Activation Of Sonic Hedgehog Pathway Research Articles

Expression of calretinin in odontogenic keratocysts and basal cell carcinomas: A study of sporadic and Gorlin-Goltz syndrome-related cases

Gorlin-Goltz syndrome (GGS), is an autosomal dominant inherited disorder related to germline mutation of PTCH1 gene, characterised by the presence of multiple developmental anomalies and tumours, mainly basal cell carcinomas (BCC) and odontogenic keratocysts (OKC).We analysed and compared the expression of calretinin in 16 sporadic OKCs, from 15 patients, and 12 syndromic OKCs from 11 patients; in 19 BCC's and 2 cutaneous keratocysts (CKC) belonging to 4 GGS patients, 15 sporadic BCCs and 3 steatocystomas (SC).Calretinin was negative in 10 of 12 syndromic OKCs, focally positive (<5% of cells) in 2; six sporadic OKCs were negative, 6 focally and 4 diffusely positive (p = .02, cases focally and diffusely positive vs. cases negative). All BCCs of 3 GGS patients were negative, the fourth patient presented two BCCs negative and 5 focally or diffusely positive; 7 sporadic BCCs were negative and 8 focally positive (p = NS). Two CKCs resulted negative in one GGS patient; 2 sporadic SCs were positive, and a third was negative.PTCH1 mutations produce an altered PTCH protein and an aberrant activation of Sonic hedgehog (SHH) pathway, leading to tumoral proliferation. It has been demonstrated that treatment of human foetal radial glia cells with SHH reduces, whereas the blockage of SHH increases calretinin expression. We found a lower expression of calretinin in syndromic OKCs compared to sporadic cases. Although calretinin's value in differential diagnosis between sporadic and syndromic tumours appears not crucial, our results shed light on the possible link between SHH dysfunction and calretinin expression in GGS-related tumours.

Identification of recurrent FHL2-GLI2 oncogenic fusion in sclerosing stromal tumors of the ovary

Sclerosing stromal tumor (SST) of the ovary is a rare type of sex cord-stromal tumor (SCST), whose genetic underpinning is currently unknown. Here, using whole-exome, targeted capture and RNA-sequencing, we report recurrent FHL2-GLI2 fusion genes in 65% (17/26) of SSTs and other GLI2 rearrangements in additional 15% (4/26) SSTs, none of which are detected in other types of SCSTs (n = 48) or common cancer types (n = 9,950). The FHL2-GLI2 fusions result in transcriptomic activation of the Sonic Hedgehog (SHH) pathway in SSTs. Expression of the FHL2-GLI2 fusion in vitro leads to the acquisition of phenotypic characteristics of SSTs, increased proliferation, migration and colony formation, and SHH pathway activation. Targeted inhibition of the SHH pathway results in reversal of these oncogenic properties, indicating its role in the pathogenesis of SSTs. Our results demonstrate that the FHL2-GLI2 fusion is likely the oncogenic driver of SSTs, defining a genotypic–phenotypic correlation in ovarian neoplasms.

Hypoxia induced Sonic Hedgehog signaling regulates cancer stemness, epithelial-to-mesenchymal transition and invasion in cholangiocarcinoma

Aberrant activation of Sonic Hedgehog (SHH) pathway has been implicated in a variety of cancers including cholangiocarcinoma (CC); however, the influencing factors are still unknown. Additionally, intratumoral hypoxia is known to contribute towards therapeutic resistance through modulatory effects on various pathways. In this study, we investigated the relationship between hypoxia and SHH pathway activation and the effect of this interplay on cancer stemness and epithelial-to- mesenchymal transition (EMT) during cholangiocarcinogenesis. Hypoxia promoted SHH pathway activation, evidenced by upregulated SHH and SMO levels, and enhanced glioma-associated oncogene homolog 1 (GLI1) nuclear translocation; whereas silencing of HIF-1α impaired SHH upregulation. Hypoxia also enhanced the expression of cancer stem cell (CSC) transcription factors (NANOG, Oct4, SOX2), CD133 and EMT markers (N-cadherin, Vimentin), thereby supporting invasion. Cyclopamine treatment suppressed hypoxia induced SHH pathway activation, consequently reducing invasiveness by downregulating the expression of CSC transcription factors, CD133 and EMT. Cyclopamine induced apoptosis in CC cells under hypoxia, suggesting that hypoxia induced activation of SHH pathway has modulatory effects on CC progression. Therefore, SHH signaling is proposed as a target for CC treatment, which is refractory to standard chemotherapy.

