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. ^

The role of erbB2 receptor in human breast cancer metastasis

Overexpression of c-erbB-2 gene-encoded p185 has been correlated with lymph node metastasis and poor prognosis in breast cancer patients. To investigate whether overexpression of c-erbB-2 can enhance metastatic potential of human breast cancer cells, we compared the metastatic phenotypes of the parental MDA-MB-435 cells and the c-erbB-2 gene transfected 435.eB cells. In vivo experimental metastasis assays demonstrated that mice injected erbB2-overexpressing 435.eB transfectants formed significantly more metastatic tumors than the mice injected with parental and control cells. The changes in metastatic potential in vivo were accompanied by increased invasiveness in vitro . The transfectants and the parental cells all had similar growth rates and transformation potential. These findings suggest that c- erbB-2 gene can enhance the intrinsic metastatic potentials of MDA-MB-435 cells without increasing their transformation abilities. ^ Homophilic adhesion may affect invasive and metastatic potential of tumor cells. We found that Heregulin-β1 (HRG-β1), a growth factor that activates receptor kinases erbB3 and erbB4, can enhance aggregation of MCF-7 and SKBR3 human breast cancer cells. While investigating the downstream signals involved in HRG-β1-increased cell aggregation, we observed that HRG-β1 increased the kinase activities of extracellular signal-regulated protein kinase (ERK) and PI3K in these cells. By using different kinase inhibitors, we found that the HRG-β1-activated MEK1-ERK pathway has no demonstrable role in the induction of cell aggregation, whereas HRG-β1-activated PI3K is required for enhancing breast cancer cell aggregation. These results have provided one mechanism by which HRG-β1-activated signaling of erbB receptors may affect invasive/metastatic properties of breast cancer cells. ^ To identify the structural motifs within the erbB2 receptor that are required for erbB2 increased metastatic potential in breast cancer cells, we injected different forms of mutated erbB2 expressing MDA-MB-435 cell line transfectants with or without the EGF-like domain of heregulin-β1 protein (HRG/egf) into ICR-SCID mice to test the metastatic survival rate. The results show that an intact kinase domain of erbB2 receptor is required for erbB2 enhanced metastatic potential in these cells. The C-terminal tyrosine 1248 residue of erbB2 may also play a role in enhancing metastatic potential. Moreover, the results suggest that HRG/egf promote the metastatic potential of human breast cancer cells in vivo. ^

Fas ligand and soluble Fas -opposing molecules in melanoma lung metastasis

Deregulation of apoptotic cell death can result in aberrant accumulation of cells and increased tumor incidence. Fas (CD95) and Fas ligand (FasL) are a receptor-ligand pair whose activation induces apoptosis in many cell types. Previously, we demonstrated that low metastatic, Fas+ K1735-P murine melanoma cells spontaneously metastasize to the lung following orthotopic injection into FasL-deficient (gld) mice compared to wild-type (wt) controls. We further demonstrated that the expression of the Fas antagonist soluble Fas (sFas) directly correlates with disease stage in patients with melanoma, breast, and colon cancer. These findings document a role for host-derived FasL, in the control of metastatic disease and suggest a role for tumor-associated sFas in acquiring metastatic potential. To directly test whether FasL expressed on lymphocytes or on lung stromal cells restricts metastasis, bone marrow chimeras were generated between C3H wt and C3H gld mice. Chimeric animals were injected subcutaneously with 5 × 105 K1735-P and the incidence and number of spontaneous lung metastases scored. The data show that wt mice receiving gld marrow had a greater number of lung metastases (median 9.5, range 2–31) than gld mice reconstituted with wt marrow (median 1, range 0–31; p < 0.016). Interestingly, both groups had fewer metastases compared to gld controls (median 18.5, range 0–46) but more than wt controls (median 2, range 0–7). These observations provide the first evidence that both hematopoietic- and nonhematopoietic-host derived FasL, are important in the control of melanoma metastasis to the lung. To directly test whether tumor-associated sFas expression can enhance metastasis, K1735-P cells were transfected with three isoforms of sFas (Exo4Del, Exo6Del, and Exo3, 4, 6Del). RT-PCR and ELISA analysis confirmed the expression of sFas RNA and protein respectively. Following intravenous injection of 5 × 104 cells, sFas transfected cells formed significantly more experimental lung metastases [Exo6Del clone 3 (median 22, range 0–36), Exo6Del clone 7 (median 31, range 4–50), Exo3, 4, 6Del (median 22.5, range 13–48)] compared to vector control cells (median 6.5, range 3–29). Together, these data provide the first evidence that sFas is sufficient to enhance the metastatic potential of Fas+ melanoma cells. ^

