Hh Inhibitors Research Articles

Inhibition of Melanoma Cell Migration and Invasion Targeting the Hypoxic Tumor Associated CAXII.

Simple SummaryMelanoma is a potential metastatic cancer with a poor prognosis and a low free-survival rate. Thus, discovering new therapeutic strategies will be helpful to fight against it. Carbonic anhydrases IX (CAIX) and XII (CAXII) along with Hedgehog pathway aberrations are already demonstrated to be involved in melanoma progression. Here we investigated the effects of either an indirect or direct CAXII inhibition on cell migration and invasion in three melanoma cell lines. First we indirectly inhibited CAXII through the smoothened antagonist cyclopamine, which resulted in a decreased CAXII protein expression and cell migration. Thereafter, we directly blocked CAXII using new small molecules. This resulted in reducing not only cell migration, but also the invasiveness ability of highly aggressive melanoma cell lines. This evidence may contribute to further exploiting the therapeutic role of CAXII in melanoma progression and invasiveness.Background: Intratumoral hypoxia contributes to cancer progression and poor prognosis. Carbonic anhydrases IX (CAIX) and XII (CAXII) play pivotal roles in tumor cell adaptation and survival, as aberrant Hedgehog (Hh) pathway does. In malignant melanoma both features have been investigated for years, but they have not been correlated before and/or identified as a potential pharmacological target. Here, for the first time, we demonstrated that malignant melanoma cell motility was impaired by targeting CAXII via either CAs inhibitors or through the inhibition of the Hh pathway. Methods: We tested cell motility in three melanoma cell lines (WM-35, SK-MEL28, and A375), with different invasiveness capabilities. To this end we performed a scratch assay in the presence of the smoothened (SMO) antagonist cyclopamine (cyclo) or CAs inhibitors under normoxia or hypoxia. Then, we analyzed the invasiveness potential in the cell lines which were more affected by cyclo and CAs inhibitors (SK-MEL28 and A375). Western blot was employed to assess the expression of the hypoxia inducible factor 1α, CAXII, and FAK phosphorylation. Immunofluorescence staining was performed to verify the blockade of CAXII expression. Results: Hh inhibition reduced melanoma cell migration and CAXII expression under both normoxic and hypoxic conditions. Interestingly, basal CAXII expression was higher in the two more aggressive melanoma cell lines. Finally, a direct CAXII blockade impaired melanoma cell migration and invasion under hypoxia. This was associated with a decrease of FAK phosphorylation and metalloprotease activities. Conclusions: CAXII may be used as a target for melanoma treatment not only through its direct inhibition, but also through Hh blockade.

The bromodomain inhibitor IBET-151 attenuates vismodegib-resistant esophageal adenocarcinoma growth through reduction of GLI signaling.

The Hedgehog/GLI (HH/GLI) signaling pathway plays a critical role in human oncogenesis. Unfortunately, the clinical use of HH inhibitor(s) has been associated with serious adverse effects and mutation-related drug resistance. Since the efficacy of SMO (Smoothened) and GLI inhibitors is limited in clinical trials, there remains a critical need for the HH/GLI pathway inhibitors with different mechanisms of action. Here, we show that esophageal adenocarcinoma (EAC) cell lines are insensitive to vismodegib (SMO inhibitor) but respond to GANT61 (GLI1 inhibitor). Furthermore, we examine the role of GLI1 in tumorigenicity of EAC and how a selective bromodomain inhibitor IBET-151 downregulates transcriptional activity of the GLI1 transcription factor in EAC. Our study demonstrates that GLI1 plays an important role in tumorigenicity of EAC and that elevated GLI1 expression in patients’ ultrasound-assisted endoscopic biopsy may predict the response to neoadjuvant chemotherapy (NAC) FOLFOX. Importantly, IBET-151 abrogates the growth of vismodegib-resistant EAC cells and downregulates HH/GLI by reducing the occupancy of BRD4 at the GLI1 locus. IBET-151 also attenuates tumor growth of EAC-PDXs and does so in an on-target manner as it reduces the expression of GLI1. We identify HH/GLI signaling as a novel druggable pathway in EAC as well as validate an ability of clinically relevant GLI inhibitor to attenuate the viability of vismodegib-resistant EAC cells. Therefore, we propose that selective bromodomain inhibitors, such as IBET-151, could be used as novel therapeutic agents for EAC patients harboring GLI-dependent tumors.

