Molecular events associated with trisomy of chromosome 7 in mouse skin chemical carcinogenesis

The primary objective of this study has been to investigate the effects at the molecular level of trisomy of mouse chromosome 7 in chemically induced skin tumors. It was previously proposed that the initiation event in the mouse skin carcinogenesis model is a heterozygous mutation of the Ha-ras-1 gene, mapped to chromosome 7. Previous studies in this laboratory identified trisomy 7 as one of the primary nonrandom cytogenetic abnormalities found in the majority of severely dysplastic papillomas and squamous cell carcinomas induced in SENCAR mice by an initiation-promotion protocol. Therefore, the first hypothesis tested was that trisomy 7 occurs by specific duplication of the chromosome carrying a mutated Ha-ras-1 allele. Results of a quantitative analysis of normal/mutated allelic ratios of the Ha-ras-1 gene confirmed this hypothesis, showing that most of the tumors exhibited overrepresentation of the mutated allele in the form of 1/2, 0/3, and 0/2 (normal/mutated) ratios. In addition, histopathological analysis of the tumors showed an apparent association between the degree of malignancy and the dosage of the mutated Ha-ras-1 allele. To determine the mechanism for loss of the normal Ha-ras-1 allele, found in 30% of the tumors, a comparison of constitutional and tumor genotypes was performed at different informative loci of chromosome 7. By combining Southern blot and polymerase chain reaction fragment length polymorphism analyses of DNAs extracted from squamous cell carcinomas, complete loss of heterozygosity was detected in 15 of 20 tumors at the Hbb locus, and in 5 of 5 tumors at the int-2 locus, both distal to Ha-ras-1. In addition, polymerase chain reaction analysis of DNA extracted from papillomas indicated that loss of heterozygosity occurs in late-stage lesions exhibiting a high degree of dysplasia and areas of microinvasion, suggesting that this event may be associated to the acquisition of the malignant phenotype. Allelic dosage analysis of tumors that had become homozygous at Hbb but retained heterozygosis at Ha-ras-1, indicated that loss of heterozygosity on mouse chromosome 7 occurs by a mitotic recombination mechanism. Overall, these findings suggest the presence of a putative tumor suppressor locus on the 7F1-ter region of mouse chromosome 7. Thus, loss of function by homozygosis at this putative suppressor locus may complement activation of the Ha-ras-1 gene during tumor progression, and might be associated with the malignant conversion stage of mouse skin carcinogenesis. ^

Antisense-mediated inhibition of papillomavirus type-18 in human squamous cell carcinoma

Numerous co-factors, genetic, environmental and physical, play an important role in development and prognosis of cancer. Each year in the USA, more than 31,000 cases of oral and 13,000 cases of cervical cancer are diagnosed. Substantial epidemiological data supports a high correlation between development of these cancers and the presence of specific types of human papillomaviruses (HPV). Molecular biological studies show that not only are several of the viral genes necessary and sufficient to cause transformation but they also function synergistically with other co-factors. Evidence suggests that prevention of infection or inhibition of viral gene expression may alter the course of malignant transition. The main objective of this project was to test the hypothesis that some human carcinoma cells, containing HPV, behave in malignant manner because the viral genes function in the maintenance of some aspect of the transformed phenotype.^ The specific aims were (1) to select oral and cervical cancer cell lines which were HPV-negative or which harbored transcriptionally active HPV-18, (2) to construct and determine the effects of recombinant sense or antisense expressing vectors, (3) to test the effects of synthetic antisense oligodeoxynucleotides on the transformed behavior of these cells.^ To screen cells, we performed Southern and Northern analysis and polymerase chain reactions. When antisense-expressing vectors were used, cells harboring low numbers of HPV-18 where unable to survive transfection but they were readily transfected with all other constructs. Rare antisense transfectants obtained from HPV-positive cells showed significantly altered characteristics including malignant potential in nude mice. The HPV-negative cells showed no differences in transfection efficiencies or growth characteristics with any construct.^ In addition, treatment of the HPV-positive cells with antisense, but not random oligodeoxynucleotides, resulted in decreased cell proliferation and even cell death. These effects were dose-dependent, synergistic and HPV-specific.^ These results suggest that expression of viral genes play an important role in the maintenance of the transformed phenotype which implies that inhibition of expression, by antisense molecules, may be therapeutic in HPV-induced tumors. ^

