Paracrine Hedgehog Signaling Research Articles

P4HA2 hydroxylates SUFU to regulate the paracrine Hedgehog signaling and promote B-cell lymphoma progression

Aberrations in the Hedgehog (Hh) signaling pathway are significantly prevailed in various cancers, including B-cell lymphoma. A critical facet of Hh signal transduction involves the dynamic regulation of the suppressor of fused homolog (SUFU)-glioma-associated oncogene homolog (GLI) complex within the kinesin family member 7 (KIF7)-supported ciliary tip compartment. However, the specific post-translational modifications of SUFU-GLI complex within this context have remained largely unexplored. Our study reveals a novel regulatory mechanism involving prolyl 4-hydroxylase 2 (P4HA2), which forms a complex with KIF7 and is essential for signal transduction of Hh pathway. We demonstrate that, upon Hh pathway activation, P4HA2 relocates alongside KIF7 to the ciliary tip. Here, it hydroxylates SUFU to inhibit its function, thus amplifying the Hh signaling. Moreover, the absence of P4HA2 significantly impedes B lymphoma progression. This effect can be attributed to the suppression of Hh signaling in stromal fibroblasts, resulting in decreased growth factors essential for malignant proliferation of B lymphoma cells. Our findings highlight the role of P4HA2-mediated hydroxylation in modulating Hh signaling and propose a novel stromal-targeted therapeutic strategy for B-cell lymphoma.

Lrig1-expressing epidermal progenitors require SCD1 to maintain the dermal papilla niche

Niche cells are widely known to regulate stem/progenitor cells in many mammalian tissues. In the hair, dermal papilla niche cells are well accepted to regulate hair stem/progenitor cells. However, how niche cells themselves are maintained is largely unknown. We present evidence implicating hair matrix progenitors and the lipid modifying enzyme, Stearoyl CoA Desaturase 1, in the regulation of the dermal papilla niche during the anagen-catagen transition of the mouse hair cycle. Our data suggest that this takes place via autocrine Wnt signalling and paracrine Hedgehog signalling. To our knowledge, this is the first report demonstrating a potential role for matrix progenitor cells in maintaining the dermal papilla niche.

Berberine inhibits colorectal tumor growth by suppressing SHH secretion.

Hedgehog plays an important role in a wide range of physiological and pathological conditions. Paracrine activation of Hedgehog pathway in stromal cells increases the expression of VEGF, which promotes neovascularization in colorectal cancer and ultimately the growth of colorectal cancer. Berberine (BBR) has anticancer activity. In this study we investigated whether BBR inhibited the growth of colon cancer through suppressing the paracrine sonic hedgehog (SHH) signaling in vitro and in vivo. We showed that BBR (1-10 μM) dose-dependently inhibited the secretion and expression of SHH protein in HT-29 and SW480 cells. BBR did not influence the transcription of SHH, but promoted the degradation of SHH mRNA, thus decreased the SHH mRNA expression in the colorectal cancer cells. In nude mice bearing HT-29 xenograft, oral administration of BBR (100 mg · kg-1 · d-1) or a positive control drug GDC-0449 (100 mg · kg-1 · d-1) for 4 weeks markedly suppressed the growth of HT-29 tumor with BBR exhibiting a better antitumor efficacy. The tumor growth inhibition caused by BBR or GDC-0449 was comparable to their respective inhibitory effect on the mouse-specific Gli mRNA expression in the tumor. However, BBR (20 μM) did not affect the expression of human transcription factor Gli1 mRNA in HT-29 and SW480 cells. In conclusion, BBR promotes the degradation of SHH mRNA in colorectal cancer cells, interrupting the paracrine Hedgehog signaling pathway activity thus suppresses the colorectal cancer growth. This study reveals a novel molecular mechanism underlying the anticancer action of BBR.

Discrete Hedgehog Factor Expression and Action in the Developing Phallus.

Hypospadias is a failure of urethral closure within the penis occurring in 1 in 125 boys at birth and is increasing in frequency. While paracrine hedgehog signalling is implicated in the process of urethral closure, how these factors act on a tissue level to execute closure itself is unknown. This study aimed to understand the role of different hedgehog signalling members in urethral closure. The tammar wallaby (Macropus eugenii) provides a unique system to understand urethral closure as it allows direct treatment of developing offspring because mothers give birth to young before urethral closure begins. Wallaby pouch young were treated with vehicle or oestradiol (known to induce hypospadias in males) and samples subjected to RNAseq for differential expression and gene ontology analyses. Localisation of Sonic Hedgehog (SHH) and Indian Hedgehog (IHH), as well as the transcription factor SOX9, were assessed in normal phallus tissue using immunofluorescence. Normal tissue culture explants were treated with SHH or IHH and analysed for AR, ESR1, PTCH1, GLI2, SOX9, IHH and SHH expression by qPCR. Gene ontology analysis showed enrichment for bone differentiation terms in male samples compared with either female samples or males treated with oestradiol. Expression of SHH and IHH localised to specific tissue areas during development, akin to their compartmentalised expression in developing bone. Treatment of phallus explants with SHH or IHH induced factor-specific expression of genes associated with bone differentiation. This reveals a potential developmental interaction involved in urethral closure that mimics bone differentiation and incorporates discrete hedgehog activity within the developing phallus and phallic urethra.

