Abstract

BACKGROUND:

Meningiomas are the most common primary intracranial tumors in adults. While a majority of meningiomas are slow growing neoplasms that may cured by surgical resection, a subset demonstrates more aggressive behavior and insidiously recurs despite surgery and radiation, without effective alternative treatment options. Elucidation of critical mitogenic pathways in meningioma oncogenesis may offer new therapeutic strategies. We performed an integrated genomic and molecular analysis to characterize the expression and function of osteoglycin (OGN) in meningiomas and explored possible therapeutic approaches for OGN-expressing meningiomas.

METHODS:

OGN mRNA expression in human meningiomas was assessed by RNA microarray and RNAscope. The impact of OGN on cell proliferation, colony formation, and mitogenic signaling cascades was assessed in a human meningioma cell line (IOMM-Lee) with stable overexpression of OGN. Furthermore, the functional consequences of introducing an AKT inhibitor in OGN-overexpressing meningioma cells were assessed.

RESULTS:

OGN mRNA expression was dramatically increased in meningiomas compared to a spectrum of other brain tumors and normal brain. OGN-overexpressing meningioma cells demonstrated an elevated rate of cell proliferation, cell cycle activation, and colony formation as compared with cells transfected with control vector. In addition, NF2 mRNA and protein expression were both attenuated in OGN-overexpressing cells. Conversely, mTOR pathway and AKT activation increased in OGN-overexpressing cells compared to control cells. Lastly, introduction of an AKT inhibitor reduced OGN expression in meningioma cells and resulted in increased cell death and autophagy, suggestive of a reciprocal relationship between OGN and AKT.

CONCLUSION:

We identify OGN as a novel oncogene in meningioma proliferation. AKT inhibition reduces OGN protein levels in meningioma cells, with a concomitant increase in cell death, which provides a promising treatment option for meningiomas with OGN overexpression.

Targeting EGFR is a validated approach in the treatment of squamous-cell cancers (SCCs), although there are no established biomarkers for predicting response. We have identified a synonymous mutation in EGFR, c.2361G>A (encoding p.Gln787Gln), in two patients with head and neck SCC (HNSCC) who were exceptional responders to gefitinib, and we showed in patient-derived cultures that the A/A genotype was associated with greater sensitivity to tyrosine kinase inhibitors (TKIs) as compared to the G/A and G/G genotypes. Remarkably, single-copy G>A nucleotide editing in isogenic models conferred a 70-fold increase in sensitivity due to decreased stability of the EGFR-AS1 long noncoding RNA (lncRNA). In the appropriate context, sensitivity could be recapitulated through EGFR-AS1 knockdown in vitro and in vivo, whereas overexpression was sufficient to induce resistance to TKIs. Reduced EGFR-AS1 levels shifted splicing toward EGFR isoform D, leading to ligand-mediated pathway activation. In co-clinical trials involving patients and patient-derived xenograft (PDX) models, tumor shrinkage was most pronounced in the context of the A/A genotype for EGFR-Q787Q, low expression of EGFR-AS1 and high expression of EGFR isoform D. Our study reveals how a 'silent' mutation influences the levels of a lncRNA, resulting in noncanonical EGFR addiction, and delineates a new predictive biomarker suite for response to EGFR TKIs.

Dopaminergic systems play a major role in reward-related behavior and dysregulation of dopamine (DA) systems can cause several mental disorders, including depression. We previously reported that dopamine D2 receptor knockout (D2R-/-) mice display increased anxiety and depression-like behaviors upon chronic stress. Here, we observed that chronic stress caused myelin loss in wild-type (WT) mice, while the myelin level in D2R-/- mice, which was already lower than that in WT mice, was not affected upon stress. Fewer mature oligodendrocytes (OLs) were observed in the corpus callosum of stressed WT mice, while in D2R-/- mice, both the control and stressed group displayed a decrease in the number of mature OLs. We observed a decrease in the number of active β-catenin (ABC)-expressing and TCF4-expressing cells among OL lineage cells in the corpus callosum of stressed WT mice, while such regulation was not found in D2R-/- mice. Administration of lithium normalized the behavioral impairments and myelin damage induced by chronic stress in WT mice, and restored the number of ABC-positive and TCF4-positive OLs, while such effect was not found in D2R-/- mice. Together, our findings indicate that chronic stress induces myelin loss through the Wnt/β-catenin signaling pathway in association with DA signaling through D2R.

Histone Deacetylase 11 (HDAC11) is highly expressed in the central nervous system where it has been reported to have roles in neural differentiation. In contrast with previous studies showing nuclear and cytoplasmic localisation, we observed synaptic enrichment of HDAC11. Knockout mouse models for HDACs 1-9 have been important for guiding the development of isoform specific HDAC inhibitors as effective therapeutics. Given the close relationship between HDAC11 and neural cells in vitro, we examined neural tissue in a previously uncharacterised Hdac11 knockout mouse (Hdac11 KO/KO). Loss of HDAC11 had no obvious impact on brain morphology and neural stem/precursor cells isolated from Hdac11 KO/KO mice had comparable proliferation and differentiation characteristics. However, in differentiating neural cells we observed decreased expression of schizophrenia-associated gene Fez1 (fasciculation and elongation protein zeta 1), a gene previously reported to be regulated by HDAC11 activity. FEZ1 has been associated with the dendritic growth of neurons and risk of schizophrenia via its interaction with DISC1 (disrupted in schizophrenia 1). Examination of cortical, cerebellar and hippocampal tissue reveal decreased Fez1 expression specifically in the hippocampus of adult mice. The results of this study demonstrate that loss of HDAC11 has age dependent and brain-region specific consequences.

