Transcript Slide 1

Progesterone receptor membrane component 1 (PGRMC1) and its role in the establishment and progression of
female cancers
Melissa L. McCallum1, Cindy A. Pru1, Hannah Balash, Bo R. Rueda2, John J. Peluso3, James K. Pru1
Department of Animal Sciences1, Washington State University, Pullman, WA 99164; Vincent Center for Reproductive Biology2, Harvard Medical School, Boston, MA
02114; Department of Cell Biology3; University of Connecticut Health Center, Farmington, CT 06030
INTRODUCTION
The second leading cause of death among women in the United
States is cancer. Approximately 40,000 women die of breast cancer
annually, and another 40,000 are diagnosed with endometrial
cancer. Often, the way endometrial cancers are treated is by
completely removing the female reproductive tract, leading to
infertility. Many cancers involving the female reproductive system
depend on hormone regulation. In order to investigate the role of
progesterone in cancer, studies in our lab are focused on a novel
membrane progestin receptor called progesterone receptor
membrane component 1, or PGRMC1. We are working to determine if
PGRMC1 is important in the growth and chemoresistance of tumors.
Here, a simple subtraction approach was taken in which PGRMC1
either remained intact in a cell population or was depleted. The
PGRMC1 knockdown cells were produced by injecting a cell colony
with a Lentivirus that eliminated the PGRMC1 gene. Two cell lines,
the MDA-MB-231 breast cancer cells and the Ishikawa (IKLV)
endometrial cancer cells were used for the experiments. Microarray
analysis was performed and this resulted in differential regulation of
many genes that was verified primarily by reverse transcriptase
polymerase chain reaction (RT-PCR). Western blot analysis was also
used to validate microarray data for some genes. Then human
xenograft tumors were developed in immunocompromised mouse
models. Six mice were injected intraperitoneally (into the body
cavity) with five million GFP-labeled IKLV cells, three with the
PGRMC1-intact cells and three with the PGRMC1-deplete cells, and
the tumors were collected 6 weeks later. Results indicate that many
genes that promote blood vessel growth and stress resistance are
up-regulated in PGRMC1-intact cell populations. For example, lysyl
oxidase (LOX) and kinase insert domain receptor (KDR) were shown
to be up-regulated by RT-PCR. The tumors collected were also
significantly larger in mice whose tumors were generated from
PGRMC1-intact cells. These results suggest that PGRMC1 impacts
tumor growth and resistance to stress. This information could be
used to help develop strategies to reduce the fatality of cancers of
the female reproductive system.
RESULTS
Fig. 1. Knockdown Experiment
A
B
C
D
Figure 1. IKLV and MDA cancer cell treatment and PGRMC1
knockdown. A) Effects of vehicle (ethanol), doxorubicin (Dox)
and/or progesterone (P4) in IKLV endometrial cancer cell death. B)
Effects of vehicle, Dox and/or P4 in MDA breast cancer cell death.
C) Knockdown of PGRMC1 in IKLV cells using lentiviral-based
shRNA. D) Knockdown of PGRMC1 in MDA cells using lentiviralbased shRNA.
*Table 1. IKLV and MDA Microarray Analysis
Data
Microarray analysis data of the top ten up-regulated and the top ten
down-regulated genes in PGRMC1-intact versus PGRMC1-deplete
IKLV and MDA cancer cells.
*Fig. 4. RT-PCR Validation
Fig. 2. IKLV and MDA s.c. Tumor Growth
HYPOTHESIS
PGRMC1-deficiency in breast and endometrial cancer cells results in
reduced tumor growth in vivo and altered gene expression in vitro.
MATERIALS AND METHODS
 IKLV and MDA-MB-231 cancer cell lines
 Lentivirus
 Microarray analysis
 RT-PCR validation
 Created primers for genes indicated by microarray
analysis and ran a PCR on cDNA
Immunocompromised mouse model
 Injected 3 mice with 5 million GFP-labeled IKLV
PGRMC1 –depleted cells and 3 mice with 5 million GFPlabeled IKLV PGRMC1-intact cells
 Allowed tumors to develop over 6 weeks
 Removed tumors, measured and embedded in paraffin
wax
Figure
2.
Subcutaneous
xenograft
tumor
growth
in
immunocompromised mice using PGRMC1-intact (control) and
PGRMC1-deplete (PGRMC1 KD) endometrial or breast cancer cells.
*Fig. 3. IKLV i.p. Tumor Volume
Figure 4. RT-PCR from cDNA generated from cell cultures of
PGRMC1-intact versus PGRMC1-deplete IKLV and MDA cells.
CONCLUSIONS
*
 The presence of PGRMC1 causes tumors to be larger in size
than if PGRMC1 is absent
 When PGRMC1 is present, genes that promote blood vessel
formation and stress resistance are more abundantly expressed
Figure 3. IKLV tumors volume 6 weeks after i.p. injection. * p < 0.05
*denotes data generated by M. McCallum