Gli Activity Research Articles

AP-1 and TGF\xdf cooperativity drives non-canonical Hedgehog signaling in resistant basal cell carcinoma

Tumor heterogeneity and lack of knowledge about resistant cell states remain a barrier to targeted cancer therapies. Basal cell carcinomas (BCCs) depend on Hedgehog (Hh)/Gli signaling, but can develop mechanisms of Smoothened (SMO) inhibitor resistance. We previously identified a nuclear myocardin-related transcription factor (nMRTF) resistance pathway that amplifies noncanonical Gli1 activity, but characteristics and drivers of the nMRTF cell state remain unknown. Here, we use single cell RNA-sequencing of patient tumors to identify three prognostic surface markers (LYPD3, TACSTD2, and LY6D) which correlate with nMRTF and resistance to SMO inhibitors. The nMRTF cell state resembles transit-amplifying cells of the hair follicle matrix, with AP-1 and TGFß cooperativity driving nMRTF activation. JNK/AP-1 signaling commissions chromatin accessibility and Smad3 DNA binding leading to a transcriptional program of RhoGEFs that facilitate nMRTF activity. Importantly, small molecule AP-1 inhibitors selectively target LYPD3+/TACSTD2+/LY6D+ nMRTF human BCCs ex vivo, opening an avenue for improving combinatorial therapies.

Yes-Associated Protein Contributes to Cell Proliferation and Migration of Gastric Cancer via Activation of Gli1.

ObjectiveIn the present study, we aimed to explore the potential oncogenic property and the internal mechanism of yes-associated protein (YAP) in gastric cancer (GC).Materials and MethodsYAP protein levels were evaluated in human GC tissues and paired normal tissues using immunohistochemistry (IHC). The role of YAP in regulating GC cell proliferation and migration was verified by genetic manipulation in vitro. Western blot analysis was used to determine the molecular signaling to explain the mechanism of the observed YAP effects in GC.ResultsNuclear YAP protein expression was upregulated in GC tissues, and high nuclear YAP level was significantly correlated with lymph node metastasis (LNM) and tumor node metastasis (TNM) stage in patients suffered from GC. YAP knockdown inhibited GC cell proliferation, migration and epithelial–mesenchymal transition (EMT) progress in vitro, whereas YAP elevation did the opposite. YAP regulated glioma-associated oncogene-1 (Gli1) expression independent of smoothened homolog (SMO). YAP modulated protein kinase B (AKT)/mechanistic target of rapamycin (mTOR) signaling pathway in GC cells.ConclusionYAP enhanced GC cell proliferation and migration potentially via its regulation of Gli1 expression through the non-classical Hedgehog pathway, indicating suppression of YAP/Gli1 signaling axis may highlight a new entry point for combination therapy of GC.

Hedgehog pathway activation in oral squamous cell carcinoma: cancer-associated fibroblasts exhibit nuclear GLI-1 localization

The purpose of this study was to evaluate the expression of Hedgehog (HH) signaling molecules (SHH and GLI-1) by cancer-associated fibroblasts (CAF) in oral squamous cell carcinoma (OSCC). Immunohistochemistry was used to detect molecular HH signaling and CAF-related protein expression, including α-SMA and S100A4, in 70 samples of human OSCC. The colocalization of α-SMA and S100A4 with SHH was also evaluated by double-staining. In vitro study was performed using primary normal oral fibroblast (NOF) and CAF through immunofluorescence and Western Blot for CAF-proteins, SHH, and GLI-1. Forty-five cases (64.28%) were positive for α-SMA exclusively in tumor stroma, and S100A4 was identified in the cytoplasm of CAFs in 94.28% (n = 66) of the cases. With respect to stromal cells, 64 (91.43%) OSCC cases were positive for SHH, and 31 were positive for GLI-1 (44.29%); positive correlations were found between SHH and α-SMA (p < 0.0001, φ = 0.51), as well as between SHH and S100A4 (p = 0.087, φ = 0.94). Protein expression of SHH and GLI-1 was observed in primary CAFs and NOFs. Although SHH was found to be localized in the cellular cytoplasm of both cell types, GLI-1 was present only in the nuclei of CAF. Our results indicate that CAFs are not only potential sources of HH ligands in tumor stroma, but may also respond to HH signaling through nuclear GLI-1 activation. We further observed that elevated SHH expression by OSCC cells was associated with higher CAF density, reinforcing the chemoattractant role played by these molecules.

