Robert J. Goulet

Constitutive activation of NF-kappaB during progression of breast cancer to hormone-independent growth.

Breast cancers often progress from a hormone-dependent, nonmetastatic, antiestrogen-sensitive phenotype to a hormone-independent, antiestrogen- and chemotherapy-resistant phenotype with highly invasive and metastatic growth properties. This progression is usually accompanied by altered function of the estrogen receptor (ER) or outgrowth of ER-negative cancer cells. To understand the molecular mechanisms responsible for metastatic growth of ER-negative breast cancers, the activities of the transcription factor NF-kappaB (which modulates the expression of genes involved in cell proliferation, differentiation, apoptosis, and metastasis) were compared in ER-positive (MCF-7 and T47-D) and ER-negative (MDA-MB-231 and MDA-MB-435) human breast cancer cell lines. NF-kappaB, which is usually maintained in an inactive state by protein-protein interaction with inhibitor IkappaBs, was found to be constitutively active in ER-negative breast cancer cell lines. Constitutive DNA binding of NF-kappaB was also observed with extracts from ER-negative, poorly differentiated primary breast tumors. Progression of the rat mammary carcinoma cell line RM22-F5 from an ER-positive, nonmalignant phenotype (E phenotype) to an ER-negative, malignant phenotype (F phenotype) was also accompanied by constitutive activation of NF-kappaB. Analysis of individual subunits of NF-kappaB revealed that all ER-negative cell lines, including RM22-F5 cells of F phenotype, contain a unique 37-kDa protein which is antigenically related to the RelA subunit. Cell-type-specific differences in IkappaB alpha, -beta, and -gamma were also observed. In transient-transfection experiments, constitutive activity of an NF-kappaB-dependent promoter was observed in MDA-MB-231 and RM22-F5 cells of F phenotype, and this activity was efficiently repressed by cotransfected ER. Since ER inhibits the constitutive as well as inducible activation function of NF-kappaB in a dose-dependent manner, we propose that breast cancers that lack functional ER overexpress NF-kappaB-regulated genes. Furthermore, since recent data indicate that NF-kappaB protects cells from tumor necrosis factor alpha-, ionizing radiation-, and chemotherapeutic agent daunorubicin-mediated apoptosis, our results provide an explanation for chemotherapeutic resistance in ER-negative breast cancers.

Paclitaxel sensitivity of breast cancer cells with constitutively active NF-κB is enhanced by IκBα super-repressor and parthenolide

The transcription factor nuclear factor-κB (NF-κB) regulates genes important for tumor invasion, metastasis and chemoresistance. Normally, NF-κB remains sequestered in an inactive state by cytoplasmic inhibitor-of-κB (IκB) proteins. NF-κB translocates to nucleus and activates gene expression upon exposure of cells to growth factors and cytokines. We and others have shown previously that NF-κB is constitutively active in a subset of breast cancers. In this study, we show that constitutive activation of NF-κB leads to overexpression of the anti-apoptotic genes c-inhibitor of apoptosis 2 (c-IAP2) and manganese superoxide dismutase (Mn-SOD) in breast cancer cells. Furthermore, expression of the anti-apoptotic tumor necrosis factor receptor associated factor 1 (TRAF1) and defender-against cell death (DAD-1) is regulated by NF-κB in certain breast cancer cells. We also demonstrate that NF-κB-inducible genes protect cancer cells against paclitaxel as MDA-MB-231 breast cancer cells modified to overexpress IκBα required lower concentrations of paclitaxel to arrest at the G2/M phase of the cell cycle and undergo apoptosis when compared to parental cells. The effect of NF-κB on paclitaxel-sensitivity appears to be specific to cancer cells because normal fibroblasts derived from embryos lacking p65 subunit of NF-κB and wild type littermate embryos were insensitive to paclitaxel-induced G2/M cell cycle arrest. Parthenolide, an active ingredient of herbal remedies such as feverfew (tanacetum parthenium), mimicked the effects of IκBα by inhibiting NF-κB DNA binding activity and Mn-SOD expression, and increasing paclitaxel-induced apoptosis of breast cancer cells. These results suggest that active ingredients of herbs with anti-inflammatory properties may be useful in increasing the sensitivity of cancers with constitutively active NF-κB to chemotherapeutic drugs.