Integrated Multi-Omics Characterization of Neurofibromatosis Type 1 Related Peripheral Nerve Sheath Tumors Identifies Two Distinct Malignant Peripheral Nerve Sheath Tumors Subtypes with Sonic Hedgehog or WNT Pathway Activation

Abstract INTRODUCTION Malignant peripheral nerve sheath tumors (MPNSTs) are highly aggressive Schwann-cell derived sarcomas and the leading cause of mortality in neurofibromatosis Type 1. In many cases, MPNSTs develop through malignant transformation of an underlying neurofibroma (NF). The genomic alterations and molecular pathways driving malignant transformation is not well understood. In this study, we leverage multi-platform genomic and epigenomic profiling of human NFs and MPNSTs to identify targetable molecular pathways that lead to malignant transformation. METHODS Fresh-frozen tumor (N = 108; 19 MPNSTs, 18 premalignant NFs, 37 benign peripheral nerve NFs, and 34 cutaneous NFs) and matched-controls were allocated for NGS platforms including methylation-profiling, RNA, whole exome and ChIP sequencing. In addition, functional validation of identified pathways was performed using CRISPR/cas9 knockout of relevant sonic hedgehog (SHH) and WNT pathway genes in immortalized neurofibroma cell lines. RESULTS Unsupervised consensus clustering of peripheral nerve tumors identified 4 distinct methylation subgroups, with MPNSTs clustering in either Methyl_Grp-2B or Methyl_Grp-3. Transcriptomic analysis of the MPNSTs, identified SHH pathway activation in Methyl_Grp-3 MPNSTs (NES = 1.9952, P &lt; .0001, FDR &lt; 0.001) and WNT pathway activation in Methyl_Grp-2B MPNSTs (NES = 1.7630, P &lt; .0001, FDR = 0.0058). Single sample gene set enrichment analysis demonstrates that there is a strong negative correlation between SHH and WNT pathway activation in MPNSTs (r = −0.78, P = .007). Methyl_Gr-3 MPNSTs harbor more PTCH1 copy number loss (62.5% vs 0%, P &lt; .05) and PTCH1 promoter hypermethylation (mean beta value: 0.2899 vs. 0.06119, P &lt; .05). PTCH1 knockout in immortalized neurofibroma cell lines increased expression of SHH pathway target genes (GLI1, GLI2, and SMO), and increased cellular proliferation and invasiveness. CONCLUSION We found that MPNSTs can be classified into two molecular subgroups driven by SHH or WNT pathway activation. Furthermore, PTCH1 loss is one mechanism that mediates SHH pathway activation. These results suggest a targeted approach should be taken for treating MPNSTs, and SHH and WNT pathway inhibition may be promising avenue for therapeutic development.

Abstract 4679: NRF2/SHH signaling cascade promotes tumor-initiating cell lineage and drug resistance in hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) is the fifth most common malignancy worldwide. The long-term prognosis of HCC remains unsatisfactory due to high recurrence rates and chemoresistance. Solid evidence shows that tumor-initiating cells (T-ICs) are the root of tumor relapse and drug resistance, which led to poor prognosis of patients with hepatocellular carcinoma (HCC). Therefore, identification of crucial transcriptional regulator in liver T-IC may improve the prognosis of these patients. Through in vitro liver T-IC enrichment approach, we identified Nuclear Factor (Erythroid-Derived 2)-Like 2 (NRF2) as transcriptions regulator be significantly upregulated in enriched liver T-IC populations. NRF2 regulated liver T-IC properties including self-renewal, tumorigenicity, and liver T-IC marker expression and significantly affected patients’ clinical outcome. Furthermore, NRF2 repression led to sensitization to doxorubicin and 5-FU treatment. Furthermore, we found that ROS-induced NRF2 activation regulates sorafenib resistance of HCC cells. Mechanistically, NRF2 was found to physically bind to promoter SHH, which triggers activation of sonic hedgehog pathway. NRF2 was significantly correlated with SHH expression, which was associated with poor patient’s survival. The effect of NRF2 knockdown was eliminated upon administration of recombinant SHH, demonstrating that NRF2 mediated the TIC function via upregulation of SHH expression. Our study suggested a novel regulatory mechanism for the canonical sonic hedgehog pathway that may function through NRF2/SHH/GLI signaling axis, thus mediating T-IC phenotypes. Targeting NRF2 mediated signaling cascade alone or in combination with other treatment modalities may be a new a potential therapeutic approach for treatment of HCC. Citation Format: Hoi Wing Leung, Eunice Yuen Ting Lau, Kin Wah Lee. NRF2/SHH signaling cascade promotes tumor-initiating cell lineage and drug resistance in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4679.

