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Stem cells are undifferentiated biological cells that can differentiate into all lineages under defined control condition. Stem cell neuronal differentiation can faithfully recapitulate stages of neural development and generate neuronal progenitors, mature neurons, and glial cells. Stem cell technology will largely allow for the replacement of animal studies and reduce costs, and will provide a new paradigm for in toxic genomics, bioinformatics, systems biology, and epigenetics studies. Here, we describe a nonadherent neuronal differentiation methodology developed in our laboratory, which can rapidly derive neurons and astrocytes from human embryonic stem cells (hESCs) and induced pluripotent stem cell (hiPSC) and use of this platform for nanoparticle neurotoxicity study.

Stem cells are undifferentiated biological cells that can differentiate into all lineages under defined control condition. Stem cell neuronal differentiation can faithfully recapitulate stages of neural development and generate neuronal progenitors, mature neurons, and glial cells. Stem cell technology will largely allow for the replacement of animal studies and reduce costs, and will provide a new paradigm for in toxic genomics, bioinformatics, systems biology, and epigenetics studies. Here, we describe a nonadherent neuronal differentiation methodology developed in our laboratory, which can rapidly derive neurons and astrocytes from human embryonic stem cells (hESCs) and induced pluripotent stem cell (hiPSC) and use of this platform for nanoparticle neurotoxicity study.

The chemotherapeutic agent temozolomide (TMZ) kills tumor cells preferentially via alkylation of the O6-position of guanine. However, cells that express the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT), or harbor deficient DNA mismatch repair (MMR) function, are profoundly resistant to this drug. TMZ is in clinical use for melanoma, but objective response rates are low, even when TMZ is combined with O6-benzylguanine (O6BG), a potent MGMT inhibitor. We used in vitro and in vivo models of melanoma to characterize the early events leading to cellular TMZ resistance. Melanoma cell lines were exposed to a single treatment with TMZ, at physiologically relevant concentrations, in the absence or presence of O6BG. Surviving clones and mass cultures were analyzed by Western blot, colony formation assays, and DNA methylation studies. Mice with melanoma xenografts received TMZ treatment, and tumor tissue was analyzed by immunohistochemistry. We found that MGMT-negative melanoma cell cultures, before any drug treatment, already harbored a small fraction of MGMT-positive cells, which survived TMZ treatment and promptly became the dominant cell type within the surviving population. The MGMT-negative status in individual cells was not stable, as clonal selection of MGMT-negative cells again resulted in a mixed population harboring MGMT-positive, TMZ-resistant cells. Blocking the survival advantage of MGMT via the addition of O6BG still resulted in surviving clones, although at much lower frequency and independent of MGMT, and the resistance mechanism of these clones was based on a common lack of expression of MSH6, a key MMR enzyme. TMZ treatment of mice implanted with MGMT-negative melanoma cells resulted in effective tumor growth delay, but eventually tumor growth resumed, with tumor tissue having become MGMT positive. Altogether, these data reveal stochastic expression of MGMT as a pre-existing, key determinant of TMZ resistance in melanoma cell lines. Although MGMT activity can effectively be eliminated by pharmacologic intervention with O6BG, additional layers of TMZ resistance, although considerably rarer, are present as well and minimize the cytotoxic impact of TMZ/O6BG combination treatment. Our results provide rational explanations regarding clinical observations, where the TMZ/O6BG regimen has yielded mostly disappointing outcomes in melanoma patients.

Despite the introduction of new therapies for multiple myeloma (MM), many patients are still dying from this disease and novel treatments are urgently needed. We have designed a novel hybrid molecule, called NEO214, that was generated by covalent conjugation of the natural monoterpene perillyl alcohol (POH), an inducer of endoplasmic reticulum (ER) stress, to rolipram (Rp), an inhibitor of phosphodiesterase-4 (PDE4). Its potential anticancer effects were investigated in a panel of MM cell lines. We found that NEO214 effectively killed MM cells in vitro with a potency that was over an order of magnitude stronger than that of its individual components, either alone or in combination. The cytotoxic mechanism of NEO214 involved severe ER stress and prolonged induction of CCAAT/enhancer-binding protein homologous protein (CHOP), a key pro-apoptotic component of the ER stress response. These effects were prevented by salubrinal, a pharmacologic inhibitor of ER stress, and by CHOP gene knockout. Conversely, combination of NEO214 with bortezomib, a drug in clinical use for patients with MM, resulted in synergistic enhancement of MM cell death. Combination with the adenylate cyclase stimulant forskolin did not enhance NEO214 impact, indicating that cyclic adenosine 3′,5′-monophosphate (AMP) pathways might play a lesser role. Our study introduces the novel agent NEO214 as a potent inducer of ER stress with significant anti-MM activity in vitro. It should be further investigated as a potential MM therapy aimed at exploiting this tumor’s distinct sensitivity to ER stress.

