A Novel Chordoma Xenograft Allows In Vivo Drug Testing and Reveals the Importance of NF-κB Signaling in Chordoma Biology

Chordoma is a rare primary bone malignancy that arises in the skull base, spine and sacrum and originates from remnants of the notochord. These tumors are typically resistant to conventional chemotherapy, and to date there are no FDA-approved agents to treat chordoma. The lack of in vivo models of chordoma has impeded the development of new therapies for this tumor. Primary tumor from a sacral chordoma was xenografted into NOD/SCID/IL-2R γ-null mice. The xenograft is serially transplantable and was characterized by both gene expression analysis and whole genome SNP genotyping. The NIH Chemical Genomics Center performed high-throughput screening of 2,816 compounds using two established chordoma cell lines, U-CH1 and U-CH2B. The screen yielded several compounds that showed activity and two, sunitinib and bortezomib, were tested in the xenograft. Both agents slowed the growth of the xenograft tumor. Sensitivity to an inhibitor of IκB, as well as inhibition of an NF-κB gene expression signature demonstrated the importance of NF-κB signaling for chordoma growth. This serially transplantable chordoma xenograft is thus a practical model to study chordomas and perform in vivo preclinical drug testing.     

LncRNA-NONHSAT024778 promote the proliferation and invasion of chordoma cell by regulating miR-1290/Robo1 axis

Chordoma is a malignant bone tumor originating from the embryonic remnants of the notochord. lncRNAs act as competing endogenous RNAs (ceRNAs) and play a critical role in tumor pathology. However, the biological role of lncRNA-NONHSAT024778 and the underlying molecular mechanism in chordoma remains unknown. qRT-PCR was used to analyze the expression changes of NONHSAT024778 and miR-1290 in chordoma tissues and cell lines. Bioinformatics analysis and luciferase reporter assay were applied to detect the targeting binding effect between NONHSAT024778 and miR-1290, and between Robo1 and miR-1290. The effect of NONHSAT024778 on chordoma cell proliferation and invasion and its regulation of miR-1290 by acting as a ceRNA were also investigated. An increased NONHSAT024778 expression was correlated with a decreased miR-1290 level in chordoma tissues. NONHSAT024778 knockdown suppressed the proliferation and invasion of chordoma cells. miR-1290 restored expression rescued the carcinogenic function of NONHSAT024778. Bioinformatics analysis showed that NONHSAT024778 acted as ceRNA to regulate Robo1 via sponging miR-1290 in chordoma cells, thereby promoting chordoma cell malignant progression. In vivo results confirmed the anti-tumor effects of NONHSAT024778 knockdown activating miR-1290 to inhibit the oncogene Robo1. NONHSAT024778 is substantially overexpressed, whereas miR-1290 is decreased in chordoma tissue. NONHSAT024778-miR-1290-Robo1 axis plays a critical role in chordoma tumorigenesis and might be a potential predictive biomarker for the diagnosis and therapeutic target among patients with chordoma.     

Characterization of the endolysosomal system in human chordoma cell lines: is there a role of lysosomes in chemoresistance of this rare bone tumor?

Chordoma is a rare tumor of the bone derived from remnants of the notochord with pronounced chemoresistance. A common feature of the notochord and chordoma cells is distinct vacuolization. Recently, the notochord vacuole was described as a lysosome-related organelle. Since lysosomes are considered as mediators of drug resistance in cancer, we were interested whether they may also play a role in chemoresistance of chordoma. We characterized the lysosomal compartment in chordoma cell lines by cytochemistry, electron microscopy (ELMI) and mutational analysis of genes essential for the physiology of lysosomes. Furthermore, we tested for the first time the cytotoxicity of chloroquine, which targets lysosomes, on chordoma. Cytochemical stainings clearly demonstrated a huge mass of lysosomes in chordoma cell lines with perinuclear accumulation. Also vacuoles in chordoma cells were positive for the lysosomal marker LAMP1 but showed no acidic pH. Genetic analysis detected no apparent mutation associated with known lysosomal pathologies suggesting that vacuolization and the huge lysosomal mass of chordoma cell lines is rather a relict of the notochord than a result of transformation. ELMI investigation of chordoma cells confirmed the presence of large vacuoles, lysosomes and autophagosomes with heterogeneous ultrastructure embedded in glycogen. Interestingly, chordoma cells seem to mobilize cellular glycogen stores via autophagy. Our first preclinical data suggested no therapeutically benefit of chloroquine for chordoma. Even though, chordoma cells are crammed with lysosomes which are according to their discoverer de Duve “cellular suicide bags”. Destabilizing these “suicide bags” might be a promising strategy for the treatment of chordoma.     

