Transforming growth factor beta signaling is essential for the autonomous formation of cartilage-like tissue by expanded chondrocytes.

Cartilage is a tissue with limited self-healing potential. Hence, cartilage defects require surgical attention to prevent or postpone the development of osteoarthritis. For cell-based cartilage repair strategies, in particular autologous chondrocyte implantation, articular chondrocytes are isolated from cartilage and expanded in vitro to increase the number of cells required for therapy. During expansion, the cells lose the competence to autonomously form a cartilage-like tissue, that is in the absence of exogenously added chondrogenic growth factors, such as TGF-βs. We hypothesized that signaling elicited by autocrine and/or paracrine TGF-β is essential for the formation of cartilage-like tissue and that alterations within the TGF-β signaling pathway during expansion interfere with this process. Primary bovine articular chondrocytes were harvested and expanded in monolayer culture up to passage six and the formation of cartilage tissue was investigated in high density pellet cultures grown for three weeks. Chondrocytes expanded for up to three passages maintained the potential for autonomous cartilage-like tissue formation. After three passages, however, exogenous TGF-β1 was required to induce the formation of cartilage-like tissue. When TGF-β signaling was blocked by inhibiting the TGF-β receptor 1 kinase, the autonomous formation of cartilage-like tissue was abrogated. At the initiation of pellet culture, chondrocytes from passage three and later showed levels of transcripts coding for TGF-β receptors 1 and 2 and TGF-β2 to be three-, five- and five-fold decreased, respectively, as compared to primary chondrocytes. In conclusion, the autonomous formation of cartilage-like tissue by expanded chondrocytes is dependent on signaling induced by autocrine and/or paracrine TGF-β. We propose that a decrease in the expression of the chondrogenic growth factor TGF-β2 and of the TGF-β receptors in expanded chondrocytes accounts for a decrease in the activity of the TGF-β signaling pathway and hence for the loss of the potential for autonomous cartilage-like tissue formation.

Susceptibility versus resistance in alveolar echinococcosis (larval infection with Echinococcus multilocularis).

Epidemiological studies have demonstrated that the majority of human individuals exposed to infection with Echinococcus spp. eggs exhibit resistance to disease as shown by either seroconversion to parasite--specific antigens, and/or the presence of 'dying out' or 'aborted' metacestodes, not including hereby those individuals who putatively got infected but did not seroconvert and who subsequently allowed no development of the pathogen. For those individuals where infection leads to disease, the developing parasite is partially controlled by host immunity. In infected humans, the type of immune response developed by the host accounts for the subsequent trichotomy concerning the parasite development: (i) seroconversion proving infection, but lack of any hepatic lesion indicating the failure of the parasite to establish and further develop within the liver; or resistance as shown by the presence of fully calcified lesions; (ii) controlled susceptibility as found in the "conventional" alveolar echinococcosis (AE) patients who experience clinical signs and symptoms approximately 5-15 years after infection, and (iii) uncontrolled hyperproliferation of the metacestode due to an impaired immune response (AIDS or other immunodeficiencies). Immunomodulation of host immunity toward anergy seems to be triggered by parasite metabolites. Beside immunomodulating IL-10, TGFβ-driven regulatory T cells have been shown to play a crucial role in the parasite-modulated progressive course of AE. A novel CD4+CD25+ Treg effector molecule FGL2 recently yielded new insight into the tolerance process in Echinococcus multilocularis infection.

In vivo definition of genetic and cellular aspects of mammalian sexual differentiation

