module 1 lectures 1 & 2 - genes and mutations, early cancer detection via molecular screens

human genome composition

1.5% genomic DNA encodes protein

2% biologically important sequences (promoters/enhancers)

96.5% "junk," non-coding DNA

2012 ENCODE data showed that most of the genome is

composed of functional elements (not junk)

human genome project

- 2000

- 3 billion base pairs DNA sequences

- approx. 21,000 genes discovered

encyclopedia of DNA elements (ENCODE)

launched in september 2003 to identify all the functional elements in the human genome

ENCODE discoveries

demonstrated that over 80% human genome serves functional purpose

- 30 research papers published Sept 2012 from 32 labs

ENCODE by the numbers

- 147 cell types studied

- 80% functional portion of human genome

- 20,687 protein-coding genes

- 18,400 RNA genes

- 1640 data sets

- 30 papers published this week

- 442 researchers

- $288 million funding for pilot, technology, model organism, and current project

polymorphisms

- functionally silent genetic differences between individuals

- phenotypically silent, but identifiable via DNA sequencing

- "fast" evolving DNA; heterozygous locus

genes are conserved over millions of years and between species: example

- last common descendant of fly and mammals = 600 million years ago

- yet, genes for eye development: eyeless (fly) and Pax 6 (mammal) are highly conserved and interchangeable!

experiment: express mouse Pax6 gene in fly embryo --> result and conclusion?

result: mouse Pax 6 gene expression able to direct formation of an eye on fly leg

conclusion: interchangeability of genes

conservation of gene function lies in

amino acid sequence

- which can remain similar even when nucleotides differ

how does conservation of gene function/amino acid sequence simplifie genetic studies?

if you want to study a human gene, find it in yeast first

gene localization

finding the chromosomal region that contains the gene

- FISH mapping with fluorescent probes (short nucleotide sequences that bind to chromosomes and light up)

- identification of 6 distinct genes on chromosome 5

why are there 2 dots represented on each chromosome?

already went through S phase so each chromatid has 2x DNA

human genome project accomplished

mapped positions based on DNA sequences --> chromosome map

- 1st sequence of human genome

karyotype

a picture of all the chromosomes in a cell arranged in pairs

autosomes

all diploid, important in cancer development

2 categories of mammalian genes

1) housekeeping genes (10,000-15,0000)

2) tissue-specific genes (1000)

housekeeping genes

- 10,000-15,000 (more numerous)

- dedicated to maintaining fundamental biological functions (always on)

- common to all cell types

tissue-specific genes

- 1000 (less fewer)

- dedicated to production of proteins required by a specific differentiated cell

what creates phenotype?

proteins create it from genotype (nucleotide sequence)

types of proteins at work

- cytoskeleton (structural)

- extracellular matrix (structural)

- intermediary metabolism (biochem rxns)

- cell-cell signaling proteins and signal transduction proteins—central to cancer formation

cytoskeleton proteins

proteins involved in cellular scaffolding

types of cytoskeleton proteins

- intermediate filaments

- microfilaments

- microtubules

roles of cytoskeleton proteins

- cell shape

- motility

- cell division

- intracellular transport

*all matter in carcinogenesis and metastasis

intermediate filaments

keratin, vimentin, laminin

- stationary; maintain cell shape within the cell

microfilaments

actin

- polymerize and depolymerize; movement and muscle contraction (w myosin)

microtubules

tubulin

cell motility involves

cytoskeleton

human vascular endothelial cell movement plot

- experiment: growth factor is added to one end of culture dish to attract cells.

- each point represents an electronically plotted 10 min time interval.

**these cellular movements are critical to the formation of new blood vessels in cancer cells

what allows cell motility?

actin filaments

extracellular matrix (ECM)

meshwork of collagen fibers, glycoproteins, hyaluronan, proteoglycans

proteins of the extracellular matrix (ECM)

secreted by fibroblast cells

if every cell in a body has the same DNA, how do hundreds of cell types, each w distinct phenotypes, exists in the human body?

cellular differentiation and selective gene expression

nuclear equivalency

nucleus of 1 cell is enough to create a whole organism

experiment to prove nuclear equivalency

cloning of Dolly from somatic cells

- cloned sheep from a somatic (diploid) cell from "mother" mammary gland = not by fertilization

gene regulation in cancer cells

the tight regulation is altered

some modes of gene regulation at level of transcription

1) enhancer and silencer gene elements

2) transcription factors—combinations

3) alternative RNA splicing

4) change in chromatin state: methylation and acetylation of DNA and histones. (the histone code)

5) RNA interference

gene functional parts

- non-tx control regions (enhancers, promoters)

- transcribed sequences (become RNA)

how do transcription factors control gene expression

by binding control regions and altering the DNA

eukaryotic enhancers

located several 1000 bps upstream and downstream of promoter

how do enhancers communicate with promoters?

