Kapaettu Satyamoorthy

Downregulation of E-cadherin and Desmoglein 1 by autocrine hepatocyte growth factor during melanoma development

During melanoma development, transformed cells evade keratinocyte-mediated control by downregulating cell adhesion molecules. This study investigated the regulation of cell adhesion by hepatocyte growth factor (HGF) in melanoma. Melanocytes and two melanoma lines, WM164 and WM35, expressed normal level E-cadherin and Desmoglein 1, whereas most melanomas (18 out of 20) expressed no E-cadherin and significantly reduced Desmoglein 1. Overexpression of dominant negative E-cadherin and Desmoglein in melanocytes demonstrated that both molecules contribute to adhesion between melanocytes and keratinocytes. In contrast to melanocytes, most melanomas expressed HGF. All melanocytic cells expressed the HGF receptor c-Met, and autocrine HGF caused constitutive activation of c-Met, MAPK and PI3K. When autocrine activation was induced with HGF-expressing adenovirus, E-cadherin and Desmoglein 1 were decreased in melanocytes, WM164 and WM35. MAPK inhibitor PD98059 and PI3K inhibitor wortmannin partially blocked the downregulation, suggesting that both pathways are involved in this process. c-Met was coimmunoprecipitated with E-cadherin, Desmoglein 1 and Plakoglobin, suggesting that they form a complex (es) that acts to regulate intercellular adhesion. Together, the results indicate that autocrine HGF decouples melanomas from keratinocytes by downregulating E-cadherin and Desmoglein 1, therefore frees melanoma cells from the control by keratinocytes and allows dissemination of the tumor mass.

Melanoma cell lines from different stages of progression and their biological and molecular analyses.

The biological and molecular characteristics of cell lines from metastatic melanomas have been extensively studied but less is known about cells from the biologically earliest stage of primary melanoma. The overall success rate of establishing permanent cell lines from such lesions is only 10% of that for biologically late primary or metastatic melanomas, although our laboratory now has eight cell lines available. The cells are immortal but show reduced or no proliferation in soft agar and immunodeficient mice when compared with primary melanomas from the biologically advanced vertical growth phase. Metastatic melanoma cell lines from patients with familial melanoma or xeroderma pigmentosum are biologically similar to those from patients with spontaneous melanomas. Irrespective of the malignant stages, deletions and mutations can occur in exons 1–3 of the p16INK4A gene. DNA fingerprinting was then employed to demonstrate the uniqueness of individual cell lines and to confirm the identity of cell lines derived from same patients. These cell lines are an excellent resource to investigate melanoma progression.

Function and regulation of melanoma-stromal fibroblast interactions: when seeds meet soil.

Melanoma development and progression not only involve genetic and epigenetic changes that take place within the melanocytic cells, but also involve processes that are determined collectively by contextual factors including intercellular adhesions and communications. In this review, we focus on melanoma–stromal fibroblast crosstalk by direct cell–cell contact and by growth factors/cytokines/chemokines interacting with their respective receptors. The interactions between melanoma cells and stromal fibroblasts create a context that promotes tumor growth, migration/invasion, and angiogenesis. An understanding of this process and developing new experimental and screening models are of great importance for the development of effective therapeutical strategies to treat melanoma.

Induction of p21 WAF1/CIP1 and Inhibition of Cdk2 Mediated by the Tumor Suppressor p16 INK4a

ABSTRACT The tumor suppressor p16 INK4a inhibits cyclin-dependent kinases 4 and 6. This activates the retinoblastoma protein (pRB) and, through incompletely understood events, arrests the cell division cycle. To permit biochemical analysis of the arrest, we generated U2-OS osteogenic sarcoma cell clones in which p16 transcription could be induced. In these clones, binding of p16 to cdk4 and cdk6 abrogated binding of cyclin D1, p27 KIP1 , and p21 WAF1/CIP1 . Concomitantly, the total cellular level of p21 increased severalfold via a posttranscriptional mechanism. Most cyclin E-cdk2 complexes associated with p21 and became inactive, expression of cyclin A was curtailed, and DNA synthesis was strongly inhibited. Induction of p21 alone, in a sibling clone, to the level observed during p16 induction substantially reproduced these effects. Overexpression of either cyclin E or A prevented p16 from mediating arrest. We then extended these studies to HCT 116 colorectal carcinoma cells and a p21-null clone derived by homologous recombination. In the parental cells, p16 expression also augmented total cellular and cdk2-bound p21. Moreover, p16 strongly inhibited DNA synthesis in the parental cells but not in the p21-null derivative. These findings indicate that p21-mediated inhibition of cdk2 contributes to the cell cycle arrest imposed by p16 and is a potential point of cooperation between the p16/pRB and p14ARF/p53 tumor suppressor pathways.

