Metastatic Murine Melanoma Cell Line Research Articles

CCN2 Expression by Tumor Stroma Is Required for Melanoma Metastasis

Metastatic melanoma has an extremely poor prognosis with few durable remissions. The secreted matricellular protein connective tissue growth factor (CCN2) is overexpressed in cancers including melanoma and may represent a viable therapeutic target. However, the mechanism underlying the contribution of CCN2 to melanoma progression is unclear. Herein, we use the highly metastatic murine melanoma cell line B16(F10) and syngeneic mice, in which CCN2 expression is knocked out in fibroblasts, to demonstrate that loss of CCN2, either in melanoma cells or in the niche, impedes the ability of melanoma cells to invade. Specifically, loss of CCN2 in melanoma cells diminished their ability to invade through collagen in vitro, and loss of fibroblast-derived CCN2 decreased spontaneous metastases of melanoma cells from the skin to the lungs in vivo. Proliferation and tumor growth were not affected by loss of CCN2. CCN2-deficient B16(F10) cells showed reduced expression of the matricellular protein periostin; addition of recombinant periostin rescued the in vitro invasion defect of these cells. Immunohistochemical analysis of CCN2-deficient mice confirmed loss of periostin expression in the absence of CCN2. CCN2 and periostin mRNA levels are positively correlated with each other and with the stromal composition of human melanoma lesions but not BRAF mutations. Thus, CCN2 promotes invasion and metastasis via periostin and should be further evaluated as a possible therapeutic target for BRAF inhibitor-resistant melanoma.

CCN2 expression and localization in melanoma cells

The matricellular protein connective tissue growth factor (CTGF, CCN2) is overexpressed in several forms of cancer and may represent a novel target in anti-cancer therapy. However, whether CCN2 is expressed in melanoma cells is unknown. The highly metastatic murine melanoma cell line B16(F10) was used for our studies. Real time polymerase chain reaction analysis was used to detect mRNA expression of CCN1, CCN2, CCN3 and CCN4 in Western blot and immunofluorescence analyses were used to detect CCN2 protein. Inhibitors of signal transduction cascades were used to probe the mechanism underlying CCN2 expression in B16(F10) cells. CCN2 was expressed in B16(F10) cells, and was reduced by the FAK/src inhibitor PP2 and the MEK/ERK inhibitor U0126 indicating that CCN2 acts downstream of these pathways in B16(F10) murine melanoma cells. Expression of CCN1, CCN3 and CCN4 was not reduced by PP2 or U0126; in fact, expression of CCN4 mRNA was elevated by PP2 or U0126 treatment. To our surprise, CCN2 protein was detected in the nuclei of B16(F10) cells, and was undetectable in the cytoplasm. CCN2 was expressed in B16(F10) melanoma cells, adding to the list of cancer cells in which CCN2 is expressed. Of the CCN family members tested, only CCN2 is downstream of the highly oncogenic MEK/ERK pathway. CCN2 should be further evaluated for a possible role in melanoma growth and progression.

Nm23-H1 Metastasis Suppressor Phosphorylation of Kinase Suppressor of Ras via a Histidine Protein Kinase Pathway

The metastasis-suppressive activity of Nm23-H1 was previously correlated with its in vitro histidine protein kinase activity, but physiological substrates have not been identified. We hypothesized that proteins that interact with histidine kinases throughout evolution may represent partners for Nm23-H1 and focused on the interaction of Arabidopsis "two-component" histidine kinase ERS with CTR1. A mammalian homolog of CTR1 was previously reported to be c-Raf; we now report that CTR1 also exhibits homology to the kinase suppressor of Ras (KSR), a scaffold protein for the mitogen-activated protein kinase (MAPK) cascade. Nm23-H1 co-immunoprecipitated KSR from lysates of transiently transfected 293T cells and at endogenous protein expression levels in MDA-MB-435 breast carcinoma cells. Autophosphorylated recombinant Nm23-H1 phosphorylated KSR in vitro. Phosphoamino acid analysis identified serine as the major target, and two peaks of Nm23-H1 phosphorylation were identified upon high performance liquid chromatography analysis of KSR tryptic peptides. Using site-directed mutagenesis, we found that Nm23-H1 phosphorylated KSR serine 392, a 14-3-3-binding site, as well as serine 434 when serine 392 was mutated. Phosphorylated MAPK but not total MAPK levels were reduced in an nm23-H1 transfectant of MDA-MB-435 cells. The data identify a complex in vitro histidine-to-serine protein kinase pathway, which may contribute to signal transduction and metastasis.

