Abeloff Clinical Oncology 2020 (Part 1)

A.
BIOLOGY AND CANCER

1 Molecular Tools in Cancer Research
Mauro W. Costa, Muneer G. Hasham, and Nadia Rosenthal
S UMMARY OF K EY P OI N T S
• Our understanding and treatment of growth and differentiation has techniques, including whole-genome
cancer have always relied heavily on revolutionized the diagnosis, analysis, expression profiling, and
parallel developments in biologic prognosis, and treatment of refined genetic manipulation in and
research. Molecular biology provides malignant disorders. use of genetically diverse animal
the basic tools to study genes • This introductory chapter relates models, providing the conceptual
involved with cancer growth patterns basic principles of molecular biology and technical background necessary
and tumor suppression. An to emerging perspectives on the to grasp the central principles and
advanced understanding of the origin and progression of cancer and new methods of current cancer
molecular processes governing cell explains newly developed laboratory research.
Since the last edition of this book was published, advances in our genetic changes confer selective advantages on tumor cell clones by
understanding of the basic mechanisms of cancer have continued to disrupting control of cell proliferation. The identification of specific
inform and refine clinical approaches to prevention and therapy. New mutations that characterize a tumor cell has proved invaluable for
prognostic and predictive markers derived from molecular biology analyzing the neoplastic progression and remission of the disease. The
can now pinpoint specific genetic changes in particular tumors or emergence of cancer cells is a byproduct of the necessity for continuous
detect occult malignant cells in normal tissues, leading to improved cell division and DNA replication to maintain organ functionality
technologies for tumor screening and early detection. Diagnostic throughout the life cycle.
approaches have expanded from morphologic criteria and single-gene The highly heterogeneous nature of tumors, each composed of
analysis to whole-genome technologies and single-cell genomics multiple cell types, led to the formulation of the “cancer stem cell”
imported from other biologic disciplines. A new systemic vision of hypothesis, which posits that only a subpopulation of cancer cells is
cancer is emerging, in which the importance of individual mutation able to maintain self-renewal, unlimited growth, and capacity for
has been superseded by an appreciation for higher-order organization differentiation into other, more specialized cancer cell types. Cancer
and individual genetic background, disrupted by complex interactions stem cells display bona fide stem cell markers, in contrast to other
of disease-associated factors and gene-environmental parameters that cancer cells present in the tumor, which do not have tumorigenic
affect tumor cell behavior. Results from these cross-disciplinary potential. In fact, fewer than 1 in 10,000 cells present in human acute
investigations underscore the complexity of carcinogenesis and have myeloid leukemia are capable of reinitiating a new tumor when
profoundly influenced the design of strategies for both cancer prevention transplanted into animals. Cancer stem cells have been identified in
and advanced cancer therapy. many solid tumors in the brain, colon, ovaries, prostate, and pancreas,
This overview will serve as a foundation of conceptual and technical suggesting that more effective cancer therapies would target these
information for understanding the exciting new advances in cancer self-renewing cells, rather than the tumor as a whole. The cancer stem
research described in subsequent chapters. Since the discovery of cell concept differs from the original clonal evolution hypothesis,
oncogenes, which provided the first concrete evidence of cancer’s which states that every cell in a tumor mass is capable of self-renewal
genetic basis, applications of advanced molecular techniques and and differentiation, and suggests that detecting and targeting subtle
instrumentation have yielded new insights into normal cell biology. genetic and epigenetic differences that distinguish cancer stem cells
A basic fluency in molecular biology and genetics has become a necessary may provide a more effective avenue to intervention in disease
prerequisite for clinical oncologists because many of the new diagnostic progression.
and prognostic tools currently in use rely on these fundamental Heterogeneity can also arise as a result of stochastic mutational
principles of gene, protein, and cell function. events that lead to cancer progression. Clastogenic insults to the genome,
or genomic instability due to aberrant gene regulation, could lead to
OUR UNSTABLE HEREDITY loss of heterozygosity (LOH) of tumor suppressor genes such as TP53,
RB1, or BRCA, and can also lead to tumor heterogeneity and change
Cancer genetics has classically relied on the candidate-gene approach, in disease progression. Furthermore, activation of DNA or RNA editing
detecting acquired or inherited changes in specific genetic loci accu- enzymes in tumors could lead to kataegis, a DNA hypermutation
mulated in a single cell, which then proliferates to produce a tumor process, and increase tumor heterogeneity. Although there are molecular
composed of its identical clonal progeny. During the early steps of biology tools currently available to detect aberrant but stable genomes,
tumor formation, mutations that lead to an intrinsic genetic instability the later processes that lead to genomic instability make diagnosis
allow additional deleterious genetic alterations to accumulate. These and prognosis more challenging.
2
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Molecular Tools in Cancer Research • CHAPTER 1 3
DETECTING CANCER MUTATIONS The functional unit of inherited information in DNA, the gene,
is most often represented by a discrete section of sequence necessary
Methods for mutation detection all rely on the manipulation of DNA, to encode a particular protein structure. Gene expression is initiated
the basic building block of heredity in the cell. DNA consists of two by forming a copy of the gene, messenger RNA (mRNA), constructed
long strands of polynucleotides that twist around each other clockwise base by base from the DNA template by a polymerase enzyme. Once
in a double helix (Fig. 1.1). Nucleic acid bases attached to the sugar transcribed, an mRNA transcript is modified and the processed product
groups of each strand face each other within the helix, perpendicular is transported out of the nucleus. In the cytoplasm, proteins are
to its axis. These comprise only four bases: the purines adenine and then synthesized, or translated, in macromolecular complexes called
guanine (A and G) and the pyrimidines cytosine and thymine (C and ribosomes that read the mRNA sequence and convert the nucleic acid
T). During assembly of the double helix, stable pairings of nucleotides code, based on three-base segments or codons, into a 20–amino acid
from either strand are made between A and T, or between G and C. code to form the corresponding protein.
Each base pair forms one of the billions of rungs in the long, unbroken Although these canonic processes drive gene expression in all normal
ladder of DNA forming a chromosome. cells, cancer cells defy the rules. For instance, uracils, which are found
Genes Cell nucleus

containing
23 pairs of
chromosomes
DNA
strand
Chromosomes
Sugar
Cytosine Bases
thymine
Bases
Adenine and
guanine
Phosphate
group P
H
H
C H
H
H C
H
P
H
H
P
H
C
H
H CH2
P P
H
CH2
H
CH2
H
P P
H
CH2
H CH2
Figure 1.1 • DNA structure. Deoxyribonucleic acid (DNA) is the cell’s genetic material, contained in single compacted strands comprising chromosomes
within the cell nucleus. In the DNA double helix, the two intertwined components of its backbone, composed of sugar (deoxyribose) and phosphate molecules,
are connected by pairs of molecules called bases. The sequence of four bases (guanine, adenine, thymine, and cytosine) in the DNA helix determines the specificity
of genetic information. The bases face inward from the sugar-phosphate backbone and form pairs with complementary bases on the opposing strand for
specific recognition. The arrangement of chemical groups is unique for each base pair, allowing base pairs to be specifically targeted by transcription factors,
polymerases, restriction enzymes, and other DNA-binding proteins. (From http://www.terrapsych.com/dna.jpg.)
4 Part I: Science and Clinical Oncology
on RNA, can be detected in the DNA of cancer cells because of their is shared by all members of a species, the recent sequencing of thousands
high mutation rates. Paradoxically, these deviations from the norm of individual human genomes has given rise to the new field of
allow the development of molecular biology tools to better diagnose genomics, providing us with new tools to reveal the more subtle
and predict tumor progression. variations that arise between individuals. These variations are critical,
both as a natural engine driving heterogeneity within a species, and
GENERATING DIVERSITY WITH as a source of predisposition to cancer types. The most common forms
ALTERNATE SPLICING of human genetic variations, or alleles, arise as single-nucleotide
polymorphisms (SNPs). Because these allelic dissimilarities are abundant,
In higher organisms, most protein coding gene sequences are interrupted inherited, and dispersed throughout the genome, SNPs can be used
by stretches of noncoding DNA sequences, called introns. In the to track racial diversity, personal traits, and susceptibility to common
nucleus, these introns are removed after mRNA transcription to produce forms of cancer (Fig. 1.3). Commercial entities have developed tools
a continuous chain of coding sequences, or exons, that subsequently that can detect thousands of SNPs with relatively little sample material.
undergo translation into protein. The splicing process requires absolute Platforms such as MegaMUGA or GigaMUGA can allow mammalian
precision because the deletion or addition of a single nucleotide at genetic mapping that can aid in a number of diagnoses and can
the splice junction would throw the three-base coding sequence out distinguish between predictive and prognostic markers.
of frame, or lead to exon skipping or addition, creating abnormal How do SNPs arise between individuals? One source of variation
proteins. in DNA sequence derives from deviations in the strict base-pairing
The dramatic increase in genetic complexity conferred by alternate rule underlying the structure, storage, retrieval, and transfer of genetic
RNA splicing is underscored by the multiple splice patterns of many information. The duplicated genetic information in the two strands
medically relevant genes, in which different combinations of exons of DNA not only permits the repair of a damaged coding sequence
are chosen for the final mRNA transcript, such that one gene can but also forms the basis for the replication of DNA. During cell
encode many different proteins (Fig. 1.2). The choice of protein isoform division, polymerase enzymes unwind the DNA strands and copy
to be expressed from a gene with multiple splicing possibilities is a them, using the base sequences as a template for constructing a new
decision that can be perturbed in disease. Errors in splicing mechanisms helix so that the dividing cell passes its entire genetic content on to
have been associated with a large group of cancers. These include its progeny. Errors in this process are rare, and person-to-person
mutations in the oncogene p53 in more then 12 different types of differences comprise only about 0.1% of the human genome. SNPs
cancer, mutL homolog 1 protein (MLH1) mutation in hereditary are inherited if they occur in the germline. Many genetically inherited
nonpolyposis colorectal cancer, and several transcription factors and variations occur in regions that do not encode protein or alter the
cell signaling and membrane proteins. When mutations in the splicing regulation of nearby genes. Given the disruptive effects even subtle
site lead to insertion of novel sequences in the mRNA, the encoded genetic changes may have on cell function, it is important to distinguish
protein can be used as a potential clinical marker, as seen for the SNPs that represent true mutations from benign polymorphisms.
transcription factor NSFR in small cell lung cancer. Owing to their Our ability to monitor hundreds of thousands of SNPs simultane-
unique expression in cancer cells, these markers can be further explored ously is one of the most important advances in modern medical genetics.
as new cancer-specific therapeutic targets. Relatively simple genotyping technologies for SNP detection rely largely
on the polymerase chain reaction (PCR). In procedures that use this
GENOMICS OF CANCER reaction, two chemically synthesized single-stranded DNA fragments,
or primers, are designed to match chromosomal DNA sequences
The complete set of DNA sequences carried on all the chromosomes flanking the segment in which an SNP is positioned. With the addition
is known as the genome. Although the general map of the genome of nucleotide building blocks and a heat-stable DNA polymerase, the
Exon Exon Exon Exon Exon

