Download ISCs regulatory signalling and cancer pathways

ISCs regulatory signalling and cancer pathways
The pathways controlling the ISC are slowly being unravelled and, if these
cells prove to be the origin of “tumour propagating cells”, should provide us with
targets at which future cancer treatments can be aimed. The proper cell fate decisions
of the ISC progeny as they migrate, proliferate and differentiate on route to assuming
their appropriate positions within the villus are organised within the mircoanatomy of
the crypt structure. These are tightly controlled processes with cells migrating at
different rates. In the small intestine the upward migrating enteroendocrine cells
migrate at the same rate as the surrounding cells turning over every approximately
four days [30, 31], however in the large intestine they migrate independently over 23
days as opposed to the 4 days of the surrounding cells [73]. Indeed it is the short life
span of these cells that negates the effects of any post differentiated mutations they
may have acquired [74]. Any perturbation of the pathways regulating the ISCs and its
daughter cells proliferation, differentiation or migration that allows accumulation and
retention of cells within the intestinal epithelium would be prerequisite to neoplastic
transformation. Further, although there is little evidence, account must also be taken
of signals from the cells that surround the crypt such as the enteric neuron [75], as it is
likely they also mediate different cell fates along the vertical crypt axis. Genetic
experiments have shown that many regulatory signals are involved in the crypt-villus
patterning, including the bone morphogenetic (BMP), Notch, PtdIns(3,4,5) kinase,
Sonic Hedgehog (in the developing mouse[76]) and Wnt pathways [77, 78, 79, 80].
Wnt signalling
Wnt signalling is critical for both the development of the intestine and the
homeostasis of the adult tissue [81, 82, 83, 84]. However it is the deregulation of this
pathway in CRC that has lead to its extensive study in the intestine [85, 83], [86]. As
a mutation in the Apc gene (usually resulting in a protein truncation [87]), a single
component of the canonical Wnt pathway, is associated with 85% of sporadic
intestinal neoplasia and almost all inherited CRCs namely familial adenomatous
polyposis (FAP). Inactivation of Apc is the earliest known genetic event in CRC [88].
The Wnt pathway (from Drosophila Wg (wingless) and Int gene [89]) starts
with a large family of 19 secreted molecules called Wnts which bind to at least 10
members of its receptor families Frizzled (Fz) and low-density lipoprotein receptorrelated protein family LRP5/6. The binding of Wnts to the Fz receptor permits an
interaction and phosphorylation of a cytoplasmic molecule called dishevelled (Dsh).
Dsh then disrupts the so call “-catenin destruction complex”, -catenin is a
signalling molecule which if allowed to accumulate in the cytoplasm will
subsequently translocate to the nucleus where it activates members of the TCF/LEF
family of transcription factors [90, 91, 92]. Intracellular levels of -catenin is
regulated via formation of a multiprotein “destruction complex” encompassing
glycogen synthase kinase β (GSK3β), casein kinase 1 (CK1), and the scaffolding
proteins Apc, Axin1 and Axin2.
This multiprotein complex of molecules
phosphorylates specific serines and threonines within -catenin, triggering
ubiquitination by TrCP and degradation in proteasomes. Consequently the presence
of Wnt ligands or any disruption of the destruction complex allows β-catenin to
accumulate and translocate to the nucleus. Once Wnt signalling has been activated
there is alteration in the expression pattern of its TCF/LEF target genes (for a list of
known Wnt target genes see www.stanford.edu/~rnusse/). A gradient of Wnt
signalling along the crypt-villus axis controls expression of the EphB/ephrinB sorting
receptors and ligands. This gradient of receptors allows the correct positioning of
intestinal epithelial cells indeed EphB receptor activity suppresses CRC [93, 94]. It is
these findings and the presence of nuclear β-catenin at the base of the crypt, adjacent
to the presumptive stem cell niche, that has suggested a critical role for Wnt signalling
in intestinal homeostasis and stem cell maintenance [95]. Deregulating the Wnt
pathway by removing the Apc gene alters the stemness, proliferation and
differentiation of the ISCs in the adult murine intestine. As Wnt activation
immediately imposes disruption of the tissue architecture and the formation of benign
growths, a phenotype mimicking tumourigenesis [95]. Further demonstrated by the
evidence that this phenotype is entirely dependent on the Wnt target gene C-Myc [96].