Genomic testing, tumor microenvironment and targeted therapy of Hedgehog-related human cancers.

Hedgehog signals are transduced through Patched receptors to the Smoothened (SMO)-SUFU-GLI and SMO-Gi-RhoA signaling cascades. MTOR-S6K1 and MEK-ERK signals are also transduced to GLI activators through post-translational modifications. The GLI transcription network up-regulates target genes, such as BCL2, FOXA2, FOXE1, FOXF1, FOXL1, FOXM1, GLI1, HHIP, PTCH1 and WNT2B, in a cellular context-dependent manner. Aberrant Hedgehog signaling in tumor cells leads to self-renewal, survival, proliferation and invasion. Paracrine Hedgehog signaling in the tumor microenvironment (TME), which harbors cancer-associated fibroblasts, leads to angiogenesis, fibrosis, immune evasion and neuropathic pain. Hedgehog-related genetic alterations occur frequently in basal cell carcinoma (BCC) (85%) and Sonic Hedgehog (SHH)-subgroup medulloblastoma (87%) and less frequently in breast cancer, colorectal cancer, gastric cancer, pancreatic cancer, non-small-cell lung cancer (NSCLC) and ovarian cancer. Among investigational SMO inhibitors, vismodegib and sonidegib are approved for the treatment of patients with BCC, and glasdegib is approved for the treatment of patients with acute myeloid leukemia (AML). Resistance to SMO inhibitors is caused by acquired SMO mutations, SUFU deletions, GLI2 amplification, other by-passing mechanisms of GLI activation and WNT/β-catenin signaling activation. GLI-DNA-interaction inhibitors (glabrescione B and GANT61), GLI2 destabilizers (arsenic trioxide and pirfenidone) and a GLI-deacetylation inhibitor (4SC-202) were shown to block GLI-dependent transcription and tumorigenesis in preclinical studies. By contrast, SMO inhibitors can remodel the immunosuppressive TME that is dominated by M2-like tumor-associated macrophages (M2-TAMs), myeloid-derived suppressor cells and regulatory T cells, and thus, a Phase I/II clinical trial of the immune checkpoint inhibitor pembrolizumab with or without vismodegib in BCC patients is ongoing.

Disruption of stromal hedgehog signaling initiates RNF5-mediated proteasomal degradation of PTEN and accelerates pancreatic tumor growth.

The contribution of the tumor microenvironment to pancreatic ductal adenocarcinoma (PDAC) development is currently unclear. We therefore examined the consequences of disrupting paracrine Hedgehog (HH) signaling in PDAC stroma. Herein, we show that ablation of the key HH signaling gene Smoothened (Smo) in stromal fibroblasts led to increased proliferation of pancreatic tumor cells. Furthermore, Smo deletion resulted in proteasomal degradation of the tumor suppressor PTEN and activation of oncogenic protein kinase B (AKT) in fibroblasts. An unbiased proteomic screen identified RNF5 as a novel E3 ubiquitin ligase responsible for degradation of phosphatase and tensin homolog (PTEN) in Smo-null fibroblasts. Ring Finger Protein 5 (Rnf5) knockdown or pharmacological inhibition of glycogen synthase kinase 3β (GSKβ), the kinase that marks PTEN for ubiquitination, rescued PTEN levels and reversed the oncogenic phenotype, identifying a new node of PTEN regulation. In PDAC patients, low stromal PTEN correlated with reduced overall survival. Mechanistically, PTEN loss decreased hydraulic permeability of the extracellular matrix, which was reversed by hyaluronidase treatment. These results define non-cell autonomous tumor-promoting mechanisms activated by disruption of the HH/PTEN axis and identifies new targets for restoring stromal tumor-suppressive functions.

Targeting the Hedgehog signalling pathway in triple negative breast cancer.

e24216Background: Paracrine Hedgehog (Hh) signalling occurs in ~30% of all triple negative breast cancers (TNBCs) and confers worse overall survival. The tumour stroma has been shown to facilitate ...

