AMBRA1 Negatively Regulates the Function of ALDH1B1, a Cancer Stem Cell Marker, by Controlling Its Ubiquitination

Abstract

Activating molecule in Beclin-1-regulated autophagy (AMBRA1), a negative regulator of tumorigenesis, is a substrate receptor of the ubiquitin conjugation system. ALDH1B1, an aldehyde dehydrogenase, is a cancer stem cell (CSC) marker that is required for carcinogenesis via upregulation of the β-catenin pathway. Although accumulating evidence suggests a role for ubiquitination in the regulation of CSC markers, the ubiquitination-mediated regulation of ALDH1B1 has not been unraveled. While proteome analysis has suggested that AMBRA1 and ALDH1B1 can interact, their interaction has not been validated. Here, we show that AMBRA1 is a negative regulator of ALDH1B1. The expression of ALDH1B1-regulated genes, including PTEN, CTNNB1 (β-catenin), and CSC-related β-catenin target genes, is inversely regulated by AMBRA1, suggesting a negative regulatory role of AMBRA1 in the expression of ALDH1B1-regulated genes. We found that the K27- and K33-linked ubiquitination of ALDH1B1 is mediated via the cooperation of AMBRA1 with other E3 ligases, such as TRAF6. Importantly, ubiquitination site mapping revealed that K506, K511, and K515 are important for the K27-linked ubiquitination of ALDH1B1, while K33-linked ubiquitination occurs at K506. A ubiquitination-defective mutant of ALDH1B1 increased the self-association ability of ALDH1B1, suggesting a negative correlation between the ubiquitination and self-association of ALDH1B1. Together, our findings indicate that ALDH1B1 is negatively regulated by AMBRA1-mediated noncanonical ubiquitination.

AMBRA1 and its role as a target for anticancer therapy

“…AMBRA1 is an intrinsically disordered protein that was associated with various tumor progressions, including autophagy, tumorigenesis, proliferation, EMT, and apoptosis ( 26 , 34 , 45 , 48 ). Studies found that AMBRA1 regulates tumorigenesis by targeting the activity of c-MYC, STAT3, and ALDH1B1 ( 23 , 26 , 33 ). In addition, AMBRA1 also regulated tumor proliferation, EMT, migration, and invasion by inhibiting cyclin D, FAK1, and Smad4 ( 34 – 36 , 38 , 45 ).…”

“…AMBRA1 is also associated with cancer development, including EMT, migration, invasion, and metastasis ( 26 , 45 , 73 ) ( Figure 4 ). Interestingly, AMBRA1 plays an oncogenic role in hepatocellular carcinoma, metastatic breast cancer, and medulloblastoma, whereas AMBRA1 seems to be a tumor suppressor in colorectal cancer cell, melanoma, and squamous cell carcinoma ( 23 , 24 , 29 , 33 , 38 , 45 , 74 , 75 ). The different effects of AMBRA1 in cancers may be due to diverse types and stages of cancer, as AMBRA1 is an autophagy-associated protein and has different roles depending on the gene context ( 76 ).…”

“…Through multifarious molecular interaction techniques, such as mass spectrum, genetic engineering technology, co-immunoprecipitation (co-IP), and yeast two-hybrid screening, numerous interaction partners of AMBRA1 have been demonstrated, as summarized in Table 1 . Therefore, not surprisingly, AMBRA1 participates in diverse physiological and pathological processes, for instance, embryogenesis, neural development, tumorigenesis and proliferation, differentiation, and epithelial–mesenchymal transition (EMT) ( 13 , 26 , 33 – 36 , 42 – 45 ).…”

The Concept of Cancer Stem Cells: Elaborating on ALDH1B1 as an Emerging Marker of Cancer Progression

“…This finding is of importance, considering that the CSCs exhibit highly elaborative and effective DNA damage response machinery [ 216 ]. Finally, Baek et al (2021) reported that AMBRA, a negative regulator of several CSC-related genes, negatively regulates ALDH1B1 through K27- and K33-associated ubiquitination [ 203 ].…”

The involvement of E3 ubiquitin ligases in the development and progression of colorectal cancer

“…Overexpression of E3 ligase TRIM25 also promotes oxaliplatin resistance and the stem cell properties in CRC by blocking the K63-linked ubiquitination and degradation of EZH2 mediated by E3 ligase TNF receptor-associated factor 6 (TRAF6) [ 29 ]. TRAF6 also suppresses CSC properties by conjugating K27- and K33-linked ubiquitination to the cancer stemness marker aldehyde dehydrogenase 1 family member B1 (ALDH1B1) and decreasing its enzyme activity [ 30 ]. The autoubiquitination of E3 ligase TRIM21 at K214 and K217 is promoted by the regulator CSN6, thereby decreasing TRIM21-mediated ubiquitination and degradation of Oct1, a transcription activator of another cancer stemness marker ALDH1A1 [ 31 ].…”