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. ^

Activation of dendritic cells from patients with epithelial ovarian cancer

Dendritic cells (DCs) are the most potent antigen-presenting cells for inducing immune responses to tumor cells. Lin−HLA-DR + DC populations in peripheral blood mononuclear cells (PBMCs) and in ascites mononuclear leukocytes (MNLs) of patients with epithelial ovarian cancer (EOC) are phenotypically immature. Lin−HLA-DR + DCs from PBMCs of normal subjects and EOC patients and MNLs from ascites cells of patients were examined for specific cell surface markers or indicators of differentiation or activation. Separating Lin− HLA-DR+ DCs into subsets based on their HLA-DR intensity provided an additional method for identifying the two major lineages of DCs, myeloid and plasmacytoid. The activation potential of these DCs following exposure to the maturation agents CD40 ligand (CD40L) and lipopolysaccharide (LPS) was examined by measurement of IL-12 and IL-10 concentrations in DC culture supernatants in addition to their ability to stimulate allogeneic T cells. DCs from PBMCs of normal subjects and EOC patients and DCs isolated from ascites MNLs of EOC patients were separated into subsets based on CD11c and CD123 cell surface marker expression identifying the major DC types. These subsets were then compared with cells sorted on the basis of HLA-DR intensity. The in vivo behavior of DCs and DC subsets in peripheral blood and ascites following treatment of peritoneal carcinoma patients with the growth factor fins-like tyrosine kinase 3 ligand (Flt3L) was also examined. Increases in proportions and total numbers of DCs from peripheral blood and ascites were associated with increased secretion of IL-12 and IL-10 following in vitro activation of cultured DCs. There were differences between DCs from PBMCs and ascites and between DC subsets in expression of cell surface markers, cytokine profile, and the ability of Lin−HLA-DR + cells to stimulate proliferation of allogeneic T cells from EOC patients. These Lin−HLA-DR+ cells have certain functional properties that suggest that they could have the potential to facilitate an adaptive anti-tumor immune response. ^

The differential staurosporine mediated G1 arrest in normal versus tumor cells is dependent on the retinoblastoma and Chk1 proteins

The ability to regulate cell cycle progression is one of the differences that separates normal from tumor cells. A protein, which is frequently mutated or deleted in a majority of tumor cells, is the retinoblastoma protein (pRb). Previously, we reported that normal cells, which have a wild-type Rb pathway, can be reversibly arrested in the G1 phase of the cell cycle by staurosporine (ST), while tumor cells were unaffected by this treatment. As a result, ST may be used to protect normal cells against the toxic affects of chemotherapy. Here we set out to determine the mechanism(s) by which ST can mediate a reversible G1 arrest in pRb positive cells. To this end, we used an isogenic cell model system of normal human mammary epithelial cells (HMEC) with either intact pRb+ (p53-) or p53+ (pRb-) treated with ST. Our results show that pRb+ cells treated with low concentrations of ST, arrested in the G1 phase of the cell cycle; however, in pRb - cells there was no response. This was verified as a true G 1 arrest in pRb+ cells by two different methods for monitoring cell cycle kinetics and in two additional model systems for Rb (i.e. pRb -/- mouse embryo fibroblasts, and downregulation of RB with siRNA). Our results indicated that ST-mediated G1 arrest required pRb, which in turn initiated a cascade of events leading to inhibition of CDK4 and CDK2 activities and up-regulation of p21 protein. Further assessment of this pathway revealed the novel finding that Chk1 expression and activity were required for the Rb-dependent, ST-mediated G1 arrest. In fact, overexpression of Chk1 facilitated recovery from ST-mediated G1 arrest, an effect only observed in RB+ cells. Collectively, our data suggest pRb is able to cooperate with Chk1 to mediate a G1 arrest in pRb+ cells, but not in pRb- cells. The elucidation of this pathway can help identify novel agents that can be used to protect cancer patients against the debilitating affects of chemotherapy, by targeting only the normal proliferating cells in the body that are otherwise destroyed. ^

