Olli Saksela

Metastatic Outgrowth Encompasses COL-I, FN1, and POSTN Up-Regulation and Assembly to Fibrillar Networks Regulating Cell Adhesion, Migration, and Growth

Although the outgrowth of micrometastases into macrometastases is the rate-limiting step in metastatic progression and the main determinant of cancer fatality, the molecular mechanisms involved have been little studied. Here, we compared the gene expression profiles of melanoma lymph node micro- and macrometastases and unexpectedly found no common up-regulation of any single growth factor/cytokine, except for the cytokine-like SPP1. Importantly, metastatic outgrowth was found to be consistently associated with activation of the transforming growth factor-beta signaling pathway (confirmed by phospho-SMAD2 staining) and concerted up-regulation of POSTN, FN1, COL-I, and VCAN genes-all inducible by transforming growth factor-beta. The encoded extracellular matrix proteins were found to together form intricate fibrillar networks around tumor cell nests in melanoma and breast cancer metastases from various organs. Functional analyses suggested that these newly synthesized protein networks regulate adhesion, migration, and growth of tumor cells, fibroblasts, and endothelial cells. POSTN acted as an anti-adhesive molecule counteracting the adhesive functions of FN1 and COL-I. Further, cellular FN and POSTN were specifically overexpressed in the newly forming/formed tumor blood vessels. Transforming growth factor-beta receptors and the metastasis-related matrix proteins, POSTN and FN1, in particular, may thus provide attractive targets for development of new therapies against disseminated melanoma, breast cancer, and possibly other tumors, by affecting key processes of metastasis: tumor/stromal cell migration, growth, and angiogenesis.

Switch to an invasive growth phase in melanoma is associated with tenascin-C, fibronectin, and procollagen-I forming specific channel structures for invasion

Malignant melanomas are characterized by their high propensity to invade and metastasize, but the molecular mechanisms of these traits have remained elusive. Our DNA microarray analyses of benign nevi and melanoma tissue specimens revealed that the genes encoding extracellular matrix proteins tenascin‐C (TN‐C), fibronectin (FN), and procollagen‐I (PCOL‐I) are highly upregulated in invasive and metastatic melanomas. The expression and distribution of these proteins were further studied by immunohistochemistry in benign nevi, radially and vertically growing melanomas, sentinel node micrometastases, and macrometastases. TN‐C was increased in all invasive tumours and metastases, especially at invasion fronts, but not in benign nevi or non‐invasive melanomas. Significantly, the intensity of TN‐C staining correlated with metastasis to sentinel lymph nodes, better than tumour thickness (Breslow). Moreover, TN‐C, FN, and PCOL‐I appeared to co‐localize in the tumours and form tubular meshworks and channels ensheathing the melanoma cells. Our data suggest that melanoma invasion is associated with the formation of special channel‐like structures, providing a new concept, structured tumour cell spreading. Altogether, these data provide potential new prognostic markers and therapeutic targets/strategies for preventing melanoma dissemination. Copyright

TGF-β Signaling, Activated Stromal Fibroblasts, and Cysteine Cathepsins B and L Drive the Invasive Growth of Human Melanoma Cells

Accumulating evidence indicates that interactions between cancer cells and stromal cells are important for the development/progression of many cancers. Herein, we found that the invasive growth of melanoma cells in three-dimensional-Matrigel/collagen-I matrices is dramatically increased on their co-culture with embryonic or adult skin fibroblasts. Studies with fluorescent-labeled cells revealed that the melanoma cells first activate the fibroblasts, which then take the lead in invasion. To identify the physiologically relevant invasion-related proteases involved, we performed genome-wide microarray analyses of invasive human melanomas and benign nevi; we found up-regulation of cysteine cathepsins B and L, matrix metalloproteinase (MMP)-1 and -9, and urokinase- and tissue-type plasminogen activators. The mRNA levels of cathepsins B/L and plasminogen activators, but not MMPs, correlated with metastasis. The invasiveness/growth of the melanoma cells with fibroblasts was inhibited by cell membrane-permeable inhibitors of cathepsins B/L, but not by wide-spectrum inhibitors of MMPs. The IHC analysis of primary melanomas and benign nevi revealed cathepsin B to be predominantly expressed by melanoma cells and cathepsin L to be predominantly expressed by the tumor-associated fibroblasts surrounding the invading melanoma cells. Finally, cathepsin B regulated TGF-β production/signaling, which was required for the activation of fibroblasts and their promotion of the invasive growth of melanoma cells. These data provide a basis for testing inhibitors of TGF-β signaling and cathepsins B/L in the therapy of invasive/metastatic melanomas.