Abstract A30: Alisertib acts synergistically with sonidegib by modulating primary cilia assembly in a pediatric RELA ependymoma cell line

Abstract Introduction: Recently we found that RELA ependymomas (EPN) demonstrate inappropriate activation of the Hedgehog (Hh) pathway. However, how this activation is modulated remains to be investigated. Primary cilia are essential to positive and negative modulation of Hh signaling and changes in ciliation seem to be linked to Hh drug resistance in pediatric cancers. In contrast, targeting cilia integrity by molecules that prevents ciliary disassembly, such as Aurora-A (AURKA), has been shown to overcome this resistance. However, the role of the Hh pathway and the prevalence and function of cilia in pediatric RELA ependymoma (EPN) subgroup have not been examined. Thus, we explored the effect of Hh inhibitor, sonidegib, alone or in combination with Alisertib, AURKA inhibitor, in EPN RELA cell line. Methods: Hh pathway activation of RELA cell line (BXD-1425) was evaluated by immunostaining using ARL13B, a marker specific for ciliary membranes, proto-oncogene Smoothened (SMO) and Hh effector, GLI, antibodies in untreated cells or after stimulation with SMO agonist (SAG). Western blot was performed for protein expression of GLI1, GLI2, and full-length (FL) and repressor form (R) of Hh transcriptional program GLI3. Cell proliferation was assessed by CCK8 assay. Cell apoptosis was detected with Annexin V-FITC by flow cytometry. Results: We showed that EPN RELA cell line is frequently ciliated. Fluorescence intensity showed SMO and GLI recruitment to cilia in both BXD untreated cells and after stimulation with SAG, implicating in activation of Hh signals. Surprisingly, sonidegib treatment reduced ciliary formation in BXD cell line. Cilia loss was followed by increase of GLI recruitment to cilia and GLI2 expression, potentially by reduction of GLI3R. Our results indicate that impaired GLI processing probably by cilia disruption could explain persistent activation of downstream Hh pathway. To test this hypothesis, we targeted the cilia integrity through the combination of sonidegib with alisertib, to see if the cilia maintenance might affect Hh function and overcome sonidegib resistance. The combination prevented the primary cilia disruption followed by upregulation of GLI3R and downregulation of GLI recruitment to cilia, GLI2, and GLI3FL expression. Our data point towards to a synergistic combination that enhances sonidegib efficiency by primary cilia preservation, reducing cell proliferation and increasing apoptosis BXD cells compared to drugs alone treatment. Conclusion: Our findings suggest a synergistic combination of sonidegib with alisertib that enhances the inhibition of Hh major effectors such as GLIs (GLI2 and GLI3FL) and induces GLI3R expression. Our data suggest that combination of these agents may represent a novel approach for treatment of ST-EPN-RELA tumors to overcome chemoresistance and these mechanisms are potentially mediated by primary cilia, a key regulator for Hh activity. Citation Format: Taciani de Almeida Magalhães, Gustavo Alencastro Veiga Cruzeiro, Kleiton Silva Borges, Cherry Liu Yulu, Graziella Ribeiro de Souza, Keteryne Rodrigues da Silva, Carlos Alberto Scridelli, Adrian Salic, Luiz Gonzaga Tone. Alisertib acts synergistically with sonidegib by modulating primary cilia assembly in a pediatric RELA ependymoma cell line [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A30.

Trp53 and Rb1 regulate autophagy and ligand-dependent Hedgehog signaling.

Ligand-dependent activation of Hedgehog (Hh) signaling in cancer occurs without mutations in canonical pathway genes. Consequently, the genetic basis of Hh pathway activation in adult solid tumors, such as small-cell lung cancer (SCLC), is unknown. Here we show that combined inactivation of Trp53 and Rb1, a defining genetic feature of SCLC, leads to hypersensitivity to Hh ligand in vitro, and during neural tube development in vivo. This response is associated with the aberrant formation of primary cilia, an organelle essential for canonical Hh signaling through smoothened, a transmembrane protein targeted by small-molecule Hh inhibitors. We further show that loss of both Trp53 and Rb1 disables transcription of genes in the autophagic machinery necessary for the degradation of primary cilia. In turn, we also demonstrate a requirement for Kif3a, a gene essential for the formation of primary cilia, in a mouse model of SCLC induced by conditional deletion of both Trp53 and Rb1 in the adult airway. Our results provide a mechanistic framework for therapeutic targeting of ligand-dependent Hh signaling in human cancers with somatic mutations in both TP53 and RB1.

