Identification and gene expression profiling of tumor-initiating cells isolated from human osteosarcoma cell lines in an orthotopic mouse model

Abstract Abstract 606 Multiple myeloma is a complex malignancy with multiple underlying genetic events. Our group has spent considerable effort over the last 15 years to elucidate the genetic underpinnings of myeloma. Most recently, we used array-based comparative genomic hybridization (aCGH) as a discovery tool in 62 myeloma patients and 46 myeloma cell lines. In that preliminary screen using the Agilent 44B aCGH platform (∼70kb resolution) we identified a diverse array of abnormalities, which resulted in constitutive activation of the NF-kB pathways. That initial analysis concentrated on the 43 genes we identified as potential targets of the 13 homozygous deletion events detected in the patient samples. A pathway analysis of these genes revealed a single pathway involving TRAF3, TRAF2, BIRC2, BIRC3, and CYLD. This first analysis focused exclusively on abnormalities present in the patient samples as we worried some abnormalities identified exclusively in the cell lines might not be relevant to the pathogenesis of myeloma in patients. However, several abnormalities were equally or more frequent overall but occurred exclusively in cell lines including CDKN2C (14 samples), CDKN1B (4 samples), KDM6A/UTX (4 samples), RB1 (3 samples), TP53 (3 samples). Given the fact that KDM6A/UTX deletions were as frequent as many of the best-described tumor suppressors it seemed like a good candidate but in the absence of patient events or a known function at the time it was not prioritized for further study. Recently, as part of the Multiple Myeloma Research Consortium (MMRC) Genomics Initiative, we have completed the analysis of a cohort of 250 myeloma patient samples by aCGH using the Agilent 244A aCGH platform (∼15kb resolution) and gene expression profiling using the Affymetrix U133Plus2.0 genechip. In this cohort with a significantly improved aCGH platform we identified 17 genes that are recurrently inactivated by homozygous deletions including DIAPH2 (15 samples), CDKN2C (14 samples), TRAF3 (11 samples), CYLD (8 samples), BIRC2/3 (7 samples), KDM6A/UTX (6 samples), and RB1 (5 samples). Based on the significant improvement in resolution and data quality achieved with the Agilent 244A aCGH platform we rescreen all of the cell lines on this improved platform. This significantly changed the frequency of several homozygous deletions in this population with the most frequently targeted genes now being CDKN2C (20 samples), KDM6A/UTX (13 samples), DIAPH2 (7 samples), RB1 (4 samples), TP53 (4 samples), CDKN1B (4 samples), and TRAF3 (4 samples). Moreover, as part of the genomic characterization of a spontaneous myeloma mouse model that we have developed, Vk*-Myc, we have identified recurrent (∼50%) homozygous deletions of Kdm6a/Utx. Therefore, one of the genes most commonly targeted by a homozygous deletion in human and mouse myeloma is KDM6A/UTX. In late 2007 after we had identified the first patients with KDM6A/UTX deletions it was shown that UTX is a functional histone demethylase that removes methyl groups from histone H3 lysine 27 (H3K27me). Given the high incidence of deletions and the fact that MMSET, the overexpressed target gene of t(4;14) in myeloma, is predicted to methylate H3K27, H3K36, and/or H4K20 by evolutionary conservation we developed the hypothesis that myeloma is characterized by abnormalities that result in excessive H3K27me (typically a repressive chromatin mark). Given the extensive whole genome sequencing occurring in the MMRC genomics initiative we elected to focus our resequencing efforts on the cell lines exclusively. These studies identified an additional 4 samples with LOH and an inactivating mutation bringing the total percentage of inactivated cell lines to 33%. Clearly, in the expanded patient and cell line cohorts the inactivation of KDM6A/UTX is not independent of MMSET overexpression suggesting they may act independently or synergistically. We are currently attempting to identify the genes controlled by KDM6A/UTX inactivation to better understand the functional consequences of this highly recurrent event. However, in the mouse model unlike the patient or cell lines, the gene expression profiling has identified a gene expression signature that differentiates UTX inactivated and functional samples suggesting an oncogenic function of inactivation. Disclosures: No relevant conflicts of interest to declare.