Murine intestine cells that lose Apc undergo rapid entry into S-phase, enhanced
apoptosis, failed migration and perturbed differentiation [95]. For these benign
growths or adenomas to progress towards carcinoma other Wnt promoting factors are
involved, such as oncogenic activation of the KRAS oncogene [97], autocrine Wnt
feedback loops [98] and paracrine modulation of epithelial Wnt signalling by the
myofibroblasts and other stromal cells [99]. The Wnts can also stimulate cellular
responses through the so called “non-canonical” Wnt pathway. These are β-catenin
independent and involve calcium trafficking or induce morphogenic changes via the
planar cell polarity pathway.
In summary the loss of Apc, activating the Wnt pathway, immediately confers
virtually all of the phenotypes associated with the very early stages of intestinal
neoplasia but deficiency of either C-Myc [100] or β-catenin [101] leads to loss of the
mutant tumour propagating cells and replacement by wild type crypts in murine
models. Targeting this pathway in CRC may give effective control of CRC and its
metastases. For example mice which are heterozygous for a mutation in Apc develop
multiple intestinal neoplasia (MIN) [102] (as in human FAP patients) which is
attenuated by loss of the methyl binding domain proteins Mbd2 and kaiso [103, 104].
Mice null for these genes show an associated reduction in expression of Wnt target
genes upon Wnt activation.
ISC therapeutics
How we approach the development of ISC therapeutics depends where we
intervene in an intestinal tumours’ development. In a sporadic occurrence if it is
detected at a late stage (which could be avoided with adequate screening of
susceptible patients) we may need to debulk the tumour (which current drugs are able
to do) and target the tumour propagating cells to prevent its recurrence. To target the
propagating cells in this situation we would need knowledge of the pathways the cells
have utilised to drive their transformation. In hereditary predispositions such as FAP
with 100% penetrance of tumours we could predict the pathway involved but it would
be beneficial for treatments to prevent the initiation of the polyps that will lead to
cancer. When approaching the problem there is an initial decision to be made
whether we want the ISC therapeutic agent to prevent tumour initiation or to remove
tumours that have developed. Either way it is likely the same signalling pathways,
namely Wnt, in the ISCs will be targeted.
Preventing tumour initiation
The earliest known genetic event in CRC is the inactivation of Apc leading to
aberrant expression of Wnt target genes FAP these tumours are benign but eventually
lead to cancer later in life. The inactivation of the remaining Apc results in nuclear βcatenin aberrantly activating Wnt target genes to drive the ISC to over proliferate and
form a polyp. A process crucial to this is the hypermethylation of gene promoters
which results in their silencing. In mice it is known that inhibition of DNA
methylation through reduction of DNA methyltransferase 1 (DNMT1) [129, 130] or
perturbation of a protein that interprets the DNA methylation signal e.g. Mbd2,
suppresses intestinal tumourigenesis in ApcMin mice [104]. We can assume
hypomethylation and gene promoter hypermethyaltion are events that silence or over
express gene essential for initial neoplastic transformation of a cell.
Large scale alterations in DNA methylation, with global hypomethylation and
promoter specific hypermethylation linked to aberrant patterns of histone
modification are common in cancer cells [131, 132] with estimates suggesting that the
average tumour will contain approximately 100–400 hypermethylated promoter
regions. Demethylating and histone deacetylase (HDAC) inhibiting drugs (e.g.
zebularine, RG108, trichostatin A, green tea extract EGCG, hydroxamic acids,
benzamides & cyclic tetrapeptides) are showing promising results in cancer where
specific hypermethylation is implicated in the etiology [133]. Some of these drugs are
in phase I or II trials and are proving to be effective in certain cancers with newer
molecules showing less toxicity.