A large-scale drug screen identifies selective inhibitors of class I HDACs as a potential therapeutic option for SHH medulloblastoma.

Medulloblastoma (MB) is one of the most frequent malignant brain tumors of children, and a large set of these tumors is characterized by aberrant activation of the sonic hedgehog (SHH) pathway. While some tumors initially respond to inhibition of the SHH pathway component Smoothened (SMO), tumors ultimately recur due to downstream resistance mechanisms, indicating a need for novel therapeutic options. Here we performed a targeted small-molecule screen on a stable, SHH-dependent murine MB cell line (SMB21). Comprehensive isotype profiling of histone deacetylase (HDAC) inhibitors was performed, and effects of HDAC inhibition were evaluated in cell lines both sensitive and resistant to SMO inhibition. Lastly, distinct mouse models of SHH MB were used to demonstrate pharmacologic efficacy in vivo. A subset of the HDAC inhibitors tested significantly inhibit tumor growth of SMB21 cells by preventing SHH pathway activation. Isotype profiling of HDAC inhibitors, together with genetic approaches suggested that concerted inhibition of multiple class I HDACs is necessary to achieve pathway inhibition. Of note, class I HDAC inhibitors were also efficacious in suppressing growth of diverse SMO inhibitor‒resistant clones of SMB21 cells. Finally, we show that the novel HDAC inhibitor quisinostat targets multiple class I HDACs, is well tolerated in mouse models, and robustly inhibits growth of SHH MB cells in vivo as well as in vitro. Our data provide strong evidence that quisinostat or other class I HDAC inhibitors might be therapeutically useful for patients with SHH MB, including those resistant to SMO inhibition.

Sonic hedgehog pathway activation regulates cervical cancer stem cell characteristics during epithelial to mesenchymal transition.

Resistance to therapy and metastasis remains one of the leading causes of mortality due to cervical cancer despite advances in detection and treatment. The mechanism of epithelial to mesenchymal transition (EMT) provides conceptual explanation to the invasiveness and metastatic spread of cancer but it has not been fully understood in cervical cancer. This study aims to investigate the mechanism by which silencing of E-cadherin gene regulates EMT leading to proliferation, invasion, and chemoresistance of cervical cancer cells through the Hedgehog (Hh) signaling pathway. We developed an in vitro EMT model by the knockdown of E-cadherin expression in cervical cancer cell lines.To understand the role of developmental pathway like Hh in the progression of cervical cancer, we investigated the expression of Hh pathway mediators by array in E-cadherin low cervical cancer cells and observed upregulation of Hh pathway. This was further validated on low passage patient-derived cell lines and cervical carcinoma tissue sections from cervical cancer patients. Further, we evaluated the role of two inhibitors (cyclopamine and GANT58) of the Hh pathway on invasiveness and apoptosis in E-cadherin low cervical cancer cells. In conclusion, we observed that inhibition of Hh pathway with GANT58 along with current therapeutic procedures could be more effective in targeting drug-resistant EMT cells and bulk tumor cells in cervical cancer.

Hedgehog pathway permissive conditions allow generation of immortal cell lines from granule cells derived from cancerous and non-cancerous cerebellum.

