Altered glutamine metabolism in platinum resistant ovarian cancer.

Ovarian cancer is characterized by an increase in cellular energy metabolism, which is predominantly satisfied by glucose and glutamine. Targeting metabolic pathways is an attractive approach to enhance the therapeutic effectiveness and to potentially overcome drug resistance in ovarian cancer. In platinum-sensitive ovarian cancer cell lines the metabolism of both, glucose and glutamine was initially up-regulated in response to platinum treatment. In contrast, platinum-resistant cells revealed a significant dependency on the presence of glutamine, with an upregulated expression of glutamine transporter ASCT2 and glutaminase. This resulted in a higher oxygen consumption rate compared to platinum-sensitive cell lines reflecting the increased dependency of glutamine utilization through the tricarboxylic acid cycle. The important role of glutamine metabolism was confirmed by stable overexpression of glutaminase, which conferred platinum resistance. Conversely, shRNA knockdown of glutaminase in platinum resistant cells resulted in re-sensitization to platinum treatment. Importantly, combining the glutaminase inhibitor BPTES with platinum synergistically inhibited platinum sensitive and resistant ovarian cancers in vitro. Apoptotic induction was significantly increased using platinum together with BPTES compared to either treatment alone. Our findings suggest that targeting glutamine metabolism together with platinum based chemotherapy offers a potential treatment strategy particularly in drug resistant ovarian cancer.

Evaluation and comparison of a new DOTA and DTPA-bombesin agonist in vitro and in vivo in low and high GRPR expressing prostate and breast tumor models.

We evaluated and compared a new bombesin analog [Tyr-Gly5, Nle(14)]-BBN(6-14) conjugated to DOTA or DTPA and radiolabeled with In-111 in low and high GRPR expressing tumor models. Both peptides were radiolabeled with high radiochemical purity and specific activity. In vitro assays on T-47D, LNCaP and PC-3 cells showed that the affinity of peptides is similar and a higher binding and internalization of DOTA-peptide to PC-3 cells was observed. Both peptides could target PC-3 and LNCaP tumors in vivo and both tumor types could be visualized by microSPECT/CT.

Prognostic and therapeutic impact of argininosuccinate synthetase 1 control in bladder cancer as monitored longitudinally by PET imaging.

Targeted therapies have yet to have significant impact on the survival of patients with bladder cancer. In this study, we focused on the urea cycle enzyme argininosuccinate synthetase 1 (ASS1) as a therapeutic target in bladder cancer, based on our discovery of the prognostic and functional import of ASS1 in this setting. ASS1 expression status in bladder tumors from 183 Caucasian and 295 Asian patients was analyzed, along with its hypothesized prognostic impact and association with clinicopathologic features, including tumor size and invasion. Furthermore, the genetics, biology, and therapeutic implications of ASS1 loss were investigated in urothelial cancer cells. We detected ASS1 negativity in 40% of bladder cancers, in which multivariate analysis indicated worse disease-specific and metastasis-free survival. ASS1 loss secondary to epigenetic silencing was accompanied by increased tumor cell proliferation and invasion, consistent with a tumor-suppressor role for ASS1. In developing a treatment approach, we identified a novel targeted antimetabolite strategy to exploit arginine deprivation with pegylated arginine deiminase (ADI-PEG20) as a therapeutic. ADI-PEG20 was synthetically lethal in ASS1-methylated bladder cells and its exposure was associated with a marked reduction in intracellular levels of thymidine, due to suppression of both uptake and de novo synthesis. We found that thymidine uptake correlated with thymidine kinase-1 protein levels and that thymidine levels were imageable with [(18)F]-fluoro-L-thymidine (FLT)-positron emission tomography (PET). In contrast, inhibition of de novo synthesis was linked to decreased expression of thymidylate synthase and dihydrofolate reductase. Notably, inhibition of de novo synthesis was associated with potentiation of ADI-PEG20 activity by the antifolate drug pemetrexed. Taken together, our findings argue that arginine deprivation combined with antifolates warrants clinical investigation in ASS1-negative urothelial and related cancers, using FLT-PET as an early surrogate marker of response.

Brn-3a transcription factor blocks p53-mediated activation of proapoptotic target genes Noxa and Bax in vitro and in vivo to determine cell fate.

