Upregulation Of Shh Research Articles

Impaired cardiomyocytes accelerate cardiac hypertrophy and fibrosis by delivering exosomes containing Shh/N-Shh/Gli1 in angiotensin II infused mice

BackgroundsHeart failure (HF) is characterized by progressive cardiac hypertrophy and fibrosis, yet the underlying pathological mechanisms remain unclear. Exosomes are pivotal in cellular communication and are key signaling carriers in HFs. This study investigated the roles of exosomes in HF. MethodsEight-week-old male mice were divided into three groups: a control group, an Ang II group receiving angiotensin II (Ang II) infusion for 4 weeks, and an Ang II + DMA group receiving Ang II and dimethyl amiloride (DMA) infusion. This study examined the associations between cardiac injury, exosomes, and their substrate Shh. Furthermore, we conducted cellular experiments to assess the effects of Ang II-induced injury in primary cardiomyocytes on other cardiomyocytes and fibroblasts, and to test the therapeutic effects of the exosome inhibitor DMA and the Shh signaling inhibitor cyclopamine (CPN). ResultsAng II-induced cardiac hypertrophy and fibrosis, which were accompanied by exosome secretion and Shh upregulation in vivo. DMA relieved these cardiac lesions. Furthermore, cellular experiments revealed that Ang II-induced cardiomyocytes hypertrophy and activated cardiac fibroblasts by promoting the release of exosomes containing Shh/N-Shh/Gli1. Both DMA and CPN nullified fibroblast activation and proliferation. ConclusionsAng II-induced cardiomyocyte injury leads to cardiac hypertrophy and fibrosis through the release of exosomes carrying Shh signaling. The suppression of exosome secretion or the Shh pathway could offer new strategies for treating HF.

HPV16 infection promotes the malignant transformation of the esophagus and progression of esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) may be correlated with HPV infection, and the mechanism underlying the ESCC formation induced by HPV16 infection remains elusive. Here, we overexpressed HPV16 E6 and E7 and coordinated the overexpression of these two genes in EPC2 and ESCC cells. We found that E7 and coordinated expression of E6 and E7 promoted the proliferation of EPC2 cells, and upregulation of shh was responsible for cell proliferation since the use of vismodegib led to the failure of organoid formation. Meanwhile, overexpression of E6 and E7 in ESCC cells promoted cell proliferation, migration, and invasion in vitro. Importantly, E6 and E7 coordinately increased the capability of tumor growth in nude mice, while vismodegib slowed the growth of tumors in NCG mice. Moreover, a series of genes and proteins changed in cell lines after overexpression of the E6 and E7 genes, the potential biological processes and pathways were systematically analyzed using a bioinformatics assay. Together, these findings suggest that the activation of the hedgehog pathway induced by HPV16 infection may initially transform basal cells in the esophagus and promote following malignant processes in ESCC cells. The application of hedgehog inhibitors may represent a therapeutic avenue for ESCC treatment.

Analysis of the occurrence and distribution of primary and recurrent basal cell carcinoma of head and neck coupled to the assessment of tumor microenvironment and Sonic hedgehog signaling.

Often, basal cell carcinoma (BCC) displays local aggressiveness, and when developed in the head and neck presents with deep tissue invasion and recurrence. Previous studies have pointed out the necessity of systematic assessment of primary and recurrent BCC based on a better understanding of the biology and function of its microenvironment. Although hedgehog-dependent tumor cells signaling to the underlying stroma, and vice versa, have been demonstrated to be implicated in the pathogenesis of BCC, little is known about peculiarities of the tumor microenvironment and the above-mentioned signaling in the head and neck. The occurrence and distribution of 79 primary and recurrent BCCs developed in the head and neck region were estimated. The data were coupled with the immunohistochemical assessment of type IV collagen, laminin, alpha-smooth muscle actin (α-SMA), and Sonic hedgehog (Shh). The frequency of the mixed BCCs and the predominance of the nose and cheek region affection by primary and recurrent tumors were demonstrated. Furthermore, the increase of peritumoral and entire stromal α-SMA immunoreactivity in the mixed recurrent BCC was confirmed using statistics. We found the increase of strong levels of Shh immunoexpression in the aggressive variants of BCC – infiltrative, mixed, and micronodular. Surprisingly, we confirmed the upregulation of Shh paralleled by the downregulation of α-SMA immunoexpression in the superficial subtype of the tumor. Our results suggest the necessity of further studies assessing the nature of the tumor along with the peculiarities of signaling in BCCs of head and neck.

