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Expression of Sonic hedgehog signaling molecules in normal, hyperplastic and carcinomatous endometrium
Abstract
The aim of the present study was to determine the expression profile of the hedgehog (Hh) signaling molecules in normal, hyperplastic, and carcinomatous uterine endometrium. For this purpose, 271 endometrial tissue samples, (62 of normal endometrium, 127 of endometrial hyperplasias, and 82 endometrial adenocarcinomas) were studied using antibodies recognizing Hh-related signaling proteins, such as, sonic hedgehog (Shh), Patched (PTCH), Smoothened (Smo), Suppressor of fused [Su(Fu)], Gli-1, Gli-2, and Gli-3 by immunohistochemistry. The mRNA expression of these molecules was also assessed on reverse transcription-polymerase chain reaction. In the normal endometrium, the expression of Hh signaling molecules was generally downregulated except for Su(Fu), Gli-2, and Shh. In particular, the expression of both PTCH and Smo was very low or almost absent. Overall expression of Hh signaling molecules increased in hyperplastic endometrium; in particular, PTCH and Smo were significantly highly expressed in complex and atypical hyperplasia. In carcinoma samples extensive alterations were observed in the expression pattern of the signaling molecules. Nuclear Gli-2, cytoplasmic Gli-3, and Su(Fu) were overexpressed, whereas Shh, PTCH, and Smo expression were significantly reduced compared with the hyperplastic endometrium. The results suggest that the alteration of Hh signaling may be implicated in tumorigenesis of the endometrium.
... alterations in the expression pattern of Hh signaling molecules also suggest that Shh signaling network functions differently in normal and hyperplasic endometrium than under the carcinomatous condition 24 . However, steroid-regulatory mechanism and signaling cascade (ligand-dependent/canonical pathway and ligand-independent/non-canonical pathway) of the Hh signaling associated towards estrogen-mediated endometrial hyperplasia progression still remain unclear. ...
... p values: a p < 0.001, b p < 0.01, c p < 0.05 and d p > 0.05 vs. control for Gsk3β expression; e p < 0.001, f p < 0.01, g p < 0.05 and h p > 0.05 vs. control for Gli1 expression. transcription-polymerase chain reaction 24 . The expression of major Hh signaling mediators, including both activators and suppressors is known to be positively correlated with ER/PR expression in normal endometrium 24 . ...
... transcription-polymerase chain reaction 24 . The expression of major Hh signaling mediators, including both activators and suppressors is known to be positively correlated with ER/PR expression in normal endometrium 24 . However, the steroid-regulatory mechanisms of Hh signaling pathway as well as its physiological and pathological role, in endometrial hyperplasia are still not known. ...
The present study was undertaken to explore the functional involvement of Hh signaling and its regulatory mechanism in endometrial hyperplasia. Differential expression of Hh signaling molecules i.e., Ihh, Shh, Gli1 or Gsk3β was observed in endometrial hyperplasial (EH) cells as compared to normal endometrial cells. Estradiol induced the expression of Hh signaling molecules and attenuated the expression of Gsk3β whereas anti-estrogen (K1) or progestin (MPA) suppressed these effects in EH cells. Cyclopamine treatment or Gli1 siRNA knockdown suppressed the growth of EH cells and reduced the expression of proliferative markers. Estradiol also induced the nuclear translocation of Gli1 which was suppressed by both MPA and K1 in EH cells. While exploring non-canonical mechanism, LY-294002 (Gsk3β activator) caused a decrease in Gli1 expression indicating the involvement of Gsk3β in Gli1 regulation. Further, Gsk3β silencing promoted the expression and nuclear translocation of Gli1 demonstrating that Gsk3β serves as a negative kinase regulator of Gli1 in EH cells. Similar attenuation of Hh signaling molecules was observed in rats with uterine hyperplasia undergoing anti-estrogen treatment. The study suggested that Hh/Gli1 cascade (canonical pathway) as well as Gsk3β-Gli1 crosstalk (non-canonical pathway) play crucial role in estrogen-dependent cell proliferation in endometrial hyperplasia.
... Acting through its receptor (PR), progesterone increases uterine expression of HH protein, which in turn regulates NR2F2 expression (Lee et al. 2006b, Simon et al. 2009a. HH is crucial to normal and abnormal uterine (and endometrial) development and is also regulated by progesterone (Feng et al. 2007, Kim et al. 2009, Wei et al. 2010, Franco & Yao 2012. Currently, however, data are lacking on the potential roles of progesterone, RA and/or SHH regulatory molecules in controlling CTNNB1 and/or NR2F2 in human myometrium and fibroid tissues. ...
... A fibroid-associated increase in CTNNB1 confirms microarray data by us and others (Luo et al. 2005a, Zaitseva et al. 2006. Aberrant expression/ mutations of CTNNB1 leading to increased nuclear localisation, and enhanced Wnt signalling, have been observed in endometrial carcinoma (Fukuchi et al. 1998, Saegusa & Okayasu 2001, Moreno-Bueno et al. 2002, Kim et al. 2009). Non-uterine smooth muscle tumours similarly demonstrate increased CTNNB1 expression and nuclear translocation during proliferation or predisposing tumour development (Gosens et al. 2010, van Veelen et al. 2011). ...
... The HH pathway is important in many normal and abnormal cellular functions, including embryonic development, stem cell proliferation and cancer genesis (reviewed by Varjosalo & Taipale (2008), Choi et al. (2011), Harris et al. (2011) and Park et al. (2011). The HH pathway is also crucial to the development of reproductive tract (Franco & Yao 2012), uterine function (Takamoto et al. 2005, Lee et al. 2006a) and uterine malignancies (Feng et al. 2007, Kim et al. 2009). Despite evidence for a role of HH in the uterus, and our results demonstrate that the HH pathway is active in cultured myometrial and fibroid cells; NR2F2 and CTNNB1 mRNA expressions were not affected by SHH treatment. ...
Uterine fibroids are the most common benign tumour afflicting women of reproductive age. Despite the large healthcare burden caused by fibroids, there is only limited understanding of the molecular mechanisms that drive fibroid pathophysiology. Although a large number of genes are differentially expressed in fibroids compared with myometrium, it is likely that most of these differences are a consequence of the fibroid presence and are not causal. The aim of this study was to investigate the expression and regulation of NR2F2 and CTNNB1 based on their potential causal role in uterine fibroid pathophysiology. We used real-time quantitative RT-PCR, western blotting and immunohistochemistry to describe the expression of NR2F2 and CTNNB1 in matched human uterine fibroid and myometrial tissues. Primary myometrial and fibroid smooth muscle cell cultures were treated with progesterone and/or retinoic acid (RA) and sonic hedgehog (SHH) conditioned media to investigate regulatory pathways for these proteins. We showed that NR2F2 and CTNNB1 are aberrantly expressed in fibroid tissue compared with matched myometrium, with strong blood vessel-specific localisation. Although the SHH pathway was shown to be active in myometrial and fibroid primary cultures, it did not regulate NR2F2 or CTNNB1 mRNA expression. However, progesterone and RA combined regulated NR2F2 mRNA, but not CTNNB1, in myometrial but not fibroid primary cultures. In conclusion, we demonstrate aberrant expression and regulation of NR2F2 and CTNNB1 in uterine fibroids compared with normal myometrium, consistent with the hypothesis that these factors may play a causal role uterine fibroid development.
... However, GLI2 has also been well documented as an important oncogene, and its overexpression has been demonstrated in a variety of tumors (3)(4)(5)(6)(7)(8)(9)(10)(11). In vivo models have shown that GLI2 overexpression alone can drive cancer development (4,10). ...
... GLI2 plays an important role in tumor development and maintenance, and it is found overexpressed in a variety of cancers. This oncogene regulates target genes that contribute to cell cycle progression, cell survival, and invasion and metastasis (3)(4)(5)(6)(7)(8)(9)(10)(11). Interestingly, genetic aberrations to explain this overexpression are uncommon. ...
