Experimental studies on ErbB targeted therapy in malignant melanoma

Malignant melanoma has one of the fastest increasing incidences among the different types of cancer in the Western world. This raise can partly be ascribed to the change in sun habits that has taken place during the last decades, since the major external risk factor for melanoma is exposure to ultraviolet radiation. In the case of patients with early stages of melanoma, the prognosis is usually good and the disease may be cured by surgery alone. However, with conventional anti-cancer treatments, patients diagnosed with unresectable or metastatic melanoma have a very low 5-year survival rate ranging from less than 10 percent to about 20 percent, depending on the location and extent of metastatic spread. Despite the development of novel promising targeted drugs, such as the immunomodulating antibody ipilimumab and the B-raf inhibitor vemurafenib, that have been shown to significantly extend patient survival, there is still an urgent need for new and improved treatment strategies which can further increase the survival of patients with advanced malignant melanoma. The aim of this thesis was to investigate the anti-tumor effect of two different tyrosine kinase inhibitors (TKIs), gefitinib and canertinib, on human malignant melanoma cell lines with wild-type BRAF and NRAS. We investigated the effect of these two drugs on cell proliferation, survival and on the ErbB1-4 receptor phosphorylation, as well as the downstream signaling molecules Akt, Erk1/2 and Stat3. We also established a melanoma cell line resistant to gefitinib treatment and studied the resistance mechanisms developed by the cells. The aim of this thesis was to investigate the anti-tumor effect of two different tyrosine kinase inhibitors (TKIs), gefitinib and canertinib, on human malignant melanoma cell lines with wild-type BRAF and NRAS. We investigated the effect of these two drugs on cell proliferation, survival and on the ErbB1-4 receptor phosphorylation, as well as the downstream signaling molecules Akt, Erk1/2 and Stat3. We also established a melanoma cell line resistant to gefitinib treatment and studied the resistance mechanisms developed by the cells. In conclusion, gefitinib and canertinib display promising anti-tumor effects on ErbB-expressing malignant melanoma and might be used in future studies in combination with conventional chemotherapy or other targeted therapies in the treatment of malignant melanoma patients not harboring BRAF or NRAS mutations

Experimental studies on ErbB targeted therapy in malignant melanoma

Malignant melanoma has one of the fastest increasing incidences among the different types of cancer in the Western world. This raise can partly be ascribed to the change in sun habits that has taken place during the last decades, since the major external risk factor for melanoma is exposure to ultraviolet radiation. In the case of patients with early stages of melanoma, the prognosis is usually good and the disease may be cured by surgery alone. However, with conventional anti-cancer treatments, patients diagnosed with unresectable or metastatic melanoma have a very low 5-year survival rate ranging from less than 10 percent to about 20 percent, depending on the location and extent of metastatic spread. Despite the development of novel promising targeted drugs, such as the immunomodulating antibody ipilimumab and the B-raf inhibitor vemurafenib, that have been shown to significantly extend patient survival, there is still an urgent need for new and improved treatment strategies which can further increase the survival of patients with advanced malignant melanoma. The aim of this thesis was to investigate the anti-tumor effect of two different tyrosine kinase inhibitors (TKIs), gefitinib and canertinib, on human malignant melanoma cell lines with wild-type BRAF and NRAS. We investigated the effect of these two drugs on cell proliferation, survival and on the ErbB1-4 receptor phosphorylation, as well as the downstream signaling molecules Akt, Erk1/2 and Stat3. We also established a melanoma cell line resistant to gefitinib treatment and studied the resistance mechanisms developed by the cells. The aim of this thesis was to investigate the anti-tumor effect of two different tyrosine kinase inhibitors (TKIs), gefitinib and canertinib, on human malignant melanoma cell lines with wild-type BRAF and NRAS. We investigated the effect of these two drugs on cell proliferation, survival and on the ErbB1-4 receptor phosphorylation, as well as the downstream signaling molecules Akt, Erk1/2 and Stat3. We also established a melanoma cell line resistant to gefitinib treatment and studied the resistance mechanisms developed by the cells. In conclusion, gefitinib and canertinib display promising anti-tumor effects on ErbB-expressing malignant melanoma and might be used in future studies in combination with conventional chemotherapy or other targeted therapies in the treatment of malignant melanoma patients not harboring BRAF or NRAS mutations

Aggregated Alpha-Synuclein Transfer Efficiently between Cultured Human Neuron-Like Cells and Localize to Lysosomes.

