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Myc interactions in intestinal cancer: Partners in crime
E XP E RI ME N T AL C E L L R E S EA RC H 31 7 ( 20 1 1) 2 7 2 5– 2 73 1
available at www.sciencedirect.com
www.elsevier.com/locate/yexcr
Review Article
Wnt/Myc interactions in intestinal cancer: Partners in crime Kevin Myant, Owen J. Sansom⁎ Beatson Institute for Cancer Research, Garscube Estate, Switchback Road, Glasgow, Scotland G61 1BD, UK
A R T I C L E I N F O R M A T I O N
A B S T R A C T
Article Chronology:
Loss of the APC (adenomatous polyposis coli) gene in colorectal cancer leads to a rapid
Received 23 June 2011
deregulation of TCF/LEF target genes. Of all these target genes, the transcription factor c-MYC
Revised version received 29 July 2011
appears the most critical. In this review we will discuss the interplay of Wnt and c-MYC signaling
Accepted 1 August 2011
during intestinal homeostasis and transformation. Furthermore, we will discuss recent data
Available online 7 August 2011
showing that further deregulation of c-MYC levels during colorectal carcinogenesis may drive tumor progression. Moreover, understanding these additional control mechanisms may allow
Keywords:
targeting of c-MYC during colorectal carcinogenesis.
APC
© 2011 Elsevier Inc. All rights reserved.
c-MYC Colorectal cancer Wnt Initiation Progression
Contents Introduction . . . . . . . . . . . . . . . . . . . Wnt/Myc signaling in the intestinal epithelium . Pathways driven by c-Myc . . . . . . . . . . . . Post transcriptional control of c-MYC expression Conclusions . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . .
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Introduction The intestinal epithelium is a simple structure whose primary function is the absorption of nutrients. The epithelium is divided into 2 distinct structures, proliferative crypts which constantly produce intestinal cells and absorptive villi that absorb nutrients (Fig. 1A). The intestinal crypt consists of a small number of stem ⁎ Corresponding author. E-mail address: [email protected] (O.J. Sansom). 0014-4827/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2011.08.001
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cells that give rise to a population of transient amplifying cells. These cells divide and differentiate as they move up the crypt– villus axis generating the different intestinal lineages. As the cells leave the crypt, they stop dividing and are eventually shed from the villi tips. In mammals the epithelium is believed to turn over every 3–5 days with this shedding of cells at the villus tips balanced by production of new cells in the crypts [1]. A number of
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signaling pathways have been shown to be crucial for efficient turnover of the intestine including hedgehog, Notch, plateletderived growth factor, bone morphogenetic protein and Wnt/Myc. This review will focus on the role of Wnt/Myc signaling in normal intestinal homeostasis and in particular the pathogenic changes associated with intestinal transformation.
is perhaps unsurprising that aberrant Wnt signaling is involved in intestinal transformation. In fact, loss of APC is recognized as the key early event in the development of colorectal cancers (CRC). Up to 80% of sporadic CRCs have mutations within the APC gene and people carrying germline mutations within APC develop a CRC disposition syndrome: Familial Adenomatous Polyposis. A number of mouse models of CRC have demonstrated that inactivation of Apc alone can drive intestinal hyperplasia [10,11] and a classical murine model of sporadic intestinal neoplasia contains a truncating mutation in the Apc gene [12]. Additionally, mice in which one allele of β-catenin lacks the GSK-3β phosphorylation develop intestinal adenomas that display stabilized nuclear β-catenin [13]. Also, human cell culture models of colorectal cancer and human tumor samples display activation of Wnt signaling and upregulation of several Wnt target genes [14]. As outlined above, the proliferative compartment of the small intestine is characterized by active Wnt signaling. The deletion of Apc in the small intestine leads to rapid enlargement of intestinal crypts as cells continue to proliferate, fail to differentiate and no longer migrate up the crypt–villus axis. The knockout tissue displays constitutive Wnt signaling that is characterized by high levels of nuclear β-catenin and overexpression of Wnt target genes such as c-Myc [11] (Fig. 1B). c-Myc is a pleiotropic transcription factor of the basic helix-loop-helix-leucine zipper family that regulates the expression of target genes involved in diverse cellular processes such as apoptosis, cell cycle progression, cell growth and DNA replication [15,16]. c-Myc is a promiscuous factor that is believed to bind to the promoter and regulate the expression of up to 15% of human genes [17,18]. c-Myc overexpression is commonly observed in human CRC samples indicating that overexpression of this Wnt target gene due to transcriptional deregulation may play a central role in intestinal transformation [19]. The control of c-Myc
Wnt/Myc signaling in the intestinal epithelium Wnt signaling proteins are highly conserved secreted signaling molecules that bind to cell surface Frizzled/LRP co-receptor complexes and activate canonical Wnt signaling [2]. In the absence of Wnt signaling β-catenin is bound to a destruction complex of adenomatous polyposis coli (APC), Axin, glycogen synthase kinase 3β (GSK-3β) and casein kinase 1 (CK1). β-catenin is sequentially phosphorylated by CK1 and GSK-3β, which leads to ubiquitination and subsequent proteolytic degradation. Activation of Wnt signaling by binding of Wnt ligand leads to dissociation of this complex and permits release and stabilization of β-catenin. Stabilized β-catenin can then enter the nucleus and via interaction with TCF/LEF transcription factors promote expression of Wnt target genes such as c-MYC, cyclin D2 and CD44 [1,2]. Wnt signaling is an essential component of intestinal homeostasis and is critical for efficient proliferation of cells within the crypt [3,4]. This is demonstrated in the accumulation of nuclear β-catenin in a number of cells at the base of the intestinal crypt [5,6]. Furthermore, key components of Wnt signaling such as Tcf4 and β-catenin are critical for maintenance of the proliferative cells within the intestinal crypt [4,7,8]. Also, a recent study has shown that Wnt ligand can strongly supplement the intestinal niche in ex vivo cultures of intestinal crypts [9]. Due to its crucial role in intestinal proliferation it
B
C
Villlus
A
APCmut Wnt activation
P53 mutation pERK
Crypt
c-MYC Wnt c-MYC
CDK4 Cyclin D2
FBXW7 miR-34c CIP2A HECTH9 c-MYC
Homeostasis
Hyperproliferation
Invasion / metastasis
Fig. 1 – A critical role for Wnt/Myc signaling during CRC progression. A. The normal intestinal epithelium consists of two distinct structures; the proliferative crypt and absorptive villus. Wnt signaling and c-MYC expression are observed in the crypt. B. Following APC mutation Wnt signaling is activated leading to increased c-MYC expression. TLR driven ERK activation may amplify this signal. Overexpression of c-MYC drives hyperproliferation in part through upregulation of cyclin D2 and CDK4. C. As tumors progress, P53 may be lost. This allows a further increase of c-MYC levels likely due to misregulation of a number of posttranscriptional control mechanisms. This may promote expression of a subset of c-MYC target genes that drive invasion and metastasis.
