Familial Cancer Predisposition Syndromes. Retinoblastoma (RB) 13q14 Familial Adenomatous Polyposis (APC) 5q21

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1 Familial Cancer Predisposition Syndromes Retinoblastoma (RB) 13q14 Familial Adenomatous Polyposis (APC) 5q21

2 Retinoblastoma

3 Eye Retinal Epithelium develops in the first five years of life Retinoblastoma is a rare childhood tumor of the retinal epithelium.

4 Retinobalstoma

5 Presentation as a function of age Presents in early childhood, by age 5 25% of cases are unilateral (affecting only one eye) 75% of cases are bilateral and or multifocal Multifocal cases usually have family history of Retinoblastoma, and osteoscarcoma Multifocal patients are at increased risk for development of other malignancies later in life

6 Bilateral patients have increased risk

7 Bilateral Patients have a Family History 50% of offspring are affected autosomal dominant inheritance pattern.

8 Knudson Two-Hit Hypothesis

9 Loss of Heterozygosity (LOH)

10 Loss of Heterozygosity (LOH)

11 LOH at the RB locus is observed in sporadic cancers

12 LOH through mitotic recombination

13 Two mutant RB alleles are observed in Familial and Sporadic Cancers

14 Somatically acquired point mutations in remaining allele of RB

15 Functions of RB Inhibitor of a transcription factor E2F Functional RB inhibits cell cycling Oncogenic Viruses all have proteins that bind to and inactivate RB

16 Lessons Learned Loss of RB function is important for cancer development. Familial cancer predisposition syndrome co-segregates with mutant RB All retinoblastomas suffer somatically acquired inactivating mutations in one or more alleles. Oncogenic viruses inactivate the RB protein Recessive at the cellular level=loss of function required for tumor formation Dominant behavior at the organism level=single mutant allele causes cancer predisposition syndrome.

17 Other tumor suppressors? Systematic survey of every somatic chromosome revealed frequent deletions on other chromosomal arms Some of these loci are linked with cancer predisposition syndromes (5q)

18 Familial Adenomatous Polyposis

19 Good Colon Large number of epithelial cells that are in constant turnover Neoplasms are uncommon in young individuals. Benign polyps typical of older individuals (Professors) 50% of individuals will have at least one colon polyp by age 70 Develop into colon cancer if not removed Easily treated by surgical removal.

20 FAP (BAD) Colon Rare syndrome in which adolescent patients present with thousands of colon polyps

21 Normal vs FAP colon

22 Normal Colon Histology

23 Adenoma Histology

24 Histology of an Adenomatous Polyp.

25 Adenomatous Polyps are cancer precursors

26 Sporadic vs Familial Polyps 50% of the population will have one or more polyps of the colon by age % of polyps, if left untreated, will develop into colorectal cancer Genetic predisposition toward polyposis of the colon reveal underlying molecular genetics of familial and sporadic polyp development

27 FAP family tree Predisposition is inherited in an autosomal dominant manner Highly penetrate, variable expressivity Susceptibility locus maps to 5q (linkage disequilibrium)

28 Map of Mutations A A mutant gene linked to FAP is called APC (Adenomatous Polyposis Coli) FAP patients inherit one inactive mutant allele and 100% of colonic neoplasms arising in FAP patients have acquired a second, inactivating mutation in the remaining wild-type allele. 80% of sporadic (nonfamilial( nonfamilial) ) colonic neoplasms have two, somatically acquired mutations in the APC gene. LOSS OF APC IS AN EARLY EVENT= APC IS A GATEKEEPER GENE

29 Biochemistry and Molecular Biology of APC

30 Association of the APC Tumor Suppressor Protein with Catenins Su, Li-Kuo Kuo; ; Vogelstein, Bert; Kinzler, Kenneth W.*

