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`Different Facets of Copy Number Changes: Permanent, Transient, and
`Adaptive
`
`Sweta Mishra, Johnathan R. Whetstine
`Massachusetts General Hospital Cancer Center and Department of Medicine, Harvard Medical School, Charlestown, Massachusetts, USA
`
`Chromosomal copy number changes are frequently associated with harmful consequences and are thought of as an underlying
`mechanism for the development of diseases. However, changes in copy number are observed during development and occur dur-
`ing normal biological processes. In this review, we highlight the causes and consequences of copy number changes in normal
`physiologic processes as well as cover their associations with cancer and acquired drug resistance. We discuss the permanent and
`transient nature of copy number gains and relate these observations to a new mechanism driving transient site-specific copy
`gains (TSSGs). Finally, we discuss implications of TSSGs in generating intratumoral heterogeneity and tumor evolution and how
`TSSGs can influence the therapeutic response in cancer.
`
`It was long thought that the DNA sequences of healthy individ-
`
`uals were 99.9% identical to each other (1). However, genome-
`wide sequencing efforts in individuals from multiple ethnicities
`have revealed more variations in the genetic architecture than
`were previously appreciated (2–4).
`These genomic alterations have been termed structural vari-
`ants, which are further classified as microscopic or submicrosco-
`pic, depending on the amount of DNA involved (5). The micro-
`scopic variations have historically been identified through
`chromosome banding techniques (6) and comprise at least 500 kb
`of DNA (7). Examples of these variants are whole-chromosome
`gain or loss (referred to as aneuploidy [7, 8]), translocation
`(change in location of a chromosomal segment [9]), deletion (de-
`letion of a DNA segment relative to the rest of the chromosome
`[10]), duplication (a chromosomal segment occurs in two or
`more copies per haploid genome [11]), and inversion (reversal in
`orientation of a DNA segment compared to the rest of the chro-
`mosome [12, 13]). A schematic of structural variants resulting in
`copy number changes is shown in Fig. 1. With the development
`of more sophisticated tools, such as array-based comparative
`genomic hybridization (GGH) arrays (14–16), smaller variants
`(submicroscopic alterations) in the size range of 1 to 500 kb can be
`detected (5). Genome sequencing has further revealed small inser-
`tions and deletions (indels) spanning from 1 to 10,000 bp across
`the human genome which could cause considerable variability in
`the human population (17, 18).
`The most common variant identified under submicroscopic
`alterations is copy number variation (CNV). CNV is defined as a
`genomic segment of more than 1 kb present at a variable copy
`number in comparison to a reference genome (19–22). The first
`studies documenting the genome-wide presence of CNVs in the
`normal human genome came from work in the laboratories of Lee
`(23) and Wigler (24). These studies described more than 200
`large-scale CNVs (LCVs; about 100 kb or greater) in normal indi-
`viduals. These studies also paved the way for the creation of the
`Database of Genomic Variants (DGV) in 2004, which catalogs all
`the human CNVs and structural variations present in healthy in-
`dividuals.
`The sequencing efforts from the International HapMap Con-
`sortium (25) and 1000 Genomes Project (26) have led to the iden-
`tification and frequency determinations of novel CNVs in the hu-
`
`man genome. CNVs are now known to contribute to 4.8% to 9.5%
`of the variability in the human genome (27, 28), which is more
`than what is accounted for by single nucleotide polymorphisms
`(SNPs; accounting for 0.1% of the variations) (29). Recently, the
`CNV map for the human genome was constructed (28), and it
`documented all the small- and large-scale CNVs present in nor-
`mal healthy individuals. CNVs can either have no phenotypic con-
`sequences in individuals (4, 23, 24) or lead to adaptive benefits
`that have been observed in a wide range of species (5).