Negative regulation of chemokine receptor CXCR4 by tumor suppressor p53 in breast cancer cells: implications of p53 mutation or isoform expression on breast cancer cell invasion

Chemokine receptor CXCR4 and its ligand CXCL12 are suggested to be involved in migration, invasion and metastasis of breast cancer cells. Mutation of the tumor suppressor gene p53 in breast cancer is associated with metastasis and aggressive clinical phenotype. In this report, we demonstrate that wild type but not the dominant-negative mutant (V143A) or cancer-specific mutants (R175H or R280K) of p53 repress CXCR4 expression. Recently described cancer-specific p53 isoform, Δ133p53, also failed to repress CXCR4 promoter activity. Short-interfering RNA-mediated depletion of p53 increased endogenous CXCR4 expression in MCF-7 breast cancer cells that contain wild-type p53. Basal CXCR4 promoter activity in HCT116 colon carcinoma cells deleted of p53 [HCT116(p53KO)] was 10-fold higher compared to that in parental HCT116 cells with functional wild-type p53. Deletion analysis of CXCR4 promoter identified a seven-base pair p53-repressor element homologous to cyclic AMP/AP-1 response (CRE/AP-1) element. Electrophoretic mobility shift and chromatin immunoprecipitation assays revealed binding of ATF-1 and cJun to the CRE/AP-1 element. The p53 rescue drug PRIMA-1 reduced CXCR4 mRNA and cell surface expression in MDA-MB-231 cells, which express R280K mutant p53. CP-31398, another p53 rescue drug, similarly reduced cell surface levels of CXCR4. PRIMA-1-mediated decrease in CXCR4 expression correlated with reduced invasion of MDA-MB-231 cells through matrigel. These results suggest a mechanism for elevated CXCR4 expression and metastasis of breast cancers with p53 mutations or isoform expression. We propose that p53 rescue drugs either alone or in combination with chemotherapeutic drugs may be effective in reducing CXCR4-mediated metastasis.

Race and Ethnicity and Breast Cancer Outcomes in an Underinsured Population

BACKGROUND The disparity in breast cancer mortality between African American women and non-Hispanic white women has been the subject of increased scrutiny. Few studies have addressed these differences in the setting of equal access to health care. We compared the breast cancer outcomes of underinsured African American and non-Hispanic white patients who were treated at a single institution. METHODS We conducted a retrospective review of medical records for breast cancer patients who were treated at Wishard Memorial Hospital from January 1, 1997, to February 28, 2006. A total of 574 patients (259 non-Hispanic whites and 315 African Americans) were evaluated. A Cox proportional hazards regression analysis for competing risks was performed. All statistical tests were two-sided. RESULTS Sociodemographic characteristics were similar in the two groups, and both racial groups were equally unlikely to have undergone screening mammography during the 2 years before diagnosis. Most (84%) of the patients were underinsured. The median time from diagnosis to operation, receipt of adequate surgery, and use of all types of adjuvant therapy were similar in the two groups. Median follow-up was 80.3 months for non-Hispanic whites and 77.9 months for African Americans. After accounting for the effect of comorbidities, African American race was statistically significantly associated with breast cancer-specific mortality (African Americans vs non-Hispanic whites: 26.0% vs 17.5%, P = .028; hazard ratio [HR] of death = 1.64, 95% confidence interval [CI] = 1.06 to 2.55). Adjustment for age at diagnosis, clinical stage, and hormone receptor status attenuated the effect, and the effect of race on breast cancer-specific survival was no longer statistically significant (HR of death from breast cancer = 1.43, 95% CI = 0.89 to 2.30). After adjustment for sociodemographic factors, the hazard ratio for race was further attenuated (HR = 1.26; 95% CI = 0.79 to 2.00). CONCLUSIONS In this underinsured population, African American patients had poorer breast cancer-specific survival than non-Hispanic white patients. After adjustment for clinical and sociodemographic factors, the effect of race on survival was no longer statistically significant.

A cancer-associated PCNA expressed in breast cancer has implications as a potential biomarker

Two isoforms of proliferating cell nuclear antigen (PCNA) have been observed in breast cancer cells. Commercially available antibodies to PCNA recognize both isoforms and, therefore, cannot differentiate between the PCNA isoforms in malignant and nonmalignant breast epithelial cells and tissues. We have developed a unique antibody that specifically detects a PCNA isoform (caPCNA) associated with breast cancer epithelial cells grown in culture and breast-tumor tissues. Immunostaining studies using this antibody suggest that the caPCNA isoform may be useful as a marker of breast cancer and that the caPCNA-specific antibody could potentially serve as a highly effective detector of malignancy. We also report here that the caPCNA isoform functions in breast cancer-cell DNA replication and interacts with DNA polymerase δ. Our studies indicate that the caPCNA isoform may be a previously uncharacterized detector of breast cancer.

