Summer Undergraduate Research Program - Fred Hutchinson ...

Alternative Functions of Telomerase in Breast Cancer
Kelea Somerton, Shibani Mukherjee, Eduardo J. Firpo, James M. Roberts
Proliferation advantage conferred by Telomerase
is necessary and sufficient to cooperate with
Her2 and form dysmorphic acini
Abstract Methods
HMECs were transduced with retroviral vectors containing either WT, NTERM, or NDAT forms
of Telomerase, or empty vector. To examine the cooperation between Telomerase and Her2,
structures were transduced with two separate vectors, one containing either WT, NDAT, or
NTERM Telomerase or empty vector, and the other containing inducible Her2. Transduced
cells were then grown in 3D culture on a Matrigel bed. After 10 days in culture, Her2 was
induced by adding AP1510.
2010 Lee Hartwell Poster Award
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Vec WT NTerm NDAT
+Her2 +Her2 +Her2 +Her2
4A. 4B. 4C.
35
70
30
**
30
60 **
25
25
50
**
20
20
40
**
15
15
30
10
20
10
5
10
5
0
0
0
Vec WT Vec WT
Vec WT NTerm NDAT
+Her2 +Her2
+Her2 +Her2 +Her2 +Her2
4D.
Average Luminal cells/ Acinus
Average Cell numbers/ Acinus
Average Cell numbers/ Acinus
Cultures were fixed at either day 15 or 19. Day 15 structures were immunostained for Ki67
(proliferation marker) and counter-stained for To-Pro-3 (DNA). Using confocal microscopy,
images of the midsections of approximately 60 acinar structures (containing more than 7
cells per midsection) for each cell type were collected. The total number of cells per acinar
midsection, as well as the number of luminal and Ki67 positive cells were counted for each
acinus. A t-test was used to analyze the results and p-values were determined.
The Telomerase protein is upregulated in almost all human cancers. Telomerase can
contribute to tumorigenesis by maintaining the telomeres cancer cells, thus enabling the cells
to avoid senescence and have an extended lifespan. We have identified additional telomereindependent
roles for Telomerase in tumorigenesis. We have seen that Telomerase can cause
proliferation of primary human mammary epithelial cells (HMECs) in growth-factor-limiting
conditions, which is a hallmark of cancer. Additionally, we have separated this proliferation
advantage function of Telomerase from its telomere maintenance and extended lifespan
functions with the use of two mutant forms of Telomerase, NTERM and NDAT. In this study,
we extend these findings using a 3-Dimensional cell culture model system in which HMECs
plated on Matrigel develop into 3D spheres of cells, called acini. The spheres resemble
Terminal Ductal Lobular units in the breast, which have growth-arrested cells and hollow
lumens. Hyperplasia and Ductal Carcinoma in situ (early stages of breast cancer) show
increased proliferation of epithelial cells and filling of the lumen. We use the more
physiologically relevant 3D culture system to model acini and study the aberrant phenotypes
associated with breast cancer initiation and progression. We also use this system to analyze
the ability of Telomerase to cooperate with the Her2 oncogene, which is upregulated in 20-
30% of breast cancers. We show that the proliferation advantage conferred by Telomerase is
sufficient to cause increased proliferation and luminal filling, resulting in acinar structures that
share properties of early stage breast cancer. We further show that Telomerase is necessary
for Her2-mediated proliferation and luminal filling. Their cooperation results in formation of
dysmorphic acini, which retain an intact basement membrane and resemble early stage, preinvasive
breast cancer. Finally, we show that the proliferation advantage function alone is
sufficient for this cooperation with Her2.
Vec+Her2 WT+Her2
NTerm+Her2 NDAT+Her2
Day 19 acinar structures were visualized using a Nikon SMZ1500 microscope. Structures
were then immunostained for Laminin V (basement membrane marker) and counter-stained
for To-Pro-3 (DNA), and imaged using confocal microscopy.
4E.
Background
154
Proliferation advantage conferred by Telomerase
is sufficient to increase cell numbers and cause
luminal filling of acini in 3D culture
Figure 4. Determining effects of WT and mutant forms of Telomerase on the ability of
Her2 to form dysmorphic acini.
4A: Cells transduced with either WT Telomerase or Her2 alone were compared to cells with
both WT Telomerase and Her2. Cells were stained with To-Pro-3. Average cell numbers per
acinar midsection are shown. **p