The Role of ENPP1 in Brain Metastasis of HER2-Positive Breast Cancer

Academic Background

Brain metastasis (BM) is a severe neurological complication in patients with HER2-positive breast cancer (HER2+ BC), with an incidence rate as high as 50%. The formation of brain metastasis is closely related to how tumor cells cross the blood-brain barrier (BBB). The BBB is a specialized endothelial cell barrier composed of tight junction proteins and adherens junction proteins, which prevents most molecules and cells from entering the brain parenchyma. However, tumor cells disrupt the integrity of the BBB by secreting specific biomolecules, thereby promoting the formation of brain metastasis. Although previous studies have shown that tumor-secreted factors play an important role in BBB dysfunction, the early mechanisms remain unclear. Therefore, understanding how HER2+ breast cancer cells disrupt the BBB through secreted factors is crucial for developing strategies to prevent and treat brain metastasis.

Source of the Paper

This paper was co-authored by Liliana Santos, Francesca Tomatis, Hugo R. S. Ferreira, and others, with the research team coming from multiple institutions, including the Faculty of Medicine at the University of Coimbra and the Coimbra Institute for Clinical and Biomedical Research (ICBR) in Portugal. The paper was published online on August 30, 2024, in the journal Neuro-Oncology, titled “ENPP1 induces blood–brain barrier dysfunction and promotes brain metastasis formation in human epidermal growth factor receptor 2-positive breast cancer.”

Research Process and Results

1. Research Process

a) In Vitro BBB Model and In Vivo Pre-Metastatic Model

The research team first constructed an in vitro BBB model by co-culturing human brain microvascular endothelial cells (ECs) with pericytes to simulate the structure and function of the BBB. Subsequently, the researchers treated the BBB model with the secretome (SCR) of HER2+ breast cancer cell lines (JIMT-1 and SUM190) and their brain-tropic variants (JIMT-1-Br and SUM190-Br) to evaluate its impact on BBB permeability. At the same time, the team established an in vivo pre-metastatic mouse model by daily intraperitoneal injection of SCR or orthotopic implantation of brain-tropic cells to observe BBB disruption.

b) Proteomic Analysis

To identify the key factors causing BBB dysfunction, the researchers performed mass spectrometry analysis on the secretome of JIMT-1-Br and SUM190-Br cells and compared it with that of the parental cells. Through the screening of differentially expressed proteins (DEPs), the researchers identified a protein called ENPP1 (Ectonucleotide Pyrophosphatase/Phosphodiesterase 1) that was significantly enriched in brain-tropic cells.

c) Functional Validation of ENPP1

To validate the role of ENPP1 in BBB disruption, the researchers treated the BBB model with an ENPP1 inhibitor (ENPP1i) and found that ENPP1i effectively prevented the increase in BBB permeability. Additionally, the researchers knocked down ENPP1 expression using siRNA, further confirming the critical role of ENPP1 in BBB dysfunction.

d) Animal Model Validation

The researchers used CRISPR/Cas9 technology to generate ENPP1 knockout (KO) JIMT-1-Br cells and injected them into mice to observe their effects on BBB permeability and brain metastasis formation. The results showed that ENPP1 knockout significantly reduced the metastatic burden and prolonged the survival of the mice.

2. Main Results

a) Impact of Secretome on BBB Permeability

In vitro experiments demonstrated that the secretome of brain-tropic cells significantly increased BBB permeability, leading to the downregulation of tight junction proteins (such as ZO-1 and Claudin-5) and adherens junction proteins (such as β-catenin). In vivo experiments further confirmed that the secretome of brain-tropic cells could disrupt BBB integrity through the systemic release of ENPP1.

b) Mechanism of ENPP1

ENPP1 induces BBB dysfunction by inhibiting the insulin signaling pathway and its downstream Akt/GSK3β/β-catenin pathway. Specifically, ENPP1 binds to the insulin receptor (InsR), inhibits Akt phosphorylation, and subsequently suppresses GSK3β activity, ultimately leading to the degradation of β-catenin and the loss of tight junction proteins.

c) Effects of ENPP1 Knockout

ENPP1 knockout significantly reduced the formation of brain metastasis and prolonged the survival of mice. Additionally, ENPP1 knockout prevented BBB disruption, indicating that ENPP1 plays a key role in the early stages of brain metastasis.

3. Conclusions and Significance

The research results indicate that ENPP1 is a key factor enabling HER2+ breast cancer cells to cross the BBB and form brain metastases. ENPP1 disrupts BBB integrity by inhibiting the insulin signaling pathway, thereby promoting brain metastasis of tumor cells. Furthermore, the secretion levels of ENPP1 are positively correlated with brain metastatic burden and negatively correlated with overall patient survival, suggesting that ENPP1 may serve as an early diagnostic marker for brain metastasis.

4. Research Highlights

• Innovative Discovery: First to reveal the critical role of ENPP1 in BBB disruption and brain metastasis formation.
• Mechanistic Insights: Detailed elucidation of the molecular mechanism by which ENPP1 induces BBB dysfunction through the inhibition of the insulin signaling pathway.
• Clinical Application Potential: ENPP1 inhibitors may become a new strategy for preventing or treating brain metastasis in HER2+ breast cancer.

5. Other Valuable Information

The research team also found that the secretion levels of ENPP1 are closely related to brain metastatic burden, suggesting that ENPP1 may serve as a prognostic marker for brain metastasis. Additionally, ENPP1 inhibitors showed significant anti-metastatic effects in mouse models, laying the foundation for future clinical research.

Summary

This study not only reveals the critical role of ENPP1 in brain metastasis of HER2+ breast cancer but also provides important theoretical support for the development of new therapeutic strategies. By targeting ENPP1, it may be possible to effectively prevent or delay brain metastasis in HER2+ breast cancer patients, thereby improving their survival rates and quality of life.