Developmental programming of tumor suppressor gene penetrance

Epidemiological studies have led to the hypothesis that major risk factors for developing diseases such as hypertension, cardiovascular disease and adult-onset diabetes are established during development. This developmental programming hypothesis proposes that exposure to an adverse stimulus or insult at critical, sensitive periods of development can induce permanent alterations in normal physiological processes that lead to increased disease risk later in life. For cancer, inheritance of a tumor suppressor gene defect confers a high relative risk for disease development. However, these defects are rarely 100% penetrant. Traditionally, gene-environment interactions are thought to contribute to the penetrance of tumor suppressor gene defects by facilitating or inhibiting the acquisition of additional somatic mutations required for tumorigenesis. The studies presented herein identify developmental programming as a distinctive type of gene-environment interaction that can enhance the penetrance of a tumor suppressor gene defect in adult life. Using rats predisposed to uterine leiomyoma due to a germ-line defect in one allele of the tuberous sclerosis complex 2 (Tsc-2) tumor suppressor gene, these studies show that early-life exposure to the xenoestrogen, diethylstilbestrol (DES), during development of the uterus increased tumor incidence, multiplicity and size in genetically predisposed animals, but failed to induce tumors in wild-type rats. Uterine leiomyomas are ovarian-hormone dependent tumors that develop from the uterine myometrium. DES exposure was shown to developmentally program the myometrium, causing increased expression of estrogen-responsive genes prior to the onset of tumors. Loss of function of the normal Tsc-2 allele remained the rate-limiting event for tumorigenesis; however, tumors that developed in exposed animals displayed an enhanced proliferative response to ovarian steroid hormones relative to tumors that developed in unexposed animals. Furthermore, the studies presented herein identify developmental periods during which target tissues are maximally susceptible to developmental programming. These data suggest that exposure to environmental factors during critical periods of development can permanently alter normal physiological tissue responses and thus lead to increased disease risk in genetically susceptible individuals. ^

High-resolution microarray analysis of chromosome 20q in human colon cancer metastasis model systems

Amplification of human chromosome 20q DNA is the most frequently occurring chromosomal abnormality detected in sporadic colorectal carcinomas and shows significant correlation with liver metastases. Through comprehensive high-resolution microarray comparative genomic hybridization and microarray gene expression profiling, we have characterized chromosome 20q amplicon genes associated with human colorectal cancer metastasis in two in vitro metastasis model systems. The results revealed increasing complexity of the 20q genomic profile from the primary tumor-derived cell lines to the lymph node and liver metastasis derived cell lines. Expression analysis of chromosome 20q revealed a subset of over expressed genes residing within the regions of genomic copy number gain in all the tumor cell lines, suggesting these are Chromosome 20q copy number responsive genes. Bases on their preferential expression levels in the model system cell lines and known biological function, four of the over expressed genes mapping to the common intervals of genomic copy gain were considered the most promising candidate colorectal metastasis-associated genes. Validation of genomic copy number and expression array data was carried out on these genes, with one gene, DNMT3B, standing out as expressed at a relatively higher levels in the metastasis-derived cell lines compared with their primary-derived counterparts in both the models systems analyzed. The data provide evidence for the role of chromosome 20q genes with low copy gain and elevated expression in the clonal evolution of metastatic cells and suggests that such genes may serve as early biomarkers of metastatic potential. The data also support the utility of the combined microarray comparative genomic hybridization and expression array analysis for identifying copy number responsive genes in areas of low DNA copy gain in cancer cells. ^

The role of SRC family kinase activation in prostate cancer growth and lymph node metastasis

