Aberrant epithelial cell interaction promotes esophageal squamous-cell carcinoma development and progression

Aberrant epithelial cell interaction promotes esophageal squamous-cell carcinoma development and progression
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As one of the most prevalent gastrointestinal cancers in the world, esophageal squamous cell carcinoma (ESCC) develops through chronic inflammation (INF), precancerous lesions including low-grade intraepithelial neoplasia (LGIN) and high-grade intraepithelial neoplasia (HGIN), and invasive ESCC.1 According to the prospective study, 74% of HGIN cases and 31% of LGIN cases developed into ESCC.2 Thus, identification of the molecular mechanisms from normal to malignancy is critical for early detection, diagnosis, and treatment of this disease. In previous studies, we have deciphered the aberrant gene expression of epithelial cells and the cellular compositions in the microenvironment of different disease stages using single-cell transcriptomic analysis. For instance, we have investigated the cell transition states in a multistep mouse model receiving 4-nitroquinoline-1-oxide (4NQO) with ESCC progression resembling the development of human ESCC, discovering a collection of important oncogenic molecule evolution of epithelial cells.3 Furthermore, we have found that human esophagus also exhibits certain transitory features observed in mouse  model.4 Additionally, we have demonstrated that epithelial cells can activate fibroblasts to promote ESCC development through reduced ANXA1-FPR2 ligand-receptor interaction.5 However, the exact in situ molecular mechanism and intercellular signaling that drive proliferation and invasion of diseased epithelial cells remain unclear. The ability to analyze the molecular changes related to tissue destruction during ESCC development has been revolutionized by the recently established spatial transcriptomic technologies, which combines tissue spatial imaging with in situ gene expression.6 In this study, we explored tissue reorganization and spatial molecular alterations during ESCC development using spatial and single-cell transcriptome analysis in both mouse and human samples.

We deciphered the spatial gene expression patterns and cellular communication networks in normal and multistage diseased esophageal tissue samples from mice and patients with ESCC. In diseased esophageal tissue samples, we identified a spatially defined region of ESCC development with significant expression of tumor hallmarks, outgrowing from the basal layer of the epithelium. We observed multiple aberrant cell interactions within these tissue regions. Remarkably, we found the enhanced interaction among epithelial cells mediated by EFNB1 and EPHB4 expanded from the basal layer to ESCC development region. Functional analysis demonstrates that the aberrant EFNB1-EPHB4 interaction in epithelial cells accelerates ESCC progression via enhanced cell cycle and epithelial-mesenchymal transition (EMT) pathways both activated by SRC/ERK/AKT signaling. Furthermore, we discovered for the first time that ΔNP63 is a transcription factor of both EFNB1 and EPHB4 genes, which is overexpressed in precancerous and cancerous lesions of the esophagus due to TP53 dysfunction. In our recent work, we analyzed 1,275 micro-biopsies from normal, precancerous and cancerous esophageal samples to decipher driver mutations that promote ESCC development and progression and found that TP53-biallelic loss or multiple mutation is the major driver, probably by causing copy number alterations of certain chromosomal regions.7 Our research further underscored the culprit role of TP53 in human ESCC tumorigenesis. These findings uncover for the first time that the TP53-TP63/∆NP63-EFNB1-EPHB4 signaling is essential in ESCC development and progression.

As members of Ephrin/Eph family, EFNB1 and EPHB4 are located on the cell membrane and their interaction can trigger short-range cell-cell communication between adjacent cells, resulting in a variety of biological processes.8 In the present study, we demonstrated that EFNB1 and EPHB4 expression levels gradually and significantly increase in both mouse and human esophageal epithelial cells along with ESCC progression. More importantly, we found that epithelial cells with aberrantly elevated EFNB1-EPHB4 interaction originate mainly from the basal layer of the epithelium and then expand to the upper layers when the disease progresses, which accelerates proliferation of diseased cells and triggers EMT, facilitating ESCC development and progression. These novel findings provide important evidence to support a broad range of pharmacological designs targeting EFNB1, EPHB4 or the Eph/Ephrin interaction to prevent or cure cancer in the future.

EMT is a highly complex and coordinated process in which epithelial cells lose multiple biological characteristics associated with epithelial differentiation while acquiring behaviors resembling mesenchymal cells concurrently.9 Despite the essential role of EMT in tumor invasion, we revealed for the first time that the aberrant EFNB1-EPHB4 interaction, occurring at INF/LGIN stage and increasing at HGIN and ESCC stages, can trigger esophageal epithelial cell EMT and proliferation. These findings highlighted that EFNB1-EPHB4 interaction induced EMT is an early event in ESCC development since it enhances the transformation of normal epithelial cells into malignant phenotypes.

