GIT1 protects against breast cancer growth through negative regulation of Notch.

Hyperactive Notch signalling is frequently observed in breast cancer and correlates with poor prognosis. However, relatively few mutations in the core Notch signalling pathway have been identified in breast cancer, suggesting that as yet unknown mechanisms increase Notch activity. Here we show that increased expression levels of GIT1 correlate with high relapse-free survival in oestrogen receptor-negative (ER(-)) breast cancer patients and that GIT1 mediates negative regulation of Notch. GIT1 knockdown in ER(-) breast tumour cells increased signalling downstream of Notch and activity of aldehyde dehydrogenase, a predictor of poor clinical outcome. GIT1 interacts with the Notch intracellular domain (ICD) and influences signalling by inhibiting the cytoplasm-to-nucleus transport of the Notch ICD. In xenograft experiments, overexpression of GIT1 in ER(-) cells prevented or reduced Notch-driven tumour formation. These results identify GIT1 as a modulator of Notch signalling and a guardian against breast cancer growth.

High Expression of PPM1D Induces Tumors Phenotypically Similar to TP53 Loss-of-Function Mutations in Mice.

PPM1D is a negative regulator of p53 and genomic aberrations resulting in increased activity of PPM1D have been observed in cancers of different origins, indicating that PPM1D has oncogenic properties. We established a transgenic mouse model overexpressing PPM1D and showed that these mice developed a wide variety of cancers. PPM1D-expressing mice developed tumors phenotypically and genetically similar to tumors in mice with dysfunctional p53. T-cell lymphoblastic lymphoma was the most frequent cancer observed in these mice (55%) followed by adenocarcinomas (24%), leukemia (12%) and other solid tumors including neuroblastoma. Characterization of T-cell lymphomas in mice overexpressing PPM1D demonstrates Pten-deletion and p53-accumulation similar to mice with p53 loss-of-function. Also, Notch1 mutations which are recurrently observed in T-cell acute lymphoblastic lymphoma (T-ALL) were frequently detected in PPM1D-transgenic mice. Hence, PPM1D acts as an oncogenic driver in connection with cellular stress, suggesting that the PPM1D gene status and expression levels should be investigated in TP53 wild-type tumors.

Inhibition of the ubiquitin-proteasome system by an NQO1-activatable compound.

Malignant cells display an increased sensitivity towards drugs that reduce the function of the ubiquitin-proteasome system (UPS), which is the primary proteolytic system for destruction of aberrant proteins. Here, we report on the discovery of the bioactivatable compound CBK77, which causes an irreversible collapse of the UPS, accompanied by a general accumulation of ubiquitylated proteins and caspase-dependent cell death. CBK77 caused accumulation of ubiquitin-dependent, but not ubiquitin-independent, reporter substrates of the UPS, suggesting a selective effect on ubiquitin-dependent proteolysis. In a genome-wide CRISPR interference screen, we identified the redox enzyme NAD(P)H:quinone oxidoreductase 1 (NQO1) as a critical mediator of CBK77 activity, and further demonstrated its role as the compound bioactivator. Through affinity-based proteomics, we found that CBK77 covalently interacts with ubiquitin. In vitro experiments showed that CBK77-treated ubiquitin conjugates were less susceptible to disassembly by deubiquitylating enzymes. In vivo efficacy of CBK77 was validated by reduced growth of NQO1-proficient human adenocarcinoma cells in nude mice treated with CBK77. This first-in-class NQO1-activatable UPS inhibitor suggests that it may be possible to exploit the intracellular environment in malignant cells for leveraging the impact of compounds that impair the UPS.

Establishment of an in vitro 3D model for neuroblastoma enables preclinical investigation of combined tumor-stroma drug targeting.