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expression in CRC has been a subject of much recent debate. It was first identified as a Wnt target gene by He and colleagues who demonstrated that its promoter contains a Wnt responsive element (WRE) [20]. Further studies have expanded our understanding of cMyc regulation by Wnt signaling. In particular, recent work has suggested that β-catenin dependent intrachromosomal interactions between WREs in the c-MYC locus control its expression [21]. Furthermore, the c-MYC enhancer region 8q24 was identified as a susceptibility locus for CRC and has recently been shown to interact with the c-MYC locus in CRC [22,23]. However, whether this interaction actually impacts on expression is unclear. If it does, it suggests that the slightly increased levels of c-MYC may be associated with increased risk of CRC development. The functional role of c-Myc in the small intestine has been examined using conditional deletion models [24,25]. These studies indicated that over the short term c-Myc is dispensable for intestinal enterocyte survival. However, the study by Muncan and colleagues demonstrated that c-Myc deficient intestinal cells proliferate slower than wild-type cells and are progressively lost over the longer term [25]. More recently, transient overexpression of a dominant negative c-Myc protein (‘omo-myc’) which blocks transcriptional activation by Myc proteins also led to reduced intestinal proliferation [26]. Although not absolutely required for intestinal homeostasis, we have demonstrated that c-Myc contributes to all the phenotypes associated with Apc loss in the small intestine [27]. Remarkably, codeletion of Apc and c-Myc gave rise to cells that proliferated, differentiated and migrated as wild-type. We were able to show that this effect is downstream of constitutive Wnt signaling as β-catenin was localized to the nuclei of double deficient cells. Expression profiling of Apc c-Myc double deficient intestines indicated that around 1/3 of genes upregulated following loss of Apc require c-Myc for their expression. This indicates that c-Myc may be the primary driving force of intestinal hyperplasia following Wnt activation. Supporting this is the finding that reduction of c-Myc levels by 50% attenuates the phenotypes of Apc loss and reduces intestinal tumorigenesis [28]. Moreover, downregulation of Myb which is commonly overexpressed in intestinal adenomas suppressed tumorigenesis due to a downregulation of c-Myc [29]. One question that is raised by these studies is whether increased c-Myc expression would be sufficient to drive the phenotypes of Apc loss. It should be noted that c-MYC is not amplified in CRC and thus one would expect that it would be cooperation between Wnt and c-Myc signaling that would confer the intestinal progenitor phenotype caused by Apc gene deletion. Two studies have addressed this. The first aimed to induce additional low levels of c-Myc in adult tissues. This was achieved by expression of exogenous c-Myc from the Rosa26 locus (which is ubiquitously expressed at a relatively low level). In most tissues, this relatively low level of c-Myc expression caused increased proliferation. However, within the colorectal epithelium where Rosa26 is expressed slightly more highly, increased apoptosis was also observed, though no other changes in intestinal homeostasis were noted [30]. A second study aimed to express very high levels of c-Myc and see if this is sufficient for transformation and found that this level of c-Myc could drive enterocyte hyperproliferation [31]. However, this study found other aspects of Apc loss such as paneth cell mislocalization were not driven by c-Myc over expression. Also, the authors observed induction of the Arf/p53 tumor suppressor pathway that does not occur following deregula-
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tion of Wnt signaling. This may indicate that other Wnt activated pathways are required to fully drive the phenotypes of Apc loss. As c-Myc levels following Apc loss are only increased approximately four-fold we believe that cooperation with nuclear β-catenin is important for induction of the full complement of intestinal Wnt target genes. This is consistent with a model where c-Myc binding sites are found within a plethora of target genes within the genome, however overexpression of c-Myc alone is not sufficient to drive transcription of these genes unless at very high, non physiological levels. One key question that still remains is whether endogenous c-Myc is required for the survival of established tumors. This has been addressed in other systems such as lung and pancreatic tumors, where inhibition of endogenous c-Myc caused tumor regression and similar work needs to be performed in intestinal cells lacking Apc [26,32]. The intestinal epithelium is a relatively exposed tissue that is commonly damaged by external factors. As such it displays a remarkable capacity to regenerate following injury induced by DNA damage, surgical resection or deletion of genes essential for intestinal homeostasis. Intestinal regeneration is characterized by increased proliferation of cells within the crypt that display Wnt activation and high levels of c-Myc expression [4,25]. We have recently characterized the role of Wnt/Myc signaling in intestinal regeneration and have observed that this process shares several characteristics with neoplastic transformation. By conditionally deleting c-Myc within the small intestine we were able to demonstrate an absolute requirement for it in intestinal regeneration following gamma irradiation [33]. Moreover, we were able to demonstrate that the precise levels of c-Myc are critical for this process as c-Myc cDNA targeted to the Rosa26 locus (and hence at near physiological levels though not upregulatable by Wnt signaling during regeneration) was unable to rescue the failed regeneration. That physiological regeneration and neoplastic transformation share the same signaling pathways is further emphasized by the demonstration that FAK signaling, which is activated downstream of c-Myc and drives AKT/ mTOR signaling, is also required for efficient hyperproliferation and tumorigenesis following Apc loss [33]. Furthermore, studies have shown that the mTOR inhibitor rapamycin can suppress intestinal tumorigenesis in ApcMin/+ mice highlighting the importance of this signaling pathway for Wnt driven transformation [34]. This interesting finding suggests that the key signaling components of intestinal transformation are pathways important for normal physiological processes ‘hijacked’ by constitutive Wnt/Myc signaling.