Previous studies demonstrated that TP63 is a major oncogene with frequent amplification and overexpression in ESCC, which can induce the expression of specific oncogenes in human malignancies to stimulate the expression of EMT markers.10 The current investigation has revealed that TP63 amplification and overexpression occurs early in precancerous lesions and increases gradually as ESCC developed. We demonstrated for the first time that ΔNP63 is the common transcription factor of both EFNB1 and EPHB4. It is noteworthy that ΔNP63 overexpression in precancerous and cancerous epithelial cells may result from copy number alteration induced by TP53 dysfunction, which can cause widespread genomic disruption such as rearrangements according to our recent work.7

In conclusion, the present study clarifies the spatiotemporal alterations of intercellular signal transductions in tissue microenvironment during mouse and human ESCC development. Specifically, we uncovered the aberrant EFNB1-EPHB4 interaction among epithelial cells originating from the basal layer that triggers EMT and cell proliferation in ESCC development and progression. We also identified that TP53 mutation-induced TP63 overexpression is the cause of aberrant EFNB1-EPHB4 interaction. These results suggest an important effect of aberrant TP53-TP63/ΔNP63-EFNB1-EPHB4 signaling axis on oncogenic disorganization of esophageal tissues. Discovering these spatiotemporal gene regulatory networks within tissue microenvironment might have clinical implication in establishing early diagnosis and therapy based on these molecules for ESCC and perhaps other epithelial cancers.

Graphic Abstract

Professors Dongxin Lin and Chen Wu are the corresponding authors of this paper, with Dr. Liping Chen and PhD candidate Shihao Zhu serving as co-first authors. Shihao Zhu is currently seeking a postdoctoral position in the UK or the EU and is open to communication.

References

1     Nagtegaal, I. D. et al. The 2019 WHO classification of tumours of the digestive system. Histopathology 76, 182-188 (2020).

2     Wang, G. Q. et al. Histological precursors of oesophageal squamous cell carcinoma: results from a 13 year prospective follow up study in a high risk population. Gut 54, 187-192 (2005).

3     Yao, J. et al. Single-cell transcriptomic analysis in a mouse model deciphers cell transition states in the multistep development of esophageal cancer. Nat Commun 11, 3715 (2020).

4     Zhang, X. et al. Dissecting esophageal squamous-cell carcinoma ecosystem by single-cell transcriptomic analysis. Nat Commun 12, 5291 (2021).

5     Chen, Y. et al. Epithelial cells activate fibroblasts to promote esophageal cancer development. Cancer Cell 41, 903-918 (2023).

6     Ståhl, P. L. et al. Visualization and analysis of gene expression in tissue sections by spatial transcriptomics. Science 353, 78-82 (2016).

7     Chang J, et al. Genomic alterations driving precancerous to cancerous lesions in esophageal cancer development. Cancer Cell. Nov 20:S1535-6108(23)00394-X (2023).

8     Kania, A. & Klein, R. Mechanisms of ephrin-Eph signalling in development, physiology and disease. Nat Rev Mol Cell Biol 17, 240-256 (2016).

9     Lambert, A. W. & Weinberg, R. A. Linking EMT programmes to normal and neoplastic epithelial stem cells. Nat Rev Cancer 21, 325-338 (2021).

10   Fisher, M. L., Balinth, S. & Mills, A. A. ΔNp63α in cancer: importance and therapeutic opportunities. Trends Cell Biol 33, 280-292 (2023).

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Gastrointestinal Cancer
Life Sciences > Biological Sciences > Cancer Biology > Cancers > Gastrointestinal Cancer
Protein-Ligand Interactions
Life Sciences > Biological Sciences > Chemical Biology > Biochemistry > Protein Biochemistry > Protein-Ligand Interactions
Oesophageal Cancer
Life Sciences > Biological Sciences > Cancer Biology > Cancers > Gastrointestinal Cancer > Oesophageal Cancer
Cancer Genetics and Genomics
Life Sciences > Biological Sciences > Cancer Biology > Cancer Genetics and Genomics
Oncogenesis
Life Sciences > Health Sciences > Biomedical Research > Pathogenesis > Oncogenesis
Oncogenes
Life Sciences > Biological Sciences > Cancer Biology > Oncogenes