The majority of anti-cancer therapies target the proliferating tumor cells, while the tumor stroma, principally unaffected, survives, and provide a niche for surviving tumor cells. Combining tumor cell and stroma-targeting therapies thus have a potential to improve patient outcome. The neuroblastoma stroma contains cancer-associated fibroblasts expressing microsomal prostaglandin E synthase-1 (mPGES-1). mPGES-1-derived prostaglandin E2 (PGE2 ) is known to promote tumor growth through increased proliferation and survival of tumor cells, immune suppression, angiogenesis, and therapy resistance, and we, therefore, hypothesize that mPGES-1 constitutes an interesting stromal target. Here, we aimed to develop a relevant in vitro model to study combination therapies. Co-culturing of neuroblastoma and fibroblast cells in 3D tumor spheroids mimic neuroblastoma tumors with regard to the cyclooxygenase/mPGES-1/PGE2 pathway. Using the spheroid model, we show that the inhibition of fibroblast-derived mPGES-1 enhanced the cytotoxic effect of doxorubicin and vincristine and significantly reduced tumor cell viability and spheroid growth. Cyclic treatment with vincristine in combination with an mPGES-1 inhibitor abrogated cell repopulation. Moreover, inhibition of mPGES-1 potentiated the cytotoxic effect of vincristine on established neuroblastoma allografts in mice. In conclusion, we established a 3D neuroblastoma model, highlighting the potential of combining stromal targeting of mPGES-1 with tumor cell targeting drugs like vincristine.

Inhibitors of ribosome biogenesis repress the growth of MYCN-amplified neuroblastoma.

Abnormal increases in nucleolar size and number caused by dysregulation of ribosome biogenesis has emerged as a hallmark in the majority of spontaneous cancers. The observed ribosome hyperactivity can be directly induced by the MYC transcription factors controlling the expression of RNA and protein components of the ribosome. Neuroblastoma, a highly malignant childhood tumor of the sympathetic nervous system, is frequently characterized by MYCN gene amplification and high expression of MYCN and c-MYC signature genes. Here, we show a strong correlation between high-risk disease, MYCN expression, poor survival, and ribosome biogenesis in neuroblastoma patients. Treatment of neuroblastoma cells with quarfloxin or CX-5461, two small molecule inhibitors of RNA polymerase I, suppressed MycN expression, induced DNA damage, and activated p53 followed by cell cycle arrest or apoptosis. CX-5461 repressed the growth of established MYCN-amplified neuroblastoma xenograft tumors in nude mice. These findings suggest that inhibition of ribosome biogenesis represent new therapeutic opportunities for children with high-risk neuroblastomas expressing high levels of Myc.

Inhibition of Microsomal Prostaglandin E Synthase-1 in Cancer-Associated Fibroblasts Suppresses Neuroblastoma Tumor Growth.

Despite recent progress in diagnosis and treatment, survival for children with high-risk metastatic neuroblastoma is still poor. Prostaglandin E2 (PGE2)-driven inflammation promotes tumor growth, immune suppression, angiogenesis and resistance to established cancer therapies. In neuroblastoma, cancer-associated fibroblasts (CAFs) residing in the tumor microenvironment are the primary source of PGE2. However, clinical targeting of PGE2 with current non-steroidal anti-inflammatory drugs or cyclooxygenase inhibitors has been limited due to risk of adverse side effects. By specifically targeting microsomal prostaglandin E synthase-1 (mPGES-1) activity with a small molecule inhibitor we could block CAF-derived PGE2 production leading to reduced tumor growth, impaired angiogenesis, inhibited CAF migration and infiltration, reduced tumor cell proliferation and a favorable shift in the M1/M2 macrophage ratio. In this study, we provide proof-of-principle of the benefits of targeting mPGES-1 in neuroblastoma, applicable to a wide variety of tumors. This non-toxic single drug treatment targeting infiltrating stromal cells opens up for combination treatment options with established cancer therapies.

Inhibition of chemerin/CMKLR1 axis in neuroblastoma cells reduces clonogenicity and cell viability in vitro and impairs tumor growth in vivo.

Pro-inflammatory cells, cytokines, and chemokines are essential in promoting a tumor supporting microenvironment. Chemerin is a chemotactic protein and a natural ligand for the receptors CMKLR1, GPR1, and CCRL2. The chemerin/CMKLR1 axis is involved in immunity and inflammation, and it has also been implicated in obesity and cancer. In neuroblastoma, a childhood tumor of the peripheral nervous system we identified correlations between high CMKLR1 and GPR1 expression and reduced overall survival probability. CMKLR1, GPR1, and chemerin RNA and protein were detected in neuroblastoma cell lines and neuroblastoma primary tumor tissue. Chemerin induced calcium mobilization, increased MMP-2 synthesis as well as MAP-kinase- and Akt-mediated signaling in neuroblastoma cells. Stimulation of neuroblastoma cells with serum, TNFα or IL-1ß increased chemerin secretion. The small molecule CMKLR1 antagonist α-NETA reduced the clonogenicity and viability of neuroblastoma cell lines indicating the chemerin/CMKLR1 axis as a promoting factor in neuroblastoma tumorigenesis. Furthermore, nude mice carrying neuroblastoma SK-N-AS cells as xenografts showed impaired tumor growth when treated daily with α-NETA from day 1 after tumor cell injection. This study demonstrates the potential of the chemerin/CMKLR1 axis as a prognostic factor and possible therapeutic target in neuroblastoma.