Systematic search for the best gene expression markers for melanoma micrometastasis detection

Melanoma is notorious for its high tendency to metastasize and its refractoriness to treatment thereafter. Metastasis is believed to occur mostly through the lymphatic system, and the status of sentinel lymph nodes is currently recognized as the best prognostic indicator. Unfortunately, the lymphatic metastatic process is still poorly understood and the occurrence of sentinel node metastases (micrometastases) may be underestimated. We performed genome‐wide gene expression analyses of melanoma lymph node micrometastases and macrometastases, and of primary melanomas and benign naevi, to characterize the early metastatic cells molecularly and to disclose the best diagnostic markers and rational targets for therapy. Significance analysis of microarrays identified 22 over‐ and five under‐expressed genes with ≥ four‐fold changes in the micrometastases. Of these genes, MLANA, TYR, MIA, ERBB3, PRAME, and SPP1 were tested as potential markers by RT‐PCR and immunohistochemistry. In a prospective study of 160 patients, our graded MLANA and TYR RT‐PCR analyses disclosed clinically significant metastases, as assessed by disease recurrence, better than histological and immunohistochemical examinations. These results strongly suggest the clinical implementation of quantifiable RT‐PCR assays to confirm and complement the pathological examination of sentinel node metastases. Furthermore, SPP1 and PRAME proved valuable as melanoma‐specific markers capable of differentiating melanoma cells from benign naevi in the sentinel lymph nodes. Importantly, these two genes may also prove to be ideal targets for drug development and therapy. Most molecular traits of the micrometastases were already present in the primary tumours, suggesting that micrometastasis to sentinel lymph nodes is a fairly non‐selective process. Copyright

Transforming Growth Factor Beta-Induced (TGFBI) Is an Anti-Adhesive Protein Regulating the Invasive Growth of Melanoma Cells

Melanoma is a malignancy characterized by high invasive/metastatic potential, with no efficient therapy after metastasis. Understanding the molecular mechanisms underlying the invasive/metastatic tendency is therefore important. Our genome-wide gene expression analyses revealed that human melanoma cell lines WM793 and especially WM239 (vertical growth phase and metastatic cells, respectively) overexpress the extracellular matrix (ECM) protein transforming growth factor β induced (TGFBI). In adhesion assays, recombinant TGFBI was strongly anti-adhesive for both melanoma cells and skin fibroblasts. TGFBI further impaired the adhesion of melanoma cells to the adhesive ECM proteins fibronectin, collagen-I, and laminin, known to interact with it. Unexpectedly, WM239 cells migrated/invaded more effectively in three-dimensional collagen-I and Matrigel cultures after knockdown of TGFBI by shRNA expression. However, in the physiological subcutaneous microenvironment in nude mice, after TGFBI knockdown, these cells showed markedly impaired tumor growth and invasive capability; the initially formed small tumors later underwent myxoid degeneration and completely regressed. By contrast, the expanding control tumors showed intense TGFBI staining at the tumor edges, co-localizing with the fibrillar fibronectin/tenascin-C/periostin structures that characteristically surround melanoma cells at invasion fronts. Furthermore, TGFBI was found in similar fibrillar structures in clinical human melanoma metastases as well, co-localizing with fibronectin. These data imply an important role for TGFBI in the ECM deposition and invasive growth of melanoma cells, rendering TGFBI a potential target for therapeutic interventions.

MMP-21 is upregulated at early stages of melanoma progression but disappears with more aggressive phenotype

The expression of matrix metalloproteinases (MMPs) is frequently altered during malignant transformation. We examined the profile of three recently cloned MMPs, MMP-21, MMP-26, and MMP-28, in melanomas in vivo and in culture. Immunohistochemistry for MMPs-21, -26, -28, and -13 in melanoma specimens (27 nonmetastatic, 26 with nodal micrometastases, and 10 in situ melanomas) from 63 patients was performed. MMP-21 was expressed in melanoma cells in 29/53 cases, being more frequent in melanoma samples without micrometastases. Six out of ten in situ melanomas were positive, while five nevus samples were negative. MMP-26 and -28 were not generally expressed in melanoma cells. MMP-13 was detected in melanoma cells in 36/53 samples. MMP-21 was not found in sentinel nodes with metastases, while MMP-13 was seen in all of them. MMP-21 messenger RNA was variably expressed in all five melanoma cell lines investigated using reverse transcriptase–polymerase chain reaction. Our results suggest that expression of MMP-21 may serve as a marker of malignant transformation of melanocytes and does not associate with the presence of micrometastases.