`One of the major challenges in the field is to distinguish benign
`CNVs (events that do not lead to phenotypic consequences) from
`pathogenic CNVs that underlie diseases (30). Pathogenic CNVs
`are often associated with deleterious consequences because of an
`imbalance in gene dosage (31) and/or aberrant chromosomal
`structure (5, 7, 32, 33). Pathogenic CNVs have been associated
`with several disorders, including the following: obesity (34), dia-
`betes (35), developmental disorders (36), psychiatric diseases (37)
`such as autism spectrum disorder (38), schizophrenia (39), and
`Alzheimer’s disease (40, 41), and cancer (42–44). In this review,
`we focus mainly on copy number alterations observed in cancer
`and their functional implications.
`CNVs can either be present in the germ line or can arise in
`phenotypically normal tissues and organs, which are referred to as
`somatic CNVs (45, 46). Instead of being randomly present in the
`genome, CNVs are preferentially found to occur in regions that
`are rich in low-copy-number repeats (segmental duplications)
`(47–50), heterochromatic areas (e.g., telomeres and centro-
`meres), and replication origins and palindromic regions (28).
`There are several proposed mechanisms that underlie the genera-
`tion of somatic CNVs: nonallelic homologous recombination
`(NAHR), nonhomologous end joining (NHEJ), defects in DNA
`
`Accepted manuscript posted online 11 January 2016
`Citation Mishra S, Whetstine JR. 2016. Different facets of copy number changes:
`permanent, transient, and adaptive. Mol Cell Biol 36:1050 –1063.
`doi:10.1128/MCB.00652-15.
`Address correspondence to Johnathan R. Whetstine,
`jwhetstine@hms.harvard.edu.
`Copyright © 2016 Mishra and Whetstine This is an open-access article distributed
`under the terms of the Creative Commons Attribution 4.0 International license.
`
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`Minireview
`
`development provide an advantage during selective pressures and
`environmental conditions (7). Here, we discuss examples from
`developmental biology and their relationships to functional im-
`pact. We also highlight the relationship between somatic CNVs
`and tissue homeostasis.
`Several lower and higher eukaryotes use gene amplification to
`respond to cellular signals (Fig. 2). Electron microscopy studies in
`the early 1970s demonstrated that ribosomal genes are amplified
`for the production of large amounts of ribosomes required during
`early embryogenesis (52). Ribosomal DNA (rDNA) amplifica-
`tions were observed during oocyte formation in amphibians such
`as Xenopus leavis (53–55), insects such as water beetles (56), mol-
`luscs (55), and in the macronuclear rDNA of Paramecium (57)
`and Tetrahymena (58). Thus, such an increase in rDNA synthesis
`to meet higher protein synthesis demands in different tissues
`highlights gene amplification as a common principle in develop-
`mental biology.
`Besides rDNA, specific chromosomal regions identified as
`“DNA puffs” are amplified and expressed to form structural pro-
`teins required for cocoon formation in the salivary gland of sciarid
`flies (59, 60). Amplification of the DNA puffs occurs in response
`to the hormone ecdysone, which is required during larval devel-
`opment (60). Another example of gene amplification triggered by
`developmental signals can be observed during eggshell formation
`in Drosophila melanogaster (61). Eggshells require amplification of
`chorion genes in the follicle cells of the ovary, and these genes are
`expressed late in differentiation (61, 62). The amplifications of
`only specific chromosomal regions and genes and not the whole
`genome highlight the specific response that can occur across or-
`ganisms. These examples suggest the ability of cellular cues to
`trigger these site-specific amplifications, which raises the question
`about what molecular mechanisms underpin this selective ampli-
`fication across species.
