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Generation of an Activin-Inducible Mouse Mammary Epithelial Model System Natalia Trikoz December 9, 2015 Abstract:

Activin and TGFβ are cytokines of the Transforming Growth Factor superfamily and are thought to have similar function. However, their temporal expression patterns and functions during pregnancy suggest otherwise. TGFβ is expressed during involution of mammary epithelium after cessation of lactation, and is responsible for cell cycle arrest and apoptosis. Activin, on the other hand, is present throughout pregnancy and peaks during lactation, and is presumed to be responsible for differentiation of mammary epithelium. Women who suffer from preeclampsia during pregnancy have higher levels of serum Activin and an increased protection against breast cancer, relative to women that have undergone a normal pregnancy. Therefore, we hypothesize that Activin promotes differentiation and maturation of the mammary gland during pregnancy and may be part of the protective effect of parity. Results from the Jerry/Dunphy lab showed that in vitro, CDβGeo cells treated with TGFβ underwent epithelial to mesenchymal transition (EMT), which is a characteristic of metastatic cancer cells, and 100% of the pre-treated cells developed into mammary tumors when transplanted into mice. In contrast, Activin-treated CDβGeo cells did not undergo EMT, but rather expressed markers of differentiated luminal cells. These differentiated Activin-treated cells had poor outgrowth potential when transplanted into mice, and tumor potential could not be defined.

This problem can be overcome by creating a doxycycline-inducible Activin construct. This way, in vivo, the cells can outgrow normally before Activin expression is turned on through doxycycline treatment in drinking water. Therefore, my project was to transduce a mouse mammary epithelial cell line with a doxycycline inducible activin construct. CDβGeo cells have been successfully infected with the Activin construct: pINDUCER-14 Activin and they appropriately express Activin A in vitro when treated with doxycycline. In the future, these cells will be transplanted into cleared fat pads of host mice, and tested for the efficiency of Activin in vivo. Development of a functional inducible Activin mouse mammary model will be useful for future experiments to test the effect of Activin on tumorigenesis in the mammary gland in vivo. Background Pregnancy and breast cancer

Parity is the most significant factor that can modify breast cancer potential. Epidemiological studies have established that there is a life-long protective effect associated with pregnancy [1]. However, parity causes a transient increase in risk for pregnancy associated breast cancer (PABC). For women under the age of 20, the long-term protective breast cancer effect can be

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as high as 50%, indicating that women have 50% fewer cancers compared to nulliparous women. This protective effect decreases with age. For ages 30 to 34 the protective effect is negligible, and after the age of 35 there is an increase in breast cancer risk associated with parity (Fig. 1).

Figure 1. Schematic of breast cancer risk in parous women relative to nulliparous women [1] TGFβ and Activin

The molecular mechanisms underlying the protective effect of parity are still unclear, but the current hypothesis is that it results from alterations in hormone serum levels, growth factors, and cytokines [1]. Two cytokines that influence mammary gland development are part of the Transforming Growth Factor Beta (TGFβ) superfamily: TGFβ and Activin. It is well established that TGFβ has a paradoxical role in breast cancer, acting as both a tumor suppressor and a tumor promoter. The role of Activin, on the other hand, is not well characterized. These two cytokines share the same intracellular signaling pathway, and are thus assumed to have similar functions. However, several characteristics indicate that they have vastly divergent functions [2]. TGFβ and Activin have diverging functions

The first reason why Activin and TGFβ are thought to have divergent functions is because of receptor specificity. Activin and TGFβ have structurally different receptors, and Activin does not bind to the TGFβ receptor, and vice versa. Activin and TGFβ share the same intracellular SMAD-dependent signaling pathways, but the fact that the two cytokines have individual receptors also allows the possibility for divergent intracellular signaling pathways.

A second reason why Activin and TGFβ are thought to have different functions is that they are expressed during distinct parts of mammary gland development. Activin is expressed during expansion of mammary tissue during pregnancy and lactation [2]. Its presumed function is differentiation of epithelium in preparation for lactation. TGFβ is expressed during involution, a process that eliminates milk-producing cells at weaning [1]. Its presumed function is elimination

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of excess luminal cells through apoptosis during mammary gland regression and re-modeling (Fig. 2).