The Role of Smoothened in Cancer.

Smoothened (SMO) belongs to the Hedgehog (HH) signaling pathway, which regulates cell growth, migration, invasion and stem cells in cancer. The HH signaling pathway includes both canonical and noncanonical pathways. The canonical HH pathway functions through major HH molecules such as HH ligands, PTCH, SMO and GLI, whereas the noncanonical HH pathway involves the activation of SMO or GLI through other pathways. The role of SMO has been discussed in different types of cancer, including breast, liver, pancreatic and colon cancers. SMO expression correlates with tumor size, invasiveness, metastasis and recurrence. In addition, SMO inhibitors can suppress cancer formation, reduce the proliferation of cancer cells, trigger apoptosis and suppress cancer stem cell activity. A better understanding of the role of SMO in cancer could contribute to the development of novel therapeutic approaches.

Acute Alcohol Intoxication Impairs Sonic Hedgehog-Gli1 Signaling and Activation of Primitive Hematopoietic Precursor Cells in the Early Stage of Host Response to Bacteremia.

Activation of hematopoietic stem cells [HSCs, lineage(lin)- stem cell growth factor receptor (c-kit)+ stem cell antigen-1(Sca-1)+ , or LKS cells in mice] is critical for initiating the granulopoietic response. This study determined the effect of alcohol exposure on sonic hedgehog (SHH) signaling in the regulation of HSC activation during bacteremia. Acute alcohol intoxication was induced in mice by intraperitoneal (i.p.) injection of 20% alcohol (5g alcohol/kg body weight). Control mice received i.p. saline. Thirty minutes later, mice were intravenously (i.v.) injected with Escherichia coli (E.coli, 1 to 5×107 CFUs/mouse) or saline. SHH expression by lineage-negative bone marrow cells (BMCs) was significantly increased 24hours after E.coli infection. Extracellular signal-regulated kinase 1/2 (ERK1/2)-specificity protein 1 (Sp1) signaling promotes SHH expression. ERK1/2 was markedly activated in BMCs 8hours following E.coli infection. Alcohol suppressed both the activation of ERK1/2 and up-regulation of SHH expression following E.coli infection. E.coli infection up-regulated GLI family zinc finger 1 (Gli1) gene expression by BMCs and increased Gli1 protein content in LKS cells. The extent of Gli1 expression was correlated with the activity of proliferation in LKS cells. Alcohol inhibited up-regulation of Gli1 expression and activation of LKS cells in response to E.coli infection. Alcohol also interrupted the granulopoietic response to bacteremia. These data show that alcohol disrupts SHH-Gli1 signaling and HSC activation in the early stage of the granulopoietic response, which may serve as an important mechanism underlying the impairment of immune defense against bacterial infection in host excessively consuming alcohol.

Fine-Tuning of GLI Activity through Arginine Methylation: Its Mechanisms and Function

The glioma-associated oncogene (GLI) family consists of GLI1, GLI2, and GLI3 in mammals. This family has important roles in development and homeostasis. To achieve these roles, the GLI family has widespread outputs. GLI activity is therefore strictly regulated at multiple levels, including via post-translational modifications for context-dependent GLI target gene expression. The protein arginine methyl transferase (PRMT) family is also associated with embryogenesis, homeostasis, and cancer mainly via epigenetic modifications. In the PRMT family, PRMT1, PRMT5, and PRMT7 reportedly regulate GLI1 and GLI2 activity. PRMT1 methylates GLI1 to upregulate its activity and target gene expression. Cytoplasmic PRMT5 methylates GLI1 and promotes GLI1 protein stabilization. Conversely, nucleic PRMT5 interacts with MENIN to suppress growth arrest-specific protein 1 expression, which assists Hedgehog ligand binding to Patched, indirectly resulting in downregulated GLI1 activity. PRMT7-mediated GLI2 methylation upregulates its activity through the dissociation of GLI2 and Suppressor of Fused. Together, PRMT1, PRMT5, and PRMT7 regulate GLI activity at multiple revels. Furthermore, the GLI and PRMT families have strong links with various cancers through cancer stem cell maintenance. Therefore, PRMT-mediated regulation of GLI activity would have important roles in cancer stem cell maintenance.

