Melanoma Metastasis Suppressor Research Articles

Functional analysis of the emerging roles for the KISS1/KISS1R signaling pathway in cancer metastasis

Cancer metastasis, a process that primary tumor cells disseminate to secondary organs, is the most lethal and least effectively treated characteristic of human cancers. Kisspeptins are proteins encoded by the KISS1 gene that was originally described as a melanoma metastasis suppressor gene. Then, Kisspeptins were discovered as the natural ligands of the G-protein-coupled receptor 54 (GPR54) that is also called KISS1R. The KISS1/KISS1R signaling is essential to control GnRH secretion during puberty and to establish mammalian reproductive function through the hypothalamic-pituitary-gonadal (HPG) axis. Although KISS1 primarily plays a suppressive role in the metastasis progression in several cancer types, emerging evidence indicates that the physiological effect of KISS1/KISS1R in cancer metastasis is tissue context-dependent and still controversial. Here, we will discuss the epigenetic mechanism involved in the regulation of KISS1 gene expression, the context-dependent role of KISS1/KISS1R, prometastasis/anti-metastasis signaling pathways of KISS1/KISS1R, and the perspective anticancer therapeutics via targeting KISS1/KISS1R.

MiR-542-3p suppresses invasion and metastasis by targeting the proto-oncogene serine/threonine protein kinase, PIM1, in melanoma

Aberrant microRNAs (miRNAs) contribute to metastasis of various cancer types, including melanoma. miR-542-3p has been characterized as a tumor suppressor in several cancers. However, the exact expression patterns of miR-542-3p and the precise molecular mechanisms underlying its role in melanoma require further exploration. In the current study, we demonstrated that miR-542-3p is significantly downregulated in melanoma cell lines and clinical specimens. Exogenous expression of miR-542-3p resulted in marked inhibition of melanoma cell migration, invasion and epithelial-mesenchymal transition (EMT) in vitro and lung metastasis in vivo. The proto-oncogene serine/threonine protein kinase, PIM1, was identified as a direct target of miR-542-3p using luciferase reporter assay, qRT-PCR and western blot analyses. Overexpression of PIM1 partially rescued miR-542-3p-mediated suppression of cell migration, invasion and EMT. Our results collectively indicate that miR-542-3p serves as a metastasis suppressor in melanoma, supporting its utility as a promising therapeutic candidate.

Dynamic interactions between cancer cells and the embryonic microenvironment regulate cell invasion and reveal EphB6 as a metastasis suppressor.

Metastatic dissemination drives the high mortality associated with melanoma. However, difficulties in visualizing in vivo cell dynamics during metastatic invasion have limited our understanding of these cell behaviors. Recent evidence has revealed that melanoma cells exploit portions of their ancestral embryonic neural crest emigration program to facilitate invasion. What remains to be determined is how embryonic microenvironmental signals influence invasive melanoma cell behavior, and whether these signals are relevant to human disease. To address these questions, we interrogated the role of the neural crest microenvironment in dictating the spatiotemporal pattern of melanoma cell invasion in the chick embryo using 2-photon time-lapse microscopy. Results reveal that both permissive and inhibitory neural crest microenvironmental signals regulate the timing and direction of melanoma invasion to coincide with the neural crest migration pattern. These cues include bidirectional signaling mediated through the ephrin family of receptor tyrosine kinases. We demonstrate that EphB6 reexpression forces metastatic melanoma cells to deviate from the canonical migration pattern observed in the chick embryo transplant model. Furthermore, EphB6-expressing melanoma cells display significantly reduced metastatic potential in a chorioallantoic membrane (CAM) metastasis assay. These data on melanoma invasion in the embryonic neural crest and CAM microenvironments identify EphB6 as a metastasis suppressor in melanoma, likely acting at the stage of intravasation. This article links cellular metastasis to behaviors observed in the ancestrally related embryonic neural crest and demonstrates the powerful influence of the embryonic microenvironment in regulating cell migratory behavior.

Abstract 3868: Identification of PHLPP1 as a novel metastasis suppressor in melanoma .

