An Epigenomic Approach to Improving Response to Neoadjuvant Cisplatin Chemotherapy in Bladder Cancer.

Bladder cancer is among the five most common cancers diagnosed in the Western world and causes significant mortality and morbidity rates in affected patients. Therapeutic options to treat the disease in advanced muscle-invasive bladder cancer (MIBC) include cystectomy and chemotherapy. Neoadjuvant cisplatin-based combination chemotherapy is effective in MIBC; however, it has not been widely adopted by the community. One reason is that many patients do not respond to neoadjuvant chemotherapy, and no biomarker currently exists to identify these patients. It is also not clear whether a strategy to sensitize chemoresistant patients may exist. We sought to identify cisplatin-resistance patterns in preclinical models of bladder cancer, and test whether treatment with the epigenetic modifier decitabine is able to sensitize cisplatin-resistant bladder cancer cell lines. Using a screening approach in cisplatin-resistant bladder cancer cell lines, we identified dysregulated genes by RNA sequencing (RNAseq) and DNA methylation assays. DNA methylation analysis of tumors from 18 patients receiving cisplatin-based chemotherapy was used to confirm in vitro results. Cisplatin-resistant bladder cancer cells were treated with decitabine to investigate epigenetic sensitization of resistant cell lines. Our results show that HOXA9 promoter methylation status is associated with response to cisplatin-based chemotherapy in bladder cancer cell lines and in metastatic bladder cancer. Bladder cancer cells resistant to cisplatin chemotherapy can be sensitized to cisplatin by the DNA methylation inhibitor decitabine. Our data suggest that HOXA9 promoter methylation could serve as potential predictive biomarker and decitabine might sensitize resistant tumors in patients receiving cisplatin-based chemotherapy.

Impact of ERBB2 mutations on in vitro sensitivity of bladder cancer to lapatinib.

Lapatinib, a dual tyrosine kinase inhibitor of ErbB1 and ErbB2, shows a clinical benefit in a subset of patients with advanced urothelial bladder cancer (UBC). We hypothesized that the corresponding gene, ERBB2, is affected by mutations in a subset of UBC and that these mutations impact ErbB2 function, signaling, UBC proliferation, gene expression, and predict response to lapatinib. We found ERBB2 mutations in 5 of 33 UBC cell lines (15%), all of which were derived from invasive or high grade tumors. Phosphorylation and activation of ErbB2 and its downstream pathways were markedly enhanced in mutated cell lines compared with the ERBB2 wild-type. In addition, the gene expression profile was distinct, specifically for genes encoding for proteins of the extracellular matrix. RT112 cells infected with ERBB2 mutants showed a particular growth pattern ("mini-foci"). Upon treatment with lapatinib, 93% of these "mini-foci" were reversed. The sensitivity to lapatinib was greatest among cell lines with ERBB2 mutations. In conclusion, ERBB2 mutations occur in a subset of UBC and impact proliferation, signaling, gene expression and predict a greater response to lapatinib. If confirmed in the clinical setting, this may lead the way toward personalized treatment of a subset of UBC.

Frequent truncating mutations of STAG2 in bladder cancer.

Here we report the discovery of truncating mutations of the gene encoding the cohesin subunit STAG2, which regulates sister chromatid cohesion and segregation, in 36% of papillary non-invasive urothelial carcinomas and 16% of invasive urothelial carcinomas of the bladder. Our studies suggest that STAG2 has a role in controlling chromosome number but not the proliferation of bladder cancer cells. These findings identify STAG2 as one of the most commonly mutated genes in bladder cancer.

Krüppel-like factor 9 and progesterone receptor coregulation of decidualizing endometrial stromal cells: implications for the pathogenesis of endometriosis.