However which genes are initially epimutated as to allow neoplastic
transformation in the ISC and which are secondary effects are unknown [134, 135].
One major concern about epigenetic drugs is their general lack of target specificity as
global demethylation can potentially activate oncogenes [136, 137]. To maximize
efficiency of epigenetic drugs, their mechanisms and targets must be more clearly
defined. Results in mice with proteins such as Mbd2 which interpret the methylation
signal but still reduce intestinal tumour burden may provide us with specific genes for
therapeutic targeting. Which specific gene(s) are being derepressed via Mbd2 loss
that prevent tumourigenesis has yet to be defined. These genes are of interest as the
loss of Mbd2 combined with specific removal of Apc in the intestine doesn’t suppress
Wnt signalling but does reduce its elevation. Another reason Mbd2 may be an
attractive therapeutic target is the possibility that it may allow therapeutics with less
cytotoxic effects. If as in mice humans can tolerate Mbd2 loss without harmful
effects. The caveats to this are that the effects of such therapeutics are transient and
the aberrant epigenetic patterns are likely to return once the treatment period
terminates. However they are quite promising chemopreventive agents in cases when
epimutations increase the risk of developing a disease in individuals who do not yet
show signs of malignancy. The results of the long term exposure to such agent may
be their undoing in a preventative measure but they may still be effective post tumour
initiation in preventing recurrence after tumour removal. We are at the dawn of
epigenetic medications but further progress is required, in particular, in the
development of compounds with higher specificity and greater efficacy to see if they
fulfil their potential.
Preventing tumour recurrence
As removal and debulking of CRC is already feasible it is the recurrence of
these tumours which represent the main challenges for ISC therapeutics in sporadic
patients. Assuming that within a metastasising tumour there are cells resistant to
cytotoxic therapy with the potential to regenerate the tumour in situ and ex situ. As
the pathways these cells have utilised to allow neoplastic transformation is likely to
differ from tumour to tumour then initially we would assume all pathways have to be
targeted. However as most other pathways all converge with Wnt signalling at some
stage this would be the likely first target. There are many molecules identified which
will down regulate Wnt signalling [138]. These molecules work at all levels of the
canonical Wnt pathway. From the top of the pathway the natural inhibitors of Wnt
such as the FRPs or Dickkopf gene family which is commonly down regulated in
gastrointestinal tumours and can inhibit invasion in vitro [139, 140] and adult mice
intestinal proliferation in vivo [81]. Through to genes involved in stabilising the βcatenin destruction complex, down regulating β-catenin, targeting the nuclear βcatenin with molecules such as ICAT and TBLR1[141] which inhibit the βcatenin/TCF4 interaction and finally targeting the Wnt target genes themselves such
as C-myc. Many of these targets have been attacked by different mechanism in vitro
and in vivo. By repairing the defective components of the β-catenin destruction
complex through introducing wild type versions [142, 143], over-expressing βcatenin/TCF inhibitors [144], antisense degradation of β-catenin [145], introduction of
β-catenin binding proteins that target for degradation [146, 147, 148] and screens
highlighting molecules to disrupt the β-catenin/TCF4 interaction [149, 150] have all
been show to inhibit tumour growth and survival.
Therapeutic specificity for the cancer ISC
For any ISC therapy it is critical that the normal somatic stem population is
not catastrophically impaired. The therapeutic agent ideally shall be tumour specific
to avoid side effects as for example the Wnt pathway is also is also a therapeutic
target for neurodegenerative, bone and cardiovascular disease [151]. Targeting of
single pathways will probably not be sufficient due to systemic toxicity limitations.
