USP35 Promotes Breast Cancer Progression by Regulating PFK-1 Ubiquitination to Mediate Glycolysis

Academic Background

Breast cancer is the most common malignant tumor affecting women worldwide and remains a leading cause of cancer-related mortality among women. Despite the development of various treatment strategies for different molecular subtypes of breast cancer, such as endocrine therapy, targeted therapy, and PARP inhibitors, 20%-30% of patients still experience recurrence and metastasis, which are the main reasons for the high mortality rate of breast cancer. Therefore, identifying new molecular markers to address the challenges of recurrence and metastasis in breast cancer is crucial.

Cancer cells exhibit high metabolic characteristics and rely on aerobic glycolysis to meet their energy demands, a phenomenon known as the “Warburg effect.” In the process of glycolysis, phosphofructokinase-1 (PFK-1) is a key rate-limiting enzyme that catalyzes the conversion of fructose-6-phosphate (F6P) to fructose-1,6-bisphosphate (F1,6BP), thereby driving glycolysis. Studies have shown that PFK-1 expression is significantly upregulated in breast cancer tissues, and its activity is closely related to tumor progression. However, the regulatory mechanisms of PFK-1 in breast cancer remain unclear.

Ubiquitin-specific protease 35 (USP35), a member of the deubiquitinating enzyme family, stabilizes downstream target proteins through deubiquitination, thereby influencing their function. USP35 has been found to be associated with tumor progression in various cancers, but its role in breast cancer remains unclear. This study aims to investigate whether USP35 affects breast cancer glycolysis and progression by regulating the ubiquitination level of PFK-1.

Source of the Paper

This paper was co-authored by Weibin Lian, Chengye Hong, Debo Chen, and Chuan Wang from the Department of Breast Surgery at Fujian Medical University Union Hospital and the Department of Breast Surgery at Quanzhou First Hospital Affiliated to Fujian Medical University. It was first published online in the journal American Journal of Physiology-Cell Physiology on December 23, 2024, with the DOI: 10.1152/ajpcell.00733.2024.

Research Workflow and Results

1. Upregulation of USP35 Expression in Breast Cancer

The study initially evaluated the mRNA and protein expression levels of USP35 in breast cancer tissues and cells using quantitative real-time PCR (qPCR) and Western blot. The results showed that USP35 expression was significantly higher in breast cancer tissues compared to adjacent normal tissues and was also markedly elevated in various breast cancer cell lines (e.g., MCF-7 and MDA-MB-231) compared to normal breast epithelial cells (MCF-10A). These findings suggest that USP35 may play an important role in the occurrence and development of breast cancer.

2. USP35 Regulates Proliferation and Apoptosis in Breast Cancer Cells

To explore the function of USP35, the research team used shRNA technology to knock down USP35 expression in MCF-7 and MDA-MB-231 cells. Cell proliferation was assessed using the 5-ethynyl-2’-deoxyuridine (EdU) assay, while apoptosis was detected via flow cytometry. The results demonstrated that knocking down USP35 significantly inhibited breast cancer cell proliferation and promoted apoptosis. This indicates that USP35 plays a critical role in maintaining the survival and proliferation of breast cancer cells.

3. USP35 Regulates Glycolysis Through PFK-1

Further investigations revealed that knocking down USP35 led to decreased mRNA and protein expression levels of PFK-1, accompanied by reductions in extracellular acidification rate (ECAR) and oxygen consumption rate (OCR). Seahorse metabolic flux analysis showed that USP35 knockdown significantly reduced the glycolytic capacity of breast cancer cells. Additionally, USP35 knockdown decreased glucose consumption, lactate production, and lactate dehydrogenase (LDH) activity. These results indicate that USP35 influences the glycolytic process in breast cancer cells by regulating PFK-1 expression.

4. USP35 Stabilizes PFK-1 Through Deubiquitination

To uncover the specific mechanism by which USP35 regulates PFK-1, the research team conducted co-immunoprecipitation (Co-IP) experiments and found that USP35 directly interacts with PFK-1. Further studies showed that USP35 stabilizes PFK-1 through deubiquitination, thereby increasing its protein expression level. Knocking down USP35 significantly increased the ubiquitination level of PFK-1, indicating that USP35 protects PFK-1 from degradation via the ubiquitin-proteasome system through deubiquitination.

5. USP35 Inhibits Tumor Growth In Vivo

To validate the role of USP35 in vivo, the research team established a subcutaneous xenograft tumor model in nude mice. The results showed that the tumor volume and weight were significantly smaller in mice injected with USP35-knockdown MCF-7 cells compared to the control group. Immunohistochemistry (IHC) analysis revealed that USP35 knockdown significantly reduced Ki-67 expression in tumor tissues, indicating that USP35 knockdown inhibited tumor cell proliferation. Furthermore, USP35 knockdown also decreased PFK-1 expression and glycolysis-related indicators (such as glucose consumption, lactate production, and LDH activity) in tumor tissues, further confirming the mechanism by which USP35 regulates glycolysis in breast cancer through PFK-1.

Conclusions and Significance

This study found that USP35 expression is significantly upregulated in breast cancer and promotes glycolysis and proliferation in breast cancer cells by stabilizing PFK-1 through deubiquitination. The USP35/PFK-1 axis provides a new target and theoretical basis for the precision treatment of breast cancer. The study not only reveals the molecular mechanism of USP35 in breast cancer but also offers important clues for the development of anti-tumor drugs targeting the USP35/PFK-1 axis.

Highlights of the Study

1. Key Discovery: For the first time, this study elucidated the molecular mechanism by which USP35 regulates breast cancer glycolysis through PFK-1 deubiquitination.
2. Innovation: Both in vitro and in vivo experiments validated the critical role of the USP35/PFK-1 axis in breast cancer, providing a new target for breast cancer treatment.
3. Clinical Application Value: The discovery of the USP35/PFK-1 axis offers new directions for precision treatment and drug development in breast cancer.

Other Valuable Information

The study also pointed out that more tissue samples are needed in the future to verify the high expression of USP35 in breast cancer and to explore its specific role in different molecular subtypes of breast cancer. Additionally, the mechanisms by which USP35 regulates other downstream signaling pathways warrant further investigation.

Through this study, the USP35/PFK-1 axis has become an important research direction in the field of breast cancer treatment, offering new hope for breast cancer patients in the future.