Cancer Drug Resistance: Unraveling Its Complexity

It’s a heartbreaking story, and one that happens too often. A patient with advanced cancer
receives a drug that helps shrink their tumors, allowing them more time with family, but then
weeks or months later the cancer comes back and the drug no longer works.

Drug resistance remains one of the biggest challenges in cancer therapy. It exists across all types
of cancer and all modes of treatment, including molecularly targeted therapy, immunotherapy,
and chemotherapy. Solving the puzzle of why cancers become resistant to therapy and how to
overcome or prevent it is a goal that NCI is pursuing on many fronts, including basic science to
understand biological mechanisms and clinical trials testing new treatment strategies.
Multiple factors, within cancer cells themselves and in the local environment in which the cancer
cells exist (the tumor microenvironment), contribute to how well a drug works. These factors
may differ from patient to patient and even among tumors in a single patient. Tumors are made
of diverse cells that may have different genetic, epigenetic, and metabolic characteristics that
have different sensitivities to treatment. Tumors also consist of immune cells, blood
vessels, fibroblasts, and other cells and components that interact with the cancer cells. These
interactions often promote tumor development, progression, and response to treatment.
Although a drug may kill some cancer cells, almost invariably a subset of them will be resistant
and survive the treatment. Cancers often have multiple mechanisms for surviving and growing,
which may change over time and in response to treatment. That is why combining treatments that
have different mechanisms of action can kill more cancer cells and reduce the chance that drug
resistance will emerge.
Most of the research on drug resistance has focused on identifying genetic mechanisms, such
as mutations that alter a protein such that it impairs the binding of a drug. Research is revealing
the importance of additional mechanisms of drug resistance, such as epigenetic factors that
regulate the activity of genes and the dynamics between diverse cells in the tumor
microenvironment. Overcoming resistance, then, requires understanding these complex
biological processes in the first place, to better anticipate and steer the dynamic,
multidimensional evolutionary process unfolding inside a patient with cancer.

Aided by advanced preclinical tools and new drug design approaches, NCI-funded researchers
are revealing a clearer picture of cancer drug resistance and developing new treatment
approaches to overcome it.
Targeting Cancer Cell Plasticity
Starting in 2006, doctors began to describe rare cases of patients with non-small cell lung cancer
(NSCLC) whose cancers transformed into small cell lung cancer after treatment with EGFR
inhibitors. This change in cell identity is one type of what scientists refer to as cell plasticity, and
NCI-funded research is piecing together the puzzle of how it may hinder cancer treatment.
Cell plasticity is a cell’s ability to undergo changes that alter its appearance and function
(its phenotype). These changes can occur in cancer cells because of genetic and nongenetic
alterations, cues from other cells in the tumor microenvironment, and/or drug treatment. A cell’s
ability to change and adapt offers it additional routes to resist treatment.
More recently, similar observations emerged from men with prostate cancer who were treated
with androgen deprivation therapy: aggressive and deadly forms of neuroendocrine prostate
cancers emerged. In addition to NSCLC and prostate cancer, scientists have described cell
plasticity in several additional cancer types, including melanoma and breast cancer.
While different cancers demonstrate their own patterns of cell plasticity, NCI-funded research
has revealed some biological mechanisms that are common across cancer types. For example,
research supported by NCI has implicated EZH2, an enzyme that regulates gene expression, in
the ability of both NSCLC and prostate cancer to change phenotypes. Thus, research on one
cancer may enrich studies of resistance patterns in other cancers, resulting in the identification of
treatments that could work for several cancer types. This example illustrates that our
understanding of the fundamental and molecular mechanisms of cancer is fueling advances
in precision oncology, including drugs approved to treat cancers with specific genetic
abnormalities rather than where in the body the cancer started.

Treating Cancers Like Evolving Ecosystems

A tumor can be thought of as an ecological system that evolves over time. Researchers are
therefore applying the concepts of evolutionary ecology to study cancer and its response to
treatment. Evolutionary ecology is a scientific field that examines how interactions among
species and between species and their environment shape species through selection and
adaptation, and the consequences of the resulting evolutionary change.

As an example, NCI-funded research led by investigators at Cleveland Clinic and Case

Comprehensive Cancer Center recently developed an "evolutionary game assay" that directly
quantifies and describes the interactions between tumor cells that are sensitive and resistant to a
targeted therapy in an experimental model of NSCLC. They found that the interactions between
cells were different under different conditions. The researchers suggest that changing the types of
interactions between cells—in other words, the "games they play"—can co-opt the cells’
evolution to better help the patient by preventing drug-resistant cells from “winning.”

