGZ4 weakened the transfer of A549 cells from the inoculation side to the opposite side (Figure f, g). SAH-JGZ4 reduced the frequency of tumor initiating cells (TIC) by six times (Figure j, k), indicating that the stability of targeting EGFR is a potential strategy to inhibit the growth of lung cancer stem cells.

Results and discussion

This study shows that elevated TRIB3 participates in the pathogenesis and progression of NSCLC by enhancing the recycling and stability of EGFR. TRIB3–PKCα–WWP1 induces the K63-linked ubiquitination of EGFR on K689, forming a positive regulatory axis for the recycling and stability of EGFR. In addition, the researchers showed the molecular details about the tumor-promoting effect of WWP1 by inducing the ubiquitination of K689 EGFR linked to K63, which subsequently promoted EGFR circulation and maintained EGFR stability.

At the same time, SAH-JGZ4 was also developed in this study, which can interfere with the interaction of TRIB3-EGFR and produce anticancer effects by inhibiting EGFR recycling and then inducing EGFR degradation. In addition, SAH-JGZ4 treatment also showed an inhibitory effect on the compensation pathway by promoting the degradation of c-Met and continuously inhibiting STAT3/5 signal transduction. EGFR and KRAS mutations are the two main driving factors in NSCLC. Contrary to EGFR targeted therapy, there is no clinically effective inhibitor for mutant KRAS protein. This study found that SAH-JGZ4 treatment of KRAS mutant A549 cells will reduce KRAS is active and inhibits the occurrence and development of tumors, which indicates that promoting EGFR degradation has the therapeutic potential of targeting “non-treatable” KRAS mutations. These characteristics of SAH-JGZ4 indicate that it is an effective alternative drug for EGFR targeted therapy (especially to overcome TKI resistance). Researchers say that the therapeutic effect of SAH-JGZ4 combined with KRAS inhibitors will also be studied in the future Continue the evaluation.
All in all, studies have shown that the synergistic expression and action of TRIB3, EGFR and PKCα establish the TRIB3-PKCa-WWP1 regulatory axis, and promote the development of NSCLC by enhancing the recycling, stability and signal transduction of EGFR. In addition, targeting the TRIB3-EGFR interaction to promote EGFR degradation is a potential treatment option for the treatment of EGFR-related NSCLC cases. Medicilon, as a new drug development CRO, will continue to pay attention to this research progress, hoping to help the development of new lung cancer drugs.

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