{\it RAS\/} is required for the expression of interleukin-1$\beta$ in two diverse leukemic cell lines

Under normal physiological conditions, cells of the hematopoietic system produce Interleukin-1$\beta$(IL-1$\beta)$ only when a stimulus is present. Leukemic cells, however, can constitutively produce this cytokine without an exogenous source of activation. In addition, IL-1$\beta$ can operate as an autocrine and/or paracrine growth factor for leukemic blasts. In order to study the cellular basis for this aberrant production, we analyzed two leukemic cell lines (B1 and W1) which express high levels of IL-1$\beta$ and use IL-1$\beta$ as an autocrine growth factor. Initial studies demonstrated: (1) lack of rearrangement and/or amplification in the IL-1$\beta$ gene and its promoter; and (2) intact responsiveness to regulators such as cycloheximide and dexamethasone, implying that the molecular defect was upstream. Analysis of the Ras inducible transcription factors by gel shift assay demonstrated constitutive transcription factor binding in the IL-1$\beta$ promoter. Furthermore, RAS mutations were found at codon 12 in the K-RAS and N-RAS genes in the B1 and W1 cells, respectively. To deduce the effects of activated Ras on IL-1$\beta$ expression, two classes of farnesyltransferase inhibitors and an adenoviral vector expressing antisense targeted to K-RAS were utilized. The farnesyltransferase inhibitors perillyl alcohol and B581 were able to reduce IL-1$\beta$ levels by 80% and 50% in the B1 cells, respectively. In W1 cells, IL-1$\beta$ was reduced by 60% with 1mM perillyl alcohol. Antisense RNA targeted to K-RAS confirmed the results demonstrating a 50% reduction in IL-1$\beta$ expression in the B1 cells. In addition, decreased binding at the crucial NF-IL6/CREB binding site correlated with decreased IL-1$\beta$ production and cellular proliferation implying that this site was a downstream effector of Ras signaling. Our data suggest that mutated RAS genes may be responsible for autocrine IL-1$\beta$ production in some leukemias by stimulating signal transduction pathways that activate the IL-1$\beta$ promoter. ^

Expression and regulation of interleukins in human head and neck squamous cell carcinoma

Patients with head and neck squamous cell carcinoma (HNSCC) demonstrate abnormal cell-mediated immunity which is most pronounced at the primary tumor site. Therefore, we tested whether this aberrant immunity could be due to tumor-derived cytokines. We investigated the presence of cytokine mRNA and protein in 8 HNSCC-derived cell lines; RT-PCR results indicated mRNA's for IL-1$\alpha$ and TGF-$\alpha$ (8/8), TGF-$\beta$ (7/8), IL-1$\beta$ (7/8), IL-4 and IL-6 (4/8). IL-2, IFN-$\gamma,$ and TNF-$\alpha$ mRNA was not detected. Supernatants from 6 of these cell lines were analyzed by ELISA and IL-1$\alpha,$ IL-1$\beta,$ and IL-6 were markedly increased compared to HPV-16 immortalized human oral keratinocytes. IL-1$\alpha$ was found in the highest concentration $>$IL-6 $>$ IL-1$\beta.$^ To approach the mechanisms of cytokine regulation, 4 cell lines were compared for HPV DNA presence, p53 status, and cytokine expression. An association between HPV DNA and cytokine expression was not found. However, cell lines secreting the most IL-6 had mutant p53 and/or HPV 16 E6/E7 expression. Further regulatory investigations revealed that exogenous IL-1$\alpha$ and/or IL-1$\beta$ minimally stimulated the proliferation of 2/3 cell lines, as well as strongly induced IL-6 production in 3/3; this effect was completely abrogated by IL-1Ra. IL-1Ra also inhibited the secretion of IL-1$\alpha$ and IL-1$\beta$ in 2/3 cell lines. These data suggest an IL-1 autocrine loop in certain HNSCC cell lines. Because IL-2 induces IL-1 and is used in therapy of HNSCC, the expression of IL-2 receptor was also investigated; IL-2 $\alpha$ and $\beta$ subunits were detected in 3/3 cell lines and $\gamma$ subunits was detected in one. Exogenous IL-2 inhibited the proliferation, but stimulated the secretion of IL-1$\alpha$ in 2/3, and IL-1$\beta$ and IL-6 in 1/3 cell lines.^ To determine if our cell line findings were applicable to patients, immunohistochemistry was performed on biopsies from 12 invasive tumors. Unexpectedly, universal intracellular production of IL-1$\alpha,$ IL-1$\beta,$ and IL-6 protein was detected. Therefore, the aberrant elaboration of biologically active IL-1 and IL-6 may contribute to altered immune status in HNSCC patients. ^