Renal cell carcinoma (RCC) is one of the most lethal urologic cancers. About one-third of RCC patients already have distal metastasis at the time of diagnosis. There is growing evidence that Hox antisense intergenic RNA (HOTAIR) plays essential roles in metastasis in several types of cancers. However, the precise mechanism by which HOTAIR enhances malignancy remains unclear, especially in RCC. Here, we demonstrated that HOTAIR enhances RCC-cell migration by regulating the insulin growth factor-binding protein 2 (IGFBP2) expression. HOTAIR expression in tumors was significantly correlated with nuclear grade, lymph-node metastasis, and lung metastasis. High HOTAIR expression was associated with a poor prognosis in both our dataset and The Cancer Genome Atlas dataset. Migratory capacity was enhanced in RCC cell lines in a HOTAIR-dependent manner. HOTAIR overexpression accelerated tumorigenicity and lung metastasis in immunodeficient mice. Microarray analysis revealed that IGFBP2 expression was upregulated in HOTAIR-overexpressing cells compared with control cells. The enhanced migration activity of HOTAIR-overexpressing cells was attenuated by IGFBP2 knockdown. IGFBP2 and HOTAIR were co-expressed in clinical RCC samples. Our findings suggest that the HOTAIR-IGFBP2 axis plays critical roles in RCC metastasis and may serve as a novel therapeutic target for advanced RCC.

Adult T-cell leukemia/lymphoma (ATL) is an aggressive T cell malignancy that occurs in HTLV-1 infected patients. Most ATL patients develop osteolytic lesions and hypercalcemia of malignancy, causing severe skeletal related complications and reduced overall survival. The HTLV-1 virus encodes 2 viral oncogenes, Tax and HBZ. Tax, a transcriptional activator, is critical to ATL development, and has been implicated in pathologic osteolysis. HBZ, HTLV-1 basic leucine zipper transcription factor, promotes tumor cell proliferation and disrupts Wnt pathway modulators; however, its role in ATL induced osteolytic bone loss is unknown. To determine if HBZ is sufficient for the development of bone loss, we established a transgenic Granzyme B HBZ (Gzmb-HBZ) mouse model. Lymphoproliferative disease including tumors, enlarged spleens and/or abnormal white cell counts developed in two-thirds of Gzmb-HBZ mice at 18 months. HBZ positive cells were detected in tumors, spleen and bone marrow. Importantly, pathologic bone loss and hypercalcemia were present at 18 months. Bone-acting factors were present in serum and RANKL, PTHrP and DKK1, key mediators of hypercalcemia and bone loss, were upregulated in Gzmb-HBZ T cells. These data demonstrate that Gzmb-HBZ mice model ATL bone disease and express factors that are current therapeutic targets for metastatic and bone resident tumors.

Hundreds of circular RNAs (circRNAs) are highly abundant in the mammalian brain, often with conserved expression. Here we show that the circRNA Cdr1as is massively bound by the microRNAs (miRNAs) miR-7 and miR-671 in human and mouse brains. When the Cdr1as locus was removed from the mouse genome, knockout animals displayed impaired sensorimotor gating-a deficit in the ability to filter out unnecessary information-which is associated with neuropsychiatric disorders. Electrophysiological recordings revealed dysfunctional synaptic transmission. Expression of miR-7 and miR-671 was specifically and posttranscriptionally misregulated in all brain regions analyzed. Expression of immediate early genes such as Fos, a direct miR-7 target, was enhanced in Cdr1as-deficient brains, providing a possible molecular link to the behavioral phenotype. Our data indicate an in vivo loss-of-function circRNA phenotype and suggest that interactions between Cdr1as and miRNAs are important for normal brain function.

Angiosarcoma is an aggressive vascular sarcoma with an extremely poor prognosis. Due to the relative rarity of this disease, its molecular drivers and optimal treatment strategies are obscure. DICER1 is an RNase III endoribonuclease central to microRNA biogenesis, and germline DICER1 mutations result in a cancer predisposition syndrome, associated with an increased risk of many tumor types. Here we show that biallelic Dicer1 deletion with aP2-Cre drives aggressive and metastatic angiosarcoma independent of other genetically engineered oncogenes or tumor suppressor loss. Angiosarcomas in aP2-Cre;Dicer1Flox/- mice histologically and genetically resemble human angiosarcoma. MicroRNA-23 target genes including the oncogenes Ccnd1 as well as Adam19, Plau, and Wsb1 that promote invasiveness and metastasis were enriched in mouse and human angiosarcoma. These studies illustrate that Dicer1 can function as a traditional loss-of-function tumor suppressor gene, and they provide a fully penetrant animal model for the study of angiosarcoma development and metastasis.

