Academic Year 2015 - 2016
LIPID METABOLISM IN COLORECTAL CANCER: ALTERATIONS, THERAPEUTIC OPPORTUNITIES, AND OUTLOOK ON
MOLECULAR DIAGNOSTICS
Stijn DE KEUKELEIRE
Promotor: Prof. Dr. Lennart Martens
Dissertation presented in the 2nd Master year in the programme of
Master of Medicine in Medicine
FACULTY OF MEDICINE AND
HEALTH SCIENCES
My gratitude goes out to my girlfriend and family, whose support and personal interest have
been a major encouragement in writing this review. I would also like to thank my supervisor,
Prof. Lennart Martens, as for each time we had an appointment, he inspired me with his
knowledge and personal interest regarding this topic.
This thesis is dedicated to my mother: her fight against injustice goes on. May my work
inspire others to investigate and develop possibilities for battling this cruel illness.
1 ABSTRACT ............................................................................................................................................... 1
2 INTRODUCTION ....................................................................................................................................... 2
3 METHOD ................................................................................................................................................. 4
4 RESULTS .................................................................................................................................................. 5
4.1 COLORECTAL CANCER ............................................................................................................................... 5 4.1.1 Epidemiology ................................................................................................................................... 5 4.1.2 Prevention and screening ................................................................................................................ 5 4.1.3 Classification method ...................................................................................................................... 6 4.1.4 Treatment ........................................................................................................................................ 6
4.1.4.1 Non-metastatic colorectal cancer ................................................................................................................ 6 4.1.4.2 Metastatic colorectal cancer ........................................................................................................................ 6
4.2 METABOLOMICS IN COLORECTAL CANCER ................................................................................................ 7 4.2.1 Current and upcoming ‘omics’ approaches for biomarkers ........................................................... 7 4.2.2 Lipidomics and its analytical technology ........................................................................................ 8
4.3 LIPID METABOLISM AND COLORECTAL CANCER:........................................................................................ 9 4.3.1 Free Fatty Acids ............................................................................................................................ 10
4.3.1.1 Pathway .................................................................................................................................................... 10 4.3.1.2 Known alterations in colorectal cancer ..................................................................................................... 13 4.3.1.3 Alterations in free fatty acid synthesis pathways and distribution ............................................................ 14
4.3.1.3.1 Enzymes and other proteins ........................................................................................................... 14 4.3.1.3.2 Fatty acid distribution..................................................................................................................... 16
4.3.1.4 Alterations in eicosanoid metabolism and distribution ............................................................................. 18 4.3.1.4.1 COX-pathway ................................................................................................................................ 18 4.3.1.4.2 LOX-pathway ................................................................................................................................. 19 4.3.1.4.3 CYT P450 epoxygenase pathway ................................................................................................... 20
4.3.2 Glycerolipids .................................................................................................................................. 21 4.3.2.1 Pathway ............................................................................................................................................... 21 4.3.2.2 Glycerolipids and colorectal cancer ..................................................................................................... 25
4.3.2.2.1 Alterations in the glycerophosphate pathway ............................................................................... 25 4.3.2.2.2 Glycerophospholipids ..................................................................................................................... 26 4.3.2.2.3 Phospholipases ............................................................................................................................... 27 4.3.2.2.4 Triglycerids ..................................................................................................................................... 28
4.3.3 Sphingolipids ................................................................................................................................. 29 4.3.3.1 Pathway ............................................................................................................................................... 29 4.3.3.2 Sphingolipids in colorectal cancer ....................................................................................................... 31
4.3.4 Sterols ............................................................................................................................................ 35 4.3.4.1 Pathway ............................................................................................................................................... 35 4.3.4.2 Alterations in colorectal cancer ........................................................................................................... 36
4.3.5 Lipoproteins and lipid droplets ...................................................................................................... 39 4.4 AN INTERPRETATION OF PREVIOUSLY OBTAINED EXPERIMENTAL DATA .................................................. 41
5 DISCUSSION .......................................................................................................................................... 42
6 SUMMARY IN DUTCH ............................................................................................................................ 44
7 REFERENCES .......................................................................................................................................... 45
8 APPENDIX ............................................................................................................................................. 55
1
1 Abstract
Colorectal cancer (CRC) is one of the most prevalent and deadliest cancers worldwide, which
has prompted scientists to search of better screening methods and adequate therapies for this
disease. Lipidomics, the study of lipid profiles in cells, tissues and other organisms, is an
under-investigated research field, but has already showed promising results in many types of
cancers, including CRC. This literature review tries to comprehensively resume all alterations
in lipid metabolism that are linked with CRC. There are four major lipid groups to be
discussed: free fatty acids, glycerolipids (including triglycerides and
glycerophosphospholipids), sphingolipids and sterol lipids. The changing distribution of lipid
bodies and lipoproteins in CRC will also be mentioned. Firstly, free fatty acids in tumour
samples showed up-regulation of de novo synthesis-related enzymes (for instance ACLY and
FASN) and significant alterations in fatty acid distribution, shifting towards more desaturated,
pro-inflammatory ω-6 fatty acids. Regarding glycerolipids, triglycerides were mainly down-
regulated and phosphatidylcholine was more augmented in CRC tissue samples, while
lysophosphatidylcholines was shown up-regulated in plasma of CRC patients. Ceramide is the
major component in sphingolipid metabolism and due to its anti-proliferative, pro-apoptotic
properties CRC cells try to evade pathways that lead to the generation of this metabolite.
Cholesterol de novo synthesis and uptake from lipoproteins was enhanced in these neoplastic
cells, which was also highlighted by their higher expression of LDL and HDL cellular
receptors. Lastly, intracellular lipid droplet augmentation has already been associated with
malignancy and indeed was present in colon cancer cell lines. These findings indicate that
lipidomics could provide many opportunities in targeted therapy and prognostic or screening
biomarkers in CRC, although more intensive research is necessary if lipid metabolism is to
be integrated in our therapeutic and screening policy, not only for CRC but also other types of
cancers.
2
2 Introduction
Colorectal cancer still remains a major health topic: last year’s data show that the yearly
incidence amounts to almost 2 million people with more than 600 000 deaths throughout the
globe. These data vary in different countries, depending on age, sex, hereditary factors and
risk-factors concerning lifestyle-issues such as alcohol consumption, smoking, red meat
intake, obesity, diabetes and inflammatory bowel diseases (IBD). (1)
The current treatment strategy and prognosis are mainly determined by the clinicopathological
staging system TNM. Based on the Dukes staging system, it provides information concerning
the local tumor growth, lymph nodal invasion and metastasis of the colorectal neoplasm. (2)
Today, surgery still remains the main curative option for non-metastatic colorectal cancer,
with adjuvant or neo-adjuvant therapy (using radiotherapy, chemotherapy or radio-
chemotherapy) conserved for higher stages or high-risk patients. (1)
Metabolomics is a new research field, attempting to profile intra- and extracellular
metabolites (sugars, amino acids, nucleotides and lipids) to acquire new biomarkers for
diagnosis and prognosis, and to gain better insight in the fundamental steps involved in
carcinogenesis. Metabolomics has shown promising results in CRC staging and metastasis
detection and some minor results were achieved when pharmaco-metabolomics were used in
cancer treatment. (3) Lipidomics is a branch of metabolomics that examines the lipid
pathways and networks by quantitatively defining lipid profiles in organelles, cells and
organisms and has taken some huge steps forward regarding applications and further
analytical technologies. Mass spectrometry remains the cornerstone for investigating these
lipidomes. (4)
Lipids are complex but unique molecules with an irreplaceable role in cell structure
maintenance, energy provision, as signaling. (5) According to the classification by Fahy et al.-
, these lipid molecules are commonly divided into eight main groups: free fatty acids,
sphingolipids, glycerolipids, phosphoglycerolipids, sterols, prenols, saccharolipids and
polyketides. (6) The first five main groups contain bioactive (signaling) lipid molecules such
as fatty acids, diacylglycerol, ceramide, sphingosine, lysophosphatic acid that interfere in the
regulation or (de)activation of different signaling lipid pathways.
3
Prenol lipids, saccharolipids and polyketides however are mainly of bacterial, fungal and
plant origin and will therefore not be discussed in this thesis. (5)
There is substantial evidence that neoplasm cells show alterations in their lipid metabolism.
(7) These alterations can cause changes in cell membrane structures, energy homeostasis and
cell signaling with defects in gene expression, protein distribution and cell functioning,
including the processes of apoptosis, autophagy, necrosis, proliferation, differentiation,
growth, drug resistance and chemotherapy response. (5) (7)
By comprehending this phenomenon of lipid alteration, we could obtain vital information
about the pathogenesis in CRC, create new biomarkers for screening or diagnosing CRC in
earlier stages, and suggest new molecular targets for anticancer therapy. (8)
In this review, we will describe current used approaches for lipid profiling and detection of
lipid deformities in CRC with an eye to biomarker discovery and potential treatment options.
Most importantly, we will give a brief description of the metabolic pathways of each lipid
subgroup and its function in the colorectal tissue, and the abnormalities in these pathways that
could lead to the development of CRC.
4
3 Method
The related articles were acquired via the PubMed database by querying results with different
MeSH-terms. The most useful MeSH-terms were ‘colorectal neoplasms’, ‘neoplasm staging’
and ‘lipids/metabolism’.
The review by Huang et al. contains a division of those lipids in different categories: fatty
acids, sterols, sphingolipids, glycerolipids and glycerophospholipids (5) and these terms were
subsequently used for further investigation in combination with the above noted MeSH terms.
This method allowed enough reviews to be retrieved to obtain a sufficient background on
these topics.
However, a more profound analysis was necessary, so the investigation was expanded by
utilizing Google Scholar and Google as additional search engines. When a sufficient amount
of relevant articles were acquired, the exploration was continued by using specialized terms
in these search engines to deepen the investigation on certain topics such as ‘epoxygenases’,
‘lipoxygenases’, ‘cyclooxygenases’, ‘eicosonaids’, ‘glycerophosphate acyltransferase’,
‘sphingomyeline’, and ‘ceramide’ all coupled with ‘colorectal cancer’. Lastly, the snowball
effect method was used based on certain articles of interest, in which the references included
in these articles led to additional articles of interest. The results were filtered by restricting the
retrieved articles to those written in English.
5
4 Results
4.1 Colorectal cancer
4.1.1 Epidemiology
CRC is one of the most diagnosed cancers and causes the majority of cancer-related deaths in
the Western world, with a global incidence of two million people, more than half a million
deaths per year and a cumulative lifetime risk of developing CRC of approximately 5%.(9)
In recent years, more and more risk factors have been discovered including age (with a peak
incidence at 70 years), male sex, family history, obesity, diabetes, IBD, alcohol consumption,
tobacco use, red or processed meat consumption, low fruit and vegetable intake and even
intestinal bacterial infections (e.g. Helicobacter pylorum). (1) A high variability in incidence
exists between countries, though these incidence rates are highest in Western European
countries, Australia and New Zealand. This suggests that a Western life style may be a
significant risk factor in CRC development. It is important to note that the increasing
prevalence can also be due to improved screening procedures, which allow detection of more
asymptomatic patients, combined with the beneficial progression in treatment, which expands
the patients’ survival.(10)
4.1.2 Prevention and screening
Knowing that CRC has such a large impact on global health, prevention is an essential part of
this medical topic. Primary prevention is the most effective strategy in avoiding CRC and can
be maintained by reducing the adjustable risk factors and promoting a healthy lifestyle. (11)
Primary prevention by intake of certain drugs such as NSAID or hormone therapies however,
has important side effects and still lacks fundamental evidence.