Aberrant expression and/or activation of Src Family of non-receptor protein tyrosine kinases (SFKs) occur frequently during progressive stages of multiple types of human malignancies, including prostate cancer. Two SFKs, Src and Lyn, are expressed and implicated in prostate cancer progression. Work in this dissertation investigated the specific roles of Src and Lyn in the prostate tumor progression, and the effects of SFK inhibition on prostate tumor growth and lymph node metastasis in pre-clinical mouse models. ^ Firstly, using a pharmacological inhibitor of SFKs in clinical trials, dasatinib, I demonstrated that SFK inhibition affects both cellular migration and proliferation in vitro. Systemic administration of dasatinib reduced primary tumor growth, as well as development of lymph node metastases, in both androgen-sensitive and -resistant orthotopic prostate cancer mouse models. Immunohistochemical analysis of the primary tumors revealed that dasatinib treatment decreased SFK phosphorylation but not expression, resulting in decreased cellular proliferation and increased apoptosis. For this analysis of immunohistochemical stained tissues, I developed a novel method of quantifying immunohistochemical stain intensity that greatly reduced the inherent bias in analyzing staining intensity. ^ To determine if Src and Lyn played overlapping or distinct roles in prostate cancer tumor growth and progression, Src expression alone was inhibited by small-interfering RNA. The resulting stable cell lines were decreased in migration, but not substantially affected in proliferation rates. In contrast, an analogous strategy targeting Lyn led to stable cell lines in which proliferation rates were significantly reduced. ^ Lastly, I tested the efficacy of a novel SFK inhibitor (KX2-391) targeting peptide substrate-binding domain, on prostate cancer growth and lymph node metastasis in vivo. I demonstrated that KX2-391 has similar effects as dasatinib, an ATP-competitive small molecular inhibitor, on both the primary tumor growth and development of lymph node metastasis in vivo, work that contributed to the first-in-man Phase I clinical trial of KX2-391. ^ In summary, studies in this dissertation provide the first demonstration that Src and Lyn activities affect different cellular functions required for prostate tumor growth and metastasis, and SFK inhibitors effectively reduce primary tumor growth and lymph node metastasis. Therefore, I conclude that SFKs are promising therapeutic targets for treatment of human prostate cancer. ^

Oncogenic activation of c-Abl tyrosine kinase in non-small cell lung cancer: FUS1 tumor suppressor down-regulates c-Abl tyrosine kinase

The FUS1 tumor suppressor gene (TSG) has been found to be deficient in many human non-small cell lung cancer (NSCLC) tissue samples and cell lines (1,2,3). Studies have shown potent anti-tumor activity of FUS1 in animal models where FUS1 was delivered through a liposomal vector (4) and the use of FUS1 as a therapeutic agent is currently being studied in clinical human trials (5). Currently, the mechanisms of FUS1 activity are being investigated and my studies have shown that c-Abl tyrosine kinase is inhibited by the FUS1 TSG.^ Considering that many NSCLC cell lines are FUS1 deficient, my studies further identified that FUS1 deficient NSCLC cells have an activated c-Abl tyrosine kinase. C-Abl is a known proto-oncogene and while c-Abl kinase is tightly regulated in normal cells, constitutively active Abl kinase is known to contribute to the oncogenic phenotype in some types of hematopoietic cancers. My studies show that the active c-Abl kinase contributes to the oncogenicity of NSCLC cells, particularly in tumors that are deficient in FUS1, and that c-Abl may prove to be a viable target in NSCLC therapy.^ Current studies have shown that growth factor receptors play a role in NSCLC. Over-expression of the epidermal growth factor receptor (EGFR) plays a significant role in aggressiveness of NSCLC. Current late stage treatments include EFGR tyrosine kinase inhibitors or EGFR antibodies. Platelet-derived growth factor receptor (PDGFR) also has been shown to play a role in NSCLC. Of note, both growth factor receptors are known upstream activators of c-Abl kinase. My studies indicate that growth factor receptor simulation along deficiency in FUS1 expression contributes to the activation of c-Abl kinase in NSCLC cells. ^

Alterations in cell adhesion and migration are transcriptionally regulated by the tumor suppressor, TP53

