OPEN Oncogene (2018) 37, 286–301 www.nature.com/onc ORIGINAL ARTICLE ING5 activity in self-renewal of glioblastoma stem cells via calcium and follicle stimulating hormone pathways F Wang1,2, AY Wang2,3, C Chesnelong2,3, Y Yang1,2,4, A Nabbi1,2, S Thalappilly1,2, V Alekseev1 and K Riabowol1,2 Stem cell-like brain tumor initiating cells (BTICs) cause recurrence of glioblastomas, with BTIC ‘stemness’ affected by epigenetic mechanisms. The ING family of epigenetic regulators (ING1-5) function by targeting histone acetyltransferase (HAT) or histone deacetylase complexes to the H3K4me3 mark to alter histone acetylation and subsequently, gene expression. Here we find that ectopic expression of ING5, the targeting subunit of HBO1, MOZ and MORF HAT complexes increases expression of the Oct4, Olig2 and Nestin stem cell markers, promotes self-renewal, prevents lineage differentiation and increases stem cell pools in BTIC populations. This activity requires the plant homeodomain region of ING5 that interacts specifically with the H3K4me3 mark. ING5 also enhances PI3K/AKT and MEK/ERK activity to sustain self-renewal of BTICs over serial passage of stem cell-like spheres. ING5 exerts these effects by activating transcription of calcium channel and follicle stimulating hormone pathway genes. In silico analyses of The Cancer Genome Atlas data suggest that ING5 is a positive regulator of BTIC stemness, whose expression negatively correlates with patient prognosis, especially in the Proneural and Classical subtypes, and in tumors with low SOX2 expression. These data suggest that altering histone acetylation status and signaling pathways induced by ING5 may provide useful clinical strategies to target tumor resistance and recurrence in glioblastoma. Oncogene (2018) 37, 286–301; doi:10.1038/onc.2017.324; published online 18 September 2017 INTRODUCTION of induced pluripotent stem cells.20 However, there are limited Glioblastoma multiforme (GBM) is the most frequent and lethal studies about specific epigenetic factors in BTIC regulation, with type of intracranial tumor with a five-year survival rate of 5.1%.1,2 most focusing on members of the polycomb group and mixed- 21,22 An intratumoral lineage hierarchy has been uncovered following lineage leukemia. the identification of tumor initiating cells in brain tumors,3,4 and The inhibitor of growth (ING) family of epigenetic regulators the progenitor-like status of brain tumor initiating cells (BTICs) is (ING1-5) affect growth, the DNA damage response, cellular 23–26 thought to make gliomas particularly refractory to standard cancer senescence and apoptosis. INGs are stoichiometric compo- treatments. Primary cultures of BTICs can establish tumors during nents of histone acetyltransferase (HAT) or histone deacetylase 27–29 serial in vivo transplantation5,6 and repopulate tumor sites after (HDAC) complexes and target them specifically to the irradiation and chemotherapy.7,8 BTICs display a combination of H3K4me3 mark with their plant homeodomain (PHD) form of zinc 30 neural stem cell phenotypes and tumorigenic properties, and may finger. Although initially identified as type II tumor suppressors 31 originate from crosstalk between deregulated developmental and intensively studied in cancer systems, INGs also function in processes and mitogenic mutations during gliomagenesis.9 Both germ cell development and cell differentiation via epigenetic – neurodevelopmental factors and mitogenic signaling pathways regulation.32 34 35,36 contribute to the ‘stemness character’ of BTICs. For example, the ING5 has been reported to regulate cell proliferation, 37 38 37,39 Notch and inhibitor of differentiation (ID) proteins promote self- apoptosis, metastasis and chemoresistance in different renewal in both neural stem cells and BTICs.10–12 EGFR and PDGFR cancer models. Like other ING family members, ING5 can bind and 40,41 activation, the most common genetic aberrations in gliomas, can acetylate p53 by associated HAT complexes, which is assumed induce de-differentiation of mature astrocytes to cause glioma- to account for most of its inhibitiory effects on proliferation seen 37,42–44 genesis in mouse models,13,14 and promote BTIC maintenance in different cancers. However, ING5 also possesses p53- in vitro.15 independent functions in promoting the proliferation of several Epigenetic alterations drive oncogenesis16 and in GBM, there is cancer cell lines, probably due to interactions with HBO128 and a disproportionately high rate of mutation in chromatin CDK2.35 ING5 is rarely mutated in cancer but the deregulation of modifiers.17 The epigenetic landscape, which is distinct between its expression has been reported in many cancer types, with BTICs and differentiated GBM cells, helps maintain BTIC identity decreased