Carlos Albizu University Medicinal Chemistry Questions

You can refer to additional scientific literature to come up with possible answers.
However, provided relevant manuscripts should be enough to help you answer these
questions. You may include scheme(s) and/or figure(s) to illustrate a particular point.
Please follow the recommended word limits (including the figure captions). Note that for
the purposes of any activity in this class, you are prohibited from posting these
questions and accessing the information provided by contract cheating sites
Question 1. Acyclovir is available as a topical, oral, or IV medication. Oral acyclovir, however,
has poor bioavailability (~15% to 30%), which necessitates high doses and frequent
administration. The development of a prodrug, valacyclovir or valaciclovir, has overcome this
limitation, with >50% of the dose being absorbed in adults and achievable plasma levels that are
comparable to the IV administration of acyclovir. Using the accompanying manuscript as a guide
(Fig. 1, MacDougall and Guglielmo, J. Antimicrob. Chemother., 2004), summarize the
pharmacokinetics of valacyclovir. (400 words max; 10 points)
Question 2. Taxol is one of the most widely used anticancer agents in the world. The antitumor
activity of Taxol results from its ability to promote the assembly and stabilization of microtubules.
A better understanding of these interactions is essential for rational drug design and the
development of novel and potent chemotherapeutic agents. Using the accompanying
manuscript as a guide, Alushin et al., Cell 2018; Figs. 5 and 7, explain the proposed mode of
action of Taxol. (400 words max; 10 points)
Question 3. Cisplatin is one of the most effective broad-spectrum anticancer drugs. However,
the resistance against cisplatin develops rapidly, resulting in relapse and therapeutic failure.
Summarize the mechanisms with which cancer cells can build (pre-target) resistance against
cisplatin (Fig. 1 and Table 1 of the attached paper, Chen and Chang, Int. J. Mol. Sci., 2019).
Note: I am looking for a general summary and not precise details of each mechanism. (400
words max; 10 points)
Question 4. In the class, we discussed HIV-1 capsid, a metastable megadalton assembly that
protects the viral genome and is responsible for the spread of viral infection. To prevent
retroviral infections, however, their mammalian hosts express a variety of proteins, termed
host-restriction factors. One of the most well-known restriction factors in this evolutionary tug of
war against retroviruses is a protein called TRIM5α. Use the accompanying manuscript as a
guide, Ganser- Pornillos and Pornillos, Nature Reviews Microbiology 2019 (Figs. 3 and 4), to
summarize the mechanism with which this protein attempts to nullify retroviral infection. Also
comment on why this protein is not that successful in preventing the spread of HIV-1; page 553
of this manuscript. (400 words max; 10 points)
Question 5. A process known as “antigenic drift” is responsible for blunting the effect of
vaccines against influenza. In addition, influenza A, the only known strain to cause pandemics,
employs a process called “antigenic shift”. Using the accompanying manuscript as a guide, Kim
et al., Viral Immunology, 2018 (pages 175–176), briefly explain these two processes and
summarize the strategies that are used by influenza virus to evade host-immune response. (400
words max; 10 points)
Considerations for Future
Vaccine Development
VIRAL IMMUNOLOGY
Volume 31, Number 2, 2018
ª Mary Ann Liebert, Inc.
Pp. 174–183
DOI: 10.1089/vim.2017.0141
Influenza Virus:
Dealing with a Drifting and Shifting Pathogen
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Hyunsuh Kim, Robert G. Webster, and Richard J. Webby
Abstract
Numerous modern technological and scientific advances have changed the vaccine industry. However, nearly
70 years of influenza vaccine usage have passed without substantial changes in the underlying principles of the
vaccine. The challenge of vaccinating against influenza lies in the constantly changing nature of the virus itself.
Influenza viruses undergo antigenic evolution through antigenic drift and shift in their surface glycoproteins.
This has forced frequent updates of vaccine antigens to ensure that the somewhat narrowly focused vaccineinduced immune responses defend against circulating strains. Few vaccine production systems have been
developed that can entertain such constant changes. Although influenza virus infection induces long-lived
immunologic memory to the same or similar strains, most people do not encounter the same strain repeatedly in
their lifespan, suggesting that enhancement of natural immunity is required to improve influenza vaccines. It is
clear that transformative change of influenza vaccines requires a rethink of how we immunize. In this study, we
review the problems associated with the changing nature of the virus, and highlight some of the approaches
being employed to improve influenza vaccines.
Keywords: influenza virus, antigenic drift, antigenic shift, vaccine
Introduction
I
nfluenza virus produces a conundrum when it comes
to successful vaccination. From a purely immunologic
standpoint, it is clear what form of immunity has the most
protective ability, which is antibody to the globular head of
the viral surface protein hemagglutinin (HA). While other
forms of immunity, both humoral and cellular, have been
shown to be protective, the HA head-specific antibodies interfere with the attachment of viruses to their cellular surface
receptors and are solely capable of neutralizing infection
(11,16,20–22,45,51,91,92,102).
Current influenza vaccine production is based on this
understanding, and vaccines are routinely designed to elicit
protective neutralizing antibody responses. The flipside of
this conundrum is that the HA globular head is the most
variable part of the virus. As such, the virus can evolve to
evade immunity generated by prior vaccination, and the
targeting of HA using conventional approaches provides
little hope for substantive improvements in how we vaccinate. Unfortunately, this flipside has proved to be a formidable obstacle to overcome. The inactivated influenza
vaccines that are the mainstay of our current influenza
vaccination programs generate a relatively narrow immune
response that is short-lived. Instead of driving different
vaccination approaches, the obstacle of antigen diversity
has historically driven the development of a truly remarkable system of global virus tracking and annual vaccine
reformulations to ensure vaccine/virus matching.
Both influenza A and B viruses, two genera of the Orthomyxoviridae family, are causes of substantial morbidity
and mortality in humans and are targets for our current influenza vaccines. Two subtypes, classified based on the antigenic properties of their surface glycoproteins HA and
neuraminidase (NA), of influenza A viruses currently circulate in humans, A(H1N1) and A(H3N2). While influenza B
viruses are not further categorized into subtypes, two genetically and antigenically distinct lineages of virus, B/Victoria/
2/87-like and B/Yamagata/16/88-like viruses, are also found
in humans (81). The World Health Organization (WHO)-led
tracking system traces the evolution of these viruses through
epidemic seasons and selects the most appropriate virus antigens to be included in the seasonal influenza vaccine.
Concomitant with the development of the WHO tracking
system has been the development of a regulatory framework
that allows for antigen changes within the context of an
approved vaccine process. So instead of overcoming the
problem of antigenic change of the target virus by clever
antigen and/or vaccine design, our most successful approach
to date has been to develop a system that can rapidly
Department of Infectious Diseases, St. Jude Children’s Research Hospital, Memphis, Tennessee.
174
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INFLUENZA VIRUS: DRIFTING AND SHIFTING
respond to the virus change itself. It is, however, clear that
this approach, while remarkable and a poster child for a
global surveillance program, is not optimal and the search
for better target antigen(s) and/or ways to deliver them is a
priority. Fueled, in part, by improvements in analysis of
single B cells and the spread of programs to estimate seasonal vaccine effectiveness, there has been a resurgence in
the drive to improve influenza vaccines. A number of novel
approaches are in various stages of preclinical and clinical
development, offering hope that improvements could be on
the way. There is little consensus, however, on what direction the field should take to offer the most improvements
and this is reflected in the multiple strategies being employed. While none of these approaches appears to fulfill all
requirements for an optimal influenza vaccine, the combined
experiences and knowledge gained will be invaluable. In
this review, we focus on the problem facing influenza vaccines and highlight some improvement strategies being explored.
The Problem, a Constantly Moving Target
The influenza virus is the classic example of a constantly
evolving pathogen and its ability to evade the most potent
immunity allows it to be a continual threat to its hosts, and
to survive in populations with considerable prior exposure.
The virus has two major mechanisms for antigenic change,
antigenic drift, and antigenic shift. While both mechanisms
have evolved to evade natural immunity, they also interfere
with successful vaccination.
Antigenic drift is the process by which minor changes are
introduced into key viral epitopes through point mutations in
the viral genome (5). Frequent mutations are introduced into
the influenza virus genome during the replication cycle due
to the lack of proofreading mechanisms associated with
the virally encoded RNA-dependent RNA polymerase (88).
While many of the mutations lead to nonviable progeny
or make minor changes to proteins or genes, some occur
in antigenic regions of the HA and/or NA glycoproteins
(27,50,93). When epitope-altering mutations occur, viruses
containing them are rapidly selected by host immunity
driving antigenic drift (99). Although there is no reason to
suggest that the inherent mutation rates are different across
viral RNA segments, the highest rate of drift appears in
antigenic regions, because viruses with antigenic changes
preferentially escape preexisting immunity (8,103).
