Mechanism of RASAL1 and PTEN Co-Alterations Promoting Cancer Aggressiveness

Academic Background

Cancer is one of the leading causes of death worldwide, and its occurrence and progression involve abnormalities in multiple genes and signaling pathways. The PI3K-AKT signaling pathway plays a crucial role in cancer, and its overactivation is closely related to tumor aggressiveness and poor prognosis. PTEN (Phosphatase and Tensin Homolog) is a key negative regulator of the PI3K-AKT pathway, and its loss of function leads to overactivation of this pathway. However, it remains unclear whether other genes also play important roles in regulating the PI3K-AKT pathway, especially when co-acting with PTEN.

RASAL1 (RAS Protein Activator Like 1) is a RAS GTPase-activating protein (RASGAP) that converts active GTP-bound RAS into inactive GDP-bound form, thereby inhibiting the activation of the PI3K pathway. Although reduced expression of RASAL1 in certain cancers is associated with aggressiveness, its broad role as a tumor suppressor gene has not been fully established. This study aims to explore the role of RASAL1 gene alterations in cancer, particularly how it cooperates with PTEN gene alterations to promote cancer aggressiveness by synergistically activating the PI3K-AKT pathway.

Source of the Paper

This paper was co-authored by Xiaopei Shen, Jie Tan, Rengyun Liu, Guangwu Zhu, Lisa Roper, and Mingzhao Xing from the Division of Endocrinology, Diabetes & Metabolism and the Department of Pathology at Johns Hopkins University School of Medicine. The paper was published online on July 20, 2024, in the journal Molecular Oncology, with the DOI: 10.¹⁰⁰²⁄₁₈₇₈-0261.13701.

Research Process and Results

1. Data Collection and Analysis

The research team obtained whole-exome mutation, gene copy number, RNA sequencing, and clinical outcome data from 9,924 patients across 33 cancer types in The Cancer Genome Atlas (TCGA) database. Gene copy number alterations were analyzed using GISTIC 2.0, and functionally irrelevant mutations (e.g., silent mutations, intron mutations) were excluded. The study also analyzed 724 known cancer genes from the Cancer Gene Census (CGC) database.

2. Prevalence of RASAL1 Gene Alterations

The study found that RASAL1 exhibits widespread genetic alterations, including copy number loss and mutations, across various cancers. The average frequency of RASAL1 alterations in 33 cancer types was 14.25%, with over 10% alteration frequency in 19 cancer types. Copy number loss and mutations were considered equally deleterious and were collectively treated as harmful genetic alterations.

3. Co-Occurrence of RASAL1 with PTEN and TP53 Alterations

The study revealed that RASAL1 alterations significantly co-occur with PTEN and TP53 alterations. Specifically, 52.97% of cancers with RASAL1 alterations also had PTEN alterations, while 64.78% had TP53 mutations. This co-occurrence suggests that RASAL1, PTEN, and TP53 may synergistically regulate the PI3K-AKT pathway in cancer.

4. RASAL1 Alterations Activate the PI3K Pathway

Analysis of reverse-phase protein array (RPPA) data showed that cancers with RASAL1 alterations exhibited significantly higher levels of AKT phosphorylation, indicating PI3K pathway activation. This association was particularly pronounced in breast cancer, prostate adenocarcinoma, and thymoma. Even in cancers without PTEN and TP53 alterations, RASAL1 alterations were associated with increased AKT phosphorylation.

5. RASAL1 Alterations and Poor Clinical Outcomes

The study found that cancer patients with RASAL1 alterations had significantly higher disease-specific mortality rates compared to those without RASAL1 alterations (27.93% vs. 20.57%). In cancers without PTEN and TP53 alterations, patients with RASAL1 alterations also had higher mortality rates (23.28% vs. 13.11%). Kaplan-Meier analysis showed that RASAL1 alterations significantly accelerated the decline in disease-specific survival.

6. Synergistic Activation of the PI3K Pathway by RASAL1 and PTEN Co-Alterations

The study demonstrated that cancers with concurrent RASAL1 and PTEN alterations exhibited significantly higher PI3K pathway activation compared to those with single gene alterations. In breast cancer, patients with both RASAL1 and PTEN alterations had the highest levels of AKT phosphorylation, as well as significantly higher disease progression and mortality rates. Kaplan-Meier analysis revealed that patients with concurrent RASAL1 and PTEN alterations had significantly shorter disease-specific survival and progression-free survival.

7. Synergistic Effects of RASAL1, PTEN, and TP53 Triple Alterations

The study found that patients with concurrent RASAL1, PTEN, and TP53 alterations had the worst clinical outcomes. In breast cancer, 68.75% of patients with triple alterations had triple-negative breast cancer (TNBC), compared to only 3.83% in patients without any gene alterations. The triple alterations significantly increased the aggressiveness of breast cancer, highlighting the importance of the synergistic interaction among RASAL1, PTEN, and TP53 in cancer progression.

8. Validation Using Genetic Knockout Mouse Models

The research team constructed a RASAL1 knockout mouse model using CRISPR-Cas9 technology and crossed it with PTEN knockout mice to generate double-knockout mice. The study found that double-knockout mice exhibited robust PI3K pathway activation and developed metastatic malignancies, whereas single-gene knockout mice only developed benign neoplasms. These results further validated the synergistic role of RASAL1 and PTEN in regulating the PI3K pathway.

Conclusions and Significance

This study provides extensive genetic, clinical, and functional evidence confirming RASAL1 as an important tumor suppressor gene that plays a critical role in cancer by regulating the PI3K-AKT pathway. Specifically, the co-alteration of RASAL1 and PTEN forms a unique genetic mechanism that synergistically activates the PI3K pathway, promoting cancer aggressiveness and poor prognosis. This discovery not only deepens our understanding of the role of the PI3K pathway in cancer but also offers new genetic markers for molecular classification and precision treatment of cancer.

Research Highlights

1. Broad Scope: The study encompassed 9,924 patients across 33 cancer types, demonstrating the widespread prevalence of RASAL1 alterations and their significant role in various cancers.
2. Synergistic Interaction: For the first time, the study revealed the synergistic interaction between RASAL1 and PTEN in regulating the PI3K pathway, providing new insights into the genetic mechanisms of cancer.
3. Clinical Implications: The triple alterations of RASAL1, PTEN, and TP53 were significantly associated with the triple-negative phenotype in breast cancer, offering important insights for molecular classification and prognosis assessment.
4. Functional Validation: The use of genetic knockout mouse models validated the functional synergy between RASAL1 and PTEN in regulating the PI3K pathway, providing robust experimental support for the findings.

Additional Valuable Information

The study also found that RASAL1 alterations are rare in papillary thyroid cancer (PTC) but more common in follicular thyroid cancer (FTC) and anaplastic thyroid cancer (ATC), consistent with the important role of the PI3K pathway in thyroid cancer. Additionally, the study explored epigenetic and post-translational abnormalities of RASAL1, further supporting its broad role as a tumor suppressor gene.

This study provides significant theoretical and practical guidance for understanding the molecular mechanisms of cancer and developing clinical treatments, holding substantial scientific value and application prospects.