Gene 1 2 3 4 5
1 2 3 4 5
RNA
Alternative splicing
RNA 1 2 3 4 5 1 2 4 5 1 2 3 5
Translation Translation Translation
Protein A Protein B Protein C
Figure 1.2 • RNA splicing. Alternate splicing produces multiple related proteins, or isoforms, from a single gene. (From Guttmacher AE, Collins F.
Genomic medicine—a primer. N Engl J Med. 2002;347:1512–1520.)
Primary tumor Metastasis SNP density

11p15.5
11p15.4
11p15.3
11p15.2
11p15.1
11p14.3
11p14.2
11p14.1
11p13
11p12
Tens to hundreds
11p11.2
of changes between >10 million SNPs 11p11.12
primary and secondary between individuals 11p11.11
tumors 11q11
11q12.1
11q12.2
11q12.3
11q13.1
11q13.2
11q13.3
Primary tumor Metastasis 11q13.4
11q13.5
11q14.1
11q14.2
11q14.3
11q21
11q22.1
11q22.2
11q22.3
11q23.1
11q23.2
11q23.3
11q24.1
11q24.2
11q24.3
11q25
Figure 1.3 • Determining cancer susceptibility with single-nucleotide polymorphisms (SNPs). Millions of SNPs exist between individuals, as depicted by
the red arrows and the SNP density map of human chromosome 11 (right). By contrast, point mutations, deletions, insertions, and rearrangements between
normal tissues and tumors or between primary and secondary tumors probably number in the tens to hundreds (or potentially thousands), as depicted by the
spectral karyotype image at the bottom of the figure. Because the constitutional genetic polymorphisms are present in all the tissues of the body, it might be
possible to distinguish differences in metastatic versus nonmetastatic tumors and in nontumor tissues before they ever happen to develop a solid tumor. (From
Hunter K. Host genetics influence tumour metastasis. Nat Rev Cancer. 2006;6:141–146.)
primer pairs, or amplicons, initiate synthesis of new DNA strands, base sequence that carries unique protein coding information. Other
using the chromosomal material as a template. Each successive copying noncoding DNA sequences are used for directing the transcription
cycle, initiated by “melting” the resulting double-stranded products of neighboring genes, through complex regulatory circuits involving
with heat, doubles the number of DNA segments in the reaction (Fig. protein binding and modification of the DNA itself, or shifting of
1.4). The technique is exceptionally sensitive; millions of identical its chromosomal packaging. Although genomic instability is generally
DNA copies can be generated in a matter of hours with PCR by using considered a consequence of tumor formation rather than the initial
a single DNA molecule as the starting material. trigger of cancer, the loss, gain, or rearrangement of chromosomal
Other novel methods for large-scale SNP detection include single- segments through deletion or translocation is a common form of
nucleotide primer extension, allele-specific hybridization, oligonucleotide neoplastic mutation, as protein coding segments from different genes
ligation assay, and invasive signal amplification, which detect poly- are combined or regulatory sequences are brought into new proximity
morphisms directly from genomic DNA without the requirement of to genes they do not normally control, as seen in chronic myeloid
PCR amplification. The International HapMap Project was established leukemia (CML). In CML, recombination events lead to the fusion
with the objective of identifying those variations (commonly thought of BCR and ABL genes (Philadelphia chromosome). This results in
to be on the order of 10 million in our genome) in the human popula- constitutive activation of the fused gene, leading to loss of proliferative
tion. This project is already in its third phase (HapMap3), now control in myeloid cells and consequently cancer. Gross changes in
including both SNPs and copy number variations observed in 1184 DNA arrangement can be detected by cytogenetic analysis of chro-
samples from 11 different human populations. Regardless of the method mosomal features on metaphase spreads. Although fluorescence in
used to characterize them, the collective SNPs in a selected genomic situ hybridization (FISH) provides greater resolution by localizing
region characterize a haplotype, or specific combination of alleles at specific chromosomal DNA sequences corresponding to fluorescently
multiple linked genetic loci along a chromosome that are inherited labeled probes (Fig. 1.5), and can be used to track specific alterations
together. in chromosomal structure where known genes are involved, spectral
Even when the SNPs within a given haplotype are not directly karyotyping (SKY) is a powerful and more general tool that could
involved in a disease, they provide markers for clonality and for the aid diagnosis of cancer genomes. With each fluorescently labeled
loss or rearrangement of specific chromosomal segments in growing chromosome assigned a specific color, translocations and additions
tumors. In the human nucleus, each of the 23 tightly compacted are revealed as multicolored chromosomes, or large deletions as pieces
chromosomes has a characteristic size and structure, and a distinctive of missing chromosomes.
Cycle 1 Cycle 2 Cycle 3 The plethora of data arising from genome-wide association studies
using currently available techniques poses particular challenges to
cancer researchers. Discerning the causal genetic variants among
genotype-phenotype associations requires extensive replication, control
for underlying genetic differences in population cohorts, and consistent
classification of clinical outcomes. New technologies must be met
Separation of strands
with equivalently sophisticated and rigorous analytic methodologies
for the true genetic cause of cancer to be teased out from our variable
and often unstable heredity.
Primers
Sequence
to be BUILDING GENE LIBRARIES
amplified
The engineering of genes by recombinant DNA technology evolved
from methods initially devised to provide sequences in amounts
sufficient for biochemical analysis. The original protocol involves
clipping the desired segment from the surrounding DNA and inserting
it into a bacterial or viral vector, which is then amplified millions of
times in a host bacterium. Using recombinant DNA technology, genetic
Primers Heat Heat Heat engineering can routinely produce industrial quantities of pure, clinically
useful products in a cost-effective way. For diagnostic purposes, it is
easier and faster to amplify a known genomic DNA sequence directly
Figure 1.4 • Amplification of DNA by polymerase chain reaction (PCR). from a patient sample with PCR, but the classic approach is still
The DNA sequence to be amplified is selected by primers, which are short, applied to the construction of recombinant DNA libraries.
synthetic oligonucleotides that correspond to sequences flanking the DNA to To be useful, a DNA library must be as complete as possible, with
be amplified. After an excess of primers is added to the DNA, together with recombinant members, or clones, sufficiently numerous to include
a heat-stable DNA polymerase, the strands of both the genomic DNA and all the sequences in an individual genome. For certain kinds of gene-
the primers are separated by heating and allowed to cool. A heat-stable
polymerase elongates the primers on either strand, thus generating two new,
linkage analysis that require long, uninterrupted stretches of DNA,
identical double-stranded DNA molecules and doubling the number of DNA special vectors, such as bacterial or yeast artificial chromosomes, can
fragments. Each cycle takes just a few minutes and doubles the number of carry foreign DNA fragments of enormous lengths. Chromosomal
copies of the original DNA fragment. segments represented in genomic DNA libraries can contain the
9 9 22 22
9 der(9) 22 der(22)
Figure 1.5 • Detection of chromosomal translocations. Fluorescence in situ hybridization (FISH) technology uses a labeled DNA segment as a probe to
search homologous sequences in interphase chromosomes for the t(9;22)(q34;q11) translocation, associated with chronic myeloid leukemia. On the left, patient
nuclei were hybridized with probes for chromosome 9 (labeled with SpectrumRed fluorophore) and chromosome 22 (labeled with SpectrumGreen). (Modified
from Varella-Garcia M. Molecular cytogenetics in solid tumors: laboratorial tool for diagnosis, prognosis, and therapy. Oncologist. 2003;8:45–58.)
structure of an entire gene, including the information that regulates The interaction between protein and DNA is increasingly
its expression, and formed the starting material for sequencing of the used to identify transcription factor binding sites in a regulatory
human genome. region. Whereas electrophoretic mobility shift assays (EMSAs), or
Many cancer-associated genes were originally identified through DNA footprinting, were once standard techniques for determining
use of partial DNA libraries, which contain only the DNA sequences protein-DNA interactions, emerging genome-wide technologies, such
transcribed by a particular tissue or type of cell. The starting mate- as chromatin immunoprecipitation on microarray chip (ChIP-chip)
rial in this case is mRNA. For cloning purposes, the enzyme reverse and chromatin immunoprecipitation on sequencing (ChIP-seq), are
transcriptase can convert mRNA into complementary DNA (cDNA). revolutionizing the way in which we see the interaction of a transcription
The number of clones in a cDNA library is much smaller than factor complex with virtually all of its potential genomic targets in
in a genomic library because a cDNA library represents only the a particular cell state. These strategies involve the use of candidate
genes expressed by the tissue of interest and contains exclusively the protein–specific antibodies to pull down DNA targets regulated by
coding portion of genes. For this particular reason, this technique them. These targets are further identified with the use of microarray
has now become obsolete for organisms whose genome has now ChIP-chip or next generation sequencing ChIP-seq technologies
been fully sequenced. New advances in PCR chemistry allowed (see Fig. 1.14).
for the direct cloning of increasingly larger cDNA fragments with Our appreciation of oncogenic perturbations, by mutation of
high specificity and low error rates. Highly accurate PCR technol- regulatory protein coding genes or by loss of controlled signaling by
ogy, coupled with the constantly evolving generation of genomic cell cycle switches or in the target sequences these proteins recognize,
sequence maps in humans and model organisms, has exponentially has recently extended to include posttranslational modifications that
expanded the availability of candidate genes to be tested in cancer control protein activity, such as phosphorylation, ubiquitylation, and
biology. SUMOylation. Tumor-associated changes in these modifications
underscore the multiple levels of control necessary to ensure correct
LOSING CONTROL OF THE GENOME gene expression that is so central to the normal function of the cell.
Mutations that lead to oncogenic transformation of a cell invariably EPIGENETICS AND CANCER
affect the expression of its genetic information that specifies functional
products, either RNA molecules or proteins used for various cellular Epigenetics refers to general control of gene expression that is inherited
functions. The primary level of gene control is the transcription of during cell division, although not part of the DNA sequence itself.
DNA into RNA. Gene regulation, or the control of RNA synthesis, Epigenetic regulation involves changes in chromatin, a higher-order
represents a complex process that is itself a frequent target of neoplastic building block of chromosomes that wraps DNA into coils with
mutation. scaffolding proteins such as histones. Histones are a necessary com-
DNA regulatory sequences do not encode a product. However, ponent of chromosomal compaction, but also play a critical role in
without them a cell could not coordinate the expression of the hundreds gene accessibility (Fig. 1.7). Active genetic loci are associated with
of thousands of genes in its nucleus, select only certain genes for loosely configured euchromatin, whereas silent loci are condensed in
expression, and activate or repress them in response to precise internal heterochromatin. The state of chromatin configuration (euchromatin
or external signals. These control centers of the genome contain binding or heterochromatin) both controls and is controlled by patterns of
sites for multiple proteins, called transcription factors, which interact histone modifications such as methylation and acetylation on specific
to form regulatory networks controlling gene transcription. Their DNA sequences. This pattern relates the underlying genetic information
function can be altered by signals that induce modifications such as to its higher-order structure that determines whether a particular gene
phosphorylation, or by interactions with other regulators such as steroid regulatory element is available to transcription factors (on or off status).
hormones. Many of the cell’s responses to a wide variety of external These epigenetic modifications of the nuclear environment that
stimuli, such as neurotransmitters, antigens, cytokines, and growth determine the accessibility of a gene can persist during cell division,
factors, are mediated through transcription factors binding to DNA because inherited epigenetic patterns provide permanent marks for
regulatory sequences. altered chromatin configuration in daughter cells. The pattern of
Certain regulatory DNA sequences common to many genes are modifications generated by the epigenetic code rivals the complexity
positioned upstream of the transcription start site (Fig. 1.6). Collectively of the DNA code itself.
called the “promoter” of a gene, these proximal sequences comprise The accessibility of genomic regions can favor mutations. Enzymes
binding sites for the RNA polymerase and its numerous cofactors. such as the APOBEC family exploit this accessibility to induce C to
Whereas the position of the promoter with regard to the transcription U mutation, which is then converted to T or staggered single-strand
start site is relatively inflexible, other DNA regulatory elements, known breaks. If not rectified, these point mutations or breaks can lead to
as enhancers, occur in unpredictable locations, often at a considerable hypermutations. Kataegis is an example wherein such hypermutation
distance from the genes they control. Some transcription factors bind occurs on the BRCA locus, generating neoplasia.
to particular regions of enhancers and drive their associated genes in Research has linked rearrangement of chromatin and associated
many types of cells, whereas others, active in only a limited variety DNA methylation with the inactivation of tumor suppressor genes and
of cells, maintain a tissue-specific pattern of gene expression. Enhancers neoplastic transformation. Defects that could lead to cancer involve
are often responsible for the aberrant expression of genes induced by perturbations in the “epigenotype” of a particular locus, through the
chromosomal translocation associated with specific forms of cancer: silencing of normally active genes or activation of normally silent
a normally quiescent gene promoting cell growth that is dislocated genes, associated with changes in DNA methylation, histone modifica-
to a position near a strong enhancer may be activated inappropriately, tion, and chromatin proteins (Fig. 1.8). Changes in the number or
resulting in loss of growth control. density of heterochromatin proteins associated with cancer-related
Enhancers and promoters have been assigned specific roles by means genes such as EZH2, or of euchromatic proteins such as trithorax in
of cell culture assays or in transgenic animals in which putative regula- leukemia, can also be associated with abnormal patterns of methyla-
tory DNA sequences are linked to test or “reporter” genes, and are tion in gene promoter regions and with higher-order chromosomal
examined for their ability to activate expression of the reporter gene structures that are only beginning to be understood. Finally, it is
in response to the appropriate signals. Through assessment of the increasingly evident that interactions among the “epigenome,” the
effects of deleting, adding, or changing DNA sequences within the genome, and the environment are common targets for mutation
regulatory element, the precise nucleotides that are critical for recogni- and can have profound effects on the gene expression readout of a
tion by transcription factors can be determined. cancer cell.
Gene structure
Exon 1 Exon 2 Exon 3
Enhancer Promoter Intron 1 Intron 2
TATAA GT AG GT AG AATAAA
Transcription Gene expression

