GANT-61 inhibits pancreatic cancer stem cell growth in vitro and in NOD/SCID/IL2R gamma null mice xenograft

Abstract

Multiple lines of evidence suggest that the Sonic Hedgehog (Shh) signaling pathway is aberrantly reactivated in pancreatic cancer stem cells (CSCs). The objectives of this study were to examine the molecular mechanisms by which GANT-61 (Gli transcription factor inhibitor) regulates stem cell characteristics and tumor growth. Effects of GANT-61 on CSC’s viability, spheroid formation, apoptosis, DNA-binding and transcriptional activities, and epithelial–mesenchymal transition (EMT) were measured. Humanized NOD/SCID/IL2R gamma^null mice were used to examine the effects of GANT-61 on CSC’s tumor growth. GANT-61 inhibited cell viability, spheroid formation, and Gli–DNA binding and transcriptional activities, and induced apoptosis by activation of caspase-3 and cleavage of Poly-ADP ribose Polymerase (PARP). GANT-61 increased the expression of TRAIL-R1/DR4, TRAIL-R2/DR5 and Fas, and decreased expression of PDGFRα and Bcl-2. GANT-61 also suppressed EMT by up-regulating E-cadherin and inhibiting N-cadherin and transcription factors Snail, Slug and Zeb1. In addition, GANT-61 inhibited pluripotency maintaining factors Nanog, Oct4, Sox-2 and cMyc. Suppression of both Gli1 plus Gli2 by shRNA mimicked the changes in cell viability, spheroid formation, apoptosis and gene expression observed in GANT-61-treated pancreatic CSCs. Furthermore, GANT-61 inhibited CSC tumor growth which was associated with up-regulation of DR4 and DR5 expression, and suppression of Gli1, Gli2, Bcl-2, CCND2 and Zeb1 expression in tumor tissues derived from NOD/SCID IL2Rγ null mice. Our data highlight the importance of Shh pathway for self-renewal and metastasis of pancreatic CSCs, and also suggest Gli as a therapeutic target for pancreatic cancer in eliminating CSCs.

Introduction

Cancer stem cells (CSCs) comprise a subset of hierarchically organized, rare cancer cells with the ability to initiate cancer in genetically modified murine models [1], [2]. CSCs may be responsible for tumor onset, self-renewal/maintenance, mutation accumulation, and metastasis [3], [4]. The existence of CSCs in pancreatic cancer could explain the high frequency of cancers relapse and resistance to chemotherapy. Several lines of evidence indicate that Sonic Hedgehog (Shh) signaling system plays a key role in CSC biology. In general, it plays an essential role in the regulation of CSCs self-renewal, differentiation, and tumorigenic potential, suggesting Shh signaling could be a promising therapeutic target in pancreatic cancer. Inhibition of Shh signaling may abrogate the resistance of CSCs to chemotherapy and could lead to the development of novel therapeutic approaches for the treatment of pancreatic cancer.

During embryonic development, conserved signaling pathways such as Hedgehog (Hh), Wingless (Wnt) and Notch regulate morphogenesis by dictating cell fate decisions such as self-renewal and differentiation. These pathways are subsequently silenced in most adult tissues but frequently reactivated in a wide range of human malignancies. Recent evidence suggests that the Shh signaling pathway [5] is aberrantly reactivated and recognized as one of the mediators in the majority of pancreatic cancers, and that hedgehog blockade has the potential to prevent disease progression and metastatic spread [6]. The binding of short-acting polypeptide ligands (namely; Shh, Ihh, or Dhh) to the Patched receptor diminishes its inhibitory effects on Smoothened, allowing signal transduction through the Shh pathway that results in activation and nuclear translocation of Gli family transcription factors (Gli1 and Gli2). Shh is a morphogen required for proper pattern formation during embryogenesis; however, deregulation of this pathway is responsible for several human cancers, syndromes and malformations [7]. Shh signaling is also critical in the regulation of cellular proliferation, cellular survival, stemness, and cell fate determination in a variety of organs [6]. Multiple lines of evidence support the idea that Shh signaling has a role in normal pancreas development and there is mounting evidence that dysregulated Shh signaling plays some role in pancreatic cancer [8]. Increased Hh signaling has been described in a wide range of other human cancers, including chronic myeloid leukemia (CML), multiple myeloma (MM), glioblastoma, prostate cancer, breast cancer, and small cell lung cancer.

The Gli family of transcription factors has dual functions such as activator and repressor, and can respond to combinatorial and cooperative Gli activity. They play critical roles in the mediation and interpretation of Shh signals [9]. Shh-driven cancers arise from a variety of mutations that affect different components, including the key transcriptional effector Gli proteins, and lead to a variety of human malignancies including medulloblastoma, rhabdomyosarcoma, melanoma, basal cell carcinoma, breast, lung, liver, stomach, and prostate cancers [10], [11], [12], [13]. However, very little is known regarding the specific role of Shh signaling in maintaining pancreatic CSC characteristics, and the downstream target genes involved in determination of cell fate. In comparison to Gli1, a little is known about the role of Gli2-mediated Shh signaling in tumors [14]. Gli2 is mostly regarded as a primary transcriptional activator of Shh signaling, even though it is also suggested to have a weak repressing activity. With Gli1 as a transcriptional target of Gli2, this may amplify Shh induced Gli2-mediated transcription of Gli1 target genes [15]. Gli1 and Gli2 also induce transcription of overlapping and distinct sets of target genes which regulate cell proliferation, cell cycle, cell survival and metastasis [16], [17], [18], [19], [20]. However, the roles of Shh pathway in maintaining pluripotency of pancreatic CSCs are unknown.

The majority of mitogenic/oncogenic signal-activated signaling pathways stimulate epithelial–mesenchymal transition (EMT), a complex reprogramming process which plays an indispensable role in tumor invasion and metastasis [21]. The main features of EMT include loss of epithelial markers (α- and γ-catenin and E-cadherin), gain of mesenchymal cell markers (fibronectin, N-cadherin and vimentin), and the acquisition of migratory and invasive properties [22]. Currently, studies show that EMT is controlled by a group of transcriptional repressors, such as Snail, Slug, Twist1 and Zeb1/2. Upon activation, these repressors recruit histone deacetylases to the E-box elements of the E-cadherin promoter, resulting in transcriptional silencing of E-cadherin expression [23].

The main objective of this paper is to examine the molecular mechanisms by which GANT-61 (Gli transcription inhibitor) regulate pancreatic cancer stem cell characteristics and tumor growth. Our data demonstrated that GANT-61 inhibited pancreatic CSC proliferation and self-renewal, and induced apoptosis. In further detailed analyzes of pancreatic CSCs, GANT-61 decreased both Gli1 and Gli2 expressions, and Gli–DNA binding and Gli-luciferase reporter activities. Furthermore, GANT-61 decreased the expression of PDGFRα concomitant with elevated levels of Fas, increased the expressions of TRAIL-R1/DR4 and TRAIL-R2/DR5, decreased Bcl-2 expression, and induced cleavage of caspase-3 and PARP. In addition, GANT-61-induced changes in gene expression related to self- renewal, proliferation and apoptosis were modulated by Gli1 plus Gli2 shRNA, thus pointing a role of Shh pathway in modulating pancreatic CSC characteristics. Most importantly, GANT-61 inhibited CSC tumor growth in the NOD/SCID IL2Rγ null mice. These data suggest that GANT-61 suppresses pancreatic CSC characteristics and tumor growth by modulating genes involved in cell proliferation, self-renewal and metastasis, and thus can be used as a potential chemotherapeutic agent for the treatment of human pancreatic cancer.