Biomarkers: the useful and the not so useful--an assessment of molecular prognostic markers for cutaneous melanoma.

Aims. No effective treatment for lung cancer exists currently. One reason for this, is the development of drug resistance, assumed to be associated with cancer stem cell (CSCs) emergence within the tumour. This pilot study aimed to identify CSCs in 121 non-small cell lung cancer (NSCLC) patient samples via detection of the expression of stem cell markers – CD133 and nestin. Material and methods. Archived paraffin blocks of 121 patient samples were prepared as Tissue Microarrays (TMA). Indirect immunohistochemical staining was used to determine the level of expression of CD133 and nestin. Double immunofluorescence staining was used to investigate the co-expression of these two markers. To determine the correlation between expression of nestin and CD133 with the length of asymptomatic period and overall patient survival we used the Kaplan-Meyer analysis. Results. CD133 expression was detected in 22 (19%), nestin in the epithelium in 74 (66%) and vasculature in 78 (70%) of patients. Co-expression of these two markers was found in 21 (17%) patients in less than 1% of positive cells without impact on disease free or overall survival. Conclusions. We identified CD133 + /nestin + cells as novel potential markers of lung cancer CSCs.

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Identification of CD133+/nestin+ putative cancer stem cells in non-small cell lung cancer.

Cutaneous malignant melanoma is the most aggressive form of skin cancer with an extremely poor survival rate for the patients diagnosed with locally invasive and metastatic disease states. Intensive research has led in last few years to an improvement of the early detection and curative treatment of primary cutaneous melanomas that are confined to the skin by tumor surgical resection. However, locally advanced and disseminated melanomas are generally resistant to conventional treatments, including ionizing radiation, systemic chemotherapy, immunotherapy and/or adjuvant stem cell-based therapies, and result in the death of patients. The rapid progression of primary melanomas to locally invasive and/or metastatic disease states remains a major obstacle for an early effective diagnosis and a curative therapeutic intervention for melanoma patients. Importantly, recent advances in the melanoma research have led to the identification of different gene products that are often implicated in the malignant transformation of melanocytic cells into melanoma cells, including melanoma stem/progenitor cells, during melanoma initiation and progression to locally advanced and metastatic disease states. The frequent deregulated genes products encompass the oncogenic B-RafV600E and N-RasQ61R mutants, different receptor tyrosine kinases and developmental pathways such as epidermal growth factor receptor (EGFR), stem cell-like factor (SCF) receptor KIT, hedgehog, Wnt/β-catenin, Notch, stromal cell-derived factor-1 (SDF-1)/CXC chemokine receptor-4 (CXCR4) and vascular endothelial growth factor (VEGF)/VEGFR receptor. These growth factors can cooperate to activate distinct tumorigenic downstream signaling elements and epithelial-mesenchymal transition (EMT)-associated molecules, including phosphatidylinositol 3’-kinase (PI3K)/Akt/ molecular target of rapamycin (mTOR), nuclear factor-kappaB (NF-κB), macrophage inhibitory cytokine-1 (MIC-1), vimentin, snail and twist. Of therapeutic relevance, these deregulated signal transduction components constitute new potential biomarkers and therapeutic targets of great clinical interest for improving the efficacy of current diagnostic and prognostic methods and management of patients diagnosed with locally advanced, metastatic and/or relapsed melanomas.

Novel biomarkers and therapeutic targets for optimizing the therapeutic management of melanomas.

Notch suppression by gamma-secretase inhibitors is a valid approach against melanoma. However, most of studies have evaluated the short-term effect of DAPT on tumor cells or even cancer stem cells. In the present study, we surveyed the short-term and long-term effects of DAPT on the stem cell properties of A375 and NA8 as melanoma cell lines. The effects of DAPT were tested both in vitro and in vivo using xenograft models. In A375 with B-raf mutation, DAPT decreased the level of NOTCH1, NOTH2, and HES1 as downstream genes of the Notch pathway. This was accompanied by enhanced apoptosis after 24 h treatment, arrest in the G2-M phase, and impaired ability of colony and melanosphere formation at the short term. Moreover, tumor growth also reduced during 13 days of treatment. However, long-term treatment of DAPT promoted tumor growth in the xenograft model and enhanced the number and size of colonies and spheroids in vitro. The gene expression studies confirmed the up-regulation of Wnt and Notch downstream genes as well as AXIN1, CSNK2A3, and CEBPA2 following the removal of Notch inhibitor in vitro and in the xenograft model. Moreover, the Gompertz-based mathematical model determined a new drug resistance term in the present study. Our data supported that the long-term and not short-term inhibition of Notch by DAPT may enhance tumor growth and motility through up-regulation of AXIN1, CSNK2A3, and CEBPA2 genes in B-raf mutated A375 cells.

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Long-Term Inhibition of Notch in A-375 Melanoma Cells Enhances Tumor Growth Through the Enhancement of AXIN1, CSNK2A3, and CEBPA2 as Intermediate Genes in Wnt and Notch Pathways.

OBJECTIVE To study the effect of lentivirus-mediated RNA interference (RNAi) of nestin on the metastatic potential of human melanoma cell line UACC903. METHODS A lentiviral vector for RNAi targeting the coding region of human nestin mRNA (nestin-RNAi-LV) and another control vector containing a nonsense sequence were constructed. The vectors were transfected into UACC903 cells, and nestin expression in the cells was detected by RT-PCR, immunofluorence assay and Western blotting. The invasive ability and migration of the transfected UACC903 cells was evaluated using Transwell and scrape assay, respectively. Fluorescence assay was used to examine the expressions of E-cadherin, N-cadherin and β-catenin in the cells. RESULTS The lentiviral vector nestin-RNAi-LV was constructed successfully. Compared with the control vector, nestin-RNAi-LV resulted in obviously reduced expression of nestin mRNA and protein, lowered migration ability of UACC903 cells, and reduced cell adhesion and invasiveness (P<0.05). CONCLUSION Lentivirus-mediated nestin RNAi can specifically inhibit nestin expression to cause decreased cell migration and invasiveness of human melanoma cell line UACC903.

[Effect of lentivirus-mediated nestin gene silencing on metastatic potential of human melanoma cell line UACC903 in vitro].

UNLABELLED Nestin is one of intermedial filaments exprimed in proliferating progenitor cells of the CNS and PNS (central and peripheral nervous system). Postnatal reexpression of the protein occures mainly in CNS tumors and correlates with a high grade of malignancy. The aim of our study is assessment of the nestin expression in benign and malignant skin melanocytic lesions with respect to presume a prognostic role of this protein. We examined 127 bioptic specimens, including 42 nodular melanomas (NM), 32 superficial spreading melanomas (SSM), 10 dysplastic nevi and 43 common intradermal or dermoepidermal nevi. We proved significant increase in nestin expression in melanoma groups, especially in nodular melanomas, where nestin was localized mainly in the peripheral, invasive areas of the tumor mass. CONCLUSION Detection of nestin expression might be used as an additional melanocytic tumour marker.

Hirnák J

Papers

The role of intermedial filament nestin in malignant melanoma progression