14-3-3 zeta overexpression in early stage breast disease and tumor progression

Recent progress in diagnostic tools allows many breast cancers to be detected at an early pre-invasive stage. Thus, a better understanding of the molecular basis of early breast cancer progression is essential. 14-3-3 is a family of highly conserved and ubiquitously expressed proteins that are expressed in all eukaryotic organisms. In mammals there are seven isoforms, which bind to phosphor-serine/threonine residues regulating essential cellular processes such as signal transduction, cell cycle progression, and apoptosis. Our laboratory has discovered that a particular 14-3-3 family member, Zeta, is overexpressed in over 40% of breast tumor tissues. Furthermore, I examined the stage of breast disease in which 14-3-3ζ overexpression occurs and found that increased expression of 14-3-3ζ begins at the stage of atypical ductal hyperplasia, a very early stage of breast disease that confers increased risk for progress toward breast cancer. To determine whether 14-3-3ζ overexpression is a decisive early event in breast cancer, I overexpressed 14-3-3ζ in MCF10A cells, a non-transformed mammary epithelial cell (MEC) line and examined its impact on acini formation in a three dimensional (3D) culture model which simulates a basic unit of structure in the mammary gland. I discovered that 14-3-3ζ overexpression severely disrupted the acini architecture resulting in the disruption of polarity and luminal filling. Both are critical morphological events in the pre-neoplastic breast disease. This thesis focuses on the molecular mechanism of luminal filling. Proper lumen formation is a result of anoikis, a specific type apoptosis of cells not attached to the basement membrane. I found that 14-3-3ζ overexpression conferred a resistance to anoikis. Additionally, 14-3-3ζ overexpression in MCF10A cells and in MECs from 14-3-3ζ transgenic mice reduced expression of p53, which is known to mediate anoikis. Mechanistically, 14-3-3ζ induced hyperactivation of the PI3K/Akt pathway which led to phosphorylation and translocation of the MDM2 to the nucleus resulting in increased p53 degradation. Ectopic expression of p53 restored luminal apoptosis in 14-3-3ζ overexpressing MCF10A acini in 3D cultures. These data suggest that 14-3-3ζ overexpression is a critical event in early breast disease and down-regulation of p53 is one of the mechanisms by which 14-3-3ζ alters MEC acini structure and may increase the risk of progression to breast cancer. ^

Molecular mechanisms of response in gastrointestinal stromal tumors treated with imatinib mesylate

Gastrointestinal stromal tumors (GIST) represent 80% of sarcoma arising from the GI tract. The inciting event in tumor progression is mutation of the kit or, rarely, platelet derived growth factor receptor-α (PDGFR) gene. These mutations encode ligand independent, constitutively active proteins: Kit or PDGFR. ^ These tumors are notoriously chemo and radio resistant. Historically, patients with advanced disease realized a median overall survival of 9 months. However, with modern management of GIST with imatinib mesylate (Novartis), a small molecule inhibitor of the Kit, PDGFR, and Abl tyrosine kinases, patients now realize a median overall survival greater than 30 months. However, almost half of patients present with surgically resectable GIST and the utility of imatinib in this context has not been prospectively studied. Also, therapeutic benefit of imatinib is variable from patient to patient and alternative targeted therapy is emerging as potential alternatives to imatinib. Thus, elucidating prognostic factors for patients with GIST in the imatinib-era is crucial to providing optimal care to each particular patient. Moreover, the exact mechanism of action of imatinib in GIST is not fully understood. Therefore, physicians find difficulty in accurately predicting which patient will benefit from imatinib, how to assess response to therapy, and the time at which to assess response. ^ I have hypothesized that imatinib is tolerable and clinically beneficial in the context of surgery, VEGF expression and kit non-exon 11 genotypes portend poor survival on imatinib therapy, and imatinib's mechanism of action is in part due to anti-vascular effects and inhibition of the Kit/SCF signaling axis of tumor-associated endothelial cells. ^ Results herein demonstrate that imatinib is safe and increases the duration of disease-free survival when combined with surgery. Radiographic and molecular (namely, apoptosis) changes occur within 3 days of imatinib initiation. I illustrate that non-exon 11 mutant genotypes and VEGF are poor prognostic factors for patients treated with imatinib. These findings may allow for patient stratification to emerging therapies rather than imatinib. I show that imatinib has anti-vascular effects via inducing tumor endothelial cell apoptosis perhaps by abrogation of the Kit/SCF signaling axis. ^

