EphA2 expression is regulated by EGFR and KRAS and promotes non-small cell lung cancer progression

Lung cancer is the leading cause of cancer deaths worldwide. The development of improved systemic therapy is needed for the most common form of the disease, non-small cell lung cancer (NSCLC). This will depend on the identification of valid molecular targets. Recent studies point to the receptor tyrosine kinase EphA2 as a novel therapeutic target. Overexpression of EphA2 has been demonstrated in a number of epithelial cancers, and its expression has been associated with more severe disease. Regulation of EphA2 in cancer is poorly understood. Recently, regulation of EphA2 by EGFR and KRAS has been reported in a number of in vitro models, but no examination of this relationship has been undertaken in patient tumors. Because of the established importance of EGFR and KRAS in NSCLC, we have investigated the relationship between these mutations and EphA2 in NSCLC patient tissues and cell lines. The significance of Epha2 expression was further examined by testing for correlation with survival, metastases, histology, and smoking status in patient tissues, and tumor cell proliferation and migration in vitro. EphA2 expression was analyzed in by immunohistochemistry in tissue microarray (TMA) format utilizing surgically resected lung cancer specimens. EGFR and KRAS mutation status was determined for the majority of specimens. EphA2 expression was detected in >90% of NSCLC tumors. High EphA2 expression was associated with decreased time to recurrence and metastases, and predicted poorer progression free and overall survival. Expression of EphA2 was positively correlated with activated EGFR and with KRAS mutation. Expression of EphA2 was also positively correlated with a history of smoking. There was no association between gender or histology and EphA2 expression. In H322 cells, activation of EGFR or KRAS resulted in an increase in EphA2 protein expression. Downregulation of EphA2 resulted in decreased proliferation in a clonal growth assay, and inhibited migration in a wound healing assay, in a panel of cell lines. The decrease in proliferation correlated with a transient decrease in the levels of phospho-ERK, a downstream effector of EGFR and KRAS. Based on these data, the potential of EphA2 as a therapeutic target for NSCLC should be further investigated. ^

HRGbeta1 inhibits metastasis of breast cancer cell lines, one of which exhibits lineage infidelity

Recent publications have questioned the origin of the MDA-MB-435 breast cancer cell line and have suggested that it is of melanocyte origin rather than breast epithelial origin. The data presented herein show unequivocally that MDA-MB-435 does express breast epithelial markers and produces milk-specific lipids. The data also indicated that MDA-MB-435 does express some melanocyte proteins but this expression occurs in the same MDA-MB-435 cells that express breast epithelial proteins. Although MDA-MB-435 does not strictly adhere to a breast lineage, it does retain breast specific markers and is thus valid as an experimental cell line in breast cancer studies. ^ Heregulinβ1 (HRGβ1) has been shown to both stimulate and inhibit breast tumorigenic and metasastasic phenotypes. Some studies used only the EGF-like domain of the extracellular domain of HRGβ1 while others used bacterially-expressed HRGβ1. Our in vitro data demonstrated that the full-length extracellular domain of human HRGβ1 reduced clonal growth of MDA-MB-435 breast cancer cells but stimulated apoptosis in MDA-MB-435 and MCF-7 breast cancer cells. In addition, mammalian-expressed HRGβ1 did not dramatically affect matrix metalloproteinase-9 activity but did inhibit cell motility of MDA-MB-435 and MCF-7 cells. Taken together, the in vitro data indicated that HRGβ1 inhibits metastasis-associated properties. ^ The in vivo data demonstrated that inducible expression of the full-length extracellular domain of human HRGβ1 in MDA-MB-435 cells reduced tumor volume and cell proliferation but increased apoptosis of cells injected at the mammary fat pad in nude mice. More importantly, HRGβ1 reduced the number of metastases observed by a spontaneous metastasis assay. Taken together, these data indicate that the full-length extracellular domain of human HRGβ1 has the net effect of inhibiting breast cancer metastasis. ^

Delta like ligand 4 is a critical regulator of bone marrow cell differentiation into pericytes/vascular smooth muscle cells and is essential for the vasculogenesis that supports the growth of Ewing’s sarcoma