Secondary prevention comprises early detection of CRC, which can be obtained by screening
with faecal occult blood tests, colonoscopy or sigmoidoscopy and even virtual colonoscopy
by using CT imaging. This latter technique has been proposed as a screening technique,
though exposion to radiation remains an important consequence, while the cost-effectiveness
of this procedure is doubted, especially for primary prevention.
Options for tertiary prevention of surviving CRC patients have not been fully explored yet.
Physical exercises, cessation of smoking and long term intake of aspirin or NSAIDs have
been recommended, but more investigation remains necessary. (10)
6
4.1.3 Classification method
Officially, there are two classification methods that are utilized for staging CRC: Dukes
stadium and the TNM system. Biopsy samples and imaging techniques
(ultrasonography,(PET-) CT, MRI) are necessary for determining which stage belongs to a
specific patient. Because the Dukes staging classification received the connotation of being
out-dated and confusing, tumors are expected to be categorized according to the latest version
of the UICC TNM-system since 2003. (2)(12) Table 1 (see appendix) provides an overview of
the Dukes and TNM classification systems, and describes the different stages according to the
findings of imaging techniques and histopathological investigation. Categorizing the stage of
the patient’s CRC is important for determining the prognosis and type of therapy this patient
should receive. (11)
4.1.4 Treatment
4.1.4.1 Non-metastatic colorectal cancer
As mentioned above, surgery remains the imperative curative option in non-metastatic CRC.
Colon surgery is performed by removing the tumor and its corresponding lymph nodes with a
clear resection margin. Treatment of rectal cancer is executed by total mesorectal excision.
These procedures were initially performed using open surgery, but laparoscopy has proven to
be equally efficient with a lower mortality, morbidity and shorter hospitalization, although it
is technically more difficult for the surgeon.
Locoregional recurrence is a risk factor after surgery and is associated with a poorer
prognosis. Due to the anatomical complexity of the pelvis, rectal cancer has a higher risk of
recurrence compared with colon cancer. Avoiding recurrence can be accomplished by
resection using wide margins and, if necessary, combined with pre-operative and/or post-
operative chemotherapy, radiotherapy or chemo-radiotherapy. Adjuvant chemotherapy can
also be advised when there is an increased risk for distant metastasis after resection. The
decision wheter surgery with or without (neo-) adjuvant therapy should be applied depends on
the CRC stage, together with the patient’s own preference. (1) (8) (11)
4.1.4.2 Metastatic colorectal cancer
The treatment policy for CRC patients with distant metastasis varies from case to case.
Surgery of liver or lung metastases can be offered if these metastases seem resectable, but
when irresectable, palliative chemotherapy should be the standard. Alongside traditional
7
chemotherapeutics, more and more new drugs can be utilized as a form of personalized
therapy. Examples are Bevacizumab and Aflibercept, targeting VEGF (vascular endothelial
growth factor) or Cetixumab and Panitumumab, targeting EPGF (epidermal growth factor).
(1)(11)
4.2 Metabolomics in colorectal cancer
4.2.1 Current and upcoming ‘omics’ approaches for biomarkers
Over the past years, genomics and proteomics have been the center of investigation for
carcinogenesis. This has provided an abundance of information that led to the development of
new insights, new therapeutic targets and strategies, and nucleotide- or protein-based
biomarkers. (13) Some of the most prevalently used and investigated biomarkers are
described in this section.
Micro-satellite Instability (MSI), a short repetitive DNA nucleotide sequence involved in
DNA repair after replication, is currently one of the most used biomarkers. The MSI status is
divided in 3 different categories: MSI high (>30%), MSI low (10-30%) or MSS
(microsatellite stable). It still remains unclear if the MSI status can be interpreted as a positive
or negative factor for the prognosis. Studies have showed that the MSI status is associated
with some clinicopathological variables and patients with a high MSI status have a higher
general five year survival rate. (8) (14)
CIMP or hypermethylation of the CpG promoter island, is a well-known transcriptional
silencer of DNA repair genes and tumor suppressor genes. It is often correlated with MSI and
CIN and CRCs are therefore increasingly divided according to their CIMP status: CIMP-high,
CIMP-low and CIMP-negative. (8)
CIN or Chromosomal instability is a characteristic found in almost 60-80% of all CRCs. It
depicts the chromosome alterations, either structural or numerical, in CRC cells and is
associated with poor prognosis and moderately differentiated types of cancer cells. (8)(12)
KRAS is a proto-oncogen that, when mutated, continuously activates the MAPK pathway
and PI3K/Akt, both of which promote carcinogenesis by cellular proliferation. (8) It is present
in 30-50% of all CRCs but still hasn’t been considered as an essential predictive biomarker.
(14)
8
BRAF is an inhibitor of the RAS/MAPK intracellular signaling pathway, encoding a
serine/threonine kinase. Mutations of this gene are correlated with the early stages of
colorectal cancer development. Moreover, KRAS and BRAF mutations have both been
associated with poor response on EPGF-inhibitors. (12) (14)
VEGF, vascular endothelial growth factor, is a pro-angiogenic factor in the development of
CRC and has been proposed as a molecular biomarker for lymph node metastasis. (8)
LOH or Loss Of Heterozygosity, especially located in chromosome 18q, is able to inactivate
tumor-suppressor genes and has a certain prognostic value, though this potential biomarker
needs further investigation. (8)
Interestingly there is also a metabolomics based biomarker under development: a prediction
model using the metabolites 2-hydroxy butyrate, aspartic acid, kynurenine and cystamine has
already been tested and proposed as an early detection method for CRC stage 1 and 2. (15)
4.2.2 Lipidomics and its analytical technology
In recent years, lipidomics has been introduced as a new chapter in metabolomics,
representing the analysis of lipid metabolism in organelles, cells, tissues and organisms, in
health and disease. (6) (13) Lipids embody essential roles in human physiological and
pathological processes such as apoptosis, proliferation and carcinogenesis, and although they
are extremely complex, more and more scientists are investing time in the development of
new techniques for separation and identification of these lipid molecules. (16)
The first step in lipid analysis comprises extraction of lipid molecules. This can be
accomplished by using a mixture of methanol, chloroform and water (the Blight and Dyer-
method). Many more techniques for lipid extraction from samples are available but will not be
further discussed here. (16)
Due to its high reproducibility, selectivity and sensitivity for detecting different lipid
components in body fluids or tissue samples, mass spectrometry (MS) is the most widespread
standard procedure for identification of lipid molecules. MS can be combined with separation
techniques such as capillary electrophoresis (CE), gas chromatography (GC) or in most cases,
high performance liquid chromatography (HP-LC). (3)(6)
9
HP-LC separates the different lipid components according to their chemical or physical
properties by introducing the lipids/liquid to a high pressurized monolithic silica tube or
column, called the stationary phase. A compound solvent, dubbed the mobile phase, is then
pushed over the column at high pressure, and this compound solvent is differentially mixed
from hydrophilic and hydrophobic primary solvents. Lipids first adhere to the column, but
dissolve into the compound solvent mixture and thus elute from the column when the
hydrophobicity is sufficiently high. This system thus effectively allows the separation of
lipids with different physiochemical properties over time.
Once the separated components have left the column, these lipids are in the MS instrument.
Initially, ionization of the lipids is performed, usually via electrospay ionization (ESI), which
introduces a charge on the lipid molecules, creating lipid ions. Next, separation according to
the mass-over-charge ratio (m/z) is performed by bringing the ionized molecules into a mass
analyzer, typically the rapidly oscillating magnetic field of a quadrupole for lipids. The m/z-
separated ionized lipid molecules then impact an electron multiplier that serves as a detector,
allowing a spectrum to be created of the number of ion impacts per m/z.. (17)
4.3 Lipid metabolism and colorectal cancer:
Lipids are apolar, hydrophobic molecules with an essential role in the thermal isolation of the
human body and the emulsification of food in the intestines using bile acids. Most importantly
for this thesis, they are also an alternative energy supply for cells, provide structural integrity
for cellular membranes, and are essential signaling components, be it as second messengers or
as hormones.
Cellular lipid metabolism contains complex processes and includes uptake, synthesis,
degradation and transport. Every lipid can be regulated by different pathways in different
tissues or cells, and activated or inhibited by physiological, pathological or therapeutic
circumstances.
Bioactive lipids transduct signals to regulate lipid metabolism using different pathways:
o G-protein coupled receptors
o Tyrosine kinases
o Integrin signaling
o Ion channel signaling
o pH changes
o oxidative stress
10
o mechanical stress
Cancer cells gain defects or alterations in their lipid metabolism, causing changes different
cellular processes such as proliferation, differentiation, motility and growth. (5) This could be
induced by the over-expression of essential lipid enzymes, altering the cell’s functions and
enhancing lipid biosynthesis. Excessive lipogenic enzyme function is correlated with
enhanced transcription of lipogenic genes, for instance by the transcription-regulator SREBP
(sterol regulating element-binding protein) by growth factors or by growth signaling
pathways. Epidermal growth factor, keratinocyte growth factor, erBb-receptors and Her2/neu
receptor tyrosine kinase have already been identified, while the PI3K/Akt pathway is
activated in multiple types of cancer and plays a central role in the expression of lipogenic
enzymes. (18)
According to the Lipid Classification and Nomenclature Committee, lipids are divided in
eight main groups: free fatty acids, glycerolipids, glycerophospholipids, sphingolipids, sterol
lipids, prenol lipids, saccharolipids and polyketides. (5) However, in this thesis, a slight
modification in this classification method will be applied. Because mono- ,di- and
triacylglyceroles as well as glycerophospholipids all originate from a glycerol-backbone and
undergo a common biosynthetic pathway (until phosphatic acid). ‘Glycerolipids’ and
‘glycerophospholipids’ will therefore be unified as one term in this thesis: glycerolipids.
In addition, lipoproteins could also be included as unique lipid entities. (19) These different
lipid classes will be discussed in more detail in the subsequent sections, with a specific focus
on their pathway, and any alterations thereof in CRC.
4.3.1 Free Fatty Acids
4.3.1.1 Pathway
The FA biosynthesis usually begins with the provision of acetyl-groups by citrate, generated
in the Krebs-cycle. Subsequently, citrate is converted into acetyl-CoA and oxaleacetate by
ATP citrate-lyase (ACLY). Acetyl-CoA carboxylase (ACC) is the rate-limiting step of fatty
acid-biosynthesis, converting acetyl-CoA into malonyl-CoA. Acetyl and malonyl groups are
attached together by fatty acid synthase (FASN). Eventually this generates the substrate
palmitic acid (16 C) due to repeated condensations of acetyl-groups. Desaturation is an
optional step in the fatty acid biosynthesis, taking place in the endoplasmatic reticulum and
executed by SCD (stearoyl-CoA desaturase). This introduces a double bond in the saturated
11
fatty acids, making it a mono-unsaturated fatty acid. Palmitic acid is the main saturated fatty
acid and can be converted into other saturated or unsaturated fatty acids such as stearic acids,
oleic acid and more. (20)
It is important to notice that the pathway described above represents the production of non-
essential fatty acids only. The essential fatty acids cannot be produced by humans and other
mammals, and can only be obtained through the diet. (21)
The eicosanoids are derived from 20-carbon essential fatty acids: prostanoids, leukotrienes
and epoxygenases are produced from ω-6 arachidonic acid (AA), while resolvins and
protectins are obtained from ω-3 PUFA (poly-unsaturated fatty acids), including
eicosapentaenoic (EPA) and docosahexaenoic acid (DHA). (22)
AA is an essential fatty acid and a key metabolite for the production of eicosanoids via the
COX, LOX and CYT P450 (EPOX) pathways. The grand majority of eicosanoids acquire
their AA from membrane glycerophospholipids by activation of PLA2, which releases the
substrate at the sn2-position of the glycerol-backbone. Another fraction of the AA is obtained
via linoleic acid (LA): firstly, γ-linoleic-CoA (GLA) can be obtained by diet or by the slow
reaction mechanism of LA-CoA with a ∆6-desaturase. When going through the microsomal
elongation pathway, GLA-CoA is exchanged for dihomo-γ-linoleoyl-CoA (DGLA) which is
converted into AA by a ∆5-desaturase. (23)
AA can eventually be metabolized through three different pathways for generating
eicosanoids: cyclooxygenase (COX)-pathway providing prostanoids, lipoxygenases,
leukotrienes and P450 epoxygenase supplying hydroxyeicosatetraenoic acid (HETE),
epoxyeicosatetraenoic acid (ETE) and hydroperoxyeicosatetraenoic (HPETE). (22) An
overview of the eicosanoid pathways and its products can be seen in the figure below.
