Racial differences in estrogen receptor staining levels and implications for treatment and survival among estrogen receptor positive, HER2-negative invasive breast cancers.

BACKGROUND: African American women (AAW) die more frequently from estrogen receptor (ER) positive breast cancer than European American women (EAW). We investigated the relationship between race, percent ER staining, treatment, and clinical outcomes. METHODS: Percent ER staining (weakly ER+: 1-10%, moderately ER+: 11-50%, strongly ER+: > 50%) was abstracted from pathology reports for 1573 women with ER+/HER2- invasive breast cancer treated at a single cancer center in Detroit, MI from 2010 to 2017. Clinical outcomes and tumor characteristics were obtained from the Metropolitan Detroit Cancer Surveillance System. Associations of ER levels with demographic and clinical characteristics were evaluated using logistic regression. Overall and breast cancer-specific (BCS) survival were evaluated using Cox proportional hazards models. RESULTS: AAW were more likely to have tumors with lower ER staining levels than EAW (weakly ER+: Odds ratio (OR) 2.19, p = 0.019; moderately ER+: OR 2.80, p = 0.005). Women with weakly compared to strongly ER+ tumors were less likely to receive endocrine therapy (ET) regardless of race (OR 0.79, p < 0.001). Mortality was predicted by both AA race (Overall hazard ratio (HR) = 1.72, p < 0.001; BCS HR 1.45, p = 0.08) and low (1-50%) ER (Overall HR 1.57, p = 0.083; BCS HR 2.11, p = 0.017) adjusting for clinic-pathologic characteristics. ET was associated with improved BCS survival in all women (1-50%: HR 0.11, p < 0.001; > 50%: HR 0.24, p < 0.001). CONCLUSION: The biology of ER+/HER2- tumors varies by race, although this does not appear to account for racial differences in survival. Although ET substantially reduces mortality among women with weakly ER+ tumors, these women are less likely to be treated with ET and have poorer outcomes.

IgA Fc-folate conjugate activates and recruits neutrophils to directly target triple-negative breast cancer cells.

PURPOSE: According to the American Cancer Society, 1 in 8 women in the U.S. will develop breast cancer, with triple-negative breast cancer (TNBC) comprising 15-20% of all breast cancer cases. TNBC is an aggressive subtype due to its high metastatic potential and lack of targeted therapy. Recently, folate receptor alpha (FRA) is found to be expressed on 80% of TNBC with high expression correlating with poor prognosis. In this study, we examined whether binding IgA Fc-folate molecules to FRA receptors on TNBC cells can elicit and induce neutrophils (PMNs), by binding their FcαR1 receptors, to destroy TNBC cells. METHODS: FRA was analyzed on TNBC cells and binding assays were performed using 3H-folate. Fc-folate was synthesized by linking Fc fragments of IgA via amine groups to folate. Binding specificity and antibody-dependent cellular cytotoxicity (ADCC) potential of Fc-folate to FcαR1 were confirmed by measuring PMN adhesion and myeloperoxidase (MPO) release in a cell-based ELISA. Fc-folate binding to FRA-expressing TNBC cells inducing PMNs to destroy these cells was determined using 51Cr-release and calcein-labeling assays. RESULTS: Our results demonstrate expression of FRA on TNBC cells at levels consistent with folate binding. Fc-folate binds with high affinity to FRA compared to whole IgA-folate and induces MPO release from PMN when bound to FcαR1. Fc-folate inhibited binding of 3H-folate to TNBC cells and induced significant cell lysis of TNBC cells when incubated in the presence of PMNs. CONCLUSION: These findings support the hypothesis that an IgA Fc-folate conjugate can destroy TNBC cells by eliciting PMN-mediated ADCC.

Variation in the use of advanced imaging at the time of breast cancer diagnosis in a statewide registry.

