Sonic Hedgehog Signaling Pathway Is Activated in ALK-Positive Anaplastic Large Cell Lymphoma

Abstract

Deregulation of the sonic hedgehog (SHH) signaling pathway has been implicated in several cancers but has not been explored in T-cell lymphomas. Here, we report that the SHH/ GLI1 signaling pathway is activated in anaplastic lymphoma kinase (ALK)-positive anaplastic large cell lymphoma (ALCL). We show that SHH, but not its transcriptional effector GLI1, is amplified in ALK+ ALCL tumors and cell lines, and that SHH and GLI1 proteins are highly expressed in ALK+ ALCL tumors and cell lines. We also show that inhibition of SHH/GLI1 signaling with cyclopamine-KAAD, as well as silencing GLI1 gene expression by small interfering (si)RNA, decreased cell viability and clonogenicity of ALK+ ALCL cells. Transfection of wild-type or mutant NPM-ALK into 293T cells showed that only wild-type NPM-ALK increased GLI1 protein levels and activated SHH/GLI1 signaling as shown by increase of CCND2 mRNA levels. Inhibition of ALK tyrosine kinase and phosphatidylinositol 3-kinase (PI3K)/AKT or forced expression of pAKT down-regulated or up-regulated SHH/GLI1 signaling, respectively. Inhibition of GSK3B in 293T cells also increased protein levels of GLI1. In conclusion, the SHH/GLI1 signaling pathway is activated in ALK+ ALCL. SHH/GLI1 activation is the result of SHH gene amplification and is further mediated by NPM-ALK through activation of PI3K/AKT and stabilization of GLI1 protein. There is a positive synergistic effect between the SHH/ GLI1 and PI3K/AKT pathways that contributes to the lymphomagenic effect of NPM-ALK.

Sonic hedgehog signaling proteins and ATP-binding cassette G2 are aberrantly expressed in diffuse large B-Cell lymphoma

“…Recently, we have shown that SHH signaling is activated in ALK-positive anaplastic large cell lymphoma and that SHH activation mediates cell proliferation, cell survival, and contributes to the oncogenic effect of NPM-ALK in this lymphoma type. 17 Here, we show that diffuse large B-cell lymphomas commonly express SHH, GLI1, and GLI2, at variable levels, and that a large percentage of these tumors express high levels of these proteins. This is not true for follicular lymphoma and chronic lymphocytic leukemia/small lymphocytic lymphoma.…”

“…Cases were considered positive if 10% or more (by estimate) of the tumor cells were stained with an antibody, as previously described. 17 Protein expression was scored as negative, low, or high depending on the staining signal intensity. Expression of ABCG2 in the tumor cells was compared with that seen in endothelial cells; if higher, then expression of ABCG2 was considered high and if lower or equal, then expression of ABCG2 was considered low.…”

Hedgehog signaling pathway is activated in diffuse large B-cell lymphoma and contributes to tumor cell survival and proliferation

“…Previously, we also showed that cyclopamine-KAAD had no or little effect on the cell viability of cell lines with low levels of expression of HH and GLI proteins, such as Jurkat cells. 19 Long-term treatment of Pfeiffer, DOHH2 and OCI-LY3 with cyclopamine-KAAD (10 mM) for 2 weeks also resulted in decrease colony formation; an B80% (Pfeiffer), 75% (DOHH2) and a 65% (OCI-LY3) decrease in clonogenicity in comparison with controls (Supplementary Figure 1B). …”

“…Transfections were performed by electroporation using the nucleofector transfection kit (Amaxa Biosystems, Gaithersberg, MD, USA) as per the manufacturer's instructions and as previously described. 19 Transfection of DOHH2 and BJAB was performed using the Nucleofector solution T and transfection of OCI-LY3 was performed using solution V (Amaxa Biosystems). Cells were harvested at 48 h after siRNA transfections and viability was tested by Trypan blue staining.…”

“…We showed that HH signaling is activated in ALK-positive anaplastic large cell lymphoma cell lines and tumors and that selective inhibition of HH signaling pathway induced a significant decrease in cell viability due to apoptosis and cell-cycle arrest in ALK-positive anaplastic large cell lymphoma cell lines. 19 We have also investigated the expression of HH signaling proteins in a series of 67 DLBCL tumors and cells lines. 20 Our study results showed HH signaling proteins to be aberrantly expressed in DLBCL in comparison with lowgrade B-cell lymphomas and reactive lymph nodes.…”