BRAF inhibitor resistance of melanoma cells triggers increased susceptibility to natural killer cell-mediated lysis.

BACKGROUND: Targeted therapies and immunotherapies are first-line treatments for patients with advanced melanoma. Serine-threonine protein kinase B-RAF (BRAF) and mitogen-activated protein kinase (MEK) inhibition leads to a 70% response rate in patients with advanced melanoma with a BRAFV600E/K mutation. However, acquired resistance occurs in the majority of patients, leading to relapse. Immunotherapies that activate immune cytotoxic effectors induce long-lasting responses in 30% of patients. In that context, combination of targeted therapies with immunotherapy (IT) is a promising approach. We considered boosting natural killer (NK) cell tumor immunosurveillance, as melanoma cells express stress-induced molecules and activate NK cell lysis. METHODS: Here we have generated vemurafenib (a BRAF inihibitor)-resistant (R) cells from BRAFV600E SK28 and M14-sensitive (S) melanoma cell lines and investigated how resistance interferes with immunogenicity to NK cells. We determined the levels of several soluble molecules including NK ligands in 61 melanoma patients at baseline and 6 months M post-treatment with targeted therapies or immunotherapies. RESULTS: Vemurafenib resistance involved activation of p-AKT in SK28R and of p-MEK/p-ERK in M14R cells and was accompanied by modulation of NK ligands. Compared with S cells, SK28R displayed an increased expression of natural killer group 2 D (NKG2D) receptor ligands (major histocompatibility complex class (MHC) I chain-related protein A (MICA) and UL16-binding protein 2 (ULBP2)) whereas M14R exhibited decreased ULBP2 . SK28R and M14R cells induced higher NK degranulation and interferon gamma secretion and were more efficiently lysed by donor and patient NK cells. SK28R showed increased tumor necrosis factor-related apoptosis-inducing ligand receptor II (TRAIL-RII) expression and TRAIL-induced apoptosis, and TRAIL-induced apoptosis of M14R was decreased. Combined BRAF/MEK inhibitors abrogated the growth of SK28S, M14S, and M14R cells, while growth of SK28R was maintained. BRAF/MEK inhibition attenuated NK activity but R cell lines activated polyfunctional NK cells and were lysed with high efficiency. We investigated the relationship of soluble NK ligands and response to treatment in a series of melanoma patients. Soluble NKG2D ligands known to regulate the receptor function have been associated to cancer progression. Serum analysis of patients treated with target therapies or IT indicates that soluble forms of NK ligands (MICA, B7H6, programmed cell death ligand 1, and carcinoembryonic antigen cell adhesion molecule 1) may correlate with clinical response. CONCLUSION: These results support strategies combining targeted therapies and NK-based immunotherapies.

Two-dimensional dynamic evaluation of natural killer cell-mediated lysis of adherent target cells.

Cell-mediated cytotoxicity is a major function of cytotoxic lymphocytes. The cytotoxic function of immune effectors requires accurate and sensitive quantification as tumor immunotherapies are actively developed to treat growing types of tumors. Various methods have been developed to quantify this function. A first approach consists in measuring the externalization of LAMP-1 (lysosomal associated membrane 1), CD107a molecules transitory expressed at the cell surface by degranulating cytotoxic cells and is determined by flow cytometry. The focus of the chapter concerns the second approach that quantifies target cell lysis resulting from the close contact interaction with cytotoxic Natural Killer (NK) lymphocytes. For long time, target cell lysis was evaluated by chromium release assay, requiring use of radioactive labeled salt (51Cr) and specific devices not compatible with repeated tests performed for immunomonitoring of patients. Other methods include fluorimetric and bioluminescence assays. Monitoring immune cell-mediated lysis of adherent targets by the dynamic measure of cell proliferation using the Real time cell analyzer (RTCA) is a good alternative. The test relies on sensitive dynamic measure of cell index values during their interactions with cytotoxic immune effectors. The cell index increasing with cell proliferation rapidly drops in presence of immune cells. The lysis is quantified in reference with targets alone and expressed as percentages of lysis curves. Moreover, the dynamic monitoring of target cell index allows the evaluation of drugs, cytokines, mAbs on target cell lysis. Here we describe a robust and sensitive method for quantification of immune cell-mediated lysis of adherent targets.

