‫التقرير النهائي‬
Administrative Information
‫المعلومات االدارية‬
03-06-10
:‫المرجع‬
Project Title - )‫عنوان المشروع (عربي وأجنبي‬
THE ROLE OF RHO GTPASES AND OTHER PROTEINS IN ASTROCYTOMA MALIGNANCY AND
METASTASIS : A THERAPEUTIC APPROACH
‫ نهج للعالج‬: ‫تحديد البروتينات المسؤولة عن غزو اورام المخ الخبيثة‬
Principal Investigator - ‫الباحث الرئيسي‬
‫رقم الهاتف‬
‫العنوان االلكتروني‬
Telephone
e-mail
03473008 Mirvat.elsibai@lau.edu.lb
‫العنوان‬
Address
Beirut
campus
Sage 303
‫الوظيفية‬
Post
Assistant
Professor
‫المؤسسة‬
Institution
LAU
‫االسم‬
Name
Mirvat ElSibai
Co-Workers - ‫الباحثون المشاركون‬
‫العنوان االلكتروني‬
e-mail
2 years
‫المؤسسة‬
Institution
‫االسم‬
Name
: Duration -‫المدة التعاقدية للمشروع‬
Scientific Information
‫العلمية‬
‫المعلومات‬
ّ
Objectives - ‫الهدف‬
AIM 1:
We have previously shown that Cdc42 is the main Rho GTPase leading to actin polymerization
and protrusion formation in EGF-stimulated breast adenocarcinoma cells, MTLn3 cells 11. This
was shown to be through the activation of the WASP family of proteins and Arp2/3
downstream of Cdc42 and upstream of actin activation 11. Recent work confirmed our work in
MTLn3 cells, showing Cdc42 to be required for protrusion and motility in melanoma cell lines
18
. StarD13 could be exerting its tumor suppressor role through the inhibition of Cdc42 activity.
Preliminary data from our laboratory showed that the total level of expression of the Cdc42
protein showed an increase in the more malignant astrocytomas, as compared to the low grade
astrocytomas (Figure 2-Appendix). This could be consistent with an oncogenic pro-invasive
role of Cdc42 in malignant astrocytomas. In order to investigate this hypothesis, we first need to
establish the role of Cdc42 in astrocytoma malignancy and motility.
We will examine the role of Cdc42 and StarD13 using two approaches: the first being a
correlation approach where we will correlate the levels of expression of the two proteins to the
levels of malignancy of the disease. This will be done using RT-PCR as well as
immunohistochemistry of whole tissues and Western Blot analysis of extracts from tissues of
patients with different grades of astrocytomas. We have obtained the approval from the
committee for human research in our institution and we have started collecting few samples.
We also plan on correlating the pattern of localization of Cdc42 and StarD13 to their possible
functions in astrocytoma cell lines. We have obtained 8 astrocytoma cell lines from our
collaborator, Dr. Marc Symons (collaboration letter enclosed) and also have obtained normal
astrocytes (Lonza) in order to compare them to the cancer cells. A first look showed that indeed
Cdc42 localizes to the leading edge of the astrocytoma cells that have a protrusion. This
experiment was done in SNB-19 cells which are grade IV astrocytoma cells (GBM) (Figure 3Appendix).
The second approach will be to manipulate the levels of StarD13 and of Cdc42 and to look at
the effects of the increase or decrease in the cellular levels of the proteins on the behavior of
cells; this includes cell motility, cell proliferation and apoptosis. The manipulation of the
protein levels is done through either the overexpression of a GFP-StarD13 fusion construct
(which we already obtained from Dr. Hitoshi Yaigsawa in Japan), the overexpression of a
constitutively active GFP-Cdc42 construct or through knocking down the proteins using
siRNA. We will run a pull-down assay, which we have established in our laboratory and have
used extensively 10,11,19,20, in order to show the effect of StarD13 overexpression or knockdown
on Cdc42 activation. We will monitor the actin makeup or the cells and the activation of
proteins that regulate actin, as well as the direct movement of the cells in culture, will reflect the
invasive ability of the cells and how it changed in response to the manipulation
(overexpression/knockdown). This is done through imaging the cells, as they translocate in
culture, frame by frame using live microscopy, and computing the net paths crossed by the cells
during the whole course of the movie 10,11.