SFRP4 regulation of Wnt7a signaling and cellular proliferation in the endometrium

In the endometrium, hormonal effects on epithelial cells are often elicited through stromal hormone receptors via unknown paracrine mechanisms. Several lines of evidence support the hypothesis that Wnts participate in stromal-epithelial cell communication and thus mediate hormone action. Characterization of specific Wnt signaling components in the endometrium was performed using cellular localization studies and evaluating hormone effects in a rat model. Wnt7a was expressed in the luminal epithelium, whereas the extracellular Wnt modulator, SFRP4, was localized to the endometrial stroma. SFRP4 expression is significantly decreased in endometrial carcinoma and aberrant Wnt7a signaling has been shown to cause uterine defects and contribute to the onset of disease. The specific Fzds and SFRPs that bind Wnt7a and the particular signal transduction pathway each Wnt7a-Fzd pair activates have not been identified. Additionally, the function of Wnt7a and SFRP4 in the endometrium has not been addressed. A survey of all Wnt signaling proteins expressed in the endometrium was conducted and Fzd5 and Fzd10 were identified as two receptors capable of transducing the Wnt7a signal. Biologically active recombinant Wnt7a and SFRP4 proteins were purified for quantitative biochemical studies. In Ishikawa cells, Wnt7a binding to Fzd5 activated β-catenin/canonical Wnt signaling and increased cellular proliferation. Wnt7a signaling mediated by Fzd10 induced a non-canonical/JNK-responsive pathway. SFRP4 suppressed Wnt7a action in both an autocrine and paracrine manner. Treatment with SFRP4 protein and overexpression of SFRP4 inhibited endometrial cancer cell growth and induced apoptosis in vitro. A split-eGFP complementation assay was developed to visually detect Wnt7a-Fzd interactions and subsequent pathway activation in cells. By employing a unique ELISA-based protein-protein binding technique, it was demonstrated that Wnt7a binds to SFRP4 and Fzd5 with equal nanomolar affinity. The development of these novel biological tools could lead to a better understanding of Wnt-protein interactions and the identification of new modulators of Wnt signaling. This study supports a mechanism by which the nature of the Wnt7a signal in the endometrium is dependent upon the Fzd repertoire of the cell and can be regulated by SFRP4. The potential tumor suppressor function of SFRP4 suggests it may serve as a therapeutic target for endometrial carcinoma. ^

Elucidating the role of mesenchymal stem cell participation in the tumor microenvironment

To meet the requirements for rapid tumor growth, a complex array of non-neoplastic vascular, fibroblastic, and immune cells are recruited to the tumor microenvironment. Understanding the origin, composition, and mechanism(s) for recruitment of these stromal components will help identify areas for therapeutic intervention. Previous findings have suggested that ex-vivo expanded bone marrow-derived MSC home to the sites of tumor development, responding to inflammatory signals and can serve as effective drug delivery vehicles. Therefore, we first sought to fully assess conditions under which MSC migrate to and incorporate into inflammatory microenvironments and the consequences of modulated inflammation. MSC delivered to animals bearing inflammatory insults were monitored by bioluminescence imaging and displayed specific tropism and selective incorporation into all tumor and wound sites. These findings were consistent across routes of tumor establishment, MSC administration, and immunocompetence. MSC were then used as drug delivery vehicles, transporting Interferon β to sites of pancreatic tumors. This therapy was effective at inhibiting pancreatic tumor growth under homeostatic conditions, but inhibition was lost when inflammation was decreased with CDDO-Me combination treatment. Next, to examine the endogenous tumor microenvironment, a series of tissue transplant experiments were carried out in which tissues were genetically labeled and engrafted in recipients prior to tumor establishment. Tumors were then analyzed for markers of tumor associated fibroblasts (TAF): α-smooth muscle actin (α-SMA), nerve glia antigen 2 (NG2), fibroblast activation protein (FAP), and fibroblast specific protein (FSP) as well as endothelial marker CD31 and macrophage marker F4/80. We determined the majority of α-SMA+, NG2+ and CD31+ cells were non-bone marrow derived, while most FAP+, FSP+, and F4/80+ cells were recruited from the bone marrow. In accord, transplants of prospectively isolated BM MSC prior to tumor development indicated that these cells were recruited to the tumor microenvironment and co-expressed FAP and FSP. In contrast, fat transplant experiments revealed recruited fat derived cells co-expressed α-SMA, NG2, and CD31. These results indicate TAF are a heterogeneous population composed of subpopulations with distinct tissues of origin. These models have provided a platform upon which further investigation into tumor microenvironment composition and tests for candidate drugs can be performed. ^