Testosterone exposure in prenatal life disrupts epithelial nuclear morphology, smooth muscle layer pattern, and FGF10 and Shh expression in prostate

The aim of this study was to evaluate whether high levels of exogenous testosterone (T) interfere in prostate morphogenesis. Pregnant females were exposed to subcutaneous injections of T cypionate (500 μg/animal) at gestational days 20 and 22. Male and female pups were euthanized at postnatal days 1 and 15. 15-day-old males had only fibroblast growth factor 10 (FGF10) immunostaining and nuclear form factor altered by the treatment, whereas treated females (T1 and T15) had almost all analyzed parameters changed. T1 females showed an increased anogenital distance (AGD), whereas T15 females had both AGD and ovary weight increased. T1 females had a higher number of epithelial buds emerging from the urethral and vaginal epithelium. We observed ectopic prostatic tissue surrounding the vagina in both T1 and T15 females. Moreover, the ectopic acini of T15 females showed delayed luminal formation, and there was a thickening of the periacinar smooth muscle layer (SML). Finally, FGF10 immunostaining intensity decreased in both T15 male and female prostates. Indeed, Sonic hedgehog (Shh) was upregulated in T15 female prostates, whereas no difference was observed between the male groups. These data showed that exogenous T changed the nuclear morphology of prostate epithelial cells in both males and females. Surprisingly, smooth muscle hyperplasia was also observed in the ectopic female prostate. Moreover, T downregulated FGF10 in both male and female prostates. Interestingly, the results suggest that FGF10 downregulation is mediated by the upregulation of Shh in females. In conclusion, exogenous T disrupts prostate development, particularly, affecting, the female.

Hedgehog signaling promotes multidrug resistance by regulation of ABC transporters in oral squamous cell carcinoma.

Multidrug resistance (MDR) is one of the reasons for treatment failure in oral squamous cancer patients; however, the MDR mechanisms remain elusive. Two human oral squamous cell carcinoma (OSCC) cell lines, CAL27 and SCC9, were analyzed by stepwise selection upon exposure to 5-fluorouracil (5-FU) and cisplatin (CDDP) for MDR cell line establishment, and cell viability was analyzed by CCK8 assays. Transcriptomes of the CAL27 and CAL27-MDR cells were analyzed by RNA-seq assay. The molecules in Sonic hedgehog (Shh) signal pathway and MDR related pathway were selected for validation by qRT-PCR and Western blot. Human OSCC tissues were applied for immunohistochemical and clinicopathological analysis. The OSCC-MDR cell lines with resistance to 5-FU and CDDP were successfully established. Protein expression levels of ATP-binding cassette (ABC) transporter ABCC1 and ABCG2 were increased 1.6-fold and 2.1-flod, respectively, in OSCC specimen from the patients with chemotherapy. The RNA-seq assay speculated the activation of Hedgehog (Hh) signaling with the upregulation of SHH, PTCH1, and SMO in OSCC-MDR cells as compared with OSCC cells. Furthermore, immunohistochemical studies confirmed that the high expression of SHH, PTCH1, and SMO in OSCC patients was significantly associated with chemotherapy. Overexpression of SHH increased the chemoresistance in OSCC cells, while the mRNA expression levels of PTCH1 and ABCG2 were increased in OSCC cells upon stimulation with recombinant SHH protein. These results revealed that the activation of Hh signaling pathway regulating ABC transporters is associated MDR in OSCC.

SUN-100 Exosome-packaged sonic hedgehog in cardiomyocyte plays a central role in type 2 cardiorenal syndrome