The differentiation of the reproductive organs is an essential developmental process required for the proper transmission of the genetic material. Müllerian inhibiting substance (MIS) is produced by testes and is necessary for the regression of the Müllerian ducts: the anlagen of the uterus, fallopian tubes and cervix. In vitro and standard transgenic mouse studies indicate that the nuclear hormone receptor Steroidogenic factor 1 (SF-1) and the transcription factor SOX9 play an essential role in the regulation of Mis. To test this hypothesis, mutations in the endogenous SF-1 and SOX9 binding sites in the mouse Mis promoter were introduced by gene targeting in embryonic stem (ES) cells. In disagreement with cell culture and transgenic mouse studies, male mice homozygous for the mutant SF-1 binding site correctly initiated Mis transcription in the fetal testes, although at significantly reduced levels. Surprisingly, sufficient Mis was produced for complete elimination of the Müllerian duct system. However, when the SF-1 binding site mutation was combined with an Mis -null allele, the further decrease in Mis levels led to a partial retention of uterine tissue, but only at a distance from the testes. In contrast, males homozygous for the mutant SOX9 binding site did not initiate Mis transcription, resulting in pseudohermaphrodites with a uterus and oviducts. These studies suggest an essential role for SOX9 in the initiation of Mis transcription, whereas SF-1 appears to act as a quantitative regulator of Mis transcript levels perhaps for influencing non-Müllerian duct tissues. ^ The Mis type II receptor, a member of the TGF- b superfamily, is also required for the proper regression of the Müllerian ducts. Mis type II receptor-deficient human males and their murine counterparts develop as pseudohermaphrodites. A lacZ reporter cassette was introduced into the mouse Mis type II receptor gene, by homologous recombination in ES cells. Expression studies, based on b -galactosidase activity, show marked expression of the MIS type II receptor in the postnatal Sertoli cells of the testis as well as in the prenatal and postnatal granulosa cells of the ovary. Expression is also seen in the mesenchymal cells surrounding the Müllerian duct and in the longitudinal muscle layer of the uterus. ^

The role of endogenous IFN-beta in cutaneous melanoma: Consequences of epidermal hyperplasia

The purpose of this study was to characterize epidermal hyperplasia overlying malignant melanoma, to determine the mitogenic factor responsible for the induction of this hyperplasia and to investigate its biological consequence. Whether increased keratinocyte proliferation overlying melanoma is due to production of growth factors by the tumor cells or to other mechanisms is unknown. Epidermal hyperplasia overlying human melanoma was found overlying thick (>4.0mm), but not thin (<1.0mm) tumors. Immunostaining of the sections for growth factors related to angiogenesis revealed that epidermal hyperplasia was associated with loss of IFN-β production by the keratinocytes directly overlying the tumors. Since previous studies from our laboratory have demonstrated that exogenous administration of IFN-β negatively regulates angiogenesis, we hypothesize that tumors are able to produce growth factors which stimulate the proliferation of cells in the surrounding tissues. This hyperplasia leads to a decrease in the endogenous negative regulator of angiogenesis, IFN-β. ^ The human melanoma cell line, DM-4 and several of its clones were studied to identify the mitogenic factor for keratinocytes. The expression of TGF-α directly correlated with epidermal hyperplasia in the DM-4 clones. A375SM, a human melanoma cell line that produces high levels of TGF-α, was transfected with a plasmid encoding full-length antisense TGF-α. The parental and transfected cells were implanted intradermally into nude mice. The extent of epidermal hyperplasia directly correlated with expression of TGF-α and decreased production of IFN-β, hence, increased angiogenesis. ^ In the next set of experiments, we determined the role of IFN-β on angiogenesis, tumor growth and metastasis of skin tumors. Transgenic mice containing a functional mutation in the receptor for IFN α/β were obtained. A375SM melanoma cells were implanted both s.c. and i.v. into IFN α/βR −/− mice. Tumors in the IFN α/β R −/− mice exhibited increased angiogenesis and metastasis. IFN α/βR −/− mice were exposed to chronic UV irradiation. Autochthonous tumors developed earlier in the transgenic mice than the wild-type mice. ^ Collectively, the data show that TGF-α produced by tumor cells induces proliferation of keratinocytes, leading to epidermal hyperplasia overlying malignant melanoma associated with loss of IFN-β and enhanced angiogenesis, tumorigenicity and metastasis. ^

Characterization of healing tissue in a tooth extraction socket in a rabbit model