Negative Regulation of Transactivation Function but Not DNA Binding of NF-κB and AP-1 by IκBβ1 in Breast Cancer Cells

The transcription factor NF-κB regulates the expression of genes involved in cancer cell invasion, metastasis, angiogenesis, and resistance to chemotherapy. In normal cells NF-κB is maintained in the cytoplasm by protein-protein interaction with inhibitor IκBs. In contrast, in cancer cells a substantial amount of NF-κB is in the nucleus and constitutively activates target genes. To understand the mechanisms of constitutive NF-κB activation, we have analyzed the function of IκBα and IκBβ in breast cancer cells. In most cases, constitutive NF-κB DNA binding correlated with reduced levels of either IκBα or IκBβ isoforms. Overexpression of IκBα but not IκBβ1 resulted in reduced constitutive DNA binding of NF-κB in MDA-MB-231 cells. Unexpectedly, IκBβ1 overexpression moderately increased 12-O-tetradecanoylphorbol-13-acetate- and interleukin-1-inducible NF-κB DNA binding. 12-O-Tetradecanoylphorbol-13-acetate- and interleukin-1-induced transactivation by NF-κB, however, was lower in IκBβ1-overexpressing cells. Mutants of IκBβ1 lacking the C-terminal casein kinase II phosphorylation sites, which form a stable complex with DNA bound NF-κB without inhibiting its transactivation in other cell types, repressed the transactivation by NF-κB in MDA-MB-231 cells. Consistent with the results of transient transfections, the expression of urokinase plasminogen activator, an NF-κB target gene, was reduced in IκBβ1-overexpressing cells. These results suggest that depending on the cell type, IκBβ1 represses the expression of NF-κB-regulated genes by inhibiting either DNA binding or transactivation function of NF-κB.

An analysis of perioperative cholangiography in one thousand laparoscopic cholecystectomies

BACKGROUND We undertook this retrospective study to ascertain the proper role of perioperative cholangiography in the management of 1002 patients undergoing laparoscopic cholecystectomy for symptomatic cholelithiasis. METHODS Nine hundred forty-one patients were categorized as being at high or low risk for choledocholithiasis according to the presence or absence of jaundice, pancreatitis, elevated bilirubin, alkaline phosphatase, serum glutamic-oxaloacetic transaminase, or radiographic evidence of common bile duct stones (CBDSs). RESULTS Intraoperative cholangiography (IOCG) and preoperative endoscopic retrograde cholangiopancreatography (ERCP) were equivalent in the detection of CBDSs, and laparoscopic common bile duct exploration (CBDE) was successful in 12 of the 21 patients (57%) in whom it was attempted. The ducts of the other 52 patients with CBDSs were successfully cleared by preoperative or postoperative ERCP. CONCLUSIONS Laparoscopic IOCG is successful in detecting CBDS in high-risk patients and half of these ducts can be cleared laparoscopically. The incidence of CBDS in low-risk patients is 1.7%, a risk that does not warrant routine cholangiography. These data suggest ERCP should be reserved for those at-risk individuals in whom IOCG or laparoscopic duct clearance has been unsuccessful.

Efficacy of the Ahmed S2 glaucoma valve compared with the Baerveldt 250-mm2 glaucoma implant.

