The Effect of Estrogen Treatment on MCF-7 Breast Cancer Cells With Various

Expressions of GDPD6 and its Impact on Abnormal Choline Metabolic Function

Shubhangy Raghavan

Fairchild Wheeler Multi-Magnet

Capstone I

Ms. Veillette

June 2018
Shubhangy Raghavan

Capstone 1 Period 4

Spring 2018

Project Proposal

Abstract:

Proliferating cells undergo metabolic reprogramming from catabolic to anabolic

metabolism, which is beneficial to proliferating cells due to the increase in biosynthesis. In non-

cancerous proliferating cells, the metabolic reprogramming is undone after proliferation. In

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

pathways affected by metabolic reprogramming is choline metabolism, which produces

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 cell lines were then treated with estrogen after a period of starvation.

Following the treatment, western blot and NMR-Spectroscopy were performed on the cell lines

to determine the level of GPC present in the samples. 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.

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

done (Cancer in the Sixteenth to Eighteenth Century, 2014).

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

the US will develop invasive breast cancer over their lifetime.

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,

specifically phospholipids, is controlled by choline metabolism, that frequently undergoes

metabolic reprogramming resulting in an increase in lipid production. Choline metabolism is

critical for cancer cells because the formation of phospholipids can go towards a new cell

membrane for a daughter cell (Glunde et al, 2011).

Metabolic changes in cells can be studied at both gene and protein expression level.

Gene expression is measured using a microassay. A microarray consists of immobilized short

oligonucleotide sequences of various genes on a glass slide. One can compare the expression

of a gene in an infected cell to a control cell by hybridizing/binding fluorescently tagged cDNA

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

gene expression (How DNA Microarrays Work, n.d.).

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

compare the expression of a protein between different samples in a similar way to

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

cancer can “hack” it and help cancer proliferate.

Lit Review:

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

Department of Biochemistry and Biophysics in Goteborg University, a historic university located

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

cells, or bio-molecules contribute to the reprogramming of metabolic pathways? Metabolic

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 is critical for treatment with chemotherapeutic drugs or radiation therapy.

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

being studied by microarray technology or by sequencing RNA called next generation

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

by immunoblotting or by nuclear magnetic resonance spectroscopy.

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 ."

(Ward & Thompson, 2012).

In proliferating cells, mitochondrial enzymes are used to facilitate anabolic metabolism,

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

excessive production of 2-hydroxyglutarate (2HG), a metabolite, can indicate a mutation in

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;

DeBerardinis et al, 2008).

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.
 This study is relevant because it explains how metabolic reprogramming is critical to

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

order to stop cancer cells from proliferating and progressing..

A study published in 2011 by Wang et al in St. Jude’s Children’s Research Hospital

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

in the metabolic activity (Wang et al, 2011). Wang applied a mass-spectroscopy-based

metabolomic approach to determine the concentration of metabolites in activated T cells (Wang

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

increased the rate of glycolysis, glutamine consumption through oxidative catabolism,

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

correlated with the reduction of glycolytic activity. (Wang et al, 2011).

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,

2011; Ward & Thompson, 2012).

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

mentioned as a gene of interest by Ward and Thompson.

Hypoxia and Hypoxia Inducible Factor

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

source is about. The second source is "Hypoxia-inducible Factors: Coupling Glucose

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,

Medicine, Oncology, Radiation Oncology, and Biological Chemistry at the McKusick-Nathans

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

metabolism to glycolytic enzymes.

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

is important in helping cells survive under hypoxic conditions.

LDHA facilitates the transition of pyruvate to lactate acid (Semenza, 2016). HIF-1 can

suppress oxidative metabolism by inhibiting fatty acid oxidation, inducing mitochondrial-selective

autophagy, and inhibiting proteins on the ETC (Semenza, 2016). Mitochondrial-selective

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

production of ROS (Semenza, 2016) .

HIF-1 also increases the production of mitochondrial antioxidants, specifically a subunit

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

here) to detect HIF-1a activity in cells. (Zhong et al, 1999)

 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

(Semenza, 2016). PHGDH is phosphoglycerate dehydrogenase, an enzyme present in

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

accumulation of biomass (Semenza, 2016).

