Delta-like 4/Notch signaling promotes Apc Min/ + tumor initiation through angiogenic and non-angiogenic related mechanisms
Activation of Notch pathway seems to promote intestinal tumorigenesis induced by Apc loss [13] and Dll4 is one of the Notch signaling pathway components found to be upregulated in these tumors [46]. Reports have shown that Dll4 inhibition delays the tumor growth by deregulating the tumor angiogenic process [24–27], but in CRC anti-DLL4 therapy may also reduce the cancer stem cell frequency [29]. Despite the advances in the understanding of Dll4/Notch signaling in cancer, most of the previous reports were focused on role of Dll4 in the tumor angiogenic process and further studies are still needed to unveil all the mechanisms by which Dll4 affects the tumor initiation and development in the gut.
Reports have shown that all Notch receptors, ligands and some of the Notch target genes are expressed in the normal gut [8, 37, 38]. However, in the Apc
Min/+ intestinal tumors their expression has been poorly described. A study indicated that expression of Notch receptors and ligands closely follows the expression in the normal crypts, while Hes1 expression was observed uniformly in the adenomas [8]. Other report showed that Jagged1 is overexpressed in the tumor tissue with concomitant Notch1 and 2 activation [39]. In the present work we analysed the protein expression pattern of most Notch pathway members in the Apc
Min/+ intestinal tumors compared with the normal WT gut. Regarding the previous gene expression analysis of Notch members in the normal gut [37, 38], we observed some differences in our analysis. These included the presence of Notch2 in the bottom of the large intestinal crypts, of Notch3 and 4 in the small and large intestinal epithelium and Hes1 not only in the small intestinal crypts [37], but diffusely expressed in the small and large intestine.
Our expression analysis in the Apc
Min/+ small and large intestinal adenomas confirmed that the Notch pathway is present and activated in intestinal adenomas harbouring Apc mutations [8, 13, 39, 46]. Dll4 and Jagged1 were more expressed in these tumors than the other members of this pathway. Comparing the adenomas with the normal WT gut we found that Dll1 and Dll3 lose their expression in the large intestine. We observed a different expression pattern of Dll4, all Notch receptors (with regional variation) and Hes5 in the tumor epithelium. The same Notch members and Hes1, instead of Hes5, seemed upregulated in the adenomas (Notch3 only in the large intestine). Thus our expression analysis indicates that in the Apc
Min/+ small and large intestinal adenomas, Dll4 is the most upregulated ligand and is present both in the tumor epithelium and endothelium.
In the normal gut Dll4 acts redundantly with Dll1 mediating the Notch signaling regulation of the intestinal stem cells proliferation and their commitment towards de secretory cell fate [6]. We found that Dll4 is expressed near the Lgr5+ stem cells also in the intestinal tumors, therefore indicating a possible role of this ligand in the maintenance of also the tumor stem cells. These stem cells are believed to be responsible for tumor initiation and progression [47, 48] and depend on proper angiogenesis to function and survive [22]. Therefore we intended to elucidate if Dll4 also regulates the fate of tumor stem cells beside its angiogenic effect in a spontaneous model of CRC, the Apc
Min/+ mouse. To address this question we compared ubiquitous with endothelial-specific Dll4 loss-of-function mouse mutants. Our results highlighted the importance of Dll4 angiogenic and epithelial effect during intestinal Apc
Min/+ tumor initiation and development rather than in maintaining the normal gut homeostasis. Pellegrinet el al. reported that in the normal gut, Dll4 intestinal epithelial-specific inhibition alone is not sufficient to promote a phenotype due to redundant Dll1-mediated Notch signaling [6]. This lack of intestinal effect after Dll4 inhibition can also be related to the fact that in the normal gut simultaneous inhibition of Notch1 and 2 is necessary to result in complete conversion of the crypt progenitors into postmitotic goblet cells [7] and it is not known whether Dll4 can activate both receptors in the gut. Nevertheless, our results show that Dll4 seems at least partially responsible for the known effects of Notch activation during intestinal tumorigenesis, as Dll4 ubiquitous deletion led to a similar, but less pronounced, epithelial phenotype as the pan-Notch/gamma-secretase inhibition in the Apc
Min/+ tumors [8]. However, as the alterations were moderated with no macroscopic repercussions (no observed increase of mucus secretion) in the ubiquitous Dll4 mutants’ gut, Dll1 may partially compensate the lack of Dll4 in this setting and/or Dll4 may not activate both Notch1 and 2 receptors. In addition, as we only analysed the Lgr5 and Bmi1 positive stem cell populations, it is not certain if this pathway can affect similarly all the stem cells present in the intestinal tumors.
We found that ubiquitous and endothelial-specific Dll4 blockade led to a similar phenotype in the small and large intestine, but a stronger effect on tumor initiation was observed in the small intestine and a greater impact on tumor growth was seen in the large intestine.
By comparing the ubiquitous mutants with the endothelial-specific knockouts we found that the observed epithelial phenotype is probably caused by the deregulation of the tumor angiogenesis but also by other important mechanisms. Both ubiquitous and endothelial-specific mutants had an equivalent angiogenic phenotype, with equally increased hypoxia and apoptosis leading to similar reduction of the tumor volume. Therefore, Dll4 deletion inhibited the intestinal tumor growth by inducing a dysfunctional and immature angiogenesis that led to hypoxia and therefore apoptosis as previously reported in other tumor models [24, 26, 27, 49].