A hallmark of influenza virus antigenic drift in humans is
the replacement of older viruses by new drifted variants,
although at rates dependent on subtype: antigenically variant
populations have been shown to co-circulate for longer periods in A(H1N1) and influenza B viruses (24), whereas
turnover is more rapid with A(H3N2) viruses (26). This
succession of strains is what makes current vaccination
strategies feasible, that is, diversity is limited at any given
time. The challenge is of course for the WHO tracking
system to correctly predict or detect the emerging lineages
at an early stage. Due to the 6 or more months required to
prepare a vaccine, the possibility exists that by the time a
vaccine is manufactured to support a global campaign, it is
no longer matched with circulating viruses (13). Any vaccination approach that targets the classic neutralizing responses to HA and/or NA must deal with antigenic drift
175
effectively. It is also possible that novel vaccines that increase the potency of immunity to other viral proteins may
lead to more rapid antigenic evolution.
The second form of antigenic change that influenza
viruses have at their disposal is antigenic shift. Rather than
the gradual changes seen with antigenic drift, antigenic
shift results in a complete exchange of HA and/or NA
genes (100). Antigenic shift only occurs among influenza A
viruses due to their extensive animal reservoirs, the sources
of antigenically distinct viruses (12). Influenza B viruses do
not have a recognized animal reservoir [although there have
been reports of virus presence in seals and horses (46,75)]
and hence do not undergo antigenic shift (72). In humans,
the result of antigenic shift is an influenza virus to which the
population has limited immunity, leading to increased
transmission, and a pandemic. To avoid pandemics, public
health entities react with vigor to detections of novel subtypes, such as A(H5N1) and A(H7N9), in humans (112). As
population immunity builds to a pandemic virus, the overall
disease burden reduces and seasonality returns. The viruses
present in humans today are all descended from prior pandemic strains that have evolved through drift in humans (90).
Pandemics have emerged infrequently, but repeatedly over
the past 100 years. The emergence of the 1918 A(H1N1)
pandemic virus occurred as a consequence of the transmission of an avian virus to humans (70). The 1957 A(H2N2)
pandemic virus was derived from a descendent of the 1918
A(H1N1) virus that had acquired the HA, NA, and polymerase
basic protein 1 (PB1) gene segments from an avian virus
source (48). The 1968 A(H3N2) pandemic virus was similarly
derived from an A(H2N2) virus that had acquired HA and PB1
genes from an avian virus (23). An A(H1N1) virus similar to
viruses that had circulated in humans in the mid-1950s reemerged in 1977 (67). The 2009 A(H1N1) pandemic virus
contained segments from avian, human, and swine viruses that
had all been circulating in swine populations before virus
emergence in humans (32,40). At present, descendants of the
1968 A(H3N2), 2009 A(H1N1), and influenza B viruses circulate in humans and all cause substantial morbidity and
mortality; any successful vaccination approach must protect, at
minimum, from these. To target a pandemic, new vaccines
require months of production. An ideal seasonal influenza
vaccine would induce immunity to seasonal strains as well as
provide some immunity to pandemic viruses, but the diversity
of viruses in animal reservoirs (Fig. 1) makes this an incredibly
difficult proposition.
The capacity for antigenic shift is a consequence of the
segmented nature of the influenza virus genome. Upon coinfection of a single cell, gene segment exchange can occur
between viruses (7,37). Although each functional virus must
have a copy of each segment, 254 theoretical combinations
of the eight gene segments are possible from a dual infection
(56). In practice, not all combinations are seen and other
factors limit the extent of viruses generated; these factors
are poorly understood and the outcomes of mixed infections
are unpredictable (57,97). Nevertheless, some patterns of
reassortment have been identified, such as the associations
of various matrix 2 (M2), HA, polymerase acidic protein
(PA), and PB2 gene segments and/or proteins in avian
viruses (15). Influenza B viruses also undergo reassortment
in humans and can be accompanied by deletions and insertions (61,62). Reassortment between influenza virus genera
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176
KIM ET AL.
FIG. 1. Influenza viruses in different hosts. There are four genera of influenza viruses: A, B, C, and D, each with a
distinctive host range as shown. Influenza A viruses are zoonotic pathogens that have a wide host range, which includes
humans and other mammalian species. However, avian species are the primary hosts of influenza A viruses; currently, 18
HA and 11 NA subtypes are known, with most maintained in wild aquatic bird populations with sporadic transmission to
other hosts. HA, hemagglutinin; NA, neuraminidase.
(i.e., between influenza A, B, C, and D) does not occur
(33,60,66). However, reassortment between chimeric influenza A and B virus gene segments has been successful and it
has shown that some of the incompatibility depends on terminal packaging and other noncoding regions (28,38).
Not only is reassortment important for the evolution of
influenza viruses but is also harnessed for the current process of influenza vaccine manufacture. The influenza A seed
viruses used to produce antigen for inactivated and live attenuated influenza vaccines are not the wild-type viruses
themselves, but rather reassortant viruses containing the HA
and NA gene segments for the target strain and the majority
of remaining segments from a master strain generated to
enhance yield and safety. The influenza B antigens in inactivated vaccines are variably derived from wild-type or
reassortant viruses produced using a similar approach. Since
the late 1990s, it has been possible to generate reassortant
influenza viruses from cloned complementary DNA (cDNA)
copies of the viral genomes by plasmid-based reverse genetics (29,69). The methods have taken some of the uncer-
tainty away from the reassortment process and they are
routinely used in the preparation of seed viruses for live attenuated seasonal vaccines and inactivated vaccines against
zoonotic viruses. Importantly, the reverse genetics systems
also allow for directed mutagenesis of the viral genome.
As the amount and speed of vaccine manufacture are directly related to the yields and growth kinetics of the
seed viruses, attempts have been made to improve vaccine
yield by introductions of mutations into the master virus
(10,49,101). The current WHO-led system provides a relatively uniform set of seed viruses to manufacturers of
inactivated vaccines. As mutations and/or genetic regions
are identified, which increase desirable properties of these
seed viruses, proprietary master strains may become more
of the norm than the exception. Improving the yields of
conventional and live attenuated seed viruses must remain a high priority and while not necessarily improving the breadth of immunity, such activities increase the
speed with which manufacturers can respond to a changing virus.
INFLUENZA VIRUS: DRIFTING AND SHIFTING
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Considerations for Improvements to the Current System
Currently available commercial influenza vaccines can be
categorized into three groups:inactivated influenza vaccines,
recombinant influenza vaccines, and live attenuated influenza vaccines. As the name suggests, the latter vaccines are
replication-competent viruses attenuated in their ability to
cause disease. The former two vaccines are administered by
the intramuscular route and the live attenuated vaccines are
administered by the intranasal route using relatively simple delivery systems. Various forms of inactivated vaccines
have been deployed, including inactivated whole virus, split
virion, and subunit.
Influenza vaccines were first commercialized in 1945 to
help prevent or reduce diseases caused by epidemics and
pandemics such as the pandemic of 1918, which killed an
estimated 20–50 million people (43), or the most recent
2009 A(H1N1) influenza pandemic (74,76). However, vaccines are far from perfect. This is particularly true in the
elderly where the inactivated vaccines (the mainstay of our
current vaccination programs) rarely reach effectiveness
estimates of 50%. The effectiveness of influenza vaccines
has been found, in some studies, to reduce influenza illness by
up to 70–95% in healthy adults, although vaccines are often
less successful (71,82,89,105). The less than optimal effectiveness levels of inactivated influenza vaccines have been
major political and scientific drivers for new vaccine development, but also dampen public opinion. Public expectation
of vaccine effectiveness may be overly optimistic, and meeting or managing this expectation is challenging. Expectations
may rise when protection is observed in small animal studies
during the preclinical development of a vaccine, but disappointments may follow in clinical trials. Small animal models
provide limited guidance as to true efficacy in either humans
or larger animal models, such as ferrets.
The development of influenza vaccines is also challenged
by poor immunologic correlates of protection. The only
currently accepted correlate is hemagglutination inhibition
(HI) serum antibody titers of 1:32 to 1:40, although there is
far from robust experimental support for this (14). It is still,
however, an important benchmark for vaccines that elicit
protection primarily through the generation of neutralizing antibodies targeting the HA. The development of next
generation influenza vaccines that may function through
different mechanisms is hampered by a complete lack of
other convincing protective immunologic correlates. Indeed,
the live attenuated vaccines have shown efficacy without
generating serum HI titers of 40, demonstrating that protection is afforded by other mechanisms. As newer targets
are identified and different components of the immune response are triggered, the lack of known correlates becomes
more problematic. A better understanding of the hallmarks
of a successful immune response to natural infection will help
guide the optimization of vaccine platforms. Animal models
are critical in this discovery path, but it is near impossible to
mimic the repeated exposures and considerable preexisiting
immunity that exist in the human population.
Incremental improvements to overcome the limitations of
the current influenza vaccine platforms have included exploring a shift away from the egg-based manufacture to cellbased systems. There are theoretical improvements afforded
by this shift, some relating to the increased flexibility of the
177
supply chain of cells compared to eggs. There are, however,
other reasons to move toward a cell-based product. One of
the most important is the fact that some influenza viruses
acquire adaptations in the HA upon growth in eggs and a
subset of these changes can impact antigenicity resulting in
a mismatch between vaccine antigen and circulating strain.
While the effect of growth substrate choice on virus antigenicity has been long recognized (47), the true impact on
vaccine effectiveness in humans is becoming more apparent [for example, Skowronski et al. (85)].