The p53 transcription factor is a tumor suppressor and a master regulator of apoptosis and the cell cycle in response to cell stress. In some advanced tumors, such as prostate cancers, the loss of p53 correlates with an increase in the occurrence of metastases. In addition, several groups have suggested that p53 status correlates with changes in cell migration and cell morphology associated with a migratory phenotype. Others have identified several genes with roles in cell migration that are directly transcriptionally regulated by p53. Even so, modulation of cell migration is not widely recognized as a p53 stress response. ^ In an effort to identify novel p53 target genes and expand our knowledge of the p53 transcriptional response, we performed Affymetrix gene expression analysis in p53-null PC3 prostate cancer cells following infection with a control virus or adenoviral construct expressing wild-type p53. Over 300 genes that had not been previously recognized as p53 target genes were identified. Of these genes, 224 were upregulated and 111 were downregulated (p<0.05). Functional over-representation analysis identified cell migration as a significantly over-represented biological function of p53. Further analysis identified two genes that are critical for the control of cell migration as potential p53 targets. One, hyaluronan mediated motility receptor (HMMR), has recently been shown to be a p53 target important for regulation of the cell cycle. Here, we show that HMMR is downregulated by p53 in several cell lines, and HMMR's regulation is dependent on the presence of the cdk inhibitor, p21, and histone deactelyase activity. The other gene, carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), itself a tumor suppressor, is shown here, for the first time, as a p53 direct target by ChIP analysis. We next determined the effect of p53 activation on cell migration and found that p53 significantly slows the rate of cell migration in Boyden chamber migration assays and digital videomicroscopy wound healing studies. Further, our studies established the specific roles of CEACAM1 and HMMR in cell migration and determine that loss of CEACAM1 and overexpression of HMMR independently contribute to increased cell migration. Taken together, these studies provide a direct mechanistic link between p53 to the regulatory control of specific target genes that mediate cell adhesion and migration. ^

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. ^

The role of reactive astrocytes in the resistance of melanoma brain metastasis to chemotherapy

Brain metastasis is resistant to chemotherapy while the leaky blood-brain-barrier in brain metastasis can not be the underlying reason. Metastatic tumor cells (“seed”) exploit the host microenvironment (“soil”) for survival advantages. Astrocytes which maintain the homeostasis of the brain microenvironment become reactive subsequent to brain damages and protect neurons from various injuries. We observed reactive astrocytes surrounding and infiltrating into brain metastasis in both clinical specimen and experimental animal model, thus raising a possibility that reactive astrocytes may protect tumor cells from cytotoxic chemotherapeutic drugs. ^ To test this hypothesis, we first generated an immortalized astrocyte cell line from H-2Kb-tsA58 mice. The immortal mouse astrocytes expressed specific markers including GFAP. Scanning electron microscopy demonstrated that astrocytes formed direct physical contact with tumor cells. Moreover, the expression of GFAP by astrocytes was up-regulated subsequent to co-culture with tumor cells, indicating that the co-culture of astrocytes and tumor cells may serve as a model to recapitulate the pathophysiological situation of brain metastasis. ^ In co-culture, astrocytes dramatically reduced apoptosis of tumor cells produced by various chemotherapeutic drugs. This protection effect was not because of culturing cells from different species since mouse fibroblasts did not protect tumor cells from chemotherapy. Furthermore, the protection by astrocytes was completely dependent on a physical contact. ^ Gap junctional communication (GJC) served as this physical contact. Tumor cells and astrocytes both expressed the major component of gap junctional channel—connexin 43 and formed functional GJC as evidenced by the “dye transfer” assay. The blockage of GJC between tumor cells and astrocytes by either specific chemical blocker carbenoxolone (CBX) or by genetically knocking down connexin 43 on astrocytes reversed the chemo-protection. ^ Calcium was the signal molecule transmitted through GJC that rescued tumor cells from chemotherapy. Accumulation of cytoplasmic calcium preceded the progress of apoptosis in tumor cells treated with chemotherapeutic drugs. Furthermore, chelation of accumulated cytoplasmic calcium inhibited the apoptosis of tumor cells treated with chemotherapeutic drugs. Most importantly, astrocytes could “shunt” the accumulated cytoplasmic calcium from tumor cells (treated with chemotherapeutic drug) through GJC. We also used gene expression micro-array to investigate global molecular consequence of tumor cells forming GJC with astrocytes. The data demonstrated that astrocytes (but not fibroblasts), through GJC, up-regulated the expressions of several well known survival genes in tumor cells. ^ In summary, this dissertation provides a novel mechanism underlying the resistance of brain metastasis to chemotherapy, which is due to protection by astrocytes through GJC. Interference with the GJC between astrocytes and tumor cells holds great promise in sensitizing brain metastasis to chemotherapy and improving the prognosis for patients with brain metastasis. ^

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. ^

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.