The histology of healing in a tooth extraction socket has been described in many studies. The focus of research in bone biology and healing is now centered on molecular events that regulate repair of injured tissue. Rapid progress in cellular and molecular biology has resulted in identification of many signaling molecules (growth factors and cytokines) associated with formation and repair of skeletal tissues. Some of these include members of the transforming growth factor-β superfamily (including the bone morphogenetic proteins), fibroblast growth factors, platelet derived growth factors and insulin like growth factors. ^ Healing of a tooth extraction socket is a complex process involving tissue repair and regeneration. It involves chemotaxis of appropriate cells into the wound, transformation of undifferentiated mesenchymal cells to osteoprogenitor cells, proliferation and differentiation of committed bone forming cells, extracellular matrix synthesis, mineralization of osteoid, maturation and remodeling of bone. Current data suggests that these cellular events are precisely controlled and regulated by specific signaling molecules. A plethora of cytokines; have been identified and studied in the past two decades. Some of these like transforming growth factor beta (TGF-β), vascular endothelial growth factor (VEGF), platelet derived growth factor (PDGF) and fibroblast growth factors (FGFs) are well conserved proteins involved in the initial response to injury and repair in soft and hard tissue. ^ The purpose of this study was to characterize the spatial and temporal localization of TGF-βl, VEGF, PDGF-A, FGF-2 and BMP-2, and secretory IgA in a tooth extraction socket model, and evaluate correlation of spatial and temporal changes of these growth factors to histological events. The results of this study showed positive correlation of histological events to spatial and temporal localization of TGF-β1, BMP-2, FGF-2, PDGF-A, and VEGF in a rabbit tooth extraction model. ^

Tumor heterogeneity dictates sensitivity to gefitinib (Iressa(TM)/ZD1839) in solid tumor models

The epidermal growth factor receptor (EGFR) and its ligands are overexpressed in many human tumors, including bladder and pancreas, correlating with a more aggressive tumor phenotype and poor patient prognosis. We initiated the present study to characterize the heterogeneity of gefitinib responsiveness in a panel of human bladder and pancreatic cancer cell lines in order to identify the biological characteristics of EGFR-dependent proliferation that could be used to prospectively identify drug-sensitive tumors. A second objective was to elucidate how to best exploit these results by utilizing gefitinib in combination therapy. To these ends, we examined the effects of the EGFR antagonist gefitinib on proliferation and apoptosis in a panel of 18 human bladder cancer cell lines and 9 human pancreatic cancer cell lines. Our data confirmed the existence of marked heterogeneity in Iressa responsiveness with less than half of the cell lines displaying significant growth inhibition by clinically relevant concentrations of the drug. Gefitinib responsiveness was found to be p27 kip1 dependent as DNA synthesis was restored following exposure to p27siRNA. Unfortunately, Iressa responsiveness was not closely linked to surface EGFR or TGF-α expression in the bladder cancer cells, however, cellular TGF-α expression correlated directly with Iressa sensitivity in the pancreatic cancer cell lines. These findings provide the potential for prospectively identifying patients with drug-sensitive tumors. ^ Further studies aimed at exploiting gefitinib-mediated cell cycle effects led us to investigate if gefitinib-mediated TRAIL sensitization correlated with increased p27kip1 accumulation. We observed that increased TRAIL sensitivity following gefitinib exposure was not dependent on p27 kip1 expression. Additional studies initiated to examine the role(s) of Akt and Erk signaling demonstrated that exposure to PI3K or MEK inhibitors significantly enhanced TRAIL-induced apoptosis at concentrations that block target phosphorylation. Furthermore, combinations of TRAIL and the PI3K or MEK inhibitors increased procaspase-8 processing above levels observed with TRAIL alone, indicating that the effects were exerted at the level of caspase-8 activation, considered the earliest step in the TRAIL pathway. ^

The role of transforming growth factor beta signaling in cardiac hypertrophy and fibrosis in heart failure

Objective. To determine whether transforming growth factor beta (TGF-β) receptor blockade using an oral antagonist has an effect on cardiac myocyte size in the hearts of transgenic mice with a heart failure phenotype. ^ Methods. In this pilot experimental study, cardiac tissue sections from the hearts of transgenic mice overexpressing tumor necrosis factor (MHCsTNF mice) having a phenotype of heart failure and wild-type mice, treated with an orally available TGF-β receptor antagonist were stained with wheat germ agglutinin to delineate the myocyte cell membrane and imaged using fluorescence microscopy. Using MetaVue software, the cardiac myocyte circumference was traced and the cross sectional area (CSA) of individual myocytes were measured. Measurements were repeated at the epicardial, mid-myocardial and endocardial levels to ensure adequate sampling and to minimize the effect of regional variations in myocyte size. ANOVA testing with post-hoc pairwise comparisons was done to assess any difference between the drug-treated and diluent-treated groups. ^ Results. There were no statistically significant differences in the average myocyte CSA measured at the epicardial, mid-myocardial or endocardial levels between diluent treated littermate control mice, drug treated normal mice, diluent-treated transgenic mice and drug-treated transgenic mice. There was no difference between the average pan-myocardial cross sectional area between any of the four groups mentioned above. ^ Conclusions. TGF-β receptor blockade using oral TGF-β receptor antagonist does not alter myocyte size in MHCsTNF mice that have a phenotype of heart failure. ^