BACKGROUND: Although national guidelines do not recommend extent of disease imaging for patients with newly diagnosed early stage breast cancer given that the harm outweighs the benefits, high rates of testing have been documented. The 2012 Choosing Wisely guidelines specifically addressed this issue. We examined the change over time in imaging use across a statewide collaborative, as well as the reasons for performing imaging and the impact on cost of care. METHODS: Clinicopathologic data and use of advanced imaging tests (positron emission tomography, computed tomography, and bone scan) were abstracted from the medical records of patients treated at 25 participating sites in the Michigan Breast Oncology Quality Initiative (MiBOQI). For patients diagnosed in 2014 and 2015, reasons for testing were abstracted from the medical record. RESULTS: Of the 34,078 patients diagnosed with stage 0-II breast cancer between 2008 and 2015 in MiBOQI, 6853 (20.1%) underwent testing with at least 1 imaging modality in the 90 days after diagnosis. There was considerable variability in rates of testing across the 25 sites for all stages of disease. Between 2008 and 2015, testing decreased over time for patients with stage 0-IIA disease (all P < .001) and remained stable for stage IIB disease (P = .10). This decrease in testing over time resulted in a cost savings, especially for patients with stage I disease. CONCLUSION: Use of advanced imaging at the time of diagnosis decreased over time in a large statewide collaborative. Additional interventions are warranted to further reduce rates of unnecessary imaging to improve quality of care for patients with breast cancer. Cancer 2017;123:2975-83. © 2017 American Cancer Society.

Associations between use of the 21-gene recurrence score assay and chemotherapy regimen selection in a statewide registry.

BACKGROUND: The 21-gene recurrence score (RS) assay predicts response to adjuvant chemotherapy in patients with early-stage, hormone receptor-positive, human epidermal growth factor receptor 2 (HER2)-negative invasive breast cancer, but to the authors' knowledge, the role of the assay in guiding the selection of chemotherapy regimen has not been established. The current study was conducted to examine patterns of use of the RS assay for selecting chemotherapy regimens across a statewide registry from 2006 through 2013. METHODS: Demographic, pathologic, and treatment data were abstracted from medical records for 16,666 women with breast cancer who were treated at 25 hospital systems across Michigan that were participating in the Michigan Breast Oncology Quality Initiative. Treatment patterns were examined based on the RS assay test result. RESULTS: Approximately 25% of patients with lymph node-negative disease who underwent testing with the RS assay and who were treated with chemotherapy received an anthracycline-based regimen, compared with 49% of patients with lymph node-negative disease who were treated with chemotherapy and who had not undergone testing with the RS assay. Of those patients with lymph node-positive disease who underwent testing with the RS assay and who received chemotherapy, 31% received an anthracycline-based regimen. In comparison, 71% of patients with lymph node-positive, chemotherapy-treated disease who did not undergo testing received an anthracycline. From 2006 through 2013, there was a statistically significant decrease in the use of anthracycline-containing regimens in both patients with lymph node-negative and lymph node-positive disease. CONCLUSIONS: Use of anthracycline-containing chemotherapy regimens in eligible patients appears to vary with use of the RS assay, despite the lack of evidence supporting use of the assay to guide regimen selection. Results of ongoing prospective trials should help to define the role of the RS assay in this setting. Cancer 2017;123:948-56. © 2016 American Cancer Society.

Riluzole synergizes with paclitaxel to inhibit cell growth and induce apoptosis in triple-negative breast cancer.

PURPOSE: One in eight women will develop breast cancer, 15-20% of whom will have triple-negative breast cancer (TNBC), an aggressive breast cancer with no current targeted therapy. We have demonstrated that riluzole, an FDA-approved drug for treating amyotrophic lateral sclerosis, inhibits growth of TNBC. In this study, we explore potential synergism between riluzole and paclitaxel, a chemotherapeutic agent commonly used to treat TNBC, in regulating TNBC proliferation, cell cycle arrest, and apoptosis. METHODS: TNBC cells were treated with paclitaxel and/or riluzole and synergistic effects on cell proliferation were quantified via MTT assay and CompuSyn analysis. Apoptosis was observed morphologically and by measuring cleaved PARP/caspase three products. Microarray analysis was performed using MDA-MB-231 cells to examine cell cycle genes regulated by riluzole and any enhanced effects on paclitaxel-mediated cell cycle arrest, determined by FACS analysis. These results were confirmed in vivo using a MDA-MB-231 xenograft model. RESULTS: Strong enhanced or synergistic effects of riluzole on paclitaxel regulation of cell cycle progression and apoptosis was demonstrated in all TNBC cells tested as well as in the xenograft model. The MDA-MB-231, SUM149, and SUM229 cells, which are resistant to paclitaxel treatment, demonstrated the strongest synergistic or enhanced effect. Key protein kinases were shown to be upregulated in this study by riluzole as well as downstream cell cycle genes regulated by these kinases. CONCLUSIONS: All TNBC cells tested responded synergistically to riluzole and paclitaxel strongly suggesting the usefulness of this combinatorial treatment strategy in TNBC, especially for patients whose tumors are relatively resistant to paclitaxel.

Riluzole mediates anti-tumor properties in breast cancer cells independent of metabotropic glutamate receptor-1.