Other NCI-funded studies are testing whether different drug doses and schedules might decrease
the likelihood that drug resistance will develop as a result of evolutionary dynamics. For
example, if drugs can be given in a way that allows a proportion of the easier-to-treat sensitive
cells to disproportionately survive, they may compete with and block the growth of the resistant
cells in the tumor. With this adaptive cancer therapy approach, it is possible that the tumor may
never be completely eradicated, but it may remain relatively stable, thereby limiting the
development of uncontrollable drug resistance.

Degrading—Not Blocking—the Target to Avoid Resistance

Using new technology to degrade proteins of interest, such as those that drive cancer cell growth,
is an emerging cancer treatment strategy. One example of this technology is called proteolysis
targeting chimera (PROTAC), in which molecules are generated that tag a specific protein for
degradation by a cell’s normal machinery for getting rid of unwanted or damaged proteins. An
advantage of this approach is that it can avoid some mechanisms of drug resistance seen with
some cancer therapies, such as mutations in the target of a drug or overexpression of the target.
For example, NCI-funded researchers at Yale University created a PROTAC molecule as a
potential treatment for advanced prostate cancer. Androgen receptor (AR) signaling plays a
pivotal role in prostate cancer initiation and growth, and drugs that inhibit ARs are the standard
of care for patients with metastatic disease. Unfortunately, most tumors treated with AR
inhibitors eventually develop drug resistance. Some mechanisms of resistance result in continued
AR signaling despite the presence of these drugs.
The PROTAC molecule the Yale team invented consists of an AR-targeting portion and a
portion that binds selectively to a protein, called E3 ligase. The E3 ligase is part of the cell’s
normal machinery that degrades proteins. With additional support from NCI’s Small Business
Innovation Research (SBIR) program, Arvinas, Inc. of New Haven, Connecticut, is further
developing and testing this PROTAC in clinical trials for patients with metastatic prostate cancer
whose cancer has progressed after AR therapy.
With the help of NCI funding, PROTAC and other similar technologies have shown promise in
addressing resistance in other cancer types, including chronic lymphocytic leukemia that has
become resistant to the drug ibrutinib (Imbruvica)
Using Advanced Preclinical Models to Address Resistance
Researchers have traditionally used cancer cell lines to study mutations and other mechanisms
that make cancer cells sensitive or resistant to therapies. But cell lines do not always share key
features of cancers found in patients. In addition, cancer cell lines lack the three-dimensional
structure of a tumor found in a person and the relationships with surrounding cells in the tumor
microenvironment.
Animal models, such as mice that carry tumors implanted from a sample of a patient’s cancer,
can more closely resemble the tumors found in humans. The tumor microenvironment and cancer
growth, progression, and treatment effects of animal models more closely mimic those found in
people. However, animal models are expensive, can take months to produce, and are not made in
large enough quantities for testing more than a few drugs at a time. They also lack an intact
immune system, making them inadequate to study the interaction between the immune system
and cancer. While current cancer models are useful for answering some research questions,
additional tools are needed.

NCI-funded researchers are leveraging new technologies and models to gain a fuller
understanding of drug resistance. They include three-dimensional human tumor cultures and
engineered platforms that support living human tissues, both of which incorporate cells that
surround and interact with tumor cells in the tumor microenvironment to mimic conditions in the
human body.

Miniature tumors called patient-derived tumor organoids are three-dimensional cancer cell
clusters grown in the lab from a sample of a patient’s tumor. Scientists are using them in the
laboratory to study various aspects of cancer biology, including mechanisms of drug resistance.
Researchers have developed organoids from a variety of cancer types, including breast, prostate,
liver, brain, and pancreatic cancer.
One example of this research is in pancreatic cancer, which is one of the most lethal cancer
types, in part because it is largely resistant to treatment and is generally detected at a late stage
after the cancer has spread. NCI-funded researchers at Cold Spring Harbor Laboratory in New
York and their collaborators have created a “living library” of pancreatic cancer organoids
derived from patient samples from multiple clinical institutions. In a retrospective analysis of a
small number of patients, the organoid’s sensitivity to chemotherapy reflected the patient’s
response to therapy. In addition, tumor sampling in a single patient over time predicted the
development of chemotherapy resistance that paralleled disease progression in the patient.
Understanding why resistance emerges and having models to help predict it could improve the
selection of treatments for patients in the future.

Scientists are developing additional cancer models that accurately represent the structure and
function of tumors in human organs and tissues. For example, tissue chips are three-dimensional
cross sections of living human tissue on a device about the size of a computer memory stick. For
cancer researchers, tissue chips and other engineered tumor systems are enhancing the
understanding of tumor physiology and aiding cancer treatment research.