Pathways driven by c-Myc It is tempting to speculate that the activation of Wnt/Myc signaling that accompanies Apc loss drives the activation of numerous signaling pathways that may be susceptible to therapeutic targeting. By co-deletion of Apc and c-Myc in the small intestine we defined Wnt driven gene expression into 2 subsets: c-Myc dependent and c-Myc independent. As c-Myc and downstream signaling pathways are required to drive the phenotypes of Apc loss this raises the question of what contributions the transcriptional targets of c-Myc play in this process. One key process that we identified as being regulated by c-Myc was that of the cell cycle in particular cyclin D2 and cyclin dependant kinase 4 (cdk4) (Fig. 1B). Cyclin proteins, in complex with cdk partners sequentially phosphorylate and inactivate retinoblastoma protein during cell cycle progression. They are
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important components of the cell cycle machinery that are commonly deregulated in human cancer [35]. As it has been shown that the cyclin D2-cdk4/6 complex is an important mediator of c-Myc signaling [36] we have recently sought to examine the role of this complex downstream of c-Myc in intestinal tumorigenesis [37]. In this study we demonstrated that in the intestine, cyclin D2 and cdk4overexpression following Apc loss depends on c-Myc expression. Also, the hyperproliferation that accompanies Apc loss is partially dependent on expression of cyclin D2 and tumor growth and development in ApcMin/+ mice is strongly reliant on cyclin D2. From a pharmacological perspective, inhibition of cdk4/6 strongly suppressed the proliferation of adenomatous cells. This study provides a proof of principle approach that pathways activated by c-MYC may be viable targets for the prevention of CRC. As well as activating the expression of genes involved in driving cell cycle progression, c-MYC has been shown to repress expression of the cell cycle inhibitor P21[38]. Acting in concert with MIZ-1, c-MYC can bind to the P21 promoter and directly block its transcription. Thus, an important function of c-MYC overexpression in CRC may be to drive proliferation and prevent cellular differentiation via repression of P21.
Post transcriptional control of c-MYC expression As it is such a strong modifier of cellular fate it is unsurprising that cells have evolved numerous ways to fine tune c-MYC expression. c-MYC expression is controlled at transcriptional, translational and post-translational levels. In this part of the review we will discuss a number of recent developments into understanding the posttranscriptional control of c-MYC expression and their implications for therapeutic treatment of CRC. One mechanism employed to control the stability of c-MYC protein is the sequential phosphorylation and ubiquitinylation of c-MYC leading to its degradation. In an elegant series of experiments the Sears laboratory uncovered a role for phosphorylation of two residues in the N-terminal portion of c-MYC for this process [39]. To summarize, serine 62 phosphorylation by Rasactivated ERK permits the phosphorylation of threonine 58 by GSK-3β. This, in turn leads to isomerization of proline 63 by the Pin1 proline isomerase, which exposes S62 for dephosphorylation by PP2A. c-MYC phosphorylated solely at T58 is then targeted for ubiquitinylation and degradation. This phosphorylation pathway can be controlled by Ras signaling through both ERK activation and GSK-3β inhibition via PI3K/Akt. Thus Ras signaling can lead to stabilization and accumulation of c-MYC through control of its phosphorylation. Interestingly, point mutations of T58 are found in some cases of Burkitt's lymphoma and this mutation increases the transformation efficiency of c-MYC in vitro indicating that increased stability of c-MYC is oncogenic. One study has already suggested that ERK is required to stabilize c-Myc following Apc loss [40] (Fig. 1B). This study showed that ERK activation downstream of Toll-Like Receptor (TLR)MyD88 is required for c-Myc stability and treatment of ApcMin/+ mice with an ERK inhibitor suppressed intestinal polyposis. These findings complement an earlier study demonstrating a requirement for MyD88 in ApcMin/+ intestinal tumorigenesis [41] and indicate a link between intestinal flora and Myc signaling via ERK activation. Given K-RAS is commonly mutated during CRC progression and as K-RAS can itself activate ERK, it will be
interesting to see if ERK activation and c-MYC stability become independent of TLR/MyD88 in these tumors. This may also explain the context specific data of the role of c-Myc downstream of Apc loss in vivo. Our studies have shown that the hyperproliferation and hepatomegaly following Apc loss within the liver are β-catenin dependent but c-Myc independent [42]. In the intestine, microflora would activate TLR signaling and then provide a signal to stabilize c-Myc in the absence of Apc, however in the liver this signal would be missing and thus c-Myc activity may not be enhanced to equivalent levels as in the intestine. Consistent with this, many of the Wnt transcriptional targets that are c-Myc dependent in the intestine are c-Myc independent in the liver [42]. Furthermore, it is tempting to speculate that the presence of microflora activated TLR signaling may explain why germline mutations of APC give rise predominantly to intestinal tumors. Deregulation of a number of other proteins that would result in stabilization of c-MYC during colorectal cancer progression have been identified. One of these is a protein termed cancerous inhibitor of PP2A (CIP2A) that was identified by mass spectrometry analysis of c-MYC interacting proteins [43]. This protein was shown to stabilize c-MYC and induce transformation in a manner similar to T58 c-MYC mutants, indicative of a role in inhibition of dephosphorylation of S62 [39]. CIP2A has been shown to be overexpressed in a number of human malignancies including colon cancer, gastric cancer [44] and is associated with breast cancer progression [45]. Following phosphorylation, c-MYC is subject to ubiquitinylation and proteosome mediated degradation. The ubiquitin ligase F-Box and WD repeat containing domain 7 (FBXW7) has