Figure 2. Normal Mouse Mammary Gland Development (Adapted from [3])

More is known about TGFβ function in the mammary gland and in mammary tumorigenesis. TGFβ has a dual role in breast cancer: it acts as both a tumor suppressor and a tumor promoter. It acts as a tumor suppressor because during tumor initiation, it causes cell death and increases the transcription of p53, a protein involved in tumor surveillance. However, after extended exposure, TGFβ functions as a tumor promoter, because it causes mammary epithelial cells to undergo Epithelial to Mesenchymal Transition (EMT). EMT is a hallmark of metastatic tumors, allowing cells to detach from the primary tumor and enter the circulation to invade distant sites. TGFβ and EMT also may increase cancer stem cells (CSCs), which self-renew, initiate metastasis, and are present in breast tumors [4]. The EMT phenotypic conversion consists of downregulation of epithelial markers (like E-cadherin), loss of intracellular junctions, and upregulation of mesenchymal proteins [5]. Unlike TGFβ, Activin does not cause mammary epithelial cells to undergo EMT, and this is a third reason why Activin and TGFβ are thought to have different functions.

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Figure 3. Epithelial to Mesenchymal Transition (EMT) [5]

Therefore, Activin and TGFβ may have diverging functions. Activin may have a role in the long-term protective effect of the reduced breast cancer risk resulting from parity. Activin is expressed during differentiation of mammary epithelium in preparation for lactation, which decreases the ratio of stem cells to differentiated luminal epithelial cells in the population. Since stem cells are considered to be potential cells of origin for breast carcinomas [6], a decreased population may correspond to a decrease in cancer potential. Additionally, women who suffer from preeclampsia during pregnancy have significantly elevated Activin serum levels, and a 2-fold decrease in breast cancer risk when compared to parous women [7,8,9]. Because of these observations, the hypothesis is that Activin could be part of the protective effect of parity. Hypothesis: Because Activin is expressed during pregnancy and lactation, when the protective effect is established, and because the correlational further decrease of breast cancer risk in women with preeclampsia who have elevated Activin serum levels, the hypothesis is that Activin may be part of the protective effect of parity. Model System

To study the effect of Activin and TGFβ, mice and associated derived cell lines are used. CD𝛽Geo mammary epithelial cells, derived from BALB/c mice, establish a cell line that responds to Activin and TGFβ. When transplanted into the mammary fat pads of BALB/c mice, CD𝛽Geo cells produce normal mammary ductal trees. Since CD𝛽Geo cells are p53 deficient, they form spontaneous tumors in 40%-50% of transplants in approximately 33 weeks. Therefore, we can compare changes in tumor frequency in response to different compounds, which makes this model system ideal for tumor studies [10]. Prior Experiments

The Dr. Jerry/Dr. Dunphy lab has previously conducted experiments with mice on the effects Activin and TGFβ. CD𝛽Geo cells were treated in vitro with Activin, TGFβ, and a Control treatment (Fig. 4). Results showed that TGFβ -treated CD𝛽Geo cells underwent persistent EMT, with a phenotypic conversion from epithelial cuboidal shape to a mesenchymal spindle-like shape, with loss of E-cadherin, and rearrangement of Actin. E-cadherin is an epithelial cell adhesion marker (Fig. 4 - green). Actin is a cytoskeletal element that is re-arranged in longitudinal spindle-like fibers that is characteristic of mesenchymal cells (Fig. 4 - red). Activin

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treated cells did not undergo EMT, because they did not lose expression of E-cadherin and did not undergo the spindle-like phenotypic change.

Figure 4. TGFβ induces EMT in CDβGeo cells, while Activin does not (Previous work in Jerry/Dunphy lab)

For the in vivo part of the study, CD𝛽Geo cells were treated with Activin and TGFβ in vitro, and then 50,000 cells were transplanted into cleared mouse mammary fat pads of BALB/c mice. A survival curve for tumor incidence was generated. TGFβ treated cells caused a significant increase in tumor potential, with 100% of mice forming tumors after 30 days. Activin treated cells had poor outgrowth in the mammary gland compared to the control, and did not form normal ductal trees. The tumor formation was similar to the tumor formation frequency of the control, but these results may have been due to failed outgrowth.