Epigenetic deregulations in chordoma

Chordoma is a rare type of malignant bone tumour arising from remnant notochord and prognosis of patients with it remains poor as its molecular and genetic mechanisms are not well understood. Increasing evidence has demonstrated that epigenetic mechanisms (DNA methylation, histone modification and nucleosome remodelling), play a crucial role in the pathogenesis of many diseases. Aberrant epigenetic patterns are present in patients with chordoma, indicating a potential role for epigenetic mechanisms inthis malignancy. Furthermore, epigenetic alterations may provide novel biomarkers for diagnosis and prognosis as well as therapeutic targets for treatment. In this review, we discuss relevant epigenetic findings associated with chordoma, and their potential application for diagnosis, prognosis and treatment.     

Pharmacologic inhibition of ALK5 causes selective induction of terminal differentiation in mouse keratinocytes expressing oncogenic HRAS

TGFβ has both tumor suppressive and oncogenic roles in cancer development. We previously showed that SB431542 (SB), a small molecule inhibitor of the TGFβ type I receptor (ALK5) kinase, suppressed benign epidermal tumor formation but enhanced malignant conversion. Here, we show that SB treatment of primary K5rTA/tetORASV12G bitransgenic keratinocytes did not alter HRASV12G-induced keratinocyte hyperproliferation. However, continuous SB treatment significantly enhanced HRASV12G-induced cornified envelope formation and cell death linked to increased expression of enzymes transglutaminase (TGM) 1 and TGM3 and constituents of the cornified envelope small proline-rich protein (SPR) 1A and SPR2H. In contrast, TGFβ1 suppressed cornified envelope formation in HRASV12G keratinocytes. Similar results were obtained in HRASV12G transgenic mice treated topically with SB or by coexpressing TGFβ1 and HRASV12G in the epidermis. Despite significant cell death, SB-resistant HRASV12G keratinocytes repopulated the primary culture that had overcome HRas-induced senescence. These cells expressed reduced levels of p16(ink4a) and were growth stimulated by SB but remained sensitive to a calcium-induced growth arrest. Together these results suggest that differential responsiveness to cornification may represent a mechanism by which pharmacologic blockade of TGFβ signaling can inhibit the outgrowth of preneoplastic lesions but may cause a more progressed phenotype in a separate keratinocyte population.     

Erlotinib Inhibits Growth of a Patient-Derived Chordoma Xenograft

Chordomas are rare primary bone tumors that occur along the neuraxis. Primary treatment is surgery, often followed by radiotherapy. Treatment options for patients with recurrence are limited and, notably, there are no FDA approved therapeutic agents. Development of therapeutic options has been limited by the paucity of preclinical model systems. We have established and previously reported the initial characterization of the first patient-derived chordoma xenograft model. In this study, we further characterize this model and demonstrate that it continues to resemble the original patient tumor histologically and immunohistochemically, maintains nuclear expression of brachyury, and is highly concordant with the original patient tumor by whole genome genotyping. Pathway analysis of this xenograft demonstrates activation of epidermal growth factor receptor (EGFR). In vitro studies demonstrate that two small molecule inhibitors of EGFR, erlotinib and gefitinib, inhibit proliferation of the chordoma cell line U-CH 1. We further demonstrate that erlotinib significantly inhibits chordoma growth in vivo. Evaluation of tumors post-treatment reveals that erlotinib reduces phosphorylation of EGFR. This is the first demonstration of antitumor activity in a patient-derived chordoma xenograft model and these findings support further evaluation of EGFR inhibitors in this disease.     

