Frizzled receptor 6 marks rare, highly tumourigenic stem-like cells in mouse and human neuroblastomas

Copyright © 2011 Cantilena et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 3.0 License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. The article was made available through the Brunel Open Access Publishing Fund.Wnt signalling is an important component of vertebrate development, required for specification of the neural crest. Ten Wnt receptors [Frizzled receptor 1-10 (Fzd1-10)] have been identified so far, some of which are expressed in the developing nervous system and the neural crest. Here we show that expression of one such receptors, Fzd6, predicts poor survival in neuroblastoma patients and marks rare, HIF1/2 α-positive cells in tumour hypoxic areas. Fzd6 positive neuroblastoma cells form neurospheres with high efficiency, are resistant to doxorubicin killing and express high levels of mesenchymal markers such as Twist1 and Notch1. Expression of Fzd6 is required for the expression of genes of the noncanonical Wnt pathway and the spheres forming activity. When transplanted into immunodeficient mice, neuroblastoma cells expressing the Fzd6 marker grow more aggressively than their Fzd6 negative counterparts. We conclude that Fzd6 is a new surface marker of aggressive neuroblastoma cells with stem cell-like features.This work was sponsored by the Wellcome Trust, the RICC cancer fund, SPARKS, the Italian Association for Cancer Research, Regione Liguria and the Italian Ministry of Health

Human γδ T-Cells: from surface receptors to the therapy of high-risk leukemias

γδ T lymphocytes are potent effector cells, capable of efficiently killing tumor and leukemia cells. Their activation is mediated by γδ T-cell receptor (TCR) and by activating receptors shared with NK cells (e.g., NKG2D and DNAM-1). γδ T-cell triggering occurs upon interaction with specific ligands, including phosphoantigens (for Vγ9Vδ2 TCR), MICA-B and UL16 binding protein (for NKG2D), and PVR and Nectin-2 (for DNAM-1). They also respond to cytokines undergoing proliferation and release of cytokines/chemokines. Although at the genomic level γδ T-cells have the potential of an extraordinary TCR diversification, in tissues they display a restricted repertoire. Recent studies have identified various γδ TCR rearrangements following either hematopoietic stem cell transplantation (HSCT) or cytomegalovirus infection, accounting for their "adaptive" potential. In humans, peripheral blood γδ T-cells are primarily composed of Vγ9Vδ2 chains, while a minor proportion express Vδ1. They do not recognize antigens in the context of MHC molecules, thus bypassing tumor escape based on MHC class I downregulation. In view of their potent antileukemia activity and absence of any relevant graft-versus-host disease-inducing effect, γδ T-cells may play an important role in the successful clinical outcome of patients undergoing HLA-haploidentical HSCT depleted of TCR αβ T/CD19+ B lymphocytes to cure high-risk acute leukemias. In this setting, high numbers of both γδ T-cells (Vδ1 and Vδ2) and NK cells are infused together with CD34+ HSC and may contribute to rapid control of infections and leukemia relapse. Notably, zoledronic acid potentiates the cytolytic activity of γδ T-cells in vitro and its infusion in patients strongly promotes γδ T-cell differentiation and cytolytic activity; thus, treatment with this agent may contribute to further improve the patient clinical outcome after HLA-haploidentical HSCT depleted of TCR αβ T/CD19+ B lymphocytes

Grb7 Upregulation Is a Molecular Adaptation to HER2 Signaling Inhibition Due to Removal of Akt-Mediated Gene Repression

