Targeting the Neurokinin-1 Receptor Compromises Canonical Wnt Signaling in Hepatoblastoma

Abstract

The substance P (SP)/NK-1 receptor (NK1R) complex represents an intriguing anticancer target for a variety of tumors, including hepatoblastoma (HB). Therefore, NK1R antagonists, such as the clinical drug aprepitant, recently have been proposed as potent anticancer agents. However, very little is known regarding the molecular basis of NK1R inhibition in cancer. Using reverse phase protein array, Western blot, Super TOP/FOP, confocal microscopy, and sphere formation ability (SFA) assays, we identified the AKT and Wnt signaling pathways as the key targets of aprepitant in three human HB cell lines (HepT1, HepG2, and HuH6). Following NK1R blockage, we observed decreased phosphorylation of p70S6K and 4E-BP1/2 and inhibition of the canonical Wnt pathway with subsequent decrease of HB cell growth. This effect was dependent of high baseline Wnt activity either by mutational status of b-catenin or extrinsic Wnt activation. Wnt inhibition seemed to be strengthened by disruption of the FOXM1-b-catenin complex. Furthermore, treatment of HB cells with aprepitant led to reduced expression of (liver) stemness markers (AFP, CD13, SOX2, NANOG, and OCT4) and SFA when grown under cancer stem cell conditions. Taken together, we show for the first time that targeting the SP/NK1R signaling cascade inhibits canonical Wnt signaling in HB cells. These findings reveal important insight into the molecular mechanisms of the SP/NK1R complex as a critical component in a model of pediatric liver cancer and may support the development of novel therapeutic interventions for HB and other Wnt-activated cancers.

Therapeutic Innovations for Targeting Childhood Neuroblastoma: Implications of the Neurokinin-1 Receptor System

“…Henssen et al reported that NK1R inhibition in neuroblastoma repressed E2F2 and induced TP53 signaling, and that this effect may be in part due to activation of SRC (26). In a recent publication regarding the downstream signaling following NK1R inhibition in hepatoblastoma, we found potent inhibition of the AKT/mTor pathway, with subsequent strong inhibition of 4E-BP1/2 and p70S6K, both downstream members of the AKT pathway (25).…”

“…There is now accumulating evidence for hepatoblastoma that NK1R may be highly expressed in CSCs of that tumor and can be readily targeted with NK1R antagonists. For example, our data show that the application of NK1R antagonists not only diminished the sphere-formation capacity of hepatoblastoma cells, but also decreased the expression of liver-specific CSC markers and selected ESC genes (25). Importantly, liver specific CSCs could be successfully targeted with NK1R antagonists such as aprepitant.…”

“…FOXM1 is a key interacting protein of the canonical Wnt pathway and here responsible for the translocation of β-catenin into the nucleus. This potent crosslink between the NK1R and the canonical Wnt pathway, until then believed to be two completely separate tumor targets, was the first time an inhibition of the Wnt pathway has been described by targeting the SP/NK1R complex (25). Certainly, this new insight opens hope for therapeutic options in which both NK1R and the canonical Wnt pathway could be targeted simultaneously to generate a strong anticancer effect.…”

“…The NK1R has become an interesting target in a large variety of childhood cancers, including several forms of leukemia, osteosarcoma, and hepatoblastoma (22)(23)(24)(25). In a recent study Henssen et al added neuroblastoma to this list (26).…”

Therapeutic Innovations for Targeting Hepatoblastoma

“…Recently, our group was able to describe the downstream mechanisms following inhibition of NK1R by aprepitant on hepatoblastoma cells (107). First, a robust inhibition of 4EBP1 and p70S6K was identified, which are both downstream members of the PI3K/AKT/mTOR pathway.…”