drp, A novel cell density regulated protein

Growing cells are continuously processing signals of all varieties and responding to these signals by changes in cellular gene expression. One signal that cells in close proximity relay to each other is cell-cell contact. Non-transformed cells respond to cell-cell contact by arrest of growth and entry into G$\sb0,$ a process known as contact inhibition. Transformed cells do not respond to contact inhibition and continue to grow to high cell density, forming foci when in cell culture and tumors in the living organism. The events surrounding the generation, transduction, and response to cellular contact are poorly understood. In the present study, a novel gene product, drp, is shown to be expressed at high levels in cultured cells at high cell density. This density regulated protein, drp, has an apparent molecular weight of 70 kDa. Northern analysis shows drp to be highly expressed in cardiac and skeletal muscle and least abundant in lung and kidney tissues. By homology to two independently derived sequence tagged sites (STSs) used in the human genome project, drp or a closely related sequence maps to human chromosome 12. Density-dependent increases in drp expression have been demonstrated in six different cell lines including NIH 3T3, Hela and a human teratocarcinoma cell line, PA-1. Cells exhibit increased drp expression both when they are plated at increasing concentrations per unit area, or plated at low density and allowed to grow naturally to higher cell density. Cells at high density can exhibit several phenotypes including growth arrest, accumulation of soluble factors in the media, and increased numbers of cell contacts. Growth arrest by serum starvation or TGF-$\beta$ treatment fails to produce an increase in drp expression. Similarly, treatment of low density cells with conditioned media from high density cells fails to elicit drp expression. These results argue that neither soluble factors accumulated or expressed at high density nor simple exit from the cell cycle is sufficient to produce an increase in drp expression. The expression of drp appears to be uniquely regulated by cell density alone. ^

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. ^

Molecular alterations in nucleotide excision repair genes in malignant glioma and their relevance to malignant progression and drug resistance

Recently, it has become apparent that DNA repair mechanisms are involved in the malignant progression and resistance to therapy of gliomas. Many investigators have shown that increased levels of O6-methyl guanine DNA alkyltransferase, a DNA monoalkyl adduct repair enzyme, are correlated with resistance of malignant glioma cell lines to nitrosourea-based chemotherapy. Three important DNA excision repair genes ERCC1 (excision repair cross complementation group 1), ERCC2 (excision repair cross complementation group 2), and ERCC6 (excision repair cross complementation group 6) have been studied in human tumors. Gene copy number variation of ERCC1 and ERCC2 has been observed in primary glioma tissues. A number of reports describing a relationship between ERCC1 gene alterations and resistance to anti-cancer drugs have been also described. The levels of ERCC1 gene expression, however, have not been correlated with drug resistance in gliomas. The expression of ERCC6 gene transcribes has been shown to vary with tissue types and to be highest in the brain. There have been no comprehensive studies so far, however, of ERCC6 gene expression and molecular alterations in malignant glioma. This project examined the ERCC1 expression levels and correlated them with cisplatin resistance in malignant glioma cell lines. We also examined the molecular alterations of ERCC6 gene in primary glioma tissues and cells and analyzed whether these alterations are related to tumor progression and chemotherapy resistance. Our results indicate the presence of mutations and/or deletions in exons II and V of the ERCC6 gene, and these alterations are more frequent in exon II. Furthermore, the mutations and/or deletions in exon II were shown to be associated with increased malignant grade of gliomas. The results on the Levels of ERCC1 gene transcripts showed that expression levels correlate with cisplatin resistance. The increase in ERCC1 mRNA induced by cisplatin could be down-regulated by cyclosporin A and herbimycin A. The results of this study are likely to provide useful information for clinical treatment of human gliomas. ^