`Examples of copy number variations have been reported in
`various tissues in mammals. Using techniques such as spectral
`karyotyping (SKY), fluorescence in situ hybdridization (FISH),
`and single-cell sequencing approaches, various groups have re-
`ported both small- and large-scale changes in chromosomal copy
`numbers in mouse and human tissues, particularly in neurons,
`liver cells, and skin fibroblasts (Fig. 2). For example, approxi-
`mately 33% of the neuroblasts in the embryonic mouse brain and
`20% of neurons in the adult mouse cerebral cortex showed aneu-
`ploidy (63). The reduction in aneuploidy in the adult brain was
`hypothesized to be due to a neuroblast programmed cell death
`mechanism during brain development (64). Westra and col-
`leagues also uncovered that 15 to 20% of neural progenitor cells
`in both mouse and human cerebella exhibited aneuploidy (65)
`(Fig. 2).
`Additionally, high levels of subchromosomal CNVs (deletion
`and duplication events) were observed in the human frontal cor-
`tex neurons. Multiple copy number changes were noted within a
`small set of neurons, suggesting that CNVs might be restricted to
`either individual cells or specific neural lineages (66). These data
`suggest that the generation of copy number changes is an impor-
`tant process for achieving diversity in the neuronal populations
`during central nervous system development. However, this possi-
`bility has yet to be proven. It was reported that the transcripts
`arising from CNVs in the mouse brain are more tightly regulated
`than are other tissues such as lung, liver, heart, kidney, and testis
`(67). It would be important to determine the rate of correlation
`
`FIG 1 Types of copy number changes. (A) Representative examples of struc-
`tural chromosomal alterations are shown, with a new sequence insertion (D),
`deletion of region AB, and duplication of sequence B (ABB). The reference
`chromosome is shown at the top. (B) Aneuploidy with whole chromosome
`gain (the extra black chromosome) and loss (of black chromosome) are de-
`picted with respect to a normal mitotic reference nucleus. (C) A part of a
`chromosome (black) can be amplified or deleted (black), giving rise to seg-
`mental aneuploidy. This is demonstrated here as involving rearrangement of
`only one chromosome. A more likely scenario is an unbalanced translocation,
`which is not shown in the figure. (D) Homogenously staining regions (HSR)
`and double minutes (DMs) are chromosomal structures that are generated as
`a consequence of gene amplification. HSRs are repeated units clustered at a
`single chromosomal locus (red), and DMs are unstable circular extrachromo-
`somal DNA structures lacking a centromere or a telomere. In addition to these
`structures, amplicons can be present at a number of loci in the genome (not
`shown).
`
`replication, and DNA damage response and repair pathways.
`These mechanisms have been extensively discussed elsewhere;
`therefore, we refer our readers to several reviews (32, 33, 51).
`In this review, we explore the relationship between copy num-
`ber changes and biological consequences, with a particular focus
`on development and tissue homeostasis under physiological as
`well as pathological conditions. This review focuses on these
`relationships, especially in the context of cancer. We further
`discuss a recently discovered process driving transient site-spe-
`cific copy number gains (TSSGs) in cancer cells and its impli-
`cations during adaptive responses such as stress and chemo-
`therapeutic sensitivity.
`
`COPY NUMBER CHANGES IN DEVELOPMENT AND
`PHYSIOLOGY
`Chromosomal copy number changes and the associated gene am-
`plifications and losses are observed during development in both
`lower and higher eukaryotes [reviewed in reference 7]. The
`appearance of CNVs during normal biology suggests that copy
`number changes can have important functional consequences.
`A common hypothesis is that increased gene dosages during
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`FIG 2 Copy number changes during normal development and physiological conditions. Representative copy number changes are shown for organisms and
`specific tissues under different developmental and physiological conditions. Please refer to text for detailed descriptions and corresponding references.
`
`between CNVs and expression changes in the human brain and
`whether there are underlying functional consequences of the af-
`fected transcripts in generating neural diversity and plasticity.
`Somatic CNVs are also observed in mammalian hepatocytes
`and skin. A study by Duncan and colleagues suggested that ap-
`proximately 50% of normal adult hepatocytes have changes in
`chromosomal numbers (gains or losses) such that genetically di-
`verse sets of cells are present in the liver (68, 69). However, single-
`cell next-generation sequencing has reported a lower level of
`aneuploidy (⬍5%) in cells of liver, skin, and human neurons (70).