While substantial resources and effort have been utilized
in this area, the lower yields of most cell-based systems
have offset any positive gain in terms of speed and flexibility of production. Cell-based platforms are still an important component of a global vaccine response, particularly
to pandemic influenza. An alternative approach to the egg
issues has been to remove the need for growth of virus altogether. As current inactivated vaccines are enriched for HA,
recombinant HA approaches have been explored. The most
commercially successful approach to date has utilized the
baculovirus system (94). These vaccines have been shown
to be protective in different animal models (6,42,44) and a
baculovirus expressed HA protein-based influenza vaccine
is licensed in the United States (18). Studies have shown
that the recombinant HA vaccines can be protective in immunologically primed healthy adults and the elderly
(19,94). A more widespread switch to recombinant platforms, however, will be challenged by a high capital investment necessitated by the need to phase in a new system.
In addition, a switch in manufacturing process without a
substantial change in antigen, still, only provides solutions
for some of the influenza vaccine issues and does not address
the primary concern of matching vaccine HA antigen with a
constantly moving target.
So, are there ways to tweak current systems to improve the
breadth and longevity of the induced response? Potential
strategies to improve the breadth of responses to vaccination
have included the addition of the other immunogenic surface
antigen, the NA. Studies have suggested that the addition of the
NA to HA-based vaccines can improve the breadth of protection (6,59). In addition, recent data from both human challenge and clinical vaccine trials have shown that antibodies to
the NA are independent correlates of protection. In influenza
human challenge models, it was shown that baseline NA inhibition antibody titers correlated with reduced measures of
disease severity (64). Similarly, analysis of data from vaccine
efficacy studies showed that increased NA inhibition antibody
titers following inactivated influenza administration was correlated with a reduced frequency of subsequent influenza virus
infection (65). As all inactivated products start with whole
viruses, there is a probability that available products contain
NAs; however, there are no specific regulations on controlling
NA concentration. NA content is generally unknown and likely
varies from manufacturer to manufacturer and probably from
batch to batch. How difficult altering the current production
systems to include standardized amounts of the NA would be
is unclear, but should be explored.
As might be expected, adjuvant technology has also been
used with influenza vaccines in the hope of increasing immunogenicity and breadth of immunity with mixed success.
Alum (aluminum salts) has been licensed as an adjuvant for
human use; however, it has had mixed results with
178
influenza vaccines and does not promote cell-mediated immunity (53). The oil-in-water emulsions AS03, MF59, and
AF03 have demonstrated a significant dose-sparing effect
when used in conjunction with influenza vaccines and have
been shown to increase the breadth of the immune response
when compared to unadjuvanted formulations (3,35). Other
approaches such as ISCOMATRIX (83) and immunopotentiating reconstituted influenza virosomes (IRIVs) (34)
have also been studied. However, there may be perception
and possibly regulatory issues associated with adjuvant use
in vaccines requiring frequent boosting.
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Toward a More Universal Influenza Vaccine
What have been discussed to date are issues and some
possible solutions to overcoming the shortfalls of current
vaccines, the most important of which is matching vaccine
HA antigens to the HA of circulating viruses. So long as the
globular head of HA remains the vaccine target, it is difficult
to imagine this ever being overcome. The search for more
conserved epitopes and antigens is critical to truly advancing influenza vaccinology and heading it in the direction of a
universal influenza vaccine.
What should a more broadly reactive influenza vaccine target? The influenza virus was initially classified as a
myxovirus (1) with later reclassification within the family of
Orthomyxoviridae (63). There are seven genera within the
Orthomyxoviridae family: influenza A, B, C, and D virus,
tick-borne Thogotovirus, infectious salmon anemia (ISA)
virus (52), and Quaranjavirus (39). Orthomyxoviruses are
segmented, single-stranded, negative-sense RNA viruses
(109). The influenza viruses are classified on the antigenic
properties of their matrix (M) and nucleoprotein (NP) (106)
KIM ET AL.
with influenza A and B viruses first isolated in 1933 and
1940, respectively (30,68); influenza C virus was first isolated in 1949 (31). An influenza virus with moderate homology to human influenza C viruses was isolated from
swine in 2011 and has been proposed as a new genus of
influenza virus named influenza D virus, and a new member
of the family Orthomyxoviridae (36). Due to their distinct
characteristics and limited impact as human pathogens, influenza C and D viruses are not vaccine targets of high
importance. Therefore, although universal influenza vaccines should provide protection against all influenza viruses,
when it comes down to vaccine development, coverage of
influenza A viruses and influenza B viruses is most important.
With this in mind, searches for more conserved sequences
within the influenza virus proteome have been explored. The
most extensive endeavors have targeted various epitopes
within the HA (Fig. 2), the ectodomain of matrix 2 protein
(M2e), and the NP. Some of the earliest hopes for a more
broadly reactive vaccine were focused on M2e. The M2
protein is a transmembrane ion channel that acts during the
influenza virus lifecycle to regulate the pH of various cellular and viral compartments. M2e contains an N-terminal
sequence highly conserved in influenza A viruses (25).
Vaccines based on this conserved domain are protective
in small animal models, particularly mice, and can induce
broadly reactive immune responses (17,80,84). The mechanism of protection with M2e approaches is antibody based,
but involves mechanisms beyond simple neutralization effects. While there is still some uncertainty about the exact
mechanisms of protection afforded by M2e-based vaccines,
antibody-dependent cell-mediated cytotoxicity has been
implicated [reviewed in Lee et al. (55)]. Some of the M2e
approaches have not shown as much promise past murine
FIG. 2. HA as a vaccine target. Representation of HA trimer showing the two major domains (globular head and stalk)
and their antigenic and immunologic characteristics.
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INFLUENZA VIRUS: DRIFTING AND SHIFTING
studies, although others have advanced into clinical trials
(95). Producing a vaccine based on a single peptide antigen
is not a feasible standalone product, and the best promise
for M2e approaches is to supplement other viral antigens/
vaccines. While the M2e epitope is relatively well conserved, focusing the immune response on it will undoubtedly lead to issues of viral escape. Determining the best way
to harness the expanded breadth of M2e immunity warrants
further investment.
The approaches to influenza vaccines discussed to date
have primarily relied on generating humoral immunity (with
the exception of live attenuated platforms, for which the
primary means of protective immunity is unclear). Other
more conventional approaches to expanded breadth of influenza vaccines have focused on eliciting cellular, particularly cytotoxic CD8+ T cell, immunity; such cells often
target epitopes on more conserved viral proteins typically
not exposed to antibody-driven selection. One example of
this is a recombinant pox vaccine expressing the NP. This
vaccine induced strong immune responses that protected
against multiple influenza virus challenges (96). Similarly,
other approaches designed to induce CD8+ T cell immunity
have been shown to be broadly protective against various
influenza viruses in animal models (77,79).
While these are but just two examples of the many that
have gone down the T cell path, most have targeted the
immunodominant epitopes present in the NP and M1 in the
context of generating heterologous immunity mediated by
cross-reactive T cells (9,54). It is somewhat unclear as to
what the future of T cell -directed influenza vaccines holds.
While animal models clearly show that CD8+ T cells can
play a critical role in protection from influenza virus infection, the role of T cell protection in influenza disease in
humans [even though there has been some rapid progress
(86,104)] is less clear. Some of this is due to the difficulty in
examining the T cell response at the site of infection as well
as the high cost and technical challenges of conducting
immunologic studies in naturally infected individuals. It is
very difficult to imagine, however, that T cells do not have a
substantial influence on the outcomes of human influenza.
The development of methods to induce memory CD8+ T cell
populations through vaccination should be encouraged. A
better understanding on the protective effects of targeting
different epitopes/proteins in the context of human immunity would help push these approaches forward.
Much of the recent activity in universal influenza vaccine
approaches has centered on focusing the immune response
to more conserved domains of the HA. While it has been
recognized for some time that more conserved antibody
epitopes exist on the HA, particularly in the stalk domain,
the development of single-cell B cell analysis tools has
uncovered an underappreciated role B cells play in response
to infection and vaccination. B cell responses to the HA
stalk domain are substantially less dominant than those to
the globular head, but they do exist and are variably neutralizing (2,20,73). An important clinical advance is the
development of broadly reactive antibodies to the HA stalk
as treatment options (78,87). Anti-influenza HA stalk-specific
broadly neutralizing antibodies, such as CT149, SFV0052G02, and MEDI8852, have been developed and are capable
of recognizing structurally constrained epitopes (45,58,110).
The challenge is to utilize this knowledge to design vaccine
179
antigens able to induce broadly neutralizing antibodies. Studies conducted during the 2009 pandemic showed that under
certain circumstances, exposure to novel antigens induced
stalk-directed antibody responses, providing more useful
guidance on how to stimulate broadly reactive responses
(108). Encouraging early results from mice immunized with
stable HA stalk vaccines (which lacked the immunodominant
globular head domain) showed that the HA stalk could induce
broad protection (41,111). Currently, a number of other approaches are in development and clinical data should be
available in the coming years. It should also be noted that
studies looking at the individual human B cell response have
identified rare antibodies that are broadly reactive across influenza virus subtypes; these antibodies bind near the receptor
binding site of the HA (22,102).
