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Electropherograms of the CUTC5, CUTC48, CUTC60 and CUTC61 cell line, PDX, and patient samples.

Kinase inhibitor sensitivity of the CUTC cell lines.

Cancer cell lines are critical models to study tumor progression and response to therapy. In 2008, we showed that approximately 50% of thyroid cancer cell lines were redundant or not of thyroid cancer origin. We therefore generated new authenticated thyroid cancer cell lines and patient-derived xenograft (PDX) models using in vitro and feeder cell approaches, and characterized these models in vitro and in vivo. We developed four thyroid cancer cell lines, two derived from 2 different patients with papillary thyroid cancer (PTC) pleural effusions, CUTC5, and CUTC48; one derived from a patient with anaplastic thyroid cancer (ATC), CUTC60; and one derived from a patient with follicular thyroid cancer (FTC), CUTC61. One PDX model (CUTC60-PDX) was also developed. Short tandem repeat (STR) genotyping showed that each cell line and PDX is unique and match the original patient tissue. The CUTC5 and CUTC60 cells harbor the BRAF (V600E) mutation, the CUTC48 cell line expresses the RET/PTC1 rearrangement, and the CUTC61 cells have the HRAS (Q61R) mutation. Moderate to high levels of PAX8 and variable levels of NKX2-1 were detected in each cell line and PDX. The CUTC5 and CUTC60 cell lines form tumors in orthotopic and flank xenograft mouse models.Implications: We have developed the second RET/PTC1-expressing PTC-derived cell line in existence, which is a major advance in studying RET signaling. We have further linked all cell lines to the originating patients, providing a set of novel, authenticated thyroid cancer cell lines and PDX models to study advanced thyroid cancer.

Continuous cell lines are widely used, but can result in invalid, irreproducible research data. Cell line misidentification is a common problem that can be detected by authentication testing; however, misidentified cell lines continue to be used in publications. Here we explore the impact of one misidentified cell line, KB (HeLa), on the scientific literature. We identified 574 articles between 2000 and 2014 that provided an incorrect attribution for KB, in accordance with its false identity as oral epidermoid carcinoma, but only 57 articles that provided a correct attribution for KB, as HeLa or cervical adenocarcinoma. Statistical analysis of 57 correct and 171 incorrect articles showed that the number of citations to these articles increased over time. Content analysis of 200 citing articles showed there was a tendency to describe the cell line in accordance with the description in the cited paper. Analysis of journal impact factor showed no significant difference between correct and incorrect groups. Articles using KB or citing that usage were most frequently published in the subject areas of pharmacology, pharmacy, oncology, and medicinal chemistry. These findings are important for science policy and support the need for journals to require authentication testing as a condition of publication. Cancer Res; 77(11); 2784–8. ©2017 AACR.

Background: A plethora of agents is in early stages of development for colorectal cancer (CRC), including those that target the insulin-like growth factor I receptor (IGFIR) pathway. In the current environment of numerous cancer targets, it is imperative that patient selection strategies be developed with the intent of preliminary testing in the latter stages of phase I trials. The goal of this study was to develop and characterize predictive biomarkers for an IGFIR tyrosine kinase inhibitor, OSI-906, that could be applied in CRC-specific studies of this agent.Methods: Twenty-seven CRC cell lines were exposed to OSI-906 and classified according to IC50 value as sensitive (≤1.5 μmol/L) or resistant (>5 μmol/L). Cell lines were subjected to immunoblotting and immunohistochemistry for effector proteins, IGFIR copy number by fluorescence in situ hybridization, KRAS/BRAF/phosphoinositide 3-kinase mutation status, and baseline gene array analysis. The most sensitive and resistant cell lines were used for gene array and pathway analyses, along with shRNA knockdown of highly ranked genes. The resulting integrated genomic classifier was then tested against eight human CRC explants in vivo.Results: Baseline gene array data from cell lines and xenografts were used to develop a k-top scoring pair (k-TSP) classifier, which, in combination with IGFIR fluorescence in situ hybridization and KRAS mutational status, was able to predict with 100% accuracy a test set of patient-derived CRC xenografts.Conclusions: These results indicate that an integrated approach to the development of individualized therapy is feasible and should be applied early in the development of novel agents, ideally in conjunction with late-stage phase I trials. Clin Cancer Res; 16(12); 3193–204. ©2010 AACR.

