Functional and mechanistic characterization of [delta]Np63[alpha] in esophageal squamous cell carcinoma

Abstract

Esophageal squamous cell carcinoma (ESCC) has provoked a major challenge to human health. In normal esophagus, the predominant p63 isoform, ΔNp63α, functions as a master regulator for epithelial renewal and homeostasis. Intriguingly, ESCC tumors often retain high expression of ΔNp63α, suggesting a crucial role in promoting tumorigenesis. However, the functional roles of ΔNp63α in ESCC have not been fully elucidated.

This study establishes a novel oncogenic role of ΔNp63α in constraining the cancer cell-autonomous tumor suppressive type I interferon (IFN) response in ESCC. Transcriptome profiling identified type I IFN response as the most significantly enriched pathway in p63-depleted ESCC cells. Perturbing type I IFN response by STAT1 ablation significantly rescued cell death induced by p63 depletion in ESCC cell lines. Moreover, type I IFN restriction by ΔNp63α is specifically present in cancer cells, but not in normal cells, revealing a cancer-specific role of ΔNp63α in ESCC.

Furthermore, this study unravels an unexpected outcome of p63 depletion on viral mimicry induction. Transposon element (TE) profiling showed upregulation of retrotransposon expression (e.g. LINE1,HERVs, LTRs) in p63-depleted ESCC cells, which was verified by RT-qPCR following dsRNA enrichment. In particular, LINE1 was significantly elevated at RNA and protein levels upon p63 depletion in ESCC cells. Moreover, dsRNA sensors, TLR3, MDA5, and RIG-I, were upregulated in response to elevated endogenous dsRNA level in p63-depleted cells. Abrogating dsRNA-sensing pathways in viral mimicry, either through inhibiting TLR3 or MAVS (common adaptor protein of MDA5 and RIG-I), partially rescued cell death induced by p63 depletion in ESCC, indicating viral mimicry response is activated in p63-depleted cells when the endogenous dsRNAs are elevated beyond the cell tolerance level to elicit tumor suppression through type I IFN response. In parallel to virus mimicry activation, p63 depletion directly activates STING to stimulate type I IFN response. A strong negative correlation between TP63 and STING1 was observed in transcriptomes of a panel of naive ESCC patient-derived organoids (PDOs). p63 depletion in ESCC cell lines also upregulated STING and phospho-STING, albeit with no significant effect on the expression of cGAS, the classic STING activator. STING inhibition partially rescued the cell death in p63-depleted ESCC cells.

Type I IFN response, once activated by p63 depletion either through elevated dsRNAs in viral mimicry or direct STING activation, exerts a tumor-suppressive role in cancer cells through inhibition of tumor formation, cancer cell proliferation, and enhanced cell death. Besides cell-intrinsic consequences, type I IFN activation modulates immune response to specifically eliminate cancer cells. Upregulation of NK cells and total macrophages was observed in xenografts deficient in p63, as shown by Eomes and AdgreI upregulation. Transcriptome analysis also showed increased HLA expression in p63-depleted cells, suggesting enhanced antigen presentation to reboot the immunosurveillance in the tumor microenvironment for cancer cell elimination.

To conclude, this study explicitly illustrates that ΔNp63α suppresses type I IFN response through viral mimicry and STING suppression, to promote ESCC tumorigenesis. This discovery opens a new avenue to exploit viral mimicry induction and STING agonists as therapeutic options for a subgroup of ESCC patients.