been identified as being required for c-MYC ubiquitinylation [46,47]. FBXW7 is a known tumor suppressor and its ubiquitinylation of c-MYC depends on T58 phosphorylation. Thus, it is likely an important player in phosphorylation dependent degradation of c-MYC. Interestingly, 2 recent studies have shown that loss of Fbxw7 rapidly accelerates intestinal tumorigenesis in the ApcMin/+ mouse and that it is mutated in human CRC [48,49]. Whether this is down to its role in controlling c-MYC expression is a matter of some debate as FBXW7 also affects a number of other pathways such as Notch, Jun and DEK. The ubiquitinylation of c-MYC is subject to an additional level of control in the form of a ubiquitin specific protease termed USP28 [50]. This protein binds to c-MYC through its interaction with FBXW7, leading to its deubiquitination and stabilization. USP28 appears to be important for c-MYC driven proliferation and is overexpressed in colon and breast carcinomas. Together, these data argue of the important role control of c-MYC protein levels via phosphorylation mediated ubiquitinylation plays in CRC. HECTH9/HUWE1 is another ubiquitin ligase that plays a role in regulating c-MYC function although its role is somewhat controversial. The Eilers laboratory identified HECTH9 as a novel c-MYC interacting protein [51]. They found that HECTH9 catalyzed ligation of K-63 ubiquitin chains onto c-MYC and that it is required for activation of a subset of c-MYC target genes. Mechanistically, they proposed that this ubiquitinylation is required for recruitment of the acetyl transferase p300 to c-MYC bound promoters. They also found HECTH9 to be overexpressed in colonic tumors. Thus they argue that HECTH9 is required for c-MYC function and that its inhibition should in turn compromise the function of c-MYC and halt proliferation in cancerous tissue. In a separate study Zhao and colleagues identified HUWE1 as an interaction partner of n-MYC in neuronal cells that catalyzes K-48 linked ubiquitinylation [52]. This ubiquitinylation leads to proteosomal degradation of n-MYC. Functionally, they
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report that HUWE1 mediated degradation of n-MYC is important for neuronal differentiation with this function inhibited in cells lacking HUWE1. These 2 differing reports would have profound implications for targeting HUWE1 as a means of disrupting MYC function. If, as appears likely, HUWE1 has strikingly different roles in MYC biology in different cellular circumstances, then the effects of inhibition would be expected to be markedly different in them also. Further functional in vivo analysis should allow us to resolve whether HECTH9/HUWE1 inhibition will be efficacious for CRC. In addition to the control of c-MYC protein stability, a pathway that controls translational stability of c-MYC is also altered in CRC. Members of the miR-34 family of microRNAs were initially identified as direct transcriptional targets of p53. The family consists of 3 members, 34a, 34b and 34c and all have anti-proliferative capacity. It has recently been reported that c-MYC translation is negatively regulated by miR-34b/c in response to DNA damage and this may prevent replication of damaged DNA [53]. A recent study has uncovered an elegant feedback loop whereby the expression of miR-34b/c is controlled by MK5-phosphorylated FOXO3a. The expression of MK5 in turn is regulated by c-MYC, thus c-MYC levels can be precisely regulated via translational inhibition by miR-34b/c [54]. The authors provide evidence that this feedback loop is disrupted during progression of CRC and that MK5 downregulation may accompany tumor metastasis. Moreover, it has been suggested that 30–90% of CRC samples demonstrate miR-34b/c promoter methylation [55] indicating that silencing of this pathway may be important to the progression of CRC. A further observation made in the study of Kress and colleagues [54] was that in cells lacking P53, c-MYC was induced even more dramatically following downregulation of components of this feedback loop. The authors suggest this may lead to expression of a subset of c-MYC target genes that drive tumor progression. Interestingly, this complements a recent study that highlighted clear thresholds of c-Myc activity in vivo [27]. In this study it was demonstrated that high levels of c-Myc lead to apoptosis in normal tissue by induction of Arf/p53 tumor suppressor pathway whereas slightly lower levels are tumorigenic. These studies indicate that loss of P53 may be permissive to allow maximal c-MYC expression and inhibition of this feedback loop may allow accumulation of c-MYC during tumor progression. The identification of this pathway, and the silencing of miR-34b/c in CRC imply an important tumor suppressive role for this micro-RNA. This may also raise an opportunity for reintroduction of miR-34b/c within CRC to inhibit c-MYC function. Importantly, the growth of non small cell lung carcinoma xenografts has been shown to be suppressed by systemic treatment of mice with liposome packaged miR-34a suggesting this might be therapeutically achievable [56].
Conclusions In summary, there is now strong evidence that increased c-MYC expression is a key component of Wnt signaling downstream of APC loss. This is coordinated at multiple levels including transcription, translation and protein stability (Fig. 1). Cooperation of Wnt/Myc signaling is central for the initiation of CRC. During CRC progression further deregulation of c-MYC occurs and mutations in K-RAS and P53 may allow increased levels of c-MYC activity (Fig. 1C). This has been proposed to drive a subset of c-MYC target genes important for invasion, angiogenesis and metastasis [32,57]. As yet it is unclear whether this increased
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activation of c-MYC correlates with increased Wnt signaling. To this end, β-catenin/TCF4 signaling is also proposed to be further deregulated during colorectal cancer, particularly at invasive fronts and where cells have undergone EMT. Further studies should allow us to elucidate the relative functional importance of the plethora of c-MYC regulators and whether any of this will allow us to target c-MYC function in CRC.