factors
RNA polymerase
Exon 1 Exon 2 Exon 3
Transcription
Transcription-initiation pre-
complex 5' 3' mRNA
Transcript processing
RNA-clipping enzyme
AAUAAA
5' cap
PolyA tail
AAAA...
Adenosine-adding
Intron lariat enzyme (terminal
transferase)
Splicing
Nucleus AAAA...
Spliceosome
Processed
transcript mRNA
Cytoplasm AAAA...
Translation into protein
Figure 1.6 • Mammalian gene structure and expression. The DNA sequences that are transcribed as RNA are collectively called the gene and include exons
(expressed sequences) and introns (intervening sequences). Introns invariably begin with the nucleotide sequence GT and end with AG. An AT-rich sequence
in the last exon forms a signal for processing the end of the RNA transcript. Regulatory sequences that make up the promoter and include the TATA box
occur close to the site where transcription starts. Enhancer sequences are located at variable distances from the gene. Gene expression begins with the binding
of multiple protein factors to enhancer sequences and promoter sequences. These factors help form the transcription-initiation complex, which includes the
enzyme RNA polymerase and multiple polymerase-associated proteins. The primary transcript (pre-mRNA) includes both exon and intron sequences. Post-
transcriptional processing begins with changes at both ends of the RNA transcript. At the 5′ end, enzymes add a special nucleotide cap; at the 3′ end, an
enzyme clips the pre-mRNA about 30 base pairs (bp) after the AAUAAA sequence in the last exon. Another enzyme adds a polyA tail, which consists of up
to 200 adenine nucleotides. Next, spliceosomes remove the introns by cutting the RNA at the boundaries between exons and introns. The process of excision
forms lariats of the intron sequences. The spliced mRNA is now mature and can leave the nucleus for protein translation in the cytoplasm. (From Rosenthal
N. Regulation of gene expression. N Engl J Med. 1994;331:931–932.)
Nucleosome
DNA
The solenoid
Figure 1.7 • Chromatin packaging of DNA. The 4 meters of DNA in every human cell must be compressed in the nucleus, reaching compaction ratios
of 1 : 400,000. This is achieved by wrapping the DNA (blue) around histone protein complexes (green), forming nucleosomes connected by a thread of free
linker DNA. Each nucleosome, together with its linker, packages about 200 bp (66 nm) of DNA. The nucleosomes are then coiled into chromatin, a rope of
nucleoprotein about 30 nm thick (bottom left electron micrograph). To allow DNA to be accessed by transcription and replication apparatus, chromatin is relaxed
(bottom right electron micrograph). (Courtesy Jakob Waterborg. www.umkc.edu/sbs/waterborg/chromat/chromatn.html. Copyright 1998 Jakob Waterborg.)
Gene X Gene X
X
Gene Y
X
Gene Y
A Normal B Epigenetic lesions

Figure 1.8 • Gene accessibility through epigenetics. Illustration depicts known and possible defects in the epigenome that could lead to disease. (A) Gene
X is a transcriptionally active gene with sparse DNA methylation (brown circles), an open chromatin structure, interaction with euchromatin proteins (green
protein complex), and histone modifications such as H3K9 acetylation and H3K4 methylation (green circles). Gene Y is a transcriptionally silent gene with
dense DNA methylation, a closed chromatin structure, interaction with heterochromatin proteins (red protein complex), and histone modifications such as
H3K27 methylation (pink circles). (B) The abnormal cell could switch its epigenotype through the silencing of normally active genes or activation of normally
silent genes, with the attendant changes in DNA methylation, histone modification, and chromatin proteins. In addition, the epigenetic lesion could include
a change in the number or density of heterochromatin proteins in gene X (such as EZH2 in cancer) or euchromatic proteins in gene Y (such as trithorax in
leukemia). There may also be an abnormally dense pattern of methylation in gene promoters (shown in gene X ), and an overall reduction in DNA methylation
(shown in gene Y ) in cancer. The insets show that the higher-order loop configuration may be altered, although such structures are currently only beginning
to be understood.
PROFILING TUMORS underlying tumor progression by following the changes in a tumor

