L1-CAM expression in ccRCC correlates with shorter patients survival times and confers chemoresistance in renal cell carcinoma cells

Conflicting data exist about the expression of L1 cell adhesion molecule (L1-CAM) in clear cell renal cell carcinoma (ccRCC). To determine the clinical usefulness of L1-CAM as a therapeutic or prognostic marker molecule in renal cancer patients, we analyzed its expression on a cohort of 282 renal cell carcinoma (RCC) patients. L1-CAM expression was found in 49.5% of 282 renal cancer tissues. Importantly, L1-CAM expression in patients with ccRCC was associated with significantly shorter patient survival time. We further present evidence that L1-CAM was involved in the resistance against therapeutic reagents like rapamycin, sunitinib and cisplatin. The downregulation of L1-CAM expression decreased renal cancer cell proliferation and reduced the expression of cyclin D1. In addition, we found out that Von Hippel-Lindau (VHL) deficiency was accompanied by a downregulation of the transcription factor PAX8 and L1-CAM. In normal renal tissue, PAX8 and L1-CAM were co-expressed in collecting duct cells. Importantly, the downregulation of PAX8 by small interfering RNA increased the expression of L1-CAM and concomitantly induced the migration of renal cancer cells. Furthermore, we observed in 65.3% of 282 RCC patients a downregulation of PAX8 expression. With chromatin immunoprecipitation analysis, we additionally demonstrate that PAX8 can bind to the promoter of L1-CAM and we further observed that the downregulation of PAX8 was accompanied by increased L1-CAM expression in a high fraction of ccRCC patients. In summary, we show that VHL and PAX8 are involved in the regulation of L1-CAM in renal cancer and L1-CAM represents an important therapeutic and prognostic marker protein for the treatment of ccRC

PAX2 Regulates ADAM10 Expression and Mediates Anchorage-Independent Cell Growth of Melanoma Cells

PAX transcription factors play an important role during development and carcinogenesis. In this study, we investigated PAX2 protein levels in melanocytes and melanoma cells by Western Blot and immunofluorescence analysis and characterized the role of PAX2 in the pathogenesis of melanoma. In vitro we found weak PAX2 protein expression in keratinocytes and melanocytes. Compared to melanocytes increased PAX2 protein levels were detectable in melanoma cell lines. Interestingly, in tissue sections of melanoma patients nuclear PAX2 expression strongly correlated with nuclear atypia and the degree of prominent nucleoli, indicating an association of PAX2 with a more atypical cellular phenotype. In addition, with chromatin immunoprecipitation assay, PAX2 overexpression and PAX2 siRNA we present compelling evidence that PAX2 can regulate ADAM10 expression, a metalloproteinase known to play important roles in melanoma metastasis. In human tissue samples we found co-expression of PAX2 and ADAM10 in melanocytes of benign nevi and in melanoma cells of patients with malignant melanoma. Importantly, the downregulation of PAX2 by specific siRNA inhibited the anchorage independent cell growth and decreased the migratory and invasive capacity of melanoma cells. Furthermore, the downregulation of PAX2 abrogated the chemoresistance of melanoma cells against cisplatin, indicating that PAX2 expression mediates cell survival and plays important roles during melanoma progression

Downregulation of PAX2 decreases the proliferation, migration and invasion of melanoma cells.

<p>Anchorage-dependent (<b>A</b>) and anchorage-independent (<b>B</b>) cell growth was investigated by using a MTT proliferation assay. Twenty-four hours after siRNA transfection, SkMel5 cells treated with transfection reagents alone (mock) or transfected with scrambled siRNA (sc-siRNA) or PAX2-specific siRNAs were seeded into uncoated anchorage dependent cell growth) or polyHEME coated (anchorage independent cell growth) 96 well plates and cell growth was measured 24, 48 and 72 hours later using a MTT-assay. 3 independent experiments have been performed and statistical analysis has been performed using Anova post-hoc analysis. ***P<0.001 considered statistically significant compared to control transfected cells (Mock). <b>###</b>P<0.001 considered statistically significant compared to scrambled-siRNA transfected cells, *P<0.01 considered statistically significant compared to scrambled-siRNA transfected cells. (<b>C</b>) Migration assay of SkMel5 cells was performed 48 h after the transfection with control siRNA (sc-siRNA) or PAX2 specific siRNA (PAX2-siRNA). ***P<0.001 considered statistically significant compared to control siRNA transfected cells (sc-siRNA). (<b>D</b>) The invasive capacity of SkMel5 cells was analyzed 48 h after the transfection of contol (sc-siRNA) or PAX2 siRNA (PAX2-siRNA) in an invasion assay as described under material and methods ***P<0.001 considered statistically significant compared to control siRNA transfected cells (sc-siRNA).</p

Immunohistochemical analysis of PAX2 expression in tissue sections of benign nevi and malignant melanoma.