Many estrogen receptor alpha (ERα)-positive breast cancers initially respond to aromatase inhibitors (AIs), but eventually acquire resistance. Here, we report that serum- and glucocorticoid-inducible kinase 3 (SGK3), a kinase transcriptionally regulated by ERα in breast cancer, sustains ERα signaling and drives acquired AI resistance. SGK3 is up-regulated and essential for endoplasmic reticulum (EnR) homeostasis through preserving sarcoplasmic/EnR calcium ATPase 2b (SERCA2b) function in AI-resistant cells. We have further found that EnR stress response down-regulates ERα expression through the protein kinase RNA-like EnR kinase (PERK) arm, and SGK3 retains ERα expression and signaling by preventing excessive EnR stress. Our study reveals regulation of ERα expression mediated by the EnR stress response and the feed-forward regulation between SGK3 and ERα in breast cancer. Given SGK3 inhibition reduces AI-resistant cell survival by eliciting excessive EnR stress and also depletes ERα expression/function, we propose SGK3 inhibition as a potential effective treatment of acquired AI-resistant breast cancer.

IκB kinase (IKK) complex is a key regulator of NF-κB pathways. Signal-induced interaction of the IKKγ (NEMO) subunit with the C-terminal IKKγ/NEMO-binding domain (γBD) of IKKβ is an essential interaction for IKK regulation. Underlying regulatory mechanism(s) of this interaction are not known. Phosphorylation of γBD has been suggested to play a regulatory role for IKK activation. However, a kinase that phosphorylates γBD has not been identified. In this study, we used a C-terminal fragment of IKKβ as substrate and purified Polo-like kinase 1 (Plk1) from HeLa cell extracts by standard chromatography as a γBD kinase. Plk1 phosphorylates serines 733, 740, and 750 in the γBD of IKKβ in vitro. Phosphorylating γBD with Plk1 decreased its affinity for IKKγ in pulldown assay. We generated phosphoantibodies against serine 740 and showed that γBD is phosphorylated in vivo. Expressing a constitutively active Plk1 in mammalian cells reduced tumor necrosis factor (TNF)-induced IKK activation, resulting in decreased phosphorylation of endogenous IκBα and reduced NF-κB activation. To activate endogenous Plk1, cells were treated with nocodazole, which reduced TNF-induced IKK activation, and increased the phosphorylation of γBD. Knocking down Plk1 in mammalian cells restored TNF-induced IKK activation in nocodazole-treated cells. Activation of Plk1 inhibited TNF-induced expression of cyclin D1. In cells in which Plk1 was knocked down, TNFα increased expression of cyclin D1 and the proportion of cells in the S phase of the cell cycle. Taken together, this study shows that phosphorylation regulates the interaction of γBD of IKKβ with IKKγ and therefore plays a critical role for IKK activation. Moreover, we identify Plk1 as a γBD kinase, which negatively regulates TNF-induced IKK activation and cyclin D1 expression, thereby affecting cell cycle regulation. Untimely activation of cyclin D1 by TNFα can provide a potential mechanism for an involvement of TNFα in inflammation-induced cancer.

