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Bowel Disease Research Foundation
of The Association of Coloproctology of Great Britain and Ireland
Advancing the cure and treatment of bowel disease
Research
Project Final Report
Principal investigator
Project Title
Start date
David Bowden
Institution
Countess of Chester
NHS FT
Evaluation Of Acid Ceramidase As A Potential Response
Biomarker And Radiosensitisation Target In Rectal Cancer
Feb 2015
Finish date
Feb 2016
Lay Summary
(max 500 words)
Please use the
following format:
a) Problem addressed,
background and
strategic significance
b) Method(s) used
c) Hoped for results
d) Actual results and
implications for
treatment/understandi
ng
Colorectal cancer is the second commonest cause of cancerrelated death in the UK, with rectal cancer comprising
approximately one third of newly diagnosed cases. Whilst
surgery remains the most efficacious treatment, radiotherapy
has become routinely used in the neoadjuvant setting for
rectal cancer, with ‘short-course’ treatment conferring a
reduction in local disease recurrence and an increase in
disease free survival post-operatively. ‘Long-course’
radiotherapy combined with chemotherapeutic sensitizers
such as capecitabine (chemoradiotherapy) is used in locally
advanced disease in an attempt to achieve down-staging of
the tumour, and to minimise the risk of involved margins at
surgical resection.
The response to chemoradiotherapy is variable. There is an
observed pathological complete response (where no tumour
cells are detectable in a resection specimen) in 15-20% of
patients, with an overall down-staging effect observed in up
to 70%. Patients with a pathological complete response to
chemoradiotherapy have significantly reduced local disease
recurrence or metastases, and improved overall and disease
free survival at 5 years, when compared with those who do
not respond.
Neo-adjuvant therapy is not without risk of local and
systemic toxicity. Examples of morbidity include long-term
ano-rectal and genito-urinary dysfunction in up to 50% of
patients, an increased risk of post-operative anastomotic leak,
and systemic risks of cardiac failure and secondary tumours.
Patients who do not respond to chemoradiotherapy are not
only exposed to its risks but also to a delay in the
commencement of the next stage of their treatment.
There is currently no way of predicting who will respond to
chemoradiotherapy. The ability to predict response would
allow positive selection of patients who will benefit from
treatment, and exclude those who would only be exposed to
the risks and delay to other treatment modalities.
Additionally, an understanding of the mechanisms behind
radio-resistance may permit the opportunity for this to be
manipulated to improve treatment efficacy, conferring
advantages in reducing local recurrence and improving
overall/disease free survival whilst potentially increasing the
proportion of patients who would benefit from treatment. An
improvement in treatment efficacy may in turn reduce
dosage requirements and subsequently unwanted off-target
effects.
Our group have previously identified one protein to be
overexpressed in relatively radioresistant patients. The aim of
this project was confirm this finding in a larger set of patients
and explore whether this protein plays a direct role in
mediating radioresistance in a colorectal cell line. The
implication of this protein, acid ceramidase, in
radioresistance is a novel one and this study has the potential
to further increase the knowledge of radioresitance in rectal
cancer. Furthermore, this protein has a known
pharmacological inhibitor and thus has translational
potential. The proposed project will explore both
pharmacological and molecular manipulation of this protein
and if time permits seek to explore whether manipulation
affects radiosensitivity.
Background (purpose
for project)
Introduction
Colorectal cancer is the second commonest cause of cancer
death in the UK. Locally advanced rectal cancer represents a
particular surgical and oncological challenge in both the
treatment of the primary tumour and also early access to
systemic therapy. Response to chemoradiotherapy is
extremely variable, and it is clearly established that there are
no reliable clinical or molecular predictors of response (1).
The overall long term survival from rectal cancer remains
fairly static with most patients succumbing to systemic
disease. The ability to predict or improve radioresistance
would ensure that unnecessary morbidity (radiotherapy side
effects, delay in systemic chemotherapy) is reduced for this
very important group of patients (2).