ApoE mimetic peptide targeted nanoparticles carrying a BRD4 inhibitor for treating Medulloblastoma in mice

Treatment of medulloblastoma (MB) is challenging due to diverse genetic make-up, chemoresistance and inefficient drug transport across the blood brain barrier (BBB). Since hedgehog (Hh) signaling regulates cancer cell proliferation and tumorigenicity, Hh inhibitors have the potential to treat sonic Hh driven MB (SHH-MB), but their repeated use develops chemoresistance due to mutations in smoothened (SMO). Herein, we aimed to overcome these problems by modulating GLI transcription using JQ1, which is a small molecule BRD4 inhibitor. JQ1 inhibited HD-MB03 and DAOY cell proliferation, with the IC50 of 402 and 4220 nM, respectively. JQ1 inhibited colony formation, but increased apoptosis in HD-MB03 and DAOY cells. Western blot analysis confirmed significant inhibition of GLI1 and c-MYC protein expression in DAOY and HD-MB03 cells, respectively. JQ1 was encapsulated into apolipoprotein (ApoE) mimetic peptide decorated nanoparticles (ApoE-NPs), with the mean particle size of 64 nm and drug loading of 10% (w/w). ApoE-NPs increased JQ1 concentration in the tumor by 5 and 8 folds at 6 and 24 h after systemic administration into orthotopic MB tumor bearing NSG mice compared to non-targeted JQ1 loaded NPs. Although there was also modest increase in JQ1 delivery to the liver, there was no hepatotoxicity as evidenced by H&E staining and little increase in serum ALT and AST after treatment with JQ1 loaded ApoE-NPs. There was also significant decrease in the orthotopic MB tumor burden after systemic administration of JQ1 loaded ApoE- NPs at the dose of 10 mg/kg every 3rd day for a total of 8 injections. In conclusion, JQ1 loaded NPs have the potential to treat Group 3 and SHH driven MB in mice.

Abstract P5-04-17: Hedgehog pathway is involved in cancer immune surveillance through PDL1 modulation

Abstract Background: Recently, immune check-point inhibitors have shown efficacy in triple-negative breast cancer (TNBC). Since Hedgehog (Hh) signalling mediates crosstalk between breast cancer cells and tumor-infiltrating immune cells, we investigated the mechanisms by which Hh can modulate PDL1 expression and the tumor-immune microenvironment. Methods: TNBC tumors from untreated patients were subjects to PDL1 and Gli1 expression analysis by IHC. The correlation of Hh pathway activation and PDL1 expression was assessed in TNBC cells treated with the SMO-inhibitor NVP-LDE225. The main aim was to study the PDL1/Gli1 cross-talk. Results: The expression of PDL1 and Gli1, the major indicator for the canonical Hh signaling activation, were assessed by IHC in a tissue microarray (TMA) of TNBC samples from 237 untreated patients. In 203/237 cases we could analyze both PDL1 and Gli1 protein expression. A significant correlation between PDL1 and Gli1 expression was found. Indeed, of 77/203 (38%) PDL1 positive tumors 42/77 (54%) expressed Gli1. In order to explore correlations between other Hh pathway members and PDL1 in TNBCs, we interrogated the open-access database TCGA; PDL1 positive TNBCs showed high levels of SMO and PTCH1, Hh pathway receptors (Q-value 0.018 and 0.010). To better understand the link between Hh pathway and PDL1, we selected a panel of TNBC cell lines; the pharmacological Hh pathway inhibition led to a reduction of PDL1 protein and mRNA. On the other hand, engineered cells harbouring Gli1 overexpression showed higher levels of PDL1. Therefore, we hypothesized that Hh pathway could be involved in the PDL1 transcriptional modulation. To address this issue, we performed a luciferase reporter assay and a ChIP analysis following by PCR amplification of the PDL1 gene promoter. We found that Gli1 binds the PDL1 promoter, indicating that Gli1 transcriptionally enhances PDL1 expression. Gli1 expression was evaluated also in 4T1 cells, a TNBC murine model; furthermore, in 4T1 cells PDL1 protein expression was inhibited by NVP-LDE225. In vivo experiment will be performed in Balb/C mice orthotopically xenografted with 4T1 cells to confirm the role of Hh pathway to modulate the PDL1 expression and the tumor microenviroment in vivo. Conclusions: Our results suggest that Hh pathway has a crucial role in cancer immune evasion trough PDL-1 modulation. Due to their ability to target both tumor cells and the tumor microenvironment, Hh inhibitors could represent promising therapeutics to be clinically investigated in Gli1 overexpressing TNBC patients. Citation Format: Pietro De Placido, Concetta Di Mauro, Daniela Esposito, Ada Pesapane, Stefania Belli, Fabiana Napolitano, Antonio Santaniello, Priscilla Cascetta, Annachiara Carratù, Eleonora Mozzillo, Roberta Marciano, Alberto Servetto, Roberto Bianco, Luigi Formisano. Hedgehog pathway is involved in cancer immune surveillance through PDL1 modulation [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-04-17.