The research was to appraise the utility of the patient-derived tumor xenografts (PDXs) as models of estrogen receptor positive (ER+HER2- and ER+HER2+) breast cancers. We compared protein expression profiles by Reverse Phase Protein Array (RPPA) in tumors that resulted in PDXs compared to those that did not. Our overall PDX intake rate for ER+ breast cancer was 9% (9/97). The intake rate for ER+HER2+ tumors (3/16, 19%) was higher than for ER+HER2- tumors (6/81, 7%). Heat map analyses of RPPA data showed that ER+HER2- tumors were divided into 2 groups by luminal A/B signature [protein expression of ER, AR, Bcl-2, Bim (BCL2L11), GATA3 and INPP4b], and this expression signature was also associated with the rate of PDX intake. Cell survival pathways such as the PI3K/AKT signaling and RAS/ERK pathways were more activated in the specimens that could be established as PDX in both classes. Expression of the ER protein itself may have a bearing on the potential success of an ER+ PDX model. In addition, HER2 and its downstream protein expressions were up-regulated in the ER+HER2+ patient tumors that were successfully established as PDX models. Moreover, the comparison of RPPA data between original and PDX tumors suggested that the selection/adaptation process required to grow the tumors in mice is unavoidable for generation of ER+ PDX models, and we identified differences between patient tumor samples and paired PDX tumors. A better understanding of the biological characteristics of ER+PDX would be the key to using PDX models in assessing treatment strategies in a preclinical setting.

Choroidal neovascularization (CNV) is a blinding complication of age-related macular degeneration that manifests as the growth of immature choroidal blood vessels through Bruch’s membrane, where they can leak fluid or hemorrhage under the retina. Here, we demonstrate that the histone deacetylase inhibitor (HDACi) trichostatin A (TSA) can down-regulate the pro-angiogenic hypoxia-inducible factor-1α and vascular endothelial growth factor (VEGF), and up-regulate the anti-angiogenic and neuro-protective pigment epithelium derived factor in human retinal pigment epithelial (RPE) cells. Most strikingly, TSA markedly down-regulates the expression of VEGF receptor-2 in human vascular endothelial cells and, thus, can knock down pro-angiogenic cell signaling. Additionally, TSA suppresses CNV-associated wound healing response and RPE epithelial-mesenchymal transdifferentiation. In the laser-induced model of CNV using C57Bl/6 mice, systemic administration of TSA significantly reduces fluorescein leakage and the size of CNV lesions at post—laser days 7 and 14 as well as the immunohistochemical expression of VEGF, VEGFR2, and smooth muscle actin in CNV lesions at post-laser day 7. This report suggests that TSA, and possibly HDACi’s in general, should be further evaluated for their therapeutic potential for the treatment of CNV.

Epigenetics has become an increasingly important area of biomedical research. Increasing evidence shows that epigenetic alterations influence common pathologic responses including inflammation, ischemia, neoplasia, aging, and neurodegeneration. Importantly, epigenetic mechanisms may have a pathogenic role in many complex eye diseases such as corneal dystrophy, cataract, glaucoma, diabetic retinopathy, ocular neoplasia, uveitis, and age-related macular degeneration. The emerging emphasis on epigenetic mechanisms in studies of eye disease may provide new insights into the pathogenesis of complex eye diseases and aid in the development of novel treatments for these diseases.

IkappaB kinase (IKK) complex is a key regulator of NF-kappaB pathways. Signal-induced interaction of the IKKgamma (NEMO) subunit with the C-terminal IKKgamma/NEMO-binding domain (gammaBD) of IKKbeta is an essential interaction for IKK regulation. Underlying regulatory mechanism(s) of this interaction are not known. Phosphorylation of gammaBD has been suggested to play a regulatory role for IKK activation. However, a kinase that phosphorylates gammaBD has not been identified. In this study, we used a C-terminal fragment of IKKbeta as substrate and purified Polo-like kinase 1 (Plk1) from HeLa cell extracts by standard chromatography as a gammaBD kinase. Plk1 phosphorylates serines 733, 740, and 750 in the gammaBD of IKKbeta in vitro. Phosphorylating gammaBD with Plk1 decreased its affinity for IKKgamma in pulldown assay. We generated phosphoantibodies against serine 740 and showed that gammaBD is phosphorylated in vivo. Expressing a constitutively active Plk1 in mammalian cells reduced tumor necrosis factor (TNF)-induced IKK activation, resulting in decreased phosphorylation of endogenous IkappaBalpha and reduced NF-kappaB activation. To activate endogenous Plk1, cells were treated with nocodazole, which reduced TNF-induced IKK activation, and increased the phosphorylation of gammaBD. Knocking down Plk1 in mammalian cells restored TNF-induced IKK activation in nocodazole-treated cells. Activation of Plk1 inhibited TNF-induced expression of cyclin D1. In cells in which Plk1 was knocked down, TNFalpha increased expression of cyclin D1 and the proportion of cells in the S phase of the cell cycle. Taken together, this study shows that phosphorylation regulates the interaction of gammaBD of IKKbeta with IKKgamma and therefore plays a critical role for IKK activation. Moreover, we identify Plk1 as a gammaBD kinase, which negatively regulates TNF-induced IKK activation and cyclin D1 expression, thereby affecting cell cycle regulation. Untimely activation of cyclin D1 by TNFalpha can provide a potential mechanism for an involvement of TNFalpha in inflammation-induced cancer.