ADHERENS JUNCTIONS AND THE ACTOMYOSIN NETWORK REGULATE ORGAN GROWTH BY MODULATING HIPPO PATHWAY ACTIVITY IN DROSOPHILA

Adherens junctions (AJs) and basolateral modules are important for the establishment and maintenance of apico-basal polarity. Loss of AJs and basolateral module members lead to tumor formation, as well as poor prognosis for metastasis. Recently, in mammalian studies it has been shown that loss of either AJ or basolateral module members deregulate Yorkie activity, the downstream transcriptional effector of the Hippo pathway. Importantly, it is unclear if AJ and basolateral components act through the same or parallel mechanisms to regulate Yorkie activity. Here, we dissect how loss of AJ and basolateral components affects Hippo signaling in Drosophila. Surprisingly, while scrib knock-down tissue displays increased reporter activity autonomously, α-cat knock-down tissue shows a cell autonomous decrease and a cell non-autonomous increase of Hippo reporter activity. We provided several lines of evidence to show the differential regulation in polarity protein localizations and oncogenic cooperative overgrowth by AJs and basolateral complexes. Finally, we show that Hippo pathway activity is induced in α-cat and scrib double knocked-down tissue. Taken together, our results provide evidence to show that basolateral modules and AJs act in parallel to modulate Hippo pathway activity. Non-muscle myosin II is an actomyosin component that interacts with the actin. Non-muscle myosin II also interacts with lgl, though the function of this interaction is not clear. Our lab demonstrated that modulating F-actin regulates Hippo pathway activity, and lgl also has been described as a Hippo pathway regulator. Therefore we suspect that myosin II is also involved in Hippo pathway regulation. We first characterized non-muscle Myosin II as a novel tumor suppressor gene by affecting Hippo pathway activity. Upstream regulators of Myosin II, members in the Rho signaling pathway, also displayed similar phenotypes as the Myosin II knock-down tissues. Apoptosis is also induced in myosin II knock-down tissues, however, blocking cell death does not affect myosin II knock-down induced Hippo activation. Our data suggested hyperactivating myosin II induced F-actin accumulation so therefore induces Hippo target activation. Unexpectedly, we also observed that reducing F-actin activity induced Hippo target activation in vivo. These controversial data indicated that actomyosin may regulate the Hippo pathway through multiple mechanisms.

The Role Of IL-8-Mediated Src Family Kinase Activation In Tumor-Tumor And Tumor-Stromal Interactions In Metastasis Of Prostate Cancer

Men with localized prostate cancer (PCa) have a 100% five-year survival rate, but this rate drops to 33% for men with metastatic disease. A better understanding of the metastatic process is needed to develop better therapies for PCa. Aberrant activation of protein tyrosine kinases, including Src Family Kinases (SFKs) contribute to metastasis through numerous functions, one of which leads to increased expression of cytokines, such as IL-8. However, the relationship between Src activity and IL-8 regulation is not completely understood. In cell line models, I determined that IL-8 activates Src and in turn Src activates IL-8 demonstrating a feed forward loop contributing to the migration and invasion of PCa cells. However, IL-8 is also produced by tumor-associated stromal cells. In bone marrow derived stromal cells (HS5), I demonstrated a feed forward loop occurs as was observed in tumor cells. HS5 conditioned media increased Src activity in PCa cells. By silencing IL-8 in HS5 cells, Src activity was decreased to control levels in PCa cells as was migration and invasion. Thus, stromal cells producing IL-8 contribute to metastatic properties of PCa by a paracrine mechanism. To examine the effect of stromal cells on tumor growth and metastatic potential of PCa in vivo, I mixed HS5 and PCa cells and co-injected them intraprostatically. I determined that tumor growth and metastases were increased. By silencing IL-8 in HS5 cells and co-injecting them with PCa cells intraprostatically, tumor growth and metastases were still increased relative to injection of PCa cells alone, but decreased relative to co-injections with PCa cells and HS5 cells. These studies demonstrated: (1) a feed forward loop in both tumor and stromal cells, whereby IL-8 activates Src, derepressing IL-8 expression in PCa cells in vitro; (2) stromal produced IL-8 activates Src and contributes to the migration and invasion of PCa cells in vitro; and (3) stromal produced IL-8 is responsible, in part, for increases in PCa tumor growth and metastatic potential. Together, these studies demonstrated that IL-8-mediated Src activity increases the metastatic potential of PCa and therapeutic agents interfering with the IL-8/SFK signaling axis may be useful for prevention and treatment of metastases.

