Table of Contents  

Markowitz: Genetic lessons from colon cancer

Lynch syndrome – capturing colon cancer genetics

My research focuses on the molecular genetics of colorectal cancer.1 This work reflects an over 20-year-long effort to understand one of my first patients, a 30-year-old accountant with metastatic colon cancer. Both his father and paternal grandfather had suffered from the same disease. Collaborative studies with Bert Vogelstein’s laboratory revealed that the cause of colon cancer in this family, and in other similar families, lay in the inheritance of germline mutations that inactivate genes involved in DNA mismatch repair.24 This discovery provided a genetic basis for the now widely recognized familial colon cancer syndrome known as Lynch syndrome/hereditary non-polyposis colorectal cancer, which accounts for approximately 5% of colorectal cancer cases.1

DNA methylation – a cancer driver and a diagnostic marker

An immediate puzzle was the finding that approximately 15% of seemingly non-familial colorectal cancers also demonstrate evidence of loss of DNA mismatch repair, as reflected by the molecular phenotype of microsatellite instability.1 The cause of these non-familial colon cancers was elucidated when we and other investigators discovered that in these tumours the hMLH1 DNA mismatch repair gene has been epigenetically silenced by aberrant DNA methylation of the gene promoter.5 We identified a wider role for aberrant gene methylation as a driver of gastrointestinal cancers by showing that, in individuals with hereditary diffuse gastric cancer who inherit one mutant copy of the CDH1 gene, the second genetic hit that directly initiates tumour development is similarly aberrant methylation and silencing of the promoter of the second wild-type CDH1 allele.6

We recognized that aberrant methylation could also have important uses in cancer diagnosis. An assay we developed to recognize methylation of hMLH1 has been widely adopted to distinguish repair-deficient colon cancers as being inherited or sporadic.7 To further explore the potential of methylated DNA in cancer diagnosis, we conducted a genome-wide analysis for aberrant methylation in human colon cancer, identifying the first exon of the vimentin gene as unmethylated in the normal colon and densely methylated in 80% of colon cancers.8 We showed that this methylated vimentin DNA can be detected in the blood of 50% of patients with early-stage colon cancer.9 As a strategy to better detect cancer, we demonstrated that the methylated vimentin DNA could also be detected in faeces in 84% of individuals with early-stage colorectal cancers. This test was brought forward for clinical use by Exact Sciences (ColoSure, Exact Sciences Corporation, Madison, WI) and provided proof of concept for a follow-on methylated DNA stool test (Cologuard, Exact Sciences Corporation, Madison, WI) that is now Food and Drug Administration approved. In broader studies of the gastrointestinal tract, we determined that aberrant vimentin methylation is even more sensitive as a marker of premalignant changes in the oesophagus, where aberrant vimentin methylation is detectable in 90% of cases of Barrett’s oesophagus, which is a precursor of oesophageal adenocarcinoma.10 This discovery opens the door to non-endoscopic screening for Barrett’s oesophagus, based on detecting vimentin DNA methylation in samples obtained from oesophageal brushings,10 an approach that we are currently testing in human clinical trials.

TGF-beta – a core tumour suppressor

Our studies of Lynch syndrome also posed the question of why a defect in DNA mismatch repair would specifically give rise to colon cancer. We hypothesized that certain key colon cancer-suppressor genes might have latent genomic instability and be dependent on DNA mismatch repair for maintenance of genomic integrity. We confirmed this hypothesis by discovering that DNA repair-deficient colon cancers ubiquitously activate a repetitive poly-A tract in the coding region of the transforming growth factor (TGF)-beta receptor type II gene (TGFBR2) to become a hotspot for frameshift mutations that destroy TGFBR2 protein function.11 This discovery provided a molecular mechanism connecting the DNA repair defect in Lynch syndrome to the development of colon cancers. Moreover, this discovery provided the first genetic evidence that TGFBR2, and by implication TGF-beta signalling, is a key colon cancer suppressor. In further studies we showed that 30% of all colorectal harbour TGFBR2 mutations. This includes 15% of repair-proficient colon cancers that also develop inactivating mutations in TGFBR2, but in these cancers the TGFBR2 mutations are scattered throughout the gene.12 In collaboration with the Vogelstein laboratory, we further showed that colon cancers in which TGFBR2 remains wild type also still commonly inactivate TGF-beta signalling, via developing mutations in the SMAD4 component of the pathway.13 The genetic finding that TGF-beta signalling components are key targets for mutations in colon cancer was again validated by our findings as members of the team that first sequenced the entire colon cancer-coding genome,14,15 and was further validated by the finding of near-universal mutations of SMAD4 in human pancreas cancers.