The modulation of GLI2, an oncogenic transcription factor commonly upregulated in cancer, is in many cases not due to genetic defects, suggesting dysregulation through alternative mechanisms. The identity of these molecular events remains for the most part unknown. Here, we identified TFII-I as a novel repressor of GLI2 expression. Mapping experiments suggest that the INR region of the GLI2 promoter is necessary for GLI2 repression. ChIP studies showed that TFII-I binds to this INR. TFII-I knockdown decreased the binding of NELF-A, a component of the promoter-proximal pausing complex at this site, and enriched phosphorylated RNAPII serine 2 in the GLI2 gene body. Immunoprecipitation studies demonstrate TFII-I interaction with SPT5, another pausing complex component. TFII-I overexpression antagonized GLI2 induction by TGFβ, a known activator of GLI2 in cancer cells. TGFβ reduced endogenous TFII-I binding to the INR and increased RNAPII SerP2 in the gene body. We demonstrate that this regulatory mechanism is not exclusive of GLI2. TGFβ-induced genes CCR7, TGFβ1 and EGR3 showed similar decreased TFII-I and NELF-A INR binding and increased RNAPII SerP2 in the gene body post-TGFβ treatment. Together these results identify TFII-I as a novel repressor of a subset of TGFβ-responsive genes through the regulation of RNAPII pausing.
... Hedgehog (Hh) signaling is essential during the embryonic development for fundamental processes including stem cell maintenance, determination of cell fate, tissue polarity, cell proliferation as well as differentiation [14][15][16]. Recently, it has been investigated that Sonic hedgehog (Shh) signaling network is implicated in pathogenesis of hyperplasic endometrium as well as in early events of endometrial tumorigenesis [17][18][19]. In human, the canonical Hh signaling pathway is initiated in the target cell by the binding of one of the Hh ligands (Sonic, Indian, or Desert) to the transmembrane receptor patched (Ptch) [20][21][22]. ...
... The enhanced ROS generation and accompanying OS indicated the involvement of defensive molecular mechanisms in hyperplasial cells. The induction in Shh signaling molecules mediated via canonical and non-canonical pathways plays important role in EH cells proliferation [18,19]. Thus, we investigated the precise molecular mechanism and explored the link, if any, between Shh signaling and the protective mechanism in primary cultured EH cells. ...
Hh/Gli1 cascade as well as Gsk3β-Gli1 crosstalk play crucial role in estrogen-dependent progression of endometrial hyperplasia (EH). However, the underlying mechanisms involved in progression of disease still remain unclear. In the present study, we explored the role of Hh signaling in protection of endometrial hyperplasial cells against oxidative stress and the underlying mechanism involved therein. EH cells were found to be more resistant towards H2O2–induced oxidative stress (IC50: ~3x) as compared with normal endometrial cells. Estrogen (E2) pre-treatment followed by cytotoxic dose of H2O2, almost rescued the EH cells from apoptosis and caused the increased expression of downstream Shh signaling molecules i.e., Smo, Ptch, and Gli1. Whereas pretreatment with cyclopamine was not able to curtail H2O2-induced effects indicating that estrogen protects these cells via activation of Shh pathway. Further, H2O2- induced ROS and lipid peroxidation alongwith decreased activities of antioxidant enzymes glutathione peroxidase and superoxide dismutase were found to be reversed in EH cells pre- exposed to E2 or rShh. The rShh suppressed H2O2-induced cell death and caused attenuation of mitochondrial apoptotic mediators and prevented disruption in mitochondrial morphology and mitochondrial membrane potential in EH cells. The functional blockage of signaling by Shh siRNA or Gli1 siRNA led to significantly increased expression of mitochondrial fission protein dynamin-like GTPase Drp1. The H2O2 -treated EH cells showed diminished Gli1 and increased Drp1 expression, concurrent with reduced p-Drp1-(serine637). Whereas rShh pre -treated EH cells presented normal mitochondrial dynamics with dense, long networks of mitochondria alongwith nuclear accumulation of Gli1 and the decreased expression of Drp1. Overall, our results implicated that Shh signaling modulates antioxidant defense system and stabilizes mitochondrial dynamics by suppressing Drp1 protein which maintains the survival of EH cells against oxidative stress.
... We used the Kim KH et al. scoring method to evaluate both the intensity of the IHC staining and the proportion of the stained epithelial cells [21]. The staining intensity was classified as 1, weak; 2, moderate; and 3, strong. ...
... These observations suggest that the SHH signaling pathway is activated in thyroid tumorigenesis. This notion is in line with several other studies showing increased expression of the members of the SHH pathway in malignant tumour, such as breast cancer, endometrial adenocarcinomas, and ovarian carcinomas [9,21,24,25]. ...
The sonic hedgehog (SHH) and STAT3 signaling 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 papillary thyroid cancer (PTC), we examined the expression of SHH signaling pathway molecules including SHH, Patched (PTCH), Smoothened (SMO) and GLI1 (glioma-associated oncogene homolog 1), as well as p-STAT3 (phosphorylation at Tyr705) by immunohistochemistry in 164 cases of PTC. In PTC, 70.12%, 64.02%, 68.90%, 64.02%, and 56.71% and in the adjacent normal thyroid tissues, 18.29%, 18.90%, 26.83%, 14.63%, and 10.98% of the specimens stained positive for SHH, PTCH, SMO, GLI1, and p-STAT3, respectively. Significant difference were found for the positive rate of SHH, PTCH, SMO, and GLI1 as well as p-STAT3 expression between PTC and adjacent normal thyroid tissues. There was a high accordance rate between SHH, PTCH, SMO, and GLI1 expression and all of them positively correlated with larger tumor size, the presence of ETE and LNM, and higher TNM stage. P-STAT3 expression positively correlated with the presence of ETE and LNM, and higher TNM stage but not age, gender, tumor size of the PTC patients. Signifi cant positive correlation between p-STAT3 and SHH, PTCH, SMO and GLI expression was found in PTC. These findings suggest that the SHH and STAT3 signaling pathways are frequently activated in PTC, interact with each other and may therefore be indicators for prognosis or potential targets for therapy against PTC.
... Sonic hedgehog (SHH) signaling was first identified as a morphogen that plays a key role in regulating pattern formation throughout embryonic limb development. 7,8 Interestingly, in addition to this canonical function, particular attention has been devoted to the noncanonical functions of SHH as a regulatory signaling molecule in various human uterine diseases, such as endometriosis 9 and endometrial hyperplasia, 10 because of its abnormal activation during the initiation and development of these diseases. Moreover, Palma et al. 11 showed that SHH regulates self-renewal and lineage differentiation of stem cells in post-natal and adult brains. ...
Local endometrial stem cells play an important role in regulating endometrial thickness, which is an essential factor for successful embryo implantation and pregnancy outcomes. Importantly, defects in endometrial stem cell function can be responsible for thin endometrium and subsequent recurrent pregnancy losses. Therefore, many researchers have directed their efforts toward finding a novel stimulatory factor that can enhance the regenerative capacity of endometrial stem cells. Sonic hedgehog (SHH) is a morphogen that plays a key role in regulating pattern formation throughout embryonic limb development. In addition to this canonical function, we identified for the first time that SHH is actively secreted as a stem cell-activating factor in response to tissue injury and subsequently stimulates tissue regeneration by promoting various beneficial functions of endometrial stem cells. Our results also showed that SHH exerts stimulatory effects on endometrial stem cells via the FAK/ERK1/2 and/or phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathways. More importantly, we also observed that endometrial stem cells stimulated with SHH showed markedly enhanced differentiation and migratory capacities and subsequent in vivo therapeutic effects in an endometrial ablation animal model.
... As we all know, endometriosis is a benign disease, but it shares certain characteristics with malignant tumors, such as invasive and unrestrained ectopic cell growth (12). The aberrant expression of GLI1 in endometrial carcinomas has also been described (13,14). Previous research reported that GLI1 is temporary increased in the glandular nuclei and cytoplasm of normal secretory phase endometrium when compared to the proliferative phase (15). ...
Background:
Endometriosis is a benign gynecological disorder which shares certain characteristics with malignant tumor like migration, invasion and proliferation. Glioma-associated oncogene homolog 1 (GLI1) has been implicated in some cancers including endometrial cancer, however, its role in endometriosis remains unknown.
