Gene expression analysis of the hedgehog signaling cascade in the chick midbrain and spinal cord.

The signaling molecule Sonic Hedgehog (SHH) plays a critical role in patterning the ventral midbrain of vertebrates. Our recent studies have established that the requirement for Hedgehog (HH) signaling in the chick midbrain is modulated spatially and temporally in a complex manner across the midbrain anlage. Unfortunately, the patterns of expression of downstream regulators that might modulate the HH signal in the midbrain are not currently known. To fill this gap, we have examined across time, the expression pattern of 14 genes that function in the HH signaling cascade in the midbrain and spinal cord. Our results suggest that SHH expression in the axial mesendoderm begins before the expression of known HH receptors/HH-binding proteins (e.g., PTC1, PTC2, HHIP, BOC, MEGALIN). In the midbrain, PTC and GLI genes are expressed and then eliminated very early from the ventral midline. However, they exhibit high and persistent expression in the midbrain region circumscribing the SHH source. Intriguingly, multiple HH-binding proteins (BOC, MEGALIN) and HH effectors (GLI1-3, SMO, SUFU, DZIP) are expressed in the dorsal midbrain and the midbrain-hindbrain boundary. Finally, we report for the first time that IHH is expressed in intermediate regions of the spinal cord, where its expression does not overlap with that of SHH.     

在乳腺癌干细胞中Hedgehog信号通路相关基因表达增强

目的 了解Hedgehog信号通路在乳腺癌发生发展中的作用.方法 用免疫磁珠法从无血清培养的乳腺癌悬浮细胞中分选CD44+CD24-细胞和非CD44+CD24-细胞,用real-time RT-PCR法检测Hedgehog信号通路主要分子$HH、PTCH1、SMO和GLI1 mRNA在细胞中的表达,用免疫组织化学法检测上述因子在乳腺癌组织中的表达.结果 分选出的CD44+CIDA-细胞约占乳腺癌悬浮细胞总数的8.25%,分选出的CD44+CD24-细胞表达干细胞标志蛋白ALDHA1和Oct-4;SHH、PTCH1、SMO和GLI1 mRNA在CD44+CD24-细胞中的表达均高于其在非CD44+CD24-细胞中的表达(P＜0.05);SMO和GLI1蛋白在三阴性乳腺癌的表达均高于非三阴性乳腺癌组织(P＜0.05).结论 在乳腺癌干细胞CD44+CD24-细胞中Hedgehog信号通路被激活,抑制癌症干细胞中Hedgehog通路的活化可能会降低或阻止乳腺癌的复发及化疗耐受.     

HIF-1α、ALDH1 和Hedgehog 信号通路协同促进三阴性乳腺癌中癌症干细胞活化的相关性研究

目的:探讨HIF-1α、ALDH1 和Hedgehog 信号通路在三阴性乳腺癌中癌症干细胞活化的相关性及其临床意义。方法:采用免疫磁珠法从三阴性乳腺癌细胞系MDA-MB-231 细胞中分选出ALDH1+乳腺癌干细胞和ALDH1-乳腺癌细胞,采用qRT-PCR 方法比较HIF-1α和Hedgehog 信号传导通路主要分子SHH、PTCH1、SMO、GLI1 在这两种细胞中的表达差异;采用免疫组化方法了解HIF-1α和ALDH1 在三阴性乳腺癌组织中的表达以及与干细胞信号传导通路Hedgehog 的相互关系。结果:HIF-1αmRNA、SMO mRNA 和GLI1 mRNA 在ALDH1+乳腺癌干细胞中的表达均明显高于ALDH1-乳腺癌细胞,差异具有显著统计学意义(P 均<0.05)。在三阴性乳腺癌与非三阴性乳腺癌组织中HIF-1α的阳性表达率分别为90.0% 和70.0%,ALDH1 的阳性表达率分别为93.3%和66.7%,差异均具有显著统计学意义(P 均<0.05)。在三阴性乳腺癌组织中HIF-1α和ALDH1 的表达呈正相关(r =0.53,P<0.01)。HIF-1α的表达与三阴性乳腺癌的淋巴结转移和TNM 分期有关(P 均<0 .05),ALDH1 的表达与组织学分级和TNM 分期有关(P 均<0.05)。HIF-1α与Hedgehog 信号通路分子SHH(r = 0.584,P<0.01)、SMO(r=0.467,P<0.01)和GLI1(r =0.439,P<0.05)的表达呈正相关,ALDH1 与SHH(r =0.426,P<0.05)和GLI1(r =0.394,P<0.05)的表达呈正相关。结论:HIF-1α和Hedgehog 信号通路在ALDH1+ 乳腺癌干细胞中活化,HIF-1α、ALDH1 与Hedgehog信号传导通路可能相互协同激活癌症干细胞从而促进三阴性乳腺癌的恶性进展。     