Decreased nm23 expression, but not Ki-67 labeling index, is significantly correlated with lymph node metastasis of breast invasive ductal carcinoma

The nm23 gene was originally identified by differential hybridization of metastatic murine melanoma cell lines. Some experimental studies demonstrated a significantly reduced metastatic potential of melanoma cell lines transfected with the nm23 gene. In this study, we clarified the relationship between lymph node status and nm23 immunoreactivity, as well as Ki-67 labeling index (LI), of human breast cancer. Of the 44 breast invasive ductal carcinomas, nm23-diffusely positive expression [nm23(+)] was detected in 17 (38.6%), and focally positive/negative expression [nm23(+/-/-)] in 27 (61.4%) cases. Lymph node metastasis was found at a significantly higher incidence in the nm23(+/-/-) cases (18/27, 66.7%) than in the nm23(+) cases (4/17, 23.5%) (p>0.001). In the lymph node metastasis-positive cases, mean LI of Ki-67 cells was 20.9% at the center of the tumors and 24.0% at the advanced margins. In the lymph node metastasis-negative cases, mean LI of Ki-67 cells was 12.4% at the center of the tumors and 27.2% at the advanced margins. Decrease of nm23 expression, but not Ki-67 LI, was significantly correlated with lymph node metastasis of breast invasive ductal carcinoma.

Loss of nm23 expression predicts distal metastases and poorer survival for breast cancer.

The nm23 gene, originally identified by differential hybridization of metastatic murine melanoma cell lines has been associated with decreased metastatic potential. In this study, we evaluated the utility of nm23 expression levels as a predictive and a prognostic biomarker for distal metastases and poor survival in a Chinese cohort of 168 breast cancer patients. Our study indicates that high nm23 expression is associated with older age (older than 35 years) and smaller tumor size. There is no statistically significant association between nm23 expression and pathologic type or axillary metastasis. High nm23 expression is associated with the absence of distal metastases. Nearly 80% of women with high nm23 expression are alive after 10 years compared to 25% with low expression; at five years the cumulative survival proportions are 86% and 46%, respectively. The Cox model for survival indicates that controlling for tumor size and presence of axillary metastases at diagnosis, the hazard for women with low nm23 expression is nearly 4 times that of women with high nm23 expression. Low nm23 expression is predictive of distal metastases and appears to be a risk factor that is independent of the presence or absence of positive axillary nodes at diagnosis.

Gelsolin functions as a metastasis suppressor in B16-BL6 mouse melanoma cells and requirement of the carboxyl-terminus for its effect.

Gelsolin, an actin-binding protein, is implicated as a critical regulator in cell motility. In addition, we have reported that cellular levels of gelsolin are decreased in various tumor cells, and overexpression of gelsolin by gene transfer suppresses tumorigenicity. We sought to assess the effects of gelsolin overexpression on metastasis and to determine the importance of a carboxyl-terminus that confers Ca(2+) dependency on gelsolin for effects of its overexpression. Expression vectors with cDNA encoding either full-length wild-type or His321 mutant form, isolated from a flat revertant of Ras-transformed cells and a carboxyl-terminal truncate, C-del of gelsolin, were transfected into a highly metastatic murine melanoma cell line, B16-BL6. Expression of introduced cDNA in transfectants was confirmed using Western blotting, 2-dimensional gel electrophoresis and reverse transcription-polymerase chain reaction (RT-PCR). We characterized phenotypes of transfectants, such as growth rate, colony formation in soft agar, cell motility and metastasis formation in vivo. Transfectants expressing the wild-type, His321 mutant and C-del gelsolin exhibited reduced growth ability in soft agar. Although expression of integrin beta1 or alpha4 on the cell surface of transfectants was not changed, wild-type and His321 mutant gelsolin, except for C-del gelsolin, exhibited retardation of cell spreading, reduced chemotatic migration to fibronectin and suppressed lung colonization in spontaneous metastasis assay. Gelsolin may function as a metastasis suppressor as well as a tumor suppressor gene. The carboxyl-terminus of gelsolin is important for retardation of cell spreading, reduced chemotasis and metastasis suppression.

Heterogeneous expression of nm23 gene product as a predictor of lymph nodal status in human breast cancer.

The nm23 gene was originally identified by differential hybridization of metastatic murine melanoma cell lines. Some experimental studies demonstrated a significantly low metastatic potential of melanoma cell lines transfected with the nm23 gene. In this study, we clarified the relationship between intracellular nm23-immunoreactivity and lymph nodal status of human breast cancer. We analyzed 82 surgically removed breast tumors including 67 invasive carcinomas (ductal, lobular and mucinous carcinomas). The nm23 expression was diffusely positive in the benign tumors and non-invasive carcinomas. Of the invasive ductal carcinomas, lymph node metastasis was found in 67.7% (21/31) of the focally positive/negative cases and in 18.2% (4/22) of the diffusely positive cases (p<0.001). Immunohistochemically, advanced margins of invasive carcinomas with lymph node metastasis were shown to be negative for nm23 expression, while intraductal carcinoma components were positive. This observation suggested that focally positive/negative nm23 expression can be a predictor of lymph node metastasis of invasive ductal breast carcinoma.

Reduction of telomeric signals in murine melanoma and human breast cancer cell lines treated with 3'-azido-2'-3'-dideoxythymidine.

