Escolar Documentos
Profissional Documentos
Cultura Documentos
Shubhangy Raghavan
Capstone I &II
Capstone 2 Period 3
Fall 2018
Abstract:
metabolism, which is beneficial to proliferating cells due to the increase in biosynthesis. In non-
cancer cells, the metabolism is not reverted back, thus leaving the cell in a constant state of
growth and division, which aids in the formation and spread of tumors. Various factors such as
hypoxia and gene profiles affect the extent of metabolic reprogramming. One of the main
phospholipids used in the cell membrane. The metabolic reprogramming of choline metabolism
is critical to the formation of tumors. GDPD6, a gene that influences the expression of GPC, a
metabolite used in choline metabolism, was overexpressed in one group, wild-type or random in
another group, and empty-vector (or silent/not-expressed) in another group of MCF-7 breast
cancer cell lines. The intent of this study was to treat cell lines with estrogen after a period of
starvation. Following the treatment, western blot and NMR-Spectroscopy would be performed
on the cell lines to determine the level of GPC present in the samples. It was expected that in
cells with an overexpression of GDPD6 treated with estrogen, there was an increase in the
amount of GPC when compared to empty-vector for GDPD6 cells. This suggests that GDPD6
regulates the amount of GPC present in cells. In reality, the transfection was not successful and
Introduction:
Since the time of the Ancient Egyptians, cancer has been a disease plaguing
humankind. According to cancer.org the word cancer was created by Hippocrates, who was
considered by many to be the father of medicine. While there was no cure for the disease then,
science has developed new techniques, such as radiation, chemotherapy, and more recently
immunotherapy. During the time of the Ancient Egyptians, there was no cure or treatment
available because the understanding of cancer was limited (Early History of Cancer, 2014).
Oncology, the study of cancer, only began in 1761 after Giovanni Morgagni conducted
autopsies related to the illness. Around this time, the only treatment available was surgery to
remove the tumor. If the tumor had already began to move, then the surgery was most likely not
Cancer is caused when cells grow and divide out of control. The acceleration of
proliferation, or cells growing and dividing, causes DNA mutations to accumulate. The
breakdown of the cell-cycle, the inhibition of proto-oncogenes such as p53, and the use of
oncogenes drives the formation of cancer (What is Cancer, 2015). Cancer cells skip
checkpoints in the cell cycle, and thus divide with mutated DNA. Without the cell cycle
regulation and without greater apoptosis around the body, these proliferating cells accumulate
and become a tumor, which is called tumorigenesis (What is Cancer, 2015). In some cases, a
cell can get into the bloodstream and travel to a different part of the body, a process called
metastasis (What is Cancer, 2015). Cancers are named for their origin, however (What is
Cancer, 2015). According to cancer.gov, 1,685,210 new cases of cancer will be diagnosed in a
year. The most common will be breast cancer. According to breastcancer.org, 1 in 8 women in
Metabolism is the total chemical reactions in a cell. Cellular respiration is the use of food
as an energy source and chemically altering it to a form of energy the body can use and one of
the most important metabolic pathways. In oxidative respiration, there are four stages.
Glycolysis is the transformation of sugar, or in some cases fat and proteins, into pyruvate
molecules, 2 NADH (an electron carrier) and 2 ATP. This occurs through series of chemical
reactions within the cell. The next stage is called the link reaction, wherein the pyruvate
molecules form into Acetyl-CoA for the third step: Krebs Cycle. During the Krebs cycle, the
Acetyl-CoA undergoes many reactions and produces 2 ATP as well as NADH and FADH2
(electron carriers). Finally, the fourth step: oxidative phosphorylation. The electron acceptors
power the membrane proteins on the inner mitochondrial membrane. All the H+ ions actively
transport from the mitochondrial matrix to the intermembrane space. The electrons are the
energy source and thus NADH is converted to NAD+ and H+, FADH2 is converted to FAD+ and
2H+, and the remaining electrons are picked up by oxygen to create water. Then, the H+ ions
diffuse from the intermembrane space to the mitochondrial matrix through a channel called ATP
Synthase,which powers the ATP synthase to create ATP from ADP and a phosphate (Cellular
Respiration, 2004).
This can only occur when oxygen is present. So what happens when there is not enough
oxygen? The cell uses anaerobic respiration. The cell will only use glycolysis to create its
energy. Under anaerobic conditions, metabolism in the cell results in lactic acid formation that
leads to muscle cramps (Cellular Respiration, 2004). Hypoxia is a condition of low oxygen
availability. Many cancer cells are under hypoxic conditions, so they use anabolic metabolism to
meet their needs (Semenza, 2016; Ward & Thompson, 2012). Anabolic metabolism is the
synthesis of molecules from smaller molecules. This is beneficial to cancer cells because it
allows them to meet the demands of proliferating cells. Metabolism results in increased
production of certain enzymes or proteins (Ward & Thompson, 2012). The production of lipids,
Metabolic changes in cells can be studied at both gene and protein expression level.
oligonucleotide sequences of various genes on a glass slide. One can compare the expression
obtained from two different sets of samples with the oligos on the slide and then scan to
measure the expression of genes. If the expression of a certain gene is higher, the area will
appear red. If it is lower, it will appear green. However, if it is equal, then it will appear yellow.
This is used by many scientists, especially when testing for amounts of proteins, enzymes, or
Western Blots, also called immunoblotting, is another tool to test for protein expression.
By treating the protein sample with a primary antibody against a specific protein, the protein is
then “highlighted” among the protein sample. By treating the protein sample with a secondary
antibody, it ensures that the protein sample is from the species being tested. Western blots will
electrophoresis. After isolating the sample protein, it is transferred onto a polyacrylamide gel
using a power source, electroporator and a gel rack. The protein is then transferred from the gel
to a nitrocellulose membrane, which is then treated with a primary antibody and a secondary
antibody that is conjugated with a peroxidase enzyme. When imaged, the nitrocellulose
membrane will show bands where the protein is concentrated and based on the molecular
weight of the protein as well as the thickness of the band, researchers can determine the type of
protein as well as the amount in comparison to another sample (Mahamood & Yang, 2012).
The first source focuses on the metabolic reprogramming of cancer, from catabolism to
anabolism and how cancer affects the reprogramming of these pathways. The second source
focuses on how cancer cells survive under hypoxic conditions. The third and fourth source focus
on the potential use of gene signatures in diagnosing and developing a prognosis for a patient,
as well as necessary treatments. The fifth source focuses on choline metabolism, and how
Metabolic Reprogramming:
One of the most popular theories on how to treat the metastasis and growth of cancer
tumors is to deprive them of nutrients. But it was discovered that cancer cells adapt their
metabolic pathways in order to meet their energy needs. Otto Warburg was a Nobel laureate
biochemist. He discovered the disparity in metabolic systems in cancer cells. The next source is
called "Metabolic Reprogramming: A Cancer Hallmark Even Warburg Did Not Anticipate" and
was published in March of 2012. It was written by Patrick Ward and Craig Thompson. Ward
works at the Cell and Molecular Biology Graduate Group in the Perelman School of Medicine at
the University of Pennsylvania. This is a respected university, adding to his credibility. Both men
work at the Cancer Biology and Genetics Program at the Memorial Sloan-Kettering Cancer
Center in New York. Thompson, who was the head of this project, has studied the metabolic
pathways before. This source is a review of research available at the time. One of the main
sources cited by Ward and Thompson was written by Jan Rydstrom, a researcher at the
in Sweden. His paper was published in 2006 and has studied metabolism before. The second
source was written by Douglas Hanahan and Robert A. Weinberg in 2011 who both have a
history of studying cancer. Hanahan was working for the Department of Biochemistry and
Biophysics at the University of California, San Francisco, a highly respected institute. Weinberg
worked for the Whitehead Institute for Biomedical Research at Massachusetts Institute of
Technology, a world-renowned university. The third source was written by Buzzai et. Al in 2005.
Monica Buzzai worked at the Abramson Family Cancer Research Institute, Department of
Cancer Biology at the University of Pennsylvania, a respected institute in the United States.
Buzzai has spent much of her career studying metabolism. The fourth source was written by
Duvel et al. in 2010. Katrin Duvel works for the Department of Genetics and Complex Diseases
at the Harvard School of Public Health in Boston, Massachusetts, a world-renowned university.
Duvel has researched the regulation of metabolic signaling pathways in yeast and mammalian
systems.
While this research team did not conduct any experiments themselves, many of the
studies they were referencing focused on the metabolic pathways, and how they differ in
proliferating cells, as well as cancer cells. The research question was how does cancer, cancer
pathways are the chemical reactions undergone by a living organism in order to produce
energy. Ward and Thompson believed that changes in metabolic pathways or the metabolism is
directly related and influenced by cancer and is a clear indicator of cancer. Cancer influences
the metabolic pathways and changes them to suit their metabolic needs.
Since there were many different sources used by the research team, the variables vary
for each source. Overall, the independent variable is the state of the cell (cancerous,
proliferating, dormant, etc.). The dependent variable is the metabolic pathways, or the metabolic
reprogramming undergone by the cell. The control is non-cancerous cells (Ward & Thompson,
2012).
None of the studies mentioned by Ward and Thompson used animal testing. All of the
testing was done on either cells, mitochondria, or metabolic pathways. This allowed for
researchers to isolate the organelle, pathway, or cell in question, and test their hypothesis in a
controlled manner with no input from external factors (Ward & Thompson, 2012).
The methods differed in each source cited by this article, but the way metabolic changes
are measured is standard throughout most experiments. Various methods have been used
understanding the metabolic changes in cancer cells in vitro and in patients in the clinic.
To study metabolic changes in tumors in patients and also their treatment, radiolabeled
glucose analog with isotope of fluorine (18F) called 18F-deoxyglucose (FDG) is frequently used
and metabolic changes analyzed using positron emission tomography (PET). Images obtained
upon injecting FDG will pick up areas of tumor where there is no available oxygen. This
Understanding of the cancer cell metabolism is achieved through altering the growth
factor requirement for cancer cells to grow in tissue culture plates in the lab. Initial studies were
performed by feeding the cells with radiolabeled glucose or growth factors and looking at the
formation of product in the form of protein at various time points. Once the various proteins and
enzymes that convert each product along the pathway was purified and inhibitors found,
metabolic alterations were confirmed upon addition of inhibitors by either western blotting or at
mRNA level changes by polymerase chain reaction (PCR). Presently, metabolic changes are
sequencing in the tumor cells under various conditions. To distinguish various metabolites,
biomolecules produced and used in metabolism (a middle molecule), in the cytosolic and
membrane compartment, cell fractionation was performed and assessed for metabolic changes
There are many notable results . Nutrient uptake and metabolism type is dependent on
the amount of nutrients in the environment (Ward & Thompson, 2012). Signaling pathways can
be activated without growth factors (Ward & Thompson, 2012). If there is a low level of growth
factors, the cell will undergo catabolic metabolism, which is the breakdown of larger molecules
into smaller molecules (Ward & Thompson, 2012). If there is a high level of growth factors, the
cell will undergo anabolic metabolism, which is the buildup of large molecules from smaller
molecules (Ward and Thompson, 2012). In proliferating cells, changes in the rate of turnover of
molecules through a metabolic pathway, also called a metabolic pathway, occurs in response to
growth factors, regardless of ATP (Ward & Thompson, 2012; DeBerardinis et al, 2008). In
proliferating cells, it is imperative that their metabolism heads towards the direction of
macromolecule synthesis in order for the cell to double its mass and then divide (Ward &
Thompson, 2012; DeBerardinis et al, 2008). That is why proliferating cells, as well as cancer
cells, need anabolic metabolism, as "proliferating cells are in much greater need of reduced
carbon and reduced nitrogen, as well as cytosolic NADPH for reductive biosynthetic reactions ."
with the aerobic metabolism occurring as a secondary method (Ward & Thompson, 2012;
DeBerardinis et al, 2008). When the pathway PI3K/Akt, which controls the cell cycle, gets
damaged, it causes cancer spontaneously (Ward & Thompson, 2012; DeBerardinis et al, 2008).
