While alterations in xenobiotic metabolism are considered causal in the development of bladder cancer (BCa), the precise mechanisms involved are poorly understood. In this study, we used high-throughput mass spectrometry to measure over 2,000 compounds in 58 clinical specimens, identifying 35 metabolites which exhibited significant changes in BCa. This metabolic signature distinguished both normal and benign bladder from BCa. Exploratory analyses of this metabolomic signature in urine showed promise in distinguishing BCa from controls, and also non-muscle from muscle-invasive BCa. Subsequent enrichment-based bioprocess mapping revealed alterations in phase I/II metabolism and suggested a possible role for DNA methylation in perturbing xenobiotic metabolism in BCa. In particular, we validated tumor-associated hypermethylation in the CYP1A1 and CYP1B1 promoters of BCa tissues by bisulfite sequence analysis and methylation-specific PCR, and also by in vitro treatment of T-24 BCa cell line with the DNA demethylating agent 5-aza-2′-deoxycytidine. Further, we showed that expression of CYP1A1 and CYP1B1 was reduced significantly in an independent cohort of BCa specimens compared to matched benign adjacent tissues. In summary, our findings identified candidate diagnostic and prognostic markers and highlighted mechanisms associated with the silencing of xenobiotic metabolism. The metabolomic signature we describe offers potential as a urinary biomarker for early detection and staging of BCa, highlighting the utility of evaluating metabolomic profiles of cancer to gain insights into bioprocesses perturbed during tumor development and progression.

Nucleophosmin 1 (NPM1) is an oligomeric, nucleolar phosphoprotein that functions as a molecular chaperone for both proteins and nucleic acids. NPM1 is mutated in approximately one-third of patients with AML. The mutant NPM1c؉ contains a 4-base insert that results in extra C-terminal residues encoding a nuclear export signal, which causes NPM1c؉ to be localized in the cytoplasm. Here, we determined the effects of targeting NPM1 in cultured and primary AML IntroductionNucleophosmin (NPM1 or B23.1) is a ubiquitously expressed, nucleolar phosphoprotein that functions as a molecular chaperone, shuttling between the nucleolus and the cytoplasm. 1-3 NPM1 plays multiple roles in cell growth and proliferation by participating in diverse biologic processes, including ribosome biogenesis and transport, centrosome duplication, DNA repair, transcriptional regulation and histone chaperoning. 4-7 Intracellular NPM1 is predominantly oligomeric and binds to other proteins, including the tumor suppressor proteins p14ARF and p53. 1,[8][9][10] Multifunctional characteristic of NPM1 appears to be dictated not only by its sub-cellular localization and its binding partners, but is also influenced by the various post translational modifications in NPM1, including acetylation, phosphorylation, poly-ubiquitination and sumoylation. [11][12][13][14] Wild-type (WT) NPM1 contains distinct structural domains that account for its ability to act as a multifunctional protein. 1,15 NPM1 has an N-terminal conserved, hydrophobic, oligomerization domain (residues, 1-110), which is common to all isoforms of NPM1 and critical for its chaperone activity. [1][2][3] Recently, NSC348884 was identified as a small molecule inhibitor that disrupts NPM1 dimer/oligomer formation, inducing apoptosis of cancer cells. 16 Oncogenic fusion proteins created by chromosomal translocation involving NPM1 gene, or mutations in NPM1 are observed in leukemia and lymphoma. 17 Notably, NPM-ALK fusion protein is found in CD30ϩ anaplastic large-cell lymphoma, 18 while leukemia related NPM1 fusion proteins include NPM-MLF1 and NPM-RAR␣. 17,19,20 These chimeric fusion proteins contain the N-terminal NPM1 oligomerization domain and a C-terminal fragment of the other protein. 17 NPM1 gene is also mutated in one third of adult acute myeloid leukemia (AML), especially those with the normal karyotype. 21 NPM1 mutations are heterozygous and, in the majority, localized to exon 12 of the gene. 21,22 Approximately 50 different types of mutations have been found, all creating the cytoplasm-dislocated mutant (Mt) NPM1 (NPM1cϩ) protein. 21,22 The most common is the type-A mutation, accounting for 75% of cases, which consists of TCTG tetranucleotide tandem duplication at position 956-959 of the NPM1 coding sequence. [22][23][24] This mutation causes the loss of tryptophans 288 and 290 (or 290 alone) from the carboxy-terminus and the creation of an additional leucine-rich nuclear export motif in the NPM1 protein, which causes the aberrant cytoplasmic dislocation of NPM1cϩ. [22][23][24] Knock...