Supplementary Figure S1. Copy number alteration profile for TERT locus in cell lines harboring homozygous TERT promoter mutations. Copy number changes are expressed as shades of red (gain) or blue (loss). Names of cell lines and log-ratio (lr) values are shown. Supplementary Figure S2. Genome-wide copy number alteration profile of PTC-derived cell lines (top) vs. ATC-derived cell lines (bottom). Chromosome numbers are shown on the top panel. Copy number changes are expressed as shades of red (gain) or blue (loss). Supplementary Figure S3. Representation of the chromosomal location for the 16 recurrent copy number alterations identified by GISTIC in 58 thyroid cancer cell lines. Supplementary Figure S4. Hierarchical clustering of gene expression profiles of thyroid cancer cell lines. Spearman correlation distance metric and Ward agglomeration method were used. Colors identify cell lines originating from tumors of different histologic subtypes. Font color indicates the histological type of the primary tumor from which these cell lines were generated, as follows: red= anaplastic; purple= poorly differentiated; green=follicular; blue= papillary thyroid cancer. Colored dots represent driver alteration: blue= BRAFV600E; orange= N-/H-/KRAS; purple= CCDC6-RET; green= NF1; yellow= MKRN1-BRAF; gray= FGFR2-OGDH; black= unknown. Supplementary Figure S5. Expression of individual genes in the thyroid differentiation signature. Supplementary Figure S6. Correlation of BRS calculated using our algorithm vs. the algorithm published by TCGA. The color of the circles indicates tumor genotype: blue- BRAFV600E, red- RAS gene mutation, green - neither BRAFV600E nor RAS mutation, orange - both BRAFV600E and RAS gene mutations present. Tumors with low BRS are BRAFV600E-like; tumors with high BRS are RAS-like. Supplementary Figure S7. Representative pictures of BAM files from MSK-IMPACT sequencing of CUTC5 specimens for three mutations: A. BRAF p.V600E; B. TP53 C135W; and C. ARID1A E1108*. Each panel shows the sequencing reads for the cell line (top) and primary tumor (bottom). The reference allele and protein translation is shown in the bottommost area, whereas the alternative allele is highlighted in the sequencing reads within each panel. Percentages on the left represent the frequency of the alternative allele in each of the samples tested. All three mutations are detected at low frequencies in the original pleural effusion sample, from which CUTC5 cell line was established. Supplementary Figure S8. Evolution of allelic frequencies from thyroid primary tumors to cell lines for A. TERT promoter; and B. TP53. Graphic representation of the alternative allele frequencies ("Alt Allele Freq"). Cell line names are color-coded on the right side of each graph. All TERT promoter mutations are the canonical c.-124C>T or c.-146C>T, whereas TP53 mutations are indicated. Supplementary Figure S9. Genome-wide copy number alteration profile of primary tumor/cell line and/or patient-derived xenograft (PDX) paired samples from 11 patients. Chromosome numbers are shown on the top panel. Copy number changes are expressed as shades of red (gain) or blue (loss). Colored dots indicate specimen types: cell line (green), primary tumor (orange) or PDX (purple). Cell line names are provided. Supplementary Figure S10. Global MSK-IMPACT copy number profile of THJ560 paired samples. Sample-centered representation of the copy number alterations, expressed as log2 ratios, for primary tumor (top panel), cell line (middle panel) and patient-derived xenograft (bottom panel). Table inserts show the top deletions, corresponding to the CDKN2A/CDKN2B locus for the cell line and xenograft, which are absent in the primary tumor.

Supplementary Table S1. Cell line authentication by Short Tandem Repeat Profiling (STR) Supplementary Table S2. Full list of genetic variants identified by MSK-IMPACT sequencing Supplementary Table S3. Details for identified high-confidence gene rearrangements resulting in in-frame fusion proteins Supplementary Table S4. Recurrent copy number alterations in thyroid cancer cell lines. Supplementary Table S5. Genes located within the recurrent copy number alteration regions. Supplementary Table S6. Differentially expressed genes, which are located within CNA regions. "Matching_CNA_type" column indicates whether the direction of the expression change corresponds to the CNA type (133 out of 134, overexpression for gene amplification and underexpression for gene deletions). Supplementary Table S7. Thyroid cancer cell lines gene expression data. Normalized gene expression values for all genes in the 44 cell lines assessed by Affymetrix expression microarray in this study ("CU" prefix, University of Colorado). Supplementary Table S8. Genes with differential expression in PTC vs. ATC cell lines. List of differentially expressed genes between PTC-derived vs. ATC-derived cell lines (limma, adjusted p-value

Contains the Supplementary Methods and the legends to accompany the Supplementary Tables.

CCR Translation for This Article from Development of an Integrated Genomic Classifier for a Novel Agent in Colorectal Cancer: Approach to Individualized Therapy in Early Development

Supplementary Methods, Supplementary Figure and Table Legends

Purpose:Thyroid cancer cell lines are valuable models but have been neglected in pancancer genomic studies. Moreover, their misidentification has been a significant problem. We aim to provide a validated dataset for thyroid cancer researchers.Experimental Design:We performed next-generation sequencing (NGS) and analyzed the transcriptome of 60 authenticated thyroid cell lines and compared our findings with the known genomic defects in human thyroid cancers.Results:Unsupervised transcriptomic analysis showed that 94% of thyroid cell lines clustered distinctly from other lineages. Thyroid cancer cell line mutations recapitulate those found in primary tumors (e.g., BRAF, RAS, or gene fusions). Mutations in the TERT promoter (83%) and TP53 (71%) were highly prevalent. There were frequent alterations in PTEN, PIK3CA, and of members of the SWI/SNF chromatin remodeling complex, mismatch repair, cell-cycle checkpoint, and histone methyl- and acetyltransferase functional groups. Copy number alterations (CNA) were more prevalent in cell lines derived from advanced versus differentiated cancers, as reported in primary tumors, although the precise CNAs were only partially recapitulated. Transcriptomic analysis showed that all cell lines were profoundly dedifferentiated, regardless of their derivation, making them good models for advanced disease. However, they maintained the BRAFV600E versus RAS-dependent consequences on MAPK transcriptional output, which correlated with differential sensitivity to MEK inhibitors. Paired primary tumor-cell line samples showed high concordance of mutations. Complete loss of p53 function in TP53 heterozygous tumors was the most prominent event selected during in vitro immortalization.Conclusions:This cell line resource will help inform future preclinical studies exploring tumor-specific dependencies.

Contains the following Supplementary material: Supplementary Table 1: Examples of KB Terminology Supplementary Table 2: "Correct" Dataset, n=57 Supplementary Table 3: "Incorrect" Dataset, n=574 Supplementary Table 4: Sample articles from "Incorrect" dataset, n=171 Supplementary Table 5: Comparing citation counts Supplementary Table 6: KB Use in Citing Articles Supplementary Table 7: Journal Impact Factors Supplementary Table 8: Journal Subject Areas

PDF file - 97K, shRNA target sequences (S1); Primers used for quantitative RT-PCR (S2).

