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Hedgehog signal is re-activated in several cancers. In this study, we examined the role of Gli3 on malignant phenotype of tumorigenicity for colorectal cancer and its relationship with p53, WNT and ERK/AKT signals. Gli3 expression was detected in HT29 and SW480 (p53-mutant) cells, but not in DLD-1 (p53-mutant) or HCT116 (p53-wild type) cells by reverse transcription-polymerase chain reaction and immunocytochemistry. Full-length Gli3 transfection increased anchor-independent growth for all cells regardless of p53 status, with upregulation of adhesion-related genes. Exogenous Sonic-Hedgehog increased activator-type of Gli3 and colony formation in Gli3-positive HT29 and SW480 cells. After implantation of Gli3-FL or mock-transfectant DLD-1 cells into SCID mice, tumor formation was highly observed in only Gli3-FL-transfectant group. In clinical specimens, Gli3 expression was detected in subsets of colorectal cancer and related with poorly-differentiated histological type, while Sonic–Hedgehog was present with high incidence. In conclusion, activator Gli3 signal augments tumorigenicity of colorectal cancer irrespective of p53 status.
Hedgehog (Hh) signal is morphogenically important for embryonic patterning and controlling growth and cell fate during neonatal development.[1-3] Three types of Hh ligands homologus, Sonic-Hedgehog (Shh), Indian-Hedgehog and Desert-Hedgehog, have been identified in mammals. In the absence of Hh ligands, Patched (Ptch), a 12-pass transmembrane receptor, suppresses Smoothened (Smo) activity, a G-protein-coupled receptor-like protein. Binding of Hh ligands to Ptch leads to the Ptch inactivation and consequent Smo activation. The signal transmits to downstream transcriptional factors: GLI proteins: Gli1, Gli2 and Gli3.[1, 5] Whereas Gli1 lacks a repressor domain, Gli2 and Gli3 possess repressor and activator domains. In the presence of Hh ligands, Gli2 and Gli3 are transmitted to nuclei as full-length activator (Gli2-FL and Gli3-FL) and transcribe the target genes of Hh signal. In contrast, when there are no or less Hh ligand stimuli, they are cleaved by the ubiquitin ligase and generate transcriptional repressor isoforms (Gli2-R and Gli3-R).[7-9] Gli2 generally functions as an activator of Hh signal but Gli3 is proposed to act as a repressor of Hh in embryonic development. Therefore, Hh ligand signal accelerates Gli1 transactivation and inhibits the formation of repressor isoforms (Gli2-R and Gli3-R), which is reflected in Hh activation.
A series of evidence has demonstrated Gli1 gene amplification/overexpression in glioma, medulloblastoma and rhabdomyosarcoma, and Shh overexpression in gastric, pancreas, and breast cancers. The Shh-Gli1 signal controls the phenotypes of invasiveness[18-21] and stemness[22-26] in various types of cancer. In colorectal cancer, Shh was reported to overexpress in tumor specimens and enhance the proliferation of colorectal cancer cells in vitro together with Gli1 mRNA upregulation. On the other hand, Gli1 transfection inhibits the proliferation of colorectal cancer cells via inactivation of WNT signal. Alternatively, Gli2 and Gli3 knockout mice exhibit the phenotype of abnormal development of intestine[29-31] and Gli3 knockdown inhibits anchor-dependent proliferation via p53 activation in colorectal cancer cells with wild type p53. However, the majority (60–70%) of colorectal cancer has mutations in p53 gene,[33-35] and the influence of Hh pathway on the proliferation and tumorigenicity of colorectal cancer remains unclear.
In this study, the transfection of full-length Gli3 (Gli3-FL), but not Gli1 or Gli2, upregulated adherence-related genes and increased anchor-independent growth and tumorigenicity for colorectal cancer cells, regardless of p53 status. Shh signal augmented Gli3-FL isoform and the growth potential in endogenous Gli3-positive colorectal cancer cells. In clinical specimens, Gli3 expression was detected in subsets of colorectal cancer together with Shh overexpression. It is concluded that Shh-enhancing Gli3 activator signal may be involved in the tumorigenicity for colorectal cancer.