CONTEXT: Endometriosis is characterized by progesterone resistance and associated with infertility. Krüppel-like factor 9 (KLF9) is a progesterone receptor (PGR)-interacting protein, and mice null for Klf9 are subfertile. Whether loss of KLF9 expression contributes to progesterone resistance of eutopic endometrium of women with endometriosis is unknown. OBJECTIVE: The aims were to investigate 1) KLF9 expression in eutopic endometrium of women with and without endometriosis, 2) effects of attenuated KLF9 expression on WNT-signaling component expression and on WNT inhibitor Dickkopf-1 promoter activity in human endometrial stromal cells (HESC), and 3) PGR and KLF9 coregulation of the stromal transcriptome network. METHODS: Transcript levels of KLF9, PGR, and WNT signaling components were measured in eutopic endometrium of women with and without endometriosis. Transcript and protein levels of WNT signaling components in HESC transfected with KLF9 and/or PGR small interfering RNA were analyzed by quantitative RT-PCR and Western blot. KLF9 and PGR coregulation of Dickkopf-1 promoter activity was evaluated using human Dickkopf-1-luciferase promoter/reporter constructs and by chromatin immunoprecipitation. KLF9 and PGR signaling networks were analyzed by gene expression array profiling. RESULTS: Eutopic endometrium from women with endometriosis had reduced expression of KLF9 mRNA together with those of PGR-B, WNT4, WNT2, and DKK1. KLF9 and PGR were recruited to the DKK1 promoter and modified each other's transactivity. In HESC, KLF9 and PGR coregulated components of the WNT, cytokine, and IGF gene networks that are implicated in endometriosis and infertility. CONCLUSION: Loss of KLF9 coregulation of endometrial stromal PGR-responsive gene networks may underlie progesterone resistance in endometriosis.

KLF9 is a novel transcriptional regulator of bortezomib- and LBH589-induced apoptosis in multiple myeloma cells.

Bortezomib, a therapeutic agent for multiple myeloma (MM) and mantle cell lymphoma, suppresses proteosomal degradation leading to substantial changes in cellular transcriptional programs and ultimately resulting in apoptosis. Transcriptional regulators required for bortezomib-induced apoptosis in MM cells are largely unknown. Using gene expression profiling, we identified 36 transcription factors that displayed altered expression in MM cells treated with bortezomib. Analysis of a publically available database identified Kruppel-like family factor 9 (KLF9) as the only transcription factor with significantly higher basal expression in MM cells from patients who responded to bortezomib compared with nonresponders. We demonstrated that KLF9 in cultured MM cells was up-regulated by bortezomib; however, it was not through the induction of endoplasmic reticulum stress. Instead, KLF9 levels correlated with bortezomib-dependent inhibition of histone deacetylases (HDAC) and were increased by the HDAC inhibitor LBH589 (panobinostat). Furthermore, bortezomib induced binding of endogenous KLF9 to the promoter of the proapoptotic gene NOXA. Importantly, KLF9 knockdown impaired NOXA up-regulation and apoptosis caused by bortezomib, LBH589, or a combination of theses drugs, whereas KLF9 overexpression induced apoptosis that was partially NOXA-dependent. Our data identify KLF9 as a novel and potentially clinically relevant transcriptional regulator of drug-induced apoptosis in MM cells.

Direct role of nucleotide metabolism in C-MYC-dependent proliferation of melanoma cells.

To identify C-MYC targets rate-limiting for proliferation of malignant melanoma, we stably inhibited C-MYC in several human metastatic melanoma lines via lentivirus-based shRNAs approximately to the levels detected in normal melanocytes. C-MYC depletion did not significantly affect levels of E2F1 protein reported to regulate expression of many S-phase specific genes, but resulted in the repression of several genes encoding enzymes rate-limiting for dNTP metabolism. These included thymidylate synthase (TS), inosine monophosphate dehydrogenase 2 (IMPDH2) and phosphoribosyl pyrophosphate synthetase 2 (PRPS2). C-MYC depletion also resulted in reduction in the amounts of deoxyribonucleoside triphosphates (dNTPs) and inhibition of proliferation. shRNA-mediated suppression of TS, IMPDH2 or PRPS2 resulted in the decrease of dNTP pools and retardation of the cell cycle progression of melanoma cells in a manner similar to that of C-MYC-depletion in those cells. Reciprocally, concurrent overexpression of cDNAs for TS, IMPDH2 and PRPS2 delayed proliferative arrest caused by inhibition of C-MYC in melanoma cells. Overexpression of C-MYC in normal melanocytes enhanced expression of the above enzymes and increased individual dNTP pools. Analysis of in vivo C-MYC interactions with TS, IMPDH2 and PRPS2 genes confirmed that they are direct C-MYC targets. Moreover, all three proteins express at higher levels in cells from several metastatic melanoma lines compared to normal melanocytes. Our data establish a novel functional link between C-MYC and dNTP metabolism and identify its role in proliferation of tumor cells.