cell’s transcriptional landscape.
Monitoring global gene expression patterns of cells represents one of With reliance on two-color fluorescence-based microarray technology
the latest breakthroughs in developing a molecular taxonomy of cancer. (DNA microarray), simultaneous evaluation of thousands of gene
Although classic blotting and probe hybridization techniques (Northern transcripts and their relative expression can provide a snapshot of the
blot) are still a reliable way to monitor expression of individual genes, “transcriptome,” the full complement of RNA transcripts produced
these techniques have limitations, such as unequal hybridization at a specific time during the progression of malignancy.
efficiency of individual probes, sensitivity for low copy or small Transcriptional profiling with microarrays typically involves screens
transcripts, and difficulty in detecting multiple RNAs simultaneously of mRNA expression from two sources (such as tumor and normal
or in simultaneously analyzing a large number of targets. For cancer cells), using cDNA or oligonucleotide libraries that are arranged in
studies, it is important to be able to compare the expression pattern extremely high density on microchips. These are probed with a mixture
of all known RNAs, including noncoding RNAs, between cancer cells of fluorescently tagged cDNAs generated from the tumor and normal
and normal cells. Thus new genome-wide analytic techniques are the samples, which results in differential staining of each gene spot. The
state-of-the-art choice to detect mRNA expression profiles at a single relative intensity of the two different colors reflects the RNA expres-
point in time or cell state. Genome-wide profiling of gene expression sion level of each gene; this is analyzed with a laser confocal scanner
in tumors delivers an unprecedented view into the biologic processes (Fig. 1.9). With microarrays, single genes that constitute diagnostic,
Tumors
Reference Tumor
RNA RNA
Genes
Statistical
cDNA
analysis
Hybridization
of probe to
microarray Multidimensional-scaling plot
A B C
Donor Recipient
paraffin block paraffin block
D E Tissue microarray
Figure 1.9 • Microarray-based expression profiling of tumor tissue. (A) Reference RNA and tumor RNA are labeled by reverse transcription with different
fluorescent dyes (green for the reference cells and red for the tumor cells) and hybridized to a cDNA microarray containing robotically printed cDNA clones.
(B) The slides are scanned with a confocal laser scanning microscope, and color images are generated with RNA from the tumor and reference cells for each
hybridization. Genes upregulated in the tumors appear red, whereas those with decreased expression appear green. Genes with similar levels of expression in
the two samples appear yellow. Genes of interest are selected on the basis of the differences in the level of expression by known tumor classes (e.g., BRCA1-
mutation–positive and BRCA2-mutation–positive). Statistical analysis determines whether these differences in the gene expression profiles are greater than
would be expected to occur by chance. (C) The differences in the patterns of gene expression between tumor classes can be portrayed in the form of a
color-coded plot, and the relations between tumors can be portrayed in the form of a multidimensional-scaling plot. Tumors with similar gene-expression
profiles cluster close to one another in the multidimensional-scaling plot. (D) Particular genes of interest can be further studied through the use of a large
number of arrayed, paraffin-embedded tumor specimens, referred to as tissue microarrays. (E) Immunohistochemical analyses of hundreds or thousands of
these arrayed biopsy specimens can be performed in order to extend the microarray findings. (From Hedenfalk I, Duggan D, Chen Y, et al. Gene expression
profiles in hereditary breast cancer. N Engl J Med. 2001;344:539–548.)
prognostic, or therapeutically relevant markers can be systematically technique relies on the generation of small fragments of cDNA from
monitored. Alternatively, the entire set of expressed genes can be any RNA sample, followed by sequencing of these expressed tags from
collectively analyzed through use of powerful statistical methods to one end (single-end sequencing) or both ends (pair-end sequencing),
classify tumors according to their transcriptional profile. Microarray resulting in fragments of 30 to 400 base pairs (bp). The resulting
analysis has already dramatically improved our ability to explore the sequences can be then mapped against the known reference genome
genetic changes associated with cancer etiology and development and or transcriptome of a certain species. Unlike microarray analysis of
is providing new tools for disease diagnosis and prognostic assessment. preselected gene sets, RNA-seq allows the unbiased identification of
For example, DNA microarray analysis of multiple primary breast all genes, or even the presence of different isoforms, expressed in the
tumor transcriptomes has revealed reproducible signature expression of sample, allowing a comprehensive comparison of transcript levels
70 associated genes. These markers have been recently cleared by the between normal and cancer cells.
US Food and Drug Administration (FDA) for PCR–based diagnostics The technologies just described can also be applied to the analysis
showing that expression analysis of a relative small gene group can of noncoding RNA species. In addition to the 20,000 protein coding
predict the prognosis of early stage breast cancers. When applied on a transcripts used to classify a wide variety of human tumors, hundreds
larger scale, these assays can predict response to chemotherapy, or opti- if not thousands of small, noncoding interference RNA species, with
mize pharmaceutical intervention by targeting therapeutic approaches critical functions in multiple biologic processes, have been discovered;
to specific patient populations and ultimately to individualized therapy. many of these RNA species are directly or indirectly involved in the
A novel high-throughput approach for global transcriptome analysis control of cell proliferation. Known as microRNAs (miRNAs), these
has been made possible by advances in strategies that allow mass short transcripts arise from primary genome-encoded transcripts of
sequencing of DNA fragments. With this technique, called RNA-seq, variable sizes that are processed into 70- to 100-nucleotide hairpin-
it is now possible to obtain a comprehensive and unbiased analysis shaped precursors, which are processed into mature miRNAs of 21 to
of all mRNA transcripts present in cells or tissues. (Fig. 1.10). The 23 bp RNA molecules (Fig. 1.11). miRNAs function by base-pairing
2x poly (A) RPKM RPKM

selection
Brain 0.0 0.0
Liver 0.0 0.0
25-bp
reads
Add standards
and shatter RNA Muscle 0.5 50.1
Make cDNA
and sequence Muscle splices
1 kb
Map 25-bp tags RNA-Seq
onto genome graph
Myf5
Myf6
Conservation
25-bpm Uniquely mappable

Calculate splices
transcript
Repeating elements by RepeatMasker
prevalence
Myf6
Conservation 20 kb
Uniquely map.
2 RPKM 1 RPKM 1 RPKM RepeatMasker
A B C
Figure 1.10 • Methods for high-throughput transcriptome analyses. (A) Schematics of regular protocol for RNA-seq sample preparation, showing poly-A
tail specific mRNA isolation followed by fragmentation of RNA into smaller regions, further used for cDNA conversion. Polymerase chain reaction (PCR)
fragments are then tethered by adaptors, sequenced by synthesis, and aligned to the reference genome or transcriptome to calculate relative prevalence of
mRNAs (RPKM). (B) Target fragments can be used to map exon-intron boundaries and thus infer present and quantify different mRNA isoforms in the
sample of interest, as shown for the muscle specific gene Myf6 in this example. (C) Data generated with this method can also be compared with analysis of
other tissues or samples, allowing assessment of relative quantification of targets, as exemplified here for a highly specific gene (red peaks) for muscle samples.
(From Mortazavi A, Williams BA, et al. Mapping and quantifying mammalian transcriptomes by RNA-seq. Nat Methods. 2008;5:621–628.)
Protein-coding gene MicroRNA gene
Transcription of pri-microRNA
Transcription
of mRNA
Pri-microRNA
OR
Drosha
DGCR8
Processing
Nucleus
of pri-microRNAs
into pre-microRNA
Pre-microRNA Ran-GTP
Exportin 5
Transport of
pre-microRNAs into
the cytoplasm
Processing of
Dicer pre-microRNA into
small RNA duplexes
Loqs/TRBP
Cytoplasm
RISC
||||||||||||||||||||
||||
|||
|||
|||
||| ||||||||||||||||||||||||||||| An
||| |||||||||
||| ||||||
||| |||| |||||
|||| |||||
|||||||||
Delivery of
RISC-microRNA
complex
mRNA degradation Translational repression
Figure 1.11 • MicroRNA production and gene regulation in animal cells. Mature functional microRNAs of approximately 22 nucleotides are generated
from long primary microRNA (pri-microRNA) transcripts. First, the pri-microRNAs, which usually contain a few hundred to a few thousand base pairs, are
processed in the nucleus into stem-loop precursors (pre-microRNA) of approximately 70 nucleotides by the RNase III endonuclease Drosha and DiGeorge
syndrome critical region gene 8 (DGCR8). The pre-microRNAs are then actively transported into the cytoplasm by exportin 5 and Ran-GTP and further
processed into small RNA duplexes of approximately 22 nucleotides by the Dicer RNase III enzyme and its partner Loqacious (Loqs), a homologue of the
human immunodeficiency virus transactivating response RNA-binding protein. The functional strand of the microRNA duplex is then loaded into the
RNA-induced silencing complex (RISC). Finally, the microRNA guides the RISC to the target messenger RNA (mRNA) target for translational repression or
degradation of mRNA. (Modified from Chen CZ. New Eng J Med. 2005;353:1768–71.)
with target mRNAs to inhibit translation and/or promote mRNA suppression. The usefulness of monitoring the expression of miRNAs
degradation. In the context of cancer, miRNAs may act in concert with in human cancer is just now being explored, but preliminary findings
other effectors such as p53 to inhibit inappropriate cell proliferation. reveal an extraordinary level of diversity in miRNA expression across
A global decrease in miRNA levels is often observed in human cancers, cancers, and the large amount of diagnostic information encoded
indicating that small RNAs may have an intrinsic function in tumor in a relatively small number of miRNAs. Significant technologic
advances facilitating the profiling of the miRNA expression patterns CANCER PROTEOME
in normal and cancer tissues hint at the unexpected greater reliability of
miRNA expression signatures than the respective signatures of protein The term proteome describes the entire complement of proteins expressed
coding genes in classifying cancer types. Along with their potential by the genome of a cell, tissue, or organism. More specifically, it is
diagnostic value, miRNAs are also being tested for their prognostic used to describe the set of all the expressed proteins at a given time
use in predicting clinical behaviors of cancer patients. point in a defined setting, such as a tumor. Like RNA transcription,
Because probe specificity in miRNA microarray analysis is prob- the synthesis of proteins is a highly regulated process that contributes
lematic owing to the small target size, hybridization can be performed to the specific proteome of a particular cell and can be perturbed in
first in solution, and then quantified with multicolor flow sorting. diseases such as cancer.
Real-time PCR can also be used to quantify specific miRNA sets, or Advances in protein analytic techniques over the last decades have
to capture a more detailed picture of their changing expression profiles progressed to the point that even small numbers of specific proteins
in tumor progression. Identification of the miRNAs involved in tumor expressed in tissues can be used to predict the prognosis of a cancer.
pathogenesis and elucidation of their action in a specific cancer will The improvement of protein-based assays has made it possible to
be the next necessary steps for their manipulation in a therapeutic identify and examine the expression of most proteins, and to envision
setting. large-scale protein analysis on the level of gene-based screens. Various
Advances in this field have revealed that miRNAs are also involved systematic methodologies have contributed to the current explosion
in cancer initiation and progression, and specific modulation of of information on the proteome. These are now being compared for
such RNAs may serve as a therapeutic strategy. Inhibition of key their suitability as platforms for the generation of databases on protein
miRNAs using antagomirs (a class of chemically modified anti-miRNA structural features, interaction maps, activity profiles, and regulatory
oligonucleotides) has been effective in suppressing tumor growth in modifications.
mouse models. It remains to be seen if these results can be extended to The yeast two-hybrid system has been a popular genetics-based
treatment of cancer in the clinic, but interference with miRNA function approach for detecting protein-protein interactions inside a cell (Fig.
is an attractive new tool for the development of cancer therapies. 1.12). One protein fused to the DNA binding domain (bait) and a
DNA-binding domain
fused to protein A A
A Promoter Reporter gene
Activator region fused to