`The differences in the reported levels of aneuploidies could reflect
`the different types of assays employed to follow copy number
`changes (i.e., FISH and SKY versus single-cell sequencing, respec-
`tively).
`The genetic variation resulting from the changes in copy num-
`ber could be a mechanism employed during tissue development in
`order to achieve diversity in cell populations. Copy number vari-
`ations may allow developing tissues to adapt to cellular and
`growth requirements during tissue expansion and organ develop-
`ment. Another advantage for the observed CNVs could be to
`adapt to encountered metabolic or toxic challenges, especially by
`hepatocytes (see the discussion in “Mammals,” below). By identi-
`fying the regulatory features for regions undergoing CNV and the
`
`affected genes in different tissues, we would be able to understand
`tissue-specific gene expression and underlying diversity within tis-
`sues.
`
`COPY NUMBER CHANGES AS AN ADAPTIVE RESPONSE
`Many studies in bacteria, yeast, and mammals have shown that
`copy number changes can arise as a consequence of selection,
`which may allow cells to exhibit an increased fitness and/or sur-
`vival advantage. In this section, we discuss the relationship be-
`tween different cellular conditions and the emergence of CNVs
`from different species (Fig. 2).
`Bacteria. Acquisition of antibiotic resistance can occur through
`the uptake of foreign DNA harboring resistance genes through the
`bacterial competence pathway (71). A recent study by Slager et al.
`demonstrated that different species of bacteria could increase the
`copy number of genes involved in the competence pathway (com
`genes) in response to antibiotics causing replication stress (72).
`These genes are located closer to the origin of replication (OriC),
`and their amplification occurs through multiple origin firing
`events at the OriC, which increases their copy number and tran-
`scription rates. In Salmonella enterica serovar Typhimurium, gene
`amplification aids in the development of antibiotic resistance. Ad-
`aptation to the antibiotic cephalosporin occurred through ampli-
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`fication and increased gene dosage/expression of the ␤-lactamase
`gene (blaTEM-1 [73]). The enzyme ␤-lactamase results in the hy-
`drolysis of cephalosporin (74, 75), which results in a reduced drug
`response.
`These highlighted examples illustrate the impact selective pres-
`sure can have on DNA amplification and gene expression in bac-
`teria (Fig. 2). Additional examples have been observed and are
`discussed in a review by Sandegren et al. (76). Taken together, the
`existing data illustrate the relationship between input signals and
`changes at distinct regions of the bacterial genome. In the future, it
`will be interesting to know if this selection is based on fitness or the
`result of targeted DNA replication in prokaryotes.
`Yeast. Similar to bacteria, yeasts also exhibit changes in DNA
`content based on selective pressure. For example, gene rearrange-
`ments and copy number changes have been observed in Candida
`albicans when it is passaged through a murine host (77). It has
`been hypothesized that these changes in ploidy could generate the
`genetic and phenotypic diversity required for adaptation in the
`new host environment. Consistent with these observations, CNV
`has been associated with antifungal drug resistance and adaptive
`benefits (78, 79). For example, fluconazole treatment for C. albi-
`cans infection results in the development of whole-chromosome
`gains and aneuploidy (80). Upon CGH analyses for the copy num-
`ber changes in 70 azole-resistant and -sensitive strains, Selmecki et
`al. found increased levels of aneuploidy in resistant strains (50%)
`compared to the sensitive ones (7.14%) (81, 82). Trisomies of
`chromosome 5, including a segmental aneuploidy consisting of an
`isochromosome (formed by the attachment of two left arms of
`chromosome 5 around a single centromere), were also associated
`with azole resistance. Gains of this isochromosome were associ-
`ated with increased expression of genes involved in drug resistance
`(82). Some of these genes encoded efflux pump proteins involved
`in resistance: an ATP-binding cassette (ABC) transporter and a
`multidrug resistance transporter (83). Other genes were ERG11 (a
`target of fluconazole [84]) and TAC1 (a transcription factor that
`upregulates ABC gene expression [82]). There is a need to identify
`other structural variations and affected genes conferring a surviv-
`al/adaptive advantage against antibiotics and whether these
`changes are conserved across other fungal species.