Tumor cell-selective regulation of NOXA by c-MYC in response to proteasome inhibition.

The proteasome controls a plethora of survival factors in all mammalian cells analyzed to date. Therefore, it is puzzling that proteasome inhibitors such as bortezomib can display a preferential toxicity toward malignant cells. In fact, proteasome inhibitors have the salient feature of promoting a dramatic induction of the proapoptotic protein NOXA in a tumor cell-restricted manner. However, the molecular determinants that control this specific regulation of NOXA are unknown. Here, we show that the induction of NOXA by bortezomib is directly dependent on the oncogene c-MYC. This requirement for c-MYC was found in a variety of tumor cell types, in marked contrast with dispensable roles of p53, HIF-1alpha, and E2F-1 (classical proteasomal targets that can regulate NOXA mRNA under stress). Conserved MYC-binding sites identified at the NOXA promoter were validated by ChIP and reporter assays. Down-regulation of the endogenous levels of c-MYC abrogated the induction of NOXA in proteasome-defective tumor cells. Conversely, forced expression of c-MYC enabled normal cells to accumulate NOXA and subsequently activate cell death programs in response to proteasome blockage. c-MYC is itself a proteasomal target whose levels or function are invariably up-regulated during tumor progression. Our data provide an unexpected function of c-MYC in the control of the apoptotic machinery, and reveal a long sought-after oncogenic event conferring sensitivity to proteasome inhibition.

Delineation of the functional domains of the extracellular region of YWK-II Protein/APLP2 of sperm membrane.

The sperm membrane protein, designated as YWK-II protein/APLP2, is a member of the amyloid precursor protein (APP) superfamily and is a type I transmembrane protein involved in fertilization. Here, the structure-function of the domains of YWK-II protein was examined. Five segments with overlapping ends encompassing the entire extracellular region of mouse YWK-II gene were prepared, cloned and separately expressed in E. coli. The recombinant YWK-II segments were fused with glutathione S-transferase (GST), purified and evaluated for their antifertility activities by measuring their capacity to block in vitro mouse sperm-egg interaction. The structural domain(s) involved in the fertilization process was identified. The polypeptide segment corresponding to position 22-207 of YWK-II-763 inhibited the early stage of fertilization when the spermatozoa interacted with zona-free eggs; whereas the polypeptide segment 201-395 (lacking 309-364) of YWK-II-763 blocked sperm-egg membrane fusion. The remaining three segments, 201-395, 389-574 and 517-704 (lacking 613-624) of YWK-II-763, did not influence the in vitro fertilization process. The present results suggest that segment 22-308 of YWK-II-763 participates in the binding and fusion of sperm and egg plasma membranes thereby promoting fertilization.

Increased CCAAT enhancer-binding protein epsilon (C/EBPepsilon) expression and premature apoptosis in myeloid cells expressing Gfi-1 N382S mutant associated with severe congenital neutropenia.

Granulocyte-colony-stimulating factor (G-CSF) stimulates the activation of multiple signaling pathways, leading to alterations in the activities of transcription factors. Gfi-1 is a zinc finger transcriptional repressor that is required for granulopoiesis. How Gfi-1 acts in myeloid cells is poorly understood. We show here that the expression of Gfi-1 was up-regulated during G-CSF-induced granulocytic differentiation in myeloid 32D cells. Truncation of the carboxyl terminus of the G-CSF receptor, as seen in patients with acute myeloid leukemia evolving from severe congenital neutropenia, disrupted Gfi-1 up-regulation by G-CSF. Ectopic expression of a dominant negative Gfi-1 mutant, N382S, which was associated with severe congenital neutropenia, resulted in premature apoptosis and reduced proliferation of cells induced to differentiate with G-CSF. The expression of neutrophil elastase (NE) and CCAAT enhancer-binding protein epsilon (C/EBPepsilon) was significantly increased in 32D cells expressing N382S. In contrast, overexpression of wild type Gfi-1 abolished G-CSF-induced up-regulation of C/EBPepsilon but had no apparent effect on NE up-regulation by G-CSF. Notably, G-CSF-dependent proliferation and survival were inhibited upon overexpression of C/EBPepsilon but not NE. These data indicate that Gfi-1 down-regulates C/EBPepsilon expression and suggest that increased expression of C/EBPepsilon as a consequence of loss of Gfi-1 function may be deleterious to the proliferation and survival of early myeloid cells.

