Probes for YAP

Background: Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer deaths with a 5-year survival rate of approximately 7%. PDAC may originate from acinar cell trans-differentiation into ductal-like cells, termed acinar-to-ductal metaplasia (ADM), triggered by chronic pancreatitis and/or mutations in K-Ras. The progression to PDAC is associated with a dense fibrotic stroma, including cancer-associated fibroblasts (CAFs). YAP is a tension-stimulated CAF activator that promotes ECM stiffening, creating a permissive microenvironment for cancer progression. We hypothesize that the Hippo pathway may coordinate fibroinflammatory signals emanating from the stromal compartment during regenerative responses to acinar cell injury and progression towards PDAC. Methods: To resolve the transcriptional changes occurring during the transition to ADM and PDAC, we mapped the in situ expression of over 1800 RNA targets in patient-derived tissues using NanoString Technologies’ Digital Spatial Profiling (DSP) technology. We also performed immune-profiling and evaluated Yap expression in human ADM by immunohistochemistry. To study the in vivo role of Hippo signaling in stromal cells, we conditionally deleted Yap/Taz in Collagen1a2-producing cells in a murine model of caerulein-induced pancreatitis, which recapitulates many of the features associated with human ADM. I will analyze the resulting phenotype by immunostaining for metaplastic, proliferative, immune and stromal markers. Results: DSP analysis revealed genes implicated in fibroblast activation, epithelial-to-mesenchymal transition (EMT), neutrophil activation and IFNγ signaling as potential key drivers of ADM. I will further evaluate the expression of candidate genes and survey Yap expression at the single cell level in human ADM tissue by multiplexed RNAscope in situ hybridization. We found up-regulation of CD4+ and CD8+ T cells in ADM, and an increasing trend of neutrophil and macrophage accumulation in the progression from normal parenchyma to ADM to PDAC. Conclusions: This work will provide an in-depth understanding of epithelial-stroma crosstalk in ADM and a foundation for the development of new therapeutic strategies for treating non-invasive precursor lesions like ADM, thereby preventing pancreatic cancer progression. Source of Funding: This research is supported by the Fonds de Recherce du Quebec - Santé (FRQS), Canadian Institutes of Health Research (CIHR) and the Research Institute of the McGill University Health Centre (RI-MUHC).

Cancer relapse after chemotherapy remains a main cause of cancer-related death. Although the relapse is thought to result from the propagation of resident cancer stem cells (CSCs)1, a lack of experimental platforms that enable prospective analysis of CSC dynamics with sufficient spatiotemporal resolution has hindered testing of this hypothesis. Here, we develop a live genetic lineage-tracing system that allows longitudinal tracking of individual cells in xenotransplanted human colorectal cancer organoids and identify LGR5+ CSCs that display a dormant behavior in a chemo-naive state. Dormant LGR5+ cells are marked by p27 expression, and intravital imaging directly demonstrates the persistence of LGR5+p27+ cells during chemotherapy, followed by clonal expansion. Transcriptome analysis reveals an upregulation of COL17A1, a cell adhesion molecule that strengthens hemidesmosome, in dormant LGR5+p27+ cells. COL17A1-knockout organoids lose the dormant LGR5+p27+ subpopulation and become sensitive to chemotherapy, suggesting a role of cell-matrix interface in dormancy maintenance. Chemotherapy disrupts COL17A1 and breaks the dormancy in LGR5+p27+ cells through FAK-YAP activation. Abrogation of YAP signaling restrains chemo-resistant cells from exiting dormancy and delays tumor regrowth, highlighting the therapeutic potential of YAP inhibition in preventing cancer relapse. These results offer a viable therapeutic approach to overcome refractoriness of human colorectal cancer to conventional chemotherapy.