Cardiorenal syndrome type 2 (CRS2) is characterized by chronic abnormalities in cardiac function causing progressive chronic kidney disease (CKD). However, the mediators connecting heart and kidney remains largely unknown. In this study, we provide evidence supporting that exosomes containing Shh generated by injured cardiomyocyte plays a vital role in mediating cardiorenal connection in mouse model of transverse aortic constriction (TAC). Four weeks after TAC, mice were randomized into three groups: 1) sham control; 2) TAC mice; 3) TAC mice injected daily with cyclopamine (CPN), a specific inhibitor of hedgehog signaling. At 8 weeks after TAC, all animals were sacrificed. Urine, blood, heart and kidney tissues were collected for analysis. Another set of mice were randomized into three groups after 4 weeks of TAC: 1) sham control mice injected with pcDNA3 plasmid; 2) TAC mice injected with pcDNA3 plasmid; 3) TAC mice injected with shRab27a plasmid, a small GTPases implicated in regulating exosome biogenesis and secretion. These mice were injected with pcDNA3 or shRab27a plasmids once a week for three weeks beginning at 4 weeks after TAC. At 8 weeks after TAC, all animals were sacrificed. Urine, blood, heart and kidney tissues were collected for analysis. Exosomes were isolated from TAC mice serum and CRS2 patients’ serum, and incubated with normal rat kidney interstitial fibroblast (NRK-49F) cells. At 8 weeks after TAC, cardiac hypertrophy and fibrosis were prominent, as evidenced by increased expression of β-myosin heavy chain, α-actin and fibronectin. Echocardiography also revealed an impaired cardiac function in TAC mice. These cardiac lesions were accompanied by an upregulation of Shh and increased production of exosomes in cardiac tissue. Electron microscopy confirmed abundant exosomes derived from the injured cardiomyocytes in the heart of TAC mice. Blockade of Shh signaling by CPN ameliorated cardiac injury and restored heart function. Moreover, TAC mice also developed kidney injury secondary to chronic heart failure, manifested by proteinuria, kidney fibrosis, podocyte injury, and renal upregulation of Shh. CPN mitigated all these lesions in the kidneys. Notably, serum derived from TAC mice or CRS patients was able to induce renal interstitial fibroblast activation in vitro, indicating the circulating factors as mediators of cardiorenal connection. Furthermore, exosomes isolated from TAC serum was sufficient to cause fibroblast activation and matrix production. Furthermore, blockade of exosome biogenesis and secretion by knockdown of Rab27a expression not only protected against cardiac hypertrophy and fibrosis but also ameliorated renal fibrotic lesions. These studies demonstrate that heart-derived, circulating exosomes are a unique and effective vehicle to deliver Shh into the kidney after TAC. Targeting exosome generation and secretion or inhibition of Shh signaling could be novel therapeutic strategies for protecting both heart and kidney in cardiorenal syndrome.

Widespread expression of Sonic hedgehog (Shh) and Nrf2 in patients treated with cisplatin predicts outcome in resected tumors and are potential therapeutic targets for HPV-negative head and neck cancer

Background:Sonic hedgehog (Shh) and Nrf2 play a critical role in chemotherapeutic resistance. These two genes have been found to be dysregulated in head and neck squamous cell carcinomas (HNSCC). The purpose of this study was to analyze the expression, function and clinical prognostic relationship of Shh and Nrf2 in HNSCC in the context of therapeutic resistance and cancer stem cells (CSCs).Methods:We analyzed a cohort of patients with HNSCC to identify potential therapeutic biomarkers correlating with overall survival (OS) as well as disease-free survival (DFS) from our own data and validated these results using The Cancer Genome Atlas dataset. Expression of Shh and Nrf2 was knocked down by siRNA and cell growth, sphere growth and chemotherapeutic resistance were evaluated.Results:Widespread abundant expression of Shh and Nrf2 proteins were associated with shorter OS and DFS. The combination of Shh and Nrf2 expression levels was found to be a significant predictor of patient DFS. The tumor stromal index was correlated with Shh expression and inversely associated with shorter OS and DFS. Inhibition of Shh by siRNA or cyclopamine resulted in the attenuation of resistant CSC self-renewal, invasion, clonogenic growth and re-sensitization to the chemotherapeutic agents. Concomitant upregulation of Shh and Nrf2 proved to be an independent predictor of poor OS and DFS in patients with HNSCC.Conclusions:These findings suggest that Shh and Nrf2 could serve as therapeutic targets as well as promising dual prognostic therapeutic biomarkers for HNSCC.

Sonic Hedgehog signaling limits atopic dermatitis via Gli2-driven immune regulation.

Hedgehog (Hh) proteins regulate development and tissue homeostasis, but their role in atopic dermatitis (AD) remains unknown. We found that on induction of mouse AD, Sonic Hedgehog (Shh) expression in skin and Hh pathway action in skin T cells were increased. Shh signaling reduced AD pathology and the levels of Shh expression determined disease severity. Hh-mediated transcription in skin T cells in AD-induced mice increased Treg populations and their suppressive function through increased active transforming growth factor–β (TGF-β) in Treg signaling to skin T effector populations to reduce disease progression and pathology. RNA sequencing of skin CD4+ T cells from AD-induced mice demonstrated that Hh signaling increased expression of immunoregulatory genes and reduced expression of inflammatory and chemokine genes. Addition of recombinant Shh to cultures of naive human CD4+ T cells in iTreg culture conditions increased FOXP3 expression. Our findings establish an important role for Shh upregulation in preventing AD, by increased Gli-driven, Treg cell–mediated immune suppression, paving the way for a potential new therapeutic strategy.

