Expression of stromal cell markers in distinct compartments of human skin cancers

BACKGROUND: The importance of changes in the supporting tumor stroma for cancer initiation and progression is well established. The characteristics of an activated tumor stroma, however, are not completely understood. In an effort to better characterize the desmoplastic response to human skin tumors, we evaluated the expression pattern of three stromal cell markers, fibroblast-activation protein (FAP), endoglyx-1, and endosialin, in a series of melanocytic and epithelial skin tumors. METHODS: Immunohistochemistry using antibodies against FAP, endoglyx-1, and endosialin was carried out in skin samples obtained from 43 patients. Furthermore, microarray data from an independent set of human skin cancers were analyzed. RESULTS: FAP-positive fibroblasts were detected in all tumor tissues tested, including cases of melanocytic nevi, melanoma metastases, basal cell carcinomas, and squamous cell carcinomas. In cutaneous melanoma metastases, we identified different compartments within the stromal response on the basis of the regions of FAP expression. Endoglyx-1 expression was confined to normal and tumor blood vessel endothelium including 'hot spots' of neoangiogenesis within the cutaneous melanoma metastases. Endosialin was selectively induced in subsets of small- and medium-sized tumor blood vessels in melanoma metastases and squamous cell carcinomas. CONCLUSIONS: These data describe novel aspects of stromal marker expression in distinct compartments of human skin tumors and may point to potential targets for novel therapeutic strategies aimed at the tumor stroma.     

The role of RhoC in growth and metastatic capacity of melanoma

Background:  RhoC overexpression in tumor cells promotes invasive and metastatic behavior. RhoC expression levels have been correlated with tumor progression and metastasis in multiple human cancers. In melanoma, RhoC is upregulated in highly metastatic tumors. Induced expression in melanoma cell lines resulted in invasion and metastasis, whereas inhibition of RhoC reversed the metastatic phenotype both in vitro and in vivo .
Methods:  RhoC mRNA and protein expression in two human melanoma cell lines (DX3aza and MeWo) and pooled primary melanocytes were investigated by means of real-time quantitative polymerase chain reaction and western blotting. RhoC protein expression was evaluated in 123 primary cutaneous melanoma samples by the use of immunohistochemistry and correlated with known prognostic features.
Results:  RhoC upregulation was observed in the highly metastatic DX3aza cell line, whereas in MeWo, only low expression levels could be detected. RhoC expression in primary cutaneous melanoma was strongly associated with thicker and ulcerated tumors. RhoC expression was associated with the presence of lymphatic metastases at the time of diagnosis and shorter disease-free and overall survival rates, without being an independent predictor.
Conclusion:  These results further support a role for RhoC in growth and metastasis of melanoma.     

Stromal Fibroblast-Specific Expression of ADAM-9 Modulates Proliferation and Apoptosis in Melanoma Cells In Vitro and In Vivo

ADAMs are members of the zinc metalloproteinase superfamily characterized by the presence of disintegrin and metalloprotease domains. In human melanoma, ADAM-9 is expressed in focalized areas of the tumor-stroma border in both melanoma and stromal cells. However, the role of ADAM-9 in melanoma progression remains elusive. To analyze the role of stromal-derived ADAM-9 for the growth and survival of melanoma cells, we have used in vitro coculture systems of melanoma cells and ADAM-9(-/-) fibroblasts. Coculture of melanoma cells in the presence of ADAM-9(-/-) fibroblasts led to increased melanoma cell proliferation and reduced apoptosis as compared with control cocultures. We identified TIMP-1 and sTNFRI as the two relevant factors expressed in increased amounts in culture supernatants from ADAM-9(-/-) fibroblasts. TIMP-1 was associated with induced melanoma cell proliferation, whereas soluble TNFR1 mediated the reduced cellular apoptosis in vitro. In vivo, injection of murine melanoma cells into the flank of ADAM-9(-/-) animals resulted in the development of significantly larger tumors than in wild-type animals as a result of increased proliferation and decreased apoptosis of melanoma cells. Taken together, stromal expression of ADAM-9 during melanoma development modulates the expression of TIMP-1 and sTNFR1, which in turn affect tumor cell proliferation and apoptosis.     