Inhibition of Hedgehog signaling suppresses proliferation and microcyst formation of human Autosomal Dominant Polycystic Kidney Disease cells

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is caused by mutation of PKD1 or PKD2, which encode polycystin 1 and 2, respectively. The polycystins localize to primary cilia and the functional loss of the polycystin complex leads to the formation and progressive growth of fluid-filled cysts in the kidney. The pathogenesis of ADPKD is complex and molecular mechanisms connecting ciliary dysfunction to renal cystogenesis are unclear. Primary cilia mediate Hedgehog signaling, which modulates cell proliferation and differentiation in a tissue-dependent manner. Previously, we showed that Hedgehog signaling was increased in cystic kidneys of several PKD mouse models and that Hedgehog inhibition prevented cyst formation in embryonic PKD mouse kidneys treated with cAMP. Here, we show that in human ADPKD tissue, Hedgehog target and activator, Glioma 1, was elevated and localized to cyst-lining epithelial cells and to interstitial cells, suggesting increased autocrine and paracrine Hedgehog signaling in ADPKD, respectively. Further, Hedgehog inhibitors reduced basal and cAMP-induced proliferation of ADPKD cells and cyst formation in vitro. These data suggest that Hedgehog signaling is increased in human ADPKD and that suppression of Hedgehog signaling can counter cellular processes that promote cyst growth in vitro.

Abstract 1334: Mesenchymal cell sub-populations selectively modulate paracrine hedgehog signaling in triple negative breast cancer

Abstract Recent studies correlate Hedgehog (Hh) signaling with reduced survival rates in triple negative breast cancer (TNBC) patients. Activation of hedgehog signaling in the adjacent mesenchyme has been shown to promote tumor growth and it is a poor prognosis factor for TNBC. We developed a novel tumor-mesenchymal in vitro model of hedgehog signaling in TNBC to evaluate the role of mesenchymal cell sub-populations in the proliferative potential and stem cell markers of breast cancer cells using a custom designed multiwell array. As a source of mesenchymal cell sub-populations we evaluated myofibroblasts, mesenchymal stem cells derived from bone marrow or adipose tissue and tumor cells that undergo epithelial-mesenchymal transition (EMT). Tumor cells were culture with 1 or 2 mesenchymal cell sub-populations in adjacent compartments +/- SHH ligand for 72-96hrs. Active Hh signaling was confirmed by up-regulation of main Hh target genes (e.g. Gli1, Patch1). Paracrine Hh signaling only in bone marrow-derived mesenchymal stem cell and EMT significantly increased proliferation (10-15%) of TNBC and normal breast cancer cell lines. Addition of adipose-derived mesenchymal stem cells or pharmacological Hh inhibitors (Cyclopamine and Gant61) partially abolished tumor growth indicating that other non-canonical signals are involve in paracrine Hh-driven tumors. Cancer stem cell markers were selectively modulated in co-cultures. Our results suggest that paracrine Hh signaling-driven by bone marrow and EMT mesenchymal are potential therapeutic targets to treating TNBC. Citation Format: Karla P. Ramos, Maribella Domenech. Mesenchymal cell sub-populations selectively modulate paracrine hedgehog signaling in triple negative breast cancer [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 1334. doi:10.1158/1538-7445.AM2017-1334

Abstract B02: Open multi-microwell array for the study of paracrine signaling in tumors

Abstract The development and progression of tumors are modulated by the interaction among multiple cell types present at the tumor adjacent tissue. Few in vitro cell culture platforms allow one to test multi-cellular interactions and those suitable require high numbers of cells limiting studies to cell lines and excluding heterogeneous patient samples, and fail to provide tunable discrete adjacent compartments for the retrieval of independent cell population readouts. Mixing of cells in adjacent compartments and cell sorting after mixed culture represent a costly and time consuming procedure that often results is lost, reduction and/or damage of the recovered sample. To overcome some of these limitations we have developed an open multi-microwell array that enables co-culture of up to 4 cell populations in discrete adjacent wells using either 2- or 3-dimension culture conditions. The circular microwells are made of poly-dimethyl siloxane (PDMS) and placed on top of glass or tissue culture plastic. The open-well format allows cells and culture media to be manually loaded and replaced using a pipette independently of passive pumping method, and without the need of vacuum-assisted filling or syringe pumps. The microwell array is aligned to a 96well plate format for automated fluorescent readouts. The cell culture surface area of each microwell is 5.7mm2 (5ul) and can accommodate 5,000-10,000 cells but the design of each microwell can be scale-down to accommodate smaller cell samples. The adjacent microwells are contained within an outer-ring of 8mm diameter and 750um height. After cell seeding, adjacent compartments can be connected by dispensing a volume of 30-40ul inside the outer ring. A model of tumor-mesenchyme paracrine Hedgehog signaling was developed and characterized to show the feasibility of this array for multi-cell soluble factor signaling studies. Activation of Hedgehog (Hh) signaling in the mesenchyme promotes proliferation of tumor cells via secreted factors. As a source of mesenchymal cell sub-types we evaluated TGF-b treated fibroblasts and tumor epithelial cells that undergo epithelial-mesenchymal transition. Myofibroblast phenotype was confirmed based on overexpression of smooth muscle actin, vimentin and fibroblast activating protein, and reduce levels of caveolin-1. Active Hh signaling and pharmacological inhibition with cyclopamine were confirmed by up- and down- regulation of canonical Hh target genes Gli1 and Patch1. Active hedgehog signaling and cell viability was maintained over 5-7days 2-dimensional and collagen type I embedded mesenchymal cells. Breast-derived human epithelial and mesenchymal cells co-cultured in microwells and transwells showed similar increase in tumor cell proliferation rates but with a reduction of 80% or more in the total cells needed compared to transwells. Flow cytometer read-outs of proliferation and tumor cell distribution of CD44/CD24 cell surface receptors were similar among transwells and microwells. Adjacent culture of mouse derived fibroblasts (NIH-3T3) and human tumor cells showed recovery of genomic sample without cell-population mixing using primers for specific for human and mouse GAPDH. Doubling of the number of microwells with mesenchymal cells modulated the observed proliferative effect in tumor cells suggesting that mesenchymal cell ratios modulate tumor response to paracrine Hh signaling. This observation could not be replicated in macro-scale platforms as active Hh signaling in the mesenchyme is achieved via cell growth arrest using a combination of low serum and cell confluence. The open multi-microwell array represents a new user-friendly multi-culture microscale array with optimized capabilities for the study of paracrine signaling among discrete cell populations compared to macroscale culture technology. Citation Format: Karla P. Ramos, Fernando Boria, Maribella Domenech. Open multi-microwell array for the study of paracrine signaling in tumors. [abstract]. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr B02.

