Falk Symposium No :30-9:00 am, Saturday, October 1, 2005 Maritim Hotel, Berlin Of mice and man: Mouse models for colon carcinogenesis

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1 Falk Symposium No :30-9:00 am, Saturday, October 1, 2005 Maritim Hotel, Berlin Of mice and man: Mouse models for colon carcinogenesis Makoto Mark Taketo, M.D., Ph.D. Department of Pharmacology, Graduate School of Medicine Kyoto University, Kyoto, Japan

2 Humans are nothing more than mice that lost hair!? =

3 Mouse Models of Colon Cancer and Polyposis 1. Mouse models of familial adenomatous polyposis (FAP) 1a Apc Min mice, Apc Δ716 mice, and Apc 1638N mice 1b Modifiers of Apc intestinal polyposis 1c Mutations in DNA mismatch repair genes 1c Stabilizing β-catenin mutant 2. Mouse models of gastrointestinal hamartoma syndromes 2a Smad4 mutant mice as juvenile polyposis model 2b Lkb1 mutant mice as Peutz-Jeghers disease model 2c Cdx2 mutant mice as colonic hamartoma model 3. Mouse models of colon cancer 3a Mouse models of hereditary nonpolyposis colon cancer (HNPCC) 3b Additional mutations in Apc Min, Apc Δ716, or Apc 1638N mice 3c Colon cancer models with mutations in other genes 3d Mouse models for colon cancer associated with IBD 4. Mouse models used for tumor chemotherapy and chemoprevention 5. Our latest results on Wnt signaling and chromosomal instability (CIN) 5a Quantitative aspects of Wnt signaling in polyposis 5b Quantitative aspects of chromosomal instability (CIN)

4 Two Genetic Origins of Colon Cancer 1. Polyposis Colon Cancer Frequent in distal colorectal axis. Caused by APC mutations. (Adenomatous Polyposis Coli) Hereditary cases: FAP (Familial Adenomatous Polyposis) 2. Nonpolyposis Colon Cancer Frequent in proximal (right side of) colon. Caused by, e.g., MSH2 mutations. (DNA mismatch repair protein) Hereditary cases: HNPCC (Hereditary Nonpolyposis Colon Cancer) (M.M. Taketo)

5 Wnt Signaling Pathway β-cat β-cat GSK3β P Axin APC Ubiqitin (M.M. Taketo)

6 Wnt Signaling Pathway β-cat M GSK3β Axin APC β-cat (M.M. Taketo) TCF β-cat β-cat β-cat CycD1, c-myc, WISP, Lamininγ2 Matrilysin, CD44, upar, CDx1, MDR1 etc.

7 Wnt Signal Activation Qualitative and Quantitative Regulations of Proliferation and Differentiation (Stem Cell Characteristics?) (Dependent on developmental stages and cell types)

8 Apc Δ716 Germ-Line Chimera (M.M. Taketo)

9 Modifiers of Apc Intestinal Polyposis 1. Mom-1 (Modifier of Min) locus on Chr 4, encoding spla 2 (secretory phospholipase A2)? AKR (w.t.; low polyp #) vs. c57bl/6 (mutant) > lower PGE 2 and polyp no. in B6 expected (?) 2. COX-2 (Ptgs2) & COX-1 (Ptgs1) genes Homozygous mutation reduces polyps by ~80%. NSAID, and aspirin show similar effects. FAP and polyposis patients were treated by COX-2 inhbitors > Heart side effects (not for prevention?). 3. Other genes; Bloom and Werner DNA helicase genes, telomerase, DNA methylase, Mdr-1, Cdx2 etc.

10 Construction of Apc/Cdx2 Compound Mutant Apc Δ716 Polyposis Cdx2 Mutant Colonic Polyposis (M.M. Taketo)

11 Significant Increase in Colon Polyp No. in Apc/Cdx2 Mice (Proximal Distal) Apc Δ716 Apc/Cdx2 Cdx2 ( Δ Hamartoma) ( Adenoma) (Aoki, K. et al. Nat. Genet. 35, , 2003)

12 Reduced CDX2 Causes Anaphase Bridges in the Colon (Apc/Cdx2) Polyps Nl. Epithelium (Wild Type) (Apc) (DAPI stain for DNA) (Cdx2 Hamartoma) (Apc/Cdx2) (Aoki, K. et al. Nat. Genet. 35, , 2003)

13 Correlation between ABI and Mitotically Unstable Chromosomes (CIN) Anaphase Bridges (Giemsa) Spectral Karyotype Analysis (SKY) (Gisselsson et al., Proc. Natl. Acad Sci. USA 97: , 2000)

