Sequential targeting of PARP with carboplatin inhibits primary tumour growth and distant metastasis in triple-negative breast cancer.

BACKGROUND: Patients with triple-negative breast cancer (TNBC) develop early recurrence. While PARP inhibitors (PARPi) have demonstrated potential in BRCA1/2-mutant (BRCAMUT) TNBC, durable responses will likely be achieved if PARPi are used in combination. It is plausible that sequential administration of a potent PARPi like talazoparib in combination with carboplatin can enhance primary tumour and metastasis inhibition in BRCAMUT and BRCA1/2 wild-type (BRCAWT) TNBCs, and decrease toxicity. METHODS: We evaluated the impact of the concurrent combination of talazoparib and carboplatin on cell survival in 13 TNBC cell lines. We compared the concurrent and sequential combination upon fork replication, migration and invasion. We also used three orthotopic xenograft models to evaluate primary tumour growth, distant metastasis, and toxicity. RESULTS: Concurrent talazoparib and carboplatin was synergistic in 92.3% of TNBC cell lines, independent of BRCA1/2-mutation status. The sequential combination decreased fork speed in normal cells, but not in TNBC cells. The talazoparib-first sequential combination resulted in a strong reduction in migration (70.4%, P < 0.0001), invasion (56.9%, P < 0.0001), lung micrometastasis (56.4%, P < 0.0001), and less toxicity in a BRCAWT model. CONCLUSION: The sequential combination of talazoparib and carboplatin is an effective approach to inhibit micrometastatic disease, providing rationale for the use of this combination in early TNBC patients.

Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons.

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their post-natal development, abundance, and accessibility, CGNs are an ideal model to study neuronal processes, including neuronal development, neuronal migration, and physiological neuronal activity stimulation. In addition, CGN cultures provide an excellent model for studying different modes of cell death including excitotoxicity and apoptosis. Within a week in culture, CGNs express N-methyl-D-aspartate (NMDA) receptors, a specific ionotropic glutamate receptor with many critical functions in neuronal health and disease. The addition of low concentrations of NMDA in conjunction with membrane depolarization to rodent primary CGN cultures has been used to model physiological neuronal activity stimulation while the addition of high concentrations of NMDA can be employed to model excitotoxic neuronal injury. Here, a method of isolation and culturing of CGNs from 6 day old pups as well as genetic manipulation of CGNs by adenoviruses and lentiviruses are described. We also present optimized protocols on how to stimulate NMDA-induced excitotoxicity, low-potassium-induced apoptosis, oxidative stress and DNA damage following transduction of these neurons.

Control of glioblastoma tumorigenesis by feed-forward cytokine signaling.

EGFRvIII-STAT3 signaling is important in glioblastoma pathogenesis. Here, we identified the cytokine receptor OSMR as a direct target gene of the transcription factor STAT3 in mouse astrocytes and human brain tumor stem cells (BTSCs). We found that OSMR functioned as an essential co-receptor for EGFRvIII. OSMR formed a physical complex with EGFRvIII, and depletion of OSMR impaired EGFRvIII-STAT3 signaling. Conversely, pharmacological inhibition of EGFRvIII phosphorylation inhibited the EGFRvIII-OSMR interaction and activation of STAT3. EGFRvIII-OSMR signaling in tumors operated constitutively, whereas EGFR-OSMR signaling in nontumor cells was synergistically activated by the ligands EGF and OSM. Finally, knockdown of OSMR strongly suppressed cell proliferation and tumor growth of mouse glioblastoma cells and human BTSC xenografts in mice, and prolonged the lifespan of these mice. Our findings identify OSMR as a critical regulator of glioblastoma tumor growth that orchestrates a feed-forward signaling mechanism with EGFRvIII and STAT3 to drive tumorigenesis.

Gene expression profiling from formalin-fixed paraffin-embedded tumors of pediatric glioblastoma.

PURPOSE: Gene expression profiling has proved crucial for understanding the biology of cancer. In rare diseases, including pediatric glioblastoma (pGBM), the lack of readily available fresh frozen (FF) material limits the feasibility of this analysis, as well as its validation, on independent data sets, a step needed to ensure relevance, mandating the use of alternate RNA sources. To overcome the limitation of material number and to validate results we obtained on FF pGBM, we did microarray analysis on RNA extracted from formalin-fixed, paraffin-embedded archival samples from pGBM and control brains, wherein we had no control on the fixation process. EXPERIMENTAL DESIGN: RNA from 16 pGBM and 3 control brains was extracted and linearly amplified. Reverse transcription-PCR on housekeeping and formerly identified tumor-associated genes and microarray analysis were done on this RNA source. Results were validated by immunohistochemistry. RESULTS: Despite extensive RNA degradation, microarray analysis was possible on 16 of 19 samples and reproduced the pattern of results obtained on FF pGBM. Gene lists and ontology subgrouping were highly concordant in both sample types. Similar to the findings on FF samples, we were able to identify two subsets of pGBM based on their association/lack of association with evidence consistent with an active Ras pathway. CONCLUSIONS: Archival formalin-fixed, paraffin-embedded tissues are an invaluable resource as they are the most widely available materials often accessible in conjunction with clinical and follow-up data. Gene expression profiling on this material is feasible and may represent a significant advance for understanding the biology of rare human diseases.