Significance of cell kinetic parameters in the prognosis of malignant melanoma: a review

Malignant melanoma has been extensively studied concerning methods of predicting progression and clinical outcome. The maximum tumor thickness as measured by Breslow's method is the cornerstone prognostic criterion, but despite this, evolution of the disease in some patients remains unpredictable, confirming that new reliable prognostic factors are awaited. Cell kinetic evaluation has been shown to be a useful tool for assessing the prognosis of breast and gastrointestinal cancer patients. Indeed, in these fields, the mitotic index and MIB-1 expression index, which are indirect estimates of the growth fraction of tumor cell population, are commonly shown to correlate with tumor grade and patient survival and presented as prognostic factors. In melanoma, results of cell kinetic investigations are conflicting: some studies have established a link between high proliferative activity and a bad prognosis, whereas other reports suggest the opposite. The aim of this review is to discuss these findings.     

Expression and function of the kallikrein-related peptidase 6 in the human melanoma microenvironment

Cutaneous malignant melanoma is an aggressive disease of poor prognosis. Clinical and experimental studies have provided major insight into the pathogenesis of the disease, including the functional interaction between melanoma cells and surrounding keratinocytes, fibroblasts, and immune cells. Nevertheless, patients with metastasized melanoma have a very poor prognosis and are largely refractory to clinical therapies. Hence, diagnostic tools to monitor melanoma development, as well as therapeutic targets, are urgently needed. We investigated the expression pattern of the kallikrein-related peptidase 6 (KLK6) in human melanoma tissue sections throughout tumor development. Although KLK6 was not detectable in tumor cells, we found strong KLK6 protein expression in keratinocytes and stromal cells located adjacent to benign nevi, primary melanomas, and cutaneous metastatic lesions, suggesting a paracrine function of extracellular KLK6 during neoplastic transformation and malignant progression. Accordingly, recombinant Klk6 protein significantly induced melanoma cell migration and invasion accompanied by an accelerated intracellular Ca(2+) flux. We could further demonstrate that KLK6-induced intracellular Ca(2+) flux and tumor cell invasion critically depends on the protease-activated receptor 1 (PAR1). Our data provide experimental evidence that specific inhibition of the KLK6-PAR1 axis may interfere with the deleterious effect of tumor-microenvironment interaction and represent a potential option for translational melanoma research.     

Tubulin expression in melanocytic skin tumors. An immunohistochemical study

The microtubulus system as a part of the cellular cytoskeleton contributes to cell movement. Microtubulus assembly and disassembly is considered to be essential for tumor invasion and serves as a target for tumor chemotherapy. Using immunohistochemical methods, we investigated the distribution of tubulin in normal skin and 34 melanocytic skin tumors. In normal skin, tubulin was strongly expressed in dermal nerves, melanocytes, fibroblasts within the papillary dermis and in myoepithelial cells. In melanocytic skin tumors, nevus cells and melanoma cells stained positive, particularly at the periphery of the lesions, where there were single cells and small nests. The main difference between benign and malignant melanocytic tumors was found in the stromal cells: In melanocytic nevi, the stromal fibroblasts were entirely tubulin negative; whereas, adjacent to the invasive edge in primary and metastatic malignant melanoma, the stroma fibroblasts were strongly positive. Our results show that tubulin is regularly expressed in melanocytic skin tumors and may serve as a prerequisite for cell movement. The pronounced expression of tubulin in fibroblasts surrounding malignant melanocytic skin lesions reflects a stromal alteration that might contribute to tumor invasion.     

黑素瘤发病机制相关蛋白的研究进展

黑素瘤是高死亡率的皮肤肿瘤,鉴定出与黑素瘤发病及进展相关的蛋白质有助于阐明黑素瘤的发病机制,临床上应早期诊断以提高疗效.近年来,比较蛋白组学、蛋白质表达谱等多种蛋白质组学研究方法应用于黑素瘤发病机制的研究,有助于明确相关细胞信号通路蛋白的功能,阐明肿瘤耐药机制、发现可能的肿瘤标记物以利于早期诊断、判断疗效和预后.     

Role of chemokines in melanoma progression

Metastasis is the main cause of death from melanoma. Chemokines are low molecular weight chemotactic cytokines that facilitate cellular migration. Thus, cells that express receptors for a given chemokine are attracted to the site of its production. As certain chemokines are found in abundance in organs that are common targets of metastasis and receptors for these chemokines are expressed by tumor cells, it was hypothesized that chemokine gradients might selectively facilitate metastasis to these organs. A later finding that these chemokines were produced by tumor cells, with evidence of autocrine effects, obliged the modification of that hypothesis. Many chemokines are also known to have opposing effects according to the type of cell they are acting on (tumor, inflammatory/immune, or endothelial cells), their functional status, or interactions with other molecules. The expression of chemokines and their receptors by melanoma cells enhances tumor progression by altering their microenvironment, stimulating angiogenesis, and inhibiting the immune response.     

