Wnt Signalling and Disease

Question 1

What happens when there is loss of the ability to regulate bet-catenin?

Answer 1

Results in unregulated Wnt signalling, leading to uncontrolled proliferation and development of cancer.

Question 2

Which Wnt signalling components act as oncogenes and tumour suppressors?

Answer 2

• Oncogenes: Wnt1 (mice mammary tumours), Beta-Catenin (CRC).
• Tumour suppressors: Axin (Hepatocellular carcinoma), APC (FAP), TCF.

Question 3

What is the consequence of Wnt1 overactivation?

Answer 3

Overexpression of Wnt1 leads to constitutive activation of the Wnt signalling pathway. Wnt1 (Int1) was first identified in mammary tumours in mice due to insertion of DNA by the mouse mammary tumour virus (MMTV).

Wnt1 overactivation leads to constitutive activation of the pathway: all of the receptors are bound and the destruction complex cannot be formed. Beta-catenin can continuously activate transcription by displacing Groucho.

Question 4

What is the consequence of loss of functional APC?

Answer 4

APC misregulation is associated with development of familial adenomatous polyposis (FAP). APC is a major scaffolding component of the destruction complex, so when functional APC is lost the destruction complex can’t form. This means that beta-catenin isn’t phophorylated and so constitutively activates transcription.

FAP is an inherited predisposition to colon cancer, 80% of colon cancers have inactivated APC, the rest are due to overactive beta-catenin.

Question 5

Outline FAP.

Answer 5

Familial adenomatous polyposis (FAP) is an inherited predisposition to colon cancer. 80% of colon cancers have inactivated APC, with the other 20% being due to overactive beta-catenin.

Many mutations are found in the Mutation Cluster Region (MCR) region, which is an area important in carcinogenesis. The MCR of APC is located within the central part of the open reading frame, overlapping with the region encoding the 20 amino acid repeats (20R) that are beta-catenin-binding sites. Each mutation in the MCR leads to the synthesis of a truncated APC product expressed in a colorectal tumour.

The two hit hypothesis means that inheritance of an APC mutant allele gives a predisposition to development of cancer as only one mutation needed on the WT APC allele.

Question 6

How does mutant APC lead to development of cancerous tissue?

Answer 6

Vili are constantly sloughed off in the intestine, with stem cells in crypt replacing the lost cells (this is maintained by a high level of Wnt). Stem cells create transit amplifying cell (TA) that can further differentiate into paneth cells, goblet cells, enterocytes, and entero-endocrine cells.

As cells replicate and move away from the crypt, they lose Wnt signals and lose the ability to divide and differentiate (Paneth, goblet etc.). Proliferation is maintained by presence of Wnt, so a mutation in APC means that beta-catenin is not degraded so constitutively enters the nucleus and activates transcription. The cells moving into the villus still transcribe Wnt target genes and constantly divide. They then fill the villus and form a cancerous mass.

Question 7

What is the consequence of beta-catenin mutations at its phosphorylation sites? Name some specific amino acids that are affected by mutations.

Answer 7

Beta-catenin is unregulated in many cancers (including 20% of CRC). Mutations are clusterred in the phosphorylation region of beta-catenin (encoded by CTNNb1), meaning it cannot be degraded and leads constant transcription of Wnt genes. This lead to uncontrolled cell proliferation and oncogenesis.

• G34: colorectal cancer, hepatocellular carcinoma
• S45: colorectal cancer, hepatocellular carcinoma
• D32: hepatocellular carcinoma

Question 8

What is the consequence of loss of functional Axin?

Answer 8

Human Axin is a tumour suppressor, and mutants identified are related to development of human hepatocellular carcinomas.

Question 9

What is the consequence of mutant TCF?

Answer 9

Tcf1 mutant mice develop adenomas in the gut and mammary glands.

Question 10

Wnt signalling leads to transcription of target genes. It is the action of these target genes that leads to cancer and other diseases. What are some of the target genes of Wnt that are upregulated in colon cancer?

Answer 10

• C-Myc
• Cyclin D
• C-Jun
• Matrix-metalloprotease MMP-7
• Endothelin-1
• Gastrin
• BMP4
• Survivin
• FGF18

Question 11

Aside from cancer, what diseases are implicated in Wnt dysregulation?

Answer 11

Question 12

Define osteoporosis. What genes are implicated in altered bone density disorders?

Answer 12

Osteoporosis is a progressive systemic skeletal disease characterized by low bone mass and micro architectural deterioration of bone tissue, with consequent increase in bone fragility and susceptibility of fracture.

• LPR5: LRP5/6 are important in bone density: Wnt involved in regulation in osteoblasts and osteoclasts. Mutants are found in both low and high bone mass disorders. Loss of function of LRP5 is associated with low bone density, gain of function of LRP5 is associated with high bone density.
  
  • Dickkopf (Dkk) is an extracellular inhibitor of Wnt signalling. Dkk binds LRP and another transmembrane receptor Kremen (Krm). This tertiary complex is internalised, thus downregulating signalling. The mutations in LRP5 in patients exhibiting high bone mass prevents Dkk binding. High bone mass in these patients could be due to unrestrained Wnt signalling, not countered by Dkk.

Question 13

Name some high bone mass disorders due to mutations in Wnt signalling proteins.

Answer 13

Question 14

Mutations in which Wnt signallnig proteins cause FEVR?

Answer 14

Familial Exudative Vitreoretinopathy (FEVR) is an inherited blinding disorder of the retinal vascular system. Mutations in Frizzled 4 (4%-20% of cases) and LRP5 (12%-18% of cases) have been shown to be involved.

Wnt signalling thus required for vascularisation of the eye.

Question 15

How can Wnt signalling be altered therapeutically?

Answer 15

• Antibody targeting Wnt to prevent binding and downregulate signalling.
• siRNAs to knock-down expression of proteins (siRNAs have homology to mRNAs of interest, which binds the transcripts and causes dsRNA degradation), e.g. reduced level of Dsh.
• Small molecule inhibitors: prevents protein-protein interactions, e.g. dishevelled complex formation antagonists. Reduced transcription of targets. Beta-Catenin/TFC interaction.
• LiCl: interferes with GS3K interaction.

Question 16

What extracellular components of Wnt signalling can be targeted for therapy?

Answer 16

• Wnts have been targeted with antibodies.
• FRPs or Dkks could be used to inhibit signalling:
  
  • Dkk-3 expression in non-small cell lung carcinoma (NSLCC) cell inhibited cell growth.
  • Dkk-3 and dominant-negative LRP5 expression in Saos-2 cells reduced invasion capacity and motility.

Question 17

What cytoplasmic components of Wnt signalling can be targeted for therapy?

Answer 17

• Introduction of wild-type Axin-1 induced apoptosis in hepatocellular and colorecetal cancers.
• A small molecule inhibitor of Dsh physical interactions has been described that down-regulates signalling in culture and increases apoptosis of cancer cell lines.
• Antisense against β-Catenin decreased expression and led to tumour regression.
• Introduction of a mutant F-box protein (part of proteolytic machinery) led to increased targetting for degredation of free (but not cadherin bound) β-Catenin.

Question 18

What nuclear components of Wnt signalling can be targeted for therapy?

Answer 18

• Structural details of the β-Catenin/Tcf complex highlight possibility of developing drugs to inhibit such interactions.
• 7000 natural compounds screened for inhibition of interaction, and several were found that also reduce β-Catenin-dependent activities.

Question 19

Name some inhibitors of Wnt signalling that can be used in anti-cancer therapy?

Answer 19

• Sulindac
• Acetyl salicylic acid (Aspirin)
• Celecoxib
• Endostatin