While there is considerable research effort ongoing, directing the immune response to target the conserved HA
stalk domain, other approaches have taken a somewhat
different route to induce more broadly reactive HA-focused
immunity by generating computationally derived ‘‘consensus’’ antigens. While there are several slight variations on
the theme, the basic approach has been to utilize the sequence of circulating strains and generate an artificial sequence that is most representative of the group. The theory
behind this is that this should minimize the average genetic,
and by inference antigenic, distance between the vaccine
antigen and circulating viruses. In animal models, this has
been shown to be an effective approach against numerous
influenza virus subtypes including, but certainly not limited
to, A(H1N1) (98) and A(H3N2) (107) viruses.
While most of the more universal approaches have concentrated on influenza A viruses, where the most genetic and
antigenic diversity exists, universal influenza B vaccines are
also being developed. An example of one approach targets
the highly conserved influenza B HA cleavage site located
within the stalk domain. Three doses of this conjugated
vaccine protected from multiple influenza B virus challenges (4). Similar to what has been explored with influenza
A viruses, conserved epitopes from influenza B viruses have
been discovered and tested in mice (16).
Concluding Remarks
While the long-term goal for influenza vaccination must
remain on a more universal vaccine, there is some debate on
exactly what the traits of such a vaccine should be. We can
all agree that a single dose of vaccine capable of protecting
against all epidemic and pandemic viruses would be ideal,
but is likely unrealistic. What then is realistic? The current
approaches suffer from narrowness and short duration of
induced immunity. Improving either of these, either in
tandem or alone, would be a major step forward and is not
outside the realm of possibility. Having a vaccine that would
cover more drift variants might mean less vaccine updates
and a reduced need for yearly vaccinations. Such vaccines
may not cover the gamut of influenza A subtypes, but would
still constitute a significant public health achievement. Of
course challenges remain even for these relatively minor
improvements. The challenges include inducing responses
to epitopes other than the immunodominant HA head, and
the tradeoff between immunity potency and breadth. Even
with a current vaccine that induces the most potent form of
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180
immunity, neutralizing antibodies to the HA globular head,
vaccine effectiveness measures can be disappointing. In the
context of pandemic vaccination where the initial end goals
of vaccination might be to reduce mortality, a less robust
protective immunity might be tolerated, but in the context of
seasonal epidemic influenza viruses, there is little room for
loss of potency. Another major focus of influenza vaccine
improvements, whether in terms of breadth, duration, or
immunogenicity, concerns the elderly. This sector of the
population is hardest hit by the severe consequences of influenza infection and is recognized as a priority vaccination
target, but has the most disappointing responses to conventional vaccines. The full-throttle forward approach to a
more universal vaccine should not come at the expense of
improving vaccines for the elderly; the improvements here
may be more readily achieved and the public health impacts
just as significant.
While there is still clearly work to do, there has been a
marked progress in influenza vaccine research over the past
decade, and vaccination has become an important means of
protection against influenza. In addition to taking steps forward to realize a perfect vaccine, overcoming the social atmosphere is also a challenge for vaccine developers and
cannot be ignored. The erosion of public trust in vaccination
could cause an important global health issue; the variable efficacy of influenza vaccines does not help this problem. Current approaches to improve influenza vaccines are diverse and
at various stages of development. While some will undoubtedly fail, the data that each will generate will be invaluable
and it is likely that different options for influenza vaccination
will be available in the coming future. Watch this space.
Author Disclosure Statement
No competing financial interests exist.
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Address correspondence to:
Dr. Richard J. Webby
Department of Infectious Diseases
St. Jude Children’s Research Hospital
262 Danny Thomas Place
Memphis, TN 38195
E-mail: richard.webby@stjude.org
Oncogene (2012) 31, 1869–1883
& 2012 Macmillan Publishers Limited All rights reserved 0950-9232/12
www.nature.com/onc
REVIEW
Molecular mechanisms of cisplatin resistance
L Galluzzi1,2,3, L Senovilla1,2,3, I Vitale1,2,3, J Michels1,2,3, I Martins1,2,3, O Kepp1,2,3, M Castedo1,2,3
and G Kroemer1,4,5,6,7
1
INSERM, U848 ‘Apoptosis, Cancer and Immunity’, Villejuif, France; 2Institut Gustave Roussy, Villejuif, France; 3Université Paris
Sud-XI, Villejuif, France; 4Metabolomics Platform, Institut Gustave Roussy, Villejuif, France; 5Centre de Recherche des Cordeliers,
Paris, France; 6Pôle de Biologie, Hôpital Européen Georges Pompidou, AP-HP, Paris, France and 7Université Paris Descartes,
Sorbonne Paris Cité, Paris, France
Platinum-based drugs, and in particular cis-diamminedichloroplatinum(II) (best known as cisplatin), are
employed for the treatment of a wide array of solid
malignancies, including testicular, ovarian, head and neck,
colorectal, bladder and lung cancers. Cisplatin exerts
anticancer effects via multiple mechanisms, yet its most
prominent (and best understood) mode of action involves
the generation of DNA lesions followed by the activation
of the DNA damage response and the induction of
mitochondrial apoptosis. Despite a consistent rate of
initial responses, cisplatin treatment often results in the
development of chemoresistance, leading to therapeutic
failure. An intense research has been conducted during the
past 30 years and several mechanisms that account for the
cisplatin-resistant phenotype of tumor cells have been
described. Here, we provide a systematic discussion of
these mechanism by classifying them in alterations (1) that
involve steps preceding the binding of cisplatin to DNA
(pre-target resistance), (2) that directly relate to DNA–
cisplatin adducts (on-target resistance), (3) concerning the
lethal signaling pathway(s) elicited by cisplatin-mediated
DNA damage (post-target resistance) and (4) affecting
molecular circuitries that do not present obvious links with
cisplatin-elicited signals (off-target resistance). As in some
clinical settings cisplatin constitutes the major therapeutic
option, the development of chemosensitization strategies
constitute a goal with important clinical implications.
Oncogene (2012) 31, 1869–1883; doi:10.1038/onc.2011.384;
published online 5 September 2011
Keywords: ATP7B; CTR1; ERCC1; glutathione; metallothioneins; TP53
Introduction
First approved by FDA (Food and Drug Administration) in 1978 for the treatment of testicular and bladder
cancer, cis-diamminedichloroplatinum(II) (best known
Correspondence: Dr G Kroemer, INSERM, U848, Institut Gustave
Roussy, Pavillon de Recherche 1, 39 rue Camille Desmoulins, F-94805,
Villejuif, France.
E-mail: kroemer@orange.fr
Received 30 June 2011; revised 26 July 2011; accepted 27 July 2011;
published online 5 September 2011
as cisplatin or CDDP) is a largely employed platinumbased compound that exerts clinical activity against a
wide spectrum of solid neoplasms, including testicular,
bladder, ovarian, colorectal, lung and head and neck
cancers (Prestayko et al., 1979; Lebwohl and Canetta,
1998; Galanski, 2006). Cisplatin often leads to an initial
therapeutic success associated with partial responses or
disease stabilization. Still, many patients (in particular
in the context of colorectal, lung and prostate cancers)
are intrinsically resistant to cisplatin-based therapies.
Moreover, an important fraction of originally sensitive
tumors eventually develop chemoresistance (this is
frequently observed in ovarian cancer patients) (Ozols,
1991; Giaccone, 2000; Koberle et al., 2010). The cytotoxicity of cisplatin (which is given intravenously as
short-term infusion in physiological saline) also affects
kidneys (nephrotoxicity), peripheral nerves (neurotoxicity) and the inner ear (ototoxicity) (Cvitkovic et al.,
1977; Kelland, 2007). Still, the main limitation to the
clinical usefulness of cisplatin as an anticancer drug is
the high incidence of chemoresistance.
In the early 1980s, second-generation platinum
compounds were developed with the speciﬁc aim of
reducing the side effects of cisplatin while retaining its
anticancer properties. These efforts led to the discovery
of cis-diammine (cyclobutane-1,1-dicarboxylate-O,O’)
platinum(II) (carboplatin), which essentially does not
display nephro- and neurotoxicity, yet forms the same
types of DNA adducts (see below) as cisplatin, although
with a reduced potency (Harrap, 1985). Carboplatin,
whose most prominent side effects concern the bone
marrow, frequently leading to reversible thrombocytopenia, was approved by the FDA for the treatment
of ovarian cancer in 1989. As the active form of
carboplatin is identical to that of cisplatin (see below),
it was not surprising to ﬁnd out that most cisplatinresistant tumors also fail to respond to carboplatin.