Methods:
The aim of this study was to explore the expression pattern of GLI1 in endometriosis, and further investigate the effect of GLI1 regulation on human endometrial stromal cells. The expression of GLI1 in normal endometrium and ectopic tissues was analyzed by immunohistochemistry, quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and western blot. The Short hairpin RNA (ShRNA) intervention technique and GLI1 inhibitor GANT-61 were used to silence GLI1. The expression levels of GLI1, MMP2 and MMP9 was detected by qRT-PCR and western blot. The migration and invasion ability of human endometrial stromal cells was determined by wound healing assay and transwell migration/invasion assay. The viability and proliferation potentiality of cells was detected by MTT assays and colony formation assay, respectively.
Results:
We found that the expression of GLI1 mRNA and protein were significantly higher in ectopic endometrium from patients with endometriosis. Our analyses also show that GLI1 downregulation attenuated cells migration, invasion and proliferation abilities. What's more, reduced expression of GLI1 inhibited the expression of matrix metalloproteinase 2 (MMP2) and matrix metalloproteinase 9 (MMP9).
Conclusions:
Our findings suggest that high levels of GLI1 may contribute to the development of endometriosis by promoting cell migration, invasion and proliferation involving regulation of MMP2 and MMP9 expression. Therefore, inhibition of GLI1 might be a novel potential therapeutic approach to the treatment of endometriosis, which sheds new light on our understanding of the pathogenesis of endometriosis.
... By binding and inactivating Patched (Ptc/PTCH), Hh signaling pathway unleashes the Smoothened (Smo) transmembrane protein to trigger downstream intracellular events. A previous study showed that SUFU expression is significantly lower in simple and complex endometrial hyperplasia than that in normal endometrium [27]. Accordingly, the expression of SUFU, Gli2, and PTCH2 should be theoretically regulated by NOMAC in the endometrial cancer; however, our RT-qPCR results showed that the mRNA levels of Gli2 and PTCH2 did not change significantly as SUFU upregulation, and they were inconsistent with cDNA microarray analysis. ...
Nomegestrol acetate (NOMAC) has been successfully used for the treatment of some gynecological disorders, and as a combined oral contraceptive with approval in many countries. In this study, we investigated the effects of NOMAC on human endometrial cancer cells in vitro and in vivo. The proliferation of human endometrial cancer cells (RL95-2 and KLE) were assessed using CCK-8 and EdU incorporation assays. Whole-genome cDNA microarray analysis was used to identify the effects of NOMAC on gene expression profiles in RL95-2 cells. RL95-2 xenograft nude mice were treated with NOMAC (50, 100, and 200 mg/kg) or medroxyprogesterone acetate (MPA; 100 and 200 mg/kg) for 28 consecutive days. The results showed that NOMAC significantly inhibited the growth of RL95-2 cells in a concentration-dependent manner, but not in KLE cells. Further investigation demonstrated that NOMAC produced a stronger inhibition of tumor growth (inhibition rates for 50, 100, and 200 mg/kg NOMAC were 24.74%, 47.04%, and 58.06%, respectively) than did MPA (inhibition rates for 100 and 200 mg/kg MPA were 41.06% and 27.01%, respectively) in the nude mice bearing the cell line of RL95-2. NOMAC altered the expression of several genes related to cancer cell proliferation, including SUFU and Wnt7a. The upregulation of SUFU and Wnt7a was confirmed using real-time quantitative polymerase chain reaction and Western blotting in RL95-2 cells and RL95-2 xenograft tumor tissues, but not in KLE cells. These data indicate that NOMAC can inhibit the proliferation of RL95-2 cell in vitro and suppress the growth of xenografts in the nude mice bearing the cell line of RL95-2 in vivo. This effect could be related to the upregulating expression of SUFU and Wnt7a.
... Immunohistochemical analysis of endometrial samples (normal, hyperplastic, neoplastic) showed progressively increasing GLI2 protein expression. Moreover malignant samples had a significantly higher amount of GLI2 localized to the nucleus (Kim et al. 2009b). Hepatocellular carcinoma (HCC) Huh7 cells were sorted and selected using markers of invasive potential (CD133-/ EpCAM-) (Fan et al. 2016). ...
... Further, treatment of Ishikawa and HHUA cells with cyclopamine, a specific inhibitor of Hh pathway suppressed growth of these cell lines by 56 and 67% respectively. Later, Kim et al. (2009) reported an overall increase in expression of Hh signaling molecules in hyperplastic endometrium as compared to normal endometrium. In carcinoma samples extensive alterations in the expression pattern of signaling molecules were observed. ...
Endometrioid endometrial carcinoma (EEC), also known as type I endometrial cancer (EC), accounts for over 70-80% of all cases that are usually associated with estrogen stimulation, and often develops in a background of atypical endometrial hyperplasia. The increased incidence of EC is mainly confined to this type of cancer. Most EEC patients present at an early stage and generally have a favorable prognosis, however, up to 30% of EEC present as high-risk tumors which have invaded deep into the myometrium at diagnosis and progressively lead to local or extra pelvic metastasis. The poor survival of advanced EC is related to the lack of effective therapies, which can be attributed to poor understanding of the molecular mechanisms underlying the progression of disease toward invasion and metastasis. Multiple lines of evidence illustrate that EMT-like events are central to tumor progression and malignant transformation, endowing the incipient cancer cell with invasive and metastatic properties. The aim of this review is to summarize the current knowledge on molecular events associated with EMT in progression, invasion and metastasis of EEC. Further, the role of epigenetic modifications and microRNA regulation, tumor microenvironment and MELF (microcystic, elongated and fragmented glands) like invasion pattern has been discussed. We believe this article may perhaps stimulate further research in this field that may aid in identifying high-risk patients within this clinically challenging patient group and also lead to the recognition of novel targets for the prevention of metastasis-the most fatal consequence of endometrial carcinogenesis.
... The status of GLI2 as an oncogene has also been well established, as outlined in the following paragraphs. Increased expression of GLI2 has been identified in a variety of tumor types, such as basal cell carcinoma, diffuse large B-cell lymphoma, endometrial carcinoma, osteosarcoma, melanoma, colon cancer, prostate cancer, transitional cell carcinoma, pancreatic adenocarcinoma, some types of breast cancer, and ovarian cancer [55][56][57][58][59][60][61][62][63][64][65] . The following sections will discuss the various roles that GLI2 plays in cancer development and progression including cell proliferation and cell cycle progression, cell survival, metastasis, and regulation of the tumor microenvironment. ...
GLI2 is an oncogene encoding a unique transcription factor with both repressor and activator functions. Vitally important in development, it is also thought to be necessary for homeostasis of adult cells. However, deregulation of the GLI2 protein can result in detrimental effects to an organism, such as congenital defects or cancer. Historically deemed an activator and effector molecule of the Hedgehog signaling pathway, GLI2 has since been shown to be a critical effector of other signaling pathways, thus positioning itself as a potent mediator of signaling crosstalk. While GLI2 activity can be modulated by a variety of signaling influences, its regulation at the gene level is less understood. Indeed, gene mutations in GLI2 have been reported, but these generally led to developmental defects and are less commonly identified in tumors as being a cause of its deregulation. While the biological importance of GLI2 overexpression in a multitude of unrelated cancers has been well established, questions about the mechanisms leading to aberrant expression have remained largely unanswered. Furthering our understanding of both the transcriptional regulation of the GLI2 gene and the target genes regulated by GLI2 may identify novel therapeutic targets for cancer treatment.
... Routine hematoxylin-eosin staining was performed to evaluate adrenal morphology. IHC studies were performed using the primary antibodies anti-SHH (sc-1194), anti-GLI1 (sc-20687), anti-GLI2 (Ab-7181), and anti-GLI3 (sc-20688), as has previously been validated (20,21). Labeling was developed with 3,3=diaminobenzine (DAB; Vector Laboratories Inc) and the sections were counterstained with Harris hematoxylin. ...
Background:
The sonic hedgehog (SHH) pathway plays a key role in rodent adrenal cortex development and is involved in tumorigenesis in several human tissues, but data in human adrenal glands are limited.
Objectives:
The objectives of the study were to analyze the involvement of the SHH pathway in human adrenal development and tumorigenesis and the effects of SHH inhibition on an adrenocortical tumor (ACT) cell line.
Patients and methods:
Expression of SHH pathway components was evaluated by immunohistochemistry in 51 normal adrenals (33 fetal) and 34 ACTs (23 pediatric) and by quantitative PCR in 81 ACTs (61 pediatric) and 19 controls (10 pediatric). The effects of SHH pathway inhibition on gene expression and cell viability in the NCI-H295A adrenocortical tumor cell line after cyclopamine treatment were analyzed.
