Ronald J. Weigel

Up-regulation of Akt3 in Estrogen Receptor-deficient Breast Cancers and Androgen-independent Prostate Cancer Lines

We measured the insulin-stimulated amount of Akt1, Akt2, and Akt3 enzymatic activities in four breast cancer cell lines and three prostate cancer cell lines. In the estrogen receptor-deficient breast cancer cells and the androgen-insensitive prostate cells, the amount of Akt3 enzymatic activity was approximately 20–60-fold higher than in the cells that were estrogen- or androgen-responsive. In contrast, the levels of Akt1 and -2 were not increased in these cells. The increase in Akt3 enzyme activity correlated with an increase in both Akt3 mRNA and protein. In a prostate cancer cell line lacking the tumor suppressor PTEN (a lipid and protein phosphatase), the basal enzymatic activity of Akt3 was constitutively elevated and represented the major active Akt in these cells. Finally, reverse transcription-PCR was used to examine the Akt3 expression in 27 primary breast carcinomas. The expression levels of Akt3 were significantly higher in the estrogen receptor-negative tumors in comparison to the estrogen receptor-positive tumors. To see if the increase in Akt3 could be due to chromosomal abnormalities, the Akt3 gene was assigned to human chromosome 1q44 by fluorescence in situ hybridization and radiation hybrid cell panel analyses. These results indicate that Akt3 may contribute to the more aggressive clinical phenotype of the estrogen receptor-negative breast cancers and androgen-insensitive prostate carcinomas.

GATA‐3 is expressed in association with estrogen receptor in breast cancer

To better understand the molecular basis for the hormone‐responsive phenotype in breast cancer, we have used a human cDNA array to compare patterns of gene expression between breast carcinoma cell lines discordant for estrogen receptor (ER) expression. These experiments indicated abundant expression of the transcription factor GATA‐3 in the ER‐positive cell lines MCF7 and T‐47D, with minimal or no expression in the ER‐negative cells lines MDA‐MB‐231 and HBL‐100. Northern blot analysis of a panel of human breast carcinoma cell lines demonstrated a correlation between ER and GATA‐3 expression. Studies of MCF7 cells grown in the absence or presence β‐estradiol indicated that GATA‐3 expression was not responsive to estradiol. Protein immunoprecipitation and gel shift analysis confirmed the presence of functional GATA‐3 protein in MCF7 but not in HBL‐100 nuclear extracts. A panel of 47 primary breast cancers was characterized for expression of ER and GATA‐3 using immunoperoxidase assay. In primary tumors, a statistically significant correlation between ER and GATA‐3 expression was established (p < 0.0001, χ2). Our results indicate that GATA‐3, in association with ER, is likely to regulate genes critical to the hormone‐responsive breast cancer phenotype. Int. J. Cancer (Pred. Oncol.) 84:122–128, 1999.

Physical and functional interactions between the Wwox tumor suppressor protein and the AP-2γ transcription factor

The WWOX gene encodes a tumor suppressor WW domain-containing protein, Wwox. Alterations of WWOX have been demonstrated in multiple types of cancer, and introduction of Wwox into Wwox-negative tumor cells has resulted in tumor suppression and apoptosis. The Wwox protein contains two WW domains that typically bind proline-rich motifs and mediate protein–protein interactions. Recently, we have described functional cross-talk between the Wwox protein and the p53 homologue, p73. To further explore the biological function of Wwox, we investigated other interacting candidates. In this report, we demonstrate a physical and functional association between AP-2γ transcription factor and the Wwox protein. AP-2γ at 20q13.2 encodes a transcription factor and is frequently amplified in breast carcinoma. We show that Wwox binds to the PPPY motif of AP-2γ via its first WW domain. Alterations of tyrosine 33 in the first WW domain of Wwox or the proline-rich motif in AP-2γ dramatically reduce this interaction. In addition, our results demonstrate that Wwox expression triggers redistribution of nuclear AP-2γ to the cytoplasm, hence suppressing its transactivating function. Our results suggest that Wwox tumor suppressor protein inhibits AP-2γ oncogenic activity by sequestering it in the cytoplasm.

Sonography in primary hyperparathyroidism: review with emphasis on scanning technique.

