Hedgehog Inhibition With the Orally Bioavailable Smo Antagonist LDE225 Represses Tumor Growth and Prolongs Survival in a Transgenic Mouse Model of Islet Cell Neoplasms
Volker FendrichDominik WieseJens WaldmannMatthias LauthAnna E. HeverhagenJohannes RehmDetlef K. Bartsch
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In Brief Background: This study was designed to evaluate the role of the hedgehog pathway in tumor progression of murine islet cell tumors. Blockade of aberrant hedgehog activation has recently been proposed as a therapeutic target, but effects in models of islet cell tumors with a new orally bioavailable Smoothened (Smo) antagonist LDE225 have not been examined. Material and Methods: To assess in vivo effects, transgenic Rip1Tag2 mice, which develop islet cell neoplasms, were treated with vehicle or LDE225 (80 mg/kg/d) from week 5 until death. The resected pancreata were evaluated macroscopically and microscopically by iummohistochemsistry. Quantitative real-time polymerase chain reaction was performed for hedgehog target genes with RNA from islet, isolated from treated and untreated Rip1Tag2 mice. Results: LDE225 significantly reduced tumor volume by 95% compared with untreated control mice. Hedgehog inhibition with LDE225 significantly prolonged median survival in the used transgenic mouse model (105 vs 116 days; P = 0.02). Quantitative real-time polymerase chain reaction for downstream hedgehog target genes demonstrated significant downregulation in the islet cell tumors of Rip1Tag2 mice treated with LDE225, confirming the ability to achieve effective pharmacologic levels of LDE225 within the desired tissue site, in vivo. Conclusion: This is the first study to show that the orally bioavailable Smo antagonist LDE225 may provide a new option for therapy of islet cell neoplasms. This study was designed to evaluate the role of the hedgehog pathway in tumor progression of murine islet cell tumors. Transgenic Rip1Tag2 mice, which develop islet cell neoplasms, were treated with the Smo antagonist LDE225, resulting in decreased tumor volume and prolonged median survival. This is the first study to show that orally bioavailable LDE225 may provide a new option for therapy of islet cell neoplasms.Keywords:
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Abstract LB-430: Hedgehog signaling is required for the survival and tumorigenicity of cancer cells.
Abstract Background: Using small molecule smoothened (SMO) antagonists, Hedgehog (HH) signaling had been implicated to play an important role in a variety of cancers (e.g. Brain, Lung, Pancreas, digestive tract etc.). Recently, based on studies in a selected set of pancreatic and colorectal cancer cells it has been suggested: a) earlier observation of HH signaling dependence of various cancers by others and us is due to non-specific effect of SMO antagonists; b) HH producing cancer cells do not have a functional HH pathway; c) HH signaling is not required for the growth or survival of cancer cells rather it affects the growth of tumor by activating HH pathway in stroma cells. Given the reported prevalence of HH signaling in cancers and the suggested implication of this latter work on devising a successful cancer therapy, we decided to investigate the role of HH signaling in cancer in a manner independent of SMO antagonist. Lung cancer, being one of the deadliest cancers with the reported higher incidence of HH signaling, was used as a test system to evaluate the basic assumptions of this recently proposed model of HH role in cancer. Objective: Define the role of HH signaling in cancer cells. Methodology: The human non-small cell lung carcinoma (NSCLC) cell lines (HOP62, A549, U1752, H23, H157, H522) were used as a model system to study the relevance of HH signaling in cancer cells. The HH signaling was modulated by shRNA mediated knockdown of various HH signaling components (e.g. SMO, GLI1, SHH). The lentiviruses expressing shRNA were made and used to deliver the shRNA in NSCLC cells. HH responsiveness (quantitative real time RT-PCR and western blotting), cell proliferation (ATP quantitation), survival (Annexin-V labeling), anchorage independent growth (soft agar assay) and tumorigenesis (xenografts in Nu/Nu nude mice) were evaluated following shRNA mediated attenuation of HH signaling. Currently, HH pathway activation in cancer and stroma cells of primary lung cancer samples is being evaluated by immunohistochemistry (IHC) and RNA in situ hybridization (ISH). Preliminary Results and Conclusions: Exogenous expression of SHH in HOP62 and A549 induced the HH target gene GLI1 and PTCH1 expression while shRNA mediated knockdown of HH pathway components (e.g. SMO, SHH) inhibited the HH target gene expression. Further more, knockdown of HH pathway components, attenuated the proliferation, increased the apoptosis while decreased the anchorage independent growth of tested lung cancer cell lines. GLI1 shRNA mediated attenuation of HH signaling in A549 cells, greatly reduced their tumorigenicity in nude mice. We anticipate seeing activated HH signaling in cancer and stroma cells of primary lung tumor samples by IHC and ISH. Together, these results would indicate that cancer cells do elaborate a functional HH signaling pathway and require HH signaling for growth, survival and tumorigenicity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-430. doi:10.1158/1538-7445.AM2011-LB-430