31 Identification of proteins associated with APC Figure 1. Identification of proteins associated with APC. (A) Coimmunoprecipitation of cellular proteins with APC. Lysates were prepared from metabolically labeled SW480 or HCT116 cells. Immuno-precipitations were performed with pab 1801, a mab specific for p53 as a negative control; CF11, a mab specific for the NH 2 -terminus of APC [7]; ; and DB1, a mab specific for the COOH-terminus of APC [7].. Positions of molecular size markers (in kilodaltons) ) are shown on the left. (B) Association of cellular proteins with GST-fused APC fragments. Lysates were prepared from metabolically labeled SW480 cells. Numbers refer to APC codons.. The CF11 lane shows p95 and p100 coimmunoprecipitated with CF11 as a positive control. The other lanes show the proteins that bound to the indicated GST-APC fragments. (C) Schematic of APC fragments expressed in bacteria as GST fusion proteins and summary of their ability to bind p95 and p100.

32 Identification of APC-associated p100 and p95 as "alpha"- and "beta"-catenin catenin, respectively (A) Association of APC with GST-fused "beta"-catenin catenin. Lysates prepared from unlabeled SW480 or HCT116 cells were incubated with GST-CTN (containing "beta"- catenin residues 75 to 781) or GST-"Delta"CTN (containing "beta"-catenin residues 536 to 781). Bound APC was detected by immunoblotting as in [22].. Unlabeled lanes contain total cell lysates.. (B) Comigration of APC-associated p100 and p95 with "alpha"- and "beta"-catenin catenin. Lysates were prepared from metabolically labeled SW480 or HCT116 cells and immunoprecipitations were carried out with pab 1801, CF11, or HECD-1, a mab specific for E-cadherin cadherin.. Comparative peptide mapping of p100 and "alpha"-catenin (C) and of p95 and "beta"-catenin (D). Proteins were isolated from metabolically labeled SW480 or HCT116 cells by immunoprecipitation with antibody CF11, by binding to GST-APC(G), or by immunoprecipitation with HECD-1. Partial proteolysis experiments were performed with V8 protease [29].

33 Identification of the catenin- binding domain of APC. A) Schematic of APC fragments used for mapping of the catenin-binding domain. Fragments F2, F3, F2a, and F2b were expressed in vitro and tested for binding to GST-CTN. Fragments R1, R2, and R3 were expressed as GST fusion proteins and tested for binding to catenins from lysates of metabolically labeled SW480 cells. G-H indicates the overlapping region between GST-APC(G) and GST-APC(H) shown in Figure 1C. The black boxes in G-H indicate the 15-amino acid repeats. (B) Sequence of the three 15-amino acid repeats in APC, with the consensus sequence shown below. Numbers refer to APC codons.. Abbreviations for the amino acids are: D, Asp; E, Glu; ; I, Ile; ; K, Lys; ; L, Leu; ; N, Asn; ; P, Pro; R, Arg; ; S, Ser; T, Thr; and Y, Tyr.. (C) Association of APC fragments containing the repeats with the "alpha"- and "beta"-catenins catenins.

34 Conclusion APC interacts with Beta Catenin

35 Beta Catenin interacts with TCF4 Two-hybrid interaction experiments using B-Catenin as the Bait revealed the DNA- binding protein TCF4 as an interaction partner. Site selection experiments identified a DNA sequence to which TCF4 binds. TCF4 has no transactivation ability.

36 Constitutive Transcriptional Activation by a β-catenin-tcf Complex in APC / Colon Carcinoma Vladimir Korinek, * Nick Barker, * Patrice J. Morin, Dick van Wichen, Roel de Weger, Kenneth W. Kinzler, Bert Vogelstein, Hans Clevers

37 Transactivational properties of β-catenin + htcf-4. Figure 3. Transactivational properties of -catenin- catenin-htcf-4. All reporter assays were performed as duplicate transfections.. For each condition, both values are shown. (A)( ) Reporter gene assays in IIA1.6 B cells. Cells were transfected by electroporation with 1 µg g of luciferase reporter plasmid, 5 µg g of -catenin- expression plasmid, and 3 µg g of htcf-4 expression plasmids. Empty pcdna was added to a total of 10 µg g of plasmid DNA. (B)( ) Reporter gene assays in SW480 colon carcinoma cells. Cells were transfected with 0.3 µg g of the indicated luciferase reporter gene, 0.7 µg of pcatcontrol as internal control, the indicated amounts of pcmvneo-apc, and empty pcdna to a total of 2.5 µg g of plasmid DNA. Control CAT values (pcatcontrol( pcatcontrol) ) are given in the right panel.