Abstract 198: MUC13 promotes pancreatic tumor-stromal interactions by influencing tumor microenvironment

Abstract Objective: Pancreatic cancer (PanCa) is the third leading cause of cancer-related deaths in the U.S. MUC13, mucin, is aberrantly expressed in PanCa and its overexpression leads to tumorigenic and metastatic behavior of cancer cells. Herein, we investigate the role of MUC13 in tumor microenvironment (TME) that favors PanCa progression/metastasis and drug resistance. Methods: MUC13 expressing (Panc-1) and knockdown PanCa cells (HPAF-II) and pancreatic stromal cells (PSCs), derived from freshly collected human pancreatic tissues, were utilized for the study. Using immunoblotting, ELISA, qPCR and RT-PCR techniques, we assessed the MUC13-induced alterations in the expression of important molecules that are involved in promotion of pancreatic tumor stromal interactions and drug resistance. Cytation 3 live cell imaging was performed for 3D tumor-stromal cell co-culture and invasion assays. Xenograft mice tumor tissues generated using ectopically expressing MUC13 and null cells were used for the study. Results: Our results demonstrate that MUC13 activates SHH signaling pathway in PanCa cells via upregulation of SHH and its related important downstream targets such as CXCR-4, Gli-1, SMO, PTCH1/2, NFκB and p-AKT in PanCa cells using immunoblotting and PCR. We also observed increased expression of SHH, Gli-1, CXCR-4 and α-SMA in MUC13-expressing xenograft tumors using Immunohistochemistry. Ectopic MUC13-expressing PanCa cells grown in co-culture with PSCs showed enhanced secretion of IL-6 and CXCL-12 (ELISA), increase in invasion (Matrigel invasion assay) and migratory (Boyden chamber assay) potential of both the cultured PanCa as well as stromal cells. Additionally, ectopically MUC13-expressing cells were resistant to treatment with gemcitabine as depicted by proliferation assay and qPCR results showed enhanced expression of ribonucleoside-diphosphate reductase (RRM1/2) and miR-21, which are known to be involved in gemcitabine resistance. Overall, our results suggest that MUC13 modulates tumor-stromal crosstalk, promoting tumor growth, invasion and drug resistance in TME. Conclusion: Our results suggest that MUC13 expression might influence signaling supportive of interactive TME, leading to increased tumor growth and drug resistance. These findings illustrate mechanisms by which MUC13 promotes tumorigenic TME and contributes to PanCa cell survival, metastasis and drug resistance. Citation Format: Sheema S. Khan, Kyle Doxtater, Sonam Kumari, Saini Setua, Mohammed Sikander, Shabnam Malik, Murali Yallapu, Stephen Behrman, Subhash Chauhan, Meena Jaggi. MUC13 promotes pancreatic tumor-stromal interactions by influencing tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 198.

The Activity of LHx2‐Associated cis Regulatory Modules

Limb outgrowth is controlled, in part, by fibroblast growth factor (FGF) and sonic hedgehog (Shh). FGF up‐regulation of SHH is essential for coordinated limb pattering; however, not much is known about this mechanism. We recently found that LHX2 is an intermediate in FGF‐mediated SHH up‐regulation. LHX2 is part of a family of genes that are known to integrate signaling events. Thus, we hypothesized that FGF would up‐regulate LHX2 through associated potential cis regulatory modules (PCRM). Multiple PCRMs were found in association with the LHX2 locus. PCRMs were screened using comparative genomics, proximity to the LHX2 promoter, and transcription factor binding sites related to FGF signaling. To exam the role of suspected PCRMs, we electroporated PCRM‐reporter constructs into chick embryos. We found one PCRM with activity in the brain coincident with LHX2 expression, but none of our initial PCRMs exhibited activity in the limbs. We reevaluated the LHX2‐associated PCRMs in light of limb‐specific chromatin activation marks and identified 5 additional PCRMs for analysis. Studies are underway to determine limb‐related PCRM activity of this new cohort. Investigations regarding the relationship between FGF and LHX2 will provide important insights into the mechanisms underlying the FGF‐SHH regulatory loop.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Proliferation and committed differentiation into dopamine neurons of neural stem cells induced by the active ingredients of radix astragali.