These observations ignited another wave of drug development that in 2002 led to the introduction of [(1R,2R)cyclohexane-1,2-diamine](ethanedioato-O,O0 ) platinum(II)
(oxaliplatin) into clinical practice. Oxaliplatin exhibits
distinct pharmacological and immunological properties
than cisplatin and carboplatin, in line with the fact that
it features the bidentate ligand 1,2-diaminocyclohexane
in place of two monodentate ammine ligands (Kidani
et al., 1978). However, in spite of the fact that cisplatin-
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L Galluzzi et al
1870
refractory cancers are generally considered to be
sensitive to oxaliplatin, clinical data suggest that there
may be some degree of cross-resistance (Stordal et al.,
2007). Oxaliplatin is currently employed against colorectal cancer in association with 5-ﬂuorouracil and
folinic acid (the so-called FOLFOX protocol) (Giacchetti
et al., 2000; de Gramont et al., 2000; Rothenberg et al.,
2003; Goldberg et al., 2004), and may also be useful
for the treatment of lung cancer (Raez et al., 2010).
Of note, other platinum derivatives that have recently
entered clinical trials, such as amminedichloro(2-methylpyridine) platinum (picoplatin) and (OC-6-43)-bis(acetato)
amminedichloro(cyclohexylamine)platinum (satraplatin),
have not yet been shown to provide signiﬁcant advantages
over cisplatin, oxaliplatin and carboplatin (Choy, 2006;
Eckardt et al., 2009). Moreover, in speciﬁc clinical settings,
cisplatin represents by far the most prominent, if not the
sole, therapeutic option (Armstrong et al., 2006).
Circumventing cisplatin resistance remains therefore a
critical goal for anticancer therapy and considerable
efforts have been undertaken to solve this problem
throughout the past three decades. Here, we brieﬂy
introduce the modes of action of cisplatin and then
systematically present the molecular mechanisms that
can account for the cisplatin-resistant phenotype.
Finally, we suggest combination strategies that might
be exploited for reverting cisplatin resistance in tumors.
Mode of action
The detailed description of the molecular mechanisms
that underlie the anticancer potential of cisplatin goes
largely beyond the scope of this review, and can be
found elsewhere (Kelman and Peresie, 1979; Sanderson
et al., 1996; Siddik, 2003). Here, we will provide key
facts that explain the molecular basis of cisplatin
resistance.
Cisplatin exerts anticancer effects via an intertwined
signaling pathway that might be separated into one
nuclear and one cytoplasmic module. As such, cisplatin is
inert and must be intracellularly activated by a series of
aquation reactions that consist in the substitution of one
or both cis-chloro groups with water molecules (elKhateeb et al., 1999; Kelland, 2000). This reaction occurs
spontaneously in the cytoplasm, owing to the relatively
low concentration of chloride ions (B2–10 mM when
compared with B100 mM in the extracellular milieu), and
leads to the generation of highly reactive mono- and biaquated cisplatin forms (Eastman, 1987a; Michalke,
2010). These molecules are prone to interact with a wide
number of cytoplasmic substrates, and in particular with
endogenous nucleophiles such as reduced glutathione
(GSH), methionine, metallothioneins and proteins (via
their cysteines) (Timerbaev et al., 2006). Thus, cytoplasmic cisplatin has the potential to deplete reduced
equivalents and to tilt the redox balance toward oxidative
stress (which facilitates DNA damage; see below), but is
also susceptible to inactivation by a number of cytoprotective antioxidant systems (Koberle et al., 2010).
Oncogene
Aquated cisplatin avidly binds DNA, with a predilection for nucleophilic N7-sites on purine bases. This leads
to the generation of protein–DNA complexes as well as
of DNA–DNA inter- and intra-strand adducts (Eastman, 1987b). Although the signaling pathways that are
triggered by protein/cisplatin/DNA complexes have
been largely ignored, great efforts have been spent to
elucidate the molecular cascades that are activated by
DNA–DNA inter- and intra-strand adducts. The
latter—and notably 1,2-intrastrand ApG and CpG
crosslinks—have been indicated as the most prominent
cisplatin-induced DNA lesions and have been suggested
to account for most, if not all, cisplatin cytotoxicity
(Kelland et al., 1993). This notion, which in the past has
generated a vivid debate, nowadays appears as an
oversimpliﬁcation, especially in consideration of the fact
that: (1) only B1% of intracellular cisplatin binds to
nuclear DNA (Gonzalez et al., 2001) and (2) cisplatin
(as well as oxaliplatin) has been shown to exert
signiﬁcant cytotoxicity in enucleated cells (cytoplasts)
(Mandic et al., 2003; Berndtsson et al., 2007; Obeid
et al., 2007). Irrespective of this, the best-characterized
mode of action of cisplatin involves the DNA-damage
response and mitochondrial apoptosis (Jamieson and
Lippard, 1999; Cohen and Lippard, 2001).
Cisplatin-induced lesions cause distortions in DNA
that can be recognized by multiple repair pathways
(Bellon et al., 1991). Among these, the nucleotide
excision repair (NER) reportedly constitutes the most
prominent mechanism for the removal of cisplatin
adducts (Chaney and Sancar, 1996; Furuta et al.,
2002). However, proteins belonging to the mismatch
repair (MMR) system also participate in the recognition
and resolution of cisplatin lesions (Kunkel and Erie,
2005). When the extent of damage is limited, cisplatin
adducts induce an arrest in the S and G2 phases of the
cell cycle, a phenomenon that exerts cytoprotective
effects by (1) allowing repair mechanisms to re-establish
DNA integrity and (2) preventing potentially abortive
or abnormal mitoses (Vitale et al., 2011). Conversely, if
DNA damage is beyond repair, cells become committed
to (most often apoptotic) death.
The major signaling cascade that bridges cisplatininduced DNA lesions to apoptosis involves the
sequential activation of the ataxia telangiectasia
mutated (ATM)- and RAD3-related protein (ATR, a
sensor of DNA damage) and checkpoint kinase 1
(CHEK1, the most prominent substrate and downstream effector of ATR), which in turn phosphorylates
the tumor suppression protein TP53 on serine 20,
allowing for its stabilization (Shieh et al., 2000; Appella
and Anderson, 2001; Damia et al., 2001; Zhao and
Piwnica-Worms, 2001). Activated TP53 exerts lethal
functions via nuclear and cytoplasmic mechanisms that
eventually lead to mitochondrial outer membrane
permeabilization or increased signaling via death receptors followed by cell death (Kroemer et al., 2007; Galluzzi
et al., 2011) In response to cisplatin, CHEK1 has also
been shown to activate various branches of the mitogenactivated protein kinase (MAPK) system, including those
mediated by extracellular signal-regulated kinases, c-JUN
Chemoresistance to cisplatin
L Galluzzi et al
1871
N-terminal kinases and stress-activated protein kinases
(Persons et al., 2000; Wang et al., 2000; Dent and Grant,
2001; Yeh et al., 2002). The relative contribution of
these signaling modules to the cytotoxic effects of
cisplatin remain to be deciphered, as contrasting reports
can be found in literature (Dent and Grant, 2001).
Intriguingly, although ATM (another important sensor
of DNA damage) appears to participate in cisplatininduced cell cycle arrest but not cell death (Sancar et al.,
2004; Wang et al., 2006), its major downstream target,
CHEK2, has been shown to convey lethal signals in
response to cisplatin in an ATM-independent fashion
(Damia et al., 2001; Pabla et al., 2008).
Thus, there appear to be multiple mechanisms that
underlie the cytotoxic and antiproliferative potential of
cisplatin (Figure 1). The cisplatin-resistant phenotype of
cancer cells can derive from alterations in any of these
molecular circuitries as well as from changes that affect
the intracellular uptake of cisplatin or the execution of
the apoptotic program.
Mechanisms of pre-target resistance
There are at least two mechanisms by which cancer cells
elude the cytotoxic potential of cisplatin before it binds
to cytoplasmic targets and DNA: (1) a reduced intracellular accumulation of cisplatin and (2) an increased
sequestration of cisplatin by GSH, metallothioneins
and other cytoplasmic ‘scavengers’ with nucleophilic
properties (Table 1).
A wide array of (mostly natural) anticancer agents is
associated with the so-called multidrug resistance, a
phenomenon whereby drugs are subjected to increased
efﬂux via relatively nonselective members of the ATPbinding cassette (ABC) family of ATPases like the
P-glycoprotein (Molnar et al., 2010). This is not the case
of cisplatin, whose limited intracellular accumulation
most often (although not always; see below) derives
from reduced uptake (Smith et al., 1993; Wada et al.,
1999; Baekelandt et al., 2000). Irrespective of the underlying mechanisms, several cisplatin-resistant cancer cells
exhibit consistent reductions in the accumulation of
cisplatin (Loh et al., 1992; Mellish et al., 1993).
For a long time, cisplatin was believed to enter cells
prominently by passive diffusion across the plasma
membrane, mainly because the uptake of cisplatin,
which is highly polar, is relatively slow when compared
with that of chemically similar anticancer agents that
are actively transported (Yoshida et al., 1994; Kelland,
2000). More recently, however, the copper transporter 1
(CTR1), a transmembrane protein involved in copper
homeostasis, turned out to play an important role in
the uptake of cisplatin. Ctr1/ mouse embryonic
ﬁbroblasts accumulate much less cisplatin than their
wild-type counterparts, and are indeed two- to threefold more resistant to its cytotoxic effects (Ishida
et al., 2002; Katano et al., 2002; Holzer et al., 2006).