Differential response of primary and metastatic melanomas to neutrophils attracted by IL‐8

IL‐8 is a strong chemoattractant for neutrophils, and it is constitutively produced by many tumors, including human melanomas. To determine the biologic importance of IL‐8 for melanoma cells from primary and metastatic lesions, we transduced selected cell lines constitutively producing low levels of IL‐8 with IL‐8 cDNA using a replication‐deficient adenoviral vector. Nontumorigenic SBcl2 primary melanoma cells formed tumors when transduced with increasing plaque‐forming units of IL‐8 per cell. However, at high IL‐8 transduction levels (100 ng/ml/105 cells in 48 hr), tumor growth was impaired due to massive neutrophil infiltration. A similar biphasic response was observed in WM115 primary melanomas, which are tumorigenic but not metastatic. Depletion of neutrophils with an antibody that blocks the accumulation of granulocytes at the site of inflammation enabled transduced primary melanomas secreting high levels of IL‐8 to survive and grow. In contrast, highly tumorigenic and metastatic 451Lu cells showed marked increases in tumor growth and number of metastatic foci in the lungs depending on the expression levels of IL‐8. Cytotoxicity assays with isolated neutrophils confirmed the preferential killing of primary over metastatic melanoma cells. SBcl2 cells stimulated by IL‐8 to form tumors in immunodeficient mice were induced to produce VEGF, suggesting that the angiogenic response is enhanced due to increased growth factor production. Our results demonstrate that nontumorigenic primary melanomas depend on IL‐8 stimulation in vivo for growth and that tumor growth depends on the level of neutrophil infiltration. Metastatic melanomas proliferate in vivo independently of infiltrating neutrophils.

Lessons from melanocyte development for understanding the biological events in naevus and melanoma formation.

Recent advances in mouse genetics have identified molecular changes that are critical for melanocyte maturation and differentiation. This review briefly summarizes the current knowledge of distinct steps in melanocyte development, and identifies for each step the most important molecules such as the growth factors stem cell factor and endothelin-3, with their respective receptors. Classical cadherins, i.e. E-cadherin, N-cadherin and P-cadherin, determine melanocyte positioning in the skin. During naevus and melanoma development, the two growth factor signalling pathways are downregulated, whereas cadherin expression shifts concomitantly with re-positioning of the naevus and melanoma cells in the skin. Loss of E-cadherin and gain of N-cadherin by melanoma cells has profound consequences for the regulatory cross-talk between various types of cells in the skin. Naevus and melanoma cells that do not express E-cadherin are resistant to control by keratinocytes and establish close communications with fibroblasts and endothelial cells. However, forced expression of E-cadherin in melanoma cells can reverse the malignant phenotype by re-establishing the control of keratinocytes over the melanoma cells. Even highly aggressive metastatic melanoma cells can be signalled to turn off the expression of genes associated with tumour invasion and metastasis, suggesting that this strategy could be utilized in the therapy of melanoma.

Induction of melanoma phenotypes in human skin by growth factors and ultraviolet B

Exposure to UV radiation likely plays a key role in melanoma development, whereas other etiologic agents remain unknown. Here we show that in normal human skin an increased expression of a combination of three growth factors, basic fibroblast growth factor, stem cell factor, and endothelin-3, along with exposure to UVB can transform normal melanocytes into a melanoma phenotype within 4 weeks. Invasion of melanoma lesions was found in skin from newborn donors, whereas melanomas in adult skin were of a noninvasive in situ type only. This suggests that susceptibility of skin to exogenous tumor promoters is dependent on age. This is the first report on human cancer initiation in vivo in which an imbalance of physiological factors combined with an environmental carcinogen can lead to transformation of normal tissue.