Acknowledgments Owen Sansom is supported by CR-UK and Kevin Myant is supported by AICR. Thanks to Jen Morton for comments.
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available at www.sciencedirect.com
www.elsevier.com/locate/yexcr
Review Article
Wnt/Myc interactions in intestinal cancer: Partners in crime Kevin Myant, Owen J. Sansom⁎ Beatson Institute for Cancer Research, Garscube Estate, Switchback Road, Glasgow, Scotland G61 1BD, UK
A R T I C L E I N F O R M A T I O N
A B S T R A C T
Article Chronology:
Loss of the APC (adenomatous polyposis coli) gene in colorectal cancer leads to a rapid
Received 23 June 2011
deregulation of TCF/LEF target genes. Of all these target genes, the transcription factor c-MYC
Revised version received 29 July 2011
appears the most critical. In this review we will discuss the interplay of Wnt and c-MYC signaling
Accepted 1 August 2011
during intestinal homeostasis and transformation. Furthermore, we will discuss recent data
Available online 7 August 2011
showing that further deregulation of c-MYC levels during colorectal carcinogenesis may drive tumor progression. Moreover, understanding these additional control mechanisms may allow
Keywords:
targeting of c-MYC during colorectal carcinogenesis.
APC
© 2011 Elsevier Inc. All rights reserved.
c-MYC Colorectal cancer Wnt Initiation Progression
Contents Introduction . . . . . . . . . . . . . . . . . . . Wnt/Myc signaling in the intestinal epithelium . Pathways driven by c-Myc . . . . . . . . . . . . Post transcriptional control of c-MYC expression Conclusions . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . .
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Introduction The intestinal epithelium is a simple structure whose primary function is the absorption of nutrients. The epithelium is divided into 2 distinct structures, proliferative crypts which constantly produce intestinal cells and absorptive villi that absorb nutrients (Fig. 1A). The intestinal crypt consists of a small number of stem ⁎ Corresponding author. E-mail address: [email protected] (O.J. Sansom). 0014-4827/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2011.08.001
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cells that give rise to a population of transient amplifying cells. These cells divide and differentiate as they move up the crypt– villus axis generating the different intestinal lineages. As the cells leave the crypt, they stop dividing and are eventually shed from the villi tips. In mammals the epithelium is believed to turn over every 3–5 days with this shedding of cells at the villus tips balanced by production of new cells in the crypts [1]. A number of
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signaling pathways have been shown to be crucial for efficient turnover of the intestine including hedgehog, Notch, plateletderived growth factor, bone morphogenetic protein and Wnt/Myc. This review will focus on the role of Wnt/Myc signaling in normal intestinal homeostasis and in particular the pathogenic changes associated with intestinal transformation.
is perhaps unsurprising that aberrant Wnt signaling is involved in intestinal transformation. In fact, loss of APC is recognized as the key early event in the development of colorectal cancers (CRC). Up to 80% of sporadic CRCs have mutations within the APC gene and people carrying germline mutations within APC develop a CRC disposition syndrome: Familial Adenomatous Polyposis. A number of mouse models of CRC have demonstrated that inactivation of Apc alone can drive intestinal hyperplasia [10,11] and a classical murine model of sporadic intestinal neoplasia contains a truncating mutation in the Apc gene [12]. Additionally, mice in which one allele of β-catenin lacks the GSK-3β phosphorylation develop intestinal adenomas that display stabilized nuclear β-catenin [13]. Also, human cell culture models of colorectal cancer and human tumor samples display activation of Wnt signaling and upregulation of several Wnt target genes [14]. As outlined above, the proliferative compartment of the small intestine is characterized by active Wnt signaling. The deletion of Apc in the small intestine leads to rapid enlargement of intestinal crypts as cells continue to proliferate, fail to differentiate and no longer migrate up the crypt–villus axis. The knockout tissue displays constitutive Wnt signaling that is characterized by high levels of nuclear β-catenin and overexpression of Wnt target genes such as c-Myc [11] (Fig. 1B). c-Myc is a pleiotropic transcription factor of the basic helix-loop-helix-leucine zipper family that regulates the expression of target genes involved in diverse cellular processes such as apoptosis, cell cycle progression, cell growth and DNA replication [15,16]. c-Myc is a promiscuous factor that is believed to bind to the promoter and regulate the expression of up to 15% of human genes [17,18]. c-Myc overexpression is commonly observed in human CRC samples indicating that overexpression of this Wnt target gene due to transcriptional deregulation may play a central role in intestinal transformation [19]. The control of c-Myc
Wnt/Myc signaling in the intestinal epithelium Wnt signaling proteins are highly conserved secreted signaling molecules that bind to cell surface Frizzled/LRP co-receptor complexes and activate canonical Wnt signaling [2]. In the absence of Wnt signaling β-catenin is bound to a destruction complex of adenomatous polyposis coli (APC), Axin, glycogen synthase kinase 3β (GSK-3β) and casein kinase 1 (CK1). β-catenin is sequentially phosphorylated by CK1 and GSK-3β, which leads to ubiquitination and subsequent proteolytic degradation. Activation of Wnt signaling by binding of Wnt ligand leads to dissociation of this complex and permits release and stabilization of β-catenin. Stabilized β-catenin can then enter the nucleus and via interaction with TCF/LEF transcription factors promote expression of Wnt target genes such as c-MYC, cyclin D2 and CD44 [1,2]. Wnt signaling is an essential component of intestinal homeostasis and is critical for efficient proliferation of cells within the crypt [3,4]. This is demonstrated in the accumulation of nuclear β-catenin in a number of cells at the base of the intestinal crypt [5,6]. Furthermore, key components of Wnt signaling such as Tcf4 and β-catenin are critical for maintenance of the proliferative cells within the intestinal crypt [4,7,8]. Also, a recent study has shown that Wnt ligand can strongly supplement the intestinal niche in ex vivo cultures of intestinal crypts [9]. Due to its crucial role in intestinal proliferation it
B
C
Villlus
A
APCmut Wnt activation
P53 mutation pERK
Crypt
c-MYC Wnt c-MYC
CDK4 Cyclin D2
FBXW7 miR-34c CIP2A HECTH9 c-MYC
Homeostasis
Hyperproliferation
Invasion / metastasis
Fig. 1 – A critical role for Wnt/Myc signaling during CRC progression. A. The normal intestinal epithelium consists of two distinct structures; the proliferative crypt and absorptive villus. Wnt signaling and c-MYC expression are observed in the crypt. B. Following APC mutation Wnt signaling is activated leading to increased c-MYC expression. TLR driven ERK activation may amplify this signal. Overexpression of c-MYC drives hyperproliferation in part through upregulation of cyclin D2 and CDK4. C. As tumors progress, P53 may be lost. This allows a further increase of c-MYC levels likely due to misregulation of a number of posttranscriptional control mechanisms. This may promote expression of a subset of c-MYC target genes that drive invasion and metastasis.