Loss of proapoptotic Bcl-2-related multidomain proteins in primary melanomas is associated with poor prognosis

Prognosis of primary melanoma is presently based on morphological parameters, mainly tumor thickness. However, more reliable prognostic markers are needed that allow a better stratification of patients, especially with regard to therapeutic options. Here, a retrospective study was performed on patients with primary superficial-spreading melanoma (SSM, n=44) or nodular melanoma (n=16) of 1.5-4 mm thickness. Thirty patients had survived the follow-up of 10 years, whereas the other 30 patients developed metastases. Tumor sections were analyzed by immunohistochemistry for the expression of regulators of the cell cycle (p21; retinoblastoma protein (pRb)), of the intrinsic or extrinsic proapoptotic pathways (p53; murine double minute gene 2 protein; tumour necrosis factor-related apoptosis-inducing ligand (TRAIL)-R1/DR4; TRAIL-R2/DR5) and of Bcl-2-related proteins (Bcl-2, Mcl-1, Bax, Bak, Bok), which regulate the common mitochondrial apoptotic pathway. In SSM, decrease of Bax and Bak was significantly correlated with a poor prognosis: high Bax was associated with 10-year survival rates of 68%, whereas low Bax resulted in only 26% survival, and high Bak was associated with 10-year survival rates of 62%, whereas low Bak resulted in only 10% survival. Regulators of apoptosis may therefore candidate for independent prognostic markers for primary melanomas. The study underlines the particular role of the mitochondrial apoptosis pathway and of proapoptotic Bcl-2-related proteins for melanoma progression.     

Influence of cultured dermal fibroblasts on human melanoma cell proliferation,matrix metalloproteinase-2 (MMP-2) expression and invasion in vitro

The dermis is the main site of melanoma invasion. Matrix metalloproteinases (MMPs), especially MMP-2, produced by melanoma or surrounding stromal cells, are essential for the destruction of dermal extracellular matrix. Here, we examined how dermal fibroblasts influenced proliferation, MMP-2 secretion and invasion of human melanoma cell lines in vitro. Human melanoma cell lines M3 Da and M1Dor were cocultured with dermal fibroblasts under non-contact and contact conditions in order to assess both soluble and insoluble factors, respectively. Zymographic analysis showed that the levels of MMP-2 and TIMP-2 in melanoma cells were not altered in non-contact cocultures when compared with those in individual cultures. However, in contact cocultures, the expression of MMP-2 in membrane extracts was enhanced. Under our coculture conditions, dermal fibroblasts failed to upregulate melanoma cell invasion through a three-dimensional type I collagen matrix. Since stromal and cancer cell contacts have been shown to occur after disruption of the extracellular matrix, we hypothesized that fibroblasts may influence melanoma cell invasion after the beginning of tumor progression through the dermis.     

Plexin C1, a receptor for semaphorin 7a, inactivates cofilin and is a potential tumor suppressor for melanoma progression

Melanocytes are progenitor cells for melanoma, which arises through step-wise progression from dysplastic to invasive, to metastatic tumor. Our previous data showed that semaphorin 7A (Sema7A), a protein involved in axon guidance, stimulates melanocyte adhesion and dendricity through opposing actions of beta1-integrin and Plexin C1 receptors. We now show that Plexin C1 is diminished or absent in human melanoma cell lines; analysis of tissue microarrays of nevi, melanoma, and metastatic melanoma showed a decrease in Plexin C1 expression in metastatic melanoma, and an inverse correlation of Plexin C1 expression with depth of invasion. We examined the signaling intermediates of Sema7A and downstream targets of Plexin C1 in human melanocytes. Sema7A activated mitogen-activated protein kinase and inactivated cofilin, an actin-binding protein involved in cell migration. When Plexin C1 expression was silenced, Sema7A failed to phosphorylate cofilin, indicating that cofilin is downstream of Plexin C1. Further, Lim kinase II, a protein that phosphorylates cofilin, is upregulated by Sema7A in a Plexin C1-dependent manner. These data identify Plexin C1 as a potential tumor suppressor protein in melanoma progression, and suggest that loss of Plexin C1 expression may promote melanoma invasion and metastasis through loss of inhibitory signaling on cofilin activation.     

