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

Giant Polypoid Gastric Heterotopia with Ectopic Thyroid Tissue: Unusual Cause of Jejuno-Jejunal Intussusception

A giant polyp was found within the proximal jejunum of a 16-year-old-girl that was originally suspected to be a hamartomatous Peutz-Jeghers polyp. Examination revealed a giant polypoid gastric heterotopia containing ectopic thyroid tissue. The presence of gastric heterotopia in the small intestine is well documented. Rare cases of ectopic thyroid tissue in the small intestine have also been reported. Ectopic thyroid tissue arising within a polypoid gastric heterotopia in the small intestine, to our knowledge, has not previously been reported

Giant Polypoid Gastric Heterotopia with Ectopic Thyroid Tissue: Unusual Cause of Jejuno-Jejunal Intussusception

A giant polyp was found within the proximal jejunum of a 16-year-old-girl that was originally suspected to be a hamartomatous Peutz-Jeghers polyp. Examination revealed a giant polypoid gastric heterotopia containing ectopic thyroid tissue. The presence of gastric heterotopia in the small intestine is well documented. Rare cases of ectopic thyroid tissue in the small intestine have also been reported. Ectopic thyroid tissue arising within a polypoid gastric heterotopia in the small intestine, to our knowledge, has not previously been reported

Pre-Clinical Study Evaluating Novel Protein Phosphatase 2A Activators as Therapeutics for Neuroblastoma

Background: Protein phosphatase 2A (PP2A) functions as an inhibitor of cancer cell proliferation, and its tumor suppressor function is attenuated in many cancers. Previous studies utilized FTY720, an immunomodulating compound known to activate PP2A, and demonstrated a decrease in the malignant phenotype in neuroblastoma. We wished to investigate the effects of two novel PP2A activators, ATUX-792 (792) and DBK-1154 (1154). Methods: Long-term passage neuroblastoma cell lines and human neuroblastoma patient-derived xenograft (PDX) cells were used. Cells were treated with 792 or 1154, and viability, proliferation, and motility were examined. The effect on tumor growth was investigated using a murine flank tumor model. Results: Treatment with 792 or 1154 resulted in PP2A activation, decreased cell survival, proliferation, and motility in neuroblastoma cells. Immunoblotting revealed a decrease in MYCN protein expression with increasing concentrations of 792 and 1154. Treatment with 792 led to tumor necrosis and decreased tumor growth in vivo. Conclusions: PP2A activation with 792 or 1154 decreased survival, proliferation, and motility of neuroblastoma in vitro and tumor growth in vivo. Both compounds resulted in decreased expression of the oncogenic protein MYCN. These findings indicate a potential therapeutic role for these novel PP2A activators in neuroblastoma

Serine-Threonine Kinase Receptor-Associated Protein (STRAP) Knockout Decreases the Malignant Phenotype in Neuroblastoma Cell Lines

Background: Serine-threonine kinase receptor-associated protein (STRAP) plays an important role in neural development but also in tumor growth. Neuroblastoma, a tumor of neural crest origin, is the most common extracranial solid malignancy of childhood and it continues to carry a poor prognosis. The recent discovery of the role of STRAP in another pediatric solid tumor, osteosarcoma, and the known function of STRAP in neural development, led us to investigate the role of STRAP in neuroblastoma tumorigenesis. Methods: STRAP protein expression was abrogated in two human neuroblastoma cell lines, SK-N-AS and SK-N-BE(2), using transient knockdown with siRNA, stable knockdown with shRNA lentiviral transfection, and CRISPR-Cas9 genetic knockout. STRAP knockdown and knockout cells were examined for phenotypic alterations in vitro and tumor growth in vivo. Results: Cell proliferation, motility, and growth were significantly decreased in STRAP knockout compared to wild-type cells. Indicators of stemness, including mRNA abundance of common stem cell markers Oct4, Nanog, and Nestin, the percentage of cells expressing CD133 on their surface, and the ability to form tumorspheres were significantly decreased in the STRAP KO cells. In vivo, STRAP knockout cells formed tumors less readily than wild-type tumor cells. Conclusion: These novel findings demonstrated that STRAP plays a role in tumorigenesis and maintenance of neuroblastoma stemness

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

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

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

Survival of nude mice with SK-N-BE(2) neuroblastoma xenografts treated with M002 and repeated XRT.

<p>Established SK-N-BE(2) right flank xenografts (N=25) were treated with vehicle (50 μL) or M002 (1 × 10⁴ PFU/50 μL) and low dose ionizing radiation (XRT) (3 Gy) at Day 0. Subsequent low doses of XRT (3 Gy) were given at 7 day intervals (black arrows) to all vehicle treated animals (n=5); the M002 treated animals were divided into four groups (n=5 per group) to receive either no further treatment or an additional 1, 2, or 3 doses of low dose XRT at 7 day intervals (black arrows). Three surviving animals, all in the M002 XRT × 4 regimen were euthanized at 53 days, with small mean tumor volume (450 ± 131.1 mm³). The mean survival time increased with each subsequent dose of XRT, reaching statistical significance after four doses. M002 with XRT at all groups showed a survival advantage over vehicle with subsequent doses of XRT. </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