14 Anaphase Bridge Index (ABI) ABI Correlates with Frequency of Mitotically Unstable Chromosomes* (borderline/low malignant tumors) *Ring Chromosomes, Dicentric Chromosomes and Telomeric Associations (RDT) (Gisselsson et al., Proc. Natl. Acad Sci. USA 97: , 2000)

15 Mutations in DNA mismatch repair genes Three types of DNA mismatch repair proteins 1. Essential proteins for cell viabiltiy; MSH2 and MLH1 2. Non-essential (redundant) proteins MSH3, 6; PMS1, MLH3, and EXO1 3. DNA replication proteins (essential) PCNA, RFC, RPA, DNA polymerase δ Single mutations of many of these genes cause adenomas, because Apc is one of the targets.

16 Intestinal Polyps in Stabilized β-catenin (Catnb Δex3 ) Mutants K19-cre ~3,000 polyps FABP-cre ~700 polyps (Harada et al., EMBO J. 18 : , 1999)

17 Mouse Models of Colon Cancer and Polyposis 1. Mouse models of familial adenomatous polyposis (FAP) 1a Apc Min mice, Apc Δ716 mice, and Apc 1638N mice 1b Modifiers of Apc intestinal polyposis 1c Mutations in DNA mismatch repair genes 1c Stabilizing β-catenin mutant 2. Mouse models of gastrointestinal hamartoma syndromes 2a Smad4 mutant mice as juvenile polyposis model 2b Lkb1 mutant mice as Peutz-Jeghers disease model 2c Cdx2 mutant mice as colonic hamartoma model 3. Mouse models of colon cancer 3a Mouse models of hereditary nonpolyposis colon cancer (HNPCC) 3b Additional mutations in Apc Min, Apc Δ716, or Apc 1638N mice 3c Colon cancer models with mutations in other genes 3d Mouse models for colon cancer associated with IBD 4. Mouse models used for tumor chemotherapy and chemoprevention 5. Our latest results on Wnt signaling and chromosomal instability (CIN) 5a Quantitative aspects of Wnt signaling in polyposis 5b Quantitative aspects of chromosomal instability (CIN)

18 Mouse models of gastrointestinal hamartoma syndromes Hamartomas are benign tumors composed of indiginous components of the normal tissues, and usually do not show signs of dysplasia. Some hamartomas, however, tend to progress to malignant cancer and now regarded as precancerous conditions. (Or, patients with harmartomas develop cancer independent of the primary hamartomas?)

19 Gastric Hamartomatous Polyps in Smad4 (+/ ) Mice (Takaku, K. et al., Cancer Res. 59: , 1999)

20 Mutations in the SMAD4/DPC4 Gene in Juvenile Polyposis James R. Howe,* Stina Roth, John C. Ringold, Robert W. Summers, Heikki J. Järvinen, Pertti Sistonen, Ian P. M. Tomlinson, Richard S. Houlston, Steve Bevan, Frank A. Mitros, Edwin M. Stone, Lauri A. Aaltonen Familial juvenile polyposis is an autosomal dominant disease characterized by a predisposition to hamartomatous polyps and gastrointestinal cancer. Here it is shown that a subset of juvenile polyposis families carry germ line mutations in the gene SMAD4 (also known as DPC4), located on chromosome 18q21.1, that encodes a critical cytoplasmic mediator in the transforming growth factor-b signaling pathway. The mutant SMAD4 proteins are predicted to be truncated at the carboxyl-terminus and lack sequences required for normal function. These results confirm an important role for SMAD4 in the development of gastrointestinal tumors. (Science 280, , 1998)

21 Gastrointestinal Polyps in Lkb1 Heterozygotes (Peutz-Jeghers disease model) Gastic Polyps A Duodenal Polyp (Miyoshi et al. Cancer Res. 62: , 2002)

22 Gastric Hamartoma in Lkb1 Heterozygotes Arborizing Smooth Muscle Bundles No Wnt Signal Activation (Miyoshi et al. Cancer Res. 62: , 2002)

23 Mouse Models of Colon Cancer and Polyposis 1. Mouse models of familial adenomatous polyposis (FAP) 1a Apc Min mice, Apc Δ716 mice, and Apc 1638N mice 1b Modifiers of Apc intestinal polyposis 1c Mutations in DNA mismatch repair genes 1c Stabilizing β-catenin mutant 2. Mouse models of gastrointestinal hamartoma syndromes 2a Smad4 mutant mice as juvenile polyposis model 2b Lkb1 mutant mice as Peutz-Jeghers disease model 2c Cdx2 mutant mice as colonic hamartoma model 3. Mouse models of colon cancer 3a Mouse models of hereditary nonpolyposis colon cancer (HNPCC) 3b Additional mutations in Apc Min, Apc Δ716, or Apc 1638N mice 3c Colon cancer models with mutations in other genes 3d Mouse models for colon cancer associated with IBD 4. Mouse models used for tumor chemotherapy and chemoprevention 5. Our latest results on Wnt signaling and chromosomal instability (CIN) 5a Quantitative aspects of Wnt signaling in polyposis 5b Quantitative aspects of chromosomal instability (CIN)