It also causes an increase in glucose uptake and glycolysis. This increases the biosynthesis of
the cell, allowing it to grow and divide (Ward & Thompson, 2012; DeBerardinis et al, 2008).
Most cancers depend on the synthesis of amino acids (Ward & Thompson, 2012). Another
factor, called hypoxia-inducible factor 1(HIF-1) converts pyruvate into lactate, and blocks
glucose carbon incorporation into mitochondrial citrate, which is important for lipid production
(Ward & Thompson, 2012; DeBerardinis et al, 2008). Another transcription factor, called Myc
transcription factor, promotes the use of glutamine, which acts as a nitrogen donor, something
important to proliferating cells (Ward & Thompson, 2012; DeBerardinis et al, 2008).
In addition, expressing the isoforms, or a different form, of metabolic enzymes can allow
cancer cells to select for metabolic reprogramming (Ward & Thompson, 2012). The isoforms
allow for anabolic metabolism, which leads to a larger scale of biosynthesis. This ends up
allowing the cell to grow and divide. Amplification of metabolic enzymes may also facilitate
anabolic metabolism (Ward & Thompson, 2012; DeBerardinis et al, 2008). Some accumulation
of metabolic enzymes can cause metabolic reprogramming that eventually facilitates anabolic
metabolism which leads to cancer (Ward & Thompson, 2012; DeBerardinis et al, 2008). The
certain genes and can lead to cancer developing (Ward & Thompson, 2012). It affects cell
differentiation. 2HG also affects the expression of certain genes (Ward & Thompson, 2012).
Metabolites such as 2HG, succinate and fumarate that help in progression of cancer are termed
as oncometabolite (Ward & Thompson, 2012; DeBerardinis et al, 2008). Proliferating cells use
these pathways, oncometabolites, and growth factors to retain the flux of carbon in order to
promote biosynthesis (Ward & Thompson, 2012; DeBerardinis et al, 2008). This promotes
growth and division, which can lead to cancer if not controlled (Ward & Thompson, 2012;
Thompson and Ward believed that cancer influences the metabolic pathways and
changes them to suit their metabolic needs. This was supported by the results because
oncogenes and cancer influences the expression of genes and growth factors which influence
metabolic pathways (Ward & Thompson, 2012). Changes in genes expression, enzyme
activities, and growth factors push for metabolic reprogramming which influences the
mitochondria to conduct anabolic metabolism (Ward & Thompson, 2012). Anabolic metabolism
is more favorable to proliferating cells due to the increase in biosynthesis (Ward & Thompson,
2012). This allows cancer cells to grow and divide and form tumors.
proliferating cells, and cancer cells use the reprogrammed metabolism to continue to proliferate
and spread. This reprogrammed metabolism is anabolic metabolism, which is more useful for
cells that need to synthesize biomolecules (Ward & Thompson, 2012). This source explains the
factors that cause metabolic reprogramming, making it relevant. The take home value is that
cancer cells select for anabolic metabolism and thus metabolic reprogramming because it is
more beneficial to them (Ward & Thompson, 2012). It is not a secondary effect, as what was
thought before. While it was already known cancer cells have a different metabolic signature
from other cells, Otto Warburg thought it was a secondary effect, and not the main driver of
cancer (Ward & Thompson, 2012). This adds to scientific knowledge on the subject by taking
Warburg's theory and disproving it with recent data. They conclude that the metabolic changes
a cell undergoes allows cancer to proliferate and thrive (Ward & Thompson, 2012). This can
allow for a future cure because scientists can focus on controlling the metabolic changes in
supported the findings of Ward and Thompson. Wang et al used isolated murine primary T cells
that were either maintained in interleukin 7 or stimulated with anti-CD3 plus anti-CD28 (Wang et
al, 2011). The cells were then used for the study after incubation for 72 hours. During this time,
the activated T cells increased in size for 24 hours. Over the next 24-72 hours, the T cells
underwent a rapid division process, providing a window during which they underwent changes
et al, 2011). T cells that had been activated for a longer time had a higher concentration of
metabolites when compared to resting T cells (Wang et al, 2011). The higher concentration of
metabolites was especially prevalent in the pathways that produce lipids, amino acids, and
nucleotides (Wang et al, 2011). There was a lower concentration of carnitines, which is used for
fatty acid oxidation (Wang et al, 2011). According to Wang et al, the activation of T cells
breakdown of glucose through the pentose phosphate pathway (PPP), and oxygen consumption
(Wang et al, 2011). Fatty acid oxidation and pyruvate oxidation through the Krebs cycle showed
a lower rate (Wang et al, 2011). After as a follow-up, Wang et al applied chemical inhibitors
targeting activation-induced signaling pathways (Wang et al, 2011). Then, they followed the
expression of Myc and HIF-1a (Wang et al, 2011). They found inhibiting any of the pathways
reduced the induction of Myc and HIF-1a after T cell activation (Wang et al, 2011). This
This supports the idea that during the proliferation of cancer cells, the metabolic
pathways are influenced, with some being repressed and others being stimulated (Wang et al,
2011; Ward & Thompson, 2012). The pathways that synthesize biomolecules such as lipids,
amino acids, and nucleotides, are stimulated and increase production of biomolecules (Wang et
al, 2011; Ward & Thompson, 2012). The fatty-acid oxidation and pyruvate oxidation through the
Krebs cycle was diminished (Wang et al, 2011; Ward & Thompson, 2012). The cell still creates
energy through glycolysis, which is heightened to meet the energy needs of the cell (Wang et al,
This section gave an overview of the metabolic changes that occur when cancer
proliferates, including the metabolites, genes, and pathways that affect the metabolic
reprogramming. These genes play an important role in the composition of the gene signature,
which will be discussed in future sources. Choline metabolism, a pathway that is affected by
metabolic reprogramming in cancer proliferation, will be explored as well. The next source looks
at an environmental factor called hypoxia, as well as its effect on one of the genes in particular
[Hypoxia Inducible Factor-1 (HIF-1)], and its effect on cell growth and cell biology. HIF-1 was
Our body needs oxygen to survive, so what happens when cancer cells are in oxygen-
deprived environments? This was the subject of years of research and is what the second
Metabolism and Redox Regulation with Induction of the Breast Cancer Stem Cell Phenotype".
This study was done by Gregg L. Semenza, a researcher at the Department of Pediatrics,
Institute of Genetic Medicine at the Institute for cell Engineering at Johns Hopkins University
School of Medicine where the study was done. He is a renowned researcher and has studied
cancer and its relationship to oxygen for decades. This article was published at the end of 2016
in The Embo Journal. This source references other articles. The first article was written by Kim
et al in 2006. He works at the Graduate Program of Pathobiology at Johns Hopkins. Kim has
written multiple papers on cancer metabolism. The second article he references is by Samanta
et al in 2016. Samanta works at the Institute for Cell Engineering at Johns Hopkins School of
Medicine. He has written many articles on cancer, as well as hypoxia and HIF.
The question this article is asking is how hypoxia inducible factors affect the metabolism
in cells as well as how they affect breast cancer stem cells. Semenza believed that HIF allows
cells to survive in hypoxia and HIF increased the production of mitochondrial antioxidants
(Semenza, 2016). The independent variable is cells induced in hypoxia (Semenza, 2016). The
dependent variable is the change in metabolism. The control is control cells, or cells that were
not exposed to hypoxia (Semenza, 2016). There were many references mentioned in this
review article. Most references used various types of cells, from breast cancer cells to general
mammalian cells (Semenza, 2016). One project cited used mammalian cells and exposed them
to hypoxic conditions. Mammalian cells were used in order to demonstrate the effect in
mammals such as mice, rats, and humans. Another reference used HIF-1a knockout mice
embryo fibroblasts (Semenza, 2016). HIF-1a knockout mice are mice that do not have hypoxia
induced factors, or HIF. Fibroblasts are cells in connective tissue that produce collagen and
other fibers. Mice embryonic fibroblasts are fibroblasts taken from mice embryo. In this
reference, HIF-1a knockout mice embryo fibroblasts were exposed to hypoxia and their
metabolisms were tested (Semenza, 2016). This was done to discover the effect of hypoxia
induced factors on the cell's ability to survive in hypoxia. They were compared to wild-type cells,
which are just control cells (Semenza, 2016). Another source used six lines breast cancer cells
that included estrogen receptor-positive (ER+) and estrogen receptor-negative (ER-) breast
cancer cells (Semenza, 2016). They used breast cancer cells to discover whether exposing
breast cancer cells to hypoxic conditions would increase antioxidants production. ER(+) MCF-7
cells and ER(-) MDA-MB 231 cells were used to test the previous hypothesis that stem cell
metabolism was designed to protect the cells against oxidant exposure (Semenza, 2016).
These cells were exposed to 1% oxygen for three days (Semenza, 2016). MDA-MB-231 cells
were then injected into the mammary fat pad of immunodeficient mice to test whether the cells
could still form tumors (Semenza, 2016). The cells were still efficient at forming tumors but in
the tumors, there were less breast cancer stem cells. If conditions were limiting (less cells were
injected), the cells were less successful at forming tumors and metastasis the tumors. The stem
cells increased oxidant exposure to assist cell survival and tumor formation. The mammary fat
pad is the breast of the mice. Immunodeficient mice are mice where their immune system is
weak or non-existent.
Breast cancer cells were used to test the effect of HIF-1 on chemotherapy resistance
(Semenza, 2016). They were divided into two groups, where one group was treated with
cytotoxic chemotherapy and the other group was not treated (control). Gene expression data
from 3,500 human breast cancers were used to test whether the effect of hypoxia on
chemotherapy was relevant to humans with breast cancer (Semenza, 2016). The level of
metabolic enzymes were tested (Semenza, 2016). Triple negative breast cancer cells were also
used as a comparison (Semenza, 2016). Breast cancer stem cells were used to test whether
HIFs affected the specification of breast cancer stem cells. (Semenza, 2016)
Cells switch from oxidative metabolism to glycolytic metabolism in order to avoid build-
up of ROS, of reactive oxygen species (Semenza, 2016). ROS are chemically reactive
components that contain oxygen. A build-up of ROS can lead to cellular dysfunction and death.