Adenosine mediated lung mast cell degranulation in the mouse

Adenosine has been implicated to play a role in inflammatory processes associated with asthma. Most notable is adenosine's ability to potentiate mediator release from mast cells. Mast cells are bone marrow derived inflammatory cells that can release mediators that have both immediate and chronic effects on airway constriction and inflammation. Most physiological roles of adenosine are mediated through adenosine receptors. Four subtypes of adenosine receptors have been identified, A1, A2A, A2B and A 3. The mechanisms by which adenosine can influence the release of mediators from lung tissue mast cells is not understood due to lack of in vivo models. Mice deficient in the enzyme adenosine deaminase (ADA) have been generated. ADA controls the levels of adenosine in tissues and cells, and consequently, adenosine accumulates in the lungs of ADA-deficient mice. ADA-deficient mice develop features seen in asthmatics, including lung eosinophilia and mucus hypersecretion. In addition, lung tissue mast cell degranulation was associated with elevated adenosine in ADA-deficient lungs and can be prevented by ADA enzyme therapy. We established primary murine lung mast cell cultures, and used real time RT-PCR and immunofluorescence to demonstrate that A 2A, A2B and A3 receptors are expressed on murine lung mast cells. Studies using selective adenosine receptor agonists and antagonists and A3 receptor deficient (A3−/−) mast cells suggested that activation of A3 receptors could induce mast cell mediator release in vitro. Furthermore, this mediator release was associated with increases in intracellular Ca++ that appeared to be mediated through a Gi and PI3K pathway. In addition, nebulized A3 receptor agonist directly induced lung mast cell degranulation in wild type mice while having no effect in A3−/− mice. These results demonstrate that the A3 receptor plays an important role in adenosine mediated murine lung mast cell degranulation. Therefore, the A3 adenosine receptor and its signaling pathways may represent novel therapeutic targets for the treatment and prevention of asthma. ^

Antitumor activity of an Ets protein, PEA3, in breast cancer cell lines MDA-MB-361DYT2 and BT474M1

Polyomavirus enhancer activator 3 (PEA3) is a member of the Ets family of transcription factors. We demonstrated in a previous study that, through down-regulating the HER-2/neu oncogene at the transcriptional level, PEA3 can inhibit the growth and tumor development of HER-2/neu-overexpressing ovarian cancer cells. Here, we established stable clones of the human breast cancer cell line MDA-MB-361DYT2 that express PEA3 under the control of a tetracycline-inducible promoter. The expression of PEA3 in this cell line inhibited cell growth and resulted in cell cycle delay in the G1 phase independently of the HER-2/neu down-regulation. In an orthotopic breast cancer model, we showed that expression of PEA3 inhibited tumor growth and prolonged the survival of tumor-bearing mice. In a parallel experiment in another breast cancer cell line, BT474M1, we were unable to obtain stable PEA3-inducible transfectants, which suggests that PEA3 possessed a strong growth inhibitory effect in this cell line. Indeed, PEA3 coupled with the liposome SN2 demonstrated therapeutic effects in mice bearing tumors induced by BT474M1. These results provide evidence that the PEA3 gene could function as an antitumor and gene therapy agent for human breast cancers. ^

Chemopreventive function of retinoid X receptors in human squamous cell carcinoma of the skin