Abstract Metastatic dissemination is the cause of nearly all melanoma-related deaths. Despite an improving understanding of genetic lesions present in human melanomas, how these alternations relate to the metastatic process is not well understood. Previously, we found that activation of the PI3K/Akt pathway is a driving force in metastatic melanomas. Here we identify PHLPP1 as a novel metastasis suppressor in melanoma. PHLPP1 was recently identified as a Ser/Thr-specific phosphatase, belonging to pleckstrin homology (PH) domain leucine-rich repeat protein phosphatase family. The two isoforms of this family, PHLPP1 and PHLPP2 control kinase signaling in the AKT family by catalyzing the dephosphorylation of the hydrophobic phosphorylation motif. PHLPP1 can specifically dephosphorylate AKT2 and AKT3, whereas PHLPP2 dephosphorylates AKT1 and AKT3. To study the role of PHLPPs in metastatic melanoma, we first examined the expression of PHLPPs in 30 melanoma metastases, 30 primary melanomas, and 9 normal skin tissues using tissue microarray analysis. We found that PHLPP1 was significantly downregulated or lost in metastatic melanoma samples, but not PHLPP2. Moreover, analysis of various human melanoma cell lines (12 highly metastatic, 14 poorly metastatic melanoma cell lines and 1 normal melanoctye cell line) demonstrated that PHLPP1 expression correlated with metastatic behavior, supporting the notion that PHLPP1 is involved in the metastatic regulation of melanoma. To confirm a functional role for PHLPP1 in metastasis, we forced the expression of PHLPP1 and PHLPP2 in B16 and A375sm melanoma cell lines. Overexpression of PHLPP1 dramatically inhibited metastasis in both cell lines. Unexpectedly, the PHLPP2 isoform had little influence on metastatic ability in both cell lines in vivo, despite the fact that both PHLPP1 and PHLPP2 were able to inhibit in vitro cell proliferation, motility, migration as well as colony formation in soft agar. Notably, introduction of PH mutant forms of PHLPP1 still blunted the metastatic potential of both cell lines, whereas a phosphatase-deficient C-terminal mutant did not affect metastasis. Interestingly, transfected AKT2 active form could rescue PHLPP1-mediaed inhibition of metastasis. Our data demonstrate at the first time that PHLPP1 plays a critical role in inhibiting metastasis through its phosphatase activity, and suggest that PHLPP1 could be a diagnostic marker and therapeutic target for metastatic melanoma. Citation Format: Yanlin Yu, Andrew Lu, Meng Dai, Glenn Merlino. Identification of PHLPP1 as a novel metastasis suppressor in melanoma . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3868. doi:10.1158/1538-7445.AM2013-3868

Erythroid Differentiation Regulator 1, an Interleukin 18-Regulated Gene, Acts as a Metastasis Suppressor in Melanoma

Erythroid differentiation regulator (Erdr1) was first discovered in mouse leukemia cell lines and functions as a stress-related survival factor. This study investigated whether Erdr1 regulates murine melanoma progression, as well as the mechanism involved in Erdr1-regulated metastasis. The expression of Erdr1 is negatively correlated with IL-18 expression, which has a pro-cancer effect in melanoma. To study the role of Erdr1 as an anti-cancer factor, cell migration, invasion, and proliferation were measured. Erdr1 overexpression markedly inhibited the level of cell migration, invasion, and proliferation in B16F10 cells in vitro. In addition, Erdr1 overexpression significantly suppressed melanoma lung colonization, metastasis, and tumor growth in vivo. To identify the factors involved in Erdr1-reduced metastasis, heat shock protein 90 (HSP90), a well-known stress protein and contributor to tumor metastasis, was examined. We found that HSP90 was significantly decreased in Erdr1-overexpressing cells. Functional analysis demonstrated that HSP90 small-interfering RNA transfection reduced the migration ability and metastasis of melanoma. In conclusion, Erdr1 shows a powerful anti-metastasis effect that leads to the ability to reduce the metastatic potential of murine malignant melanoma cells. Erdr1 is an anti-metastatic factor that may be a possible therapeutic target for treatment of melanoma.