NKG2D/NKG2-Ligand Pathway Offers New Opportunities in Cancer Treatment.

The antitumor functions of NK cells are regulated by the integration of positive and negative signals triggered by numerous membrane receptors present on the NK cells themselves. Among the main activating receptors, NKG2D binds several stress-induced molecules on tumor targets. Engagement of NKG2D by its ligands (NKG2D-Ls) induces NK cell activation leading to production of cytokines and target cell lysis. These effects have therapeutic potential as NKG2D-Ls are widely expressed by solid tumors, whereas their expression in healthy cells is limited. Here, we describe the genetic and environmental factors regulating the NKG2D/NKG2D-L pathway in tumors. NKG2D-L expression is linked to cellular stress and cell proliferation, and has been associated with oncogenic mutations. Tumors have been found to alter their to NKG2D-L expression as they progress, which interferes with the antitumor function of the pathway. Nevertheless, this pathway could be advantageously exploited for cancer therapy. Various cancer treatments, including chemotherapy and targeted therapies, indirectly interfere with the cellular and soluble forms of NKG2D-Ls. In addition, NKG2D introduced into chimeric antigen receptors in T- and NK cells is a promising tumor immunotherapy approach.

CD16+NKG2Ahigh Natural Killer Cells Infiltrate Breast Cancer-Draining Lymph Nodes.

Tumor-draining lymph nodes (TD-LNs) are the first site of metastasis of breast cancer. Natural killer (NK) cells that infiltrate TD-LNs [including noninvaded (NI) or metastatic (M)-LNs from breast cancer patients] and NK cells from healthy donor (HD)-LNs were characterized, and their phenotype analyzed by flow cytometry. Low percentages of tumor cells invaded M-LNs, and these cells expressed ULBP2 and HLA class I molecules. Although NK cells from paired NI and M-LNs were similar, they expressed different markers compared with HD-LN NK cells. Compared with HD-LNs, TD-LN NK cells expressed activating DNAM-1, NKG2C and inhibitory NKG2A receptors, and exhibited elevated CXCR3 expression. CD16, NKG2A, and NKp46 expression were shown to be increased in stage IIIA breast cancer patients. TD-LNs contained a large proportion of activated CD56brightCD16+ NK cells with high expression of NKG2A. We also showed that a subset of LN NK cells expressed PD-1, expression of which was correlated with NKp30 and NKG2C expression. LN NK cell activation status was evaluated by degranulation potential and lytic capacity toward breast cancer cells. NK cells from TD-LNs degranulated after coculture with breast cancer cell lines. Cytokine-activated TD-LN NK cells exerted greater lysis of breast cancer cell lines than HD-LN NK cells and preferentially lysed the HLA class Ilow MCF-7 breast cancer cell line. TD-LNs from breast cancer patients, thus, contained activated lytic NK cells. The expression of inhibitory receptor NKG2A and checkpoint PD-1 by NK cells infiltrating breast cancer-draining LNs supports their potential as targets for immunotherapies using anti-NKG2A and/or anti-PD-1.

Shifting the Balance of Activating and Inhibitory Natural Killer Receptor Ligands on BRAFV600E Melanoma Lines with Vemurafenib.