We have previously shown that vasculogenesis, the process by which bone marrow-derived cells are recruited to the tumor and organized to form a blood vessel network de novo, is essential for the growth of Ewing’s sarcoma. We further demonstrated that these bone marrow cells differentiate into pericytes/vascular smooth muscle cells(vSMC) and contribute to the formation of the functional vascular network. The molecular mechanisms that control bone marrow cell differentiation into pericytes/vSMC in Ewing’s sarcoma are poorly understood. Here, we demonstrate that the Notch ligand Delta like ligand 4 (DLL4) plays a critical role in this process. DLL4 is essential for the formation of mature blood vessels during development and in several tumor models. Inhibition of DLL4 causes increased vascular sprouting, decreased pericyte coverage, and decreased vessel functionality. We demonstrate for the first time that DLL4 is expressed by bone marrow-derived pericytes/vascular smooth muscle cells in two Ewing’s sarcoma xenograft models and by perivascular cells in 12 out of 14 patient samples. Using dominant negative mastermind to inhibit Notch, we demonstrate that Notch signaling is essential for bone marrow cell participation in vasculogenesis. Further, inhibition of DLL4 using either shRNA or the monoclonal DLL4 neutralizing antibody YW152F led to dramatic changes in blood vessel morphology and function. Vessels in tumors where DLL4 was inhibited were smaller, lacked lumens, had significantly reduced numbers of bone marrow-derived pericyte/vascular smooth muscle cells, and were less functional. Importantly, growth of TC71 and A4573 tumors was significantly inhibited by treatment with YW152F. Additionally, we provide in vitro evidence that DLL4-Notch signaling is involved in bone marrow-derived pericyte/vascular smooth muscle cell formation outside of the Ewing’s sarcoma environment. Pericyte/vascular smooth muscle cell marker expression by whole bone marrow cells cultured with mouse embryonic stromal cells was reduced when DLL4 was inhibited by YW152F. For the first time, our findings demonstrate a role for DLL4 in bone marrow-derived pericyte/vascular smooth muscle differentiation as well as a critical role for DLL4 in Ewing’s sarcoma tumor growth.

Stromal derived growth factor alpha (SDF-1α) induces the differentiation of bone marrow progenitor cells (BMCs) into pericytes by regulating platelet derived growth factor B (PDGF-B) transcription

We previously demonstrated that bone marrow cells (BMCs) migrate to TC71 and A4573 Ewing’s sarcoma tumors where they can differentiate into endothelial cells (ECs) and pericytes and, participate in the tumor vascular development. This process of neo-vascularization, known as vasculogenesis, is essential for Ewing’s sarcoma growth with the soluble vascular endothelial growth factor, VEGF165, being the chemotactic factor for BMC migration to the tumor site. Inhibiting VEGF165 in TC71 tumors (TC/siVEGF7-1) inhibited BMC infiltration to the tumor site and tumor growth. Introducing the stromal-derived growth factor (SDF-1α) into the TC/siVEGF7-1 tumors partially restored vasculogenesis with infiltration of BMCs to a perivascular area where they differentiated into pericytes and rescued tumor growth. RNA collected from the SDF-1α-treated TC/siVEGF7-1 tumors also revealed an increase in platelet-derived growth factor B (PDGF-B) mRNA levels. PDGF-B expression is elevated in several cancer types and the role of PDGF-B and its receptor, PDGFR-β, has been extensively described in the process of pericyte maturation. However, the mechanisms by which PDGF-B expression is up-regulated during vascular remodeling and the process by which BMCs differentiate into pericytes during tumor vasculogenesis remain areas of investigation. In this study, we are the first to demonstrate that SDF-1α regulates the expression of PDGF-B via a transcriptional mechanism which involves binding of the ELK-1 transcription factor to the pdgf-b promoter. We are also first to validate the critical role of the SDF-1α/PDGF-B pathway in the differentiation of BMCs into pericytes both in vitro and in vivo. SDF-1α up-regulated PDGF-B expression in both TC/siVEGF7-1 and HEK293 cells. In contrast, down-regulating SDF-1α, down-regulated PDGF-B. We cloned the 2 kb pdgf-b promoter fragment into the pGL3 reporter vector and showed that SDF-1α induced pdgf-b promoter activity. We used chromatin immunoprecipitation (ChIP) and demonstrated that the ELK-1 transcription factor bound to the pdgf-b promoter in response to SDF-1α stimulation in both TC/siVEGF7-1 and HEK293 cells. We collected BMCs from the hind femurs of mice and cultured the cells in medium containing SDF-1α and PDGF-B and found that PDGFR-β+ BMCs differentiated into NG2 and desmin positive pericytes in vitro. In contrast, inhibiting SDF-1α and PDGF-B abolished this differentiation process. In vivo, we injected TC71 or A4573 tumor-bearing mice with the SDF-1α antagonist, AMD3100 and found that inhibiting SDF-1α signaling in the tumor microenvironment decreased the tumor microvessel density, decreased the tumor blood vessel perfusion and, increased tumor cell apoptosis. We then analyzed the effect of AMD3100 on vasculogenesis of Ewing’s sarcoma and found that BMCs migrated to the tumor site where they differentiated into ECs but, they did not form thick perivascular layers of NG2 and desmin positive pericytes. Finally, we stained the AMD3100-treated tumors for PDGF-B and showed that inhibiting SDF-1α signaling also inhibited PDGF-B expression. All together, these findings demonstrated that the SDF-1α/PDGF-B pathway plays a critical role in the formation of BM-derived pericytes during vasculogenesis of Ewing’s sarcoma tumors.

