Mary E. Miele

KISS1 metastasis suppression and emergent pathways.

Metastatic disease is the most critical impediment to cancer patient survival. However, comparatively little is known concerning the intricate pathways which govern the complex phenotypes associated with metastasis. The KISS1 metastasis suppressor gene inhibits metastasis in both in vivo melanoma and breast carcinoma models. Despite its clear physiological activity, the mechanism of KISS1 remains unclear. Recent identification of a 54 amino acid peptide of KISS1, termed metastin or kisspeptin-54, and its cognate G-protein coupled receptor (hOT7T175, AXOR12, GPR54) have provided additional clues and avenues of research. While studies have attributed KISS1 with modulation of NFκB regulation, experiments with metastin and its receptor implicate MAP kinase pathways and also suggest the potential of autocrine, paracrine and endocrine roles. Impacts on motility, chemotaxis, adhesion and invasion have each been documented in disparate cell lines and conflicting observations require resolution. Nevertheless, mounting clinical evidence, particularly the loss of KISS1 in metastases, correlates KISS1 and metastin receptor expression with human tumor progression. Together, the data substantiate roles for these molecules in metastasis regulation.

Metastasis suppressed, but tumorigenicity and local invasiveness unaffected, in the human melanoma cell line MelJuSo after introduction of human chromosomes 1 or 6

Progression of human melanoma toward increasingly malignant behavior is associated with several nonrandom chromosomal aberrations, most commonly involving chromosomes 1, 6, 7, 9, and 10. We previously showed that introduction of human chromosome 6 into the highly metastatic human malignant melanoma cell line C8161 completely suppressed metastasis without altering tumorigenicity (Welch DR, Chen P, Miele ME, et al., Oncogene 9:255–262, 1994). Alterations of chromosome 1 are the most frequent chromosome abnormality observed in melanomas, and they frequently arise late in tumor progression. The purpose of the study presented here was to compare the effects of chromosomes 1 and 6 on malignant melanoma metastasis. By using microcell‐mediated chromosome transfer, single copies of neo‐tagged human chromosomes 1 or 6 were introduced into the human melanoma cell line MelJuSo. The presence of the added chromosome was verified by G banding of karyotypes, fluorescence in situ hybridization, and screening for polymorphic markers on each chromosome. The incidence and number of metastases per lung after intravenous or intradermal injection of parental MelJuSo cells was significantly (P < 0.01) greater than those of hybrids containing either chromosome 1 or chromosome 6, although chromosome 1 was a less potent inhibitor of metastasis than chromosome 6. Cultures established from primary tumors and metastases remained neomycin resistant, suggesting that portions of the added chromosomes were retained. These results strengthen the evidence for the presence of a melanoma metastasis suppressor gene on chromosome 6. neo6/MelJuSo hybrids expressed 2.4‐ to 3.4‐fold more of the melanoma differentiation‐associated gene mda‐6 (previously shown to be identical to WAF1/CIP1/Sdi1/CAP20) than parental metastatic cells. mda‐6/WAF1 is among the candidate genes on chromosome 6. These results also demonstrate, for the first time, the existence of metastasis suppressor genes on human chromosome 1, although these genes appear to be less potent than the one encoded on chromosome 6.

A human melanoma metastasis-suppressor locus maps to 6q16.3-q23

Loss, deletion or rearrangement along large portions of the long arm (q‐arm) of chromosome 6 occurs in >80% of late‐stage human melanomas, suggesting that genes controlling malignant characteristics are encoded there. Metastasis, but not tumorigenicity, was completely suppressed in the human melanoma cell line C8161 into which an additional intact chromosome 6 had been introduced by microcell‐mediated chromosome transfer. Our objective was to refine the location of a putative metastasis suppressor gene. To do this, we transferred an intact (neo6) and a deletion variant [neo6qdel; neo6(del)(q16.3‐q23)] of neomycin‐tagged human chromosome 6 into metastatic C8161 subclone 9 (C8161.9) by MMCT. Single cell hybrid clones were selected in G‐418 and isolated. Following verification that the hybrids retained the expected regions of chromosome 6 using a panel of polymorphic sequence‐tagged sites, the hybrids were tested for tumorigenicity and metastasis in athymic mice. As reported previously, intact, normal chromosome 6 suppressed metastasis whether tumor cells were injected i.v. or into an orthotopic (i.e., intradermal) site. In contrast, metastasis was not suppressed in the neo6qdel hybrids. Tumorigenicity was unaffected in hybrids prepared with either chromosome 6 donor. These data strongly suggest that a human melanoma metastasis suppressor locus maps between 6q16.3‐q23 (≈40 cM). Int. J. Cancer 86:524–528, 2000.

