OCT4SOX2

NANOG

Totipotent Pluripotent

NANOGOCT4

SOX2

BRYMESP

OCT4 MESP

BRYNKX25

OCT4 MESP

BRYNKX25

ISL1

NKX25

NKX25

ISL1

?

?

HIF

GATA4

HOXB5Mesodermal Progenitors

First Heart Field Progenitors

Second Heart Field ProgenitorsEndothelial Cells

Postnatal cardiac progenitors

Cardiac Muscle Cells

Cardiac Conduction Cells

Differentiation

Affects of Hypoxia on Stem Cell Phenotypes Mediated by mTOR Inhibition Andrew Schroeder Jr, Kajal Patel, Scott Findlay, Michael Jewer, Krista Vincent, Lynne-Marie Postovit

WORLDWIDE, BREAST CANCER IS THE MOST PROMINENT INVASIVE CANCER in women. It comprises 22.9% of invasive cancers in women and 16% of all female cancers. A considerable amount of the current knowledge on breast cancer is acquired from in vitro and in vivo studies performed with cell lines derived from breast cancer.

THE HALLMARKS OF CANCER DESCRIBE SIX BIOLOGICAL CAPABILITIES acquired through the multistep progression of cancer. They include sustaining proliferative signaling, evading growth suppressors, resisting cell death, enabling replicative immortality, inducing angiogenesis (blood-vessel forming), and activating invasion and metastasis (spreading of cancer).

To determine the affect of these conditions on invasion and metastasis, a soft agar assay for colony formation was used which measures the anchorage dependence of cells. Anchorage dependence describes cells that must attach to a solid surface before they can divide: they fail to grow in agar gel. When the cells are transformed, as in cancer, they become anchorage independent and can grow in gel, making them more dangerous in cancer because they are more versatile. We predict that both hypoxia and INK128 will increase anchorage independence in T-47D cells.

To determine the affect of hypoxia and INK-128 inhibition on replicative immortality and the expression of stem cell like phenotypes, real-time reverse transcriptase PCR was employed. Cancer stem cells (CSC) are thought to drive uncontrolled tumor growth. OCT4, NANOG, and SOX2 are three genes that are expressed in CSC. They play a critical role in maintaining pluripotency (ability to divide into different specialized cells) and self-renewal properties in CSC. See Figure 0. By measuring the expression of these genes using real time reverse transcriptase PCR (Polymerase Chain Reaction) we can determine which conditions make cells more stem cell like, and thus more dangerous. Both hypoxia and INK-128 are hypothesized to increase the expression of these genes.

Hypoxia and INK-128 are inhibitors of a key cell pathway called mTOR, which is vital in many cellular functions. Inhibition of mTOR increases the pluripotency of cells and increases their capacity for anchorage independent growth, making these cells more dangerous. By using a specific inhibitor of mTOR along with hypoxia, we can better understand how hypoxia affects mTOR and thus how it affects cancer progression.

1. T-47D CELLS WERE TREATED UNDER HYPOXIA (2% O2 at 37° C), 20nM of INK-128 (21% O2 at 37°C), or normoxia (21% O2 at 37°C) and incubated for 48 hours.

2. Base level of agarose was prepared by combining 6mL of liquefied 1% agarose with 6mL of complete 2X RPMI. Each well of the six-welled plate was then plated with 2mL of the mixture and allowed to solidify. This was repeated three times for hypoxia, normoxia, and ink plates.

3. After 48 hours, cells were aspirated, washed with PBS, trypsinized, neutralized, and strained before cell suspension.

4. Cells were then counted using a hemocytometer and concentrations were calculated. Volume of cells were then calculated for 1000 cells/well at 1.5mL media per well.

5. Top layer of agarose was then prepared with 4.5mL 0.7% agarose and 4.5mL 2X RPMI combined with calculated cell volume and 1.5mL was plated onto each well.

6. Plates were held in incubation for 1-2 weeks and media was replaced every second day. Following this incubation period formed colonies were analyzed morphologically using cell stain and quantified by counting the number of colonies formed per well.

1. T-47D CELLS WERE TREATED UNDER HYPOXIA (2% O2 at 37° C), 20nM of INK-128 (21% O2 at 37°C), and normoxia (21% O2 at 37°C) and incubated for 48 hours.