The purpose of this study was to determine, using fluorescence in situ hybridization (FISH), whether treatment of cancer cells with 3'-azido-2'-3'-dideoxythymidine (AZT) has any effect on telomere length as determined by the telomeric signal intensity. To do so, we treated a metastatic murine melanoma cell line (K-1735 clone X-21) and a human breast cancer cell line (MCF-7) with three concentrations of AZT for 72 h at 37ûC. FISH preparations processed using an all-human telomeric DNA probe showed a significantly reduced, concentration-dependent telomeric signal intensity in interphase and metaphase spreads of AZT-treated cells as compared with the signal intensity in untreated controls, which showed no reduction. We conclude from these preliminary results that AZT has the potential of targeting the telomeric ends of chromosomes in cancer cells and promoting cell death and could well be tested along with other chemotherapy drugs given to cancer patients for this purpose.

Nm23-H1: Genetic Alterations and Expression Patterns in Tumor Metastasis

Nm23-H1 originally was discovered on the basis of its reduced RNA expression in very highly metastatic murine melanoma cell lines. Since that time, it has been shown to reduce the metastatic potential of highly aggressive tumors and to be an indicator of poor patient prognosis in many cancer types. Although LOH of NME1 is prevalent in many tumors, this event does not predict reliably the overall levels of Nm23-H1 expression and therefore cannot be used as a genetic screen for the aggressive potential of a cancer. In addition, mutant forms of the gene exist; however, expression patterns seem to be the key to the differences observed between tumor cells of high and low metastatic potential. Differential Nm23-H1 expression may reflect transcriptional regulation and/or the stability of the Nm23-H1 transcript. The mechanism of action of the protein is still unknown; however, the protein does possess a unique kinase activity that is essential in other organisms. The histidine protein-kinase activity is the sole known activity that correlates tightly with the ability of Nm23-H1 to suppress cell motility. The identification of other components comprising this signaling pathway will be the next step toward unraveling the mysteries of this metastasis suppressor.

Dolastatin-10 induces polyploidy, telomeric associations and apoptosis in a murine melanoma cell line.

Our purpose was to study the effects of dolastatin-10 (Dol-10) on chromosome morphology, telomeric associations, induction of polyploidy and cell death in a metastatic murine melanoma cell line, K1735 clone X-21. Murine melanoma cells were treated with various concentrations (10 ng/ml, 100 ng/ml and 1000 ng/ml) of Dol-10 for 4, 24 and 72 h continuously and harvested immediately without recovery. In another set of experiments, cells were treated for 4 h with the same concentrations, washed with prewarmed medium and then allowed to recover in drug-free medium for 24 h and subsequently harvested. Our preliminary results indicated: i) a drug-mediated increase in the frequency of metaphases, with telomeric associations resulting in multicentric and ring configurations; ii) induction of clumping in metaphase chromosomes; iii) induction of polyploidy as a result of endoreduplication; iv) formation of micronucleated cells; and v) induction of cell death. These observations indicated that Dol-10 could be a potent antineoplastic drug against malignant melanoma. In addition to its reported interaction with cell microtubules, the mechanism of action of Dol-10 may be mediated through the loss of telomeric repeats and induction of chromosome aberrations.

Asn-linked oligosaccharide-dependent interaction between laminin and gp120/140. An alpha 6/beta 1 integrin.

Receptor-mediated recognition and adhesion to laminin, a specific glycoprotein from basement membranes, exert an important role in many biological phenomena. Studying cell surface proteins of B16-F10, a metastatic murine melanoma cell line, we identified a 120-140 kDa glycoprotein (gp120/140) that binds laminin. This glycoprotein was recognized by a polyclonal antibody raised against the human fibronectin receptor beta 1-integrin chain, as well as immunoprecipitated by an anti-alpha 6 chain (monoclonal antibody GoH3), characterizing it as an alpha 6/beta 1-integrin. Its binding to laminin was specific and displayed moderate affinity, as its apparent dissociation constant was 18 nM. To characterize the influence of carbohydrate moieties on the laminin-gp120/140 interaction, metaperiodate oxidation, metabolic inhibition of glycosylation, and enzymatic deglycosylation studies were performed. Our results indicate that gp120/140 Asn-linked oligosaccharides play a part in this interaction. Reciprocally, both metaperiodate and N-glycanase treatment of native laminin reduced its binding to gp120/140, characterizing the latter as a lectin-like molecule. These results point to glycosylation processes as a possible mechanism for variable binding specificity profiles among integrins.

Influence of collagen substrata on glycosaminoglycan production by B16 melanoma cells.

A cloned metastatic murine melanoma cell line exhibited similar growth characteristics when propagated on either type I collagen, type IV collagen, or plastic. However, cells grown on both types of collagen exhibited an altered cellular morphology and on type IV collagen only, an increased substrate adhesiveness, relative to those maintained on a plastic substratum. Incorporation of [3H]glucosamine and [35S]sulfate into glycosaminoglycans (GAGs) of cells grown on collagen substrates was 20% and 40% less, respectively, than cells grown on plastic, whereas degradation of cell-associated [35S]sulfate-labeled GAGs was similar in cells grown on collagen or plastic. Although the composition of GAGs was similar in all cultures, consisting of approximately 60% chondroitin and 40% heparin or heparan sulfate, the degree of sulfation of the heparin or heparan sulfate molecules was markedly decreased in cultures grown on collagen. The results indicate that the composition of the extracellular matrix influences the biological behavior of B16 melanoma cells, in part by altering the amount and nature of the GAG molecules produced.