Our initial pilot data was obtained from eight patients
undergoing chemoradiation followed by surgical resection in
a single UK centre (Chester). Tumour samples prior to
chemoradiotherapy and post surgery were subjected to
isobaric tagging for relative quantification (iTRAQ). A total of
3359 unique proteins were identified, all of which were
present in at least half of the samples and therefore taken
forwards for analysis. The data were acquired over 3
experimental runs, with a mean (SD) of 7 (21.2) peptides used
to identify the proteins of origin. This corresponds to a mean
(SD) coverage of 15.18% (15.72%) for identified proteins. A
total of 8 proteins were differentially expressed between the
initial diagnostic samples of the responders and nonresponders (which were classified according to histological
regression). Of these proteins, 3 were downregulated and 5
upregulated. On pathway analysis using Ingenuity software,
the ceramide degradation pathway was identified as being
the most significantly downregulated pathway in those
tumours responding to chemoradiotherapy.
Acid ceramidase is a sphingolipid - a group of biomolecules
known to be responsible for important signalling functions in
the control of cell growth and differentiation (3). Importantly,
a recent study has convincingly demonstrated that
downregulation of the corresponding gene with siRNA in a
prostate cancer cell line confers radiosensitivity (4). This in
vitro study in PPC-1 cells assessed radiation response by
clonogenic and cytotoxic assays, and also demonstrated that
upregulation of acid ceramidase decreased sensitivity to
radiation and created cross-resistance to chemotherapy. The
small molecule acid ceramidase inhibitor, LCL385, was also
sufficient to sensitize PPC-1 cells to radiation. These data
support our finding of low levels of acid ceramidase in those
tumours responding to radiotherapy. Carmofur, in common
to the widely used radiosensitiser capecitabine, is a
fluorouracil analogue currently in widespread clinical use in
the US in the adjuvant setting for colorectal cancer. In
addition to its presumed primary mode of action as a
thymidylate synthetase inhibitor it has also independently
been shown to inhibit acid ceramidase in the human tumour
derived cell lines SW403 (colorectal) and LNCaP (prostate), a
property not shared by fluorouracil or capecitabine (5).
Methods
1. Construction of a tissue microarray (TMA) of rectal cancer
patients undergoing long course chemoradiotherapy prior to
surgical resection.
2. Immunohistochemical analysis of TMA and scoring of
staining along with correlation with clinical (as assessed by
radiological response) and pathological response.
3. Molecular inhibition of AC expression using both
pharmacological manipulation with the chemotherapy agent
Carmofur and molecular manipulation with siRNA.
Confirmation of inhibition to confirmed using Western
blotting and also analysis of AC activity using a propriety
activity assay (courtesy of University of Barcelona)
Results and discussion
1. Immunohistochemical analysis of a tissue microarray of
111 patients undergoing chemoradiation followed by surgical
resection in a single institution (COCH). This confirmed that
high AC expression in normal colonic epithelium was
associated with poor response (p=0.012). Within the tumour
diagnostic biopsies, AC expression in the stroma was also
strongly associated with response with low expression in
good responders (p=0.012) and complete pathological
response (p=0.003). Conversely, high stromal expression was
associated with poor response (p=0.017)
During the study we were also fortunate to collaborate with
the Sanger Institute at the University of Cambridge who were
able to analyse AC protein expression across a wide range of
cell lines (n=49) in order to support our proteomic findings.
This confirmed a wide range of differential levels of AC
expression (-2.06 to +1.6, compared with normalised control).
Analysis of mRNA gene expression across 1000+ cancer cell
lines (from Affymetrix U219 gene expression array) confirms
differential expression at this level also.
Table 1: AC protein expression across 49 colorectal cell lines (courtesy of
Sanger Institute, University of Cambridge (scores normalised to an internal
control)
2. Inhibition of AC expression in the colorectal cancer cell
line HCT116 (p53+/+) was assessed using western blotting
after molecular and pharmacological treatment. siRNA
inhibition reduced AC expression to a maximum to 15.5%
across a variety of concentrations after 48 hours. Inhibition
with carmofur (2uM) appears to achieve maximum effect
between 2 and 3 hours with a maximum reduction in
expression to 32.4%.
