Cancer occurs in various organs after adolescence, suggesting that cancers are derived from cells harboring mutations in common genes in each organ after the embryonic stage. Such mutation generally occurs in genes that control embryonic morphogenesis. In Drosophila, morphogenesis signaling is classified in maternal effect genes that determine the longitudinal axis, gap genes that roughly determine the segmentation along the axis, pair rule genes that determine the location of somite, homeotic genes that characterize somite, and segment polarity genes that contribute to the embodiment of somite. After the embryonic stage, this morphogenesis signaling ceases; however, part of this signaling is believed to continue, mainly in the stem cells, to maintain homeostasis. We selected the segment polarity genes that regulate Hedgehog (Hh) signaling for the present study because these genes are functional even after the embryonic stage. This pathway is reactivated in various types of cancer and in cancerous stem cells. In this review, we summarize recent efforts to assess the contribution of the Hh signaling pathway in various types of cancer.
Hedgehog (Hh) signaling is an important factor in growth and patterning during embryonic development. A mutation in Patched, Smoothened or Gli1, which regulate the Hh signaling pathway, might lead to the onset of glioblastoma, basal cell carcinoma, medulloblastoma and rhabdomyosarcoma. Recently, Hh signaling has been reported to be activated in a ligand-dependent manner, contributing to carcinogenesis and cancer progression. Hedgehog signaling is reactivated in various types of cancer, and this contributes to cancer progression by facilitating proliferation, invasion and cell survival. Moreover, Hh signaling is associated with several other signaling pathways that contribute to cancer progression. These observations indicate that controlling Hh signaling might become a target for novel molecular targeting therapy. (Cancer Sci 2011; 102: 1756–1760)
Hedgehog signaling pathway
The Hh signaling pathway, which is a morphogenesis signaling pathway, is crucial for the growth and patterning of various tissues during embryonic development,(1,2) and disruption of this pathway induces congenital anomalies in the central nervous system, axial skeleton, limbs and other organs. Although the activation mechanism of this pathway has not been completely elucidated (Fig. 1), this pathway is sequestered in the cytoplasm and is a highly coordinated network of Hh proteins (Sonic Hh, Indian Hh and Desert Hh), the 12-transmembrane receptors (Patched1 [Ptch1] and Patched2 [Ptch2]), the seven-transmembrane protein Smoothened (Smo), and the transcription factor Gli family (Gli1, Gli2 and Gli3).(3–5) In the absence of Hh ligands, Gli2 and Gli3 (Gli) form a large protein complex with other proteins, such as the kinesin-like Costal2, the serine–threonine kinase Fused, and suppressor of Fused (SUFU). Furthermore, Ptch suppresses the signaling activity of Smo.(3–5) Costal2 might promote the degradation or proteolysis of Gli, which generates the repressor form of Gli (Rep-Gli2/3). The translocation of Rep-Gli2/3 to the nucleus inhibits the transcription of target genes, including Gli1 and Ptch, and cancer proliferation- and invasion-related genes, such as cyclin D, Myc, Snail and Bcl2.(6) In the presence of Hh, it binds to Ptch, activating Smo, and both degradation of Gli2 and proteolytic processing of Gli3 into its repressive form are inhibited,(7,8) thereby permitting Gli2 to function as a strong activator of the Hh signaling (Act-Gli2) and allowing full-length Gli3 to serve as an activator (Act-Gli3).(9,10) Recent reports indicate that Hh signaling occurs in primary cilia.(11–13) Primary cilia are cell surface projections found on most vertebrate cells that function as sensory “antennae” for signal transduction.(14) The extension of the cilium is maintained by the transport of particles along the axoneme, which is mediated by the intraciliar transport machinery.(15) Several components of the Hh pathway, including Smo, Ptch and the Gli proteins, accumulate in primary cilia, and Smo is enriched in cilia following stimulation with Hh(16) (Fig. 1). Therefore, drugs that inhibit ciliogenesis might also act as Hh signaling inhibitors. Among members of the Gli family (Gli1, Gli2 and Gli3), Gli2 plays an important role in mediating Hh signaling in the neural tube;(17) however, Gli1 and Gli2 double-mutant embryos display more severe deficits in ventral neuronal subtype specification than embryos lacking Gli2 alone.(18) This result indicates that Gli1 can activate the Hh signaling pathway.(19) Therefore, the nuclear translocation of Gli1 is considered to be a marker for Hh pathway activation.(20,21)
Hedgehog signaling pathway activation by mutations
The relation between Hh pathway activation and tumor initiation was first reported in malignant tumors of the central nervous system and skin. Initially, a high amplification of Gli1 was reported in glioblastomas.(22) Later, Ptch and Smo mutations were confirmed in basal cell carcinomas,(23,24) medulloblastomas(25) and rhabdomyosarcomas.(26) A Ptch1 mutation was also identified in Gorlin syndrome, in which patients form a high-risk group for basal cell carcinoma.(27,28) Moreover, in non-familial basal cell carcinoma, a frequent mutation of Smo as well as Ptch1 activates the Hh signaling pathway.(29) Collectively, the amplification or mutation of genes downstream of Ptch is believed to contribute to activation of the Hh signaling pathway in these malignancies.
