Delineating the mechanism(s) of BDNF/TrkB mediated proliferation in Neuroblastoma

Delineating the mechanism(s) of BDNF/TrkB mediated proliferation in Neuroblastoma Timothy Christopher Graham, B.S. Supervisory Professor: Patrick Zweidler-McKay, MD/PhD Neuroblastoma is the most common extra-cranial solid tumor in children, arising from neural crest precursor cells. The neurotrophin receptors (TrkA/B/C) have been implicated as important prognostic markers, linking the biology of the tumor to patient outcome. High expression of TrkA and TrkC receptors have been linked to favorable biological features and high patient survival, while TrkB is expressed in unfavorable, aggressive tumors. Several studies suggest that high levels and activation of TrkB by its ligand brain-derived neurotrophic factor (BDNF) stimulates tumor cell survival, proliferation, and chemoresistance. However, little is known about the molecular mechanisms that regulate proliferation. The TrkB signaling pathway in neuroblastoma cells has been difficult to evaluate due to the loss of TrkB expression when the cells are used in vitro. Here we determined the role of proximal signaling pathways downstream of TrkB on neuroblastoma proliferation. By analyzing a panel of neuroblastoma cell lines, we found that the SMS-KCN cells express detectable levels of protein and mRNA levels of TrkB as analyzed by western, RT-PCR, and surface expression by flow cytometry. By the addition of exogenous human recombinant BDNF, we showed that activation of TrkB is important in the proliferation of the cells and can be repressed by inhibiting TrkB kinase function. By BDNF stimulation and use of specific kinase inhibitors, the common pathways involving PLCg, PI3K/AKT, and MAPK were initially investigated in addition to PI3K/MTOR and FYN pathways. We demonstrate for the first time that Fyn plays a critical role in TrkB mediated proliferation in neuroblastoma. Constitutively active and over-expressed Fyn reduced neuroblastoma proliferation, as measured by PCNA expression. Knockdown of Fyn by shRNA was shown to cooperate with activated TrkB for an enhanced proliferative response. Although TrkB activation has been implicated in the proliferation of neuroblastoma cells, little is known about its effects on cell cycle regulation. Protein levels of pRB, CDK2, CDK4, CDC25A, cyclin D1, and cyclin E were analyzed following BDNF stimulation. We found that BDNF mediated activation of TrkB induces multiple common proximal signaling pathways including the anti-proliferative Fyn pathway and drives cell cycle machinery to enhance the proliferation of neuroblastoma cells.

Enhanced estrogen-induced proliferation in obese rat endometrium.

OBJECTIVE: We tested the hypothesis that the proliferative estrogen effect on the endometrium is enhanced in obese vs lean animals. STUDY DESIGN: Using Zucker fa/fa obese rats and lean control, we examined endometrial cell proliferation and the expression patterns of certain estrogen-regulated proproliferative and antiproliferative genes after short-term treatment with estradiol. RESULTS: No significant morphologic/histologic difference was seen between the obese rats and the lean rats. Estrogen-induced proproliferative genes cyclin A and c-Myc messenger RNA expression were significantly higher in the endometrium of obese rats compared with those of the lean control. Expression of the antiproliferative gene p27Kip1 was suppressed by estrogen treatment in both obese and lean rats; however, the decrease was more pronounced in obese rats. Estrogen more strongly induced the antiproliferative genes retinaldehyde dehydrogenases 2 and secreted frizzled-related protein 4 in lean rats but had little or no effect in obese rats. CONCLUSION: Enhancement of estrogen-induced endometrial proproliferative gene expression and suppression of antiproliferative gene expression was seen in the endometrium of obese vs lean animals.

