Theodore S. Nowicki

Inhibition of uPAR and uPA reduces invasion in papillary thyroid carcinoma cells

We analyzed the expression of urokinase plasminogen activator (uPA) and its receptor (uPAR) in papillary thyroid carcinoma (PTC) and normal thyroid tissue and examined in vitro how uPA and uPAR contribute to an invasive/metastatic phenotype, and the functional consequences of inhibiting this system.

Arachidonate 5 Lipoxygenase Expression in Papillary Thyroid Carcinoma Promotes Invasion via MMP-9 Induction

Arachidonate 5‐lipoxygenase (ALOX5) expression and activity has been implicated in tumor pathogenesis, yet its role in papillary thyroid carcinoma (PTC) has not been characterized. ALOX5 protein and mRNA were upregulated in PTC compared to matched, normal thyroid tissue, and ALOX5 expression correlated with invasive tumor histopathology. Evidence suggests that PTC invasion is mediated through the induction of matrix metalloproteinases (MMPs) that can degrade and remodel the extracellular matrix (ECM). A correlation between MMP‐9 and ALOX5 protein expression was established by immunohistochemical analysis of PTC and normal thyroid tissues using a tissue array. Transfection of ALOX5 into a PTC cell line (BCPAP) increased MMP‐9 secretion and cell invasion across an ECM barrier. The ALOX5 product, 5(S)‐hydroxyeicosatetraenoic acid also increased MMP‐9 protein expression by BCPAP in a dose‐dependent manner. Inhibitors of MMP‐9 and ALOX5 reversed ALOX5‐enhanced invasion. Here we describe a new role for ALOX5 as a mediator of invasion via MMP‐9 induction; this ALOX5/MMP9 pathway represents a new avenue in the search for functional biomarkers and/or potential therapeutic targets for aggressive PTC. J. Cell. Biochem. 113: 1998–2008, 2012.

Rituximab therapy to prevent relapse in chronic relapsing thrombotic thrombocytopenic purpura (TTP) in a child.

Our patient first developed thrombotic thrombocytopenic purpura (TTP) at age 10 years with an initial platelet count of 10,000/μL. She achieved remission with plasmapheresis (PE), but suffered 2 relapses in the next 2 years, each approximately 1 year from PE, with ADAMTS13 levels of <5%. Early in her third remission, with vincristine (weekly × 4 doses) and prednisone (for 2 weeks) her ADAMTS13 increased to 99% in 24 weeks, but decreased to <4% in the next 38 weeks. After 4 weekly doses of rituximab (375 mg/m2), her ADAMTS13 level reached 101% in 9 weeks and has remained consistently above 97% on bimonthly monitoring for more than a year. She remains in continuous clinical and hematologic remission with an ADAMTS13 level of 108% at 60 weeks from rituximab therapy and 124 weeks from her second relapse. This case report suggests that monitoring ADAMTS13 level at regular intervals in recurrent TTP may help us identify patients at risk for further relapse; and such a relapse may be prevented, or at least delayed with timely rituximab therapy, thus reducing morbidity from relapsed TTP and its treatment.

Early recognition of renal toxicity of high-dose methotrexate therapy: a case report.

A 10-year-old boy with osteosarcoma and normal renal function manifested laboratory evidence of impending renal toxicity and extreme elevation of aspartate aminotrasferase and alanine aminotransferase within 2 hours after the completion of a 4-hour infusion of high-dose methotrexate (MTX) (12 g/m2), and went on to develop acute renal failure with life-threatening hyperkalemia 29 hours later. Although his renal function recovered completely with high-dose leucovorin, hemodialysis, charcoal hemoperfusion, and carboxypeptidase G2, we present this case to emphasize that signs of renal toxicity may be present as early as 2 hours after the completion of a 4-hour MTX infusion, and to suggest that monitoring for MTX toxicity should perhaps begin within a few hours after the completion of 4-hour MTX infusion.

The Urokinase Plasminogen Activator System in Metastatic Papillary Thyroid Carcinoma: A Potential Therapeutic Target

Although distant metastases occur in a minority of papillary thyroid carcinoma (PTC) patients, their impact on disease course and survival rates is profound, with aggregate 10-yr survival rates of 40% (1). Limited treatment options further complicate management of metastatic PTC. Radioiodine (RAI) remains the standard of care for most distant metastases in PTC. Of particular concern, however, is the significant prevalence of PTC metastases unable to trap RAI; such metastases are termed RAI-refractory (RAIR). Because thyroid cancer loses differentiated features of normal thyroid tissue, one feature commonly lost is sodium iodide symporter expression, which is the entire basis for the use of RAI therapy in thyroid malignancies. This phenomenon can occur in a heterogeneous manner: in a given patient, RAI uptake can vary between individual metastases, and it can even vary within a metastasis at the cellular level.

Abstract 1502: Increased expression of KLK7 and KLK10 in papillary thyroid cancer

The American Cancer Society estimates that there will be approximately 44,670 new cases of thyroid cancer and 1,690 deaths due to the disease in the United States in 2010. Approximately 80% of these cancers are papillary thyroid carcinoma (PTC), representing the most common endocrine malignancy. Kallikreins are serine proteases that have many physiological functions, including the remodeling of extracellular matrix (ECM). As PTC has the ability to degrade and remodel the ECM to facilitate penetration through the thyroid capsule and invasion of extra-thyroidal tissues, we investigated the expression of kallikrein 7 (KLK7) and kallikrein 10 (KLK10) in PTC. RNA expression of KLK7 and KLK10 in PTC was measured using real-time RT-PCR (normalized to GAPDH) (n = 26) and found to be significantly upregulated, with a mean fold change of 28.8 and 27.8 (P Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1502. doi:10.1158/1538-7445.AM2011-1502

