Protein kinase C inhibitor Gö6976 but not Gö6983 induces the reversion of E- to N-cadherin switch and metastatic phenotype in melanoma: identification of the role of protein kinase D1

Abstract

BackgroundMelanoma is a highly metastatic type of cancer that is resistant to all standard anticancer therapies and thus has a poor prognosis. Therefore, metastatic melanoma represents a significant clinical problem and requires novel and effective targeted therapies. The protein kinase C (PKC) family comprises multiple isoforms of serine/threonine kinases that possess distinct roles in cancer development and progression. In this study, we determined whether inhibition of PKC could revert a major process required for melanoma progression and metastasis; i.e. the E- to N-cadherin switch.MethodsThe cadherin switch was analyzed in different patient-derived primary tumors and their respective metastatic melanoma cells to determine the appropriate cellular model (aggressive E-cadherin-negative/N-cadherin-positive metastasis-derived melanoma cells). Next, PKC inhibition in two selected metastatic melanoma cell lines, was performed by using either pharmacological inhibitors (Gö6976 and Gö6983) or stable lentiviral shRNA transduction. The expression of E-cadherin and N-cadherin was determined by western blot. The consequences of cadherin switch reversion were analyzed: cell morphology, intercellular interactions, and β-catenin subcellular localization were analyzed by immunofluorescence labeling and confocal microscopy; cyclin D1 expression was analyzed by western blot; cell metastatic potential was determined by anchorage-independent growth assay using methylcellulose as semi-solid medium and cell migration potential by wound healing and transwell assays.ResultsGö6976 but not Gö6983 reversed the E- to N-cadherin switch and as a consequence induced intercellular interactions, profound morphological changes from elongated mesenchymal-like to cuboidal epithelial-like shape, β-catenin translocation from the nucleus to the plasma membrane inhibiting its oncogenic function, and reverting the metastatic potential of the aggressive melanoma cells. Comparison of the target spectrum of these inhibitors indicated that these observations were not the consequence of the inhibition of conventional PKCs (cPKCs), but allowed the identification of a novel serine/threonine kinase, i.e. protein kinase Cμ, also known as protein kinase D1 (PKD1), whose specific inhibition allows the reversion of the metastatic phenotype in aggressive melanoma.ConclusionIn conclusion, our study suggests, for the first time, that while cPKCs don’t embody a pertinent therapeutic target, inhibition of PKD1 represents a novel attractive approach for the treatment of metastatic melanoma.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-3007-5) contains supplementary material, which is available to authorized users.

PKD1 is a potential biomarker and therapeutic target in triple-negative breast cancer

“…Pharmacological inhibition or siRNA-mediated depletion of PKD1 has previously shown cytotoxicity in melanoma, breast cancer, prostate cancer and pancreatic cancer cells [ 11 , 13 , 19 , 25 , 27 ]. The CRT0066101 compound was also able to inhibit tumor growth of MCF7 chemoresistant xenografts in vivo [ 13 ].…”

“…It is an atypical protein kinase C (PKC) activated by growth factors, mitogenic neuropeptides, as well as oxidative stress [ 9 ]. PKD1 regulates a variety of biological processes such as cell proliferation, survival, motility, organization of the Golgi apparatus and membrane trafficking [ 10 , 11 ]. Hotspot activating mutations of PRKD1 have recently been identified in polymorphous low-grade adenocarcinomas of salivary glands and likely constitute oncogenic drivers in these tumors [ 12 ].…”

Deciphering the Role of Protein Kinase D1 (PKD1) in Cellular Proliferation

“…This hypothesis does not however provide an explanation as to why the prodifferentiative PKD2 would be the most abundantly expressed in actively dividing cells. In hyperplastic human skin disorders, such as melanoma (40), basal cell carcinoma, and psoriasis (37), PKD1 was found to be upregulated. Taken together, these data suggest that, despite the fact that PKD1 seems not to be of primary importance in normal skin homeostasis (35), it has, whatever the species models, an evident proproliferative role in the context of skin carcinogenesis, wound healing, and other skin hyperproliferative diseases like psoriasis.…”

“…Consequently, these data also suggest why targeting PKD1, by relatively selective inhibitors, has to be considered as an option for the treatment and prevention of epidermal tumorigenesis, and for other hyperproliferative diseases such as psoriasis. To this goal, we showed that inhibition of PKD1 in melanoma cells (i) decreased their colony-forming capacities probably through the regulation of the ERK, JNK, and NFkB signaling pathways, and (ii) induced the relocation of b-catenin from nucleus to plasma membrane, and the subsequent expression decrease of some proproliferative target genes such as cyclin D1 (40).…”

(+)‐Usnic Acid Inhibits Migration of c‐KIT Positive Cells in Human Colorectal Cancer

“…Exogenous SCF was employed to induce phosphorylation of c-KIT (T703 site) and PKC-A (Thr638) [ 31 ]. Go-6976, an inhibitor of PKC [ 32 ], was employed to simulate PKC suppression. HCT116 cells were divided into seven groups: (1) DSMO as control, (2-3) (+)-UA (8 μ M, 24 or 48 hours), (4) SCF (100 ng/ml, 30 minutes), (5-6) (+)-UA (8 μ M, 24 or 48 hours) + SCF (100 ng/ml, 30 minutes), and (7) SCF (100 ng/ml, 30 min) + Go-6976 (6 μ M, 30 min).…”