Christina K. Speirs

Haploinsufficiency of Six3 Fails to Activate Sonic hedgehog Expression in the Ventral Forebrain and Causes Holoprosencephaly

Holoprosencephaly (HPE), the most common forebrain malformation, is characterized by an incomplete separation of the cerebral hemispheres. Mutations in the homeobox gene SIX3 account for 1.3% of all cases of human HPE. Using zebrafish-based assays, we have now determined that HPE-associated Six3 mutant proteins function as hypomorphs. Haploinsufficiency of Six3 caused by deletion of one allele of Six3 or by replacement of wild-type Six3 with HPE-associated Six3 mutant alleles was sufficient to recapitulate in mouse models most of the phenotypic features of human HPE. We demonstrate that Shh is a direct target of Six3 in the rostral diencephalon ventral midline (RDVM). Reduced amounts of functional Six3 protein fail to activate Shh expression in the mutant RDVM and ultimately lead to HPE. These results identify Six3 as a direct regulator of Shh expression and reveal a crossregulatory loop between Shh and Six3 in the ventral forebrain.

Prostaglandin Gβγ signaling stimulates gastrulation movements by limiting cell adhesion through Snai1a stabilization

Gastrulation movements form the germ layers and shape them into the vertebrate body. Gastrulation entails a variety of cell behaviors, including directed cell migration and cell delamination, which are also involved in other physiological and pathological processes, such as cancer metastasis. Decreased Prostaglandin E2 (PGE2) synthesis due to interference with the Cyclooxygenase (Cox) and Prostaglandin E synthase (Ptges) enzymes halts gastrulation and limits cancer cell invasiveness, but how PGE2 regulates cell motility remains unclear. Here we show that PGE2-deficient zebrafish embryos, impaired in the epiboly, internalization, convergence and extension gastrulation movements, exhibit markedly increased cell-cell adhesion, which contributes to defective cell movements in the gastrula. Our analyses reveal that PGE2 promotes cell protrusive activity and limits cell adhesion by modulating E-cadherin transcript and protein, in part through stabilization of the Snai1a (also known as Snail1) transcriptional repressor, an evolutionarily conserved regulator of cell delamination and directed migration. We delineate a pathway whereby PGE2 potentiates interaction between the receptor-coupled G protein βγ subunits and Gsk3β to inhibit proteasomal degradation of Snai1a. However, overexpression of β-catenin cannot stabilize Snai1a in PGE2-deficient gastrulae. Thus, the Gsk3β-mediated and β-catenin-independent inhibition of cell adhesion by Prostaglandins provides an additional mechanism for the functional interactions between the PGE2 and Wnt signaling pathways during development and disease. We propose that ubiquitously expressed PGE2 synthesizing enzymes, by promoting the stability of Snai1a, enable the precise and rapid regulation of cell adhesion that is required for the dynamic cell behaviors that drive various gastrulation movements.

Inhibition of JAK2 signaling by TG101209 enhances radiotherapy in lung cancer models.

Introduction: Persistent STAT3 activation contributes to lung carcinogenesis. Survivin, one of STAT3-regulated genes, is antiapoptotic and confers cancer radioresistance. Methods: We tested whether TG101209, a small-molecule inhibitor of JAK2 (a STAT3-activating tyrosine kinase), affected survivin expression and sensitized lung cancer to radiation. We investigated whether inhibition of JAK2 signaling with TG101209 can be used to reduce survivin expression and enhance radiosensitivity of lung cancer cells in vitro and tumor growth delay in vivo. JAK2 downstream signaling, including PI3-K/Akt and Ras/MAPK/ERK pathways, was also explored. Results: TG101209 inhibited STAT3 activation and survivin expression and sensitized HCC2429 (dose enhancement ratio = 1.34, p = 0.002) and H460 (dose enhancement ratio = 1.09, p = 0.006) cells to radiation in clonogenic assays. Radiation promoted phospho-Akt and phospho-ERK in H460 cells, while their levels were unchanged in HCC2429. After treatment with TG101209, phospho-ERK protein levels were reduced in both HCC2429 and H460 cells. HCC2429 cells transfected with KRAS-12V mutant were more resistant to radiation- and TG101209-induced apoptosis than wild-type control cells. In vivo, addition of TG101209 to radiation in lung xenografts produced a significant tumor growth delay (>10 days) compared with radiation alone and was well tolerated. Immunohistochemistry staining of tumor sections showed that TG101209 increased apoptosis and decreased cell proliferation and vascular density, suggesting that TG101209 also has antiangiogenic effects. Conclusions: TG101209 enhanced the effects of radiation in lung cancer in vitro and in vivo. This study suggests the potential utility of selecting lung cancer patients according to KRAS mutation status for future clinical trials testing combination of TG101209 and radiotherapy.

Zebrafish model of tuberous sclerosis complex reveals cell-autonomous and non-cell-autonomous functions of mutant tuberin

SUMMARY Tuberous sclerosis complex (TSC) is an autosomal dominant disease caused by mutations in either the TSC1 (encodes hamartin) or TSC2 (encodes tuberin) genes. Patients with TSC have hamartomas in various organs throughout the whole body, most notably in the brain, skin, eye, heart, kidney and lung. To study the development of hamartomas, we generated a zebrafish model of TSC featuring a nonsense mutation (vu242) in the tsc2 gene. This tsc2vu242 allele encodes a truncated Tuberin protein lacking the GAP domain, which is required for inhibition of Rheb and of the TOR kinase within TORC1. We show that tsc2vu242 is a recessive larval-lethal mutation that causes increased cell size in the brain and liver. Greatly elevated TORC1 signaling is observed in tsc2vu242/vu242 homozygous zebrafish, and is moderately increased in tsc2vu242/+ heterozygotes. Forebrain neurons are poorly organized in tsc2vu242/vu242 homozygous mutants, which have extensive gray and white matter disorganization and ectopically positioned cells. Genetic mosaic analyses demonstrate that tsc2 limits TORC1 signaling in a cell-autonomous manner. However, in chimeric animals, tsc2vu242/vu242 mutant cells also mislocalize wild-type host cells in the forebrain in a non-cell-autonomous manner. These results demonstrate a highly conserved role of tsc2 in zebrafish and establish a new animal model for studies of TSC. The finding of a non-cell-autonomous function of mutant cells might help explain the formation of brain hamartomas and cortical malformations in human TSC.