The Brn-3a POU transcription factor is associated with survival and the differentiation of sensory neuronal cells during development. Brn-3a mediates its effects either by the direct regulation of target genes or indirectly upon interaction with proteins such as p53. Brn-3a differentially regulates p53-mediated gene expression and modifies its effect on cell fate. Here we show that, like Bax, Brn-3a antagonizes p53-mediated transcription of another proapoptotic target, Noxa, significantly reducing transactivation of the Noxa promoter by p53. This effect requires the p53 binding site, and electrophoretic mobility shift assay studies suggest that Brn-3a is associated with p53 when it is bound to its site in the Noxa promoter. The wild type but not the mutant promoter can be immunoprecipitated with Brn-3a in chromatin immunoprecipitation assays. Thus, Brn-3a may act by preventing the recruitment of cofactors required for p53 to transactivate this promoter. The co-expression of Brn-3a and p53 results in decreased endogenous Noxa protein in the neuronal cell line, ND7, suggesting a direct functional effect of this interaction. Moreover, there is a significant elevation of both proapoptotic Bax and Noxa proteins in sensory neuronal tissue taken from Brn-3a-/- embryos during development, compared with wild type controls. Striking changes occurred at embryonic day 14.5, a time that precedes a significant loss of specific neurons in the mutant embryos, but not at embryonic day 16.5 when Brn-3a-expressing cells are already lost by apoptosis. Therefore, the lack of antagonism by Brn-3a on activation of proapoptotic p53 target genes may contribute to the increased apoptosis seen in the Brn-3a-/- embryos. These results support a crucial role for Brn-3a in determining the pathway taken by p53 when co-expressed during development and thus in controlling the fate of these cells.

Coexpression of Brn-3a POU protein with p53 in a population of neuronal progenitor cells is associated with differentiation and protection against apoptosis.

The Brn-3a transcription factor is critical for survival and differentiation of sensory neurons derived from neural crest cells (NCC). Interaction of Brn-3a with p53 results in differential effects on target gene expression, which profoundly affects fate of neuronal cells. Here we demonstrate colocalization of p53 in a subset of Brn-3a-positive NCC-derived cells fated for the sensory neuronal lineage. The distinct morphology of Brn-3a/p53-coexpressing cells suggested a differentiated neuronal cell type, and this was confirmed by colocalization of p53 with differentiation marker NF-160. Functional effects of Brn-3a/p53 coexpression were analyzed in NCC cultured from Brn-3a -/- embryos, which showed significantly increased apoptosis upon induction of p53 compared with wild-type NCC, suggesting that Brn-3a modulates the p53-mediated fate of NCC that coexpress both factors. Thus, p53 is expressed in neuronal cells undergoing differentiation as well as apoptosis. Interaction with Brn-3a in sensory neurons may be critical for modulating p53-mediated gene expression and hence cell fate.

ATM mutations are rare in familial chronic lymphocytic leukemia.

It is now recognized that a subset of B-cell chronic lymphocytic leukemia (CLL) is familial. The genetic basis of familial CLL is poorly understood, but recently germ line mutations in the Ataxia Telangiectasia (ATM) gene have been proposed to confer susceptibility to CLL. The evidence for this notion is, however, not unequivocal. To examine this proposition further we have screened the ATM gene for mutations in CLLs from 61 individuals in 29 families. Truncating ATM mutations, including a known ATM mutation, were detected in 2 affected individuals, but the mutations did not cosegregate with CLL in the families. In addition, 3 novel ATM missense mutations were detected. Common ATM missense mutations were not overrepresented. The data support previous observations that ATM mutation is associated with B-CLL. However, ATM mutations do not account for familial clustering of the disease.

Relationship between glutathione S-transferase M1, T1, and P1 polymorphisms and chronic lymphocytic leukemia.

Interindividual differences in susceptibility to hematologic malignancies may be mediated in part through polymorphic variability in the bioactivation and detoxification of carcinogens. The glutathione S-transferases (GSTs) have been implicated as susceptibility genes in this context for a number of cancers. The aim of this study was to examine whether polymorphic variation in GSTs confers susceptibility to chronic lymphocytic leukemia (CLL). GSTM1, GSTT1, and GSTP1 genotypes were determined in 138 patients and 280 healthy individuals. The frequency of both GSTM1 and GSTT1 null genotypes and the GSTP1-Ile allele was higher in cases than in controls. There was evidence of a trend in increasing risk with the number of putative "high-risk" alleles of the GST family carried (P =.04). The risk of CLL associated with possession of all 3 "high-risk" genotypes was increased 2.8-fold (OR = 2.8, 95% confidence interval: 1.1-6.9). Our findings suggest that heritable GST status may influence the risk of developing CLL.

Analysis of the Fanconi anaemia complementation group A gene in acute myeloid leukaemia.

Acute myeloid leukaemia (AML) is the most common acute leukaemia in adults. Around 10-15% of individuals with recessively inherited Fanconi anaemia (FA) develop AML. FA is one of a group of recessive syndromes characterized by excessive spontaneous chromosomal breakage in which heterozygote carriers appear to display an increased risk of cancer and there is some indirect evidence that FA carriers may also be at increased risk of AML. This suggests that FA genes may play a role in the development of AML in the wider context. To examine this proposition, further, we have screened samples from 79 AML patients for mutations in the major FA gene, FANCA. No truncating FANCA mutations were detected. One missense mutation previously designated as pathogenic and five novel missense mutations causing non-conservative amino acid substitutions were detected. The data suggests that while FANCA mutations are rare, FANCA mutations may contribute to the development of the disease in a subset of AML.