Probes for YAP

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.