Characterization of tumor-associated Foxp3+ regulatory T cells and de-novo induction by the tumor microenvironment

Regulatory T cells expressing the fork-head box transcription factor 3 (Foxp3) play a central role in the dominant control of immunological tolerance. Compelling evidence obtained from both animal and clinical studies have now linked the expansion and accumulation of Foxp3+ regulatory T cells associated with tumor lesions to the failure of immune-mediated tumor rejection. However, further progress of the field is hampered by the gap of knowledge regarding their phenotypic, functional, and the developmental origins in which these tumor-associated Foxp3+ regulatory T cells are derived. Here, we have characterized the general properties of tumor-associated Foxp3+ regulatory T cells and addressed the issue of tumor microenvironment mediated de-novo induction by utilizing a well known murine tumor model MCA-205 in combination with our BAC Foxp3-GFP reporter mice and OT-II TCR transgenic mice on the RAG deficient background (RAG OT-II). De-novo induction defines a distinct mechanism of converting non-regulatory precursor cells to Foxp3+ regulatory T cells in the periphery as opposed to the expansion of pre-existing regulatory T cells formed naturally during thymic T cell development. This mechanism is of particularly importance to how tumors induce tumor-antigen-specific suppressor cells to subvert anti-tumor immune responses. Our study has found that tumor-associated Foxp3+ regulatory T cells are highly activated, undergo vigorous proliferation, are more potent by in-vitro suppression assays, and express higher levels of membrane-bound TGF-β1 than non-tumor regulatory T cells. With Foxp3-GFP reporter mice or RAG OT-II TCR transgenic mice, we show that tumor tissue can induce detectable de-novo generation of Foxp3+ regulatory T cells of both polyclonal or antigen specific naïve T cells. This process was not only limited for subcutaneous tumors but for lung tumors as well. Furthermore, this process required the inducing antigen to be co-localized within the tumor tissue. Examination of tumor tissue revealed an abundance of myeloid CD11b+ antigen-presenting cells that were capable of inducing Foxp3+ regulatory T cells. Taken together, these findings elucidate the general attributes and origins of tumor-associated Foxp3+ regulatory T cells in the tumor microenvironment and in their role in the negative regulation of tumor immunity.^

Mechanotransduction pathway activation in familial thoracic aortic aneurysms and dissections

Thoracic aortic aneurysms and dissections (TAAD) are autosomal dominantly inherited in 19% of patients. Mapping studies determined that the disease is genetically heterogeneous with multiple loci and genetic mutations accounting for familial TAAD. However, regardless of the specific mutation, resulting pathology is consistently medial degeneration, characterized by increased proteoglycans and loss of elastic fibers. We tested the hypothesis that genetic mutations leading to familial TAAD alter common pathways in aortic smooth muscle cells (SMCs). Identification of mutations at R460 in TGFBR2 reveals a 5% contribution to TAAD, however downstream analysis of Smad2 phosphorylation in the TGF-β pathway is not commonly altered in familial or sporadic disease when compared to controls. Expression profiling using Illumina's Sentrix HumanRef 8 Expression Beadchip array was done on RNA isolated from SMCs explanted from 6 patients with inherited TAAD with no identified mutation and 3 healthy controls obtained from the International Institute for the Advancement of Medicine. Significant increases in expression of proteoglycan genes in patients' SMCs, specifically lumican, podocan, and decorin were confirmed using Q-PCR and tissue immunofluorescence. NCI's Ingenuity Pathway Analysis predicted alterations in the ERK, insulin receptor and SAPK/JNK pathways (p<0.001), which SMCs activate in response to cyclic stretch. Immunoblotting indicated increased phosphorylation of ERK and GSK-3β, a protein from the insulin receptor pathway, in explanted patient SMCs, also confirmed by increased immunoreactivity against phosphorylated ERK and GSK-3β in the sub-intimal SMCs from patient tissue compared to controls. To determine if mechanotransduction pathway activation was responsible for the medial degeneration a specific inhibitor of GSK-3β, SB216763 was incubated with control cells and significantly increased the expression levels of proteoglycans. Mechanical strain was also applied to control SMCs confirming pathways stimulation with stretch. Incubation with pathway inhibitors against insulin receptor and ERK pathways identify, for the first time that stretch induced GSK-3β phosphorylation may increase proteoglycan expression, and ERK phosphorylation may regulate the expression of MMP2, a protein known to degrade elastic fibers. Furthermore, specific mutations in SMC-specific β-myosin heavy chain and α-actin, in addition to upregulation of pathways activated by cyclic stretch suggest that SMC response to hemodynamic factors, play a role in this disease. ^