protein B
B
B Promoter Reporter gene

Figure 1.12 • Exploring protein-protein interactions with the yeast two-hybrid
system. Two-hybrid technology exploits the fact that transcriptional activators are Activator region fused to
modular in nature. Two physically distinct functional domains are necessary to get protein B
transcription: a DNA-binding domain that binds to the DNA of the promoter and DNA-binding domain
an activation domain that binds to the basal transcription apparatus and activates fused to protein A A B Transcription
transcription. (A) The known gene encoding protein A is cloned into the “bait”
vector, fused to the gene encoding a DNA-binding domain from some transcription
factor. When placed into a yeast system with a reporter gene, this fusion protein
can bind to the reporter gene promoter, but it cannot activate transcription. C Promoter Reporter gene
(B) Separately, a second gene (or a library of cDNA fragments encoding potential
interactors), protein B, is cloned into the “prey” vector, fused to an activation domain
of a different transcription factor. When placed into a yeast strain containing the 96 Bait strains 1 Prey strain
reporter gene, it cannot activate transcription because it has no DNA-binding domain.
(C) When the two vectors are placed into the same yeast, a transcription factor is
formed that can activate the reporter gene if protein B, made by the second plasmid,
binds to protein A. (D) Screening a yeast two-hybrid library. The plate on the left
holds 96 different yeast strains in patches (or colonies), each of which expresses a
different bait protein (top). The plate on the right holds 96 patches, each of the
same yeast strain (prey strain) that expresses a protein fused to an activation domain
(prey). The plate of bait strains and the plate of prey strains are pressed to the same
replica velvet, and the impression is lifted with a plate containing yeast extract
peptone dextrose (YPD) medium. After 1 day of growth on the YPD plate, during
which time the two strains mate to form diploids, the YPD plate is pressed to a Replicate velvet
new replica velvet, and the impression is lifted with a plate containing diploid Diploids
selection medium and an indicator such as X-Gal. Blue patches (dark spots) on the
X-Gal plate indicate that the lacZ reporter is transcribed, suggesting that the prey YPD
interacts with the bait at that location. (C from http://www.nature.com/…/journal/
v403/n6770/full/403601a0_r.html. D from Bartel PL, Fields S, eds. The Yeast Replicate velvet
Two-Hybrid System. New York: Oxford University Press; 1997; Finley RL Jr, Brent
R: Two-hybrid analysis of genetic regulatory networks. Retrieved from http://www. D X-Gal
genetics.wayne.edu/finlab/YTHnetworks.html.)
different protein fused to the activation domain of a transcriptional as phosphorylation, SUMOylation, or ubiquitination. These LC-MS/
activator (prey) are expressed together in yeast cells. If the bait MS systems, such as the iTRAQ, allow for a more precise and indi-
and prey interact, transcription of a reported gene is induced and vidualized diagnosis of cancer.
detected typically by a color reaction that reflects the transactiva- Monoclonal antibodies (mAbs) have been a cornerstone of protein
tion of the reporter gene, and by proxy, the interaction of the two analysis in cancer research, and more recently have risen to prominence
test proteins. The method can also be used for large-scale protein as cancer therapeutics based on their exquisite specificity for protein
interactions, determination of RNA-protein interactions, and protein- targets and their potent interference with protein function. Novel
ligand binding. strategies have been developed that target not only antigens highly
As a complementary proteomics tool, mass spectrometry (MS) is expressed in cancer cells but also to enhance the innate immune
an accurate mass measurement of charged peptides isolated by two- response against cancer cells. These antibodies can act via several
dimensional gel electrophoresis, producing a mass-to-charge ratio of mechanisms, including antibody-dependent cellular cytotoxicity
charged samples under vacuum that can be used to determine the (ADCC), complement-mediated cytotoxicity (CMC), and antibody-
sequence identity of peptides. Combined with a specific proteolytic dependent cellular phagocytosis (ADCP) (Fig. 1.13). Laboratory mice
cleavage step, mass spectroscopy can be used for peptide mass mapping. have been the animal model of choice for generating a ready source
Automation of this process has made mass spectroscopy the analytic of diverse, high-affinity and high-specificity mAbs; however, the use
tool of choice for many proteomics projects. For diagnostic purposes, of rodent antibodies as therapeutic agents has been restricted by the
liquid chromatography and mass spectrometry (LC-MS/MS) have inherent immunogenicity of mouse proteins in a human setting. The
been combined to detect not only a single–amino acid change in the more recent application of transgenic mouse technology to introduce
whole proteome, but also posttranslational protein modification such variable regions encoded by human sequences into the corresponding
Signal Bispecific
BiTE
perturbation Toxin
cytotoxicity
cytotoxic cells
Direct
Nonrestricted
activation of
Radionuclide
Drug
TrioMab
Tumor cell
CMC death
MAC
inhibitory signaling
Fc-mediated immune
effector engagement
Blockade of
ADCC
Phagocytosis
ADCP
APC Helper T cell Cytotoxic T cell

IC uptake MHC class II presentation MHC class I cross-presentation
Introduction of adaptive immune responses
Antigen BiTE MHC TCR T cell

class I Phagocytic
APC
Monoclonal Immunotoxin MHC Fc receptor Tumor cell
antibody class II
Perforin and
Bispecific Compliment Innate granzymes
CD3 KIR
antibody (C1q) effector
Figure 1.13 • Mechanisms for antibody-based therapies used against cancer cells. Multiple current approaches involve direct cytotoxicity, Fc-mediated
immune effector engagement, nonrestricted activation of cytotoxic T cells, and blockade of inhibitory signaling. The diverse spectrum of action of these therapies
will allow the inclusion of various anticancer targets in the near future (From Weiner LM, Murray JC, Shuptrine CW. Antibody-based immunotherapy of
cancer. Cell. 2012;148:1081–1084.)
mouse immunoglobulin genes has enabled the generation of “human- being developed that have increased specificity toward individualized
ized” therapeutic mAbs with reduced immunogenicity. In addition, cancers.
bispecific antibodies (bsAbs) with dual affinity for tumor antigens, From an epigenetic perspective, new techniques are enabling the
such as TriomAb, have been shown to effectively kill tumor cells by genome-wide characterization of protein-DNA interactions that can
inducing memory T-cell protective immunity. In addition to the uncover novel transcription factor targets, histone modifications,
expected use of mAbs directed at extracellular epitopes (protein regions and DNA methylation patterns within a cancer cell. Combining
recognized by the antibody), evidence from mouse models has raised chromatin immunoprecipitation (ChIP) with microarray (ChIP-chip)
the possibility of using antibodies targeting intracellular epitopes for allows genome-wide screening for the binding position of protein
anticancer therapies. Targeting such antigens would enrich immuno- factors to their gene targets. In ChIP-chip assays or ChIP-seq, a
therapy, allowing the use of tumor-specific intracellular mediators of cross-linking reagent is applied in vivo to proteins associated with
cell survival and proliferation. Numerous mAb-based agents are currently DNA in the nucleus, which then can be coimmunoprecipitated
in trial or in use as therapeutics for cancer, and the potential for with specific antibodies to the protein under analysis. The bound
further optimization of mAbs through genetic engineering promises DNA and appropriate controls are then fluorescently labeled and
to open new avenues for in vivo therapy. applied to microscopic slides for microarray analysis, or directly
A recent advancement in mAb-based cancer therapy is the generation sequenced, rendering a simultaneous profile of all the binding posi-
of chimeric antigen receptor (CAR) T lymphocytes to target tumors tions of specific proteins in the cancer cell’s genome (Fig. 1.14). The
in vivo. These are effector T-lymphocytes engineered to express a mAb global profiling of promoter occupancy of specific cancers, wherein
that recognizes specific groups of cancer cells. The receptors are chimeric, protein-DNA interaction profiles discriminate patients with tumors
composed of engineered molecules from diverse sources. The first from those presenting different clinical outcomes, is a promising
generation of CAR-modified T cells (CAR T cells) showed success in predictive method.
preclinical trials and have entered phase I clinical trials in ovarian After a decade of development, proteomics is still primarily a
cancer, neuroblastoma, and various types of leukemia and lymphoma. basic research activity, yet in the near future this technology is likely
Newer generations of these therapeutic lymphocytes are currently to have a profound impact on medicine. By defining the collective
Analysis and visualization Peak calling Alignment
Chr1:13456-13486
Chr1:24323-24293
2
Chr1:45678-45708 Sequencing
Chr1:54321-54351
Information content
1.5
Chr1:55679-55709
1
etc...
0.5
ChlP-Seq 0
1 2 3 4 5 6 7 8 9 10
Position
Library
construction
Antibody
Binding sites
TF TF
105
4
P < 0.001
log2 enrichment
20Kb
104
Cy3 intensity
0
–1
1000
ChlP-on-chip 200bp
Binding sites
ChlP replicate 1
100
Peak
ChlP replicate 2
Peak
10
10 100 1000 104 105
Cy5 intensity
Binding site identification Array data analysis Genomic arrays
Figure 1.14 • Methods for unbiased identification of transcription factor binding sites. Chromatin immunoprecipitation on sequencing (ChIP-seq) and
chromatin immunoprecipitation on microarray chip (ChIP-chip) can provide location, isolation, and identification of the DNA sequences occupied by specific
DNA-binding proteins in cells. Proteins capable of DNA interactions are targeted with specific antibodies. DNA and the associated proteins are cross-linked;
DNA is fragmented into 150 to 500 bp and immunoprecipitated. After reversion of the cross-link, DNA is isolated and either mass-sequenced (ChIP-seq)
or used as probes in a genomic array (ChIP-chip), and binding sites occupied by the proteins can be identified in the genome. These binding sites may indicate
functions of various transcriptional regulators and help identify their target genes during development and disease progression. The types of functional elements
identified with these techniques include promoters, enhancers, repressor and silencing elements, insulators, boundary elements, and sequences that control
DNA replication. (From Kim TH, Ren B. Annu Rev Genomics Hum Genet. 2006;7:81–102 and Liu et al. BMC Biol. 2010;8:56.)
Human Drosophila Yeast C. elegans Arabidopsis
Transcriptional Virus-host Metabolic Protein-protein Disease