15-PGDH – a tumour suppressor

To identify candidate mediators of TGF-beta suppression of colon cancer, we used whole-transcriptome profiling and informatics to interrogate for genes that TGF-beta turns on in a normal colon but that become turned off in colon cancers. The best such exemplar proved to be HPGD, a gene encoding a prostaglandin-degrading enzyme, 15-hydroxyprostaglandin dehydrogenase (15-PGDH).16 No literature connected 15-PGDH with cancer; however, we rapidly confirmed that 15-PGDH is highly expressed in the normal human colon but is lost in > 80% of human colon cancers.16 Restoring 15-PGDH expression (by gene transfection) blocked the ability of colon cancer cells to grow in mice.16 Conversely, knocking out the 15-PGDH gene in mice doubled colon levels of prostaglandin E2 (PGE2) and increased colon tumour induction by over sevenfold.17 These findings define a mechanism by which TGF-beta, via 15-PGDH, acts as a metabolic antagonist of the COX-2 oncogene, specifically acting via 15-PGDH-catalysed degradation of the PGE2 onco-metabolite.

15-PGDH – a predictor for cancer prevention

Pharmaceutical inhibition of COX-2, via aspirin and other non-steroidal anti-inflammatory drugs (NSAIDs), is a key strategy for chemoprevention of colon cancer, with individuals who chronically use aspirin reducing their colon cancer risk by approximately one-third.18 We hypothesized that 15-PGDH might physiologically partner with NSAIDs in regulating tissue PGE2. We found that 15-PGDH-deficient mice become resistant to the NSAID celecoxib, which in these mice is both markedly attenuated in ability to reduce colon PGE2 and ineffective in preventing colon tumours.19 In a pilot analysis, we found that celecoxib-resistant humans who developed colon adenomas while on celecoxib treatment similarly showed below average colon 15-PGDH.19 Most compellingly, in a large observational study, we found that aspirin use was associated with a 50% reduction in colon cancers in individuals who have above average colon 15-PGDH levels, whereas aspirin was essentially inactive in reducing colon cancer risk in persons with below average colon 15-PGDH.20 These findings now point the way to ‘personalized chemoprevention’ by enabling identification of individuals who will or will not benefit from taking aspirin for colon cancer prevention. These findings also address the conundrum posed by the US Preventative Services Task Force, which in 2016 endorsed using aspirin for preventing colon cancer but, not being able to identify in whom aspirin works, restricted that endorsement to individuals already taking the drug to prevent cardiac disease.21

15-PGDH – a therapeutic target for treating colitis

Finding that many individuals have low colon 15-PGDH levels suggested that although high 15-PGDH may protect from colon tumours, in other contexts low 15-PGDH might also confer an advantage. Indeed, studies of 15-PGDH knockout mice showed that they are almost completely resistant to dextran sulfate sodium (DSS)-induced colitis, and enabled us to show that this is due to 15-PGDH negatively regulating the proliferation of colon stem cells after tissue injury.22 Specifically, although in wild-type mice DSS suppresses colonocyte proliferation in the lower crypts, inducing mucosal ulceration and colitis, in 15-PGDH knockout mice DSS induces increased colonocyte proliferation at the base of the crypts, which nearly completely prevents induction of colon ulcers and colitis.22 To take therapeutic advantage of this finding, we developed SW033291, a potent (Ki 0.1 nM) small-molecule 15-PGDH inhibitor, showing SW033291 doubles PGE2 in the mouse colon and markedly protected from DSS colitis.22 Moreover, 15-PGDH regulation of injury responses is conserved across multiple tissues.22 After partial hepatectomy, mice livers regrow twice as fast in 15-PGDH knockout or in SW033291-treated mice as in controls.22 Furthermore, after a bone marrow transplant, SW033291-treated mice recover peripheral blood counts a week faster than control mice.22 In partnership with the National Cancer Institute, an SW033291 analogue is now undergoing safety studies in preparation for potential human trials.

In overview, the TGF-beta/15-PGDH pathway plays a key role in suppressing colon cancer, in regulating individual responses to colon cancer prevention with aspirin and other NSAIDs, and in regulating colon stem cell response to injury. Moreover, 15-PGDH is a therapeutic target, with small-molecule 15-PGDH inhibitors able to promote colon epithelial healing as a novel method for treating ulcerative colitis.

References

1. 

Markowitz SD, Bertagnolli MM. Molecular basis of colorectal cancer. N Engl J Med 2009; 361:2449–60. http://dx.doi.org/10.1056/NEJMra0804588

2. 

Liu B, Nicolaides NC, Markowitz S, et al. Mismatch repair gene defects in sporadic colorectal cancers with microsatellite instability. Nat Genet 1995; 9:48–55. http://dx.doi.org/10.1038/ng0195-48

3. 

Liu B, Parsons RE, Hamilton SR, et al. hMSH2 mutations in hereditary nonpolyposis colorectal cancer kindreds. Cancer Res 1994; 54:4590–4.

4. 