38 Constitutive presence of β-catenin-htcf- 4 complexes in APC -/- cells Fig. 4. Constitutive presence of -catenin- catenin-htcf-4 complexes in APC -/- cells. Gel retardation assays were performed on nuclear extracts from the indicated cell lines before and after a 20-hour exposure to Zn 2+. Samples in lanes 1, 4, 7, and 10 were incubated under standard conditions. Anti--catenin (0.25 µg) was added to the samples in lanes 2, 5, 8, and 11. A control antibody (human CD4, 0.25 µg) was added to the samples in lanes 3, 6, 9, and 12. NS, nonspecific band also observed with mutant (nonbinding) probe (lane Mt)

39 Conclusion-B-catenin catenin and TCF4 interact and transactivate gene expression

40 Activation of β-catenin-tcf Signaling in Colon Cancer by Mutations in - Catenin or APC Patrice J. Morin, * Andrew B. Sparks, * Vladimir Korinek,, Nick Barker, Hans Clevers, Bert Vogelstein, Kenneth W. Kinzler

41 Effects of APC mutations on CRT Effects of APC mutations on CRT. (A)( ) Schematic of wild-type (WT) and mutant APC. APC is a 2843-aa protein (23) that contains Armadillo (ARM) repeats in the NH 2 - terminus (24), 15- and 20-aa -catenin-binding repeats in the central region (5, 6), and a basic region in the COOH-terminus (23). The COOH-terminus also contains a TXV sequence, which mediates DLG binding (9). (B)) Effects of WT and mutant APC on CRT. SW480 cells containing endogenous mutant APC were transfected with the APC expression vectors shown in (A) and CRT was measured (25). Cells were transfected with increasing amounts of WT APC (0, 0.15, and 0.5 µg, indicated by the black wedge) or 0.5 µg g of mutant APC.. CRT reporter activities are expressed relative to assays containing no WT APC and are the means of three replicates. Error bars represent standard deviations

42 Evaluation of CRT in colorectal cancer cell lines with WT APC. Evaluation of CRT in colorectal cancer cell lines with WT APC. (A)( Immunoblot of endogenous APC in the SW480, DLD1, HCT116, SW48, and 293 cell lines, developed with APC monoclonal antibody FE9 (26( 26). (B)( ) Effects of exogenous WT APC on CRT in cell lines with endogenous mutated or WT APC. Cells were transfected with increasing amounts (0, 0.15, and 0.5 µg g for DLD1 and SW48; 0, 0.5, and 5 µg g for HCT116) of WT APC or APC1309 mutant (0.5 µg g for DLD1 and SW48; 5 µg g for HCT116), and CRT was assessed as in Fig. 1.. CRT reporter activities are expressed relative to activity in extracts without exogenous APC and are the means of three replicates. Error bars represent standard deviations.

43 Evaluation of β-catenin in colorectal cancer cell lines with WT APC Evaluation of -catenin- in colorectal cancer cell lines with WT APC. (A)( Immunoblot of the cell lines used in this study, developed with -catenin- monoclonal antibody C19220 (Transduction Laboratories, Lexington, Kentucky) (22( 22). (B)( ) Sequence of CTNNB1 in HCT116 and SW48 (27( 27). The left panel (nucleotides 121 to 143 from HCT116) reveals the presence of a deletion in addition to the WT sequence. The middle panel (antisense( strand of nucleotides 156 to 113 of the WT and deleted alleles of HCT116) reveals the 3-bp deletion (TCT) that removed codon 45 in half the clones. The right panel (nucleotides 80 to 113 from SW48) reveals a C-to-A transition affecting codon 33 (TCT to TAT). (C)( Schematic of -catenin- illustrating the Armadillo repeats (24( 24) and negative regulatory domain. The residues in larger type fit the consensus sequence for GSK-3 phosphorylation (20)) and those in bold have been demonstrated to affect down-regulation of -catenin- through GSK-3 phosphorylation in Xenopus embryos (17( 17). The five mutations found in human colon cancers are indicated at the top. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; ; D, Asp; E, Glu; ; F, Phe; ; G, Gly; ; H, His; I, Ile; ; K, Lys; ; L, Leu; ; M, Met; N, Asn; ; P, Pro; Q, Gln; ; R, Arg; ; S, Ser; T, Thr; ; V, Val; W, Trp; ; and Y, Tyr.

