CDKN2A overexpression linked to radioresistance and immunosuppression in colorectal cancer

Colorectal cancer remains one of the world’s most formidable health challenges, ranking as the second leading cause of cancer-related mortality and the third most frequently diagnosed malignancy. With an annual incidence of 35.1 per 100,000 people and a mortality rate of 12.9 per 100,000, the disease claims a devastating toll, particularly as rates climb among younger adults. Between 5 and 30 percent of patients experience recurrence after surgical tumor resection, underscoring the urgent need for better therapeutic strategies. Now, a team of researchers from Fujian Medical University Union Hospital and collaborating institutions across China has identified a single gene that may explain why many colorectal tumors shrug off both radiation and chemotherapy while simultaneously building a fortress against the immune system. The gene, CDKN2A, emerges as a master regulator of treatment resistance and immune evasion, offering a tantalizing new target for intervention.

The investigation, published in Frontiers in Cell and Developmental Biology, took a comprehensive approach to understanding CDKN2A’s role in colorectal cancer. The researchers began by mining vast transcriptomic and clinical datasets from The Cancer Genome Atlas and the Gene Expression Omnibus databases. They focused on 688 human radiation response-related genes, identified by integrating five GEO datasets that compared gene expression profiles between radiosensitive and radioresistant cancer cells across multiple malignancies. From this pool, they pinpointed 495 genes differentially expressed between tumor and normal tissues and 36 genes significantly linked to overall survival. The intersection yielded 28 key genes, and among these, CDKN2A stood out as the hub gene, exhibiting the most extensive interactions with other prognosis-related radiation response genes in the protein-protein interaction network.

What makes CDKN2A particularly intriguing is its paradoxical nature. The gene encodes the p16 protein, which normally functions as a tumor suppressor by inhibiting CDK4/6 kinase activity, thereby maintaining cell cycle arrest in the G1 phase. It can also initiate DNA repair or apoptosis by activating p53. Yet in colorectal cancer, the researchers found that CDKN2A is consistently overexpressed rather than deleted or mutated. Bulk and single-cell transcriptome analyses, along with immunohistochemistry, revealed elevated CDKN2A expression in tumor specimens. The mutation frequency of CDKN2A in these tumors was remarkably low, approximately 1 percent, suggesting that overexpression rather than genetic alteration drives the gene’s malignant role. Single-cell analysis identified 21 cell clusters comprising 12 cell types in colorectal cancer tissue, with three distinct tumor cell subtypes showing heterogeneous CDKN2A expression, particularly high in the C6 and C9 subtypes.

The clinical implications are striking. The researchers obtained tumor samples from 46 patients with rectal cancer prior to treatment, all of whom received neoadjuvant long-course chemoradiotherapy at a radiation dose of 50.4 Gy in 28 fractions combined with fluorouracil and leucovorin calcium chemotherapy, followed by standardized surgery. Patients whose tumors failed to respond to neoadjuvant chemoradiotherapy had significantly higher CDKN2A expression than those who responded, with a P value of 0.039. The team then reviewed postoperative pathology and follow-up data from 290 rectal cancer patients who underwent surgery and 5-fluorouracil-based adjuvant treatment. Those who experienced recurrence showed elevated CDKN2A expression compared with those who did not, at P equal to 0.037. These findings establish CDKN2A overexpression as a clinical marker of treatment failure and disease return.

To probe the biological function of CDKN2A, the researchers turned to laboratory experiments using the human colorectal cancer cell line HCT 116. They deployed short hairpin RNA to knock down CDKN2A expression, with the most effective construct reducing mRNA levels dramatically as confirmed by reverse-transcription PCR and Western blot. The results were unequivocal: CDKN2A knockdown led to a marked decrease in proliferation, invasion, and migration capabilities. When combined with X-ray irradiation at 4 Gy, the effect was amplified. Clonogenic assays demonstrated that the clone formation rate following combined irradiation was significantly diminished in the CDKN2A knockdown group compared with controls. Wound-healing and transwell assays further confirmed that reducing CDKN2A expression impaired the migratory and invasive capacity of irradiated cells, supporting a contributory role for CDKN2A in radiation resistance.

The researchers extended their findings into living systems by constructing subcutaneous tumor models in female BALB/c mice. Tumors with CDKN2A knockdown grew more slowly after irradiation than control tumors. Immunohistochemistry revealed that the knockdown group had significantly more necrosis after irradiation, with P values below 0.05. These in vivo results corroborate the in vitro data, demonstrating that suppressing CDKN2A enhances the radiosensitivity of colorectal cancer tumors in living organisms, not just in culture dishes.

Beyond radiation resistance, CDKN2A overexpression carries a dire prognostic signal. Patients with high CDKN2A expression demonstrated inferior overall survival, progression-free survival, recurrence-free survival, and disease-free survival. The gene’s expression correlated with multiple aggressive clinicopathological features, including age at or below 65 years, lymph node metastasis, distant metastasis, and high TNM stage. These associations position CDKN2A as both a prognostic biomarker and a participant in malignant progression, linking its overexpression to the most invasive and lethal forms of the disease.