Exposing cells to oxygen leads to the production of ROS, but exposing cells to not enough
oxygen also leads to the production of ROS. Thus, cells must be exposed to a certain range of
oxygen, which for most mammalian cells is 20-65 mmHg (Semenza, 2016). Hypoxia-induced
factor, also called HIF-1, induces genes encoding glycolytic enzymes in cells that are under
hypoxia (Semenza, 2016; Ziello et al, 2007). HIF-1 activates the expression of PDK1 (pyruvate
dehydrogenase kinase) and LDHA (lactate dehydrogenase A) to switch cells from oxidative
LDHA converts a form of lactate and NAD to pyruvate and NADH in anaerobic
respiration. PDK1 becomes pyruvate dehydrogenase which facilitates the change from pyruvate
to Acetyl CoA and entry into the Krebs Cycle (Kim et al, 2006). PDK1 is also targeted by Myc
transcription factor (Kim et al, 2006). In Hif1a-/- mice exposed to hypoxic conditions, PDK1 did
not increase in levels as it did in wild type mice (Kim et al, 2006). This shows that the activation
of PDK1 relies on HIF-1a. In HIF1a-/- mice that were exposed to hypoxic conditions and had
forced overexpression of PDK1, proliferation was able to continue in contrast to when there was
no forced expression (Kim et al, 2006). In addition to helping proliferation, it also stopped
hypoxia induced apoptosis, or programmed cell death (Kim et al, 2006). This shows that HIF-1a
LDHA facilitates the transition of pyruvate to lactate acid (Semenza, 2016). HIF-1 can
autophagy is the breakdown of the mitochondria (Semenza, 2016) . Thus, it can be concluded
that HIF-1 regulates glucose metabolism based on oxygen levels to avoid the risk of excess
of HIF-1 called HIF-1a, increases the production of mitochondrial antioxidants (Semenza, 2016;
Zhong et al, 1999; Kim et al, 2006). HIF-1a was found to be more important in regulating
metabolism in comparison to HIF-1b (Kim et al, 2006; Semenza, 2016; Zhong et al, 1999). It
induced the expression of mRNAs encoding enzymes important to the serine synthesis pathway
and mitochondrial one-carbon metabolism in four out of six human breast cancer cells analyzed,
with PHGDH and SHMT2 found in all six lines. These are the enzymes used in the first reaction
for the serine synthesis pathway and the mitochondrial-one carbon metabolism (mito1CM),
respectively. In a 1999 paper published by Hua Zhong, they created an antibody against HIF-1a
protein, which can be useful in determining the activity of the protein, and tested it in cancer
cells. The antibody functioned properly in those cells so they tested the antibody in tissues
(Zhong et al, 1999). The immunoblots from these test showed that the antibody could pick up
the activity of the HIF-1a protein where it was present (Zhong et al, 1999). This was a huge deal
for researchers because they could now use immunohistochemistry (the process described
Exposing ER+ MCF-7 cells and ER-MDA-MB-231 cells to low levels of oxygen for three
days caused an increase in breast cancer stem cells, but was repealed by PHGDH knockdown
glycolysis that helps catalyze the change from one metabolite to another, with the end result
being pyruvate molecules (Semenza, 2016). It did not affect the ability to form a tumor in the
latter cells, but the resulting tumors were reduced about four fold (Semenza, 2016). When
BCSC were limited, tumors formed in only six out of fourteen mice injected with PHGDH-
knockdown breast cancer cells (Semenza, 2016). In mice injected with control cells, seven out
of seven mice formed tumors (Semenza, 2016). PHGDH-knockdown inhibits metastasis. Breast
cancer cell lines that have PHGDH gene amplified is impaired by PHGDH knockdown
(Semenza, 2016). The loss of this gene does not impair the growth of cells without PHGDH
amplification (Semenza, 2016). PHGDH had no effect on the growth of ER+ MCF-7 cells and
increased the growth of ER- MDA-MB-231 cells (Semenza, 2016). This suggests the serine
synthesis pathway, which produces amino acids and proteins, is not important for the
the production of new blood vessels) therapy decreases tumor growth but increases tumor
hypoxia, leading to the induction of HIF-1 activity (Ziello et al, 2007; Semenza, 2016). This
activity promotes cancer growth. Combination with a HIF-1 inhibitor prevented the induction of
HIF-1 activity and increased survival in a mouse with metastatic breast cancer (Ziello et al,
2007; Semenza, 2016). An analysis of 3,500 breast cancers revealed that additional expression
of PHGDH mRNA or mRNA encoding enzymes for serine synthesis pathway and mito1CM. This
shows that exposure of breast cancer cells to hypoxia induces the glucose metabolites that are
The hypothesis was supported because he hypothesized that HIF increased the
production of mitochondrial antioxidants, and that HIF allows cells to survive in hypoxia
(Semenza, 2016). This was proven in the results of the papers he cited. HIF-1 regulates glucose
metabolism and oxidative metabolism in order to allow the cell to survive in hypoxia or
hyperopia (Ziello et al, 2007; Kim et al, 2006; Zhong et al, 1999; Semenza, 2016). HIF
enhances the flux through the serine synthesis pathway as well as mito1CM pathway, which
results in the production of antioxidants (Ziello et al, 2007; Semenza, 2016; Zhong et al, 1999).
HIFs also contribute to the specification of breast cancer stem cells by increasing the
expression of genes coding for pluripotency (cell diversity) factors. (Semenza, 2016)
This study is relevant because it is a very recent study which summarizes the effect HIF
and hypoxia has on breast cancer cells and metabolism. The take home value is that HIF-1
regulates glucose metabolism in order for the cell to survive in abnormal oxygen levels (Ziello et
al, 2007; Semenza, 2016; Zhong et al, 1999; Kim et al, 2006). HIF also increases the flux of the
pathways that produce antioxidants (Zhong et al, 1999). This study adds to the scientific
knowledge on the subject by showing how HIF can influence the metabolism of the cell in
hypoxia.
Semenza examined the effect of HIF and hypoxia on cancer proliferation, something that
was mentioned in Thompson and Ward’s article. Semenza mentioned the various proteins and
biomolecules affected by HIF-1. While these sources examined the effect of one specific gene,
HIF-1, on cancer cell proliferation in hypoxic environments, the next two sources will examine
how the total expression of genes can be used in breast cancer research and treatment.
It is no secret that the total composition of genes in cancer cells can determine the
composition of a tumor. The use of gene signature has been examined by researchers in both a
research and clinical setting, in a New England Journal of Medicine article from March 2009
called "Gene-Expression Signatures in Breast Cancer.” It was written by Christos Sotiriou and
Lajos Pusztai. Sotiriou is a tumor biologist in the Universite Libre de Bruxelles, a private and
prestigious college in Brussels. He has worked on tumors and cancers for thirteen years. Lajos
fellowship at the University of Texas. This study was conducted at the Universite Libre de
Bruxelles and the Department of Breast Medical Oncology at the University of Texas, Anderson
Cancer Center. This source references other articles. One such article is by Vijver et al. which
will be used as the next source. The second source is by Buyse et al in 2006. Buyse is a cancer
researcher at the International Drug Development Institute in Belgium. He has written many
articles about gene signatures in breast cancer. The third source is by Bueno-de-Mesquita et al
in 2007. He works at the Netherlands Cancer Institute and has published many articles on the
use of gene sequencing to treat breast cancer. The fourth reference is authored by Haibe-Kains
et al in 2008. He works at Universite Libre de Bruxelles and has authored other papers on
cancer and gene sequencing. The fifth source is by Habel et al in 2006. Habel works at the
Division of Research, Kaiser Permanente, in Oakland, California. Habel has written many
articles on breast cancer. The sixth source is by Ayers et al in 2004. He works at Millennium
Pharmaceuticals Inc. He has authored many papers on genes and gene sequencing and
signatures. The seventh source is by Hess et al in 2006. He works at the University of Texas
and has authored many papers on breast cancer. The eighth reference is by Loi et al in 2007.
Loi works at Universite Libre de Bruxelles and has authored many papers on breast cancer.
The question they are asking is how can gene expression signatures be used in cancer
treatment and research. They hypothesized that using gene expression signatures can lead to
better classification system which will lead to a better and more specified or catered treatment
options and more accurate prognosis. The independent variable is the gene profile and the
prognosis. The dependent variable is the treatment called for and the accuracy of the prognosis.
The constant is the type of cancer they are using, as well as the animal (humans).
Some references this source cites used humans in their experiment. In one, 78 patients
with node-negative breast cancer who received no systemic adjuvant therapy were measured
for the expression of 70 genes. The assay then calculates a prognosis score that categorizes
patients into good or poor outlooks. 130 patients received systemic adjuvant chemotherapy or
hormonal therapy. In a validation study, 307 patients who had received no systemic therapy
were used in the same way. The assay tested the patients for the expression of 70 genes and
calculated a prognosis score based on that. In both cases, the patients were followed up on
once a year for 10 years. Patients with node-negative breast cancer were used in these cases
because if they had node-positive breast cancer, they would have cancer in their lymph nodes
as well, possibly affecting their treatment. Mainly for this reason, using gene signatures was
only approved by the FDA for node-negative patients. Some patients received no systemic
adjuvant therapy in order to test the probability of a cancer relapse and to test the accuracy of
the prognosis score.In another reference, 650 patients with ER-(+ve) breast cancer were
selected as long as the patient had no prior treatment or were only treated with tamoxifen. An
assay tested for 97 gene signatures in these cases in order to discriminate between low grade
and high grade tumors. ER-(+ve) breast cancers respond to estrogen and were chosen in this
reference because ER-(-ve) breast cancer is treated differently. In 668 patients with ER(+ve),
node-negative breast cancer who were treated with tamoxifen, the assay tested for the
probability of a relapse in cancer. Each patient was followed up with for 10 years to determine
the accuracy of this probability. 651 patients with ER-(+ve), node negative, tamoxifen treated
breast cancer were used to examine the association of the recurrence score (probability of
cancer relapse) with adjuvant chemotherapy treatment. The difference in adjuvant treatment
was used to see if the recurrence score was affected by the adjuvant treatment. Biopsy samples
from 133 patients with stage 1, 2, or 3 breast cancer who received a weekly treatment of
paclitaxel combined with fluorouracil, doxorubicin, and cyclophosphamide were used to test
whether gene expression profiles of cancers that readily respond to chemotherapy differs from
cancers that are slightly more resistant to chemotherapy. 82 of those selected were used to
create a multigene signature predictive of pathologic complete response and 51 patients were
used to test the accuracy. Publicly available gene expression data from 3000 breast tumors
were used to examine the relationship between risk of recurrence and molecular subtype. This
was used because the authors wanted to test the gene signatures in breast cancer tumors and
molecules are extracted and converted into complementary DNA and each sample is labeled
with a florescent dye for distinguishing. It is then mixed with a control sample, with normal gene
expression. The sample is then allowed to bind to the microarray slide and the slide is then
scanned to measure the expression of genes. If the expression of a certain gene is higher, the
area will appear red. If it is lower, it will appear green. However, if it is equal, then it will appear
yellow.
According to the paper (Sotiriou & Pusztai, 2009) based on molecular signature,there
are four main classes of breast cancer, which have been distinguished through gene expression
profiling (Goldhirsh et al., 2011). Basal-like breast cancers are also known as triple negative
breast cancers because they have three types of receptors that are nonfunctional. Luminal-A
cancers are ER-(+ve) and are low grade. They are associated with low expression of
proliferating genes (Sotiriou & Pusztai, 2009; Goldhirsh et al., 2011). Luminal B cancers are ER-
(+ve) but express low levels of hormone receptors and are high grade (Sotiriou & Pusztai, 2009;
Goldhirsh et al., 2011). HER2-positive cancers show amplification of ERBB2 gene, which
codes for a tyrosine kinase receptor in the Epidermal growth factor family (Sotiriou & Pusztai,
Luminal types of cancer express high amounts of luminal cytokeratins, keratins found in
cytoskeleton, as well as the genetic markers of luminal epithelial cells of normal breast tissue
(Sotiriou & Pusztai, 2009). Basal-like tumors have a dysfunctional BRCA1 pathway, which helps
in DNA repair and activation of cell cycle checkpoints (Sotiriou & Pusztai, 2009). In the different
subtypes of breast cancer, there is a difference in copy numbers of particular genes (Sotiriou &
Pusztai, 2009). Basal-like cancers have a lot of variation, indicating greater genetic complexity.
