Demonstration of a Melanoma-Specific CD44 Alternative Splicing Pattern That Remains Qualitatively Stable,but Shows Quantitative Changes during Tumour Progression

The role of CD44 in the progression of human melanoma has mostly been characterised by qualitative changes in expression of its individual variable exons. These exons however, may be expressed to form a number of molecules, the alternative splice variants of CD44, which may be structurally and functionally different. Using real-time PCR measurements with variable exon specific primers we have determined that all are expressed in human melanoma. To permit comparison between different tumours we identified a stable CD44 variable exon (CD44v) expression pattern, or CD44 ‘fingerprint’. This was found to remain unchanged in melanoma cell lines cultured in different matrix environments. To evaluate evolution of this fingerprint during tumour progression we established a scid mouse model, in which the pure expression pattern of metastatic primary tumours, circulating cells and metastases, non-metastatic primary tumours and lung colonies could be studied. Our analyses demonstrated, that although the melanoma CD44 fingerprint is qualitatively stable, quantitative changes are observed suggesting a possible role in tumour progression.     

microRNA-214 contributes to melanoma tumour progression through suppression of TFAP2C

Malignant melanoma is fatal in its metastatic stage. It is therefore essential to unravel the molecular mechanisms that govern disease progression to metastasis. MicroRNAs (miRs) are endogenous non-coding RNAs involved in tumourigenesis. Using a melanoma progression model, we identified a novel pathway controlled by miR-214 that coordinates metastatic capability. Pathway components include TFAP2C, homologue of a well-established melanoma tumour suppressor, the adhesion receptor ITGA3 and multiple surface molecules. Modulation of miR-214 influences in vitro tumour cell movement and survival to anoikis as well as extravasation from blood vessels and lung metastasis formation in vivo. Considering that miR-214 is known to be highly expressed in human melanomas, our data suggest a critical role for this miRNA in disease progression and the establishment of distant metastases.     

Systematic classification of melanoma cells by phenotype-specific gene expression mapping

There is growing evidence that the metastatic spread of melanoma is driven not by a linear increase in tumorigenic aggressiveness, but rather by switching back and forth between two different phenotypes of metastatic potential. In vitro these phenotypes are respectively defined by the characteristics of strong proliferation/weak invasiveness and weak proliferation/strong invasiveness. Melanoma cell phenotype is tightly linked to gene expression. Taking advantage of this, we have developed a gene expression-based tool for predicting phenotype called Heuristic Online Phenotype Prediction. We demonstrate the predictive utility of this tool by comparing phenotype-specific signatures with measurements of characteristics of melanoma phenotype-specific biology in different melanoma cell lines and short-term cultures. We further show that 86% of 536 tested melanoma lines and short-term cultures are significantly associated with the phenotypes we describe. These findings reinforce the concept that a two-state system, as described by the phenotype switching model, underlies melanoma progression.     

Comparative proteomic analysis of matched primary and metastatic melanoma cell lines

Identification of the biochemical pathways involved in the transformation from primary to metastatic melanoma is an area under intense investigation. A 2DE proteomics approach has been applied herein to the matched patient primary and metastatic melanoma cell lines WM-115 and WM-266-4, respectively, to better understand the processes that underlie tumor progression. Image analysis between samples aligned 470 common gel spots. Quantitative gel analysis indicated 115 gel spots of greater intensity in the metastatic line compared with the primary one, leading to the identification of 131 proteins via database searching of nano-LC-ESI-Q-TOF-MS/MS data. This more than tripled the number of proteins previously shown to be of higher abundance during melanoma progression. Also observed were 22 gel spots to be of lesser intensity in the metastatic line with respect to the primary one. Of these gel spots 15 proteins could be identified. Numerous proteins from both groups had not been reported previously to participate in melanoma progression. Further analysis of one protein, cyclophilin A, confirmed that this protein is expressed at higher levels in metastatic melanoma compared with primary melanoma and normal fibroblasts. Overall, this study expands our knowledge of protein modulation during melanoma stages, and suggests new targets for inhibitor development.     