AIM 2:
Other than Cdc42, StarD13 is a GAP for Rho. Indeed when we transfected T98G cells (grade
IV astrocytoma cells) with GFP-StarD13, the cells showed a complete loss of stress fibers,
which confirms the inhibitory effect of StarD13 on the Rho/ROCK pathway. We have
previously shown that RhoA is a positive regulator of breast adenocarcinoma motility and that
there is an area of RhoA activation towards the leading edge as MTLn3 cells underwent cell
motility. ROCK has been described to be a negative regulator of cell motility in astrocytoma
cell lines 21. We were able to confirm those observations in our system and indeed inhibiting
ROCK led to an increase in cell motility in grade IV astrocytoma cells (data not shown). We
also obtained FRET data showing that total cellular RhoA activation is actually higher in grade
IV astrocytoma than in grade I (Figure5-Appendix). Immunohistochemical analysis also
revealed an increase in the total protein level of Rho in grade IV astrocytoma as compared to
grade I (Figure 6-Appendix). The increase in RhoA activation was consistent with the decrease
in StarD13 expression in malignant astrocytomas. RhoA signals to ROCK and to mDia (the
actin nucleator) downstream. The inhibition of ROCK alone led to an increase in cell motility
but it remains to elucidate the effect of RhoA inhibition on cell motility in astrocytoma cell
lines.
We will also examine the role of RhoA by manipulating the levels of RhoA and ROCK and
looking at the effects of the increase or decrease in the cellular levels of the proteins on the
behavior of cells; this includes cell motility, cell proliferation and apoptosis. The manipulation
of the protein levels is done through either the overexpression of active RhoA, inhibition of
ROCK using Y27632 and ROCK1/2 siRNA, and inhibiting RhoA using RhoA siRNA. We will
monitor the direct movement of the cells in culture, will reflect the invasive ability of the cells
and how it changed in response to the manipulation (overexpression/knockdown). This is done
through imaging the cells, as they translocate in culture, frame by frame using live microscopy,
and computing the net paths crossed by the cells during the whole course of the movie 10,11.
AIM 3:
We also aim to apply the 2D cell culture model in animals. Our collaborator, Dr. Symons, is
working on an animal model for brain tumor and we plan on collaborating in order to image for
activations of RhoA 10,22, Cdc42 11,23, and possibly Rac 24 in animals with brain tumor
xenografts. Imaging in live animals will be made possible through intravital imaging 25. This
system will enable us to determine the roles of these RhoGTPases, as well as their regulators, in
a 3D environment. This will also enable us to study cell invasion, as opposed to 2D movement.
Tumor cells will be visualized after being transfected with biosensors for RhoA, Rac and
Cdc42. The cells will also be manipulated for the proteins of interest and then injected into the
animal in order to be traced and imaged for their invasive characteristics.
Achievements -‫أالنجازات المحققة‬
During the last 2 years, we examined signaling pathways governing malignancy and invasion in
the brain tumor, astrocytoma. We focused on the protein Stard13 which regulates key proteins
involved in cell invasion and proliferation. IHC analysis on Grade I-IV brain tissues from
patients showed StarD13 to be overexpressed in grade III and IV astrocytoma tumors when
compared to grade I and II. However, when we mined the REMBRANDT data, we found that
the mRNA levels of StarD13 are indeed higher in the higher grades but much lower than the
normal tissues. The overexpression of a GFP-StarD13 construct in astrocytoma cells led to the
increase in cell death and a decrease of cell viability. Knocking down StarD13 using siRNA led
to a decrease in cell death and an increase in cell viability. When looking at the mechanism, we
found that the tumor suppressor effect of StarD13 is through the inhibition of the cell cycle and
not through the activation of apoptosis. When knocking down StarD13, we also saw an increase
in p-ERK, uncovering a potential link between Rho GTPases and ERK activation. Our future
interests would be to determine which Rho GTPase is responsible for this effect and to elucidate
the direct link between the Rho GTPase and ERK.
Another branch of the work was to examine the role of StarD13 and its downstream proteins,
including RhoA and Rac, in brain tumor invasion. Our results showed that RhoA, Rac1, and
StarD13 are essential for astrocytoma cell migration. Rac1 plays a role in the formation of focal
complexes, which ultimately mature into focal adhesions under the action of RhoA. StarD13
plays a role in the inhibition of RhoA in focal complexes, a process that seems indispensible for
astrocytoma cell migration
In conclusion, we identified a new tumor suppressor in brain tumor. Targeting of this tumor
suppressor would be a new target for therapeutics.
Perspectives - ‫آفاق البحث‬
The completion of this project has many benefits, the work will help defining the role of a
very prominent tumor suppressor in brain tumor. This disease remains an obscure disease
and its mode of malignancy and invasion poorly understood. Deciphering the molecular
pathways that lead to its malignancy presents a tremendous value and a very promising
therapeutic target. Targeting the tumor suppressor/overexpression, will lead to selective
inhibition of invasion in cancer cells. This is due to the fact that, following this approach,
we would be re-instating a normal mechanism, which will not harm normal cells.
