Metastatic renal cancer is responsible for a large number of renal cancer-related deaths worldwide. A major challenge in the treatment of those patients is resistance to Sunitinib and everolimus-based therapies[23]. Therefore, It is therefore essential to gain profound understanding of the mechanisms underlying drug resistance in renal cancer and to develop effective therapeutic strategies to overcome it. In this study, we demonstrated that MICAL-L2 expression level correlated with various clinicopathological parameters of KIRC, including pathological stage, histological type, and TNM stages. Bioinformatic analysis and MICAL-L2 staining in KIRC patient specimens revealed that MICAL-L2 was associated with a poor prognosis for KIRC patients. We found that MICAL-L2 significantly promotes aggressive proliferation, migration, Sunitinib and Everolimus resistance of KIRC. We identified ACTN4 as an interacting protein with MICALL2, and showed that MICAL-L2 stabilises ACTN4 by repressing its ubiquitination level in the presence of Rab13. The MICALL2/ACTN4 complex activates vimentin expression and promotes the malignant progression of KIRC.
Previous studies have reported that MICAL-L2 is a susceptibility gene which plays an important role in executive inhibition deficiency associated with hyperactive-impulsive behavior[24]. MICAL-L2 deficiency also impairs pulmonary fibrogenesis and restrains EMT in response to bleomycin treatment[25]. In addition, MICAL-L2 has been frequently studied in tumors. MICAL-L2 has been shown to be a promising prognostic biomarker in colorectal cancer[3]. MICAL-L2 was also found to be highly expressed in advanced ovarian cancer tissues and could inhibit the autophagy process of ovarian cancer cells[4]. Mechanistically, MICAL-L2 interacts with Rab13 to direct cancer cell migration[26]. MICAL-L2 is also acts as a key regulator of c-Myc stability to promote NSCLC cell proliferation[6]. However, the role of MICAL-L2 in KIRC remains unclear. In this study, we demonstrate that MICAL-L2 acts as a pro-oncogene to promote proliferation, migration and drug resistance in renal cancer.
In our study, we identified ACTN4 as a leading candidate for the protein that interacts with MICAL-L2 through bioinformatics analysis and Co-IP experiments. Therefore, we further investigated ACTN4 as a target protein of MICAL-L2. ACTN4 is an actin-binding protein normally expressed in non-muscle cells. It plays a role in the regulation of the cytoskeleton, cell motility and tumor development[27]. Copy number gain of ACTN4 has been identified as an independent risk factor for poor prognosis in patients with urothelial carcinoma[28]. In a study of lung adenocarcinoma, ACTN4 gene amplification was identified as a biomarker for adjuvant chemotherapy with tegafur/uracil in patients[29]. Sun et al. found that in GLUT4 exocytosis, insulin increased the binding of MICAL-L2 to ACTN4 and then localised GLUT4 vesicles to the periphery of muscle cells[30]. Consistently, our experiments showed that knockdown of ACTN4 partially abrogated the role of MICAL-L2 in promoting vimentin expression, cell proliferation and cell migration in KIRC, suggesting that ACTN4 is a downstream target protein of MICALL2 associated with vimentin expression and KIRC carcinogenesis.
In our experiments, we found that MICAL-L2 was positively correlated with the vimentin expression which is normally considered as a marker of EMT. Then, we experimentally verified that MICAL-L2 promotes the vimentin expression by stabilising ACTN4, thereby promoting proliferation, migration, and drug resistance of KIRC. Interestingly, it underscores the growing recognition of vimentin as a pivotal therapeutic target in cancer treatment, with a primary focus on mitigating metastasis and overcoming drug resistance[31]. Moreover, tumor-derived vimentin represents a biomarker for predicting resistance to adjuvant chemotherapy and the T-cell-inflamed phenotype in small cell lung cancer[32]. It is therefore worth exploring whether MICALL2/ACTN4, the regulator of vimentin, can be used as a target to alleviate drug resistance of KIRC cells.
Next, we investigated the specific mechanism underlying the interaction between MICAL-L2 and ACTN4. In this report, we found that the binding of MICAL-L2 to ACTN4 is dependent on the Rab13. Rab13 is the only activator of MICAL-L2 that has been identified to date, which competitively binds to the CC domain of MICAL-L2 and opens the MICAL-L2 spatial structure[33]. Interestingly, the addition of proteasome inhibitors, MG132 and Velcade, inhibited ACTN4 degradation induced by Rab13 depletion, suggesting that Rab13 may inhibit ACTN4 degradation via the proteasome pathway. Meanwhile, Rab13 inhibits the ubiquitinated degradation of ACTN4, thereby enhancing the protein stability of ACTN4. Together, Rab13 depletion impairs ACTN4 stability and promotes ACTN4 degradation via the ubiquitin-proteasome pathway, leading to inhibition of renal cancer cell migration and proliferation.
In this study, we identified MICAL-L2 as an independent diagnostic and prognostic biomarker for KIRC. Furthermore, MICAL-L2 is resistant to multiple drugs according to bioinformatics analysis. However, the role of MICAL-L2 in the therapy of renal cancer is rarely investigated. Sunitinib and everolimus-based therapy is the main drug therapy approach for metastatic renal cell carcinoma and is frequently combined with other agents in clinical practice[21, 34, 35]. Everolimus is a derivative of rapamycin and acts similarly to rapamycin as an mTOR inhibitor. Sunitinib is a multi-targeted inhibitor of receptor tyrosine kinases (RTKs) such as PDGF-R and VEGF-R. Both are effective in improving the prognosis of KIRC patients. Although targeted therapy has significantly improved the prognosis of renal carcinoma patients, the development of drug resistance remains a significant barrier to effective treatment[23, 36]. We observed that after Sunitinib and Everolimus treatment, MICAL-L2 levels were increased in KIRC cell lines. Meanwhile, knockdown of Sunitinib and Everolimus combined with MICAL-L2 knockdown greatly reduced ACTN4 and vimentin expression, renal cancer cell proliferation and cell migration capacity, suggesting that MICALL2/ACTN4/vimentin confers resistance to KIRC cells. CCG-1423, an inhibitor of MICAL2, has been reported to inhibit tumourigenesis in head and neck cancer[37]. Hence, exploring whether the effect of MICAL-L2 on resistance to Sunitinib and Everolimus could provide a new target for combating poor outcome in KIRC.
In conclusion, the identification of the MICALL2/ACTN4/vimentin axis provides a complementary and comprehensive understanding of the pathogenesis of KIRC and offers an opportunity to translate basic research into potential treatments in clinical practice (Fig. 10).
We elucidate novel biological roles of MICAL-L2 in driving KIRC carcinogenesis. Our study elucidates the mechanism by which the MICALL2/ACTN4/vimentin axis controls the malignant biological behavior and drug resistance of KIRC cells. These findings highlight the potential of targeting MICAL-L2 and related pathways as a strategy to increase the sensitivity of targeted therapy in KIRC.