Results:
SHH pathway proteins were present in fetal and postnatal normal adrenals and showed distinct patterns of spatiotemporal expression throughout development. Adult adrenocortical carcinomas presented with higher expression of PTCH1, SMO, GLI3, and SUFU compared with normal adult adrenal cortices. Conversely, pediatric ACTs showed lower mRNA expression of SHH, PTCH1, SMO, GLI1, and GLI3 compared with normal pediatric adrenal cortices. In vitro treatment with cyclopamine resulted in decreased GLI3, SFRP1, and CTNNB1 mRNA expression and β-catenin staining as well as decreased cell viability.
Conclusions:
The SHH pathway is active in human fetal and postnatal adrenals, up-regulated in adult adrenocortical carcinomas, and down-regulated in pediatric ACTs. SHH pathway antagonism impaired cell viability. The SHH pathway is deregulated in ACTs and might provide a new target therapy to be explored.
... 16,17 In gynecological cancers, only one study has reported high expression of Gli3 in endometrial adenocarcinoma. 18 Although both miR-506 and Gli3 have been implicated in cancer progression, the molecular mechanisms by which they affect cancer development, particularly cervical carcinoma, remain unclear. ...
Although significant advances have recently been made in the diagnosis and treatment of cervical carcinoma, the long-term survival rate for advanced cervical cancer remains low. Therefore, an urgent need exists to both uncover the molecular mechanisms and identify potential therapeutic targets for the treatment of cervical cancer. MicroRNAs (miRNAs) have important roles in cancer progression and could be used as either potential therapeutic agents or targets. miR-506 is a component of an X chromosome-linked miRNA cluster. The biological functions of miR-506 have not been well established. In this study, we found that miR-506 expression was downregulated in approximately 80% of the cervical cancer samples examined and inversely correlated with the expression of Ki-67, a marker of cell proliferation. Gain-of-function and loss-of-function studies in human cervical cancer, Caski and SiHa cells, demonstrated that miR-506 acts as a tumor suppressor by inhibiting cervical cancer growth in vitro and in vivo. Further studies showed that miR-506 induced cell cycle arrest at the G1/S transition, and enhanced apoptosis and chemosensitivity of cervical cancer cell. We subsequently identified Gli3, a hedgehog pathway transcription factor, as a direct target of miR-506 in cervical cancer. Furthermore, Gli3 silencing recapitulated the effects of miR-506, and reintroduction of Gli3 abrogated miR-506-induced cell growth arrest and apoptosis. Taken together, we conclude that miR-506 exerts its anti-proliferative function by directly targeting Gli3. This newly identified miR-506/Gli3 axis provides further insight into the pathogenesis of cervical cancer and indicates a potential novel therapeutic agent for the treatment of cervical cancer.Oncogene advance online publication, 10 March 2014; doi:10.1038/onc.2014.9.
... It has been shown that mRNA levels do not always correspond to protein levels. 32 The low number of patients examined in the later study might also explain the discrepancy. Additionally, in our study, Gli1, although a transcriptional target of Hh pathway activation, did not follow Ptch expression level modulations in primary prostate carcinomas and bone marrow metastases. ...
The hedgehog (Hh) signalling pathway has been implicated in the pathogenesis and aggressiveness of prostate cancer through epithelial-mesenchymal interactions. The aim of this study was to elucidate the cell-type partitioned expression of the Hh pathway biomarkers in the non-neoplastic and tumour microenvironments and to correlate it with the grade and stage of prostate cancer.
Expression of the Hh pathway components (Shh, Smo, Ptch, Gli1) in the microenvironment of non-neoplastic peripheral zone (n = 119), hormone-naive primary prostate carcinoma (n = 141) and castrate-resistant bone marrow metastases (n = 53) was analysed using immunohistochemistry in tissue microarrays and bone marrow sections. Results showed that epithelial Shh, Smo and Ptch expression was up-regulated, whereas stromal Smo, Ptch, and Gli1 expression was down-regulated in prostate carcinomas compared to non-neoplastic peripheral zone tissue. Ptch expression was modulated further in high-grade and high-stage primary tumours and in bone marrow metastases. Hh signalling correlated with ki67 and vascular endothelial growth factor (VEGF) but not with CD31 expression.
Our results highlight the importance of Hh-mediated epithelial-mesenchymal interactions in the non-neoplastic prostate and imply that shifting the balance from paracrine towards autocrine signalling is important in the pathogenesis and progression of prostate carcinoma.
... Many studies have found that the abnormal activation of Gli2 could greatly enhance the transcription of controlled molecules downstream the Hedgehog signaling pathway, leading to a Table 4 The genes at the core of the network. Degree denotes the number of the genes that interact with other genes (that is the number of the edges in the network), Indegree denotes the degree that other genes regulate the gene, and Outdegree denotes the degree that the gene regulates other genes Gene ID Gene Degree Indegree Outdegree Style 54361 WNT4 11 3 8 down 5468 PPARG 10 0 1 down 2776 GNAQ 7 2 0 up 7157 TP53 6 1 5 up 1499 CTNNB1 6 1 3 up 2763 GLI2 6 1 3 up Fig. 3 Quantitative real-time PCR analysis of LOX in the PA and normal tissue samples variety of tumors [17][18][19][20]. In this study, we detected overexpression of Gli2 in PA using cDNA microarray and validated this by real-time PCR. ...
Recent studies have determined that gene expression profiling using microarray technology can be used to identify tumor-related molecules. The objective of this study was to screen the differentially expressed genes between pleomorphic adenoma (PA) and the normal tissue adjacent to PA using cDNA microarrays and to further validate the differentially expressed genes by real-time PCR. In this study, we selected five pairs of PA and the surrounding normal salivary gland tissues. The total RNA was isolated from tumor and normal tissues and purified to mRNA. The mRNA was reverse-transcribed to cDNA with the incorporation of fluorescent-labeled dUTP to prepare the hybridization probes. The mixed probes were hybridized to Whole Human Gene Expression Microarrays by Agilent. Tumor-related genes were screened by analyzing the fluorescence intensity. As a result, a total of 447 genes were found to be differentially expressed between PA and normal tissue adjacent to PA. Among them, 185 genes were up-regulated and 262 genes were down-regulated in PA. By constructing a network from the differentially expressed genes, some genes, such as Gli2 and CTNNB1, were identified as being at the core of the network. In addition, differential gene expression was validated for 2 up-regulated genes, Gli2 and LOX, using real-time PCR and the results were consistent with those of the cDNA microarray analysis thus verifying the credibility of the microarray data. Therefore, our microarray data may provide clues for finding novel genes involved in the development of PA, and shed light on finding new targets for diagnosis and therapy of PA. Further characterization of these differentially expressed genes will be useful in understanding the genetic basis for PA.
... to identify high-risk patients with hepatoblastoma, we also documented a heterogeneous protein expression pattern of Hh signal components in the cancer group. our results also suggest that Patched and gli1 protein overexpression were correlated with poor prognosis of patients, which were also suggested in other cancers[19,20]. gli-1 nuclear expression might therefore be a useful and reliable biomarker for the screening and management of high-risk patients with poorer prognosis. More importantly, the association between Patched and gli1 protein overexpression with poor survival of hepatoblastoma patients was demonstrated for the first time. ...
Activation of hedgehog (Hh) pathway has been implicated in the development of human malignancies. Hh as well as related downstream target genes has been extensively studied in many kinds of malignant tumours for clinical diagnostic or prognostic utilities. This study aimed at investigating whether Hh molecules provides a molecular marker of hepatoblastoma malignancy.
We obtained tissue sections from 32 patients with hepatoblastoma as well as cholestasis and normal control. Immunohistochemical analysis were performed to determine Hh signal components in human hepatoblastoma. The prognostic significance of single expression of Hh signal components were evaluated using Cox proportional hazards regression models and Kaplan-Meier survival analysis for statistical analysis.
Expression of Hh signal components showed an increase in hepatoblastoma compared with cholestasis and normal tissues. There was a positive correlation between Smo or Gli1 expression and tumor clinicopathological features, such as histological type, tumor grade, tumor size and clinical stage. Both Smo or Gli1 protein high expression was significantly associated with poor prognosis by univariate analyses and multivariate analyses.