24 Mutations in DNA Mismatch Repair Genes Single mutations of many of these genes cause adenomas, because Apc is one of the targets. Interestingly, compound mutations of these genes often show severer phenotypes. Some develop adenocarcinomas, although none of them are metastatic.

25 Malignant Progression of Apc Δ716 Polyps by Additional Mutation in Smad4 Gene Apc Δ716 Polyposis Smad4 / Mutation Invasive Aenocarcinoma w/ desmoplasia

26 Desmoplastic Colonic Adenomacarcinoma In the Cis-Apc Δ716 /Smad4 Compound Mutant

27 Colon Cancer Model Mice with Mutations in Other Genes 1. TGF-β1 gene (Tgfb1) mutation in Rag2 mice > colon adenocarcinoma with local invasions 2. Smad3 homozygotes (1/3 reports) > colon cancer that metastasizes to LN But, penetrance is low, and other groups could not repeat the results. 3. K-ras* transgenic mice (with villin promoter) > microadenoma to adenocarcinoma that is locally invasive, but not metastatic. 4. Mucin gene Muc2 KO > adenocarcinomas in the intestines; low incidence, but invasive w/o metastasis And many others!!

28 Mouse models for colon cancer associated with IBD* 1. IL-10 deficient mice > UC*-like disease; aberrant cytokines as IFNγ > invasive adenocarcinoma in 60% (6 mo) 2. IL-2 / β2-microblobulin compound mutant mice > UC-like disease > colon adenocarcinoma in ~1/3 of mice (Cf.) N-cadherin dominant negative mutant Tg mice > IBD with adenomas, but no carcinomas *IBD, inflammatory bowel diseas; UC, ulcerative colitis

29 Mouse Models of Colon Cancer and Polyposis 1. Mouse models of familial adenomatous polyposis (FAP) 1a Apc Min mice, Apc Δ716 mice, and Apc 1638N mice 1b Modifiers of Apc intestinal polyposis 1c Mutations in DNA mismatch repair genes 1c Stabilizing β-catenin mutant 2. Mouse models of gastrointestinal hamartoma syndromes 2a Smad4 mutant mice as juvenile polyposis model 2b Lkb1 mutant mice as Peutz-Jeghers disease model 2c Cdx2 mutant mice as colonic hamartoma model 3. Mouse models of colon cancer 3a Mouse models of hereditary nonpolyposis colon cancer (HNPCC) 3b Additional mutations in Apc Min, Apc Δ716, or Apc 1638N mice 3c Colon cancer models with mutations in other genes 3d Mouse models for colon cancer associated with IBD 4. Mouse models used for tumor chemotherapy and chemoprevention 5. Our latest results on Wnt signaling and chromosomal instability (CIN) 5a Quantitative aspects of Wnt signaling in polyposis 5b Quantitative aspects of chromosomal instability (CIN)

30 COX-2 Inhibition Suppresses Polyposis Apc Δ716 Polyposis COX-2 -/- Mutant Suppression of Polyposis Apc Δ716 Polyposis F F O O O S O COX-2 Inhibitor Suppression of Polyposis (M.M. Taketo)

31 Mouse Models of Colon Cancer and Polyposis 1. Mouse models of familial adenomatous polyposis (FAP) 1a Apc Min mice, Apc Δ716 mice, and Apc 1638N mice 1b Modifiers of Apc intestinal polyposis 1c Mutations in DNA mismatch repair genes 1c Stabilizing β-catenin mutant 2. Mouse models of gastrointestinal hamartoma syndromes 2a Smad4 mutant mice as juvenile polyposis model 2b Lkb1 mutant mice as Peutz-Jeghers disease model 2c Cdx2 mutant mice as colonic hamartoma model 3. Mouse models of colon cancer 3a Mouse models of hereditary nonpolyposis colon cancer (HNPCC) 3b Additional mutations in Apc Min, Apc Δ716, or Apc 1638N mice 3c Colon cancer models with mutations in other genes 3d Mouse models for colon cancer associated with IBD 4. Mouse models used for tumor chemotherapy and chemoprevention 5. Our latest results on Wnt signaling and chromosomal instability (CIN) 5a Quantitative aspects of Wnt signaling in polyposis 5b Quantitative aspects of chromosomal instability (CIN)