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expression in CRC has been a subject of much recent debate. It was first identified as a Wnt target gene by He and colleagues who demonstrated that its promoter contains a Wnt responsive element (WRE) [20]. Further studies have expanded our understanding of cMyc regulation by Wnt signaling. In particular, recent work has suggested that β-catenin dependent intrachromosomal interactions between WREs in the c-MYC locus control its expression [21]. Furthermore, the c-MYC enhancer region 8q24 was identified as a susceptibility locus for CRC and has recently been shown to interact with the c-MYC locus in CRC [22,23]. However, whether this interaction actually impacts on expression is unclear. If it does, it suggests that the slightly increased levels of c-MYC may be associated with increased risk of CRC development. The functional role of c-Myc in the small intestine has been examined using conditional deletion models [24,25]. These studies indicated that over the short term c-Myc is dispensable for intestinal enterocyte survival. However, the study by Muncan and colleagues demonstrated that c-Myc deficient intestinal cells proliferate slower than wild-type cells and are progressively lost over the longer term [25]. More recently, transient overexpression of a dominant negative c-Myc protein (‘omo-myc’) which blocks transcriptional activation by Myc proteins also led to reduced intestinal proliferation [26]. Although not absolutely required for intestinal homeostasis, we have demonstrated that c-Myc contributes to all the phenotypes associated with Apc loss in the small intestine [27]. Remarkably, codeletion of Apc and c-Myc gave rise to cells that proliferated, differentiated and migrated as wild-type. We were able to show that this effect is downstream of constitutive Wnt signaling as β-catenin was localized to the nuclei of double deficient cells. Expression profiling of Apc c-Myc double deficient intestines indicated that around 1/3 of genes upregulated following loss of Apc require c-Myc for their expression. This indicates that c-Myc may be the primary driving force of intestinal hyperplasia following Wnt activation. Supporting this is the finding that reduction of c-Myc levels by 50% attenuates the phenotypes of Apc loss and reduces intestinal tumorigenesis [28]. Moreover, downregulation of Myb which is commonly overexpressed in intestinal adenomas suppressed tumorigenesis due to a downregulation of c-Myc [29]. One question that is raised by these studies is whether increased c-Myc expression would be sufficient to drive the phenotypes of Apc loss. It should be noted that c-MYC is not amplified in CRC and thus one would expect that it would be cooperation between Wnt and c-Myc signaling that would confer the intestinal progenitor phenotype caused by Apc gene deletion. Two studies have addressed this. The first aimed to induce additional low levels of c-Myc in adult tissues. This was achieved by expression of exogenous c-Myc from the Rosa26 locus (which is ubiquitously expressed at a relatively low level). In most tissues, this relatively low level of c-Myc expression caused increased proliferation. However, within the colorectal epithelium where Rosa26 is expressed slightly more highly, increased apoptosis was also observed, though no other changes in intestinal homeostasis were noted [30]. A second study aimed to express very high levels of c-Myc and see if this is sufficient for transformation and found that this level of c-Myc could drive enterocyte hyperproliferation [31]. However, this study found other aspects of Apc loss such as paneth cell mislocalization were not driven by c-Myc over expression. Also, the authors observed induction of the Arf/p53 tumor suppressor pathway that does not occur following deregula-
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tion of Wnt signaling. This may indicate that other Wnt activated pathways are required to fully drive the phenotypes of Apc loss. As c-Myc levels following Apc loss are only increased approximately four-fold we believe that cooperation with nuclear β-catenin is important for induction of the full complement of intestinal Wnt target genes. This is consistent with a model where c-Myc binding sites are found within a plethora of target genes within the genome, however overexpression of c-Myc alone is not sufficient to drive transcription of these genes unless at very high, non physiological levels. One key question that still remains is whether endogenous c-Myc is required for the survival of established tumors. This has been addressed in other systems such as lung and pancreatic tumors, where inhibition of endogenous c-Myc caused tumor regression and similar work needs to be performed in intestinal cells lacking Apc [26,32]. The intestinal epithelium is a relatively exposed tissue that is commonly damaged by external factors. As such it displays a remarkable capacity to regenerate following injury induced by DNA damage, surgical resection or deletion of genes essential for intestinal homeostasis. Intestinal regeneration is characterized by increased proliferation of cells within the crypt that display Wnt activation and high levels of c-Myc expression [4,25]. We have recently characterized the role of Wnt/Myc signaling in intestinal regeneration and have observed that this process shares several characteristics with neoplastic transformation. By conditionally deleting c-Myc within the small intestine we were able to demonstrate an absolute requirement for it in intestinal regeneration following gamma irradiation [33]. Moreover, we were able to demonstrate that the precise levels of c-Myc are critical for this process as c-Myc cDNA targeted to the Rosa26 locus (and hence at near physiological levels though not upregulatable by Wnt signaling during regeneration) was unable to rescue the failed regeneration. That physiological regeneration and neoplastic transformation share the same signaling pathways is further emphasized by the demonstration that FAK signaling, which is activated downstream of c-Myc and drives AKT/ mTOR signaling, is also required for efficient hyperproliferation and tumorigenesis following Apc loss [33]. Furthermore, studies have shown that the mTOR inhibitor rapamycin can suppress intestinal tumorigenesis in ApcMin/+ mice highlighting the importance of this signaling pathway for Wnt driven transformation [34]. This interesting finding suggests that the key signaling components of intestinal transformation are pathways important for normal physiological processes ‘hijacked’ by constitutive Wnt/Myc signaling.