Epigenetic deregulation of TCF21 inhibits metastasis suppressor KISS1 in metastatic melanoma

Metastatic melanoma is a fatal disease due to the lack of successful therapies and biomarkers for early detection and its incidence has been increasing. Genetic studies have defined recurrent chromosomal aberrations, suggesting the location of either tumor suppressor genes or oncogenes. Transcription factor 21 (TCF21) belongs to the class A of the basic helix-loop-helix family with reported functions in early lung and kidney development as well as tumor suppressor function in the malignancies of the lung and head and neck. In this study, we combined quantitative DNA methylation analysis in patient biopsies and in their derived cell lines to demonstrate that TCF21 expression is downregulated in metastatic melanoma by promoter hypermethylation and TCF21 promoter DNA methylation is correlated with decreased survival in metastatic skin melanoma patients. In addition, the chromosomal location of TCF21 on 6q23-q24 coincides with the location of a postulated metastasis suppressor in melanoma. Functionally, TCF21 binds the promoter of the melanoma metastasis-suppressing gene, KiSS1, and enhances its gene expression through interaction with E12, a TCF3 isoform and with TCF12. Loss of TCF21 expression results in loss of KISS1 expression through loss of direct interaction of TCF21 at the KISS1 promoter. Finally, overexpression of TCF21 inhibits motility of C8161 melanoma cells. These data suggest that epigenetic downregulation of TCF21 is functionally involved in melanoma progression and that it may serve as a biomarker for aggressive tumor behavior.

Calcium homeostasis in human melanocytes: role of transient receptor potential melastatin 1 (TRPM1) and its regulation by ultraviolet light

Transient receptor potential melastatin (TRPM) is a subfamily of ion channels that are involved in sensing taste, ambient temperature, low pH, osmolarity, and chemical ligands. Melastatin 1/TRPM1, the founding member, was originally identified as melanoma metastasis suppressor based on its expression in normal pigment cells in the skin and the eye but not in aggressive, metastasis-competent melanomas. The role of TRPM1 and its regulation in normal melanocytes and in melanoma progression is not understood. Here, we studied the relationship of TRPM1 expression to growth and differentiation of human epidermal melanocytes. TRPM1 expression and intracellular Ca(2+) levels are significantly lower in rapidly proliferating melanocytes compared to the slow growing, differentiated melanocytes. We show that lentiviral short hairpin RNA (shRNA)-mediated knockdown of TRPM1 results in reduced intracellular Ca(2+) and decreased Ca(2+) uptake suggesting a role for TRPM1 in Ca(2+) homeostasis in melanocytes. TRPM1 knockdown also resulted in a decrease in tyrosinase activity and intracellular melanin pigment. Expression of the tumor suppressor p53 by transfection or induction of endogenous p53 by ultraviolet B radiation caused repression of TRPM1 expression accompanied by decrease in mobilization of intracellular Ca(2+) and uptake of extracellular Ca(2+). These data suggest a role for TRPM1-mediated Ca(2+) homeostasis, which is also regulated by ultraviolet B, in melanogenesis.

Biomarkers of Metastatic Melanoma

Melanoma, one of the most aggressive forms of human cancer, has undergone an alarming increase in incidence in recent years. Early detection is a prerequisite for proper diagnosis and therapy orientation. Soluble biomarkers are an important tool for early diagnosis. Markers that are associated with melanocyte functions imply the enzymes involved in melanin synthesis and the melanin-related metabolites. Proteins such as autocrine melanocyte cell growth factor and melanoma metastasis suppressor have gained attention in the biomarkers domain. The antimelanoma immune response elicited in patients can not only provide new biomarkers but important therapeutic approaches in specific treatments. All the molecules generated during the metastasis process, invasion of neighboring tissue, angiogenesis, invading lymphatic/blood vessels and establishing new tumors at a distant site, are targets for biomarker discovery.

Stepping on the GAS: a brake pedal for melanoma metastasis?

Stepping on the GAS: a brake pedal for melanoma metastasis?

A genome-wide shRNA screen identifies GAS1 as a novel melanoma metastasis suppressor gene

Metastasis suppressor genes inhibit one or more steps required for metastasis without affecting primary tumor formation. Due to the complexity of the metastatic process, the development of experimental approaches for identifying genes involved in metastasis prevention has been challenging. Here we describe a genome-wide RNAi screening strategy to identify candidate metastasis suppressor genes. Following expression in weakly metastatic B16-F0 mouse melanoma cells, shRNAs were selected based upon enhanced satellite colony formation in a three-dimensional cell culture system and confirmed in a mouse experimental metastasis assay. Using this approach we discovered 22 genes whose knockdown increased metastasis without affecting primary tumor growth. We focused on one of these genes, Gas1 (Growth arrest-specific 1), because we found that it was substantially down-regulated in highly metastatic B16-F10 melanoma cells, which contributed to the high metastatic potential of this mouse cell line. We further demonstrated that Gas1 has all the expected properties of a melanoma tumor suppressor including: suppression of metastasis in a spontaneous metastasis assay, promotion of apoptosis following dissemination of cells to secondary sites, and frequent down-regulation in human melanoma metastasis-derived cell lines and metastatic tumor samples. Thus, we developed a genome-wide shRNA screening strategy that enables the discovery of new metastasis suppressor genes.