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Hedgehog (Hh) signal plays a crucial role for organ development and homeostasis in embryonic and postnatal phases, and is re-activated in several types of cancers.[1, 2, 12-17] In this study, we investigated the biological significance of Hh signal in tumorigenicity for colorectal cancer. Surprisingly, we found that the Gli3 activator signal, which was emerged by Gli3-FL transfection or the increase in non-cleaved Gli3-FL isoform by Shh stimuli, enhanced the anchor-independent growth of colorectal cancer irrespective of p53 status. Furthermore, the tumorigenicity in vivo was gained by Gli3 overexpression, and Gli3 and Shh expressions were observed in subsets of clinical specimens of colorectal cancer. The current study has firstly demonstrated the significance of Gli3 activator signal on the malignant phenotype of tumorigenicity for colorectal cancer.
It has been shown that Shh increases the growth of colorectal cancer and the Shh-Gli1 signal contributes to the enhancement in stemness and xenograft tumor growth. In our previous study and Figure S2, Gli1 transfection deteriorated the WNT activity and growth of colorectal cancer cells, while Shh increased the proliferation and Gli1 expression. These could be explained by the idea that Gli1 may augment the in vivo tumor formation of colorectal cancer with stem-like phenotype, while Shh may stimulate the growth via other Hedgehog pathways. Thus, we elucidated the effect of Gli2 and Gli3 transfection (overexpression) on the colony formation and proliferation for colorectal cancer cells. Gli2 transfection reduced the anchor-independent growth in some cell lines, but it did not affect the anchor-dependent growth. The degree of Gli1 upregulation by Gli2 transfection was only a few folds (data not shown), and the inhibitory effect of Gli1 on growth might be obscured by a Gli2 overexpression induced effect. In contrast, the growth phenotype of colorectal cancer was enhanced by Gli3-FL transfection in all cells. The effect of Gli3-FL on proliferation was weaker than that on colony formation, suggesting that Gli3-FL may mainly affect anchor-independent growth for colorectal cancer cells. Gli3-FL transfection might override the inhibitory effect of Gli2 on anchor-independent growth although it also upregulated Gli2, because the degree of Gli2 upregulation by Gli3-FL transfection was only several folds, which was much less than that by Gli2 transfection (data not shown). These results suggest that the enhancement in anchor-independent and -dependent growth is mediated through the Gli3-specific and Gli1/Gli2 independent signal pathway.
Gli3-FL protein is eventually cleaved to Gli3-R by proteolytic reaction, and we further examined whether Gli3-R contributed to the growth phenotype by transfection of Gli3-R expression plasmid. Despite the inhibition in Gli1 expression, Gli3-R overexpression did not affect either anchor-independent or anchor-dependent growth in these cells. It may be because the degree of Gli1 inhibition by Gli3-R transfection was insufficient to change the growth phenotype biologically. We then examined the effect of Gli3 silencing on the both types of growth in endogenous Gli3-positive cells harboring p53-mutation. The proliferation was downregulated by Gli3 knockdown for these cells, which is consistent with a previous report using colorectal cancer cells with p53-wild type. The anchor-independent growth was also impaired by Gli3 knockdown. These results indicate that the activator type of Gli3-FL protein may be important for the growth of colorectal cancer through p53-independent mechanisms.
The colony formation ability was accelerated with rhSHH in endogenous Gli3-positive cells, where the increase in activator type of Gli3-FL isoform was confirmed by Western blot analysis. Even though rhSHH increased Gli1 expression, rhSHH induced Gli3 activator isoform might override the inhibitory effect of Gli1 against colony formation based on our data that co-transfection of Gli1 and Gli3-FL increased anchor-independent growth. In contrast, the colony formation of Gli3-negative cells was not changed with rhSHH. To verify the linkage of Shh/Smo signal to Gli3-induced anchor-independent growth of colorectal cancer, we used cyclopamine, an inhibitor of Smo. The administration of cyclopamine suppressed the colony formation for endogenous Gli3-positive cells, which is consistent with a previous study, but not for Gli3-negative cells. To exclude the possibility for non-specific effect of chemical inhibitor, we also used Smo-targeting siRNA and confirmed the specific linkage of Smo to the Gli3-mediated growth signal. These results indicate that the Shh/Smo activation could retain Gli3 protein as a full-length activator, which may in turn accelerate the colony formation ability for colorectal cancer.
