BIOL133/HIST222

FREE

NATIONAL CENTER FOR CASE STUDY TEACHING IN SCIENCE

In Search of a Cure for Breast Cancer by Jolanta Skalska Division of Biology Alfred University, Alfred, NY

Emily was a biology major who had been accepted for a summer internship program at the University of Auckland, New Zealand. She was to join a research group whose work focussed on cancer. Tis disease had interested Emily ever since her grandmother passed away from breast cancer. Emily had learned that breast cancer is the most frequent cancer among American women. According to the Centers for Disease Control and Prevention (CDC), annually more than 200,000 women get breast cancer, and for more than 40,000 of them, it is the cause of cancer-related death. Tis disease not only afects women, but also men; each year close to 1% of breast cancer cases are detected in males. Te inherited mutations in BRCA1 and BRCA2 genes are associated with an increased risk of breast cancer, but 85% of breast cancers are detected in women without a family history of this disease.

Because of her grandmother’s experience, Emily had been genetically tested. Te results revealed she had not inherited any of the mutations known to be responsible for familial breast cancer. It seemed to Emily that the main risks for the disease involved being a woman (increased estrogen levels) and aging, which upset her greatly. Rather than simply ac- cepting her possible fate, she was determined to do something about it, and had decided to pursue a career in medical research. Bench work was appealing to her and she wanted to follow the steps of great women in science like Maria Sklodowska-Quire or Rosalind Franklin. Te prospect of joining a real research lab seemed fantastic; perhaps it would give her an opportunity to witness the process of discovery and see what a real research laboratory looked like and how it functioned.

As you work through the four parts of this case study, you will join Emily in her journey to see that fnding a cure for cancer requires incremental steps in understanding this group of diseases.

Part I – Hormones and Breast Cancer Emily arrived at the laboratory of professor Baguley early in the morning. She was welcomed by Lena a young lab technician.

“Hi, Emily! You’re the frst one today! Te rest of the team will show up for our weekly doughnuts lab meeting.”

“Oh, am I too early?” replied Emily.

“Not at all. I’ll show you around. Put the lab coat on and follow me,” said Lena, and she led Emily to the cell culture room. “Here we keep the breast cancer cell line MCF-7. Do you know much about them?”

“Not really,” said Emily.

“No worries, Eric will teach you what you need to know. Tese cells are his life!” replied Lena. Just then Eric Baguley appeared in the cell culture room.

“Hi! Are you ready for some doughnuts? Our meeting will start shortly. Today we’ll meet in the conference room.”

“In a minute boss, I want to show Emily the MCF-7,” said Lena.

“Oh yes,” Eric nodded. “Tis is our model for breast cancer. Emily, have you heard about MCF-7 cells before?”

“A little,” replied Emily.

Case copyright held by the National Center for Case Study Teaching in Science, University at Bufalo, State University of New York. Originally published October 23, 2019. Please see our usage guidelines, which outline our policy concerning permissible reproduction of this work.

NATIONAL CENTER FOR CASE STUDY TEACHING IN SCIENCE

“OK, not a problem, you’re here to learn and help us solve their mystery,” said Eric with a big smile before continuing. “MCF-7 is a breast cancer cell line. It’s described as a luminal-like, epithelial carcinoma. Carcinoma is a type of cancer which develops from epithelial cells. In this case, the epithelial cells are lining the ducts or lobules of mammary glands.”

“Oh yes, we learned about that in preparation for the interview for this internship,” interrupted Emily.

“Tat’s great,” said Eric as he picked up a dry erase marker and came up to a white board. While he was still talking, he started to draw cells on the board. “Tis type of tumor is sensitive to estrogen. MCF-7 cells are endocrine-receptor positive (estrogen positive ER+ and progesterone positive PR+), which means that they have a signifcant number of estrogen and progesterone receptors. ER+ and PR+ breast cancer is just one subtype of the breast cancer. Te growth of the cells depends on these hormones. Normally, progesterone and estrogen infuence breast development during the menstrual cycle, and also during adolescence.”

Emily was reminded of what she had learned before …

Questions 1. What is a hormone? Where in the human body are progesterone and estrogen produced? What are the efects of

these hormones on the mammary tissue? Why do you think estrogen can increase the risk of breast cancer?

2. Based on their structure, how are progesterone and estrogen classifed? Which type of a receptor do they interact with? What is the consequence of this interaction?

