Cereblon (CRBN) Suppresses Metastatic Progression through Distinct Mechanisms across Cancer Types

Abstract

Metastatic progression is a major determinant of cancer-related mortality and is driven by complex interactions between altered proteostasis, metabolic reprogramming, and stress-responsive signaling. Cereblon (CRBN), a substrate receptor of the CRL4CRBN E3 ubiquitin ligase complex, has been extensively studied as the molecular target of immunomodulatory drugs and proteolysis-targeting chimeras. However, its endogenous tumor- suppressive functions and cancer type-specific mechanisms remain incompletely understood. In this thesis, I investigated the role of CRBN in metastatic progression across multiple cancer types and identified distinct downstream mechanisms through which CRBN restrains malignant phenotypes. Preliminary pan-cancer analyses revealed that CRBN expression is reduced in several tumor types compared with corresponding normal tissues, and that low CRBN expression is associated with unfavorable overall survival in selected cancer cohorts. These observations suggested that CRBN downregulation may contribute to cancer progression in a context-dependent manner. To define the underlying mechanisms, I examined CRBN function in prostate cancer, lung adenocarcinoma, and renal cell carcinoma using public transcriptomic datasets, genetic perturbation models, biochemical assays, metabolic analyses, and functional assays of migration, invasion, and metastatic dissemination. In prostate cancer, CRBN suppressed metastatic progression by regulating 6-phosphogluconate dehydrogenase (6PGD), a key enzyme in the oxidative pentose phosphate pathway. CRBN directly interacted with a conserved C-terminal α-helix of 6PGD and promoted its polyubiquitination and proteasomal degradation independently of immunomodulatory drugs. Loss of CRBN stabilized 6PGD, increased the NADPH/NADP+ ratio, and enhanced migratory and invasive phenotypes, whereas re-expression of wild-type CRBN reversed these effects. Transcriptomic and functional analyses further demonstrated that CRBN regulates epithelial–mesenchymal transition-associated genes, including CDH1 and MMP1, through 6PGD-dependent metabolic control. In vivo, CRBN suppressed metastatic dissemination in an intra-splenic xenograft model, supporting the CRBN–6PGD axis as a functional metabolic checkpoint in prostate cancer metastasis. In lung adenocarcinoma, CRBN acted through a distinct metabolic mechanism involving cystathionine β- synthase (CBS), the rate-limiting enzyme of the transsulfuration pathway. CRBN directly interacted with CBS and promoted its polyubiquitination and proteasomal degradation. CRBN depletion increased CBS protein abundance without altering CBS mRNA expression, indicating post-translational regulation. Functionally, CRBN loss enhanced migration and invasion of lung adenocarcinoma cells in a CBS-dependent manner. Mechanistically, CBS accumulation increased intracellular glutathione availability and altered sulfur amino acid metabolism, thereby regulating expression of the epithelial–mesenchymal transition transcription factor ZEB1. These findings identify a CRBN–CBS–ZEB1 axis that links dysregulated protein quality control to metabolic adaptation and invasive behavior in lung adenocarcinoma. In renal cell carcinoma, CRBN expression was reduced in tumor tissues compared with adjacent normal kidney tissues, and low CRBN expression was associated with poorer overall survival. Unlike the prostate and lung cancer models, CRBN loss in renal cell carcinoma was associated primarily with activation of stress kinase signaling rather than a clearly defined metabolic enzyme substrate. CRBN deletion increased wound closure in VHL-wild-type Caki-1 and ACHN cells without enhancing proliferation, whereas this phenotype was not observed in VHL-mutant 786-O cells. CRBN deficiency increased TAK1 and p38 phosphorylation, and pharmacological inhibition of either kinase suppressed the enhanced wound closure phenotype. Oxidative stress- responsive transcripts were induced despite no detectable changes in basal reactive oxygen species levels or mitochondrial respiration. These results suggest that CRBN restrains a TAK1–p38 signaling axis associated with a migratory phenotype in VHL-wild-type renal cell carcinoma cells. Collectively, this thesis establishes CRBN as a context-dependent suppressor of metastatic progression across cancer types. Rather than acting through a single universal pathway, CRBN restrains malignant phenotypes through distinct downstream mechanisms: the CRBN–6PGD axis in prostate cancer, the CRBN–CBS–ZEB1 axis in lung adenocarcinoma, and the CRBN–TAK1–p38 stress kinase axis in renal cell carcinoma. These findings expand the biological significance of CRBN beyond its pharmacological role as a drug-binding substrate receptor and suggest that CRBN downregulation may create cancer type-specific vulnerabilities in metabolic and stress-responsive pathways. Therapeutic strategies aimed at targeting CRBN-regulated downstream effectors may therefore provide a rational approach for CRBN-low or metabolically rewired cancers.