Activation of c -Met contributes to growth and metastasis of human colorectal carcinoma

c-Met is the protein tyrosine kinase receptor for hepatocyte growth factor/scatter factor (HGF/SF) and mediates several normal cellular functions including proliferation, survival, and migration. Overexpression of c-Met correlates with progression and metastasis of human colorectal carcinoma (CRC). The goals of this study were to determine if overexpression of c-Met directly contributes to tumorigenicity and liver metastatic potential of colon cancer, and what are the critical pathways regulated by c-Met in this process. The studies used two colon tumor cell lines, KM12SM and KM20, which express high levels of constitutively active c-Met and are highly metastatic in nude mice. To examine the effects of c-Met overexpression, subclones of theses lines with reduced c-Met expression were obtained following transfection with a c-Met specific targeting ribozyme. Reduction of c-Met in KM12SM cells abolished liver metastases when cells were injected intrasplenically in an experimental metastasis assay. However, c-Met downregulation in theses clones was unstable. Three stable KM20 clones with a 25–35% reduction in c-Met protein levels but 60–90% reduction in basal c-Met autophosphorylation and kinase activity were obtained. While HGF increased c-Met kinase activity in the clones with reduced c-Met, the activity was less than that observed in parental or control transfected cells. Correlating with the reduction in c-Met kinase activity, subclones with reduced c-Met expression had significantly reduced in vitro growth rates, soft-agar colony forming abilities, and increased apoptosis. HGF/SF treatment did not affect anchorage-dependent growth or soft-agar colony forming abilities. Further, c-Met downregulation significantly impaired the ability of HGF/SF to induce migration. To examine the effects of reduced c-Met on tumor formation, parental and c-Met reduced KM20 cells were grown subcutaneously and intrahepatically in nude mice. c-Met downregulation delayed, but did not abolish growth at the subcutaneous site. When these cells were injected intrahepatically, both tumor incidences and size were significantly reduced. To further understand the molecular basis of c-Met in promoting tumor growth, the activation of several signaling intermediates that have been implicated in c-Met mediated growth, survival and migration were compared between KM20 parental cells and subclones with reduced c-Met expression levels. The expression and activity (as determined by phosphorylation) of AKT and Erk1/2 were unaltered. In contrast, Src kinase activity, as measured by immune complex kinase assay, was reduced 2–5 fold following c-Met downregulation. As Src has been implicated in growth, survival and migration, Src activation in c-Met overexpressing lines is likely contributing to the tumorigenic and metastatic capabilities of colon tumor cell lines that overexpress c-Met. Collectively, these results suggest that c-Met overexpression plays a causal role in the development of CRC liver metastases, and that c-Src and c-Met inhibitors may be of potential therapeutic benefit for late-stage colon cancer. ^

Heregulin -beta 1 directly associates with RhoA via the immunoglobulin -like domain thereby modulating breast cancer metastasis-related properties

Heregulins constitute a family of growth factors belonging to the epidermal growth factor (EGF) family. Breast cancers that overexpress specific members of the EGF receptor family (EGFR, ErbB2, ErbB3, ErbB4) have increased metastatic potential, and Heregulin-β1 (HRGβ1), a ligand for ErbB3 and ErbB4, has also been shown to induce metastasis-related properties in breast cancer cells in vitro. The secreted form of the HRGβ1 is composed of five distinct structural domains, including the N-terminal domain, an immunoglobulin-like domain (IgG-like), a glycosylation domain, an EGF-like domain, and a β1-specific domain. Of these, the EGF-like domain is well characterized for its function in metastasis-related properties as well as its structure. However, the contributions of the other HRGβ1 domains in breast cancer metastasis remains unclear. ^ To investigate this, HRGβ1 proteins with targeted domain deletions were purified and subjected to assays for metastasis-related properties, including aggregation, invasion, activation of EGFR family members, and motility of breast cancer cells. These assays showed that retaining the EGF-like domain of HRGβ1 is important for activation of EGFRs. Interestingly, the HRGβ1 protein lacking the IgG-like domain (NGEB) led to a decrease in breast cancer cell motility, indicating the IgG-like domain modulates cell motility, an important step in cancer metastasis. ^ To understand the underlying mechanisms, I performed protein sequence and structural analysis of HRGβ1 and identified that the IgG-like domain of HRGβ1 shares sequence homology and three-dimensional structural similarity with the IgG-like domain of TRIO. TRIO is a cytoplasmic protein that directly associates with RhoA, a GTPase involved in cell reorganization and cell motility. Therefore, I hypothesized that HRGβ1 may translocate inside the breast cancer cells through receptor mediated endocytosis and bind to RhoA via its IgG-like domain. I show wild type HRGβ1 but not NGEB binds RhoA in vitro and in vivo, leading to RhoA activation. Inhibition of HRG-β1 internalization via endocytosis disrupted HRGβ1 binding to RhoA. Additionally, breast cancer cell motility induced by HRG-β1 is reduced after treatment with inhibitors to both endocytosis and RhoA function, similar to levels seen with NGEB treatment. ^ Thus, in addition to the well-known role of HRGβ1 as an extracellular stimulator of the EGFR family members, HRGβ1 also functions within the cell as a binding partner and activator of RhoA to modulate cancer cell motility. ^