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The hedgehog (Hh) signaling pathway is activated in various types of cancer including pancreatic ductal adenocarcinoma. It has been shown that extremely low oxygen tension (below 1% O 2 ) is found in tumor tissue including pancreatic ductal adenocarcinoma cells (PDAC) and increases the invasiveness of PDAC. To investigate the contribution of the Hh pathway to hypoxia‐induced invasiveness, we examined how hypoxia affects Hh pathway activation and the invasiveness of PDAC. In the present study, three human PDAC lines were cultured under normoxic (20% O 2 ) or hypoxic (1% O 2 ) conditions. Hypoxia upregulated the transcription of Sonic hedgehog ( Shh ), Smoothened ( Smo ), Gli1 and matrix metalloproteinase9 ( MMP9 ) and increased the invasiveness of PDAC. Significantly, neither the addition of recombinant Shh (rhShh) nor the silencing of Shh affected the transcription of these genes and the invasiveness of PDAC. On the other hand, silencing of Smo decreased the transcription of Gli1 and MMP9 and PDAC invasiveness. Silencing of Gli1 or MMP9 decreased PDAC invasiveness. These results suggest that hypoxia activates the Hh pathway of PDAC by increasing the transcription of Smo in a ligand‐independent manner and increases PDAC invasiveness. ( Cancer Sci 2011; 102: 1144–1150)
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The hedgehog pathway plays a critical role in the development of the foregut. However, the role of the hedgehog pathway in primary esophageal cancers is not well studied. Here, we report that elevated expression of hedgehog target genes occurs in 14 of 22 primary esophageal cancers. The hedgehog signaling activation is not associated with tumor subtypes, stages, or differentiation. While the sonic hedgehog (Shh) transcript is localized to the tumor tissue, expression of Gli1 and PTCH1 is observed both in the tumor and in the stroma. We discovered that 4 esophageal squamous cell carcinomas, which overexpress Shh, have genomic amplification of the Shh gene. Treatment of esophageal cancer cells with smoothened antagonist, KAAD-cyclopamine, or the neutralizing antibodies of Shh reduces cell growth and induces apoptosis. Overexpression of Gli1 under the CMV promoter renders these cells resistant to the treatments. Thus, our results indicate that elevated expression of Shh and its target genes is quite common in esophageal cancers. Our data also indicate that downregulation of Gli1 expression may be an important mechanism by which KAAD-cyclopamine inhibits growth and induces apoptosis in esophageal cancer cells.
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Inhibitors of the Hedgehog (Hh) pathway transducer Smoothened (Smo) have been approved for cancer treatment, but Smo mutations often lead to tumor resistance and it remains unclear how Smo is regulated. In this study, we identified the small GTPase Arl13b as a novel partner and regulator of Smo. Arl13b regulated Smo stability, trafficking, and localization, which are each crucial for Hh signaling. In gastric cancer cells, Arl13b stimulated proliferation, migration, and invasion
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Malignant melanoma is the leading cause of death among skin cancer patients due to its tendency to metastasize. Alterations at the molecular level are often evident, which is why melanoma biology has garnered increasing interest. The hedgehog (Hh) pathway, which is essential for embryonic development, is aberrantly re-activated in melanoma and may represent a promising therapeutic target. In addition, carbonic anhydrase XII (CAXII) represents a poor prognostic target for hypoxic tumors, such as melanoma, and is involved in cell migration. Thus, we decided to investigate whether and how the Hh pathway and CAXII may control melanoma cell migration and invasiveness.The migratory and invasive capabilities of SK-MEL-28 and A375 cell lines, either un-transfected or transiently transfected with Smoothened (SMO), GLI1, or CAXII siRNA, were studied under normoxic or hypoxic conditions.For the first time, we showed that SMO and GLI1 silencing resulted in the downregulation of CAXII expression in both moderately and highly invasive melanoma cells under hypoxia. The Hh pathway as well as CAXII inhibition by siRNA resulted in impaired malignant melanoma migration and invasion.Our results suggest that CAXII and the Hh pathway are relevant in melanoma invasion and may be novel and promising therapeutical targets for melanoma clinical management.
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Hedgehog signaling is aberrantly activated in glioma, medulloblastoma, basal cell carcinoma, lung cancer, esophageal cancer, gastric cancer, pancreatic cancer, breast cancer, and other tumors. Hedgehog signals activate GLI family members via Smoothened. RTK signaling potentiates GLI activity through PI3K-AKT-mediated GSK3 inactivation or RAS-STIL1-mediated SUFU inactivation, while GPCR signaling to Gs represses GLI activity through adenylate cyclase-mediated PKA activation. GLI activators bind to GACCACCCA motif to regulate transcription of GLI1, PTCH1, PTCH2, HHIP1, MYCN, CCND1, CCND2, BCL2, CFLAR, FOXF1, FOXL1, PRDM1 (BLIMP1), JAG2, GREM1, and Follistatin. Hedgehog signals are fine-tuned based on positive feedback loop via GLI1 and negative feedback loop via PTCH1, PTCH2, and HHIP1. Excessive positive feedback or collapsed negative feedback of Hedgehog signaling due to epigenetic or genetic alterations leads to carcinogenesis. Hedgehog signals induce cellular proliferation through upregulation of N-Myc, Cyclin D/E, and FOXM1. Hedgehog signals directly upregulate JAG2, indirectly upregulate mesenchymal BMP4 via FOXF1 or FOXL1, and also upregulate WNT2B and WNT5A. Hedgehog signals induce stem cell markers BMI1, LGR5, CD44 and CD133 based on cross-talk with WNT and/or other signals. Hedgehog signals upregulate BCL2 and CFLAR to promote cellular survival, SNAI1 (Snail), SNAI2 (Slug), ZEB1, ZEB2 (SIP1), TWIST2, and FOXC2 to promote epithelial-to-mesenchymal transition, and PTHLH (PTHrP) to promote osteolytic bone metastasis. KAAD-cyclopamine, Mu-SSKYQ-cyclopamine, IPI-269609, SANT1, SANT2, CUR61414 and HhAntag are small-molecule inhibitors targeted to Smoothened, GANT58, GANT61 to GLI1 and GLI2, and Robot-nikinin to SHH. Hedgehog signaling inhibitors should be used in combination with RTK inhibitors, GPCR modulators, and/or irradiation for cancer therapy.