Tumor development, growth characteristics and spectrum of genetic aberrations in the TH-MYCN mouse model of neuroblastoma.

BACKGROUND: The TH-MYCN transgenic neuroblastoma model, with targeted MYCN expression to the developing neural crest, has been used to study neuroblastoma development and evaluate novel targeted tumor therapies. METHODS: We followed tumor development in 395 TH-MYCN (129X1/SvJ) mice (125 negative, 206 hemizygous and 64 homozygous mice) by abdominal palpations up to 40 weeks of age. DNA sequencing of MYCN in the original plasmid construct and mouse genomic DNA was done to verify the accuracy. Copy number analysis with Affymetrix® Mouse Diversity Genotyping Arrays was used to characterize acquired genetic aberrations. RESULTS: DNA sequencing confirmed presence of human MYCN cDNA in genomic TH-MYCN DNA corresponding to the original plasmid construct. Tumor incidence and growth correlated significantly to transgene status with event-free survival for hemizygous mice at 50%, and 0% for homozygous mice. Hemizygous mice developed tumors at 5.6-19 weeks (median 9.1) and homozygous mice at 4.0-6.9 weeks (5.4). The mean treatment window, time from palpable tumor to sacrifice, for hemizygous and homozygous mice was 15 and 5.2 days, respectively. Hemizygous mice developing tumors as early as homozygous mice had a longer treatment window. Age at tumor development did not influence treatment window for hemizygous mice, whereas treatment window in homozygous mice decreased significantly with increasing age. Seven out of 10 analysed tumors had a flat DNA profile with neither segmental nor numerical chromosomal aberrations. Only three tumors from hemizygous mice showed acquired genetic features with one or more numerical aberrations. Of these, one event corresponded to gain on the mouse equivalent of human chromosome 17. CONCLUSION: Hemizygous and homozygous TH-MYCN mice have significantly different neuroblastoma incidence, tumor growth characteristics and treatment windows but overlap in age at tumor development making correct early genotyping essential to evaluate therapeutic interventions. Contrasting previous studies, our data show that TH-MYCN tumors have few genetic aberrations.

Effects of small molecule inhibitors of PI3K/Akt/mTOR signaling on neuroblastoma growth in vitro and in vivo.

Activation of the PI3K/Akt signaling pathway is correlated with poor prognosis in neuroblastoma, the most common and deadly extracranial tumor of childhood. In this study, we show that the small-molecule inhibitors of phosphoinositide-dependent protein kinase-1 (PDK1) OSU03012 and the dual class I PI3K/mTOR inhibitor PI103 have profound effects on neuroblastoma survival in vitro and in vivo. Both OSU03012 and PI103 inhibited neuroblastoma growth in vitro. In treated cells, OSU03012 induced apoptosis and an S phase cell cycle arrest, whereas only minor apoptosis was detected in PI103 treated cells together with a G1 arrest. Both OSU03012 and PI103 downregulated phosphorylation of Akt and inhibited the downstream targets glycogen synthase kinase-3ß (GSK3ß) and p70 S6 kinase-1 (S6K1), as well as downregulated the expression of cyclin D1 and Mycn protein. Neuroblastoma cells expressing high levels of Mycn were more sensitive to OSU03012 or PI103 compared with cells expressing low Mycn levels. Both compounds significantly inhibited the growth of established, subcutaneous MYCN-amplified neuroblastoma xenografts in nude NMRI nu/nu mice. These results suggest that inhibition of the PI3K/Akt signaling pathway represent a clinical relevant target for the treatment of patients with high-risk MYCN-amplified neuroblastoma.

NSAIDs in neuroblastoma therapy.