Growth hormone (GH) resistance may develop as a consequence of inflammation during conditions such as inflammatory bowel disease, encompassing ulcerative colitis (UC). However, the specific role of the GH-insulin growth factor (IGF)-1-axis and/or the functional consequences of GH resistance in this condition are unclear. In situ hybridization targeting the GH receptor (GHR) and relevant transcriptional analyses were performed in patients with UC and in IL-10 knock-out mice with piroxicam accelerated colitis (PAC). Using cultured primary epithelial cells, the effects of inflammation on the molecular mechanisms governing GH resistance was verified. Also, the therapeutic potential of GH on mucosal healing was tested in the PAC model. Inflammation induced intestinal GH resistance in UC and experimental colitis by down-regulating GHR expression and up-regulating suppressor of cytokine signalling (SOCS) proteins. These effects are driven by pro-inflammatory mediators (tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6) as confirmed using primary epithelial cells. Treatment of experimental colitis with GH increased IGF-1 and body weight of the mice, but had no effects on colonic inflammation or mucosal healing. The high transcriptional similarity between UC and experimental colitis accentuates the formation of intestinal GH resistance during inflammation. Inflammation-induced GH resistance not only impairs general growth but induces a state of local resistance, which potentially impairs the actions of GH on mucosal healing during colitis when using long-acting GH therapy.

Outcomes of Zika virus (ZIKV) infection in pregnant women vary from the birth of asymptomatic offspring to abnormal development and severe brain lesions in fetuses and infants. There are concerns that offspring affected in utero and born without apparent symptoms may develop mental illnesses. Therefore, animal models are important to test interventions against in utero infection and health sequelae in symptomatic and likely more widespread asymptomatic offspring. To partially reproduce in utero infection in humans, we directly inoculated selected porcine conceptuses with ZIKV. Inoculation resulted in rapid trans-fetal infections, persistent infection in conceptuses, molecular pathology in fetal brains, fetal antibody and type I interferon responses. Offspring infected in utero showed ZIKV in their fetal membranes collected after birth. Some in utero affected piglets were small, depressed, had undersized brains, showed seizures and potentially increased activity. Our data suggest that porcine model of persistent in utero ZIKV infection has a strong potential for translational research and can be used to test therapeutic interventions in vivo.

Deep penetrating nevus (DPN) is characterized by enlarged, pigmented melanocytes that extend through the dermis. DPN can be difficult to distinguish from melanoma but rarely displays aggressive biological behavior. Here, we identify a combination of mutations of the β-catenin and mitogen-activated protein kinase pathways as characteristic of DPN. Mutations of the β-catenin pathway change the phenotype of a common nevus with BRAF mutation into that of DPN, with increased pigmentation, cell volume and nuclear cyclin D1 levels. Our results suggest that constitutive β-catenin pathway activation promotes tumorigenesis by overriding dependencies on the microenvironment that constrain proliferation of common nevi. In melanoma that arose from DPN we find additional oncogenic alterations. We identify DPN as an intermediate stage in the step-wise progression from nevus to melanoma. In summary, we delineate specific genetic alterations and their sequential order, information that can assist in the diagnostic classification and grading of these distinctive neoplasms.Deep penetrating nevi (DPN) are unusual melanocytic neoplasms with unknown genetic drivers. Here the authors show that majority of DPN harbor activating mutations in the β-catenin and the MAP-kinase pathways; this characteristic can help in the classification and grading of these distinctive neoplasms.

Zika virus (ZIKV) infection can cause fetal developmental abnormalities and Guillain-Barré syndrome in adults. Although progress has been made in understanding the link between ZIKV infection and microcephaly, the pathology of ZIKV, particularly the viral reservoirs in human, remains poorly understood. Several studies have shown that compared to serum samples, patients' urine samples often have a longer duration of ZIKV persistency and higher viral load. This finding suggests that an independent viral reservoir may exist in the human urinary system. Despite the clinical observations, the host cells of ZIKV in the human urinary system are poorly characterized. In this study, we demonstrate that ZIKV can infect renal proximal tubular epithelial cells (RPTEpiCs) in immunodeficient mice in vivo and in both immortalized and primary human renal proximal tubular epithelial cells (hRPTEpiCs) in vitro. Importantly, ZIKV infection in mouse kidneys caused caspase-3-mediated apoptosis of renal cells. Similarly, in vitro infection of immortalized and primary hRPTEpiCs resulted in notable cytopathic effects. Consistent with the clinical observations, we found that ZIKV infection can persist with prolonged duration in hRPTEpiCs. RNA-Seq analyses of infected hRPTEpiCs revealed a large number of transcriptional changes in response to ZIKV infection, including type I interferon signaling genes and anti-viral response genes. Our results suggest that hRPTEpiCs are a potential reservoir of ZIKV in the human urinary system, providing a possible explanation for the prolonged persistency of ZIKV in patients' urine.