Pathways driven by c-Myc It is tempting to speculate that the activation of Wnt/Myc signaling that accompanies Apc loss drives the activation of numerous signaling pathways that may be susceptible to therapeutic targeting. By co-deletion of Apc and c-Myc in the small intestine we defined Wnt driven gene expression into 2 subsets: c-Myc dependent and c-Myc independent. As c-Myc and downstream signaling pathways are required to drive the phenotypes of Apc loss this raises the question of what contributions the transcriptional targets of c-Myc play in this process. One key process that we identified as being regulated by c-Myc was that of the cell cycle in particular cyclin D2 and cyclin dependant kinase 4 (cdk4) (Fig. 1B). Cyclin proteins, in complex with cdk partners sequentially phosphorylate and inactivate retinoblastoma protein during cell cycle progression. They are
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important components of the cell cycle machinery that are commonly deregulated in human cancer [35]. As it has been shown that the cyclin D2-cdk4/6 complex is an important mediator of c-Myc signaling [36] we have recently sought to examine the role of this complex downstream of c-Myc in intestinal tumorigenesis [37]. In this study we demonstrated that in the intestine, cyclin D2 and cdk4overexpression following Apc loss depends on c-Myc expression. Also, the hyperproliferation that accompanies Apc loss is partially dependent on expression of cyclin D2 and tumor growth and development in ApcMin/+ mice is strongly reliant on cyclin D2. From a pharmacological perspective, inhibition of cdk4/6 strongly suppressed the proliferation of adenomatous cells. This study provides a proof of principle approach that pathways activated by c-MYC may be viable targets for the prevention of CRC. As well as activating the expression of genes involved in driving cell cycle progression, c-MYC has been shown to repress expression of the cell cycle inhibitor P21[38]. Acting in concert with MIZ-1, c-MYC can bind to the P21 promoter and directly block its transcription. Thus, an important function of c-MYC overexpression in CRC may be to drive proliferation and prevent cellular differentiation via repression of P21.
Post transcriptional control of c-MYC expression As it is such a strong modifier of cellular fate it is unsurprising that cells have evolved numerous ways to fine tune c-MYC expression. c-MYC expression is controlled at transcriptional, translational and post-translational levels. In this part of the review we will discuss a number of recent developments into understanding the posttranscriptional control of c-MYC expression and their implications for therapeutic treatment of CRC. One mechanism employed to control the stability of c-MYC protein is the sequential phosphorylation and ubiquitinylation of c-MYC leading to its degradation. In an elegant series of experiments the Sears laboratory uncovered a role for phosphorylation of two residues in the N-terminal portion of c-MYC for this process [39]. To summarize, serine 62 phosphorylation by Rasactivated ERK permits the phosphorylation of threonine 58 by GSK-3β. This, in turn leads to isomerization of proline 63 by the Pin1 proline isomerase, which exposes S62 for dephosphorylation by PP2A. c-MYC phosphorylated solely at T58 is then targeted for ubiquitinylation and degradation. This phosphorylation pathway can be controlled by Ras signaling through both ERK activation and GSK-3β inhibition via PI3K/Akt. Thus Ras signaling can lead to stabilization and accumulation of c-MYC through control of its phosphorylation. Interestingly, point mutations of T58 are found in some cases of Burkitt's lymphoma and this mutation increases the transformation efficiency of c-MYC in vitro indicating that increased stability of c-MYC is oncogenic. One study has already suggested that ERK is required to stabilize c-Myc following Apc loss [40] (Fig. 1B). This study showed that ERK activation downstream of Toll-Like Receptor (TLR)MyD88 is required for c-Myc stability and treatment of ApcMin/+ mice with an ERK inhibitor suppressed intestinal polyposis. These findings complement an earlier study demonstrating a requirement for MyD88 in ApcMin/+ intestinal tumorigenesis [41] and indicate a link between intestinal flora and Myc signaling via ERK activation. Given K-RAS is commonly mutated during CRC progression and as K-RAS can itself activate ERK, it will be
interesting to see if ERK activation and c-MYC stability become independent of TLR/MyD88 in these tumors. This may also explain the context specific data of the role of c-Myc downstream of Apc loss in vivo. Our studies have shown that the hyperproliferation and hepatomegaly following Apc loss within the liver are β-catenin dependent but c-Myc independent [42]. In the intestine, microflora would activate TLR signaling and then provide a signal to stabilize c-Myc in the absence of Apc, however in the liver this signal would be missing and thus c-Myc activity may not be enhanced to equivalent levels as in the intestine. Consistent with this, many of the Wnt transcriptional targets that are c-Myc dependent in the intestine are c-Myc independent in the liver [42]. Furthermore, it is tempting to speculate that the presence of microflora activated TLR signaling may explain why germline mutations of APC give rise predominantly to intestinal tumors. Deregulation of a number of other proteins that would result in stabilization of c-MYC during colorectal cancer progression have been identified. One of these is a protein termed cancerous inhibitor of PP2A (CIP2A) that was identified by mass spectrometry analysis of c-MYC interacting proteins [43]. This protein was shown to stabilize c-MYC and induce transformation in a manner similar to T58 c-MYC mutants, indicative of a role in inhibition of dephosphorylation of S62 [39]. CIP2A has been shown to be overexpressed in a number of human malignancies including colon cancer, gastric cancer [44] and is associated with breast cancer progression [45]. Following phosphorylation, c-MYC is subject to ubiquitinylation and proteosome mediated degradation. The ubiquitin ligase F-Box and WD repeat containing domain 7 (FBXW7) has been identified as being required for c-MYC ubiquitinylation [46,47]. FBXW7 is a known tumor