CELL POLARITY REGULATES ORGAN GROWTH THROUGH THE HIPPO PATHWAY

Defects in apical-basal cell polarity and abnormal expression of cell polarity determinants are linked to human cancer. Loss of polarity is highly correlated with malignancy. In Drosophila, perturbation of apical-basal polarity, including overexpressing the apical determinant Crumbs, can lead to uncontrolled tissue growth. Cells mutant for the basolateral determinant scribble overproliferate and can form neoplastic tumors. Interestingly, scribble mutant clones that arise in wild-type tissues are eliminated and therefore do not manifest their tumorigenic potential. However, the mechanisms by which cell polarity coordinates with growth control pathways in developing organs to achieve appropriate organ size remain obscure. To investigate the function of apical determinants in growth regulation, I investigated the mechanism by which the apical determinant Crumbs affects growth in Drosophila imaginal discs. I found that crumbs gain and loss of function cause overgrowth and induction of Hippo target genes. In addition, Crumbs is required for the proper localization of Expanded, an upstream component of the Hippo pathway. Furthermore, we uncoupled the cell polarity and growth control function of Crb through structure-functional analysis. Taken together, our data identify a role of Crb in growth regulation specifically through modulation of the Hippo pathway. To further explore the role of polarity in growth control, I investigated how cells mutant for basolateral determinants are eliminated by using patches of cells mutant for scribble (scribble mutant clones) as a model system. We found that competitive cell-cell interactions eliminate tumorigenic scribble cells by modulation of the Hippo pathway. The regulation of Hippo signaling is required and sufficient to restrain the tumorous growth of scribble mutant cells. Artificially increasing the relative fitness of scribble mutant cells unleashes their tumorigenic potential. Therefore, we have identified a novel tumor-suppression mechanism that depends on signaling between normal and tumorigenic cells. These data identify evasion of cell competition as a critical step toward malignancy and illustrate a role for wild-type tissue in eliminating abnormal cells and preventing the formation of tumors.