Regulation of chromatin structure, Smad binding and AFP expression by the Forkhead box transcription factor: Foxa1

Transcription factors must be able to access their DNA binding sites to either activate or repress transcription. However, DNA wrapping and compaction into chromatin occludes most binding sites from ready access by proteins. Pioneer transcription factors are capable of binding their DNA elements within a condensed chromatin context and then reducing the level of nucleosome occupancy so that the chromatin structure is more accessible. This altered accessibility increases the probability of other transcription factors binding to their own DNA binding elements. My hypothesis is that Foxa1, a ‘pioneer’ transcription factor, activates alpha-fetoprotein (AFP) expression by binding DNA in a chromatinized environment, reducing the nucleosome occupancy and facilitating binding of additional transcription factors.^ Using retinoic-acid differentiated mouse embryonic stem cells, we illustrate a mechanism for activation of the tumor marker AFP by the pioneer transcription factor Foxa1 and TGF-β downstream effector transcription factors Smad2 and Smad4. In differentiating embryonic stem cells, binding of the Foxa1 forkhead box transcription factor to chromatin reduces nucleosome occupancy and levels of linker histone H1 at the AFP distal promoter. The more accessible DNA is subsequently bound by the Smad2 and Smad4 transcription factors, concurrent with activation of transcription. Chromatin immunoprecipitation analyses combined with siRNA-mediated knockdown indicate that Smad protein binding and the reduction of nucleosome occupancy at the AFP distal promoter is dependent on Foxa1. In addition to facilitating transcription factor binding, Foxa1 is also associated with histone modifications related to active gene expression. Acetylation of lysine 9 on histone H3, a mark that is associated active transcription, is dependent on Foxa1, while methylation of H3K4, also associated with active transcription, is independent of Foxa1. I propose that Foxa1 potentiates a region of chromatin to respond to Smad proteins, leading to active expression of AFP.^ These studies demonstrate one mechanism whereby a transcription factor can alter the accessibility of additional transcription factors to chromatin, by altering nucleosome positions. Specifically, Foxa1 exposes DNA so that Smad4 can bind to its regulatory element and activate transcription of the tumor-marker gene AFP.^

S100A4 is a molecular mediator of endometrial carcinoma invasion

The molecular mechanisms of endometrail cancer invasion are poorly understood. S100A4, a member of the S100 Ca2+-binding protein family, was identified by oligonucleotide microarray qRT-PCR, and IHC, to be highly overexpressed in invasive endometrial carcinomas compared to non-invasive tumors. HEC-1A endometrial cancer cells transfected with S100A4 siRNA had undetectable S100A4 protein, decreased migration and invasion. The mechanism of S100A4 upregulation in endometrial cancer remains unclear. Methylation of the S100A4 gene was detected in benign endometrial glands and grade 1 tumors with no S100A4 expression. In contrast, grade 3 endometrioid tumors with high S100A4 expression showed no methylation of the gene. 5-Aza-2'-deoxycytidine, an inhibitor of DNA methyltransferase, induced the expression of S100A4 in the less invasive EC cell line, KLE, in which the S100A4 gene is hypermethylated and minimally expressed. S100A4 was induced during TGF-β1-triggered cell scattering in HEC-1A cells, in which S100A4 was demethylated. Transfection of HEC-1A cells with S100A4 siRNA significantly reduced the effect of TGF-β1 on basal migration and invasion. Our preliminary data suggested that this upregulation was mediated by the transcription factor Snail. One Snail binding consensus site was found in the region where DNA methylation was closely correlated with S100A4 gene expression. Chromatin immunoprecipitation assay confirmed the binding of Snail to this consensus site in HEC-1A cells. In SPEC2 endometrial cancer cells, loss of Snail leads to repressed S100A4 gene expression. Similar to S100A4, Snail was overexpressed in aggressive endometrial tumors. Our study suggested that the S100A4 gene was demethylated and further upregulated by the TGF-β1 and Snail pathway in invasive endometrial cancer. S100A4 could potentially serve as a good molecular marker for invasiveness and a target for therapeutic intervention for advanced endometrial cancer. ^