Objective. To review the sonographic features and focused sonographic scanning techniques that may assist in the preoperative localization of parathyroid adenomas in patients with primary hyperparathyroidism. Methods. The sonographic findings were reviewed in 54 of 58 consecutive patients with pathologically proven parathyroid adenomas. A systematic scanning approach including real‐time gray scale, color and power Doppler, and graded compression gray scale imaging was used in all patients. Results. Fifty‐four (93%) of 58 proven adenomas were correctly identified by sonography. Gray scale imaging alone was sufficient for identifying 26 (100%) of 26 large (≥1‐cm) and 3 (11%) of 25 small (<1‐cm) parathyroid adenomas. However, for 25 (89%) of 28 small adenomas, a combination of color and power Doppler and graded compression real‐time gray scale imaging was required for sonographic localization and identification. Conclusions. Knowledge of typical locations and characteristic imaging features, as well as a systematic scanning approach, can result in accurate preoperative sonographic localization of parathyroid adenomas.

TFAP2C Controls Hormone Response in Breast Cancer Cells through Multiple Pathways of Estrogen Signaling

Breast cancers expressing estrogen receptor-alpha (ERalpha) are associated with a favorable biology and are more likely to respond to hormonal therapy. In addition to ERalpha, other pathways of estrogen response have been identified including ERbeta and GPR30, a membrane receptor for estrogen, and the key mechanisms regulating expression of ERs and hormone response remain controversial. Herein, we show that TFAP2C is the key regulator of hormone responsiveness in breast carcinoma cells through the control of multiple pathways of estrogen signaling. TFAP2C regulates the expression of ERalpha directly by binding to the ERalpha promoter and indirectly via regulation of FoxM1. In so doing, TFAP2C controls the expression of ERalpha target genes, including pS2, MYB, and RERG. Furthermore, TFAP2C controlled the expression of GPR30. In distinct contrast, TFAP2A, a related factor expressed in breast cancer, was not involved in estrogen-mediated pathways but regulated expression of genes controlling cell cycle arrest and apoptosis including p21(CIP1) and IGFBP-3. Knockdown of TFAP2C abrogated the mitogenic response to estrogen exposure and decreased hormone-responsive tumor growth of breast cancer xenografts. We conclude that TFAP2C is a central control gene of hormone response and is a novel therapeutic target in the design of new drug treatments for breast cancer.

Identification of Primary Gene Targets of TFAP2C in Hormone Responsive Breast Carcinoma Cells

The TFAP2C transcription factor is involved in mammary development, differentiation, and oncogenesis. Previous studies established a role for TFAP2C in the regulation of ESR1 (ERα) and ERBB2 (Her2) in breast carcinomas. However, the role of TFAP2C in different breast cancer phenotypes has not been examined in detail. To develop a more complete characterization of TFAP2C target genes, ChIP‐seq with anti‐TFAP2C antibody and expression arrays with TFAP2C knock down were analyzed in MCF‐7 breast carcinoma cells. Genomic sequences common to the ChIP‐seq data set defined the consensus sequence for TFAP2C chromatin binding as the nine base sequence SCCTSRGGS (S = G/C, r = A/G), which closely matches the previously defined optimal in vitro binding site. Comparing expression arrays before and after knock down of TFAP2C with ChIP‐seq data demonstrated a conservative estimate that 8% of genes altered by TFAP2C expression are primary target genes and includes genes that are both induced and repressed by TFAP2C. A set of 447 primary target genes of TFAP2C was identified, which included ESR1 (ERα), FREM2, RET, FOXA1, WWOX, GREB1, MYC, and members of the retinoic acid response pathway. The identification of ESR1, WWOX, GREB1, and FOXA1 as primary targets confirmed the role of TFAP2C in hormone response. TFAP2C plays a critical role in gene regulation in hormone responsive breast cancer and its target genes are different than for the Her2 breast cancer phenotype.

Determinants of survival in patients with calciphylaxis: a multivariate analysis.