Transcriptional regulation of lung tumor suppressor GPRC5A in malignant and normal cells

Chronic exposure of the airways to cigarette smoke induces inflammatory response and genomic instability that play important roles in lung cancer development. Nuclear factor kappa B (NF-κB), the major intracellular mediator of inflammatory signals, is frequently activated in preneoplastic and malignant lung lesions. ^ Previously, we had shown that a lung tumor suppressor GPRC5A is frequently repressed in human non-small cell lung cancers (NSCLC) cells and lung tumor specimens. Recently, other groups have shown that human GPRC5A transcript levels are higher in bronchial samples of former than of current smokers. These results suggested that smoking represses GPRC5A expression and thus promotes the occurrence of lung cancer. We hypothesized that cigarette smoking or associated inflammatory response repressed GPRC5A expression through NF-κB signaling. ^ To determine the effect of inflammation, we examined GPRC5A protein expression in several lung cell lines following by TNF-α treatment. TNF-α significantly suppressed GPRC5A expression in normal small airway epithelial cells (SAEC) as well as in Calu-1 cells. Real-time PCR analysis indicated that TNF-α inhibits GPRC5A expression at the transcriptional level. NF-κB, the major downstream effectors of TNF-α signaling, mediates TNF-α-induced repression of GPRC5A because over-expression of NF-κB suppressed GPRC5A. To determine the region in the GPRC5A promoter through which NF-κB acts, we examined the ability of TNF-α to inhibit a series of reporter constructs with different deletions of GPRC5A promoter. The luciferase assay showed that the potential NF-κB binding sites containing region are irresponsible for TNF-α-induced suppression. Further analysis using constructs with different deletions in p65 revealed that NF-κB-mediated repression of GPRC5A is transcription-independent. Co-immunoprecipitation assays revealed that NF-κB could form a complex with RAR/RXR heterodimer. Moreover, the inhibitory effect of NF-κB has been found to be proportional to NF-κB/RAR ratio in luciferase assay. Finally, Chromatin IP demonstrated that NF-κB/p65 bound to GPRC5A promoter as well as RAR/RXR and suppressed transcription. Taken together, we propose that inflammation-induced NF-κB activation disrupts the RA signaling and suppresses GPRC5A expression and thus contributes to the oncogenesis of lung cancer. Our studies shed new light on the pathogenesis of lung cancer and potentially provide novel interventions for preventing and treating this disease. ^

Regulation of estrogen receptor alpha by the tumor suppressor p53 in breast cancer cells

Estrogen receptor (ER) and the tumor suppressor p53 are key prognostic indicators in breast cancer. Estrogen signaling through its receptor (ER) controls proliferation of normal as well as transformed mammary epithelial cells, and the presence of ER is established as a marker of good prognosis and response to therapy. The p53 tumor suppressor gene is often referred to as the "cellular gatekeeper" due to its extensive control of cell proliferation and apoptosis. Loss of functional p53 is a negative prognostic indicator and is correlated with lack of response to antiestrogens, reduced disease-free interval and increased chance of disease recurrence. Clinical studies have demonstrated that tumors with mutated p53 tend to be ER negative, while ER positive tumors tend to have wild type p53. ^ Recent studies from our lab indicate that p53 genotype correlates with estrogen receptor expression in mammary tumors in vivo. We therefore hypothesized that p53 regulates ER expression in mammary cancer cells by recruitment of specific cofactors to the ER promoter. To test this, MCF-7 cells were treated with doxorubicin or ionizing radiation, both of which stimulated significant increases in p53 expression, as expected, but also increased ER expression in a p53-dependent manner. Furthermore, in cells treated with siRNA targeting p53, both p53 and ER protein levels were significantly reduced. P53 was also demonstrated to transcriptionally regulate the ER promoter in luciferase assays and chromatin immunoprecipitation assays showed that p53 was recruited to the ER promoter along with CARM1, CBP, c-Jun and Sp1 and that this multifactor complex was formed in a p53-dependent manner. The regulation of ER by p53 has therapeutic implications, as the treatment of breast cancer cells with doxorubicin sensitized these cells to tamoxifen treatment. Furthermore, response to tamoxifen as well as to estrogen was dependent on p53 expression in ER positive human breast cancer cells. Taken together, these data demonstrate that p53 regulates ER expression through transcriptional control of the ER promoter, accounting for their concordant expression in human breast cancer and identifying potentially beneficial therapeutic strategies for the treatment of ER positive breast cancers. ^