Role of Insulin-Like Growth Factor-1 Signaling Pathway in Cisplatin-Resistant Lung Cancer Cells

PURPOSE The development of drug-resistant phenotypes has been a major obstacle to cisplatin use in non-small-cell lung cancer. We aimed to identify some of the molecular mechanisms that underlie cisplatin resistance using microarray expression analysis. METHODS AND MATERIALS H460 cells were treated with cisplatin. The differences between cisplatin-resistant lung cancer cells and parental H460 cells were studied using Western blot, MTS, and clonogenic assays, in vivo tumor implantation, and microarray analysis. The cisplatin-R cells were treated with human recombinant insulin-like growth factor (IGF) binding protein-3 and siRNA targeting IGF-1 receptor. RESULTS Cisplatin-R cells illustrated greater expression of the markers CD133 and aldehyde dehydrogenase, more rapid in vivo tumor growth, more resistance to cisplatin- and etoposide-induced apoptosis, and greater survival after treatment with cisplatin or radiation than the parental H460 cells. Also, cisplatin-R demonstrated decreased expression of insulin-like growth factor binding protein-3 and increased activation of IGF-1 receptor signaling compared with parental H460 cells in the presence of IGF-1. Human recombinant IGF binding protein-3 reversed cisplatin resistance in cisplatin-R cells and targeting of IGF-1 receptor using siRNA resulted in sensitization of cisplatin-R-cells to cisplatin and radiation. CONCLUSIONS The IGF-1 signaling pathway contributes to cisplatin-R to cisplatin and radiation. Thus, this pathway represents a potential target for improved lung cancer response to treatment.

Outcomes of iodine-125 plaque brachytherapy for uveal melanoma with intraoperative ultrasonography and supplemental transpupillary thermotherapy

PURPOSE To assess the impact on local tumor control of intraoperative ultrasonographic plaque visualization and selective application of transpupillary thermotherapy (TTT) in the treatment of posterior uveal melanoma with iodine-125 (I-125) episcleral plaque brachytherapy (EPB). METHODS AND MATERIALS Retrospective analysis of 526 patients treated with I-125 EPB for posterior uveal melanoma. Clinical features, dosimetric parameters, TTT treatments, and local tumor control outcomes were recorded. Statistical analysis was performed using Cox proportional hazards and Kaplan-Meier life table method. RESULTS The study included 270 men (51%) and 256 women (49%), with a median age of 63 years (mean, 62 years; range, 16-91 years). Median dose to the tumor apex was 94.4 Gy (mean, 97.8; range, 43.9-183.9) and to the tumor base was 257.9 Gy (mean, 275.6; range, 124.2-729.8). Plaque tilt >1 mm away from the sclera at plaque removal was detected in 142 cases (27%). Supplemental TTT was performed in 72 patients (13.7%). One or 2 TTT sessions were required in 71 TTT cases (98.6%). After a median follow-up of 45.9 months (mean, 53.4 months; range, 6-175 months), local tumor recurrence was detected in 19 patients (3.6%). Local tumor recurrence was associated with lower dose to the tumor base (P=.02). CONCLUSIONS Ultrasound-guided plaque localization of I-125 EPB is associated with excellent local tumor control. Detection of plaque tilt by ultrasonography at plaque removal allows supplemental TTT to be used in patients at potentially higher risk for local recurrence while sparing the majority of patients who are at low risk. Most patients require only 1 or 2 TTT sessions.

The Zinc Ionophore PCI-5002 Radiosensitizes Non-small Cell Lung Cancer Cells by Enhancing Autophagic Cell Death

Introduction: A major focus of cancer research is to identify compounds that sensitize resistant cancer cells to radiation treatment. Lung cancer cells, in particular, have high rates of radioresistance that lead to treatment failure. We have previously shown that the autophagy induced in the context of decreased apoptosis confers radiosensitivity to prostate and lung cancer cells. Zinc supplementation has antiapoptotic effects in cell culture. In addition, the accumulation of zinc in response to oxidative stress has been associated with increased autophagy in astrocyte and breast cancer cells. Methods: In this study, we hypothesized that the zinc ionophore PCI-5002 radiosensitizes lung cancer cells by inducing autophagic cell death. To test this hypothesis, we used a combination of in vitro and in vivo approaches, including clonogenic assays to test for radiosensitivity, biochemical analyses of apoptosis and autophagy, and a xenograft mouse model of tumor growth. Results: We found that PCI-5002 reduced clonogenic survival in treated cells compared with untreated cells (0.03% versus 0.1% surviving fraction, p < 0.001). The increased radiosensitive fraction of PCI-5002-treated cells was accompanied by increased autophagy. PCI-5002 treatment also reduced caspase-3 cleavage. In an irradiated xenograft mouse model, the tumor growth of irradiated, PCI-5002-treated mice was slower than untreated, irradiated mice (25 days versus 22 days to reach a 1.0 cm3 tumor size). Conclusions: PCI-5002 treatment sensitizes lung cancer cells to radiation, both in vitro and in vivo. This data suggest that PCI-5002 could potentially treat radioresistant/locally advanced lung cancer by amplifying the effects of radiotherapy.