Publications & Communications - ‫المنشورات والمساهمات في المؤتمرات‬
Defended Masters thesis:
- Bassem Khalil (August 2009-June 2011). Currently PhD student at Albert Einstein
College of Medicine in the laboratory of Dr. Jonathan Backer. Thesis title: The
Regulation of RhoA by StarD13 in Focal Adhesions is Essential for Astrocytoma Cell
Motility
-
Sally El-Sitt (August 2009-May 2011). Thesis title: Reaching for the Star: Oncogene
or tumor suppressor?
-
Samer Hanna (2010-2012). Currently PhD student at Albert Einstein College of
Medicine in the laboratory of Dr. Dianne Cox. Thesis title: Studying the role of
StarD13 in breast cancer malignancy: Effect on proliferation and apoptosis and
metastasis.
Peer-reviewed journal articles:
1. Hanna S., Khalil B.D., Saykali B., Nasrallah A., Sobh R., Nasser S. and El-Sibai M.
StarD13, a tumor suppressor needed for motility. Under preparation (2012).
2. Hanna S., Khalil B.D., Sobh R., Nasser S. and El-Sibai M. StarD13 regulates
RhoA/Rac activation in focal adhesions leading to cell motility. Under preparation
(2012).
3.
Khalil B.D., Hanna S., El-Sitt S., El-Sabban M., Hahn K.M., Symons M., and El-Sibai M.
The regulation of RhoA in focal adhesions by StarD13 is essential for astrocytoma cell
motility. Submitted to the Journal of Cellular and Molecular Biology (2012).
4. El-Sitt S., Khalil B.D., Hanna S., El-Sabban M., Fakhreddine N., and El-Sibai M.
DLC2/StarD13 plays a role of a tumor suppressor in astrocytoma. Oncol Rep. 2012
Aug;28(2):511-8. doi: 10.3892/or.2012.1819. Epub 2012 May 17
Abstracts in international Conferences:
1. Hanna S., Khalil B.D. and El-Sibai M. The Role of StarD13 in Breast Cancer
Proliferation and Cell Motility. The American Society of Cell Biology (ASCB), San
Francisco, USA, December 15-19 2012.
2. Hanna S., Khalil B.D. and El-Sibai M. The Role of StarD13 in Breast Cancer
Proliferation and Cell Motility. The European Molecular Biology Organization
(EMBO), Nice, France, September 22-25 2012.
3. Nasrallah A. and El-Sibai M. The Effect of StarD13 on Colorectal Cancer Malignancy
and Invasion. The 14 t h Int e rnat i onal Bi enni al C ongr ess of t he Met ast asi s
R es earch S o ci et y, Brisbane, Australia, September 2-6 2012.
4. Hanna S. and El-Sibai M. StarD13 a tumor suppressor needed for breast cancer cell
metastasis. 4ème colloque du club adhérence cellulaire (Interdisciplinary Institute for
Neuroscience), Bordeaux, France May 24-25 2012
5. Khalil B., El-Sitt S., El-Sabban M., Backer JM and El-Sibai M. The Regulation of
RhoA by Stard13 in Focal Adhesions is Essential for Astrocytoma Cell Motility. The
American Society for Cell Biology (ascb), Denver, Colorado, USA, December 3-7
2011.
6. El-Sitt S., Khalil B., Backer J. and El-Sibai M. The role of StarD13 in astrocytoma
malignancy: Tumor suppressor or Oncogene. EORTC (European Organization for
Research and Treatment of Cancer) the -NCI (National Cancer Institute)-ASCO Annual
Meeting on "Molecular Markers in Cancer", Brussels, Belgium, October 27-29 2011.
7. Khalil B., El-Sitt S., El-Sabban M. Backer J. and El-Sibai M. The Regulation of RhoA
by Stard13 in Focal Adhesions is Essential for Astrocytoma Cell Motility. The 3rd
EMBO meeting (European Molecular Biology Organization), Vienna, Austria,
September 10-13, 2011.
8. Khalil B., El-Sitt S., El-Sabban M. Backer J. and El-Sibai M. The Regulation of RhoA
by Stard13 in Focal Adhesions is Essential for Astrocytoma Cell Motility. Mechanisms
& Models of Cancer Meeting at the Salk Institute, San Diego, CA, USA, August 10-14,
2011.
9. Khalil B., El-Sitt S., El-Sabban M. Backer J. and El-Sibai M. The role of StarD13 and
RhoA in astrocytoma cell motility. Gordon research conference on motile and
contractile systems, 07/31/2011 - 08/05/2011 at Colby-Sawyer College in New
London, NH, USA.
Abstracts in local Conferences:
1. Hanna S. and El-Sibai M. The Role of StarD13 in Breast Cancer Proliferation and Cell
Motility. 5th Annual Poster Conference in Biology & Biomedical Sciences at LAU,
Byblos on May 18 2012. Chosen as best poster.