“In Search of a Cure for Breast Cancer” by Jolanta Skalska Page 2

NATIONAL CENTER FOR CASE STUDY TEACHING IN SCIENCE

Part II – Receptors Eric was focusing on the estrogen receptors in his drawing. “You see Emily, this breast cancer we are trying to fnd a cure for depends on the activation of estrogen receptors. We know that estrogen receptors are a group of proteins found not only inside the cell, but also on the cell membrane.”

“Even though estrogen is a steroid hormone, can it also have specifc membrane receptors?” asked Emily. She remem- bered that steroid hormones have intracellular receptors, because they can easily cross a plasma membrane.

“Yes, the membrane estrogen receptors are mostly G-protein coupled receptors,” answered Eric, drawing the G-protein coupled receptor’s activation pathway on the board.

Question 3. Draw a diagram showing the typical signaling pathway involving the G-protein coupled receptors. Share your

thoughts with other groups, and discuss the diferences between your diagrams. Reproduce your fnal diagram here and note what changes you made as a result of your discussion.

“In Search of a Cure for Breast Cancer” by Jolanta Skalska Page 3

NATIONAL CENTER FOR CASE STUDY TEACHING IN SCIENCE

“Well,” said Emily, “If the survival of the cancer cell line MCF-7 (ER+) depends heavily on the presence of estrogens in the environment, can deprivation of estrogens induce a cell cycle arrest and eventually lead to cell death?”

“You’re on the right track, Emily!” replied Eric.

Question 4. What are some possible mechanisms that could be used to target breast cancer with ER+ phenotype?

“Emily, have you heard of tamoxifen?” asked Eric.

“Yes, but I don’t see the connection between tamoxifen and breast cancer.”

“Well, initially this drug was used in contraceptive pills. Currently it is used in the treatment of ER+ breast cancer, as a competitive inhibitor for estrogen receptors.”

“OK guys,” interrupted Lena, “it’s time for our lab meeting!”

“OK, let’s go,” replied Eric. “Dr. Mendoza promised to show up for our meeting, so we’d better not be late!”

Question 5. Tamoxifen is used in treatment of ER+ breast cancer as a competitive inhibitor for estrogen receptors. What is

competitive inhibition?

“In Search of a Cure for Breast Cancer” by Jolanta Skalska Page 4

NATIONAL CENTER FOR CASE STUDY TEACHING IN SCIENCE

MCF-7

TamC3 TamR3

Part III – Generating TamC3 and TamR3 Cell Lines As Emily, Eric, and Lena entered the conference room, they found Dr. Mendoza helping himself to cofee and a doughnut, seated at a conference table with a couple of graduate students.

“Dr. Mendoza, I would like you to meet Emily, our internship student from Alfred, New York. Dr. Mendoza is an oncologist, and he works at the clinic. We collaborate,” said Eric.

“Hi Emily! Nice to meet you. Would you like a cup of cofee?”

“Hello Dr. Mendoza, yes. Tank you.”

“You’ve already met the rest of the group. I gather they took you out last night. Did you guys have fun?” Eric smiled as he looked at Mike and Jen, the graduate students in his lab.

“Yes boss! We need more time of like this,” replied Mike.

“OK, who is presenting frst today?” asked Eric.

“I’m up,” said Mike coming up to the white screen, where the PowerPoint presentation with the experimental results would be displayed. Lena dimmed the lights and Mike started his presentation.

“Some breast cancer cells can develop resistance to the tamoxifen treatment and can survive the estrogen deprivation conditions. Te mechanisms of the drug and hormone deprivation resistance are not known. To investigate this phenomenon, we took the MCF-7 cells into the laboratory”

“Did you try to make the cell resistant to tamoxifen?” asked Dr. Mendoza.

“Yes. We divided them into two groups. One batch of the cells was exposed to tamoxifen for some period of time, which we did to mimic the clinical condition for the tamoxifen treatment in patients. Some of the cells died during the course of the experiment, but those that survived were selected and cultivated further.”

“Wow!” interrupted Eric. “So in this way you generated a new cell line. What did you call it?”

“We’re calling it TamC3. Tese cells were resistant to tamoxifen. Te other batch of the MCF-7 cells was subjected to conditions without estrogen.”

“Very clever!” this time Dr. Mendoza interrupted. “Tat’s mimick- ing the condition in patients subjected to hormone deprivation therapy. And what happened?”

“Te cells that survived the estrogen deprivation established another cell line, TamR3. Tose cells are estrogen independent. Look at Figure 1. Generation of tamoxifen resistant (TamC3) this graph,” replied Mike (Figure 1). and estrogen-independent (TamR3) cell lines.