Cerebellar granule cell progenitors (GCPs) undergo proliferation in the post-natal cerebellum that is dependent on sonic hedgehog (SHH) signalling. Deregulated SHH signalling leads to type 2 medulloblastoma (MB). In this work, a novel cell culture protocol is described, which is suitable for the establishment and long-term maintenance of GCP-derived cells. This method is first applied to SHH pathway active MB cells from Atoh1-cre;Ptch1FL/FL tumours, which leads to the generation of neurosphere-like cell lines expressing GCP markers and an active SHH signalling pathway. These cells also show high sensitivity to the Smoothened inhibitor vismodegib, therefore recapitulating the SHH pathway requirement for survival shown by type 2 MB. Analysis of culture supplements reveals that bFGF and fetal bovine serum act as inhibitors of the SHH pathway and therefore preclude generation of cell lines that are relevant to the study of the SHH pathway. Consequently, these insights are transferred from the context of MB to non-transformed, post-natal day 7 cerebellum-derived cellular explants. In contrast to other, previously used methods, these GCP cultures proliferate indefinitely and depend on SHH pathway activation, either by means of the small molecule SAG or through genetic ablation of Ptch1. This culture method therefore leads to the generation of immortal neurosphere-like cell lines, that are named murine SAG-dependent spheres (mSS). Despite long-term culture, mSS cells remain dependent on continuous stimulation of the SHH pathway. Further, mSS cells maintain their lineage after extensive periods in vitro, as demonstrated by their differentiation towards the neural lineage. Herein a simple method for the generation of immortal cell lines from murine cerebella is defined. These lines can be maintained indefinitely through hedgehog pathway activation and maintain the GCP lineage.

Sonic hedgehog pathway activation is associated with cetuximab resistance and EPHB3 receptor induction in colorectal cancer.

A major problem of colorectal cancer (CRC) targeted therapies is relapse caused by drug resistance. In most cases of CRC, patients develop resistance to anticancer drugs. Cetuximab does not show many of the side effects of other anticancer drugs and improves the survival of patients with metastatic CRC. However, the molecular mechanism of cetuximab resistance is not fully understood.Methods: EPHB3-mediated cetuximab resistance was confirmed by in vitro western blotting, colony-forming assays, WST-1 colorimetric assay, and in vivo xenograft models (n = 7 per group). RNA-seq analysis and receptor tyrosine kinase assays were performed to identify the cetuximab resistance mechanism of EPHB3. All statistical tests were two-sided.Results: The expression of EFNB3, which upregulates the EPHB3 receptor, was shown to be increased via microarray analysis. When resistance to cetuximab was acquired, EPHB3 protein levels increased. Hedgehog signaling, cancer stemness, and epithelial-mesenchymal transition signaling proteins were also increased in the cetuximab-resistant human colon cancer cell line SW48R. Despite cells acquiring resistance to cetuximab, STAT3 was still responsive to EGF and cetuximab treatment. Moreover, inhibition of EPHB3 was associated with decreased STAT3 activity. Co-immunoprecipitation confirmed that EGFR and EPHB3 bind to each other and this binding increases upon resistance acquisition, suggesting that STAT3 is activated by the binding between EGFR and EPHB3. Protein levels of GLI-1, SOX2, and Vimentin, which are affected by STAT3, also increased. Similar results were obtained in samples from patients with CRC.Conclusion: EPHB3 expression is associated with anticancer drug resistance.

Patched1–ArhGAP36–PKA–Inversin axis determines the ciliary translocation of Smoothened for Sonic Hedgehog pathway activation

The Sonic Hedgehog (Shh) pathway conducts primarily in the primary cilium and plays important roles in cell proliferation, individual development, and tumorigenesis. Shh ligand binding with its ciliary membrane-localized transmembrane receptor Patched1 results in the removal of Patched1 from and the translocation of the transmembrane oncoprotein Smoothened into the cilium, leading to Shh signaling activation. However, how these processes are coupled remains unknown. Here, we show that the Patched1-ArhGAP36-PKA-Inversin axis determines the ciliary translocation of Smoothened. We find that Patched1 interacts with and stabilizes the PKA negative regulator ArhGAP36 to the centrosome. Activating the Shh pathway results in the removal of ArhGAP36 from the mother centriole and the centrosomal PKA accumulation. This PKA then phosphorylates Inversin and promotes its interaction with and the ciliary translocation of Smoothened. Knockdown of Inversin disrupts the ciliary translocation of Smoothened and Shh pathway activation. These findings reveal a regulatory molecular mechanism for the initial step of Shh pathway activation.

Integrative Analysis of Global Gene Expression Identifies Opposite Patterns of Reactive Astrogliosis in Aged Human Prefrontal Cortex.