`Consistent with gene amplification conferring a selective ad-
`vantage, Saccharomyces cerevisiae cells exposed to nutrient depri-
`vation exhibited gene amplifications that provided a cellular ben-
`efit (85). For example, glucose limitation in cultures resulted in
`the amplification of genes encoding glucose transporters (HXT6
`and HXT7), while sulfate limitation resulted in the amplification
`of SUL1, a gene that encodes a high-affinity sulfate transporter
`(Fig. 2). The question remains as to whether these physiological
`input signals are able to drive selective DNA gains through a hard-
`wired mechanism, as observed in mammalian cells (discussed in
`“TSSGs, Tumor Heterogeneity, and Cancer Evolution,” below),
`or are the result of random selection. Resolution of this issue could
`have a profound impact on our understanding of cellular fitness
`and responses to antibiotics.
`Mammals. Mammals are no exception to selective pressures
`promoting copy number changes or copy number alterations that
`impact biological consequences. For example, the copy number of
`the human salivary amylase gene AMY1, which encodes an en-
`zyme that aids in the hydrolysis of starch, is increased in popula-
`tions that have a higher starch content in their diets compared to
`low-starch-consuming populations (86). The increased copy
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`number of AMY1 also correlated with increased salivary amylase
`protein levels. This illustrates how diet-induced selective pres-
`sures could influence copy number polymorphisms in mammals.
`Other examples and the role of copy number polymorphisms in
`human adaptation have been reviewed elsewhere (33, 87, 88).
`While these studies are correlative and suggest that the environ-
`ment impacts selection, they have yet to be shown to be causal.
`Increased or decreased copy numbers of certain genes can pre-
`dispose an individual to diseases. For example, susceptibility of
`individuals to HIV/AIDS infection is increased in populations
`with a decreased copy number of the chemokine gene CCL3L1.
`This chemokine serves as a ligand for HIV coreceptor CCR5,
`which inhibits viral entry by binding to CCR5. However, HIV-
`resistant individuals show duplications of the CCL3L1 locus
`(17q21.1) and increased CCL3L1 copies imparting resistance to
`HIV infection (89). Other examples of CNVs promoting suscep-
`tibility to diseases can be found with psoriasis (associated with a
`copy number gain of the ␤-defensin gene [90, 91]), pancreatitis (a
`copy number gain of PRSS1 [92]), and Crohn’s disease (a copy
`number loss of HBD-2 [93]), among others (20, 94). The question
`remains as to whether there are mechanisms that would allow
`such changes to occur immediately in response to stimuli in the
`population or whether they reflect some mutation that was se-
`lected over time.
`Somatic mosaicism for CNVs within tissues can provide an
`adaptive response as well. CNVs within the liver can provide pro-
`tection against tissue injury. Duncan et al. demonstrated in a
`chronic liver injury model that selective gene loss could provide
`resistance to liver injury (95). Deficiency of fumaryl acetoacetate
`hydrolase (encoded by FAH; the enzyme is required in tyrosine
`catabolism) causes a buildup of fatty acids and toxic metabolites
`that result in liver failure, known as tyrosinemia. Conversely, de-
`letion of the genes encoding enzymes that function upstream of
`FAH (e.g., homogentisic acid dioxygenase [HGD]) is found to be
`protective for tyrosinemia. Mice deficient for FAH and heterozy-
`gous for a mutation in HGD can generate healthy normal hepato-
`cytes. These injury-resistant, aneuploid hepatocytes (character-
`ized by the loss of chromosome 16) are present in the liver and
`undergo expansion only when the liver is exposed to injury, dem-
`onstrating an adaptive response of cells to metabolic or toxic chal-
`lenges.