Riluzole, the only drug approved by the FDA for treating amyotrophic lateral sclerosis, inhibits melanoma proliferation through its inhibitory effect on glutamatergic signaling. We demonstrated that riluzole also inhibits the growth of triple-negative breast cancer (TNBC) and described a role for metabotropic glutamate receptor-1 (GRM1) in regulating TNBC cell growth and progression. However, the role of GRM1 in mediating riluzole's effects in breast cancer has not been fully elucidated. In this study, we seek to determine how much of riluzole's action in breast cancer is mediated through GRM1. We investigated anti-tumor properties of riluzole in TNBC and ER+ cells using cell growth, invasion, and soft-agar assays and compared riluzole activity with GRM1 levels. Using Lentiviral vectors expressing GRM1 or shGRM1, these studies were repeated in cells expressing high or low GRM1 levels where the gene was either silenced or overexpressed. Riluzole inhibited proliferation, invasion, and colony formation in both TNBC and ER+ cells. There was a trend between GRM1 expression in TNBC cells and their response to riluzole in both cell proliferation and invasion assays. However, silencing and overexpression studies had no effect on cell sensitivity to riluzole. Our results clearly suggest a GRM1-independent mechanism through which riluzole mediates its effects on breast cancer cells. Understanding the mechanism by which riluzole mediates breast cancer progression will be useful in identifying new therapeutic targets for treating TNBC and in facilitating stratification of patients in clinical trials using riluzole in conjunction with conventional therapy.

Infertility: A common target of antivaccine misinformation campaigns.

Misinformation, disinformation, and conspiracy theories about vaccines are key drivers of vaccine hesitancy. A repeated false claim about COVID-19 vaccines is that the vaccines cause female infertility. Dating back decades, various conspiracy theories have linked vaccination programs with infertility and thus harmed vaccination programs in Africa, Asia, and Central America, particularly against polio and tetanus. In the United States, Europe, and Australia, human papilloma virus (HPV) vaccines have been falsely blamed for infertility and primary ovarian insufficiency (POI). After distribution of COVID-19 vaccines began in December 2020, almost immediately there arose conspiracy theories claiming that these vaccines cause menstrual irregularities, miscarriages, and infertility, promoted by noted antivaccine activists Robert F. Kennedy, Jr. and Andrew Wakefield among others. Here we will explore the history of this antivaccine narrative, how it has been promulgated in the past and repurposed to COVID-19 vaccines, and strategies to counter it.

Short-term changes in ultrasound tomography measures of breast density and treatment-associated endocrine symptoms after tamoxifen therapy.

Although breast density decline with tamoxifen therapy is associated with greater therapeutic benefit, limited data suggest that endocrine symptoms may also be associated with improved breast cancer outcomes. However, it is unknown whether endocrine symptoms are associated with reductions in breast density after tamoxifen initiation. We evaluated treatment-associated endocrine symptoms and breast density change among 74 women prescribed tamoxifen in a 12-month longitudinal study. Treatment-associated endocrine symptoms and sound speed measures of breast density, assessed via novel whole breast ultrasound tomography (m/s), were ascertained before tamoxifen (T0) and at 1-3 (T1), 4-6 (T2), and 12 months (T3) after initiation. CYP2D6 status was genotyped, and tamoxifen metabolites were measured at T3. Using multivariable linear regression, we estimated mean change in breast density by treatment-associated endocrine symptoms adjusting for age, race, menopausal status, body mass index, and baseline density. Significant breast density declines were observed in women with treatment-associated endocrine symptoms (mean change (95% confidence interval) at T1:-0.26 m/s (-2.17,1.65); T2:-2.12 m/s (-4.02,-0.22); T3:-3.73 m/s (-5.82,-1.63); p-trend = 0.004), but not among women without symptoms (p-trend = 0.18) (p-interaction = 0.02). Similar declines were observed with increasing symptom frequency (p-trends for no symptoms = 0.91; low/moderate symptoms = 0.03; high symptoms = 0.004). Density declines remained among women with detectable tamoxifen metabolites or intermediate/efficient CYP2D6 metabolizer status. Emergent/worsening endocrine symptoms are associated with significant, early declines in breast density after tamoxifen initiation. Further studies are needed to assess whether these observations predict clinical outcomes. If confirmed, endocrine symptoms may be a proxy for tamoxifen response and useful for patients and providers to encourage adherence.

Metabotropic glutamate receptor-1: a potential therapeutic target for the treatment of breast cancer.