Perhaps most remarkably, CDKN2A appears to sculpt the tumor immune microenvironment into an immunosuppressive state. Using xCell, CIBERSORT, and EPIC computational methods to assess immune cell infiltration, the researchers found that CDKN2A expression was positively correlated with the infiltration levels of M2-type macrophages, monocytes, and regulatory T-cells, all of which promote tumor progression and immune escape. Conversely, CDKN2A expression was negatively linked to CD8-positive T-cell infiltration, the core effector cells of antitumor immunity. Single-cell transcriptomics revealed that tumor cell subtypes with high CDKN2A expression had weak interactions with CD8-positive T-cells but strong interactions with fibroblasts and endothelial cells, further reinforcing the immunosuppressive architecture. Molecular analysis showed that levels of immune inhibitors such as VTCN1, TGFB1, PDCD1, and CD274 were positively correlated with CDKN2A expression, along with cytokine ligands and receptors including CCL17, CXCL16, and CXCR5.

The immunotherapy prediction results add another layer of complexity. Analysis using The Cancer Immunome Atlas database suggested that patients with elevated CDKN2A expression showed a greater likelihood of responding to anti-PD-L1 therapy but a diminished likelihood of responding to anti-PD-1 therapy, while response to CTLA-4 was unrelated to CDKN2A expression level. The researchers speculate that in tumors with high CDKN2A expression, low CD8-positive T-cell infiltration combined with a highly activated PD-1/PD-L1 pathway leads to T-cell depletion. Anti-PD-1 treatment does not reverse this depletion, whereas PD-L1 inhibitors may reverse the immunosuppressive microenvironment. Additionally, when using PD-L1 inhibitors, PD-L2 can be retained to combine with repulsive guidance molecule B, thereby promoting T-cell expansion. In patients with low CDKN2A expression, the antitumor immune microenvironment is relatively favorable, and PD-1 inhibitors can continuously activate the tumor-killing effect of CD8-positive T-cells. These findings suggest that CDKN2A may eventually serve as a biomarker to guide clinical selection of immunotherapeutic drugs.

Functional enrichment analysis through Gene Ontology and KEGG databases illuminated the biological pathways through which CDKN2A exerts its effects. Elevated CDKN2A expression was linked to transcriptional regulation, amino acid metabolism, lipid metabolism, protein folding, and the TGF-beta signaling pathway. The gene’s function relates to intermediate filament cytoskeleton organization, regulation of macromolecule biosynthetic processes, DNA-templated regulation of transcription, MHC class I receptor activity, and N-methyltransferase activity. Drug sensitivity analysis revealed that tumors with high CDKN2A expression are more likely to be resistant to oxaliplatin and 5-fluorouracil, the two representative chemotherapy drugs used in colorectal cancer, but may be sensitive to dasatinib, lapatinib, and GDC-0879. The resistance to 5-fluorouracil may stem from the G1 phase arrest caused by CDKN2A, rendering the S-phase-specific drug ineffective. The resistance to oxaliplatin may relate to cell cycle arrest and enhanced DNA damage repair. The researchers propose that the p16 protein encoded by CDKN2A causes G1 phase arrest under stress, reducing DNA damage in adverse environments and providing time for repair, which might explain why patients with high CDKN2A expression are insensitive to radiotherapy, which mainly acts on cells in the G2/M phases undergoing mitosis.

The study’s conclusions carry profound implications for clinical management. Targeted inhibition of CDKN2A enhances the sensitivity of tumor cells to radiotherapy and may modulate the immunosuppressive state, potentially providing a new strategy for improving treatment effectiveness in colorectal cancer patients. The combination of radiation therapy with CDKN2A-targeted intervention represents a promising therapeutic avenue that could transform unresectable tumors into resectable ones and boost the pathological complete response rate in rectal cancer. However, the researchers acknowledge important limitations. The main conclusions are based on retrospective analysis of public databases, and additional prospective clinical studies, gain-of-function and rescue experiments in colorectal cancer cell lines with low endogenous CDKN2A expression are needed. The use of a single cell line limits generalizability, and the dual-tumor animal models may lead to mutual influences between tumors through cytokines and other mechanisms. The molecular mechanisms by which CDKN2A affects radiotherapy sensitivity and the immune microenvironment have not been fully elucidated, and the TGF-beta pathway remains a bioinformatic prediction without experimental validation. Moreover, the assertion that colorectal tumors with high CDKN2A expression may respond to agents such as dasatinib and lapatinib is based on algorithm predictions requiring clinical verification. More experimental and clinical studies are still needed to distinguish the similarities and differences between colon cancer and rectal cancer, as the correlation between CDKN2A and radiotherapy sensitivity has only been validated in the rectal cancer cohort. Nevertheless, this investigation provides compelling evidence supporting CDKN2A as a potential therapeutic target and prognostic biomarker, opening a new frontier in the fight against one of humanity’s deadliest cancers.

Subject of Research: Association of CDKN2A overexpression with poor prognosis, radioresistance, and immunosuppression in colorectal cancer

Article Title: Association of CDKN2A overexpression with poor prognosis, radioresistance, and immunosuppression in colorectal cancer

Article References: Lin, Y., Wang, Z., Ju, G., Zhang, Y., Huang, Z., Liu, S., Xu, B., & Zhan, Z. (2026). Association of CDKN2A overexpression with poor prognosis, radioresistance, and immunosuppression in colorectal cancer. Frontiers in Cell and Developmental Biology, 14, Article 1955910. https://doi.org/10.3389/fcell.2026.1955910

Image Credits: AI Generated

DOI: 10.3389/fcell.2026.1955910

Keywords: Association, CDKN2A, overexpression, poor, prognosis, radioresistance, immunosuppression, colorectal, cancer, scientific research