Natural Killer-Derived Exosomal miR-186 Inhibits Neuroblastoma Growth and Immune Escape Mechanisms.

In neuroblastoma, the interplay between immune cells of the tumor microenvironment and cancer cells contributes to immune escape mechanisms and drug resistance. In this study, we show that natural killer (NK) cell-derived exosomes carrying the tumor suppressor microRNA (miR)-186 exhibit cytotoxicity against MYCN-amplified neuroblastoma cell lines. The cytotoxic potential of these exosomes was partly dependent upon expression of miR-186. miR-186 was downregulated in high-risk neuroblastoma patients, and its low expression represented a poor prognostic factor that directly correlated with NK activation markers (i.e., NKG2D and DNAM-1). Expression of MYCN, AURKA, TGFBR1, and TGFBR2 was directly inhibited by miR-186. Targeted delivery of miR-186 to MYCN-amplified neuroblastoma or NK cells resulted in inhibition of neuroblastoma tumorigenic potential and prevented the TGFß1-dependent inhibition of NK cells. Altogether, these data support the investigation of a miR-186-containing nanoparticle formulation to prevent tumor growth and TGFß1-dependent immune escape in high-risk neuroblastoma patients as well as the inclusion of ex vivo-derived NK exosomes as a potential therapeutic option alongside NK cell-based immunotherapy.Significance: These findings highlight the therapeutic potential of NK cell-derived exosomes containing the tumor suppressor miR-186 that inhibits growth, spreading, and TGFß-dependent immune escape mechanisms in neuroblastoma.

Expression of Five Neuroblastoma Genes in Bone Marrow or Blood of Patients with Relapsed/Refractory Neuroblastoma Provides a New Biomarker for Disease and Prognosis.

Purpose: We determined whether quantifying neuroblastoma-associated mRNAs (NB-mRNAs) in bone marrow and blood improves assessment of disease and prediction of disease progression in patients with relapsed/refractory neuroblastoma.Experimental Design: mRNA for CHGA, DCX, DDC, PHOX2B, and TH was quantified in bone marrow and blood from 101 patients concurrently with clinical disease evaluations. Correlation between NB-mRNA (delta cycle threshold, ΔCt, for the geometric mean of genes from the TaqMan Low Density Array NB5 assay) and morphologically defined tumor cell percentage in bone marrow, 123I-meta-iodobenzylguanidine (MIBG) Curie score, and CT/MRI-defined tumor longest diameter was determined. Time-dependent covariate Cox regression was used to analyze the relationship between ΔCt and progression-free survival (PFS).Results: NB-mRNA was detectable in 83% of bone marrow (185/223) and 63% (89/142) of blood specimens, and their ΔCt values were correlated (Spearman r = 0.67, P < 0.0001), although bone marrow Ct was 7.9 ± 0.5 Ct stronger than blood Ct When bone marrow morphology, MIBG, or CT/MRI were positive, NB-mRNA was detected in 99% (99/100), 88% (100/113), and 81% (82/101) of bone marrow samples. When all three were negative, NB-mRNA was detected in 55% (11/20) of bone marrow samples. Bone marrow NB-mRNA correlated with bone marrow morphology or MIBG positivity (P < 0.0001 and P = 0.007). Bone marrow and blood ΔCt values correlated with PFS (P < 0.001; P = 0.001) even when bone marrow was morphologically negative (P = 0.001; P = 0.014). Multivariate analysis showed that bone marrow and blood ΔCt values were associated with PFS independently of clinical disease and MYCN gene status (P < 0.001; P = 0.055).Conclusions: This five-gene NB5 assay for NB-mRNA improves definition of disease status and correlates independently with PFS in relapsed/refractory neuroblastoma. Clin Cancer Res; 23(18); 5374-83. ©2017 AACR.

A limited screen for protein interactions reveals new roles for protein phosphatase 1 in cell cycle control and apoptosis.