Exposure of breast cancer cells to chemotherapy drugs induced HIF-1 activity. In

PHGDH-knockdown, exposure to chemotherapy drugs lead to cell death. Anti-angiogenic (stop

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

a part of serine synthesis pathway (Ziello et al, 2007; Semenza, 2016).

 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.

Gene Signatures in Breast Cancer

 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

Pusztai is an oncologist at Yale University, a prestigious college in Connecticut. He did his

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

find the risk of recurrence in breast cancer.

As evident above, all references used gene expression testing as their method. mRNA

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,

2009; Goldhirsh et al., 2011).

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

progenitor cells (Sotiriou & Pusztai, 2009) .

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

tumors more accurately (Sotiriou & Pusztai, 2009).

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

MammaPrint and clinical guidelines changed secondary treatment recommendations in 26% of

patients out of 427 patients compared to modern standard analysis (Sotiriou & Pusztai, 2009).

Oncotype DX measures the expression of estrogen receptors and HER2, ER regulated

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%

(Sotiriou & Pusztai, 2009).

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

cancer survival, expanding on Sotiriou and Pusztai’s point.

Gene Expression Predicting Survival

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

need for chemotherapy. The fourth source is "Gene-Expression Signature As A Predictor of

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

done at the Academic Medical Center.

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,

2002), the patients were chosen based on the following criteria:

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

modified radical mastectomy or breast-conserving surgery, including dissection of the axillary

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

establish the prognosis profile.

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.

A microarray was used to establish a preliminary 70-gene prognosis profile. mRNA

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

in on every year for 10 years (Vijver et al, 2002).

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.

A study was published in June 2018 by Sparano et al titled “Adjuvant Chemotherapy

Guided by a 21-Gene Expression Assay in Breast Cancer.” Sparano et al. tested the ability of a

21-gene assay in predicting the effectiveness of chemotherapy based on recurrence risk.

10,273 women with hormone-receptor-positive, HER2-negative, axillary node-negative breast

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.

The importance of genes in cancer research-especially when discussing proliferation

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

responsible for the production of lipids. Choline metabolism undergoes metabolic

reprogramming, a topic discussed in Ward and Thompson’s article.

Choline Metabolism

Choline metabolism is responsible for the production of lipids, specifically phospholipids

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

proliferation, a topic discussed in “Choline Metabolism in Malignant Transformation.” It was

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

Comprehensive Cancer Center in Baltimore, Maryland. Sabrina M. Ronen works at the

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

second reference was by Katz-Brull et al in 1996. Katz-Brull worked at Department of Chemical

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

2005. Ramirez de Molina worked at Unidad de Oncología Traslacional, Instituto de

Investigaciones Biomédicas, Consejo Superior de Investigaciones Científicas-UAM, Spain.

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-

cancerous cells (Glunde et al., 2011).

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

cancer. Increased Phosphocholine (PCho) and total choline-containing compounds (tCho) is a

characteristic of activated choline metabolism (Glunde et al., 2011). Phosphocholine is a

precursor of phosphatidylcholine and a breakdown product. It, along with other lipids such as

phosphatidylethanolamine (PtdCho), form the cellular membrane (Glunde et al., 2011). In

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

are beneficial to cells (Glunde et al., 2011).

In regions with hypoxia, there is a higher level of tCho, which is due to the regulation of

choline-kinase-a expression by HIF-1 (Glunde et al., 2011). Products of choline phospholipid

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

(Glunde et al., 2011).

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

compared to mammalian epithelial cells (Glunde et al., 2011).

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,

also controls the expression of CHK (Glunde et al., 2011).

CTP:phosphocholine cytidylytrasnferase (CCT) catalyzes the synthesis of CDP-choline

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

expression (Glunde et al., 2011). Phosphatidylethanolamine N-methyltransferase (PEMT)

activity and mRNA expression were decreased (Glunde et al., 2011).

Elevated activity of PtdCho-specific phospholipase D(PC-PLD) was shown in gastric and

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).

PtdCho-specific phospholipase C (PC-PLC) catalyzes the hydrolysis of PtdCho, which

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-

overexpressing cells (Glunde et al., 2011).

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

detected by noninvasive methods such as magnetic resonance radioscopy, increased levels of

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

where future research will head to.

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

choline metabolism (Glunde et al., 2011).