Suppression of peripheral blood mononuclear cell proliferation by a periodontopathic bacteria {\it Actinobacillus actinomycetemcomitans\/}

Actinobacillus actinomycetemcomitans (Aa) is a gram-negative coccobacillus implicated as a major pathogen in juvenile periodontitis. The immunosuppressive activity of a sonic extract (designated 100SN) derived from Aa was investigated. 100SN suppressed spontaneous proliferation as well as proliferative response to the mitogens, PHA and PWM, of human peripheral blood mononuclear cells (PBMC). 100SN-induced suppression of PHA-stimulated proliferation was heat-sensitive, inactivated by pronase and trypsin, dose-dependent and non-cytotoxic. There were no significant changes in the CD4$\sp+$ or CD8$\sp+$ subsets of PBMC after 7-day incubation with 100SN. There was a trend toward increased levels of the CD4$\sp+$CD45R$\sp{\rm hi}$CDw29$\sp{\rm lo}$ (naive cells, associated with suppressor-inducer activity) and CD4$\sp+$CDw29$\sp{\rm hi}$CD45R$\sp{\rm lo}$ (memory cells, associated with helper-inducer activity) subsets. The target of 100SN appeared to be the non-adherent cells and suppression by 100SN could not be reversed by indomethacin (IDM), the cyclo-oxygenase inhibitor of prostaglandin (PG) synthesis. The mechanism of 100SN-induced suppression was studied in terms of inhibition involving IL-2-regulated T cell proliferation and the results point to the possibility that suppression occurred subsequent to IL-2 receptor binding.^ The suppressive activity observed could occur through multiple mechanisms including cell-cell; contact or release of soluble factors. Supernatants derived from 7-day cultures of PBMC and 100SN (designated CSN-A) were able to suppress proliferative response of PBMC to PHA without affecting cell viability. Analysis of CSN-A showed that it contained PGE2 and soluble IL-2 receptors. Suppression by CSN-A could be partially overcome by either IDM or exogenous IL-2. Significant suppression was also maintained when both IDM and exogenous IL-2 were added at the same time. These findings suggest that PGE2 and soluble IL-2 receptors contribute to the suppression observed but other suppressive cytokine(s) may be involved. Collectively, the data indicate that a factor derived from oral bacteria associated with juvenile periodontitis have profound effects on cellular immune responses, and that these effects may be partially mediated by secondary factors produced by the host in response to the bacteria. ^

A study of factors regulating the autonomous proliferation of the steroid hormone-induced LJ6195 murine reproductive tract tumor

Neonatal estrogen treatment of BALB/c mice results in the unregulated proliferation of the cervicovaginal epithelium and eventually tumorigenesis. The conversion of the normally estrogen responsive cyclic proliferation of the vaginal epithelium to a continuous estrogen-independent pattern of growth is a complex phenomenon. The aim of this study was to gain an understanding of the mechanism(s) by which steroid hormone administration during a critical period of development alters the cyclic proliferation of vaginal epithelium, ultimately leading to carcinogenesis in the adult animal.^ The LJ6195 murine cervicovaginal tumor was induced by treating newborn female BALB/c mice with 20 $\mu$g 17$\beta$-estradiol plus 100 $\mu$g progesterone for the first 5 days after birth. In contrast to proliferation of the normal vaginal epithelium, proliferation of LJ6195 is not regulated by estradiol. Northern blot analysis of RNA from vaginal tracts of normal mice, neonatal-estrogen treated mice, and LJ6195 indicate that there is an alteration in the expression of several genes such as the estrogen receptor, c-fos, and HER2/neu. In response to neonatal estrogen treatment, the estrogen receptor is down regulated in the murine vaginal tract. Therefore, the estrogen-independent nature of this tissue is established as early as 3 months after treatment. There is strong evidence that the proliferation of LJ6195 is regulated through an autocrine growth pathway. The LJ6195 tumor expresses mRNA for the epidermal growth factor receptor. In addition, conditioned medium from the LJ6195 tumor cell line contains a growth factor(s) with epidermal growth factor-like activity. Conditioned medium from the LJ6195 cell line stimulated the proliferation of the EGF-dependent COMMA D mouse mammary gland cell line in a dose-dependent manner. The addition of an anti-mEGF-antibody to LJ6195 cell cultures significantly decreased growth. These results suggest that the EGF-receptor mediated growth pathway may play a role in regulating the estrogen-independent proliferation of the LJ6195 tumor. ^

Mechanism of dendritic epidermal T cell-mediated tolerance induction and inhibition of proliferation