Large isoform of MRJ (DNAJB6) reduces malignant activity of breast cancer

IntroductionMammalian relative of DnaJ (MRJ [DNAJB6]), a novel member of the human DnaJ family, has two isoforms. The smaller isoform, MRJ(S), is studied mainly for its possible role in Huntington's disease. There are no reports of any biologic activity of the longer isoform, MRJ(L). We investigated whether this molecule plays any role in breast cancer. Our studies were prompted by interesting observations we made regarding the expression of MRJ in breast cancer cell lines and breast cancer tissue microarrays, as described below.MethodsExpression of MRJ(L) from several breast cancer cell lines was evaluated using real-time PCR. Relative levels of the small and large isoforms in breast cancer cell lines were studied using Western blot analysis. A breast cancer progression tissue microarray was probed using anti-MRJ antibody. MRJ(L) was ectopically expressed in two breast cancer cell lines. These cell lines were evaluated for their in vitro correlates of tumor aggressiveness, such as invasion, migration, and anchorage independence. The cell lines were also evaluated for in vivo tumor growth and metastasis. The secreted proteome of the MRJ(L) expressors was analyzed to elucidate the biochemical changes brought about by re-expression of MRJ(L).ResultsWe found that MRJ(L) is expressed at a significantly lower level in aggressive breast cancer cell lines compared with normal breast. Furthermore, in clinical cases of breast cancer expression of MRJ is lost as the grade of infiltrating ductal carcinoma advances. Importantly, MRJ staining is lost in those cases that also had lymph node metastasis. We report that MRJ(L) is a protein with a functional nuclear localization sequence. Expression of MRJ(L) via an exogenous promoter in breast cancer cell line MDA-MB-231 and in MDA-MB-435 (a cell line that metastasizes from the mammary fat pad) decreases their migration and invasion, reduces their motility, and significantly reduces orthotopic tumor growth in nude mice. Moreover, the secreted proteome of the MRJ(L)-expressing cells exhibited reduced levels of tumor progression and metastasis promoting secreted proteins, such as SPP1 (osteopontin), AZGP1 (zinc binding α2-glycoprotein 1), SPARC (osteonectin), NPM1 (nucleophosmin) and VGF (VGF nerve growth factor inducible). On the other hand, levels of the secreted metastasis-suppressor KiSS1 (melanoma metastasis suppressor) were increased in the secreted proteome of the MRJ(L)-expressing cells. We confirmed by quantitative RT-PCR analysis that the secreted profile reflected altered transcription of the respective genes.ConclusionCollectively, our data indicate an important role for a totally uncharacterized isoform of DNAJB6 in breast cancer. We show that MRJ(L) is a nuclear protein that is lost in breast cancer, that regulates several key players in tumor formation and metastasis, and that is functionally able to retard tumor growth.

Dermatopathological indicators of poor melanoma prognosis are significantly inversely correlated with the expression of NM23 protein in primary cutaneous melanoma

Some dermatopathological parameters are recognized as dominant indicators of high metastatic potential in melanoma, especially Breslow thickness, ulceration, Clark's level of invasion and mitotic rate. Because NM23 protein is the product of a melanoma metastasis suppressor gene, the aim of this study was to compare such dermatopathological indicators of melanoma prognosis with NM23 protein expression in primary cutaneous melanoma. The immunohistochemical NM23 expression was semiquantitatively assessed in 30 primary cutaneous melanomas. Ten dermatopathological parameters were evaluated and compared with NM23 expression. A significant inverse correlation was found for NM23 expression in comparison with Breslow thickness (p < 0.01), ulceration (p < 0.05), Clark's level (p < 0.01), mitotic rate (p < 0.05), and vertical growth phase (p < 0.05). By contrast, no significant correlation was found for NM23 expression in comparison with cell morphology, presence of adjacent nevus, pigmentation, tumor-infiltrating lymphocytes, and regression was impossible to evaluate. The expression of NM23 protein in primary cutaneous melanoma is significantly inversely correlated with dermatopathological parameters currently recognized as powerful indicators of melanoma prognosis. NM23 may be therefore considered in the dermatopathological evaluation of primary cutaneous melanoma.