`Taken together, these few examples illustrate the CNVs present
`within populations and individual tissues and how these are asso-
`ciated with phenotypes. These data also emphasize the variations
`in the genome and how the environment and selective pressures
`can impact genetics. However, the question remains as to whether
`these genetic events occur after random selection or are the result
`of unidentified mechanisms that selectively alter the genetic land-
`scape in response to external stimuli and, in turn, drive targeted de
`novo genetic changes.
`
`COPY NUMBER ALTERATIONS IN CANCER AND THEIR
`IMPLICATIONS IN ACQUIRED DRUG RESISTANCE
`Copy number alterations involving whole chromosomes and/or
`specific chromosomal segments are frequently observed in cancer
`(96, 97). Gains/amplifications of oncogenes and loss/deletion of
`tumor suppressor genes have been historically found to be major
`drivers of tumor development. For example, amplifications of
`EGFR in gliomas (98), MYCN in neuroblastoma (99), MYC in
`acute myeloid leukemia (100), and ERBB2 in breast (101), ovarian
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`TABLE 1 Partial list of amplified genes that impact drug resistancea
`
`Cancer type
`
`Therapeutic agent(s)
`
`Gene(s) implicated in
`resistance (reference[s])
`
`Multiple myeloma
`
`Bortezomib, cisplatin
`Melphalan, cisplatin, vincristine
`Dexamethasone
`
`CKS1B (111, 121, 126)
`PDZK1 (115)
`FGFR3 (127)
`
`Cisplatin, CDK2 inhibitors
`Paclitaxel
`
`CCNE1 (128, 142)
`MDR1 (129, 130)
`
`Ovarian
`
`Lung
`
`Gefitinib
`Paclitaxel
`Crizotinib
`
`Breast
`
`Trastuzumab
`
`MET (123, 125)
`MDR1 (129, 130, 131)
`ALK, KIT (132)
`
`MET (133), IQGAP1
`(134)
`
`Minireview
`
`(102), and lung cancers (103) have been reported. Similarly, loss/
`deletions in tumor suppressor genes such as PTEN (104), TP53
`(105), and VHL (106) have been observed in a variety of tumors.
`The dependence of tumors on specific oncogenes for their prolif-
`eration and survival is referred to as oncogene addiction (107). By
`targeting these oncogenes, tumor cell growth becomes limiting or
`abrogated. For example, clinical success has been observed with
`the ERBB2 antibody trastuzumab (Herceptin) in the treatment of
`ERBB2-amplified breast cancer (108), crizotinib in the treatment
`of MET-amplified non-small cell lung cancer (109), and the epi-
`dermal growth factor receptor (EGFR) inhibitors gefitinib and
`erlotinib (which blocks the catalytic activity of EGFR) in lung
`cancer patients with EGFR mutations (110).
`In addition to oncogene amplifications, copy number altera-
`tions of different chromosomal regions have been observed in
`cancer. A genome-wide analysis of copy number alterations in
`cancer demonstrated a total of 76,000 gains and 55,000 losses
`across the 3,131 cancer samples analyzed (96). A typical tumor
`type is comprised of 17% amplifications and 16% deletions, com-
`pared to less than 0.5% in normal samples (96). These data suggest
`that somatic copy number alterations are a frequent feature in
`cancer cells. Analyses across 17 tumor types demonstrated that
`25% of the genome is affected by whole chromosome alterations
`and 10% of the genome by short chromosomal changes (focal
`events) in a typical tumor (96). Interestingly, the focally amplified
`regions often harbor known oncogenes (e.g., MYC, CCND1,
`EGFR, NKX2-1, and KRAS), while the focally deleted genomic loci
`contain tumor suppressor genes (TP53, CDKN2A/B, and Rb1).