Abnormal Hh signaling activation plays important roles in the malignant potential of hepatoblastoma. Gli1 expression is an independent prognostic marker.
Background:
The hedgehog pathway (HH) is one of the key regulators involved in many biological events. Malfunction of this pathway is associated with a variety of diseases including several types of cancers.
Methods:
We collected data from public databases and conducted a comprehensive search linking the HH pathway with female cancers. In addition, we overviewed clinical trials of targeting HH pathway in female cancers.
Results:
The activation of HH pathway and its role in female cancers, including breast cancer, ovarian cancer, cervical cancer, endometrial cancer, and uterine leiomyosarcoma were summarized. Treatment options targeting SMO and GLI in HH pathway were reviewed and discussed.
Conclusions:
The hedgehog pathway was shown to be activated in several types of female cancers. Therefore, targeting HH pathway may be considered as a therapeutic option to be acknowledged in the treatment of female cancers.
Oestrogen–progesterone signalling is highly versatile and critical for the maintenance of healthy endometrium in humans. The genomic and nongenomic signalling cascades initiated by these hormones in differentiated cells of endometrium have been the primary focus of research since 1920s. However, last decade of research has shown a significant role of stem cells in the maintenance of a healthy endometrium and the modulatory effects of hormones on these cells. Endometriosis, the growth of endometrium outside the uterus, is very common in infertile patients and the elusiveness in understanding of disease pathology causes hindrance in selection of treatment approaches to enhance fertility. In endometriosis, the stem cells are dysfunctional as it can confer progesterone resistance to their progenies resulting in disharmony of hormonal orchestration of endometrial homeostasis. The bidirectional communication between stem cell signalling pathways and oestrogen–progesterone signalling is found to be disrupted in endometriosis though it is not clear which precedes the other. In this paper, we review the intricate connection between hormones, stem cells and the cross-talks in their signalling cascades in normal endometrium and discuss how this is deregulated in endometriosis. Re-examination of the oestrogen–progesterone dependency of endometrium with a focus on stem cells is imperative to delineate infertility associated with endometriosis and thereby aid in designing better treatment modalities.
Context:
Identifying novel molecular markers for assessing endometrial receptivity is necessary for understanding human implantation and may help in improving the clinical outcome of in vitro fertilization.
Objective:
We aimed to compare the gene expression profiles of the pre-receptive vs receptive phases of the natural cycle in human endometrial biopsies.
Design:
The design of this study was detecting the global gene expression profile of human endometrial receptivity by RNA-Seq.
Setting:
This study was conducted at a university reproductive center.
Participants:
Twelve women with normal menstrual cycles participated in the study.
Intervention:
Study interventions included endometrial biopsies.
Main outcome measures:
The endometrial transcriptomes were determined by RNA-Seq, and the expression of selected differentially expressed genes (DEGs) was validated by quantitative RT-PCR.
Results:
A total of 2372 DEGs were identified by RNA-Seq. Of these genes, 1099 were up-regulated at LH+7 versus LH+2, whereas 1273 were down-regulated. Nineteen selected genes were confirmed by quantitative RT-PCR. We first demonstrated that metallothionein (MT) family members, MT1E, F, G, H, M, X, and 2A, and four novel transcripts, HAP1, ZCCHC12, MRAP2, and OVGP1, which were not previously linked to endometrial physiology, showed significant expression changes during implantation. Mineral absorption was the most enriched pathway for up-regulated genes, and cell cycle was enriched for down-regulated genes. Gene co-expression network analysis identified five core regulatory factors (GLI2, CDC25A, TLR9, MT1G, and SLC5A1) that are involved in endometrial receptivity during implantation. Examination of the promoter regions of the DEGs identified AP2 and SP1 binding sites, suggesting a potential regulatory role in endometrial gene expression for these two transcription factors.
Conclusions:
This study provides the first RNA-Seq-based transcriptome comparison of pre-receptive and receptive human endometria. Many novel candidate genes, which have not been previously studied in human endometrium, may have functional significance during implantation and serve as molecular markers for endometrial receptivity.
It was the aim of this study to evaluate the diagnostic utility of Notch-1 immunocytochemistry in distinguishing endometrial glandular and stromal breakdown (EGBD) from endometrial adenocarcinoma in endometrial cytology.
Samples of normal endometrium, EGBD and endometrial adenocarcinoma were subjected to immunocytochemical staining for Notch-1, and we examined the labeling index (LI) of Notch-1 (the ratio of intranuclear Notch-1-positive cells to total cells). We compared (1) the Notch-1 LI in normal endometrium, (2) the Notch-1 LI between normal endometrium and endometrial adenocarcinoma, and (3) the Notch-1 LI in normal endometrium, EGBD and endometrial adenocarcinoma.
In analysis item 1, the LI of Notch-1 was 32.9 ± 8.4, 19.4 ± 8.2 and 12.5 ± 7.5% in proliferative endometrium, secretory endometrium and atrophic endometrium, respectively. In analysis item 2, the LI of Notch-1 in endometrial adenocarcinoma was 45.2 ± 7.4%, which was significantly higher than that in normal endometrium. In analysis item 3, the LI of Notch-1 in EGBD was 31.3 ± 8.3%, which was significantly lower than that in endometrial adenocarcinoma.
In conclusion, Notch-1 immunocytochemistry is a useful method for distinguishing between EGBD and endometrial carcinoma in endometrial cytology.
The role of hedgehog (HH) signaling in reproductive tract development was studied in mice in which a dominant active allele of the signal transducer smoothened (SmoM2) was conditionally expressed in the Müllerian duct and ovary. Mutant females are infertile, primarily because they fail to ovulate. Levels of mRNA for targets of HH signaling, Gli1, Ptch1, and Hhip, were elevated in reproductive tracts of 24-day-old mutant mice, confirming overactivation of HH signaling. The tracts of mutant mice developed abnormally. The uterine luminal epithelium had a simple columnar morphology in control mice, but in mutants contained stratified squamous cells typical of the cervix and vagina. In mutant mice, the number of uterine glands were reduced and the oviducts were not coiled. Expression of genes within the Hox and Wnt families that regulate patterning of the reproductive tract were altered. Hoxa13, which is normally expressed primarily in the vagina and cervix, was expressed at 12-fold higher levels in the uterus of mutant mice compared with controls. Wnt5a, which is required for development of the cervix and vagina and postnatal differentiation of the uterus, was expressed at higher levels in the oviduct and uterus of mutant mice compared with controls. Mating mutant females with fertile or vasectomized males induced a severe inflammatory response in the tract. In summary, overactivation of HH signaling causes aberrant development of the reproductive tract. The phenotype observed could be mediated by ectopic expression of Hoxa13 in the uterus and elevated levels of Wnt5a in the oviducts and uterus.
Cytotoxic therapy and surgery have improved outcomes for patients with gynecologic malignancies over the last twenty years, but women's cancers still account for over ten percent of cancer related deaths annually. Insights into the pathogenesis of cancer have led to the development of drugs that target molecular pathways essential to tumor survival including angiogenesis, DNA repair, and apoptosis. This review outlines several of the promising new biologically targeted drugs currently being tested to treat gynecologic malignancies.
Explants from nonmenstrual endometria cultured in the absence of ovarian hormones undergo tissue breakdown. Addition of estradiol and progesterone (EP) prevents proteolysis. Explants include stromal and epithelial compartments which play different but complementary roles in endometrial physiology, including tissue remodeling and hormonal response. In order to characterize the cell type-specific contribution to regulation of tissue breakdown, we characterized the transcriptomes of microdissected stromal and glandular areas from endometrial explants cultured with or without EP. The datasets were also compared to other published endometrial transcriptomes. Finally, the contribution of proteolysis, hypoxia, and MAPKs to the regulation of selected genes was further investigated in explant culture. This analysis identified distinct gene expression profiles in stroma and glands, with differential response to EP, but functional clustering underlined convergence in biological processes, further indicating that endometrial remodeling requires cooperation between the two compartments through expression of cell type-specific genes. Only partial overlaps were observed between lists of genes involved in different occurrences of endometrial breakdown, pointing to a limited number of potentially crucial regulators but also to the requirement for additional mechanisms controlling tissue remodeling. We identified a group of genes differentially regulated by EP in stroma and glands among which some were sensitive to MAPKs and/or aspartic proteinases and were not induced by hypoxia. In conclusion, MAPKs and/or aspartic proteinases likely act in concert with EP to locally and specifically control differential expression of genes between degrading and preserved areas of the human endometrium.