Clinical aspects of disrupted Hedgehog signaling (Review)

The Hedgehog (HH) signaling pathway is involved in patterning and development of a variety of organ systems, including the nervous system, the skeletal system, the craniofacial structures, and the gastrointestinal tract. Recent evidence also implicates this signaling pathway in the postembryonic regulation of stem-cell number in epithelia and blood. The family of HH proteins consists of at least three different members, i.e., sonic HH (SHH), Indian HH (IHH), and desert HH (DHH). SHH is the most broadly expressed member of this family and is probably responsible for the major effects of this signaling pathway. The HH signal is received and transduced via a specific receptor complex composed of patched (PTCH) and smoothened (SMOH) transmembrane proteins. Abnormalities in this signaling cascade have been found in various developmental pathologies and neoplasms such as basal cell carcinoma. The abnormalities are associated with congenital or sporadic genetic alteration affecting function of different components of the HH signaling pathway, including SHH, PTCH, SMOH and GLI proteins.     

大鼠骨髓间充质干细胞成骨分化过程中Hedgehog信号分子的表达

背景：Hedgehog信号通路是一个在胚胎阶段调控多种组织器官发育的重要信号通路,在成骨发育方面具有重要的作用。但Hedgehog信号分子在大鼠骨髓间充质干细胞体外诱导分化为成骨细胞过程中的作用尚未清楚。 目的：体外诱导大鼠骨髓间充质干细胞向成骨分化,检测Hedgehog信号分子在成骨诱导分化过程中的变化。 方法：从大鼠骨髓中分离得到骨髓间充质干细胞,进行地塞米松成骨诱导,通过免疫组化方法鉴定成骨的情况,Western Blot方法检测 Hedgehog信号分子SHH和IHH在骨髓间充质干细胞成骨分化过程中的表达。 结果与结论：成功分离得到骨髓间充质干细胞,地塞米松诱导培养7,14,21 d 后,Ⅰ型胶原的表达量逐渐增加;在诱导成骨分化过程中,SHH蛋白表达升高,诱导组的表达明显高于未诱导组的表达(P < 0.05),而IHH蛋白的表达降低,诱导组的表达明显低于未诱导组的表达(P < 0.05)。结果提示,Hedgehog信号分子参与地塞米松诱导骨髓间充质干细胞分化为成骨细胞的过程,且SHH和IHH在间充质干细胞诱导成骨过程中的作用有差异。     

Constitutive hedgehog signaling in chondrosarcoma up-regulates tumor cell proliferation

Chondrosarcoma is a malignant cartilage tumor that may arise from benign precursor lesions, such as enchondromas. Some cases of multiple enchondromas are caused by a mutation that results in constitutive activation of Hedgehog-mediated signaling. We found that chondrosarcomas expressed high levels of the Hedgehog target genes PTCH1 and GLI1. Treatment with parathyroid hormone-related protein down-regulated Indian Hedgehog (IHH) expression in normal growth plates but not in chondrosarcoma or enchondroma organ cultures. Treatment of the chondrosarcoma organ cultures with Hedgehog protein increased cell proliferation rate, whereas addition of chemical inhibitors of Hedgehog signaling decreased the proliferation rate. Chondrosarcoma xenografts from 12 different human tumors were established in NOD-SCID mice. Treatment with triparanol, an inhibitor of Hedgehog signaling, resulted in a 60% decrease in tumor volume, a 30% decrease in cellularity, and a 20% reduction in proliferation rate. These results show that Hedgehog signaling is active in chondrosarcoma and benign cartilage tumors and regulates tumor cell proliferation. Our data raise the intriguing possibility that Hedgehog blockade could serve as an effective treatment for chondrosarcoma, a tumor for which there are currently no universally effective nonsurgical management options.     