Reversed cellular polarity in primary cutaneous mucinous carcinoma: A study on tight junction protein expression in sweat gland tumors

Primary cutaneous mucinous carcinoma (PCMC) is a rare sweat gland tumor characterized by the presence of abundant mucin around the tumor islands, but the molecular mechanisms for this structure are not well elucidated. Because mucin is epithelial in nature, it is likely to be produced by epithelial tumor cells, not by surrounding stromal cells. We hypothesized that the abundant mucin is a result of reversed cellular polarity of the tumor. To test this hypothesis, we conducted an immunohistological study to investigate expression of tight junction (TJ) proteins occludin and ZO‐1 in PCMC, as well as in normal sweat glands and other sweat gland tumors. Dot‐like or linear expression of TJ proteins was observed at ductal structures of sweat glands, and ductal or cystic structures of related tumors. In PCMC, however, TJ protein expression was clearly visible at the edges of tumor cell islands. This study provides evidence to show that the characteristic histological structure of PCMC is caused by inverse polarization of the tumor cells, and that TJ proteins are useful markers of ductal differentiation in sweat gland tumors.     

Engineering melanoma progression in a humanized environment in vivo

To overcome the lack of effective therapeutics for aggressive melanoma, new research models closely resembling the human disease are required. Here we report the development of a fully orthotopic, humanized in vivo model for melanoma, faithfully recapitulating human disease initiation and progression. To this end, human melanoma cells were seeded into engineered human dermo-epidermal skin substitutes. Transplantation onto the back of immunocompromised rats consistently resulted in the development of melanoma, displaying the hallmarks of their parental tumors. Importantly, all initial steps of disease progression were recapitulated, including the incorporation of the tumor cells into their physiological microenvironment, transition of radial to vertical growth, and establishment of highly vascularized, aggressive tumors with dermal involvement. Because all cellular components can be individually accessed using this approach, it allows manipulation of the tumor cells, as well as of the keratinocyte and stromal cell populations. Therefore, in one defined model system, tumor cell-autonomous and non-autonomous pathways regulating human disease progression can be investigated in a humanized, clinically relevant context.     

Fas ligand reduces viability in primary melanoma short-term cell cultures more than in metastatic melanoma short-term cell cultures

BACKGROUND: Apoptotic pathway aberrations are reported as important tumor progression factors in melanoma. OBJECTIVE: Effect of soluble Fas ligand (sFasL) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) on short-term cultured melanoma cell viability from different stages of melanoma. RESULTS: Recombinant human FasL reduced viability after 18 h in a dose-dependent manner in 4 of 5 cell cultures from primary tumors and 1 of 9 cell cultures from metastatic melanoma (67.5 vs. 96.4%, p = 0.007). DNA fragmentation on flow cytometry confirmed apoptosis. Incubation with TRAIL had no effect on melanoma cell viability. Immunohistochemistry showed Fas in 3 of 4 primary and in 6 of 7 metastatic lesions, no FasL in primary lesions, and FasL in 5 of 7 metastatic lesions. CONCLUSION: Melanoma short-term cell cultures from primary tumors show decreased viability under FasL, but not TRAIL stimulation rather than short-term cell cultures derived from metastases.     

Alpha-smooth muscle actin expression in tumor and stromal cells of benign and malignant human pigment cell tumors.

We examined the altered expression of alpha-smooth muscle actin (alpha-Sm) in human benign, pre-malignant, and malignant pigment cell tumors by immunohistochemical as well as biochemical (Western blot) analysis using anti-alpha-Sm monoclonal antibody (anti-alpha-Sm MoAb). The expression of alpha-Sm has been revealed immunohistochemically to be associated with mesodermal cells rather than with pigment cells. Western blot analysis using anti-alpha-Sm MoAb detected alpha-Sm expression as a 43-kD band in the extracts from normal papillary dermis, nevus cell nevus, and metastatic melanoma with stromal tissues, but not from primary melanoma with stromal tissues examined. The above findings of alpha-Sm expression by Western blot analysis were further characterized immunohistochemically in terms of the localization at the cellular level as follows. 1) In normal papillary dermis, pericytes encircling capillary vessels showed only positive staining with anti-alpha-Sm MoAb. 2) In nevus tissues, nevus cells were not shown to be positively stained, despite similar positivity of pericytes in normal papillary dermis. 3) In melanoma tissues, alpha-Sm expression of metastatic melanoma detected by Western blot analysis was found to be derived from fibroblasts with smooth-muscle differentiation (myofibroblasts), but not from melanoma cells. Such myofibroblastic stromal changes could not be found on primary melanoma tissue sections, which showed no reactivity in Western blot analysis. We conclude that the major sources of alpha-Sm in benign and pre-malignant pigment cell tumors are capillary pericytes, whereas alpha-Sm found in malignant melanoma tissue is primarily from melanoma-surrounding stromal fibroblasts that were changed to myofibroblasts by some cytokine factor(s), presumably secreted from melanoma cells.     