脊索瘤的临床治疗进展

脊索瘤是一种罕见的低度恶性肿瘤,临床治愈率低,复发率较高。手术切除是治疗脊索瘤的首选方法,目前公认的脊索瘤治疗方法是完整(或大块)切除。术后辅助放射治疗越来越多的应用于临床,而新药物的研发、术前动脉栓塞的应用,以及射频消融等都被用于脊索瘤的临床治疗。本文综述了目前临床上应用的诊疗进展,包括手术治疗、放射治疗、药物治疗及射频消融治疗等方法。     

脊索瘤的临床治疗进展

脊索瘤是一种罕见的低度恶性肿瘤,临床治愈率低,复发率较高。手术切除是治疗脊索瘤的首选方法,目前公认的脊索瘤治疗方法是完整（或大块）切除。术后辅助放射治疗越来越多的应用于临床,而新药物的研发、术前动脉栓塞的应用,以及射频消融等都被用于脊索瘤的临床治疗。本文综述了目前临床上应用的诊疗进展,包括手术治疗、放射治疗、药物治疗及射频消融治疗等方法。     

Advances in the development of chordoma models for drug discovery and precision medicine

Chordomas are malignant bone tumors that arise from remnants of the notochord. These tumors are generally slow-growing, locally aggressive, and invasive. Chordomas are typically resistant to conventional chemo- and radiotherapy. The clinical management of this disease is very challenging, usually, treatment is surgical resection, which may be combined with radiotherapy. Although chordomas have undergone histologic and genetic analysis, the molecular mechanisms that drive their pathogenesis and resistance are still largely unknown. For many years this could be attributed to the lack of accurate and reliable in vitro and in vivo tumor models. Yet, over the past decade, many efforts have been made to prioritize the generation of useful chordoma cell lines, and tumor models that have shed more light on this malignancy and have made efficacious drug discovery a greater possibility. This review summarizes and discusses recent enhancements and improvements made to generate useful chordoma models and their applications in drug discovery and precision medicine.     

DEPDC1B regulates the progression of human chordoma through UBE2T-mediated ubiquitination of BIRC5

Chordoma is a rare bone malignancy with a high rate of local recurrence and distant metastasis. Although DEP domain-containing protein 1B (DEPDC1B) is implicated in a variety of malignancies, its relationship with chordoma is unclear. In this study, the biological role and molecular mechanism of DEPDC1B in chordoma were explored. The function of DEPDC1B in chordoma cells was clarified through loss-of-function assays in vitro and in vivo. Furthermore, molecular mechanism of DEPDC1B in chordoma cells was recognized by RNA sequencing and Co-Immunoprecipitation (Co-IP) assay. The malignant behaviors of DEPDC1B knockdown chordoma cells was significantly inhibited, which was characterized by reduced proliferation, enhanced apoptosis, and hindered migration. Consistently, decreased expression of DEPDC1B suppressed tumor growth in xenograft mice. Mechanically, DEPDC1B affected the ubiquitination of baculoviral inhibitor of apoptosis repeat-containing 5 (BIRC5) through ubiquitin-conjugating enzyme E2T (UBE2T). Simultaneous downregulation of BIRC5 and DEPDC1B may exacerbate the inhibitory effects of chordoma. Moreover, BIRC5 overexpression reduced the inhibitory effects of DEPDC1B knockdown in chordoma cells. In conclusion, DEPDC1B regulates the progression of human chordoma through UBE2T-mediated ubiquitination of BIRC5, suggesting that it may be a promising candidate target with potential therapeutic value.Subject terms: CNS cancer, Oncogenes     