Dendritic epidermal T cells (DETC) comprise a unique population of T cells that reside in mouse epidermis and whose function remains unclear. Most DETC express a $\gamma\delta$ TCR, although some, including our DETC line, AU16, express an $\alpha\beta$ TCR. Additionally, AU16 cells express CD3, Thy-1, CD45, CD28, B7, and AsGM-1. Previous studies in our laboratory demonstrated that hapten-conjugated AU16 could induce specific immunologic tolerance in vivo and inhibit T cell proliferation in vitro. Both these activities are antigen-specific, and the induction of tolerance is non-MHC-restricted. In addition, AU16 cells are cytotoxic to a number of tumor cell lines in vitro. These studies suggested a role for these cells in immune surveillance. The purpose of my studies was to test the hypothesis that these functions of DETC (tolerance induction, inhibition of T cell proliferation, and tumor cell killing) were mediated by a cytotoxic mechanism. My specific aims were (1) to determine whether AU16 could prevent or delay tumor growth in vivo; and (2) to determine the mechanism whereby AU16 induce tolerance, using an in vitro proliferation assay. I first showed that AU16 cells killed a variety of skin tumor cell lines in vitro. I then demonstrated that they prevented melanoma growth in C3H mice when both cell types were mixed immediately prior to intradermal (i.d.) injection. Studies using the in vitro proliferation assay confirmed that DETC inhibit proliferation of T cells stimulated by hapten-bearing, antigen-presenting cells (FITC-APC). To determine which cell was the target, $\gamma$-irradiated, hapten-conjugated AU16 were added to the proliferation assay on d 4. They profoundly inhibited the proliferation of naive T cells to $\gamma$-irradiated, FITC-APC, as measured by ($\sp3$H) TdR uptake. This result strongly suggested that the T cell was the target of the AU16 activity because no APC were present by d 4 of the in vitro culture. In contrast, the addition of FITC-conjugated splenic T cells (SP-T) or lymph node T cells (LN-T) was less inhibitory. Preincubation of the T cells with FITC-AU16 cells for 24 h, followed by removal of the AU16 cells, completely inhibited the ability of the T cells to proliferate in response to FITC-APC, further supporting the conclusion that the T cell was the target of the AU16. Finally, AU16 cells were capable of killing a variety of activated T cells and T cell lines, arguing that the mechanism of proliferation inhibition, and possibly tolerance induction is one of cytotoxicity. Importantly, $\gamma\delta$ TCR$\sp+$ DETC behaved, both in vivo and in vitro like AU16, whereas other T cells did not. Therefore, these results are consistent with the hypothesis that AU16 cells are true DETC and that they induce tolerance by killing T cells that are antigen-activated in vivo. ^

Mechanisms of liposomal all-{\it trans\/} retinoic acid and vitamin D3 induced inhibition of cell proliferation and stimulation of apoptosis in aggressive B-cell non-Hodgkin's lymphoma

The Non-Hodgkin's Lymphoma (NHLs) are neoplasms of the immune system. Currently, less than 1% of the etiology of the 22,000 newly diagnosed lymphoma cases in the U.S.A. every year is known. This disease has a significant prevalence and high mortality rate. Cell growth in lymphomas has been shown to be an important parameter in aggressive NHL when establishing prognosis, as well as an integral part in the pathophysiology of the disease process. While many aggressive B cell NHLs respond initially to chemotherapeutic regimens such as CHOP-bleo (adriamycin, vincristine and bleomycin) etc., relapse is common, and the patient is then often refractory to further salvage treatment regimens.^ To assess their potential to inhibit aggressive B cell NHLs and induce apoptosis (also referred to as programmed cell death (PCD)), it was proposed to utilize the following biological agents-liposomal all-trans retinoic acid (L-ATRA) which is a derivative of Vitamin A in liposomes and Vitamin D3. Preliminary evidence indicates that L-ATRA may inhibit cell growth in these cells and may induce PCD as well. Detailed studies were performed to understand the above phenomena by L-ATRA and Vitamin D3 in recently established NHL-B cell lines and primary cell cultures. The gene regulation involved in the case of L-ATRA was also delineated. ^

Effects of galectin-1 and galectin-3 expression on prostate cancer cell adhesion, differentiation, proliferation, and tumorigenicity