Papadopoulos N, Nicolaides NC, Liu B, et al. Mutations of GTBP in genetically unstable cells. Science 1995; 268:1915–17. http://dx.doi.org/10.1126/science.7604266

5. 

Veigl M, Kasturi L, Olechnowicz J, et al. Biallelic inactivation of hMLH1 by epigenetic gene silencing, a novel mechanism causing human MSI cancers. Proc Natl Acad Sci USA 1998; 95:8698–702. http://dx.doi.org/10.1073/pnas.95.15.8698

6. 

Grady W, Willis J, Guilford P, et al. Methylation of the CDH1 promoter as the second genetic hit in hereditary diffuse gastric cancer. Nature Genet 2000; 26:16–17. http://dx.doi.org/10.1038/79120

7. 

Grady WM, Rajput A, Lutterbaugh JD, Markowitz SD. Detection of aberrantly methylated hMLH1 promoter DNA in the serum of patients with microsatellite unstable colon cancer. Cancer Res 2001; 61:900–2.

8. 

Chen WD, Han ZJ, Skoletsky J, et al. Detection in fecal DNA of colon cancer-specific methylation of the nonexpressed vimentin gene. J Natl Cancer Inst 2005; 97:1124–32. http://dx.doi.org/10.1093/jnci/dji204

9. 

Li M, Chen WD, Papadopoulos N, et al. Sensitive digital quantification of DNA methylation in clinical samples. Nat Biotechnol 2009; 27:858–63. http://dx.doi.org/10.1038/nbt.1559

10. 

Moinova H, Leidner RS, Ravi L, et al. Aberrant vimentin methylation is characteristic of upper gastrointestinal pathologies. Cancer Epidemiol Biomarkers Prev 2012; 21:594–600. http://dx.doi.org/10.1158/1055-9965.EPI-11-1060

11. 

Markowitz S, Wang J, Myeroff L, et al. Inactivation of the type II TGF-β receptor in colon cancer cells with microsatellite instability. Science 1995; 268:1336–8. http://dx.doi.org/10.1126/science.7761852

12. 

Grady W, Myeroff L, Swinler S, et al. Mutational inactivation of transforming growth factor β recepor type II in microsatellite stable colon cancers. Cancer Res 1999; 59:320–4.

13. 

Thiagalingam S, Lengauer C, Leach F, et al. Evaluation of candidate tumour suppressor genes on chromosome 18q loss in colorectal cancers. Nature Genet 1996; 13:343–6. http://dx.doi.org/10.1038/ng0796-343

14. 

Sjoblom T, Jones S, Wood LD, et al. The consensus coding sequences of human breast and colorectal cancers. Science 2006; 314:268–74. http://dx.doi.org/10.1126/science.1133427

15. 

Wood LD, Parsons DW, Jones S, et al. The genomic landscapes of human breast and colorectal cancers. Science 2007; 318:1108–13. http://dx.doi.org/10.1126/science.1145720

16. 

Yan M, Rerko R, Platzer P, et al. 15-Hydroxyprostaglandin dehydrogenase, a COX-2 oncogene antagonist, is a TGF-β induced suppressor of human gastrointestinal cancers. Proc Natl Acad Sci USA 2004; 101:17468–73. http://dx.doi.org/10.1073/pnas.0406142101

17. 

Myung SJ, Rerko RM, Yan M, et al. 15-Hydroxyprostaglandin dehydrogenase is an in vivo suppressor of colon tumorigenesis. Proc Natl Acad Sci USA 2006; 103:12098–102. http://dx.doi.org/10.1073/pnas.0603235103

18. 

Markowitz SD. Aspirin and colon cancer – targeting prevention? N Engl J Med 2007; 356:2195–8. http://dx.doi.org/10.1056/NEJMe078044

19. 

Yan M, Myung SJ, Fink SP, et al. 15-Hydroxyprostaglandin dehydrogenase inactivation as a mechanism of resistance to celecoxib chemoprevention of colon tumors. Proc Natl Acad Sci USA 2009; 106:9409–13. http://dx.doi.org/10.1073/pnas.0902367106

20. 

Fink SP, Yamauchi M, Nishihara R, et al. Aspirin and the risk of colorectal cancer in relation to the expression of 15-hydroxyprostaglandin dehydrogenase (HPGD). Science Translat Med 2014; 6:233re2.

21. 

Bibbins-Domingo K, U.S. Preventive Service Task Force. Aspirin use for the primary prevention of cardiovascular disease and colorectal cancer: U.S. Preventive Services Task Force recommendation statement. Ann Intern Med 2016; 164:836–45. http://dx.doi.org/10.7326/M16-0577

22. 

Zhang Y, Desai A, Yang SY, et al. Tissue regeneration. Inhibition of the prostaglandin-degrading enzyme 15-PGDH potentiates tissue regeneration. Science 2015; 348:aaa2340. http://dx.doi.org/10.1126/science.aaa2340




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