To account for failed outgrowth, Activin-treated cells were transplanted at a higher density (500,000 cells). Tumor occurrence and size were both decreased for the Activin treatment compared to control. However, even at this increased amount of transplanted cells, there was failed outgrowth in many of the mice and it is still unknown whether the reduced tumorigenesis was due to the Activin treatment or to the reduced outgrowth potential. To definitively determine if Activin promotes mammary tumor resistance, we need a system where the CD𝛽Geo cells could establish growth of the normal-like ductal trees in the fat pad prior to treating them with Activin in vivo.

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Objective Therefore, my project was to transduce a mouse mammary epithelial cell line with a doxycycline inducible Activin construct and test Activin production in vitro. These cells will later be transplanted into cleared fat pads of host mice, allowed to expand in the fat pad, and then tested for the efficiency of Activin expression in vivo. Development of a functional inducible Activin mouse mammary model will be useful for future experiments to test the effect of Activin on tumorigenesis in the mammary gland in vivo.

Generate an inducible Activin expression system in the transplantable CD𝜷Geo Mouse Epithelial Cell line:

Aim 1: Transduce CDβGeo cells with a plasmid with a doxycycline inducible Activin gene • Transduce cells with pINDUCER14-Activin Lentiviral vector • Induce expression of Activin with doxycycline and test induction effectiveness in vitro

-­‐ Test function with CAGA-Luciferase -­‐ Verify appropriate expression using qPCR

Aim 2: Transplant CDβGeo-Activin cells into mice mammary glands • Transplant 25,000 CDβGeo-Activin cells and 25,000 CD𝛽Geo-Control cells in contra-

lateral glands of 3-week-old host mice and allow for epithelial expansion • Induce expression of Activin with doxycycline-drinking water • Assess responses in mammary gland

-­‐ Perform whole mount analysis of mammary gland morphology -­‐ Assess Activin expression and induction of Activin target genes with qPCR

Methods Cell Culture

CD𝛽Geo cells were maintained in regular Mammary Epithelium Cell Line (MECL) Media: DMEM:F12 with 2.4g/L of HEPES and 1.2g/L of sodium bicarbonate supplemented with 2% adult bovine serum (Gibco), 10 ug/ml insulin (Sigma), 20 ng/ml mouse Epidermal Growth Factor (mEGF), 1% Antibiotic/Antimicotic (Life Technologies), and 0.03% Gentamycin (Life Technologies). 293T cells were maintained in DMEM:F12 with 2.4g/L of HEPES and 1.2g/L of sodium bicarbonate supplemented with 10% Fetal Bovine Serum (Gibco), 1% Antibiotic/Antimicotic ( Life Technologies), and 0.03% Gentamycin (Life Technologies). The 293T cell line is a human cells line that is used as packaging cells for infecting CD𝛽Geo cells. pINDUCER-14 Lentiviral System

To make an Activin-inducible CD𝛽Geo cell line I used the doxycycline-inducible Lentiviral pINDUCER-14 construct (Fig. 5) developed by Meebrey KL et al [11]. The human Activin A gene was exchanged into the tRFP/miR30/shRNA/miR3 region of the construct (Fig. 6). Jeff Kane and Shawn Hallett had previously done the insertion of the human Activin gene into pINDUCER-14 to make the pINDUCER-14 Activin construct. In the pINDUCER-14 Activin

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construct, EF1a is a transcription promoter that constitutively expresses rtTA3 and eGFP. The eGFP product causes cells containing the construct to fluoresce green, indicating that insertion was successful. The rtTA3 protein product combines with Doxycycline, when present, to form a complex that binds to the TRE2 promoter, which then transcribes the Activin A product. This is the inducible portion of pINDUCER-14 Activin. Therefore, Activin is only produced in the presence of Doxycycline.

Figure 5. pINDUCER-14 control construct [11]

Figure 6. The pINDUCER14-Activin construct inducible by Doxycycline (Adapted from [11]) Transfection and Infection:

In order to infect CdβGeo cells with the pINDUCER-14 control and pINDUCER-14 Activin plasmid constructs, the 293T human cell line needs to be used as packaging cells (Fig. 7). The 293T cells are transfected by adding the construct plasmid to the cell media, along with components for Lentiviral packaging and envelope components. The 293T cells take up these components, and generate Lentiviral particles, containing the pINDUCER-14 plasmid, which are then released into the media. The media from 293T cells, containing Lentiviral particles, is added to CdβGeo cells. The virus then infects the CdβGeo cells and inserts the target DNA (Activin gene) into the cell genome. Transfection of 293T cells:

293T cells were plated at a density of 2,500,000 cells/ 60mm dish with 293T media, 2 dishes per expression plasmid: GFP (as a transfection control), pINDUCER-14 (negative control), and pINDUCER-14 Activin. The next day, the 293T cells were washed with phosphate buffered saline (PBS) and transfected, using Lipofectamine 2000 (Invitrogen) with one of the expression plasmids (3μg /mL GFP; 4 μg /mL pINDUCER-14; and 4 μg /mL pINDUCER-14 Activin), along with 3 μg of psPAX2 (Lentiviral packaging components) and 2 μg of pMD2.G (envelope components). Lipofectamine increases transfection efficiency by forming a lipid layer around the plasmid, for more effective fusion with the cell membrane [12].

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The transfected 293T cells were incubated overnight in the BSL2+ room. In preparation for transduction, T25 flasks of CD𝛽Geo cells were plated with 600,000 cells/flask with MECL media. Infection of CdβGeo cells:

Prior to producing virus, the media on the 293T cells was changed. The next morning the media from each 60mm dish of 293T cells, containing Lentiviral particles, was removed, filter sterilized with a 0.45μm filter (Krackler Scientific), and diluted 1:1 with MECL media with 2μg/mL of Polybrene. Polybrene increases transfection efficiency by decreasing charge repulsion between the Lentiviral particles and components on the cell surface [13]. The mix was then added to the corresponding flasks of CD𝛽Geo cells. This process was repeated once more in the afternoon (approximately 6 hours after the initial infection).

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Figure 7. Transfection of 293 Packaging cells and Infection of CDβGeo target cells with the pINDUCER14 control and pINDUCER-14 Activin plasmids (modified from [14]).

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UV Microscope Transfection and Infection Verification The day after the infection, the CD𝛽Geo cells were washed with MECL media, and 3mL of

fresh MECL media was added to each. The UV microscope (model) was used for verification of transfection in 293T cells and Infection of CD𝛽Geo cells. As mentioned previously, the constitutionally expressed eGFP caused the cells to fluoresce green under the UV microscope when the cells were successfully infected with the virus. CAGA-Luciferase To test for Activin expression of CD𝛽Geo in response to Doxycycline treatment

Each of the two sets of three transduced CDbGeo cells conditions (GFP, pIND-14, pIND-14 Activin) were treated with either 0ug/ml (negative control) or 0.25ug/ml of doxycycline to induce Activin expression. Media was collected 48 hours after doxycycline addition, filter sterilized with 0.45μm filter (Krackler Scientific), and stored at 4℃ until 293T cells were ready to be transfected with CAGA-Luciferase. The 293T cells were plated at a density of 170,000 cells/well in triplicate in 24 well plates with 1mL of 293T media. The next day, the 293T cells were transfected with 0.5 μg CAGA-luciferase plasmid, 0.02 μg of Renilla plasmid (pRL-CMV) (Life Technology), 0.02 μg/mL of Lipofectamine, 200 μL of DMEM: F12 base media, and 300 μL of DMEM: F12 base media with 10% FBS. The next day (6-24 hours post transfection), 1mL of corresponding media previously collected from CD𝛽Geo cells was added to the 293T cells in the 24 well plate for 24-48 hours. Then the Dual Luciferase Reporter Assay (Promega) was performed with a 20/20!

Luminomer (Turner Biosystems) to assess for Activin induced luciferase.

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Results Transfection of 293T Packaging cells The 293T cells were successfully transfected with the GFP, pINDUCER-14 and pINDUCER-14 Activin plasmids, because GFP expression was detected with a fluorescent microscope (Fig. 8)

Figure 8: successful 293T transfection

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Infection of CD𝛃Geo Target Cells The CDβGeo cells were successfully transfected with the GFP, pINDUCER-14 and

pINDUCER-14 Activin plasmids, because GFP expression was detected with a fluorescent microscope (Fig.9). The infection was persistent because eGFP was still expressed for 12 days after initial infection. The negative control did not have GFP expression.