The prefrontal cortex (PFC) is one of the brain regions with more prominent changes in human aging. The molecular processes related to the cognitive decline and mood changes during aging are not completely understood. To improve our knowledge, we integrated transcriptomic data of four studies of human PFC from elderly people (58–80 years old) compared with younger people (20–40 years old) using a meta-analytic approximation combined with molecular signature analysis. We identified 1817 differentially expressed genes, 561 up-regulated and 1256 down-regulated. Pathway analysis revealed down-regulation of synaptic genes with conservation of gene expression of other neuronal regions. Additionally, we identified up-regulation of markers of astrogliosis with transcriptomic signature compatible with A1 neurotoxic astrocytes and A2 neuroprotective astrocytes. Response to interferon is related to A1 astrocytes and the A2 phenotype is mediated in aging by activation of sonic hedgehog (SHH) pathway and up-regulation of metallothioneins I and genes of the family ERM (ezrin, radixin, and moesin). The main conclusions of our study are the confirmation of a global dysfunction of the synapses in the aged PFC and the evidence of opposite phenotypes of astrogliosis in the aging brain, which we report for the first time in the present article.

Cadmium-induced neurodegeneration and activation of noncanonical sonic hedgehog pathway in rat cerebellum.

Cadmium is a nonessential toxic heavy metal, which enters the body easily and damages the cellular system. The sonic hedgehog (Shh) signaling pathway is one of the key regulatory pathways, which define neural growth and development. This study aimed to explore how cadmium exposure affects neural activities, Shh signaling cascade, and its downstream target genes. Total 18 male Wistar rats were randomly divided into two groups, control and test groups. Test rats were administered with 3 mg cadmium/kg body weight, while the control rats were treated with vehicle continuously for 28 days. Thereafter, rats were killed and the isolated brain samples were examined using oxidative stress assessment, histological and immunohistological behavioral assessment, polymerase chain reaction (PCR), and the comet assay. A disturbed oxidative balance, DNA damage, and an upregulated Shh signaling pathway were observed in cadmium-treated samples. Loss of structural integrity in cerebellum and loss of motor activity were observed in cadmium-treated rats.

Abstract 3077: Krüppel-like factor 5 and its connection to Sonic Hedgehog signaling in Barrett's esophagus and esophageal adenocarcinoma

Abstract Aims: We sought to determine whether KLF5 was involved, at either phenotypic or functional levels, in the genesis and progression of Barrett's esophagus (BE) to esophageal adenocarcinoma (EAC), and whether this involvement was related to sonic hedgehog (SHH) signaling. In human adults, KLF5 is expressed in the less differentiated cells of the intestinal crypts, where its activity promotes cell proliferation, contributes to intestinal epithelial homeostasis, and is necessary for villus formation. KLF5 has been found to be highly active in a number of other cancer types, including gastric cancer; moreover, the known role of KLF5 in intestinal epithelial differentiation may contribute to metaplasia and dysplasia in BE and EAC development. We also sought to ascertain whether KLF5 activity was connected to the sonic hedgehog (SHH) pathway, since this pathway is already known to be highly active in BE and EAC. Methods and results: Quantitative reverse transcriptase PCR (qRT-PCR) performed on RNA obtained from BE cell lines (GhTRT and QhTRT) and EAC cell lines (SKGT4 and OE33) revealed that KLF5 is underexpressed in BE cell lines and overexpressed in EAC cell lines relative to normal primary squamous esophageal epithelial cells (HEEpic). Moreover, qRT-PCR performed on clinical BE and EAC tissue specimens showed the same pattern, with downregulation in BE tissues and upregulation in EAC tissues (relative to matched normal esophageal control tissues). Next, we determined KLF5 functional effects in EAC in vitro. The EAC cell line SKGT4 was transfected with KLF5 siRNA, and WST-1 cell proliferation assays revealed significantly lower cell proliferation at 72 hours (p&amp;lt;0.00005) and 96 hours (p&amp;lt;0.05) after transfection, relative to mock-transfected SKGT4. We then assayed KLF5 downstream effects on esophageal adenocarcinogenesis as well as SHH signaling in vitro. qRT-PCR performed on KLF5-silenced SKGT4 RNA revealed significantly diminished expression (relative to mock-transfected SKGT4) of the following known EAC-related genes: CDX1 (p&amp;lt;0.005), VIL1 (p&amp;lt;0.05), MUC2 (p&amp;lt;0.005), and MUC5ac (p&amp;lt;0.0005); moreover, GLI1, a known SHH pathway target gene, was significantly downregulated by KLF5 inhibition (p&amp;lt;0.005), while PTCH1, a known SHH pathway-inhibitory gene, was significantly upregulated (p&amp;lt;0.05). Conclusion: These tissue phenotypic and in vitro functional data identify KLF5 as a potential oncogene in esophageal adenocarcinogenesis. KLF5 is a potential therapeutic and/or chemopreventive target that can be evaluated in future studies of EAC or BE, such as with metformin, which is known to inhibit KLF5 activity. We also identify a potential new connection between KLF5 and SHH pathway activity in the development of EAC. This potential connection deserves further exploration in other malignancies, as well as in physiologic functions such as human development or adult stem cell maintenance. Citation Format: Christopher K. Ng, Yulan Cheng, John M. Abraham, Stephen J. Meltzer. Krüppel-like factor 5 and its connection to Sonic Hedgehog signaling in Barrett's esophagus and esophageal adenocarcinoma [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 3077.