Accordingly, cells pre-treated with copper (the main
CTR1 substrate) are protected from cisplatin cytotoxicity
(More et al., 2010), whereas copper chelators result in
Figure 1 Modes of action of cisplatin. Because of the relatively
low (compared with the extracellular microenvironment) concentration of chloride ions, intracellular cisplatin quickly becomes
aquated and hence highly reactive. Aquated cisplatin can indeed
bind a plethora of nucleophilic species, including cysteine and
methionine residues on proteins and DNA bases. In the nucleus,
this leads to the generation of inter- and intra-strand adducts that
are recognized by the DNA damage-sensing machinery. If the
extent of damage is beyond repair, cisplatin adducts trigger the
activation of a DNA damage response (DDR) that frequently
involves the ATR kinase, CHEK1 and CHEK2 and the tumorsuppressor protein TP53. In turn, TP53 transactivates several genes
whose products facilitate mitochondrial outer membrane permeabilization (MOMP), thereby triggering intrinsic apoptosis, as well
as genes that encode for components of the extrinsic apoptotic
pathway. MOMP (alone or with the contribution of death
receptor-ignited, BID-transduced signals) sets off the caspase
cascade as well as multiple caspase-independent mechanisms that
eventually seal the cell fate. Several other signaling pathways link
cisplatin-induced DNA damage to MOMP and cell death (not
shown, see the main text for further details). In the cytoplasm,
the interaction between cisplatin and GSH, metallothioneins
or mitochondrial proteins like the VDAC results in the depletion
of reducing equivalents and/or directly sustains the generation of
reactive oxygen species (ROS). ROS can directly trigger MOMP or
exacerbate cisplatin-induced DNA damage, thereby playing a dual
role in cisplatin cytotoxicity.
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L Galluzzi et al
1872
Table 1 Mechanisms of pre-target resistance
Factor
Mode of action
Relevance
Reference
Plasma membrane copper
transporter.
Downregulated in CDDP-resistant cancer
cell lines.
CTR1 depletion increases CDDP resistance.
Copper chelators enhance the uptake and
efﬁcacy of CDDP in vitro and in vivo.
Ishida et al., 2002; Katano et al.,
2002; Holzer et al., 2006;
Ishida et al., 2010
ATP7A/ATP7B
Copper-extruding P-type ATPases
involved in the regulation of ion
homeostasis.
Upregulated in CDDP-resistant cancer
cell lines.
ATP7B expression levels may predict the
efﬁcacy of CDDP chemotherapy in patients
with ovarian cancer.
Nakayama et al., 2002; Nakayama
et al., 2004;Safaei et al., 2004;
Aida et al., 2005
MRP2
Member of the ABC family of
plasma membrane transporters.
Mediates the ATP-dependent
cellular efﬂux of CDDP.
Overexpressed in CDDP-resistant cancer
Koike et al., 1997; Cui et al., 1999;
cell lines.
Liedert et al., 2003; Korita et al.,
Modulation by antisense cDNA enhances
2010; Yamasaki et al., 2011
CDDP sensitivity.
Expression levels affect the efﬁcacy of CDDP
regimens in ESCC and HCC patients.
Reduced uptake
CTR1
Increased efﬂux
Increased inactivation
GSH/g-GCS/GST GSH scavenges electrophiles and
CDDP-resistant cells often exhibit elevated
ROS. g-GCS catalyzes GSH synthesis. levels of GSH, g-GCS and GST.
GST conjugates GSH to CDDP,
No conclusive clinical evidence.
thus facilitating its extrusion.
Metallothioneins
Intracellular thiol-containing proteins
involved in the detoxiﬁcation of
metal ions.
May bind and inactivate CDDP.
No conclusive clinical evidence.
Lewis et al., 1988;
Chen and Kuo, 2010
Kelley et al., 1988;
Kasahara et al., 1991
Abbreviations: ABC, ATP-binding cassette; CDDP, cisplatin; cDNA, complementary DNA; CTR1, copper transporter 1; ESCC, esophageal
squamous cell carcinoma; g-GCS, g-glutamylcysteine synthetase; GSH, reduced glutathione; GST, glutathione S-transferase; HCC, hepatocellular
carcinoma; MRP2, multidrug resistance protein 2; ROS, reactive oxygen species.
increased cisplatin accumulation and exacerbate cytotoxicity (Ishida et al., 2010). Of note, clinically relevant
concentrations of cisplatin reportedly downregulate
CTR1, owing to internalization followed by proteasome-mediated degradation (Holzer and Howell, 2006).
This mechanism may account (at least in part) for
multiple instances of acquired cisplatin resistance.
Early reports suggested that ABC ATPases like
multidrug resistance protein (MRP)1, MRP2, MRP3
and MRP5 would also mediate some extent of cisplatin
resistance by increasing cisplatin export (Borst et al.,
2000). In particular, results from genetic manipulations
(that is, overexpression, RNA interference) pointed to
MRP2 as the major ATPase responsible for an increased
efﬂux of cisplatin in resistant cells (Koike et al., 1997;
Cui et al., 1999; Liedert et al., 2003). Recent reports
reinforced the notion that MRP2 expression levels might
predict the responsiveness of tumors to platinum-based
therapies (Korita et al., 2010; Yamasaki et al., 2011).
Following the discovery of the role of CTR1 in cisplatin
uptake, however, attention was attracted by two copperextruding P-type ATPases, ATP7A and ATP7B. These
proteins are upregulated in cisplatin-resistant cancer cell
lines (Safaei et al., 2004), and their transfection-enforced
overexpression has been shown to drive the acquisition
of the cisplatin-resistant phenotype (Samimi et al.,
2004). Importantly, clinical studies indicate that ATP7B
Oncogene
expression levels might predict the sensitivity of ovarian
and endometrial cancers to cisplatin chemotherapy
(Nakayama et al., 2002, 2004; Aida et al., 2005). Still,
in line with the multifactorial nature of cisplatin efﬂux
(and resistance; see below), small molecules that inhibit
speciﬁc ABC transporters (for example, the P-glycoprotein-speciﬁc inhibitor 5-bromotetrandrine) appear to be
unable per se to restore cisplatin accumulation and
sensitivity (Jin et al., 2005).
Aquated cisplatin avidly binds to cytoplasmic nucleophilic species, including GSH, methionine, metallothioneins and other cysteine-rich proteins. On one hand, this
may underlie at least part of the cytoplasmic effects of
cisplatin, resulting in the depletion of antioxidant
reserves and in the establishment of oxidative stress
(Slater et al., 1995). On the other hand, nucleophilic
species act as cytoplasmic scavengers, thereby limiting
the amount of reactive cisplatin (Kasahara et al., 1991;
Sakamoto et al., 2001). Thus, elevated levels of GSH,
of the enzyme that catalyzes GSH synthesis (that is,
g-glutamylcysteine synthetase), or of the enzyme that
mediates the conjugation between cisplatin and GSH
(that is, glutathione S-transferase) have been observed
in the context of cisplatin resistance, both in vitro and
ex vivo (in cancer cell lines that were derived from one
ovarian carcinoma patient before and after the development of resistance) (Lewis et al., 1988). Of note,
Chemoresistance to cisplatin
L Galluzzi et al
1873
glutathione S-conjugates are readily extruded by cells
via MRP1 or MRP2, possibly explaining why the latter
has been more robustly associated with cisplatin
resistance than other ABC ATPases (Ishikawa, 1992).
Genetic manipulations of human and murine cells have
also linked increased levels of metallothioneins, a class
of low-molecular-weight thiol-containing proteins that
are involved in the binding and detoxiﬁcation of heavy
metal ions, to the cisplatin-resistant phenotype (Kelley
et al., 1988; Kasahara et al., 1991). However, conclusive
clinical data on this correlation are missing.
Mechanisms of on-target resistance
The recognition of inter- and intra-strand DNA
adducts and the consequent generation of an apoptotic
signal is often impaired in cisplatin-resistant cancer cells
because of a variety of defects. Alternatively, cisplatinresistant cells acquire the ability to repair adducts at an
increased pace, or become able to tolerate unrepaired
DNA lesions, thanks to a particular class of DNA
polymerases that mediate the so-called translesion
synthesis (Table 2).
The majority of cisplatin lesions are removed from
DNA by the NER system (Wood et al., 2000; Shuck
et al., 2008). In this setting, damaged nucleotides are
excised from DNA upon incision on both sides of the
lesion, followed by DNA synthesis to reconstitute
genetic integrity (Gillet and Scharer, 2006). At least 20
proteins participate in NER, including excision repair
cross-complementing rodent repair deﬁciency, complementation group 1 (ERCC1), a single-strand DNA
endonuclease that forms a tight heterodimer with
ERCC4 (also known as xeroderma pigmentosum
complementation group F (XPF)) and incises DNA on
the 50 side of bulky lesions such as cisplatin adducts
(Biggerstaff and Wood, 1992; Sijbers et al., 1996;
Ahmad et al., 2008). Early reports pointed to a
correlation between NER proﬁciency and cisplatin
resistance in multiple preclinical models (Li et al.,
1998, 2000; Metzger et al., 1998), and subsequent
studies supported this notion at the clinical level. Thus,
ERCC1 expression (be it measured at the mRNA or
protein level) has been negatively correlated with
survival and/or responsiveness to cisplatin-based regimens in several human neoplasms including bladder
(Bellmunt et al., 2007), colorectal (Shirota et al., 2001),
gastric (Metzger et al., 1998), esophageal (Kim et al.,
2008), head and neck (Handra-Luca et al., 2007; Jun
et al., 2008) and ovarian cancers (Dabholkar et al.,
1992), as well as non-small cell lung cancer (NSCLC)
(Olaussen et al., 2006). ERCC1 also participates in
interstand crosslink repair (ICR), and ICR proﬁciency
appears to be reduced and augmented in cisplatinsensitive and cisplatin-resistant tumor cells, respectively
(Zhen et al., 1992; Usanova et al., 2010).