For example, NCI is funding research projects that are developing and using engineered tumor
systems to study drug response and resistance in cancers of the brain, ovaries, and breast. In
addition, NCI’s SBIR program has supported several companies developing cancer chips,
including one developed by researchers at the University of Virginia and HemoShear
Therapeutics, LLC of Charlottesville, Virginia, to assess drug sensitivity and resistance in
pancreatic cancer.

Mechanisms of drug resistance in

cancer chemotherapy

Chemotherapy Resistance
What Is Chemotherapy Resistance?
Chemotherapy resistance occurs when cancers that have been responding to a
therapy suddenly begin to grow. In other words, the cancer cells are resisting
the effects of the chemotherapy. You may hear statements like the "cancer
chemotherapy failed." When this occurs, the drugs will need to be changed.

There are several possible reasons for chemotherapy resistance:

 Some of the cells that are not killed by the chemotherapy mutate (change)
and become resistant to the drug. Once they multiply, there may be more
resistant cells than cells that are sensitive to the chemotherapy.
  Gene amplification. A cancer cell may produce hundreds of copies of a
particular gene. This gene triggers an overproduction of protein that
renders the anticancer drug ineffective.
 Cancer cells may pump the drug out of the cell as fast as it is going in
using a molecule called p-glycoprotein.
 Cancer cells may stop taking in the drugs because the protein that
transports the drug across the cell wall stops working.
 The cancer cells may learn how to repair the DNA breaks caused by some
anti-cancer drugs.
 Cancer cells may develop a mechanism that inactivates the drug.

Research is underway to investigate ways of reducing or

preventing chemotherapy resistance.

The development of drug resistance is one reason that drugs are often given in
combination. It is thought that this may reduce the incidence of developing
resistance to any one drug. Often, if a cancer becomes resistant to one drug or
group of drugs, it is more likely that the cancer may be resistant to other drugs.
This is why it is very important to select the best possible treatment protocol
FIRST. In other words, when treating cancer, one should use the best weapon
when there is the smallest possibility of chemotherapy resistance.

Cancer Drug Resistance

One of the main causes of failure in the treatment of cancer is the development of drug resistance by the
cancer cells. This is a very serious problem that may lead to recurrence of disease or even death. This
section is intended to introduce some of the main ways in which cancer cells can resist treatments. It is
possible that more than one of these resistance mechanisms can occur in any given case. This page
contains information about:

 Selection of Resistant Cells

 Gene Amplification
 Multiple Drug Resistance (MDR)
 Blood-Brain Barrier
 Changes in Target Molecules

A Closer Look at Drug Resistance:

Methotrexate
Understanding the functions of methotrexate has led to a better understanding of the development of
drug resistance. There are three known ways in which a cell may acquire immunity to the effects of this
folate antagonist.
 1. Decreased concentration of the drug in the cell: The concentration of methotrexate in the cell can
be diminished by a change in the transport system that moves the drug into the cell. If there is
reduction in the number of channels through which methotrexate can move, less will be found
within the cell. Also, the concentration of the drug in the cell can be regulated by the altered
rates of metabolism. When the drug is metabolized it is more easily removed from the cell,
decreasing its concentration and activity within the cell. 1
2. Increased concentration of DHFR in the cell: Amplification of the DHFR gene causes an increase in
the amount of DHFR present and has been shown to correlate with reduced response to
methotrexate treatment.1
3. Mutations in DHFR that reduce DHFR:methotrexate binding: methotrexate must bind to DHFR to
prevent its activity. If a genetic change alters the binding region of DHFR in a way that reduces
methotrexate binding, DHFR will continue to activate folates and the effectiveness of the
treatment will decrease.1
All of these outcomes have been implicated in the increased resistance to methotrexate. Acquired
resistance to methotrexate is one of the primary complications of treatment with the drug. 2
Resistance to chemotherapy drugs is a key factor in the failure of many treatments.
Learn how cancer drugs are discovered and developed.

The Selection of Resistant Cells (Overview)

Selection for cancer cells that are resistant to a particular drug: While it is thought that the majority of
cancers arise from a single precursor cell, it would be an error to view a tumor as consisting of a
collection of genetically identical cells. One of the hallmarks of cancer is an increase in genetic instability
and mutation rates. These changes mean that dividing cancer cells acquire genetic changes (mutations) at
a high rate. Practically, this means that the cells in a tumor, while similar, are NOT identical. When
exposed to a cancer drug, those cells that are sensitive to the effects of the drug are killed. Those that are
resistant will survive and multiply. The result is the re-growth of a tumor that is not sensitive to the
original drug. Several reasons for the existence of the initial drug-resistant cells in the original tumor are
described below. For this reason, and others, chemotherapy drugs are often given in combination. While
the likelihood of a particular tumor cell being resistant to several drugs, especially those that attack
different cellular processes, is unlikely the large number of cancer cells in a tumor make that a real
possibility.3

