Human Cancer and mTOR Ronald Crandall. Overview Background Hypothesis Experimental Design &...

Human Cancer and mTOR

Ronald Crandall

Overview

BackgroundHypothesis Experimental Design & Expected ResultsConclusion

Cancer

National Cancer Institute

“Cancer is the uncontrolled growth of abnormal cells in the body. Cancerous cells are also called malignant cells.” - NCBI

Mutation inactivates DNA repair gene

Mutation of proto-oncogene creates an oncogene

Mutation inactivates several more tumor suppressor genes

Stages of Carcinogenesis

Grade 1 - Initiation

Grade 2 -Promotion

Grade 3 - Progression

Shah, K.V. and Howley, P.M.,“Papillomaviruses” in Virology (Fields, B.N., ed.),1996, p. 2090.

mTOR in Cancer

Constitutive activation of mTOR

can cause cancer

Occurs through mutations in mTOR or upstream signals.

Study QuestionWhat stage in cancer does mTOR most effect?

Shah, K.V. and Howley, P.M.,“Papillomaviruses” in Virology (Fields, B.N., ed.),1996, p. 2090.

Overview

BackgroundHypothesis Experimental Design & Expected ResultsConclusion

Hypothesis

Constitutively active mTOR acts as a promoter of skin epithelium cancer carcinogenesis.

Overview

BackgroundHypothesis Experimental Design & Expected ResultsConclusion

Experiment Overview

1. Establish mTOR as a cancer promoter by inducing tumors.

2. Remove tumors and analyze downstream signal expression

Model Organism - Mouse

ClustalW2 Tree

Human and Mouse mTOR share 98.9% amino acid similarity.

Experiment 1 Steps

1) Generate transgenic mice with constitutively active expression of mTOR in basal epithelial cells – Using human keratin 14 transcriptional promoter

2) Use chemical cancer initiator and promoter through skin painting to determine role of mTOR in cancer progression.

k14 promoter mTOR

Generate transgenic mice steps

Create vector with human keratin 14 promoter and mTOR

Microinject DNA into mouse ES cells and put in blastocyst.

Biopsy mice and genotype for vector product, if established mate founder mice to create transgenic line

Experiment 1 Steps

1) Generate transgenic mice with constitutively active expression of mTOR in basal epithelial cells – Using human keratin 14 transcriptional promoter

2) Use chemical cancer initiator and promoter through skin painting to determine role of mTOR in cancer progression.

k14 promoter mTOR

Determining the role mTOR plays in cancer progression

DMBA – mutagen can only initiate

TPA – growth promoter

Shah, K.V. and Howley, P.M.,“Papillomaviruses” in Virology (Fields, B.N., ed.),1996, p. 2090.

Treatment Groups

DMBA TPADMBA + TPA

Skin painting on both the transgenic and wild type mice for each treatment group.

Expected Results

8 10 12 14 16 180

2

4

6

8

10

12

14

16

.03μm DMBA + TPA

Transgenic MiceNontransgeneic Mice

Weeks

Tum

ors (

grad

e 2

or 3

)

8 10 12 14 16 180

2

4

6

8

10

12

14

16

.03μm DMBA

Transgenic MiceNontransgenic Mice

Weeks

Tum

ors (

grad

e 2

or 3

)

8 10 12 14 16 180

2

4

6

8

10

12

14

16

.03μm TPA

Transgenic MiceNontransgenic Mice

Weeks

Tum

ors (

grad

e 2

or 3

)

Experiment Overview

1. Establish mTOR as a cancer promoter by inducing tumors.

2. Remove tumors and analyze downstream signal expression

Experiment 2 Steps

• Surgically remove tumors from treatment groups– If available include cancers of each stage from

treatments

• Analyze Transcriptome and proteome– MudPIT– Ribosomal Profiling

Expression levels of downstream signals from mTOR would increase in tumors with

constitutively active mTOR

http://dx.doi.org/10.1016/j.cell.2012.03.017

Proteins

S6K14E-BP1eIF4ESREBP1

Full proteome and transcriptome profiling categories

http://dx.doi.org/10.1016/j.cell.2012.03.017

Ribosomal profiling of stage 3 tumors

Protein SynthesisCell invasion/metastasisMetabolismSignal TransductionCellular transportPost-translational modificationsRNA synthesis and processingDevelopmentApoptosisDNA repairDNA methylationAmino acid biosynthesis

Ribosomal profiling of control epidermal tissue

Protein SynthesisCell invasion/metastasisMetabolismSignal TransductionCellular transportPost-translational modificationsRNA synthesis and processingDevelopmentApoptosisDNA repairDNA methylationAmino acid biosynthesis

Conclusions

mTOR is a promoter in the progression of cancer.

Constitutively active mTOR shows increased expression of immediate downstream signaling of S6K1, 4E-BP1, eIF4E, and SREBP1.

Ribosomal profiling of constitutively active mTOR shows increased cell growth, lipid synthesis, metabolism and proliferative signaling.

mTOR future researchTest potential mTOR inhibitor drug candidates on the cell lines at each stage in cancer progression. GWAS on mTOR polymorphisms and cancer

Questions?