Fibroblast-derived Gremlin1 localises to epithelial cells at the base of the intestinal crypt

Louise R Dutton,Owen P Hoare,Victoria Bingham,Amy M.B Mccorry,Shannon Canamara,Paul B Mullan,Mark Lawler,Derek P Brazil,Philip D Dunne,Keara L Redmond,Noor Eisa Adam

doi:10.18632/oncotarget.27050

Louise R Dutton, Owen P Hoare + Show 9 more

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https://doi.org/10.18632/oncotarget.27050

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Abstract

Gremlin1 (GREM1) is a secreted glycoprotein member of the differential screening-selected gene in aberrant neuroblastoma (DAN) family of bone morphogenetic protein (BMP) antagonists, which binds to BMPs preventing their receptor engagement. Previous studies have identified that stage II colorectal cancer (CRC) patients with high levels of GREM1 gene expression in their tumour tissue have a poorer prognosis. Using a series of in silico and in situ methodologies, we demonstrate that GREM1 gene expression is significantly higher (p < 0.0001) in CRC consensus molecular subtype 4 (CMS4), compared to the other CMS subtypes and correlates (p < 0.0001) with levels of cancer-associated fibroblasts (CAFs) within the CRC tumour microenvironment (TME). Our optimised immunohistochemistry protocol identified endogenous GREM1 protein expression in both the muscularis mucosa and adjacent colonic crypt bases in mouse intestine, in contrast to RNA expression which was shown to localise specifically to the muscularis mucosa, as determined by in situ hybridisation. Importantly, we demonstrate that cells with high levels of GREM1 expression display low levels of phospho-Smad1/5, consistent with reduced BMP signalling. Taken together, these data highlight a novel paracrine signalling circuit, which involves uptake of mature GREM1 protein by colonic crypt cells following secretion from neighbouring fibroblasts in the TME.

Highlights

Gremlin1 (GREM1) is a conserved 184 aa glycoprotein antagonist of bone morphogenetic protein (BMP) signalling which regulates organogenesis and differentiation [1, 2]
This predisposition is very rare, we analysed GREM1 mRNA levels across five non-hereditary mixed polyposis syndrome (HMPS) colorectal cancer (CRC) transcriptional datasets that had been stratified into consensus molecular subtypes (CMS) subtypes [12], and identified significantly higher quantities of GREM1 mRNA in the fibroblast-rich consensus molecular subtype 4 (CMS4) subtype compared to CMS1-3 (Figure 1B, Supplementary Figure 1)
We and others have previously highlighted the association between CMS4, high levels of cancerassociated fibroblasts (CAFs), and poor prognosis in CRC [22,23,24,25] we set out to delineate the cellular-specific source of GREM1 from within the tumour microenvironment (TME) of stromal-rich CMS4 tumours

Summary

Introduction

Gremlin (GREM1) is a conserved 184 aa glycoprotein antagonist of bone morphogenetic protein (BMP) signalling which regulates organogenesis and differentiation [1, 2]. GREM1 overexpression has been linked to a range of cancers, including colorectal cancer (CRC) [5,6,7,8,9,10], with mutations affecting BMP signalling occurring in a large number of CRCs [11]. GREM1 overexpression has been identified in epithelial cells of more common sporadic colorectal traditional serrated adenomas (TSAs) [18] at the desmoplastic invasive front [13]

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