Induction of clusterin Expression by Neuronal Cell Death in Zebrafish

Clusterin, a protein associated with multiple functions, is expressed in a wide variety of mammalian tissues. Although clusterin is known to be involved in neurodegenerative diseases, ageing, and tumorigenesis, a detailed analysis of the consequences of gain- or loss-of-function approaches has yet to be performed to understand the underlying mechanisms of clusterin functions. Since clusterin levels change in neurological diseases, it is likely that clusterin contributes to cell death and degeneration in general. Zebrafish was investigated as a model system to study human diseases. During development, zebrafish clusterin was expressed in the notochord and nervous system. Embryonic overexpression of clusterin by mRNA microinjection did not affect axis formation, whereas its knock-down by anti-sense morpholino treatment resulted in neuronal cell death. To analyze the function of clusterin in neurodegeneration, a transgenic zebrafish was investigated, in which nitroreductase expression is regulated under the control of a neuron-specific huC promoter which is active between the stages of early neuronal precursors and mature neurons. Nitroreductase turns metronidazole into a cytotoxic agent that induces cell death within 12 h. After metronidazole treatment, transgenic zebrafish showed neuron-specific cell death. Interestingly, we also observed a dramatic induction of clusterin expression in the brain and spinal cord in these fish, suggesting a direct or indirect role of clusterin in neuronal cell death and thus, more generally, in neurodegeneration.     

Morphogenetic mechanisms forming the notochord rod: The turgor pressure-sheath strength model

The notochord is a defining feature of chordates. During notochord formation in vertebrates and tunicates, notochord cells display dynamic morphogenetic movement, called convergent extension, in which cells intercalate and align at the dorsal midline. However, in cephalochordates, the most basal group of chordates, the notochord is formed without convergent extension. It is simply developed from mesodermal cells at the dorsal midline. This suggests that convergent extension movement of notochord cells is a secondarily acquired developmental attribute in the common ancestor of olfactores (vertebrates + tunicates), and that the chordate ancestor innovated the notochord upon a foundation of morphogenetic mechanisms independent of cell movement. Therefore, this review focuses on biological features specific to notochord cells, which have been well studied using clawed frogs, zebrafish, and tunicates. Attributes of notochord cells, such as vacuolation, membrane trafficking, extracellular matrix formation, and apoptosis, can be understood in terms of two properties: turgor pressure of vacuoles and strength of the notochord sheath. To maintain the straight rod-like structure of the notochord, these parameters must be counterbalanced. In the future, the turgor pressure-sheath strength model, proposed in this review, will be examined in light of quantitative molecular data and mathematical simulations, illuminating the evolutionary origin of the notochord.     

Cadherin-7 function in zebrafish development

Cadherin cell adhesion molecules play crucial roles in vertebrate development. Most studies have focused on examining the functions of classical type I cadherins (e.g., cadherin-2) in the development of vertebrates. Little information is available concerning the function of classical type II cadherins (e.g., cadherin-7) in vertebrate development. We have previously shown that cadherin-7 mRNA exhibits a dynamic expression pattern in the central nervous system and notochord in embryonic zebrafish. To gain insight into the role of cadherin-7 in the formation of these structures, we analyzed their formation in zebrafish embryos injected with cadherin-7-specific antisense morpholino oligonucleotides (MO). Notochord development was severely disrupted in MO-injected embryos, whereas gross defects in the development of the central nervous system were not detected in MO-injected embryos. Our results thus demonstrate that cadherin-7 plays an important role in the normal development of the zebrafish notochord.     

斑马鱼NUP98基因的克隆及其时空表达图谱

目的：斑马鱼NUP98基因的克隆及其在个体早期发育过程中的表达情况研究。方法：提取斑马鱼胚胎的总RNA,制备地高辛标记的NUP98RNA反义探针,WISH（整体胚胎原位杂交）研究NUP98在斑马鱼早期发育过程中的表达;提取斑马鱼胚胎各时相和成鱼各组织的RNA,实时定量PCR检测斑马鱼胚胎各时相和成鱼各组织中的表达。结果：成功克隆斑马鱼NUP98基因,通过实时定量RT-PCR和原位杂交,获得NUP98基因在斑马鱼早期发育过程中的表达情况：NUP98在2-cell、32．cell、oblong、shield期、12h前普遍性表达（0．75h、1．7h、3．7h、6h、12h）;24h以后在眼部、头部表达较多,特别是在脊索表达较高;斑马鱼NUP98在0、0．5h、6h、12h、24h、48h表达逐渐降低,到72h和96h表达有所增加,但是仍低于24h其表达水平;NUP98在成鱼眼、脑、鳔、肾、肝、睾丸、胆囊、卵巢、鳍、心、肠、肌肉、腮、皮肤的表达中,眼的表达最高,明显高于其他组织,腮、卵巢、肠的表达次之,肌肉、鳔、胆囊、睾丸、皮肤、脑的表达紧随其后,鳍、肝、心、肾的表达最低。结论：NUP98基因可能在个体脑部、脊索及眼部的早期发育过程中起到了重要作用;NUP98基因可能具有抑制肿瘤发生的作用,该基因的调节异常对白血病的发生发展可能有重要影响。这些研究结果为进一步研究NUP98基因在造血系统中的作用,评估其是否适合作为血液系统恶性肿瘤的新的治疗靶点等奠定了理论基础。     