12
Fig. 1: overview of arachidonic acid metabolism
13
As mentioned above, a small portion of the eicosanoids are derived from the ω-3 PUFA, EPA
and DHA, obtained by the α-linolenic acid-metabolism or by diet, and may as well go through
the three oxidative pathways in the same way as arachidonic acid. This creates variant series
of (benign) prostaglandins, leukotriens, resolvins, protectins and other bioactive lipid
mediators as depicted in the figure below. The red marked compounds represent moderately
pro-inflammatory products while the green marked compounds represent anti-inflammatory
and anti-tumorigenic products. (24)
Fig.2: ω-3 fatty acids metabolism
4.3.1.2 Known alterations in colorectal cancer
When manufacturing new structural components, healthy cells commonly rely on dietary
circulating fatty acids or exogenous intake of carbons, which can be converted into FA’s by
liver or adipose tissue. With the exception of some metabolically active tissues such as fetal
lung tissue, hepatocytes, adipocytes, lactating breasts and the cycling endometrium, de novo
lipogenesis-pathway is reduced in most healthy cells. (18) (25)
Cancer cells require sufficient molecular building blocks as a response for their enhanced
proliferation and growth capabilities. (20) In these cases, ‘de novo’ synthesis of FA’s is
essential for building lipid membranes and signaling molecules, although some tumors are
able to capture FA’s from their environment (e.g. using FABP4). Enhanced influx of carbons,
introduced as glucose or glutamine, supports the anabolic pathways for building these new
fatty acids. Consequently, blocking FA de novo synthesis by limiting its supplies, enhancing
14
FA degradation, pushing FA’s into storage, and/or downsizing FA release from storage could
offer new therapeutic strategies in cancer therapy. (18) (26) (27)
4.3.1.3 Alterations in free fatty acid synthesis pathways and distribution
4.3.1.3.1 Enzymes and other proteins
The simplest way to obstruct synthesis is the down-regulation of the conversion of
carbohydrates to citrate in the Krebs cycle, blocking the mitochondrial citrate carrier (CIC or
protein SLC25A1) or inhibiting the enzymes that take part in the enhanced de novo
lipogenesis in neoplastic cells. (20) The mitochondrial citrate carrier is an essential transporter
in de novo FA synthesis: citrate exits the mitochondria and is cleaved into oxaloacetate and
two acetyl-CoA molecules. Some studies reported that these citrate carriers are up-regulated
in colon cancer. (28)
ACLY is a homotetrameric cytosolic enzyme that converts mitochondrial citrate into acetyl-
CoA, and thus may provide essential substrates for the FA synthesis pathway and the
mevalonate-pathway. A significant increase in ACLY expression and activity has been shown
in colonic cancer cells. (25) Another study noted that ACLY was up-regulated in CRC
compared to normal tissue, as well in chemo-resistant CRC cells compared to chemo-naive
variants. In addition, activation of the PI3K/Akt-pathway, a signal-transduction pathway that
promotes cell survival and growth as a response to extracellular factors, is correlated with
ACLY activity: enhanced glucose uptake and metabolism leads to upregulated PI3/Akt which
phosphorylates and triggers ACLY, increasing FA synthesis and being partially responsible
for carcinogenesis. (29) (30)
These findings suggest that inhibition of ALCY may be introduced as a new therapy for
targeting cancer cells. RNAi (RNA interference), or pharmacological inhibitors could be
functional for down-regulating ALCY, resulting in a growth arrest of those tumor cells. (25)
Experiments with the citrate analogues (+) and (-) 2,2-difluorocitrate, on rat livers resulted in
a reduction of ACLY-activity. This outcome was also reached with (-) hydroxycitrate in
HepG2cells (a cell-line of polar human hepatocytes), although a restricted membrane
transport and necessity of high concentrations of this citrate analogue were profound
limitations. Radicicol, an antifungal macrolide, also inhibits the enzyme in rat liver cells,
whereas SB-204990 restricts proliferation in lung cancer cells. Also, combining the ALCY
15
inhibitors with a statin seems to have profound anti-tumorigenic effects. These findings are
still somewhat controversial and further investigation is desirable. (25)
As in many cancers, fatty acid synthase (FASN) is over-expressed in CRC, with the
metastatic variant taking account for the highest expression and correlating with a poor
prognosis. The theory behind this relationship remains unclear. (31) FASN knockdown
neutralizes CRC cells by restraining the cells’ energy metabolism and with it, the mTOR-
pathway, a serine/threonine kinase member of the PI3K pathway that enhances expression of
different lipogenic enzymes . (32)
It seems that the clinical stage of CRC is significantly correlated with the serum FASN of the
patient: concentrations were higher in stage 3 and 4 than in stage 1 and 2. (33) Another study
suggested that the FASN serum level is associated with the TNM-stage: patients with a tumor
extent T1 and T2 without lymph node metastasis nor distant metastasis have a significantly
lower FASN serum than patients with a T3 or T4 tumor extent and lymph node metastasis or
distant metastasis. Furthermore, the disease-free interval and the five-year overall survival
rate was smaller for patients with an elevated serum FASN level. This suggests that FASN
could be a potential biomarker, informing us about the clinical state of the tumor and patient
prognosis. (34)
FASN could also be a potential anti-angiogenetic target: knockdown of this enzyme decreases
the microvascular density and inhibits release of VEGF-A. This stabilization in secretion of
anti- and pro-angiogenetic factors, could inhibit the tumor vascularisation and by
consequence, reduce the probability of metastasis. (31) Inhibitors of FASN, cerulenin, orlistat
and C75, induce apoptosis in several cancer cell lines, including colorectal cancer, and could
be a new effective treatment strategy. (35)
Acetyl-CoA carboxyl (ACC) has two subtypes in the human body, both having different
metabolic functions: ACCα and ACCβ. (20) TOFA(5-tetradecyloxy-2-furoic acid), an
allosteric inhibitor of ACCα and a cytotoxic agent, disturbs de novo lipogenesis, induces
apoptosis in cells, and thus may be of use as a therapeutic in colon cancer. (36)
Stearoyl COA-desaturase (SCD) has a clear role in the lipid metabolism and growth pathways
of cancer cells: by increasing lipogenesis through inhibition of FA oxidation and alteration of
several intracellular pathways, activation of Akt-pathway and deactivation of AMPK-
16
pathways, it creates a benevolent environment for cell survival and proliferation. Inhibition is
known to cause apoptosis in many cancer types, including colonic tissue, and could be a
promising new therapeutic target in cancer interventions. (20) Although a higher expression
and activity of SCD1 were found in some colonic carcinoma tissues, there is still insufficient
evidence regarding this enzyme in CRC. (37) One study measured the SCD1-activity by
calculating the 16:1 n-7/16:0 ratio, which showed a lower mean activity in colorectal cancer
than in normal mucosa. The SCD2 activity on the other hand, measured by the 18:1 n-9/18:0
ratio, showed an augmentation of this enzyme in CRC tissue. (38)
Despite the proof that blockage of lipogenic enzymes could reduce tumor growth in colorectal
cancer cells in vitro, more research has to be performed to examine if this could be a long
term strategy in CRC treatment in vivo, especially because more evidence has been revealed
that an alternative mitochondrial pathway for de novo lipogenesis in mammalian cells exists.
(39)
The CPT (carnitine palmitoyl transferase) 1-transporter, transferring the long FA chains into
the mitochondria where β-oxidation takes place, seems to be significantly decreased in
colorectal and breast carcinoma. (40) CPT-1 participates during the metabolic transformation
(solid) cancer cells undergo when faced with metabolic stress. Continuous tumor growth
deprives cancer cells from their oxygen and nutrient (glucose) supply, forcing them to search
for an alternative, anaerobic form of energy. Apparently, cancer cells up-regulate their
CPT1C-gene, leading to promoted FFA-oxidation and generation of ATP as energy source.
Indeed, experiments on colorectal cancer xenografts showed a decrease in tumor growth when
CPT1C was depleted. (41)
4.3.1.3.2 Fatty acid distribution
FA distribution of healthy and CRC tissues illustrated that there is a definite correlation
between the colorectal cancer stage and FA profiles. Both samples contained non-essential
palmitic acid, stearic acid, oleic acid, and the essential linolic acid as the most profuse FA.
(38)
CRC tissue showed a shift in FA distribution with a higher rate of saturated FA. While a 50%
increase of stearic acid was noted , the amount of myristic and palmytic acid remained
relatively stable in CRC. On the contrary, the mono-unsaturated FA palmitoleic and oleic acid
were respectively 50 and 20% lower in CRC than in normal mucosa. (38)
17
The ratio in ω-6/ ω-3 PUFA was increased in CRC with the total ω-3 PUFA being decreased
compared to normal samples.(21) Dihomo-γ linolic acid (DGLA) and AA showed an
augmentation of respectively 34% and 38%. (38) One study proved that linolic acid levels
were downsized: this finding combined with the elevated values of AA could indicate that
there is a higher turnover into this pro-inflammatory product. (21) This hypothesis was
contradicted by another study, where linolic acid levels were slightly elevated together with
arachidonic acid. Furthermore, AA/LA, AA/DGLA, and AA/ -total ω-6 PUFA ratios were
higher, thus indicating that desaturase-enzymes may be up-regulated in CRC.(38)
The concentrations of EPA and DHA, described as percentages of total FA concentration,
were respectively 88% and 37% decreased in CRC samples compared to healthy samples.
These two products are anti-inflammatory and decrease the levels of colorectal biomarkers.
(38)
The relationship between FA distribution and clinicopathological stages seems to remain
unclear. When comparing the saturated FA and mono unsaturated fatty acids (MUFA), there
were no significant differences between colorectal tissues with Duke B and C stages. Only
palmitoyl acid and 18:1 ω-9 were almost half the amount in the C-stadium than in the B-
stadium. Concerning the PUFA, the ratio ω-6/ ω-3 was strongly increased in the Dukes C
stadium compared with the B stadium. AA levels in stage C were six times higher than stage
B, but the linoleic acid level was reduced by almost 66% in stage C compared with stage B.
Another study observed no differences in SFA, MUFA and PUFA between the
clinicopathological stages (I, II and III-IV) or lymph node metastasis.(21).
