Investigating CancerKRAS Activity

What is cancer?

• All cancers derive from single cells that have acquired the characteristics of continually dividing in an unrestrained manner and invading surrounding tissues.

• Cancer cells behave in this abnormal manner because of changes in the DNA sequence of key genes, which are known as cancer genes. Therefore all cancers are genetic diseases.

Human melanoma cell undergoing cell divisionCredit: Paul Smith & Rachel Errington, Wellcome Images

Cancer information• One in three people in the Western world develop cancer and

one in five die of the disease

• There are approximately 200 types of cancer, each with different causes, symptoms and treatments

• In 2007, 297,991 people were newly diagnosed with cancer in the UK

• An individual's risk of developing cancer depends on many factors, including age, lifestyle and genetic make-up

Cancer Research UK http://info.cancerresearchuk.org/cancerstats/incidence/?a=5441

The 20 most common causes of death from cancer, UK, 2008

Cancer Research UK. Accessed July 2010http://info.cancerresearchuk.org/cancerstats/mortality/cancerdeaths /

Cancer cells have altered genomes

Karyotype illustrating structural abnormalities in cancer

Credit : Mira Grigorova and Paul Edwards, Department of Pathology, University of Cambridge, unpublishedSource: www.path.cam.ac.uk/~pawefish/BreastCellLineDescriptions/HCC38.html

What is a mutation?

• Germline mutation– A change in the DNA sequence that can be

inherited from either parent

• Somatic mutation – A change in the DNA sequence in cells other

than sperm or egg– The mutation is present in the cancer cell and its

offspring, but not in the patient’s healthy cells

Mutations & cancer genes • Cancer genes are causally implicated in oncogenesis• Mutations in cancer genes can occur somatically or can

be inherited.• Mutations in some cancer genes can be inherited from

parents, in which case they are present in every cell of the body. Such people are at a higher risk of developing cancer.

• Somatic mutations can occur in any of the cells of the body except the germ cells (sperm and egg) and therefore are not passed on to children.

Importance of somatic DNA changes in human cancer

Somatic

Inherited

Both

Only 5 –10% of cancer cases have a clear hereditary component, e.g. BRCA1 and BRCA2 in breast cancer

Even in those cases where susceptibility is clearly inherited, somatic changes are required for cancer to develop

Cancer genes

• There are two types of cancer genes: – Tumour suppressor genes – Oncogenes

• To date, we know of approximately 400 somatic “cancer genes” * but there are almost certainly more to be found

• COSMIC is a catalogue of somatic mutations found in cancer genes in human tumours and is available at: http://www.sanger.ac.uk/genetics/CGP/cosmic/

*(COSMIC v47release. July 2010)

Tumour suppressor gene

TS

Cancer

These genes normally function to PREVENT cell growth/division

Oncogene

Cancer

Ras

Genes which normally function to PROMOTE cell growth/division in a controlled manner

Examples of mutations

Sequence 1 Sequence 2 Type

ACTCGTTAGGCA Substitution

ACTCGTTAGGCA ACTCGGCA Deletion

ACTCGTTAGGCA ACTCGTTATCAGGCA Insertion

ACTCGTTAGGCA ACTTTGCAGGCA Inversion

ACTCGTTAGGCA Duplication

ACTCCTTAGGCA

ACTCGTTAGTTAGGCA

Cancer progression

Benign Tumour

In situ cancer

Invasive cancer

Metastatic cancer

Mutations in multiple cancer genes are required for the development and progression of a single cancer

www. flickr.com: lastexit

External causes of cancer: ultraviolet radiation

External causes of cancer: tobacco smoke

Lifestyle factor: diet

Biological factor: virus

• HPV is a cause of cervical cancer

• Proteins from the virus activate and deactivate cancer genes

• The role of HPV in cervical cancer has led to the development of vaccines

HPV in cervical epithelium Credit: MRC NIMR, Wellcome Images

ActivityActivity

• The KRAS gene codes for a signalling molecule

• Mutations in KRAS are present in many cancers, including pancreatic cancer

• You have to look for the mutations by comparing healthy DNA sequence with tumour DNA sequence

• Not all of you will find a mutation

Your Worksheets

If you find a mutation

EXAMPLE ONLY

How to use the codon wheel

Start from the centre and move outwards

Mark up your sequence

Heterozygous mutations

DNA change in cancer

Normal DNAsequence

BRAF gene mutation Nature 417, 906-7 (June 2002)

A double peak indicates a mutation on one chromosome and not the other i.e.

a heterozygous mutation

T → A

A

Results

Amino Acid Number

Healthy DNA Sequence

Tumour DNA Sequence

Healthy Amino Acid

Tumour Amino Acid

12 GGT GTT G (glycine) V (valine)

13 GGC GAC G (glycine) D (aspartic acid)

30 GAC GAT D (aspartic acid) D (Aspartic acid)

61 CAA CGA Q (glutamine) R (arginine)

146 GCA CCA A (alanine) P (proline)

173 GAT GAC D (Aspartic acid) D (Aspartic acid)

Amino Acid Number

Healthy DNA Sequence

Tumour DNASequence

Healthy AminoAcid

Tumour Amino Acid

12 GGT GTT G (glycine) V (valine)

13 GGC GAC G (glycine) D (aspartic acid)

30 GAC GAT D (aspartic acid) D (aspartic acid )

61 CAA CGA Q (glutamine) R (arginine)

146 GCA CCA A (alanine) P (proline)

173 GAT GAC D (aspartic acid ) D (aspartic acid)

Significant mutations

Amino Acid Number

Healthy DNA Sequence

Tumour DNA Sequence

Healthy Amino Acid

Tumour Amino Acid

12 GGT GTT G (glycine) V (valine)

13 GGC GAC G (glycine) D (asparatic acid)

61 CAA CGA Q (glutamine) R (arginine)

146 GCA CCA A (alanine) P (proline)

How common?

AA 1213,894

AA 132,111

AA 61212

AA 14633

Source: COSMIC July 2010

RB1: tumour suppressor gene

Source: COSMIC July 2010

How does this affect the KRAS protein?

Amino acid 12

Amino acid 13

Amino acid 61

Amino acid 146

Amino acid 146

What’s the impact?• KRAS helps to transmit external growth signals to the cell nucleus,

driving normal cell growth. It is:– Activated when it binds GTP– Inactivated or “switched off” when GTP is hydrolysed to GDP