With the realization that many proto-oncogenes and tumor suppressor genes are expressed and have important functions during mammalian development, it is clear that cancer often involves the inappropriate activation of genetic pathways used during normal development. A signaling cascade that has been of considerable interest to both developmental and cancer biologists involves the Hedgehog (Hh) family of secreted proteins. To date, the only transcription factors shown to be directly downstream of Hh are the zinc-finger containing proteins Cubitus interruptus (Ci) and Gli, in flies and vertebrates, respectively. The identification of many of the genes and proteins involved in Hh signaling has come largely from genetic and biochemical studies in Drosophila. Ci mediates Hh signaling through a Hh-dependent set of protein modifications that alter the activity of Ci on Hh target genes. Recent evidence suggests vertebrate Gli proteins may be similarly regulated. The interest in this pathway has taken on added importance with the identification of mutations in Hh pathway genes, including Gli genes, in several human developmental disorders and cancers. We discuss models for how Gli proteins mediate Hh signaling in both vertebrate development and cancers.
Do tumours arise from stem cells, or are they derived from more differentiated cells that, for some reason, begin to recapitulate developmental programmes? Inappropriate activation of the Sonic hedgehog-Gli signalling pathway occurs in several types of tumour, including those of the brain and the skin. Studies in these and other systems suggest that inappropriate function of the Gli transcription factors in stem or precursor cells might lead to the onset of a tumorigenic programme and that these factors are prime targets for anticancer therapies.
The Hedgehog (Hh) signaling pathway functions as an organizer in embryonic development. Genetic analysis has demonstrated a critical role for the Hh pathway in mammary gland morphogenesis. Disruption of Patched1, a component of the Hh pathway, results in abnormal growth of mammary duct. Recent studies have shown constitutive activation of the Hh pathway in various types of malignancies. However, it remains unclear whether this pathway is activated in human breast cancer. Here, we determined the expression of the components, including Sonic Hh, Patched1, and Gli1, of the Hh pathway by immunohistochemical staining in a series of 52 human breast carcinomas. All of 52 tumors display staining of high intensity for Gli1 when compared with adjacent normal tissue. The nuclear staining ratio of Gli1 correlates with expression of estrogen receptor and histologic type. Exposure to cyclopamine, a steroidal alkaloid that blocks the Hh pathway, suppresses expression of Gli1 and the growth of the Hh pathway-activated breast carcinoma cells. These data indicate that the Hh pathway is a new candidate for therapeutic target of breast cancer.
The hedgehog (Hh) signaling pathway plays an important role in foregut development, and its activity is increased in various tumors, including those of the digestive tract. Our objective in the present study was to determine the pattern and extent of Sonic hedgehog (Shh) expression in gastric cancer and related lesions as well as the methylation status of its promoter region, in an attempt to clarify the regulatory mechanism of Shh expression. A total of 237 gastric cancers and related lesions (89 carcinomas, 22 high-grade dysplasia, 21 low-grade dysplasia, 47 intestinal metaplasia, 38 chronic gastritis, and 20 normal epithelia) were subjected to immunohistochemical analysis with the Shh monoclonal antibody. The methylation status of Shh was determined by methylation-specific PCR (MS PCR), involving bisulfate treatment of DNA from 150 tissues followed by amplification using specific primer pairs designed by our group. Shh was completely absent in the upper part of normal gastric epithelia (gastric pit cells), and no significant differences were observed among the lower parts of normal epithelia, chronic gastritis, and intestinal metaplasia. However, Shh expression was significantly elevated in neoplastic lesions, such as carcinoma and high low-grade dysplasia, compared to non-neoplastic lesions. In carcinomas, Shh expression was associated with clinical stage, direct tumor invasion, and differentiation of tumor cells. Methylation of the Shh promoter region was frequent in normal gastric pit cells (11/18, 61.1%), but very rare in gastritis (0/18), intestinal metaplasia (0/19), dysplasia (0/10), and carcinoma (1/63, 1.6%), and correlated significantly with expression (P<0.001). Our results suggested that the increased and constitutive Shh expression is implicated in gastric carcinogenesis, and that promoter methylation may be an important regulatory mechanism of Shh expression.
Mammalian Gli proteins are important transcription factors involved in the regulation of Sonic hedgehog signal transduction pathway. Association of Gli2 with mammalian development and human disease led us to study the structure and expression of the human GLI2.
We show that the region encoding GLI2 repressor domain is subject to alternative splicing in the gonadal tissues and different cell lines. Two major alternatively spliced forms of GLI2 mRNA arise from skipping exon 3 (GLI2Delta3) or exons 4 and 5 (GLI2Delta4-5). Both forms contain premature translational stop codons in the GLI2 open reading frame (ORF) starting from exon 2. Translation of GLI2Delta3 and GLI2Delta4-5 in vitro, initiated from downstream AUG codons, produced N-terminally truncated proteins. In Gli-dependent transactivation assay, expression of GLI2Delta3 induced activation of the reporter gene similar to that of the full-length construct (GLI2fl) containing complete ORF. However, expression of the GLI2Delta4-5 resulted in about 10-fold increase in activation, suggesting that deletion of the major part of repressor domain was responsible for the enhanced activation of GLI2 protein.
Our data suggest that in addition to proteolytic processing, alternative splicing may be another important regulatory mechanism for the modulation of repressor and activator properties of GLI2 protein.
The hedgehog (Hh)-signaling pathway plays an essential role in normal development. Deregulation of this pathway is responsible for several types of cancers. The aim of this study was to determine the expression pattern and the extent of Hh-signaling molecules in squamous cell carcinoma of uterine cervix and its precursor lesions. A total of 106 uterine cervical cancers and related lesions (37 squamous cell carcinomas, 23 cervical intraepithelial neoplasia (CIN) III, 10 CIN II, four CIN I, 32 normal cervical epithelia) were immunohistochemically analyzed with anti-Shh, Indian Hh (Ihh), Patched (PTCH), Smoothened (Smo), Gli-1, Gli-2, Gli-3 antibodies on paraffin blocks. The results showed that the expression of all the Hh-signaling molecules was greatly enhanced in uterine cervical tumors, including carcinoma and its precursor lesions. The staining pattern was mainly cytoplasmic except for Gli-1/2, whose expression was observed in both cytoplasm and nucleus. In case of Ihh, PTCH, Smo and Gli-1, their expression in normal epithelium was completely absent or rare. The expression of all the seven Hh-signaling molecules mentioned above was significantly increased in CIN II/III and carcinoma, compared with that in normal epithelium (P < 0.05). The expression of Shh was increased by double; the first increase occurred in normal epithelium-CIN transition, and the second, during the progression of CIN to carcinoma. These results strongly suggest that the Hh-signaling pathways were extensively activated in carcinoma and CIN of uterine cervix. In conclusion, the Hh-signaling pathways may be involved in carcinogenesis of squamous cell carcinoma of uterine cervix and can be considered as a potential therapeutic target.
Research has revealed abnormal activation of the hedgehog pathway in human malignancies. The present study was undertaken to examine the expression and functional involvement of the hedgehog pathway in endometrial tissues.
The expression of sonic hedgehog (Shh), patched (Ptch), Smoothened (Smo), and Gli1 was examined in various endometrial tissues and endometrial carcinoma cell lines. The effect of hedgehog signaling on the proliferation of endometrial carcinoma cell lines was also examined.
The expression of Shh, Ptch, Smo, and Gli1 was very weak in normal endometrium, but was increased in endometrial hyperplasia and carcinoma stepwisely with significant differences. There was no marked difference in the expression of these molecules in carcinomas according to stages and histologic grades. Treatment with cyclopamine, a specific inhibitor of the hedgehog pathway, for endometrial carcinoma Ishikawa and HHUA cells suppressed growth by 56% and 67%, respectively, compared with the control. The addition of recombinant Shh peptide to HHUA cells enhanced their proliferation by 41%. The silencing of Gli1 using small interfering RNA (siGli1) resulted in the growth suppression and down-regulation of Ptch expression. In addition, the cyclopamine/siGli1-induced growth suppression was associated with the down-regulation of cyclins D1 and A and N-myc. No somatic mutations for ptch and smo genes were detected in the endometrial carcinoma cases examined.
