Probes for LGR5

Ewing sarcomas are characterized by the presence of EWS/ETS fusion genes in the absence of other recurrent genetic alterations and mechanisms of tumor heterogeneity that contribute to disease progression remain unclear. Mutations in the Wnt/beta-catenin pathway are rare in Ewing sarcoma but the Wnt pathway modulator LGR5 is often highly expressed, suggesting a potential role for the axis in tumor pathogenesis. We evaluated beta-catenin and LGR5 expression in Ewing sarcoma cell lines and tumors and noted marked intra- and inter-tumor heterogeneity. Tumors with evidence of active Wnt/beta-catenin signaling were associated with increased incidence of tumor relapse and worse overall survival. Paradoxically, RNA sequencing revealed a marked antagonism of EWS/ETS transcriptional activity in Wnt/beta-catenin activated tumor cells. Consistent with this, Wnt/beta-catenin activated cells displayed a phenotype that was reminiscent of Ewing sarcoma cells with partial EWS/ETS loss of function. Specifically, activation of Wnt/beta-catenin induced alterations to the actin cytoskeleton, acquisition of a migratory phenotype and up regulation of EWS/ETS-repressed genes. Notably, activation of Wnt/beta-catenin signaling led to marked induction of tenascin C (TNC), an established promoter of cancer metastasis, and an EWS/ETS-repressed target gene. Loss of TNC function in Ewing sarcoma cells profoundly inhibited their migratory and metastatic potential. Our studies reveal that heterogeneous activation of Wnt/beta-catenin signaling in subpopulations of tumor cells contributes to phenotypic heterogeneity and disease progression in Ewing sarcoma. Significantly, this is mediated, at least in part, by inhibition of EWS/ETS fusion protein function that results in de-repression of metastasis-associated gene programs.

Abstract

BACKGROUND & AIMS:

The intestinal epithelium is maintained by long-lived intestinal stem cells (ISCs) that reside near the crypt base. Above the ISC zone, there are short-lived progenitors that normally give rise to lineage-specific differentiated cell types but can dedifferentiate into ISCs in certain circumstances. However, the role of epithelial dedifferentiation in cancer development has not been fully elucidated.

METHODS:

We performed studies with Bhlha15-CreERT, Lgr5-DTR-GFP, Apcflox/flox, LSL-Notch (IC), and R26-reporter strains of mice. Some mice were given diphtheria toxin to ablate Lgr5 mRNA-positive cells, irradiated, or given 5-fluorouracil, hydroxyurea, doxorubicin, or dextran sodium sulfate to induce intestinal or colonic tissue injury. In intestinal tissues we analyzed the fate of progeny that expressed Bhlha15 mRNA. We used microarrays and reverse-transcription PCR to analyze gene expression patterns in healthy and injured intestinal tissues and in tumors. We analyzed gene expression patterns in human colorectal tumors using the TCGA dataset.

RESULTS:

Bhlha15 identified Paneth cells and short-lived secretory precursors (including pre-Paneth label-retaining cells) located just above the ISC zone in the intestinal epithelium. Bhlha15+ cells had no plasticity after loss of Lgr5-positive cells or irradiation. However, Bhlha15+ secretory precursors started to supply the enterocyte lineage after doxorubicin-induced epithelial injury in a Notch-dependent manner. Sustained activation of Notch converts Bhlha15+ secretory precursors to long-lived enterocyte progenitors (EPs). Administration of doxorubicin and expression of an activated form of Notch resulted in a gene expression pattern associated with EPs, whereas only sustained activation of Notch altered gene expression patterns in Bhlha15+ precursors, towards that of ISCs. Bhlha15+ EPs with sustained activation of Notch formed intestinal tumors with serrated features in mice with disruption of Apc. In the colon, Bhlha15 marked secretory precursors that became stem-like, cancer-initiating cells following dextran sodium sulfate-induced injury, via activation of Src and YAP signaling. In analyses of human colorectal tumors, we associated activation of Notch with chromosome instability-type tumors with serrated features in the left colon.

CONCLUSION:

In mice, we found that short-lived precursors can undergo permanent reprogramming by activation of Notch and YAP signaling. These cells could mediate tumor formation, in addition to traditional ISCs.