44 Functional evaluation of β-catenin mutants Functional evaluation of -catenin- mutants. (A)( ) Constitutive nuclear complex of -catenin- and Tcf in HCT116 cells. The presence of nuclear -catenin- catenin-tcf complexes was assessed by gel shift assays (12( 12). Lanes 1 to 3, the optimal Tcf retardation probe shifted with nuclear extract from HCT116 cells when no antibody was added (lane 1) or with addition of antibody to -catenin- (0.25 µg, lane 2) or an irrelevant antibody (0.25 µg, lane 3); lane 4, mutant Tcf retardation probe shifted with nuclear extract from HCT116 cells; n.s., nonspecific shifting seen with the mutant probe. (B)( ) Effects of the -catenin- mutations on CRT. Human 293 cells were transfected with WT or mutant (45, S33Y) -catenin- and CRT was assessed (28( 28). CRT reporter activities are expressed relative to WT -catenin- and are the means of three replicates. Error bars represent standard deviations. DN Tcf refers to dominant-negative htcf-4.

45 Conclusion-mutant B-catenin is a constitutively active transcriptional activator

46 β-catenin regulates expression of cyclin D1 in colon carcinoma cells OSAMU TETSU 1 AND FRANK MCCORMICK 1

47 β-catenin activates the cyclin D1 promoter Figure 1 -Catenin activates the cyclin D1 promoter. a, HeLa cells were transfected with promoters from cyclin D1, cdc2 or cyclin A driving luciferase transcription, and 0.5 or 1 µg g of wild- type (WT) or mutant-type (MT) -catenin- (CA) expression plasmids. Two forms of cyclin D1 promoter were used, one consisting of 1,748 bp from the transcriptional start (-1748CD1), the other of 962 bp (-962CD1). Empty pcdna3 (Invitrogen) was added to 2 µg g of plasmid DNA. 50 ng of prl-tk renilla luciferase reporter construct (Promega( Promega) ) was co-transfected to each sample to normalize transfection efficiency. The ratio of reporter luciferase activity to control renilla luciferase activity is indicated. The assay was performed 24 h after transfection. All experiments are expressed as mean s.d. of triplicate cultures. b,, Effects of dominant-negative TCF-4E on -catenin- catenin-dependent cyclin D1 promoter activity. Full-length (FL) TCF-4E complementary DNA was subcloned into Myc-tagged pcdna3 from TCF-4E pcdnai.. N-terminal-deleted (N) Myc-tagged TCF-4E pcdna3 was generated by PCR-based site-directed deletion mutagenesis.

48 Expression of β-catenin, cyclin D1, cdc2 and cyclin A in HeLa cells expressing mutant β-catenin Expression of -catenin- catenin, cyclin D1, cdc2 and cyclin A in HeLa cells expressing mutant -catenin- catenin. a,, Left, Quantitative RT-PCR analysis of non-transfected HeLa cells (lanes 1, 4), empty pcdna3-transfected HeLa cells (lanes 2, 5) and MT -catenin- catenin-transfected transfected stable HeLa cells (lanes 3, 6). Lanes are with reverse transcriptase; lanes are without reverse transcriptase. 50 ng of total RNA-derived cdna was used for PCR reactions. Right, Quantitation of data is shown on the left. Expression levels relative to control HeLa cells are shown. b,, Left, western blot analysis of non-transfected HeLa cells (lane 1), empty pcdna3-transfected stable HeLa cells (lane 2) and MT -catenin- catenin-transfected transfected stable HeLa cells (lane 3), 10 µg g of protein was loaded. Right, Quantified representation of western blot data. Expression levels relative to control HeLa cells are shown.