32 5. Quantitative Aspects of Wnt Signaling in Intestinal Polyposis 5a. Quantitative Wnt Regulation (Hypomorphic Apc Alleles) 5b. Chromosomal Instability Caused bywnt Signaling

33 Quantitative Aspects of Wnt Signaling Intestinal polyposis in Apc mutant mice Apc allele Apc Δ716 >> Apc Min >> Apc 1638N Polyp No. ~300 ~30 ~3 Q. Are they different in Wnt signalling stimulation? Q. Is there a threshold for polyp formation in the Wnt signal activation level? New Strategy: Hypomorphic Apc alleles

34 Apc NeoF Hypomorphic Apc alleles (Insersion of neo to an intronic enhancer) Wild-type allele Apc + Targeting vector papc-neof Targeted allele Apc neof Sa Sa Probe DT 6.5 kb E12 E11 E kb PCR PCR Sa neo Sa neo Sa Exon14 Sa Sa Exon15 1 kb Wild-type allele Apc + Sa 6.5 kb E12 E11 E13 Probe PCR Sa Exon14 Exon15 1 kb Apc NeoR Targeting vector papc-neor DT Sa neo Sa Targeted allele Apc neor Sa 4.5 kb Sa neo Sa PCR (Li, Q., Cancer Res. 65: in press, 2005)

35 Reduced Polyp Multiplicity in Hypomorphic Apc Mice Apc 716 Apc neof Apc neor (Li, Q., Cancer Res. 65: in press, 2005)

36 Small intestinal polyposis in Apc neof and Apc neor mice, compared with that in Apc Δ716 Genotype Age Incidence Mean polyp no. Mean polyp size Polyp size distribution (months) affected/total (%) (mm) < 0.5 mm > 0.5 mm Apc Δ /11 (100) 99.4± ± ± ±18.3 Apc neof 15 5/10 (50) 1.09± ± ± /15 (0) NA NA NA NA Apc neor 15 6/31 (19) 0.26± ± ± /10 (0) NA NA NA NA WT 15 0/6 (0) NA NA NA NA NA, not applicable (Li, Q., Cancer Res. 65: in press, 2005)

37 Nuclear β-catenin is Reduced in Hypomorphic Apc Mouse Polyps Apc Δ716 Apc neof Apc neor (Li, Q., Cancer Res. 65: in press, 2005)

38 Adenoma Cell Proliferation Rates Are Similar between Apc Δ716 and Hypomorphic Apc Mutants Apc Δ716 Apc neof Apc neor (Ki-67 stainig) Labeling Index (%) ±10 60±10 53± Apc Δ716 Apc neof Apc neor (Li, Q., Cancer Res. 65: in press, 2005)

39 COX-2 Expression and Akt Phosphorylation are Similar between Apc Δ716 and Hypomorphic Apc Mutants Apc Δ716 Apc neof Apc neor COX-2 pakt (Li, Q., Cancer Res. 65: in press, 2005)

40 Quantification of APC and β-catnin in the Polyps of Apc Δ716 and Hypomorphic Apc Mutants (Western) APC β-catenin (75%) R Wild-type Mutant mice Littermate Normal Polyp C1 C2 N1 N2 T1 T2 APC β-actin R Mutant mice Normal Polyp N1 N2 T1 T2 β-catenin β-actin (65%) F APC β-actin F β-catenin β-actin (50%) ² APC β-actin ² β-catenin β-actin (Ratio to the WT level in the Normal Epithelium) R, Apc NeoR ; F, Apc NeoF ; Δ, Apc Δ716 (Li, Q., Cancer Res. 65: in press, 2005)

41 APC and β-cantinin in ES Cells with Apc Δ716 and the Hypomorphic Apc Alleles (Western Blot Analysis) APC β-catenin 20% 10% 0% +/+ R/R F/F ²/² APC /+ R/R F/F ² /² β-catenin β-actin β-actin ±0.03 APC +/ β-catenin +/+ Density (Ratio to β actin) /+ 0.06±0.02 R/R 0.03±0.01 F/F 0.00±0.00 ²/² R/R F/F ²/² Density (Ratio to β actin) ± ± ± ±0.02 +/+ R/R F/F ² /² R/R F/F ²/² (Li, Q., Cancer Res. 65: in press, 2005)

