FAK Inhibition Decreases Hepatoblastoma Survival Both In Vitro and In Vivo

AbstractHepatoblastoma is the most frequently diagnosed liver tumor of childhood, and children with advanced, metastatic or relapsed disease have a disease-free survival rate under 50%. Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase that is important in many facets of tumor development and progression. FAK has been found in other pediatric solid tumors and in adult hepatocellular carcinoma, leading us to hypothesize that FAK would be present in hepatoblastoma and would impact its cellular survival. In the current study, we showed that FAK was present and phosphorylated in human hepatoblastoma tumor specimens. We also examined the effects of FAK inhibition upon hepatoblastoma cells using a number of parallel approaches to block FAK including RNAi and small molecule FAK inhibitors. FAK inhibition resulted in decreased cellular survival, invasion, and migration and increased apoptosis. Further, small molecule inhibition of FAK led to decreased tumor growth in a nude mouse xenograft model of hepatoblastoma. The findings from this study will help to further our understanding of the regulation of hepatoblastoma tumorigenesis and may provide desperately needed novel therapeutic strategies and targets for aggressive, recurrent, or metastatic hepatoblastomas

FAK Inhibition Decreases Neuroblastoma Cell Invasion, Migration and Metastasis

Neuroblastoma, the most common extracranial solid tumor of childhood, is responsible for over 15% of pediatric cancer deaths. We have shown that neuroblastoma cell lines overexpress focal adhesion kinase (FAK), a non-receptor protein tyrosine kinase that controls a number of tumorigenic pathways. In this study, we hypothesized that inhibition of FAK would result in decreased cellular migration and invasion in neuroblastoma cell lines, and decrease metastasis in a murine model. We utilized multiple human neuroblastoma cell lines and three different methods of FAK inhibition. Cell viability, migration, and invasion assays were employed to assess the effects of FAK inhibition in vitro. A nude mouse model was utilized to determine the effects of FAK inhibition on in vivo liver metastasis. Abrogation of FAK with two small molecule inhibitors, 1,2,4,5-benzenetetraamine tetrahydrochloride (Y15) and NVP-TAE226 (TAE), resulted in decreased cell survival, migration and invasion in neuroblastoma cell lines. FAK knockdown with siRNA also resulted in decreased invasion and migration in neuroblastoma cell lines. Furthermore, the effects of FAK inhibition were more pronounced in the MYCN amplified cell lines. Finally, inhibition of FAK with Y15 or TAE in a nude mouse model resulted in a significant decrease in tumor burden in SK-N-BE(2) injected animals. We believe that FAK plays an important role in the metastatic phenotype of neuroblastoma cells that is exaggerated in cell lines that overexpress MYCN. Inhibition of FAK in these cell lines significantly decreased the malignant potential of these cells. FAK inhibition warrants further investigation as a potential therapeutic target in the treatment of aggressive neuroblastoma

Preclinical Evaluation of Engineered Oncolytic Herpes Simplex Virus for the Treatment of Pediatric Solid Tumors

<div><p>Recently, investigators showed that mice with syngeneic murine gliomas that were treated with a neuroattenuated oncolytic herpes simplex virus-1 (oHSV), M002, had a significant increase in survival. M002 has deletions in both copies of the γ₁34.5 gene, enabling replication in tumor cells but precluding infection of normal cells. Previous studies have shown antitumor effects of other oHSV against a number of adult tumors including hepatocellular carcinoma and renal cell carcinoma. The purpose of the current study was to investigate the oncolytic potential of M002 against difficult to treat pediatric liver and kidney tumors. We showed that the oHSV, M002, infected, replicated, and decreased cell survival in hepatoblastoma, malignant rhabdoid kidney tumor, and renal sarcoma cell lines. In addition, we showed that in murine xenografts, treatment with M002 significantly increased survival and decreased tumor growth. Finally, these studies showed that the primary entry protein for oHSV, CD111 (nectin-1) was present in human hepatoblastoma and malignant rhabdoid kidney tumor specimens. We concluded that M002 effectively targeted these rare aggressive tumor types and that M002 may have potential for use in children with unresponsive or relapsed pediatric solid tumors.</p></div

Preclinical Evaluation of Engineered Oncolytic Herpes Simplex Virus for the Treatment of Neuroblastoma

<div><p>Despite intensive research efforts and therapeutic advances over the last few decades, the pediatric neural crest tumor, neuroblastoma, continues to be responsible for over 15% of pediatric cancer deaths. Novel therapeutic options are needed for this tumor. Recently, investigators have shown that mice with syngeneic murine gliomas treated with an engineered, neuroattenuated oncolytic herpes simplex virus-1 (oHSV), M002, had a significant increase in survival. M002 has deletions in both copies of the γ_<i>1</i>34.5 gene, enabling replication in tumor cells but precluding infection of normal neural cells. We hypothesized that M002 would also be effective in the neural crest tumor, neuroblastoma. We showed that M002 infected, replicated, and decreased survival in neuroblastoma cell lines. In addition, we showed that in murine xenografts, treatment with M002 significantly decreased tumor growth, and that this effect was augmented with the addition of ionizing radiation. Importantly, survival could be increased by subsequent doses of radiation without re-dosing of the virus. Finally, these studies showed that the primary entry protein for oHSV, CD111 was expressed by numerous neuroblastoma cell lines and was also present in human neuroblastoma specimens. We concluded that M002 effectively targeted neuroblastoma and that this oHSV may have potential for use in children with unresponsive or relapsed neuroblastoma. </p> </div

M002 treatment of renal Ewing sarcoma xenografts.

