Targeting PIM kinase as a therapeutic strategy in human hepatoblastoma

Abstract

Increasing incidence coupled with poor prognosis and treatments that are virtually unchanged over the past 20 years have made the need for the development of novel therapeutics for hepatoblastoma imperative. PIM kinases have been implicated as drivers of tumorigenesis in multiple cancers, including hepatocellular carcinoma. We hypothesized that PIM kinases, specifically PIM3, would play a role in hepatoblastoma tumorigenesis and that PIM kinase inhibition would affect hepatoblastoma in vitro and in vivo. Parameters including cell survival, proliferation, motility, and apoptosis were assessed in human hepatoblastoma cells following PIM3 knockdown with siRNA or treatment with the PIM inhibitor AZD1208. An in vivo model of human hepatoblastoma was utilized to study the effects of PIM inhibition alone and in combination with cisplatin. PIM kinases were found to be present in the human hepatoblastoma cell line, HuH6, and in a human hepatoblastoma patient-derived xenograft, COA67. PIM3 knockdown or inhibition with AZD1208 decreased cell survival, attachment independent growth, and motility. Additionally, inhibition of tumor growth was observed in a hepatoblastoma xenograft model in mice treated with AZD1208. Combination therapy with AZD1208 and cisplatin resulted in a significant increase in animal survival when compared to either treatment alone. The current studies showed that PIM kinase inhibition decreased human hepatoblastoma tumorigenicity both in vitro and in vivo, implying that PIM inhibitors may be useful as a novel therapeutic for children with hepatoblastoma.

Targeting PIM Kinases Affects Maintenance of CD133 Tumor Cell Population in Hepatoblastoma

“…As we have previously shown that PIM kinase inhibition decreases tumorigenicity in hepatoblastoma [22] , we wished to determine whether the selective pan-PIM inhibitor, AZD1208 [26] , may have an effect on the SCLCC phenotype. Treatment of HuH6 and COA67 cells with AZD1208 decreased CD133 expression as measured by Western blotting ( Figure 2 A ) and flow cytometry for CD133 expression in permeabilized cells ( Figure 2 , B and C ).…”

“…We examined the phenotypic effects of PIM inhibition on the SCLCC versus non-SCLCC populations to determine whether hepatoblastoma SCLCCs exhibited resistance to PIM kinases. We have previously demonstrated that PIM kinases, specifically PIM3, decreased hepatoblastoma proliferation and motility and induced apoptosis [22] . We showed in the current study that both SCLCCs and non-SCLCCs exhibited decreased proliferation and motility and increased apoptosis with PIM inhibition.…”

“…HuH6 cells are reported as male. The human hepatoblastoma patient-derived xenograft (PDX), COA67, was developed as described previously from a male patient [22] . Subcutaneous COA67 xenografts were harvested under sterile conditions and dissociated to a single cell solution using the Tumor Dissociation Kit, human (Miltenyi, Bergisch Gladbach, Germany) according to the manufacture's protocol and maintained in Dulbecco's Modified Eagle's Medium/Ham's F12 supplemented with 2 mmol/L l-glutamine (Thermo Fisher Scientific), 1 μg/mL penicillin/streptomycin (Gibco), 20 ng/mL epidermal growth factor (EMD Millipore, Billerica, MA), 20 ng/mL beta-fibroblast growth factor (EMD Millipore), 2% B27 supplement (Gibco), and 2.5 μg/mL amphotericin B (HyClone).…”

“…Proviral Integration site for Maloney murine leukemia virus (PIM) kinases are a family of serine/threonine kinases that promote tumorigenesis in a multitude of cancer types [15] , [16] , [17] , [18] , [19] , [20] , [21] . We have previously demonstrated that PIM inhibition decreased tumorigenesis in hepatoblastoma [22] . These data led us to hypothesize that PIM kinases may play a role in maintaining the SCLCC phenotype in hepatoblastoma and that targeting PIM kinases would decrease the phenotype in vitro and decrease SCLCC-enriched hepatoblastoma tumor growth in vivo .…”

Focal Adhesion Kinase Inhibition Contributes to Tumor Cell Survival and Motility in Neuroblastoma Patient-Derived Xenografts

“…An additional piece of the surgical specimen was placed in formalin and embedded in paraffin for immunohistochemistry. To establish the COA3 and COA6 PDXs, fresh tissue was kept in Roswell Park Memorial Institute (RPMI) 1640 medium on ice for transport and 27 mm 3 chunks were transplanted in a sterile fashion subcutaneously in the flank of female NOD SCID mice (Envigo, Prattville, AL) under anesthesia with 3% inhalational isoflurane as previously described 26,27 . Mice were maintained in specific pathogen free housing.…”