PEGylated PLGA Nanoparticle Delivery of Eggmanone for T Cell Modulation: Applications in Rheumatic Autoimmunity.

BackgroundHelper T cell activity is dysregulated in a number of diseases including those associated with rheumatic autoimmunity. Treatment options are limited and usually consist of systemic immune suppression, resulting in undesirable consequences from compromised immunity. Hedgehog (Hh) signaling has been implicated in the activation of T cells and the formation of the immune synapse, but remains understudied in the context of autoimmunity. Modulation of Hh signaling has the potential to enable controlled immunosuppression but a potential therapy has not yet been developed to leverage this opportunity.MethodsIn this work, we developed biodegradable nanoparticles to enable targeted delivery of eggmanone (Egm), a specific Hh inhibitor, to CD4+ T cell subsets. We utilized two FDA-approved polymers, poly(lactic-co-glycolic acid) and polyethylene glycol, to generate hydrolytically degradable nanoparticles. Furthermore, we employed maleimide-thiol mediated conjugation chemistry to decorate nanoparticles with anti-CD4 F(ab’) antibody fragments to enable targeted delivery of Egm.ResultsOur novel delivery system achieved a highly specific association with the majority of CD4+ T cells present among a complex cell population. Additionally, we have demonstrated antigen-specific inhibition of CD4+ T cell responses mediated by nanoparticle-formulated Egm.ConclusionThis work is the first characterization of Egm’s immunomodulatory potential. Importantly, this study also suggests the potential benefit of a biodegradable delivery vehicle that is rationally designed for preferential interaction with a specific immune cell subtype for targeted modulation of Hh signaling.

Phase 2 study of vismodegib, a hedgehog inhibitor, combined with gemcitabine and nab-paclitaxel in patients with untreated metastatic pancreatic adenocarcinoma

BackgroundThe Hedgehog (Hh) signalling pathway is overexpressed in pancreatic ductal adenocarcinoma (PDA). Preclinical studies have shown that Hh inhibitors reduce pancreatic cancer stem cells (pCSC), stroma and Hh signalling.MethodsPatients with previously untreated metastatic PDA were treated with gemcitabine and nab-paclitaxel. Vismodegib was added starting on the second cycle. The primary endpoint was progression-free survival (PFS) as compared with historical controls. Tumour biopsies to assess pCSC, stroma and Hh signalling were obtained before treatment and after cycle 1 (gemcitabine and nab-paclitaxel) or after cycle 2 (gemcitabine and nab-paclitaxel plus vismodegib).ResultsSeventy-one patients were enrolled. Median PFS and overall survival (OS) were 5.42 months (95% confidence interval [CI]: 4.37–6.97) and 9.79 months (95% CI: 7.85–10.97), respectively. Of the 67 patients evaluable for response, 27 (40%) had a response: 26 (38.8%) partial responses and 1 complete response. In the tumour samples, there were no significant changes in ALDH + pCSC following treatment.ConclusionsAdding vismodegib to chemotherapy did not improve efficacy as compared with historical rates observed with chemotherapy alone in patients with newly diagnosed metastatic pancreatic cancer. This study does not support the further evaluation of Hh inhibitors in this patient population.Trial registrationClinicalTrials.gov Identifier: NCT01088815.