DNA bends --> contact between initiation complex proteins and enhancer bound proteins

power of transcription factors

combinatorial action

pleiotropic action

combinatorial action

multiple transcription factors act in combination to create an expression program

pleiotropic action

a single type of transcription factor can elicit multiple changes within a cell by signaling a large cohort of responder genes.

what might happen if a pleiotropically acting tx factor malfunctions?

potential for launching a cancer program, where a mutated tx factor signals "gene on", affecting many responder genes

alternative RNA splicing

a single pre-mRNAs may be alternatively spliced to form distinct mRNAs and distinct proteins

- introns spliced out and exons ligated to form mature mRNA

tissue specific alternative splicing of α-tropomyosin pre-mRNA

one RNA may encode several proteins.

- alternative splicing allows different proteins (like a family of proteins) to be expressed from the same gene, depending on cell type

chromatin state affects gene expression

histones: positively charged octamer of proteins which (-) DNA wraps around --> packs DNA

post-translational modification of histone tails

occurs via covalent attachment of methyl, acetyl, phosphate or ubiquitin groups

- "open" or "close" access of DNA to transcription factors by changing charge

RNA interference

control gene regulation

- short noncoding (functional) RNA sequences bind to specific target mRNAs to destroy the message.

- result: down regulation of gene product

example of RNA interference

microRNAs control level of mRNA in cytoplasm or efficiency of translating mRNAs

microRNAs and cancer

overexpression or loss of > dozen miRNA species has been associated with the formation of a variety of human cancers = "oncoMiRs"

chromosomal alterations in cancer cells

cancer cells accumulate various DNA mutations

- aberrant chromosomal number

- aberrant chromosomal structure:

• translocations (inversions, reciprocal)

• deletions

- amplification of chromosome

- extra copy of chromosome

- loss of entire chromosome

normal chromosomal complement

- giemsa stain binds phosphate groups on DNA, creates G-banding patterns.

- giemsa binds tighter to highly condensed DNA, creating a darker band.

- lighter bands=gene rich regions

- G-banding pattern is a chromosome identifier.

chromosome painting

- hybridize chromosomal specific, fluorescently labeled DNA probes to chromosomes

euploid

normal diploid karyotype = 22 autosomes and XX or XY

aneuploid karyotype

chromosomes present in:

- inappropriate numbers &/or

- structural abnormalities

present in >85% solid tumors

abnormal karyotype in cancer cells is characterized by

genomic instability

aneuploidy: how do incorrect chromosome #'s form in the cell?

normal cell in mitotic metaphase:

- chromosomes line up

- attach to microtubules of spindle (organized by centrosome)

cancer cells exhibit

chromosomal instability (CIN)

- in vivo & in vitro

- consequential or causal?

what can lead to changes in chromosome #?

chromosome mis-segregation during mitosis

ex) non-disjunction

non-disjunction events

both sister chromatids of a chromosome pulled to one cell; other cell does not receive a copy

chromosome mis-segregation during mitosis can lead to

changes in chromosome #

- improperly assembled spindles

spindles in normal cells vs. breast cancer cells

normal cells:

- 1 centrosome

cancer cells:

- multiple centrosomes

- multipolar spindles

- example of CIN

translocations

fusion of two chromosomal segments that are not normally attached

types of translocations

- interstitial deletions

- reciprocal translocations

- inversions

chromosomal inversion

when part of the chromosome becomes oriented in the reverse of its usual direction

- note specificity achieved: break location and inversion clearly identifiable

example of chromosomal inversion

FISH labeled intrachromosomal subregions reveals chromosome 5 inversion in a plutonium worker's cells