Untangling Sources of Phenotypic Variation Characterizing the Craniofacial Disease Holoprosencephaly

Holoprosencaphly (HPE)—a craniofacial disorder in which the forebrain fails to develop into two hemispheres and the midface does not fully form—is characterized by a broad spectrum of severity. Phenotypes range from very mild midfacial narrowing to cyclopia. Mutations in Sonic hedgehog (SHH) and in SHH signaling pathway members are known to underlie HPE; however, the source of the highly variable craniofacial morphology manifesting in the HPE patient population is not understood. Previous research in a chick model suggests that reduced SHH‐signaling in the brain correlates with a continuous distribution of HPE‐like phenotypes, and that very small modifications in ligand concentration can result in highly heterogeneous morphologies. Here, we are probing the origins of morphological variation in a genetic mouse model. Although humans heterozygous for SHH mutations exhibit the full spectrum of HPE, mice heterozygous for Shh do not exhibit even mild HPE although homozygous mutants are cyclopic. Mice homozygous for mutations in the SHH‐signaling pathway member low‐density lipoprotein receptor‐related protein (Lrp) 2 however exhibit mild to severe forms of HPE, and are therefore a better model for examining sources of variation. LRP2 is an endocytic receptor required to induce proper SHH signaling in the forebrain. We used geometric morphometric analysis to quantify the shape of embryos with sequential, genetic loss of Shh and Lrp2 alleles, and qPCR was used to assess SHH signaling pathway activation. We found that there is no statistically significant difference in shape among wild‐type, Lrp2+/−, Shh+/−, and Lrp2+/−;Shh+/− embryos, while Lrp2−/− and Lrp2−/−;Shh+/− embryos diverged from the group, exhibiting a more narrow midface. Interestingly, there was increased phenotypic variance in Lrp2−/− and Lrp2−/−;Shh+/− embryos in comparison to the other genotypes, with a greater increase in variance seen in the Lrp2−/−;Shh+/− group. Our results support the hypothesis that very slight modifications to SHH pathway activation can yield large amounts of phenotypic variation.Support or Funding InformationThis work is supported by NIH/NIDCR‐R01DE018234, ‐R01DE019638, ‐T32DE730619, ‐F31DE02579001.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cigarette smoke stimulates the stemness of renal cancer stem cells via Sonic Hedgehog pathway

Cancer stem cells (CSCs) are essentially responsible for tumor initiation, growth, progression, metastasis and recurrence, and cigarette smoke (CS) is closely involved in the occurrence and development of kidney cancer. However, the effect of CS on renal CSCs has not been elucidated yet. In the present study, tumorsphere formation assay was used to enrich renal CSCs from 786-O and ACHN cells. We illustrated that CS effectively promoted renal CSCs stemness by enhancing tumorsphere formation, increasing the expression of renal CSCs markers (CD133, CD44, ALDHA1, Oct4, and Nanog) and elevating CD133+ cell population. Moreover, our results showed that CS triggered the activation of Sonic Hedgehog (SHH) pathway, while inhibition of SHH pathway dampened the promotive effects of CS on renal CSCs. Finally, higher levels of renal CSCs markers and SHH pathway-related proteins were observed in kidney cancer tissues from smokers than non-smoking cancer tissues. Taken together, these results demonstrated the important role of SHH pathway in regulating CS-induced renal CSCs stemness augment. Findings from this study could provide new insight into the molecular mechanisms of CS-elicited stemness of renal CSCs.

