Natural killer lytic-associated molecule plays a role in controlling tumor dissemination and metastasis

Abstract

Natural killer lytic-associated molecule (NKLAM) is an E3 ubiquitin ligase that plays a major role in the cytolytic activity of NK cells. NKLAM is rapidly synthesized and then targeted to the granule membranes of NK cells upon NK activation. Previous studies have shown an essential role for NKLAM in NK killing activity in vitro. These findings were extended to an in vivo model of NK-mediated tumor killing in which NKLAM-deficient knockout (KO) mice injected with B16 melanoma cells were found to have significantly higher numbers of pulmonary tumor nodules than wild-type (WT) mice. To further investigate the role of NKLAM and NK function in tumor immunity in vivo, we utilized additional tumor models to compare tumor development and progression in NKLAM KO and WT mice. Primary tumor growth, dissemination, and metastasis of RMA-S lymphoma cells and E0771 breast cancer cells were evaluated. Both tumor cell lines were stably transfected with constructs that allow expression of green fluorescent protein (GFP), which serves as a tumor-specific marker. Intravenous injection of NK-sensitive RMA-S lymphoma cells resulted in greater dissemination of lymphoma cells in NKLAM KO mice than in WT mice. Lymphoma cells were found in the lymph nodes and bone marrow (BM) of NKLAM KO mice 2 weeks after injection; few detectable tumor cells remained in WT mice. E0771 syngeneic breast cancer cells were injected into the mammary pads of NKLAM KO and WT mice. Primary tumor growth was greater in NKLAM KO than in WT mice. More significantly, there were 4–5-fold more tumor cells in the blood and lungs of NKLAM KO than in WT mice 2 weeks after injection of tumor cells into the mammary pad. These results indicate that NKLAM plays a role in tumor development in vivo, especially in controlling tumor dissemination and metastasis to distant sites.

EO771, is it a well‐characterized cell line for mouse mammary cancer model? Limit and uncertainty

“…The use of EO771 cells in mouse models has the advantage of having tumor uptake close to 100%, in particular when an orthotopic mammary implantation is used 33 . This model is really close to human breast cancer (Figure 1).…”

“…Despite their isolation in the late 1940s, characterization of hormonal receptors and classification of EO771 line remains controversial. Indeed, its classification diverges according to the authors, which is considered as triple negative in 10 publications 6,12,16‐23 and as ERα+ in 19 publications 13,24‐41 . Some authors prefer to mention the unclear status of this lineage 42,43 .…”

“…Indeed, the advantage of having a tumor line from the most used mouse strain, C57BL/6, makes it possible to have a lot of genetically modified models of this strain. Thus, EO771 cell tumor growth can be evaluated i) in KO mice for E2 (estradiol), 23 apolipoprotein E and aromatase, 42 IFN‐γ receptor, 48 natural killer lytic‐associated molecule (NKLAM), 33 MMP13, 49 calcium‐independent phospholipase A2 β, 50 IGFBP‐3, 21,51,52 DUSP1, 53 GDF2, 54 BMP10, 54 and mKIAA1462 55 but also ii) in transgenic models such as mice overexpressing IL‐15, 56 obese, 13,35 NKC KD (exhibiting silenced Ly49 expression in NK cells), 46 GFP‐LC3 (LC3 linked to GFP expressing mice), 57 mice transgenically modified to express the HER2 proto‐oncogene (ERBB2) 58 or FAT‐1 (gene leading to the endogenous formation of ω3‐Polyunsaturated fatty acid from ω6‐Polyunsaturated fatty acid) 44,59 …”

E3 ubiquitin ligase NKLAM ubiquitinates STAT1 and positively regulates STAT1-mediated transcriptional activity

“…Studies from our laboratory have demonstrated that NK cells from NKLAM-knock out (NKLAM-KO) mice have diminished anti-tumor activity [5]. Additionally, we have shown that NKLAM plays a role in controlling tumor metastasis [6]. As a family, RBR members have been shown to play a role in neurological diseases as well as innate immunity.…”

E3 ubiquitin ligase NKLAM positively regulates macrophage inducible nitric oxide synthase expression

“…NK cells from NKLAM-deficient (KO) mice have diminished anti-tumor cytolytic activity (Hoover, et al, 2009). In vivo , NKLAM plays a role in controlling tumor metastasis (Hoover, et al, 2012). Recent studies from our laboratory have also demonstrated a role for NKLAM in macrophage bacterial killing.…”