Mutual Zonated Interactions of Wnt and Hh Signaling Are Orchestrating the Metabolism of the Adult Liver in Mice and Human

The Hedgehog (Hh) and Wnt/β-Catenin (Wnt) cascades are morphogen pathways whose pronounced influence on adult liver metabolism has been identified in recent years. How both pathways communicate and control liver metabolic functions are largely unknown. Detecting core components of Wnt and Hh signaling and mathematical modeling showed that both pathways in healthy liver act largely complementary to each other in the pericentral (Wnt) and the periportal zone (Hh) and communicate mainly by mutual repression. The Wnt/Hh module inversely controls the spatiotemporal operation of various liver metabolic pathways, as revealed by transcriptome, proteome, and metabolome analyses. Shifting the balance to Wnt (activation) or Hh (inhibition) causes pericentralization and periportalization of liver functions, respectively. Thus, homeostasis of the Wnt/Hh module is essential for maintaining proper liver metabolism and to avoid the development of certain metabolic diseases. With caution due to minor species-specific differences, these conclusions may hold for human liver as well.

Mesenchymal Cells Support the Oncogenicity and Therapeutic Response of the Hedgehog Pathway in Triple-Negative Breast Cancer.

The paracrine interaction between tumor cells and adjacent stroma has been associated with the oncogenic activity of the Hedgehog (Hh) pathway in triple-negative breast tumors. The present study developed a model of paracrine Hh signaling and examined the impact of mesenchymal cell sources and culture modalities in the oncogenicity of the Hh pathway in breast tumor cells. Studies consisted of tumor cell monocultures and co-cultures with cancer-associated and normal fibroblasts, tumor cells that undergo epithelial–mesenchymal transition (EMT), or adipose-derived mesenchymal stem cells (ADMSCs). Hh ligand and pathway inhibitors, GANT61 and NVP-LDE225 (NVP), were evaluated in both cell cultures and a mouse xenograft model. Results in monocultures show that tumor cell viability and Hh transcriptional activity were not affected by Hh inhibitors. In co-cultures, down-regulation of GLI1, SMO, and PTCH1 in the stroma correlated with reduced tumor growth rates in xenografted tumors and cell cultures, confirming a paracrine interaction. Fibroblasts and EMT cells supported Hh transcriptional activity and enhanced tumor cell growth. Mixed and adjacent culture modalities indicate that tumor growth is supported via fibroblast-secreted soluble factors, whereas enriched tumor stemness requires close proximity between tumor and fibroblasts. Overall this study provides a tumor–mesenchymal model of Hh signaling and highlights the therapeutic value of mesenchymal cells in the oncogenic activity of the Hh pathway.

A Smo/Gli Multitarget Hedgehog Pathway Inhibitor Impairs Tumor Growth.

Pharmacological Hedgehog (Hh) pathway inhibition has emerged as a valuable anticancer strategy. A number of small molecules able to block the pathway at the upstream receptor Smoothened (Smo) or the downstream effector glioma-associated oncogene 1 (Gli1) has been designed and developed. In a recent study, we exploited the high versatility of the natural isoflavone scaffold for targeting the Hh signaling pathway at multiple levels showing that the simultaneous targeting of Smo and Gli1 provided synergistic Hh pathway inhibition stronger than single administration. This approach seems to effectively overcome the drug resistance, particularly at the level of Smo. Here, we combined the pharmacophores targeting Smo and Gli1 into a single and individual isoflavone, compound 22, which inhibits the Hh pathway at both upstream and downstream level. We demonstrate that this multitarget agent suppresses medulloblastoma growth in vitro and in vivo through antagonism of Smo and Gli1, which is a novel mechanism of action in Hh inhibition.

Suppressing Hedgehog signaling reverses drug resistance of refractory acute myeloid leukemia.