Ω-3 fatty acids (EPA and DHA) suppress AA-derived eicosanoid production by replacing the
membrane bound-AA by ω-3 PUFA, competing with desaturases and elongases and inhibiting
formation of LA to AA. Altogether this makes AA less available and consequently,
suppresses AA-derived eicosanoid production. (42) These ω-3 PUFA replace pro-
inflammatory substrates in the COX-2-pathway while EPA acts as the preferred substrate for
the LOX-pathway, blocking pro-angiogenetic and inflammatory prostaglandins or
leukotrienes. (28)(42) In addition, EPA and DHA seem to have a protective effect against
CRC by enhancing the caspase-dependent apoptosis pathway through down-regulation of
two regulatory elements, FLIP and XIAP. Another encouraging finding is that EPA and DHA
do not trigger apoptosis in healthy colorectal cells, which could make these potential and
effective adjuvants with other chemotherapeutic agents. (44)
18
These results suggest that the ω-6 PUFA, AA in particular, promote carcinogenesis in
colorectal tissue and that ω-3 PUFA, DHA and EPA, attempt to restrain it.
4.3.1.4 Alterations in eicosanoid metabolism and distribution
4.3.1.4.1 COX-pathway
There are 3 variants of COX-enzymes: COX-1, COX-2 and COX-3. COX-1 is a house-keeper
enzyme of gastric mucosa, renal bloodflow and platelet activation that remains stable under
physiological and pathological circumstances. COX-2 has an unmistakable role in
inflammation and other pathophysiological processes. COX-3 is a variant of COX-2 with a
currently unclear function.
It is well well-known that COX-2 expression is augmented in CRC: 50% of adenoma and
85% of adenocarcinoma had elevated values. (45)
The prostaglandin PGE2 seems to be a key factor in colorectal tumorigenesis, promoting cell
growth, angiogenesis (via VEGF) and tumor survival. (31) PGE2 was strongly up-regulated in
metastatic tumors, thus enhancing invasion and urine samples with elevated PGE2 values
gave an increased risk in colorectal cancer development. (22) (46) It is the most dominant
prostaglandin present in CRC tissue. (45) Lung, head and neck and breast cancer also had an
elevated quantity of PGE2 and were all associated with poor prognosis. (28,29) PGE2
prostaglandins are degraded by 15-PGDH, which if knocked out in murine subject animals,
induced a 7.6 times increase in risk for developing colon tumor with a doubling of PGE2
values. Transgenic mice deficient in prostaglandin target receptors EP (1-4), display a
significant reduction in colon cancer incidence. (22)
Prostacycline- or PGI2-values appear to be decreased in CRC tissue, meaning that this
prostaglandin may not be essential in colorectal carcinogenesis, though a study showed that
addition of PGI2 analogues had a protective effect against metastasis. (22) It may have a role
in tumor progression due to the fact that it activates PPARδ, thus accelerating intestinal tumor
growth in mice subjects. Further investigation will hopefully clarify the specific role of PGI2.
(45)
Although prostanoid receptors DP2 gradually stop being expressed during the adenoma-
carcinoma sequence, PGD2s function still remains unclear. Inhibition of CRC cell
proliferation has been observed when treated with PGD2, but its metabolites (PGJ2 and
15dPGJ2) induced cancer cell proliferation and survival. (22) One proposed hypothesis is that
15dPGJ2 may inhibit tumor cell growth through binding of the PPARγ-receptor. (47) Another
19
theory is that over-expression of PGD-synthase could lead to a metabolic shift in which less
PGE2 is produced and PGH2 is predominantly converted into PGD2, suppressing tumor
growth. More investigation about PGD2 and its effects in CRC and other cancer cells seems
necesarry. (45)
Prostaglandin F2α has no known role in carcinogenesis of CRC and will not be further
discussed. (48)
TxA2 is derived from the COX-1 and COX-2 pathway and contributes to tumor growth and
angiogenesis promotion. (45) Targeting COX-1 with aspirin proved to have an anti-
carcinogenic effect. Furthermore, a direct addition of TxA2 after deletion of tromboxane
synthase erased cell arrest in CRC and promoted proliferation. The tromboxane synthase
seems to gain some expression in CRC tissue compared to normal mucosa, with TxB2
metabolites and their urinary excretion being significantly raised in these patients. (22) Also,
a TxA2 synthase-inhibitor was shown to be able to block liver metastasis in CRC. (45)
NSAIDs have proven their effects in blocking inflammation, but could also offer anti-
carcinogenic effects thus reducing the risk of colon cancer and other prevalent solid tumors
(breast, prostate, lung) via COX-2 inhibition. (47) A quantity of studies confirmed that intake
of aspirin or other NSAID on a regular basis during a 10-15years period gives a relative risk
reduction of 40-50% for the development of colorectal adenoma. (45) As mentioned before,
the question thus arises if NSAIDs or aspirins could be part of the colorectal cancer
prevention strategy, albeit in combination with proper exercise, healthy diet and avoidance of
other carcinogenic risk factors. (49)
4.3.1.4.2 LOX-pathway
CRC patients often gain enhanced activity of 5-LOX and 12-LOX. With 5-LOX being over-
expressed in human tubular adenoma, villous adenoma and colorectal adenocarcinoma, it may
be associated with chronic inflammation and carcinogenesis. 12-LOX over-expression could
be more of an incentive for metastasis, while also participating in angiogenesis and cancer
cell proliferation. (42) Eicosanoid profiling illustrated that not only PGE2 and AA, but also
12-HETE was significantly altered in CRC tissue compared to normal mucosa, though
without any correlation with Dukes stadium for this metabolite. (48) Even though 15-LOX-2
didn’t show any significant alterations in expression, a 125-patient prospective study found
down-regulated 13-S-HODE (a 15-LOX-1 product from linoleic acid) concentrations when
going through the mucosa-adenoma-carcinoma sequence of the colon. (22) Indeed, 13-HODE
20
showed significantly augmented values in normal colorectal mucosa compared to colorectal
adenoma. (50) 13-S-HODE has a tumor-suppressive role, inducing cell cycle arrest in cancer
cells and restoring apoptosis via PPAR-δ activation. (51) Other LOX products effects are also
induced via the superficial G-protein cell receptors or activation of the PPAR family. (22)
Both COX-2 and LOX-5 have pro-carcinogenic effects and blocking these pathways could
enhance a shift of free AA to another, producing more pro-tumorigenic metabolites. Dual
blockade of both enzymes could provide as a new chemo-preventive strategy: it has already
been found safe and effective as treatment in osteoporosis. (42)
An inverse relation between 15-LOX-1 and COX-2 has also been noted: this was confirmed
in a study where metabolism of LA shifted towards the COX-2 and away from the 15-LOX-1
pathway when progressing through the adenoma-carcinoma sequence: while 96% of low-
grade adenomas expressed the 15-LOX-1 gene, this was only the case for 43% of carcinoma-
in-adenoma lesion subjects. Regarding COX-2, 2% of low-grade adenoma expressed the
gene, whereas this was 71% for the carcinoma-in-adenoma and even 92% of the advanced
carcinomas. (52) Furthermore, products that up-regulate 15-LOX-1 and down-regulate COX-
2, such as sulforaphane or Honokiol, inhibit intestinal polyp formation and gastric
carcinogenesis, respectively. Development of new chemotherapeutic agents and using these
metabolites as molecular biomarkers could become valuable in colorectal cancer screening,
prevention or treatment. (46)
4.3.1.4.3 CYT P450 epoxygenase pathway
The explicit role of this pathway in cancer development has not been cleared up yet, though
CYP2J2 and other CYP enzymes seem to be over-expressed in a variety of cancer cell lines,
including colon cancer.(42) It is supposed that that this pathway metabolizes free AA,
inhibiting ceramide production and apoptosis (see 4.3.3.2), but also gaining 14-,15-EET,
inhibitors of apoptosis via the PI3K-Akt pathway. (53) Though there is little known about the
role of EETs in CRC progression, patients with these types of solid tumors have elevated
levels of these compounds in blood and urine. Treatment of endothelial cells with 14-,15-EET
induced tumor growth and metastasis promotion. On the other hand, inhibitors of
epoxygenases or EET-antagonists were capable of containing tumor development in some
cancer types in rat subjects (for example glioblastoma), thus prolonging the animals’ survival.
(54) Whether these findings could be similar for CRC in humans needs to be further
investigated.
21
As mentioned, DHA is in competition with AA for being processed along the epoxygenase
pathway. This generates epoxydocosapentaenoic acids (EDPs), mediators with anti-
angiogenic and anti-tumorigenic effects. In mice, administration of 19,20-EDP blocked
VEGF- and FGF2 –induced angiogenesis, restrained primary tumor growth by 50-90% and
gave a 70% reduction of lung metastasis foci and weight. The question arises if DHA or other
ω-3 PUFA can be administered to CRC patients with high epoxygenase expression profiles to
reduce EET levels and increase EDP levels in patients, thus contributing to a better prognosis.
(24)
4.3.2 Glycerolipids
4.3.2.1 Pathway
The triglycerides (TG) and glycerophospholipids are glycerolipids that share a common
biosynthetic pathway: the glycerophosphate pathway. (5) Both products can be obtained via
the intermediate phosphatidic acid (PA), though all products have a completely differing FA
composition. (55)
TG are the first type of glycerolipids, consisting of a glycerol backbone and three esterified
FA’s, all different in length and saturation level. TG can be stored in lipoproteins (e.g. LDL,
HDL chylomicrons…) as well as in lipid droplets, present within every cell-type, but most
commonly found in adipocytes. (55)(56)
Glycerophospholipids are also built of a glycerol backbone with two esterified FA at the sn1-
and sn2-position but with a polar head group attached to position sn3. These are the cell
membrane’s most abundant building blocks, for which the composition varies from each type
of cell, organelle, inner or outer membrane. Each composition in turn shows dissimilarities in
cellular functions such as vesicular transport, membrane viscosity and signal transduction.
Due to their FA remodeling system (see further), glycerophospholipids are an extremely
important source of lipid mediators such as lysophospholipids and saturated or unsaturated
FA’s, which can be converted into eicosanoids and other lipids. (57)
The glycerophospholipids are classified according to the structure of their polar head groups.
