Laura A. Marlow

Deoxyribonucleic Acid Profiling Analysis of 40 Human Thyroid Cancer Cell Lines Reveals Cross-Contamination Resulting in Cell Line Redundancy and Misidentification

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, and the NPA87 and its derivatives matched the M14/MDA-MB-435S melanoma cell line. TTF-1 and Pax-8 mRNA levels were determined in the unique cell lines. CONCLUSIONS Many of these human cell lines have been widely used in the thyroid cancer field for the past 20 yr and are not only redundant, but not of thyroid origin. These results emphasize the importance of cell line integrity, and provide the short tandem repeat profiles for a panel of thyroid cancer cell lines that can be used as a reference for comparison of cell lines from other laboratories.

Anaplastic thyroid cancer: molecular pathogenesis and emerging therapies.

Anaplastic thyroid cancer (ATC) is a rare malignancy. While external beam radiation therapy has improved locoregional control, the median survival of approximately 4 months has not changed in more than half a century due to uncontrolled systemic metastases. The objective of this study was to review the literature in order to identify potential new strategies for treating this highly lethal cancer. PubMed searches were the principal source of articles reviewed. The molecular pathogenesis of ATC includes mutations in BRAF, RAS, catenin (cadherin-associated protein), beta 1, PIK3CA, TP53, AXIN1, PTEN, and APC genes, and chromosomal abnormalities are common. Several microarray studies have identified genes and pathways preferentially affected, and dysregulated microRNA profiles differ from differentiated thyroid cancers. Numerous proteins involving transcription factors, signaling pathways, mitosis, proliferation, cell cycle, apoptosis, adhesion, migration, epigenetics, and protein degradation are affected. A variety of agents have been successful in controlling ATC cell growth both in vitro and in nude mice xenografts. While many of these new compounds are in cancer clinical trials, there are few studies being conducted in ATC. With the recent increased knowledge of the many critical genes and proteins affected in ATC, and the extensive array of targeted therapies being developed for cancer patients, there are new opportunities to design clinical trials based upon tumor molecular profiling and preclinical studies of potentially synergistic combinatorial novel therapies.

Novel high-affinity PPARγ agonist alone and in combination with paclitaxel inhibits human anaplastic thyroid carcinoma tumor growth via p21WAF1/CIP1

Peroxisome proliferator-activated receptor gamma (PPARγ) agonists demonstrate antitumor activity likely through transactivating genes that regulate cell proliferation, apoptosis, and differentiation. The PAX8/PPARγ fusion oncogene, which is common in human follicular thyroid carcinomas appears to act via dominant negative suppression of wild-type PPARγ, suggesting that it may be a tumor suppressor gene in thyroid cells. We have identified a novel high-affinity PPARγ agonist (RS5444) that is dependent upon PPARγ for its biological activity. This is the first report of this molecule and its antitumor activity. In vitro, the IC50 for growth inhibition is ∼0.8 nM while anaplastic thyroid carcinoma (ATC) tumor growth was inhibited three- to fourfold in nude mice. siRNA against PPARγ and a pharmacological antagonist demonstrated that functional PPARγ was required for growth inhibitory activity of RS5444. RS5444 upregulated the cell cycle kinase inhibitor, p21WAF1/CIP1. Silencing p21WAF1/CIP1 rendered cells insensitive to RS5444. RS5444 plus paclitaxel demonstrated additive antiproliferative activity in cell culture and minimal ATC tumor growth in vivo. RS5444 did not induce apoptosis but combined with paclitaxel, doubled the apoptotic index compared to that of paclitaxel. Our data indicate that functional PPARγ is a molecular target for therapy in ATC. We demonstrated that RS5444, a thiazolidinedione (Tzd) derivative, alone or in combination with paclitaxel, may provide therapeutic benefit to patients diagnosed with ATC.

Pathway Signature and Cellular Differentiation in Clear Cell Renal Cell Carcinoma

Background Clear cell renal cell carcinoma (ccRCC) is the most common kidney cancer. The purpose of this study is to define a biological pathway signature and a cellular differentiation program in ccRCC. Methodology We performed gene expression profiling of early-stage ccRCC and patient-matched normal renal tissue using Affymetrix HG-U133a and HG-U133b GeneChips combined with a comprehensive bioinformatic analyses, including pathway analysis. The results were validated by real time PCR and IHC on two independent sample sets. Cellular differentiation experiments were performed on ccRCC cell lines and their matched normal renal epithelial cells, in differentiation media, to determine their mesenchymal differentiation potential. Principal Findings We identified a unique pathway signature with three major biological alterations—loss of normal renal function, down-regulated metabolism, and immune activation–which revealed an adipogenic gene expression signature linked to the hallmark lipid-laden clear cell morphology of ccRCC. Culturing normal renal and ccRCC cells in differentiation media showed that only ccRCC cells were induced to undergo adipogenic and, surprisingly, osteogenic differentiation. A gene expression signature consistent with epithelial mesenchymal transition (EMT) was identified for ccRCC. We revealed significant down-regulation of four developmental transcription factors (GATA3, TFCP2L1, TFAP2B, DMRT2) that are important for normal renal development. Conclusions ccRCC is characterized by a lack of epithelial differentiation, mesenchymal/adipogenic transdifferentiation, and pluripotent mesenchymal stem cell-like differentiation capacity in vitro. We suggest that down-regulation of developmental transcription factors may mediate the aberrant differentiation in ccRCC. We propose a model in which normal renal epithelial cells undergo dedifferentiation, EMT, and adipogenic transdifferentiation, resulting in ccRCC. Because ccRCC cells grown in adipogenic media regain the characteristic ccRCC phenotype, we have indentified a new in vitro ccRCC cell model more resembling ccRCC tumor morphology.