Renal Cell Carcinoma: Molecular Aspects.

Renal cell carcinoma is the most common form of the kidney cancer accounting for more than 85% of the cases of which clear cell renal cell carcinoma (ccRCC) is the major histological subtype. The central molecular signature for ccRCC pathogenesis is the biallelic inactivation of VHL gene due to thepresence of mutations/hyper-methylation/complete gene loss, which results in the downstream HIF activation. These events lead to increased tyrosine kinase receptor signalling pathways (RAS/MEK/ERK pathway, PI3K/AKT/mTOR pathway and NF-κB pathway), which through their downstream effector proteins causes the cell to proliferate and migrate. Recent studies have shown that VHL inactivation alone is not sufficient to induce the tumor. Mutations in numerous other genes that codes for chromatin modifiers (PBRM1, SETD2 and BAP1) and signalling proteins (PTEN and mTOR) have been identified along with activation of alternate signalling pathways like STAT and Sonic Hedgehog (SHH) pathway. It has also been shown that STAT pathway also works cooperatively with HIF to enhance the tumor progression. However, SHH pathway reactivation resulted in tumor regardless of the VHL status, indicating the complex nature of the tumor at the molecular level. Therefore, understanding the complete aetiology of ccRCC is important for future therapeutics.

Membrane-bound full-length Sonic Hedgehog identifies cancer stem cells in human non-small cell lung cancer.

The mechanism of Sonic Hedgehog (Shh) pathway activation in non-small cell lung cancer (NSCLC) is poorly described. Using an antibody against the Shh C-terminal domain, we found a small population of Shh-positive (Shh+) cells in NSCLC cells. The objective of this study was to characterize these Shh+ cells. Shh+ and Shh- cells were sorted by using Fluorescence Activated Cell Sorting (FACS) on 12 commercial NSCLC cell lines. Functional analyses on sorted cells were performed with gene expression assays (qRT-PCR and microarray) and cells were treated with cytotoxic chemotherapy and a targeted inhibitor of Shh signaling (GDC0449). We used in vivo models of nude mice inoculated with Shh+ and Shh- sorted cells and drug-treated cells. Finally, we confirmed our results in fresh human NSCLC samples (n=48) paired with normal lung tissue. We found that Shh+ cells produced an uncleaved, full-length Shh protein detected on the membranes of these cells. Shh+ cells exerted a paracrine effect on Shh- cells, inducing their proliferation and migration. Shh+ cells were chemo-resistant and showed features of cancer stem cells (CSCs) in vitro and in vivo. Pharmacological inhibition of the Shh pathway suppressed their CSC features. A high percentage of Shh+ cells was associated with poor prognosis in early-stage NSCLC patients. In conclusion, we describe for the first time the presence of an abnormal membrane-bound full-length Shh protein in human cancer cells that allows the identification of CSCs in vitro and in vivo.

αE-Catenin Is a Positive Regulator of Pancreatic Islet Cell Lineage Differentiation

SUMMARYThe development and function of epithelia depend on the establishment and maintenance of cell-cell adhesion and intercellular junctions, which operate as mechanosensor hubs for the transduction of biochemical signals regulating cell proliferation, differentiation, survival, and regeneration. Here, we show that αE-catenin, a key component of adherens junctions, functions as a positive regulator of pancreatic islet cell lineage differentiation by repressing the sonic hedgehog pathway (SHH). Thus, deletion of αE-catenin in multipotent pancreatic progenitors resulted in (1) loss of adherens junctions, (2) constitutive activation of SHH, (3) decrease in islet cell lineage differentiation, and (4) accumulation of immature Sox9+ progenitors. Pharmacological blockade of SHH signaling in pancreatic organ cultures and in vivo rescued this defect, allowing αE-catenin-null Sox9+ pancreatic progenitors to differentiate into endocrine cells. The results uncover crucial functions of αE-catenin in pancreatic islet development and harbor significant implications for the design of β cell replacement and regeneration therapies in diabetes.