2. RNA was extracted according to kit protocol and concentration was determined using photospectometry at 260nM.

3. Master mix was prepared for 5 reactions of cDNA at 20µL per reaction.

4. Master mix, calculated amount of RNA, and Nuclease-free H2O were combined and added to individual Eppendorf tubes for each sample as well as a no-template control.

5. Tubes were positioned in Thermal Cycler, where complementary DNA transcription took place.

6. Real time PCR master mix was prepared for RPLPO, NANOG, SOX2, and OCT4 and 19µL was placed into each well with 1µL of cDNA

7. A micro-seal was then placed over the plate and centrifuged before loading the BIO-RAD real-time PCR apparatus.

ABSTRACT

INTRODUCTION SOFT AGAR ASSAY

REAL-TIME RT PCR

RESULTS

Moving Forward

8. After PCR, data was transferred to an excel spreadsheet where values were normalized and compiled into graphs of log 2 representing the expression of the three genes in INK-128 and hypoxia samples.

Diagram 1: Polymerase chain reaction (PCR) allows the exponential copying of part of a DNA molecule. DNA strands are separated at approximately 94°C . Specific primers are annealed that are complementary to a site on each strand. The heat-stable Taq DNA polymerase extends the DNA from the primers.

Diagram 2: In real time PCR, we measure the cycle number at which the increase in fluorescence (and therefore cDNA) is exponential. This is shown by the thick blue horizontal line in the figure (known as the threshold) and is set by the user. The point at which the fluorescence crosses the threshold is called the Ct and is extremely important for determining the expression of a gene.

The summer research project was focused specifically on effecting replicative immortality and invasion and metastasis in T-47D breast cancer cells by subjecting them to conditions of hypoxia (low oxygen: 2%) and an INK-128 mTOR inhibitor with normoxia (normal oxygen: 21%) as a control.

Figure 1 : mTOR reduces pluripotency in cells.

Figure 2: Both hypoxia and INK128 inhibit mTOR which results in an increase in pluripotency.

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Graph 2: The MCF-7 cell line that was obtained from a colleague performing the same experiment shows an increase in anchorage independent growth for both samples of hypoxia and INK-128. This is in agreement with our earlier hypothesis and confirms that hypoxia and INK128 increase cancer progression.

Graph 1: The T-47D cell line experienced an increase in anchorage independent growth when exposed to INK-128 while anchorage independent growth was slightly less than the normoxia sample when exposed to hypoxia. From these results we can deduce that INK128 is a potent supporter of anchorage independent growth.

Graph 3: Within the real time reverse transcriptase PCR, the T-47D line experienced an unexpected and dramatic decrease in NANOG, OCT4, and SOX2 in the hypoxia sample. INK-128 experiences a slight increase in OCT4 levels however it appears that overall both hypoxia and Ink reduce the expression of these genes in T-47D cells.

Graph 4: The MCF7 line experienced an increase in all three stem-cell biomarkers in both INK-128 and hypoxic conditions which was in agreement with our earlier hypothesis. These increases represent more pluripotent cells and thus are more tumorigenic. Because INK-128 and hypoxia both inhibit mTOR we know that this pathway’s deactivation is an essential part of cancer progression.

THE RESULTS SHOWN ABOVE IN THE T47D CELL LINE SHOW THAT INK-128 HIGHLY supports anchorage independent growth while hypoxia slightly opposes it. Thus future research should be targeted toward up-regulating mTOR signaling to inhibit this type of growth and slow metastasis. In the real time PCR assay, almost all of the stem cell biomarkers in the T-47D line were down regulated in both hypoxia and ink which could mean that pluripotency is reduced under these conditions. The MCF7 cell line achieved higher anchorage independent growth in both hypoxia and INK-128 conditions, which could lead to therapies specifically targeted to releasing mTOR from hypoxia inhibition. The MCF7 cells in both INK-128 and hypoxia experienced an increase in stem cell biomarkers, increasing pluripotency and the malignancy of cancer. In the future, more research will be needed to determine exactly how the mTOR pathway is being affected so that the data generated can contribute to a viable therapy.

Figure 0:

Proliferative Signaling

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Induction of Angiogenesis

Invasion and Metastasis

The Hallmarks of Cancer

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