Hh pathway expression in human gut tissues and in inflammatory gut diseases

Sonic hedgehog (Shh) directs early gut patterning via epithelial-mesenchymal signaling and remains expressed in endoderm-derived tissues into the adult period. In human adult gut epithelium SHH/SHH expression is strongest in basal layers, which suggests that SHH may function in the maintenance of gut epithelial stem or progenitor cells. Recent publications suggest a role for aberrant SHH/SHH expression in gut epithelial neoplasias. We hypothesized that the regenerating gut epithelium in inflammatory gut disorders would show an upregulation of SHH/SHH signaling and this abnormal signal may explain the increased incidence of neoplasia in these diseases. Archived healthy gut and inflammatory gut diseased tissues were analyzed by RNA in situ hybridization and immunohistochemistry to describe location and levels of SHH signaling. We show that SHH/SHH and its receptor PTCH1/PTCH1 expression is restricted to the glandular epithelium of the gut, in an antiluminal pattern (strongest in basal layers and weak to absent in luminal epithelium). Inflammatory diseases of the gut show dramatic increases in epithelial SHH signaling. Expression increases in inflamed glandular epithelium (including metaplastic glandular epithelium), losing its radial (crypt-villous) polarity, and expression appears upregulated and present in all epithelial cells. We also describe strong SHH/SHH and PTCH1/PTCH1 expression in intraepithelial and mucosal inflammatory cells. We suggest that SHH signaling in inflammatory diseases of the gut acts to ensure stem cell restitution of damaged mucosal epithelium. However, such signaling may also present a risk for neoplastic transformation.     

Peripheral chondrosarcoma progression is accompanied by decreased Indian Hedgehog signalling

Hedgehog (HH) signalling is important for specific developmental processes, and aberrant, increased activity has been described in various tumours. Disturbed HH signalling has also been implicated in the hereditary syndrome, Multiple Osteochondromas. Indian Hedgehog (IHH), together with parathyroid hormone-like hormone (PTHLH), participates in the organization of growth plates in long bones. PTHLH signalling is absent in osteochondromas, benign tumours arising adjacent to the growth plate, but is reactivated when these tumours undergo malignant transformation towards secondary peripheral chondrosarcoma. We describe a gradual decrease in the expression of Patched (PTCH) and glioma-associated oncogene homologue 1 (GLI1) (both transcribed upon IHH activity), and GLI2 with increasing malignancy, suggesting that IHH signalling is inactive and PTHLH signalling is IHH independent in secondary peripheral chondrosarcomas. cDNA expression profiling and immunohistochemical studies suggest that transforming growth factor-beta (TGF-beta)-mediated proliferative signalling is active in high-grade chondrosarcomas since TGF-beta downstream targets were upregulated in these tumours. This is accompanied by downregulation of energy metabolism-related genes and upregulation of the proto-oncogene jun B. Thus, the tight regulation of growth plate organization by IHH signalling is still seen in osteochondroma, but gradually lost during malignant transformation to secondary peripheral chondrosarcoma and subsequent progression. TGF-beta signalling is stimulated during secondary peripheral chondrosarcoma progression and could potentially regulate the retained activity of PTHLH.     