REGULATION OF FOXC2 EXPRESSION AND FUNCTION DURING EPITHELIAL-MESENCHYMAL TRANSITION BY GSK-3β

Metastasis is the ultimate cause for the majority of cancer-related deaths. The forkhead box transcription factor FOXC2 is known to be involved in regulating metastasis as well as a variety of developmental processes, including the formation of lymphatic and cardiovascular systems. Previous studies have shown that FOXC2 protein is localized either in the nucleus and/or in the cytoplasm of human breast tumor cells. This pattern of localization is similar to that of another forkhead family member, FOXO3a. Additionally, localization of FOXO3a is known to be differentially regulated by upstream kinase AKT. Therefore, I investigated whether FOXC2 localization could also be regulated by upstream kinases. Analysis of FOXC2 protein sequence revealed two potential phosphorylation sites for GSK-3β. Furthermore, inhibition of GSK-3βsignificantly reduces FOXC2 protein. In addition, exposure of HMLE Twist cells expressing endogenous FOXC2 to the GSK-3β inhibitor, TWS119, results in accumulation of FOXC2 protein in the cytoplasm with concomitant decrease in the nucleus in a time-dependent manner. Furthermore, continued treatment with TWS119 eventually induces epithelial morphology and decreased stem cell properties including sphere formation in these cells. Further characterization of FOXC2- GSK-3β interaction and the associated signaling cascade are necessary to determine the effect of FOXC2 phosphorylation by GSK-3β on EMT and metastasis.

14-3-3 ZETA OVEREXPRESSION SERVES AS A NOVEL MOLECULAR SWITCH TURNING TGF-BETA FROM TUMOR SUPPRESSOR TO TUMOR PROMOTER

TGF-β plays an important role in differentiation and tissue morphogenesis as well as cancer progression. However, the role of TGF-β in cancer is complicate. TGF-β has primarily been recognized as tumor suppressor, because it can directly inhibit cell proliferation of normal and premalignant epithelial cell. However, in the last stage of tumor progression, TGF-β functions as tumor promoter to enhance tumor cells metastatic dissemination and expands metastatic colonies. Currently, the mechanism of how TGF-β switches its role from tumor suppressor to promoter still remains elusive. Here we identify that overexpression of 14-3-3ζ inhibits TGF-β’s cell cytostatic program through destabilizing p53 in non-transformed human mammary epithelial cells. Mechanistically, we found that 14-3-3ζ overexpression leads to 14-3-3σ downregulation, thereby activates PI3K/Akt signaling pathway and degrades p53, and further inhibits TGF-β induced p21 expression and cell cytostatic function. In addition, we found that overexpression of 14-3-3ζ promotes TGF-β induced breast cancer cells bone metastatic colonization through stabilizing Gli2, which is an important co-transcriptional factor for p-smad2 to activate PTHrP expression and bone osteolytic effect. Taken together, we reveal a novel mechanism that 14-3-3ζ dictates the tumor suppressor or metastases promoter activities of TGF-β signaling pathway through switching p-smad2 binding partner from p53 to Gli2. The expected results will not only provide us the better understanding of the important role of 14-3-3ζ in the early stage of breast cancer development, but also deeply impact our knowledge of signaling mechanisms underlying the complex roles of TGF-β in cancer, which will give us a more accurate strategy to determine when and how anti-TGF-β targeted therapy might be effective.