Abstract 2436: Non canonical activation of Sonic Hedgehog pathway in uterine leiomyosarcoma

Abstract Background: Uterine leiomyoma (ULM) and leiomyosarcoma (ULMS) are smooth muscle tumors with distinct clinical and biological behavior. They can be a cause of infertility and even death. Little is known about the factors that can to influence these tumors behavior and biology. Sonic Hedgehog (SHH) pathway components were previously implicated in the ULMS malignancy risk; however its activation mechanism is unknown. In this study, we investigated the gene expression profile of Sonic Hedgehog Signaling and pathway upstream genes in myometrium (MM), ULM and LMS cell lines. Methods: Total RNA obtained from cell lines were submitted to cDNA synthesis and quantitative real time PCR (qRT-PCR), using 7500 System (Life Technologies, USA). Gene expression of SHH, PTCH1, SMO, SUFU, GLI 1-3, CCND1, BCL-2 and BMP4 were assessed. Results: Gene expression data showed an upregulation of SMO, SUFU, GLI1, BCL-2 and BMP4 in LMS cells when compared to MM. GLI1 (RQ=11) and BCL-2 (RQ=20) genes had higher expression. In the other side, SHH, PTCH1, GLI2 and GLI3 showed upregulation in MM cells. Conclusion: Our results showed that the Sonic hedgehog pathway is activated in LMS cells but by SHH independent form (non-canonical pathway). This could be explained by the downexpression of SHH ligand and the PTCH1 receptor, and by the upexpression of SMO, GLI1 and pathway target genes as BCL-2 and BMP4 in uterine LMS cells compared to LM and MM. These results suggest a new therapeutic perspective for LMS by target drugs to inhibit SMO and GLI1 molecules. Citation Format: Natalia Garcia, Bianca C. Oliveira, Kelly P. Ferreira, Edmund C. Baracat, Katia C. Carvalho. Non canonical activation of Sonic Hedgehog pathway in uterine leiomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2436. doi:10.1158/1538-7445.AM2017-2436

Abstract 2435: Smoothened a new perspective for uterine leiomyoma treatment

Abstract Background: Uterine leiomyomas (LM) are the most common gynecologic tumors occurring in women of reproductive age. Patients may present clinical complications such as bleeding, pain and infertility. Additionally, some researchers believe that a leiomyosarcoma can arise from a degenerated LM. Efficient treatments for LM are still limited. Identification of gene markers can help to elucidate these tumors pathogenesis and to reveal molecules that can be used as therapeutic targets. Objective: To assess gene and miRNAs expression, and DNA methylation profiles of genes related to the Sonic Hedgehog (SHH) signaling pathway in uterine leiomyoma samples in order to look for potential therapeutic targets. Methods: 80 samples of LM and 20 myometrium (MM) were obtained in the Department of Obstetrics and Gynecology at HCFMUSP/Sao Paulo - Brazil. Expression profile of 106 genes and 84 miRNAs sequences related to SHH regulation, and the percentage of methylation in the promoter region of the SHH, PTCH1, SMO, SUFU, GLI and GLI3 genes were performed by quantitative Real Time PCR. Results: Among 106 genes evaluated, 16 showed hyper- and 11 showed hypoexpression in LM samples, compared to MM. Only SMOOTHENED (SMO) was found hyperegulated in LM. miRNAs analysis showed 16 sequences with different regulation profiles between LM and MM. None of them is involved in the regulation of SMO expression. DNA methylation profile showed a higher percentage of methylation in LM samples, but this difference did not exceed 2% of methylation for the PTCH1, SMO, GLI1 and GLI3 genes. Conclusion: Our results suggest an activation of SHH pathway in LMs by hyperexpression of SMO. These results are very interesting because there are SMO specific drugs approved by FDA for other tumors types. Citation Format: Natalia Garcia, Laura Gonzalez Anjos, Giovana De Nardo Maffazioli, Nilo Bozzini, Edmund Chada Baracat, Katia Candido Carvalho. Smoothened a new perspective for uterine leiomyoma treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2435. doi:10.1158/1538-7445.AM2017-2435