Colorectal cancer (CRC) is the third leading type of cancer in the United States. CRC can take several years to develop and occurs through what is known as the polyp-to-cancer sequence. Cancer is not the result of one event, but an accumulation of several critical events leading to cell immortalization and uncontrolled cell growth. During this process, the cells accumulate mutations and/or epigenetic modifications in key genes leading to their deregulation. The genes involved in these events are commonly named oncogenes (whose increased activity will favor cancer development) or tumor suppressors (whose loss of activity will favor cancer development). In CRC, TGFβ-RII (Transforming Growth Factor Beta Receptor, Type 2) exhibits a tumor suppressive function and is frequently inactivated. However, impaired TGFβ signaling is only one event among many other deregulations leading to CRC. Many of these additional mutations that cooperate with TGFβ-RII deregulation still have to be identified in order to develop effective treatments.
A study recently published in the International Journal of Cancer by Dr. Shelli Morris, a staff scientist and Dr. William Grady, principal investigator (Clinical Research Division), along with several colleagues from the Fred Hutch identified candidate genes contributing to CRC progression in association with impaired TGFβ signaling. In this work, the authors used a transposon system called Sleeping Beauty. Transposons are fragments of mobile DNA that can randomly move and integrate within the genome, in the presence of a transposase enzyme. Sleeping Beauty’s name originates from its discovery as a non-functional transposon in the genome of salmon fish. Researchers combine this transposon with a functional transposase enzyme to generate a tool that can cause random integrations within genes and deregulate normal gene function.
Dr. Morris explained “the Sleeping Beauty transposon system provides an unbiased screening tool to identify biologically relevant pathways altered in cancer”. Indeed, the researchers used Sleeping Beauty in combination with a transposase enzyme whose expression is specific to intestinal epithelial cells. Following the random integration of the transposon into critical genes, the mice developed tumors. Two groups of mice, containing either a functional or an inactivated TGFβ-RII were used. Interestingly, the mice with a defective TGFβ signaling pathway developed fewer, but larger and more advanced stage tumors, suggesting that TGFβ signaling deregulation might be more involved in cancer progression than cancer initiation. Additionally, even though not significant, 50% of the TGFβ-RII impaired mice died two months sooner than the mice with a functional TGFβ-RII, suggesting that the tumors may be of a more aggressive type. These observations are of importance to understand gene functions and their involvement in tumor initiation and progression.
130 tumors from the TGFβ-RII inactivated and functional mice were collected and the extracted genomic DNA was sequenced and analyzed using TAPDANCE and Gene-centric CIS (Common Insertion Sites), two independent methods to identify the transposon insertion sites and thus the defective candidate genes associated with that tumor. More than 80% of the identified genes have human orthologs whose mutations were previously reported in colorectal cancer. Researchers focused the analysis on the genes identified by both methods of analysis and that were specific to the tumors from the TGFβ-RII deficient mice, resulting in seventeen candidate genes. Analysis of these genes indicated that the Wnt/β-catenin and Hippo pathways were the most significantly deregulated in these tumors. Intriguingly, mutations in APC (adenomatous polyposis coli), a tumor suppressor gene often involved in CRC, were less frequent in mice with TGFβ-RII mutations, suggesting that loss of TGFβ signaling may decrease the selective pressure for loss of APC. Other pathways were altered in both TGFβ-RII functional and impaired tumors, but at different frequencies further supporting that not the same actors are necessary for tumor development when TGFβ-RII is functional or deficient.
Cancer development represents an accumulation of successive genes deregulations synergizing or antagonizing each other’s actions. By looking at the big picture these data highlight this concept and are valuable to understand which combination of genes is involved. “We were pleased to see how much overlap there was between genes identified in our mouse model and genes with known mutations in human colorectal cancer. Further analysis of these novel candidate genes and pathways may provide insight into novel treatment approaches for specific subtypes of colorectal cancer harboring these mutations” concluded Dr. Morris.
Funding for this study was provided by the National Cancer Institute, the Burroughs Wellcome Fund, a Fred Hutchinson/University of Washington Cancer Consortium Shared Resource Access Award, the Masonic Cancer Center and the NIH/FHCRC Interdisciplinary Training Program.
Morris SM,Davison J,Carter KT,O'Leary RM,Trobridge P,Knoblaugh SE,Myeroff LL,Markowitz SD,Brett BT,Scheetz TE,Dupuy AJ,Starr TK,Grady WM. 2016. Transposon mutagenesis identifies candidate genes that cooperate with loss of Transforming Growth Factor-beta signaling in mouse intestinal neoplasms. International Journal of Cancer [Epub ahead of print].