Research Background

Bladder cancer is a highly prevalent malignant tumor worldwide, ranking as the fourth most common malignancy in men. According to the 2016 report by the National Cancer Center of China (NCCR), bladder cancer ranks sixth in incidence among male malignancies. Despite continuous improvements in medical diagnostic methods and surgical techniques, there has been no significant improvement in the recurrence rate and progression of bladder cancer. The insufficient understanding of its pathophysiological characteristics and biological mechanisms is a major challenge in current diagnosis and treatment. Therefore, studying the biological mechanisms of the occurrence, recurrence, and progression of bladder cancer is of great significance to improve its diagnosis, treatment, and prognosis.

Stearoyl-CoA desaturase (SCD) in lipid metabolism has become an important target in recent research. As an endoplasmic reticulum transmembrane protein, SCD plays a crucial role in the conversion of saturated fatty acids to monounsaturated fatty acids (MUFA). Studies have shown that MUFA is associated with proliferation, invasion, and survival pathways in tumors. Stearic acid is the substrate of SCD, while oleic acid (OA) is its product. Increased SCD activity leads to a higher intracellular MUFA content, promoting tumor cell growth and invasion. Especially in bladder cancer stem cells (CSCs), abnormally high expression of SCD may play a significant role in cancer recurrence and treatment resistance. However, the reasons for the aberrant expression of SCD and its specificity in response to inhibitors in CSCs are currently unclear. Therefore, the study by Li et al. aims to uncover the transcriptional regulation mechanisms of SCD in bladder cancer CSCs and the role of endoplasmic reticulum stress/unfolded protein response in drug sensitivity.

Research Source

This study was conducted by Yuchen Li, Chiyuan Piao, Chuize Kong, and other researchers from the Department of Urology, The First Hospital of China Medical University, and published in the 2024 issue of the journal Cancer Cell International.

Research Process

Research Design and Experimental Procedures

1. Cell Culture and CSC Induction: The study utilized the bladder cancer cell line UMUC3 to induce bladder cancer stem cells (UMUC3-CSCs) through serum-free suspension culture, and conducted gene chip detection to confirm the significant expression of SCD in bladder cancer CSCs.

2. Analysis of SCD Transcription Start Sites: The UALCAN database was used to analyze the methylation levels of the SCD gene promoter region. The results showed that the methylation levels of the SCD promoter were relatively low in bladder cancer tissues compared to normal bladder mucosa tissues, suggesting that its transcriptional activation might not be significantly inhibited by DNA methylation.

3. Screening and Validation of Transcription Factors: By analyzing transcription factor binding sites in the SCD core promoter region using multiple databases (such as JASPAR, TRANSFAC, etc.), 11 candidate transcription factors were identified. Further analysis and experiments confirmed that the transcription factor TFAP2A might be a key regulator of SCD transcription. Chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assays further validated the binding site of TFAP2A to the SCD promoter.

4. Effect of SCD Inhibitors on Bladder Cancer CSCs: Using the SCD inhibitor A939572, the study demonstrated that inhibiting SCD in UMUC3-CSCs induced significant apoptosis, whereas similar apoptotic phenomena were not observed in non-induced UMUC3 cells. Additionally, RNA-Seq and lipid metabolism analysis revealed that the SCD inhibitor mainly caused lipid metabolism changes and endoplasmic reticulum stress responses in bladder cancer CSCs.

5. Cell Proliferation and Drug Sensitivity Experiments: The study further investigated the sensitivity of UMUC3-CSCs to cisplatin treatment in the presence of the SCD inhibitor. The results showed that the SCD inhibitor significantly increased the sensitivity of bladder cancer CSCs to cisplatin, and this effect was more pronounced upon the addition of the DNA double-strand repair protein RAD51 inhibitor.

6. Effect of Nervonic Acid Supplementation on Cell Proliferation: In vitro and in a mouse subcutaneous xenograft model, the study reversed the inhibitory effect of the SCD inhibitor on bladder cancer cell proliferation by supplementing with nervonic acid. It was also found that abnormally large fat granules appeared in animals treated with the SCD inhibitor.

Data Analysis and Statistics

Data analysis in this study primarily used GraphPad Prism 9 software. For comparisons between two groups, a two-tailed Student’s t-test or Mann-Whitney U test was used. For comparisons among multiple groups, one-way ANOVA combined with Dunnett’s test was used, while non-normally distributed data were analyzed using the Kruskal-Wallis test followed by Dunn’s test.

Research Results

Transcriptional Regulation Mechanism of SCD in Bladder Cancer

The study found that the methylation level of the SCD promoter region was low, not affected by DNA methylation inhibition. Further analysis identified TFAP2A as a key factor regulating SCD expression. Through ChIP and dual-luciferase reporter assays, the study confirmed the direct binding of TFAP2A to the SCD promoter region and demonstrated the transcription factor’s role in activating SCD transcription.

Effect of SCD Inhibitors on Bladder Cancer CSCs

In UMUC3-CSCs, the SCD inhibitor A939572 significantly induced apoptosis, while similar effects were not observed in non-induced UMUC3 cells. Additionally, experimental verification showed that this effect was primarily achieved via endoplasmic reticulum stress/unfolded protein responses triggered by fatty acid accumulation, while unsaturated fatty acids could partially reverse this effect.

Enhanced Drug Sensitivity of Bladder Cancer CSCs

In bladder cancer CSCs, the SCD inhibitor significantly enhanced the therapeutic effect of cisplatin, and this effect was related to the inhibition of DNA repair protein RAD51 expression, suggesting that the SCD inhibitor increased drug sensitivity of CSCs by inhibiting the DNA damage repair pathway.

Reversal Effect of Nervonic Acid

Upon supplementation with nervonic acid, the inhibitory effect of the SCD inhibitor on bladder cancer cell proliferation was significantly weakened. In vivo experimental results also indicated that tumor mass significantly increased after nervonic acid supplementation, and the formation of abnormal fat granules was observed in the SCD inhibitor group.

Research Conclusions

This study unveiled the transcriptional regulatory mechanism of SCD in bladder cancer, identified TFAP2A as a critical regulatory factor of SCD, and revealed for the first time the selective apoptosis induction mechanism of SCD inhibitors in bladder cancer CSCs. The study showed that SCD inhibitors induce apoptosis in CSCs through endoplasmic reticulum stress/unfolded protein response while inhibiting the DNA damage repair protein RAD51 to increase CSCs’ sensitivity to cisplatin. Furthermore, nervonic acid may play a crucial role in regulating cell proliferation and lipid metabolism.

Research Significance and Prospects

This research delves into the biological functions of SCD and its transcriptional regulation mechanism in bladder cancer, revealing the specific response mechanism of bladder cancer stem cells to SCD inhibitors and providing a new insight for targeted therapy of bladder cancer. Specifically, SCD inhibitors have a selective killing effect on bladder cancer CSCs, indicating that it might be a potential target for bladder cancer stem cell therapy. Future research can further optimize SCD inhibitor treatment regimens, extend experimental periods, and enhance its application effect in tumor therapy through more potent inhibitors. Additionally, the function of nervonic acid in tumor biology deserves in-depth investigation to find new therapeutic methods for bladder cancer and other tumor types.