Figure 1: Suppression of AC expression via biological (siRNA) and
pharmacological manipulation (M: untreated culture medium, V: DMSO
vehicle control, siRNA: transfected RNA knockdown cells, NC-siRNA: non
coding transfected cells, Can: AC postive rectal cancer specimen)
3. AC Activity was investigated in the same cell line with an
assay employing the substrate Rbm14-12 (University of
Barcelona). Inhibition of activity reached a maximum of
27.2% after only 30 minutes incubation with 2.5uM carmofur.
This effect was not observed with the standard
radiosensitiser 5FU. siRNA inhibition achieved similar levels
of suppression (24.4%), but this was only evident after 24
hours of incubation.
es
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Acid Ceramidase Activity ASAH1 siRNA
20
40
Time (Hours)
60
80
Acid Ceramidase Activity (%)
Acid Ceramidase Activity Pro ile - 2.5µM
Carmofur
100
80
60
40
20
0
0
1
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Time (Hours)
Figure 2: Suppression of AC activity via siRNA and carmofur: time course
after incubation versus umbelliferone fluorescence (adjusted according to
background fluorescence and normalised to time point 0)
Conclusion
Our project has confirmed our initial proteomic finding that
acid ceramidase does appear to play a role in response of
rectal cancer to chemoradiotherapy. We have furthermore
been able to demonstrate successful molecular and
pharmacological manipulation of both protein expression
and protein activity. We have also demonstrated that there
appears to be a wide range of expression of acid ceramidase
at protein and mRNA level in 49 colorectal cell lines.
Recommendations for
future work
Our initial investigation into the effects of manipulation of
acid ceramidase on radiosensitivity using a clonogenic assay
have so far not yielded any positive results. However, we
have only analysed one cell line (HT 116) which has
relatively low levels of expression. Having established a
collaboration with the Sanger Institute we have a research
fellow in place to investigate this further looking at 6 cell
lines compromising relatively high and low expressers of the
protein. Little is known about why AC may be implicated in
radioresistance and we will attempt to elucidate this further
by exploring the relationship between AC expression and
know markers of radioresistance such as DNA repair
proteins.
References
(author, title, date of
publication)
1. Ryan JE, Warrier SK, Lynch AC, Ramsay RG, Phillips,
Heriot AG. Predicting pathological complete response
to neoadjuvant chemoradiotherapy in locally advanced
rectal cancer: a systematic review. Col Dis 2015 (18):
234-246
2. Tiernan J, Cook A, Geh I, George B, Magill L,
Northover J, Verjee A, Wheeler J, Fearnhead N. Use of
a modified Delphi approach to develop research
priorities for the Association of Coloproctology of
Great Britain and Ireland. Col Dis 2014 (16): 965-970
3. García-Barros, M., Coant, N., Truman, J., Snider, A.
and Hannun, Y. (2014). Sphingolipids in colon cancer.
Biochimica et Biophysica Acta, 1841(5), 773-782.
4. Mahdy, A., Cheng, J., Li, J., Elojeimy, S., Meacham,
W., Turner,
L., Bai,
A., Gault,
C., McPherson,
A., Garcia, N., Beckham, T., Saad, A., Bielawska,
A., Bielawski, J., Hannun, Y., Keane, T., Taha,
M., Hammouda, H., Norris, J. and Liu, X. (2009). Acid
ceramidase upregulation in prostate cancer
cells
confers resistance to radiation: AC inhibition, a
potential radiosensitizer. Molecular Therapy, 17(3), 430438.
5. Realini, N., Solorzano, C., Pagliuca, C., Pizzirani,
D., Armirotti, A., Luciani, R., Costi, M., Bandiera, T.
and Piomelli, D. (2013). Discovery of highly potent acid
ceramidase inhibitors with
in
vitro
tumor
chemosensitizing activity. Scientific Reports, 3, 1035.