Therapeutic targeting of signalling synergy between Wnt and other important
pathways in the cancer stem cell may provide the angle for controlling CRC. In vitro
studies using tyrosine kinase inhibitors, and combinations of agents against the EGF
and Wnt pathways all show potential for inhibiting CRCs [152, 153]. Targeting the
cross talk between the regulatory pathways and mesenchyme in the ISC niche may
also provide the therapeutics to specifically target the cancer cells rather than normal
cells. BMP targeting may be attractive due to findings that BMP signalling in the
stroma results in Wnt suppression. Aside from the previously described pathways
there are other potential targets for drugs against colorectal cancer. The paracrine
hormone hypothesis of colorectal cancer (described by [154]) implicates the
dysregulation of the guanyl cyclase C (GCC) and its paracrine ligands. Deletion of
GCC causes an increase in the mutation rate and, presumably from the ISC, defective
lineage specific differentiation, increases the size of the proliferating cell
compartment, the number of proliferating cells in that compartment and their cell
cycle kinetics [155, 156]. This loss of the paracrine hormones is associated with overexpression of the receptors in tumours as compared to normal cells in human intestine
[157] however it is unknown if they mark the stem cells so its use in preventing
recurrence is unknown until the cancer stem cell is identified.
The requirement for tumour cell specific therapeutics is due to the concern
over destruction of the normal ISC population. This may be less of a concern once
we have characterised the ISC and is proposed quiescent versions. These are the stem
cell described earlier which allow repopulation of the crypt-villus following
destruction of the active ISC. If it exists it is the quiescent stem cell in whatever
shape or form it comes which may allow therapies targeting all active ISC to be
successful. The location of this quiescent stem cell is of great interest, as potentially
in a process called transdifferentiation a cell can extricate it self from foreign tissues,
transform and migrate to contribute to adult stem cells within other tissues. This
process is of great interest, indeed adult bone marrow cells can contribute to adult cell
lineages in several non-haematopoietic tissues, including the gastro-intestinal tract.
Further evidence is needed to show if potentially bone marrow cells can form ISCs
[158, 159], or even a mesenchymal stem cell in the lamina propria which could then
rise to an ISC. If possible all ISC could be eliminated therapeutically safe in the
knowledge that the organ will be replenished from the protected undamaged quiescent
stem cells.
Summary
To summarise for the past half century, oncologists have had systemic drugs
available that are able to induce tumour responses in patients with colorectal cancer.
However, in cases of advanced colorectal cancer, these regimens are almost never
curative. The recently introduced concept that cancer stem cells drive tumour growth
suggests a reason for these therapeutic failures. Current chemotherapeutics target
rapidly dividing cells not their controlling pathways missing the slow dividing
cytotoxic resistant cancer SCs. The failure also suggests a solution the development
of therapeutics that target cancer SCs. If we can identify the ISC and characterise the
differences in its tumour propagating counterpart we can exploit their differences.
We will then be able to treat sporadic tumours with current drugs, essential to debulk
the tumours and with the tumour propagating cell agent to prevent their recurrence.
In the last 20 years extensive information about the mechanisms by which SC
populations are maintained has been obtained without actually identifying the ISC.
Although we have still to identify the ISC we may have identified how they are
controlled as has been described earlier there are many pathways and molecules
which are capable of altering the homeostasis of the intestine. Much of the work
described here has yielded markers for cells showing characteristics of being the ISC.
We are getting nearer the goal of identifying the active, quiescent and potential ISCs
which lead to CSCs. The current proposed markers have yet to be categorically
proven but until definitive identification of the ISC they are being used to study the
effects of DNA damaging agents and chemotherapeutic agents in vivo on these
potential ISC. The value for CRC therapeutics using the targets proposed by the ISC
regulatory pathways and the various methods of attacking them (e.g. anti-sense, RNA
interference and protein knockdown strategies [160]) can only be assessed when the
proposed CSC have been identified. At that juncture we will finally be able to assess
the roles of the ISC in drug discovery.