42 Nuclear β-catenin in ES Cell Lines Homozyogous for the Apc Alleles Wild Type Apc NeoR Apc NeoF Apc Δ ±3.2 Nuclear β-catenin (%) ± ± ±0.2 +/+ R/R F/F Δ/Δ (Li, Q., Cancer Res. 65: in press, 2005)

43 Luciferase Reporter Activities of β-catnin/tcf Transcription in the ES Cell Lines with Apc Alles +/+ R/R F/F Relative ratio (TOP/FOP) FOPFLASH TOPFLASH Δ/Δ Relative light units (Li, Q., Cancer Res. 65: in press, 2005)

44 Correlation of APC Protein Level and Polyp Number Polyp number (Log) ²/² F/F R/R +/² +/F +/R +/ TOP/FOP ratio APC level (%) (Li, Q., Cancer Res. 65: in press, 2005)

45 5. Quantitative Aspects of Wnt Signaling in Intestinal Polyposis 5a. Quantitative Wnt Regulation (Hypomorphic Apc Alleles) 5b. Chromosomal Instability Caused bywnt Signaling

46 Chromosomal Instability (CIN) in Cancer 1) Many cancer cells show aneuploidy. 2) Aneuploidy is caused mostly by CIN. 3) Extent of CIN is severer in advanced cancers. Molecular mechanisms that cause CIN? Multiple mechanisms are involved! 1) Cell cycle studies in yeast G2-M & DNA repair genes 2) Mutations in cancers? Only ~10% of CIN cancers. 3) Mutations in other types of genes? Colonic polyposis in Apc/Cdx2 compound mutants. (Aoki et al., Nat.Genet., 35, , 2003) CIN caused by Wnt signaling (Polyps & ES Cells)

47 CIN by APC Mutations Truncation APC mutant protein binds to the kinetochore. (Kaplan et al., Nat.Cell Biol. 3: 429, 2001) Truncation APC mutant protein causes polyploidy. (Fodde et al., Nat.Cell Biol. 3: 433, 2001) Q. How do APC mutant proteins cause CIN? Q. Is Wnt signaling (β-catenin/tcf transcription) involved in CIN? Strategy: To determine ABI not only in Apc but also in Catnb mutants (polyps and ES cells).

48 Clinical Relevance of CIN Caused by Wnt Signaing? Aim: To determine ABI and compare between Wntactivated and non-activated cancer cases. But, most human colon cancer tissues contain mutations in genes in Wnt signaling; APC, CATNNB, AXIN, etc. Thus, use gastric cancer tissues > some show nuclear β-catenin whereas others don t.

49 Clinical Signifcance of CIN in Cancer: Cause or Result? Debated: CIN is associated with early adenomas. But massive CIN is found mostly in later stages. Phenomena: CIN is a quantitative trait, rather than all or nothing. Mutiple mechanisms can cause CIN. Lessons from mouse models: Wnt signaling can make early adenomas CIN, which may accelerate further progression. Other factors as reduced CDX2 expression can cause CIN, and increase colon polyposis.

50 Summary (Part I) Many mouse models have been established that are useful for investigations of the initiation and expansion of colon polyps, and their progression to colon cancer. They are also valuable tools to evaluate various pharmaceutical and biological agents for colon polyposis treatment and prevention of their progression to cancer. Yet, one of the key outstanding issues in this field of research is to construct practical models of colon cancer metastasis to the liver, lungs, and lymph nodes. Such models should greatly help us find novel measures to overcome colon cancer in the 21st century. (M.M. Taketo)

51 Summary (Part II) 1. We constructed mutant ES cells and mice carrying hypomorphic Apc alleles. Their polyp multiplicities were much lower than Apc null mice. In the homozyogous ES cells, the APC level was inversely correlated with both β- catenin accumulation and TCF transcriptional activity. The threshold APC level for one polyp/mouse is ~15% of WT level. 2. We scored ABI in polyps and ES cells in Apc mutant, and β-catenin mutant, respectively. High ABI was found in both types of mutants, which can be suppressed by dominant-negative TCFs. These results suggest that Wnt signaling itself also contributes to CIN. (M.M. Taketo)

52 Acknowledgments (New Results) Hypomorphic Apc Department of Pharmacology Qin Li Tomo-o Ishikawa Masanobu Oshima CIN by Wnt Signal Department of Pharmacology Koji Aoki Masahiro Aoki Hiroyuki Miyoshi Masanobu Oshima Center for Moecular Biology and Genetics Manabu Sugai Aichi Cancer Center Tetsuya Tsukamoto Tsutomu Mizoshita Masae Tatematsu Dept. of Gastroenterology & Hematology Hiroshi Seno Tsutomu Chiba Banyu Tsukuba Research Institute (Merck) Naomoto Harada