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. 2018;12(5):447-463.
doi: 10.1080/19336918.2018.1471323. Epub 2018 May 21.

Oncogenic PKC-ι activates Vimentin during epithelial-mesenchymal transition in melanoma; a study based on PKC-ι and PKC-ζ specific inhibitors

Wishrawana S Ratnayake  1 , Christopher A Apostolatos  1 , André H Apostolatos  1 , Ryan J Schutte  2 , Monica A Huynh  2 , David A Ostrov  2 , Mildred Acevedo-Duncan  1
Affiliations

Oncogenic PKC-ι activates Vimentin during epithelial-mesenchymal transition in melanoma; a study based on PKC-ι and PKC-ζ specific inhibitors

Wishrawana S Ratnayake et al. Cell Adh Migr. 2018.

Abstract

Melanoma is one of the fastest growing cancers in the United States and is accompanied with a poor prognosis owing to tumors being resistant to most therapies. Atypical protein kinase Cs (aPKC) are involved in malignancy in many cancers. We previously reported that aPKCs play a key role in melanoma's cell motility by regulating cell signaling pathways which induce epithelial-mesenchymal Transition (EMT). We tested three novel inhibitors; [4-(5-amino-4-carbamoylimidazol-1-yl)-2,3-dihydroxycyclopentyl] methyl dihydrogen phosphate (ICA-1T) along with its nucleoside analog 5-amino-1-((1R,2S,3S,4R)-2,3-dihydroxy-4-methylcyclopentyl)-1H-imidazole-4-carboxamide (ICA-1S) which are specific to protein kinase C-iota (PKC-ι) and 8-hydroxy-1,3,6-naphthalenetrisulfonic acid (ζ-Stat) which is specific to PKC-zeta (PKC-ζ) on cell proliferation, apoptosis, migration and invasion of two malignant melanoma cell lines compared to normal melanocytes. Molecular modeling was used to identify potential binding sites for the inhibitors and to predict selectivity. Kinase assay showed >50% inhibition for specified targets beyond 5 μM for all inhibitors. Both ICA-1 and ζ-Stat significantly reduced cell proliferation and induced apoptosis, while ICA-1 also significantly reduced migration and melanoma cell invasion. PKC-ι stimulated EMT via TGFβ/Par6/RhoA pathway and activated Vimentin by phosphorylation at S39. Both ICA-1 and ζ-Stat downregulate TNF-α induced NF-κB translocation to the nucleus there by inducing apoptosis. Results suggest that PKC-ι is involved in melanoma malignancy than PKC-ζ. Inhibitors proved to be effective under in-vitro conditions and need to be tested in-vivo for the validity as effective therapeutics. Overall, results show that aPKCs are essential for melanoma progression and metastasis and that they could be used as effective therapeutic targets for malignant melanoma.

Keywords: PKC-ι; apoptosis; invasion; melanoma; metastasis; migration; vimentin.