HER2-positive and luminal b tumors have higher levels of amplification (Sotiriou & Pusztai,
2009). All of this indicates that these variants may arise from different transformed stem or
Gene expression profiling can be used to predict clinical outcomes more accurately
(Sotiriou and Pusztai, 2009; Goldhirsh et al., 2011). Out of 302 patients, 87 had disagreeing
results from the clinical prediction and the gene profile. 59 had tumors that were low risk
according to gene signature, but high risk according to the clinical criteria. 28 had tumors that
were low risk based on the clinical result, but high risk according to their gene signature. The
genomic test appeared to predict the outcome more accurately (Sotiriou & Pusztai, 2009). In
this group, those who had a high risk rating had a 10 year survival rate of 69%. Those who had
a low risk grade based on the genetic assay had a 10 year survival rate of 89% (Sotiriou &
Pusztai, 2009). 97-gene signatures were used to distinguish between low and high grade
In addition, Sotiriou and Pusztai state, "MammaPrint, the genomic-grade signature, and
a 76-gene outcome signature appear to quantify mainly tumor grade and proliferation. When the
three assays were analyzed in the same population of patients who had received no systemic
adjuvant therapy, they had similar performance, suggesting that genes controlling tumor
differentiation and proliferation account for a large proportion of these classifiers." Use of
patients out of 427 patients compared to modern standard analysis (Sotiriou & Pusztai, 2009).
transcripts and several proliferation-related genes, which is used to estimate the probability of
recurrence (Sotiriou & Pusztai, 2009). It also identifies tumors that will respond to adjuvant
therapy (Sotiriou & Pusztai, 2009; Goldhirsh et al., 2011). However, a unique trend was found:
higher recurrence scores were associated with greater benefit from adjuvant therapy and lower
recurrence scores were associated with lower benefit from chemotherapy (Sotiriou & Pusztai,
2009).
In preliminary studies, there were reports of a strong association between the absence
of residual cancer (pathological complete response, or the complete removal of the tumor) and
long term cancer free survival (Sotiriou & Pusztai, 2009). The gene expression profile of cancers
can differ based on tumor sensitivity to chemotherapy (Sotiriou & Pusztai, 2009). In 133 patients
with stage 1, 2, or 3 breast cancer who received a weekly treatment of paclitaxel combined with
fluorouracil, doxorubicin, and cyclophosphamide (all chemotherapy drugs), the gene predictor
correctly identified the 92% that had a pathologic complete response (Sotiriou & Pusztai, 2009).
The positive predictive value, the number of people who truly have the disease, was only 52%,
but the negative predictive value, the number of people who don’t have the disease, was 96%
The hypothesis was supported in this source, because Soritiou and Piztai gene
expression signatures can lead to better classification system which will lead to a better and
more specified or catered treatment options and more accurate prognosis. The use of gene
profiles led to altered treatment recommendations and allowed for more accurate prognosis.
This study is relevant because the use of gene profiling is not widely used, especially in
America, but this study shows that using gene signatures can lead to a more accurate prognosis
and catered treatment response. Using this method can allow for more accurate information to
be used by the doctor and the patient. This adds to the scientific knowledge on this topic by
demonstrating how gene profiles can be used to create more accurate prognosis and catered
treatment responses.
Sotiriou and Pusztai examined the use of gene signatures in properly classifying breast
cancer, as well as its technological use in biomedical research. This gene signature is
composed of various genes that affect cancer proliferation, mentioned in Ward and Thompson’s
article. Sotiriou and Pusztai also briefly touched on the implications of gene signature in clinical
settings. Dr. Marc Vijver wrote an article about its use in a clinical setting when predicting
Gene expression has been used to properly classify cancer, but in recent years,
researchers have examined its use in a clinical setting when determining cancer survival and
Survival in Breast Cancer". It was written by Vijver et al. in 2002 and was published in The New
England Journal of Medicine. Marc Vijver is a doctor at the Department of Pathology, Academic
Medical Center, in Amsterdam. He has published other works relating to cancer. This study was
The question this paper was asking was whether gene expression signatures can be
used to provide a more accurate prognosis. The team hypothesized that gene expression
signatures can be used in a clinical setting to provide a more accurate prognosis catered to the
individual. The independent variable is the prognosis rating for each patient (microarray result)
and the dependent variable was the survival rate for each group. The control was the type of
cancer (breast cancer), stage of cancer (stage 1 and stage 2), and treatment. This paper used
295 patients with primary breast carcinomas for this study. According to the authors (Vijver et al,
Tumors from a series of 295 consecutive women with breast cancer were selected from
the fresh-frozen–tissue bank of the Netherlands Cancer Institute according to the following
criteria: the tumor was primary invasive breast carcinoma that was less than 5 cm in diameter at
pathological examination (pT1 or pT2); the apical axillary lymph nodes were tumor-negative, as
determined by a biopsy of the infraclavicular lymph nodes; the age at diagnosis was 52 years or
younger; the calendar year of diagnosis was between 1984 and 1995; and there was no
previous history of cancer, except nonmelanoma skin cancer. All patients had been treated by
lymph nodes, followed by radiotherapy if indicated. Among the 295 patients, 151 had lymph-
node–negative disease (results on pathological examination, pN0) and 144 had lymph-node–
positive disease (pN+). Ten of the 151 patients who had lymph-node–negative disease and 120
of the 144 who had lymph-node–positive disease had received adjuvant systemic therapy
consisting of chemotherapy (90 patients), hormonal therapy (20), or both (20). Sixty-one of the
patients with lymph-node–negative disease were also part of the previous study used to
This was done to test the effectiveness of using gene expression signaling as a way to
format treatment plans and prognoses in multiple types of breast cancer, treated or not. The
patients needed to be young, stage 1 or 2 breast cancer, and no previous cancer, as that would
have skewed the results. When one gets older, multiple factors can lead to death. Stage 1 and 2
breast cancers are cancers that are caught early on, which gives doctors more treatment time
and gives someone a longer life expectancy. Previous cancers would also affect treatment and
life expectancy.
molecules are extracted and converted into complementary DNA and each sample is labeled
with a florescent dye for distinguishing. It is then mixed with a control sample, with normal gene
expression. The sample is then allowed to bind to the microarray slide and the slide is then
scanned to measure the expression of genes. If the expression of a certain gene is higher, the
area will appear red. If it is lower, it will appear green. However, if it is equal, then it will appear
yellow. Each patient was then classified as either good prognosis (longer life expectancy) and
poor prognosis (poor life expectancy) based on the gene profile. Each patient was then checked
Out of 295 patients, 180 had a poor prognosis signature and 115 had a good prognosis
signature (Vijver et al, 2002). Those with poor prognosis signatures had a 10 year survival rate
of 54.5 (+/- 4.4) percent (Vijver et al, 2002). Those with good prognosis signatures had a 10
year survival rate of 94.5 (+/- 2.6) percent (Vijver et al, 2002). These signatures also affected
the chances of remaining free of distance metastases (cancer that has spread from place of
origin). Those with a poor prognosis signature has a 50.6 (+/- 4.5) percent chance of remaining
distant metastasis free (Vijver et al, 2002). Those with good prognosis scores had a 85.2 (+/-
4.3) percent chance of remaining distant metastasis free (Vijver et al, 2002). The estimated
hazard ratio (ratio of the hazard rates corresponding to the conditions described by two levels of
an explanatory variable) between the poor prognosis group and the good prognosis group was
5.1 with a 95% interval (Vijver et al, 2002). This shows that by using gene signatures, one can
accurately predict the chances of survival in those with breast cancer (Vijver et al, 2002).
The hypothesis was supported. The team originally believed that gene expression
signature can be used to accurately predict the prognosis of a cancer patient. The results
indicated that the preliminary belief was right, since those with a good prognosis result had a
higher survival rate over 10 years than those who had poor prognosis results (Vijver et al,
2002).
This study is relevant because it shows how gene expression can be used to predict the
survival chance in people with breast cancer. This will give doctors and patients a more
accurate prediction. Using the signature to predict the survival of breast cancer was not
common in those days. This contributed to the scientific research already out there by providing
us a tool that was familiar and can be use to predict the survival outcomes in patients with
breast cancer.
Guided by a 21-Gene Expression Assay in Breast Cancer.” Sparano et al. tested the ability of a
cancer were selected, with only 9719 patients eligible with follow up information. 6711, or 69%,
had a midrange recurrence score of 11 to 25 and were randomly selected to receive endocrine
therapy or chemoendocrine therapy (Sparano et al, 2018). Both groups had similar rates of
invasive cancer-free survival and overall survival. This shows that both treatment options were
equally effective in this group. In a broader sense, this shows that some women (about 70%
according to Sparano et al) who are prescribed chemotherapy with breast cancer might not
actually need it. However, this test is expensive (about 2,500 pounds in England), and questions
have been raised on whether it is really necessary or is just an extra expense to the patient,
especially in countries without free healthcare. Questions have also been raised on whether
their findings about the overuse of chemotherapy is an accurate conclusion based on their
methods. Nevertheless, more research is needed to solidify this claim, but it is a promising
result.
and metabolic changes- has been discussed in Ward and Thompson, Semenza, and Sotiriou
and Pusztai’s articles. While Vijver et al examined the use of genes in a clinical setting, Glunde
et al. examined the genes and metabolites in choline metabolism, a type of metabolism
which is paramount to creating a new cell membrane for a daughter cell. Choline metabolism
undergoes metabolic reprogramming that aids in creating more phospholipids for cancer cell
published in the Nature Review Cancer in 2011. The article was authored by Kristine Glunde,
Zaver M. Bhujwalla, and Sabrina M. Ronen. Kristine Glunde and Zaver M. Bhujwalla work at the
Johns Hopkins University In Vivo Cellular and Molecular Imaging Center, The Russell H.
Morgan Department of Radiology and Radiological Science and the Sidney Kimmel
Department of Radiology at the University of California San Francisco School of Medicine. All
three women have a history of studying cancer, with Glunde specifically studying breast cancer
and metabolic reprogramming. This study was done in Baltimore. This article references other
articles. One reference was authored by Price et al in 1989. He works at Institute of Cancer
Research, Chester Beatty Laboratories and has written many papers on cellular biology. The
Physics, Weizmann Institute of Science and authored many papers on choline metabolism. The
third reference is by Bell et al in 1998. Bell worked at MR Unit, Hammersmith Hospital, London,
UK and has published work on cancer. The fourth source is by Wu et al in 1997. Wu works at
Key lab of Glycoconjugate Research, Ministry of Public Health, Shanghai Medical University
and has published work on cancer and cell biology. The fifth source is by Ramirez de Molina in
Molina has published work on choline kinase as well as tumors and cancer.