Issues affecting molecular staging in the management of patients with melanoma

Prediction of metastatic potential remains one of the main goals to be pursued in order to better assess the risk subgroups of patients with melanoma. Detection of occult melanoma cells in peripheral blood (circulating metastatic cells [CMC]) or in sentinel lymph nodes (sentinel node metastatic cells [SNMC]), could significantly contribute to better predict survival in melanoma patients. An overview of the numerous published studies indicate the existence of several drawbacks about either the reliability of the approaches for identification of occult melanoma cells or the clinical value of CMC and SNMC as prognostic factors among melanoma patients. In this sense, characterization of the molecular mechanisms involved in development and progression of melanoma (referred to as melanomagenesis) could contribute to better classify the different subsets of melanoma patients. Increasing evidence suggest that melanoma develops as a result of accumulated abnormalities in genetic pathways within the melanocytic lineage. The different molecular mechanisms may have separate roles or cooperate during all evolutionary phases of melanocytic tumourigenesis, generating different subsets of melanoma patients with distinct aggressiveness, clinical behaviour, and response to therapy. All these features associated with either the dissemination of occult metastatic cells or the melanomagenesis might be useful to adequately manage the melanoma patients with different prognosis as well as to better address the different melanoma subsets toward more appropriate therapeutic approaches.     

Modeling cancer progression via pathway dependencies

Cancer is a heterogeneous disease often requiring a complexity of alterations to drive a normal cell to a malignancy and ultimately to a metastatic state. Certain genetic perturbations have been implicated for initiation and progression. However, to a great extent, underlying mechanisms often remain elusive. These genetic perturbations are most likely reflected by the altered expression of sets of genes or pathways, rather than individual genes, thus creating a need for models of deregulation of pathways to help provide an understanding of the mechanisms of tumorigenesis. We introduce an integrative hierarchical analysis of tumor progression that discovers which a priori defined pathways are relevant either throughout or in particular steps of progression. Pathway interaction networks are inferred for these relevant pathways over the steps in progression. This is followed by the refinement of the relevant pathways to those genes most differentially expressed in particular disease stages. The final analysis infers a gene interaction network for these refined pathways. We apply this approach to model progression in prostate cancer and melanoma, resulting in a deeper understanding of the mechanisms of tumorigenesis. Our analysis supports previous findings for the deregulation of several pathways involved in cell cycle control and proliferation in both cancer types. A novel finding of our analysis is a connection between ErbB4 and primary prostate cancer.     

Cell adhesion and metastasis.

There is no single phenotypic trait whose exclusive expression correlates universally with metastatic behaviour. Nevertheless, several lines of evidence point towards adhesive phenomena as mediating some crucial steps in the malignant spread of particular tumour types. These steps include detachment from the primary, invasion of the ECM including include detachment from the primary, invasion of the ECM including structures such as the basement membrane and organ capsules, and lodgement in the vessels of remote organs. Furthermore, there is evidence that adhesive phenomena can contribute at least in part to the selective patterns of spread shown by a variety of tumour cell types. The molecular dissection of the adhesive events involved in these processes has only just begun. It is clear already, however, that several different determinants are likely to be involved and the clinical application of this knowledge is unlikely to be immediate.     

Constitutive aberrant endogenous interleukin-1 facilitates inflammation and growth in human melanoma

