Probes for LGR5

Abstract

BACKGROUND:
In celiac disease (CD), intestinal epithelium damage occurs secondary to an immune insult and is characterized by blunting of the villi and crypt hyperplasia. Similarities between Hedgehog (Hh)/BMP4 downregulation, as reported in a mouse model, and CD histopathology, suggest mechanistic involvement of Hh/BMP4/WNT pathways in proliferation and differentiation of immature epithelial cells in the context of human intestinal homeostasis and regeneration after damage. Herein we examined the nature of intestinal crypt hyperplasia and involvement of Hh/BMP4 in CD histopathology.

METHODS AND FINDINGS:
Immunohistochemistry, qPCR and in situ hybridization were used to study a cohort of 24 healthy controls (HC) and 24 patients with diagnosed acute celiac disease (A-CD) intestinal biopsies. In A-CD we observed an increase in cells positive for Leucin-rich repeat-containing G protein-coupled receptor 5 (LGR5), an epithelial stem cell specific marker and expansion of WNT responding compartment. Further, we observed alteration in number and distribution of mesenchymal cells, predicted to be part of the intestinal stem cells niche. At the molecular level we found downregulation of indian hedgehog (IHH) and other components of the Hh pathway, but we did not observe a concurrent downregulation of BMP4. However, we observed upregulation of BMPs antagonists, gremlin 1 and gremlin 2.

CONCLUSIONS:
Our data suggest that acute CD histopathology partially recapitulates the phenotype reported in Hh knockdown models. Specifically, Hh/BMP4 paradigm appears to be decoupled in CD, as the expansion of the immature cell population does not occur consequent to downregulation of BMP4. Instead, we provide evidence that upregulation of BMP antagonists play a key role in intestinal crypt hyperplasia. This study sheds light on the molecular mechanisms underlying CD histopathology and the limitations in the use of mouse models for celiac disease.

Abstract

Cancer associated fibroblasts (CAFs) are the dominant cell population in the cancer stroma. Gremlin 1 (GREM1), an antagonist of the bone morphogenetic protein pathway, is expressed by CAFs in a variety of human cancers. However, its biological significance for cancer patients is largely unknown. We applied RNA in situ hybridization (ISH) to evaluate the prognostic value of stromal GREM1 expression in a large cohort of 670 colorectal cancers (CRCs). Overall GREM1 expression in CRCs was lower than that of the matched normal mucosa, and GREM1 expression had a strong positive correlation with BMI1 and inverse correlations with EPHB2 and OLFM4. RNA ISH localized the GREM expression to smooth muscle cells of the muscularis mucosa, fibroblasts around crypt bases and in the submucosal space of a normal colon. In various colon polyps, epithelial GREM1 expression was exclusively observed in traditional serrated adenomas. In total, 44% of CRCs were positive for stromal GREM1, which was associated with decreased lymphovascular invasion, a lower cancer stage, and nuclear β-catenin staining. Stromal GREM1 was significantly associated with improved recurrence-free and overall survival, although it was not found to be an independent prognostic marker in multivariate analyses. In addition, for locally advanced stage II and III CRCs, it was associated with better, stage-independent clinical outcomes. In summary, CRCs are frequently accompanied by GERM1-expressing fibroblasts, which are closely associated with low lymphovascular invasion and a better prognosis, suggesting stromal GREM1 as a potential biomarker and possible candidate for targeted therapy in the treatment of CRCs.

One of the most encouraging developments in oncology has been the success of BRAF inhibitors in BRAF-mutant melanoma. However, in contrast to its striking efficacy in BRAF-mutant melanomas, BRAF inhibitor monotherapy is ineffective in BRAF-mutant colorectal cancer (CRC). While many studies on BRAF inhibitor resistance in CRC have focused on mechanisms underlying the reactivation of the EGFR/RAS/RAF/MEK/ERK pathway, the current study focuses on identifying novel adaptive signaling mechanisms, a fresh angle on CRC resistance to BRAF inhibition. We found that treatment with BRAF inhibitors (both current and next generation BRAF inhibitors) upregulated the Wnt/β-catenin pathway in BRAFV600E-mutant CRC cell lines through activating the cytoplasmic tyrosine kinase FAK (focal adhesion kinase). The results showed that FAK activation upon BRAF inhibitor treatment did not require EGFR (Epidermal Growth Factor Receptor) or ERK1/2 (extracellular-signal-regulated kinases1/2) activation, implying that BRAF inhibitor treatment-induced hyperactivation of Wnt signaling is "pathway reactivation"-independent. BRAF inhibition-induced Wnt pathway activation was further validated in preclinical models of BRAFV600E-mutant CRC including cell line xenograft model and a PDX (patient-derived xenograft) model. Combined inhibition of BRAF/Wnt pathways or BRAF/FAK pathways exerted strong synergistic antitumor effects in cell culture model and mouse xenograft model. Overall, the current study has identified activation of the Wnt/β-catenin pathway as a novel fundamental cause of colon cancer resistance to BRAF inhibition. Our results suggest that while complete vertical pathway blockade is pivotal for effective and durable control of BRAF-mutant CRC, co-targeting parallel adaptive signaling-the Wnt/β-catenin pathway-is also essential.