To examine whether the Gli3-FL activator signal reflects for in vivo tumorigenicity of colorectal cancer, we implanted the Gli3-FL transfectant colorectal cancer cells into SCID mice. In HT29 cells, which have endogenous Gli3 and Shh, the tumorigenicity was not different between Gli3-FL and mock transfectants. However, in DLD-1, which has no endogenous Gli3 or Smo, a small number of Gli3-FL-transfectants formed tumors but not the mock. These results suggest that the tumorigenic potential of colorectal cancer may be gained if a certain level of full-length Gli3 isoform is present.
With respect to the responsible factors for the tumorigenicity, we postulated the WNT signal as a candidate because of frequent mutations in WNT components for colorectal cancer, which participate in cell growth, survival and migration. Actually, the HCT116 cell has gain-of-function mutation of β-catenin, and HT29, SW480 and DLD-1 have loss-of-functional mutations in adenomatous polyposis coli (APC) genes. However, Gli3-FL transfection had no effect on WNT activity in colorectal cancer cells (Fig. S5). Alternatively, mutations in Ras are also reported in about 40–50% of colorectal cancers.[44, 45] We thus analyzed the related MAPK/ERK and PI3K/AKT activities, but the increase in both activities was not detected in Gli3-FL transfectants (Fig. S6). These results suggest that the Gli3-induced tumorigenic phenotype may not be mediated by WNT, MAPK/ERK or PI3K/AKT pathway in colorectal cancer. Therefore, we tried to seek the possible factors using gene microarray analysis. In Gli3-FL transfectant HCT116 and DLD-1 cells, adherence-related genes were picked up as the candidates. Moreover, Gli3 expression upregulated several genes in “tumorigenicity” and “cancer stem cell” categories, although the common gene in the categories was only ASS1 in both cells, suggesting that these genes might synergistically contribute to the Gli3-induced tumorigenicity. Further investigation in future should be of great interest to determine which molecules are direct mediators for Gli3/Smo-induced tumorigenicity.
Relevant to clinical significance of Gli3/Shh/Smo signal of colorectal cancer specimens from patients, the positivity of Gli3 protein was 24% of total samples and the pattern of staining exhibited heterogeneity in adenocarcinoma cells. 58% of Gli3 positive samples were diagnosed as poorly differentiated type and the relationship between the two parameters was shown. Though a poorly differentiated cancer is usually recognized to lose adhesive activity, Mallin et al. have shown the upregulated expression of the adhesion-related gene: GDF15, in poorly differentiated colorectal cancer. Taken together, Gli3 expression was detected with a heterogenic pattern in the tumor region in the present study, suggesting that the expression of adhesion-related genes may not directly conflict with the loss of differentiation of colorectal cancer. Gli3 expression did not significantly correlate with TNM stage and lymph node metastasis, indicating that other factors, in addition to Gli3, are involved in malignant phenotypes for tumor progression. On the other hand, Shh was stained at the whole area of adenocarcinoma cells in the almost specimens, but not normal mucosa, as previously reported, and almost Gli3 positive specimens were Shh positive. These results suggest that the isoform of Gli3 we detected seems to be Gli3-FL type dominant, although the anti-Gli3 antibody we used reacts with both Gli3-FL and Gli3-R. The Hh target therapy may improve the outcome for patients with Gli3/Smo activated colorectal cancer. A randomized controlled study with a large number of patients should be addressed in future.
In conclusion, Hedgehog Gli3 activator signal, but not Gli1 or Gli2, is involved in anchor-independent growth and tumorigenicity for colorectal cancer through upregulation of adherence-related genes irrespective of p53 status. Taken together, histological examination of patient specimens indicates that Gli3 and Shh are expressed in subsets of colorectal cancer and may be therapeutic targets for colorectal cancer.