Dimethyl fumarate promotes the degradation of HNF1B and suppresses the progression of clear cell renal cell carcinoma

Clear cell renal cell carcinoma (ccRCC) is the most lethal subtype of renal cancer, and its treatment options remain limited. Therefore, there is an urgent need to discover therapeutic

agents for ccRCC treatment. Here, we demonstrate that dimethyl fumarate (DMF), an approved medication for multiple sclerosis [1] and psoriasis, can inhibit the proliferation of ccRCC cells.

Mechanistically, hepatocyte nuclear factor 1β (HNF1B), a transcription factor highly expressed in ccRCC, is succinated by DMF at cysteine residues, leading to its proteasomal degradation.

Furthermore, HNF1B interacts with and stabilizes Yes-associated protein (YAP), thus DMF-mediated HNF1B degradation decreases YAP protein level and the expression of its target genes,

resulting in the suppression of ccRCC cell proliferation. Importantly, oral administration of DMF sensitizes ccRCC to sunitinib treatment and enhances its efficacy in mice. In summary, we

provide evidences supporting DMF as a potential drug for clinical treatment of ccRCC by targeting HNF1B and reveal a previously unrecognized role of HNF1B in regulating YAP in ccRCC.

Renal cell carcinoma (RCC) which arises from renal tubular epithelial cells, is one of the malignant tumors of the urinary system. According to its pathological features, RCC primarily

consists of three subtypes: chromophobe renal cell carcinoma (chRCC), clear cell renal cell carcinoma (ccRCC) and papillary renal cell carcinoma (pRCC). Among these, ccRCC is the most

prevalent subtype and is characterized by the presence of abundant cytoplasmic glycogen and lipids [2, 3]. ccRCC accounts for ~75–80% of all RCC cases, and has a poorer prognosis compared to

other subtypes [4]. Over the past few decades, various therapeutic strategies have been employed for ccRCC treatment, including surgical management, chemotherapy and radiotherapy [5]. Among

these treatments, surgery remains the primary treatment option for ccRCC due to its limited response to chemotherapy and radiotherapy. Since early-stage ccRCC often presents with few

noticeable clinical symptoms, and in several cases, metastasis is already present at the time of diagnosis, which contributes to a poor prognosis with the 5-year survival rate is only 10%

[6,7,8]. Therefore, it is crucial to develop new strategies for ccRCC treatment.

HNF1B, a 65-kDa protein, is a member of the superfamily of transcription factors that includes homeodomain proteins such as Pit-1, Oct-1/2, and POU. HNF1B consists of three functional

regions: an amino-terminal domain, a DNA-binding domain and a carboxy-terminal domain. The gene encoding HNF1B is located on the long arm of chromosome 17 [9,10,11]. During the early

embryonic development, the expression of HNF1B is expressed in several organs, including liver, kidney, pancreas, lung and urinary tract. Heterozygous mutations in HNF1B are associated with

several congenital diseases including renal cysts, pancreatic hypoplasia, abnormal liver function tests, and urogenital tract abnormalities, suggesting that HNF1B is crucial for the normal

development of these organs [1, 12]. Although research on HNF1B has increased in recent years, its precise role in carcinogenesis remains insufficiently understood. Interestingly, HNF1B

functions as a protooncogene in some tumors, while it acts as a tumor suppressor in others, and whether it behaves as an oncogene or tumor suppressor appears to depend on the type and

histogenesis of the tumor [13, 14].

Dimethyl fumarate (DMF) is a small molecule compound, a dimethyl ester of fumaric acid, which was first approved by the U.S. Food and Drug Administration in 2013 under the brand name

Tecfidera [15]. It is registered as an anti-inflammatory drug for the treatment of autoimmune diseases, such as multiple sclerosis and psoriasis [16, 17]. Several studies have demonstrated

that the molecular mechanisms underlying these effects can be mainly attributed to two aspects. First, DMF disrupts the interaction between Kelch-like ECH-associated protein 1 (KEAP1) and

erythroid 2–related factor 2 (Nrf2), leading to activation of Nrf2 and subsequent expression of antioxidant genes, which protects cells from reactive oxygen species that is generated during

inflammation [16, 18, 19]. Second, DMF inhibits NF-κB signaling pathway by suppressing the translocation of NF-κB family members into the nucleus, resulting in a significant decrease in the

production of proinflammatory cytokines [16, 17, 20, 21]. However, the function of DMF in ccRCC and its underlying mechanisms remain unknown. The present study uncovers a previously unknown

role of DMF in ccRCC, embodying the concept of “old drugs, new uses”, which offers a more time- and cost-efficient alternative compared with the new drug discovery [22, 23]. We demonstrate

that DMF targets HNF1B for ubiquitin-dependent proteasomal degradation and effectively suppresses the proliferation of ccRCC cells by impairing HNF1B mediated stabilization of Yes-associated

protein (YAP). Notably, oral administration of DMF decreases HNF1B levels in mice and enhances the sensitivity of ccRCC to sunitinib treatment. Overall, our study unveils a new regulatory

axis involving DMF, HNF1B and YAP, and demonstrates that DMF is a potential candidate for clinical treatment of ccRCC.

To investigate the role of HNF1B in tumorigenesis, we utilized the GEPIA and HPA databases to determine the expression level of HNF1B in various types of tumors. Notably, HNF1B shows the

highest expression in ccRCC compared to other cancers across both databases (Fig. 1A, B). Consistently, the protein level of HNF1B is also highest in 786-O ccRCC cell line compared to other

cancer cell lines, such as SW620 (colorectal cancer), HeLa (cervical carcinoma), KTC-1 (thyroid cancer), T24 (bladder cancer), A375 (melanoma), H1299 and A549 (non-small cell lung cancer)

(Fig. 1C). A previous study reported that HNF1B functions as a tumor suppressor and inhibits cell proliferation in prostate cancer [24]. However, another study suggested that HNF1B may act

as an oncogene, even though the underlying mechanism remains unclear [25]. Given these findings, we wanted to explore what role did HNF1B play in the cell proliferation of ccRCC. HNF1B

knockout cells were generated using the CRISPR/Cas9 system (Fig. 1D), and we found that HNF1B knockout significantly suppressed both proliferation and colony formation of ccRCC cells (Fig.

1E, F). Together, these results indicate that HNF1B positively regulates cell proliferation in ccRCC.

A HNF1B expression analysis of different cancers using GEPIA database. B HNF1B expression analysis of different cancers using The Human Protein Atlas database. C Western blot analysis of

HNF1B in various cancer cell lines. D CRISPR/Cas9 system was employed to knock out HNF1B in 786-O cells and HNF1B KO efficiency was verified by Western blot analysis. E The proliferation of

786-O cells with HNF1B knockout was determined using CCK-8 assays. The data represent the mean ± SD of n = 3 independent experiments. Statistical differences were determined by one-way

ANOVA. **P