Retinoid therapy has been successful for the treatment of skin squamous cell carcinoma (SCC). A suppression of the predominant retinoid X receptor expressed in skin, retinoid X receptor α (RXRα), has been reported in skin SCC. These observations have led to the hypothesis that retinoid receptor loss contributes to the tumorigenic phenotype of epithelial cancers. To test this hypothesis, the RXRα gene was mapped in order to generate a targeting construct. Additionally the transcriptional regulation of the human RXRα a gene in keratinocytes was characterized after identifying the transcription initiation sites, the promoter, and enhancer regions of this gene. The structure is highly conserved between human and mouse. A nontumorigenic human skin-derived cell line called near diploid immortalized keratinocytes (NIKS) has the advantage of growing as organotypic raft cultures, under physiological conditions closely resembling in-vivo squamous stratification. We have exploited the raft culture technique to develop an in-vitro model for skin SCC progression that includes the NIKS cells, HaCaT cells, a premalignant cell line, and SRB 12-p9 cells, a tumorigenic SCC skin cell line. The differentiation, proliferation and nuclear receptor ligand response characteristics of this system were studied and significant and novel results were obtained. RXRs are obligate heterodimerization partners with many of the nuclear hormone receptors, including retinoic acid receptors (RARs), vitamin D3 receptors (VDR), thyroid hormone receptors (T3 R) and peroxisome proliferator activate receptors (PPARs), which are all known to be active in skin. Treatment of the three cell lines in raft culture with the RXR specific ligand BMS649, BMS961 (RARγ-specific), vitamin D3 (VDR ligand), thryoid hormone (T3R ligand) and clofibrate (PPARa ligand), and the combination of BMS649 with each of the 4 receptor partner ligands, resulted in distinct effects on differentiation, proliferation and apoptosis. The effects of activation of RXRs in each of the four-receptor pathways; in the context of skin SCC progression, with an emphasis on the VDR/RXR pathway, are discussed. These studies will lead to a better understanding of RXRα action in human skin and will help determine its role in SCC tumorigenesis, as well as its potential as a target for the prevention, treatment, and control of skin cancer. ^

Molecular and genetic analyses of Fgf signaling during cardiac outflow tract development

Epithelial-mesenchymal tissue interactions regulate the development of derivatives of the caudal pharyngeal arches (PAs) to govern the ultimate morphogenesis of the aortic arch and outflow tract (OFT) of the heart. Disruption of these signaling pathways is thought to contribute to the pathology of a significant proportion of congenital cardiovascular defects in humans. In this study, I tested whether Fibroblast Growth Factor 15 (Fgf15), a secreted signaling molecule expressed within the PAs, is an extracellular mediator of tissue interactions during PA and OFT development. Analyses of Fgf15−/− mouse embryonic hearts revealed abnormalities primarily localized to the OFT, correlating with aberrant cardiac neural crest cell behavior. The T-box-containing transcription factor Tbx1 has been implicated in the cardiovascular defects associated with the human 22q11 Deletion Syndromes, and regulates the expression of other Fgf family members within the mouse PAs. However, expression and genetic interaction studies incorporating mice deficient for Tbx1, its upstream regulator, Sonic Hedgehog (Shh), or its putative downstream effector, Fgf8, indicated that Fgf15 functions during OFT development in a manner independent of these factors. Rather, analyses of compound mutant mice indicated that Fgf15 and Fgf9, an additional Fgf family member expressed within the PAs, genetically interact, providing insight into the factors acting in conjunction with Fgf15 during OFT development. Finally, in an effort to further characterize this Fgf15-mediated developmental pathway, promoter deletion analyses were employed to isolate a 415bp sequence 7.1Kb 5′ to the Fgf15 transcription start site both necessary and sufficient to drive reporter gene expression within the epithelium of the PAs. Sequence comparisons among multiple mammalian species facilitated the identification of evolutionarily conserved potential trans-acting factor binding sites within this fragment. Subsequent studies will investigate the molecular pathway(s) through which Fgf15 functions via identification of factors that bind to this element to govern Fgf15 gene expression. Furthermore, targeted deletion of this element will establish the developmental requirement for pharyngeal epithelium-derived Fgf15 signaling function. Taken as a whole, these data demonstrate that Fgf15 is a component of a novel, Tbx1-independent molecular pathway, functioning within the PAs in a manner cooperative with Fgf9, required for proper development of the cardiac OFT. ^