Inhibition of Pancreatic Cancer Cell-Induced Paracrine Hedgehog Signaling by Liver X Receptor Agonists and Oxy16, a Naturally Occurring Oxysterol

The widespread involvement of the Hedgehog (Hh) signaling pathway in human malignancies has driven efforts to develop Hh pathway inhibitors as anti-cancer agents. The majority of these agents antagonize Smoothened (Smo), a plasma membrane-associated signal transducer molecule. However, several such Smo antagonists have failed in clinical trials to benefit patients with cancers that arise from aberrant Hh signaling (which often bypasses Smo). In this study, we report that a naturally occurring oxysterol, 20α, 22(R)-dihydroxycholesterol (Oxy16), a known metabolite in the biosynthesis of steroid hormones, strongly inhibits Hh signaling induced in C3H10T1/2 embryonic fibroblasts and NIH3T3-E1 fibroblasts through a mechanism that is independent of liver X receptor (LXR) activation. We demonstrate that Oxy16 inhibits Hh signaling in Suppressor of Fused (Sufu) null mouse embryonic fibroblast (MEF) cells, indicating that its inhibitory effect on Hh signaling is epistatic to Sufu. We further demonstrate that Oxy16 inhibits Gli1 transcriptional activity in NIH3T3-E1 cells overexpressing Gli1 and a Gli-dependent reporter construct. Altogether, data presented here suggest that Oxy16 may be a suitable starting point for the development of new drugs that inhibit Hh signaling downstream of Smo. By targeting aberrant Hh signaling, such novel Hh pathway inhibitors could significantly broaden the range of clinical applications compared to existing Smo antagonists. Furthermore, the present study adds a new facet to the spectrum of Hh pathway modulation that naturally occurring oxysterol derivatives are capable of, ranging from allosteric activation of the pathway via Smo binding to inhibition of the pathway downstream of Smo. J. Cell. Biochem. 118: 499-509, 2017. © 2016 Wiley Periodicals, Inc.

Genetic ablation of Smoothened in pancreatic fibroblasts increases acinar-ductal metaplasia.

The contribution of the microenvironment to pancreatic acinar-to-ductal metaplasia (ADM), a preneoplastic transition in oncogenic Kras-driven pancreatic cancer progression, is currently unclear. Here we show that disruption of paracrine Hedgehog signaling via genetic ablation of Smoothened (Smo) in stromal fibroblasts in a Kras(G12D) mouse model increased ADM. Smo-deleted fibroblasts had higher expression of transforming growth factor-α (Tgfa) mRNA and secreted higher levels of TGFα, leading to activation of EGFR signaling in acinar cells and increased ADM. The mechanism involved activation of AKT and noncanonical activation of the GLI family transcription factor GLI2. GLI2 was phosphorylated at Ser230 in an AKT-dependent fashion and directly regulated Tgfa expression in fibroblasts lacking Smo Additionally, Smo-deleted fibroblasts stimulated the growth of Kras(G12D)/Tp53(R172H) pancreatic tumor cells in vivo and in vitro. These results define a non-cell-autonomous mechanism modulating Kras(G12D)-driven ADM that is balanced by cross-talk between Hedgehog/SMO and AKT/GLI2 pathways in stromal fibroblasts.