(58) Phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS),
phosphatidylinositol (PI), phosphatidylglycerol (PG) and diphosphatidylglycerol (DPG) or
22
cardiolipin are the major classes. These phospholipids can be further subclassified according
to the type of chemical bond with the FA at the sn1-position: diacyl, alkylacyl and
alkenylacyl. (55) (56) The heterogenity of glycerophospholipids is further increased due to
the different FA-groups at the sn1 and sn2-position: while saturated FA’s are more common
on the sn1-position, unsaturated FA’s are mainly present on the sn2-position. (55)
In tissues other than adipocytes, synthesis of PA commences with the glycerol-backbone,
which is activated by phosphorylation at position C3. The glycerol-3-phosphate is
incorporated with a FA-CoA via the Glycerol-3-Phosphate acyltransferase (GPAT),
converting it into lysophosphatic acid (LPA). This is followed by 1-acylglycerol-3-phosphate
acyltransferase (AGPAT), creating phosphatic acid (PA), the key intermediate in triglyceride
and glycerophospholipid synthesis. This metabolite can also be produced via a different
pathway that is mainly used in adipocytes, which are lacking the glycerol-kinase enzyme. (56)
Consequently, 1-alkyl LPA is gained through use of dihydroxyaceton-phosphate (DHAP), a
metabolite produced during glycolysis, by acylation with DHAP-acyltransferase (DHAPAT)
and reduction by DHAP oxido-reductase. Eventually, AGPAT becomes involved in the
process and leads to the formation of 1-alkyl PA. (55) Lastly, PA can also be obtained
through phosphorylation of DAG, using the DAG-kinase enzyme. (58) The mitochondrial
GPAT is more likely to utilize saturated FA’s, while the microsomal GPAT generally uses
unsaturated FA’s. (59) There also seems to be a correlation between the type of mitochondrial
GPAT and AGPAT and the selection of fatty acids as substrates, although the mechanism
behind this remains uncertain. (60)
When PA follows the TAG pathway, PA-phosphatase (PAP) removes the phosphate group,
gaining 1,2-diacylglycerol (1,2-DAG). Eventually, the last fatty acid group is esterified by
DAG acyltransferase (DGAT), recovering TAG. (60) Though this pathway is commonly
present in liver and adipose tissue, intestines use the mono-acyl glycerol pathway in gaining
TAG, where absorbed dietary MAG can be sequentially converted into TAG by MGAT and
DGAT. (55)
23
Fig. 3: Overview of glycerolipid metabolism; adapted from (58)
Homeostasis of glycerophospholipids is extremely complex and the mechanisms concerning
de novo synthesis, degradation, fatty acid remodeling and interorganelle transport are not yet
fully understood. (58) Briefly summarized, glycerophospholipids can be gained via two
different pathways. Initially, after dephosphorilation of PA by PAP, DAG is converted into
PC and PE: this is the CDP-choline and CDP-ethanolamine pathway, also referred to as the
Kennedy pathway. The CDP-DAG pathway on the other hand, produces PI, PS and PG
through the reaction of PA with inositol, serine and glycerol respectively, while being aided
by CDP-DAG-synthase. (5)(55)(61) Furthermore, glycerophospholipids are able to convert
themselves into another type of phospholipid: PG can be irreversibly converted into
cardiolipin, while PS can alter into PE and vice versa. (61) A more detailed description of the
synthesis of glycerophospholipids is comprehensively described in a review about
glycerophospholipid homeostasis in mammalian cells. (58)
Newly synthesized glycerophospholipids have an altered fatty acid composition compared to
mature phospholipids. (61) All glycerophospholipids are submitted to fatty acid remodeling,
also called the Land’s cycle, thus creating a deacylation-reacylation reaction between
phospholipids and lysophospholipids. Deacylation is catalyzed by PLA1 or PLA2, depending
on whether it takes place at the sn1 or sn2 positions respectively. Lysophospholipid acyl
transferase (LPAT) substitutes the original FA by another FA on the glycerol backbone and
24
are divided in two protein families according to their substrate specificity: AGPAT (also
mentioned in the glycerophosphate pathway) and membrane bound O-acyltransferase
(MBOAT). (55)
Fig. 4: Fatty acid remodeling in glycerophospholipids; adapted from (55)
Degradation is another important step in the glycerophospholipid homeostasis process and it
is maintained via different lysosomal and non-lysosomal phospholipases. Alongside PLA1
and PLA2, PLC and PLD may also participate in the glycerophospholipid homeostasis. How
the coordination between biosynthesis and degradation of glycerophospholipids is realized,
still remains unclear. (58)
Fig. 5: Action points of phospholipases on the glycerol backbone; adapted
from (5)
25
4.3.2.2 Glycerolipids and colorectal cancer
4.3.2.2.1 Alterations in the glycerophosphate pathway
When starting with the first step in the glycerophosphate pathway, an enhanced uptake of
glycerol in HCT-15 colon cancer cells was observed. (62) Glycerol, an important metabolite
in gluconeogenesis, oxidation and lipogenesis , is transported via a specific Na+-dependent
carrier. This glycerol transporter may be a new target for drug development by reducing the
rate of the glycerophospholipid pathway, though more research is necessary on this subject.
(62) (63)
While GPAT seems to deliver no contribution to the carcinogenesis process, some evidence
indicated that AGPAT is over-expressed in several human cancers. AGPAT2 inhibition
induces growth arrest, necrosis or apoptosis and blocks RAS/RAF/Erk and PI3kinase/Akt
pathways. In addition, the expression of AGPAT11/LPCAT2 and AGPAT9/LPCAT1 were
found to be up-regulated in CRC tissue in comparison with normal mucosa. (64) This could
imply that there is a particular role for PA, being involved in cancer cell progression by
amplifying the Ras signal and activating the pro-survival MAPK and PI3K/Akt pathways and
thus the survival of tumors. (61)
LPA is an essential intermediate, influencing many pathological processes such as fibrosis,
inflammation, asthma, atherosclerosis and cancer. LPA is produced after Lyso-PLC reacts
with Lyspophospholipase D or when PA is deacylated by PLA1 or PLA2. The effects of LPA
are maintained by LPA receptors, which are coupled with specific G-proteins for regulating
downstream receptors. (65) Although LPA is a mitogen capable of enhancing proliferation in
CRC via interference with the APC/β-catenine pathway, this specific pathway is mutated in
the majority of tumor cells. There are hypotheses that LPA compensates this by over-
expression of Krüppel-like factor, a transcription factor that enhances intestinal crypt cell
proliferation. (66) Either way, the proliferation effects of LPA in an affected colon cancer cell
line depends on the type of LPA receptor. For instance, proliferation of DLD1 colon cancer
cells is promoted by LPA1, while HCT116 cells require LPA2 and LPA3. Moreover,
activation of LPA2 would deliver anti-apoptotic signals to colon cancer cells. (67) LPA2 also
showed elevated levels in CRC, while LPA1 receptors were down-regulated in colorectal
adenocarcinoma compared with normal colonic mucosa. The fact that these receptors are
aberrantly expressed in CRC and with LPA demonstrated to have proliferating, anti-apoptotic
26
effects could mean these play a certain role in the development of this disease. (68) Lastly,
cyclic Phosphatidic acid (cPA) is a structural analogue of LPA but instead, it has anti-
proliferative effects on DLD1 colon cancer cells. It inhibits cyclin D expression, blocks the
PI3K pathway and may have some potential as a therapeutic inhibitor of CRC progression.
(69)
4.3.2.2.2 Glycerophospholipids
The fatty acid remodeling enzymes, LPCAT, are able catalyze alterations in the colorectal
lipid profile, consequently contributing to cell malignancy. (67) Phosphatidyl choline (PC) is
an important structural element in cell membranes and plays a key role in cell cycle
regulation, proliferation and apoptosis by providing lipid second messengers. (27) Augmented
values of PC (16/0:16/1) were observed in CRC samples with more advanced stages.
Moreover, the ratio of PC (16/0:16/1)/LPC (16/0:16/1) was increased in colon cancer cell
membranes, implying an up-regulated activity of LPCAT4. Yet it still remains unclear if other
factors also play a role in this PC increase. (70)
Distribution of choline containing phospholipids in plasma were compared between healthy
subjects, individuals with adenomatous polyps and CRC patients. A detailed description of
these data can be read in Li, S. et al.
Shortly summarized, LPC plasma levels were gradually decreased through the colorectal
adenoma-carcinoma sequence, with the lowest concentrations present in healthy individuals.
Instead of colonoscopies, different species of LPC could be utilized as new detection
biomarkers for CRC. These could even be combined with other lipid metabolites that are
typically decreased in patient plasma such as sphingomyeline or sphingophosphocholine. (71)
PI molecules are best known for their roles in the PI3K-Akt/mTOR pathways: PI3Ks are
important kinases in gaining inactive PIP, PIP2 and active PIP3, which act as second
messengers and transduct signals from trans-membrane receptors to the cytosol. Since these
interfere in different cellular processes, failing of this pathway may result in continuous
activation with up-regulated angiogenesis, proliferation and cell survival in tumors due to
enhanced lipid metabolism and protein synthesis. Deletion of or mutations in the PTEN tumor
suppressor gene, which codes for a phosphatase that degenerates PIP3 was observed in CRC
tissues. Methods for inhibition of PI3K or conversion of active PIP3 to PIP2 or PIP, could be
valuable as new therapies in many different cancers, including CRC. Recent years, different
27
small molecule PI3K inhibitors have already been tested in clinical trials, with promising
results, but more research still remains necessary. (72) (73) (74)
4.3.2.2.3 Phospholipases
Findings suggest that PLA’s may be important regulators of cell growth.
Glycerophospholipids are known to be catabolized by PLA’s into FFA and lysolipids. Using
PC as example, these metabolites has potential proliferative effects on the one hand, but can
also re-enhance PC synthesis on the other hand by integrating these catabolized products
again in the CDP-choline pathway. This mechanism could be similar for the catabolism of
other types of glycerophospholipids. (27)
PS-PLA1 is a phospholipase that acts specifically on PS (phosphatidylserine) to produce
lysophosphatidylserine (LPS). Expression of PS-PLA1 was significantly higher in CRC
samples with increased tumor size, deeper invasion and presence of hematogenous metastasis.
Higher PS-PLA1 levels were also correlated with a decrease in disease-free survival. (75)
PLA2 is essential for releasing FFA at the sn2 position of glycerophospholipids. This includes
AA, a metabolite of the COX-1 and, more important, COX-2 pathways. As described earlier,
this is the source of pro-tumorigenic prostaglandins, for example PGE2. Defects in PLA2
could therefore be a protective factor in colorectal tumorigenesis. Cytoplasmic PLA2 was
already showed to be augmented in almost 50% of all types of colon cancer cell lines and
indeed, COX-2 expression was significantly correlated with cPLA2 expression. cPLA2
expression could enhance colon cancer development, although possibly in a less determining
way in comparison to COX-2, which is over-expressed in 80% of all colon cancer cell lines.
(76) In addition, it seems that (groupIIA) PLA2 positive cancer cells are correlated with a
lower disease free survival and patients live significantly shorter than those with PLA2
negative tumors. Furthermore, PLA2 is most abundantly expressed in stage II colorectal
cancer and the colorectal tumor’s position showed some correlation with the PLA2
expression: almost 70% of right-sided tumors were completely negative for PLA2 while this
was the case for only 42% of the left sided tumors. (77)
Platelet activating factor (PAF) does not only play a role in coagulation and immunology, but
is also suspected to take part in the carcinogenesis of colorectal and other tissue. PAF is
generated from a membrane-bound glycerophosphocholine by PLA2, gaining Lyso-PAF
28
which is followed by acetylation of the Lyso-group. Lastly, PAF can be catabolised by
acetylhydrolase (AHA). Remarkably, a worsening prognosis correlates with the amount of
catabolic products and enzymes of this pathway that are present in CRC tissue: T1-T4N0M0
had up-regulated activity of PLA2 and AHA with elevated tissue levels of LysoPAF and PAF,
TxN1M0 CRC showed the same profile but lacks elevation of PAF, while distant metastasis
(TxNxM1) CRC only showed an augmentation of AHA levels. (78) Furthermore, plasma
levels of sPLA2 and AHA were significantly, though modestly, increased in patients with
CRC. (79) These results imply that PAF de novo synthesis is increased in CRC, maybe
causing amplification of angiogenesis by VEGF production. Much still remains unclear
however, and the question thus arises if these findings are relevant. In particular,
information about this topic has apparently not evolved since 2003, which may indicate that
further investigation has not been successful, or that PAF has no interesting prospective in
CRC. (78)(79)
PLCδ1, a variant of phospolipase C, had a significantly reduced expression in CRC cell lines.
