Targeting TRA2B in Cancer: A Novel Therapeutic Strategy

Abstract

TRA2B (Transformer 2 Beta Homolog), also known as TRA2β, is a serine/arginine-rich (SR) RNA-binding protein that plays a pivotal role in regulating alternative splicing, a key post-transcriptional process enabling the production of multiple protein isoforms from a single gene. In cancer, dysregulation of TRA2B—often through overexpression—contributes to tumor initiation, progression, proliferation, migration, and therapy resistance by altering splicing patterns that suppress pro-apoptotic events, such as the inclusion of ultraconserved poison exons (PEs). These PEs introduce premature termination codons (PTCs), leading to nonsense-mediated mRNA decay (NMD) and reduced protein expression, acting as intrinsic regulatory switches to prevent abnormal cell growth. Recent advancements in antisense oligonucleotide (ASO) technology, such as ASO-1570, offer a promising precision medicine approach to selectively force PE inclusion in TRA2B transcripts, thereby downregulating TRA2B protein levels, inducing transcriptomic changes, and triggering apoptosis specifically in cancer cells while sparing normal cells. This strategy has shown efficacy in preclinical models across various cancers, including breast, lung, ovarian, and glioblastoma, and highlights TRA2B-PE's dual role as both a protein regulator and a functional long noncoding RNA (lncRNA) with anti-tumor effects. Low PE inclusion correlates with poor patient survival in multiple tumor types, underscoring its prognostic value and therapeutic potential.

1. Introduction

1.1 Overview of Alternative Splicing

Alternative splicing is a fundamental eukaryotic mechanism that enhances proteomic diversity by allowing a single pre-mRNA to generate multiple mature mRNA isoforms through selective inclusion or exclusion of exons. This process is orchestrated by the spliceosome—a large ribonucleoprotein complex—and modulated by RNA-binding proteins (RBPs) such as SR proteins and heterogeneous nuclear ribonucleoproteins (hnRNPs). Dysregulated alternative splicing is a hallmark of cancer, contributing to all major oncogenic processes, including sustained proliferation, evasion of apoptosis, invasion, metastasis, and therapeutic resistance. Aberrant splicing can arise from mutations in splicing factors, altered expression of RBPs, or cis-regulatory changes in pre-mRNA sequences.

1.2 Role of TRA2B in Normal Cellular Function

TRA2B, a member of the Transformer-2 protein family, is an essential RBP that binds to purine-rich exonic splicing enhancers (ESEs) to promote exon inclusion. In normal cells, TRA2B maintains cellular homeostasis by regulating splicing of genes involved in cell signaling, proliferation, genome stability, and apoptosis. It autoregulates its own expression via an ultraconserved poison exon (TRA2B-PE) located between exons 2 and 3, which contains a PTC. Inclusion of this PE targets the transcript for NMD, reducing TRA2B protein levels in a feedback loop. This autoregulation is cross-modulated by other splicing factors, ensuring balanced expression during development and differentiation.

1.3 TRA2B Dysregulation in Cancer

Overexpression of TRA2B has been documented in numerous aggressive malignancies, driven by mechanisms such as gene amplification (e.g., on chromosome 3q26), transcriptional upregulation by oncogenes like MYC, or post-transcriptional evasion of NMD through increased skipping of the poison exon. Elevated TRA2B levels promote oncogenic splicing isoforms that enhance cell survival and proliferation. Specific cancers associated with TRA2B overexpression include:

• Triple-negative breast cancer (TNBC), where it correlates with poor prognosis and resistance to chemotherapy
• Glioblastoma multiforme (GBM), linked to increased invasion and stemness
• Colorectal carcinoma, promoting metastasis via splicing of targets like CD44
• Lung adenocarcinoma (LUAD), associated with proliferation and survival
• Ovarian cancer, enhancing migration and therapy resistance
• Cervical squamous cell carcinoma (CESC), correlating with reduced survival
• Prostate cancer, predicting relapse and poor outcomes
• Endometrial carcinoma, increasing cell viability and proliferation

In these cancers, low inclusion of TRA2B-PE (measured as percent spliced-in, PSI) is a biomarker of poor overall survival, as observed in TCGA datasets for BRCA, CESC, LAML, LUAD, SKCM, and UVM. TRA2B's oncogenic effects involve splicing alterations in pathways like apoptosis (e.g., BCL2, BIM), cell cycle (e.g., MKI67, CHEK1), and epithelial-mesenchymal transition (EMT; e.g., RON, CD44).