Background and Purpose: Despite recent progress in our endocrine therapy of hormone receptor positive (HR+) breast cancers, a significant number of patients with primary breast cancer continue to relapse, and those with stage IV disease face a median overall survival of ∼ 3.5 years. Primary or acquired resistance to anti-estrogen-based therapies is an overarching challenge. To guide our treatment selection, there is an essential need to improve our understanding of the biology of HR+ breast tumors responsive to and those resist to anti-estrogens or aromatase inhibitors (AIs). The application of patient-derived xenografts (PDXs) in preclinical studies has begun to open the door to mimicking human disease on the research bench. However, HR+ breast cancer PDXs are difficult to establish. Although preclinical data from DeRose et al [Nat. Med. 2011: 17:1514-1520] indicate that the rate of engraftment serves as an independent predictor for poor outcome, the question which has not yet been adequately addressed is: "why some tumors can grow in mice, and some don't, even when their clinical, pathological stage and subtype (i.e. ER positivity) are same?" Here, we hypothesize that the molecular characteristics of patient HR+ tumors are key determinants to the tumor intake rate in NOD/SCID/interleukin-2 receptor gamma chain null (NSG) mice. Hence, reverse phase protein array (RPPA) analysis has be performed using human patient tumors to identify driver-pathways that impact tumor intake in NSG mice. Results and Discussion: We compared the protein expression profile of six HR+ patient tumors (four HR+ and two HR+ HER2+), which were successfully engrafted into NSG mice and established as PDX models, with the patient tumors which we were unable to establish as PDX. Of 90 patient HR+ tumors which failed to transplant, 21 tumors were picked to match the tumor type (all of them were invasive ductal carcinoma or its metastases), clinical stage and pathological grade of engrafted tumors [Table 1]. In addition to patient tumors, six established HR+ PDXs were also submitted for analysis. Quantified expressions of 272 cancer-related proteins and phospho-proteins by RPPA have been performed on these specimens. Pathways identified as predictors of intake rate of PDXs in NSG mice, and tissues from paired PDX from mice with different passages, will be evaluated for the protein expression changes to elucidate the passage effects and generate therapeutic models based on protein expression and tumor growth. Table 1. Characteristics of the patient tumors which were successfully established as PDX modelsERPgRHER2AgePatient ethnicityClinical stageNottingham histologic scoreSource++-63Hispanic3IIIBreast tumor+--71Hispanic2IIIBreast tumor+--52African-american4N/ABrain mets+--63Caucasian4N/AChest wall mets+-+34Caucasian2IIBreast tumor+++72Caucasian4IIIChest wall metsmets: metastases Citation Format: Kanaya N, Somlo G, Wu J, Frankel P, Wu SV, Nguyen D, Kai M, Chan N, Meng-Yin H, Kirschenbaum M, Kruper L, Vito C, Yuan Y, Hurria A, Mortimer J, Chen S. Identification of molecular pathways to define the intake rate of patient-derived hormone receptor positive (HR+) breast cancer xenografts (PDXs) in NOD/SCID/interleukin-2 receptor gamma chain null (NSG) mice. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-03-02.

Inhibition of DNA replication in an Escherichia coli dnaB-22 mutant failed to block quinolone-mediated lethality. Inhibition of protein synthesis by chloramphenicol inhibited nalidixic acid lethality and, to a lesser extent, ciprofloxacin lethality in both dnaB-22 and wild-type cells. Thus, major features of quinolone-mediated lethality do not depend on ongoing replication.