The abnormal activation of this pathway is involved in the proliferation of endometrial carcinoma cells possibly in an auto-/paracrine fashion, suggesting the possibility of the hedgehog pathway being a novel candidate for molecular targeting.
The association of endometrial carcinoma (EC) with endocrinopathies manifested by obesity, nulliparity, and/or increased estrogen levels of exogenous or endogenous estrogens is now well-known. EC is also seen in patients without these findings. Are these different cancers? Seventy-four cases of EC were reviewed and classified into two groups: group I, with associated adenomatous hyperplasia (AH), 31 cases; and group II, without associated AH, 43 cases. Group I included more well-differentiated and less invasive carcinomas; histologically, the pattern was glandular in all cases. In Group II, the EC were less well-differentiated, more invasive, and included, besides adenocarcinomas, clear-cell, papillary, and anaplastic carcinomas with giant tumor cells. Squamoid features were found in both groups. The possible existence of two types of EC, a hormonal-dependent EC associated with AH (which is believed to result from hyperestrogenism, and to have a better clinical prognosis), and an "independent" EC, not associated with AH, is discussed.
Apoptosis or programmed cell death represents a mechanism by which cells possessing DNA damage can be deleted. The bcl-2 proto-oncogene is a known inhibitor of apoptosis that may allow the accumulation and propagation of cells containing genetic alterations. To determine if and when the bcl-2 gene is activated during colorectal tumorigenesis and its relationship to p53, we analyzed normal mucosa, hyperplastic and dysplastic epithelial polyps, and carcinomas for the expression of these markers using immunohistochemistry. Whereas bcl-2 staining was restricted to basal epithelial cells in normal and hyperplastic mucosa, bcl-2 expression was detected in parabasal and superficial regions in dysplastic polyps and carcinomas. An inverse correlation was found between bcl-2 and p53 expression in adenomas, suggesting that these markers may regulate a common cell death pathway. Furthermore, carcinomas with a high percentage of bcl-2-positive cells were significantly more likely to have low rates of spontaneous apoptosis, as determined histologically, than those cancers with low or absent bcl-2 expression. Abnormal activation of the bcl-2 gene appears to be an early event in colorectal tumorigenesis that can inhibit apoptosis in vivo and may facilitate tumor progression.
Gli family zinc finger proteins are mediators of Sonic hedgehog (Shh) signaling in vertebrates. The question remains unanswered, however, as to how these Gli proteins participate in the Shh signaling pathway. In this study, regulatory activities associated with the Gli2 protein were investigated in relation to the Shh signaling. Although Gli2 acts as a weak transcriptional activator, it is in fact a composite of positive and negative regulatory domains. In cultured cells, truncation of the activation domain in the C-terminal half results in a protein with repressor activity, while removal of the repression domain at the N terminus converts Gli2 into a strong activator. In transgenic mouse embryos, N-terminally truncated Gli2, unlike the full length protein, activates a Shh target gene, HNF3beta, in the dorsal neural tube, thus mimicking the effect of Shh signal. This suggests that unmasking of the strong activation potential of Gli2 through modulation of the N-terminal repression domain is one of the key mechanisms of the Shh signaling. A similar regulatory mechanism involving the N-terminal region was also found for Gli3, but not for Gli1. When the Shh signal derived from the notochord is received by the neural plate, the widely expressed Gli2 and Gli3 proteins are presumably converted to their active forms in the ventral cells, leading to activation of transcription of their target genes, including Gli1.
A surprising number of apparently unrelated human diseases, including familial and sporadic cancers and a number of syndromes and malformations, seem to be associated with abnormal function of the Hedgehog (Hh) signaling pathway. Zinc-finger transcription factors of the Gli family play critical roles in the mediation and interpretation of Hh signals. Elucidating how Gli proteins work will enable us to further our knowledge of how cells proliferate, differentiate or survive in response to Hh signals, as well as to design rational therapies for these Hh-signaling related diseases (HSDs).
Developmental pathways first elucidated by genetic studies in the fruit fly, Drosophila melanogaster, are conserved in vertebrates, and disruption of these pathways has been associated with various human congenital anomalies. Many developmental genes continue to play an important role in regulation of cell growth and differentiation after embryogenesis, and mutations in some of these genes can result in cancer. Basal cell carcinoma (BCC) of the skin is the most common type of cancer in humans. Although most BCCs are sporadic, in rare cases, individuals have a hereditary disease, Gorlin syndrome, that predisposes to multiple skin tumors as well as a variety of birth defects. Mutations in the human homolog of a Drosophila gene, patched, underlie Gorlin syndrome. Genetic studies in Drosophila show that patched is part of the hedgehog signaling pathway, important in determining embryonic patterning and cell fate in multiple structures of the developing embryo. Human patched is mutated in sporadic as well as hereditary BCCs, and inactivation of this gene is probably a necessary if not sufficient step for tumor formation. Delineation of the biochemical pathway in which patched functions may lead to rational medical therapy for skin cancer and possibly other tumors.
Tumors of the central nervous system (CNS) can be devastating because they often affect children, are difficult to treat, and frequently cause mental impairment or death. New insights into the causes and potential treatment of CNS tumors have come from discovering connections with genes that control cell growth, differentiation, and death during normal development. Links between tumorigenesis and normal development are illustrated by three common CNS tumors: retinoblastoma, glioblastoma, and medulloblastoma. For example, the retinoblastoma (Rb) tumor suppressor protein is crucial for control of normal neuronal differentiation and apoptosis. Excessive activity of the epidermal growth factor receptor and loss of the phosphatase PTEN are associated with glioblastoma, and both genes are required for normal growth and development. The membrane protein Patched1 (Ptc1), which controls cell fate in many tissues, regulates cell growth in the cerebellum, and reduced Ptc1 function contributes to medulloblastoma. Just as elucidating the mechanisms that control normal development can lead to the identification of new cancer-related genes and signaling pathways, studies of tumor biology can increase our understanding of normal development. Learning that Ptc1 is a medulloblastoma tumor suppressor led directly to the identification of the Ptc1 ligand, Sonic hedgehog, as a powerful mitogen for cerebellar granule cell precursors. Much remains to be learned about the genetic events that lead to brain tumors and how each event regulates cell cycle progression, apoptosis, and differentiation. The prospects for beneficial work at the boundary between oncology and developmental biology are great.
The Suppressor of Fused [Su(fu)] protein plays a conserved role in the regulation of Gli transcription factors of the hedgehog (Hh) signaling pathway that controls cell fate and tissue patterning during development. In both Drosophila and mammals, Su(fu) represses Gli-mediated transcription, but the mode of its action is not completely understood. Recent evidence suggests that Su(fu) physically interacts with the Gli proteins and, when overexpressed, sequesters Gli in the cytoplasm. However, Su(fu) also traverses into the nucleus under the influence of a serine-threonine kinase, Fused (Fu), and has the ability to form a DNA-binding complex with Gli, suggesting that it has a nuclear function. Here we report that the mouse homolog of Su(fu) [mSu(fu)] specifically interacts with SAP18, a component of the mSin3 and histone deacetylase complex. In addition, we demonstrate that mSu(fu) functionally cooperates with SAP18 to repress transcription by recruiting the SAP18-mSin3 complex to promoters containing the Gli-binding element. These results provide biochemical evidence that Su(fu) directly participates in modulating the transcriptional activity of Gli.
Genes encoding components of the hedgehog signaling pathway are dynamically expressed in the mouse uterus preparing for implantation. Indian hedgehog (Ihh), patched (Ptc), and Gli3 are expressed at low levels in the endometrial epithelium on day 1 of pregnancy. Transcription of Ihh increases dramatically in the luminal epithelium and glands from day 3, reaching very high levels on day 4. Over the same period, Ptc, Gli1, Gli2, and noggin are strongly upregulated in the underlying mesenchymal stroma. Transcription of Ihh in ovariectomized mice is induced by progesterone but not by estrogen. Lower induction of Ihh, Ptc, and Hoxa10 is seen in response to progesterone in the uteri of Pgr(-/-) mutant mice lacking progesterone nuclear steroid receptor. This finding suggests that the hormone may regulate Ihh through both nuclear receptor-dependent and -independent pathways. We describe a method for culturing uterine explants in the absence of epithelium. Under these conditions, recombinant N-SHH protein promotes the proliferation of mesenchyme cells and the expression of noggin. We propose that IHH made by the epithelium normally functions as a paracrine growth factor for stromal cells during the early stages of pregnancy.