Increased levels of misfolded polypeptides in the endoplasmic reticulum (ER) triggers the dissociation of glucose-regulated protein 78 (GRP78) from the three transmembrane ER-stress mediators, i.e., protein kinase RNA-like ER kinase (PERK), activating transcription factor-6 (ATF6), and inositol-requiring enzyme 1α, which results in the adaptive unfolded protein response (UPR). In the present studies, we determined that histone deacetylase-6 (HDAC6) binds and deacetylates GRP78. Following treatment with the pan-histone deacetylase inhibitor panobinostat (Novartis Pharmaceuticals), or knockdown of HDAC6 by short hairpin RNA, GRP78 is acetylated in 11 lysine residues, which dissociates GRP78 from PERK. This is associated with the activation of a lethal UPR in human breast cancer cells. Coimmunoprecipitation studies showed that binding of HDAC6 to GRP78 requires the second catalytic and COOH-terminal BUZ domains of HDAC6. Treatment with panobinostat increased the levels of phosphorylated-eukaryotic translation initiation factor (p-eIF2α), ATF4, and CAAT/enhancer binding protein homologous protein (CHOP). Panobinostat treatment also increased the proapoptotic BIK, BIM, BAX, and BAK levels, as well as increased the activity of caspase-7. Knockdown of GRP78 sensitized MCF-7 cells to bortezomib and panobinostat-induced UPR and cell death. These findings indicate that enforced acetylation and decreased binding of GRP78 to PERK is mechanistically linked to panobinostat-induced UPR and cell death of breast cancer cells. Mol Cancer Ther; 9(4); 942-52. ©2010 AACR.

Prostate cancer is the second leading cause of cancer related death in American men. Development and progression of clinically localized prostate cancer is highly dependent on androgen signaling. Metastatic tumors are initially responsive to anti-androgen therapy, however become resistant to this regimen upon progression. Genomic and proteomic studies have implicated a role for androgen in regulating metabolic processes in prostate cancer. However, there have been no metabolomic profiling studies conducted thus far that have examined androgen-regulated biochemical processes in prostate cancer. Here, we have used unbiased metabolomic profiling coupled with enrichment-based bioprocess mapping to obtain insights into the biochemical alterations mediated by androgen in prostate cancer cell lines. Our findings indicate that androgen exposure results in elevation of amino acid metabolism and alteration of methylation potential in prostate cancer cells. Further, metabolic phenotyping studies confirm higher flux through pathways associated with amino acid metabolism in prostate cancer cells treated with androgen. These findings provide insight into the potential biochemical processes regulated by androgen signaling in prostate cancer. Clinically, if validated, these pathways could be exploited to develop therapeutic strategies that supplement current androgen ablative treatments while the observed androgen-regulated metabolic signatures could be employed as biomarkers that presage the development of castrate-resistant prostate cancer.

Purpose: Bortezomib induces unfolded protein response (UPR) and endoplasmic reticulum stress, as well as exhibits clinical activity in patients with relapsed and refractory mantle cell lymphoma (MCL). Here, we determined the molecular basis of the improved in vitro and in vivo activity of the combination of the panhistone deacetylase inhibitor panobinostat and bortezomib against human, cultured, and primary MCL cells.Experimental Design: Immunoblot analyses, reverse transcription-PCR, and immunofluorescent and electron microscopy were used to determine the effects of panobinostat on bortezomib-induced aggresome formation and endoplasmic reticulum stress in MCL cells.Results: Treatment with panobinostat induced heat shock protein 90 acetylation; depleted the levels of heat shock protein 90 client proteins, cyclin-dependent kinase 4, c-RAF, and AKT; and abrogated bortezomibinduced aggresome formation in MCL cells. Panobinostat also induced lethal UPR, associated with induction of CAAT/enhancer binding protein homologous protein (CHOP). Conversely, knockdown of CHOP attenuated panobinostat-induced cell death of MCL cells. Compared with each agent alone, cotreatment with panobinostat increased bortezomib-induced expression of CHOP and NOXA, as well as increased bortezomib-induced UPR and apoptosis of cultured and primary MCL cells. Cotreatment with panobinostat also increased bortezomib-mediated in vivo tumor growth inhibition and improved survival of mice bearing human Z138C MCL cell xenograft.Conclusion: These findings suggest that increased UPR and induction of CHOP are involved in enhanced anti-MCL activity of the combination of panobinostat and bortezomib.