Cyclooxygenases (COX) catalyse the conversion of arachidonic acid to prostaglandins. COX-2 is upregulated in several adult epithelial cancers. In neuroblastoma it has been shown that the majority of primary tumours and cell lines express high levels of COX-2, whereas normal adrenal medullas from children do not express COX-2. Treatment of neuroblastoma cells with nonsteroidal anti-inflammatory drugs (NSAIDs), inhibitors of COX, induces caspase-dependent apoptosis via the intrinsic mitochondrial pathway. Established neuroblastoma xenografts in nude rats treated with the dual COX-1/COX-2 inhibitor, diclofenac, or the COX-2 specific inhibitor, celecoxib significantly inhibits neuroblastoma growth in vivo. In vitro, arachidonic acid and diclofenac synergistically induces neuroblastoma cell death. This effect is further pronounced when lipoxygenases is inhibited simultaneously. Proton MR-spectroscopy (1H MRS) of neuroblastoma cells treated with COX-inhibitors demonstrates accumulation of polyunsaturated fatty acids and depletion of choline compounds. Thus, 1H MRS, which can be performed with clinical MR-scanners, is likely to provide pharmacodynamic markers of neuroblastoma response to COX-inhibition. Taken together, these data suggest the use of NSAIDs as a novel adjuvant therapy for children with neuroblastoma.

Cyclooxygenase-2 is expressed in neuroblastoma, and nonsteroidal anti-inflammatory drugs induce apoptosis and inhibit tumor growth in vivo.

Neuroblastoma is the single most common and deadly tumor of childhood and is often associated with therapy resistance. Cyclooxygenases (COXs) catalyze the conversion of arachidonic acid to prostaglandins. COX-2 is up-regulated in several adult epithelial cancers and is linked to proliferation and resistance to apoptosis. We detected COX-2 expression in neuroblastoma primary tumors and cell lines but not in normal adrenal medullas from children. Treatment of neuroblastoma cells with nonsteroidal anti-inflammatory drugs, inhibitors of COX, induced caspase-dependent apoptosis via the intrinsic mitochondrial pathway. Treatment of established neuroblastoma xenografts in nude rats with the dual COX-1/COX-2 inhibitor diclofenac or the COX-2-specific inhibitor celecoxib significantly inhibited tumor growth in vivo (P < 0.001). In vitro, arachidonic acid and diclofenac synergistically induced neuroblastoma cell death. This effect was further pronounced when lipooxygenases were simultaneously inhibited. Proton magnetic resonance spectroscopy ((1)H MRS) of neuroblastoma cells treated with COX inhibitors demonstrated accumulation of polyunsaturated fatty acids and depletion of choline compounds. Thus, (1)H MRS, which can be performed with clinical magnetic resonance scanners, is likely to provide pharmacodynamic markers of neuroblastoma response to COX inhibition. Taken together, these data suggest the use of nonsteroidal anti-inflammatory drugs as a novel adjuvant therapy for children with neuroblastoma.

No transmission of porcine endogenous retrovirus after transplantation of adult porcine islets into diabetic nude mice and immunosuppressed rats.

BACKGROUND: The aim of this study was to investigate whether transmission of porcine endogenous retrovirus (PERV) occurs in a model of diabetes reversal by the xenotransplantation of adult porcine islets (APIs) into immunoincompetent diabetic rodents. METHODS: Black-6 nu/nu mice and Lewis rats were immunosuppressed with cyclosporin A (CsA) and FTY 720, and rendered diabetic with streptozotocin. Purified APIs were transplanted into the renal subcapsular space; 5,000 islet equivalents (IEQs) were used in the nude mice (n = 4) and 40,000 IEQs in the rats (n = 4). The nude mice were sacrificed at 75 days after transplantation. In order to confirm chronic xenograft function, the graft-bearing kidney was removed prior to sacrifice. The rats were followed until xenograft rejection, at which time they were sacrificed. Immediately after sacrifice, tissue samples (liver, spleen, and small intestine) were taken for analysis. Quantitative polymerase chain reaction (PCR) was used to assess evidence of PERV transmission, and porcine cell chimerism. RESULTS: All animals became normoglycemic within 48 h of transplantation. The nude mice remained normoglycemic during the 75-day study period, with removal of the graft-bearing kidney resulting in prompt hyperglycemia. The rats remained normoglycemic until xenograft rejection, which occurred at 66 +/- 28 days. Despite the evidence of porcine cell microchimerism in recipients, real-time PCR detected no evidence of PERV transmission in any of the tissue specimens tested. CONCLUSIONS: There was no evidence of PERV transmission following transplantation of pig islets into diabetic nude mice and immunosuppressed rats.