Abstract 115: Hedgehog signaling pathway promotes breast cancer adaptation to hypoxia

Abstract Breast cancer is the most commonly diagnosed cancer in women in the US. Metastasis accounts for the majority of breast cancer-related deaths. Rapidly proliferating primary tumors experience oxygen levels that are lower in comparison to the normal tissue. The proportions of these hypoxic tumor cells in breast cancer strongly correlate to the incidence of metastasis. Around 70% of breast cancers present with bone metastasis. Tumor cells that metastasize to the bone can lead to an imbalance in bone homeostasis, often leading to osteolysis. Our lab has previously reported that the activation of Hedgehog (Hh) signaling, one of the embryonic developmental pathways found to be abnormally activated in breast cancer, leads to increased osteolysis in vivo. The incidence of osteolytic metastasis at the tibio-femoral junction was significantly reduced in the Hh signaling-deficient mice. Since a hypoxic microenvironment is prevalent in bone, it is imperative to understand how Hh signaling drives tumor cell adaptation to hypoxia. The hypoxic tumor microenvironment in the primary tumor might select for metastatic cells that are better able to metastasize to the bone. We hypothesize that Hh signaling enables the adaptation of breast cancer cells to hypoxia and consequently their metastatic potential. Hh signaling can be canonically activated through smoothened (SMO)-dependent activation of the transcriptional regulator GLI. Cellular response to hypoxia is mediated by the hypoxia-inducible factors (HIFs). To understand the importance of Hh signaling activation in hypoxic condition, we inhibited the Hh signaling pathway using GLI and SMO inhibitors. Additionally, we knocked-down GLI1 in breast cancer cell lines. We have identified a feed-forward mechanism between Hh signaling and HIF signaling in hypoxic conditions leading to sustained hypoxia response by stabilization of HIF-1α. Future investigations will address the significance of Hh signaling inhibition in a murine model of TNBC. This study will help determine the therapeutic benefit of hedgehog inhibitors in controlling hypoxia-mediated metastasis and tumorigenesis. Citation Format: Tshering D. Lama-Sherpa, Sarah C. Kammerud, Lalita A. Shevde. Hedgehog signaling pathway promotes breast cancer adaptation to hypoxia [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 115.

Abstract 6336: Non-canonical activation of Gli-2 by TGF-β/Smad3 signaling pathway in modulation of chemoresistance of hepatocellular carcinoma

