Elevated GLI3 expression in germinal center diffuse large B cell lymphoma

Diffuse large B cell lymphoma (DLBCL) is a heterogeneous and aggressive malignancy that accounts for 30% of non-Hodgkin lymphoma (NHL). Gene expression profiling further classified this disease into germinal center B cell (GCB) and activated B cell (ABC) subgroups [1] with different clinical outcomes. In general, the GCB DLBCL responds better to conventional chemotherapy, whereas patients with ABC type tend to be more refractory [2]. With standard treatment, a significant number of patients with DLBCL have been cured with Rituximab CHOP therapy. However, more than 40% of patients relapse and succumb to disease progression. Therefore, the identification of new molecular targets in DLBCL subtypes is needed for the development of new treatment options with better outcome. We and others have shown that ABC DLBCL is characterized by deregulation of several oncogenic signaling pathways such as JAK/STAT3 and NF-kB [3–5] and epigenetic modulators such as histone deacetylase 3 [6]. The hedgehog (HH) signaling pathway has also been shown to be activated in DLBCL and its inhibition reduces DLBCL growth and survival [7]. HH signaling is mediated by the GLI family of transcription factors. GLI proteins have also been implicated in a variety of malignancies including solid tumors and hematological neoplasms where they have been shown to modulate both the malignant compartment [8–10] and the tumor microenvironment [11,12]. Three members of the GLI family have been described: GLI1, GLI2 and GLI3. These transcription factors are characterized by a conserved DNAbinding domain consisting of five consecutive and symmetric zinc finger motifs [10] and recognize a specific DNA sequence on their target promoters [10]. These proteins were originally identified as effectors of the HH signaling pathway, where their activities are modulated by signaling through the smoothened (SMO) receptor (canonical HH signaling) [10]. Once activated, GLI family members mediate HH signaling to regulate embryonic development and adult tissue homeostasis through the control of cell fate specification and pattern formation. Previous studies have shown that inhibition of GLI1 or GLI2 in DLBCL using pharmacological and genetic methods results in an inhibition of cell survival suggesting the importance of HH signaling in DLCBL survival [7]. However, little is known about the role of GLI3 in DLBCL biology and whether GLI expression differs in DLBCL subtypes remains unknown. GLI3 plays an important role in normal development and mutations in the Gli3 gene have shown it to cause Pallister-Hall syndrome and Greig cephalopolysyndactyly syndrome. The role of GLI3 in cancer has been overshadowed by the GLI1 and GLI2 family members, particularly in the context of HH signaling. Upon activation of HH signaling, the GLI2 family member acts as a transcriptional activator of GLI1, while GLI3 acts as a transcriptional repressor of HH signaling. This, however, is a context and cell-specific response [13]. Therefore, most studies of GLI3 have focused on its transcriptional repressor role. However, both GLI2 and GLI3 null mice are embryonically lethal, owing to their important role during embryonic development [14]. This has also hindered our understanding of the potential role of these proteins in adult homeostasis. We examined the expression of GLI, GLI2 and GLI3 genes in human DLBCL cell lines of ABC and GCB origins. RNA was extracted from five ABC (HBL-1, OCLI-Ly3, RC-K8, RIVA and U2932) and 5 GCB (DOHH2, OCI-Ly1, OCI-Ly19, Su-DHL6 and VAL) DLBCL cell lines and quantitative realtime polymerase chain reaction (PCR) (qPCR) was performed to quantify GLI expression in these cells. We found an increase in the expression of GLI3 gene in GCB lines as compared to ABC DLBCL; however, expression of GLI1 and GLI2 was very similar between ABC and GCB subtypes (Figure 1(A)). We next compared GLI1-3 expression in DLBCL patient samples using publically available gene expression microarray data and clinical information for DLBCL downloaded from the Gene Expression