Neural stem cells (NSCs) are important cellular sources of transplantation therapies for Parkinson’s disease. This study aimed to determine the effects of extracts of radix astragali on the proliferation and differentiation into dopamine (DA) neurons in NSCs. NSCs were dealt with astragaloside IV (ASI), astragalus polysaccharide (APS), and astraisoflavan (ASF), the main active ingredients of radix astragali. First, the results from cell-count kit-8 (CCK-8) assay showed that ASI, ASF, and APS had positive effects on the proliferation of NSCs. Next, we also confirmed the effects of ASI, APS, and ASF on BrdU and nestin by immunocytochemistry. Moreover, results from quantitative RT-PCR showed ASI, APS, and ASF could promote the expressions of tyrosine hydroxylase and dopamine transporter mRNA, which are specifically expressed in DA neurons. Simultaneously, sonic hedgehog (Shh), orphan nuclear hormone 1 (Nurr1), and pituitary homeobox 3 (Ptx3) are considered to motivate the formation of DA neurons. Our result showed ASI, APS, and ASF can also promote the expressions of Shh, Nurr1, and Ptx3 mRNAs. In conclusion, our study verifies that the active ingredients of radix astragali can promote the proliferation of NSCs and induce NSC differentiation toward DA neurons in vitro. These phenomena may occur through upregulation of Shh, Nurr1, and Ptx3 in the process of drug treatment.

Salvianolic Acids for Injection (SAFI) promotes functional recovery and neurogenesis via sonic hedgehog pathway after stroke in mice

There is a pressing need of developing approaches for delayed post-stroke therapy for patients who fail to receive thrombolysis within the narrow time window. Neuroprotection of Salvianolic Acids for Injection (SAFI) for cerebral ischemia-reperfusion injury in acute phase has been well documented. The current study was to determine the influence of SAFI at the subacute phase after stroke in mice, and to elucidate the underlying mechanisms. Adult male C57BL/6 mice were subjected to permanent occlusion of the distal middle cerebral artery (dMCAO), followed by daily intraperitoneal injection of SAFI 24 h after stroke for 14 days. Motor behavior was measured by neurological function evaluations weekly, and proliferation, migration, survival and differentiation of neural progenitor cells (NPCs) were examined with immunohistochemistry. Sonic hedgehog (Shh) inhibitor cyclopamine (CYC) was injected to determine the involvement of Shh pathway in the therapeutic effects of SAFI. The results showed that SAFI led to dramatic brain functional improvement, elevated NPCs proliferation, and prompted long-term survival of newborn neurons in the subventricular zone (SVZ). Up-regulation of Shh, Ptch and nuclear translocation of Gli1 were observed in the peri-infarct region, accompanied with robust production of Brain derived neurotrophic factor (BDNF) and Nerve growth factor (NGF). Simultaneous administration with CYC strikingly attenuated the beneficial outcomes of SAFI as well as abolished SAFI induced BDNF and NGF production. Collectively, our study demonstrated SAFI significantly promoted long-term functional recovery and neurogenesis, which might be dependent on Shh signaling mediated BDNF and NGF production. Therefore, SAFI might serve as a potential clinically translatable therapy during recovery stage after stroke.

Abstract 450: Brg1 Attenuates Cardiac Dysfunction by Activating Sonic Hedgehog Pathway in Acute Myocardial Infarction