Abstract BACKGROUND: It has been demonstrated in our previous study that poorly-differentiated hepatoma cells with low α-feto-protein (AFP) expression and a CD133-/EpCAM- surface profile exhibited aberrant hedgehog signaling (Hh) activity, epithelial-mesenchymal transition and chemoresistance. However, expression of canonical Hh molecules (Sonic ligand and SMO) was not increased in poorly-differentiated hepatoma cells in contrast to well-differentiated hepatoma cells, indicating that Hh transcription factors Gli-1/2 may be activated through a non-classic pathway. The present study aims to investigate whether the TGF-β/Smad3 pathway is responsible for Gli-2 activation through a non-canonical pathway. METHODS: Expression of Sonic Hh (Shh) and Smad3 was assessed in both hepatoma cells and HCC specimens. Gli-2 and Smad3 expression was suppressed by lentiviral vector-mediated RNA interference in poorly-differentiated hepatoma Huh-7-DN and HLE cells. Chemosensitivity was evaluated in hepatoma cells with a Gli-2 or Smad3 inhibitor. RESULTS: Significantly increased Gli-1 and Gli-2 protein levels were observed in 78% (11 out of 14) and 50% (7 out of 14) HCC specimens. In 7 HCC patients with a normal serum AFP value (&amp;lt;20 μg/L), Gli-1/2 protein levels were obviously higher than their pericancerous tissue. Moreover, increased Gli-2 and ABCC1 protein levels were accompanied with increased TGF-β1 and p-Smad3 protein levels in 5 out 7 HCC specimens; whereas Shh protein levels were similar to paired pericancerous tissue. Consistent with the findings from HCC specimens, significantly higher Gli-2 and p-Smad3 nuclear protein levels were associated with increased ABCC1 protein levels in poorly-differentiated hepatoma cells (Huh-7-trans, Huh-7-DN, HLE and HLF); whereas Shh and SMO protein levels were not significantly different from well-differentiated hepatoma Huh-7 and Hep3B cells. Suppressing Gli-2 expression by specific shRNA caused marked decreased expression of Gli-2 and ABCC1 in Huh-7-DN, Huh-7-trans and HLE cells. In addition, expression of Smad3, Gli-2 and ABCC1 was significantly down-regulated in HLE cells after infected with a lentiviral vector harboring shSmad3, indicating that Gli-2 is a downstream target of Smad3. Sorafenib IC50 was significantly reduced from 35.76 to 17.45 μM or 14.04 μM in HLE cells when treated with a GLI inhibitor (GANT-61) or a Smad3 inhibitor (SIS3) at 5 μM. A potential GLI-binding site in the ABCC1 promoter was identified. ABCC1-Gli-luciferase reporter activity was increased up to 39-fold in HLE cells when transfected with constructs containing the potential GLI-binding site, compared to the construct that lacked a GLI-binding site. CONCLUSION: TGF-β/Smad3 activated Gli-2 and its direct transcriptional target ABCC1 in poorly-differentiated and chemoresistant hepatoma cells. Suppressing TGF-β/Smad3/Gli-2 signaling activity confers a potential molecular target in partially alleviating Sorafenib resistance for the treatment of refractory hepatoma. Citation Format: Jia Ding, Hui-yan Li, Li Zhang, Xiao-tian Zhou, Jian Wu. Non-canonical activation of Gli-2 by TGF-β/Smad3 signaling pathway in modulation of chemoresistance of hepatocellular carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6336.

Abstract LB-222: Aberrant activation of GLI1 and Notch1 contributes to racial disparity in triple-negative breast cancer progression

Abstract Background and Aim: Triple-negative breast cancer (TNBC) is a very aggressive subtype of breast cancer characterized by the absence of estrogen receptor (ER), progesterone receptor (PR) and HER2 receptor expression. Mortality from TNBC is significantly higher in African American (AA) women in comparison to European American (EA) women (5-year relative survival of only 14% for AA in comparison to 36% for EA) even though the incidence rates are lower in AA women. Irrespective of stage at diagnosis, AA-TNBC is more aggressive with higher metastasis and a poorer survival than EA-TNBC; therefore, it is imperative to understand the molecular determinants that drive aggressive progression of AA-TNBC. Overall aim of this study is to decipher the alterations in the molecular circuitry underlying racial disparity in TNBC progression in AA women compared to EA women. Results: AA-TNBC cells (HCC1806 and HCC1569) exhibited increased growth and higher migration potential; higher expression of stemness factors, increased number of mammospheres and higher CD44+/CD49f+ population in comparison to EA-TNBC (Hs578t, BT549, HCC1937 and HCC1187) cells. We analyzed RNA sequencing data of multiple AA and EA TNBC cell lines for self-renewal pathways (Wnt/βCatenin, GLI1/Shh and YAP-TAZ) and observed significantly higher levels of GLI1 in AA-TNBC cell lines while no significant alterations were observed in other pathway components. Further analysis of TCGA dataset revealed a positive correlation between GLI1 and Notch1 in AA-TNBC with a negative correlation in EA-TNBC. Increased expression of components of GLI1 and Notch1 pathway (SHH, Jagged, cleaved-Notch (NICD), Hes1 and FOXM1) were noted in AA-TNBC compared to EA-TNBC cells. AA-TNBC cells showed increased nuclear localization of GLI1 and NICD as compared to EA-TNBC cells. We observed that GLI and NICD co-localize in AA-TNBC. High expression of GLI1 and Notch1 correlated with poor overall survival in TNBC patients. AA TNBC Tumor samples show higher expression of GLI1 and NICD in comparison to TNBC tumors from EA patients. Concomitant inhibition of GLI1 and Notch1 using respective small molecule inhibitors, GANT61 and DAPT, along with standard chemotherapeutic agents (Doxorubicin and carboplatin) effectively inhibited AA TNBC growth and progression in mice; growth, proliferation, migration and invasion of ex vivo tumor cells and downregulated CD44+/CD49f+ and ADLH1A1+ population in a synergistic manner. Combined treatment with GANT61+ DAPT+ Carboplatin effectively reduced stem cell frequency of AA-TNBC tumors in in vivo limiting dilution assay. Conclusions: In conclusion, these results show that AA-TNBC cells are inherently aggressive with increased growth, migration and stemness potential. We found aberrant activation of GLI and Notch pathway and a crosstalk between GLI1 and NICD whose inhibition effectively inhibits AA-TNBC and sensitizes AA-TNBC to standard chemotherapy. Citation Format: Sumit Siddharth, Anirudh Saxena, Nethaji Muniraj, Sheetal Parida, Arumugam Nagalingam, Dipali Sharma. Aberrant activation of GLI1 and Notch1 contributes to racial disparity in triple-negative breast cancer progression [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-222.