Microtubule-associated protein 2, a marker of neuronal differentiation, induces mitotic defects, inhibits growth of melanoma cells, and predicts metastatic potential of cutaneous melanoma.

Dynamic instability of microtubules is critical for mitotic spindle assembly and disassembly during cell division, especially in rapidly dividing tumor cells. Microtubule-associated proteins (MAPs) are a family of proteins that influence this property. We showed previously that MAP2, a neuron-specific protein that stabilizes microtubules in the dendrites of postmitotic neurons, is induced in primary cutaneous melanoma but is absent in metastatic melanomas. We proposed that induction of a microtubule-stabilizing protein in primary melanoma could disrupt the dynamic instability of microtubules, inhibit cell division and prevent or delay tumor progression. Here we show, by Kaplan-Meier survival and multivariate Cox regression analysis, that patients diagnosed with MAP2+ primary melanomas have significantly better metastatic disease-free survival than those with MAP2- disease. Investigation of the mechanisms that underlie the effect of MAP2 on melanoma progression showed that MAP2 expression in metastatic melanoma cell lines leads to microtubule stabilization, cell cycle arrest in G2-M phase and growth inhibition. Disruption of microtubule dynamics by MAP2 resulted in multipolar mitotic spindles, defects in cytokinesis and accumulation of cells with large nuclei, similar to those seen in vivo in MAP2+ primary melanomas cells. These data suggest that ectopic activation of a neuronal differentiation gene in melanoma during early tumor progression inhibits cell division and correlates with inhibition or delay of metastasis.

Interleukin-8 overexpression is present in pyoderma gangrenosum ulcers and leads to ulcer formation in human skin xenografts.

Interleukin-8 (IL-8) is a potent chemotactic polypeptide for neutrophils. However, the role of this cytokine during inflammation remains unclear. Skin specimens from patients with pyoderma gangrenosum demonstrated IL-8 overexpression in skin ulcers, which suggests a role for IL-8 in the development of the disease. We therefore constructed a recombinant adenovirus expressing the complementary deoxyribonucleic acid encoding human IL-8 (IL-8/Ad5) that induces a 2000-fold increase in IL-8 expression of infected human fibroblasts in vitro. Human skin engrafted to severe combined immunodeficiency mice and then injected with the recombinant virus demonstrated erythema, an intense perivascular infiltration of neutrophils, and extravasation of erythrocytes after 8 hours. By 12 hours after injection, neutrophils had accumulated beneath the epidermis, which then necrotized, and one or more ulcers that remained for approximately 2 weeks were observed. Clinically and histologically, the ulcers resembled pyoderma gangrenosum. These clinical and experimental findings suggest an etiologic role of IL-8 in the pathogenesis of pyoderma gangrenosum.

Mel-CAM-specific genetic suppressor elements inhibit melanoma growth and invasion through loss of gap junctional communication

Normal human melanocytes are interspersed singly among keratinocytes along the basement membrane of the epidermis, whereas melanoma cells readily adhere to each other during invasion of the dermis or distant organs. The tumorigenic and metastatic phenotype of melanoma cells often correlates with increased expression of cell–cell and cell-matrix adhesion receptors. Mel-CAM (MCAM, MUC 18, CD146) is a cell–cell adhesion receptor highly expressed by melanoma cells but not normal melanocytes. We show here that inhibition of Mel-CAM expression in metastatic melanoma cells using genetic suppressor elements of Mel-CAM cDNA leads to inhibition of adhesion between melanoma cells and to downregulation of the tumorigenic phenotype. Growth was not inhibited in genetic suppressor elements-transduced melanoma cells cultured in monolayers but was inhibited when cells were maintained anchorage-independently in soft agar and greatly reduced in immunodeficient mice. A three-dimensional epidermal skin equivalent model demonstrated that Mel-CAM allows melanoma cells to separate from the epidermis and invade the basement membrane zone and dermis. However, melanoma cells with little or no Mel-CAM were poorly invasive, possibly due to their loss of gap junctional communication. These results suggest the multifunctional role of a melanoma-associated cell–cell adhesion receptor in tumor progression.