Question 6. Te conditions employed in laboratory settings could mimic the outcomes of prolonged usage of tamoxifen and

hormone deprivation therapy. We don’t know much about the phenotypic profle of these newly emerged cancer cells. How might you explain the lack of response of TamC3 to tamoxifen treatment and TamR3 to estrogen deprivation?

“In Search of a Cure for Breast Cancer” by Jolanta Skalska Page 5

NATIONAL CENTER FOR CASE STUDY TEACHING IN SCIENCE

100

MCF-7

MCF-7 TamC3

TamC3

TamR3

TamR3

- OnM

D lllnM

- P-Akt

- - - TotalAkt

I P-p7056K

~========::::: - - -1 Totalp7056K

- - -l •ctln

“Now it’s time to show what I got from these cells,” said Jen as she replaced Mike. “I compared the response of MCF-7, TamC3, and TamR3 cells to tamoxifen. Look at the results.” (Figure 2.)

“Oh yes, we do see resistance of these cell lines to tamoxifen,” said Eric. “It is known that MCF-7 cells are sensitive to inhibitors of mTOR (mammalian target of rapamycin); rapamycin. Te incubation with rapamycin is toxic to MCF-7 cells. But how will TamC3 and TamR3 respond to that drug?” asked Eric.

“Hold on,” interrupted Dr. Mendoza: “I don’t un- derstand it… why rapamycin?” Emily was glad this question had been asked; she was wondering the same thing.

“Well,” started Eric and grabbed the marker as he approached the white board to draw, “the mTOR is an evolutionary conserved transduction pathway, which conveys signals from epidermal growth factor receptors (EGFR) on the cell membrane into the cell through phosphatidylinositol 3-kinase (PI3K) pathway and Akt/protein kinase B. Did you guys check the phosphorylation status of signaling molecules?” Eric asked his graduate students.

“Yes, we did a western blot to check the key proteins’ phosphorylation status,” replied Jen, and she contin- ued showing her new results. “Te phosphorylation level of Akt kinase and its downstream protein p70S6K is illustrated in the picture (Figure 3).”

“Good job! But now, the question is how the cells will respond to rapamycin? What do you think?”

Question 7. Look at the data provided and predict how

the TamC3 and TamR3 would respond to the rapamycin treatment. Predict the results of the cells’ growth rate in the presence and absence of rapamycin for all three cell lines: MCF-7, TamC3 and TamR3. Draw a graph and share it with

Figure 2. Efects of tamoxifen on cell growth. Te cells were exposed to 0nM or 111nM concentration of tamoxifen for four days, and the growth rate was estimated with thymidine incorporation assay. (Adapted from: Leung, E. et al., 2014, Frontiers in Oncology 4, cc by 3.0, <https:// doi.org/10.3389/fonc.2014.00221>.)

Figure 3. Te relative level of phosphorylated Akt and p70S6K in MCF-7, in TamC3 and TamR3 cells. Actin is shown as a loading control. (Adapted from: Leung, E. et al., 2014, Frontiers in Oncology 4, cc by 3.0, <https:// doi.org/10.3389/fonc.2014.00221>.)

other groups. Do your predictions correlate? Write an explanation of your graphical prediction using knowledge about mTOR pathway. What does the phosphorylation of kinases mean for the cell signaling pathways?

“In Search of a Cure for Breast Cancer” by Jolanta Skalska Page 6

NATIONAL CENTER FOR CASE STUDY TEACHING IN SCIENCE

> ~ 1/\ 0 C:

-~ 0

0.3

1~ .!: .g 0.15 ..c: l:l

i s

T

Camptothecin (µM)

l9 o,_._ .............. ...._ ...... ---'_.....1._.,___ MCF-7 TamC3 TamR3

> 800 H2O2 (µM) ~ 1/\ 0 C:

-~ 0 .0 ·-·-..., ..c: ro C: ~

400 ·- C: ..c: <II +-' u 3 C:

0 0 (9 u

0 MCF-7 TamC3 TamR3

> ~ 1/\ 0 C:

:B :8 :c ~ C: ...,

·- C: ..c: <II

~ ~ e 8 l9

> ~ 1/\ 0 C: ~ 0 :e ~ ..c: ~ C: ..., ·- C: ..c: <II +-' u 3 C:

e 0 u

l9

400 T Doxorubicin (nM)

200

0 ............... ......1._J.-..&--J.-.J....._

MCF-7 TamC3 TamR3

6 5-fluorouracil (µM)

3

0 MCF-7 TamC3 TamR3

Part IV – Killing TamC3 and TamR3 Cell Lines Time passed quickly as the members of the research team discussed their interesting results, and it was soon time for lunch. Dr. Mendoza turned to Emily and asked, “After lunch would you like to see our clinic?”