GLI1 activation is a key mechanism of erlotinib resistance in human non-small cell lung cancer.

Lung cancer is the leading cause of cancer-associated death worldwide. In recent years, the advancement of epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) targeted therapies has provided clinical benefits for lung cancer patients with EGFR mutations. The response to EGFR-TKI varies in patients with lung cancer, and resistance typically develops during the course of the treatment. Therefore, understanding biomarkers which can predict resistance to EGFR-TKI is important. Overexpression of GLI causes activation of the Hedgehog (Hh) signaling pathway and plays a critical role in oncogenesis in numerous types of cancer. In the present study, the role of GLI1 in erlotinib resistance was investigated. GLI1 mRNA and protein expression levels were determined using reverse transcription-quantitative PCR and immunohistochemistry (IHC) in lung cancer cell lines and tumor specimens, respectively. GLI1 mRNA expression levels were found to be positively correlated with the IC50 of erlotinib in 15 non-small cell lung cancer (NSCLC) cell lines. The downregulation of GLI1 using siRNA sensitized lung cancer cells to the erlotinib treatment, whereas the overexpression of GLI1 increased the survival of lung cancer cells in the presence of erlotinib, indicating that Hh/GLI activation may play a critical role in the development of TKI resistance in lung cancer. Combined treatment with erlotinib and a GLI1 inhibitor reduced the cell viability synergistically. A retrospective study of patients with NSCLC treated with erlotinib revealed that those with a high IHC score for GLI1 protein expression had a poorer prognosis. These results indicated that GLI1 is a key regulator for TKI sensitivity, and patients with lung cancer may benefit from the combined treatment of TKI and GLI1 inhibitor.

Ankmy2 Prevents Smoothened-Independent Hyperactivation of the Hedgehog Pathway via Cilia-Regulated Adenylyl Cyclase Signaling.

The mechanisms underlying subcellular targeting of cAMP-generating adenylyl cyclases and processes regulated by their compartmentalization are poorly understood. Here, we identify Ankmy2 as a repressor of the Hedgehog pathway via adenylyl cyclase targeting. Ankmy2 binds to multiple adenylyl cyclases, determining their maturation and trafficking to primary cilia. Mice lacking Ankmy2 are mid-embryonic lethal. Knockout embryos have increased Hedgehog signaling and completely open neural tubes showing co-expansion of all ventral neuroprogenitor markers, comparable to the loss of the Hedgehog receptor Patched1. Ventralization in Ankmy2 knockout is completely independent of the Hedgehog pathway transducer Smoothened. Instead, ventralization results from the reduced formation of Gli2 and Gli3 repressors and early depletion of adenylyl cyclase III in neuroepithelial cilia, implicating deficient pathway repression. Ventralization in Ankmy2 knockout requires both cilia and Gli2 activation. These findings indicate that cilia-dependent adenylyl cyclase signaling represses the Hedgehog pathway and promotes morphogenetic patterning.

Prostaglandin E1 Inhibits GLI2 Amplification-Associated Activation of the Hedgehog Pathway and Drug Refractory Tumor Growth.