Cancer arises when a cell accumulates multiple genetic changes that allow it to elude the highly regulated balance between proliferation and apoptosis that an organism employs to suppress inappropriate growth. It has become evident that malignant transformation of a cell or group of cells often involves pathways that are active during normal development but are inappropriately regulated in neoplastic proliferation. Signaling via the Sonic hedgehog pathway is critical to vertebrate development and also appears to play an integral role in the initiation and propagation of some tumors of the muscle, skin and nervous system. Analyses of human tumors have revealed mutations in various components of the Sonic hedgehog signaling pathway that appear to result in the activation of this pathway, as inferred by the increased expression of the transcription factor, Gli1. Interestingly, a proportion of the human tumors and most of those arising in mouse models continue to express the normal Patched allele, suggesting the involvement of additional molecular events in the transformation of the haploinsufficient cells.
Embryonic signalling pathways regulate progenitor cell fates in mammalian epithelial development and cancer. Prompted by the requirement for sonic hedgehog (Shh) signalling in lung development, we investigated a role for this pathway in regeneration and carcinogenesis of airway epithelium. Here we demonstrate extensive activation of the hedgehog (Hh) pathway within the airway epithelium during repair of acute airway injury. This mode of Hh signalling is characterized by the elaboration and reception of the Shh signal within the epithelial compartment, and immediately precedes neuroendocrine differentiation. We reveal a similar pattern of Hh signalling in airway development during normal differentiation of pulmonary neuroendocrine precursor cells, and in a subset of small-cell lung cancer (SCLC), a highly aggressive and frequently lethal human tumour with primitive neuroendocrine features. These tumours maintain their malignant phenotype in vitro and in vivo through ligand-dependent Hh pathway activation. We propose that some types of SCLC might recapitulate a critical, Hh-regulated event in airway epithelial differentiation. This requirement for Hh pathway activation identifies a common lethal malignancy that may respond to pharmacological blockade of the Hh signalling pathway.
Sonic hedgehog (Shh) controls critical cellular decisions between distinct fates in many systems, particularly in stem cells. The Shh network functions as a genetic switch, and we have theoretically and computationally analyzed how its structure can endow it with the ability to switch fate choices at a threshold Shh concentration. The network is composed of a positive transcriptional feedback loop embedded within a negative signaling feedback loop. Specifically, positive feedback by the transcription factor Gli, which upregulates its own expression, leads to a switch that can adopt two distinct states as a function of Shh. However, Gli also upregulates the signaling repressor Patched, negative feedback that reins in the strong Gli autoregulatory loop. Mutations that have been associated with cancer are predicted to yield an irreversible switch to a high Gli state. Finally, stochastic simulation reveals the negative Patched feedback loop serves a critical function of dampening Gli fluctuations to reduce spontaneous state switching and preserve the network's robust, switch-like behavior. Tightly linked positive and negative feedback loops are present in many signaling systems, and the Shh system is therefore likely representative of a large set of gene regulation networks that control stem cell fate throughout development and into adulthood.
The Hedgehog (Hh) signalling pathway is crucial for normal development and patterning of numerous human organs including the gut. Hh proteins are also expressed during gastric gland development and gastric epithelial differentiation in adults. Recently, dysregulation of these developmentally important genes has been implicated in cancer, leading to the present study of the expression of Hh signalling proteins in colon cancer. In this study, normal colon and colonic lesions (hyperplastic polyp, adenoma, and colonic adenocarcinoma) were examined by immunohistochemistry using antibodies against Hh signalling molecules: the secreted protein Sonic hedgehog (SHH), its receptor Patched (PTCH), and the PTCH-associated transmembrane protein Smoothened (SMOH). The study shows that Hh signalling pathway members are expressed in normal colonic epithelium. SHH was expressed at the top of the crypts and in a few basally located cells, while PTCH was detected in the neuroendocrine cells and SMOH at the brush border of superficial epithelium. RT-PCR analysis of laser-microdissected crypts from normal human colon confirmed that mRNAs encoding these proteins were expressed in colonic epithelium. Expression of SHH, PTCH, and SMOH was up-regulated in hyperplastic polyps, adenomas, and adenocarcinomas of the colon, and SHH expression correlated with increased expression of the proliferation marker Ki-67 in all lesions examined. To address whether the Hh signalling pathway is functional in the gut, the effect of Shh on epithelial cells in vitro was explored by treating primary murine colonocytes with either Shh peptide or neutralizing anti-Shh antibody. The proportion of cells in the S-phase was assessed by bromodeoxyuridine (BrdU) incorporation. It was found that exogenous Shh promotes cell proliferation in colonocytes, while anti-Shh inhibits proliferation, suggesting that Shh is required during proliferation of epithelial cells in vitro. It is suggested that SHH is required during epithelial proliferation in the colon and that there is a possible role for Hh signalling in epithelial colon tumour progression in vivo.
To investigate proliferation in disease free postmenopausal endometrium and that harbouring endometrial adenocarcinoma-is there a dynamic, yet lurking, potential for atrophic endometrium to give rise to endometrial adenocarcinoma?
The study comprised 84 disease free endometria from asymptomatic postmenopausal women who had undergone hysterectomy for prolapse, and 50 endometrioid cell type endometrial adenocarcinomas with adjacent uninvolved postmenopausal endometrium. The non-neoplastic tissues were separated histologically into active, inactive, and mixed forms, although only the first two categories were studied immunohistochemically for oestrogen and progesterone receptors (ERs, PRs), epidermal growth factor receptor (EGFR), Ki-67, and angiogenic activity.
All postmenopausal endometria were atrophic, but only 42 were inactive; of the remaining samples, 22 were weakly proliferative and 20 were mixed active and inactive. In contrast, the non-neoplastic component of 43 of the 50 endometrial adenocarcinomas examined was of the active form; four specimens were of the pure and 39 of the mixed form. Interestingly, high ER and PR expression was seen in active and inactive endometria, but only the former were EGFR positive and had high proliferative (Ki-67) and angiogenic activity. A similar trend was also shown by the non-neoplastic atrophic endometrium adjacent to endometrial adenocarcinoma.
At least half of the disease free postmenopausal atrophic endometria show a weak proliferative pattern, either diffuse or focal, probably as a response to continuous low level oestrogenic stimulation. These tissues have a latent, although very small, carcinogenic potential, as demonstrated by the immunohistochemical profile and their frequent association with adjacent endometrial adenocarcinoma.
Cancer is increasingly being viewed as a stem cell disease, both in its propagation by a minority of cells with stem-cell-like properties and in its possible derivation from normal tissue stem cells. But stem cell activity is tightly controlled, raising the question of how normal regulation might be subverted in carcinogenesis. The long-known association between cancer and chronic tissue injury, and the more recently appreciated roles of Hedgehog and Wnt signalling pathways in tissue regeneration, stem cell renewal and cancer growth together suggest that carcinogenesis proceeds by misappropriating homeostatic mechanisms that govern tissue repair and stem cell self-renewal.
Estimates of the worldwide incidence, mortality and prevalence of 26 cancers in the year 2002 are now available in the GLOBOCAN series of the International Agency for Research on Cancer. The results are presented here in summary form, including the geographic variation between 20 large "areas" of the world. Overall, there were 10.9 million new cases, 6.7 million deaths, and 24.6 million persons alive with cancer (within three years of diagnosis). The most commonly diagnosed cancers are lung (1.35 million), breast (1.15 million), and colorectal (1 million); the most common causes of cancer death are lung cancer (1.18 million deaths), stomach cancer (700,000 deaths), and liver cancer (598,000 deaths). The most prevalent cancer in the world is breast cancer (4.4 million survivors up to 5 years following diagnosis). There are striking variations in the risk of different cancers by geographic area. Most of the international variation is due to exposure to known or suspected risk factors related to lifestyle or environment, and provides a clear challenge to prevention.
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