Expression of neurotrophins and their receptors in pigment cell lesions of the skin

The neurotrophins (NTs) are a group of growth factors involved in the development of the nervous system and presumed to play a role in neural crest-derived tumours. The expression of three NTs (NGF, BDNF, and NT-3) and their receptors (NTRs; i.e. low-affinity pan-NT receptor p75, Trk-B, and Trk-C) was studied in frozen sections of benign and malignant cutaneous pigment cell lesions, using immunohistochemistry. In order to understand the possible role of these growth factors and their receptors in the progression of primary cutaneous malignant melanomas (PCMMs), their distribution in the radial (RGP) and vertical (VGP) growth phases was particularly studied. While most of the common acquired naevi were unreactive, Spitz and blue naevi showed scattered immunoreactive cells, especially for the p75 NTR. Dysplastic naevi, but not common naevi, expressed NT-3 in their junctional component. PCMM and melanoma metastases often showed a diffuse pattern of immunostaining. NT-3 was significantly more frequently expressed in the RGP of PCMMs than in the junctional component of benign naevi, whereas more extensive immunoreactivity for NGF was found in the VGP of PCMMs, compared with the RGP; metastases more frequently expressed NGF, BDNF, and Trk-B than PCMMs. Interestingly, neurotropic melanoma expressed all NTs/NTRs except Trk-B. These immuunohistochemical data confirm suggestions from previous in vitro studies that autocrine loops of certain NTs and their respective receptors may be involved in melanoma progression; in addition, NT-3 may be involved in the junctional growth of dysplastic naevi. The precise role of these growth factors in melanoma, however, will await further functional studies.     

RSK1 Activation Promotes Invasion in Nodular Melanoma

The two major melanoma histologic subtypes, superficial spreading and nodular melanomas, differ in their speed of dermal invasion but converge biologically once they invade and metastasize. Herein, we tested the hypothesis that distinct molecular alterations arising in primary melanoma cells might persist as these tumors progress to invasion and metastasis. Ribosomal protein S6 kinase, 90 kDa, polypeptide 1 (RSK1; official name RPS6KA1) was significantly hyperactivated in human melanoma lines and metastatic tissues derived from nodular compared with superficial spreading melanoma. RSK1 was constitutively phosphorylated at Ser-380 in nodular but not superficial spreading melanoma and did not directly correlate with BRAF or MEK activation. Nodular melanoma cells were more sensitive to RSK1 inhibition using siRNA and the pharmacological inhibitor BI-D1870 compared with superficial spreading cells. Gene expression microarray analyses revealed that RSK1 orchestrated a program of gene expression that promoted cell motility and invasion. Differential overexpression of the prometastatic matrix metalloproteinase 8 and tissue inhibitor of metalloproteinases 1 in metastatic nodular compared with metastatic superficial spreading melanoma was observed. Finally, using an in vivo zebrafish model, constitutive RSK1 activation increased melanoma invasion. Together, these data reveal a novel role for activated RSK1 in the progression of nodular melanoma and suggest that melanoma originating from different histologic subtypes may be biologically distinct and that these differences are maintained as the tumors invade and metastasize.Superficial spreading melanoma (SSM) and nodular melanoma (NM) represent the two most common primary melanoma histologic subtypes, accounting for 70% and 15% to 20% of cases, respectively.