The Role of Type I Insulin-Like Growth Factor Receptor Signaling in Breast Cancer Brain Metastasis

Brain metastasis is a common cause of mortality in cancer patients. Approximately 20-30% of breast cancer patients acquire brain metastasis, yet potential therapeutic targets remain largely unknown. The type I insulin-like growth factor receptor (IGF- IR) is known to play a role in the progression of breast cancer and is currently being investigated in the clinical setting for various types of cancer. The present study demonstrates that the IGF-IR signaling axis is constitutively active in brain-seeking sublines of breast cancer cells, driving an increase in in vitro metastatic properties. We demonstrate that IGF-IR signaling is activated in an autocrine manner as a result of IGFBP3 overexpression in brain-seeking cells. Transient and stable knockdown of IGF-IR results in a downregulation of IGF-IR downstream signaling through phospho-AKT, as well as decreased in vitro migration and invasion of MDA- MB-231Br brain-seeking cells. Using an in vivo experimental brain metastasis model, we show that IGF-IR ablation attenuates the establishment of brain metastases and prolongs survival. Finally, we demonstrate that the malignancy of brain-seeking cells is attenuated by pharmacological inhibition with picropodophyllin, an IGF-IR-specific tyrosine kinase inhibitor. Together, our data suggest that the IGF-IR is an important mediator of brain metastasis and its ablation delays the onset of brain metastases in our model system.

A Genomic Approach to Identify The Notch Pathway as a Putative Tumor Suppressor in Endometrial Cancer

Endometrial cancer is the most common gynecological malignancy and the fourth most frequently diagnosed cancer among women. The molecular changes that distinguish normal endometrium from endometrial carcinoma are not thoroughly understood. Identification of these changes could potentially aid in identifying at-risk women who are especially prone to develop endometrial cancer, such as obese women and women with Lynch Syndrome. A microarray analysis was performed using normal endometrium from thin and obese women and cancerous endometrium from obese women. We validated the differential expression of ten genes whose expression was significantly up-regulated or down-regulated using qRT-PCR. All of the genes had distinct expression levels depending on the endometrial carcinoma histotype. As a result, they could serve as molecular markers to distinguish between normal endometrium and endometrial cancer, as well as between low grade endometrial carcinomas and high grade endometrial carcinomas. Two of the ten genes validated, HEYL and HES1, are down-stream targets of the Notch signaling pathway. HEYL and HES1 were identified by microarray and qRT-PCR to have a significant decrease in expression in endometrial carcinomas compared to normal endometrium. We further analyzed the differential expression of other components of the Notch signaling pathway, Notch4 and Jagged1. They were also identified by qRT-PCR to be significantly down-regulated in endometrial carcinomas compared to normal endometrium. Therefore, we believe the Notch signaling pathway to act as a tumor suppressor in endometrial carcinomas.