`These observations suggest that the selective pressures associated
`with tumorigenesis might influence targeted amplification or de-
`letion of specific regions within tumor cells instead of occurring
`randomly, which would be reminiscent of the observations seen in
`bacteria and yeasts (Fig. 2).
`Focal amplifications can also harbor oncogenes or prosurvival
`genes that can influence drug responses. For example, ⬃10% of
`cancers have a focal amplification of chromosome 1q21.2 that
`contains the antiapoptotic gene MCL1 (96). Another focally am-
`plified antiapoptotic gene that is observed in cancer is BCL2L1 on
`chromosome 20q11.21 (96). Both of these genes are important for
`cell survival; hence, their amplification within tumors could con-
`fer a distinct survival advantage. Consistent with this notion, Ber-
`oukhim et al. demonstrated that increased expression of these
`genes protected tumor cells from chemotherapy (96).
`Chromosomal alterations in several distinct regions also influ-
`ence pathogenesis in different tumor types. For example, in mul-
`tiple myeloma (MM), disease progression is characterized partly
`by the focal amplifications of a proximal region of chromosome
`1q (chr 1q). Several studies have identified a region of 10 to 15 Mb
`that corresponds to a chr 1q12-23 amplicon in MM. This region
`contains a large number of genes with amplifications or deregu-
`lated expression involved in myeloma pathogenesis, including
`CKS1B (111, 112), MUC1 (113), MCL1 (114), PDZK1 (115),
`IL-6R (116), BCL9 (117), and UBE2Q1 (118). The amplification of
`a drug-resistant oncogene, CKS1B, and the proximal chr 1q21
`region has been reported in ⬃40% of newly diagnosed MM cases
`and in 70% of patients with tumor relapse (119, 120). The gains
`observed in CKS1B are in the range of one to three copies (111,
`112). These focal amplifications are associated with poor progno-
`sis and reduced response to cisplatin therapy (111) (Table 1).
`Studies in cell cultures have further demonstrated that overex-
`
`Colorectal
`
`Gefitinib
`5-Fluorouracil
`
`MET (124)
`TMYS (135)
`
`CML
`Melanoma
`
`Imatinib
`Vemurafinib
`
`BCR-ABL (136)
`BRAF (137, 138),
`BCL2A1 (139)
`DHFR (140, 141)
`Leukemia
`Methotrexate
`a We apologize for not being able to cite or include all studies related to gene
`amplification and drug resistance.
`
`pression of CKS1B confers a reduced response to cancer chemo-
`therapeutics (121). Similarly, amplification of the PDZK1 gene
`within the chr 1q12-q22 region has been observed in primary cases
`of MM, and the overexpression of PDZK1 in cells conferred resis-
`tance to melphalan-, vincristine-, and cisplatin-induced cell
`deaths (115) (Table 1).
`Gene amplifications are associated with drug resistance in sev-
`eral tumors (122–141) (Table 1). For example, ovarian cancer
`patients with a chr 1q12-21 amplification are more resistant to
`cisplatin treatment (142, 143). Amplifications of cyclin E1
`(CCNE1) are present in 25% of high-grade serous ovarian cancers
`and are associated with poor survival and impart resistance to
`CDK2 inhibitors (144) (Table 1). In the case of non-small cell lung
`cancer cells, an 11- to 13-fold-higher copy number of chr 7q21.12
`was detected by CGH in an acquired paclitaxel-resistant lung can-
`cer model (study NCI-H460/PTX250) compared with the paren-
`tal cell line (study NCI-H460). Most of the genes within this re-
`gion were also highly expressed, including a multidrug transporter
`gene, MDR1/ABCB1 (131). These examples highlight how distinct
`regions in the genome are focally amplified and relate to altered
`patient outcome and cancer cell drug responses. Whether selective
`chromosomal alterations and gene amplifications in cells result
`from a stochastic process or occur in a directed manner in conse-
`quence to therapeutic pressure is yet to be determined.