Hedgehog signaling pathway activation in a ligand-dependent manner
Ligand-dependent Hh signaling activation, but not genomic mutation, has been reported in pancreatic cancer, lung cancer, esophageal cancer, prostate cancer, breast cancer, gastric cancer, colon cancer, ovarian cancer and hepatocellular carcinoma.(21,30–37) Namely, Hh signaling is activated in an autocrine manner, in which Hh is produced by the cancer cells themselves (Fig. 2).
Recently, a paracrine paradigm for Hh pathway-mediated carcinogenesis has been the focus of various studies including those of pancreatic, lung, esophageal, gastric and prostate cancers.(38,39) In brief, Tian et al. reported that pancreatic cancer cells secrete Hh ligands, including Shh, to induce tumor-promoting Hh target genes in the adjacent stroma cells.(38) They also reported that the pancreatic epithelium is not receptive to tumor cell-derived Hh ligands; instead, Hh ligands promote pancreatic cancer through a paracrine signaling mechanism and these signals are received by stromal tumor cells (Fig. 2). In addition, Yamasaki et al.(40) demonstrated that tumor-infiltrating monocytes or macrophages secrete Shh following an inflammatory stimulus; this Shh activates Hh signaling in the cancer cell itself (Fig. 2). Hedgehog signaling is activated in several ways in various types of cancers, indicating that the Hh signaling pathway might be a suitable target for cancer therapy.
Hedgehog ligand overexpression and tumor initiation
The mechanism of Shh overexpression by cancer cells was first demonstrated in pancreatic cancer. Kasperczyk et al.(41) demonstrated that the expression of transcription factors nuclear factor-κB (NF-κB) and Shh was closely related and that Shh activated through NF-κB pathway activation contributed to the proliferation of pancreatic cancer and resistance to TNF related apoptosis inducing ligand (TRAIL)-induced apoptosis by analyzing Shh promoter activation. Hedgehog signaling activation in cancer tissue has also been demonstrated in breast cancer.(21) Moreover, Kameda et al.(42) showed that Shh expression increases through the estrogen receptor (ER) signaling pathway, and that the Hh signaling pathway is activated in a ligand-dependent manner. Consistent with in vitro results, ER and Shh expressions are positively correlated in gastric cancer tissue.
Hedgehog signaling pathway and cancer stem cells (CSC)
Data from human tumors including glioblastoma, breast cancer, pancreatic adenocarcinoma, multiple myeloma and chronic myeloid leukemia have suggested that Hh signaling regulates cancer stem cells.(43–49) Liu et al.(47) demonstrated that the Hh signaling components Ptch1, Gli1 and Gli2 are highly expressed in normal human mammary stem/progenitor cells and that these genes are downregulated when differentiation is induced in these cells. In multiple myeloma, CSC have been found to display relatively higher levels of Hh signaling than the mature plasma cells,(46) suggesting Hh signaling can act through multiple signaling modes within the same cancer and can mediate interactions between CSC, differentiated tumor cells and the microenvironment.(49) Accordingly, the Hh pathway might play an important role in the continuous self-renewal of tissues from stem cells, which persists into post-natal and adult life.
Contribution of the Hh signaling pathway to cancer progression and invasiveness
Recently, the Hh signaling pathway was shown to mediate the progression of non-invasive breast cancer to invasive breast cancer.(50) In this report, a serial increase was found in the percentage of Shh- and Gli1-positive cases that transformed from ductal carcinoma in situ to invasive ductal carcinoma. Contribution of the Hh pathway to invasiveness was first demonstrated in prostate cancer.(51) In this report, the overexpression of Gli1 in AT2.1 cells promoted lung metastasis. Recently, matrix metalloproteinase-9 (MMP-9) and ER were shown to increase invasiveness through activation of the Hh pathway.(50,52) Nagai et al.(52) showed that Gli1 contributes to the invasiveness of pancreatic cancer. Moreover, we reported that Hh signaling contributes to invasiveness under hypoxic conditions, which is observed in regions that are distant from the supporting tumor vasculature and is characteristic of pancreatic cancer.(53,54) In this report it is demonstrated that hypoxia-induced invasiveness is caused by increased transcription of Smo and that this invasiveness is ligand independent. Collectively, Hh signaling is believed to significantly contribute to cancer invasiveness.