The vertebrate $\beta$-galactoside-binding lectins galectin-1 and galectin-3 have been proposed to function in diverse cellular processes such as adhesion, proliferation, differentiation, and tumorigenesis. Experiments were initiated to further study the functional properties of these molecules. A prostate cancer cell line, LNCaP, was identified which expressed neither galectin. This line was stably transfected with cDNA for either galectin-1 or galectin-3. The resultant clones were used to study effects on critical cell processes. LNCaP cells expressing galectin-1 on the surface were found to bind more rapidly than control lines to the human extracellular matrix proteins laminin and fibronectin, although overall binding was not increased. To analyze effects on differentiation, LNCaP cells were studied which had either been transfected with galectin-1 or which had been induced to express endogenous galectin-1 by treatment with the differentiation agent sodium butyrate. In both cases, cells displayed a slower rate of growth and increased rate of apoptosis. A transient decrease in expression of prostate specific antigen was seen in the butyrate treated cells but not in the transfected cells. To investigate the role of galectins in the process of malignant transformation and progression, immunohistochemical analysis was performed on formalin-fixed, paraffin-embedded sections of human prostate tissue, the premalignant lesion prostatic intraepithelial neoplasia, primary adenocarcinoma of the prostate, and foci of metastatic prostate cancer. Galectin-1 expression was relatively constant throughout in contrast to galectin-3 which demonstrated significantly less expression in primary and metastatic tumors. LNCaP cells transfected with galectin-3 cDNA displayed lower proliferation rates, increased spontaneous apoptosis, and G1 growth phase arrest compared to controls. Four of six galectin-3 lines tested were less tumorigenic in nude mice than controls. The following conclusions are drawn regarding the role of galectin-1 and galectin-3 expression in the context of prostate cancer: (1) galectin-1 may participate in the early stages of cancer cell adhesion to extracellular matrix proteins; (2) galectin-1 expression results in a differentiated phenotype and may contribute to differentiation induction by butyrate; (3) galectin-3 expression correlates inversely with prostate cell tumorigenesis and prostate cancer metastasis. ^

PHENOTYPIC CHARACTERIZATION OF SPONTANEOUS AND IN VITRO-MAINTAINED AKR LYMPHOMAS

T-cell lymphomas from AKR mice were studied to determine their potential as a model of T-cell differentiation. Homogeneous tumor cell lines have been used as model to study normal lymphocyte subpopulations, including differentiation lineages, functional properties, and the inducibility to maturation. The underlying concept is that each lymphoid tumor represents a monoclonal neoplastic proliferation of a discrete lymphoid subpopulation arrested at a particular differentiation stage.^ Individual tumors were analyzed to determine the extent of intertumor heterogeneity, and to determine whether lymphomas represented different thymocyte subsets, by determining the cell-surface antigenic phenotype, PNA-binding capacity, and terminal deoxynucleotidyl transferase (TdT) activity. Splenic and thymic tumor cells were compared to determine if the particular lymphoid microenvironment influenced T-cell marker expression. Several of the lymphomas were passaged in syngeneic hosts to verify the original tumor phenotype and to assess the stability of the cell surface and TdT phenotype after transplantation.^ Lymphomas were adapted to in vitro culture to determine whether the T-cell phenotype was maintained in the absence of the host microenvironment. Clonal progeny were analyzed and compared with each other and with parent cell lines to determine the extent of intratumor heterogeneity in this lymphoma system. Parent and cloned cell lines were passaged in vivo to determine whether alterations in surface phenotype occurred after transplantation.^ Our investigation has verified that most spontaneous AKR lymphomas phenotypically resemble known T-cell subsets, including both immature and mature thymic subpopulations. The in vitro lines, however, expressed a highly unstable phenotype in culture that included loss of Ly-1 and Ly-2 antigen expression. After transplantation in vivo, the in vitro lines exhibited alterations in phenotype, including re-expression of Ly antigen on some lymphomas. The inducibility of T-cell antigen markers on tumor cell lines passaged in vivo suggests that the in vitro lines may serve as a possible model system to study the molecular events involved in gene expression in the T-cell system. ^

