Abstract 1233: Depletion of Apc gene in Cdx2 gastro-intestinal cells facilitates metastasis of colorectal adenocarcinoma Lgr5 cells to distant organs
Authors: Bakheet Ahmed , Gao Wen , Cai Dan , Berger Nathan , Tse William , Han Xiaonan 
Published: April 2023

Background: 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 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 CRC or CRC hepatic-metastatic patients, CDX2, CTNNB1, α-SMA, APC and LGR5 expressions were examined. Apc flox mice were crossed with Lgr5CreER and/or Cdx2CreER mice to inactivate Apc gene in Lgr5 ISCs, Cdx2 IECs or stromal cells. Cdx2CreER;Apc mice were then crossed with tgfr2 flox mice to deplete tgfr2 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 analyses. The colorectal organoids were differentiated and transplanted into abdominal cavity of recipient mice. Meanwhile, HT-29 cells were transfected with APC and/or CDX2 gRNAs.

Results: CTNNB1 and LGR5 expression were increased in human CRC and hepatic-metastatic CRC while interstitial CDX2 and SMA colocalization were robustly pronounced compared to normal colorectum. TCGA analysis showed the positive correlation of APC lower and CDX2 higher with metastatic rectal cancer. Apc depletion in Lgr5 cells in 6-week-old mice led to Lgr5+ rectal adenocarcinoma and Lgr5 crypts in liver while depletion of Apc in Lgr5 cells in 3-month-old mice only resulted in Lgr5 intestinal adenoma. Apc depletion in Cdx2 cells led to rectal fibroma and intestinal adenoma as well as undifferentiated metastatic Lgr5 cells in liver. Notably, Apc depletion 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 epithelial-mesenchymal transition (EMT) markers (Fibronectin and Vimentin). Finally, 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. Intra-abdominal xenotransplantation 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 EMT mechanisms.

Citation Format: Ahmed Bakheet, Wen Gao, Dan Cai, Nathan Berger, William Tse, Xiaonan Han. Depletion of Apc gene in Cdx2 gastro-intestinal cells facilitates metastasis of colorectal adenocarcinoma Lgr5 cells to distant organs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1233.