OBJECTIVE To compare the efficacy of the Ahmed S2 Glaucoma Valve with the Baerveldt 250-mm(2) Glaucoma Implant in the treatment of adult glaucoma. DESIGN Comparative case series. PARTICIPANTS Fifty-nine eyes of 59 patients who received the Ahmed S2 Glaucoma Valve and 133 eyes of 133 patients who received the Baerveldt 250-mm(2) Glaucoma Implant by the Indiana University Glaucoma Service from 1996 to 2003. METHODS Eyes that had previous drainage implant procedures were excluded from both groups. If both eyes of a single patient received an implant, the second eye to undergo implantation was excluded from the study. MAIN OUTCOME MEASURES Kaplan-Meier survival with success defined as intraocular pressure (IOP) > 5 mmHg and < 22 mmHg and at least 20% reduction from preoperative IOP (with or without antiglaucoma medications) and without loss of light perception. Secondary outcome measures included intraocular pressure, visual acuity, number of glaucoma medications, and surgical complications. RESULTS The 2 groups were similar with regards to age, gender, race, neovascular glaucoma diagnosis, number of prior ocular surgeries, preoperative IOP, and number of preoperative glaucoma medications. Mean durations of follow-up were 20.0 months for Ahmed eyes and 22.9 months for Baerveldt eyes. Cumulative successes in the Ahmed group were 0.73 at 1 year and 0.62 at 2 years, whereas cumulative successes in the Baerveldt group were 0.92 at 1 year and 0.85 at 2 years (Kaplan-Meier survival functions: P = 0.03, log rank test). Male gender, African descent, neovascular glaucoma, and Ahmed implantation were found to be significant predictors of failure. At last follow-up visit, eyes in the Ahmed group had a significantly higher mean IOP (19.8+/-9.5 vs. 15.8+/-7.9 mmHg, P = 0.003, t test) and more antiglaucoma medications (1.4+/-1.2 vs. 0.9+/-1.1 medications, P = 0.008, Mann-Whitney test) than eyes in the Baerveldt group. Two methods for avoiding hypotony after Baerveldt 250-mm(2) implantation had similar outcomes. CONCLUSIONS Our study suggests that the Ahmed S2 Glaucoma Valve may be less effective at lowering IOP than the Baerveldt 250-mm(2) Glaucoma Implant.

Classification and treatment of local septic complications in acute pancreatitis

BACKGROUND An international symposium on acute pancreatitis recently developed a clinical classification system for severe acute pancreatitis that classifies all local septic complications into three groups: infected necrosis (IN), sterile necrosis (SN), and pancreatic abscess (PA). Despite the appeal of having three distinct, well-defined labels for this complex process, the clinical utility of this schema has yet to be determined. The purpose of this study was to investigate the prognostic and therapeutic utility of applying this clinical classification system to a large group of surgical patients with local septic complication from acute pancreatitis. PATIENTS AND METHODS We reviewed the cases of 62 patients with complicated pancreatitis, classifying them into IN (n = 20), SN (n = 14), or PA (n = 28) groups. Ransons score, APACHE II score, and computed tomography grading were calculated within the first 48 hours of admission. Information on patient demographics, etiology of pancreatitis, operative procedures, timing of intervention, bacteriology, blood loss, intensive care unit days, ventilator days, and morbidity and mortality were also accrued and analyzed. RESULTS Despite similar demographics and etiology of pancreatitis, patients with necrosis, both IN and SN, were more critically ill than were patients with PA (APACHE II score > 15, 21% versus 0%, respectively), required earlier operative intervention (mean 14 days versus 29 days, P = 0.02), required necrosectomy with drainage (65% versus 4%, P < 0.001) rather than simple drainage (3% versus 86%, P < 0.001), more reoperations (2.3 versus 1.1, P < 0.05), and had a significantly higher mortality rate (35% versus 4%, P < 0.05). In addition, patients with IN required significantly more hospital days, ventilator days, and blood transfusions than either patients with SN or PA (P < 0.05). CONCLUSIONS We conclude that this classification system allows for the stratification of patients into three distinct groups--infected necrosis, sterile necrosis, and pancreatic abscess--and has both therapeutic and prognostic usefulness.

Hepatic Resection of Metastatic Testicular Carcinoma: A Further Update

Background: The goal of this study was to update the multidisciplinary review of patients who underwent resection of hepatic metastases of nonseminomatous germ cell esticular carcinoma at the Indiana University Medical Center.Methods: The study involved retrospective chart review for 57 patients who underwent hepatic resection for treatment of metastatic nonseminomatous testicular carcinoma between June 1974 and May 1996. Patients were categorized ccording to the worst postchemotherapy pathologic diagnosis.Results: Chemotherapy has been highly effective in curing testicular carcinoma. However, even with platinum-based hemotherapy, one-third of patients either do not achieve complete cures or experience relapses. Since 1965, only 57 of the 2219 patients who underwent postchemotherapy retroperitoneal lymph node dissections for treatment of testicular carcinoma at our center underwent hepatic resection for treatment of metastatic disease. Because teratomas have the propensity to degenerate into sarcomas and can cause symptoms resulting from compression, these lesions must be resected to achieve cures. In addition, the only chance for survival for patients with active disease but normal serum marker levels is with complete resection of the tumor burden.Conclusions: We conclude that hepatic resection for treatment of metastatic testicular carcinoma is safe and efficacious for all patients except those with elevated marker levels after preoperative chemotherapy.