It should be noted that increased levels of ERCC1
does not necessarily (and have never been formally
shown to) correspond to increased NER and ICR
proﬁciency in patients (as methods that reliably measure
NER and ICR activity in patient material are missing),
although ERCC1 constitutes one of the rate-limiting
factors for NER (Niedernhofer et al., 2001, 2004;
Ahmad et al., 2008). Moreover, although the absence
of ERCC1 consistently correlates with cisplatin responsiveness, both in vitro and in vivo (in patients), the same
does not hold true for ERCC1 overexpression, which in
some instances resulted in increased, rather than
decreased, sensitivity (Bramson and Panasci, 1993).
This might be because of disequilibria in the components
of complex DNA repair pathways such as NER
(Coquerelle et al., 1995). However, it remains formally
possible that ERCC1 levels affect cisplatin resistance via
hitherto uncharacterized NER- and/or ICR-independent pathways. Irrespective of this issue, ERCC1
expression constitutes a very promising biomarker for
the prediction of cisplatin responsiveness in patients
(Olaussen, 2009), and has already begun to be exploited
in this sense in clinical settings.
Cisplatin-induced DNA lesions can be detected (but
not repaired) by the MMR system, which normally
handles erroneous insertions, deletions and mis-incorporations of bases that can arise during DNA replication and recombination (Vaisman et al., 1998; Kunkel
and Erie, 2005). MMR-related proteins that participate
in the recognition of GpG interstrand adducts include
MSH2 and MLH1 (Mello et al., 1996; Vaisman et al.,
1998). According to accepted viewpoints, MMR proteins would attempt to repair cisplatin adducts, fail, and
hence transmit a proapoptotic signal (Vaisman et al.,
1998). In line with this model, MSH2 and MLH1 are
often mutated or underexpressed in the context of
acquired cisplatin resistance (Aebi et al., 1996; Drummond et al., 1996; Brown et al., 1997; Fink et al., 1998),
although NSCLC patients with high MSH2 expression
who do not undergo cisplatin treatment upon tumor
resection have a better prognosis than patients with low
MSH2 levels (Kamal et al., 2010). This apparent
discrepancy simply reﬂects the intrinsic difference
between naive, previously untreated tumors (for which
high MSH2 levels constitute a good prognostic indicator) and cancers that have acquired resistance upon
cisplatin exposure (which are often associated with
reduced MSH2 expression). Thus, at least in some
clinical settings, a high DNA repair capacity appears to
protect against tumor relapse (Kamal et al., 2010) but
may prevent patients to beneﬁt from DNA-damaging
agents. Of note, defects in MLH1 and MSH6 (another
component of the MMR system) are associated with
increased level of translesion synthesis, the phenomenon
whereby DNA synthesis is not blocked but proceeds
beyond cisplatin adducts (Bassett et al., 2002). Translesion synthesis, which is also known as replicative bypass,
is mediated by the concerted activity of a speciﬁc group
of DNA polymerases including POLH, POLI, POLK,
REV1, REV3 and REV7 (Shachar et al., 2009). POLH
and the REV3–REV7 heterodimer appear to be
involved in the replicative bypass of GpG adducts (Alt
et al., 2007; Shachar et al., 2009). Defects in POLH and
REV3 have been linked to increased sensitivity to
cisplatin in multiple tumor cell lines, in vitro (WittschieOncogene
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L Galluzzi et al
1874
Table 2 Mechanisms of on-target resistance
Factor
Mode of action
Relevance
Reference
Single-strand endonuclease that—
in association with ERCC4/XPF—
incises DNA on the 50 side of bulky
lesions (such as CDDP adducts).
Also implicated in the ICR.
ERCC1 expression negatively correlates
with CDDP clinical responses in multiple
human cancers.
Proposed predictor of CDDP-based
chemotherapy sensitivity in multiple
clinical settings.
Dabholkar et al., 1992; Metzger et al.,
1998; Shirota et al., 2001; Olaussen
et al., 2006; Handra-Luca et al., 2007;
Bellmunt et al., 2007; Kim et al., 2008;
Jun et al., 2008; Olaussen, 2009
MLH1
Component of a multiprotein
complex that excides and repairs
DNA mismatches.
Implicated in DNA damage
signaling and apoptosis.
MLH1 deﬁciency is sometimes
associated with CDDP resistance
(and increased TLS).
MLH1 promoter methylation predicts
poor survival in relapsing ovarian
cancer patients.
Aebi et al., 1996; Drummond et al.,
1996; Brown et al., 1997; Fink et al.,
1998; Gifford et al., 2004
MSH2
Forms MSH2–MSH6 and MSH2–
MSH3 heterodimers that detect
DNA lesions including base–base
mismatches.
When repair cannot be accomplished,
signals for the activation of cell death.
Mutated or underexpressed in some
tumors with acquired CDDP resistance.
Low MSH2 levels predict CDDP beneﬁts
in patients with resected lung cancer.
High MSH2 levels are a positive
prognostic factor for untreated lung
cancer patients.
Aebi et al., 1996; Brown et al., 1997;
Fink et al., 1998; Kamal et al., 2010
POLH
DNA polymerase that substitutes
stalled replicative polymerases and
includes nucleotides opposite to the
DNA lesion.
Implicated in the bypass of CDDP
adducts.
POLH upregulation correlates with
Alt et al., 2007; Ceppi et al., 2009;
shorter survival in CDDP-treated NSCLC Shachar et al., 2009
patients.
REV3/REV7
Catalytic (REV3) and structural
(REV7) subunits of the TLS DNA
polymerase z.
Implicated in the bypass of CDDP
adducts.
REV3 defects correlate with increased
CDDP sensitivity in cancer cell lines.
REV overexpression is associated with
CDDP resistance, in vitro.
Conclusive clinical data are missing.
Increased NER proﬁciency
ERCC1
MMR deﬁciency
Increased TLS
Wittschieben et al., 2006; Shachar
et al., 2009; Roos et al., 2009
Increased HR proﬁciency
BRCA1/BRCA2
Critical components of the
HR DNA repair system.
Also involved in the regulation
of transcription and cell cycle
progression.
BRCA1/2-deﬁcient tumors respond better Narod and Foulkes, 2004; Edwards
to CDDP.
et al., 2008; Sakai et al., 2008
Secondary mutations that restore BRCA
function favor acquired chemoresistance.
CDDP-binding proteins
VDAC
Protein of the OM that mediates
vital functions but also participates
into the PTPC.
Aquated CDDP binds VDAC.
Depletion/or inhibition of VDAC
increases CDDP resistance.
Might also be involved in post-target
resistance.
Yang et al., 2006; Kroemer et al., 2007;
Tajeddine et al., 2008
Abbreviations: BRCA1, breast cancer 1, early onset; BRCA2, breast cancer 2, early onset; CDDP, cisplatin; ERCC1, excision repair cross-complementing
rodent repair deﬁciency, complementation group 1; HR, homologous recombination; ICR, interstrand crosslink repair; MMR, mismatch repair;
NER, nucleotide excision repair; NSCLC, non-small cell lung cancer; OM, mitochondrial outer membrane; PTPC, permeability transition pore
complex; TLS, translesion synthesis; VDAC, voltage-dependent anion channel; XPF, xeroderma pigmentosum complementation group F.
ben et al., 2006; Roos et al., 2009), whereas REV3
overexpression reportedly increases cisplatin resistance
(Wang et al., 2009). Moreover, POLH expression levels
correlate with overall survival in lung cancer patients
(Ceppi et al., 2009), REV3 was found to be upregulated
in glioma samples, correlating with tumor grade (Wang
et al., 2009), and the methylation-dependent silencing of
MLH1 has been shown to predict poor survival in
ovarian cancer patients (Gifford et al., 2004). Taken
Oncogene
together, these observations suggest that the expression
levels of components of the MMR and translesion
synthesis systems may constitute useful predictors of
cisplatin responsiveness in clinical settings, although
compelling data on this issue have not yet been reported.