Human melastatin 1 (TRPM1) is regulated by MITF and produces multiple polypeptide isoforms in melanocytes and melanoma

Melastatin 1 (MLSN1), originally identified as melanoma metastasis suppressor, represents a TRPM subfamily of transient receptor potential (TRP) proteins which serve diverse biological roles in a wide variety of cell types. Down-regulation of MLSN1 expression in human cutaneous melanoma, as indicated by in situ hybridization, appears to be a prognostic marker for melanoma metastasis. However, the exact physiological function(s) of MLSN1, the mechanism(s) involved in the regulation of its expression and its role in melanoma tumour progression are not yet clear. In this study, we identified a 654 bp upstream sequence of MLSN1, containing four E boxes (E1-E4), including an 11 bp M box (E4), that is sufficient for melanocyte-specific transcription and activation by the melanocyte transcription factor MITF (a bHLH-zip factor). Deletion analysis showed that the two distal E boxes (E3 and E4) in the MLSN1 promoter are required for both its activation by MITF and its constitutive activity in melanoma cells. Western blot analysis using polyclonal rabbit anti-human MLSN1 antibodies identified several polypeptides, presumably generated by both alternative splicing of MLSN1 messenger RNA (mRNA) and proteolytic cleavage, in both melanocytes and metastatic melanoma cells. Thus, multiple mechanisms appear to regulate MLSN1 expression in melanocytes and melanoma cells.

A bioinformatics approach to identifying fetal development genes

Gene regulation of fetal development is not well understood. In part, insulin and insulin-like growth factors (IGF) modulate placental steroid synthesis (PSS), which in turn modulates fetal growth. However, many of the genes that participate in this function remain to be identified. To find such genes, we examined the expression patterns of known IGF and placental steroid synthesis (IGF/PSS) genes in 1176 human cDNA libraries. We found a set of eight known IGF/PSS genes (PL-4, hCG, PAPP-A, EMBP, PLAP, P450 aromatase, P450scc, and 3-beta-HSD) that shared a highly similar expression profile across these libraries. We used these eight as bait in a search for other genes that showed very similar expression, and that might thus be related in function. We found ten genes closely co-expressed with the eight bait genes, but not previously reported as linked to IGF/PSS. Of these ten, six were previously reported as associated with cell growth in fetal and/or cancer tissues (malignant melanoma metastasis suppressor, PLAC-1, PSG10, PSG-beta1, serine palmitoyl transferase, and TONDU). Four are EST sequences, here named PLAC2, PLAC3, PLAC4, and PLAC5. Co-expression provides a method to identify which human genes are promising candidates for further experiments to determine their roles in fetal development.

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 ( approximately 40 cM).

The relationship of BRMS1 and RhoGDI2 gene expression to metastatic potential in lineage related human bladder cancer cell lines.

We have recently characterized a human bladder cancer cell line T24 and a more aggressive lineage related variant of it, T24T. To gain further insights, we have studied their metastatic ability in an in vivo model system. Results show that T24 forms significantly fewer [4/12 (1/11) mice had metastases with 1-2 lesions/mouse] metastasis in SCID/bg mice than T24T [14/14 (6/6) mice had metastases with a mean of 24-28 lesions/mouse]. To begin exploring the mechanisms underlying this difference, we evaluated the mRNA and protein expression levels of metastasis-suppressor genes, known to be important in the progression of other cancers, in our model of bladder cancer progression. A higher mRNA expression of BRMS1, a metastasis suppressor in breast cancer, was observed in T24 cells. In addition, RhoGDI2 mRNA expression was only observed in T24 when compared to T24T, suggesting that Rho activation might play a significant role in the metastatic cascade. However, a basal level mRNA expression of KISS1, described as metastasis suppressor in melanoma and breast, was observed in both the lines and had slightly higher expression in T24T. No difference of Nm23-H1, KAI1, MKK4/SEK1 and E-Cadherin protein levels were noted between these two lines. In summary, it appears that the T24/T24T paired cell lines constitute a useful model for the study of human bladder cancer metastasis that will allow both the discovery and mechanistic evaluation of genes potentially involved in this process.

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 increasing 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.