Stearoyl-CoA Desaturase 1 Is a Novel Molecular Therapeutic Target for Clear Cell Renal Cell Carcinoma

Purpose: We set out to identify Stearoyl-CoA desaturase 1 (SCD1) as a novel molecular target in clear cell renal cell carcinoma (ccRCC) and examine its role in tumor cell growth and viability in vitro and in vivo independently as well as in combination with current U.S. Food and Drug Administration (FDA)-approved regimens. Experimental Design: Patient normal and ccRCC tissue samples and cell lines were examined for SCD1 expression. Genetic knockdown models and targeted inhibition of SCD1 through use of a small molecule inhibitor, A939572, were analyzed for growth, apoptosis, and alterations in gene expression using gene array analysis. Therapeutic models of synergy were evaluated utilizing pharmacologic inhibition of SCD1 with the tyrosine kinase inhibitors (TKI) sunitinib and pazopanib, and the mTOR inhibitor temsirolimus. Results: Our studies identify increased SCD1 expression in all stages of ccRCC. Both genetic knockdown and pharmacologic inhibition of SCD1 decreased tumor cell proliferation and induced apoptosis in vitro and in vivo. Upon gene array, quantitative real-time PCR, and protein analysis of A939572-treated or SCD1 lentiviral knockdown samples, induction of endoplasmic reticulum stress response signaling was observed, providing mechanistic insight for SCD1 activity in ccRCC. Furthermore, combinatorial application of A939572 with temsirolimus synergistically inhibited tumor growth in vitro and in vivo. Conclusions: Increased SCD1 expression supports ccRCC viability and therefore we propose it as a novel molecular target for therapy either independently or in combination with an mTOR inhibitor for patients whose disease cannot be remedied with surgical intervention, such as in cases of advanced or metastatic disease. Clin Cancer Res; 19(9); 2368–80. ©2013 AACR.

p120 catenin induces opposing effects on tumor cell growth depending on E-cadherin expression

p120 catenin regulates the activity of the Rho family guanosine triphosphatases (including RhoA and Rac1) in an adhesion-dependent manner. Through this action, p120 promotes a sessile cellular phenotype when associated with epithelial cadherin (E-cadherin) or a motile phenotype when associated with mesenchymal cadherins. In this study, we show that p120 also exerts significant and diametrically opposing effects on tumor cell growth depending on E-cadherin expression. Endogenous p120 acts to stabilize E-cadherin complexes and to actively promote the tumor-suppressive function of E-cadherin, potently inhibiting Ras activation. Upon E-cadherin loss during tumor progression, the negative regulation of Ras is relieved; under these conditions, endogenous p120 promotes transformed cell growth both in vitro and in vivo by activating a Rac1–mitogen-activated protein kinase signaling pathway normally activated by the adhesion of cells to the extracellular matrix. These data indicate that both E-cadherin and p120 are important regulators of tumor cell growth and imply roles for both proteins in chemoresistance and targeted therapeutics.

Lipid rafts play an important role in Aβ biogenesis by regulating the β-secretase pathway

The Alzheimer’s amyloid β protein (Aβ) precursor (APP) is proteolytically cleaved by β-secretase to N- and C-terminal fragments sAPPβ and CTFβ, respectively. Subsequently, CTFβ is cleaved by γ-secretase to generate Aβ. We previously showed that the levels of secreted Aβ and sAPPβ were significantly reduced upon removal of glycosylphosphatidylinositol (GPI)-anchored proteins from either primary brain cells or Chinese hamster ovary cultures. The results indicated that GPI-anchored proteins facilitated β-secretase activity. In this report, we strengthen the previous findings by demonstrating that CTFβ, like a sAPPβ, is also reduced upon removal of GPI-anchored proteins and that sAPPβ does not accumulate in an intracellular compartment. This facilitation pathway does not appear to be important for the processing of a disease-linked mutant form of APP (670NL), known to be a superior β-secretase substrate. A novel aspartyl protease, BACE, responsible for β-secretase activity in the brain is not GPI-anchored. However, BACE in brain membranes accumulate in lipid rafts, a compartment marked by the accumulation of GPI-anchored proteins. This finding is consistent with the hypothesis that BACE interacts with GPI-anchored proteins that facilitate its activity possibly by chaperoning it into lipid rafts.

