TY - JOUR
T1 - Genetic editing of colonic organoids provides a molecularly distinct and orthotopic preclinical model of serrated carcinogenesis
AU - Lannagan, Tamsin R.M.
AU - Lee, Young K.
AU - Wang, Tongtong
AU - Roper, Jatin
AU - Bettington, Mark L.
AU - Fennell, Lochlan
AU - Vrbanac, Laura
AU - Jonavicius, Lisa
AU - Somashekar, Roshini
AU - Gieniec, Krystyna
AU - Yang, Miao
AU - Ng, Jia Q.
AU - Suzuki, Nobumi
AU - Ichinose, Mari
AU - Wright, Josephine A.
AU - Kobayashi, Hiroki
AU - Putoczki, Tracey L.
AU - Hayakawa, Yoku
AU - Leedham, Simon J.
AU - Abud, Helen E.
AU - Yilmaz, Ömer H.
AU - Marker, Julie
AU - Klebe, Sonja
AU - Wirapati, Pratyaksha
AU - Mukherjee, Siddhartha
AU - Tejpar, Sabine
AU - Leggett, Barbara A.
AU - Whitehall, Vicki L.J.
AU - Worthley, Daniel L.
AU - Woods, Susan L.
PY - 2019/4/1
Y1 - 2019/4/1
N2 - Objective Serrated colorectal cancer (CRC) accounts for approximately 25% of cases and includes tumours that are among the most treatment resistant and with worst outcomes. This CRC subtype is associated with activating mutations in the mitogen-Activated kinase pathway gene, BRAF, and epigenetic modifications termed the CpG Island Methylator Phenotype, leading to epigenetic silencing of key tumour suppressor genes. It is still not clear which (epi-)genetic changes are most important in neoplastic progression and we begin to address this knowledge gap herein. Design We use organoid culture combined with CRISPR/Cas9 genome engineering to sequentially introduce genetic alterations associated with serrated CRC and which regulate the stem cell niche, senescence and DNA mismatch repair. Results Targeted biallelic gene alterations were verified by DNA sequencing. Organoid growth in the absence of niche factors was assessed, as well as analysis of downstream molecular pathway activity. Orthotopic engraftment of complex organoid lines, but not Braf V600E alone, quickly generated adenocarcinoma in vivo with serrated features consistent with human disease. Loss of the essential DNA mismatch repair enzyme, Mlh1, led to microsatellite instability. Sphingolipid metabolism genes are differentially regulated in both our mouse models of serrated CRC and human CRC, with key members of this pathway having prognostic significance in the human setting. Conclusion We generate rapid, complex models of serrated CRC to determine the contribution of specific genetic alterations to carcinogenesis. Analysis of our models alongside patient data has led to the identification of a potential susceptibility for this tumour type.
AB - Objective Serrated colorectal cancer (CRC) accounts for approximately 25% of cases and includes tumours that are among the most treatment resistant and with worst outcomes. This CRC subtype is associated with activating mutations in the mitogen-Activated kinase pathway gene, BRAF, and epigenetic modifications termed the CpG Island Methylator Phenotype, leading to epigenetic silencing of key tumour suppressor genes. It is still not clear which (epi-)genetic changes are most important in neoplastic progression and we begin to address this knowledge gap herein. Design We use organoid culture combined with CRISPR/Cas9 genome engineering to sequentially introduce genetic alterations associated with serrated CRC and which regulate the stem cell niche, senescence and DNA mismatch repair. Results Targeted biallelic gene alterations were verified by DNA sequencing. Organoid growth in the absence of niche factors was assessed, as well as analysis of downstream molecular pathway activity. Orthotopic engraftment of complex organoid lines, but not Braf V600E alone, quickly generated adenocarcinoma in vivo with serrated features consistent with human disease. Loss of the essential DNA mismatch repair enzyme, Mlh1, led to microsatellite instability. Sphingolipid metabolism genes are differentially regulated in both our mouse models of serrated CRC and human CRC, with key members of this pathway having prognostic significance in the human setting. Conclusion We generate rapid, complex models of serrated CRC to determine the contribution of specific genetic alterations to carcinogenesis. Analysis of our models alongside patient data has led to the identification of a potential susceptibility for this tumour type.
KW - cancer genetics
KW - colorectal cancer
KW - gene mutation
KW - methylation
KW - oncogenes
UR - http://www.scopus.com/inward/record.url?scp=85049146061&partnerID=8YFLogxK
UR - http://purl.org/au-research/grants/nhmrc/1081852
UR - http://purl.org/au-research/grants/nhmrc/1140236
U2 - 10.1136/gutjnl-2017-315920
DO - 10.1136/gutjnl-2017-315920
M3 - Article
C2 - 29666172
AN - SCOPUS:85049146061
SN - 0017-5749
VL - 68
SP - 684
EP - 692
JO - Gut
JF - Gut
IS - 4
ER -