Molecular Mechanisms of Vascular Disease in Patients with Rare Variants in MYH11

Thoracic aortic aneurysms and dissections (TAAD) are the primary disease affecting the thoracic ascending aorta, with an incidence rate of 10.4/100,000. Although about 20% of patients carry a mutation in a single gene that causes their disease, the remaining 80% of patients may also have genetic factors that increase their risk for developing TAAD. Many of the genes that predispose to TAAD encode proteins involved in smooth muscle cell (SMC) contraction and the disease-causing mutations are predicted to disrupt contractile function. SMCs are the predominant cell type in the ascending aortic wall. Mutations in MYH11, encoding the smooth muscle specific myosin heavy chain, are a rare cause of inherited TAAD. However, rare but recurrent non-synonymous variants in MYH11 are present in the general population but do not cause inherited TAAD. The goal of this study was to assess the potential role of these rare variants in vascular diseases. Two distinct variants were selected: the most commonly seen rare variant, MYH11 R247C, and a duplication of the chromosomal region spanning the MYH11 locus at 16p13.1. Genetic analyses indicated that both of these variants were significantly enriched in patients with TAAD compared with controls. A knock-in mouse model of the Myh11 R247C rare variant was generated, and these mice survive and reproduce normally. They have no structural abnormalities of the aorta or signs of aortic disease, but do have decreased aortic contractility. Myh11R247C/R247C mice also have increased proliferative response to vascular injury in vivo and increased proliferation of SMCs in vitro. Myh11R247C/R247C SMCs have decreased contractile gene and protein expression and are dedifferentiated. In fibroblasts, myosin force generation is required for maturation of focal adhesions, and enhancers of RhoA activity replace enhancers of Rac1 activity as maturation occurs. Consistent with these previous findings, focal adhesions are smaller in Myh11R247C/R247C SMCs, and there is decreased RhoA activation. A RhoA activator (CN03) rescues the dedifferentiated phenotype of Myh11R247C/R247C SMCs. Myh11R247C/R247C mice were bred with an existing murine model of aneurysm formation, the Acta2-/- mouse. Over time, mice carrying the R247C allele in conjunction with heterozygous or homozygous loss of Acta2 had significantly increased aortic diameter, and a more rapid accumulation of pathologic markers. These results suggest that the Myh11 R247C rare variant acts as a modifier gene increasing the risk for and severity of TAAD in mice. In patients with 16p13.1 duplications, aortic MYH11 expression is increased, but there is no corresponding increase in smooth muscle myosin heavy chain protein. Using SMCs that overexpress Myh11, we identified alterations in SMC phenotype leading to excessive protein turnover. All contractile proteins, not just myosin, are affected, and the proteins are turned over by autophagic degradation. Surprisingly, these cells are also more contractile compared with wild-type SMCs. The results described in this dissertation firmly establish that rare variants in MYH11 significantly affect the phenotype of SMCs. Further, the data suggests that these rare variants do increase the risk of TAAD via pathways involving altered SMC phenotype and contraction. Therefore, this study validates that these rare genetic variants alter vascular SMCs and provides model systems to explore the contribution of rare variants to disease.

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.