- power of FISH in determining aneuploidy

reciprocal translocations

chromosomal segments exchanged between nonhomologous chromosomes

- ex) exchange between 9 and 22 forms CML, chronic myelongenous leukemia

interstitial deletions

gene segment between arrows deleted and the flanking ends rejoined

- cancer ex. loss of inhibitory gene

what would not count as an interstitial deletion?

a total loss of a chromosomal segment since no segment rejoins to itself.

amplifications

increase in gene copy number

2 types of amplifications

1) HSR: homogeneously staining region

2) DMs: double minutes

HSR amplification

repeated rounds of chromosomal reduplication result in an elongated chromosome with head to tail repeats of a particular segment

ex) giemsa stain reveals banding of a typical streched appearance, as well as highly abnormal karyotype

double minutes

extrachromosomal, autonomously replicating segments that were originally part of the chromosome

ex) breast cancer cells with amplified HER2/neu oncogene borne on DMs—causes a great increase in the dosage of that gene

2 types of cancer genes

oncogenes and tumor suppressor genes

oncogenes

genes that have the potential to cause cancer once mutated; can enhance growth

tumor suppressor genes

a gene whose protein product inhibits cell division --> when mutated causes cancer

besides oncogenes and tumor surpressors, cancer cells also have __________ mutations

passenger

passenger mutation

mutation in a cancer that does not contribute to tumorgenesis

can a tumor have HSR and DMs in the same cell?

yes, COLO320 tumor cells

paranchymal cells

functional cell type within a tissue that performs the specific tasks associated with that tissue's role

- tumor cells may obstruct or replace them, disrupting normal tissue function

somatic vs. germline mutations

germline mutations are only 5-10% and can be passed down/inherited

somatic mutations

- not inherited by offspring

- form a clonal population from a single progenitor

germline mutations

- occur in sperm, eggs or their precursor cells

- affect offspring

how can mutations occur with cellular repair mechanisms in place?

while cellular repair mechanisms in place to maintain genomic integrity, they are not infallible

early detection methods

screening for cancers

driver mutations

protein biomarkers

effective cancer screening tests (FDA approved testing)

- mammography (breast cancer)

- colonoscopy (colorectal cancer)

- PAP Smear (cervical cancer)

- PSA test (prostate cancer, efficacy?)

genomics and oncology in cancer testing

using DNA sequencing and RNA transcripts to determine the structure and function of genomes

genomics and oncology testing methods

1) karyotype & FISH

2) microarrays

3) GWAS single nucleotide polymorphism screen

4) NGS next generation sequencing – paired comparison of patient’s normal tissue and tumor tissue

5) genomic assays for circulating tumor cells DNA and protein biomarkers (CANCERSEEK)

2) microarrays reveal

selective gene expression

gene expression microarray analysis

allows evaluation of thousands of gene expression patterns in a given cell type

- small red bars = high gene exp

- small green bars = low gene exp

- vertical axis= exp levels of 1800 genes in this array

- horizontal axis = mRNAs from 142 different human tumors

oncotyping

like normal tissues, each class of tumor has its own characteristic spectrum of gene expression

- allows identification of tumor of unknown origin and individualized treatment

ex) tumor of unknown origin was lung cancer

3) GWAS: genome-wide association studies

genetic epidemiology

- phenotype first studies

- examine a group with particular disease -> screen 100,000s of loci simultaneously for single nucleotide polymorphisms (SNPs)

- NCI: PLCO =prostate, lung, colorectal, ovarian GWAS

pros of GWAS

- find common gene trends among patients

- identification of at-risk population (screening for SNP biomarkers)

- allows for potential causal relationships

- tailoring of treatment options (gene-drug interactions)

cons of GWAS

- may not be predictive of disease

- cannot assess rare genetic variants

- associations represent small effect size

- causality and patient risk not clear

- genes and other risk factors not known (environment, diet)

4) NGS next generation sequencing

classify cancers according to genomic characteristics, potential to guide therapy

- paired sample testing reveals genetic differences between normal and malignant tumor tissue

types of NGS

- WGS whole genome sequencing

- WES whole exome sequencing

- RNA-seq

WGS (whole genome sequencing)

- sequence entire genome in one day, $1000 cost

- detects point mutations, indels, copy number variation, rearrangements and translocations