Neogenin regulates Sonic Hedgehog pathway activity during digit patterning

Background : Digit patterning integrates signaling by the Sonic Hedgehog (SHH), fibroblast growth factor (FGF), and bone morphogenetic protein (BMP) pathways. GLI3, a component of the SHH pathway, is a major regulator of digit number and identity. Neogenin (encoded by Neo1) is a cell surface protein that serves to transduce signals from several ligands, including BMPs, in various developmental contexts. Although neogenin is implicated in BMP signaling, it has not been linked to SHH signaling and its role in digit patterning is unknown. Results : We report that Neo1 mutant mice have preaxial polydactyly with low penetrance. Expression of SHH target genes, but not BMP target genes, is altered in Neo1 mutant limb buds. Analysis of mice carrying mutations in both Neo1 and Gli3 reveals that, although neogenin plays a role in constraint of digit numbers, suppressing polydactyly, it is also required for the severe polydactyly caused by loss of GLI3. Furthermore, embryo fibroblasts from Neo1 mutant mice are sensitized to SHH pathway activation in vitro. Conclusions : Our findings indicate that neogenin regulates SHH signaling in the limb bud to achieve proper digit patterning. Developmental Dynamics 241:627–637, 2012. © 2012 Wiley Periodicals Inc.     

STAT3 activation and aberrant ligand-dependent sonic hedgehog signaling in human pulmonary adenocarcinoma

The Sonic hedgehog (SHH) signaling and STAT3 pathways play important roles during carcinogenesis with possible interaction. To determine the association of the activation of SHH signaling pathway and STAT3 pathway in carcinogenesis of human non-small cell lung carcinomas (NSCLC), firstly we examined the expression of SHH signaling molecules including SHH, Gli1(glioma-associated oncogene homolog 1) and HHIP (Hh interacting protein), as well as p-STAT3 (phosphorylation at Tyr705) by immunohistochemistry in 87 cases of NSCLC, 12 atypical adenomatous hyperplasia (AAH) and 20 adjacent normal lung tissues. The expression of SHH, Gli1, HHIP and p-STAT3 was detected respectively in 87/87(100%), 74/87(85.1%), 75/87(86.2%) and 54/87(62.1%) of the NSCLC cases, but not in the adjacent normal lung parenchyma. Ligand-dependent SHH pathway activation and STAT3 signaling activation were observed in most cases of NSCLC, and the high SHH pathway activation rate and STAT3 activation rate were significantly higher in adenocarcinoma compared with squamous cell carcinomas and large cell carcinomas (P<0.05). Both SHH and STAT3 pathway activation level correlated with histological grade in adenocarcinoma, being higher in well-differentiated types (P<0.05). Furthermore, high SHH pathway activation and p-STAT3 expression were also detected in 10/12(83.3%) of AAH cases as well as in most cases of early-stage adenocarcinoma - adenocarcinoma in situ (AIS) and minimally invasive adenocarcinomas (MIA). Correlation analysis showed that p-STAT3 protein expression level was correlated positively with ligand-dependent activation level of SHH signaling in adenocarcinoma (r(s)=0.585, P=0.000) and AAH (r(s)=0.996, P=0.000). We speculated that activation of STAT3 could up-regulate SHH gene expression directly or indirectly, and thereby activated SHH signaling resulting in lung tumor cell ontogeny. To explore the interactional mechanism, we then performed serial transient co-transfection assay in human pulmonary adenocarcinoma cell line H441 cells, and the results confirmed STAT3 activation can up-regulate SHH gene expression indirectly. In conclusion, aberrant ligand-dependent SHH signaling activation occurs frequently in NSCLC. The signaling plays a more active role in adenocarcinoma, and is an early event in carcinogenesis of lung adenocarcinoma. The involvement of STAT3 pathway activation might function in inducing the process.     

The sonic hedgehog signaling network in development and neoplasia

The sonic hedgehog (SHH) pathway was first defined genetically in fruit flies. Subsequently, the SHH network has been shown to be critical for normal mammalian development, by mediating interactions between stromal and epithelial cells. Recent evidence suggests that, deregulation of SHH signaling is important in the pathogenesis of cancer. Further, some observations suggest that a SHH paracrine mechanism mediating tumor-mesenchymal interactions may contribute to the metastatic capacity of cancer. Preclinical studies demonstrate that tumor cells in which SHH is deregulated are dependent on signaling through this pathway for the maintenance of proliferation and viability. SHH antagonists have been identified and show promise in inhibiting tumor growth in preclinical studies. The utility of these agents in the management of cancer patients awaits the outcome of ongoing and future clinical trials.