Abstract 1406: Downregulation of uPAR inhibits migration, invasion, proliferation, FAK/PI3K/Akt signaling, and induces senescence in papillary thyroid carcinoma cells

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Papillary thyroid carcinoma (PTC) is the most common endocrine and thyroid malignancy. The urokinase plasminogen activator receptor (uPAR) plays an important role in cancer pathogenesis, including breakdown of the extracellular matrix, invasion, and metastasis. Additionally, there is increasing evidence that uPAR also promotes tumorigenesis via the modulation of multiple signaling pathways. BRAFV600E, the most common initial genetic mutation in PTC, leads to ERK1/2 hyperphosphorylation, which has been shown in numerous cancers to induce uPAR. Treatment of the BRAFV600E-positive PTC cell line, BCPAP, with the MEK/ERK inhibitor U0126 reduced uPAR RNA levels by 90%. siRNA-mediated down-regulation of uPAR in BCPAP cells resulted in drastically reduced proliferation rates and decreased clonigenic survival, as well as demonstrated senescence-associated nuclear morphology and induction of β-galactosidase activity. uPAR-knockdown BCPAP cells also displayed greatly reduced migration and invasion rates, as well as a complete loss of the cells’ plasminogen-enhanced invasiveness. Additionally, uPAR down-regulation lead to greatly decreased activity in the focal adhesion kinase (FAK)/phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway, as well as a modest reduction in ERK1/2 signaling (which remained constitutively active secondary to BRAFV600E activation), which suggests the existence of a uPAR signaling feedback loop capable of affecting the various phenotypic changes exhibited by uPAR-knockdown cells. Taken together, these data provide new evidence of a novel role for uPAR induction as a central component in PTC pathogenesis, and highlight the potential of uPAR as a therapeutic target. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1406. doi:10.1158/1538-7445.AM2011-1406

Abstract 1063: BRAFV600E status predicts higher uPA levels in papillary thyroid cancer

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Of the 44,670 new cases of thyroid cancer predicted for this year in the US by the American Cancer Society, 80% will be papillary thyroid carcinoma (PTC). PTC typically arises from a gain-of-function mutation in the RET, RAS, or BRAF genes which make up a linear signaling cascade for ERK activation in over 90% of PTC cases. PTCs possessing the BRAFV600E mutation have been associated with greater local invasion and regional metastatic potential. Urokinase plasminogen activator (uPA) has been shown to be an important mediator of invasion and metastasis in several cancers. To determine the extent to which BRAFV600E mutation status predicts up-regulation of the uPA in PTC, a study using patient thyroid tissue samples obtained over a 6-year period at a single tertiary care center was undertaken. DNA and RNA were obtained from patient PTC and matched, normal thyroid tissue samples using the Trizol method. BRAFV600E mutational status of the DNA was determined using the TaqMan SNP genotyping assay, while RNA was analyzed for differences in uPA transcription levels (relative to matched, normal thyroid tissue) by qRT-PCR. Fifty-four percent of the PTC samples possessed the BRAFV600E mutation. PTC samples bearing the BRAFV600E mutation displayed significantly higher uPA RNA levels (relative to matched control tissue) than those samples with wild-type BRAF (5.883 vs. 1.27-fold, p<0.05, Wilcoxon signed-rank test). Additionally, uPA RNA levels were significantly higher in patients with nodal metastasis (p<0.05). These data provide new evidence of the roles of BRAFV600E and uPA in PTC invasive pathology and demonstrate for the first time that BRAFV600E status is able to predict higher uPA levels in PTC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1063. doi:10.1158/1538-7445.AM2011-1063

Abstract 520: Inhibition of uPAR and uPA reduces invasion and degradative potential in papillary thyroid carcinoma cells

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Papillary thyroid cancer (PTC) is the most prevalent endocrine and thyroid malignancy. The ability of PTC to invade and migrate great distances contributes to distant metastases, which represent the most common cause of PTC-related death. The urokinase plasminogen activator (uPA) and the urokinase plasminogen receptor (uPAR) are key mediators of tumor invasion. Upon binding to uPAR, pro-uPA is converted to its active form, which is then capable of cleaving plasminogen to plasmin. Plasmin can then degrade components of the basement membrane and extracellular matrix, a prerequisite for tumor cell invasion and metastasis. The binding of uPA by uPAR also seems to mediate several signaling events that contribute to a migratory phenotype, as well as various growth signals. In this study, we analyzed uPA and uPAR expression in PTC and normal thyroid tissue, and examined in vitro how uPA and uPAR contribute to an invasive/metastatic phenotype, as well as the functional consequences of inhibiting this system. uPA and uPAR RNA were also significantly higher in patients with metastatic disease. Casein-plasminogen zymography and western blotting demonstrated increased active uPA secreted by BCPAP cells compared to NTHY-Ori-3-1. Fluorimetric assays revealed that BCPAP CM was able to activate plasminogen, resulting in measureable casein hydrolysis. This casein hydrolysis was prevented by the addition of several specific uPA inhibitors. The in vitro invasion phenotype of BCPAP cells was augmented by the addition of plasminogen, and this augmentation was reversed by inhibitory anti-uPA and anti-uPAR antibodies. Cells transfected with siRNA against uPAR demonstrated decreased migratory and invasive potentials as measured by Matrigel invasion assays. Finally, uPAR-knockdown cells exhibited decreased proliferation when compared to non-targeting siRNA transfectant cells. These data provide new functional evidence of the uPA/uPAR systems role in PTC invasion/metastasis and demonstrate the attractiveness of uPA and uPAR as molecular biomarkers and therapeutic targets. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 520.