Interleukin (IL)-1-mediated inflammation is proposed to contribute to the development and progression of some cancers. IL-1 family member proteins are known to be expressed constitutively in many melanoma tumor cells, and we hypothesize that these support molecular pathways of inflammation and facilitate tumor growth. To investigate the expression of IL-1α and IL-1β in melanoma patients, and their association with disease progression, immunohistochemical staining was carried out on tissues from 170 patients including benign nevi, primary melanomas, and metastatic melanomas. IL-1β levels were low (or zero) in benign nevi and higher in primary and metastatic melanomas (P < 0.0001). IL-1α was expressed in about 73% of nevi and 55% of metastatic melanomas, with levels significantly higher in primary tumors (P < 0.0001); most (98%) primary melanoma samples were positive for IL-1α. In vitro studies with seven human melanoma cell lines showed that five cell lines expressed IL-1α and IL-1β proteins and mRNA. We identified for the first time several important downstream signaling pathways affected by endogenous IL-1, including reactive oxygen and nitrogen species, COX-2, and phosphorylated NF-κB inhibitor (IκB) and stress-activated protein kinase/c-jun-NH(2)-kinase; all of which were decreased by siRNA to IL-1s. Downregulation of IL-1α, IL-1β, or MyD88 substantially increased p21 and p53 levels. Treatment with IL-1 receptor type I neutralizing antibody or IL-1 pathway-specific siRNAs led to growth arrest in IL-1-positive melanoma cells. Furthermore, blocking the IL-1 pathway increased autophagy in IL-1-positive melanoma cells. These results indicate that the endogenous IL-1 system is functional in most human melanoma and interrupting its signaling inhibits the growth of IL-1-positive melanoma cells.     

Pathobiology of cancer metastasis: a short account

ABSTRACT: Cancer-initiating cells display aberrant functional and phenotypic characteristics of normal stem cells from which they evolved by accumulation of multiple cytogenetic and/or epigenetic alterations. Signal transduction pathways which are essential for normal stem cell function are abnormally expressed by cancer cells, with a cancer cell phenotype playing an essential role in cancerization and metastasis.Local tumour progression, metastasis and metastatic tumour growth are mediated by direct cell-to-cell and paracrine reciprocal interactions between cancer cells and various stromal cells including fibroblasts, macrophages, bone marrow derived stem cells and progenitor cells. These interactions mediate breakdown of basement membrane barriers and angiogenesis both locally at the invasive front of the primary tumour and at the distant metastatic site; attract primary tumour cells to the candidate metastatic site; and promote proliferation, survival and growth of primary tumour cells and of metastatic cells at their distant site.It is the purpose of this article to highlight the analogies between some of the genetic programs of normal stem cells, and of cancer cells participating in the process of metastasis.     

Metabolic strategies of melanoma cells: Mechanisms,interactions with the tumor microenvironment,and therapeutic implications

Melanomas are metabolically heterogeneous, and they are able to adapt in order to utilize a variety of fuels that facilitate tumor progression and metastasis. The significance of metabolism in melanoma is supported by growing evidence of impact on the efficacy of contemporary therapies for this disease. There are also data to support that the metabolic phenotypes of melanoma cells depend upon contributions from both intrinsic oncogenic pathways and extrinsic factors in the tumor microenvironment. This review summarizes current understanding of the metabolic processes that promote cutaneous melanoma tumorigenesis and progression, the regulation of cancer cell metabolism by the tumor microenvironment, and the impact of metabolic pathways on targeted and immune therapies.     

A cross-cancer metastasis signature in the microRNA–mRNA axis of paired tissue samples

In the progression of cancer, cells acquire genetic mutations that cause uncontrolled growth. Over time, the primary tumour may undergo additional mutations that allow for the cancerous cells to spread throughout the body as metastases. Since metastatic development typically results in markedly worse patient outcomes, research into the identity and function of metastasis-associated biomarkers could eventually translate into clinical diagnostics or novel therapeutics. Although the general processes underpinning metastatic progression are understood, no clear cross-cancer biomarker profile has emerged. However, the literature suggests that some microRNAs (miRNAs) may play an important role in the metastatic progression of several cancer types. Using a subset of The Cancer Genome Atlas (TCGA) data, we performed an integrated analysis of mRNA and miRNA expression with paired metastatic and primary tumour samples to interrogate how the miRNA–mRNA regulatory axis influences metastatic progression. From this, we successfully built mRNA- and miRNA-specific classifiers that can discriminate pairs of metastatic and primary samples across 11 cancer types. In addition, we identified a number of miRNAs whose metastasis-associated dysregulation could predict mRNA metastasis-associated dysregulation. Among the most predictive miRNAs, we found several previously implicated in cancer progression, including miR-301b, miR-1296, and miR-423. Taken together, our results suggest that metastatic samples have a common cross-cancer signature when compared with their primary tumour pair, and that these miRNA biomarkers can be used to predict metastatic status as well as mRNA expression.

