Marianne Lähde1, Sarika Heino1, Jenny Högström2, Seppo Kaijalainen3, Andrey Anisimov1, Dustin Flanagan4, Pauliina Kallio1, Veli-Matti Leppänen5, Ari Ristimäki6, Olli Ritvos7, Katherine Wu8, Tuomas Tammela9, Michael Hodder10, Owen J Sansom10, Kari Alitalo11. 1. Translational Cancer Medicine Program, Research Programs Unit, Faculty of Medicine, University of Helsinki, Helsinki, Finland; iCAN Digital Precision Cancer Medicine Flagship, University of Helsinki and Helsinki University Hospital, Helsinki, Finland. 2. Translational Cancer Medicine Program, Research Programs Unit, Faculty of Medicine, University of Helsinki, Helsinki, Finland; iCAN Digital Precision Cancer Medicine Flagship, University of Helsinki and Helsinki University Hospital, Helsinki, Finland; Cancer Research Institute, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts. 3. Translational Cancer Medicine Program, Research Programs Unit, Faculty of Medicine, University of Helsinki, Helsinki, Finland. 4. Cancer Research UK Beatson Institute, Glasgow, United Kingdom. 5. Translational Cancer Medicine Program, Research Programs Unit, Faculty of Medicine, University of Helsinki, Helsinki, Finland; Wihuri Research Institute, Biomedicum Helsinki, Helsinki, Finland. 6. Department of Pathology, HUSLAB, HUS Diagnostic Center, Helsinki University Hospital; Medicum and Applied Tumor Genomics, Research Programs Unit, Faculty of Medicine, University of Helsinki, Helsinki, Finland. 7. Department of Physiology, Faculty of Medicine, University of Helsinki, Helsinki, Finland. 8. Cancer Biology and Genetics Program, Memorial Sloan Kettering Cancer Center, New York, New York. 9. Cancer Biology and Genetics Program, Memorial Sloan Kettering Cancer Center, New York, New York; Cell and Developmental Biology, Weill-Cornell Medical College, New York, New York. 10. Cancer Research UK Beatson Institute, Glasgow, United Kingdom; Institute of Cancer Sciences, University of Glasgow, Glasgow, United Kingdom. 11. Translational Cancer Medicine Program, Research Programs Unit, Faculty of Medicine, University of Helsinki, Helsinki, Finland; iCAN Digital Precision Cancer Medicine Flagship, University of Helsinki and Helsinki University Hospital, Helsinki, Finland; Wihuri Research Institute, Biomedicum Helsinki, Helsinki, Finland. Electronic address: kari.alitalo@helsinki.fi.
Abstract
BACKGROUND & AIMS: Mutations in the APC gene and other genes in the Wnt signaling pathway contribute to development of colorectal carcinomas. R-spondins (RSPOs) are secreted proteins that amplify Wnt signaling in intestinal stem cells. Alterations in RSPO genes have been identified in human colorectal tumors. We studied the effects of RSPO1 overexpression in ApcMin/+ mutant mice. METHODS: An adeno associated viral vector encoding RSPO1-Fc fusion protein, or control vector, was injected into ApcMin/+mice. Their intestinal crypts were isolated and cultured as organoids. which were incubated with or without RSPO1-Fc and an inhibitor of transforming growth factor beta receptor (TGFBR). Livers were collected from mice and analyzed by immunohistochemistry. Organoids and adenomas were analyzed by quantitative reverse-transcription PCR, single cell RNA sequencing, and immunohistochemistry. RESULTS: Intestines from Apc+/+ mice injected with the vector encoding RSPO1-Fc had significantly deeper crypts, longer villi, with increased EdU labeling, indicating increased proliferation of epithelial cells, in comparison to mice given control vector. AAV-RSPO1-Fc-transduced ApcMin/+ mice also developed fewer and smaller intestinal tumors and had significantly longer survival times. Adenomas of ApcMin/+ mice injected with the RSPO1-Fc vector showed a rapid increase in apoptosis and in the expression of Wnt target genes, followed by reduced expression of messenger RNAs and proteins regulated