Background: Brahma-related gene 1 (Brg1), the core ATPase subunit of a large chromatin remodeling complex, plays critical role in the regulation of gene expression during cardiac growth, differentiation. In adults, Brg1 is turned off in cardiomyocytes and reactivated by cardiac stress. How Brg1 in myocardial infarction (MI) is poorly understood. The sonic hedgehog (Shh) pathway was activated to protect the injured cardiomyocytes during the development of MI. However, by which Shh activation is mediated in MI still remains to be determined. Methods and results: Adult male C57BL/6 mice were subjected to ligation of the left anterior descending coronary artery for MI model. We investigated Brg1 activation and how it regulated the gene expressions of Shh pathway in peri-infarct zone in MI mice. Our data demonstrated that in peri-infarct zone, the protein of Brg1 was significantly increased 7 days after MI compared with the sham group in control mice, accompanied by upregulation of shh, patched-1 (Ptch1), and glioma-associated oncogene-1(Gli1). We further revealed that with adenoviral intramyocardial injection, the Brg1 overexpression reduced the percentage myocardial infarct, improved cardiac dysfunction, decreased the number of apoptotic cells and upregulated the ratio of Bcl-2/Bax in MI mice. Conversely, shRNA-mediated knockdown of Brg1 enlarged the percentage myocardial infarct, exacerbated cardiac dysfunction, increased the apoptotic cells and downregulated the ratio of Bcl-2/Bax. More importantly, the Brg1 overexpression increased Shh, Ptch1 and Gli1 expressions, whereas Brg1 knockdown suppressed Shh, Ptch1 and Gli1 expressions. Chromatin immunoprecipitation confirmed that Brg1 were recruited to the regulatory elements of Shh, Ptch1 and Gli1. This recruitment was associated with transcriptional upregulation of these three genes. Conclusions: This study indicated that in MI mice, the myocardial Brg1 is upregulated and that the upregulated Brg1 contributes to protection of cardiac function and activation of Shh pathway. Strategies that are aimed at augment the Brg1-Shh pathway may offer useful means for preventing and treating cardiac dysfunction in patients with MI. Key words: Acute myocardial infarction, Brg1, Shh pathway

Hedgehog pathway overexpression in pancreatic cancer is abrogated by new-generation taxoid SB-T-1216.

The Hedgehog pathway is one of the major driver pathways in pancreatic ductal adenocarcinoma. This study investigated prognostic importance of Hedgehog signaling pathway in pancreatic cancer patients who underwent a radical resection. Tumors and adjacent non-neoplastic pancreatic tissues were obtained from 45 patients with histologically verified pancreatic cancer. The effect of experimental taxane chemotherapy on the expression of Hedgehog pathway was evaluated in vivo using a mouse xenograft model prepared using pancreatic cancer cell line Paca-44. Mice were treated by experimental Stony Brook Taxane SB-T-1216. The transcript profile of 34 Hedgehog pathway genes in patients and xenografts was assessed using quantitative PCR. The Hedgehog pathway was strongly overexpressed in pancreatic tumors and upregulation of SHH, IHH, HHAT and PTCH1 was associated with a trend toward decreased patient survival. No association of Hedgehog pathway expression with KRAS mutation status was found in tumors. Sonic hedgehog ligand was overexpressed, but all other downstream genes were downregulated by SB-T-1216 treatment in vivo. Suppression of HH pathway expression in vivo by taxane-based chemotherapy suggests a new mechanism of action for treatment of this aggressive tumor.

Gli promotes epithelial-mesenchymal transition in human lung adenocarcinomas.

Adenocarcinoma is the most common type of lung cancer. Epithelial-mesenchymal transition (EMT) is required for tumor invasion/metastasis and the components that control this process are potential therapeutic targets. This study we examined the role of Gli in lung adenocarcinoma and whether its activation regulates metastasis through EMT in lung adenocarcinoma. We found that tumors with high Gli expression had significantly lower E-Cadherin expression in two independent cohorts of patients with lung adenocarcinoma that we studied. In vitro up-regulation of SHh resulted in increased cell migration while small molecule inhibitors of Smo or Gli significantly reduced cell mobility both in a wound healing assay and in a 3D cell invasion assay. Inhibition of Gli in vivo decreased tumor growth and induced an increase in E-Cadherin expression. Our results indicate that Gli may be critical for lung adenocarcinoma metastasis and that a novel Gli inhibitor shows promise as a therapeutic agent by preventing cell migration and invasion in vitro and significantly reducing tumor growth and increasing E-Cadherin expression in vivo.