     

Differential Expression of Prognostic Proteomic Markers in Primary Tumour,Venous Tumour Thrombus and Metastatic Renal Cell Cancer Tissue and Correlation with Patient Outcome

Renal cell carcinoma (RCC) is the most deadly of urological malignancies. Metastatic disease affects one third of patients at diagnosis with a further third developing metastatic disease after extirpative surgery. Heterogeneity in the clinical course ensures predicting metastasis is notoriously difficult, despite the routine use of prognostic clinico-pathological parameters in risk stratification. With greater understanding of pathways involved in disease pathogenesis, a number of biomarkers have been shown to have prognostic significance, including Ki67, p53, vascular endothelial growth factor receptor 1 (VEGFR1) and ligand D (VEGFD), SNAIL and SLUG. Previous pathway analysis has been from study of the primary tumour, with little attention to the metastatic tumours which are the focus of targeted molecular therapies. As such, in this study a tissue microarray from 177 patients with primary renal tumour, renal vein tumour thrombus and/or RCC metastasis has been created and used with Automated Quantitative Analysis (AQUA) of immunofluorescence to study the prognostic significance of these markers in locally advanced and metastatic disease. Furthermore, this has allowed assessment of differential protein expression between the primary tumours, renal vein tumour thrombi and metastases. The results demonstrate that clinico-pathological parameters remain the most significant predictors of cancer specific survival; however, high VEGFR1 or VEGFD can predict poor cancer specific survival on univariate analysis for locally advanced and metastatic disease. There was significantly greater expression of Ki67, p53, VEGFR1, SLUG and SNAIL in the metastases compared with the primary tumours and renal vein tumour thrombi. With the exception of p53, these differences in protein expression have not been shown previously in RCC. This confirms the importance of proliferation, angiogenesis and epithelial to mesenchymal transition in the pathogenesis and metastasis of RCC. Importantly, this work highlights the need for further pathway analysis of metastatic tumours for overcoming drug resistance and developing new therapies.     

Loss of XRCC1 confers a metastatic phenotype to melanoma cells and is associated with poor survival in patients with melanoma

Ultraviolet (UV) radiation‐induced DNA damage and genomic instability is one of the leading causes for melanoma. X‐ray repair cross‐complementary protein 1, XRCC1, plays a critically important role in base excision repair pathway. This study was therefore performed to analyze the correlation between XRCC1 expression, melanoma progression, and patient survival. Using a tissue microarray with a total of 119 patients with melanoma, we demonstrate that loss of XRCC1 expression is associated with the progression of disease from dysplastic nevi to primary melanoma and to metastatic melanoma. We found that the loss of XRCC1 was correlated with the progression of melanoma from AJCC stage II to stage III and with worse overall and disease‐specific 5‐yr and 10‐yr survival of patients with melanoma. Furthermore, we also illustrate the inhibitory effect of XRCC1 on melanoma cell invasion and migration, which are the regulatory events in melanoma metastasis.     

Mesenchymal transition and dissemination of cancer cells is driven by myeloid-derived suppressor cells infiltrating the primary tumor

In order to metastasize, cancer cells need to acquire a motile phenotype. Previously, development of this phenotype was thought to rely on the acquisition of selected, random mutations and thus would occur late in cancer progression. However, recent studies show that cancer cells disseminate early, implying the existence of a different, faster route to the metastatic motile phenotype. Using a spontaneous murine model of melanoma, we show that a subset of bone marrow-derived immune cells (myeloid-derived suppressor cells or MDSC) preferentially infiltrates the primary tumor and actively promotes cancer cell dissemination by inducing epithelial-mesenchymal transition (EMT). CXCL5 is the main chemokine attracting MDSC to the primary tumor. In vitro assay using purified MDSC showed that TGF-β, EGF, and HGF signaling pathways are all used by MDSC to induce EMT in cancer cells. These findings explain how cancer cells acquire a motile phenotype so early and provide a mechanistic explanation for the long recognized link between inflammation and cancer progression.