The question they are asking is how can choline metabolism be used in cancer
treatments, research, and screening? The team hypothesized that choline metabolism can
provide biomarkers for screening and be used as a guide for future cancer treatments and
research. The independent variable is whether the cell is cancerous or not. The dependent
variable is the changes in genes and proteins that affect the choline metabolism. The controls
are the types of cells used, the animal from which the cells came from, and regular, non-
This source references other sources that use various breast cancer cells and
mammalian epithelial cells (Glunde et al., 2011). This is to determine genes that are expressed
in cancer compared to regular cells. Knockout mice were used to determine which gene loss is
lethal. Various cancer cell lines were used as well to determine which genes and proteins were
active in cancers. Mouse fibroblasts were used to examine the overexpression certain genes
would have. In this paper, the research team was looking for the expression of certain genes or
proteins, which can be found using microarrays or western blots (Glunde et al., 2011).
Choline metabolism is the production of lipids and has recently emerged as a hallmark of
precursor of phosphatidylcholine and a breakdown product. It, along with other lipids such as
proliferating cells, PCho and tCho levels are raised, but not to extent which they are in cancer
(Glunde et al., 2011). The levels of glycerophosphocholine (GPC), a metabolite found in choline
metabolism, is inversely related to the levels of PCho. Although both increase in certain
cancers, including breast cancer, the increase in PCho is more drastic than the increase in
GPC. This indicates that although GPC and PCho make up tCho signal, higher levels of GPC
metabolism may function as second messengers that are essential for signal transduction in
proliferating cells (Glunde et al., 2011). Growth factors, cytokines, and oncogenes also regulate
choline metabolism (Glunde et al., 2011). The increased PCho and tCho levels in cancers are
caused by the interaction of multiple enzymes which are at the core of choline metabolism
There are four types of choline transporting transmembrane systems that have been
shown to have a role in cancer (Glunde et al., 2011). High affinity choline transporters (CHT) are
for choline less than 10 micromoles (Glunde et al., 2011). Choline transporter like proteins
(CTL) are responsible for intermediate affinity and sodium independent lipid transportation
(Glunde et al., 2011). There are six variants of the gene controlling this protein and this gene
undergoes alternative splicing, meaning there are many variants of this one transporter (Glunde
et al., 2011). Organic cation transporters (OTC) have three subtypes and locate organic cations
in a sodium independent and reversible manner (Glunde et al., 2011). Organic cation/carnitine
transporters (OTCN) have two human isoforms (Glunde et al., 2011). OTCs and OTCNs move
low affinity lipids and other organic cations (Glunde et al., 2011). CTL1, an isotope of CTL, was
shown to be expressed in various cancers, including breast cancer (Glunde et al., 2011).
Increased lipid transport and expression of CTL1 was shown in breast cancer cells when
Choline kinase catalyzes the phosphorylation of choline using ATP and produces PCho
(Glunde et al., 2011). There are three isoforms of choline kinase encoded by two genes: choline
kinase-a (CHKA) and choline kinase-B (CHKB) (Glunde et al., 2011). There are only two
functional forms: CHKa1 and CHKa2 (Glunde et al., 2011). The loss of CHKa is lethal in
embryos in knockout mice. Thus, the increase in CHK activity in cancer results from an increase
in CHKa expression (Glunde et al., 2011). Overexpression of CHKa has been reported in
several various human derived cancer cell lines (Glunde et al., 2011). The activity of CHKa was
also shown to increase in cancer (Glunde et al., 2011). CHKa is affected by the P13K-AKT
signaling pathway, which regulates the cell cycle and has been identified as an important
pathway in cancer proliferation (Glunde et al., 2011). The overexpression of CHKa resulted in
the expression of genes that are associated with proliferation, but partial inhibition of CHKa
resulted in apoptosis (Glunde et al., 2011). Knocking down CHKa reduced P13K-AKT signaling
and stopped cell proliferation. CHKa also inhibits tumor growth independent of this pathway
(Glunde et al., 2011). Transcriptional factors such as HIF1 and MYC, present in the pathway,
and inorganic phosphate from PCho and cytidine triphosphate (CTP) (Glunde et al., 2011).
CDP-choline is one of the most used intermediaries in the lipid synthesis pathway, called the
Kennedy pathway, and is used to form PtdCho (Glunde et al., 2011). In liver cancer, the
production of cancers was shown to be associated with an increase in CCT activity and mRNA
ovarian cancers (Glunde et al., 2011). Phospholipase D1 (PLD1) and 2 activity, genes that code
for phospholipase D, was shown to activate in response to extracellular stimuli (Glunde et al.,
2011). They hydrolyze PtdCho to phosphatidic acid and choline (Glunde et al., 2011). This is
one of the breakdown pathways in PtdCho metabolism (Glunde et al., 2011). It occurs as three
splice variants. PLD1 activity increased in breast cancer and melanoma (Glunde et al., 2011).
PLD2 activity increased in renal cancer (Glunde et al., 2011). PLD1 or PLD2 expression was
able to transform cells that overexpressed tyrosine kinase (Glunde et al., 2011). A higher PLD2
activity in breast cancer was shown to imply multidrug resistance (Glunde et al., 2011).
Increased PC-PLD activity correlated with a loss of estrogen receptor expression in breast
cancer cells (Glunde et al., 2011). It has also been implicated in tumor invasion (Glunde et al.,
2011).
produces PCho and second messengers which start signal transduction cascades (Glunde et
al., 2011). In ovarian cancer, elevated PCho levels are partially caused by PC-PLC activation
(Glunde et al., 2011). In breast cancer cells, PC-PLC accumulates on membrane of HER2-
An increase in tCho levels in indicative of cancer (Glunde et al., 2011). Since many
choline containing compounds, as well as the enzymes or proteins mentioned here, can be
these biomolecules can be indicative of cancer and provide a noninvasive biomarker (Glunde et
al., 2011). Chemotherapy drugs results in a decrease of tCho levels in responding tumors
(Glunde et al., 2011). There is still work needed in how choline metabolism can be used in
therapeutic responses (Glunde et al., 2011). Inhibiting metabolites in choline metabolism might
be a possible treatment option (Glunde et al., 2011). A CHKa inhibitor is undergoing clinical
trials in cancer patients (Glunde et al., 2011). However, there is still work to be done, which is
The hypothesis was supported. The team hypothesized choline metabolism can provide
biomarkers for screening and be used as a guide for future cancer treatments and research
(Glunde et al., 2011). It can be used as a biomarker and future research is focusing on inhibiting
associated with it, and how it can be used in future research as a treatment option. The
implications of this could be a cure for cancer, or a way to stop cancer from growing or
spreading. By inhibiting choline metabolism and thus lipid production, it is thus inhibiting the
potential for proliferation. This study adds to the scientific knowledge by introducing the
enzymes associated with it and how researchers can inhibit choline metabolism and stop cancer
proliferation.
In a recently published article by Cao et al. in 2016, the authors studied the effect of two
genes, called GDPD5 and GDPD6, on cancer cell proliferation by the production of
231 breast cancer cell lines were used in the experiment. MCF-7 is estrogen sensitive and
weakly metastatic (the spreading of cancer beyond its origin) in comparison to MDA-MB-231
which is estrogen independent and highly metastatic (Cao et al., 2015). Using small interfering
RNA (siRNA), an RNA which interferes with the expression of genes, that targets and silences
the expression of both GDPD5 and GDPD6 (Cao et al., 2015). Non-targeted siRNA was used
as a control. Each treated group had three samples. The RNA was isolated and 1 microgram of
RNA was used as a template for preparing the cDNA (complementary DNA derived from an
RNA sample) (Cao et al., 2015). Expression of these genes were then determined in the cDNA
from control and silenced samples using PCR, a lab technique that uses Taq polymerase to
make copies of the gene (Cao et al., 2015). Magnetic resonance spectroscopy (MRS) was also
performed on both control and transfected cells (Cao et al., 2015). Cell proliferation was
monitored over a period of 48 hours and was then manually counted (Cao et al., 2015). Cell
migration and invasion was investigated through a cell invasion assay, which works similarly to
amount of GPC from control cells (Cao et al., 2015). This would lead to more tCho (Cao et al.,
2015). PC and free choline levels did not change (Cao et al., 2015), indicating that the cancer
was not proliferating or spreading. In MCF-7 cells with GDPD5 knocked down, there was a
decrease in proliferation and cell viability, but was not present in MDA-MB-231 (Cao et al.,
2015). In cells with GDPD6 knock-down, there was no change in cell proliferation or viability
(Cao et al., 2015). In MDA-MB-231 cells with GDPD5 knock- down, there was a greater
decrease in cell migration or invasion than in MDA-MB-231 cells with GDPD6 knocked down
(Cao et al., 2015). MCF-7 cells did not migrate at all (Cao et al., 2015). However, when
examined by a scratch assay, the MCF-7 cells that had either gene knocked down showed a
decrease in migration and invasion in comparison to the control (Cao et al., 2015).
This supports the idea that choline metabolism affects the proliferation of cancer cells. It
also provides the regulatory genes for GPC levels in the cell, which are beneficial (Cao et al.,
2015). In future studies, these genes can be examined and manipulated to decrease the
amount of GPC and decrease the rate of cell proliferation and migration and invasion (Cao et
Ward and Thompson examined the metabolic changes that occur in cancer cells during
proliferation, especially in how they use glycolysis to make enough energy but switch to
anabolic metabolism to keep up with the growing biomolecular needs of the cell. This goes hand
in hand with the mutation of the cell cycle, where the excess of biomolecules prompts the cell
for cell division and thus increases proliferation. But the increase in cells in a given area
increases the cell density and prompts hypoxia. In order to survive in the hypoxic conditions, the
cell uses a gene called HIF-1 which also affects cancer proliferation, as stated by Semenza.
The amount of gene activity in the cell in Sotiriou and Pustai and Vijver et al.’s articles were
measured by microarrays. The amount of protein in the cell in Glunde et al.’s article was
Conclusion
Metabolic reprogramming and changes in gene expression can affect the proliferation of
cancer (Ward & Thompson, 2012). Gene signatures can provide more information on the cancer
tumor and can provide a more accurate prognosis (Sotiriou & Pusztai, 2009; Vijver et al., 2002).
Most cancer cells are under hypoxic conditions, and thus switch to anabolic metabolism to keep
up with their energy and synthesis needs. HIFs allow cells to survive under hypoxic conditions
(Semenza, 2016). All the articles were well written and straightforward, although the article
describing the metabolic changes occurring in cancer cells began to include unimportant and
irrelevant information by the end, detailing pathways that did not affect metabolic
reprogramming and genes that were only loosely related and not pertinent to the subject matter
of metabolic changes in cancer cells. Many of the works referenced in the article describing
hypoxia and hypoxia inducible factor were carried out by the author, Gregg Semenza, but it was
not compromising, due to the fact the research team was not the same and those articles were
peer reviewed and highly accredited. The sources describing the use of metabolic signatures for
breast cancer research and prognoses were extremely similar but Sotiriou and Pusztai’s article
looked at how doctors and researchers can use gene profiles in a broader way than Vijver et al.