Suppression of human melanoma metastasis following introduction of chromosome 6 is independent of NME1 (Nm23)

Metastasis is suppressed more than 95% following microcell-mediated transfer of a single copy of neomycin-tagged human chromosome 6 (neo6) into the human melanoma cell lines C8161 and MelJuSo. Concomitant with metastasis suppression is upregulation of NME1 (Nm23-H1) mRNA and protein expression. The purposes of this study were to determine whether NME1 expression was responsible for metastasis suppression in neo6/melanoma hybrids, and whether genes on chromosome 6 regulate NME1. Using neo6/C8161 cells, transfection of CAT reporter constructs linked to the NME1 promoter failed to consistently induce CAT. Therefore, it does not appear that genes on chromosome 6 directly control transcription of NME1. Transfection and overexpression of NME1 in MelJuSo, under the control of the CMV promoter, resulted in 40-80% inhibition of lung metastasis following i.v. inoculation of 2´10 cells. Only one transfectant of C8161 subclone 9 (C8161cl.9) cells was suppressed for metastasis. Control transfections with pCMVneo or pSV2neo did not suppress metastasis in either cell line. Taken together, these data suggest that NME1 can reduce metastatic potential of some human melanoma cells; but, this inhibitory activity appears to be independent of the metastasis suppression following introduction of chromosome 6 into C8161 and MelJuSo human melanoma cell lines.

Modulation of TNF-α mRNA production in rat C6 glioma cells by TNF-α, IL-1β, IL-6, and IFN-α: In vitro analysis of cytokine-cytokine interactions

Cytokines regulate the expression of other cytokines in the centrally derived rat C6 glioma cell line. However, the modulation of tumor necrosis factor-α (TNF-α, a pivotal proinflammatory cytokine) in C6 cells is unknown. Here we investigated the expression of TNF-α mRNA in C6 glioma cells in response to TNF-α, interleukin-1β (IL-1β), IL-1 receptor antagonist (IL-1Ra), interleukin-6 (IL-6), and interferon-α (IFN-α). The data show that (1) IL-1β induced a significant upregulation of TNF-α mRNA; (2) the effect of IL-1β on TNF-α mRNA expression was completely blocked by the concomitant application of IL-1Ra, which suggests specificity of IL-1β action through the IL-1 signaling receptor; (3) no detectable modulation of TNF-α mRNA expression was observed with the individual applications of TNF-α, IL-6, or IFN-α; (4) the concomitant treatments of TNF-α + IL-1β or TNF-α + IL-1β + IL-6 strongly upregulated TNF-α mRNA expression, whereas the concomitant application of TNF-α + IL-6 or IL-1β + IL-6 induced a moderate increase; and (5) IFN-α significantly attenuated induction of TNF-α mRNA by TNF-α + IL-1β + IL-6. Thus, IL-1β, TNF-α and IL-6 interact to upregulate TNF-α mRNA expression synergistically, and IFN-α acts as an inhibitory cytokine in C6 glioma cells. These findings also suggest that the rat C6 glioma cell line may be used as an in vitro model to characterize cytokine-cytokine interactions.

Brain tumor development in rats is associated with changes in central nervous system cytokine and neuropeptide systems

Cytokines have roles in tumor biology and induce neurological manifestations. Cytokines produced in response to a brain tumor may generate neurological manifestations via paracrine action. We investigated cytokine modulation in an in vivo brain tumor model with behavioral, morphological, and molecular approaches. Rat C6 glioma cells were implanted into the third cerebral ventricle of Wistar rats, their behavior was monitored, and the development of an intracranial tumor of astrocytic origin was confirmed by histology and positive immunostaining for vimentin, S-100 protein, and glial fibrillary acidic protein. Sensitive and specific RNase protection assays were used to analyze cytokine messenger RNA (mRNA) in brain regions from anorexic brain tumor-bearing animals. Brain tumor formation was associated with significant increased levels of interleukin (IL)-1beta, IL-1 receptor antagonist, IL-1 receptor type I, tumor necrosis factor (TNF)-alpha, and transforming growth factor (TGF)-beta1 mRNAs in the cerebellum, hippocampus, and hypothalamus. IL-1 receptor accessory proteins I and II mRNAs were increased in the cerebellum and hypothalamus. We also examined hypothalamic feeding-associated components: neuropeptide Y and proopiomelanocortin mRNAs were down-regulated, glycoprotein 130 mRNA levels were up-regulated, and leptin receptor (OB-R) mRNA levels were unchanged. These dissimilar profiles of mRNA expression suggest specificity of brain tumor-induced transcriptional changes. The data implicate cytokines as important factors in brain tumor-host interactions in vivo. The data also show that the C6 cell-induced glioma can be used as a behavioral-molecular model to study cytokine and neuropeptide modulation and action during the host biochemical and physiological responses to brain tumor development. Paracrine interactions seem pivotal because cytokine modulation was observed in various brain regions. These results also suggest that cytokine and neuropeptide changes during brain tumor progression are involved in brain tumor-associated neurological and neuropsychiatrical manifestations.