<p><b>A</b> SK-NEP-1 cells, (1.5×10⁶ cells) were injected into the subcapsular space of the left kidney in 6 week old female athymic nude mice. After three weeks, the renal tumors were directly injected with either control vehicle (PBS +10% glycerol/50 µL, n = 6) or M002 oncolytic herpes simplex virus (1×10⁷ PFU/50 µL, n = 7). Two weeks following treatment, the animals were euthanized and the kidney tumors were harvested, measured with a caliper and weighed. <b>B</b> Tumor volumes in the animals treated with M002 were half those of animals treated with vehicle only (<i>bar = mean</i>). <b>C</b> Tumor weights following M002 treatment were also nearly half of those of vehicle treated tumors (<i>bar = mean</i>). <b>D</b> Formalin-fixed, paraffin embedded samples of vehicle treated SK-NEP-1 tumor specimens were stained for hematoxylin and eosin. Examination of these kidney tumor specimens revealed native tumor architecture consisting of sheets of small round blue cells. Representative photomicrographs at 10× with enlarged area of detail at 40×. <b>E</b> Formalin-fixed, paraffin embedded samples of M002 treated SK-NEP-1 tumor specimens were stained for hematoxylin and eosin. These specimens showed tumor necrosis (<i>open arrow</i>) and hemorrhage (<i>pink area</i>), and inflammatory cell infiltrates (<i>closed arrow</i>). Representative photomicrographs at 10× with enlarged area of detail at 40×.</p

Survival of immunocompetent AJ mice with Neuro-2a murine neuroblastoma tumors treated with R3659 or M002.

<p>Neuro-2a murine neuroblastoma tumor cells (5 × 10⁵ cells) were injected into the right flank of immunocompetent syngeneic AJ mice. Once tumors reached 100 mm³, animals received an intra-tumoral injection of R3659 [parent virus, 1 × 10⁷ PFU / 50μL (n=8)] or M002 [IL-12 engineered virus, 1 × 10⁷ PFU / 50μL (n=7)] and animals were followed for survival. The median survival of the animals with tumors treated with R3659 was 7 days and was significantly decreased from the median survival of animals with M002-treated tumors (10 days, p = 0.02). </p

Immunohistochemical staining for HSV in SK-NEP-1 tumor xenografts.

<p>Formalin-fixed, paraffin embedded samples of SK-NEP-1 tumor xenografts (those presented in the data in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086843#pone-0086843-g005" target="_blank">Figure 5</a>) were stained for HSV using immunohistochemistry, and representative photomicrographs presented. <b>A</b> No HSV was detected by immunohistochemical staining in SK-NEP-1 tumors treated with vehicle alone. Negative controls (rabbit IgG) were included with each run (<i>small box insert</i>). <b>B</b> HSV immunohistochemical staining revealed HSV present in the M002 treated SK-NEP-1 xenografts (<i>brown stain</i>). There was no viral staining seen in the negative controls (rabbit IgG) (<i>small box insert</i>).</p

M002 infection and cell survival.

<p><b>A</b> To further verify infectivity, murine IL-12 production was determined in HuH6, G401 and SK-NEP-1 cell lines following treatment with M002 oHSV. Cell lines were infected with M002 at 0, 0.01 or 0.1 PFU/cell. After 48 hours, the supernates were collected and concentrations of murine IL-12 determined by ELISA. Data reported as mean ± standard error of the mean. There was a significant increase in murine IL-12 production in all cell lines even at the lower MOI of 0.01 PFU/cell. <b>B</b> HuH6, G401 and SK-NEP-1 cell lines were treated with M002 at increasing MOI. After 72 hr of treatment, cell viability was measured with alamarBlue™ assays. Data reported as mean ± standard error of the mean. There was a significant decrease in viability in all cell lines following M002 treatment. The LD₅₀ was calculated for each cell line for M002 (PFU/cell). <b>C</b> A comparison was made between percent cells staining for CD111 and the sensitivity of the cell lines to M002. There was a non-linear, inverse correlation between the two variables. The straight lines represent the slope between two points and the curved line the correlation calculated with the three points, represented by the polynomial equation. The bars represent the SEM for the data points in both the x and y axis.</p

M002 infection resulted in murine IL-12 production in human neuroblastoma cell lines.

<p>Murine IL-12 production was determined in SK-N-AS <b>A</b> and SK-N-BE(2) B cell lines following treatment with M002 oHSV. Cell lines were infected with M002 at 0, 0.15 or 0.30 PFU/cell. At 24, 48, and 72 hours post-infection, the supernates were collected and concentrations of murine IL-12 determined by ELISA. Data reported as mean ± standard error of the mean. There was a significant increase in murine IL-12 production in both cell lines as early as 24 hours post-infection.</p