regulatory network network network interaction network
Alzheimer’s
disease
Hypertension
Pseudohypo-
Atherosclerosis aldosteronism
Asthma
Figure 1.15 • Interactome networks and human disease. Networks are integrated sources of information obtained from biochemical, molecular, proteomic,
and other high-throughput analyses. Different networks can be obtained for each organism, organ, or cell. In the first instance, central regulatory “nodes”
identify important components in the network. These networks and their data can then be integrated and compared with healthy and disease models, allowing
an integrative view of events that is much more powerful than isolated networks. (Modified from Vidal M, Cusick ME, Barabási A. Interactome networks
and human disease. Cell. 2011;144:986–998.)
protein-protein interactions in a cancer cell (its “interactome”), recently, PDX models have been further optimized with the use of
functional relationships between disease-promoting genes may be humanized host mice that are modified to contain human immune
revealed that provide novel candidates for intervention (Fig. 1.15). systems.
Networks of disorder-gene associations are already being built that
offer a platform for describing all known phenotype and disease-gene TRANSGENIC MODELS OF CANCER
associations, often indicating the common genetic origin of many
diseases. A precise diagnosis of cancer through use of proteomics Integrating an oncogene that causes malignancy into the genome of
can be envisioned, based on highly discriminating patterns of a mouse without altering the mouse’s own genes generates a transgenic,
proteins in easily accessible patient samples. Proteomics informa- cancer-prone mouse that transmits this trait to its offspring with a
tion also promises to provide sophisticated mathematical models of dominant pattern of inheritance. The technology for producing
the molecular events underlying a process as complex as neoplastic transgenic mice joins recombinant DNA methodology with standard
transformation, which will capture the dynamics of the disease with techniques that are used today by in vitro fertilization clinics, relying
unprecedented power. on the understanding of mammalian reproduction and the development
of protocols to harvest, manipulate, and reimplant eggs and early
MODELING CANCER IN VIVO embryos (Fig. 1.17). The transgene is constructed so that the gene
product will be expressed under appropriate spatial and temporal
Once the mechanistic underpinnings of a particular cancer have been control. In addition to all the standard signals necessary for efficient
described, creating an animal model to test that mechanism becomes transcription and translation of the gene, transgenes contain a promoter,
critical to understanding the pathophysiology and to design therapeutic or regulatory region, that drives transcription in either a ubiquitous
strategies for treatment. Advances in manipulation of the mouse genome or a tissue-restricted pattern. This requires an extensive knowledge of
have resulted in more sophisticated models of human cancer. These genetic regulation in the target cells. A recent advance that circumvents
methodologies can circumvent embryonic death by targeted alteration this requirement involves embedding the transgene inside another
of gene expression only after a critical period in development, and gene locus that is expressed in the desired pattern. Held in a bacterial
reduce the complexity of gene functional analysis by restricting its artificial chromosome (BAC) for easier manipulation, this long stretch
pattern of activation. Inducible gene expression or silencing also allows of DNA surrounding the host gene is likely to carry all the necessary
acute, as opposed to chronic, effects to be assessed. Although species regulatory information to guarantee a predictable expression pattern
differences in tumor susceptibility and disease remission exist between of the introduced transgene.
mouse and man, the tools for genetic manipulation in mouse are The transgene DNA is then injected into the male pronucleus of
superior to those in other mammals, and useful information about a fertilized mouse egg, obtained from a female mouse in which
the function of oncogenes can be gained by targeted expression of hyperovulation has been hormonally induced. The injected eggs are
mutant protein products in mouse tissues. cultured to the two-cell stage and then implanted in the oviduct of
A major hurdle in generation of clinically relevant mouse models another recipient female mouse. Transgenic pups are identified by the
to develop cancer treatments stems from the lack of patient tailoring. presence of the transgene in their genomic DNA (obtained from the
Cancer cells present a highly heterogeneous population that varies tip of the tail and analyzed with PCR assay). Typically, several copies
with the genetic makeup of the individual patient. This shortcoming of the transgene are incorporated in a head-to-tail orientation into a
has been addressed with the advent of patient-specific avatars, also single random site in the mouse genome. About 30% percent of the
known as personalized mouse models or patient-derived xenograft resulting pups will have integrated the transgene into their germline
(PDX) models (Fig. 1.16). Implanting patient biopsy specimens into DNA and constitute the founders of the transgenic lines. RNA analysis
immunodeficient mice allows growth of the tumor, generating in vivo of their progeny determines the level of transgene expression, and
precision models without further in vitro manipulation of the tumor whether the transgene is being expressed in the desired location or at
tissue. These models show great promise for designing treatment and the appropriate time. Given the variability in transgene number and
drug tests that should best target the patient-specific tumor. Most chromosomal location, transgene expression patterns and levels can
A B
Figure 1.16 • Mouse avatar (PDX) models. (A) Patient-derived xenograft (PDX) mice are generated by implanting patient tumors into immunodeficient/
humanized mice. The tumors can then be propagated for several passages in fresh mice for a number of generations. (B) Usually, after the third generation
the tumors can be isolated and characterized for further study. These mice can potentially be used for patient drug-specific testing and molecular characterization,
therefore allowing for personalization of cancer treatment. (From http://www.the-scientist.com/?articles.view/articleNo/42470/title/My-Mighty-Mouse/.)
diverge considerably among different founder lines carrying the same The general scheme involves two mouse lines, one carrying the
transgene. recombinase either as a transgene driven by inducible regulatory
In general, transgenesis is optimal for modeling oncogenic mutations elements or knocked into one allele of a gene expressed in the desired
that cause a gain of function, producing disease even when they occur tissue. The other mouse line harbors a modified gene target including
in only one of a gene’s two alleles. For example, an activating mutation recognition sequences. Mating the two lines results in progeny carrying
in a growth factor that causes abnormal cell proliferation can be both the target gene and the recombinase, which interacts with the
mimicked by introducing a transgenic version of the mutated growth target gene only in the desired tissue.
factor gene under the control of an appropriate regulatory sequence A popular conditional methodology is based on the activation of
for expression in the tissue of interest. The relative susceptibility of nuclear hormone receptors to control gene expression. Two current
such a transgenic mouse to tumorigenesis can help distinguish between systems involve activation of a mammalian estrogen receptor, estrogen
a primary and secondary role of the mutant factor, and established analogue 4-hydroxy-tamoxifen, or an insect hormone receptor with
lines of these animals can be used for testing new therapeutic the corresponding ligand ecdysone. Although several variations on
protocols. these hormone-receptor systems are currently in use, the underlying
principle is the same. The Cre recombinase gene, or another regulatory
CONDITIONAL CONTROL OF protein, such as a transcription factor, is fused with the ligand-binding
ONCOGENE ACTIVATION domain (LBD) from a nuclear hormone receptor protein. The resulting
chimeric transgene is placed under the control of a promoter that
The genetic construction of cancer-prone transgenic mice with the directs expression to the tissue of interest, and transgenic animals are
capacity to induce oncogene expression in vivo provides a new avenue generated. In the absence of the hormone or an analogue, the fusion
to modeling the role of oncogenes in tumor generation and mainte- protein accumulates in the desired tissue but is rendered inactive
nance. This technology relies on conditional mutagenesis. Producing through its association with resident heat shock proteins. Hormone,
conditional mutations in mice requires a DNA recombinase enzyme administered either systemically or topically, binds to the LBD moiety
that does not recognize any mouse sequence, but rather targets short, of the fusion protein, dissociates it from the heat shock protein, and
foreign recognition sequences to catalyze recombination between them. allows the transcriptional regulatory component to find its natural
By strategic placement of these recognition sequences in appropriate DNA targets and promote lox-P mediated recombination, or in the
orientations either beside or within a mouse gene, the recombination case of an inducible transcription factor, activate expression of the
results in deletion, insertion, inversion, or translocation of associated corresponding genes. If the LBD is fused to a recombinase, administra-
genomic DNA (Fig. 1.18). Two recombinase systems are currently in tion of hormone leads to the rearrangement of target sequences. This
use: the Cre-loxP system from bacteriophage P1, and the Flp-FRT reaction is not reversible, but lends additional temporal control over
system from yeast. The 34 bp loxP or FRT recognition sequences do recombinase-based mutation. If the LBD is fused to a transcription
not occur in the mouse genome, and both Cre and Flp recombinases factor, removal of hormone leads to inactivation of the fusion protein
function autonomously, without the need for cofactors. Cre- or Flp- and gene downregulation.
mediated recombination is not distance or cell-type dependent, and Another inducible method in use is the tetracycline (tet) regula-
can occur in proliferating or differentiated tissues. tory system. In the classic design (tTA or tet-off ), a fusion protein
Figure 1.17 • Generation of transgenic mice. The transgene containing

3' Flanking the DNA sequences necessary for the expression of a functional protein is
Promoter 5' UT Coding region 2' UT
region injected into the male (larger) pronucleus of uncleaved fertilized eggs through
Transgene
a micropipette. The early embryos are then transferred into the reproductive
tract of a mouse rendered “pseudopregnant” by hormonal therapy. The resulting
pups (founders) are tested for incorporation of the transgene by assaying
genomic DNA from their tails. Founder animals that have incorporated the
transgene (+) are mated with nontransgenic mice, and their offspring are mated
with each other to confirm germline integration and to establish a line of
homozygous transgenic mice. Several transgenic lines that have incorporated
Collection of fertilized eggs from different numbers of transgenes at different integration sites (and thus express
a superovulated donor mouse various amounts of the protein of interest) are usually studied. UT, Untranslated.
(From Schuldiner AR. Transgenic animals. N Engl J Med. 1996;334:653–655.)
Cell type specific

promoter Cre loxP Target gene loxP
X
Injection
of transgene into
male pronucleus of
uncleaved fertilized egg
Transfer of early embryos into reproductive