Abstract A40: Genetic ablation of Smoothened in tumor-associated fibroblasts promotes pancreatic tumorigenesis

Abstract Purpose of Study: Pancreatic cancer is an overwhelming fatal disease with less than 5% of patients surviving beyond 5 years. The most prominent histopathological hallmark of pancreatic cancer is its uniquely dense stromal reaction as evidenced by recent reports that highlight the significant role of stromal fibroblasts on pancreatic tumor cell biology. We used novel mouse models to show that genetic inactivation of Smoothened (Smo) in stromal fibroblasts accelerated Kras-initiated tumorigenesis. Research Method: We used a genetically engineered mouse model of pancreatic cancer that relies on constitutive activation of the Kras oncogene in the epithelium. We simultaneously employed cre-loxP technology to conditionally delete Smo exclusively in the fibroblast compartment of the pancreas, thus disrupting the crucial hedgehog paracrine signaling loop between pancreatic tumor cells and fibroblasts. Novel Findings: We showed that deletion of Smo in stromal fibroblasts accelerated pancreatic tumorigenesis through a mechanism involving destabilization of fibroblast PTEN protein. Down-regulation of PTEN enhanced TGF-α production in stromal fibroblasts, and increased epithelial cell transformation and proliferation through epithelial growth factor receptor (EGFR). A selective SMO inhibitor also decreased PTEN in a Kras mouse model as well as in human primary pancreatic cancer associated fibroblasts. Importantly, in pancreatic ductal adenocarcinoma (PDAC) patient samples, low PTEN expression correlated with low SMO expression and with reduced overall survival. These results define a pathway that reprograms stromal fibroblasts from a tumor suppressive phenotype to a tumor promoting phenotype, thus highlighting the dual functions of stromal fibroblasts in pancreatic cancer and the molecular consequences of loss of the hedgehog pathway. Thus, a more comprehensive understanding of tumor-stroma interactions is required to assure effective implementation of targeted therapies. Conclusions and Implications: Recent pre-clinical reports suggest the pancreatic tumor microenvironment functions predominantly to inhibit tumor growth, challenging the concept of tumor stroma as a therapeutic target. Our results provide molecular insight into how the balance between the opposing activities of tumor stromal fibroblasts is maintained, and potentially identifies targets for restoring stromal tumor suppressive functions. In summary, we demonstrate that ablation of paracrine hedgehog signaling in SMA-positive fibroblasts leads to proteasome-mediated degradation of the PTEN tumor suppressor protein and subsequent activation of oncogenic pathways. Citation Format: Jason R. Pitarresi, Michael C. Ostrowski. Genetic ablation of Smoothened in tumor-associated fibroblasts promotes pancreatic tumorigenesis. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr A40.

Abstract 2883: Genetic ablation of Smoothened in tumor-associated fibroblasts promotes pancreatic tumorigenesis

Abstract Purpose of Study Pancreatic cancer is an overwhelming fatal disease with less than 5% of patients surviving beyond 5 years. The most prominent histopathological hallmark of pancreatic cancer is its uniquely dense stromal reaction as evidenced by recent reports that highlight the significant role of stromal fibroblasts on pancreatic tumor cell biology. We used novel mouse models to show that genetic inactivation of Smoothened (Smo) in stromal fibroblasts accelerated Kras-initiated tumorigenesis. Research Method We used a genetically engineered mouse model of pancreatic cancer that relies on constitutive activation of the Kras oncogene in the epithelium. We simultaneously employed cre-loxP technology to conditionally delete Smo exclusively in the fibroblast compartment of the pancreas, thus disrupting the crucial hedgehog paracrine signaling loop between pancreatic tumor cells and fibroblasts. Novel Findings We showed that deletion of Smo in stromal fibroblasts accelerated pancreatic tumorigenesis through a mechanism involving destabilization of fibroblast PTEN protein. An unbiased genetic screen revealed the ubiquitin E2 conjugating enzyme UBE2K as a PTEN destabilizer and knockdown of UBE2K blocked the degradation of PTEN protein. Down-regulation of PTEN enhanced TGF-α production in stromal fibroblasts, and increased epithelial cell transformation and proliferation through epithelial growth factor receptor (EGFR). A selective SMO inhibitor also decreased PTEN in a Kras mouse model as well as in human primary pancreatic cancer associated fibroblasts. Importantly, in pancreatic ductal adenocarcinoma (PDAC) patient samples, low PTEN expression correlated with low SMO expression and with reduced overall survival. These results define a pathway that reprograms stromal fibroblasts from a tumor suppressive phenotype to a tumor promoting phenotype, thus highlighting the dual functions of stromal fibroblasts in pancreatic cancer and the molecular consequences of loss of the hedgehog pathway. Thus, a more comprehensive understanding of tumor-stroma interactions is required to assure effective implementation of targeted therapies. Conclusions & Implications Recent pre-clinical reports suggest the pancreatic tumor microenvironment functions predominantly to inhibit tumor growth, challenging the concept of tumor stroma as a therapeutic target. Our results provide molecular insight into how the balance between the opposing activities of tumor stromal fibroblasts is maintained, and potentially identifies targets for restoring stromal tumor suppressive functions. In summary, we demonstrate that ablation of paracrine hedgehog signaling in SMA-positive fibroblasts leads to proteasome-mediated degradation of the PTEN tumor suppressor protein and subsequent activation of oncogenic pathways. Citation Format: Jason R. Pitarresi, Jinghai Wu, Xin Liu, Veronica Bravo, Maria C. Cuitiño, Raleigh D. Kladney, Sarah Woelke, Sarmila Majumder, Gustavo Leone, Michael C. Ostrowski. Genetic ablation of Smoothened in tumor-associated fibroblasts promotes pancreatic tumorigenesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2883. doi:10.1158/1538-7445.AM2015-2883