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Reference gene expression in human osteosarcoma cell lines treated by EGB and CTX

TURKISH JOURNAL OF BIOLOGY ◽

10.3906/biy-1607-15 ◽

2017 ◽

Vol 41 ◽

pp. 439-447

Author(s):

Ming REN ◽

Qiwei YANG ◽

Yuanyuan SONG ◽

Ao WANG ◽

Qingyu WANG ◽

...

Keyword(s):

Gene Expression ◽

Cell Lines ◽

Reference Gene ◽

Osteosarcoma Cell ◽

Human Osteosarcoma ◽

Human Osteosarcoma Cell ◽

Reference Gene Expression

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Gene expression profiling analysis of osteosarcoma cell lines

Molecular Medicine Reports ◽

10.3892/mmr.2015.3958 ◽

2015 ◽

Vol 12 (3) ◽

pp. 4266-4272 ◽

Cited By ~ 11

Author(s):

LU SUN ◽

JIE LI ◽

BING YAN

Keyword(s):

Gene Expression ◽

Gene Expression Profiling ◽

Cell Lines ◽

Expression Profiling ◽

Osteosarcoma Cell ◽

Profiling Analysis

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Effect of retinoblastoma tumor suppressor gene expression on chemosensitivity of human osteosarcoma cell lines

Oncology Reports ◽

10.3892/or.10.6.1961 ◽

2003 ◽

Author(s):

Koji Iida ◽

Tsutomu Nobori ◽

Akihiko Matsumine ◽

Akihiko Isaka ◽

Masashi Seto ◽

...

Keyword(s):

Gene Expression ◽

Tumor Suppressor ◽

Tumor Suppressor Gene ◽

Cell Lines ◽

Suppressor Gene ◽

Osteosarcoma Cell ◽

Human Osteosarcoma ◽

Retinoblastoma Tumor Suppressor ◽

Human Osteosarcoma Cell ◽

Retinoblastoma Tumor

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Analysis of Gene Expression in Cyclooxygenase-2-Overexpressed Human Osteosarcoma Cell Lines

Genomics & Informatics ◽

10.5808/gi.2014.12.4.247 ◽

2014 ◽

Vol 12 (4) ◽

pp. 247 ◽

Cited By ~ 14

Author(s):

Jeong A. Han ◽

Ji-Yeon Kim ◽

Jong-Il Kim

Keyword(s):

Gene Expression ◽

Cell Lines ◽

Cyclooxygenase 2 ◽

Osteosarcoma Cell ◽

Human Osteosarcoma ◽

Human Osteosarcoma Cell

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381 Identifying Tumor-initiating Cells in Human Osteosarcoma Cell Lines – the Function Matters

European Journal of Cancer ◽

10.1016/s0959-8049(12)71066-3 ◽

2012 ◽

Vol 48 ◽

pp. S92

Author(s):

S. Martins-Neves ◽

B.E. van den Akker ◽

W.E. Corver ◽

A.M. Cleton-Jansen ◽

C.M. Gomes

Keyword(s):

Cell Lines ◽

Osteosarcoma Cell ◽

Tumor Initiating Cells ◽

Human Osteosarcoma ◽

Human Osteosarcoma Cell

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Anti-Metastatic and Anti-Angiogenic Effects of Curcumin Analog DK1 on Human Osteosarcoma Cells In Vitro

Pharmaceuticals ◽

10.3390/ph14060532 ◽

2021 ◽

Vol 14 (6) ◽

pp. 532

Author(s):

Muhammad Nazirul Mubin Aziz ◽

Nurul Fattin Che Rahim ◽

Yazmin Hussin ◽

Swee Keong Yeap ◽

Mas Jaffri Masarudin ◽

...