Co‐Incident Upregulation of Shh and Hoxa 13 by Fgf in The Developing Limb Bud

Sonic hedgehog (Shh) is secreted from the zone of polarizing activity (ZPA) within the distal‐posterior limb bud mesoderm and regulates pattern and proliferation along the anterior‐posterior limb axis. Fibroblast growth factor 8 (Fgf8) is expressed in the distal limb bud from a rope‐like thickening called the apical ectodermal ridge (AER) and regulates proximal‐distal outgrowth and patterning. Once established, Shh and Fgf8 maintain each other's expression via a reciprocal loop, tightly regulating progressive limb development. Intermediates between Fgf and Shh are still being characterized. Gene arrays following application of Fgf2 to the posterior margin of developing chick wings showed up‐regulation of not only Shh, but also Hoxa13. Hoxa13 expression is a marker of the developing autopod or handplate. In the absence of Hoxa13, there is increased anterior apoptosis and disruption of the normal anterior‐posterior patterning. We hypothesized that the upregulation of Hoxa13 by Fgf in the posterior margin of the limb bud is important for the up‐regulation of Shh.To test this hypothesis Fgf2 or PBS soaked beads were implanted in the posterior limb margin of stage 23 chick wing buds. Embryos were harvested 24 hours after bead implantation for Hoxa13 whole mount in‐situ hybridization (WMISH).The results showed that Fgf induced ectopic Hoxa13 expression 24 hours after bead implantation. This confirmed the gene array data that showed a 13 fold increase in Hoxa13 following Fgf2 application. Limb‐specific Shh expression is regulated by the ZPA regulatory sequence (ZRS) which contains Hox binding sites important for full regulatory activity. Future studies are needed to establish whether Hoxa13 binding to the ZRS can facilitate Shh up‐regulation.

Cathepsin K induces platelet dysfunction and affects cell signaling in breast cancer - molecularly distinct behavior of cathepsin K in breast cancer.

BackgroundBreast cancer comprises clinically and molecularly distinct tumor subgroups that differ in cell histology and biology and show divergent clinical phenotypes that impede phase III trials, such as those utilizing cathepsin K inhibitors. Here we correlate the epithelial-mesenchymal-like transition breast cancer cells and cathepsin K secretion with activation and aggregation of platelets. Cathepsin K is up-regulated in cancer cells that proteolyze extracellular matrix and contributes to invasiveness. Although proteolytically activated receptors (PARs) are activated by proteases, the direct interaction of cysteine cathepsins with PARs is poorly understood. In human platelets, PAR-1 and −4 are highly expressed, but PAR-3 shows low expression and unclear functions.MethodsPlatelet aggregation was monitored by measuring changes in turbidity. Platelets were immunoblotted with anti-phospho and total p38, Src-Tyr-416, FAK-Tyr-397, and TGFβ monoclonal antibody. Activation was measured in a flow cytometer and calcium mobilization in a confocal microscope. Mammary epithelial cells were prepared from the primary breast cancer samples of 15 women with Luminal-B subtype to produce primary cells.ResultsWe demonstrate that platelets are aggregated by cathepsin K in a dose-dependent manner, but not by other cysteine cathepsins. PARs-3 and −4 were confirmed as the cathepsin K target by immunodetection and specific antagonists using a fibroblast cell line derived from PARs deficient mice. Moreover, through co-culture experiments, we show that platelets activated by cathepsin K mediated the up-regulation of SHH, PTHrP, OPN, and TGFβ in epithelial-mesenchymal-like cells from patients with Luminal B breast cancer.ConclusionsCathepsin K induces platelet dysfunction and affects signaling in breast cancer cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2203-7) contains supplementary material, which is available to authorized users.

Cooperatively transcriptional and epigenetic regulation of sonic hedgehog overexpression drives malignant potential of breast cancer.

Sonic hedgehog (Shh), a ligand of Hedgehog signaling pathway, is considered an important oncogene and an exciting potential therapeutic target in several cancers. Comprehensive understanding of the regulation mechanism of Shh in cancer cells is necessary to find an effective approach to selectively block its tumorigenic function. We and others previously demonstrated that nuclear factor-kappa B (NF-κB) activation and promoter hypomethylation contributed to the overexpression of Shh. However, the relationship between transcriptional and epigenetic regulation of Shh, and their roles in the malignant phenotype of cancer cells are still not clearly elucidated. In the present study, our data showed that the level of Shh was higher in breast cancer tissues with positive NF-κB nuclear staining and promoter hypomethylation. In addition, survival analysis revealed that Shh overexpression, but not hypomethylation and NF-κB nuclear staining, was a poor prognosis indicator for breast cancers. Moreover, in vitro data demonstrated that both NF-κB activation and hypomethylation in promoter region were positively associated with the overexpression of Shh. Mechanistically, the hypomethylation in Shh promoter could facilitate NF-κB binding to its site, and subsequently cooperate to induce transcription of Shh. Furthermore, the biological function data indicated that overexpressed Shh enhanced the self-renewal capacity and migration ability of breast cancer cells, which could be augmented by promoter demethylation and NF-κB activation. Overall, our findings reveal multiple and cooperative mechanisms of Shh upregulation in cancer cells, and the roles of Shh in tumor malignant behavior, thus suggesting a new strategy for therapeutic interventions to reduce Shh in tumors and improve patients’ prognosis.