Cisplatin-induced inter-strand adducts can lead to the
so-called double-strand breaks, DNA lesions that are
normally repaired in the S phase of the cell cycle (or
shortly after) by the machinery for homologous
Chemoresistance to cisplatin
L Galluzzi et al
1875
recombination (HR) (Smith et al., 2010). Two critical
components of the HR system are encoded by BRCA1
and BRCA2, two genes that are frequently mutated in
familial breast and ovarian cancers (Venkitaraman,
2002; Narod and Foulkes, 2004). Notably, HR-deﬁcient
cancers have a different phenotype and are often more
sensitive to crosslinking agents including cisplatin than
their HR-proﬁcient counterparts (Bryant et al., 2005;
Farmer et al., 2005; Ratnam and Low, 2007). For
instance, BRCA1/2-deﬁcient ovarian cancers metastasize to viscera more frequently than sporadic epithelial
ovarian cancers (which most often remain conﬁned to
the peritoneum), yet are generally more responsive to
platinum compounds and associated with better prognosis (Ben David et al., 2002; Chetrit et al., 2008;
Gourley et al., 2010). Moreover, it has been shown that
cisplatin resistance can develop in initially cisplatinsensitive tumors because of secondary mutations that
compensate for BRCA1/2 deﬁciency and re-establish
HR (Edwards et al., 2008; Sakai et al., 2008).
Altogether, these observations suggest that the HR
status, at least in speciﬁc clinical settings, has an
important prognostic and predictive value.
As the catalog of cisplatin interactors that may be
implicated in its cytotoxicity has not yet been entirely
elucidated, cytoplasmic proteins may also be responsible
for (at least part of) the cisplatin-resistant phenotype. With
regard to this, cisplatin has been shown to bind
mitochondrial DNA as well as the voltage-dependent
anion channel (VDAC) (Yang et al., 2006), a mitochondrial protein with vital and lethal functions (Kroemer et al.,
2007). Notably, VDAC-depleted cancer cells are highly
resistant to CDDP treatment (Tajeddine et al., 2008), yet it
is not clear whether this constitutes an example of ontarget resistance or whether in this context VDAC simply
transduces upstream proapoptotic signals (and hence
would be involved in a post-target resistance mechanism).
Mechanism of post-target resistance
Post-target resistance to cisplatin can result from a
plethora of alterations including defects in the signal
transduction pathways that normally elicit apoptosis in
response to DNA damage as well as problems with the
cell death executioner machinery itself. Nonrepairable
cisplatin-induced DNA damage leads to the activation
of a multibranched signaling cascade with proapoptotic
outcomes (see above). Genetic and epigenetic alterations
in the components of this complex signaling network
have been associated with variable levels of resistance to
cisplatin, presumably reﬂecting the relative relevance of
speciﬁc proteins (and the underlying pathways) in
different cellular and experimental settings (Table 3).
One of the most predominant mechanisms of posttarget resistance involves the inactivation of TP53
(Vousden and Lane, 2007), which occurs in approximately half of all human neoplasms (Kirsch and Kastan,
1998). This has been documented in vitro, by comparing
the sensitivity to cisplatin of a wide panel of TP53-
proﬁcient and -deﬁcient tumor cell lines (O’Connor
et al., 1997; Branch et al., 2000), and also in vivo, in the
clinical setting (Hengstler et al., 2001). Thus, ovarian
cancer patients harboring wild-type TP53 reportedly
have a higher probability to beneﬁt from cisplatin-based
chemotherapy than patients with TP53 mutations
(Gadducci et al., 2002; Feldman et al., 2008). Moreover,
testicular germ cell tumors, which are particularly
sensitive to cisplatin, are one of the few cancers in
which TP53 is rarely, if ever, inactivated (Peng et al.,
1993). Other members of the TP53 protein family,
notably the transactivation-incompetent TP63 variant
DNp63a, have recently been shown to transduce
prosurvival signals in response to cisplatin (Yuan
et al., 2010), but the putative clinical implications of
these observations remain to be established.
Intriguingly enough, tetraploid cancer cells have been
shown to endure DNA-damaging agents (including
cisplatin and oxaliplatin) far better (410-fold) than
their diploid counterparts (Castedo et al., 2006; Vitale
et al., 2007). This phenomenon can be reverted by the
depletion/inhibition of TP53, its target ribonucleotide
reductase M2 B (RRM2B) or CHEK1 (Castedo et al.,
2006; Vitale et al., 2007), implying that the cisplatinresistant phenotype of tetraploid cancer cells relies on
complex mechanisms that go beyond a merely stoichiometric (on-target) process whereby the double amount
of DNA entirely accounts for resistance. Altogether,
these observations suggest that multiple factors, including ploidy, are likely to affect the molecular mechanisms
that underlie cisplatin resistance.
Preclinical studies suggest that other proapoptotic signal
transducers such as MAPK family members might
contribute to the cisplatin-resistant phenotype (Mansouri
et al., 2003; Brozovic and Osmak, 2007). In particular, it
has been proposed that cisplatin-resistant cells would fail
to activate MAPK1 (also known as p38MAPK) and c-JUN
N-terminal kinase in a sustained fashion in response to
cisplatin (Mansouri et al., 2003; Brozovic et al., 2004). This
would limit signaling through the FAS/FASL system (an
inducer of extrinsic apoptosis) and hence confer cytoprotection (Spierings et al., 2003). Contrarily to the case of
TP53, so far no correlation has been found between the
levels of MAPKs or MAPK-related proteins and cisplatin
sensitivity in patients.
Alterations in any of the factors that regulate and
execute apoptosis, be it triggered by DNA damage or
oxidative stress via the mitochondrial pathway or be it
mediated by the extrinsic route, have the potential to
inﬂuence cisplatin sensitivity. Several dozens of proteins
(including death receptors, cytoplasmic adaptors, pro- and
antiapoptotic members of the BCL-2 protein family,
caspases, calpains, mitochondrial intermembrane proteins
and many others) participate in these lethal cascades and
most of them have been shown to modulate the response
to cisplatin (as well as to a plethora of other chemotherapeutic agents, drugs, toxins and stressors) in vitro (de La
Motte Rouge et al., 2007; Sakai et al., 2008; Tajeddine
et al., 2008; Wang et al., 2010; Janson et al., 2011).
However, only some of these proteins predict cisplatin
responsiveness in the clinical setting.
Oncogene
Chemoresistance to cisplatin
L Galluzzi et al
1876
Table 3 Mechanisms of post-target resistance
Factor
Mode of action
Relevance
Reference
BAX-like
proteins
Proapoptotic members of the BCL-2
protein family.
Castedo et al., 2006; Kroemer et al., 2007;
Tajeddine et al., 2008
BCL-2-like
proteins
Antiapoptotic members of the BCL-2
protein family.
BIRC5
(Survivin)
Caspase inhibitor of the IAP family that is
often upregulated in response to PI3K
signaling.
Component of CPC, a complex involved
in the regulation of chromosome
segregation.
Non-caspase proteases that participate
in the execution of multiple cell death
subroutines.
Mediate the initiator (caspase-9 and -8)
and executioner (caspase-3, -6 and -7)
phase of apoptosis.
Members of the JNK, ERK and SAPK
family transmit pro- and/or anti-apoptotic
signals in response to CDDP, with a
high degree of variability in different
experimental settings.
TP53 protein family member.
BAX/BAK-deﬁciency confers resistance
to CDDP and to several other stressors,
in vitro.
Conclusive clinical data are missing.
Overexpression of BCL-2, BCL-XL and
MCL-1 confers resistance to several
stressors, in vitro.
Clinical data link the expression levels of
antiapoptotic BCL-2 proteins with CDDP
resistance and recurrent disease.
Chemical inhibitors of BCL-2-like proteins are being clinically tested to overcome resistance.
BIRC5 overexpression is associated with
chemoresistance and poor prognosis in
multiple types of cancer.
BIRC5 inhibitors are currently being
evaluated in clinical trials.
Calpains
Caspases
MAPKs
DNp63a
TP53
XAF1
Tumor-suppressive protein that controls
DNA repair and apoptosis in response
to stress.
Also implicated in senescence, autophagy
and genomic stability.
Nuclear protein that antagonizes
the activity of IAPs, thus acting
as a proapoptotic factor.
In vitro, galectin-3 inhibition exacerbates
CDDP responses by enhancing calpain
activation.
In vitro, acquired resistance to CDDP is
link to modiﬁcations in the caspase
activation cascade.
JNK, ERK and SAPK inhibition has been
associated with both increased and decreased sensitivity to CDDP, depending
on the experimental setting.
Conclusive data are missing.
In vitro, transduces prosurvival signals in
response to CDDP.
CDDP-resistant tetraploid cells exhibit an
increased transcription of speciﬁc TP53
target genes.
Tumors harboring wild-type TP53 respond
better to CDDP-based chemotherapy.
High levels of XAF1 correlate with
improved progression-free survival in
advanced bladder cancer patients.
Han et al., 2003; Williams et al., 2005;
Erovic et al., 2005; Michaud et al.,
2009; Jain and Meyer-Hermann, 2011
(http://clinicaltrials.gov)
Kato et al., 2001; Nakamura et al., 2004;
Karczmarek-Borowska et al., 2005;
Altieri, 2008; Ryan et al., 2009
(http://clinicaltrials.gov)
Wang et al., 2010
Janson et al., 2011
Persons et al., 2000; Wang et al., 2000;
Dent and Grant, 2001; Yeh et al., 2002;
Mansouri et al., 2003; Brozovic et al.,
2004
Yuan et al., 2010
Peng et al., 1993; Gadducci et al., 2002;
Castedo et al., 20…

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