tract of a pseudopregnant mouse Cre Cre
A Special cell type All other cells
CMV-β actin
– promoter
– βgeo 3PA EGFP
+ loxP loxP
+ Cre
–
CMV-β actin
promoter
+ EGFP
Assay of genomic DNA from tails of founder
animals for incorporation of the transgene B loxP
Figure 1.18 • Conditional mutagenesis schemes. (A) Two mouse lines are
required for conditional gene deletion: first, a conventional transgenic mouse
line with Cre targeted to a specific tissue or cell type; and second, a mouse
strain that embodies a target gene (endogenous gene or transgene) flanked by
two loxP sites in a direct orientation (“floxed gene”). Recombination (excision
and consequently inactivation of the target gene) occurs only in those cells
expressing Cre recombinase. Hence, the target gene remains active in all cells
and tissues that do not express the Cre recombinase. (B) The Z/EG double
reporter system. These transgenic mice constitutively express lacZ under the
Sequential matings to determine control of the cytomegalovirus enhancer/chicken actin promoter. Expression
germline integration is widespread, with notable exceptions being liver and lung tissue. Expression
is observed throughout all embryonic and adult stages. When crossed with a
Cre recombinase-expressing strain, lacZ expression is replaced with enhanced
Study of phenotype green fluorescent protein expression in tissues expressing Cre. This double
reporter system makes it possible to distinguish a lack of reporter expression
from a lack of Cre recombinase expression while providing a means to assess
Cre excision activity in live animals and cells. (A Courtesy Kay-Uwe Wagner,
National Institutes of Health; B from Novak A, Guo C, Yang W, Nagy A,
Lobe CG. Z/EG, a double reporter mouse line that expresses enhanced green
fluorescent protein upon Cre-mediated excision. Genesis. 2000;28:147–155.)
combining a bacterial tet repressor and a viral transactivation domain Overlapping genetic functions can also be defined by crossbreeding
drives expression of the target transgene by binding to upstream tet mice with mutations in different genes. In this way it is possible to
operator sequences flanking the transgene transcription start site. In the study the combinatorial effects of oncogene and tumor suppressor
presence of the antibiotic inducer, the fusion protein is dissociated from gene mutations.
the operator sequences, inactivating the transgene. In a complementary Several caveats are important in considering the use of knockout
design, called reverse tetracycline-controlled transactivator (rtTA or technology in modeling cancer. Most knockouts generate loss-of-
tet-on), structural modification of the tet repressor makes the antibiotic function (null) germline mutations. Inactivation of widely expressed
an active requirement for binding of the fusion protein to the operator genes with multiple functions may have complex phenotypes. Con-
sequences, such that its administration activates transgene expression at versely, if the functions of two genes overlap, a mutation in one of
any time during the life span of the mouse, whereas withdrawal results in the genes may not produce an abnormal phenotype, owing to compensa-
downregulation of the gene. It is important that the transgene integrate tion by the unaltered partner.
into a genomic locus that permits proper tTA or rtTA regulation so that Perhaps the greatest drawback of conventional knockout technology
the system exhibits minimal “intrinsic leakiness” and good antibiotic derives from the disruption of gene function at the earliest stage
responsiveness. of its expression. If the gene has a vital developmental role, the
Conditional expression systems have already been developed to identification of functions later in development can be occluded.
generate hematopoietic, leukemogenic, and lymphomagenic mutations Therefore, although the generation of a null mutation is an excel-
in the mouse, as well as solid tumors. These inducible cancer models lent starting point for analysis, it is far from being functionally
can be exploited to identify oncogenic signals that influence host-tumor exhaustive. For these reasons, conditional mutagenesis is the emerg-
interactions, to establish the role of a given oncogenic lesion in advanced ing method of choice for the elucidation of the gene functions
tumors, and to evaluate therapies targeted toward cancer-causing that exert pleiotropic effects in a variety of cell types and tissues
mutations. Potential clinical application of inducible systems include throughout the life of the animal, which is particularly relevant
targeting virally delivered transgene expression to malignant tissues for the generation of mouse models of adult-onset diseases such
by the use of specific inducible regulatory elements, restricting the as cancer.
expression of transgenes exclusively to affected tissues, and increasing Use of recombinase-mediated gene mutation as described earlier
the therapeutic index of the vectors, particularly in the context of for conditional transgenesis, conditional knockout mutations can be
solid tumors. In all cases, a basic knowledge of the specific mutations designed to disrupt the function of a target gene in a specific tissue
involved in the molecular genetics of malignancies is required because (spatial control) and/or life stage (temporal control). Depending on
it is often unclear that the causal mutation underlying the genesis of the design of the experiment, recombinase action can delete an entire
neoplasia continues to play a central role in the progression to the gene, remove blocking sequences to induce gene expression, or rearrange
fully transformed state. This is particularly important in modeling chromosomal segments. With the advent of recent internationally
cancers characterized by genetic plasticity, wherein drug resistance can coordinated systematic mutagenesis programs aiming to place a
arise subsequent to primary tumor formation. conditional inactivating mutation in each of the 20,000 genes in the
mouse genome, the possibilities for modeling cancer are limited only
MODELS OF RECESSIVE GENE MUTATIONS by a researcher’s choice of the gene loci to test. The constantly evolving
IN CANCER techniques for gene manipulation in vivo constitute a major advance
in cancer research.
In contrast to dominantly acting oncogenes, recessive genetic disorders, Genetically modified mice are of great value in dissecting the
such as loss-of-function mutations in tumor suppressor genes, require pathogenesis of many tumor types. In some knockout studies, the
both copies (alleles) of a gene to be inactivated. The methods needed phenotype of the mutated gene is anticipated by prior knowledge
to produce animal models of recessive genetic disease differ from those of the gene’s function. However, unexpected mutant phenotypes
used in studying dominant traits. Gene knockout technology has been may help clarify the mechanism of the underlying neoplasia.
developed to generate mice wherein one allele of an endogenous gene Pharmacologic manipulation of transgenic, knockout, diversified
is removed or altered in a heritable pattern (Fig. 1.19). Gene disruption animal models of cancer will prove useful in screening therapeutic
or replacement is first engineered in pluripotential cells, termed agents with potential for study in clinical trials. Therapy involving
embryonic stem cells (ESCs), which are genetically altered by introduc- gene or cell replacement can be also tested in genetically engineered
tion of a replacement gene that is inactive or mutant. disease models.
To reduce random integration of the foreign DNA, the replacement
gene is embedded into a long stretch DNA from its native locus in EXPLOITING MOUSE DIVERSITY FOR
the mouse, which targets the recombination event to the homologous CANCER RESEARCH
position in the ESC genome. Inclusion of selectable markers along
with the replacement gene allows selection of the cells in which A novel in vivo tool has emerged that aids in understanding the etiology
homologous recombination has taken place. Site-specific recombinase of cancers, by more accurately reflecting the broad genetic variability
systems combined with gene targeting techniques in ESCs can also in the human population. Cancer research performed with mice has
be used to induce recessive single point mutations or site-specific largely focused on a few individual highly inbred strains with limited
chromosomal rearrangements in a tissue- and time-restricted pattern. genetic diversity, which would equate to single individuals in the
In a variation on this theme called knockin, a foreign gene, such as population. Yet drugs designed to treat one individual are often not
one encoding a marker or a mutated gene, can be placed in the locus effective in other patients. The Collaborative Cross (CC) was created
of an endogenous gene. The engineered ESCs are then microinjected to provide mouse models that better represent the diversity seen in
into the cavity of an intact mouse blastocyst sufficiently early in gestation natural human populations while still retaining the broad power of
that they can, in principle, populate all the tissues of the developing genetic analysis seen in mice. The CC resource is a large panel of
chimeric embryo. This is rarely the case, so contribution of ESCs to recombinant inbred (RI) strains generated by randomly mixing the
the resulting animal is most often assessed with use of ESCs and genetic diversity of eight extant inbred mouse lines, and can be used
blastocysts whose genes for coat color differ. to test the impact of treatments in a diverse genetic pool akin to the
If the ESCs contribute to the germ cells of the founder mouse, human population (Fig. 1.20). A related resource, the Diversity Outcross
their entire haploid genome can be passed on to subsequent generations. (DO), offers higher mapping resolution by randomized outcrossing
Through mating together of subsequent progeny of the founder mouse, of partially inbred CC strains, which segregates the same allelic variants
both alleles of the mutated gene can be passed to a single animal. but embeds them in a distinct population architecture in which each
Embryonic stem cell
Tumor suppressor gene
5' Homologous 3' Homologous

region region
Intron
Cellular
gene
Embryonic stem
Plasmid
cell culture pgk-neo pgk-tk DNA
Knockout
vector
Homologous
recombination
Cellular gene
replaced
Injection of embryonic stem Implantation of chimeric