Molecular profiling identifies prognostic subgroups of pediatric glioblastoma and shows increased YB-1 expression in tumors.

PURPOSE: Pediatric glioblastoma (pGBM) is a rare, but devastating brain tumor. In contrast to GBM in adults (aGBM), little is known about the mechanisms underlying its development. Our aim is to gain insight into the molecular pathways of pGBM. MATERIALS AND METHODS: Thirty-two pGBM and seven aGBM samples were investigated using biochemical and transcriptional profiling. Ras and Akt pathway activation was assessed through the phosphorylation of downstream effectors, and gene expression profiles were generated using the University Health Network Human 19K cDNA arrays. Results were validated using real-time polymerase chain reaction and immunohistochemistry and compared with existing data sets on aGBM. RESULTS: There are at least two subsets of pGBM. One subset, associated with Ras and Akt pathway activation, has very poor prognosis and exhibits increased expression of genes related to proliferation and to a neural stem-cell phenotype, similar to findings in aggressive aGBM. This subset was still molecularly distinguishable from aGBM after unsupervised and supervised analysis of expression profiles. A second subset, with better prognosis, is not associated with activation of Akt and Ras pathways, may originate from astroglial progenitors, and does not express gene signatures and markers shown to be associated with long-term survival in aGBM. Both subsets of pGBM show overexpression of Y-box-protein-1 that may help drive oncogenesis in this tumor. CONCLUSION: Our work, the first study of gene expression profiles in pGBM, provides valuable insight into active pathways and targets in a cancer with minimal survival, and suggests that these tumors cannot be understood exclusively through studies of aGBM.

In vitro osteogenic response of rat bone marrow cells to bFGF and BMP-2 treatments.

Basic fibroblast growth factor (bFGF) and bone morphogenetic protein-2 (BMP-2) are actively pursued for stimulation of bone formation. To assess their promise for systemic therapy of osteoporosis, we ascertained the effects of bFGF and BMP-2 on bone marrow cells in vitro. Bone marrow cells were obtained from young (8 weeks) and adult (32 weeks) rats by femoral aspiration and were exposed to osteogenic medium (ie, basal medium with 10 mM beta-glycerolphosphate and 100 nM dexamethasone) containing the growth factors. The cell viability in osteogenic medium was reduced after 3 weeks but not if the concentration of beta-glycerolphosphate/dexamethasone was reduced to 3 mM/30 nM. Unlike BMP-2, bFGF at 2-50 ng/mL was capable of enhancing long-term cell viability. Continuous treatment of bone marrow cells for 3 weeks resulted in dose-dependent stimulation of mineralization by BMP-2, but not by bFGF, whose activity was optimal at 2-10 ng/mL. To explore the effect of short-term exposure, bone marrow cells were treated with growth factors for 1 week and subsequent mineralization was investigated. BMP-2 exposure increased the extent of mineralization, but bFGF was not effective after the short exposure. We concluded bFGF was more potent (ie, required lower concentration) for stimulating osteogenic parameters, but BMP-2 effects were lasting on the bone marrow cells.

Bone marrow cells from normal and ovariectomized rats respond differently to basic fibroblast growth factor and bone morphogenetic protein 2 treatment in vitro.

The protein growth factors basic fibroblast growth factor (bFGF) and bone morphogenetic protein 2 (BMP-2) are being actively pursued for bone tissue engineering. Although both proteins are capable of stimulating osteogenic activity of bone marrow cells (BMCs), no studies have addressed the effect of estrogen deficiency on the growth factor responsiveness of BMCs. This study investigated the osteogenic response of BMCs from normal and ovariectomized (OVX) rats to bFGF and BMP- 2. In the absence of growth factors, a higher number of total colony-forming units (t-CFU) and alkaline phosphatase-expressing CFU (ALP-CFU) were obtained with BMCs derived from OVX rats. The percentage of ALP-CFU, however, was not significantly different between BMCs from the two groups of rats. Whereas BMP-2 did not influence the t-CFU and percentage of ALP-CFU, bFGF decreased t-CFU in BMCs derived from OVX rats and reduced the percentage of ALP-CFU in BMCs from both types of rats. Consistent with the higher t-CFU, the number of mineralized colonies (min-CFU) was also higher for BMCs derived from OVX rats. The number of min-CFU was not influenced by BMP-2 treatment, but was reduced with bFGF treatment. Comparison of the growth factor effects on a per-cell (DNA) basis confirmed the expected stimulatory effect of BMP-2 on ALP activity and mineralization in BMCs from normal rats, but these two parameters were not unequivocally stimulated in BMCs from OVX rats. We conclude that BMCs derived from normal and OVX rats exhibited significant differences in their osteogenic response to bFGF and BMP-2 treatment.