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The epithelial–mesenchymal transition (EMT) and its reversal, mesenchymal–epithelial transition (MET), are fundamental processes involved in tumor cell invasion and metastasis. SEMA3F is a secreted semaphorin and tumor suppressor downregulated by TGF-β1 and ZEB1-induced EMT. Here, we report that neuropilin (NRP)-2, the high-affinity receptor for SEMA3F and a coreceptor for certain growth factors, is upregulated during TGF-β1–driven EMT in lung cancer cells. Mechanistically, NRP2 upregulation was TβRI dependent and SMAD independent, occurring mainly at a posttranscriptional level involving increased association of mRNA with polyribosomes. Extracellular signal—regulated kinase (ERK) and AKT inhibition blocked NRP2 upregulation, whereas RNA interference-mediated attenuation of ZEB1 reduced steady-state NRP2 levels. In addition, NRP2 attenuation inhibited TGF-β1–driven morphologic transformation, migration/invasion, ERK activation, growth suppression, and changes in gene expression. In a mouse xenograft model of lung cancer, NRP2 attenuation also inhibited locally invasive features of the tumor and reversed TGF-β1–mediated growth inhibition. In support of these results, human lung cancer specimens with the highest NRP2 expression were predominantly E-cadherin negative. Furthermore, the presence of NRP2 staining strengthened the association of E-cadherin loss with high-grade tumors. Together, our results demonstrate that NRP2 contributes significantly to TGF-β1–induced EMT in lung cancer. Cancer Res; 73(23); 7111–21. ©2013 AACR.

PDF file - 1864K, NRP2 is stably up-regulated by TGFβ in lung cancer cells without increasing the halflife of mRNA or protein (S1); NRP2 up-regulation is blocked by inhibition of TβRI, AKT, ERK and ZEB1, but not by expression of inhibitory SMAD7 (S2); NRP2 knockdown blunts TGFβ-mediated transcriptional responses, ERK and AKT phosphorylation but does not influence SMAD activation (S3); NRP2 knockdown blocks cell scattering induced by TGFβ (S4); Immunohistochemical detection of NRP2 and cytokeratin in TGFβ exposed xenograft tumors with NRP2 or control knockdowns (S5); E-cadherin and NRP2 immunostaining of samples from a tumor microarray (S6); Predicted miRNA target sites in the 3' UTR of NRP2 and up-regulated NRP2 protein levels in hnRNP E1 knockdown cells (S7).

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Cell lines are essential models for biomedical research. However, they have a common and important problem that needs to be addressed. Cell lines can be misidentified, meaning that they no longer correspond to the donor from whom the cells were first obtained. This problem may arise due to cross-contamination: the accidental introduction of cells from another culture. The contaminant, which is often a rapidly dividing cell line, will overgrow and replace the original culture. The end result is a false cell line, also known as a misidentified or imposter cell line. False cell lines may come from an entirely different species, tissue, or cell type than the original donor. If undetected, false cell lines produce unreliable and irreproducible results that pollute the biomedical literature and threaten the development of reliable drug discovery and meaningful patient treatments.The goal of this study was to ascertain how widespread this problem is and how it affects the literature, as well as to estimate how much funding has been used to produce pools of scientific literature of questionable value. We focus on HEp-2 [HeLa] and Intestine 407 [HeLa], two false cell lines that are widely used in the scientific literature but were shown to be cross-contaminated in 1967. These two cell lines have been used in 8497 and 1397 published articles and extensively described as laryngeal cancer and normal intestine, respectively, rather than their true identity: the cervical cancer cell line HeLa. Discussed are tools, approaches, and resources that can address this issue-both retrospectively and prospectively.

Purpose: Thyroid cancer cell lines are valuable models but have been neglected in pancancer genomic studies. Moreover, their misidentification has been a significant problem. We aim to provide a validated dataset for thyroid cancer researchers. Experimental Design: We performed next-generation sequencing (NGS) and analyzed the transcriptome of 60 authenticated thyroid cell lines and compared our findings with the known genomic defects in human thyroid cancers. Results: Unsupervised transcriptomic analysis showed that 94% of thyroid cell lines clustered distinctly from other lineages. Thyroid cancer cell line mutations recapitulate those found in primary tumors (e.g., BRAF, RAS, or gene fusions). Mutations in the TERT promoter (83%) and TP53 (71%) were highly prevalent. There were frequent alterations in PTEN, PIK3CA, and of members of the SWI/SNF chromatin remodeling complex, mismatch repair, cell-cycle checkpoint, and histone methyl- and acetyltransferase functional groups. Copy number alterations (CNA) were more prevalent in cell lines derived from advanced versus differentiated cancers, as reported in primary tumors, although the precise CNAs were only partially recapitulated. Transcriptomic analysis showed that all cell lines were profoundly dedifferentiated, regardless of their derivation, making them good models for advanced disease. However, they maintained the BRAFV600E versus RAS-dependent consequences on MAPK transcriptional output, which correlated with differential sensitivity to MEK inhibitors. Paired primary tumor-cell line samples showed high concordance of mutations. Complete loss of p53 function in TP53 heterozygous tumors was the most prominent event selected during in vitro immortalization. Conclusions: This cell line resource will help inform future preclinical studies exploring tumor-specific dependencies.

A variety of analytical approaches have indicated that melanoma cell line UCLA-SO-M14 (M14) and breast carcinoma cell line MDA-MB-435 originate from a common donor. This indicates that at some point in the past, one of these cell lines became misidentified, meaning that it ceased to correspond to the reported donor and instead became falsely identified (through cross-contamination or other means) as a cell line from a different donor. Initial studies concluded that MDA-MB-435 was the misidentified cell line and M14 was the authentic cell line, although contradictory evidence has been published, resulting in further confusion. To address this question, we obtained early samples of the melanoma cell line (M14), a lymphoblastoid cell line from the same donor (ML14), and donor serum preserved at the originator's institution. M14 samples were cryopreserved in December 1975, before MDA-MB-435 cells were established in culture. Through a series of molecular characterizations, including short tandem repeat (STR) profiling and cytogenetic analysis, we demonstrated that later samples of M14 and MDA-MB-435 correspond to samples of M14 frozen in 1975, to the lymphoblastoid cell line ML14, and to the melanoma donor's STR profile, sex and blood type. This work demonstrates conclusively that M14 is the authentic cell line and MDA-MB-435 is misidentified. With clear provenance information and authentication testing of early samples, it is possible to resolve debates regarding the origins of problematic cell lines that are widely used in cancer research.