by the Wnt pathway, reduced cell proliferation, and less crypt branching than adenomas of mice given the control vector. Addition of RSPO1 reduced the number of adenoma organoids derived from ApcMin/+ mice and suppressed expression of Wnt target genes but increased phosphorylation of SMAD2 and transcription of genes regulated by SMAD. Inhibition of TGFBR signaling in organoids stimulated with RSPO1-Fc restored organoid formation and expression of genes regulated by Wnt. The TGFBR inhibitor restored apoptosis in adenomas from ApcMin/+ mice expressing RSPO1-Fc back to the same level as in the adenomas from mice given the control vector. CONCLUSIONS: Expression of RSPO1 in ApcMin/+ mice increases apoptosis and reduces proliferation and Wnt signaling in adenoma cells, resulting in development of fewer and smaller intestinal tumors and longer mouse survival. Addition of RSPO1 to organoids derived from adenomas inhibits their growth and promotes proliferation of intestinal stem cells that retain the APC protein; these effects are reversed by TGFB inhibitor. Strategies to increase the expression of RSPO1 might be developed for the treatment of intestinal adenomas.
BACKGROUND & AIMS: Mutations in the APC gene and other genes in the Wnt signaling pathway contribute to development of colorectal carcinomas. R-spondins (RSPOs) are secreted proteins that amplify Wnt signaling in intestinal stem cells. Alterations in RSPO genes have been identified in humancolorectal tumors. We studied the effects of RSPO1 overexpression in ApcMin/+ mutant mice. METHODS: An adeno associated viral vector encoding RSPO1-Fc fusion protein, or control vector, was injected into ApcMin/+mice. Their intestinal crypts were isolated and cultured as organoids. which were incubated with or without RSPO1-Fc and an inhibitor of transforming growth factor beta receptor (TGFBR). Livers were collected from mice and analyzed by immunohistochemistry. Organoids and adenomas were analyzed by quantitative reverse-transcription PCR, single cell RNA sequencing, and immunohistochemistry. RESULTS: Intestines from Apc+/+ mice injected with the vector encoding RSPO1-Fc had significantly deeper crypts, longer villi, with increased EdU labeling, indicating increased proliferation of epithelial cells, in comparison to mice given control vector. AAV-RSPO1-Fc-transduced ApcMin/+ mice also developed fewer and smaller intestinal tumors and had significantly longer survival times. Adenomas of ApcMin/+ mice injected with the RSPO1-Fc vector showed a rapid increase in apoptosis and in the expression of Wnt target genes, followed by reduced expression of messenger RNAs and proteins regulated by the Wnt pathway, reduced cell proliferation, and less crypt branching than adenomas of mice given the control vector. Addition of RSPO1 reduced the number of adenoma organoids derived from ApcMin/+ mice and suppressed expression of Wnt target genes but increased phosphorylation of SMAD2 and transcription of genes regulated by SMAD. Inhibition of TGFBR signaling in organoids stimulated with RSPO1-Fc restored organoid formation and expression of genes regulated by Wnt. The TGFBR inhibitor restored apoptosis in adenomas from ApcMin/+ mice expressing RSPO1-Fc back to the same level as in the adenomas from mice given the control vector. CONCLUSIONS: Expression of RSPO1 in ApcMin/+ mice increases apoptosis and reduces proliferation and Wnt signaling in adenoma cells, resulting in development of fewer and smaller intestinal tumors and longer mouse survival. Addition of RSPO1 to organoids derived from adenomas inhibits their growth and promotes proliferation of intestinal stem cells that retain the APC protein; these effects are reversed by TGFB inhibitor. Strategies to increase the expression of RSPO1 might be developed for the treatment of intestinal adenomas.