This study is relevant because it explains what choline metabolism is, the enzymes

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 2015, the authors studied the effect of two

genes, called GDPD5 and GDPD6, on cancer cell proliferation by the production of

glycerophosphocholine (GPC), a metabolite found in choline metabolism. MCF-7 and MDA-MB-

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

the assays described before (Cao et al., 2015).

In cells with GDPD5 or GDPD6 knocked down (silent), there was an increase in the

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

al., 2015).

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

measured by a western blot.

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

radioactive labeling to detect changes in metabolism.

The two sources that examined the use of gene profiling in research and clinical settings

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

integral role in causing the metabolic reprogramming.

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

metabolic reprogramming of cancer cells says:

"Although there is increasing attention to the concept of personalized medicine based on

tumor genotype, it appears likely that effective therapy will depend on targeting cancer based on

metabolic phenotype as well as genotype."

 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

abuse or cruelty. Future research is needed to determine better cancer treatment.

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

to the patient than traditional standards.

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

undertreatment of cancer. It would appear to be beneficial to incorporate genetic signatures in

the medical decisions made, especially when related to cancer.

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

have under clinical guidelines.

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.).

Cells undergoing proliferation experience metabolic reprogramming from catabolic metabolism

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

impacts the expression of glycerophosphocholine (GPC), a metabolite used to produce

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

estrogen, it could possibly lead to a decrease in proliferation through another metabolic

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 have 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. 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, 2018). 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. The data will be gathered

as the average of each group.

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

radioimmunoprecipitation assay (RIPA) buffer with protease inhibitors. An equal amount of

protein from both control and treated cells were separated on a polyacrylamide gel and resolved

in a buffer. Proteins on the gel were transferred to a nitrocellulose membrane overnight at 4

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

visualized (Kim et al., 2006; Mahmood and Yang, 2018).

The western blots from the control sample were compared to the sample from MCF-7

breast cancer cells overexpressing GDPD-6. In immunoblotting, certain bands correspond to

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 are indicated a higher protein count, which here 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

GDPD-6 can be determined.

NMR-Spectroscopy:

Both control and treated cells were washed with phosphate buffered saline (PBS),

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

reduce the risk of radiation.

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

MestReNova software (Cao et al., 2015).

These methods are commonplace in laboratory settings to determine and identify

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. 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 had the same effect. There were

no biases to affect the results.

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

lines for 48-72 hours.

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

as a facemask, might be necessary to ensure a sterile environment.

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 14 days. To conduct the immunoblotting for all the samples will take about 5

days. To conduct the NMR-spectroscopy for all the samples will take about 3 days. Data

analysis is expected to take 7 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 id 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: don’t 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

disposed of in an approved location.

Data Analysis and Collection:

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

the other subgroups and groups in order to draw the conclusion.

Conclusion:

This experiment was done to see the effect of estrogen treatment on MCF-7 breast

cancer cells overexpressing GDPD6. 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 are GDPD 6 empty vector cell lines. If the hypothesis

is correct, then in MCF-7 breast cancer cells overexpressing GDPD6, there will be a lower

amount of GPC than the amount of GPC in MCF-7 cell lines that are empty vector for GDPD6 or

wild-type. On the western blot, the band for GPC in MCF-7 cells empty-vector for GDPD6 will be
thicker than the band for GPC in MCF-7 cells overexpressing GDPD6 and the band for GPC in

MCF-7 cells wild-type for GDPD6. In the NMR-spectroscopy, the peak, indicative of GPC, will

be higher in MCF-7 cells empty-vector for GDPD6 than in MCF-7 cells overexpressing GDPD6

or wild-type.

In order to analyze the amount of GPC present in the average cell sample for each sub-

experimental group from the NMR-spectroscopy, the equation of (area of peak)/curve will be

used. This will give a numeric value. The average numeric value for the amount of GPC in cells

with GDPD6 overexpressed will be compared to the average numeric value for the amount of

GPC in cells with empty-vector GDPD6 and the average numeric value for the amount of GPC

in wild-type cells. In order to analyze the western blots, a side-by-side comparison is used. The

bands in the wells corresponding the cells with overexpression of GDPD6 are compared to the

bands in the wells corresponding to the cells with empty-vector GDPD6 and wild-type cells. The

blots for each subgroup will be compared as a whole and will then be compared across the

experimental groups. The band thickness is indicative of GPC levels, with higher GPC levels

having thicker bands and lower GPC levels having thinner bands.