Tissue inhibitors of metalloproteinases (TIMPs) are multifaceted molecules that exhibit properties beyond their classical proteinase inhibitory function. Although TIMP-1 is a known inhibitor of apoptosis in mammalian cells, the mechanisms by which it exerts its effects are not well-established. Our earlier studies using H2009 lung adenocarcinoma cells, implanted in the CNS, showed that TIMP-1 overexpressing H2009 cells (HB-1), resulted in more aggressive tumor kinetics and increased vasculature. The present study was undertaken to elucidate the role of TIMP-1 in the context of apoptosis, using the same lung cancer cell lines. Overexpressing TIMP-1 in a lung adenocarcinoma cell line H2009 resulted in an approximately 3-fold increased expression of Bcl-2, with a marked reduction in apoptosis upon staurosporine treatment. This was an MMP-independent function as a clone expressing TIMP-1 mutant T2G, lacking MMP inhibition activity, inhibited apoptosis as strongly as TIMP1 overexpressing clones, as determined by inhibition of PARP cleavage. Immunoprecipitation of Bcl-2 from cell lysates also co-immunoprecipitated TIMP-1, indicative of an interaction between these two proteins. This interaction was specific for TIMP-1 as TIMP-2 was not present in the Bcl-2 pull-down. Additionally, we show a co-dependency of TIMP-1 and Bcl-2 RNA and protein levels, such that abrogating Bcl-2 causes a downregulation of TIMP-1 but not TIMP-2. Finally, we demonstrate that TIMP-1 dependent inhibition of apoptosis occurs through p90RSK, with phosphorylation of the pro-apoptotic protein BAD at serine 112, ultimately reducing Bax levels and increasing mitochondrial permeability. Together, these studies define TIMP-1 as an important cancer biomarker and demonstrate the potential TIMP-1 as a crucial therapeutic target.

BackgroundPediatric patients with high-risk neuroblastoma (HR NB) often fail to respond to upfront intensive multimodal therapy. Tumor-acquired suppression of apoptosis contributes to therapy resistance. Many HR NB tumors depend on the anti-apoptotic protein Bcl-2 for survival, through Bcl-2 sequestration and inhibition of the pro-apoptotic protein, Bim. Bcl-2 dependent xenografts derived from aggressive human NB tumors are cured with a combination of cyclophosphamide and ABT-737, a Bcl-2/Bcl-XL/Bcl-w small molecule antagonist. The oral analogue to ABT-737, Navitoclax (ABT-263), clinically causes an immediate drop in peripheral platelet counts as mature platelets depend on Bcl-xL for survival. This led to the creation of a Bcl-2 selective inhibitor, ABT-199 (Venetoclax). A Phase I trial of ABT-199 in CLL showed remarkable antitumor activity and stable patient platelet counts. Given Bcl-XL does not play a role in HR NB survival, we hypothesized that ABT-199 would be equally potent against HR NB.MethodsCytotoxicity and apoptosis were measured in human derived NB cell lines exposed to ABT-199 combinations. Co-Immunoprecipitation evaluated Bim displacement from Bcl-2, following ABT-199. Murine xenografts of NB cell lines were grown and then exposed to a 14-day course of ABT-199 alone and with cyclophosphamide.ResultsBcl-2 dependent NB cell lines are exquisitely sensitive to ABT-199 (IC50 1.5–5 nM) in vitro, where Mcl-1 dependent NBs are completely resistant. Treatment with ABT-199 displaces Bim from Bcl-2 in NB to activate caspase 3, confirming the restoration of mitochondrial apoptosis. Murine xenografts of Mcl-1 and Bcl-2 dependent NBs were treated with a two-week course of ABT-199, cyclophosphamide, or ABT-199/cyclophosphamide combination. Mcl-1 dependent tumors did not respond to ABT-199 alone and showed no significant difference in time to tumor progression between chemotherapy alone or ABT-199/cyclophosphamide combination. In contrast, Bcl-2 dependent xenografts responded to ABT-199 alone and had sustained complete remission (CR) to the ABT-199/cyclophosphamide combination, with one recurrent tumor maintaining Bcl-2 dependence and obtaining a second CR after a second course of therapy.ConclusionHR NB patients are often thrombocytopenic at relapse, raising concerns for therapies like ABT-263 despite its HR NB tumor targeting potential. Our data confirms that Bcl-2 selective inhibitors like ABT-199 are equally potent in HR NB in vitro and in vivo and given their lack of platelet toxicity, should be translated into the clinic for HR NB.

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