Aberrant activation of the Hedgehog (HH) signaling pathway underlines the initiation and progression of a multitude of cancers. The effectiveness of the leading drugs vismodegib (GDC-0449) and sonidegib (LDE225), both Smoothened (SMO) antagonists, is compromised by acquisition of mutations that alter pathway components, notably secondary mutations in SMO and amplification of GLI2, a transcriptional mediator at the end of the pathway. Pharmacologic blockade of GLI2 activity could ultimately overcome these diversified refractory mechanisms, which would also be effective in a broader spectrum of primary tumors than current SMO antagonists. To this end, we conducted a high-content screening directly analyzing the ciliary translocation of GLI2, a key event for GLI2 activation in HH signal transduction. Several prostaglandin compounds were shown to inhibit accumulation of GLI2 within the primary cilium (PC). In particular, prostaglandin E1 (PGE1), an FDA-approved drug, is a potent GLI2 antagonist that overcame resistance mechanisms of both SMO mutagenesis and GLI2 amplification. Consistent with a role in HH pathway regulation, EP4 receptor localized to the PC. Mechanistically, PGE1 inhibited HH signaling through the EP4 receptor, enhancing cAMP-PKA activity, which promoted phosphorylation and degradation of GLI2 via the ubiquitination pathway. PGE1 also effectively inhibited the growth of drug refractory human medulloblastoma xenografts. Together, these results identify PGE1 and other prostaglandins as potential templates for complementary therapeutic development to circumvent resistance to current generation SMO antagonists in use in the clinic. SIGNIFICANCE: These findings show that PGE1 exhibits pan-inhibition against multiple drug refractory activities for Hedgehog-targeted therapies and elicits significant antitumor effects in xenograft models of drug refractory human medulloblastoma mimicking GLI2 amplification.

140 AP-1 and TGFß cooperativity drives non-canonical Hedgehog signaling in resistant basal cell carcinoma

Tumor heterogeneity and lack of knowledge about resistant cell states remain significant barriers to effective targeted cancer therapies. Basal cell carcinomas (BCCs) uniformly depend on Hedgehog (Hh)/Gli signaling for cell growth. We previously identified a nuclear myocardin-related transcription factor (nMRTF) resistance pathway that amplifies Gli1 activity, but definition of the nMRTF cell state, key factors driving its accumulation, and a therapeutic strategy targeting this cancer cell state, remain unknown. Here, we use single cell sequencing of patient tumors to demonstrate that the nMRTF cell state resembles transit-amplifying cells of the hair follicle matrix, and identify three surface markers (LYPD3, TACSTD2, and LY6D) which reliably correlate with activity and serve as robust biomarkers to highlight Smo-inhibitor-resistant tumor cell populations. Epigenetic analysis of isolated nMRTF human tumor subpopulations demonstrates that cooperative AP-1 and TGFß signaling drive nMRTF activation. Mechanistically, JunD/AP-1 signaling drives changes in chromatin accessibility leading to differential Smad3 DNA binding and a transcriptional program of upstream activators of Rho, including RhoGEFs that facilitate nMRTF activity. Importantly, we observe that small molecule AP-1 inhibitors selectively target LYPD3+/TACSTD2+/ LY6D+ nMRTF human BCCs explants. Overall our work for the first time defines tumor subpopulations based on sensitivities, opening an avenue for improved combinatorial therapies.

GLI3 resides at the intersection of hedgehog and androgen action to promote male sex differentiation.

Urogenital tract abnormalities are among the most common congenital defects in humans. Male urogenital development requires Hedgehog-GLI signaling and testicular hormones, but how these pathways interact is unclear. We found that Gli3XtJ mutant mice exhibit cryptorchidism and hypospadias due to local effects of GLI3 loss and systemic effects of testicular hormone deficiency. Fetal Leydig cells, the sole source of these hormones in developing testis, were reduced in numbers in Gli3XtJ testes, and their functional identity diminished over time. Androgen supplementation partially rescued testicular descent but not hypospadias in Gli3XtJ mutants, decoupling local effects of GLI3 loss from systemic effects of androgen insufficiency. Reintroduction of GLI3 activator (GLI3A) into Gli3XtJ testes restored expression of Hedgehog pathway and steroidogenic genes. Together, our results show a novel function for the activated form of GLI3 that translates Hedgehog signals to reinforce fetal Leydig cell identity and stimulate timely INSL3 and testosterone synthesis in the developing testis. In turn, exquisite timing and concentrations of testosterone are required to work alongside local GLI3 activity to control development of a functionally integrated male urogenital tract.