`
`DNA AMPLIFICATION AND CANCER CHEMOTHERAPEUTIC
`RESISTANCE
`Gene amplification serves as a biochemical basis for drug resis-
`tance in mammalian cells. This relationship to resistance was first
`documented in seminal work by Hakala (145–147) and Fischer
`(148) in the 1950s. They isolated highly resistant tumor cells under
`the presence of increasing concentrations of the drug methotrex-
`ate (MTX). MTX competitively inhibits the enzyme dihydrofolate
`reductase (DHFR), which catalyzes the conversion of dihydrofo-
`late to active tetrahydrofolate, which is required for the de novo
`
`1054 mcb.asm.org
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`Molecular and Cellular Biology
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`April 2016 Volume 36 Number 7
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`FOUNDATION EXHIBIT 1048
`IPR2019-00634
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`Page 1054
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`amplified DHFR genes were associated with DMs in unstable
`MTX-resistant cells (158).
`A large body of work has contributed to our understanding of
`the generation of DMs and HSRs (159–162). For example, Stor-
`lazzi et al. investigated the structures of MYCN amplifications by
`using eight neuroblastoma and two small cell carcinoma cell lines
`(162). The study provided evidence of generation of HSRs from
`DMs by an episome model wherein DNA segments were excised
`from a chromosome and then circularized and amplified to form
`DMs and chromosomally integrated to form HSRs. DMs are un-
`stable and can be eliminated after drug treatment (163, 164); how-
`ever, HSRs are more stable (165) (Fig. 1D and 3A). Amplified
`genes present on extrachromosomal DNA have been frequently
`observed in different tumor types (159, 166–168). The reversion
`of a malignant phenotype and cellular differentiation by the elim-
`ination of DMs has been shown extensively in a variety of tumors
`and cancer cell lines (167, 169, 170). Taken together, these obser-
`vations demonstrate that transient gene amplifications can be an
`effective strategy for quick adaptation to selective pressures in
`tumor cells (Fig. 3A).
`In a recent study by Nathanson et al., another example of drug-
`induced transient gene selection was demonstrated (Fig. 3B). In
`that study, oncogenes maintained on extrachromosomal DNA
`were transiently gained/lost in response to drug treatment (171).
`Glioblastoma patients harbor a constitutively active oncogenic
`variant of epidermal growth factor receptor (EGFR-vIII) that is
`formed by the in-frame deletion of exons 2 to 7 in the EGFR gene
`and found on extrachromosomal DNA (171, 172). The presence
`of EGFR-vIII makes tumor cells more sensitive to EGFR tyrosine
`kinase inhibitors (TKIs) (173). The continued treatment with
`EGFR TKIs (e.g., erlotinib) resulted in a loss of extrachromosomal
`EGFR-vIII, thus conferring resistance to the TKI. When the drug
`was withdrawn for a short period of time, there was an increase in
`EGFR-vIII on extrachromosomal DNA and, in turn, the cells were
`resensitized to erlotinib treatment (Fig. 3B). These data reiterate
`the reversibility of copy number gains and how transient copy
`number changes could impact chemotherapeutic response.
`Furthermore, Nathanson and colleagues have suggested that
`instead of a continuous therapeutic regimen, a drug holiday dur-
`ing therapy might be a more effective mechanism to restore the
`sensitivity of tumor cells to drugs (171). These studies raise the
`possibility that chemotherapy could result in the selection of cells
`with gene amplifications, which allow them to survive under this
`drug-induced stress (Fig. 3). Therefore, understanding the mech-
`anisms that result in transient or nonpermanent amplifications of
`DHFR, EGFR, and alike in cancer (Table 1) will have a profound
`impact on how we view copy number control as well as how we
`identify novel biomarker