The multiplicity of tumors was also reduced in the mutants relatively to their controls and this effect was more pronounced in the ubiquitous than in the endothelial-specific mutants, associated to a stronger reduction of tumor cell proliferation and tumor stem cell density in the first mutants. The stronger inhibitory effect on tumor cell proliferation through Dll4 ubiquitous deletion may have therefore prevented the accumulation of more mutations that lead to tumor initiation, promote the transition of microadenomas to macroadenomas and favor the neoplastic transformation. Therefore in the intestinal adenomas, Dll4 seems to promote proliferation and maintain the stem cells through angiogenic, but also non-angiogenic related mechanisms. Indeed we observed decreased expression of Myc, cyclin D1 and D2, independently of ?-catenin activation, only in the ubiqDll4
-/-
tumors.
The Wnt signaling has been considered a crucial player in the initiation of CRC associated to inactive mutations in the APC gene [3]. Nuclear accumulation of ?-catenin promotes neoplastic conversion by triggering the cell cycle-regulators Cyclin D1 and D2 and Myc and, consequently, uncontrolled cell proliferation contributing to tumor progression [40, 50, 51]. Notch signaling seems to cooperate with Wnt signaling to trigger intestinal tumorigenesis, as activation of Notch in Apc mutant mice led to a significant increase in the number of adenomas developed [13]. Additionally, a previous study indicated that Jagged1 was the link between Wnt and Notch pathways in the Apc
Min/+ tumorigenesis, where ?-catenin seems to transcriptionally activate Jagged1 [39]. However, it has been shown that the Mastermind-like 1 co-activator of Notch pathway can bind to the promoters of Cyclin D1 and Myc in colon cancer cell lines [52] and these molecules are activated directly by Notch1 in other types of cancer [53–57] and possibly by Cyclin D1 in CRC [58]. It has been also demonstrated that Cyclin D2 and Myc are also induced by Notch1 to promote stem cell renewal in another setting [59]. Additionally, the overexpression of Dll4 in a leukemia cell line led to increased protein expression of Myc [60]. Therefore, during Apc
Min/+ tumorigenesis Dll4/Notch signaling may directly upregulate the expression of Cyclin D1 and D2 and Myc. We observed that Dll4 deletion reduced tumorigenesis without affecting ?-catenin nuclear accumulation and thus Wnt activation. Therefore, Dll4/Notch activation may promote intestinal tumorigenesis by angiogenic and non-angiogenic mediated mechanisms in a ?-catenin independent manner. The non-angiogenic related regulation may include a synergistic effect of Dll4/Notch with Wnt signaling to promote tumorigenesis by increasing the transcription of important Wnt target genes.
In addition, Dll4 ubiquitous inhibition upregulated the zinc finger-containing transcription factor KLF4 in the Apc
Min/+ tumors. KLF4 is a cell proliferation inhibitor and can act as a tumor suppressor, being normally downregulated in Apc
Min/+ tumors and in early stages of human CRC [61]. The loss of one of its alleles increases Apc
Min/+ tumorigenesis, possibly by derepressing ?-catenin mediated gene expression [62]. A previous work indicated that Notch signaling supresses KLF4 expression in intestinal tumors and colorectal cancer cells [62]. Our results indicate that Dll4 seems to be the ligand responsible for this Notch-mediated phenotype. Therefore, Dll4/Notch may promote carcinogenesis by upregulating the transcription of Wnt target genes through KLF4 downregulation in the Apc mutated tumors. Previous work indicated that Hes1 downregulation by Notch inhibition derepresses Atoh1, which seems to induce KLF4 upregulation to promote goblet cell differentiation in a redundant manner [11, 63]. However, it seems that Hes1 may act both upstream and downstream of Atoh1 to negatively regulate KLF4 [11]. We found that in the ubiquitous, but not in the endothelial-specific, Dll4 knockouts, the reduction of Lgr5 and Bmi1 positive tumor stem cell density was accompanied with increased tumor epithelium differentiation with a moderate deviation towards the secretory lineages, probably due to the observed Atoh1 and Klf4 overexpression by Hes1 downregulation as it occurs when Notch signalling is inhibited [8, 63]. This indicates that besides the effect on angiogenesis, Dll4/Notch signaling seems to have an additional role maintaining the tumor stem cells undifferentiated.
Additionally, Dll4/Notch ubiquitous inhibition promoted the transcription of the cell cycle regulators cyclin-dependent kinase (CDK) inhibitors Cdkn1b and Cdkn1c in the Apc
Min/+ tumors. A previous report showed that inactivation of Notch1 and 2 in the normal gut is associated with derepression of these CDK inhibitors [7]. This phenotype was completely abrogated in the absence of Atoh1 [7, 64], a molecule that is also considered to act as a tumor suppressor in CRC [65]. Therefore, Dll4/Notch inhibition may also negatively affect the tumor stem cell populations through Atoh1 derepression-mediated upregulation of the CDK inhibitors Cdkn1b and Cdkn1c.