Targeting AKT/mTOR Signaling Pathways During Murine Skin Tumor Promotion and the Impact of Dietary Energy Balance Manipulation

The prevalence of obesity has continued to rise over the last several decades in the United States lending to overall increases in risk for chronic diseases including many types of cancer. In contrast, reduction in energy consumption via calorie restriction (CR) has been shown to be a potent inhibitor of carcinogenesis across a broad range of species and tumor types. Previous data has demonstrated differential signaling through Akt and mTOR via the IGF-1R and other growth factor receptors across the diet-induced obesity (DIO)/CR spectrum. Furthermore, mTORC1 is known to be regulated directly via nutrient availability, supporting its role in the link between epithelial carcinogenesis and diet-induced obesity. In an effort to better understand the importance of mTORC1 in the context of both positive and negative energy balance during epithelial carcinogenesis, we have employed the use of specific pharmacological inhibitors, rapamycin (mTORC1 inhibitor) and metformin (AMPK activator) to target mTORC1 or various components of this pathway during skin tumor promotion. Two-stage skin carcinogenesis studies demonstrated that mTORC1 inhibition via rapamycin, metformin or combination treatments greatly inhibited skin tumor development in normal, overweight and obese mice. Furthermore, mechanisms by which these chemopreventive agents may be exerting their anti-tumor effects were explored. In addition, the effect of these compounds on the epidermal proliferative response was analyzed and drastic decreases in epidermal hyperproliferation and hyperplasia were found. Rapamycin also inhibited dermal inflammatory cell infiltration in a dose-dependent manner. Both compounds also blocked or attenuated TPA-induced signaling through epidermal mTORC1 as well as several downstream targets. In addition, inhibition of this pathway by metformin appeared to be, at least in part, dependent on AMPK activation in the skin. Overall, the data indicate that pharmacological strategies targeting this pathway offset the tumor-enhancing effects of DIO and may serve as possible CR mimetics. They suggest that mTORC1 contributes significantly to the process of skin tumor promotion, specifically during dietary energy balance effects. Exploiting the mechanistic information underlying dietary energy balance responsive pathways will help translate decades of research into effective strategies for prevention of epithelial carcinogenesis.

Fibulin-2 stabilizes tumor extracellular matrix and drives malignant progression of lung adenocarcinoma

The ECM of epithelial carcinomas undergoes structural remodeling during periods of uncontrolled growth, creating regional heterogeneity and torsional stress. How tumors maintain ECM integrity in the face of dynamic biophysical forces is still largely unclear. This study addresses these deficiencies using mouse models of human lung adenocarcinoma. Spontaneous lung tumors were marked by disorganized basement membranes, dense collagen networks, and increased tissue stiffness. Metastasis-prone lung adenocarcinoma cells secreted fibulin-2 (Fbln2), a matrix glycoprotein involved in ECM supra-molecular assembly. Fibulin-2 depletion in tumor cells decreased the intra-tumoral abundance of matrix metalloproteinases and reduced collagen cross-linking and tumor compressive properties resulting in inhibited tumor growth and metastasis. Fbln2 deposition within intra-tumoral fibrotic bands was a predictor of poor clinical outcome in patients. Collectively, these findings support a feed-forward model in which tumor cells secrete matrix-stabilizing factors required for the assembly of ECM that preferentially favors malignant progression. To our knowledge, this is the first evidence that tumor cells directly regulate the integrity of their surrounding matrix through the secretion of matrix-stabilizing factors such as fibulin-2. These findings open a new avenue of research into matrix assembly molecules as potential therapeutic targets in cancer patients.