TH and XFKBP12 interact and regulate proliferation and differentiation in neural ectoderm through the TOR signaling pathway

During development, embryos must carefully integrate the processes of cell proliferation and differentiation. TH has been identified in Xenopus laevis as a gene product that functions in regulating differentiation of the neural ectoderm through its effect on cell proliferation. However, the mechanism and molecular pathway through which TH functions are not known. We identified the Xenopus FK506 binding protein homolog (XFKBP12) as a protein that interacted with TH in a yeast two-hybrid screen with TH as the bait. The direct and specific interaction between TH and XFKBP12 was supported by several tests including CO-IP, drug competence assay and mutagenesis analysis. To investigate the function of XFKBP12 during embryogenesis, we created an XFKBP12 loss of function embryo using antisense morpholino oligonucleotides (MO). XFKBP12 MO injected embryos displayed similar phenotypes as TH depleted embryos. We also demonstrated that both TH and XFKBP12 functioned through the TOR signaling pathway which is a target for cancer therapies. The interaction between TH and XFKBP 12 was required to regulate the proliferation of neural cells. Therefore, our study indicates that TH represents the endogenous ligand of XFKBP12 and together they coordinate neural cell proliferation and differentiation through the conserved rapamycin sensitive TOR pathway. Thus, understanding how this pathway functions in development will not only provide us important insights into the relationship between proliferation and differentiation, but help design rational cancer therapies targeting this pathway. ^

Role of the cyclin dependent kinase-4 in normal and neoplastic proliferation

In the last few years, our laboratory has studied the regulatory mechanisms of proliferation and differentiation in epidermal tissues. Our results showed differences in the roles of cyclin dependent-kinases 4 and 6, and the three D-type cyclins, during normal epidermal proliferation and neoplastic development. Thus, to elucidate the role of the different cell cycle regulators, we developed transgenic mice that overexpress CDK4 (K5-CDK4), or their cognate D-type cyclins, in epithelial tissues. The most severe phenotype was observed in K5-CDK4 animals that developed dermal fibrosis, epidermal hyperplasia and hypertrophy. Forced expression of CDK4 in the epidermal basal cell layer increased the malignant conversion of skin papillomas to squamous cell carcinomas (SCC). Contrastingly, lack of CDK4 completely inhibited tumor development, suggesting that CDK4 is required in this process. Biochemical studies demonstrated that p21 Cip1 and p27Kip1 inhibitors are sequestered by CDK4 resulting in indirect activation of Cyclin E/CDK2, implicating the non-catalytic activity of CDK4 in deregulation of the cell cycle progression. ^ It has been proposed that the proliferative and oncogenic role of Myc is linked to its ability to induce the transcription of CDK4, cyclin D1, and cyclin D2 in vitro. Deregulation of Myc oncogene has been found in several human cancers. Also it has been demonstrated that CDK4 has the ability to functionally inactivate the product of the tumor suppressor gene Rb, providing a link between Myc and the CDK4/cyclin D1/pRb/p16 pathway in some malignant tumors. Here, we sought to determine the role of CDK4 as a mediator of Myc activities by developing a Myc overexpressing mouse nullizygous for CDK4. We demonstrated that lack of CDK4 results in reduced keratinocyte proliferation and epidermal thickness in K5-Myc/CDK4-null mice. In addition, complete reversion of tumor development was observed. All together, this work demonstrates that CDK4 acts as an oncogene independent of the D-type cyclin levels and it is an important mediator of the tumorigenesis induced by Myc. In addition, we showed that the sequestering activity of CDK4 is critical for the development of epidermal hyperplasia during normal proliferation, malignant progression from papillomas to squamous cell carcinomas, and tumorigenesis induced by Myc. ^