Studies on regulation of skeletal muscle differentiation by growth factors

Skeletal muscle differentiation involves sequential events in which proliferating undifferentiated myoblasts withdraw from the cell cycle and fuse to form multinucleated myotubes. The process of fusion is accompanied by the disappearance of proteins associated with cell proliferation and the coordinate induction of a battery of muscle-specific gene products, which includes the muscle isoenzyme of creatine kinase, nicotinic acetylcholine receptor, and contractile proteins such as alpha-actin. The molecular events associated with myogenesis are particularly amenable to experimental analysis because the events which occur in vivo can be recapitulated in vitro using established muscle cell lines. Initiation of myogenic differentiation in vitro can be achieved by removing serum from the culture medium. Myogenesis, therefore, can be considered to be regulated through a repression-type of mechanism by components in serum. The objectives of this project were to identify the components involved in regulation of myogenesis and approach the mechanism(s) whereby these components achieve their regulatory function. Initially, the effects of a series of polypeptide growth factors on myogenesis were examined. Among them TGF$\beta$ and FGF were found to be potent inhibitors of myogenic differentiation which did not affect cell proliferation. The inhibitory effects of these growth factors on differentiation requires their persistent presence in the culture medium. After myoblasts have undergone fusion, they become refractory to the inhibitory effects of TGF$\beta$, FGF, and serum. When fusion is inhibited by the presence of EGTA, a Ca$\sp{2+}$ chelator, muscle-specific genes are expressed reversibly upon removal of inhibitory growth factors. Subsequent exposure of biochemically differentiated cells to serum or TGF$\beta$ leads to down-regulation of muscle-specific genes. Stimulation with serum also leads to reentry of myocytes into the cell cycle, whereas fused myotubes are irreversibly and terminally differentiated. Measurement of levels of TGF$\beta$ receptors reveals that under non-fusing conditions, TGF$\beta$ receptor levels in biochemically differentiated myocytes remained as high as in undifferentiated myoblasts, while during terminal differentiation, TGF$\beta$ receptors decreased at least five-fold. Thus, down-regulation of TGF$\beta$ receptors is coupled to irreversible differentiation, but not reversible differentiation in the absence of fusion. The possible involvement of second messenger systems, such as cAMP and protein kinase C, in the pathway(s) by which TGF$\beta$, FGF, or serum factors transduce their signals from the cell surface to the nucleus was also examined. The results showed that myogenic differentiation is subject to negative regulation through cAMP elevation-dependent and cAMP elevation-independent pathways and that serum mitogens, TGF$\beta$ and FGF inhibit differentiation through a mechanism independent of cAMP-elevation or protein kinase C activation. ^

Molecular mechanisms of prostatic carcinoma growth and progression

Prostate cancer represents the most commonly diagnosed malignancies in American men and is the second leading cause of male cancer deaths. The overall objectives of this research were designed to understand the cellular and molecular mechanisms of prostatic carcinoma growth and progression. This dissertation was divided into two major parts: (1) to clone and characterize soluble factor(s) associated with bone that may mediate prostatic carcinoma growth and progression; (2) to investigate the roles of extracellular matrix in prostatic carcinogenesis.^ The propensity of prostate cancer cells to metastasize to the axial skeleton and the subsequent osteoblastic reactions observed in the bone indicate the possible reciprocal cellular interaction between prostate cancer cells and the bone microenvironment. To understand the molecular and cellular basis of this interaction, I focused on the identification and cloning of soluble factor(s) from bone stromal cells that may exert direct mitogenic action on cultured prostate cells. A novel BPGF-1 gene expressed specifically by bone and male accessory sex organs (prostate, seminal vesicles, and coagulating gland) was identified and cloned.^ The BPGF-1 was identified and cloned from a cDNA expression library prepared from a human bone stromal cell line, MS. The conditioned medium (CM) of this cell line contains mitogenic materials that induce human prostate cancer cell growth both in vivo and in vitro. The cDNA expression library was screened by an antibody prepared against the mitogenic fraction of the CM.^ The cloned BPGF-1 cDNA comprises 3171 nucleotides with a single open reading frame of 1620 nucleotides encoding 540 amino acids. The BPGF-1 gene encodes two transcripts (3.3 and 2.5 kb) with approximately equal intensity in human cells and tissues, but only one transcript (2.5 kb) in rat and mouse tissues. Southern blot analysis of human genomic DNA revealed a single BPGF-1 gene. The BPGF-1 gene is expressed predominantly in bone and seminal vesicles, but at a substantially lower level in prostate. Polyclonal antibodies generated from synthetic peptides that correspond to the nucleotide sequences of the cloned BPGF-1 cDNA reacted with a putative BPGF-1 protein with an apparent molecular weight of 70 kDa. The conditioned media isolated from COS cells transfected with BPGF-1 cDNA stimulated the proliferation and increased the anchorage-independent growth of prostate epithelial cells. These findings led us to hypothesize that BPGF-1 expression in relevant organs, such as prostate, seminal vesicles, and bone, may lead to local prostate cancer growth, metastasis to the seminal vesicles, and subsequently dissemination to the skeleton.^ To assess the importance of extracellular matrix in prostatic carcinogenesis, the role of extracellular matrix in induction of rat prostatic carcinoma growth in vivo was evaluated. NbE-1, a nontumorigenic rat prostatic epithelial cell line, was induced to form carcinoma in athymic nude hosts by coinjecting them with Matrigel and selected extracellular matrix components. Induction of prostatic tumor formation by laminin and collagen IV was inhibited by their respective antibodies. Prostatic epithelial cells cloned from the tumor tissues were found to form tumors in athymic nude hosts in the absence of exogenously added extracellular matrix. These results suggest that extracellular matrix induce irreversibly prostatic epithelial cells that behave distinctively different from the parental prostatic epithelial cell line. ^