“Yes!” Emily responded.

“Good, I want to show you how we take care of our cancer patients who take chemo. We administer to them a cocktail of drugs like camptothecin, doxorubicin, cisplatin, and fuorouracil.”

Te meeting was over. Te whole group went to the cafeteria to grab a bite. After lunch Emily went with Dr. Mendoza to see the oncology patients. She learned that some of them sufered from side efects of chemotherapy.

Te following weeks at Eric’s lab were very productive. Emily learned new techniques, and performed experiments with some help from Lena, Jen and Chris. Tey exposed the MCF-7 and TamC3 to diferent chemotherapeutic agents.

Question 8. Look at the data provided in Figure 4 and describe the efects of drugs and oxidative stress on the cell proliferation

(growth) inhibition. What are the implications of the data for patient treatment?

Figure 4. Te efects of camptothecin, doxorubicin, H2O2 and 5-fuorouracil on MCF-7, TamC3 and TamR3 growth inhibition. Te data shows the drugs’ concentrations at which 50% of the cells subjected to treatment stopped grow- ing as measured by a cell proliferation assay. (Adapted from: Leung, E. et al., 2014, Frontiers in Oncology 4, cc by 3.0, <https://doi.org/10.3389/fonc.2014.00221>.)

“In Search of a Cure for Breast Cancer” by Jolanta Skalska Page 7

NATIONAL CENTER FOR CASE STUDY TEACHING IN SCIENCE

~ A 8

"- V

isoo T 1.4 T .. ~ 15.

:, .s QI

c3400 !0.7 V .. :, t: t .. ... 0 QI 0 0 ;'.!; MCF-7 TamC3 TamR3 MCF-7 TamC3 TamR3

'i

QI ., V V

D C C C: QI ., V V

~ t 60 ~ 2. 0 0 :, :E <= QI <3 ~ 3000 ~1 .2 ~

0 .. E 0 ..

C: .. .g QI 0 MCF-7 TamC3 TamR3 0 TamC3 TamR3 ·~ .. MCF-7 a: .. .; a:

9. Eric and his research group were trying to understand why daughter cell lines TamC3 and TamR3 have “diferent” responses to chemotherapeutic agents and oxidative stress relative to the parental cell line (MCF-7). Indicate the relative response of the cell lines (more/less sensitive or resistant) to the drugs/conditions listed:

MCF-7 TamC3 and TamR3 Tamoxifen Rapamycin Doxorubicin, fuorouracil, camptothecin Oxidative stress

From that point on, Emily was very busy. Te metabolic profle in the three cell lines was investigated. Te uptake of glucose, the level of reactive oxygen species (DCFDA fuores- cence) and production of lactate acid was measured in MCF-7, TamC3 and TamR3 cells. Te mitochondrial activity was also assessed with the alamar blue fuorescence assay. Emily was pleased. She had obtained good results to report during the next lab meeting (Figure 5)!

Question 10. Based on the results (Figure 5), what can

we conclude about the metabolic profle of these three cell lines? Explain why the level of lactic acid was measured. Support your answer with an equation/diagram of the appropriate chemical reaction.

Figure 5. Te H3 2-deoxyglucose uptake (A), lactate production (B), mitochon- drial activity (C), and reactive oxygen species generation (D) in MCF-7, TamC3 and TamR3 cell lines. (Adapted from: Leung, E. et al., 2014, Frontiers in Oncology 4, cc by 3.0, <https://doi.org/10.3389/fonc.2014.00221>.)

Short Essay Based on the data presented in this case study, what conclusion can be drawn about sub-lines of the MCF-7 breast cancer cell lines, TamC3 and TamR3? What is the molecular mechanism of their phenotypic profle? How could their resistance to rapamycin and sensitivity to chemotherapeutic drugs and oxidative stress be explained? Discuss your thoughts with the data provided. Your response should be at least 250 words and be modelled on the format of the

“Discussion” section of a standard scientifc research paper.

“In Search of a Cure for Breast Cancer” by Jolanta Skalska Page 8