Cytoplasmic Sequestration of p27 via AKT Phosphorylation in Renal Cell Carcinoma

Purpose: p27 localization and expression has prognostic and predictive value in cancer. Little is known regarding expression patterns of p27 in renal cell carcinoma (RCC) or how p27 participates in disease progression or response to therapy. Experimental Design: RCC-derived cell lines, primary tumors, and normal renal epithelial cells were analyzed for p27 expression, phosphorylation (T157 of the NLS), and subcellular localization. RCC-derived cell lines were treated with phosphatidylinositol 3-kinase (PI3K) and mammalian target of rapamycin (mTOR) inhibitors and effects on p27 localization were assessed. The potential contribution of cytoplasmic p27 to resistance to apoptosis was also evaluated. Results: p27 was elevated in tumors compared with matched controls, and cytoplasmic mislocalization of p27 was associated with increasing tumor grade. Cytoplasmic localization of p27 correlated with phosphorylation at T157, an AKT phosphorylation site in the p27 NLS. In RCC cell lines, activated PI3K/AKT signaling was accompanied by mislocalization of p27. AKT activation and phosphorylation of p27 was associated with resistance to apoptosis, and small interfering RNA knockdown of p27 or relocalization to the nucleus increased apoptosis in RCC cells. Treatment with the PI3K inhibitors LY294002 or wortmannin resulted in nuclear relocalization of p27, whereas mTOR inhibition by rapamycin did not. Conclusions: In RCC, p27 is phosphorylated at T157 of the NLS, with increasing tumor grade associated with cytoplasmic p27. PI3K inhibition (which reduces AKT activity) reduces T157 phosphorylation and induces nuclear relocalization of p27, whereas mTOR inhibition does not. Clinical testing of these findings may provide a rational approach for use of mTOR and PI3K/AKT pathway inhibitors in patients with RCC.

Reactivation of Suppressed RhoB is a Critical Step for the Inhibition of Anaplastic Thyroid Cancer Growth

Anaplastic thyroid carcinoma (ATC) is a highly aggressive form of the disease for which new therapeutic options are desperately needed. Previously, we showed that the high-affinity peroxisome proliferator-activated receptor gamma (PPARgamma) agonist, RS5444, inhibits cell proliferation of ATC cells via induction of the cyclin-dependent kinase inhibitor p21(WAF1/CIP1) (p21). We show here that up-regulation of RhoB is a critical step in PPARgamma-mediated activation of p21-induced cell stasis. Using multiple independently derived ATC cell lines, we found that treatment with RS5444 leads to the up-regulation of RhoB and subsequent activation of p21, and that silencing of RhoB by RNAi blocks the ability of RS5444 to induce p21 and to inhibit cell proliferation. Our results show that transcriptional regulation of RhoB by the nuclear transcription factor PPARgamma is responsible for the induction of p21 mRNA and protein. We further implicate RhoB as a key signaling effector for the growth inhibition of ATC, as treatment with a histone deacetylase inhibitor shown to increase RhoB expression in lung cancer cells caused the up-regulation of RhoB in ATC cells accompanied by increased expression of p21 and inhibition of cell proliferation; this effect occurred even in ATC cells that were unresponsive to RS5444 due to a lack of expression of PPARgamma. Our results implicate RhoB as a novel intermediate in critical signaling pathways and as an additional target for therapeutic intervention in ATC.

Efatutazone, an Oral PPAR-γ Agonist, in Combination With Paclitaxel in Anaplastic Thyroid Cancer: Results of a Multicenter Phase 1 Trial

PURPOSE A phase 1 study was initiated to determine the safety, potential effectiveness, and maximal tolerated dose and recommended phase 2 dose of efatutazone and paclitaxel in anaplastic thyroid cancer. EXPERIMENTAL DESIGN Patients received efatutazone (0.15, 0.3, or 0.5 mg) orally twice daily and then paclitaxel every 3 weeks. Patient tolerance and outcomes were assessed, as were serum efatutazone pharmacokinetics. RESULTS Ten of 15 patients were women. Median age was 59 years. Seven patients received 0.15 mg of efatutazone, 6 patients received 0.3 mg, and 2 patients received 0.5 mg. One patient receiving 0.3 mg of efatutazone had a partial response from day 69 to day 175; 7 patients attained stable disease. Median times to progression were 48 and 68 days in patients receiving 0.15 mg of efatutazone and 0.3 mg of efatutazone, respectively; corresponding median survival was 98 vs 138 days. The median peak efatutazone blood level was 8.6 ng/mL for 0.15-mg dosing vs 22.0 ng/mL for 0.3-mg twice daily dosing. Ten patients had grade 3 or greater adverse events (Common Terminology Criteria for Adverse Events), with 2 of these (anemia and edema) related to efatutazone. Thirteen events of edema were reported in 8 patients, with 2 of grade 3 or greater. Eight patients had ≥1 serious adverse event, with 1 of these (anemia) attributed to efatutazone and 1 (anaphylactic reaction) related to paclitaxel. The maximal tolerated dose was not achieved. Angiopoietin-like 4 was induced by efatutazone in tissue biopsy samples of 2 patients. CONCLUSIONS Efatutazone and paclitaxel in combination were safe and tolerated and had biologic activity.