Continuous cell lines are widely used, but can result in invalid, irreproducible research data. Cell line misidentification is a common problem that can be detected by authentication testing; however, misidentified cell lines continue to be used in publications. Here we explore the impact of one misidentified cell line, KB (HeLa), on the scientific literature. We identified 574 articles between 2000 and 2014 that provided an incorrect attribution for KB, in accordance with its false identity as oral epidermoid carcinoma, but only 57 articles that provided a correct attribution for KB, as HeLa or cervical adenocarcinoma. Statistical analysis of 57 correct and 171 incorrect articles showed that the number of citations to these articles increased over time. Content analysis of 200 citing articles showed there was a tendency to describe the cell line in accordance with the description in the cited paper. Analysis of journal impact factor showed no significant difference between correct and incorrect groups. Articles using KB or citing that usage were most frequently published in the subject areas of pharmacology, pharmacy, oncology, and medicinal chemistry. These findings are important for science policy and support the need for journals to require authentication testing as a condition of publication. Cancer Res; 77(11); 2784–8. ©2017 AACR.

Research in toxicology relies on in vitro models such as cell lines. These living models are prone to change and may be described in publications with insufficient information or quality control testing. This article sets out recommendations to improve the reliability of cell-based research.

A variety of analytical approaches have indicated that melanoma cell line UCLA‐SO‐M14 (M14) and breast carcinoma cell line MDA‐MB‐435 originate from a common donor. This indicates that at some point in the past, one of these cell lines became misidentified, meaning that it ceased to correspond to the reported donor and instead became falsely identified (through cross‐contamination or other means) as a cell line from a different donor. Initial studies concluded that MDA‐MB‐435 was the misidentified cell line and M14 was the authentic cell line, although contradictory evidence has been published, resulting in further confusion. To address this question, we obtained early samples of the melanoma cell line (M14), a lymphoblastoid cell line from the same donor (ML14), and donor serum preserved at the originator's institution. M14 samples were cryopreserved in December 1975, before MDA‐MB‐435 cells were established in culture. Through a series of molecular characterizations, including short tandem repeat (STR) profiling and cytogenetic analysis, we demonstrated that later samples of M14 and MDA‐MB‐435 correspond to samples of M14 frozen in 1975, to the lymphoblastoid cell line ML14, and to the melanoma donor's STR profile, sex and blood type. This work demonstrates conclusively that M14 is the authentic cell line and MDA‐MB‐435 is misidentified. With clear provenance information and authentication testing of early samples, it is possible to resolve debates regarding the origins of problematic cell lines that are widely used in cancer research., What's new? A variety of analytical approaches have indicated that melanoma cell line M14 and breast carcinoma cell line MDA‐MB‐435 originate from a common donor, but there is ongoing debate regarding which is the misidentified cell line. Here, authentication testing of M14 from 1975 (prior to the establishment of MDA‐MB‐435), with comparison to donor serum and lymphoblastoid cell line ML14, shows that M14 is the authentic cell line and MDA‐MB‐435 is a misidentified derivative. With clear provenance information and authentication testing of early samples, debates regarding the origins of problematic cell lines that are widely used in cancer research can be resolved.

The epithelial to mesenchymal transition (EMT) enables epithelial cells with a migratory mesenchymal phenotype. It is activated in cancer cells and is involved in invasion, metastasis and stem-like properties. ZEB1, an E-box binding transcription factor, is a major suppressor of epithelial genes in lung cancer. In the present study, we show that in H358 non-small cell lung cancer cells, ZEB1 downregulates EpCAM (coding for an epithelial cell adhesion molecule), ESRP1 (epithelial splicing regulatory protein), ST14 (a membrane associated serine protease involved in HGF processing) and RAB25 (a small G-protein) by direct binding to these genes. Following ZEB1 induction, acetylation of histone H4 and histone H3 on lysine 9 (H3K9) and 27 (H3K27) was decreased on ZEB1 binding sites on these genes as demonstrated by chromatin immunoprecipitation. Of note, decreased H3K27 acetylation could be also detected by western blot and immunocytochemistry in ZEB1 induced cells. In lung cancers, H3K27 acetylation level was higher in the tumor compartment than in the corresponding stroma where ZEB1 was more often expressed. Since HDAC and DNA methylation inhibitors increased expression of ZEB1 target genes, targeting these epigenetic modifications would be expected to reduce metastasis.