Hedgehog Signaling in Malignant Pleural Mesothelioma.

Malignant pleural mesothelioma (MPM) is a cancer associated with exposure to asbestos fibers, which accumulate in the pleural space, damage tissue and stimulate regeneration. Hedgehog signaling is a pathway important during embryonic mesothelium development and is inactivated in adult mesothelium. The pathway is reactivated in some MPM patients with poor clinical outcome, mainly mediated by the expression of the ligands. Nevertheless, mutations in components of the pathway have been observed in a few cases. Data from different MPM animal models and primary culture suggest that both autocrine and paracrine Hedgehog signaling are important to maintain tumor growth. Drugs inhibiting the pathway at the level of the smoothened receptor (Smo) or glioma-associated protein transcription factors (Gli) have been used mostly in experimental models. For clinical development, biomarkers are necessary for the selection of patients who can benefit from Hedgehog signaling inhibition.

Abstract PR01: Paradoxical acceleration of pancreatic tumorigenesis through Smoothened inhibition: A post-clinical trial in genetically engineered mice

Abstract Over the past several years, our group has studied the role of tumor stroma in limiting drug delivery and promoting primary chemoresistance in pancreatic ductal adenocarcinoma (PDA). A key player in this process is the Hedgehog (Hh) pathway which forms a paracrine signal to promote desmoplasia. Overexpression of the secreted ligand Sonic Hedgehog begins early in PDA tumorigenesis and acts to promote the proliferation of nearby stromal cells, recapitulating a role of the Hh pathway in mammalian development. By intervening in this axis through pharmacological inhibition with a Smoothened (Smo) inhibitor, we previously demonstrated increased delivery of chemotherapy to tumors arising in a genetically engineered model of PDA, resulting in improved overall survival. However, subsequent clinical trials in metastatic PDA found that those patients treated with chemotherapy + placebo lived longer than those with chemotherapy + Smo inhibitor. To better understand this outcome, we performed a series of “post-clinical trials” in genetically engineered mice. Surprisingly, we found that early and prolonged Smoothened inhibition led to a dramatic acceleration of pancreatic tumorigenesis. This contrasted starkly with our previous findings of prolonged survival using a short treatment course in large, late-stage tumors. Moreover, Smoothened inhibitor treated tumors were poorly differentiated, highly vascularized, and lacking in desmoplastic stroma. During pancreatic development, signals from nearby stromal cells designate the exocrine fate of epithelial progenitor cells, and support exocrine growth. Tumor stromal cells may play a similar pro-differentiation role in pancreatic cancer, resulting in the well- to moderately-differentiated ductal morphology that is characteristic of human PDA. To directly test the hypothesis that Smo inhibition and subsequent stromal depletion causes change in differentiation state, we employed a newly developed abdominal laparotomy procedure to acquire pre- and post-treatment tumors samples from mice treated with Smo inhibitor. Data from the first 8 animals evaluated show clear indications of de-differentiation and trans-differentiation. Ongoing molecular and genomic analyses will determine whether this reflects a change in the underlying molecular class of the tumor, and will aid in our understanding of the basic molecular mechanisms responsible for this dramatic alteration in phenotype. Reporter alleles are being utilized to identify the stromal cell types involved in the paracrine Hedgehog signalling axis in PDA. These data will also be informative for our understanding of the many stroma-targeted therapies that are currently undergoing clinical development. This abstract is also presented as Poster B22. Citation Format: Paul E. Oberstein, Dafydd H. Thomas, Carmine F. Palermo, Stephen A. Sastra, Kenneth P. Olive. Paradoxical acceleration of pancreatic tumorigenesis through Smoothened inhibition: A post-clinical trial in genetically engineered mice. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr PR01.

