Introduction

The contribution of the tumor microenvironment and extracellular matrix to the aggressive biology of Gastric Cancer (GC) has been recently characterized (1). During GC progression striking changes in the lymphatic vessels (LVs) occur and lymphatic metastasis is the commonest feature of GC spread, representing the most significant prognostic factor determining the clinical outcomes (2). Recent evidence supporting a functional link among EMILIN-1, an Extra Cellular Matrix component (ECM), tumour growth and lymph-angiogenesis has been provided. EMILIN-1 represents a key structural element in the maintenance and function of LVs and displays anti-proliferative effects through the interaction with α4β1 integrin, as demonstrated in skin and colon cancer (3,4,5). Interestingly, α4 integrin is transcriptionally repressed in GC, but not in the normal gastric epithelium (7), suggesting a crucial mechanism exerted by the integrin/EMILIN1 recognition also in this context.

Aims & Methods

The aim of this study is to analyze the possible role of EMILIN-1 as a molecular regulator in in the context of GC development and progression.
We performed Immunofluorescence staining of human gastric biopsy samples from normal and neoplastic tissues collected during the endoscopy procedures. Biopsies from pre-malignant lesions were also included. For in vivo experiments, we used the syngeneic YTN16 gastric cancer cell line (7) which was injected subcutaneously and intraperitoneally in genetically modified EMILIN-1 mice. In alternative, carcinogenesis was induced in mice using N-methyl-N-nitrosourea (MNU). GES-1 (normal stomach mucosa), and several GC cell lines representative of different GC stages and histotypes (i.e. AGS, Hs746.T, NCI-N87 and KATO-III) were employed for in vitro experiments.

Results

EMILIN-1 is diffusely deposited in normal gastric mucosa and is significantly decreased in malignant and pre-neoplastic samples; this finding correlates with the presence of aberrant LVs. EMILIN-1 mutant mice (knock out and E955A knock in) developed tumors earlier compared to WT animals: 20 days post-injection of YTN16 cells, tumors developed in EMILIN-1 mutant mice were larger and displayed a significant increase of lymphangiogenesis. Treatment of transgenic mice with MNU associated with an increased number of tumors, exacerbated aggressive lesions and higher levels of LV abnormalities.
To unveil the main mechanisms responsible for EMILIN-1 reduction we treated in vitro fibroblasts and LECs (the major cellular sources of EMILIN1 in the gastric microenvironment) with conditioned media derived from GC cells lines; dramatic effects on EMILIN1 expression were observed, both at the mRNA and protein levels. Small-RNAseq analysis on culture media allowed us to identify miR125a-5p as a possible molecular regulator of EMILIN-1 levels in this context.

Conclusion

This study demonstrates for the first time that loss of EMILIN-1 in GC leads to lymphatic dysfunction and proliferative advantages that sustain tumorigenesis, and assess the use of our animal model as a valuable tool to verify the fate of GC upon loss of EMILIN-1.

References

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