2. Mechanism of TRA2B in Cancer

2.1 Poison Exons as Regulatory Elements

Poison exons are highly conserved non-coding sequences that, when included in mRNA, introduce PTCs, triggering NMD and degrading the transcript to prevent production of dysfunctional proteins. In normal cells, they function as "safety switches" to autoregulate splicing factor expression, including TRA2B, SRSF1, and other SR proteins. During cellular stress or abnormal growth, PE inclusion limits excessive protein accumulation, promoting apoptosis if unchecked.

2.2 Autoregulation and Cross-Regulation of TRA2B

TRA2B's poison exon is autoregulated: high TRA2B protein levels bind to intronic splicing silencers (ISSs) near the PE, promoting its inclusion and subsequent NMD, thus maintaining homeostasis. This loop is influenced by cross-regulation with paralog TRA2A and other RBPs like SRSF1, forming a coordinated network that modulates SR protein expression during cell differentiation and tumorigenesis. In cancer, mutations or epigenetic changes disrupt this balance, leading to PE skipping, TRA2B overexpression, and downstream oncogenic splicing.

2.3 Impact on Cancer Hallmarks

Overactive TRA2B in malignant cells skips poison exons in target genes, producing isoforms that evade apoptosis, sustain proliferation, and confer resistance. For instance, it alters splicing of CHEK1 to promote genome instability, BCL2 to inhibit cell death, and ERα in breast cancer to drive hormone-independent growth. Recent studies reveal TRA2B-PE transcripts also function as lncRNAs, interacting with RBPs like DDX10 and RBM15 to exert anti-tumor effects, adding a layer of complexity to its role.

3. Antisense Oligonucleotide Therapy

3.1 Principles of ASO Technology

Antisense oligonucleotides (ASOs) are synthetic, single-stranded DNA or RNA analogs (typically 15-25 nucleotides) that hybridize to complementary RNA sequences via Watson-Crick base pairing. They modulate gene expression by inducing RNase H-mediated degradation, blocking translation, or altering splicing. For splicing modulation, splice-switching ASOs (SSOs) target ISSs or enhancers to redirect splice site selection.

3.2 Targeting TRA2B with ASOs

ASO-1570, a 2'-O-methoxyethyl (MOE)-modified SSO, binds to an ISS downstream of TRA2B-PE, blocking repressive factors and forcing PE inclusion. This leads to:

• Introduction of a PTC, triggering NMD and reducing TRA2B protein by up to 50-70%
• Transcriptomic shifts, including differential splicing of >500 events (e.g., cassette exons, retained introns) and gene expression changes in apoptosis and proliferation pathways
• Selective apoptosis in cancer cells via caspase-3/7 activation, with minimal off-target effects (predicted <7 mismatches via GGGenome)

The dual mechanism—protein downregulation and lncRNA-mediated anti-tumor activity—enhances efficacy. Normal cells remain unaffected due to balanced TRA2B regulation.

3.3 Preclinical Evidence

In vitro studies across 10 cancer types (e.g., breast MDA-MB-231, glioma U87MG) show ASO-1570 reduces viability, proliferation (EdU assay), and induces cell death. In 3D organoids and PDX models (e.g., breast and GBM), intratumoral ASO delivery shrinks tumors, increases PE inclusion (RT-qPCR), and alters splicing (RNA-seq). CRISPR knockouts confirm specificity, with no effects in ASO-site-deleted cells.

4. Clinical Implications and Future Directions

4.1 Potential as Precision Medicine

TRA2B-targeted ASOs represent a gene-specific therapy for cancers with high TRA2B expression or low PE PSI, offering low toxicity compared to chemotherapy. Biomarkers like PE inclusion could guide patient selection.

4.2 Challenges and Optimizations

Key hurdles include ASO delivery (e.g., lipid nanoparticles for tumor targeting), stability (chemical modifications like MOE, LNA), and specificity (minimizing off-targets). Preclinical toxicity is low, but clinical trials must monitor visual/ocular effects seen in other splicing modulators.

4.3 Combination Therapies and Ongoing Research

Synergies with chemotherapy, immunotherapy, or other splicing inhibitors (e.g., SF3B1 modulators like H3B-8800) are promising. Future directions include:

• Phase I/II trials for TNBC and GBM
• AI-driven ASO design for enhanced potency
• Exploring lncRNA functions for novel targets
• Integrating with CRISPR for permanent splicing edits

This approach exemplifies how manipulating alternative splicing can yield targeted therapies with superior efficacy and safety profiles.

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