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Figures

Figure 1.
Figure 1.
Inhibitor structures, molecular docking and kinase activity. Fig. 1A, 1B and 1C represent molecular structures of ICA-1T (MW = 336.24 g/mol), ICA-1S (MW = 256.26 g/mol) and ζ-Stat (MW = 384.34 g/mol), respectively. Fig. 1D shows the molecular surface of PKC-ι bound to ATP (3A8W) with docked ICA-1T. The molecular surface of residues that differ between PKC-ι and PKC-ζ are colored based on sequence similarity (using the Blosum62 scoring matrix). Blosum62 similarity values are: blue, 40–50, cyan, 50–60, green, 60–70, yellow, 70–90, orange and 90–100, red. Residues that are identical between PKC-ι and PKC-ζ are colored red. ATP is depicted as sticks with yellow for carbon, red for oxygen, blue for nitrogen. ICA-1T is depicted as sticks colored by element with white, red, and blue representing carbon, oxygen, and nitrogen respectively. Fig. 1E shows 3 enlarged pictures of lowest energy docking conformations of ICA-1T, ICA-1S, and ζ-Stat docked in the identified allosteric docking sites. Table in Fig. 1F is a summary of molecular docking scores (ΔG°) in kcal/mol for 3 inhibitors against PKC-ι and PKC-ζ with and without ATP. Fig. 1G represent the effect of ICA-1T, ICA-1S and ζ-Stat on PKC-ι and PKC-ζ activity. Recombinant active PKC-ζ or PKC-ι were incubated with MBP in the presence or absence of inhibitors (0.1- 20 µM) and percentage kinase activity was plotted against inhibitor concentration. N = 3 experiments were performed for each experiment and mean ± SD are plotted. Statistical significance is indicated by asterisks as **P < 0.01.
Figure 2
Figure 2
. The effects of aPKC inhibitors (ICA-1T, ICA-1S and ζ-stat) on cell proliferation, cell viability and cytotoxicity for melanoma and normal melanocyte cells. Results depict the effect of inhibitors on MEL-F-NEO (Fig. 2A), SK-MEL-2 (Fig. 2B) and MeWo (Fig. 2C) cell proliferation based on cell proliferation assay. Approximately 4 × 104 were cultured in T25 flasks and treated with either equal volume of sterile water (control) or inhibitors (0.1- 10 µM). Additional doses of sterile water or inhibitors were supplied every 24 h during a 3 day incubation period. Subsequently, cells were lifted and counted. Cytotoxicity of aPKC inhibitors was measured using WST-1 assay for MEL-F-NEO (Fig. 2D), SK-MEL-2 (Fig. 2E) and MeWo (Fig. 2F) cells. The absorbance at 450 nm is due to production of water soluble formazan and was measured as a function of time. The absorbance is directly proportional to the number of cells. Experimental concentrations in WST-1 assay for ICA-1T, ICA-1S and ζ-Stat were 1 μM, 2.5 μM and 5 μM, respectively for all three cell lines. The absorbance at 450 nm against time is plotted. Experiments (N = 3) were performed for each cell line and mean ± SD are plotted. Statistical significance is indicated by asterisk as *P < 0.05 and **P < 0.01.
Figure 3.
Figure 3.
Inhibitors decrease melanoma cell migration and invasion. Fig. 3A and 3B represent the effect of aPKC inhibitors (2.5 µM of ICA-1S, 1 μM of ICA-1T and 5 μM of ζ-Stat) on melanoma cell migration in wound healing assay and Fig. 3C and 3D represent the effect of inhibitors on melanoma cell invasion in Boyden chamber assay with basement extract (BME). In the wound healing assay, microscopic photographs (40 ×) of scratches on cells at the beginning (day 0) were compared with the photographs taken after 3 days. The effect of inhibitors are shown (Fig. 3A) compared to their controls. Experiments (N = 3) were performed for each cell line and randomly picked photographs are shown. Fig. 3B represents a comparison of calculated percent wound closure for the photographs taken using ImageJ (NIH, Rockville, MD, USA). For Boyden chamber assay (Fig. 3C), invaded cells in the bottom surface of transwell insert were stained with 0.5% crystal violet and microscopic pictures were taken (100 ×). Subsequently, crystal violet was dissolved in 70% ethanol and absorbance was measured at 590 nm which directly proportional to the number of invaded cells. Mean ± SD are plotted. Statistical significance is indicated by asterisk as **P < 0.01.
Figure 4.
Figure 4.
Effect of inhibitors (ICA-1S, ICA-1T and ζ-Stat) on aPKC expression, apoptosis, and signaling pathways related to EMT in melanoma cells. Expression of the protein levels of phosphorylated PKC-ι, total PKC-ι, phosphorylated PKC-ζ, total PKC-ζ, Caspase-3, cleaved PARP, total PARP, Bcl-2, β-catenin, Vimentin, phosphorylated Vimentin, Par6, phosphorylated PTEN, RhoA, E-cadherin, phosphorylated AKT and NF-κB p65, IκB, phosphorylated IκB and phosphorylated IKKα/β for the inhibitor treatments (2.5 µM of ICA-1S, 1 μM of ICA-1T and 5 μM of ζ-Stat) are shown in Fig. 4A. 40–80 µg of protein was loaded in to each well and β-actin was used as the loading control in each Western blot. Fig. 4B represents the densitometry values for Western blots in Fig. 4A. Experiments (N = 3) were performed in each trial and representative bands are shown.