<p>(<b>A</b>) In normal sweat glands, PAX2 is expressed in gland epithelial cells (black arrows) while intermingled stromal cells only show very weak or absent nuclear PAX2 expression (green arrow) Bar represent 100 µm. (<b>B, C</b>) Normal appearing epidermal cell layers adjacent to (<b>B,</b> bar represent 100 µm) nevi or (<b>C,</b> bar represent 200 µm) malignant melanoma show a differentially PAX2 expression with strongest PAX2 levels in germinal basal cell layers (black arrows) decreasing in higher differentiated keratinocytes and finally being absent in corneocytes (green arrows). (<b>D</b>) Malignant melanoma cells constantly exhibit a heterogeneous nuclear PAX2 expression. Strongest expression is observed in large atypical nuclei with prominent nucleoli (black arrows). Bar represent 50 µm. (<b>E, F</b>) PAX2 expression in intradermal nevi was heterogeneous and did not correlate with histological features (Original magnification: A–C: 20×; D–F: 40×). Bars represent 50 µm.</p

PAX2 and ADAM10 expression in melanocytes, keratinocytes and melanoma cells.

<p>(<b>A</b>) Western Blot analysis was performed to determine the PAX2 and ADAM10 expression in melanocytes (Mel43), keratinocytes and melanoma cells (A375, G361, IPC298, MeWo, NW1539 and SKMel5). Notably, 5 of 6 melanoma cell line show PAX2 and ADAM10 expression. β-Actin Western Blot analysis was performed to control equal protein loading. (<b>B</b>) Immunofluorescence staining of primary melanocytes Mel43 (left image) and the melanoma cell lines IPC298 (middle image) and G361 (right image) was performed to investigate the localisation of ADAM10 and PAX2. Cells were incubated with monoclonal ADAM10 and polyclonal PAX2 specific antibodies, followed by Alex488 coupled secondary antibodies (green) and Cy3 coupled secondary antibodies (red). The cells were stained with DAPI to visualize nuclei (blue). (<b>C</b>) The relative immunofluorescence intensity of ADAM10 and PAX2 expression in the melanocytes Mel43 and the melanoma celllines IPC298 and G631 were determined and depicted in a graph. ***P<0.001 PAX2 immunofluorescence intensity considered statistically significant compared to the PAX2 immunofluorescence intensity of melanocytes. <b>###</b>P<0.001 ADAM10 immunofluorescence intensity considered statistically significant compared to ADAM10 immunofluorescence intensity. (<b>D</b>) The specificity of ADAM10 and PAX2 immunofluorescence staining was controlled by using isotype specific control (control IgG) antibodies.</p

PAX2 regulates ADAM10 expression in melanoma cells.

<p>(<b>A</b>) Chromatin immunoprecipitation (ChIP) assay was performed with SKMel5 cells as described in material and methods. One representative experiment of three independently performed experiments is shown. (<b>B</b>) SKMel5 cells were transfected with pcDNA3.1 plasmid alone or with PAX2-pcDNA3.1 plasmid DNA. The expression of PAX2 and ADAM10 was determined by Western Blot analysis. β-actin was used to determine equal protein loading. (<b>C</b>) SkMel5 were transfected with 10 nM scrambled siRNA (sc-siRNA) or with 10 nM of two different PAX2-siRNAs (PAX2-siRNA1-2). 48 hours and 72 hours after the transfection, cells were lysed and the protein expression level of PAX2 and ADAM10 was investigated by Western Blot analysis. β-actin was used to determine equal protein loading. (<b>D</b>) SkMel5 were transfected with 10 nM scrambled siRNA (sc-siRNA) or with 10 nM of two different PAX2-siRNAs (PAX2-siRNA1-2). 48 hours and 72 hours after the transfection, cells were lysed and the protein expression level of PAX8 was investigated by Western Blot analysis. β-actin was used to determine equal protein loading. (<b>E</b>) Immunofluorescence analysis with ADAM10 and PAX2 specific antibodies were performed in sc-siRNA (left image) and PAX2- siRNA (right image) transfected SkMel-5 cells. ADAM10 expression was visualized by Cy3 coupled goat anti-mouse secondary antibodies, whereas PAX2 expression was detected with Alexa488 coupled goat anti-rabbit antibodies. (<b>F</b>) In the graphs the quantification of ADAM10 and PAX2 immunofluorescence intensity is shown. ***P<0.001 considered statistically significant compared to the sc-siRNA transfected SkMel5 cells.</p

PAX2 is expressed in melanocytes of benign nevi and melanoma cells of patients with malignant melanoma.

<p>Tissue sections of benign nevi (<b>A</b>) and malignant melanoma (<b>B</b>) were investigated by double immunofluorescence analysis with S100 (melanocyte marker) and PAX2 specific antibodies. S100 expression was visualized by Cy3 coupled secondary antibodies (red) and PAX2 expression was detected with Alexa488 coupled goat anti-rabbit secondary antibodies (green). White arrows in the higher magnified insets indicate PAX2 expression in nucleoli of melanocytes of benign nevi (<b>A</b>), yellow arrows in the higher magnified insets specify PAX2 expression in nucleoli of melanoma cells (<b>B</b>).</p

ADAM10 and PAX2 are co-expressed in melanocytes and melanoma cells in tissue sections of benign nevi and malignant melanoma.

<p>To determine if ADAM10 and PAX2 are co-expressed in melanocytes of benign nevi or in melanoma cells of patients with malignant melanoma, double immunofluorescence analysis on tissue sections has been performed. ADAM10 (<b>green</b>) and PAX2 (<b>red</b>) expression is detectable in melanocytes of benign nevi (<b>A</b> insets represent higher magnification of the single channels and the merged image of all 3 channels) and in melanoma cells of patients with malignant melanoma (<b>B</b> and <b>C</b> insets represent higher magnification of the single channels and the merged image of all 3 channels).</p