Anaplastic thyroid cancer represents one of the most aggressive cancers. The active form of vitamin D, 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), has been shown to have antiproliferative and/or redifferentiating properties in several malignancies, including thyroid cancer. The objective of this study was to investigate the effects of 1,25(OH)(2)D(3) and the superagonistic analog CD578 in anaplastic thyroid cancer, alone or in combination with paclitaxel, a taxane, and suberoylanilide hydroxamic acid (SAHA), a potent histone deacetylase inhibitor with promising effects in undifferentiated thyroid cancer. Four human thyroid cancer cell lines (FTC-133, C643, 8505C and HTh74) were treated with 1,25(OH)(2)D(3) or CD578, alone or in combination with paclitaxel or SAHA. Effects on cell growth and differentiation were evaluated. Clear effects on growth arrest were observed in a clonogenic assay, and absolute cell counts demonstrated a 24-36% reduction in all cell lines after 72h treatment with 1,25(OH)(2)D(3) (10(-6)M) and a 60% inhibition after 120h in the most sensitive cell line HTh74. A similar growth inhibition was shown after treatment with a 1000-fold lower concentration of analog CD578. This growth arrest was explained by antiproliferative effects, further supported by an increased % of cells in the G(0)-G(1) phase of the cell cycle and by a decreased transcription factor E2F1 mRNA expression. Combination treatments of 1,25(OH)(2)D(3) or CD578 with paclitaxel or SAHA resulted in an additive and in some conditions a synergistic effect on the inhibition of proliferation. Redifferentiation analysis revealed only a modest increase in sodium iodide symporter and thyroglobulin mRNA expression after treatment with 1,25(OH)(2)D(3), without additive effect after combination treatment. No effects were observed on TSH-receptor or thyroid peroxidase mRNA expression. Our in vitro findings demonstrate that the superagonistic vitamin D analog CD578 holds promise as adjuvant antiproliferative therapy of anaplastic thyroid cancer, especially in combination with other drugs such as paclitaxel or SAHA.

Osteosarcoma (OS) remains an incurable and ultimately fatal disease in many patients, and novel forms of therapy are needed. Improved models of OS that more closely mimic human disease would provide more robust information regarding the utility of novel therapies. Spontaneous OS in dogs may provide such a model. Pharmacologic inhibition of histone deacetylase (HDAC) enzymes has a variety of anti-tumor effects but may demonstrate the most utility when utilized in combination with standard cytotoxic therapies. We sought to determine the in vitro and in vivo effects of the HDAC inhibitor valproic acid (VPA) on doxorubicin (DOX) sensitivity in canine and human OS.We evaluated the in vitro anti-proliferative and apoptotic effects of VPA/DOX combination treatment, alterations in histone acetylation and nuclear DOX accumulation resulting from VPA treatment, and the in vivo efficacy of combination therapy in a xenograft model.Treatment of canine and human OS cell lines with clinically achievable VPA concentrations resulted in increased histone acetylation but modest anti-proliferative effects. Pre-incubation with VPA followed by doxorubicin (DOX) resulted in significant growth inhibition and potentiation of apoptosis, associated with a dose-dependent increase in nuclear DOX accumulation. The combination of VPA and DOX was superior to either monotherapy in a canine OS xenograft model.These results demonstrate a rationale for the addition of HDAC inhibitors to current protocols for the treatment of OS and illustrate the similarities in response to HDAC inhibitors between human and canine OS, lending further credibility to the canine OS model.

Context: Cell lines derived from human cancers provide critical tools to study disease mechanisms and develop novel therapies. Recent reports indicate that up to 36% of cell lines are cross- contaminated. Objective: We evaluated 40 reported thyroid cancer-derived cell lines using short tandem repeat and single nucleotide polymorphism array analysis. Results: Only 23 of 40 cell lines tested have unique genetic profiles. The following groups of cell lines are likely derivatives of the same cell line: BHP5-16, BHP17-10, BHP14-9, and NPA87; BHP2-7, BHP10-3, BHP7-13, and TPC1; KAT5, KAT10, KAT4, KAT7, KAT50, KAK1, ARO81-1, and MRO87-1; and K1 and K2. The unique cell lines include BCPAP, KTC1, TT2609-C02, FTC133, ML1, WRO82-1, 8505C, SW1736, Cal-62, T235, T238, Uhth-104, ACT-1, HTh74, KAT18, TTA1, FRO81-2, HTh7, C643, BHT101, and KTC-2. The misidentified cell lines included the DRO90-1, which matched the melanoma-derived cell line, A-375. The ARO81-1 and its derivatives matched the HT-29 colon cancer cell line,...

Studying the molecular basis of familial renal cell carcinoma (RCC) has allowed identification of novel RCC genes involved in the pathogenesis of both inherited and sporadic RCC. We describe a constitutional balanced t(3;8)(p14;q24.1) translocation found in a brother and sister with bilateral clear cell RCC (CC-RCC) diagnosed in their forties. Consistent with a prior report, we demonstrated by RT-PCR of RNA from lymphoblastoid cells fusion mRNAs derived from the fragile histidine triad (FHIT) at 3p14 and TRC8 at 8q24.1 in both affected siblings. Cytogenetic analysis of a CC-RCC tumor from the affected sister from short-term tumor cell culture showed both diploid and pseudotetraploid populations containing the translocation and normal appearing chromosomes 3 and 8. Fluorescent in situ hybridization using bacterial artificial chromosomes containing sequences from the FHIT and TRC8 genes demonstrated normal FHIT signals and TRC8 signals on nontranslocated chromosomes in the constitutional blood sample, but the TRC8 signal was absent in a subset of diploid and pseudotetraploid cells from the tumor. The tumor also contained a heterozygous VHL frameshift somatic mutation. These results confirm that balanced translocations disrupting the TRC8 and FHIT genes result in an increased genetic susceptibility for bilateral CC-RCC. The presence of diploid and tetraploid tumor cells with and without TRC8 deletions on the nontranslocated chromosome suggest that loss of the remaining normal allele of TRC8 may contribute to tumor development at later stages. © 2007 Wiley-Liss, Inc.