All five sources used microarrays or immunoblotting to find certain genes or enzymes active in
cells. The two sources on gene signatures and gene profiles used microarrays exclusively, while
the source on hypoxia and HIFs as well as the source on choline metabolism leaned more on
immunoblotting to conduct their studies. The source describing metabolic reprogramming used
looked at humans with breast cancer and the other three sources looked at cancer cells,
tumors, cancer cell lines, or mammalian non-cancer cells. The source on hypoxia and HIFs
mentioned animal testing on mice. The two sources that examined the use of gene profiling in
research and clinical settings looked at the accuracy of gene signatures in a ten year span,
while the other three did not have a set length of time for the study. However, since the other
three did not include human testing, we can assume that the studies were carried out in a
couple of months.
The researchers in the two sources that examined the use of gene profiling in research
and clinical settings set out to discover whether gene signatures can be used in treating cancer.
The researchers in the articles describing the metabolic changes, including the choline
metabolic changes, that occur in cancer cells examined the changes in metabolism that can
cause cancer proliferation or the changes caused by cancer. The researchers in the article
describing hypoxia and HIFs examined the effect of hypoxia on cancer cells and how HIFs allow
the cell to survive under hypoxic conditions. All articles agreed with the belief that cancer cells
undergo a metabolic reprogramming that helps those cells proliferate, and HIF-1 plays an
All of these sources are important to understanding the capstone project. The first
source talks about metabolic reprogramming, which is the topic of the capstone project. The
second source talks about hypoxia and HIFs, which are widely found in cancer and important to
understand. The articles that talk about the use of gene signatures in breast cancer, from
treatment to prognosis, is important because breast cancer is the cancer the Capstone project
will focus on. The sources on choline metabolism and the genes and metabolites encompassing
it examined a common metabolic reprogramming that allows for cancer cell proliferation, which
is the metabolic pathway of choice for this project. Future work needs to be done to understand
the degree to which metabolic reprogramming is done and how it is controlled or reversed, as
well as how gene signatures can be implemented in treatment options in a clinical setting.
Potential Impacts
Ethical Impact
The use of animals in future research will have an ethical impact. The creation of
medicine to reverse metabolic reprogramming will have to be tested on mice first. According to
the source on choline metabolism reprogramming, "studies investigating the molecular basis
underlying these changes are urgently needed." This shows that there is a need for future
research and those future research would most likely include animal testing. The source on the
tumor genotype, it appears likely that effective therapy will depend on targeting cancer based on
This shows that future cancer therapy will be different from today. While some say that
animal testing is unethical, many researchers make it a point to inflict no pain on the animal.
These procedures need to first be evaluated by an independent ethics committee before they
are approved. The ethics committee will only approve procedures that demonstrate no animal
Health Impact
The use of genetic signatures in clinical settings will have a health impact. Genetic
signatures can be used to provide a more accurate prognosis. The article by Sotiriou and
Pusztai says, "Gene-expression profiling has been used to develop genomic tests that may
provide better predictions of clinical outcome that the traditional clinical and pathological
standards." This shows that there is a benefit in using gene expression profiling: it is more
accurate than traditional standards and can provide a more accurate prediction. The article by
Vijver et al. says, "The gene-expression profile we studied is a more powerful predictor of the
outcome of disease in young patients with breast cancer than standard systems based on
clinical and histological data." This shows that using gene expression profiling is more beneficial
Gene signatures can also be used to determine more appropriate treatment options
based on the patient. The article by Vijver et al. says, "A more accurate means of
prognostication in breast cancer will improve the selection of patients for adjuvant therapy." This
shows that your gene signature can determine whether a patient needs this secondary
treatment or not. While some will argue that the focus needs to shift into finding a cure, it is also
imperative that all the tools necessary are provided to keep cancer patients alive until that time.
It is also imperative to make sure that each patient receives the best care for themselves and
not the standard care that might not work for them. By incorporating gene profiles into treatment
decisions, the patient is receiving the best care for themselves and the clinic is saving resources
that can be used for another patient. In the article by Vijver et al., it says, “[Gene profiling]
should also improve the selection of patients who would benefit from adjuvant systemic
treatment, reducing the rate of both overtreatment and undertreatment.” This shows that by
incorporating gene profiling into healthcare decisions, it would also prevent overtreatment and
Economic Impact:
The use of microarrays will also have an economic value, on both patients and the
clinics. The clinics will need more money to buy the array, as trays cost between $100-$300.
This will provide more business to the company selling the array, a point raised by some
researchers. The use of microarrays will also save patients money by not recommending
unneeded chemotherapy treatment. The article by Vijver et al. says, "The gene-expression
profile we studied is a more powerful predictor of the outcome of disease in young patients with
breast cancer than standard systems based on clinical and histological criteria." This shows that
one’s gene signature can determine whether a patient needs this secondary treatment or not,
which can save the patient money, as well as predicting the outcome of the disease more
accurately. The article by Sotiriou and Pusztai says, "The use of microarrays in combination with
clinical guidelines led to altered adjuvant treatment recommendations in 26% of patients." This
shows that gene signatures can change treatment recommendations given by doctors and can
cause a patient not to spend thousands on an unneeded treatment when the same patient might
Problem Statement:
Around 1.7 million people worldwide are diagnosed with breast cancer in a year, with
40,920 women in the US expected to die due to breast cancer (Breast Cancer Statistics, n.d.).
to anabolic metabolism in order to keep up with the biosynthesis needs of the cell. Cells then
revert back to catabolic metabolism when proliferation is done. In cancer cells, the metabolic
reprogramming is not reversed, leaving the cell in a constant state of proliferation that increases
tumorigenesis (Ward & Thompson, 2012). One of the metabolic pathways that is heightened is
choline metabolism, which is responsible for the production of phospholipids used in the cell
membrane (Glunde et al., 2011). GDPD6 is a gene found in the choline metabolism which
phospholipids (Glunde et al., 2011). When GDPD6 is silent, more GPC is produced which
means there is less phosphocholine and less tumor invasion (Glunde et al., 2011). Estrogen
acts as a growth factor in breast cells, and by treating cells where GDPD6 is overexpressed with
pathway.
Methodology:
Methods:
If estrogen is given to a breast cancer cell line that has an overexpression of GDPD 6,
then the amount of glycerophosphocholine (GPC) will be less than the amount in a breast
cancer cell line that has basal (normal) expression. To test this hypothesis, three types of MCF-
7 breast cancer cell lines were grown in cell culture flasks (T75 and T175). One over-expressed
GDPD-6, a critical gene in choline metabolism that affects the expression of GPC. Another was
an empty-vector for GDPD-6 (will not express the gene). These groups of cells will act as the
independent variable. The third cell line will be wild-type, or random, gene expression and will
act as the control. This cell line will not be genetically modified as opposed to the other two cell
lines and will represent GDPD6 expression present in the average person. After growing the cell
lines, all the cells were starved and deprived of nutrients, hormones, and growth factors for 48-
72 hours. Each type of MCF-7 breast cancer cell line was then split into three groups, with three
subgroups or sub-experimental groups. One group in each line was treated with 50 nM of
estrogen in 20 mL of water (three sub-experimental groups; this is to ensure more accurate data
as the average can be taken and it minimizes the risk of outliers in the data). The second group
was treated with 100 nM of estrogen in 20 mL of water (three sub-experimental groups). The
third group in each line was treated with 250 nM of estrogen in 20 mL of water (three sub-
experimental groups). Pinton et al. suggested targeting estrogen receptors in certain cancers,
such as ovarian cancer. After the duration of treatment was completed (48-72 hours), a western
blot, also called immunoblotting, was performed to examine the levels of GPC, the dependent
variable. An NMR-spectroscopy, which measures the amount of a protein, especially a
metabolite, was used to measure the amount of GPC present in the cell samples. The data will
Tissue Culture:
Tissue culture is extremely imperative to this experiment, but it is easy to get wrong. To
begin the cell culture, plate 1 mL of MCF-7 breast cancer cells into the T75 flask with 15 mL of
MEM media with 10% FBS. Media in the flasks must be aspirated and replenished every other
day. Once cells reach 70-80% confluency, transfer the cells to a T175 flask and grow. Media
must be stored in a 4 degree Celsius freezer. Before replenishing media, the media must be
heated in a water bath. The media should not be left open for long. When using the pipette tips,
do not “double dip”, or put the tip in the media, then the flask, and back into the media; use a
new pipette tip for each flask to reduce the risk of contamination. When changing the media in
the flasks under the hood, the hood should have a “vacuum” in place that sucks the media in the
cell flasks into a central container, where at the end of the day or when it gets filled, it can be
disposed of.
Western Blots:
A sonicator was used to lyse the cells and a centrifuge was used to separate the protein.
Whole cell protein extract was obtained from both control and treated cells by using
protein from both control and treated cells were separated on a polyacrylamide gel and resolved
degrees Celsius. The transferred protein was exposed to a specific antibody for GPC, which in
this case is CD236. Immunoblots thus obtained after probing was detected using a secondary
human antibody conjugated with horseradish peroxidase (HRP). Later, using a western blot
imager, the signal was detected and the difference between the control and treated protein
The western blots from the control sample were compared to the sample from MCF-7
certain proteins. From the molecular weight of GPC, it can be determined which band signifies
GPC. GPC should be around the 40 kDa marker, which will be slightly lower than the middle.
Thicker bands in this indicate a higher protein count, which here also indicates a higher GPC
count. From the size and location of the band, the amount of GPC present in both the empty-
vector for GDPD-6 MCF-7 breast cancer cells and MCF-7 breast cancer cells over-expressing
NMR-Spectroscopy:
Both control and treated cells were washed with phosphate buffered saline (PBS), and
hydrolyzed using 5% trypsin. The GPC was extracted using a dual-phase extraction method
using methanol, chloroform, and water in a 1:1:1 ratio. After the samples were dissolved in a
deuterium oxide solution, an NMR spectrometer was used to measure the samples (Cao et al.,
2015). The results are displayed on the graph. While measuring using the spectrometer, it is
important to stand a good distance away or watch behind the window unless properly trained to
Quantification of the GPC present in the cells were done by using the equation of area of
peak/curve on the graph and measured in nM. Quantification can also be done using
proteins found in cells and cell extract. NMR-spectroscopy is commonly used to determine the
exact metabolite found from the pattern emitted. Daly et al. used NMR spectroscopy to monitor
the addition of choline, ethanolamine, and hemicholinium-3 (a choline inhibitor) in breast cancer
cells. By using NMR spectroscopy, they were able to detect changes in the breast cancer cells
signaling due to the addition of these molecules (Daly et al., 1987). Cao et al. used MRS to
determine the metabolite found in MCF-7 cells with either GDPD5 or GDPD6 silenced or
repressed (Cao et al., 2015). Another study used western blotting to determine the prevalence
of PDK1 protein (Kim et al., 2006). Zhong et al. used immunoblotting to determine the
prevalence of HIF-1a by using the antibody protein MAb H1α67 (Zhong et al., 1999).
