human cancer and mtor ronald crandall. overview background hypothesis experimental design &...
TRANSCRIPT
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?