Epidermal growth factor receptor (EGFR) and tumour growth factor alpha (TGFalpha) are frequently overexpressed in renal cell carcinoma (RCC) yet responses to single-agent EGFR inhibitors are uncommon. Although von Hippel-Lindau (VHL) mutations are predominant, RCC also develops in individuals with tuberous sclerosis (TSC). Tuberous sclerosis mutations activate mammalian target of rapamycin (mTOR) and biochemically resemble VHL alterations. We found that RCC cell lines expressed EGFR mRNA in the near-absence of other ErbB family members. Combined EGFR and mTOR inhibition synergistically impaired growth in a VHL-dependent manner. Iressa blocked ERK1/2 phosphorylation specifically in wt-VHL cells, whereas rapamycin inhibited phospho-RPS6 and 4E-BP1 irrespective of VHL. In contrast, phospho-AKT was resistant to these agents and MYC translation initiation (polysome binding) was similarly unaffected unless AKT was inhibited. Primary RCCs vs cell lines contained similar amounts of phospho-ERK1/2, much higher levels of ErbB-3, less phospho-AKT, and no evidence of phospho-RPS6, suggesting that mTOR activity was reduced. A subset of tumours and cell lines expressed elevated eIF4E in the absence of upstream activation. Despite similar amounts of EGFR mRNA, cell lines (vs tumours) overexpressed EGFR protein. In the paired cell lines, PRC3 and WT8, EGFR protein was elevated post-transcriptionally in the VHL mutant and EGF-stimulated phosphorylation was prolonged. We propose that combined EGFR and mTOR inhibitors may be useful in the subset of RCCs with wt-VHL. However, apparent differences between primary tumours and cell lines require further investigation.

From tissue culture to final analysis, cell culture is a group of techniques used in the majority of life science laboratories. There are numerous standard methodologies, but many of the techniques used are continuously changing and evolving. From cell preparation and growth to final analysis, the cell culture workflow encompasses many areas of expertise, making it difficult for a researcher to be an authority in all of them. Properly optimizing workflows and utilizing the latest best practices are the keys to obtaining consistent results and advancing scientific research. Join this webinar to learn the latest tips and tricks of the trade from the experts.

The use of dideoxynucleotide triphosphates labeled with different fluorescent dyes (dye terminators) is the most versatile method for automated DNA sequencing. However, variation in peak heights reduces base-calling accuracy and limits heterozygous allele detection, favoring use of dye-labeled primers for this purpose. We have discovered that the addition of a manganese salt to the PE Applied Biosystems dye-terminator sequencing kits overcomes these limitations for the older rhodamine dyes as well as the more recent dichloro-rhodamine dyes (dRhodamine and BigDyes). Addition of manganese to reactions containing dRhodamine-based dye terminators produced the highest base-calling accuracy. This combination resulted in the most uniform electropherogram profiles, superior to those produced by BigDye terminators and published for dye primers, and facilitated detection of heterozygous alleles.

Two-component histidine kinases recently have been found in eukaryotic organisms including fungi, slime molds, and plants. We describe the identification of a gene, COS1 , from the opportunistic pathogen Candida albicans by using a PCR-based screening strategy. The sequence of COS1 indicates that it encodes a homolog of the histidine kinase Nik-1 from the filamentous fungus Neurospora crassa. COS1 is also identical to a gene called CaNIK1 identified in C. albicans by low stringency hybridization using CaSLN1 as a probe [Nagahashi, S., Mio, T., Yamada-Okabe, T., Arisawa, M., Bussey, H. & Yamada-Okabe, H. (1998) Microbiol. 44, 425–432]. We assess the function of COS1/CaNIK1 by constructing a diploid deletion mutant. Mutants lacking both copies of COS1 appear normal when grown as yeast cells; however, they exhibit defective hyphal formation when placed on solid agar media, either in response to nutrient deprivation or serum. In constrast to the Δnik-1 mutant, the Δcos1/Δcos1 mutant does not demonstrate deleterious effects when grown in media of high osmolarity; however both Δnik-1 and Δcos1/Δcos1 mutants show defective hyphal formation. Thus, as predicted for Nik-1, Cos1p may be involved in some aspect of hyphal morphogenesis and may play a role in virulence properties of the organism.

The epithelial–mesenchymal transition (EMT) and its reversal, mesenchymal–epithelial transition (MET), are fundamental processes involved in tumor cell invasion and metastasis. SEMA3F is a secreted semaphorin and tumor suppressor downregulated by TGF-β1 and ZEB1-induced EMT. Here, we report that neuropilin (NRP)-2, the high-affinity receptor for SEMA3F and a coreceptor for certain growth factors, is upregulated during TGF-β1–driven EMT in lung cancer cells. Mechanistically, NRP2 upregulation was TβRI dependent and SMAD independent, occurring mainly at a posttranscriptional level involving increased association of mRNA with polyribosomes. Extracellular signal—regulated kinase (ERK) and AKT inhibition blocked NRP2 upregulation, whereas RNA interference-mediated attenuation of ZEB1 reduced steady-state NRP2 levels. In addition, NRP2 attenuation inhibited TGF-β1–driven morphologic transformation, migration/invasion, ERK activation, growth suppression, and changes in gene expression. In a mouse xenograft model of lung cancer, NRP2 attenuation also inhibited locally invasive features of the tumor and reversed TGF-β1–mediated growth inhibition. In support of these results, human lung cancer specimens with the highest NRP2 expression were predominantly E-cadherin negative. Furthermore, the presence of NRP2 staining strengthened the association of E-cadherin loss with high-grade tumors. Together, our results demonstrate that NRP2 contributes significantly to TGF-β1–induced EMT in lung cancer. Cancer Res; 73(23); 7111–21. ©2013 AACR.