cells into host blastocyst blastocyst in foster mother
Selection by neomycin and ganciclovir
Germline offspring Chimeric offspring
Figure 1.19 • Gene knockout strategies. Embryonic stem cells (upper left panel) contain the tumor suppressor cellular gene (upper right panel), which
consists of exon 1 (olive green, a 5′ noncoding region), an intron, and exon 2 (red, a protein coding region, and yellow, a 3′ noncoding region). A knockout
vector—consisting of a collinear assembly of a DNA flanking segment 5′ to the cellular gene (blue), the phosphoglycerate kinase–bacterial neomycin gene
(pgk-neo, violet), a 3′ segment of the cellular gene (yellow), a DNA flanking segment 3′ to the cellular gene (green), and the phosphoglycerate kinase–viral
thymidine kinase gene (pgk-tk, orange)—is created and introduced into the embryonic–stem cell culture. Double recombination occurs between the cellular gene
and the knockout vector in the 5′ homologous regions and the 3′ homologous regions (dashed lines), resulting in the incorporation of the inactive knockout
vector, including pgk-neo but not pgk-tk, into the cellular genomic locus of the embryonic stem cell. The presence of pgk-neo and the absence of pgk-tk in
these replaced genes will allow survival of these embryonic stem cells after positive-negative selection with neomycin and ganciclovir. The clone of mutant
embryonic stem cells is injected into a host blastocyst, which is implanted into a pseudopregnant foster mother and subsequently develops into a chimeric
offspring (bottom). The contribution of the embryonic stem cells to the germ cells of the chimeric mouse results in germline transmission of the embryonic
stem cell genome to offspring that are heterozygous for the mutated tumor suppressor allele. The heterozygotes are mated to produce mutant, cancer-prone
mice homozygous for tumor suppressor deficiency. (Modified from Mazjoub JA, Muglia LJ. Knockout mice. N Engl J Med. 1996;334:904–906.)
Founder strains Diversity Outbred (DO) formation and holds great promise for improved treatment of
A/J
human cancer.
Chemotherapy still has numerous side effects. As a number of
C57BL/6J
Outbreeding oncologists may testify, patients sometimes forgo treatment because
129S1/SvImJ of its toxic nature. In a number of cases of chronic lymphocytic
leukemia, if the symptoms of the disease are “under control,” treatment
NOD/ShiLtJ is not prescribed. Therefore it is important to find alleviation strategies
NZO/H1LtJ Collaborative Cross (CC) and cures that minimize the side effects. The goal should be to cure
Inbreeding the patient without devastating the person. One future avenue, apart
CAST/EiJ from finding treatment regimens that reduce side effects, is the discovery
PWK/PhJ of chemotherapies with minimal side effects. The nature of several
generations of chemotherapy was to attack cellular processes, such as
WSB/EiJ DNA replication, metabolism, and cell division, with brute force, and
A by trial and error find a balance between alleviating the neoplastic
growth and not interfering with the normal processes. It may be time
to rethink that premise. We now see examples wherein a less potent
chemotherapy elicits a similar effect on a cancer as a potent one,
although the less potent compound may take longer to achieve its
effect. However, because of its low potency, the side effects are minimal.
Therefore, in the future, the success of a therapy needs to be measured
B not only in terms of how quickly a patient is cancer free, but how
well the patient is during and after the treatment. With emerging
Figure 1.20 • Generation and characteristics of Collaborative Cross (CC) technologies we will be able to fine-tune current successful therapies
and Diversity Outbred (DO) mice. (A) Each CC line originates from intercrosses so that treatment is not so burdensome.
obtained from eight founder lines. Individual unique independent line is In any field of medicine, resistance to the therapy occurs. Evolution-
inbred for at least 15 generations, creating individual CC lines. Together, those ary processes show that predation leads to natural selection of species
lines represent a much broader genetic variation when compared with the
parental lines and therefore are a better representation of natural populations with mutations that avoid their elimination. Similarly, in cancers,
but contain a high degree of homozygosity. Random mating from early 144 during the predation—chemotherapy—clones arise that become
CC crosses leads to a highly genetic heterogeneous outbred population that resistant to the therapy. With emerging technologies such as single-cell
more closely resembles the diversity found in human populations. (B) Images deep sequencing, we will be able to not only detect the rare subclone
of mice representing CC lines. (From Centre for Diabetes Research, Harry that could give rise to resistance, but also predict the probability of
Perkins Institute of Medical Research, Nedlands 6009, W.A Australia” and the development of resistance by sequencing certain markers. For
“School of Medical and Health Sciences, Edith Cowan University, Joondalup instance, clones with mutations in DNA repair pathways have a higher
W.A. 6027 Australia.) probability for genomic instability, which is a precursor for the rise
of resistant clones.
While we pursue new technologies to diagnose patients and
understand the molecular nature of the cancers, an emerging trend
will be to reexamine previous formulated hypotheses or treatments
that could not be tackled before because of the lack of technologies
animal has a high degree of heterozygosity and carries a unique or resources. For instance, it has been long thought that genetic variation
combination of alleles. plays an important role in cancer treatment. This premise was observed
These diversified mice have been shown to be a powerful tool in in human clinical trials, wherein conditions in some populations were
determining the etiology of cancers. In a recent study, analysis of mice refractory to certain treatments. In the future, we would be able to
generated by crossing CC strains with a mouse line carrying a mutated predict the extent to which a treatment would either work or fail in
tumor suppressor APCmin showed that colon cancer frequency and certain population by using diverse mice, and to map alleles that
spectrum varied predictably with genetic background. Identification confer resistance or susceptibility of a tumor before reaching human
of these genetic modifiers of cancer genesis or suppression would clinical trials. Another example is processes that lead to neoplasticity,
inform the design of novel mouse cancer models, potentially yielding such as LOH, which have been studied in cell lines that are homo-
genetic markers that can predict human cancers. geneous in nature. Attempts to use primary cells were not possible
because of polyclonality of cells and the short life span of tumor cells
FUTURE VIEW in vitro.
With all the aforementioned technologies, we now can, and
Recent discoveries that cancer stem cells share essential signaling nodes need to, reexamine older hypotheses with the appropriate type of
with normal stem cells suggest that targeting critical steps in these primary cells. We will probably uncover novel processes of cancer
pathways may lead to improved alternative cancer therapies. However, that were masked, and resolve decades-old controversies and
every human tumor is subtly different. We are now fast approaching competing hypotheses, leading to better understanding and cures
a new era in medicine: creating “tailored” treatments to individual for cancer.
tumors by obtaining integrative “personal omics profiles” (Fig. 1.21). In the future, fields of medicine will continue to marry and
The generation of novel mouse strains that represent the diversity merge; this has been exemplified in numerous examples, some
seen in human populations will likely lead to a more tailored approach mentioned in this chapter—for instance, the use of viruses to deliver
in understanding cancer diversity and therefore will allow the develop- chemotherapy, or the use of engineered T cells to attack cancer. As
ment of more efficient treatments. emerging molecular tools uncover novel processes in different fields
Understanding of underlying molecular biologic principles of of medicine, the cross talk between oncology and these other fields
malignancy, with pathophysiologic consequences, will generate an will continue, enabling discovery of new avenues to alleviate or even
invaluable resource for resolution of complex genetics of tumor cure cancers.
SAMPLE TYPE METHOD ANALYSES
Whole-genome
Variant calling/phasing
sequencing
Heteroallelic and variant
expression
Whole-transcriptome
PBMC sequencing RNA editing
(mRNA and miRNA)
Quantitative differential
Integrated personal OMICS

expression & dynamics
Variant confirmation in
Proteome profiling
RNA and protein
Untargeted proteome Quantitative differential

profiling expression and dynamics
Targeted proteome
Quantitative expression
profiling (cytokines)
Serum Metabolome profiling Dynamics
AutoAntibodyome Differential reactivity

profiling
Medical/lab tests Glucose, HbA1c, CRP,

Telomere length
A
3 4
5
2
Serpina 1
E366K
RNA edits
6
Heteroallelic SNVs
1
Protein-downregulated
7
(HRV vs healthy)
Protein-upregulated
Y
(HRV vs healthy)
RNA-downregulated
8
(HRV vs healthy)
X
RNA-upregulated
(HRV vs healthy)
Indels
9
22
SV-duplications
21
SV-deletions
20
10
Chr. ideogram
19
18
11
17 14 Chr. number
16 12
B 15
14 13
Figure 1.21 • Integrative personal omics profiles (iPOP). (A) Integrative analysis of iPOP experimental design, indicating tissues and techniques analyzed
in healthy and diseased individuals. (B) Circos plot summarizing iPOP. From outer to inner rings: chromosome ideogram; genomic data (pale blue ring),
structural variants (deletions [blue tiles], duplications [red tiles]), indels (green triangles); transcriptomic data (yellow ring); proteomic data (light purple ring).
(Modified from Chen R, Mias GI, et al. Personal omics profiling reveals dynamic molecular and medical phenotypes. Cell. 2012;148:1293–1307.)
SUGGESTED READINGS
Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter Hunter K. Host genetics influence tumour metastasis. The Complex Trait Consortium. The Collaborative
P. Molecular Biology of the Cell. 4th ed. London, UK: Nat Rev Cancer. 2006;6:141–146. Cross, a community resource for the genetic analysis
Taylor and Francis Group; 2002. Malaney P, Nicosia SV, Davé V. One mouse, one patient of complex traits. Nat Genet. 2004;36:1133–1137.
Chen CZ. MicroRNAs as oncogenes and tumor suppres- paradigm: new avatars of personalized cancer therapy. Weinberg RA. Biology of Cancer. Garland Science; 2006.
sors. New Eng. J. Med. 2005;353:1768–1771. Cancer Lett. 2014;344:1–12. Rosenthal N, Brown S. The mouse ascending: per-
Feinberg AP. Phenotypic plasticity and the epigenetics of Pecorino L. Molecular Biology of Cancer: Mechanisms, spectives for human-disease models. Nat Cell Biol.
human disease. Nature. 2007;447:433–440. Targets, and Therapeutics. USA: Oxford University 2007;9:993–999.
Frese KK, Tuveson DA. Maximizing mouse cancer models. Press; 2005. Wu J, Smith LT, Plass C, Huang TH. ChIP-chip comes
Nat Rev Cancer. 2007;7:654–658. Svenson KL, Gatti DM, Valdar W, Welsh CE, Cheng R, of age for genome-wide functional analysis. Cancer
Goh KI, Cusick ME, Valle D, Childs B, Vidal M, Barabasi Chelser EJ, et al. High resolution genetic mapping Res. 2006;66:6899–6902.
AL. The human disease network. Proc Natl Acad Sci using the mouse diversity outbred population. Genetics.
USA. 2007;104:8685–8690. 2012;190:437–447.

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A.

BIOLOGY AND CANCER

Genes Cell nucleus

Exon Exon Exon Exon Exon

Translation Translation Translation

Protein A Protein B Protein C

Primary tumor Metastasis SNP density

Transcription Gene expression

Translation into protein

A Normal B Epigenetic lesions

PROFILING TUMORS underlying tumor progression by following the changes in a tumor

2x poly (A) RPKM RPKM

Liver 0.0 0.0

25-bpm Uniquely mappable

Protein-coding gene MicroRNA gene

mRNA degradation Translational repression

A Promoter Reporter gene

Activator region fused to

B Promoter Reporter gene

APC Helper T cell Cytotoxic T cell

Antigen BiTE MHC TCR T cell

Analysis and visualization Peak calling Alignment

Binding site identification Array data analysis Genomic arrays

Human Drosophila Yeast C. elegans Arabidopsis

Transcriptional Virus-host Metabolic Protein-protein Disease

Figure 1.17 • Generation of transgenic mice. The transgene containing

Cell type specific

Transfer of early embryos into reproductive

A Special cell type All other cells

Embryonic stem cell

Tumor suppressor gene

5' Homologous 3' Homologous

Injection of embryonic stem Implantation of chimeric

Germline offspring Chimeric offspring

SAMPLE TYPE METHOD ANALYSES

Integrated personal OMICS

Untargeted proteome Quantitative differential

Serum Metabolome profiling Dynamics

AutoAntibodyome Differential reactivity

Medical/lab tests Glucose, HbA1c, CRP,

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