Abstract 3603: A novel approach to enhance delivery and sensitivity of gemcitabine in pancreatic cancer by suppression of desmoplasia

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is an extremely fatal disease due to lack of efficient therapy and resistance to current standard therapy, gemcitabine (GEM). Numerous studies implicate the intimate reciprocal interactions due to paracrine Hedgehog (Hh) signaling between epithelia and underlying stroma that leads to desmoplasia and causes chemoresistance in PDAC. We report for the first time that a non-steroidal drug, ormeloxifene (ORM) has anticancer properties and depletes tumor-associated stromal tissue by inhibiting the Hh cellular signaling pathway in PDAC. This provoked us to investigate the combinatorial effects of ORM with GEM and we observed that ORM disrupts the stroma of pancreatic tumors, alters vascular network and thereby facilitates delivery and sensitivity of GEM. Methods: Using cell line models, Western blotting and QPCR were used to investigate the treatment effects of ORM, GEM and their combination on Hh signaling pathway and the related proteins involved in PDAC. To determine the effect of ORM on anti-tumorigenic sensitivity of GEM, we developed PDAC xenograft mice models using PanCa cells, BXPC3. Tumor tissue sections were stained for SHH (sonic hedgehog), cygb/STAP (stellate cell selective markers), α-SMA (PSC activation), FSP-1 (Fibroblast surface protein-1), collagen-1 by immunoflorescence, F4/80 (macrophage), CD31 (vasularization) through immunohistochemistry and microRNAs through in situ hybridization. ELIZA was performed for SDF1/CXCL12 and SHH secretion. Results: ORM inhibits cell proliferation and induces apoptosis in pancreatic cancer cells. ORM alone and in combination with GEM inhibits the Hh signaling pathway by downregulating both the protein and mRNA levels of SHH and its related important downstream targets: GLI1, PTCH1/2, NFκB, p-AKT and Cyclin D1. ORM potentiates the anti-tumorigenic effect of GEM by 75% in PDAC xenograft mice. Investigations of treated xenograft tumor tissues show that ORM depletes tumor-associated stromal tissue by inhibiting the Hh cellular signaling pathway and both mouse/human collagen-1, a hallmark of desmoplastic reaction. These xenograft tumors treated with ORM in combination with GEM also show the inhibition of oncogenic miR-21 and restoration of tumor suppressor, miR-132. The combination therapy inhibits stromal cell populations infiltrating into the tumor tissue and produces increased intratumoral vascular density and concentration of GEM, leading to transient stabilization of tumor growth. Tumor cells, co-cultivated with TGFβ stimulated human pancreatic stromal cells, displayed increased invasive capabilities which were inhibited on treatment with ORM alone or in combination with GEM. Conclusion: We propose that ORM has a high therapeutic index and represents a great promise for a combination therapy with GEM against stroma and neoplastic cells as a treatment of choice Citation Format: Sheema Khan, Mara Ebeling, A. Ansarullah, Neeraj Chauhan, Rishi Kumar Gara, Meena Jaggi, Haotian Zhao, Subhash C. Chauhan. A novel approach to enhance delivery and sensitivity of gemcitabine in pancreatic cancer by suppression of desmoplasia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3603. doi:10.1158/1538-7445.AM2014-3603

Novel Neutralizing Hedgehog Antibody MEDI-5304 Exhibits Antitumor Activity by Inhibiting Paracrine Hedgehog Signaling

The hedgehog pathway has been implicated in the tumorigenesis, tumor progression, and metastasis of numerous human cancers. We generated the first fully human hedgehog antibody MEDI-5304 and characterized its antitumor activity and preclinical toxicology. MEDI-5304 bound sonic hedgehog (SHH) and Indian hedgehog (IHH) with low picomolar affinity and neutralized SHH and IHH activity in cellular mGLI1 reporter assays. The antibody inhibited transcription of hedgehog target genes and osteoblast differentiation of C3H10T1/2 cells. We evaluated the activity of MEDI-5304 in vivo in model systems that allowed us to evaluate two primary hypotheses of hedgehog function in human cancer, paracrine signaling between tumor and stromal cells and cancer stem cell (CSC) self-renewal. MEDI-5304 displayed robust pharmacodynamic effects in stromal cells that translated to antitumor efficacy as a single agent in an HT-29/MEF coimplantation model of paracrine hedgehog signaling. MEDI-5304 also improved responses to carboplatin in the HT-29/MEF model. The antibody, however, had no effect as a single agent or in combination with gemcitabine on the CSC frequency or growth of several primary pancreatic cancer explant models. These findings support the conclusion that hedgehog contributes to tumor biology via paracrine tumor-stromal signaling but not via CSC maintenance or propagation. Finally, the only safety study finding associated with MEDI-5304 was ondontodysplasia in rats. Thus, MEDI-5304 represents a potent dual hedgehog inhibitor suitable for continued development to evaluate efficacy and safety in human patients with tumors harboring elevated levels of SHH or IHH.