BackgroundHedgehog (Hh) signaling is involved in the pathogenesis of tumors. By performing gene chip analysis, we predicted that Hh signaling might regulate multiple downstream pathways in acute myeloid leukemia (AML).MethodsIn this study, the potential role of the Hh pathway in refractory AML, and the impact of Hh expression on clinical prognosis were examined. We also investigated the role of the Hh inhibitor NVP-LDE225 in reversing drug resistance of refractory primary AML cells in vitro and the roles of multiple drug-resistant HL60/Adriamycin-resistant cells in vitro and in vivo (in a xenograft mouse model). Finally, we explored the underlying mechanisms.ResultsHh pathway was highly active in chemotherapy-resistant AML cells; by contrast, activation was less pronounced in chemosensitive cells and non-refractory primary cells. Strong activation of this pathway was associated with higher recurrence rates and poorer relapse-free and overall survival. NVP-LDE225 inhibited MRP1 protein expression, increased intracellular accumulation of Adriamycin, and reversed chemotherapeutic resistance. These effects were likely mediated through inhibition of the IGF-1R/Akt/MRP1 pathway. In the AML xenograft mouse model, NVP-LDE225 plus Adriamycin resulted in marked tumor regression.ConclusionThese findings suggest that targeting the Hh pathway might be a therapeutic avenue for overcoming MDR resistance and preventing refractory AML.

The Hedgehog Signaling Pathway: A Viable Target in Breast Cancer?

The hedgehog (Hh) pathway plays a key role in embryonic development and stem cell programs. Deregulation of the Hh pathway is a key driver of basal cell carcinoma, and therapeutic targeting led to approval of Hh inhibitor, vismodegib, in the management of this cancer. The Hh pathway is implicated in other malignancies including hormone receptor (HR+) positive and triple negative breast cancer (TNBC). Hh signaling, which is activated in human mammary stem cells, results in activation of glioma-associated oncogene (GLI) transcription factors. High GLI1 expression correlates with worse outcomes in breast cancer. Non-canonical GLI1 activation is one mechanism by which estrogen exposure promotes breast cancer stem cell proliferation and epithelial–mesenchymal transition. Tamoxifen resistant cell lines show aberrant activation of Hh signaling, and knockdown of Hh pathway inhibited growth of tamoxifen resistant cells. As in other cancers Hh signaling is activated by the PI3K/AKT pathway in these endocrine resistant cell lines. Hh pathway activation has also been reported to mediate chemotherapy resistance in TNBC via various mechanisms including paracrine signaling to tumor micro-environment and selective proliferation of cancer stem cells. Co-activation of Hh and Wnt signaling pathways is a poor prognostic marker in TNBC. Early phase clinical trials are evaluating the combination of smoothened (SMO) inhibitors and chemotherapy in TNBC. In addition to SMO inhibitors like vismodegib and sonidegib, which are in clinical use for basal cell carcinoma, GLI1 inhibitors like GANT58 and GANT61 are in preclinical drug development and might be an effective mechanism to overcome drug resistance in breast cancer. Gene signatures predictive of Hh pathway activation could enrich for patients likely to respond to these agents.

Paclitaxel and Itraconazole Co‐Encapsulated Micelle Prolongs the Survival of Spontaneous LSL‐KrasG12D/+, LSL‐Trp53R172H/+, Pdx‐1‐Cre Genetically Engineered Mouse Model of Pancreatic Cancer

AbstractPancreatic ductal adenocarcinoma (PDAC) is one of the most malignant gastrointestinal cancers with an extremely complex tumor microenvironment and a poor prognosis. The upregulation of Hedgehog (Hh) signaling pathway contributes to the abundance of stroma and barren blood vessels in the PDAC tumor microenvironment, creating a physical and biological barrier leading to strong inherent resistance to drugs. Therefore, inhibition of the Hh signaling pathway combined with chemotherapy has been explored in anti‐PDAC chemotherapy. In this study, the paclitaxel (PTX) and itraconazole (ITA) co‐encapsulated poly (ethylene glycol)‐b‐poly (d, l‐lactide) (PEG‐PLA) micelle (PIM) shows superior therapeutic outcome in PDAC. Pharmaceutically, PIM demonstrates optimized systemic pharmacokinetics and increases tumor drug accumulation due to its serum stability. Pharmacologically, through Hh inhibition and blood vessel normalization contributed by ITA and cytotoxicity contributed by PTX, PIM not only significantly inhibits the tumor growth of the human MIA PaCa‐2 cell‐derived orthotopic pancreatic cancer model but also significantly prolongs the survival of LSL‐KrasG12D/+; LSL‐Trp53R172H/+; Pdx‐1‐Cre (KPC) genetic engineered, spontaneous PDAC mice, a clinically relevant PDAC model with high similarity as human PDAC in terms of cancer histology and biology.