Esx1 is an X-chromosome-imprinted regulator of placental formation and fetal growth

Placental formation and genomic imprinting are two important features of embryonic development in placental mammals. Genetic studies have demonstrated that imprinted genes play a prominent role in regulating placental formation. In marsupials, mice and humans, the paternally derived X chromosome is preferentially inactivated in the placental tissues of female embryos. This special form of genomic imprinting may have evolved under the same selective forces as autosomal imprinted genes. This chromosomal imprinting phenomenon predicts the existence of maternally expressed X-linked genes that regulate placental development.^ In this study, an X-linked homeobox gene, designated Esx1 has been isolated. During embryogenesis, Esx1 was expressed in a subset of placental tissues and regulates formation of the chorioallantoic placenta. Esx1 acted as an imprinted gene. Heterozygous female mice that inherit an Esx1-null allele from their father developed normally. However, heterozygous females that inherit the Esx1 mutation from their mother were born 20% smaller than normal and had an identical phenotype to hemizygous mutant males and homozygous mutant females. Surprisingly, although Esx1 mutant embryos were initially comparable in size to wild-type controls at 13.5 days post coitum (E13.5) their placentas were significantly larger (51% heavier than controls). Defects in the morphogenesis of the labyrinthine layer were observed as early as E11.5. Subsequently, vascularization abnormalities developed at the maternal-fetal interface, causing fetal growth retardation. These results identify Esx1 as the first essential X-chromosome-imprinted regulator of placental development that influences fetal growth and may have important implications in understanding human placental insufficiency syndromes such as intrauterine growth retardation (IUGR). ^

HETEROGENEITY IN VIRUS EXPRESSION, TUMORIGENICITY, AND IN VITRO GROWTH PROPERTIES OF DRUG-RESISTANT CLONES OF AN RIII MOUSE MAMMARY TUMOR CELL LINE

Four 8-azaguanine (AG)-resistant and 5-bromodeoxyuridine (BUdR)-resistant clones of a mouse mammary adenocarcinoma cell line, RIII 7387, were developed and analyzed for their tumorigenic properties, in vitro characteristics, and virus expression. These characteristics were analyzed for relationships of any of the cellular parameters and the ability of these lines to produce tumors in syngeneic animals.^ The results of this study demonstrated that the parental line consists of a heterogeneous population of cells. Doubling times, saturation densities, and 2-deoxy-D-glucose uptake varied between sublines. In addition, while all sublines were found to express both B-type and C-type viral antigenic markers, levels of the major B-type and C-type viral proteins varied in the subclones. The sublines also differed markedly in their response to the presence of dexamethasone, glutathione, and insulin in the tissue culture medium.^ Variations in retrovirus expression were convirmed by electron microscopy. Budding and extracellular virus particles were seen in the majority of the cell lines. Virus particles in one of the BUdR-resistant lines, BUD9, were found however, only in inclusions and vacuoles. The AG-resistant subline AGE11 was observed to be rich in intracytoplasmic A particles. The examination of these cell lines for the presence of retroviral RNA-dependent DNA polymerase (RT) activity revealed that some B-type RT activity could be found in the culture fluid of most of the cell lines but that little C-type RT activity could be found suggesting that the C-type virus particles expressed by these RIII clones contain a defective RT.^ Tumor clones also varied in their ability to form tumors in syngeneic RIII mice. Tumor incidence ranged from 50% to 100%. The majority of the tumors regressed within 30 days post infection.^ Statistical analysis indicated that while these clones varied in their characteristics, there was no correlation between the ability of these cell lines to form tumors in syngeneic mice and any of the other characteristics examined.^ These studies have confirmed and extended the growing evidence that tumors, regardless of their natural origin, consist of heterogeneous subpopulations of cells which may vary widely in their in vitro growth behavior, their antigenic expression, and their malignant properties. ^