From the GDPD6 overexpressed cells, there will be more GPC produced than in the

wild-type and empty-vector cells. There will also be more GPC the less estrogen there is, as

estrogen acts as a growth factor. In the wild-type MCF-7 cells, there will be more GPC than in

the empty vector cells, but less than in the GDPD6 overexpressed cells. Within the wild-type

experimental group, there will be more GPC produced in the 50 mL of estrogen in 20 mL of

solution in comparison to the 250 mL of estrogen in 20 mL of solution. The empty-vector

GDPD6 MCF-7 cells will produce the least amount of GPC compared to the MCF-7 cells

overexpressing GDPD6 and the wild-type cells. Within the empty vector experimental group,

cells exposed to lesser amounts of estrogen will produce more GPC than cells exposed to

higher levels of estrogen.

 Unexpected outcome would be the cells overexpressing GDPD6 had an increase in

GPC whereas the cells with empty-vector GDPD6 had a decrease in GPC. This would mean

that estrogen does trigger an offsetting pathway that affects the expression of GDPD6 or the

amount of GPC.

In the western blot, the band for GPC in cells overexpressing GDPD6 and treated with

estrogen was weaker than the band for GPC in cells that were transfected with an empty-vector

GDPD6 or were wild-type control. In the NMR-Spectroscopy, the peak for GPC in cells that

overexpress GDPD6 was lower than the peak for cells that were empty vector GDPD6 or wild-

type control. The results are similar to the findings in Cao et al. (2014) article in which, the cells

were silenced for either GDPD5 or GDPD6. The findings indicate that GDPD6 plays a role in

regulating the amount of GPC present in breast cancer cells regardless of the presence of

estrogen. This also means that estrogen does not influence GPC expression in spite of GDP6

overexpression. In a clinical setting, this would mean that for breast cancer cells overexpressing

GDPD6, estrogen treatment would not be beneficial for treatment.

GDPD6 has been highlighted as a potential target for targeted gene therapy. This result

does not change that, since it could still be useful in cells where GDPD6 is overexpressed. In

cells with an overexpression of GDPD6 estrogen treatment would not be effective, since it did

not change the levels of GPC in the cell samples treated with estrogen in this experiment.
Citations:

5X RIPA Buffer, with Protease Inhibitor Cocktail (2017) Retrieved from

https://www.cellbiolabs.com/sites/default/files/AKR-190-ripa-buffer_0.pdf

Breast Cancer Statistics (n.d.) Retrieved from https://www.bcrf.org/breast-cancer-statistics

Cancer in the Sixteenth to Eigtheenth Centuries (2014) Retrieved from https://www.cancer.org/cancer/cancer-

basics/history-of-cancer/sixteenth-to-eighteenth-centuries.html

Cancer Statistics (2018) Retrieved from https://www.cancer.gov/about-cancer/understanding/statistics

Cao, M. D., Cheng, M., Rizwan, A., Jiang, L., Krishnamachary, B., Bhujwalla, Z. M., … Glunde, K. (2016). Targeting

choline phospholipid metabolism: GDPD5 and GDPD6 silencing decrease breast cancer cell proliferation, migration,

and invasion. NMR in Biomedicine, 29(8), 1098–1107. http://doi.org/10.1002/nbm.3573

Cellular Respiration (2004) Retrieved from

https://www.biology.iupui.edu/biocourses/N100/2k4ch7respirationnotes.html

Daly, P., Lyon, R., Faustino, P., Cohen, J. (1987). Phospholipid metabolism in cancer cells monitored by 31P NMR

spectroscopy. Journal of Biological Chemistry, 262(31), 14875-14878. Doi: 3667610

DeBerardinis et al. (2008) The Biology of Cancer: Metabolic Reprogramming Fuels Cell Growth and Proliferation.

ScienceDirect, 7(1), 11-20. Doi: https://doi.org/10.1016/j.cmet.2007.10.002

Early History of Cancer (2014) Retrieved from https://www.cancer.org/cancer/cancer-basics/history-of-cancer/what-is-

cancer.html

Glunde, K., Bhujwalla, Z. M., & Ronen, S. M. (2011). Choline metabolism in malignant transformation. Nature

Reviews. Cancer, 11(12), 835–848. http://doi.org/10.1038/nrc3162

Goldhirsch, A., Wood, W. C., Coates, A. S., Gelber, R. D., Thürlimann, B., Senn, H.-J., & Panel members. (2011).