The role of suppressor factors in the regulation of immune responses by ultraviolet radiation -induced suppressor T lymphocytes

The purpose of these studies was to determine the role of suppressor factors (TsF) in the regulation of immune responses by ultraviolet radiation-induced suppressor T lymphocytes (Ts). The Ts were induced following epicutaneous sensitization with contact allergens to an unirradiated site on mice irradiated five days earlier with 40 kJ/m$\sp2$ UVB (280-320 nm) radiation. The spleens of such mice contain afferent, hapten-specific, Thy-1$\sp+$, Lyt-1$\sp+$,2$\sp-$ Ts that suppress in vivo contact hypersensitivity (CHS) and antibody responses and the in vitro generation of cytotoxic T lymphocytes (CTL). Four approaches were used to determine the role of TsF. First, lysates produced from sonically-disrupted Ts were injected i.v. into normal animals; they inhibited CHS in vivo in a nonspecific manner. The lysates suppressed the induction and elicitation of CHS, and they inhibited the in vitro generation of CTL. Lysates prepared from splenocytes obtained from unirradiated mice or UV-irradiated, unsensitized mice failed to inhibit either response. Second, supernatants from cultures containing Ts, normal syngeneic responder lymphocytes, and hapten-modified stimulator cells were injected i.v. into normal recipients. They inhibited the induction of CHS and did so in a hapten-specific manner. Cellular and kinetic requirements were observed for the generation of suppressive activity. Splenocytes from mice treated with Ts supernatants suppressed CHS when transferred into normal animals. The supernatants also suppressed the in vitro generation of specific CTL. Third, the TsF-specific B16G monoclonal antibody was tested for its ability to modulate the effects of UV radiation in vivo. The i.v. injection of B16G into UV-irradiated mice reduced the suppression of CHS. Splenocytes of B16G-treated mice transferred into normal recipients, and they suppressed CHS, indicating that the Ts were not depleted. Fourth, B16G was used to isolate a putative TsF by antibody immunoadsorbance. When the B16G-bound fraction was eluted and injected i.v. into normal animals, it suppressed CHS and represented a 900-fold enrichment of activity over the starting material, based on specific activity. By SDS-PAGE, the B16G-bound material contained nondisulfide-linked 45- and 50-kDa components. These results suggest that TsF may play an immunoregulatory role in CHS. The isolation of a UV radiation-induced TsF lends credence to the involvement of such molecules. ^

The structural and functional relationship of the p53 tumor suppressor protein

The tumor-suppressing function of p53 can be affected in a variety of manners. Here, we describe a novel mechanism of transformation by mutant p53. Previously, it had been believed that mutant p53 molecules transform cells by oligomerizing with wild-type p53 and inactivating it. However, we demonstrated that there exists an additional mechanism of inactivation of p53 available to p53 mutants. It involves sequestration of cofactors necessary to p53, and subsequent interruption of its transactivation and tumor suppression functions. The p53 amino or carboxyl termini, known to interact with a large number of cellular factors, can affect wild-type p53 in this manner. Although they are unable to oligomerize with wild-type p53, they transform cells containing p53, and inhibit its transactivation ability. In addition, they interrupt growth suppression by p53, but not RB, confirming that they specifically affect p53 function, rather than having a general growth-stimulatory phenomenon. Also, we have cloned a p53 tumor mutation which results in expression of the amino terminus of p53. This provides a means to study the factor-sequestration transforming mechanism in vivo. Additionally, we found that the published sequence of the mdm2 gene is in error. mdm2 is a gene intimately involved with p53, blocking its ability to transform cells. Finally, previous data had established the influence of cell-cycle status on p53 function. In growth-arrested cells, wild-type p53 expressed by a transgene cannot activate transcription, but if these cells are forced to cycle by addition of cyclin E, p53 once again becomes functional. In this study, we extend these findings by examining only those cells successfully transfected, using fluorescence-activated cell sorting. Our results support the previous data, that cyclin E pushes growth-arrested cells back into the cell cycle. In summary, we have demonstrated the potential importance of cofactor association and protein modification to the abilities of p53 to cause transcription activation and repression, inhibition of DNA replication and induction of DNA repair, and initiation of cell-cycle arrest and apoptosis. Further elucidation of these processes and their roles in tumor suppression will prove fascinating indeed. ^