This project does not answer what metabolic changes are present in cells
overexpressing GDPD-6 in the presence of estrogen pellets. Estrogen pellets are more
concentrated than a diluted solution of estrogen, but both work the same in effect. Insufficient
time limits this experiment, as the cells take a while to grow and if there is a mistake, it takes
time away from the time used to do western blots and NMR-spectroscopy. The cells take about
two weeks to grow and there is a timeframe of only 2 months for the entire experiment. A
mistake in the growth of the cell can push the project back and make the experiment more
difficult to complete by the deadline. An acceptable amount of time would be about another
month (3 months total). It was not possible to test whether using other growth factors, such as
insulin-like growth hormone and other paracrine hormones, had the same effect. There were no
Materials:
To properly conduct this experiment, around 27 cell lines are needed, 9 lines of human
MCF-7 ER+ breast cancer cells overexpressing GDPD6, 9 cell lines of the human MCF-7 ER+
empty-vector GDPD6 breast cancer cells, and 9 lines of the control wild-type human MCF-7
ER+ breast cancer cells. These cell lines will originate from a single cell line in a T75 flask and
will be given enough MEM media with FBS (a solution that contains the nutrients needed for cell
growth) needed to proliferate, making sure that the cell density does not get too dense or go
over 70-80% density. After reaching this density, the cells will be transferred over to a larger
T175 flask and again grown to 70-80% density. The flasks will then be split in a 1:4 ratio,
meaning that the contents in one flask will be evenly split into four flasks; this enables more cell
growth for the experiment. 2 million cells will then be plated in 100 mm petri dishes, using
trypsin to break up the cells and transfer them. The cells will then be grown to 70-80%
confluency again. At this point, the cells will be switched from MEM media to MEM/F12 phenol
red free, hormone free media supplemented with 5% charcoal-dextran stripped FBS and L-
glutamine for 48-72 hours. Estrogen pellets will also be needed and will be washed with PBS
and then fixed in 75% ethanol at 4 degrees Celsius. This mixture will be used to treat the cell
A microscope would be beneficial to see the progress of the cells while they are growing.
Media must be stored at 4 degrees Celsius and needs to be warmed in a water-bath. The flasks
containing the cells must be stored in a carbon dioxide chamber. All cell culture work has to be
done in a hood, where excess waste can be properly collected and thrown away, in order to
ensure a sterile environment. Sleeves and gloves must be worn and should be sprayed with
70% ethanol before being put under the hood. The hood surface must be sprayed with 70%
ethanol before and after use. Anything entering the hood - including cell flasks, media bottles,
trypsin, and pipette tips- must be sprayed with ethanol beforehand. Additional precautions, such
To run the western blot, a sonicator is needed to lyse the cell and expose the cytosol
and all its components. A centrifuge will be used to separate the protein and other cellular
molecules. RIPA Buffer with protease inhibitors is needed to ensure the lysing of the cell while
maintaining protein integrity. To make the RIPA buffer with protease inhibitor, RIPA buffer needs
to be mixed with protease inhibitors to dilute the inhibitors in a 1:100 ratio. (Cell Biolabs, 2017).
A stacking gel solution is used which consists of 11.4 g of Tris, to 150 mL of water. A rack is
used and assembled for gel solidification. An electroporator is also used to hold the rack used
for gel solidification as well as the electrophoresis that occurs in the next steps. Running buffer,
made of 30 grams of Tris, 144 grams of glycine, and 10 grams of SDS (a chemical compound
used in cleaning products) was added to 1000 mL of water. Polyacrylamide gel is useful due to
its low affinity for most protein stains which makes it useful for protein separation. A power
supply is needed for the proteins to begin traveling down the gel. The proteins were then
transferred to a nitrocellulose membrane due to its ability to immobilize proteins while still
having a high affinity for protein (Thermo Fisher Science, n.d.). A transfer sandwich is needed to
transfer to the nitrocellulose membrane and consist of a wet sponge and filter paper in transfer
buffer, gel, polyvinylidene fluoride, and three more filter papers soaked in transfer buffer. The
transfer buffer is made from 28.8 grams of glycine, 6.04 grams of Tris, 200 ml of methanol and
dissolved in 1.6 L of water. The polyvinylidene fluoride is a synthetic resin used due to its affinity
for amino acids. The transfer needs to be done on ice to maintain 4 degrees Celsius.
The nitrocellulose membrane with the protein was blocked with 5% skim milk in TBST
(TBS with tween, a fatty-acid). Skim milk is used to prevent the antibody from sticking to the
membrane. TBST is used by mixing 137 mM of salt, 2.7 mM of KCl, and 19 mM of tris in water
and then adding 1 mL of tween (polysorbate 20) for every liter of the mixture. (Cold Springs
Harbor Protocol, 2013). The nitrocellulose membrane with the primary antibody needs to stay in
a freezer at 4 degrees Celsius overnight to immobilize the protein. CD236 antibody was used
because it is a GPC antibody, and will be considered the primary antibody. The antibody was
added to bovine serum albumin due to its binding capacities. Bovine serum albumin binds to
nonspecific sites. After, a secondary antibody for humans in conjunction with horseradish
peroxidase was used. The secondary antibody is specific for the species and detects different
regions of the primary antibody. Here, a human secondary antibody needs to be used. This
secondary antibody is mixed with horseradish peroxidase to produce color on the western blot.
It is then washed with TBST and then using a western blot imager, it is possible to determine
the amount and type of protein present in the sample. (Mahamood & Yang, 2012).
For the NMR spectrometer, both samples of cells were washed with PBS and
hydrolyzed using 5% trypsin because trypsin breaks down proteins into smaller polypeptides.
Methanol, chloroform, and water are all needed in a 1:1:1 ratio for the dual phase extraction
method from what Cao et al. used in their 2015 report (Cao et al., 2015). Chloroform is a solvent
for the dual extraction method. Deuterium oxide is then needed as a solvent for the samples to
dissolve in. Deuterium is just heavy hydrogen, so it is a combination of deuterium and oxygen. A
NMR spectrometer is needed to get the reading and quantification of the amount of GPC in the
cells.
Gantt Chart:
Figure 1: Gantt Chart showing tasks to be done and estimated amount of time
Figure 1 shows the tasks to be done when completing the experiment and the estimated
amount of time it will take. Cell culture, starving the cells, and then the estrogen treatment will
each take about 20 days. To conduct the immunoblotting for all the samples will take about 3
days. To conduct the NMR-spectroscopy for all the samples will take about 1 day. Data analysis
is expected to take 5 days. The estimated time is the time it is expected to take conservatively.
Safety Precautions:
To complete this experiment, gloves must be worn at all times to make sure infections
do not spread between the cell to the person and vice versa. Face masks should be worn (but
are not required) in order to reduce the spread of bacteria and moisture. If not wearing a face
mask, coughing, sneezing, laughing, and talking must also be limited to reduce the risk of
contamination. Thus, it is highly encouraged to wear a face mask. All lab equipment will be
sprayed with 70% ethanol before use. After completing lab experiments for the day, it is
imperative to maintain good hygiene to stop the spread of germs from inside the lab to the
outside environment. While doing the NMR-spectroscopy, machine users must stand a safe
distance away from the machine to reduce the risk of radiation. Electronics and magnets must
be kept away from the machine a well to not impair its function.
As long as proper lab procedure and cell culture procedure is followed, the cells do not
pose a risk to humans. Millions of cells would need to be injected for it to be an issue, and that is
only in immunocompromised people. The chances of this occurring are slim to none as long as
proper lab procedure is followed. In order to dispose of the cells and media if needed, 10% bleach
solution must be poured and the liquids in the containers must turn clear. After that, the media
and the cells with the bleach solution can be poured into the sink with a drain while water is
running to dilute it. For the chemicals used in the western blot, standard safety precautions apply:
do not ingest any chemicals, sniff any chemicals, or touch the chemicals with bare hands (always
use gloves). Do not touch sensitive parts of the skin, namely the face, with the gloves being used
and hands should be washed after finishing working with the chemicals. Many of these chemicals
are labeled as irritant, meaning that if it comes in contact with the skin, eyes, nose or mouth, it can
cause some irritation. Keep chemicals away from open flame or heat source, unless confirmed it
is safe.
If the chemicals somehow get into the eye, find the nearest eye wash station and
thoroughly wash the eye for about 10 seconds. If the chemicals come in contact with the skin
(the chemicals are spilled), wash the exposed skin with cold water and monitor for any adverse
reactions. Refer to the emergency room, immediate health care center, or the primary physician
for further check-up. If the chemicals are ingested, call the local poison control center, the lab’s
emergency number for this situation (which should be posted in the lab), or go directly to the
emergency room. When using methanol and chloroform, it is IMPERATIVE not to sniff, breathe,
or ingest it. Do not ingest methanol or chloroform; if ingested go to the local emergency room
immediately. Chloroform has been used as a anesthetic in the past, but in high doses can be
lethal. Methanol is extremely lethal, and even small doses can lead to permanent damage. The
effects will not be noticeable for hours, but effective treatment can lower the risk of permanent
damage. Methanol is also extremely flammable, so it must not be kept near an open flame or
heat source.
When disposing of biohazards, such as pipette tips, media bottles, and flasks, a “trash
box” with a red biohazard bag should be present: this is where the biohazards should be
disposed of. After the bag is filled, the top of the bag should be tied and the bag should be
For the western blot, each sub-experimental group will be taken and tested. A western
blot will be produced for each one. Each blot will then be analyzed for GPC. Since GPC is a
protein with a certain weight, it is possible to estimate where on the blot it should be based on
the running time of one hour, which is around 40 kda, or a little bit above the middle in the blot.
The thickness of the band will be compared throughout the samples, as the thicker the band,
the more protein there is. The average of each subgroup (such as the 50 mL of estrogen in 20
mL of water) will be taken and used during the NMR-spectroscopy and will produce a graph.
The graph will be analyzed using the equation of area of the peak/curve and will be compared to
Figure 1: Various Choline Metabolism Genes In MCF-7 Breast Cancer Cell Lines vs. Gene Expression, measured by
Discussion:
Unfortunately, the experiment could not be completed as planned. The data in Figures 1-
4 is from a preliminary test to test for the expression of GDPD6 in the MCF-7 breast cancer cell
lines. Before going further and treating the cell lines with estrogen, the cell’s gene expression for
GDPD6 was checked in order to ensure that the cells were properly expressing GDPD6 in the
way the experiment was set up. Unfortunately, the preliminary trial showed that the cells were
not expressing GDPD6 properly and the transfection (the process of introducing genes into the
Figure 1 displays the expression of genes in the choline metabolic pathway for three
different cell lines, MCF-7 wild-type (green bar), MCF-7 GDPD6 empty-vector (blue bar), and
MCF-7 GDPD6 overexpression (red bar). The fold change in mRNA is indicative of the genes’
activity, where the higher the fold change in mRNA is indicative of more gene expression and
the lower the fold change in mRNA is indicative of less gene expression. This is because in
order to get mRNA, the gene must be expressed and transcription must occur. If there is a
higher fold change in mRNA, this means that the gene was expressed and transcription
occurred. In Figure 1, it is shown that for GDPD6 expression, the cell line MCF-7 GDPD6
empty-vector has a higher gene expression, whereas the cell lines MCF-7 wild-type and MCF-7
GDPD6 overexpression have relatively the same level of gene expression. This result shows
that there was a problem with the transfection, and the genes are not being properly expressed.