Over 60% of human melanoma tumors bear a mutation in the BRAF gene. The most frequent mutation is a substitution at codon 600 (V600E), leading to a constitutively active BRAF and overactivation of the MAPK pathway. Patients harboring mutated BRAF respond to kinase inhibitors such as vemurafenib. However, these responses are transient, and relapses are frequent. Melanoma cells are efficiently lysed by activated natural killer (NK) cells. Melanoma cells express several stress-induced ligands that are recognized by activating NK-cell receptors. We have investigated the effect of vemurafenib on the immunogenicity of seven BRAF-mutated melanoma cells to NK cells and on their growth and sensitivity to NK-cell-mediated lysis. We showed that vemurafenib treatment modulated expression of ligands for two activating NK receptors, increasing expression of B7-H6, a ligand for NKp30, and decreasing expression of MICA and ULBP2, ligands for NKG2D. Vemurafenib also increased expression of HLA class I and HLA-E molecules, likely leading to higher engagement of inhibitory receptors (KIRs and NKG2A, respectively), and decreased lysis of vemurafenib-treated melanoma cell lines by cytokine-activated NK cells. Finally, we showed that whereas batimastat (a broad-spectrum matrix metalloprotease inhibitor) increased cell surface ULBP2 by reducing its shedding, vemurafenib lowered soluble ULBP2, indicating that BRAF signal inhibition diminished expression of both cell-surface and soluble forms of NKG2D ligands. Vemurafenib, inhibiting BRAF signaling, shifted the balance of activatory and inhibitory NK ligands on melanoma cells and displayed immunoregulatory effects on NK-cell functional activities. Cancer Immunol Res; 5(7); 582-93. ©2017 AACR.

Patient's Natural Killer Cells in the Era of Targeted Therapies: Role for Tumor Killers.

Natural killer (NK) cells are potent antitumor effectors, involved in hematological malignancies and solid tumor immunosurveillance. They infiltrate various solid tumors, and their numbers are correlated with good outcome. The function of NK cells extends their lytic capacities toward tumor cells expressing stress-induced ligands, through secretion of immunoregulatory cytokines, and interactions with other immune cells. Altered NK cell function due to tumor immune escape is frequent in advanced tumors; however, strategies to release the function of NK infiltrating tumors are emerging. Recent therapies targeting specific oncogenic mutations improved the treatment of cancer patients, but patients often relapse. The actual development consists in combined therapeutic strategies including agents targeting the proliferation of tumor cells and others restorating functional antitumor immune effectors for efficient and durable efficacy of anticancer treatment. In that context, we discuss the recent results of the literature to propose hypotheses concerning the potential use of NK cells, potent antitumor cytotoxic effectors, to design novel antitumor strategies.

EGF/EGFR axis contributes to the progression of cholangiocarcinoma through the induction of an epithelial-mesenchymal transition.

BACKGROUND & AIMS: Epithelial-mesenchymal transition (EMT) is a cellular process involved in cancer progression. The first step of EMT consists in the disruption of E-cadherin-mediated adherens junctions. Cholangiocarcinoma (CCA), a cancer with a poor prognosis due to local invasion and metastasis, displays EMT features. EGFR, a receptor tyrosine kinase, plays a major role in CCA progression. The aim of the study was to determine if EMT is induced by EGFR in CCA cells. METHODS: In vivo, the expression of E-cadherin was analysed in CCA tumours of 100 patients and correlated with pathological features and EGFR expression, and in a xenograft model in mice treated with gefitinib, an inhibitor of EGFR. In vitro, the regulation of EMT by EGFR was investigated in CCA cell lines. RESULTS: In human CCA, a cytoplasmic localization of E-cadherin occurred in 50% of the tumours was associated with the peripheral type of CCA, tumour size, the presence of satellite nodules and EGFR overexpression. In xenografted tumours, E-cadherin displayed a cytoplasmic pattern whereas the treatment of mice with gefitinib restored the membranous expression of E-cadherin. In vitro, EGF induced scattering of CCA cells that resulted from the disruption of adherens junctions. Internalization and decreased expression of E-cadherin, as well as nuclear translocation of ß-catenin, were observed in EGF-treated CCA cells. In these cells, EMT-transcription factors (i.e., Slug and Zeb-1) and mesenchymal markers (i.e., N-cadherin and α-SMA) were induced, favoring cell invasiveness through cytoskeleton remodeling. All these effects were inhibited by gefitinib. CONCLUSIONS: The EGF/EGFR axis triggers EMT in CCA cells highlighting the key role of this pathway in CCA progression.