Metabotropic glutamate receptors are G-protein-coupled receptors normally expressed in the central nervous system where they mediate neuronal excitability, synaptic plasticity, and feedback inhibition of neurotransmitter release. However, recent data suggest that these receptors are also expressed and functional in some cancers, most notably melanoma. We detected the expression of metabotropic glutamate receptor-1 (gene: GRM1; protein: mGluR1) in triple negative breast cancer cells and evaluated its role in regulating the pro-proliferative phenotype of these cells. mGluR1 inhibitors (Riluzole or BAY36-7620) inhibited the proliferation of triple negative breast cancer cells in a time- and dose-dependent manner and this inhibition correlated with increased apoptosis as demonstrated by increase in PARP cleavage products and Annexin V staining. mGluR1 knockdown using Lentiviral constructs expressing shRNA targeting GRM1 also inhibited proliferation compared to non-silencing controls. In addition, treatment of mice bearing MDA-MB-231 xenografts with Riluzole or BAY36-7620, by intraperitoneal injection, resulted in a significant reduction in tumor volume of up to 80%. Moreover, Riluzole was effective against triple negative breast cancer xenografts in mice at doses equivalent to those currently being used in humans for the treatment of amyotrophic lateral sclerosis. Our observations implicate mGluR1 and glutamate signaling as a promising new molecular target for the treatment of breast cancer. Even more promising, Riluzole, because it is an oral drug that can be administered with low toxicity, represents a promising approach in the treatment of triple negative breast cancer.

Rapid Reductions in Breast Density following Tamoxifen Therapy as Evaluated by Whole-Breast Ultrasound Tomography.

PURPOSE: Women whose mammographic breast density declines within 12-18 months of initiating tamoxifen for chemoprevention or adjuvant treatment show improved therapeutic responses compared with those whose density is unchanged. We tested whether measuring changes in sound speed (a surrogate of breast density) using ultrasound tomography (UST) could enable rapid identification of favorable responses to tamoxifen. METHODS: We evaluated serial density measures at baseline and at 1 to 3, 4 to 6, and 12+ months among 74 women (aged 30-70 years) following initiation of tamoxifen for clinical indications, including an elevated risk of breast cancer (20%) and diagnoses of in situ (39%) or invasive (40%) breast carcinoma, enrolled at Karmanos Cancer Institute and Henry Ford Health System (Detroit, MI, USA). For comparison, we evaluated an untreated group with screen negative mammography and frequency-matched on age, race, and menopausal status (n = 150), at baseline and 12 months. Paired t-tests were used to assess differences in UST sound speed over time and between tamoxifen-treated and untreated patients. RESULTS: Sound speed declined steadily over the 12 month period among patients receiving tamoxifen (mean (SD): -3.0 (8.2) m/s; p = 0.001), whereas density remained unchanged in the untreated group (mean (SD): 0.4 (7.1) m/s; p = 0.75 (relative change between groups: p = 0.0009)). In the tamoxifen group, we observed significant sound speed reductions as early as 4-6 months after tamoxifen initiation (mean (SD): -2.1 (6.8) m/s; p = 0.008). Sound speed reductions were greatest among premenopausal patients (P-interaction = 0.0002) and those in the middle and upper tertiles of baseline sound speed (P-interaction = 0.002). CONCLUSIONS: UST can image rapid declines in sound speed following initiation of tamoxifen. Given that sound speed and mammographic density are correlated, we propose that UST breast imaging may capture early responses to tamoxifen, which in turn may have utility in predicting therapeutic efficacy.

The ethics of innovation for Alzheimer's disease: the risk of overstating evidence for metabolic enhancement protocols.

Medical practice is ideally based on robust, relevant research. However, the lack of disease-modifying treatments for Alzheimer's disease has motivated "innovative practice" to improve patients' well-being despite insufficient evidence for the regular use of such interventions in health systems treating millions of patients. Innovative or new non-validated practice poses at least three distinct ethical questions: first, about the responsible application of new non-validated practice to individual patients (clinical ethics); second, about the way in which data from new non-validated practice are communicated via the scientific and lay press (scientific communication ethics); and third, about the prospect of making new non-validated interventions widely available before more definitive testing (public health ethics). We argue that the authors of metabolic enhancement protocols for Alzheimer's disease have overstated the evidence in favor of these interventions within the scientific and lay press, failing to communicate weaknesses in their data and uncertainty about their conclusions. Such unmeasured language may create false hope, cause financial harm, undermine informed consent, and frustrate the production of generalizable knowledge necessary to face the societal problems posed by this devastating disease. We therefore offer more stringent guidelines for responsible innovation in the treatment of Alzheimer's disease.