^1,2 SSM is characterized by a radial growth phase (RGP) consisting of an intraepidermal component. Whereas SSM can proceed from a RGP to a vertical growth phase (VGP) and finally to distant metastases, NM grows rapidly in a vertical manner (VGP), with no horizontal growth phase.³To date, the prognostic and therapeutic impact of melanoma histologic subtypes has been relatively limited. The American Joint Committee on Cancer staging system uses tumor thickness, ulceration, mitotic index, and lymph node status, but not histologic subtype, in the recurrence/metastasis risk assessment of patients with primary localized melanoma.⁴ This is, in part, due to the current linear model of melanoma progression, which dictates that melanoma begins with the transformation of epidermal melanocytes and an initial RGP, followed by a subsequent transition to a VGP and distant metastasis.^5–7 Hence, it is generally accepted that the speed of dermal invasion is the only aspect that differentiates the NM and SSM subtypes.Recent discoveries in other solid tumor types emphasize the potential role of histology-driven molecular characterization to assist in the diagnosis and treatment of cancer.^8–11 Indeed, the utility of histologic classification in melanoma has been demonstrated with acral lentiginous melanoma, which composes approximately 10% of primary melanomas. The prevalence of molecular alterations in the c-KIT oncogene in this histologic melanoma subtype has defined acral lentiginous melanoma as a distinct and useful subclassification of melanoma, and a phase 2 trial of the c-KIT inhibitor imatinib validated the rationale of subtype-specific therapy for this group of melanoma patients.¹² In contrast, the clinical relevance of the SSM and NM subtypes has been largely overlooked.Recent reports by our groups and others suggest that primary SSM and NM might be distinct biological entities.^13–19 Unbiased, high-throughput genetic techniques, such as comparative genomic hybridization, single nucleotide polymorphism arrays, and microarrays, have revealed the presence of recurrent SSM-specific deletions that are present or even amplified in NM and, thus, cannot be reconciled with the linear progression model, even when epigenetic modifications are taken into account. Similarly, significant alterations in mRNA and miRNA gene expression are observed when comparing SSM with nevi and NM, and these alterations cannot be explained by the existing stepwise (linear) model.^16,17 Together, these findings suggest that distinct molecular alterations between SSM and NM might underlie the biological differences between these subtypes. However, it is unclear whether differences that exist between primary SSM and NM are retained during progression to invasion and metastasis.Herein, we tested the hypothesis that subtype-specific differences between SSM and NM persist throughout progression of primary melanoma to metastatic disease. We used a combination of human melanoma cell lines representing SSM and NM, human tissues from metastatic SSM and NM, and a zebrafish model of melanoma to demonstrate the role of protein S6 kinase, 90 kDa, polypeptide 1 (RSK1; official name RPS6KA1) in melanoma invasion in vitro and in vivo. These data suggest that metastatic melanoma originating from different histologic subtypes might be biologically distinct and reveal differences that are maintained from the primary tumor to metastatic disease.     