Inhibition of Wee1 is emerging as a novel therapeutic strategy for cancer, and some data suggest that cells with dysfunctional p53 are more sensitive to Wee1 inhibition combined with conventional chemotherapy than those with functional p53. We and others found that Wee1 inhibition sensitizes leukemia cells to cytarabine. Thus, we sought to determine whether chemosensitization by Wee1 inhibition is dependent on p53 dysfunction and whether combining Wee1 inhibition is tolerable and effective in vivo. Synergistic inhibition of proliferation with a Wee1 inhibitor in clinical development, MK1775, and cytarabine was observed in all acute myelogenous leukemia (AML) cell lines tested, regardless of p53 functionality. Mechanistic studies indicate that inhibition of Wee1 abrogates the S-phase checkpoint and augments apoptosis induced by cytarabine. In AML and lung cancer cell lines, genetic disruption of p53 did not alter the cells' enhanced sensitivity to antimetabolites with Wee1 inhibition. Finally, mice with AML were treated with cytarabine and/or MK1775. The combination of MK1775 and cytarabine was well tolerated in mice and enhanced the antileukemia effects of cytarabine, including survival. Thus, inhibition of Wee1 sensitizes hematologic and solid tumor cell lines to antimetabolite chemotherapeutics, whether p53 is functional or not, suggesting that the use of p53 mutation as a predictive biomarker for response to Wee1 inhibition may be restricted to certain cancers and/or chemotherapeutics. These data provide preclinical justification for testing MK1775 and cytarabine in patients with leukemia. Mol Cancer Ther; 12(12); 2675–84. ©2013 AACR.

The purpose of this study was to explore the role of transcription factor Ets1 in estrogen receptor α (ERα)-positive breast cancer progression. We expressed human Ets1 or empty vector in four human ERα-positive breast cancer cell lines and observed increased colony formation. Further examination of cellular responses in stable Ets1-expressing MCF7 clones displayed increased proliferation, migration, and invasion. Ets1-expressing MCF7 tumors grown in the mammary fat pads of nude mice exhibited increased rates of tumor growth (7.36±2.47 mm(3)/day) compared to control MCF7 tumors (2.52±1.70 mm(3)/day), but maintained their dependence on estradiol for tumor growth. Proliferation marker Ki-67 staining was not different between control and Ets1-expressing tumors, but Ets1-expressing tumors exhibited large necrotic centers and elevated apoptotic staining. Ets1 was shown to cooperate with ERα and the p160 nuclear receptor coactivator (NCOA/SRC) family to increase activation of a consensus estrogen response element luciferase reporter construct. Ets1-expressing MCF7 cells also exhibited elevated expression of the ERα target genes, progesterone receptor and trefoil factor 1. Using GST-pulldown assays, Ets1 formed stable complexes containing both ERα and p160 nuclear receptor coactivators. Taken together, these data suggest that the Ets1-dependent estradiol sensitization of breast cancer cells and tumors may be partially due to the ability of Ets1 to cooperate with ERα and nuclear receptor coactivators to stimulate transcriptional activity of estrogen-dependent genes.

The functional significance of decreased RAP1GAP protein expression in human tumors is unclear. To identify targets of RAP1GAP downregulation in the thyroid gland, RAP1 and RAP2 protein expression in human thyroid cells and in primary thyroid tumors were analyzed. RAP1GAP and RAP2 were co-expressed in normal thyroid follicular cells. Intriguingly, RAP1 was not detected in normal thyroid cells, although it was detected in papillary thyroid carcinomas, which also expressed RAP2. Both RAP proteins were detected at the membrane in papillary thyroid tumors, suggesting that they are activated when RAP1GAP is downregulated. To explore the functional significance of RAP1GAP depletion, RAP1GAP was transiently expressed at the lowest level that is sufficient to block endogenous RAP2 activity in papillary and anaplastic thyroid carcinoma cell lines. RAP1GAP impaired the ability of cells to spread and migrate on collagen. Although RAP1GAP had no effect on protein tyrosine phosphorylation in growing cells, RAP1GAP impaired phosphorylation of focal adhesion kinase and paxillin at sites phosphorylated by SRC in cells acutely plated on collagen. SRC activity was increased in suspended cells, where it was inhibited by RAP1GAP. Inhibition of SRC kinase activity impaired cell spreading and motility. These findings identify SRC as a target of RAP1GAP depletion and suggest that the downregulation of RAP1GAP in thyroid tumors enhances SRC-dependent signals that regulate cellular architecture and motility.

Cell lines derived from human ovarian and endometrial cancers, and their immortalized non-malignant counterparts, are critical tools to investigate and characterize molecular mechanisms underlying gynecologic tumorigenesis, and facilitate development of novel therapeutics. To determine the extent of misidentification, contamination and redundancy, with evident consequences for the validity of research based upon these models, we undertook a systematic analysis and cataloging of endometrial and ovarian cell lines.Profiling of cell lines by analysis of DNA microsatellite short tandem repeats (STR), p53 nucleotide polymorphisms and microsatellite instability was performed.Fifty-one ovarian cancer lines were profiled with ten found to be redundant and five (A2008, OV2008, C13, SK-OV-4 and SK-OV-6) identified as cervical cancer cells. Ten endometrial cell lines were analyzed, with RL-92, HEC-1A, HEC-1B, HEC-50, KLE, and AN3CA all exhibiting unique, uncontaminated STR profiles. Multiple variants of Ishikawa and ECC-1 endometrial cancer cell lines were genotyped and analyzed by sequencing of mutations in the p53 gene. The profile of ECC-1 cells did not match the EnCa-101 tumor, from which it was reportedly derived, and all ECC-1 isolates were genotyped as Ishikawa cells, MCF-7 breast cancer cells, or a combination thereof. Two normal, immortalized endometrial epithelial cell lines, HES cells and the hTERT-EEC line, were identified as HeLa cervical carcinoma and MCF-7 breast cancer cells, respectively.Results demonstrate significant misidentification, duplication, and loss of integrity of endometrial and ovarian cancer cell lines. Authentication by STR DNA profiling is a simple and economical method to verify and validate studies undertaken with these models.