Keyword(s):

Cell Lines ◽

Safety Profile ◽

Quantitative Polymerase Chain Reaction ◽

Tube Formation ◽

Osteosarcoma Cell ◽

Curcumin Analog ◽

Human Osteosarcoma ◽

Human Osteosarcoma Cells ◽

Human Osteosarcoma Cell

Osteosarcoma (OS) is a life-threatening malignant bone tumor associated with poor prognosis among children. The survival rate of the patient is still arguably low even with intensive treatment provided, plus with the inherent side effects from the chemotherapy, which gives more unfavorable outcomes. Hence, the search for potent anti-osteosarcoma agent with promising safety profile is still on going. Natural occurring substance like curcumin has gained a lot of attention due to its splendid safety profile as well as it pharmacological advantages such as anti-metastasis and anti-angiogenesis. However, natural curcumin was widely known for its poor cellular uptake, which undermines all potential that it possesses. This prompted the development of synthetically synthesized curcuminoid analog, known as (Z)-3-hydroxy-1-(2-hydroxyphenyl)-3-phenylprop-2- en-1-one (DK1). In this present study, in vitro scratch assay, transwell migration/invasion assay, HUVEC tube formation assay, and ex vivo rat aortic ring assays were performed in order to investigate the anti-metastatic and anti-angiogenic potential of DK1. For further comprehension of DK1 mechanism on human osteosarcoma cell lines, microarray gene expression analysis, quantitative polymerase chain reaction (qPCR), and proteome profiler were adopted, providing valuable forecast from the expression of important genes and proteins related to metastasis and angiogenesis. Based on the data gathered from the bioassays, DK1 was able to inhibit the metastasis and angiogenesis of human osteosarcoma cell lines by significantly reducing the cell motility, number of migrated and invaded cells as well as the tube formation and micro-vessels sprouting. Additionally, DK1 also has significantly regulated several cancer pathways involved in OS proliferation, metastasis, and angiogenesis such as PI3K/Akt and NF-κB in both U-2 OS and MG-63. Regulation of PI3K/Akt caused up-regulation of genes related to metastasis inhibition, namely, PTEN, FOXO, PLK3, and GADD45A. Meanwhile, NF-κB pathway was regulated by mitigating the expression of NF-κB activator such as IKBKB and IKBKE in MG-63, whilst up-regulating the expression of NF-κB inhibitors such as NFKBIA and NFKBIE in U-2 OS. Finally, DK1 also has successfully hindered the metastatic and angiogenic capability of OS cell lines by down-regulating the expression of pro-metastatic genes and proteins like MMP3, COL11A1, FGF1, Endoglin, uPA, and IGFBP2 in U-2 OS. Whilst for MG-63, the significantly down-regulated oncogenes were Serpin E1, AKT2, VEGF, uPA, PD-ECGF, and Endoglin. These results suggest that curcumin analog DK1 may serve as a potential new anti-osteosarcoma agent due to its anti-metastatic and anti-angiogenic attributes.

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Assessing Various Control Samples for Microarray Gene Expression Profiling of Laryngeal Squamous Cell Carcinoma

Biomolecules ◽

10.3390/biom11040588 ◽

2021 ◽

Vol 11 (4) ◽

pp. 588

Author(s):

Adam Ustaszewski ◽

Magdalena Kostrzewska-Poczekaj ◽

Joanna Janiszewska ◽

Malgorzata Jarmuz-Szymczak ◽

Malgorzata Wierzbicka ◽

...

Keyword(s):

Gene Expression ◽

Squamous Cell Carcinoma ◽

Epithelial Cells ◽

Cell Carcinoma ◽

Gene Expression Profiling ◽

Cell Lines ◽

Squamous Cell ◽

Expression Profiling ◽

Laryngeal Squamous Cell Carcinoma ◽

Control Samples

Selection of optimal control samples is crucial in expression profiling tumor samples. To address this issue, we performed microarray expression profiling of control samples routinely used in head and neck squamous cell carcinoma studies: human bronchial and tracheal epithelial cells, squamous cells obtained by laser uvulopalatoplasty and tumor surgical margins. We compared the results using multidimensional scaling and hierarchical clustering versus tumor samples and laryngeal squamous cell carcinoma cell lines. A general observation from our study is that the analyzed cohorts separated according to two dominant factors: “malignancy”, which separated controls from malignant samples and “cell culture-microenvironment” which reflected the differences between cultured and non-cultured samples. In conclusion, we advocate the use of cultured epithelial cells as controls for gene expression profiling of cancer cell lines. In contrast, comparisons of gene expression profiles of cancer cell lines versus surgical margin controls should be treated with caution, whereas fresh frozen surgical margins seem to be appropriate for gene expression profiling of tumor samples.

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