Abstract 3740: Ionizing radiation-induced transcriptomic changes in triple-negative breast cancer cells reveal putative mechanisms of treatment resistance

Abstract Breast cancer is the most common malignancy and the second leading cause of cancer-associated deaths in women. Of these, triple-negative breast cancers (TNBCs) remain the most difficult to treat. Unlike hormone-driven breast cancers, TNBCs that remain in remission past the five-year mark rarely relapse and are considered functionally cured. Thus, investigating mechanisms of treatment resistance in order to improve remission rates is of paramount importance. In this study, we use RNA-Seq to analyze global transcriptomic changes in TNBC cells in response to DNA damage induced by ionizing radiation (IR). Applying gene set enrichment analysis (GSEA), we have identified a number of expression signatures that represent putative mechanisms of resistance to treatment. In prior work, we have demonstrated the role of the transcription factor Gli1, the terminal effector of the Hedgehog (Hh) signaling pathway, in regulating the expression of DNA repair proteins in response to genotoxic damage with cisplatin. We have previously shown that Hh inhibition blunts the expected upregulation of specific proteins involved in single-strand DNA repair in ovarian cancer cells. Here, we expand on our earlier studies by examining TNBC cells with high levels of Hh activity and querying how global transcriptomic changes in response to IR are altered by Hh inhibition. Our data suggest that rational manipulation of these pathways may sensitize TNBCs to treatment and improve the functional cure rate of these tumors. Citation Format: Victor T. Lin, Tshering D. Lama-Sherpa, Rajeev S. Samant, Lalita A. Shevde. Ionizing radiation-induced transcriptomic changes in triple-negative breast cancer cells reveal putative mechanisms of treatment resistance [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 3740.

The E6/E7 oncogenes of human papilloma virus and estradiol regulate hedgehog signaling activity in a murine model of cervical cancer

Human papilloma virus oncogenes and estradiol are major etiologic factors associated with cervical cancer. In order to understand the mechanism by which these two factors promote carcinogenesis, the role of the Hedgehog (Hh) signaling pathway was evaluated during the normal growth of cervical epithelium and in the presence of E6/E7 oncogenes and exogenous estradiol. Hh signaling activity was determined in live animals (i.e., Gli-Luc reporter levels) during the estrous cycle and was found to be higher in the cervical area during the major growth phases, proestrus-estrus, in comparison to the diestrus phase. The same pattern was observed in transgenic mice expressing the E6/E7 oncogenes, though with notably higher levels than in control mice. Adding estradiol also markedly increased Gli activity in the cervix and the skin. In agreement with the correlation between high bioluminescence and tissue growth in different context, cervical cell proliferation was reduced upon Hh signaling inhibition in mice. Treatment with itraconazole, a putative novel Hh inhibitor, at an early stage of cervical carcinogenesis, did not decrease Hh signaling but it did reduce growth. Therefore, Hh signaling likely contributes to cervical carcinogenesis and itraconazole is effective to reduce growth but by a mechanism involving additional signaling pathways.

The Combination of Particle Irradiation With the Hedgehog Inhibitor GANT61 Differently Modulates the Radiosensitivity and Migration of Cancer Cells Compared to X-Ray Irradiation.

Due to the advantages of charged particles compared to conventional radiotherapy, a vast increase is noted in the use of particle therapy in the clinic. These advantages include an improved dose deposition and increased biological effectiveness. Metastasis is still an important cause of mortality in cancer patients and evidence has shown that conventional radiotherapy can increase the formation of metastasizing cells. An important pathway involved in the process of metastasis is the Hedgehog (Hh) signaling pathway. Recent studies have demonstrated that activation of the Hh pathway, in response to X-rays, can lead to radioresistance and increased migratory, and invasive capabilities of cancer cells. Here, we investigated the effect of X-rays, protons, and carbon ions on cell survival, migration, and Hh pathway gene expression in prostate cancer (PC3) and medulloblastoma (DAOY) cell lines. In addition, the potential modulation of cell survival and migration by the Hh pathway inhibitor GANT61 was investigated. We found that in both cell lines, carbon ions were more effective in decreasing cell survival and migration as well as inducing more significant alterations in the Hh pathway genes compared to X-rays or protons. In addition, we show here for the first time that the Hh inhibitor GANT61 is able to sensitize DAOY medulloblastoma cells to particle radiation (proton and carbon ion) but not to conventional X-rays. This important finding demonstrates that the results of combination treatment strategies with X-ray radiotherapy cannot be automatically extrapolated to particle therapy and should be investigated separately. In conclusion, combining GANT61 with particle radiation could offer a benefit for specific cancer types with regard to cancer cell survival.

