EFEMP1 suppresses malignant glioma growth and exerts its action within the tumor extracellular compartment.

PURPOSE: There are conflicting reports regarding the function of EFEMP1 in different cancer types. In this study, we sought to evaluate the role of EFEMP1 in malignant glioma biology. EXPERIMENTAL DESIGN: Real-time qRT-PCR was used to quantify EFEMP1 expression in 95 glioblastoma multiforme (GBM). Human high-grade glioma cell lines and primary cultures were engineered to express ectopic EFEMP1, a small hairpin RNA of EFEMP1, or treated with exogenous recombinant EFEMP1 protein. Following treatment, growth was assayed both in vitro and in vivo (subcutaneous (s.c.) and intracranial (i.c.) xenograft model systems). RESULTS: Cox regression revealed that EFEMP1 is a favorable prognostic marker for patients with GBM. Over-expression of EFEMP1 eliminated tumor development and suppressed angiogenesis, cell proliferation, and VEGFA expression, while the converse was true with knock-down of endogenous EFEMP1 expression. The EFEMP1 suppression of tumor onset time was nearly restored by ectopic VEGFA expression; however, overall tumor growth rate remained suppressed. This suggested that inhibition of angiogenesis was only partly responsible for EFEMP1's impact on glioma development. In glioma cells that were treated by exogenous EFEMP1 protein or over-expressed endogenous EFEMP1, the EGFR level was reduced and AKT signaling activity attenuated. Mixing of EFEMP1 protein with cells prior to s.c. and i.c. implantations or injection of the protein around the established s.c. xenografts, both significantly suppressed tumorigenicity. CONCLUSIONS: Overall, our data reveals that EEFEMP1 suppresses glioma growth in vivo, both by modulating the tumor extracellular microenvironment and by altering critical intracellular oncogenic signaling pathways.

PAX6 suppression of glioma angiogenesis and the expression of vascular endothelial growth factor A.

We reported that PAX6 suppresses glioblastoma cell growth in vivo and anchorage-independent growth without significant alteration of cell proliferation in vitro, suggesting that PAX6 may alter the tumor microenvironment. Because we found that PAX6 downregulates expression of the gene encoding vascular endothelial growth factor A (VEGFA) in glioma cells, we used a subcutaneous xenograft model to verify PAX6 suppression of VEGFA-induced angiogenesis based on CD31-immunostaining of endothelial cells. The results showed a significant reduction of VEGFA at the transcription level in PAX6-transfected cells in xenografts and PAX6 has a suppressive effect on the microvascular amplification typically seen in glioblastoma. We showed that PAX6 suppression of VEGFA expression requires its DNA binding-domain. The C-terminal truncation mutant of PAX6, however, did not show the dominant negative function in regulating VEGFA expression that it showed previously in regulating MMP2 expression. In the glioma cell line U251HF, we further determined that blocking the PI3K/Akt signaling pathway with either adenoviral-mediated PTEN expression or LY294002 enhanced PAX6-mediated suppression of VEGFA in an additive manner; thus, PAX6-mediated suppression of VEGFA is not via the canonical pathway through HIF1A. These two VEGFA-regulatory pathways can also be similarly modulated in another malignant glioma cell line, U87, but not in LN229 where the basal VEGFA level is low and PTEN is wild-type. PAX6 suppression of VEGFA appears to be blocked in LN229. In conclusion, our data showed that PAX6 can initiate in glioma cells a new signaling pathway independent of PI3K/Akt-HIF1A signaling to suppress VEGFA expression.

Transient Green's Tensor for a Layered Solid Half-Space With Different Interface Conditions.

Pulsed ultrasonic techniques can be and have been used to examine the interface conditions of a bonded structure. To provide a theoretical basis for such testing techniques we model the structure as a layer on top of a half-space, both of different elastic properties, with various interface bonding conditions. The exact dynamic Green's tensor for such a structure is explicitly derived from the three-dimensional equations of motion. The final solution is a series. Each term of the series corresponds to a successive arrival of a "generalized ray" and each is a definite line integral along a fixed path which can be easily computed numerically. Willis' method is used in the derivation. A new scheme of automatic generation of the arrivals and ray paths using combinatorial analysis, along with the summation of the corresponding products of reflection coefficients is presented. A FORTRAN code is developed for computation of the Green's tensor when both the source and the detector are located on the top surface. The Green's tensor is then used to simulate displacements due to pulsed ultrasonic point sources of known time waveform. Results show that the interface bonding conditions have a great influence on the transient displacements. For example, when the interface bonding conditions vary, some of the first few head waves and regular reflected rays change polarities and amplitudes. This phenomenon can be used to infer the quality of the interface bond of materials in ultrasonic nondestructive evaluation. In addition the results are useful in the study of acoustic microscopy probes, coatings, and geo-exploration.