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Hepatocellular carcinoma (HCC) is one of the leading causes of cancer‑associated mortality worldwide. Hepatocarcinogenesis involves numerous interlinked factors and processes, including the Sonic hedgehog (Shh) signaling pathway, which participates in the carcinogenesis, progression, invasiveness, recurrence and cancer stem cell maintenance of HCC. The Shh signaling pathway is activated by ligands that bind to their receptor protein, Protein patched homolog (Ptch). The process of Shh ligand binding to Ptch weakens the inhibition of smoothened homolog (SMO) and activates signal transduction via glioma‑associated oncogene homolog (Gli) transcription factors. The overexpression of Shh pathway molecules, including Shh, Ptch‑1, Gli and SMO has been indicated in patients with HCC. It has also been suggested that the Shh signaling pathway exhibits cross‑talk between numerous other signaling pathways. The inactivation of the Shh signaling pathway reduces HCC growth, increases radio‑sensitivity and increases the beneficial effect of chemotherapy in HCC treatment. Therefore, inhibition of the Shh pathway may be an effective target therapy that can be used in the treatment of HCC.
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Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest human malignancies, with a dismal six month median survival. Currently there is inadequate understanding of the molecular mechanisms behind PDAC initiation and progression from pancreatic intraepithelial neoplasia (PanINs), the most common precursor lesions, to full blown carcinoma. Thus, there is a lack of effective treatment modalities. The Hedgehog (Hh) signaling pathway plays an important role in PDAC; 75% of PDAC cases display increased expression of Hh ligands. In fact, Hh signaling is found in early PanIN lesions and promotes tumor growth, and persists as the cancer progresses. Interestingly, Hh ligands do not act on the tumor cells themselves, but instead function in a paracrine manner, signaling to the surrounding tumor stroma, which in turn promotes tumor progression. PDAC is characterized by a highly desmoplastic stroma, however the mechanisms that activate Hh signaling in the stroma are currently not well understood, and represent potential therapeutic targets. Hh pathway activation involves the binding of Hh ligands to the canonical receptor, Patched1 (Ptch1). This terminates Ptch1-mediated repression of Smoothened (Smo) and furthers downstream effects through activation of the Gli family of transcription factors. Recently, new co-receptors were identified that play an essential role in Hh pathway function. CAMrelated/down-regulated by oncogenes (Cdo), Brother of Cdo (Boc), and Growth arrest-specific 1 (Gas1) all cooperate with Ptch1 to promote Hh signaling during development. A central question is to what degree these receptors act to mediate Hh pathway function in adult tissues, especially in Hh-driven diseases, such as PDAC. Our data reveals that in the healthy pancreas, Boc and Gas1 are expressed in a perivascular and periductal manner in both fibroblasts and stellate cells as shown by co-stains with αSMA and vimentin. We did not detect Cdo expression. Interestingly, during cancer progression, Gas1 and Boc expression is significantly increased throughout the cancer stroma. This finding is notable as expression of Gli2, a vital transcription factor in the Hh pathway, is also significantly increased in the stroma during tumorigenesis. Functional studies on Boc-/-;Gas1-/- mouse embryonic fibroblasts (MEFs) treated with Hh ligand showed a drastic reduction in Hh response compared to wild-type MEFs, indicating that these co-receptors are important for Hh signal transduction. To further functionally characterize these receptors, we performed co-injection experiments with wild-type pancreatic fibroblasts and tumor cells or Boc-/-;Gas1-/- fibroblasts and tumor cells to determine the effect of co-receptor loss on tumor growth. Strikingly, tumors co-injected with Boc-/-;Gas1-/- fibroblasts formed significantly larger tumors. Histological analysis revealed that these tumors had a higher degree of vascularity. Future studies include dissecting the manner in which mutant fibroblasts support increased vascularity and whether this increase renders these tumors more susceptible to chemotherapy. This abstract is also presented as poster B37. Citation Format: Esha Mathew, Benjamin L. Allen, Marina Pasca di Magliano. Novel hedgehog co-receptors in pancreatic cancer progression. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr PR10.
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