49 Repression of cyclin D1 expression by a dominant-negative TCF-4E Repression of cyclin D1 expression by a dominant-negative TCF-4E. Cycling colon cancer cells were transfected with either 7.5 µg g pcdna3 or N TCF-4E pcdna3 expression vector with 2.5 µg pmacs K k (Miltenvi Biotec). 48 h after transfection, cells expressing the truncated H 2KH k were collected by beads coated with an antibody to mouse H 2KH k according to its instruction manual. Total cellular protein was isolated immediately and subjected to immunoblotting with cyclin D1 monoclonal antibody. Subsequently, the identical membranes were blotted with -actin- to indicate that equal amounts of protein were loaded in each lane.

50 G1 growth arrest in HCT116 colon cancer cells by dominant negative TCF-4E, and rescue by ectopic expression of cyclin D1. Figure 6 G1 growth arrest in HCT116 colon cancer cells by dominant negative TCF-4E, and rescue by ectopic expression of cyclin D1. Cell-cycle analysis was performed after N TCF-4E retrovirus infection of HCT116 and of HCT116 cells transfected with His-tagged cyclin D1 cdna (Invitrogen) expressed from the cytomegalovirus promoter (Cyclin( D1 HCT116). Exponentially growing cells were infected with LXSN (Clontech), empty vector retrovirus (control) or retrovirus expressing N TCF-4E. a,, Western blot analysis of cells infected with dominant-negative TCF-4E. Cell extracts were prepared 24 h after infection and the blot was first probed with a monoclonal antibody against cyclin D1. The blot was then probed with antibodies against cyclins D2, D3 and E and cdk2 and cdk4 and cdk6. Cyclin D3 was not detectable in these extracts. b, Western blot of HCT116 cells expressing cyclin D1 from the cytomegalovirus (cmv( cmv) ) promoter. Extracts were prepared from exponentially growing HCT116 cells or cells expressing cyclin D1 from the cytomegalovirus promoter (cmv( cyclic D1), and blotted with antibodies specific for cyclin D1, His-tag (Invitrogen) or cyclin E. c,, Colony-formation assay on cells infected with dominant-negative TCF-4E and selected with G single cells from HCT116 and Cyclin D1 HCT116 were infected with LXSN or N TCF-4E LXSN, respectively. These vectors express the neomycin gene. 14 days after infection and growth in G418, cells were stained with 0.5% crystal violet containing 20% ethanol. d,, Viable cell number after infection. Cells were collected at the indicated times (0, 24, 48 h) after infection, stained with trypan blue and counted with a haemocytometer. e,, Flow cytometric cell-cycle analysis. Cells were stained with propidium iodide to show cell-cycle distribution by DNA content. 10,000 cells were tested. G0/G1, S and G2/M phases of the cell cycle are indicated by arrows.

51 Conclusion-Cyclin Cyclin D1 is regulated by b-catenin catenin/tcf4, which in turn controls the G1-S transition

52 Functions of APC-Wnt Pathway APC functions to inhibit the wnt pathway by inactivation of β-catenin Rare colon tumors with no mutation in APC has mutant β-catenin Wnt signalling pathway promotes transactivation of anti-apoptotic/survival genes

53 The Wnt Pathway in Stem Cell Biology

54 Targeted Disruption of TCF4 disrupts intestinal stem cells

55 TCF4 protein is Absent

56 Intestinal Epithelium is disrupted Stem cells are absent

57 Proliferating Stem Cells are Absent

58 Cancer is interrupted Stem Cell Development

59 Lessons Learned Familial Cancer Predisposition Syndromes are rare disorders that give insight into the genetic mechanisms applicable to common, non-familial cancers. Physical and genetic interactions of tumor suppressors with other proteins provide clues to their roles in normal cell biology