The efficacy of anti-HER2 therapeutics, such as lapatinib and trastuzumab, is limited by primary and acquired resistance. Cellular adaptations that allow breast cancer cell to survive prolonged HER2 inhibition include de-repression of the transcription factor FOXO3A with consequent estrogen receptor activation, and/or increased HER3 signaling. Here, we used low-density arrays, quantitative PCR, and western blotting to determine how HER2 signaling inhibition with lapatinib or PI3K inhibitors affects the expression of genes involved in breast cancer metastatic spread and overall prognosis. Retroviral transgenesis was used to express constitutively active forms of Akt in the HER2+ breast cancer cell line SKBR3, and Grb7 in MCF7 cells. Specific gene silencing was obtained by siRNAs transfection. A murine BT474 xenograft cancer model was used to assess the effect of lapatinib on gene expression in vivo. We found that lapatinib induces upregulation of Grb7, an adaptor protein involved in receptor tyrosine kinase signaling and promoting cell survival and cell migration. Grb7 upregulation induced by lapatinib was found to occur in cancer cells in vitro and in vivo. We demonstrate that Grb7 upregulation is recreated by PI3K inhibitors while being prevented by constitutively active Akt. Thus, Grb7 is repressed by PI3K signaling and lapatinib-mediated Akt inhibition is responsible for Grb7 de-repression. Finally, we show that Grb7 removal by RNA-interference reduces breast cancer cell viability and increases the activity of lapatinib. In conclusion, Grb7 upregulation is a potentially adverse consequence of HER2 signaling inhibition. Preventing Grb7 accumulation and/or its interaction with receptor tyrosine kinases may increase the benefit of HER2-targeting drugs

PD-L1 expression in metastatic neuroblastoma as an additional mechanism for limiting immune surveillance

The prognosis of high-risk neuroblastoma (NB) remains poor, although immunotherapies with anti-GD2 antibodies have been reported to provide some benefit. Immunotherapies can be associated with an IFNγ storm that induces in tumor cells the “adaptive immune resistance” characterized by the de-novo expression of Programmed Death Ligands (PD-Ls). Tumor cells can also constitutively express PD-Ls in response to oncogenic signaling. Here, we analyze the constitutive and the inducible surface expression of PD-Ls in NB cells. We show that virtually all HLA class Ipos NB cell lines constitutively express PD-L1, whereas PD-L2 is rarely detected. IFNγ upregulates or induces PD-L1 both in NB cell lines in vitro and in NB engrafted nude/nude mice. Importantly, after IFNγ stimulation PD-L1 can be acquired by NB cell lines, as well as by metastatic neuroblasts isolated from bone marrow aspirates of high-risk NB patients, characterized by different MYCN amplification status. Interestingly, in one patient NB cells were poorly responsive to IFNγ stimulation, pointing out that responsiveness to IFNγ might represent a further element of heterogeneity in metastatic neuroblasts. Finally, we document the presence of lymphocytes expressing the PD-1 receptor in NB-infiltrated bone marrow of patients. PD-1pos cells are mainly represented by αβ T cells, but also include small populations of γδ T cells and NK cells. Moreover, PD-1pos T cells have a higher expression of activation markers. Overall, our data show that a PD-L1-mediated immune resistance mechanism occurs in metastatic neuroblasts and provide a biological rationale for blocking the PD-1/PD-Ls axis in future combined immunotherapeutic approaches

A promyelocytic leukemia protein-thrombospondin 2 axis and the risk of relapse in neuroblastoma

Purpose. Neuroblastoma is a childhood malignancy originating from the sympathetic nervous system with a complex biology, prone to metastasize and relapse. High-risk, metastatic cases are explained in part by amplification or mutation of oncogenes such as MYCN and ALK and loss of tumour suppressor genes in chromosome band 1p. However, it is fundamental to identify other pathways responsible for the large portion of neuroblastomas with no obvious molecular alterations. Experimental design. Neuroblastoma cell lines were used for assessment of tumour growth in vivo and in vitro. Protein expression in tissues and cells was assessed using immunofluorescence and immunohistochemistry. The association of PML expression with neuroblastoma outcome and relapse was calculated using log-rank and Mann-Whitney tests, respectively. Gene expression was assessed using chip microarrays. Results: PML is detected in the developing and adult sympathetic nervous system, whereas it is not expressed or low in metastatic neuroblastoma tumours. Reduced PML expression in patients with low-risk cancers - i.e. localized and negative for the MYCN protooncogene - is strongly associated with tumour recurrence. PML-I, but not PML-IV, isoform suppresses angiogenesis via upregulation of thrombospondin-2 (TSP-2), a key inhibitor of angiogenesis. Finally, PML-I and TSP-2 expression inversely correlates with tumour angiogenesis and recurrence in localized neuroblastomas. Dvorkina et al. A promyelocytic leukaemia protein-thrombospondin 2 axis and the risk of relapse in neuroblastoma 3 Conclusions: Our work reveals a novel PML-I-TSP2 axis for regulation of angiogenesis and cancer relapse, which could be used to identify patients with low-risk, localized tumours that might benefit from chemotherapy