Significance of signal transducer and activator of transcription 3 during multistage mouse skin carcinogenesis

Signal transducer and activator of transcription 3 (Stat3) is a signaling molecule that transduces signal from cell surface receptors, itself translocates into the nucleus, binds to consensus promoter sequences and activates gene transcription. Here, we showed that Stat3 is constitutively activated in both premalignant tumors (papillomas) and squamous cell carcinomas of mouse skin that is induced by topical treatment with an initiator 7,12-dimethylbenz[a]anthracene (DMBA) followed by a tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Additional data demonstrated that epidermal growth factor signaling contributes to the activation of Stat3 in this model. Using mice where Stat3 function is abrogated in keratinocytes via the Cre-LoxP system (K5Cre.Stat3 flox/flox), we demonstrated that Stat3 is required for de novo carcinogenesis since Stat3 deficiency leads to a complete abrogation of skin tumor development induced by DMBA and TPA. We subsequently showed that Stat3 plays a role in both the initiation and promotion stages of carcinogenesis. During initiation, Stat3 functions as an anti-apoptotic molecule for maintaining the survival of DNA-damaged keratinocyte stem cells. During promotion, Stat3 functions as a critical regulator for G1 to S phase cell cycle progression to confer selective clonal expansion of initiated cells into papillomas. On the other hand, using transgenic mice over-expressing a constitutively dimerized form of Stat3 (Stat3C) in keratinocytes (K5.Stat3C), we revealed a role for Stat3 in tumor progression. After treatment with DMBA and TPA, K5.Stat3C transgenic mice developed skin tumors with a shorter latency when 100% bypassed the premalignant stage and became carcinoma in situ. Histological and immunohistochemical analysis revealed these tumors as highly vascularized and poorly differentiated. More strikingly, these tumors exhibited invasion into surrounding mesenchymal tissue, some of which metastasized into lung. The tumor-mesenchymal front was characterized by partial loss of E-cadherin and elevation of vimentin, markers characterizing epithelial-mesenchymal transition. On the other hand, inhibition of Stat3 via a decoy oligonucleotide led to a significant reduction of tumor size in approximately 50% of all papillomas tested. In conclusion, we demonstrated that Stat3 plays a critical in all three stages (initiation, promotion and progression) of skin carcinogenesis, and it may potentially become a good target for cancer prevention and anti-cancer therapy. ^

Insulin-like growth factor binding protein 2 (IGFBP2) promotes glioma development and progression

Overexpression of insulin-like growth factor binding protein 2 (IGFBP2) is associated with progression and poor survival in many types of human cancer (such as prostate, ovarian, adrenocortical, breast, colorectal carcinomas, leukemia, and high-grade gliomas). We therefore hypothesize that IGFBP2 is a key regulator of tumor progression. We tested our hypothesis in gliomas using the somatic gene transfer RCAS-tva mouse model system, which permits the introduction of specific genes into specific, cell lineages, in this case glial cells (RCAS: Replication competent avian sarcomavirus, tv-a: avian RCAS virus receptor). Mice are transgenic and harbor the tv-a receptor under the control of a glial-specific promoter and study genes are cloned into the RCAS vector for post-natal intracranial delivery. For these experiments, the study genes were IGFBP2, platelet-derived growth factor B (PDGFB), K-Ras, Akt, and IIp45 (invasion inhibitory protein 45 kDa; known to bind and block IGFBP2 activity), which were delivered separately and in combination. Our results show that PDGFB signaling leads exclusively to the formation of low-grade (WHO grade II) oligodendrogliomas. PDGFB delivered in combination with IGFBP2 results in the formation of anaplastic oligodendrogliomas (WHO grade III), which are characterized by increased cellularity, vascular proliferation, small regions of necrosis, increased mitotic activity, and increased activation of the Akt pathway. IIp45 injected in combination with PDGFB and IGFBP2 ablates IGFBP2-induced tumor progression, which results in formation of low-grade oligodendrogliomas, and an overall reduction in tumor incidence. K-Ras expression was required to form astrocytomas with either IGFBP2 or Akt, indicating the activation of two separate pathways is necessary for gliomagenesis. In ex vivo experiments, blockade of Akt by an inhibitor led to decreased viability of cells co-expressing IGFBP2 versus PDGFB expression alone. This study provides definitive evidence, for the first time, that: (1) IGFBP2 plays a role in activation of the Akt pathway, (2) IGFBP2 collaborates with K-Ras or PDGFB in the development and progression of two major types of glioma, and (3) IGFBP2-induced tumor progression can be ablated by IIp45 or by specific inhibition of the Akt pathway. ^