Figure 9. Successful infection of CDβGeo cells 1 and 12 days after infection: persistent expression of GFP. CAGA-Luciferase Results

Firefly Luciferase is induced in pINDUCER-14 Activin with Doxycycline treatment, indicating Activin expression (Fig. 10). Activin expression was not present in pINDUCER-14 Activin without doxycycline treatment, indicating that no unintended expression occurred. Renilla was the transfection control and normalizer, and it remained stable in all treatments. The samples were normalized to Renilla, and the results showed that pINDUCER-14 Activin with Doxycycline treatment induced Activin expression efficiently, and this occurred only in the pINDUCER Activin cells treated with Doxycycline.

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Figure 10: Activin expression induced with Doxycycline (CAGA-Luciferase assay)

0 10 20 30 40 50 60 70 80

GFP (- dox)

GFP (+dox)

pIND-14 (-dox)

pIND-14 (+dox)

pIND-14 ACTIVIN

(-dox)

pIND-14 ACTIVIN (+dox)

GFP

Firefly

0  

0.5  

1  

1.5  

2  

2.5  

GFP  (-­‐  dox)  

GFP  (+dox)  

pIND-­‐14  (-­‐dox)    

pIND-­‐14  (+dox)  

pIND-­‐14  ACTIVIN  (-­‐dox)  

pIND-­‐14  ACTIVIN  (+dox)  

GFP  

Renilla  

0  5  10  15  20  25  30  35  

GFP  (-­‐  dox)  

GFP  (+dox)  

pIND-­‐14  (-­‐dox)    

pIND-­‐14  (+dox)  

pIND-­‐14  ACTIVIN  (-­‐dox)  

pIND-­‐14  ACTIVIN  (+dox)  

GFP  

Ratio  (Fire-ly  to  Renilla)  :  Activin  Expression  

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Discussion

Understanding the role of Activin in mammary gland development and tumorigenesis is important because it may provide a potential mechanism for decreasing breast cancer risk. Previous work in the Jerry/Dunphy lab has shown that TGFβ treated cells in vitro form tumors 100% of the time when transplanted into mice, while Activin treated cells fail to outgrow. Since Activin is present in mammary gland development during differentiation of mammary epithelium to the lactating state, the reason for the failed outgrowth may be that the cells are already terminally differentiated. The purpose of this project was to overcome this problem by creating a doxycycline inducible activin construct. This way, in vivo the cells can outgrow normally before Activin expression is turned on through doxycycline treatment in drinking water. This Activin-inducible system will allow the assessment of tumorigenesis in response to Activin.

The 293T packaging cells were successfully transfected and the CD𝛽Geo cells were successfully infected, as seen from the fluorescent microscope images. Doxycycline successfully induced Activin expression only in the pINDUCER-Activin plasmid, as demonstrated by the CAGA-Luciferase assay, indicating that the inducible portion of the plasmid was operative. Upon induction with doxycycline, the CDbGeo cells were able to transcribe mRNA to produce a functional Activin protein, which was secreted into the medium, dimerized, and signaled through the Activin receptor on the cell surface of the 293T cells to stimulate the CAGA-luc reporter.

The next objective is to verify appropriate expression of Activin using qPCR, and to quantify the amount of Activin protein produced. After that, the CDβGeo-Activin cells will be transplanted into mice mammary glands and allowed to expand before inducing expression of Activin with doxycycline-drinking water. After 14-day activin induction we will assess responses in mammary gland, including morphology and Activin expression. Once this has been completed, a tumor study can be conducted to observe the effect of Activin on tumorigenesis.

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References

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2) Dunphy, K. A., et al. "The Role of Activin in Mammary Gland Development and Oncogenesis." Journal of mammary gland biology and neoplasia 16.2 (2011): 117-26. Print.

3) Hennighausen, L., and G. W. Robinson. "Information Networks in the Mammary Gland." Nature reviews.Molecular cell biology 6.9 (2005): 715-25. Print.

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12) Dalby, B., et al. "Advanced Transfection with Lipofectamine 2000 Reagent: Primary Neurons, siRNA, and High-Throughput Applications." Methods (San Diego, Calif.) 33.2 (2004): 95-103. Print.

13) Davis, H. E., J. R. Morgan, and M. L. Yarmush. "Polybrene Increases Retrovirus Gene Transfer Efficiency by Enhancing Receptor-Independent Virus Adsorption on Target Cell Membranes." Biophysical chemistry 97.2-3 (2002): 159-72. Print.

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