Cathepsin E (CTSE) is an enzyme belonging to the aspartic protease family, meaning it cleaves proteins using a specific chemical mechanism involving aspartic acid residues. While cathepsins are most commonly known for their role in normal protein breakdown inside cells, aberrant cathepsin activity has been increasingly linked to cancer progression.
This study begins by demonstrating that CTSE is significantly overexpressed in colorectal cancer (CRC) tissues compared to normal colon tissue. Critically, higher CTSE expression correlates with more advanced TNM staging and worse patient prognosis, suggesting that CTSE is not just a bystander but an active driver of disease progression.
Despite the growing evidence of CTSE's clinical relevance in CRC, the molecular mechanisms by which CTSE promotes cancer growth, metastasis, and immune escape had not been fully understood. This study sets out to unravel those mechanisms using a combination of cellular, biochemical, and in vivo experiments.
A key discovery of this study is that CTSE physically interacts with ZNF326, a zinc finger transcription factor. Using co-immunoprecipitation and other binding assays, the researchers confirmed that these two proteins bind together inside CRC cells, setting off a chain of molecular events that ultimately promotes cancer.
The critical mechanistic step is that CTSE promotes ADP-ribosylation of ZNF326 at the glutamate 338 position. ADP-ribosylation is a post-translational modification where an ADP-ribose molecule is attached to a protein, altering its function or stability. In this case, this modification stabilizes ZNF326, preventing its normal breakdown inside the cell.
Stabilized ZNF326 in turn activates the TGF-beta/SMAD signaling pathway, a well-established driver of epithelial-to-mesenchymal transition (EMT). EMT is the process by which cancer cells lose their epithelial character, become more mobile and invasive, and gain the ability to migrate through tissues and establish metastases. The CTSE-ZNF326-TGF-beta axis thus provides a molecular explanation for how CTSE drives invasion and spread in CRC.
Beyond promoting invasiveness, this study reveals that CTSE also helps colorectal cancer evade the immune system. Specifically, CTSE was found to enhance the interaction between PD-L1 on tumor cells and PD-1 on CD8+ T cells, a mechanism known as the immune checkpoint.
PD-L1 (Programmed Death-Ligand 1) is a protein on the surface of tumor cells that, when it binds to PD-1 on cytotoxic T cells, effectively puts those T cells to sleep, preventing them from killing the tumor. By boosting this interaction, CTSE reduces the ability of CD8+ T cells, the main anti-cancer immune cells, to recognize and destroy CRC cells.
Experiments confirmed that when CTSE was knocked out or inhibited, CD8+ T cell killing activity was significantly restored, meaning the tumors became more susceptible to immune attack. This establishes CTSE as a key enabler of immune evasion in colorectal cancer, opening a new avenue for therapeutic intervention by targeting CTSE to restore anti-tumor immunity.
The study also investigates the upstream regulation of CTSE, asking why CTSE is overexpressed in cancer in the first place. A key finding is that DNMT3A, an enzyme responsible for adding methyl groups to RNA bases (a process called m5C RNA methylation), acts as a negative regulator of CTSE expression.
In normal colon cells, DNMT3A-mediated m5C methylation of CTSE mRNA suppresses CTSE protein production. However, in colorectal cancer, DNMT3A activity may be reduced or disrupted, leading to a loss of this methylation-based silencing and a resulting increase in CTSE levels.
This discovery adds an epigenetic layer to the CTSE story. Epigenetic regulation refers to changes in gene expression that do not involve altering the DNA sequence itself but instead involve chemical modifications to DNA, histones, or RNA. Understanding how CTSE is epigenetically unleashed in cancer cells provides another potential target for therapeutic intervention.
Given that CTSE both promotes tumor invasion and suppresses immune activity, the researchers tested whether blocking CTSE could enhance the effectiveness of existing immunotherapy drugs. They combined HY-P0018, a CTSE inhibitor, with HY-19745, a PD-1/PD-L1 checkpoint inhibitor, in mouse tumor models.
The combination treatment showed synergistic anti-tumor effects, meaning the two drugs together were more effective than either drug alone. Tumor growth was substantially more suppressed with the combination, and immune parameters within the tumors, including CD8+ T cell numbers and activity, were significantly improved.
These in vivo results suggest that CTSE inhibition could serve as a valuable combinatorial strategy alongside checkpoint immunotherapy in colorectal cancer patients. Patients whose tumors express high levels of CTSE might be particularly good candidates for this dual approach, representing a personalized medicine opportunity.
This study establishes CTSE as a multifunctional oncogene in colorectal cancer, operating through at least two distinct pathways: the CTSE-ZNF326-TGF-beta axis driving invasion and metastasis, and the CTSE-PD-L1/PD-1 axis enabling immune evasion. Both pathways converge to make CTSE a highly attractive therapeutic target.
From a clinical perspective, CTSE expression levels could serve as a prognostic biomarker to identify CRC patients at higher risk of metastasis or poor response to standard therapy. Pathological testing for CTSE could be integrated into existing tumor profiling workflows using standard immunohistochemical techniques.
Looking ahead, the authors call for further translational studies to validate CTSE inhibitors in clinical settings. Understanding whether CTSE inhibition is safe and effective in humans, and which patient populations would benefit most, will be important next steps before this approach can move into clinical trials for colorectal cancer.
The conclusions in this study are supported by a comprehensive set of experiments. In cell line studies, CTSE was overexpressed or knocked down in multiple human CRC cell lines, and the effects on proliferation, migration, invasion, and immune evasion were measured. Knockdown of CTSE consistently reduced cancer cell aggressiveness.
Co-immunoprecipitation and mass spectrometry were used to confirm the physical binding between CTSE and ZNF326, and to precisely identify the ADP-ribosylation site at glutamate 338 on ZNF326. These biochemical approaches provide strong mechanistic evidence for the proposed molecular pathway.
In mouse xenograft and syngeneic tumor models, CTSE-expressing tumors grew faster and were more resistant to immune attack compared to CTSE-knockout tumors. The combination therapy experiments were also conducted in these mouse models, confirming the therapeutic potential of dual CTSE and checkpoint inhibition in a living organism before any human testing.