If the transfection was a success, the results would show that the cell line MCF-7 GDPD6
overexpression would have a higher fold change in mRNA and the cell line MCF-7 GDPD6
empty-vector would have little to no fold change in mRNA, however, this is not the case. The
cell line MCF-7 wild-type expressed GDPD6 to a level as expected, most likely because this cell
Figures 2-4 are of the western blot conducted for each cell line, with Figure 2 being Trial
1, Figure 3 being Trial 2, and Figure 4 being Trial 3. The bands in Figures 2-4 are indicative of
the level of GPC produced by the cell line, where the thicker and darker the band, the more
GPC is produced and vice versa. As shown in Figure 2, the cell lines MCF-7 GDPD6 empty
vector and MCF-7 GDPD6 overexpression have the same level of band thickness. This
indicated that the two cell lines have the same level of GPC, which should not be the case if the
transfection worked. If the transfection worked, the band thickness on the MCF-7 GPDP6
empty-vector cell line would be greater than the band thickness on the MCF-7 GDPD6
overexpression, based on the research done by Cao et al in 2016. This pattern is repeated in
Figure 3 and Figure 4, where the cell lines MCF-7 GDPD6 empty vector and MCF-7 GDPD6
In Figure 2, the cell line MCF-7 wild-type had less band thickness than the cell lines
MCF-7 GDPD6 empty vector and MCF-7 GDPD6 overexpression. In Figure 3, the cell line MCF-
7 wild-type had slightly thicker bands than the cell lines MCF-7 GDPD6 empty vector and MCF-
7 GDPD6 overexpression. In Figure 4, the cell line MCF-7 wild-type had slightly thinner bands
than the cell lines MCF-7 GDPD6 empty vector and MCF-7 GDPD6 overexpression. In Figure 3
and Figure 4 the MCF-7 wild-type had similar band thickness in comparison to the cell lines
MCF-7 GDPD6 empty vector and MCF-7 GDPD6 overexpression. The band thickness for MCF-
7 wild-type is not unexpected, as this cell line was not genetically modified, so the cell line is
This conclusion is supported by the research done in a paper published in 2016 by Cao
et al. on the effect silencing GDPD5 and GDPD6 has on GPC production and breast cancer
proliferation. In the article (which is referenced in the literature review), Cao et al used siRNA to
silence the genes in two cell lines of MCF-7 and MDA-MB-231 breast cancer cells, with one cell
line for each type of breast cancer cell remaining interference-free, acting as the control, similar
to how this experiment was originally set up. While the intended experiment would have also
tested the effect of MCF-7 cell lines with various levels of GDPD6 expression - including
GDPD6 overexpression - exposed to estrogen on GPC production, this experiment tested the
effect of silencing GDPD6 on MCF-7 breast cancer cell lines. Cao et al found that in MCF-7
breast cancer cell lines where either GDPD6 or GDPD5 was silenced, there was an increase in
GPC and total choline. There was no change in phosphocholine. The increase in GPC and the
no change in phosphocholine levels indicates that cells did not proliferate and metastasize,
which upon further inquiry was confirmed by the group (Cao et al., 2016). Another article by
Wijnen et al. in 2014 tested the effects of silencing GDPD5 on the amount of phospholipid
metabolites found in MDA-MB-231 breast cancer cells and saw similar results to the ones
displayed by Cao et al., which help support the findings in the 2016 Cao et al. paper. Another
article by Cheng et al. in 2016 supported the results found by Cao et al.; however, it is important
to note that the articles share a few collaborators, which may have impacted the objectiveness
of the results.
The research done by Cao et al. supports the conclusions reached from Figures 2-4, as
if the transfection truly worked, the band thickness of the cell lines MCF-7 GDPD6 empty vector
and MCF-7 GDPD6 overexpression would not be the same. It can be predicted that if the
transfection truly worked, the band thickness of the cell line MCF-7 GDPD6 empty vector would
The hypothesis was that if estrogen is given to a breast cancer cell line that has an
overexpression of GDPD6, then the amount of glycerophosphocholine (GPC) will be less than
the amount in a breast cancer cell line that has basal (normal) expression. While the cells could
not be treated with estrogen as planned, the results from Figures 2-4 should have showed that
type and empty-vector MCF-7 breast cancer cells if the transfection had worked. Additionally, a
study published in 2010 by Moestue et al. showed a link between GPC and estrogen levels.
According to the article, “GPC concentration has been shown to be negatively correlated with
estrogen receptor content in breast carcinomas…” which indicates that as estrogen levels
increase, GPC levels decrease. Alternatively, if estrogen levels decrease in a cell sample, GPC
levels increase. Their research also showed that GPC levels were higher and phosphocholine
levels were lower in the luminal-like animal model, which would support the findings and
research done by Sotiriou & Pusztai (Moestue et al., 2010; Sotiriou & Pusztai, 2009). Another
study done in 2016 by Jia et al. showed that estrogen increases breast cancer proliferation by
reprogramming choline metabolism. The study showed that Estrogen Receptor-a (ERa) is a
critical factor and regulator in breast cancer proliferation. ERa regulates Cho
phosphotransferase (CHPT1), which is responsible for the increase in PtdCho production, which
is a critical choline metabolic product that goes on to form the cell membrane (Jia et al., 2016;
Glunde et al., 2011). An increase in estrogen causes ERa to activate PtdCho production, which
Based on these papers, it can be inferred that if the transfection had worked and the
cells were treated with estrogen, the cell line MCF-7 empty vector breast cancer cells would
have the greatest amount of GPC, regardless of estrogen dosage. This is supported by the
research done by Cao et al. where they found silencing GDPD6 increases GPC and decreases
breast cancer proliferation. In regards to the estrogen dosage, however, the MCF-7 empty
vector cells treated with 50 nM in 20 mL would have more GPC present than the MCF-7 empty
vector cells treated with 250 nM in 20 mL. The cell line MCF-7 GDPD6 overexpression breast
cancer cells would have the least amount of GPC when compared to the other two cell lines. In
regards to the estrogen dosage the MCF-7 GDPD6 overexpression cells treated with 50 nM in
20 mL would have more GPC present than the MCF-7 GDPD6 overexpression cells treated with
250 nM in 20 mL. This is supported by the research done by Moestue et al., Sotiriou & Pusztai,
Jia et al. and Glunde et al., where they found estrogen increases breast cancer proliferation.
This would theoretically make the hypothesis true, as the MCF-7 GDPD6 overexpression cell
line had less GPC present than the MCF-7 wild-type cell line when all the cell lines were treated
with estrogen.
If these results prove to be accurate, this would solidify GDPD6 being a gene of interest
in decreasing breast cancer proliferation. In terms of overall health, particularly women’s health,
this result implies that estrogen is a contributor to breast cancer proliferation and drugs given to
treat Luminal A breast cancer (which is ER+) should not have estrogen. It also implies that
silencing GDPD6 in cells can potentially decrease breast cancer proliferation. It would be
interesting to broaden the experiment to include basal cell lines. MCF-7 is a Luminal A cell line
that is estrogen receptor positive and is generally responsive to hormones produced by the
endocrine system (Holliday & Speirs, 2011). It would be intriguing to include a cell line that is
estrogen receptor negative, such as the MDA-MB cell lines - specifically the MDA-MB-231 cell
During the time this experiment was being conducted, many things went wrong. The cell
lines were growing at a slower rate than what was customary, taking nearly 3 weeks to get to
even 80% confluency when this limit should have occurred in under one week. There was a
bacterial infection in the cell incubator, which contaminated the cells in the flasks and caused
apoptosis, or cell death. After cleaning the cell incubator and restarting the tissue culture with
more care and caution, the cells were growing well to the point there would be enough cells to
run the experiment within 3 weeks of starting the tissue culture. Unfortunately, the cells once
again got a bacterial contamination and died despite efforts to save the cells. The source of the
bacterial contamination was unknown, but it was still costly. In order to ensure success next
time this experiment is conducted, the transfection of GDPD6 must be redone into a new line of
MCF-7 cells. This will help ensure that the cells are not contaminated. This will also allow for
standard, new media will be needed. Alternatively, the cells could be treated with antibiotics, but
it is unknown how that will affect the metabolism of the cell and the results would not be
trustworthy.
Conclusion:
In this study, the effect of estrogen treatment on MCF-7 breast cancer cells with varying
levels of GDPD6 gene expression was studied. If estrogen is given to a breast cancer cell line
that has an overexpression of GDPD6, then the amount of glycerophosphocholine (GPC) will be
less than the amount in a breast cancer cell line that has basal (normal) expression. There were
three cell lines: MCF-7 GDPD6 overexpression, MCF-7 GDPD6 empty-vector, and MCF-7 wild-
type/control. There were three subdivisions within these cell lines and each division was treated
with different levels of estrogen. One group was treated with 50 nM of estrogen in 20 mL of
solution. Another group was treated with 100 nM of estrogen in 20 mL of solution. The third
The results of this study showed that the transfection did not work and must be re-
completed in order to move further with the study. However, supporting studies did show that
had the experiment gone unimpaired, the hypothesis would be supported. That is to say, MCF-7
breast cancer cells with an overexpression of GDPD6 would have a lower level of GPC than
MCF-7 breast cancer cells with a basal level of GDPD6 expression and MCF-7 breast cancer
cells with a basal level of GDPD6 expression would have a lower amount of GPC than MCF-7
breast cancer cells where GDPD6 is silent. In addition, cells treated with 50 nM of estrogen in
20 mL of solution will produce less GPC than cells treated with 100 nM of estrogen in 20 mL of
solution. Cells treated with 100 nM of estrogen in 20 mL of solution will produce less GPC than
cells treated with with 250 nM of estrogen in 20 mL of solution. This implies that cells that have
choline metabolism and cancer cell proliferation. It also shows the silencing of GDPD6 is a
potential pathway for stopping cancer cell proliferation in the future. The results presented in this
study adds to the current body of research, as research on the effect of estrogen on GPC levels
in combination with GDPD6 are limited. This study shows the effect of GDPD6 in combination
with estrogen on GPC levels, choline metabolism, and breast cancer cell proliferation,
They were proliferating at a slower rate than normal and were continuously getting
contaminated and dying. The experiment could not be thoroughly conducted and the cells could
not be treated with estrogen. This is obviously a limitation of the experiment, as the results and
conclusions are hypothetical and are not supported with concrete data. In the future, a new line
of MCF-7 breast cancer cells should be transduced. Extra precaution should be taken when
conducting tissue culture. This will ensure the cells will be viable for treatment with estrogen. In
future studies, more trials should be conducted in order to ensure validity of results. Future
studies could also broaden their cell lines to include other breast cancer cell lines, such as
MDA-MB-231. Future studies could also determine the effect of estrogen treatment on other
genes crucial to choline metabolism, such as GDPD5. Future studies will also need to evaluate
choline metabolism and other metabolic enzymes within cancer. Choline metabolic enzymes will
need to be studies further in order to develop a more sturdy understanding of cancer and how
proliferation can be halted. The enzymes structure and cooperation within choline metabolism
must be studied as well. New therapies can also be created that target metabolic enzymes.
These new treatments might be more affordable for cancer patients, allowing for better care to
be readily available. A more conclusive understanding of the genes and enzymes related to
metabolism will allow for a better gene-expression profile for the patient, which will in turn allow
the treatment for the patient to be more suited for their specific needs. Gene-expression profiling
will allow doctors to be more accurate with their diagnosis and treatment suggestions which will
allow for better care of cancer patients. Future studies will need to test potential treatments in a
mouse model.
Acknowledgement:
Special thanks to Johns Hopkins University and Johns Hopkins School of Medicine for the
opportunity to collaborate. Special thanks to Dr. Kristine Glunde, Dr Kanchan Sonkar, Dr. Caitlin
Tressler, and Dr. Balaji Krishnamachary for their guidance, advice, and help in completing this
experiment. Thank you to Ms. Keli Veillette and Ms. Cynthia Crudale for their help, support, and
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