A Novel Mechanism of Skin Tumor Promotion Involving Interferon-gamma (IFNγ)/Signal Transducer and Activator of Transcription-1 (Stat1) Signaling in Epidermis

The JAK-STAT pathway is a major signaling pathway involved in many biological processes including proliferation, apoptosis, and differentiation. Aberrant expression of STATs has been reported in multiple human cancers and murine mouse models of tumorigenesis. Previous studies from our lab and others have established a critical role for Stat3 in epithelial tumorigenesis, but the role of Stat1 is largely unknown. The current study was designed to explore the role of Stat1 during multistage skin carcinogenesis. Topical treatment with both TPA and the anthrone derivative chrysarobin (CHRY) led to rapid phosphorylation of Stat1 on both tyrosine (Tyr701) and serine (Ser727) residues in epidermis. CHRY treatment also led to upregulation of unphosphorylated Stat1 (uStat1) at later time points. In addition, CHRY treatment also led to upregulation of IRF-1 mRNA and protein which was dependent on Stat1. Further analyses demonstrated that topical treatment with CHRY but not TPA upregulated interferon-gamma (IFNg) mRNA in the epidermis and that the induction of both IRF-1 and uStat1 was dependent on IFNg signaling. Stat1 deficient (Stat1-/-) mice were highly resistant to skin tumor promotion by CHRY. In contrast, the tumor response (in terms of both papillomas and squamous cell carcinomas) was similar in Stat1-/- mice and wild-type littermates with TPA as the promoter. Histological evaluation of the proliferative response confirmed the data obtained from the tumor study for both TPA and CHRY. In addition, maximal induction of both cyclooxygenase-2 and inducible nitric oxide synthase in epidermis following treatment with CHRY was also dependent on the presence of functional Stat1. Following CHRY treatment, Stat1-/- mice exhibited reduced macrophage infiltration and reduced production of many immune cell derived chemokines/cytokines. These studies define a novel mechanism associated with skin tumor promotion by the anthrone class of tumor promoters involving upregulation of IFNg signaling in the epidermis and downstream signaling through activated (phosphorylated) Stat1 and subsequent upregulation of IRF-1 and uStat1.

ROLE OF MIR-19A RELEASED BY INFLAMMATORY BREAST CANCER CELLS IN THE REGULATION OF DENDRITIC CELL FUNCTIONS: IN VITRO MODEL OF CROSSTALK IN THE TUMOR MICROENVIRONMENT OF INFLAMMATORY BREAST CANCER

Inflammatory breast cancer (IBC) is a rare but very aggressive form of locally advanced breast cancer (1-6% of total breast cancer patients in United States), with a 5-year overall survival rate of only 40.5%, compared with 85% of the non-IBC patients. So far, a unique molecular signature for IBC able to explain the dramatic differences in the tumor biology between IBC and non-IBC has not been identified. As immune cells in the tumor microenvironment plays an important role in regulating tumor progression, we hypothesized that tumor-associated dendritic cells (TADC) may be responsible for regulating the development of the aggressive characteristics of IBC. MiRNAs can be released into the extracellular space and mediate the intercellular communication by regulating target gene expression beyond their cells of origin. We hypothesized that miRNAs released by IBC cells can induce an increased activation status, secretion of pro-inflammatory cytokines and migration ability of TADC. In an in vitro model of IBC tumor microenvironment, we found that the co-cultured of the IBC cell line SUM-149 with immature dendritic cells (iDCSUM-149) induced a higher degree of activation and maturation of iDCSUM-149 upon stimulation with lipopolysaccharide (LPS) compared with iDCs co-cultured with the non-IBC cell line SUM-159 (iDCSUM-159), resulting in: increased expression of the costimulatory and activation markers; higher production of pro-inflammatory cytokines (TNF-a, IL-6); and 3) higher migratory ability. These differences were due to the exosome-mediated transfer of miR-19a and miR-146a from SUM-149 and SUM-159, respectively, to iDCs, causing the downregulation of the miR-19a target genes PTEN, SOCS-1 and the miR-146a target genes IRAK1, TRAF6. PTEN, SOCS-1 and IRAK1, TRAF6 are important negative and positive regulator of cytokine- and TLR-mediated activation/maturation signaling pathway in DCs. Increased levels of IL-6 induced the upregulation of miR-19a synthesis in SUM-149 cells that was associated with the induction of CD44+CD24-ALDH1+ cancer stem cells (CSCs) with epithelial-to-mesenchymal transition (EMT) characteristics. In conclusion, in IBC tumor microenvironment IL-6/miR-19a axis can represent a self-sustaining loop able to maintain a pro-inflammatory status of DCs, leading to the development of tumor cells with high metastatic potential (EMT CSCs) responsible of the poor prognosis in IBC patients.