The association between vitamin D and thyroid cancer is unclear. It is unknown if CYP27A1 or CYP2R1 are present in normal thyroid or cancer cells and there is limited information regarding response to treatment with vitamin D. SV40 immortalized follicular cells (N-thy) and six thyroid cancer cell lines were treated with 10 µm vitamin D3, 0.1 µm 1,25(OH)2D3 or vehicle × 24 h. CYP27A1, CYP2R1, CYP27B1 and CYP24A1 mRNA were measured using quantitative real-time-PCR before and after treatment. Cell proliferation was also evaluated in TPC1 and C643 cells after treatment with D3, 25(OH)D3 and 1,25(OH)2D3. Baseline CYP27A1 and CYP27B1 mRNA were present in all cells, CYP2R1 was higher and CYP24A1 mRNA was lower in cancer cell lines versus N-thy. TPC1 cells had increased CYP24A1 mRNA levels when treated with both D3 (3.49, p < 0.001) and 1,25(OH)2D3 (5.05, p < 0.001). C643 cells showed increased CYP24A1 mRNA expression when treated with 1,25(OH)2D3 (5.36, p < 0.001). D3, 25(OH)D3 and 1,25(OH)2D3 all significantly decreased cell proliferation in TPC1 and C643 cells. Overall, both cancerous and N-thy cell lines express CYP27A1 and CYP2R1 in addition to CYP27B1, establishing the potential to metabolize D3 to 1,25(OH)2D3. Additionally, vitamin D3, 25(OH)D3 and 1,25(OH)2D3 all had an antiproliferative effect on two thyroid cancer cell lines.

Increases in Rap activity have been associated with tumor progression. Although activating mutations in Rap have not been described, downregulation of Rap1GAP is frequent in human tumors including thyroid carcinomas. In this study, we explored whether endogenous Rap1GAP expression could be restored to thyroid tumor cells. The effects of deacetylase inhibitors and a demethylating agent, individually and in combination, were examined in four differentiated and six anaplastic thyroid carcinoma (ATC) cell lines. Treatment with the structurally distinct histone deacetylase (HDAC) inhibitors, sodium butyrate and trichostatin A, increased Rap1GAP expression in all the differentiated thyroid carcinoma cell lines and in four of the six ATC cell lines. The demethylating agent, 5-aza-deoxycytidine, restored Rap1GAP expression in one anaplastic cell line and enhanced the effects of HDAC inhibitors in a second anaplastic cell line. Western blotting indicated that Rap2 was highly expressed in human thyroid cancer cells. Importantly, treatment with HDAC inhibitors impaired Rap2 activity in both differentiated and anaplastic tumor cell lines. The mechanism through which Rap activity is repressed appears to entail effects on the expression of multiple Rap regulators, including RapGEFs and RapGAPs. These results suggest that HDAC inhibitors may provide a tractable approach to impair Rap activity in human tumor cells.

Background: A plethora of agents is in early stages of development for colorectal cancer (CRC), including those that target the insulin-like growth factor I receptor (IGFIR) pathway. In the current environment of numerous cancer targets, it is imperative that patient selection strategies be developed with the intent of preliminary testing in the latter stages of phase I trials. The goal of this study was to develop and characterize predictive biomarkers for an IGFIR tyrosine kinase inhibitor, OSI-906, that could be applied in CRC-specific studies of this agent. Methods: Twenty-seven CRC cell lines were exposed to OSI-906 and classified according to IC50 value as sensitive (≤1.5 μmol/L) or resistant (>5 μmol/L). Cell lines were subjected to immunoblotting and immunohistochemistry for effector proteins, IGFIR copy number by fluorescence in situ hybridization, KRAS/BRAF/phosphoinositide 3-kinase mutation status, and baseline gene array analysis. The most sensitive and resistant cell lines were used for gene array and pathway analyses, along with shRNA knockdown of highly ranked genes. The resulting integrated genomic classifier was then tested against eight human CRC explants in vivo. Results: Baseline gene array data from cell lines and xenografts were used to develop a k-top scoring pair (k-TSP) classifier, which, in combination with IGFIR fluorescence in situ hybridization and KRAS mutational status, was able to predict with 100% accuracy a test set of patient-derived CRC xenografts. Conclusions: These results indicate that an integrated approach to the development of individualized therapy is feasible and should be applied early in the development of novel agents, ideally in conjunction with late-stage phase I trials. Clin Cancer Res; 16(12); 3193–204. ©2010 AACR.

Rap1GAP expression is decreased in human tumors. The significance of its downregulation is unknown. We show that Rap1GAP expression is decreased in primary colorectal carcinomas. To elucidate the advantages conferred on tumor cells by loss of Rap1GAP, Rap1GAP expression was silenced in human colon carcinoma cells. Suppressing Rap1GAP induced profound alterations in cell adhesion. Rap1GAP-depleted cells exhibited defects in cell/cell adhesion that included an aberrant distribution of adherens junction proteins. Depletion of Rap1GAP enhanced adhesion and spreading on collagen. Silencing of Rap expression normalized spreading and restored E-cadherin, beta-catenin, and p120-catenin to cell/cell contacts, indicating that unrestrained Rap activity underlies the alterations in cell adhesion. The defects in adherens junction protein distribution required integrin signaling as E-cadherin and p120-catenin were restored at cell/cell contacts when cells were plated on poly-l-lysine. Unexpectedly, Src activity was increased in Rap1GAP-depleted cells. Inhibition of Src impaired spreading and restored E-cadherin at cell/cell contacts. These findings provide the first evidence that Rap1GAP contributes to cell/cell adhesion and highlight a role for Rap1GAP in regulating cell/matrix and cell/cell adhesion. The frequent downregulation of Rap1GAP in epithelial tumors where alterations in cell/cell and cell/matrix adhesion are early steps in tumor dissemination supports a role for Rap1GAP depletion in tumor progression.