TAK-441, a novel investigational smoothened antagonist, delays castration-resistant progression in prostate cancer by disrupting paracrine hedgehog signaling

Hedgehog (Hh) signaling is a highly conserved intercellular and intracellular communication mechanism that governs organogenesis and is dysregulated in cancers of numerous tissues, including prostate. Up-regulated expression of the Hh ligands, Sonic (Shh) and Desert (Dhh), has been reported in androgen-deprived and castration-resistant prostate cancer (CRPC). In a cohort of therapy naive, short- and long-term neoadjuvant hormone therapy-treated (NHT), and CRPC specimens, we observed elevated Dhh expression predominantly in long-term NHT specimens and elevated Shh expression predominantly in CRPC specimens. Together with previously demonstrated reciprocal signaling between Shh-producing prostate cancer (PCa) cells and urogenital mesenchymal fibroblasts, these results suggest that castration-induced Hh expression promotes CRPC progression through reciprocal paracrine signaling within the tumor microenvironment. We tested whether the orally available Smoothened (Smo) antagonist, TAK-441, could impair castration-resistant progression of LNCaP PCa xenografts by disrupting paracrine Hh signaling. Although TAK-441 or cyclopamine did not affect androgen withdrawal-induced Shh up-regulation or viability of LNCaP cells, castration-resistant progression of LNCaP xenografts was significantly delayed in animals treated with TAK-441. In TAK-441-treated xenografts, expression of murine orthologs of the Hh-activated genes, Gli1, Gli2 and Ptch1, was substantially suppressed, while expression of the corresponding human orthologs was unaffected. As androgen-deprived LNCaP cells up-regulate Shh expression, but are not sensitive to Smo antagonists, these studies indicate that TAK-441 leads to delayed castration-resistant progression of LNCaP xenografts by disrupting paracrine Hh signaling with the tumor stroma. Thus, paracrine Hh signaling may offer unique opportunities for prognostic biomarker development, drug targeting and therapeutic response monitoring of PCa progression.

Abstract 4952: A novel smoothened antagonist, TAK-441, delays castration-resistant progression in prostate cancer by disrupting paracrine hedgehog signaling.

Abstract Hedgehog (Hh) signaling is a highly conserved inter- and intracellular communication mechanism that governs organogenesis and is dysregulated in cancers of numerous tissues, including prostate. Up-regulated expression of the Hh ligands, Sonic (Shh) and Desert (Dhh), has been reported in androgen-deprived and castration-resistant prostate cancer (CRPC). In a cohort of therapy naive, short- and long-term neoadjuvant hormone therapy-treated (NHT), and CRPC specimens, we observed elevated Dhh expression predominantly in long-term NHT specimens and elevated Shh expression predominantly in CRPC specimens. Together with previously demonstrated reciprocal signaling between Shh-producing prostate cancer (PCa) cells and urogenital mesenchymal fibroblasts, these results suggest that castration-induced Hh expression promotes CRPC progression through reciprocal paracrine signaling within the tumor microenvironment. We tested whether the orally available Smoothened (Smo) antagonist, TAK-441, could impair castration-resistant progression of LNCaP PCa xenografts by disrupting paracrine Hh signaling. While TAK-441 or cyclopamine did not affect androgen withdrawal-induced Shh up-regulation or viability of LNCaP cells, castration-resistant progression of LNCaP xenografts was significantly delayed in animals treated with TAK-441. In TAK-441-treated xenografts, expression of murine orthologues of the Hh-activated genes, Gli1, Gli2, and Ptch1, was substantially suppressed, while expression of the corresponding human orthologues was unaffected. Since androgen-deprived LNCaP cells up-regulate Shh expression, but are not sensitive to Smo antagonists, these studies indicate that TAK-441 leads to delayed castration-resistant progression of LNCaP xenografts by disrupting paracrine Hh signaling with the tumor stroma. Thus, paracrine Hh signaling may offer unique opportunities for prognostic biomarker development, drug targeting and therapeutic response monitoring of PCa progression. Citation Format: Mazyar Ghaffari, Naokazu Ibuki, Irene Iu, Mitali Pandey, Ladan Fazli, Masahide Kashiwagi, Hideaki Tojo, Osamu Nakanishi, Martin E. Gleave, Michael E. Cox. A novel smoothened antagonist, TAK-441, delays castration-resistant progression in prostate cancer by disrupting paracrine hedgehog signaling. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4952. doi:10.1158/1538-7445.AM2013-4952