Strategies for subtypes—dealing with the diversity of breast cancer: highlights of the St Gallen International Expert

Consensus on the Primary Therapy of Early Breast Cancer 2011. Annals of Oncology, 22(8), 1736–1747.

http://doi.org/10.1093/annonc/mdr304

How Common Is Breast Cancer? (2017) Retrieved from https://www.cancer.org/cancer/breast-cancer/about/how-

common-is-breast-cancer.html

How DNA Microarrays Work (n.d.) Retrieved from

http://www.pbs.org/wgbh/nova/education/activities/3413_genes_02.html

Kim et al. (2006). HIF-1-mediated expression of pyruvate dehydrogenase kinase: A metabolic switch required for

cellular adaptation to hypoxia. Cell Press, 3(3), 177-185. https://doi.org/10.1016/j.cmet.2006.02.002

Mahmood, T., & Yang, P.-C. (2012). Western Blot: Technique, Theory, and Trouble Shooting. North American

Journal of Medical Sciences, 4(9), 429–434. http://doi.org/10.4103/1947-2714.100998

Patrick S. Ward, Craig B. Thompson, (2012). Metabolic Reprogramming: A Cancer Hallmark Even W arburg Did Not

Anticipate, Cancer Cell, Volume 21, Issue 3, Pages 297-308, ISSN 1535-6108,

https://doi.org/10.1016/j.ccr.2012.02.014.

Pinton et al. (2018). Targeting estrogen receptor beta (ERβ) for treatment of ovarian cancer: importance of KDM6B

and SIRT1 for ERβ expression and functionality. Nature, Oncogenesis (15). https://doi.org/10.1038/s41389-018-

0027-9DO

Overview of Western Blotting (n.d.) Retrieved from https://www.thermofisher.com/us/en/home/life-science/protein-

biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/overview-western-

blotting.html

Semenza, Gregg (2016). Hypoxia-inducible Factors: Coupling Glucose Metabolism and Redox Regulation with

Induction of the Breast Cancer Stem Cell Phenotype, Embo Journal, Volume 36, Issue 3, doi:

10.15252/embj.201695204

Sotiriou, C., Pusztai, L. (2009). Gene-Expression Signatures in Breast Cancer, New England Journal of Medicine,

360;8, doi: 10.1056/NEJMra0801289

Sparano et al. (2018). Adjuvant Chemotherapy Guided by a 21-Gene Expression Assay in Breast Cancer, New

England Journal of Medicine (ePrint) doi: 10.1056/NEJMoa1804710

TBST for Western Blotting (2013) Retrieved from

http://cshprotocols.cshlp.org/content/2013/3/pdb.rec074104.full?text_only=true
U.S. Breast Cancer Statistics (2018) Retrieved from http://www.breastcancer.org/symptoms/understand_bc/statistics

Vijver et al (2002). A Gene-Expression Signature as a Predictor of Survival in Breast Cancer, New England Journal of

Medicine, 347:1999-2009, doi:10.1056/NEJMoa021967

Wang, R., Dillon, C. P., Shi, L. Z., Milasta, S., Carter, R., Finkelstein, D., … Green, D. R. (2011). The transcription

factor Myc controls metabolic reprogramming upon T lymphocyte activation. Immunity, 35(6), 871–882.

http://doi.org/10.1016/j.immuni.2011.09.021

What is Cancer? (2015) Retrieved from https://www.cancer.gov/about-cancer/understanding/what-is-cancer

Zhong et al. (1999). Overexpression of hypoxia-inducible factor 1alpha in common human cancers and their

metastases. American Association for Cancer Research, 59(22), 5830-1835. Doi: 10582706

Ziello, J. E., Jovin, I. S., & Huang, Y. (2007). Hypoxia-Inducible Factor (HIF)-1 Regulatory Pathway and its Potential

for Therapeutic Intervention in Malignancy and Ischemia. The Yale Journal of Biology and Medicine, 80(2), 51–60.

Doi: PMC2140184