2.
Nasrallah A. and El-Sibai M. The effect of StarD13 on colorectal cancer malignancy
and invasion. 5th Annual Poster Conference in Biology & Biomedical Sciences at LAU,
Byblos on May 18 2012.
3. Khalil B. and El-Sibai M. The Regulation of RhoA in Focal Adhesions by StarD13 is
Essential for Astrocytoma Cell Motility. The Lebanese Association for the
Advancement of Science (LAAS) “18th International Science Meeting”, March 2012.
4. Hanna S. and El-Sibai M. The Role of StarD13 in Breast Cancer Proliferation and Cell
Motility. The Lebanese Association for the Advancement of Science (LAAS), “18th
International Science Meeting”, March 2012.
5. Nasrallah A. and El-Sibai M. the effect of StarD13 on colorectal cancer malignancy and
invasion. The Lebanese Association for the Advancement of Science (LAAS), “18th
International Science Meeting”, March 2012.
6. Hanna S., Rizk S. and El-Sibai M. The role of StarD13 in Breast Cancer proliferation
and metastasis. AUST “Enhancing Graduate Research in the Biomedical Sciences in
Lebanon: Role of National and International Collaborations” conference, May 30, 31
2011.
7. Khalil B. and El-Sibai M. The regulation of RhoA in focal adhesions by StarD13 is
essential for astrocytoma cell motility. AUST “Enhancing Graduate Research in the
Biomedical Sciences in Lebanon: Role of National and International Collaborations”
conference, May 30, 31 2011.
8.
El-Sitt S., Khalil B., El-Sabban M., Backer J.M. and El-Sibai M. StarD13 is a tumor
suppressor in Brain astrocytoma. AUST “Enhancing Graduate Research in the
Biomedical Sciences in Lebanon: Role of National and International Collaborations”
conference, May 30, 31 2011.
9.
Khalil B., El-Sitt S., El-Sabban M., Backer J.M. and El-Sibai M. The roles of RhoA
and StarD13 in astrocytoma motility. The 44th Middle East medical Assembly (MEMA),
AUB on May 7th2011.
10.
Hanna S., Rizk S. and El-Sibai M. The role of StarD13 in Breast Cancer proliferation
and metastasis. 4th Annual Poster Conference in Biology & Biomedical Sciences at
LAU, Byblos on May 4th2011. Chosen as second best poster.
11. Khalil B. and El-Sibai M. The regulation of RhoA in focal adhesions by StarD13 is
essential for astrocytoma cell motility. 4th Annual Poster Conference in Biology &
Biomedical Sciences at LAU, Byblos on May 4th2011.
12.
El-Sitt S., Khalil B., El-Sabban M., Backer J.M. and El-Sibai M. StarD13 is a tumor
suppressor in Brain astrocytoma. 4th Annual Poster Conference in Biology &
Biomedical Sciences at LAU, Byblos on May 4th2011.
13.
El-Sitt S., Khalil B., El-Sabban M., Backer J.M. and El-Sibai M. The role of StarD13
in Astrocytoma Malignancy. The Lebanese Association for the Advancement of Science
(LAAS) “The 17th International Scientific Conference”, November 2010.
14.
Khalil B., El-Sitt S., El-Sabban M., Backer J.M. and El-Sibai M. The role of StarD13,
a RhoA and Cdc42 GAP, in astrocytoma cell motility. The Lebanese Association for the
Advancement of Science (LAAS) “The 17th International Scientific Conference”,
November 2010.
15.
El-Sitt S., Khalil B., El-Sabban M., Backer J.M. and El-Sibai M. The role of StarD13
in Astrocytoma Malignancy. The third annual Molecular Biology Poster Conference at
LAU Byblos) May 2010.
16.
Khalil B., El-Sitt S., El-Sabban M., Backer J.M. and El-Sibai M. The role of StarD13,
a RhoA and Cdc42 GAP, in astrocytoma cell motility. The third annual Molecular
Biology Poster Conference at LAU Byblos) May 2010. Was chosen as best poster
(http://www.lau.edu.lb/news-events/news/archive/biology_graduate_students_comp/)
Abstract - ‫موجز عن نتائج البحث‬
In the past 2 years our work that has been funded by LAU and NCSR was very productive and
resulted in several posters at national conferences (see at the end of the document), as well as in
international conferences. The work also resulted in the completion of 3 Masters Thesis
projects, where the students defended at LAU in June 2011 and 2012. This work also resulted in
an article that was published in 2012 in the Journal of Oncology Reports and in another paper
that we just submitted to the Molecular and Cellular Biology journal. In both articles NCSR is
acknowledged for its support in funding our work.