Breast cancer risk with aromatase inhibition and phytoestrogen-free diet

In this thesis a mouse model was used to examine the effect of pubertal estrogen inhibition and a phytoestrogen-free diet on the development of mammary glands. The study question was does treatment with aromatase inhibitor during puberty increase susceptibility to breast cancer among cohorts that consumed a diet free of phytoestrogens. The study design consisted of a cohort of mice treated with aromatase inhibitor, letrozole, during puberty and a vehicular group that was used as a control. Both groups were fed a diet free of phytoestrogens from the time of weaning until sacrifice during adulthood. The study aimed to assess mammary gland development in terms of breast cancer risk. The methods employed in this research included morphological and histological analysis of mammary glands, as well as estradiol, RNA and protein analysis. The main finding of the study was that mice exposed to aromatase inhibitor during puberty developed mammary glands with specific characteristics suggestive of vulnerability to oncogenesis such as increased lateral branching, increased number of glands, increase ductal hyperplasia, and diminished expression of TGFβ and p27 protein levels. The conclusions suggest that puberty is a critical period in which the mammary gland is susceptible to environmental threats that may result in deleterious epigenetic effects leading to an increased breast cancer risk in adulthood. This study has several public health implications; the most significant is that environmental threats during puberty may result in adverse mammary gland development and that phytoestrogen sources in the diet are necessary for normal maturation of the mammary glands.^

CHARACTERIZATION OF DIFFERENTIATION AND PROGNOSTIC BIOMARKERS ON CD8+ TUMOR-INFILTRATING LYMPHOCYTES IN METASTATIC MELANOMA

CD8+ cytotoxic T lymphocytes (CTL) frequently infiltrate tumors, yet most melanoma patients fail to undergo tumor regression. We studied the differentiation of the CD8+ tumor-infiltrating lymphocytes (TIL) from 44 metastatic melanoma patients using known T-cell differentiation markers. We also compared CD8+ TIL against the T cells from matched melanoma patients’ peripheral blood. We discovered a novel subset of CD8+ TIL co-expressing early-differentiation markers, CD27, CD28, and a late/senescent CTL differentiation marker, CD57. This CD8+CD57+ TIL expressed a cytolytic enzyme, granzyme B (GB), yet did not express another cytolytic pore-forming molecule, perforin (Perf). In contrast, the CD8+CD57+ T cells in the periphery were CD27-CD28-, and GBHi and PerfHi. We found this TIL subset was not senescent and could be induced to proliferate and differentiate into CD27-CD57+, perforinHi, mature CTL. This further differentiation was arrested by TGF-β1, an immunosuppressive cytokine known to be produced by many different kinds of tumors. Therefore, we have identified a novel subset of incompletely differentiated CD8+ TIL that resembled those found in patients with uncontrolled chronic viral infections. In a related study, we explored prognostic biomarkers in metastatic melanoma patients treated in a Phase II Adoptive Cell Therapy (ACT) trial, in which autologous TIL were expanded ex vivo with IL-2 and infused into lymphodepleted patients. We unexpectedly found a significant positive clinical association with the infused CD8+ TIL expressing B- and T- lymphocyte attentuator (BTLA), an inhibitory T-cell receptor. We found that CD8+BTLA+ TIL had a superior proliferative response to IL-2, and were more capable of autocrine IL-2 production in response to TCR stimulation compared to the CD8+BTLA- TIL. The CD8+BTLA+ TIL were less differentiated and resembled the incompletely differentiated CD8+ TIL described above. In contrast, CD8+BTLA- TIL were poorly proliferative, expressed CD45RA and killer-cell immunoglobulin-like receptors (KIRs), and exhibited a gene expression signature of T cell deletion. Surprisingly, ligation of BTLA by its cognate receptor, HVEM, enhanced the survival of CD8+BTLA+ TIL by activating Akt/PKB. Our studies provide a comprehensive characterization of CD8+ TIL differentiation in melanoma, and revealed BTLA as a novel T-cell differentiation marker along with its role in promoting T cell survival.

