Academic Background and Problem Statement

Prostate Cancer (PCA) is one of the most common malignancies in men, particularly in advanced stages, where patients typically receive Androgen Deprivation Therapy (ADT) as the standard treatment. However, despite initial treatment efficacy, patients eventually develop Castration-Resistant Prostate Cancer (CRPC), leading to treatment failure. In recent years, the use of second-generation Androgen Receptor Signaling Inhibitors (ARSIs) such as Enzalutamide has significantly improved patient survival rates. However, this has also introduced new challenges: some patients develop resistance to Enzalutamide. Studies suggest that the activation of the Glucocorticoid Receptor (GR) may be a key mechanism underlying Enzalutamide resistance, but the specific molecular mechanisms remain unclear.

Previous research has shown that the transcription factor TBX2 is overexpressed in CRPC and may influence prostate cancer progression by regulating the activity of the Androgen Receptor (AR) and GR. However, how TBX2 regulates the switch between AR and GR, and how this switch leads to Enzalutamide resistance, remains an unresolved question. This study aims to elucidate the molecular mechanisms of the TBX2-driven AR-to-GR signaling switch and explore the potential of reversing Enzalutamide resistance by disrupting TBX2-GR and TBX2-LSD1 protein interactions.

Paper Source and Author Information

This paper was co-authored by Sayanika Dutta, Hamed Khedmatgozar, Girijesh Kumar Patel, and other researchers from institutions such as Texas Tech University Health Sciences Center and Rutgers School of Health Professions. The paper was published in 2024 in the journal Oncogene, with the DOI: https://doi.org/10.1038/s41388-024-03252-5.

Research Process and Experimental Design

1. Negative Correlation Between TBX2 and AR

The study began with RNA sequencing (RNA-seq) analysis of TBX2 expression in PC3 cells. The results showed that inhibition of TBX2 significantly increased the expression of AR and its downstream target genes. Further quantitative PCR (qRT-PCR) and Western Blot experiments confirmed that TBX2 inhibition led to a significant upregulation of AR mRNA and protein levels, while TBX2 overexpression reduced AR expression. These results indicate a negative correlation between TBX2 and AR.

2. TBX2 Directly Represses AR Transcription

To verify whether TBX2 directly binds to the AR promoter to repress its transcription, the research team conducted Chromatin Immunoprecipitation (ChIP) experiments. The results showed significant enrichment of TBX2 at two sites on the AR promoter (-82 bp and -3598 bp), indicating that TBX2 directly binds to the AR promoter and represses its transcription. Further Site-Directed Mutagenesis (SDM) experiments confirmed that mutating the TBX2 binding sites significantly enhanced AR transcriptional activity, further supporting the conclusion that TBX2 directly represses AR transcription.

3. TBX2 Represses AR Coactivator GATA2

GATA2 is a key coactivator of the AR signaling pathway. The study found that TBX2 inhibition significantly upregulated GATA2 mRNA and protein levels, while TBX2 overexpression reduced GATA2 expression. ChIP experiments further confirmed that TBX2 directly binds to the GATA2 promoter and represses its transcription. These results indicate that TBX2 indirectly suppresses the AR signaling pathway by directly repressing GATA2.

4. Positive Correlation Between TBX2 and GR

The study also found that TBX2 inhibition led to downregulation of GR expression, while TBX2 overexpression increased GR expression. ChIP experiments showed that TBX2 directly binds to the GR promoter and activates its transcription. Additionally, Co-Immunoprecipitation (Co-IP) experiments confirmed a protein-protein interaction between TBX2 and GR. These results suggest that TBX2 upregulates GR expression through both direct transcriptional activation and protein-protein interactions.

5. TBX2-Driven AR-to-GR Signaling Switch Confers Enzalutamide Resistance

Through cell viability assays, the research team found that TBX2 inhibition restored Enzalutamide sensitivity in PC3 cells, while TBX2 overexpression reduced Enzalutamide sensitivity in LNCaP cells. Further experiments showed that GR knockdown restored Enzalutamide sensitivity in TBX2-overexpressing cells, while the GR agonist Dexamethasone restored Enzalutamide resistance in TBX2-inhibited cells. These results indicate that the TBX2-driven AR-to-GR signaling switch is a key mechanism underlying Enzalutamide resistance.

6. SP2509 Reverses Resistance by Disrupting TBX2-GR and TBX2-LSD1 Interactions

The research team discovered that the LSD1 inhibitor SP2509 could disrupt TBX2-GR and TBX2-LSD1 protein-protein interactions, thereby reversing Enzalutamide resistance. Further experiments showed that SP2509 significantly reduced the protein levels of TBX2, GR, and LSD1 and inhibited the proliferation of TBX2-overexpressing cells. These results suggest that SP2509, by disrupting TBX2-GR and TBX2-LSD1 interactions, offers a potential therapeutic strategy.

Main Results and Conclusions

This study reveals the critical role of TBX2 in prostate cancer, where it drives Enzalutamide resistance by repressing AR and activating GR. Specifically, TBX2 represses AR and GATA2 transcription by directly binding to their promoters, while activating GR through direct promoter binding and protein-protein interactions. This AR-to-GR signaling switch leads to Enzalutamide resistance. Additionally, the study found that the LSD1 inhibitor SP2509 can reverse resistance by disrupting TBX2-GR and TBX2-LSD1 interactions, providing new insights for CRPC treatment.

Research Highlights and Significance

1. TBX2 as a Molecular Switch: This study is the first to reveal TBX2 as a molecular switch driving the AR-to-GR signaling switch in prostate cancer, elucidating its key role in Enzalutamide resistance.
2. Dual Regulatory Mechanisms: TBX2 regulates AR and GR expression through both direct transcriptional regulation and protein-protein interactions, providing new molecular insights.
3. Unique Role of SP2509: The study found that SP2509 not only inhibits LSD1’s demethylase function but also disrupts TBX2-GR and TBX2-LSD1 interactions, offering a new drug target for CRPC treatment.
4. Clinical Relevance: The findings were validated in multiple prostate cancer patient cohorts, suggesting that the transcriptional activity relationships among TBX2, AR, and GR could serve as predictive markers for Enzalutamide resistance.

Summary

This study not only elucidates the critical role of the TBX2-driven AR-to-GR signaling switch in prostate cancer resistance but also proposes a potential therapeutic strategy to reverse resistance by disrupting TBX2-GR and TBX2-LSD1 interactions. This discovery provides new directions for CRPC treatment, with significant scientific and clinical implications.