Hedgehog Signaling is a Possible Therapeutic Target in Cd24-Negative Breast Cancer Cells

ABSTRACT Aim: Breast cancer still continues to be a leading cause of cancer-related death among women worldwide. Consequently, the development of a new effective therapeutic strategies for breast cancer is urgently required. Previously, we have shown that Hedgehog (Hh) signaling is activated in CD24-CD44+ breast cancer cells which are candidates of cancer stem-like cells and it may play an important role in these cells. However, the precise mechanim of Hh signaling activation in CD24-CD44+ breast cancer cells is still unclear. In this study, we focused on the CD24 molecule and investigated the mechanism of Hh signaling activation in CD24-negative breast cancer cells. Methods: Breast cancer cell lines, MCF-7 and HCC-1937 were used in this study. CD24 and Hh ligand; sonic Hh (Shh) were inhibited using small interfering RNA (siRNA). Expressions of CD24 and Hh signaling-related molecules were assessed by real time RT-PCR, flow cytometry and fluorescent immunochemistry. Invasiveness and colony formation were estimated by matrigel invasion assay and soft agar colony formation assay, respectively. Five-week-old NOD/SCID mice were used in xenograft experiment. Gli1 expression was used as an index of Hh signaling activation. Results: 1) CD24 siRNA transfected cells significantly increased invasiveness and colony formation compared with controls. 2) CD24 siRNA transfected cells significantly increased Hh signaling componens; Shh and Gli1 expresions compared with controls. 3) Invaded cells in matrigel invasion assay showed lower CD24 expression and higher Shh expression than non-invaded cells. 4) Shh siRNA and CD24 siRNA concurrent transfected cells significantly decreased Gli1 expression, invasiveness and colony formation compared to CD24 siRNA transfected cells in vitro. 5) Tumor volume and tumorigenesis in Shh siRNA and CD24 siRNA concurrent transfected cells was significantly lower than those in CD24 siRNA transfected cells in the xenograft mice model. 6) In fluorescent immunohistochemical staining using surgically resected specimens from cancer patients, Shh expression was negatively correlated with CD24 expression. Conclusions: Our results suggest that CD24 negative breast cancer cells may induce proliferation and invasiveness through upregulation of Shh. Therefore, Hh signaling may be a suitable therapeutic target in CD24-negative breast cancer. Disclosure: All authors have declared no conflicts of interest.

Sonic Hedgehog Signaling Pathway Mediates Cerebrolysin-Improved Neurological Function After Stroke

Cerebrolysin, a mixture of neurotrophic peptides, enhances neurogenesis and improves neurological outcome in experimental neurodegenerative diseases and stroke. The Sonic hedgehog (Shh) signaling pathway stimulates neurogenesis after stroke. The present study tests whether the Shh pathway mediates cerebrolysin-induced neurogenesis and improves neurological outcome after stroke. Rats subjected to embolic stroke were treated with cerebrolysin with or without cyclopamine. Using neural progenitor cells derived from the subventricular zone of the lateral ventricle of adult rats, we found that cerebrolysin significantly increased neural progenitor cells proliferation and their differentiation into neurons and myelinating oligodendrocytes, which were associated with upregulation of Shh and its receptors patched and smoothened. Blockage of the Shh signaling pathway with a pharmacological smoothened inhibitor, cyclopamine, abolished cerebrolysin-induced in vitro neurogenesis and oligodendrogenesis. In the ischemic rats, treatment with cerebrolysin starting 24 hours after stroke significantly increased neural progenitor cell proliferation in the subventricular zone and enhanced neurogenesis, oligodendrogenesis, and axonal remodeling in the peri-infarct area. Moreover, profound neurological function improvements were observed in rats treated with cerebrolysin from week 3 to week 5 after stroke onset compared with vehicle-treated rats. However, in vivo inhibition of the Shh pathway with cyclopamine completely reversed the effects of cerebrolysin on neurorestoration and functional recovery. These results demonstrate that the Shh pathway mediates cerebrolysin-enhanced neurogenesis and white matter remodeling and improves functional recovery in rats after stroke.