The data indicated that this enzyme inhibits tumor development, motility and invasiveness.
(80)
Phospholipase D degrades PC into PA and choline, while being aided by cofactor PIP2.
phospholipase D has a dual function: it provides structural integrity to (intra)cellular
membranes but also has a signaling function via PA and protein-protein interactions with
GTPases, kinases and phosphatases. Over-expression of phospholipase D has been found in a
variety of tumor types including CRC and is directly associated with angiogenesis, tumor
survival, cell migration and metastasis. Small molecules that are capable of inhibiting PLD
could be a promising new strategy in many types of cancers. (81)
4.3.2.2.4 Triglycerids
Serum and tissue concentrations of triglycerides have also been intensively investigated in
healthy and CRC patients. When serum and tissue lipid levels were compared between normal
and malignant colorectal tissue, tissue triglycerides were significantly down-regulated in
cancerous tissue but no significant correlation was noted for serum triglycerides. The clinical
TNM stages of colorectal cancer did show a correlation for both tissue as serum triglycerides,
where a progression in reduced triglycerides levels was noted in stage 3 and 4. Triglyceride
levels were lower in all patients with lymph node metastasis than those without lymph node
29
metastasis.(82) Furthermore, a dose-dependent positive association between adenoma
occurrence and triglyceride levels, with the distal colonic adenoma having the highest
significance , suggests a location dependent mechanism of lipid metabolism in colon cancer
development. (82) (83)
4.3.3 Sphingolipids
4.3.3.1 Pathway
Sphingolipids are composed of a sphingoid base backbone, most commonly sphingosine,
sphinganine or dihydrosphingosine, connected to a FFA via an amide-bond and a head group
(hydrogen, choline, serine, ethanolamine…) via an oxygen-bond. Depending on the type of
head group, the sphingolipids are divided in three main groups: sphingomyeline (SM),
ceramide and glycosphingolipids. (84)
Fig. 6: overview of sphingolipid metabolism
Ceramide is the central molecule in the sphingolipid metabolism. De novo synthesis
commences with palmitoyl-CoA and serine, followed by the intermediate products
sphinganine and dihydroceramide and eventually, synthesis of ceramide. (85) Ceramide can
be converted into ceramide-1-P, SM, glycerosphingolipids and sphingosine. A phosphate-
30
group can be added to this latter product, creating sphingosine-1-phosphate (S1P). Up to this
point, all mentioned steps can also be reversed for generating ceramide. Lastly, S1P-lyase
irreversibly divides S1P into ethanolamine-phosphate and palmitaldehyde.
It is important to notice that SM is commonly obtained by release from the cell membrane due
to sphingomyelinase (SMase). (86)
Glycosphingolipids are an extremely complex subgroup and it falls beyond the reach of this
thesis to characterize these. Briefly summarized, ceramide can be converted into
galactosylceramide or glucosylceramide, which in their turn can be elongated with other
saccharides such as mannose, fucose, galactose, N-acetylgalactosamine and N-
acetylneuraminic acid. The complexity of this process is illustrated in the figure below, which
represents the synthesis of glycosphingolipids in the brain. (87)
31
Fig.7: Overview of glycosphingolipid synthesis; adapted from (87)
4.3.3.2 Sphingolipids in colorectal cancer
Sphingolipids are abundant in the gut system, with twice the amount located in the small
intestine compared to the colon. (84) Not only are these compounds crucial structural
components in cell membranes, but these also provide important bioactive metabolites for
intracellular signaling and regulation of countless cellular processes including apoptosis,
growth, differentiation, proliferation, angiogenesis, cell adhesion, migration, inflammation
and lymphocyte traffic. (43)
Even though ceramide, sphingosine and S1P are considered the most important bioactive
sphingolipids, other metabolites also have their role. (89) An important remark is that
sphingolipids are quite restricted in traveling to other cellular compartments. Therefore,
effects of sphingolipids will largely take place within the compartment where they are
normally settled. (90)
As sphingolipids encompass many cellular functions, it is not improbable that alterations in
their metabolism could trigger processes that promote colorectal carcinogenesis or that grant
them resistance against certain (chemo)therapies. (90) Discrepancies in CRC concerning the
metabolism of every sphingolipid subgroup will be described in the next paragraphs with
some suggestions for therapy or screening opportunities. (86)
Regarding the SMnases, there are three subtypes of this enzyme: neutral SMase, alkaline
SMase and acidic SMase. A 1997 study showed distinct alterations in activity or presence of
these subtypes in CRC patients, who were ordered according to their Dukes stadium (A,B or
C). Alkaline SMase and neutral SMase had a decrease of respectively 75% and 50% in CRC
tissue compared with normal colorectal mucosa. Both enzymes were significantly lower in
stage B and C than in stage A. Acid SMase on the other hand, had no significant activities in
32
CRC. An important issue is that this study contained only 18 subjects, so these results should
be interpreted cautiously. (91)
Introducing 1,2-dimethylhydrazine, a DNA-methylating carcinogenic agent, to rat colon
mucosa gave a significant SM increase and SMase decrease, just before the stage when
colonic adenoma developed. (86) (88) This strongly suggested that a reduction in hydrolysis
of SM prior to the malignancy process takes place. (9)(88) In addition, Dillehay et al.
discovered that a SM-rich diet introduced to mice, who were again treated with 1,2-
dimethylhydrazine, restricted formation of aberrant crypts (pre-stadium lesions of colon
cancer). However, colon cancer incidence was not significantly reduced in these mice. (92)
Plasma levels of SM were also decreased in patients with adenomatosa polyposis (AP)
compared with healthy subjects and CRC patients, meaning that this could be a useful
biomarker for detecting AP. (71) Altogether, these results point out that SM could play its
part in the early stages of colorectal cancer development.(86) (93) (94)
Ceramide is a bioactive molecule that inhibits cell proliferation and activates apoptosis by
modification of different molecules and pathways (AKT, Bcl-2, PKCα…) (88) It can undergo
glycolysation, hydrolysis or phosphorylation, which determines whether this sphingolipid
metabolite will obtain apoptotic or mitogenic capacities. Additionally, an abundant quantity
of ceramide is generated when the colorectal cancer cells are stressed by radiotherapy,
chemotherapy, hypoxia or nutrient deprivation which activates apoptotic pathways. (95)
Investigation of primary and metastatic colon cancer in human subjects, pointed out that
ceramide levels were less than half the amount found in normal colon mucosa. (93)
Ceramidase, which also is divided in a neutral, acid and alkaline subtype, is an important
regulator of sphingosine and S1P. However, the ceramidase activity did not appear altered in
CRC patients, implying that low ceramide levels are predominantly caused by reduced SMase
activity. (91) This theory however, still requires more evidence because other factors may be
responsible for the changes in SM and ceramide levels such as alterations in de novo
sphingolipid synthesis, ceramide glycolysation and other processes. (86)
Here again, administration of ceramide analogues, C2 and C6, or inhibitors of ceramidase, D-
MAPP and B13, induced cell death in human CRC cell lines (SW403). In particular B13 had
astounding effects when introduced to mice, who had their liver injected with human colon
33
cancer cells . 70% of the animals with the SW403 and 100% of the animals with the Lovo cell
line remained tumor-free after treatment with B13. (93)
Furthermore, treatment of CRC with chemotherapy combined with an adjuvant that blocked
de novo ceramide synthesis (Fumonisin B1) or GlcCer production (1-phenyl- 2-
palmitoylamino-3-morpholino-1-propanol or PPMP) was observed. Fumonisin B1 as adjuvant
significantly decreased the rate of CRC cell apoptosis and increased the GlcCer levels in
comparison with chemotherapy alone. PPMP administration on the other hand doubled the
ceramide concentrations and increased the cell death ratio by 88%. (96) All of these
molecules that interfere with the ceramide metabolism by augmenting ceramide
concentrations, may prove useful as adjuvant chemotherapeutic agents against colorectal
cancer. (97)
Breakdown of ceramide by ceramidase offers sphingosine, a sphingoid base. Literature
claimed this molecule is quantitatively restricted in comparison to its phosphorylated variant,
S1P. Sphingosine has been put forward as a signaling molecule with pro-apoptotic and tumor-
suppressive properties by regulating Protein Kinase C-isomorfs and acidic nuclear
phospoproteins. (98) When sphingosine and sphinganine were added to a human colon cancer
cell line, this induced cell arrest at G2/M and augmented the rate of apoptosis in the
neoplasmic cells. This effect was similar for C2-ceramide, a short chain ceramide analogue,
but didn’t emerge for C2-dihydroceramide, a short chain dihydroceramide analogue,
indicating that the effects have something to do with the 4,5-trans double bond in ceramide,
which is absent in dihydroceramide. (99)
S1P is more likely to be a pro-carcinogenic factor, with mitogenic and pro-angiogenic
properties, inhibiting apoptosis and promoting maturation of colorectal neoplasm cells via
S1P-receptors. (86) It neutralizes the pro-apoptotic effects of ceramide and sphingosine when
added to tumor cells under stress reactions. (98) S1P also appears to be the missing
connection between colorectal inflammation and CRC development: by continuously
activating NF-kB and STAT-3, it provides proliferative and survival advantages to colorectal
cells. Moreover, STAT-3 again activates the S1P-R, creating a vicious circle in the
promotion of cell clonal expansion. (100) New therapeutic strategies with specific anti-S1P
antibodies already appear quite promising in different tumor lineages, including CRC. (101)
34
Deficiency of sphingosine-kinase-1 (SphK1) in mice inhibits colon polyp formation and
furthermore , is generally over-expressed in human colon cancer. (100)
Observation of SphK in colorectal tissue showed no expression in normal mucosa, negative or
moderately positive expression in adenoma, but positive expression in 89% of
adenocarcinoma and significantly higher expression in all metastatic adenocarcinoma. There
was, however, no correlation between SphK-expression and tumor invasion. (89)
Furthermore, S1P-lyase and Sphingosine-Posphatase were under-expressed in human colon
cancer cell lines, thus blocking S1P catabolism and apoptosis. The fact that S1P-lyase was
under-expressed could even suggest that sphingolipids obtained by diet, are not metabolized
in colon cancer. (102)
The S1P-pathway and the COX-2 pathway have been shown to share a connection: SphK
shutdown decreased COX-2 and PGE2 production, while S1P enhances COX-2 expression
and PGE2 production. (88) Then again, ceramide kinase and ceramide-1-P are necessary for
translocation and activation of cPLA2, which provides substrates (arachidonic acid) for the
COX-2 inflammatory and pro-carcinogenic pathway. Both these sphingolipid metabolites
significantly augment PGE2-values via two different but completely coordinated and
synergistic pathways than when given separately and in the same quantity. (103) Interestingly,
tissue samples of colorectal adenocarcinoma and their metastatic variants with an elevated
COX-2 expression were also positive for SphK, while a positive expression of SphK was not
always linked to positive COX-2 expression. (89)
As previously mentioned, glycospingolipids are extremely diverse, although changes in the
glycolysation pattern of glycolipids is a familiar phenomenon in a large quantity of cancers,
including CRC. (104) As an illustration: sialidation is often over-expressed in colon cancer
cells. Indeed, human Neu3 activity is elevated in neoplastic colons, blocking programmed cell
death and even modulating cell differentiation. (105) These findings are partially confirmed
by the augmentation of lactosylceramide (2 to 5 times higher than in normal mucosa) and a
sialidase product in colon cancer cells . (106) Another study revealed that glycosphingolipids
in CRC obtained an increased fucosylation, a decreased glycan acetylation, sulfation and
ganglioside disialylation. (104) Furthermore, the higher the Dukes stage or metastatic
potential of the patient’s CRC, the higher the sulfogalactosylceramide levels. (107) Lastly,
chemosensitizers combined with GCS-inhibitors induced cytotoxicity and therefore, apoptosis
in colon cancer. (97) Sphingolipid glycolysation could lead us to new insights on colorectal
35
carcinogenesis, new strategies for therapy or new biomarkers for screening. However, a real
pattern in abberant glycolysation has not actually been determined yet, because these differ in
every type of cancer and make use of different glycan-groups. (104)
4.3.4 Sterols
4.3.4.1 Pathway Cholesterol is an extremely important molecule in the human body: it is an essential
component in cell membranes, in which the majority is non-esterified cholesterol, and
intracellular membranes, where the greater part consists of esterified cholesterol. Furthermore,
it is a precursor of bile acids, Vitamin D and steroid hormones. (108)
Fig. 8: chemical structure of cholesterol
Cholesterol is a polycyclic structure containing a total of 27 carbon atoms. The hydroxyl-
group on C3 can be replaced by an ester-bond. Both cholesterol forms are transported in
lipoprotein-particles (chylomicrons, VLDL, LDL, IDL and HDL) due to its lack of solubility
in water or plasma. Cholesterol can be obtained through diet, but is mainly de novo
synthesized. Cholesterol biosynthesis is a complex process : aided by the mevalonate
pathway, it commences with condensation of acetyl-CoA and acetoacetyl-CoA, producing
HMG-CoA. Eventually, this product is reduced to mevalonate by HMGCoA-reductase, the
rate-limiting step of cholesterol biosynthesis. (7) Afterwards, two kinase reactions
sequentially take place, directly followed by a decarboxylation, forming
isopentenylpyrophosphate (IPP). This latter product can be converted into
dimethylallylpyrophosphate (DMPP) by isomerisation. Condensation of IPP and DMPP
produces geranylpyrophosphate (GPP), which sequentially undergoes a condensation reaction
with IPP, gaining farnesylpyrophosphate (FPP). Both these condensations are sustained by
36
geranylpyrophosphate-synthase (GPPs) and farnesylpyrophosphate-synthase (FPPs). Squalene
is synthesized in the next step, derived from FPP. With help of the squalene-epoxidase and
lanosterol-synthase, it is converted into lanosterol. From here on, 19 different reactions aided
by nine different enzymes, are necessary to gain the final product, cholesterol. (108)
In addition, cholesterol can be esterified with a long FFA by acyl-coenzyme cholesterol
acyltransferase (ACAT), gaining cholesteryl esters. This is the main form in which cholesterol
can be stored in cells, and transported through the blood via lipoproteins.