H1N1 Influenza Virus Cross-Activates Gli1 to Disrupt the Intercellular Junctions of Alveolar Epithelial Cells

Influenza A virus (IAV) primarily infects the airway and alveolar epithelial cells and disrupts the intercellular junctions, leading to increased paracellular permeability. Although this pathological change plays a critical role in lung tissue injury and secondary infection, the molecular mechanism of IAV-induced damage to the alveolar barrier remains obscure. Here, we report that Gli1, a transcription factor in the sonic hedgehog (Shh) signaling pathway, is cross-activated by the MAP and PI3 kinase pathways in H1N1 virus (PR8)-infected A549 cells and in the lungs of H1N1 virus-infected mice. Gli1 activation induces Snail expression, which downregulates the expression of intercellular junction proteins, including E-cadherin, ZO-1, and Occludin, and increases paracellular permeability. Inhibition of the Shh pathway restores the levels of Snail and intercellular junction proteins in H1N1-infected cells. Our study suggests that Gli1 activation plays an important role in disrupting the intercellular junctions and in promoting the pathogenesis of H1N1 virus infections.

Small Molecule Intervention in a Protein Kinase C–Gli Transcription Factor Axis

Aberrations in the Hedgehog (Hh) signaling pathway are responsible for a broad range of human cancers, yet only a subset rely on the activity of the clinical target, Smoothened (Smo). Emerging cases of cancers that are insensitive to Smo-targeting drugs demand new therapeutic targets and agents for inhibition. As such, we sought to pursue a recently discovered connection between the Hedgehog pathway transcription factors, the glioma-associated oncogene homologues (Glis), and protein kinase C (PKC) isozymes. Here, we report our assessment of a structurally diverse library of PKC effectors for their influence on Gli function. Using cell lines that employ distinct mechanisms of Gli activation up- and downstream of Smo, we identify a PKC effector that acts as a nanomolar Gli antagonist downstream of Smo through a mitogen-activated protein kinase kinase (MEK)-independent mechanism. This agent provides a unique tool to illuminate crosstalk between PKC isozymes and Hh signaling and new opportunities for therapeutic intervention in Hh pathway-dependent cancers.

Bicyclic Imidazolium Inhibitors of Gli Transcription Factor Activity.

Gli transcription factors within the Hedgehog (Hh) signaling pathway direct key events in mammalian development and promote a number of human cancers. Current therapies for Gli-driven tumors target Smoothened (SMO), a G protein-coupled receptor-like protein that functions upstream in the Hh pathway. Although these drugs can have remarkable clinical efficacy, mutations in SMO and downstream Hh pathway components frequently lead to chemoresistance. In principle, therapies that act at the level of Gli proteins, through direct or indirect mechanisms, would be more efficacious. We therefore screened 325 120 compounds for their ability to block the constitutive Gli activity induced by loss of Suppressor of Fused (SUFU), a scaffolding protein that directly inhibits Gli function. Our studies reveal a family of bicyclic imidazolium derivatives that can inhibit Gli-dependent transcription without affecting the ciliary trafficking or proteolytic processing of these transcription factors. We anticipate that these chemical antagonists will be valuable tools for investigating the mechanisms of Gli regulation and developing new strategies for targeting Gli-driven cancers.

Hedgehog Inhibitors Suppress Osteoclastogenesis in In Vitro Cultures, and Deletion of Smo in Macrophage/Osteoclast Lineage Prevents Age-Related Bone Loss.

