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.

BACKGROUND: Melanoma 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. METHODS: The 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 ß-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. RESULTS: Gö6976 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, ß-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. CONCLUSION: In 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.

Febrile neutropenia prophylaxis, G-CSF physician preferences: discrete-choice experiment.

OBJECTIVES: Febrile neutropenia (FN) commonly occurs during cancer chemotherapy. Prophylaxis with granulocyte colony-stimulating factors (G-CSFs) is known to reduce the severity and incidence of FN and infections in patients with cancer. Despite the proven efficacy, G-CSFs are not always prescribed as recommended. We performed a discrete-choice experiment (DCE) to determine what factors drive the physician preference for FN prophylaxis in patients with cancer undergoing chemotherapy. METHODS: Attributes for the DCE were selected based on literature search and on expert focus group discussions and comprised pain at the injection site, presence of bone pain, associated fever/influenza syndrome, efficacy of prophylaxis, biosimilar availability, number of injections per chemotherapy cycle and cost. Oncologists, in a national database, were solicited to participate in an online DCE. The study collected the responses to the choice scenarios, the oncologist characteristics and their usual prescriptions of G-CSFs in the context of breast, lungs and gastrointestinal cancers. RESULTS: Overall, the responses from 205 physicians were analysed. The physicians were mainly male (61%), with ≤20 years of experience (76%) and working only in public hospitals (73%). The physicians prescribe G-CSF primary prophylaxis for 32% of patients: filgrastim in 46% and pegfilgrastim in 54%. The choice of G-CSF for primary and secondary prophylaxis was driven by cost and number of injections. Biosimilars were well accepted. CONCLUSION: Cost and convenience of G-CSF drive the physician decision to prescribe or not G-CSF for primary and secondary FN prophylaxes. It is important that these results be incorporated in the optimisation of G-CSF prescription in the clinical setting.

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

Protein Kinase D1 (PKD1) is a serine/threonine kinase encoded by the PRKD1 gene. PKD1 has been previously shown to be a prognostic factor in ERα+ tamoxifen-resistant breast tumors and PKD1 overexpression confers estrogen independence to ERα+ MCF7 cells. In the present study, our goal was to determine whether PKD1 is a prognostic factor and/or a relevant therapeutic target in breast cancer. We analyzed PRKD1 mRNA levels in 527 primary breast tumors. We found that high PRKD1 mRNA levels were significantly and independently associated with a low metastasis-free survival in the whole breast cancer population and in the triple-negative breast cancer (TNBC) subtype specifically. High PRKD1 mRNA levels were also associated with a low overall survival in TNBC. We identified novel PKD1 inhibitors and assessed their antitumor activity in vitro in TNBC cell lines and in vivo in a TNBC patient-derived xenograft (PDX) model. Pharmacological inhibition and siRNA-mediated depletion of PKD1 reduced colony formation in MDA-MB-436 TNBC cells. PKD1 inhibition also reduced tumor growth in vivo in a TNBC PDX model. Together, these results establish PKD1 as a poor prognostic factor and a potential therapeutic target in TNBC.