Role and regulation of c-KIT gene expression in tumor growth and metastasis of human melanoma

The purpose of this study was to investigate the role of the c-KIT receptor in the progression of human melanoma and the mechanism(s) for the regulation of c-KIT gene expression in human melanoma.^ The molecular changes associated with the transition of melanoma cells from radial growth phase (RGP) to vertical growth phase (VGP) (metastatic phenotype) are not well-defined. Expression of the tyrosine-kinase receptor c-KIT progressively decreases during local tumor growth and invasion of human melanomas. To provide direct evidence that the metastasis of human melanoma is associated with the loss of c-KIT expression, highly metastatic A375SM cells, which express very low or undetectable levels of c-KIT, were tranduced with the human c-KIT gene. We demonstrated that enforced c-KIT expression in highly metastatic human melanoma cells significantly suppressed their tumorigenicity and metastatic propensity in nude mice. In addition, we showed that the ligand for c-KIT, SCF, induces apoptosis in human melanoma cells expressing c-KIT under both in vitro and in vivo conditions. These results suggest that loss of c-KIT receptor may allow malignant melanoma cells to escape SCF/c-KIT-mediated apoptosis, thus contributing to tumor growth and eventually metastasis.^ Furthermore, we investigated the possible mechanism(s) for the down-regulation of c-KIT gene expression in malignant melanoma. Sequence analysis of the c-KIT promoter indicated that this promoter contains several consensus binding-site sequences including three putative AP2 and two Myb sites. Although Myb was shown to be associated with c-KIT expression in human hemotopoietic cells, we found no correlation between c-KIT expression and Myb expression in human melanoma cell lines. In contrast, we showed that c-KIT expression directly correlates with expression of AP2 in human melanoma cells. We found that highly metastatic cells do not express the transcription factor AP2. Expression of AP2 in A375SM cells (c-KIT-negative and AP2-negative) was enough to restore luciferase activity driven by the c-KIT promoter in a dose-dependent manner. On the other hand, co-expression of the dominant-negative form of AP2 (AP2B) in Mel-501 cells (c-KIT-positive and AP2-positive) resulted in two-fold reduction in luciferase activity. Electrophoretic mobility shift assays revealed that the c-KIT promoter contains functional AP2 binding sites which could associate with AP2 protein. Endogenous c-KIT gene expression levels were elevated in AP2 stably-transfected human melanoma A375SM cells. Expression of exogenous AP2 in A375SM cells inhibited their tumorigenicity and metastatic potential in nude mice. The c-KIT ligand, SCF, also induced apoptosis in the AP2 stably-transfected A375SM cells. The identification of AP2 as an important regulator for c-KIT expression suggests that AP2 may have tumor growth and metastasis inhibitory properties, possibly mediated through c-KIT/SCF effects on apoptosis of human melanoma cells. Since AP2 binding sites were found in the promoters of other genes involved in the progression of human melanoma, such as MMP2 (72 kDa collagenase), MCAM/MUC18 and P21/WAF-1, our findings suggest that loss of AP2 expression might be a crucial event in the development of malignant melanoma. ^