Essential role of lysyl oxidases in notochord development

Recent studies reveal a critical role for copper in the development of the zebrafish notochord, suggesting that specific cuproenzymes are required for the structural integrity of the notochord sheath. We now demonstrate that beta-aminopropionitrile, a known inhibitor of the copper-dependent lysyl oxidases, causes notochord distortion in the zebrafish embryo identical to that seen in copper deficiency. Characterization of the zebrafish lysyl oxidase genes reveals eight unique sequences, several of which are expressed in the developing notochord. Specific gene knockdown demonstrates that loss of loxl1 results in notochord distortion, and that loxl1 and loxl5b have overlapping roles in notochord formation. Interestingly, while notochord abnormalities are not observed following partial knockdown of loxl1 or loxl5b alone, in each case this markedly sensitizes developing embryos to notochord distortion if copper availability is diminished. Likewise, partial knockdown of the lysyl oxidase substrate col2a1 results in notochord distortion when combined with reduced copper availability or partial knockdown of loxl1 or loxl5b. These data reveal a complex interplay of gene expression and nutrient availability critical to notochord development. They also provide insight into specific genetic and nutritional factors that may play a role in the pathogenesis of structural birth defects of the axial skeleton.     

A Novel Zinc Finger Protein 219-like (ZNF219L) is Involved in the Regulation of Collagen Type 2 Alpha 1a (col2a1a) Gene Expression in Zebrafish Notochord

The notochord is required for body plan patterning in vertebrates, and defects in notochord development during embryogenesis can lead to diseases affecting the adult. It is therefore important to elucidate the gene regulatory mechanism underlying notochord formation. In this study, we cloned the zebrafish zinc finger 219-like (ZNF219L) based on mammalian ZNF219, which contains nine C2H2-type zinc finger domains. Through whole-mount in situ hybridization, we found that znf219L mRNA is mainly expressed in the zebrafish midbrain-hindbrain boundary, hindbrain, and notochord during development. The znf219L morpholino knockdown caused partial abnormal notochord phenotype and reduced expression of endogenous col2a1a in the notochord specifically. In addition, ZNF219L could recognize binding sites with GGGGG motifs and trigger augmented activity of the col2a1a promoter in a luciferase assay. Furthermore, in vitro binding experiments revealed that ZNF219L recognizes the GGGGG motifs in the promoter region of the zebrafish col2a1a gene through its sixth and ninth zinc finger domains. Taken together, our results reveal that ZNF219L is involved in regulating the expression of col2a1a in zebrafish notochord specifically.     

Notochord vacuoles are lysosome-related organelles that function in axis and spine morphogenesis

The notochord plays critical structural and signaling roles during vertebrate development. At the center of the vertebrate notochord is a large fluid-filled organelle, the notochord vacuole. Although these highly conserved intracellular structures have been described for decades, little is known about the molecular mechanisms involved in their biogenesis and maintenance. Here we show that zebrafish notochord vacuoles are specialized lysosome-related organelles whose formation and maintenance requires late endosomal trafficking regulated by the vacuole-specific Rab32a and H⁺-ATPase–dependent acidification. We establish that notochord vacuoles are required for body axis elongation during embryonic development and identify a novel role in spine morphogenesis. Thus, the vertebrate notochord plays important structural roles beyond early development.