Depletion of Apc gene in Cdx2 gastro-intestinal cells facilitates metastasis of colorectal adenocarcinoma Lgr5 cells to distant organs

Abstract Background: Caudal-type homeobox 2 (CDX2) is a transcription factor expressed in the gastro-intestinal (GI) epithelial (IEC) and stromal cells. CDX2 along with APC can regulate Lgr5 intestinal stem cell (ISC) differentiation to control GI development or neoplasia. CDX2 or APC loss function is associated with colorectal cancer (CRC) advance. However, whether APC directly regulates CRC metastasis is not clear. We aim to determine the role of inactive Apc gene in Cdx2 GI cells on Lgr5 ISC metastasis. Methods: Tissue sections were collected from colorectal adenocarcinoma or CRC hepatic-metastatic patients, CDX2, CTNNB1, α-SMA, APC and LGR5 expressions were determined. Apc flox mice were crossed with Lgr5CreER and/or Cdx2CreER mice and Apc gene in the Lgr5 ISCs, Cdx2 IECs or stromal cells was inactivated. Cdx2CreER;Apc mice were then crossed with tgfr2 flox mice, tgfr2 was depleted in the Apc-deficient Cdx2 cells. GI tract and liver histology was evaluated and the expression of Lgr5, APC, α-SMA, β-catenin, and CDX2 were examined. The colorectal polyps induced by Apc inactivation were dissected, total RNA was extracted to perform RNA-Seq and quantitative PCR (qPCR) analyses. The colorectal organoids were differentiated. These differentiated organoids were then under intra-abdominal xenotransplantation. Meanwhile, HT-29 cells were transfected with APC and/or CDX2 gRNAs. Results: CTNNB1 and LGR5 expression are increased in human colorectal adenocarcinoma and hepatic-metastatic CRC while interstitial CDX2 and SMA colocalization are robustly pronounced compared to normal colorectum. Induction of Apc depletion in Lgr5 cells in 6-week-old mice leads to Lgr5+ rectal adenocarcinoma and Lgr5 crypts in liver while depletion of Apc in Lgr5 cells in 3-month-old mice results in only Lgr5 intestinal adenoma. 10 day-induction of Apc depletion in Cdx2 cells leads to rectal fibroma and intestinal adenoma as well as undifferentiated metastatic Lgr5 cells in liver. Notably, Apc depletion led to decreased crypt CDX2 while increased colocalization of CDX2 and α-SMA in the stromal cells of adenocarcinoma, and increased expression of TGFBβr2, β-catenin, and EMT markers. Induction of Apc depletion in both Cdx2 and Lgr5 cells led to signet ring cell carcinomas in colorectum and liver. Finally, inducing tgfr2 depletion in Apc-inactive Cdx2 cells impaired GI cancer cell metastasis to liver. Colorectal orgnoids exhibited cancer phenotypes upon Apc depletion in CDX2 or Lgr5 cells. Surprisingly, intra-abdominal xenotransplantation of colorectal organoids exhibited that organoids with Apc deficiency in Lgr5 cells appeared no colonization on or invasion of liver tissues whereas the organoids with Apc deficiency in Cdx2 cells invaded liver. Conclusion: Reduced Apc in Cdx2 cells are required for Lgr5 cell transformation to Lgr5- cancer stem cells. Inactive intestinal Apc increases Cdx2-α-SMA stromal niche cells and facilitates Lgr5 ISC metastasis to liver partially through TGF-β and/or wnt-dependent epithelial-mesenchymal transition (EMT) mechanisms. Citation Format: Xiaonan Han, Ahmed Bakheet, Wen Gao. Depletion of Apc gene in Cdx2 gastro-intestinal cells facilitates metastasis of colorectal adenocarcinoma Lgr5 cells to distant organs [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr A033.