Anteroposterior patterning of the zebrafish ear through Fgf- and Hh-dependent regulation of hmx3a expression

In the zebrafish, Fgf and Hh signalling assign anterior and posterior identity, respectively, to the poles of the developing ear. Mis-expression of fgf3 or inhibition of Hh signalling results in double-anterior ears, including ectopic expression of hmx3a. To understand how this double-anterior pattern is established, we characterised transcriptional responses in Fgf gain-of-signalling or Hh loss-of-signalling backgrounds. Mis-expression of fgf3 resulted in rapid expansion of anterior otic markers, refining over time to give the duplicated pattern. Response to Hh inhibition was very different: initial anteroposterior asymmetry was retained, with de novo duplicate expression domains appearing later. We show that Hmx3a is required for normal anterior otic patterning, and that otic patterning defects in hmx3a-/- mutants are a close phenocopy to those seen in fgf3-/- mutants. However, neither loss nor gain of hmx3a function was sufficient to generate full ear duplications. Using our data to infer a transcriptional regulatory network required for acquisition of otic anterior identity, we can recapitulate both the wild-type and the double-anterior pattern in a mathematical model.

Inhibiting Hedgehog: An Update on Pharmacological Compounds and Targeting Strategies

Important steps in embryonic development are governed by the Hedgehog (Hh) signaling pathway, an evolutionary conserved signal transduction cascade. However, Hh activity not only is crucial during embryo formation but also is involved in adult tissue repair and in several malignancies. Particularly due to its link to cancer, small molecule Hh pathway inhibitors have been developed and the first compounds have been approved for use in Hh-driven basal cell carcinoma. Almost all advanced Hh inhibitors target the critical signaling component Smoothened (SMO), but preclinical research has identified additional compounds that can block the Hh pathway along its entire signaling cascade, which, in light of emerging drug resistance occurring with SMO inhibitors, is of high importance. Herein we give an overview on currently known Hh pathway inhibitors, delineating their respective strengths and weaknesses and describing potential drug targeting strategies to interfere with Hh signaling in different cancer settings.

L-4, a Well-Tolerated and Orally Active Inhibitor of Hedgehog Pathway, Exhibited Potent Anti-tumor Effects Against Medulloblastoma in vitro and in vivo.

Inhibition of aberrant Hedgehog (Hh) pathway had been proved to be a promising therapeutic intervention in cancers like basal cell carcinoma (BCC), medulloblastoma (MB), and so on. Two drugs (Vismodegib, Sonidegib) were approved to treat BCC and more inhibitors are in clinical investigation. However, the adverse effects and drug resistance restricted the use of Hh inhibitors. In the present study, 61 synthesized compounds containing central backbone of phthalazine or dimethylpyridazine were screened as candidates of new Hh signaling inhibitors by performing dual luciferase reporter assay. Among the compounds, L-4 exhibited an IC50 value of 2.33 nM in the Shh-Light II assay. L-4 strongly inhibited the Hh pathway in vitro and blocked the Hh pathway by antagonizing the smoothened receptor (Smo). Remarkably, L-4 could significantly suppress the Hh pathway activity provoked by Smo mutant (D473H) which showed strong resistant properties to existing drugs such as Vismodegib. Orally administered L-4 exhibited prominent dose-dependent anti-tumor efficacy in vivo in Ptch+/-; p53-/- MB allograft model. Furthermore, L-4 showed good tolerance in acute toxicity test using ICR mice. These evidences indicated that L-4 was a potent, well-tolerated, orally active inhibitor of Hedgehog pathway, and might be a promising candidate in development of Hh-targeted anti-cancer drugs.