Gene Amplification
As described in the previous sections, cancer drugs work by a variety of mechanisms. How is it that cancer
cells can become resistant to these different drugs? Listed below are some of the common ways in which
cancer cells avoid cell death in the face of chemotherapy and other treatments.
Increased expression of target proteins: Some cancer drugs, such as methotrexate, are designed to
inhibit particular enzymes in key pathways controlling cell growth and division. Increased expression
(transcription) of the gene that controls levels of the target molecule can cause a large increase in the
amount of that target molecule in the cell. Since the drug concentration in the cell is limited by the
dosages that can be given, the increased numbers of target molecules means that many of the targets do
not get affected by the drug. There are just too many for the number of drug molecules present. In the
amination below you can see that in the first situation, all of the 'target' molecules (green) are bound by
the drug (red). In the second situation, there are too many target molecules present. In a cell, this would
mean that the drugs effectiveness would decrease, perhaps to a point where it no longer slowed cell
growth
One mechanism by which the expression of the target genes can become elevated is through the process
of gene amplification. This process involves the selective replication of a region of a chromosome. The
process can be repeated many times, making many copies of that particular region. The gene
amplification event is diagrammed schematically below. The normal replication process is depicted on the
left and the amplification of a portion of the chromosome is shown on the right

The genes within the amplified portion of the chromosome can each be transcribed, leading to the
production of a large amount of the proteins encoded by those genes

Multiple Drug Resistance (MDR)

Failure of the drugs to enter the target cell and/or drug ejection: There are several reasons why drugs
may not reach therapeutic levels within cancer cells. One of the most frequent problems is the
amplification of a gene commonly known as MDR1 for multiple drug resistance. Another common name
for this same protein is the P-glycoproteinA protein that has been modified by the addition of one or more sugar
molecules (carbohydrates). The addition of the sugar molecules alters the properties/function of the protein. Many
proteins located on the outside of cell membranes are glycoproteins. . This gene encodes a large transmembrane
protein that has the ability to a) stop certain drugs from entering a cell and b) eject drugs from the cell
once they have entered. This combination of capabilities makes the MDR protein very effective at
reducing intracellular concentrations of a variety of chemotherapy agents. While the normal function of
this protein has nothing to do with chemotherapy drugs, it is quite often the reason for chemotherapy
drug failure. The prevention of drug entry and drug ejection are depicted in the animation below
The importance of MDR mediated drug resistance is underscored by the fact that several drugs designed
to inhibit the activity of this protein are currently under investigation. 4In addition, MDR is being examined
as a gene therapy treatment that could be used to increase drug tolerance in individuals undergoing
chemotherapy. The MDR gene is being inserted into bone marrow stem cells which are then placed back
into the patient. The increased expression of MDR in the bone marrow cells renders them less susceptible
to the harmful effects of the drugs and allows the patient to tolerate higher doses of the chemotherapy
drugs. It is hoped that the increased levels of the drugs will more effectively eliminate the cancer

Blood-Brain Barrier
Depending on the size and location of the tumor, it is possible that the treatments being used may not be
able to gain access to the target cells. In large tumors, the central portions may be hard to reach due to
limited blood supplies in the tumor. A different problem is encountered in the treatment of cancers
located in the brain cavity. The brain is supplied with nutrients by a network of blood vessels. These
vessels are constructed in a manner slightly different from the majority of the circulatory system. The
changes in these vessels make it difficult for many different types of molecules to cross into the space
surrounding the brain. The restricted movement of molecules across these vessels is termed the blood-
brain barrier. For this reason, certain drugs are ineffective against brain tumors. The process of selective
movement across the blood:brain barrier is shown below
Changes in Target Molecules
The target molecule is no longer present: It is possible that the target of a particular treatment is lost
during the progression of cancer development. An example would be the loss of the estrogen
receptor (ER) from breast or ovarian cancer cells. This change would theoretically render the use of the
anti-estrogen drug tamoxifen much less effective. The loss of the ER from these cells is an indication that
the cells are no longer dependent on the presence of estrogen as a growth stimulator. For this reason,
the status of the ER is often determined during the initial phase of breast and ovarian cancer diagnosis. 3
The target molecule is altered: Gene mutation is common in cancer cells. Exposure to chemotherapy
drugs can kill cells that have a normal version of a particular target while sparing those that have acquired
a modifed version of the gene. While the slightly altered version of the gene may still function in the cell,
it can no longer be inhibited by that particular drug. The process is depicted below.
An example of the above process is the selection for drug resistance in patients treated with
the kinase inhibitor Gleevec®. Recent research has identified specific mutations in the target gene that
render the protein resistant to the drug