Faculty Bibliography

BACKGROUND:Papillary thyroid cancer (PTC) recurrence risk is difficult to predict. No current risk classification system incorporates BRAF mutational status. Here, we assess the incremental value of BRAF mutational status in predicting PTC recurrence relative to existing recurrence risk algorithms. METHODS:Serial data were collected for a historical cohort having undergone total thyroidectomy for papillary thyroid carcinoma (PTC) during a 5-year period. Corresponding BRAF(V600E) testing was performed and Cox proportional hazard regression modeling, with and without BRAF status, was used to evaluate existing recurrence risk algorithms. RESULTS:The 5-year cumulative PTC recurrence incidence within our 356 patient cohort was 15%. A total of 205 (81%) of associated archived specimens were successfully genotyped, and 110 (54%) harbored the BRAF(V600E) mutation. The 5-year cumulative recurrence incidence among BRAF(V600E) patients was 20% versus 8% among BRAF wild type. BRAF(V600E) was significantly associated with time to recurrence when added to the following algorithms: AMES (hazard ratio [HR] 2.43 [confidence interval 1.08-5.49]), MACIS category (HR 2.46 [1.09-5.54]), AJCC-TNM (HR 2.51 [1.11-5.66]), and ATA recurrence-risk category (HR 2.44 [1.08-5.50]), and model discrimination improved (incremental c-index range 0.046-0.109). CONCLUSION/CONCLUSIONS:The addition of BRAF mutational status to established risk algorithms improves the discrimination of risk recurrence in patients undergoing total thyroidectomy for PTC.

MicroRNAs (miRs) are important regulators of gene expression in normal physiology and disease, and are widely misexpressed in cancer. A number of studies have identified miR-21 as an important promoter of oncogenesis. However, as is true of most miRs, the mechanisms behind the aberrant expression of miR-21 in cancer are poorly understood. Herein, we examine the regulation of miR-21 expression in colorectal cancer (CRC) cells by the oncogenic epidermal growth factor (EGF)/Ras pathway and by Ets transcription factors, modulators of epithelial oncogenesis that are frequently misexpressed in CRC. We show that EGF/Ras efficiently induces the miR-21 primary transcript, but this does not rapidly and simply translate into higher mature miR-21 levels. Rather, induction of mature miR-21 by constitutive activation of this pathway is slow, is associated with only minimal activation of mitogen-activated protein kinase, and may involve stimulation of post-transcriptional processing by mechanisms other than Dicer stabilization. We further identify Ets transcription factors as modifiers of miR-21 expression in CRC. The effects of Ets factors on miR-21 expression are cell context-dependent, and appear to involve both direct and indirect mechanisms. The Ets factor Pea3 emerges from our studies as a consistent repressor of miR-21 transcription. Overall, our studies identify a complex relationship between oncogenic pathways and steady-state miR-21 levels in CRC, and highlight the need for greater understanding of the control of miR expression in cancer and other disease states.

BACKGROUND:The ETS family transcription factor ESE-1 is often overexpressed in human breast cancer. ESE-1 initiates transformation of MCF-12A cells via a non-transcriptional, cytoplasmic process that is mediated by a unique 40-amino acid serine and aspartic acid rich (SAR) subdomain, whereas, ESE-1's nuclear transcriptional property is required to maintain the transformed phenotype of MCF7, ZR-75-1 and T47D breast cancer cells. RESULTS:To map the minimal functional nuclear localization (NLS) and nuclear export (NES) signals, we fused in-frame putative NLS and NES motifs between GFP and the SAR domain. Using these GFP constructs as reporters of subcellular localization, we mapped a single NLS to six basic amino acids (242 HGKRRR 247) in the AT-hook and two CRM1-dependent NES motifs, one to the pointed domain (NES1: 102 LCNCALEELRL 112) and another to the DNA binding domain (DBD), (NES2: 275 LWEFIRDILI 284). Moreover, analysis of a putative NLS located in the DBD (316 GQKKKNSN 323) by a similar GFP-SAR reporter or by internal deletion of the DBD, revealed this sequence to lack NLS activity. To assess the role of NES2 in regulating ESE-1 subcellular localization and subsequent transformation potency, we site-specifically mutagenized NES2, within full-length GFP-ESE-1 and GFP-NES2-SAR reporter constructs. These studies show that site-specific mutation of NES2 completely abrogates ESE-1 transforming activity. Furthermore, we show that exclusive cytoplasmic targeting of the SAR domain is sufficient to initiate transformation, and we report that an intact SAR domain is required, since block mutagenesis reveals that an intact SAR domain is necessary to maintain its full transforming potency. Finally, using a monoclonal antibody targeting the SAR domain, we demonstrate that the SAR domain contains a region accessible for protein - protein interactions. CONCLUSIONS:These data highlight that ESE-1 contains NLS and NES signals that play a critical role in regulating its subcellular localization and function, and that an intact SAR domain mediates MEC transformation exclusively in the cytoplasm, via a novel nontranscriptional mechanism, whereby the SAR motif is accessible for ligand and/or protein interactions. These findings are significant, since they provide novel molecular insights into the functions of ETS transcription factors in mammary cell transformation.