B cells in ADA deficiency

Deficiency of the enzyme adenosine deaminase (ADA) results in severe lymphopenia in humans. Mice with an inactivating mutation in the ADA gene also exhibit profound lymphopenia, as well as pulmonary insufficiency and ribcage abnormalities. In fact, the mouse model has a phenotype that is remarkably similar to that of the human disease, making the mice valuable tools for unraveling the mechanism of lymphocyte destruction in absence of this housekeeping gene. T cell deficiency in ADA deficiency has been extensively studied by others, revealing a block in early thymocyte development. In contrast, our studies revealed that early B cell development in the bone marrow is normal. ADA-deficient mice, however, exhibit profound defects in germinal center formation, preventing antigen-dependent B cell maturation in the spleen. ADA-deficient spleen B cells display significant defects in proliferation and activation signaling, and produce more IgM than their normal counterparts, suggesting that extrafollicular plasmablasts are overrepresented. B cells from ADA-deficient mouse spleens undergo apoptosis more readily than those from normal mouse spleens. Levels of ADA's substrates, adenosine and 2′-deoxyadenosine, are elevated in both bone marrow and spleen in ADA-deficient mice. S ′-adenosyihomoeysteine hydrolase (SAH hydrolase) activity is significantly inhibited in both locales, as well. dATP levels, though, are only elevated in spleen, where B cell development is impaired, and not in bone marrow, where B cell ontogeny is normal. This finding points to dATP as the causative agent of lymphocyte death in ADA deficiency. ADA deficiency results in inhibition of the enzyme ribonucleotide reductase, thereby depleting nucleoside pools needed for DNA repair. Another mouse model that lacks a functional gene encoding a protein involved in DNA repair and/or cell cycle checkpoint regulation, p53-binding protein 1, exhibits blocks in T and B cell development that are similar to those seen in ADA-deficient mice. Unraveling the mechanisms of lymphocyte destruction in ADA deficiency may further understanding of lymphocyte biology, facilitate better chemotherapeutic treatment for lymphoproliferative diseases, and improve gene and enzyme therapy regimens attempted for ADA deficiency. ^

Ontogeny and regulation of interleukin-7 expressing thymic epithelial cells

T cell development is a multistage process of differentiation that depends on proper thymocyte-thymic epithelial cell (TEC) interactions. Epithelial cells in the thymus are organized in a three-dimensional network that provides support and signals for thymocyte maturation. Concurrently, proper TEC differentiation in the adult thymus relies on thymocyte-derived signals. TECs produce interleukin-7 (IL-7), a non-redundant cytokine that promotes the survival, differentiation, and proliferation of thymocytes. We have identified IL-7 expressing TECs throughout ontogeny and in the adult thymus by in situ hybridization analysis. IL-7 expression is initiated in the thymic fated domain of the thymic primordium by embryonic day 11.5, in a Foxn1 independent pathway. Marked changes occur in the localization and regulation of IL-7 expressing TECs during development. Whereas IL-7 expressing TECs are present throughout the early thymic rudiment, the majority of IL-7 producing TECs are concentrated in the adult thymic medulla. By analyzing mouse strains that sustain blocks at different stages of thymocyte development, we show that IL-7 expression is initiated independently of hematopoietic-derived signals during thymic organogenesis. However, thymocyte-derived signals play an essential role in regulating IL-7 expression in the adult TEC compartment. Furthermore, distinct thymocyte subsets regulate the expression of IL-7 and keratin 5 in adult cortical epithelium. Intraperitoneal injection of Recombination Activating Gene deficient mice (RAG-2−/−) with anti-CD3ϵ monoclonal antibody (mAb) induces CD4− 8− double negative thymocytes to undergo β-selection and differentiate into CD4+8+ cells. Analysis of the thymic stromal compartment reveals that progression through β-selection renders thymocytes competent to alter the pattern of IL-7 expression in the cortical TEC compartment. RAG-2−/− mice do not generate mature T cells and therefore the RAG-2−/− thymus is devoid of organized medullary regions. Histological examination of RAG-2−/− thymus following anti-CD3ϵ stimulation reveals the emergence of mature thymic medullary regions, as assessed by H & E staining and expression of thymic stromal medullary markers. Stromal medullary reorganization occurs in the absence of T cell receptor αβ expression, suggesting that activation of RAG-2−/− thymocytes by CD3ϵ ligation generates thymocyte-derived signals that induce thymic epithelial reorganization, generating a mature medullary compartment. This model provides a tool to assess the mechanisms underlying thymic medullary development. ^