suppressor and its ubiquitinylation of c-MYC depends on T58 phosphorylation. Thus, it is likely an important player in phosphorylation dependent degradation of c-MYC. Interestingly, 2 recent studies have shown that loss of Fbxw7 rapidly accelerates intestinal tumorigenesis in the ApcMin/+ mouse and that it is mutated in human CRC [48,49]. Whether this is down to its role in controlling c-MYC expression is a matter of some debate as FBXW7 also affects a number of other pathways such as Notch, Jun and DEK. The ubiquitinylation of c-MYC is subject to an additional level of control in the form of a ubiquitin specific protease termed USP28 [50]. This protein binds to c-MYC through its interaction with FBXW7, leading to its deubiquitination and stabilization. USP28 appears to be important for c-MYC driven proliferation and is overexpressed in colon and breast carcinomas. Together, these data argue of the important role control of c-MYC protein levels via phosphorylation mediated ubiquitinylation plays in CRC. HECTH9/HUWE1 is another ubiquitin ligase that plays a role in regulating c-MYC function although its role is somewhat controversial. The Eilers laboratory identified HECTH9 as a novel c-MYC interacting protein [51]. They found that HECTH9 catalyzed ligation of K-63 ubiquitin chains onto c-MYC and that it is required for activation of a subset of c-MYC target genes. Mechanistically, they proposed that this ubiquitinylation is required for recruitment of the acetyl transferase p300 to c-MYC bound promoters. They also found HECTH9 to be overexpressed in colonic tumors. Thus they argue that HECTH9 is required for c-MYC function and that its inhibition should in turn compromise the function of c-MYC and halt proliferation in cancerous tissue. In a separate study Zhao and colleagues identified HUWE1 as an interaction partner of n-MYC in neuronal cells that catalyzes K-48 linked ubiquitinylation [52]. This ubiquitinylation leads to proteosomal degradation of n-MYC. Functionally, they
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report that HUWE1 mediated degradation of n-MYC is important for neuronal differentiation with this function inhibited in cells lacking HUWE1. These 2 differing reports would have profound implications for targeting HUWE1 as a means of disrupting MYC function. If, as appears likely, HUWE1 has strikingly different roles in MYC biology in different cellular circumstances, then the effects of inhibition would be expected to be markedly different in them also. Further functional in vivo analysis should allow us to resolve whether HECTH9/HUWE1 inhibition will be efficacious for CRC. In addition to the control of c-MYC protein stability, a pathway that controls translational stability of c-MYC is also altered in CRC. Members of the miR-34 family of microRNAs were initially identified as direct transcriptional targets of p53. The family consists of 3 members, 34a, 34b and 34c and all have anti-proliferative capacity. It has recently been reported that c-MYC translation is negatively regulated by miR-34b/c in response to DNA damage and this may prevent replication of damaged DNA [53]. A recent study has uncovered an elegant feedback loop whereby the expression of miR-34b/c is controlled by MK5-phosphorylated FOXO3a. The expression of MK5 in turn is regulated by c-MYC, thus c-MYC levels can be precisely regulated via translational inhibition by miR-34b/c [54]. The authors provide evidence that this feedback loop is disrupted during progression of CRC and that MK5 downregulation may accompany tumor metastasis. Moreover, it has been suggested that 30–90% of CRC samples demonstrate miR-34b/c promoter methylation [55] indicating that silencing of this pathway may be important to the progression of CRC. A further observation made in the study of Kress and colleagues [54] was that in cells lacking P53, c-MYC was induced even more dramatically following downregulation of components of this feedback loop. The authors suggest this may lead to expression of a subset of c-MYC target genes that drive tumor progression. Interestingly, this complements a recent study that highlighted clear thresholds of c-Myc activity in vivo [27]. In this study it was demonstrated that high levels of c-Myc lead to apoptosis in normal tissue by induction of Arf/p53 tumor suppressor pathway whereas slightly lower levels are tumorigenic. These studies indicate that loss of P53 may be permissive to allow maximal c-MYC expression and inhibition of this feedback loop may allow accumulation of c-MYC during tumor progression. The identification of this pathway, and the silencing of miR-34b/c in CRC imply an important tumor suppressive role for this micro-RNA. This may also raise an opportunity for reintroduction of miR-34b/c within CRC to inhibit c-MYC function. Importantly, the growth of non small cell lung carcinoma xenografts has been shown to be suppressed by systemic treatment of mice with liposome packaged miR-34a suggesting this might be therapeutically achievable [56].
Conclusions In summary, there is now strong evidence that increased c-MYC expression is a key component of Wnt signaling downstream of APC loss. This is coordinated at multiple levels including transcription, translation and protein stability (Fig. 1). Cooperation of Wnt/Myc signaling is central for the initiation of CRC. During CRC progression further deregulation of c-MYC occurs and mutations in K-RAS and P53 may allow increased levels of c-MYC activity (Fig. 1C). This has been proposed to drive a subset of c-MYC target genes important for invasion, angiogenesis and metastasis [32,57]. As yet it is unclear whether this increased
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activation of c-MYC correlates with increased Wnt signaling. To this end, β-catenin/TCF4 signaling is also proposed to be further deregulated during colorectal cancer, particularly at invasive fronts and where cells have undergone EMT. Further studies should allow us to elucidate the relative functional importance of the plethora of c-MYC regulators and whether any of this will allow us to target c-MYC function in CRC.
Acknowledgments Owen Sansom is supported by CR-UK and Kevin Myant is supported by AICR. Thanks to Jen Morton for comments.
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