Distinct MHC gene expression patterns during progression of melanoma

Abnormal expression of major histocompatibility complex (MHC) molecules in melanoma has been reported previously. However, the MHC molecule expression patterns in different growth phases of melanoma and the underlying mechanisms are not well understood. Here, we demonstrate that in vertical growth phase (VGP) melanomas, MHC genes are subject to increased rates of DNA copy number gains, accompanied by increased expression, in comparison to normal melanocytes. In contrast, MHC expression in metastatic melanomas drastically decreased compared to VGP melanomas, despite still prevalent DNA copy number gains. Subsequent investigations found that the master transactivator of MHC genes, CIITA, was also significantly downregulated in metastatic melanomas when compared to VGP melanomas. This could be one of the mechanisms accounting for the discrepancy between DNA copy number and expression level in metastatic melanomas, a potentially separate mechanism of gene regulation. These results infer a dynamic role of MHC function in melanoma progression. We propose potential mechanisms for the overexpression of MHC molecules in earlier stages of melanoma as well as for its downregulation in metastatic melanomas. © 2009 Wiley‐Liss, Inc.     

Comparison of the prognostic value of matrix metalloproteinases 2 and 9 in cutaneous melanoma

Previously, gelatinases matrix metalloproteinase (MMP)-2 and MMP-9 have been shown to be involved in melanoma invasion and progression. Also, overexpression of MMP-2 has been suggested to be linked to hematogenous metastasis in melanoma. This study was conducted to study the prognostic value of MMP-2 and MMP-9 in human melanoma. The expression of MMP-2 and MMP-9 immunoreactive protein was evaluated in 157 cases of primary melanomas. An immunohistochemical analysis using specific monoclonal antibodies for MMP-2 and MMP-9 on paraffin-embedded tissues was performed, and the immunostaining results were compared with the clinical course of melanoma. Overexpression of MMP-2 (>20% of malignant cells positive) was an independent prognostic marker for melanoma related death, with odds ratio of 2.6 (95% confidence interval, 1.32-5.07). The 10-year disease-specific survival rate was only 51% in patients with overexpression of MMP-2 protein compared with 79% in patients with a primary melanoma with low expression for MMP-2 (P = .001). Interestingly, male patients with a melanoma with overexpression of MMP-2 showed a 10-year disease-specific survival of only 41% compared with 77% in other male patients (P = .003). It is notable that the immunoreactive protein for MMP-9 in primary melanoma was not found to be of any prognostic importance. In the future, MMP-2 could be acknowledged as a new prognostic factor in melanoma. MMP-9, on the other hand, was not associated with the clinical course of melanoma. Based on the current data, MMP-2 could be evaluated as an inclusion factor for adjuvant studies especially in male melanoma patients.     

Metastatic conversion of chemically transformed human cells

A linear model for human cell metastasis has been developed in vitro from chemically transformed normal human cells. The chemically transformed cells are nontumorigenic in nude mice, but can be converted to a tumorigenic phenotype by transfection with a nondirectional cDNA library or antisense cDNA to the ML-1 gene. The primary transfected cell line (TR1T) forms localized, progressively growing tumors in nude mice that do not invade into the surrounding tissue. This tumorigenic TR1T cell line could be advanced into a metastatic stage following an additional transfection (TR2M cell line) with the cDNA expression library or antisense cDNA to the ML-1 gene. Metastatic cells, selected from tumors that were attached to internal organs, exhibited an increase in invasiveness as measured in vitro using an invasion chamber. The metastatic cells also exhibited an increased expression of matrix metalloproteinase-1 (MMP-1), although MMP-1 was not part of the cDNA that was transfected into either the TR1T cells or the doubly transfected metastatic TR2M cells. These data suggest that the increase in MMP-1 expression was a secondary downstream event responding to an upstream genetic change that initiated the conversion of cells from a tumorigenic to a metastatic stage. In summary, human cell lines representing premalignant, malignant, and metastatic phenotypes have been established in culture that can be used to identify gene changes that occur as normal human cells progress to a metastatic stage during tumor development. One gene, ML-1, that is found in the expression library appears to be involved in malignant progression, because ML-1 antisense cDNA will convert chemically transformed cells to both tumorigenic and metastatic stages, and cells from both local and metastatic tumors have a reduced or complete loss of expression of the ML-1 gene.     

Upregulation of IL4-induced gene 1 enzyme by B2 cells during melanoma progression impairs their antitumor properties

B cells present in human cutaneous melanoma have been associated with protective or detrimental effects on disease progression according to their phenotype. By using the RET model of spontaneous melanoma and adoptive transfer of B16 melanoma cells, we show that immature and follicular B2 (B2-FO) cells exert a protective effect on melanoma progression by promoting the generation of effector memory T cells and limiting the recruitment of polymorphonuclear myeloid-derived suppressor cells. Unfortunately, this beneficial effect progressively wanes as a consequence of enhanced expression of the IL4-induced gene 1 (IL4I1) enzyme by immature B cells and B2-FO cells. Endogenous IL4I1 selectively decreases CXCR5 expression in splenic immature B cells, subverting their trafficking to primary tumors and enhancing the production of IL-10 by B2 cells, thereby promoting an immunosuppressive microenvironment. Accordingly, B2 cells from RET IL4I1^KO mice more efficiently controlled B16 melanoma growth than B2 cells from IL4I1-competent RET mice. Collectively, immature B cells and B2-FO cells are key actors in the control of melanoma growth, but their mobility and functions are differently impaired by IL4I1 overexpression during melanoma progression. Thus, our present data strongly urge us to associate an IL4I1 antagonist with current immunotherapy to improve the treatment of metastatic melanoma.