Protein phosphatase 1 (PP1) catalytic subunits typically combine with other proteins that modulate their activity, direct them to distinct substrates, or serve as substrates for PP1. More than 50 PP1-interacting proteins (PIPs) have been identified so far. Given there are approximately 10 000 phosphoproteins in mammals, many PIPs remain to be discovered. We have used arrays containing 100 carefully selected antibodies to identify novel PIPs that are important in cell proliferation and cell survival in murine fetal lung epithelial cells and human A549 lung cancer cells. The antibody arrays identified 31 potential novel PIPs and 11 of 17 well-known PIPs included as controls, suggesting a sensitivity of at least 65%. A majority of the interactions between PP1 and putative PIPs were isoform- or cell type-specific. We confirmed by co-immunoprecipitation that 9 of these proteins associate with PP1: APAF-1, Bax, E-cadherin, HSP-70, Id2, p19Skp1, p53, PCNA, and PTEN. We examined two of these interactions in greater detail in A549 cells. Exposure to nicotine enhanced association of PP1 with Bax (and Bad), but also induced inhibitory phosphorylation of PP1. In addition to p19Skp1, PP1alpha antibodies also coprecipitated cullin 1, suggesting that PP1alpha is associated with the SCF1 complex. This interaction was only detectable during the G1/S transition and S phase. Forced loss of PP1 function decreased the levels of p27Kip1, a well-known SCF1 substrate, suggesting that PP1 may rescue proteins from ubiquitin/proteasome-mediated destruction. Both of these novel interactions are consistent with PP1 facilitating cell cycle arrest and/or apoptosis.

Protein phosphatase 1alpha activity prevents oncogenic transformation.

Cyclin-dependent kinase 2 (Cdk2) phosphorylates Thr320 of protein phosphatase 1alpha (PP1alpha) in late G(1), thereby inhibiting its activity. Phosphorylation-resistant PP1alphaT320A, acting as a constitutively active (CA) mutant, causes a late G(1) arrest by preventing the phosphorylation and inactivation of the retinoblastoma protein (pRb). Both PP1alpha-mediated G(1) arrest and PP1alpha phosphorylation in late G(1) require the presence of pRb, indicating that PP1alpha is a crucial regulator of the pRb pathway, which is almost invariably mutated in human cancer. These findings prompted us to investigate whether PP1alpha interferes with oncogenic transformation. The ability of NIH 3T3 cells to form foci after transformation with ras/cyclin D1 was significantly inhibited by co-transfection with PP1alphaT320A, but not PP1alpha. Likewise, cells expressing PP1alphaT320A or PP1alphaT320A fused to green fluorescent protein (GFP) were unable to form colonies in soft agar, regardless of whether PP1alpha constructs were co-transfected with ras/cyclin D1 or transfected into stably transformed cells. Overexpressed wild-type (Wt) PP1alpha and GFP-PP1alpha were phosphorylated in Thr320, most likely explaining its lack of effect. Expression of GFP-PP1alphaT320A was associated with caspase-cleaved pRb in Western blots (WB) and morphological signs of cell death. These findings demonstrate that PP1alpha activity can override oncogenic signaling by causing cell-cycle arrest and/or apoptosis rather than restoring contact inhibition or anchorage dependence.

Protein phosphatase 1alpha is required for murine lung growth and morphogenesis.

Protein phosphatase 1 (PP1) plays important roles in cell cycle control and apoptosis, two processes that impinge on morphogenesis and differentiation. Following the precedent set by other molecules regulating the cell cycle and apoptosis, we hypothesized that PP1 may have context-specific roles in development. Therefore, we have studied the spatial and temporal expression of PP1alpha during murine lung development and determined the consequences of loss of PP1alpha function on branching morphogenesis. By using an immunohistochemical approach, we show here that PP1alpha was expressed throughout the epithelium and mesenchyme upon the emergence of the lung primordium on embryonic day 10, with immunostaining exclusively extranuclear. During the late pseudoglandular stage, PP1alpha was predominantly expressed in the distal lung epithelium, whereas the mesenchyme contained very little or no PP1alpha protein. Peri- and postnatally, PP1alpha immunostaining was mostly nuclear in apparently differentiated cells, as judged by colocalization with well-known markers for lung differentiation. Exposure of fetal lung explants to antisense oligodeoxynucleotides against PP1alpha, resulted in decreased overall size of the cultured lung, a defect in forming new airways, lack of expression of surfactant protein C, and histologic signs of poor differentiation. These data suggest that PP1alpha is required for branching morphogenesis and differentiation.