The functional role of the Hedgehog (Hh)-signaling pathway has been widely investigated in bone physiology/development. Previous studies have, however, focused primarily on Hh functions in bone formation, while its roles in bone resorption have not been fully elucidated. Here, we found that cyclopamine (smoothened (Smo) inhibitor), GANT-58 (GLI1 inhibitor), or GANT-61 (GLI1/2 inhibitor) significantly inhibited RANKL-induced osteoclast differentiation of bone marrow-derived macrophages. Although the inhibitory effects were exerted by cyclopamine or GANT-61 treatment during 0–48 h (early stage of osteoclast differentiation) or 48–96 h (late stage of osteoclast differentiation) after RANKL stimulation, GANT-58 suppressed osteoclast formation only during the early stage. These results suggest that the Smo-GLI1/2 axis mediates the whole process of osteoclastogenesis and that GLI1 activation is requisite only during early cellular events of osteoclastogenesis. Additionally, macrophage/osteoclast-specific deletion of Smo in mice was found to attenuate the aging phenotype characterized by trabecular low bone mass, suggesting that blockage of the Hh-signaling pathway in the osteoclast lineage plays a protective role against age-related bone loss. Our findings reveal a specific role of the Hh-signaling pathway in bone resorption and highlight that its inhibitors show potential as therapeutic agents that block osteoclast formation in the treatment of senile osteoporosis.

Developing Novel Means to Inhibit Metastasis Through Targeting Tumor Heterogeneity and EMT

It is well established that a subset of cells within primary breast cancers can undergo an epithelial to mesenchymal transition (EMT), although the role of EMT in metastasis remains controversial. We previously demonstrated a novel mechanism by which EMT contributes to metastasis, via mediating tumor cell crosstalk. In this seminar, I will discuss how breast cancer cells that have undergone an oncogenic EMT can increase metastasis of neighboring epithelial cancer cells via non‐canonical paracrine‐mediated activation of GLI activity that is dependent on Six1 expression in the EMT cancer cells. I will discuss how VEGF‐C, secreted by breast cancer cells that have undergone an EMT, promotes paracrine‐mediated increases in proliferation, migration, invasion, and metastasis of epithelial breast cancer cells, via non‐canonical activation of GLI‐signaling. Importantly, the aggressive phenotypes imparted by EMT cells on adjacent epithelial cancer cells can be disrupted by either inhibiting VEGF‐C in EMT cells or by knocking down NRP2, which interacts with VEGF‐C, in neighboring epithelial cancer cells both in vitro and in vivo. Interrogation of TCGA and GEO public datasets supports the relevance of this pathway in human breast cancer, as VEGF‐C and GLI pathway genes significantly positively correlate. Our study suggests that the VEGF‐C/NRP2/GLI axis may be a novel and conserved paracrine means by which EMT cells enhance metastasis, and provides potential targets for therapeutic intervention in this heterogeneous disease. In addition, because SIX1 activates VEGF‐C expression which leads to enhancement of metastasis via cell autonomous and non‐cell autonomous means, we have developed an inhibitor that targets SIX1 action and thereby inhibits metastatic disease. We describe results using this inhibitor in cell culture, where it inhibits SIX1 function and EMT, and in animals, where it inhibits metastasis without an effect on primary tumor growth.Support or Funding InformationColorado Cancer Translational Research Accelerator (CCTRA) PI Heide L. Ford, Co‐I Rui ZhaoNCI R01 CA224867 PIs Heide L. Ford and Michael T. Lewis

GLI1 Inhibitor SRI-38832 Attenuates Chemotherapeutic Resistance by Downregulating NBS1 Transcription in BRAFV600E Colorectal Cancer.

Resistance to radiation and chemotherapy in colorectal cancer (CRC) patients contribute significantly to refractory disease and disease progression. Herein, we provide mechanistic rationale for acquired or inherent chemotherapeutic resistance to the anti-tumor effects of 5-fluorouracil (5-FU) that is linked to oncogenic GLI1 transcription activity and NBS1 overexpression. Patients with high levels of GLI1 also expressed high levels of NBS1. Non-canonical activation of GLI1 is driven through oncogenic pathways in CRC, like the BRAFV600E mutation. GLI1 was identified as a novel regulator of NBS1 and discovered that by knocking down GLI1 levels in vitro, diminished NBS1 expression, increased DNA damage/apoptosis, and re-sensitization of 5-FU resistant cancer to treatment was observed. Furthermore, a novel GLI1 inhibitor, SRI-38832, which exhibited pharmacokinetic properties suitable for in vivo testing, was identified. GLI1 inhibition in a murine BRAFV600E variant xenograft model of CRC resulted in the same down-regulation of NBS1 observed in vitro as well as significant reduction of tumor growth/burden. GLI1 inhibition could therefore be a therapeutic option for 5-FU resistant and BRAFV600E variant CRC patients.