expression in lung cancer and acute myeloid and prevents interconversion between these cell types.18,19 This leukemia36,44 and increased expression in breast cancer and epigenetic barrier between BTICs and non-stem GBM cells can be glioblastoma.42,43 Overall, the role of ING5 in cancer appears to be traversed via epigenetic remodeling that resembles reprogramming highly tissue specific and a discrepancy between its nuclear and 1Department of Biochemistry & Molecular Biology, University of Calgary, Calgary, AB, Canada; 2Department of Oncology, Arnie Charbonneau Cancer Institute, Cumming School of Medicine, University of Calgary, Calgary, AB, Canada; 3Hotchkiss Brain Institute, Department of Cell Biology and Anatomy, University of Calgary, Calgary, AB, Canada and 4Department of Obstetrics and Gynecology, The First Affiliated Hospital of Harbin Medical University, Heilongjiang, China. Correspondence: Professor K Riabowol, Department of Biochemistry & Molecular Biology, University of Calgary, Calgary, AB, Canada or Department of Oncology, University of Calgary, Arnie Charbonneau Cancer Institute, Cumming School of Medicine, #311 HMRB, 3330 Hospital Dr. NW, Calgary T2N 4N1, AB, Canada. E-mail: [email protected] Received 15 May 2017; revised 10 July 2017; accepted 28 July 2017; published online 18 September 2017 ING5 promotes BTIC stemness F Wang et al 287 cytoplasmic levels noted in previous studies complicates the markers, which strongly correlate with the stemness properties investigation of its functions. ING5 appears to be more multi- and tumor initiating abilities of glioma cells.53,54 We used a functional than other ING proteins, being incorporated into the transposon-based PiggyBac system (iPB) for stable and inducible HBO1, MOZ and MORF HAT complexes.28,45 ING5 serves as a ING5 overexpression.55 iPB-ING5 cells treated by the inducer critical epigenetic regulator for epidermal stem cell maintenance cumate showed a higher percentage of CD44 and C133 positive with global co-occupancy with H3K4me3 at genes actively cells compared to the control (iPB-ctr) (Figure 2a, upper panels). expressed in stem cells and silenced during differentiation.46 High Conversely, shR-ING5 reduced CD44 positive cells (Figure 2a, lower ING5 levels were also seen in pluripotent cells compared to panels), indicating that ING5 increased the stem cell pool in BTICs. differentiated lineages,47–49 suggesting it may function as a Self-renewal is achieved in tissue stem cells through uneven common factor for cell plasticity in stem cell systems. distribution of fate determinants for stemness via asymmetric cell Since ING5–HAT complexes were reported to function in the division.56 In neural stem cells, CD133 specifically marks the apical maintenance of pluripotent cells (HBO1),50 hematopoietic stem membrane initiating subsequent asymmetric division.57 Since cells (MOZ)51 and neural stem cells (MORF),52 we asked whether ING5 reciprocally altered CD133 levels, we tested if ING5 also ING5 affected stem cell properties of BTICs and indeed, found that altered symmetry of cell division. In our mitotic pair analysis, the it does. ING5 is highly expressed in stem cells where it promotes distribution of Nestin was used as an indicator for three division BTIC self-renewal most strikingly in the absence of growth factors. modes, symmetric proliferating, symmetric differentiating and Pathway analyses indicate ING5 promotes stemness by maintain- asymmetric division (Figure 2b). Despite a high proportion of ing intracellular calcium levels and activating the follicle stimulat- symmetric division probably due to perturbed asymmetric ing hormone (FSH) pathway, which co-operatively contribute to machinery in tumor initiating cells,58 ING5 induced a more than BTIC self-renewal, providing novel candidates for targeting BTICs two-fold increase of asymmetric divisions (highlighted by the in GBMs. white arrowhead in Figure 2b), indicating a function in the intrinsic self-renewal machinery of this BTIC strain. Ectopic expression of ING5 also interfered with lineage RESULTS commitment. iPB-ING5 cells slowed FBS-induced morphological ING5 maintains BTIC self-renewal changes compared to control cells (Figure 2c) and immunostain- E14, R1 and D3 mouse embryonic stem cell lines showed an ing indicated a higher level of the neural stem cell marker Nestin immediate reduction of ING5 mRNA (Supplementary Figure S1a) and reduced level of Tubb3 in iPB-ING5 cells (Supplementary and protein (Supplementary Figures S1b, c) as pluripotent cells Figures S2a, b). Conversely, shR-ING5 knockdown cells displayed differentiated. The intrinsic stem cell properties of primary BTIC higher levels of Tubb3 and less Nestin (Figure 2d), which was cultures are best preserved in serum-free medium supplemented