Figure 5.
Figure 5.
Effect of TNF-α and TGFβ on aPKC expression, apoptosis, and signaling pathways related to EMT in melanoma cells. Expression of the protein levels of phosphorylated PKC-ι, total PKC-ι, phosphorylated PKC-ζ, total PKC-ζ, Caspase-3, cleaved PARP, total PARP, Bcl-2, β-catenin, Vimentin, phosphorylated Vimentin, Par6, phosphorylated PTEN, RhoA, E-cadherin, phosphorylated AKT and NF-κB p65, IκB, phosphorylated IκB and phosphorylated IKKα/β for the TNF-α (20 ng/ml) and TGFβ (250 pg/ml) treatments are shown in Fig. 5A for malignant melanoma cell lines (SK-MEL-2 and MeWo) are shown after the end of third day of treatments with respect to their controls. 40–80 µg of protein was loaded in to each well and β-actin was used as the loading control in each Western blot. Fig. 5B represents the densitometry values for Western blots in Fig. 5A. Experiments (N = 3) were performed in each trial and representative bands are shown.
Figure 6.
Figure 6.
PKC-ι activates Vimentin and both aPKCs stimulate nuclei translocation of NF-κB in melanoma cells. The effects of on siRNA knockdown (20 nM of PKC-ι and PKC-ζ siRNA) on the expression of aPKCs, Vimentin activation and NF-κB translocation are shown in Fig. 6A. 40 µg of protein was loaded in to each well and β-actin was used as the loading control in each Western blot. Fig. 6B is shown the association between PKC-ι and Par6. Whole cell lysates (100 µg) of malignant cells (Sk-Mel-2 and MeWo) were immunoprecipitated separately for PKC-ι and Par6 using specific antibodies. First two lanes in Western blot represents the (+) control which contained 40 µg of Sk-MEL-2 and MeWo whole cell extracts, respectively, applied to ensure that bands appeared for the specific proteins in Western blots. Western blots of PKC-ι immunoprecipitation showed an association with Par6. Reverse-immunoprecipitation of Par6 confirmed the association with PKC-ι while no association was observed with PKC-ζ. Fig. 6C represents the immunofluorescence staining of nuclei (blue panel), PKC-ι (green panel) and Vimentin (red panel) for melanoma cells (SK-MEL-2 and MeWo) treated with ICA-1T (1 μM) against controls. The images were captured at 200X magnification. Experiments (N = 3) were performed in each trial.
Figure 7.
Figure 7.
Quantitative real time PCR data of ICA-1T (1μM) treatments for PKC-ι and Vimentin for melanoma cell lines. Fig. 7A and 7C represent the amplification plots of PKC-ι and Vimentin for SK-MEL-2 cell line. Fig. 7E and 7G represent the amplification plots of PKC-ι and Vimentin for MeWo cell line. Fig. 7B and 7D represent the melt curve plots of PKC-ι and Vimentin for SK-MEL-2 cell line. Fig. 7F and 7H represent the melt curve plots of PKC-ι and Vimentin for SK-MEL-2 cell line. Fig. 7I and 7J demonstrate the mRNA level change of PKC-ι and Vimentin for ICA-1T treated MeWo and SK-MEL-2 cell lines, respectively. The ΔΔCT values were plotted with respect to the mRNA levels of control samples of each cell line. Statistical significance is indicated by asterisk as *P< 0.05. Experiments (N = 3) were performed in each trial.
Figure 8.
Figure 8.
A schematic summary of the involvement of PKC-ι and PKC-ζ in melanoma progression. Upon extra cellular stimulation with TNF-α and TGFβ, PKC-ι activates Par6, which leads to the degradation of RhoA and stimulates EMT by changing the cell integrity, loss of E-cadherin and gain of Vimentin. During this process, cadherin junctions will be destabilized as a result of loss of E-cadherin and β-catenin will be translocated to nucleus to upregulate the production of some proteins such as CD44 which further stimulate migration and EMT. Importantly PKC-ι tightly binds to Vimentin to activate them by phosphorylation and this activated Vimentin changes the cell polarity to maintain the mesenchymal phenotype. Activated Vimentin can also induce the phosphorylation of PTEN leading to inactivation of inhibitory action of PTEN on PIP3 [40]. This may result in activation of AKT through PIP3 and activated AKT pathway leading to cell survival, rapid proliferation and differentiation which are critical parts of melanoma progression. AKT could indirectly stimulate β-catenin translocation and activate NF-κB pathway in which PKC-ζ is known to play a stimulatory role on IKK-α/β. It is reported that activated NF-κB can inhibit PTEN [35].
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