CD44 is the principal mediator of hyaluronic-acid-induced melanoma cell proliferation

Interactions of the extracellular matrix component hyaluronic acid and its cellular receptors CD44 and RHAMM/IHABP have been linked to tumor progression and metastasis formation. We investigated the expression and hyaluronic-acid-dependent functions of CD44 and RHAMM/IHABP in human melanoma. Immunohistochemistry of tumor specimens at different stages of melanoma progression revealed an increased expression of CD44 and RHAMM/IHABP. High mRNA expression of CD44 was found in three highly tumorigenic melanoma cell lines compared with less tumorigenic melanoma cells or nontransformed melanocytes. RHAMM/IHABP expression was upregulated in all cell lines analyzed but not in melanocytes. In contrast to the cell surface localization of CD44, RHAMM/IHABP was detected exclusively within the cytoplasm of melanoma cells. Binding and adhesion of melanoma cells to hyaluronic acid is mainly CD44 dependent as it was inhibited to 60%--80% by an anti-CD44 monoclonal antibody whereas anti-RHAMM/IHABP sera had no effect. Culture of melanoma cells in the presence of hyaluronic acid resulted in a dose-dependent, CD44-mediated increase of melanoma cell proliferation and enhanced release of basic fibroblast growth factor and transforming growth factor beta 1. We conclude that (i) the expression of CD44 and RHAMM/IHABP is increased during melanoma progression, (ii) CD44 is the principal hyaluronic acid surface receptor on melanoma cells, and (iii) the hyaluronic-acid-induced increase of the proliferative capacity of melanoma cells is mainly dependent on CD44--hyaluronic acid interactions.     

High-frequency microsatellite instability is associated with defective DNA mismatch repair in human melanoma

Hereditary nonpolyposis colorectal cancers and a steadily increasing number of sporadic tumors display microsatellite instability. In colorectal tumors, high-frequency microsatellite instability is strictly associated with inactivation of the DNA mismatch repair genes hMSH2, hMLH1, or hPMS2, whereas mutations in the mismatch repair gene hMSH6 have been identified in a subset of tumors with low-frequency microsatellite instability. In addition to epithelial tumors of the colon, endometrium, and ovary, microsatellite instability has been reported to occur also in sporadic melanoma. The relationship between microsatellite instability and mismatch repair in melanoma cells, however, has not been investigated so far. In this study, we analyzed microsatellite instability, mismatch repair activity, and expression of the hMSH2, hMSH6, hMLH1, and hPMS2 proteins in five melanoma cell lines and in tumor specimens from which the cells were derived. Four cell lines displayed normal levels of mismatch repair activity and expressed all the mismatch repair proteins. The extracts of the fifth cell line lacked the hMLH1 and hPMS2 proteins, and were correspondingly deficient in the repair of DNA mismatches. This line displayed high-frequency microsatellite instability, whereas the four mismatch-repair-proficient cell lines displayed either no or low-frequency microsatellite instability. These findings could be confirmed in the tumor specimens, in that only the tumor that did not express hMLH1 and hPMS2 displayed high-frequency microsatellite instability. Our data are consistent with the hypothesis that in melanoma, similarly to epithelial tumors, only the high-frequency microsatellite instability phenotype is strictly dependent on a defective mismatch repair system. Further studies on a large series of tumor specimens are required to establish the frequency of mismatch repair loss in human melanoma.     

Unraveling mysteries of the multifunctional protein SPARC

The matricellular protein SPARC (secreted protein acidic and rich in cysteine) has diverse functions in melanoma cells. Because this secreted protein is produced not only in tumor cells but also in stromal cells, the question has been asked whether paracrine effects of stroma-derived SPARC contribute to melanoma progression. In this issue, Prada et al. (2007) begin to answer this question by demonstrating that SPARC produced by melanoma, but not stromal cells, is essential to regulate melanoma cell growth.