Crosstalk between the Hh pathway and other pathways
In colorectal cancer, the Wnt signaling pathway, not the Hh signaling pathway, is activated to induce cell survival. Recently, Gli1 was demonstrated to inhibit the proliferation of colon cancer cells by suppressing activation of the Wnt signaling pathway.(55,56) Expression of β-catenin, an activation marker for the Wnt signaling pathway, and Gli1 correlated inversely in 40 resected colon cancer tissues, and the transcriptional activity of the Wnt signaling pathway was reduced by the overexpression of Gli1. Moreover, Akiyoshi et al.(56) demonstrated that Gli1 can suppress Wnt signaling activation, even in colon cancer with a β-catenin mutation. This result indicates that Hh signaling can suppress Wnt signaling and that Hh signaling might be implicated in colon cancer. Recently, many researchers have demonstrated crosstalk between the Hh signaling pathway and other pathways, including the NF-κB,(41) ERα,(43) KRAS,(57) PI3k-AKT,(58) EWS/FLI1,(59) PKC-delta(60) and p53(61) pathways (Fig. 3). Consequently, Gli1 would be a suitable target for cancer therapy because it is located downstream of several signaling pathways.
Cancer therapy targeting hedgehog signaling
Because Hh signaling is activated in various types of cancer and contributes to cancer proliferation, progression and invasiveness, the Hh signaling pathway is anticipated to provide a new target for cancer therapy. Cyclopamine (C27H41NO2) and Jervine (C27H39NO3) were discovered and in particular, a series of studies with the Hh pathway inhibitor, cyclopamine, has brought about this expectation.(62)
Several one-eyed lambs (cyclopia) were borne by the sheep that grazed on the wild corn lily, oxysepalum, during a short period at a farm in Idaho, USA.(63) In 1957, the US Department of Agriculture started an 11-year investigation that led to the identification of a plant alkaloid, cyclopamine (named after cyclopia), and the condition it induced was the cause of the birth defect.(64) Cyclopamine suppresses the Hh signaling pathway through direct interaction with Smo.(65–67) Subsequently, a cyclopamine derivate, which is easier to produce, was analyzed as a candidate therapeutic agent. Cyclopamine with improved solubility (IPI-926), Smo inhibitors that considerably differ in structure from cyclopamine (GDC-0499, LDE225, BMS-833923, XL-139, PF-0449913), inhibitors of the transformation of inactive Smo into active Smo (SANT 74-75), and inhibitors of the transport of cytoplasmic inactive Smo to cilia (SANT 1-4) have been developed to date.(16,68–73) Some of these are under clinical study. For example, the results of a Phase I/II study and pilot study on basal cell carcinoma and medulloblastoma using the Smo inhibitor GDC-0449 have recently been reported.(69) One study produced promising results, demonstrating that two of 33 cases showed complete tumor reduction, and 16 of 33 cases showed partial tumor reduction in basal cell carcinoma.(74) Another study showed that GDC-0449 had a dramatic effect on a patient with medulloblastoma with multiple metastatic lesions.(69) However, in a clinical study using GDC-0499, a patient with medulloblastoma improved dramatically following treatment with GDC-0499 but relapsed a month later.(69) In a recent study, the mutational status of Hh signaling genes in the tumor after disease progression was determined.(75) An amino acid substitution was identified at a conserved aspartic acid residue of Smo. However, this substitution had no effect on Hh signaling, but disrupted the ability of GDC-0449 to bind to Smo and suppress this pathway.(75) IPI-926 has been reported to induce angiogenesis and raise the chemo-drug concentration in cancer tissue.(76) Combined therapy with Smo inhibitors and a chemo-drug or a small molecular modulator is underway in a Phase I study. These Smo inhibitors are generally administered orally once a day, because they have a short half-life.
As mentioned above, recent drug development programs have focused on Smo inhibitors. Figure 4 shows structural formula of Smo inhibitor under clinical trials that companies have already announced. Recently, Lauth et al.(77) showed that the small-molecule antagonists (GANT 58 and GANT 61) of Gli-mediated transcription, which constitutes the final step in the Hh pathway, could selectively inhibit Gli-mediated gene transactivation. More recently, robotnikinin was identified as a compound that binds to Shh and blocks its ability to induce pathway activity at the level of Ptch.(78) Ptch Ab is also being developed as a Hh signaling inhibitor.(79)Figure 5 summarizes the Hh signaling inhibitors currently being developed.(80) These results indicate that Hh signaling is a suitable target for cancer therapy.
In this review we focused on the contribution of the Hh signaling pathway to the development and progress of cancer. The Hh signaling pathway plays a pivotal role in the initiation, proliferation, invasion and metastasis of various cancers. Therefore, the development of therapy targeting Hh signaling is an important subject for future research. However, we should remember that disruption of the Hh signaling pathway induces proliferation in certain types of cancer, affects normal stem cells, and causes congenital anomalies.
This study was supported by General Scientific Research Grants (21390363) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. We thank Ms Kaori Nomiyama, Nobuhito Torada and Miyuki Omori (Kyushu University) for their skillful technical assistance.
The authors declare no financial or commercial conflicts of interest.