Molecular interactions of the central conserved domain of p53

p53 mutations are the most commonly observed genetic alterations in human cancers to date. A majority of these point mutations cluster in four evolutionarily conserved domains spanning amino acids 100-300. This region of p53 has been called its central conserved, or conformational domain. This domain of p53 is also targeted by the SV40 T antigen. Mutation, as well as interaction with SV40 T antigen results in inactivation of p53. We hypothesized that mutations and SV40 T antigen disrupt p53 function by interfering with the molecular interactions of the central conserved domain. Using a chimeric protein consisting of the central conserved domain of wild-type p53 (amino acids 115-295) and a protein A affinity tail, we isolated several cellular proteins that interact specifically with this domain of p53. These proteins range in size from 30K to 90K M$\rm\sb{r}.$ We also employed the p53 fusion protein to demonstrate that the central conserved domain of p53 possesses sequence-specific DNA-binding activity. Interestingly, the cellular proteins binding to the central conserved domain of p53 enhance the sequence-specific DNA-binding activity of full length p53. Partial purification of the individual proteins binding to the conformational domain of p53 by utilizing a sodium chloride step-gradient enabled further characterization of two proteins: (1) a 42K M$\rm\sb{r}$ protein that eluted at 0.5M NaCl, and bound DNA nonspecifically, and (2) a 35K M$\rm\sb{r}$ protein eluting into the 1.0M NaCl fraction, capable of enhancing the sequence-specific DNA-binding activity of p53. In order to determine the physiologic relevance of the molecular interactions of the conformational domain of p53, we examined the biochemical processes underlying the TNF-$\alpha$ mediated growth suppression of the NSCLC cell line H460. While growth suppression was accompanied by enhanced sequence-specific p53-DNA binding activity in TNF-$\alpha$ treated H460 nuclei, there was no increase in p53 protein levels. Furthermore, p35 was upregulated in TNF-$\alpha$ treated H460 cells, suggesting that the enhanced p53-DNA binding seen in these cells may be mediated by p35. Our studies define two novel interactions involving the central conserved domain of p53 that appear to be functionally relevant: (1) sequence-specific DNA-binding, and (2) interaction with other cellular proteins. ^

A distinct element involved in the lipopolysaccharide activation of the tumor necrosis factor -alpha promoter in a promonocytic cell line, THP-1

TNF-α is a pleiotropic cytokine involved in normal homeostasis and plays a key role in defending the host from infection and malignancy. However when deregulated, TNF-α can lead to various disease states. Therefore, understanding the mechanisms by which TNF-α is regulated may aid in its control. In spite of the knowledge gained regarding the transcriptional regulation of TNF-α further characterization of specific TNF-α promoter elements remains to be elucidated. In particular, the T&barbelow;NF-α A&barbelow;P-1/C&barbelow;RE-like (TAC) element of the TNF-α promoter has been shown to be important in the regulation of TNF-α in lymphocytes. Activating transcription factor-2 (ATF-2) and c-Jun were shown to bind to and transactivate the TAC element However, the role of TAC and transcription factors ATF-2 and c-Jun in the regulation of TNF-α in monocytes is not as well characterized. Lipopolysaccharide (LPS), a potent activator of TNF-α in monocytes, provides a good model to study the involvement of TAC in TNF-α regulation. On the other hand, all-tram retinoic acid (ATRA), a physiological monocyte-differentiation agent, is unable to induce TNF-α protein release. ^ To delineate the functional role of TAC, we transfected the wildtype or the TAC deleted TNF-α promoter-CAT construct into THP-1 promonocytic cells before stimulating them with LPS. CAT activity was induced 17-fold with the wildtype TNF-α promoter, whereas the CAT activity was uninducible when the TAC deletion mutant was used. This daft suggests that TAC is vital for LPS to activate the TNF-α promoter. Electrophoretic mobility shift assays using the TAC element as a probe showed a unique pattern for LPS-activated cells: the disappearance of the upper band of a doublet seen in untreated and ATRA treated cells. Supershift analysis identified c-Jun and ATF-2 as components of the LPS-stimulated binding complex. Transient transfection studies using dominant negative mutants of JNK, c-Jun, or ATF-2 suggest that these proteins we important for LPS to activate the TNF-α promoter. Furthermore, an increase in phosphorylated or activated c-Jun was bound to the TAC element in LPS-stimulated cells. Increased c-Jun activation was correlated with increased activity of Jun N-terminal kinase (JNK), a known upstream stimulator of c-Jun and ATF-2, in LPS-stimulated monocytes. On the other hand, ATRA did not induce TNF-α protein release nor changes in the phosphorylation of c-Jun or JNK activity, suggesting that pathways leading to ATRA differentiation of monocytic cells are independent of TNF-α activation. Together, the induction of TNF-α gene expression seems to require JNK activation, and activated c-Jun binding to the TAC element of the TNF-α promoter in THP-1 promonocytic cells. ^