malignant melanoma and cdkn2a

Malignant Melanoma and CDKN2A

Kate Coffin

http://www.liuding.com/blog/uploaded_images/fig8-794407.jpg

MelanomaGenetics and Environment

Skin cancer of melanocytes

Pre-existing nevus or new nevus

Different areas of skin

ABCDE assessment system of skin

http://www.aad.org/skin-conditions/dermatology-a-to-z/melanoma/signs-symptoms/melanoma-signs-and-symptoms

Dangers of Melanoma

Leading cause of death due to skin disease

2012 estimates in US76,250 new cases9,180 deaths

National Cancer Institute http://www.cancer.gov/cancertopics/types/melanomahttp://www.cancer.gov/aboutnci/servingpeople/cancer-statistics/snapshots

Metastasis

Spreading to other areas of skin and other tissues

14% 5 year survival rate

Miller, A et al. (2006). Melanoma. The New England Journal of Medicine. 355(1), 51-65. PMID:16822996

http://www.ncbi.nlm.nih.gov/pubmed/16822996

CDKN2A Cyclin-dependent kinase inhibitor 2A

Inhibits CDK4

Tumor Suppressor

>30 germline mutations

http://en.wikipedia.org/wiki/File:Protein_CDKN2A_PDB_1a5e.png

CDKN2A Domains – Ankyrin repeats

Pfam SMART

Found in archaea, bacteria, eukarya, and poxviruesIn other proteins associated with human disease

http://smart.embl-heidelberg.de/http://pfam.sanger.ac.uk/

http://smart.embl-heidelberg.de/

http://pfam.sanger.ac.uk/

CDKN2A Gene OntologyBiological Processes

- cell cycle arrest - cell cycle checkpoint- induction of apoptosis - negative regulation of cell growth

Cellular Components- nucleus - cytoplasm

Molecular Functions- cyclin-dependent kinase inhibitor - protein kinase binding

http://amigo.geneontology.org/cgi-bin/amigo/go.cgi

CDKN2A Protein Phylogeny

http://www.ebi.ac.uk/Tools/msa/clustalw2/

Human CDKN2A Interaction Network

http://string-db.org/

Human CDK4 Interaction Network


CDK4 Domains – Protein Kinase


Pfam

Conserved from E. coli to humansCatalytic component of Serine/Threonine kinases


CDK4 Gene OntologyBiological Processes

- regulation of cell cycle - regulation of cell proliferation- signal transduction

Cellular Components- cyclin-dependent protein kinase holoenzyme complex- transcription factor complex

Molecular Functions- cyclin-dependent protein kinase activity - ATP bindinghttp://amigo.geneontology.org/cgi-bin/amigo/go.cgi

Model of CDKN2A and CDK4

CDK4CDKN2A


CDK4CDKN2A

http://chungkitblog.files.wordpress.com/2012/01/ellen-family-visual-medium.jpg

Regulated Growth


CDK4CDKN2A


CDK4CDKN2A

http://upload.wikimedia.org/wikipedia/commons/thumb/6/6c/Melanoma.jpg/250px-Melanoma.jpg

UnregulatedGrowth


CDK4CDKN2A

?


CDK4CDKN2A

?

http://chungkitblog.files.wordpress.com/2012/01/ellen-family-visual-medium.jpg

Regulated Growth

QuestionCan I find a compound that mimics

CDKN2A by inhibiting CDK4?



HypothesisIf a compound were known to interact with and

inhibit CDK4, it could be used as a drug for melanoma

Experiment – Identify Compound

http://pubchem.ncbi.nlm.nih.gov/

Results – Identify Compound

2-Methyl-5-[(4-methylphenyl)amino]benzothiazole-4,7-dione


Cdk4 Inhibitor III

Results – Identify Compound




HypothesisIf a compound were known to interact with and

inhibit CDK4, it could be used as a drug for melanoma

HypothesisIf Cdk4 were inhibited by Cdk4 Inhibitor III in a Cdkn2a CKO mouse, the tumor cells would not proliferate and

metastasize

Why choose a mouse?

Skin and vascular systems

Previous cancer model

Cdkn2a – Cdk4 interaction

http://info.criver.com/flex_content_area/images/nude_mouse-2.jpg

Why choose a mouse?




https://www.aalaslearninglibrary.org/fileupload_content/Course245/14_Nude_tumor.jpg

Why choose a mouse?





Experiment – Treatment GroupsCdkn2a +/+

http://info.criver.com/flex_content_area/images/nude_mouse-2.jpghttp://www.miketiffee.com/uploaded_images/chemoIV-739375.jpg

1


Experiment – Treatment GroupsCdkn2a +/+ Cdkn2a -/- (CKO)


1 2



Cdkn2a +/+& Cdk4 Inhibitor III


1 2

3




Cdkn2a -/- (CKO)& Cdk4 Inhibitor III


1

43

2


Experiment – Graft Human MelanomaCdkn2a +/+ Cdkn2a -/- (CKO)


Cdkn2a -/- (CKO)& Cdk4 Inhibitor III

https://www.aalaslearninglibrary.org/fileupload_content/Course245/14_Nude_tumor.jpghttp://www.miketiffee.com/uploaded_images/chemoIV-739375.jpg

1

3 4

2

Data Collection

Microarray analysis ofCdkn2a and Cdk4expression in cells of mice

https://commons.wikimedia.org/wiki/File:DNA_microarray.svg

Expected Microarray Results1) Cdkn2a +/+ 2) Cdkn2a -/- (CKO)

3) Cdkn2a +/+& Cdk4 Inhibitor III

4) Cdkn2a -/- (CKO)& Cdk4 Inhibitor III

Left Well: Cdkn2a Right Well: Cdk4

Expected Metastasis Results1) Cdkn2a +/+ 2) Cdkn2a -/- (CKO)






LEAST METASTASIS




MOST METASTASIS

LEAST METASTASIS




MOST METASTASIS

LEAST METASTASIS

MODERATE METASTASIS

MODERATE METASTASIS

Conclusion

CDK4 Inhibitor III could be used as an antimelanoma drug by mimicking the tumor suppressor function of CDKN2A in patients with CDKN2A loss of function mutations

Future DirectionsTest other modes of drug administration

Topical cream vs Intravenous solution

Different dosages/times of administration

Study other proteins in interaction networkLook for other tumor suppressors

Study effects of UV exposure on CDKN2AHow does SPF affect this interaction?

Look for compounds in other mutated melanoma pathways

Questions?

http://www.liuding.com/blog/uploaded_images/fig8-794407.jpg

malignant melanoma and cdkn2a

Documents

compound http

cdk4cdk4cdkn2amodel

cdk4cdk4cdkn2a http

jpg250 of cdkn2a

cgi14model of cdkn2a

cdkn2a malignant melanoma

germline mutations http

cell cycle regulation