Multinucleate cell angiohistiocytoma: a report of four cases in Finland.

Sir, Multinucleate cell angiohistiocytoma (MCAH) was first described in 1985 by Smith & Wilson-Jones (1). This condition is an unusual skin disorder most commonly affecting middle-aged women. Clinically, the lesions appear as painless multiple small erythematous papules usually located on the limbs, mainly on the dorsum of the hands, but also on the face, legs and chest. Typically, the slowly progressing MCAH papules reach the size of 2 – 15 mm and may become dome-shaped or flat (2 – 4). No constant associations with other cutaneous or systemic diseases have been reported. The differential diagnosis includes cutaneous vascular proliferations such as angiofibromas or Kaposi’s sarcomas as well as sarcoidosis, lupus erythematosus, insect bite, lichen planus, lymphocytoma and granuloma annulare (5, 6). We present four cases of MCAH diagnosed at the Helsinki University Central Hospital.

Detecting field cancerization using a hyperspectral imaging system

Field cancerization denotes subclinical abnormalities in a tissue chronically exposed to UV radiation. These abnormalities can be found surrounding the clinically visible actinic keratoses.

Osteopontin Promotes the Invasive Growth of Melanoma Cells by Activating Integrin αvβ3 and Down-Regulating Tetraspanin CD9

Overexpression of osteopontin (OPN) is strongly associated with the invasiveness/metastasis of many cancers, including melanomas. However, the molecular mechanisms of OPN in these processes remain poorly understood. We found that forced expression of OPN in early vertical-growth-phase melanoma cells dramatically increased their migration/invasion and growth/survival in a three-dimensional collagen I gel. Neutralizing antibodies to OPN, integrin β1, and integrin αvβ3, but not to CD44, negated the effects of OPN. Conversely, knocking down OPN in metastatic melanoma cells abrogated the invasive growth. OPN overexpression activated and OPN knockdown inactivated αvβ3 and αvβ5 integrins, negligibly affecting their expression. We further found OPN expression to inversely correlate with tetraspanin CD9 expression. Early-stage melanoma cells displayed low OPN and high CD9 expression, and conversely, metastatic cells displayed high OPN and low CD9 expression. Overexpression of OPN in vertical-growth-phase melanoma cells induced down-regulation of CD9, and knockdown of OPN in metastatic melanoma cells up-regulated CD9. Reversion of these CD9 changes abolished the effects of OPN. Furthermore, knockdown of CD9 in early-stage melanoma cells stimulated their invasive capacity in three-dimensional collagen. Similarly, microarray analyses of benign nevi and primary melanomas from different stages revealed an inverse correlation between OPN and CD9. These data suggest that OPN promotes melanoma cell invasion by activating integrin αvβ3 and down-regulating CD9, a putative metastasis suppressor.

FGF-2 blocks TGF-β1-mediated suppression of Bcl-2 in normal melanocytes

Abstract:u2002 Normal melanocytes require growth support provided by the adjacent basement membrane. In contrast, nevus cells and melanoma cells survive in the dermis, and in vitro on a soft collagen gel. Transforming growth factor‐β1 (TGF‐β1) produced by melanocytes themselves induces apoptosis in normal melanocytes cultured on collagen gel, an effect that can be counteracted by fibroblast growth factor‐2 (FGF‐2). The purpose of this study was to investigate the mechanisms by which FGF‐2 counteracts the apoptotic signals from TGF‐β1 in melanocytes cultured on collagen gel. We report that FGF‐2 did not interfere with the signal transduction from the TGF‐β1 receptors to SMAD2/3 proteins. Instead, TGF‐β1 decreased the level of Bcl‐2 in normal melanocytes cultured on collagen gel, and FGF‐2 reversed the TGF‐β1‐mediated reduction in the level of Bcl‐2. In nevus and melanoma cells, TGF‐β1 was unable to induce a decrease in the level of Bcl‐2, and treatment with FGF‐2 did not cause an increase in the level of Bcl‐2 in nevus or melanoma cells. In conclusion, our results suggest that a reduction in the level of the anti‐apoptotic Bcl‐2 is involved in the execution of apoptosis induced by TGF‐β1 in normal melanocytes cultured on collagen gel and that FGF‐2 can prevent TGF‐β1 from causing this reduction.