BACKGROUND:Remedial surgery for patients with persistent or recurrent primary hyperparathyroidism (1 degrees HPT) remains a significant challenge. Cervical reexploration is technically difficult; reoperative neck anatomy is distorted by fibrosis and, as a result, remedial 1 degrees HPT patients carry an increased risk of injury to the recurrent (RLN) and superior laryngeal nerve(s) as well as to normal residual parathyroid tissue. Causative hyperfunctioning parathyroid tissue is also more frequently ectopic in the remedial setting and can thus be difficult to localize. METHODS:This report assimilates the current data underlying preoperative, intraoperative and postoperative remedial 1 degrees HPT management and presents an evidence-based algorithm for the management of remedial parathyroid disease. Recommendations are graded according to the quality of supporting data using the system initially developed by Sackett (Chest 95:2S-4S, 1989) and subsequently modified by Heinrich et al. (Ann Surg 243:154-168, 2006). RESULTS:Recent advances in preoperative localization and intraoperative adjuncts have lead to substantial improvements in outcomes after remedial surgery. Preoperative localization techniques, including sestamibi scintigraphy (MIBI), high resolution ultrasound (US), US-guided fine needle aspiration (FNA) and selective venous sampling (SVS), coupled with intraoperative adjuncts such as the rapid parathyroid hormone (PTH) assay have lead to reoperative cure rates as high as 96 percent. Nonetheless, management of remedial 1 degrees HPT varies significantly between surgeons and no formal recommendations standardizing the care of these patients have been published. CONCLUSIONS:Despite the significant challenges associated with remedial surgery for 1 degrees HPT, excellent outcomes can be reproducibly achieved when proper pre-, intra-, and postoperative management is employed.

Several different transcription factors, including estrogen receptor, progesterone receptor, and ETS family members, have been implicated in human breast cancer, indicating that transcription factor-induced alterations in gene expression underlie mammary cell transformation. ESE-1 is an epithelium-specific ETS transcription factor that contains two distinguishing domains, a serine- and aspartic acid-rich (SAR) domain and an AT hook domain. ESE-1 is abundantly expressed in human breast cancer and trans-activates epithelium-specific gene promoters in transient transfection assays. While it has been presumed that ETS factors transform mammary epithelial cells via their nuclear transcriptional functions, here we show (i) that ESE-1 protein is cytoplasmic in human breast cancer cells; (ii) that stably expressed green fluorescent protein-ESE-1 transforms MCF-12A human mammary epithelial cells; and (iii) that the ESE-1 SAR domain, acting in the cytoplasm, is necessary and sufficient to mediate this transformation. Deletion of transcriptional regulatory or nuclear localization domains does not impair ESE-1-mediated transformation, whereas fusing the simian virus 40 T-antigen nuclear localization signal to various ESE-1 constructs, including the SAR domain alone, inhibits their transforming capacity. Finally, we show that the nuclear localization of ESE-1 protein induces apoptosis in nontransformed mammary epithelial cells via a transcription-dependent mechanism. Together, our studies reveal two distinct ESE-1 functions, apoptosis and transformation, where the ESE-1 transcription activation domain contributes to apoptosis and the SAR domain mediates transformation via a novel nonnuclear, nontranscriptional mechanism. These studies not only describe a unique ETS factor transformation mechanism but also establish a new paradigm for cell transformation in general.

ESX is an epithelial-restricted member of a large family of transcription factors known as the Ets family. ESX expression has been shown to be correlated with Her2/neu proto-oncogene amplification in highly aggressive breast cancers and induced by Her2/neu in breast cell lines, but its role in tumorigenesis is unknown. Previously, we have shown that ESX enhances breast cell survival in colony-formation assays. In order to determine whether ESX can act as a transforming gene, we stably transfected MCF-12A human mammary epithelial cells with the ESX expression vector, pCGN2-HA-ESX. The MCF-12A cell line is immortalized, but nontransformed, and importantly, these cells fail to express endogenous ESX protein. We used pCGN2-HA-Ets-2 and pSVRas expression vectors as positive controls for transformation. Like HA-Ets-2 and V12-Ras, stable expression of ESX induced EGF-independent proliferation, serum-independent MAPK phosphorylation and growth in soft agar. Additionally, stable ESX expression conferred increased cell adhesion, motility and invasion in two-dimensional and transwell filter assays, and an epithelial to mesenchymal morphological transition. In three-dimensional cultures, parental and vector control (pCGN2) cells formed highly organized duct-like structures with evidence of cell polarity, ECM adhesion-dependent proliferation and cell survival, and lack of cellular invasion into surrounding matrix. Remarkably, the ESX stable cells formed solid, disorganized structures, with lack of cell polarity, loss of adhesion junctions and cytokeratin staining and loss of dependence on ECM adhesion for cell proliferation and survival. In addition, ESX cells invaded the surrounding matrix, indicative of a transformed and metastatic phenotype. Taken together, these data show that ESX expression alone confers a transformed and in vitro metastatic phenotype to otherwise normal MCF-12A cells.