Fig.9: Overview of cholesterol biosynthesis
4.3.4.2 Alterations in colorectal cancer
Cholesterol and cholesterol precursors are known to be important metabolites in
carcinogenesis: cancer cells require higher concentrations during their development and
further growth. Targeting this pathway could supply new therapeutic methods against CRC,
although changes in this pathway may also contribute to chemotherapy resistance. (109)
Cholesterol transport also showed some discrepancies in malignant cells, but this will be
described in the next section on lipoproteins.
37
Oxysterols are catabolic products of cholesterol, produced by cytP450-enzymes or by
reactions with reactive oxygen and nitrogen. (110) They are able to connect with LXR-
receptors and activate different cholesterol efflux pump and other regulatory proteins.
Oxysterols are also involved in LDL-receptor degradation. (109) Some oxysterols are capable
of initiating colorectal tumorigenesis by augmenting ROS/RNS activity, thus activating pro-
carcinogenic proteins like COX-2 and even supporting tumor progression by promoting
migration of neoplastic cells. (110) While it can therefore be stated that oxysterols have a
protective influence during carcinogenesis by inhibiting cholesterol abundance in cells, the
fact that some oxysterols have pro-inflammatory and pro-oxidative capacities (thus being pro-
carcinogenic), contests the former theory. (109) (110)
The cellular mechanism of cholesteryl ester (CE) homeostasis remains uncertain, but findings
indicate that many cancer cell lines are submitted to higher CE synthesis and increasing
uptake of CE delivered by LDL and HDL.(111) In breast cancer, CE cell accumulation has
already been associated with a malignant phenotype and proliferative effects, but the effects
on CRC specifically have not yet been investigated. (112)
Regarding the mevalonate pathway, an elevation of HMGCoA-reductase, FPP synthase and
farnesyltransferase have been noticed in different types of cancer, especially in colorectal
cancer.
HMGCoA-reductase is the rate limiting step in cholesterol biosynthesis, meaning this
enzyme has a major impact on pathway progression. (113) Its product, mevalonate acid, is
known to rapidly initiate DNA-replication, enhancing control loss of the synthesis pathway
and contributing to malignant transformation. (114) Although most CRC samples have
increased levels of surface LDL-receptors, HMGCoA-reducate activity in CRC was more
significantly elevated in LDL-R negative colorectal tumors, as absence of LDL-R in
malignant cells imply they are solely dependent on endogenous synthesis of cholesterol
instead on environmental uptake. (115) Quite contradictory with the former findings, absence
of LDL-R in colorectal tumors had a negative impact on patient survival, but expression of
HMGCoA-reductase is more up-regulated in TNM stage I and II than in stage III and IV, thus
giving patients a better prognostic value. (116) (117)
It also seems that HMGCoA-activity is correlated with the tumor localization in the colon:
tumors on the left side of the colon had three times the enzyme activity compared with rectal
tumors. (115) As the greatest risk in colon cancer is metastasis and in rectal cancer,
38
recurrence, these arguments feed the theory that different tumor localizations are associated
with different molecular mechanisms in carcinogenesis of which the cholesterol pathway has
its own unique function. (113)
Besides elevated mevalonate levels, many studies indicate that isoprenoid compounds FPP
and GPP are elevated in malignancy, activating oncogenes and promoting tumorigenesis.
(114)(115) Indeed, FFPs activity was enhanced in CRC tissue compared to normal mucosa.
Furthermore, FPP is the essential substrate for farnesylation, a protein isoprenylation process.
These protein alterations provide them with unique capacities considering proliferation,
differentiation and cell survival, properties that could be altered in neoplasmic cells. An up-
regulation of farnesyl transferase was noted in CRC hence confirming this theory. (114)
Inhibitors of HMGCoA-reductases, statins, are widely used for lowering cholesterol serum
levels as a part of cardiovascular risk prevention but might prove themselves worthy as
chemo-preventive agents in colorectal cancer. More and more evidence suggests that statin
intake may also interfere in tumor proliferation, growth and metastasis. (118) Besides down-
regulation of cholesterol, it reduces-PP and geranylgeranyl-PP concentrations, inhibiting
prenylation of proteins and hereby restricting essential physiological functions in tumor
development. (119) Although the role of simvastatin remains somewhat unclear: it showed
promotion of apoptosis while reducing human cancer cell proliferation in vitro. (115) (117)
Another study showed no effects in cancer cell proliferation or apoptosis, but administration
inhibited cell migration and thus metastasis probability. However,
geranylgeranylpyrophosphate or mevalonate addition reduced statins inhibitory effects on cell
migration. (120) Conclusively, a meta-analysis reported a small but significant protective
effect of statins in CRC by decreasing invasiveness, metastatic properties, and even
chemosensitizing the neoplastic cells for other chemotherapeutics. (119)
Biphosphonates are commonly used for the treatment of osteoporosis, but since these are
capable of targeting the FPPs in the mevalonate pathway, they could be valuable in blocking
cholesterol synthesis, thus restricting further CRC development.
(114) A meta-analysis concluded that oral administration of biphosphonates clearly reduces
the risk of CRC, depending on the dose and duration of drug administration. (121)
39
Total cholesterol (TCH) and free cholesterol (FCH) were measured in patients with CRC and
compared with healthy patients or benign colorectal lesions. A drop in serum TCH was
observed while serum FCH was significantly elevated. Furthermore, only serum TCH
concentrations were significantly and progressively reduced in CRC stage III and IV. The
fact that neoplasm cells have higher need of cholesterol, could explain the drop in cholesterol
serum levels due to higher uptake, while the low levels of tissue cholesterol may be caused
by the cells limited cholesterol synthesis and transport capacities, while trying to keep up with
the enhanced cholesterol need. This hypothesis remains vague and the mechanism behind this
theory hasn’t been elucidated yet. The only conclusion that can be made is that the
physiological homeostasis of cholesterol is diminished in CRC, which could offer new
therapeutic targets or predictive biomarkers. One final remark is that these alterations in lipid
levels could have been present before the patients illness, as they are independent risk factors
in the development of CRC. (82)
Besides alterations in biosynthesis, cholesterol homeostasis can also be deregulated by
deficiencies in the cholesterol efflux pumps of cells. An example is the ABCA1 efflux
function in mitochondria: inhibited ABCA1 expression in colon cancer cells increases the
mitochondrial cholesterol concentrations and reduces the release of cell death promoting
molecules. Consequently, these tumor cells are able to have prolonged survival rates. (122)
Although cholesterol is the essential precursor molecule of Vitamin D, bile acids and sterols,
alterations in their metabolism and/or the association with CRC falls beyond the reach of this
thesis.
4.3.5 Lipoproteins and lipid droplets
Lipoproteins mainly comprise molecules that are capable of transporting lipids such as
cholesterol, cholesteryl esters, TG and vitamins, to all cells. The tend to occur in a structure
that is composed of a phospholipid monolayer stabilized by neutral apolipoprotein, with a
central hydrophobic core within. Lipoproteins are classified into four classes according to
their progressing density: chylomicrons, VLDL, IDL, LDL, and HDL. Additionally, the
hydrophobic core is an ideal environment for the transportation of lipophilic agents such as
certain chemotherapeutic agents, as these remain stabilized within this core and are not
directly removed from the bloodstream, offering an elevated half-life for these compounds.
(118)
40
Many types of cancers over-express HDL- and LDL-receptors on their cell surface, with
serum HDL showing decreased values and augmented quantities of tissue HDL in cancer
patients. Indeed, CRC patients had reduced serum HDL and LDL values while tissue HDL
was increased in CRC. These values were even more highlighted when progressing through
the TNM stages due to the fact that higher tumor grade is correlated with higher demand for
cholesterol, and thus enhanced cholesterol transport. (82) (118) As Apolipoproteins form the
fundamental basis in the synthesis of lipoproteins, over-expression of these structures could
be playing a role in tumor development. Apo-A1 and Apo-B may have an altered expression
that could be associated with the HDL and LDL shifts seen in CRC. (82) Apolipoprotein E1
gene polymorphisms have already been correlated with higher CRC risk. (123)
Lipid droplets or lipid bodies are small, dynamic organelles, consisting of a lipid monolayer
and capable of stocking all cytosolic lipids in human cells. Besides lipids, these organelles
also contain a significant amount of proteins that are involved in lipid metabolism, but also
other processes that could enhance carcinogenesis. Well known examples are PI3K, MAPK,
cPLA2, COX, and LOX-enzymes, of which the latter three enzymes claim essential roles in
the pro-inflammatory eicosanoid pathway while using AA as substrate. (124) Lipid bodies
were significantly increased in colon carcinoma cell lines in comparison to normal mucosa
when investigated using Raman microscopy. (125)
Also, expression of COX-2 was fortified in lipid droplets of colon cancer cells, which was
correlated with enhanced production of PGE2. As this is a pro-inflammatory and pro-
tumorigenic factor, as described above, lipid droplets may contribute to the development of
CRC and could be suggested as a new therapeutic target, while the amount of lipid droplets in
CRC cells could be brought forward as a prognostic biomarker. (124)
41
4.4 An interpretation of previously obtained experimental data
Dr J.Foster performed data analysis for his PhD dissertation at Cambridge University, UK of
comparative lipidomic profiling data of CRC samples with normal mucosa samples. His
analysis not only aimed for identifying alterations of general lipid subclasses, but also of
individual lipids that were organized according to their specific acyl bond.
