Download Dr. Bulent Mutus “Inhibition of Neutral Sphingomyelinase 2 Can

A program of the Windsor Essex County Cancer Centre Foundation
2010 Seeds4Hope Grant Recipient
Dr. Bulent Mutus
“Inhibition of Neutral Sphingomyelinase 2 Can Lead to Breast
Cancer Cell Survival Through Plasma Membrane Steroid
Receptor Destabilization on Cholesterol Rafts”
SUMMARY OF RESEARCH PROJECT
Recent studies indicate that the use of cholesterol lowering drugs or statins
inhibits the growth and progression of prostate cancer as well as reducing breast
cancer incidence by up to 18%. Despite these exciting correlations, the manner in
which increased cholesterol leads to breast cancer initiation and/or progression is not well understood.
In this study, Drs. Bulent Mutus and Lisa Porter from the Departments of Chemistry and
Biochemistry and Biological Sciences at the University of Windsor, are focusing on an enzyme, termed neutral
sphingomyelinase (NSMase), found in the thin membranes surrounding all cells in the body. These researchers
have found that this enzyme is essentially missing in subsets of breast cancer, which currently lack effective drug
treatments. They have also observed that when NSMase is missing or inactivated this causes a drastic increase in
the amount of cholesterol in the cell membranes, which can help in the initiation and progression of breast cancer.
Drs. Mutus and Porter are trying to solve the manner in which NSMase inactivation triggers cancer and helps
cancer cells survive chemotherapy. This enzyme could represent new therapeutic directions in the treatment of
this aggressive subset of breast cancers.
HOW THIS RESEARCH HELPS ADVANCE QUALITY CANCER CARE IN OUR COMMUNITY
The findings from this research have the potential for development of novel therapeutic
strategies to improve in the treatment and/or the detection of aggressive forms of breast cancer. Such
advancements would have a tremendous impact on survival rates; thereby having high priority for
advancing cancer care both locally, regionally and beyond. In addition, future direction of this work will
promote collaborative research with the Windsor Regional Cancer Center. Strengthening University
- Hospital ties in the Windsor community is very important; not only for training, recruitment and
retention of the medical expertise but also for attracting future funds necessary to expand the
innovative technologies ongoing in the Windsor area, technologies essential to keep Windsor cancer care at the
forefront in Canada.
A program of the Windsor Essex County Cancer Centre Foundation
2010 Seeds4Hope Grant Recipient - Dr. Bulent Mutus
“Inhibition of Neutral Sphingomyelinase 2 Can Lead to Breast Cancer Cell Survival
Through Plasma Membrane Steroid Receptor Destabilization on Cholesterol Rafts”
PROGRESS REPORT
Our study examined the role of neutral sphingomyelinase (NSMase), an enzyme found in the membranes
that surround all cells. Enzymes are a class of proteins that facilitate-or catalyze- chemical reactions to take
place in cells and tissues. The reaction that NSMase catalyzes is the conversion of sphingomyelin, an important
component of cell membranes, to ceramide and phosphocholine. NSMase is central to cell survival. Under favorable
conditions NSMase has low activity and produces small amounts of ceramide. Under unfavorable conditions
such as when cells, are exposed to toxic compounds i.e. chemotherapeutic agents, the NSMase activity increases
and large amounts of ceramide are produced. Ceramide is a death signal for the cell and when present in large
amounts turns on the self-destruct or apoptotic machinery of the cell causing the cells to die.
Our hypothesis in this study was that cancer cells evade the self-destruct signal by lowering the activity of NSMase. As a result less ceramide is produced and the apoptotic signal is not initiated. These conditions are one
of the ways cancer cells are able to survive in the presence of toxic compounds. Our Results showed that, as
predicted, the breast cancer cells exhibit decreased NSMase activity when exposed to unfavorable conditions such
as in the presence of H2O2 Figure 1 (below).
FIGURE 1
But this did not affect the survival of the cancel cells i.e. they were escaping the self-destruct signal.
Figure 2 (below).
FIGURE 2
A program of the Windsor Essex County Cancer Centre Foundation
Another aspect of our study concerned the effect of NSMase on cell membrane structures termed cholesterol
microdomains or rafts (Figure 3). These rafts are very important to how the cells communicate the external
environment to the cell. For example, the raft size might be related to the ability of the cells to resist chemical
onslaughts.
FIGURE 3
We also predicted that a consequence of NSMase inhibition would be an increase in cholesterol microdomains or
rafts in the cell membranes since these rafts are composed of ~1:1 mixtures of sphingomyelin and cholesterol. If
the activity of NSMase goes down then less sphygomyelin is being destroyed as a results more rafts stay intact
and more cell protective proteins can now attach to these membrane rafts enabling cancer cells to escape death.