BACKGROUND Our study aims to assess the factors affecting survival in patients with calciphylaxis. METHODS We identified 26 patients with biopsy-proven calciphylaxis treated between 1995 and 2007. Clinical and follow-up data were obtained from medical records. Cox proportional hazards models were used to assess the factors affecting survival. RESULTS The study group consisted of 23 women and 3 men with a mean age of 56.4 +/- 12.9 years. All patients had multiple comorbidities/risk factors including coronary artery disease (58%), diabetes mellitus (58%), and peripheral vascular disease (23%). Mean laboratory values were: calcium, 9.0 mg/dL (range, 6.8-11.6); albumin, 2.8 mg/dL; phosphate, 4.5 mg/dL (range, 2.5-7.5); Ca *phosphate, 35.9; and parathyroid hormone, 320.9 pg/mL (range, 4.6-2,419). Parathyroidectomy was performed in 9 of 26 patients (35%). Of our patients, 19% underwent revascularization procedures and 58% underwent debridement. In multivariate analyses, factors associated with poor survival were female gender ( P = .01), increased weight ( P = .01), and need for vascular procedures ( P = .06). Improved survival was associated with operative debridement ( P = .01). Parathyroidectomy alone did not emerge as a determinant of patient survival, although there was a trend to improved survival when debridement and parathyroidectomy were combined ( P = .09). CONCLUSION Rather than a single intervention such as parathyroidectomy, a multidisciplinary approach involving early diagnosis, aggressive medical management, operative debridement, and parathyroidectomy may improve survival in calciphylaxis.

Surgical treatment of primary hyperaldosteronism.

ObjectiveA retrospective review of patients with primary hyperaldosteronism treated at the Duke University Medical Center was performed. Summary Background DataThe management of patients with primary hyperaldosteronism has changed dramatically in the past 20 years. The outcome of surgical treatment was examined to optimize the management of these patients. MethodsAll patients who were evaluated for primary hyperaldosteronism before operation and who had adrenal surgery at Duke University Medical Center from 1970 to 1993 were included in the study. ResultsA ratio of plasma aldosterone concentration to plasma renin activity greater than 250 was predictive of hyperaldosteronism in 94% of patients. This ratio also predicted the size of the adenoma. Surgical treatment cured hypokalemia in 90% of the patients. Hypertension was cured or greatly improved, with an average decrease of 50.4 mmHg and 25.2 mmHg for systolic and diastolic pressures, respectively. Patients who had hypertension for less than 5 years achieved a satisfactory blood pressure response more often (13 of 14 patients) than did patients who had hypertension for 5 or more years (15 of 20 patients). ConclusionsPreoperative evaluation of patients with primary hyperaldosteronism using current modes allows a posterior approach to be used in most patients. Adrenalectomy cures hypokalemia and hypertension; however, patients with a history of hypertension for less than 5 years may be more likely to be cured by adrenal resection.

Major EB virus-specific cytoplasmic transcripts in a cellular clone of the HR-1 burkitt lymphoma line during latency and after induction of viral replicative cycle by phorbol esters

We have estimated by the Northern blotting technique the size and genome location of major viral RNA transcripts found in the same cell line when the Epstein-Barr virus (EBV) genome was latent, and 48 hr after it was induced to replicate by treatment with 12-O-tetradecanoylphorbol acetate (TPA). A cellular subclone of the P3J-HR-1 line designated GG68-13 made these studies possible. Less than 1% of GG68-13 cells spontaneously synthesize viral antigens, whereas more than 80% of the cells enter the viral replicative cycle after exposure to TPA. In the absence of TPA six clearly resolved mRNAs, derived from scattered regions of the genome, are seen and at least four poorly resolved mRNAs map to BamHI fragment W, the internal repeat. Following treatment with the drug, 54 mRNAs have been identified, 28 of which are prominent. The mRNAs identified during latency are also synthesized, but in greater amounts, during viral replication. However, EBV-encoded small RNAs seem to be more abundant during latency than during viral synthesis.

GATA-3 as a Marker of Hormone Response in Breast Cancer

GATA-3 is a transcription factor that orchestrates gene expression profiles during embryogenesis of a variety of human tissues, including hematopoietic cells, skin, kidney, mammary gland, and the central nervous system. Among several other roles, GATA-3 has recently been identified as a key player of luminal cell differentiation in the mammary gland. The majority of breast cancers arise from luminal epithelial cells and hence GATA-3 appears to control a set of genes involved in the differentiation and proliferation of breast cancer. The expression of GATA-3 has a strong association with the expression of estrogen receptor-alpha (ER) in breast cancer, and there is mounting evidence that GATA-3 can be used as a clinical marker to determine response to hormonal therapy and to refine the prognosis of breast cancer patients. Here, we review the literature from the past 10 y on GATA-3 in normal and pathological states of the mammary gland. Conclusions from the literature are confirmed using meta-analyses performed by the Oncomine Research Platform.