YWK-II protein/APLP2 in mouse gametes: potential role in fertilization.

YWK-II protein is a sperm membrane component, structurally related to human placenta amyloid precursor protein homolog (APPH) and rat amyloid precursor-like protein 2 (APLP2). Its transmembrane-cytoplasmic domain has high homology (70.6%) with that of betaA4-amyloid precursor protein (APP) found in brain plaques of subjects with Alzheimer's disease. The gene encoding the YWK-II protein is expressed in various mammalian cells and tissues. In the present study, splicing patterns of YWK-II mRNA and the content of YWK-II mRNA in mouse testes, eggs, and cumulus cells were determined. Three different YWK-II transcripts were found in testes and eggs, while cumulus cells contained an additional transcript. In mouse eggs, the content of YWK-II transcript exceeded that of APP. An alternative splicing region was located in the vicinity of the kunitz protease inhibitor (KPI) domain, which may be the basis for the formation of multiple transcripts. YWK-II protein was immunolocated in male and female gametes. It was localized in the plasma membrane of mouse eggs and spermatozoa. In the male reproductive system of the mouse, the YWK-II gene was expressed in germ cells at various stages of differentiation. In mature spermatozoa, the YWK-II protein occurred in the plasma membrane enveloping the acrosome. Triggering the acrosome reaction incited a release of the YWK-II protein attached to the liberated membrane vesicles. The occurrence of the YWK-II protein in the plasma membranes of mouse gametes suggests its involvement in sperm-egg interaction.

The G-CSF receptor carboxyl terminus, truncated in AML/SCN, is required for induction of a Stat5 protease activity.

Granulocyte colony-stimulating factor (G-CSF) has been shown to stimulate the activation of the signal transducer and activator of transcription 5 (Stat5). We show here that G-CSF-stimulated activation of Stat5 was attenuated when myeloid cells were induced to differentiate with G-CSF. Attenuated activation of Stat5 correlated with reduced Stat5 protein levels, which was associated with upregulation of a Stat5 protease activity. Carboxyl terminal truncation of the G-CSF receptor or expression of leukemogenic proteins Bcr-Abl and Tel-Jak2 abolished the upregulation of the Stat5 protease activity by G-CSF. These data add to our understanding of the roles of G-CSF and Stat5 in normal granulopoiesis and leukemogenesis.

Tyrosine 729 of the G-CSF receptor controls the duration of receptor signaling: involvement of SOCS3 and SOCS1.

Mutations in the granulocyte-colony stimulating factor receptor (G-CSF-R) gene resulting in carboxy terminal truncation have been associated with acute myeloid leukemia (AML). The truncated G-CSF-R from AML patients mediate enhanced and prolonged activation of signal transducer and activator of transcription 5 (Stat5). It has been shown that Src homology-2 (SH2)-containing tyrosine phosphatase-1 attenuates the intensity of G-CSF-induced Stat5 activation through interacting with the carboxy terminus of the G-CSF-R. Using a series of tyrosine-to-phenylalanine substitution mutants, we show here that tyrosine (Tyr) 729, located in the carboxy terminus of the G-CSF-R, controls the duration of G-CSF-stimulated activation of Stat5, Akt, and extracellular signal-regulated kinase 1/2. It is interesting that activation of these signaling molecules by G-CSF was prolonged by pretreating cells with actinomycin D or cyclohexamide, suggesting that de novo protein synthesis is required for appropriate termination of G-CSF-R signaling. The transcripts for suppressor of cytokine signaling 3 (SOCS3) and SOCS1 were up-regulated rapidly upon G-CSF stimulation. Expression of SOCS3 or SOCS1, but not SOCS2 and cytokine-inducible SH2 domain-containing protein, completely suppressed G-CSF-induced Stat5 activation but had only a weak effect on Stat5 activation mediated by the receptor mutant lacking Tyr 729. SOCS1 and SOCS3 also inhibited G-CSF-dependent cell proliferation, but the inhibitory effect of the two SOCS proteins on cell proliferation was diminished when Tyr 729 of the G-CSF-R was mutated. These data indicate that Tyr 729 of the G-CSF-R is required for SOCS1- and SOCS3-mediated negative regulation of G-CSF-R signaling and that the duration and intensity of G-CSF-induced Stat5 activation are regulated by two distinct mechanisms.