Generally, tumor samples showed a significant increase (P = 0.0025) in the total sum of lipid
concentrations when compared to normal samples. This phenomenon repeated itself as the
total amount of lipids corresponding to a specific subclass was significantly altered in CRC
samples (P < 0.05) in all except three subclasses; only LPC, SM and PA did not show
significant alteration. Furthermore, the concentrations of lipid substrates within these
subclasses were increased in all tumor samples compared to normal mucosa, but subclasses
LPA, monoacylglycerols and TG were decreased.
Glycerolipids TG, PC and PE were altered most frequently in CRC and were mainly down-
regulated. These tumor samples showed a clear preference for long chain desaturated PS
(phosphatidylserine) species, while short chain saturated PS species were moderately down-
regulated. The total amount of all PE species concentrations was absolutely increased in
tumor tissues, with evidence that some reactions shifted towards the production of PE, by
using substrates PC, PS and DAG (diacylglycerol). LPC was also strongly altered in tumor
tissue in comparison with normal mucosa.
Other important findings were the shifts that manifested when lipid profiles were compared
between tumor and normal mucosa samples, tumors with different stages and different tumor
sizes. Ceramide 16:0 levels were increased in tumor size 1 samples compared to other
samples, while ceramide 18:0 levels were decreased. Additionally, PE species of patients who
received radiotherapy prior to tissue sample collection were evaluated: this highlighted that
PE 36:1 and PE 36:2 were reduced, while PE 38:4 was augmented in pre-radio samples in
comparison with all other samples. Lastly, PI and PS values of tumor size 2 samples were put
side by side with those of tumor size 3 samples. While PI 34:1 PI 36:1 and PI 36:2 were
elevated, PI 38:4 was down-regulated in tumor size 3 samples compared to tumor size 2.
Furthermore, tumor size 3 shifted towards lower PS 44:7 and PS 44:8 values. (126)
42
5 Discussion
Alterations of lipid metabolism in CRC cover an enormous quantity of information due to its
extreme complexity. Lipidomics remain a somewhat under-investigated area and the findings
that have been published so far are likely to reveal only the tip of the iceberg.
A lot of opportunities for therapy and screening methods lay within this territory, of which
some have already been mentioned above.
Regarding the FFA, ACLY and FASN have been intensively examined enzymes.
Experiments with ACLY and FASN-inhibiting medicines already showed promising results,
while serum FASN values could provide predictive and prognostic information. The shift in
FFA distribution in CRC tissue compared to healthy colorectal cells is another interesting
finding. What mechanisms drive the cancer cells in producing more ‘aggressive’ ω-6 FFA,
AA in particular, in comparison with more ‘protective’ ω-3 FFA, EPA and DHA? Although a
lot of attention has been devoted to the production of pro-inflammatory eicosanoids (e.g.
PGE2, TxA2, 12-HETE) and their role in colorectal carcinogenesis, decreasing the amount of
substrates used for these pathways may be of exceptional value for inhibiting further
development of CRC.
Glycerolipids, and mainly glycerophospholipids, have an unmistakable role in CRC
pathogenesis. In literature as well as in Foster’s thesis, TG levels were demonstrated to be
altered in different CRC stages, although it is still questionable what clinical benefits could
be acquired from these findings in CRC. LPA, PC and PLA2 have also been intensively
examined throughout the years and showed more potential for future use. PI and moreover,
PIP3, is part of a key regulator in many intracellular pathways that augment pro-tumorigenic
pathways. Suppressing PI3K/Akt/mTOR pathways have already been tested with
pharmaceuticals in different cancer types. This offered promising results at the beginning of
the trial, but chronic treatment forced the cancer cells to adapt and use other growth-
stimulating pathways, making these medicines unable to fully exterminate the cancer cells.
(127) There could therefore be room for different approaches in targeting these pathways in
CRC, for instance by inhibiting synthesis of PI-molecules.
Ceramide is likely to be at the epicenter of spingolipid metabolism alterations in CRC cells.
All results indicate that cancer cells try to avoid pathways that favor ceramide production,
43
thereby evading its apoptotic and anti-proliferative capacities. Developing methods for
enhancing ceramide production, or inhibit pathways that cut off ceramide supplies could be of
therapeutic value.
In cholesterol metabolism, all evidence points out that de novo synthesis is up-regulated in
CRC, together with enhanced uptake of cholesterol provided by lipoproteins LDL or HDL.
Not only enhanced cholesterol synthesis, but also the increased isoprenylation activity from
its intermediates FPP and GPP could be interesting targets. For decades, cholesterol
metabolism has been a major topic in cardiovascular diseases while data are showing some
significance in malignancy processes. This reinforces speculations that pharmaceuticals that
lower cholesterol uptake or that inhibit de novo synthesis could also be of use in the therapy
or prevention of CRC.
I conclude that there is plenty of information that suggests an essential role for lipid
metabolism in CRC pathogenesis. This may lead to new opportunities in treatment and
prevention of CRC, granting the patient a better prognosis due to improved and more
personalized medicines and earlier detection of the tumor, preferably during curable stages.
Nowadays, CRC is still classified according to the TNM or TNM clinical staging system, an
instrument that is primarily focused on surgical treatment of CRC. As every CRC covers a
specific, heterogeneous metabolic profile, the question rises if lipidomics (and other ‘omics’)
approaches could become the new standard in adequately categorizing CRC on a molecular
basis. This molecular classification could offer patients a personalized therapy-schedule,
depending on the type of molecular defects their colorectal tumor acquired. Still, this thesis
confirms that lipidomics is standing in its infancy, as we know so much and yet so little about
the normal biological processes in mammals that interfere with lipids, let alone in
pathological processes such as CRC. The majority of the literature that I have investigated
mainly focused on the alterations of lipid molecules in CRC regarding absolute concentrations
within a lipid sub-class. A handful of articles, including Foster’s thesis, were the only ones
who attempted to examine lipid metabolism alterations in CRC by observing each individual
lipid from each lipid sub-class on their quantity, distribution and composition in comparison
to normal mucosa. Indeed, this is an expensive and time consuming method of investigation,
but this may be the only way to fully understand the mechanisms behind CRC pathogenesis.
A long road still lays ahead of us if we want to efficiently progress within this specific
territory as much work remains to be done.
44
6 Summary in Dutch
Colorectale darmkanker blijft één van de meest prevalente, incidentrijkste en dodelijkste
maligniteiten ter wereld. Wetenschappers testen steeds nieuwe technieken te uit om
efficiëntere screeningsmethoden en therapieën te ontwikkelen . Lipidomics is onderdeel van
het netwerk van de metabolomics en bestudeert de distributie van lipiden in menselijke cellen,
weefsels, maar ook andere organismen. Lipidomics blijft vooralsnog een weinig onderzocht
terrein, doch heeft al veelbelovende resultaten geboekt in verscheidene soorten maligniteiten.
In deze literatuurstudie werd getracht om zoveel mogelijk informatie samen te vatten die tot
op heden gekend is inzake het verband tussen lipidenmetabolisme en colorectale darmkanker.
Naast de lipoproteïnen en de intracellulaire lipidendruppels, zullen de meest significante
veranderingen binnen de grootste lipidengroepen worden besproken. Dit betreft de vrije
vetzuren, glycerolipiden (met name de triglyceriden en de fosfoglycerolipiden),
sphingolipiden en sterolen. Er werd alvast gestegen activiteit waargenomen van enzymes die
de synthese van vrije vetzuren reguleren, evenals significante veranderingen in de
samenstelling van vrije vetzuren in colorectale tumor specimens. Er werd alsook een gedaalde
hoeveelheid triglyceriden en phosphaticylcholine glycerophospholipiden genoteerd in deze
specimens. Gezien ceramide de centrale molecule is binnen het sphingolipidenmetabolisme
en beschikt over pro-apoptosis en anti-proliferatieve eigenschappen, pogen colorectale
maligne cellen de productie van dit specifiek metaboliet te vermijden. Een gestegen de novo
synthese van cholesterol werd waargenomen, evenals een toegenomen opname van
cholesterol vanuit de omgeving via lipoproteïnen, wat duidelijk kon aangetoond worden
gezien de gestegen cellulaire expressie van HDL- en LDL-receptoren. Tot slot is maligniteit
al enige tijd geassocieerd aan een verhoogde aanwezigheid van intracellulaire lipidendruppels
en dit fenomeen werd inderdaad bevestigd in colorectale darmkanker specimens. Desondanks
de bovenvermelde bevindingen die aangeven dat er weldegelijk een associatie bestaat tussen
colorectale darmkanker en lipidenmetabolisme en er voldoende opportuniteiten bestaan om
nieuwe therapieën en biomerkers te ontwikkelen, staat dit gebied nog steeds in haar
kinderschoenen. Heel wat onderzoek zal nog moeten verricht worden willen we het
lipidenmetabolisme integreren in het huidige screenings-en therapiebeleid, niet alleen voor
colorectale kanker maar ook voor andere soorten maligniteiten.
45
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8 Appendix
Dukes classificiation
Stage A: invading submucosa
Stage B1: invading into muscularis propria
Stage B2: invading through muscularis propria
Stage B3: equals T4N0M0
Stage C1: as B1 but with lymph node invasion
Stage C2: as B2 but with lymph node invasion
Stage C3: as B3 but with lymph node invasion
Stage D: distant metastasis
TNM classification
T=tumor
T0= no primary tumor present
Tis: tumor in situ (located intra-epithelial or invading lamina propria)
T1: tumor invading submucosa
T2: tumor invading muscularis propria
T3: tumor invading subserosa or non-peritoneal pericolic or perirectal
structures.
T4a: tumor invading visceral peritoneal surface
T4b: tumor invading or adherent to surrounding organs
Tx: primary tumor cannot be identified
N: lymph node invasion
N0: no lymph node metastasis
N1a: metastasis in one regional lymph node
N1b: metastasis in 2-3 regional lymph nodes
N2a: metastasis in 4-6 regional lymph nodes
N2b: metastasis in 7 or more regional lymph nodes
Nx: lymph node invasion cannot be identified
M= distant metastasis
M0: no distant metastasis
56
M1a: distant metastasis to organ or other site
M1b: distant metastasis to multiple organs, sites or peritoneum
Mx: distant metastasis cannot be identified
TNM disease stages
Stage 1: T1-T2N0M0
Stage 2A: T3, N0, M0
Stage 2B: T4a, N0, M0
Stage 2C: T4b, N0, M0
Stage 3A: T1-T2, N1, M0 and T1, N2a, M0
Stage 3B: T1-T2, N2b, M0; T2-T3, N2a, M0 and T3-T4a, N1, M0
Stage 3C: T3-T4a, N2b, M0 and T4b, N1-N2, M0 and T4a, N2a, M0
Stage 4A: M1a with any T and any N
Stage 4B: M1b with any T and any N