Our predictions were substantiated: as NSMse activity decreased in the presence of a NSMase inhibitor (GW4869)
the amount of cell membrane rafts increased (Figure 4).
FIGURE 4
These results, although very intriguing were not pursued further with respect to the changes in raft-protein binding
and the affect of these raft-protein interactions on cell survival. These studies will be examined in future
proposals.
In the current study we decided to more closely investigate the mode of NSMAse inhibition.
A program of the Windsor Essex County Cancer Centre Foundation
Many studies indicate that nitric oxide (NO) overproduction leads to cytoprotection in breast tumours. We
hypothesized that the elevation of NO could be the source of NSMase inhibition observed in triple negative breast
cancer cells (Figure 1). It is well established that increased NO production can lead to inactivation of proteins
and enzymes by a process termed S-nitrosylation where the NO+ moiety is attached to thiol groups of cysteine
residues found in proteins. NSMase is an ideal candidate for S-nitrosylation as it is a thiol rich protein (Figure 5:
where c represents thiol-containing cysteine residues in the amino acid sequence of NSMase)
FIGURE 5
The exposure of the breast cancer cells to an NO+ releasing compound (GSNO) resulted in the inhibition of
NMSase activity (Figure 6).
FIGURE 6
In addition, the NO+-donor gave rise to an increase in cholesterol (Figure 7) rafts as was also observed with an
inhibitor of NSMAse (Figure 4).
A program of the Windsor Essex County Cancer Centre Foundation
In view of the fact that NSMase expression is very low in triple negative breast cencer cells, we decided to
clone and express large amounts of NSMase to identify its residues susceptible to modification/inhinbition by Snitrosylation. To this end, a his tagged construct of human NSMase and expressed the protein in E coli and
purified (Figure 8A, B, C).
FIGURE 7
FIGURE 8A
FIGURE 8B
FIGURE 8C
We then exposed the NSMase to GSNO and subjected it to proteolysis. The resultant peptides were analyzed by
MALDI-TOF-TOF mass spectrometry.
As can be seen from Figure 9, we were able to identify 6 cysteine residues, in purple, in NSMase that are susceptible to blockage by NO+.
We are currently attempting to overexpress NSMase in breast cancer cells to probe the in vitro phosphorylation
and S-nitrosylation status/activity of the protein in cancer cells in response to chemotherapeutic agents such as
doxorubicin.
A program of the Windsor Essex County Cancer Centre Foundation
FIGURE 9
Summary: Based on the results obtained to date, we are postulating that when normal cells experience
unfavorable conditions, NSMase activity is increased by phosphorylation. As a result large levels of ceramide
are produced leading cells to undergo apoptosis and die. In cancer cells the enzyme is also expected to be
phosphorylated but the concomitant elevation of NO by the cancer cells results in the inactivation by
S-nitrosylation of NSMase and as a result the large levels of ceramide, that trigger apoptosis, are not reached and
the cancer cell escapes apoptotic signaling and survives. The results of this S4H supported study are very novel
and could make significant contributions in the targeting of NSMase activity in the progression and survival of
breast cancer. One potential therapeutic intervention we envisage is the use of hydrogen sulfide donors along with
chemotherapeutic agents to tumours. In this manner NSmase that has been inhibited by S-nitrosylation can be
reactivated by hydrogen sulfide restoring the full activity of NSmase. The fully active enzyme will then produce
large amounts of ceramide as in normal cells thereby inducing the death signal in the cancer cells.
A program of the Windsor Essex County Cancer Centre Foundation
Conference Presentations:
•
First International Conference on Nitrosylation in Oncology and Immunology- Feb 2-3, 2012, Dijon France.
•3rd International Workshop on Nitric Oxide in Cancer therapy, Queen’s University, May 31-June 2, 2013.
Book Chapter:
We have been invited to contribute a book-chapter on our NSMase work, to a Book in preparation “Nitric Oxide
in Cancer Therapy” expected publication date: Aug, 2104.
Manuscripts:
A manuscript describing the regulation of NSMase by S-nitrosylation is in preparation.
Highly Qualified Personnel Training:
The following students received training under this S4H supported research:
•
Milica Krstic Undergradate Student, Mutus lab, 2012-2013;
•
Nahia Sharma Undergradate Student, Mutus lab 2013-2014;
•
Bei-Lee Sun, PhD Candidate, Mutus Lab, 2010- present;
•
Martin Crosier, PhD Candidate, Porter Lab, 2010- present;