Inflammatory Breast Cancer: The Immune Perspective

Inflammatory breast cancer (IBC) is the most insidious form of locally advanced disease. Although rare and less than 2% of all breast cancer, IBC is responsible for up to 10% of all breast cancer deaths. Despite the name, very little is known about the role of inflammation or immune mediators in IBC. Therefore, we analyzed blood samples from IBC patients and non-IBC patients, as well as healthy donor controls to establish an IBC-specific profile of peripheral blood leukocyte phenotype and function of T cells and dendritic cells and serum inflammatory cytokines. Emerging evidence suggests that host factors in the microenviromement may interact with underlying IBC genetics to promote the aggressive nature of the tumor. An integral part of the metastatic process involves epithelial to mesenchymal transition (EMT) where primary breast cancer cells gain motility and stem cell-like features that allow distant seeding. Interestingly, the IBC consortium microarray data found no clear evidence for EMT in IBC tumor tissues. It is becoming increasingly evident that inflammatory factors can induce EMT. However, it is unknown if EMT-inducing soluble factors secreted by activated immune cells in the IBC microenvironment canπ account for the absence of EMT in studies of the tumor cells themselves. We hypothesized that soluble factors from immune cells are capable of inducing EMT in IBC. We tested the ability of immune conditioned media to induce EMT in IBC cells. We found that soluble factors from activated immune cells are able to induce the expression of EMT-related factors in IBC cells along with increased migration and invasion. Specifically, the pro-inflammatory cytokines TNF-α, IL-6 and TGF-β were able to induce EMT and blocking these factors in conditioned media abated the induction of EMT. Surprisingly, unique to IBC cells, this process was related to increased levels of E-cadherin expression and adhesion, reminiscent of the characteristic tightly packed tumor emboli seen in IBC samples. This data offers insight into the unique pathology of IBC by suggesting that tumor immune interactions in the tumor microenvironment contribute to the aggressive nature of IBC implying that immune induced inflammation can be a novel therapeutic target. Specifically, we showed that soluble factors secreted by activated immune cells are capable of inducing EMT in IBC cells and may mediate the persistent E-cadherin expression observed in IBC. This data suggests that immune mediated inflammation may contribute to the highly aggressive nature of IBC and represents a potential therapeutic target that warrants further investigation.

Regulation of myogenesis by growth signals -growth factors, oncogenes and protein kinases

Establishment of a myogenic phenotype involves antagonism between cell proliferation and differentiation. The recent identification of the MyoD family of muscle-specific transcription factors provides opportunities to dissect at the molecular level the mechanisms through which defined cell type-specific transcription factors respond to environmental cues and regulate differentiation programs. This project is aimed at elucidation of the molecular mechanism whereby growth factors repress myogenesis. Initial studies demonstrated that nuclear oncogenes such as c-fos, junB and c-jun are immediate early genes that respond to serum and TGF-$\beta$. Using the muscle creatine kinase (MCK) enhancer linked to the reporter gene CAT as a marker for differentiation, we showed that transcriptional function of myogenin can be disrupted in the presence of c-Fos, JunB and cjun. In contrast, JunD, which shares DNA-binding specificity with JunB and c-Jun but is expressed constitutively in muscle cells, failed to show the inhibition. The repression by Fos and Jun is targeted at KE-2 motif, the same sequence that mediates myogenin-dependent activation and muscle-specific transactivation. Deletion analysis indicated that the transactivation domain of c-Jun at the N-terminus is responsible for the repression. Considering that myogenin is a phosphoprotein and cAMP and TPA are able to regulate myogenesis, we examined whether constitutively active protein kinase C (PKC) and protein kinase A (PKA) could substitute for exogenous growth factors and prevent transcription activation by myogenin. Indeed, the basic region of myogenin is phosphorylated by PKC at a threonine that is conserved in all members of the MyoD family. Phosphorylation at this site attenuates DNA binding activity of myogenin. Protein kinase A can also phosphorylate myogenin in a region adjacent to the DNA binding domain. However, phosphorylation at this site is insufficient to abrogate myogenin's DNA binding capacity, suggesting that PKA and PKC may affect myogenin transcriptional activity through different mechanisms. These findings provide insight into the mechanisms through which growth factor signals negatively regulate the muscle differentiation program and contribute to an understanding of signal transducing pathways between the cell membrane and nucleus. ^