Smooth muscle is an essential component of the intestine, both to maintain its structure and produce peristaltic and segmentation movements. However, very little is known about other putative roles that smooth muscle cells may have. Here, we show that smooth muscle cells may be the dominant suppliers of BMP antagonists, which are niche factors essential for intestinal stem cell maintenance. Furthermore, muscle-derived factors render epithelium reparative and fetal-like, which includes heightened YAP activity. Mechanistically, we find that the membrane-bound matrix metalloproteinase MMP17, which is exclusively expressed by smooth muscle cells, is required for intestinal epithelial repair after inflammation- or irradiation-induced injury. Furthermore, we propose that MMP17 affects intestinal epithelial reprogramming after damage indirectly by cleaving diffusible factor(s) such as the matricellular protein PERIOSTIN. Together, we identify an important signaling axis that establishes a role for smooth muscle cells as modulators of intestinal epithelial regeneration and the intestinal stem cell niche.

Circadian rhythms are daily physiological oscillations driven by the circadian clock: a 24-hour transcriptional timekeeper that regulates hormones, inflammation, and metabolism. Circadian rhythms are known to be important for health, but whether their loss contributes to colorectal cancer is not known.We tested the non-redundant clock gene, Bmal1, in intestinal homeostasis and tumorigenesis, using the Apcmin model of colorectal cancer.Bmal1 mutant, epithelium-conditional Bmal1 mutant, and photoperiod-disrupted mice bearing the Apcmin allele were assessed for tumorigenesis. Tumors and normal non-transformed tissue were characterized. Intestinal organoids were assessed for circadian transcription rhythms by RNA-sequencing, and in vivo and organoid assays were used to test Bmal1-dependent proliferation and self-renewal.Loss of Bmal1 or circadian photoperiod increases tumor initiation. In the intestinal epithelium the clock regulates transcripts involved in regeneration and intestinal stem cell signaling. Tumors have no self-autonomous clock function and only weak clock function in vivo. Apcmin clock-disrupted tumors exhibit high Yap (Hippo signaling) activity but exhibit low Wnt activity. Intestinal organoid assays reveal that loss of Bmal1 increases self-renewal in a Yap-dependent manner.Bmal1 regulates intestinal stem cell pathways, including Hippo signaling, and the loss of circadian rhythms potentiates tumor initiation.

Background & aims Fibrosis and tissue stiffening are hallmarks of the inflammatory bowel disease (IBD). We have hypothesized that the increased stiffness directly contributes to the dysregulation of the epithelial cell homeostasis in IBD. Here, we aim to determine the impact of tissue stiffening on the fate and function of the intestinal stem cells (ISCs). Methods We developed a long-term culture system consisting of 2.5-dimensional intestinal organoids grown on a hydrogel matrix with tunable stiffness. Single-cell RNA sequencing provided stiffness-regulated transcriptional signatures of the ISCs and their differentiated progeny. YAP-knockout and YAP-overexpression mice were used to manipulate YAP expression. In addition, we analyzed colon samples from murine colitis models and human IBD samples to assess the impact of stiffness on ISCs in vivo. Results We demonstrated that increasing the stiffness potently reduced the population of LGR5+ ISCs and KI-67+ proliferating cells. Conversely, cells expressing the stem cell marker, OLFM4, became dominant in the crypt-like compartments and pervaded the villus-like regions. Concomitantly, stiffening prompted the ISCs to preferentially differentiate toward goblet cells. Mechanistically, stiffening increased the expression of cytosolic YAP, driving the extension of OLFM4+ cells into the villus-like regions, while it induced the nuclear translocation of YAP, leading to preferential differentiation of ISCs towards goblet cells. Furthermore, analysis of colon samples from murine colitis models and IBD patients demonstrated cellular and molecular remodeling reminiscent of those observed in vitro. Conclusions Collectively, our findings highlight that matrix stiffness potently regulates the stemness of ISCs and their differentiation trajectory, supporting the hypothesis that fibrosis-induced gut stiffening plays a direct role in epithelial remodeling in IBD.