Parkinson's disease and other alpha-synucleinopathies are progressive neurodegenerative diseases characterized by aggregates of misfolded alpha-synuclein spreading throughout the brain. Recent evidence suggests that the pathological progression is likely due to neuron-to-neuron transfer of these aggregates between neuroanatomically connected areas of the brain. As the impact of this pathological spreading mechanism is currently debated, we aimed to investigate the transfer and subcellular location of alpha-synuclein species in a novel 3D co-culture human cell model based on highly differentiated SH-SY5Y cells. Fluorescently-labeled monomeric, oligomeric and fibrillar species of alpha-synuclein were introduced into a donor cell population and co-cultured with an EGFP-expressing acceptor-cell population of differentiated neuron-like cells. Subsequent transfer and colocalization of the different species were determined with confocal microscopy. We could confirm cell-to-cell transfer of all three alpha-synuclein species investigated. Interestingly the level of transferred oligomers and fibrils and oligomers were significantly higher than monomers, which could affect the probability of seeding and pathology in the recipient cells. Most alpha-synuclein colocalized with the lysosomal/endosomal system, both pre- and postsynaptically, suggesting its importance in the processing and spreading of alpha-synuclein

OPPONENT

Cover picture: adopted from www.biooncology.com. Original picture copyright of Genentech, Inc

Colocalization of α-syn species and selected intracellular targets in donor cells before transfer.

<p>Monomeric, oligomeric or fibrillar Cy3-labeled (red) α-syn was added to RA-differentiated SH-SY5Y cells and incubated for 3 h. After 24 h, the cells were fixed and then subjected to immunocytochemistry using Alexa Fluor 488-conjugated secondary antibody (green) showing subcellular targets. (A) Confocal microscopy images showing α-syn (red) colocalization (yellow) with LAMP2, VAMP2, KDEL, SV2 or TSG101 (green). Graphs show quantification of the total proportions of the intracellular α-syn monomers (B), oligomers (C), and fibrils (D) that are colocalized with the respective subcellular targets. The results are presented as the mean percentage of colocalization ± SEM. ** p<0.01, *** p<0.001, one-way ANOVA with Bonferroni correction. (Scale bar = 10 μm).</p

Colocalization of α-syn species and intracellular selected targets in acceptor cells after transfer.

<p>(A) Confocal images of donor and acceptor cells after 24 h co-culture show colocalization (magenta) of Cy3-labeled α-syn (red) with LAMP2, VAMP2, KDEL, SV2 or TSG101 using the Alexa Fluor 405-conjugated secondary antibody (blue) in GFP-expressing acceptor cells (green). (B-D) Quantification of the total proportions of the intracellular α-syn monomers (B), oligomers (C), and fibrils (D) that are colocalized with the respective subcellular targets. The results are presented as the mean percentage of colocalization ± SEM. * p<0.05, one-way ANOVA with Bonferroni correction. (Scale bar = 10 μm).</p

Transfer of Cy3-labeled α-syn between neuronal cells.

<p>Co-culture of neuronally differentiated donor cells containing Cy3-labeled α-syn (red) and acceptor cells transfected with EGFP-tagged endosomal Rab5a (green). (A) Confocal images show transfer (black and white arrows) of monomeric, oligomeric and fibrillar α-syn from donor cells to acceptor cells after 24 h of co-culture. (B) Quantification of acceptor cells with successfully transferred α-syn isoforms. Data are presented from an average of 460 counted acceptor cells from an average of 83 pictures per treatment. The results are presented as the mean percentage of positive transfer ± SEM. *** p<0.001, Chi-test with Bonferroni correction (Scale bar = 20 μm).</p

Uptake of α-synuclein in differentiated SH-SY5Y donor cells.

<p>SH-SY5Y cells differentiated for 7 d with 10 μM retinoic acid were treated for 3 h with 1 μM monomeric, oligomeric or fibrillar Cy3-labeled α-synuclein and reseeded onto cover slips. After 24 h or 48 h of incubation, the cells were fixed and mounted onto slides with ProLong antifade reagent containing DAPI. (A) Confocal images showing cellular uptake of monomeric, oligomeric and fibrillar species of α-synuclein (red) and DAPI (blue) staining for the nuclei. (B) The Cy3 fluorescence intensity at different concentrations of α-syn species. (C) Quantification of α-syn uptake. Data are presented from an average of 1855 counted donor cells from an average of 67 pictures per treatment and time point. The results are presented as the mean percentage of positive transfer ± SEM. n.s. not significant, *** p<0.001, Chi-test with Bonferroni correction (Scale bar = 10 μm).</p