Using Whole Breast Ultrasound Tomography to Improve Breast Cancer Risk Assessment: A Novel Risk Factor Based on the Quantitative Tissue Property of Sound Speed.

Mammographic percent density (MPD) is an independent risk factor for developing breast cancer, but its inclusion in clinical risk models provides only modest improvements in individualized risk prediction, and MPD is not typically assessed in younger women because of ionizing radiation concerns. Previous studies have shown that tissue sound speed, derived from whole breast ultrasound tomography (UST), a non-ionizing modality, is a potential surrogate marker of breast density, but prior to this study, sound speed has not been directly linked to breast cancer risk. To that end, we explored the relation of sound speed and MPD with breast cancer risk in a case-control study, including 61 cases with recent breast cancer diagnoses and a comparison group of 165 women, frequency matched to cases on age, race, and menopausal status, and with a recent negative mammogram and no personal history of breast cancer. Multivariable odds ratios (ORs) and 95% confidence intervals (CIs) were estimated for the relation of quartiles of MPD and sound speed with breast cancer risk adjusted for matching factors. Elevated MPD was associated with increased breast cancer risk, although the trend did not reach statistical significance (OR per quartile = 1.27, 95% CI: 0.95, 1.70; ptrend = 0.10). In contrast, elevated sound speed was significantly associated with breast cancer risk in a dose-response fashion (OR per quartile = 1.83, 95% CI: 1.32, 2.54; ptrend = 0.0003). The OR trend for sound speed was statistically significantly different from that observed for MPD (p = 0.005). These findings suggest that whole breast sound speed may be more strongly associated with breast cancer risk than MPD and offer future opportunities for refining the magnitude and precision of risk associations in larger, population-based studies, including women younger than usual screening ages.

The Epidemiology of Cancer Among Homeless Adults in Metropolitan Detroit.

BACKGROUND: Homeless individuals suffer and die disproportionately from chronic diseases and disorders. We describe the epidemiology of cancer among homeless persons in metropolitan Detroit. METHODS: A retrospective cohort study was performed using 1973-2014 data from the Metropolitan Detroit Cancer Surveillance System, a population-based cancer registry and member of the National Institutes of Health-National Cancer Institute's Surveillance, Epidemiology, and End Results program. Homeless adults were identified through address at diagnosis listed as a homeless shelter, hospital, or supplemental field indicating homelessness. Age-adjusted, sex-specific proportional incidence ratios (PIR) compared cancer incidence proportions by primary tumor site of homeless patients to the nonhomeless referent population. Kaplan-Meier curves depicted unadjusted survival differences in a propensity score matched sample. Differences in 10-year survival were assessed using the score test with a sandwich estimator accounting for matched cluster effects. Statistical tests were two-sided. RESULTS: A total of 388 individuals experienced homelessness at first primary invasive cancer diagnosis. Statistically significantly higher proportions of respiratory system (PIR = 1.51; 95% confidence interval = 1.28 to 1.79) and female genital system (PIR = 1.83; 95% confidence interval = 1.31 to 2.55) cancers were observed among homeless men and women, respectively. Homeless persons had poorer overall and cancer-reported survival compared with a propensity score matched referent population (median: overall survival, 20.0 vs 38.0 months, respectively, P < .001; cancer-reported survival, 38.0 vs 64.0 months, respectively, P < .001). CONCLUSION: Disparities in disease burden exist between adults who are experiencing homelessness compared with the nonhomeless population at cancer diagnosis. These findings provide clinically relevant information to understand the cancer burden in this medically underserved population and suggest an urgent need to develop cancer prevention and intervention programs to reduce disparities and improve the health of homeless persons.

Metabotropic glutamate receptor-1 regulates inflammation in triple negative breast cancer.

Breast cancer remains a major cause of death among women. 15% of these cancers are triple negative breast cancer (TNBC), an aggressive subtype of breast cancer for which no current effective targeted therapy exists. We have previously demonstrated a role for mGluR1 in mediating tumor cell growth, endothelial cell proliferation, and tumor-induced angiogenesis in TNBC. In this study, we explore a role for mGluR1 in regulating inflammation in TNBC. GRM1 expression was silenced in MDA-MB-231 cells to study changes in expression of inflammatory genes regulated by mGluR1. Results were confirmed by ELISA using GRM1-silenced and overexpressed cells and mGluR1 inhibitors. A functional role for these differentially expressed genes was determined in vitro and in vivo. 131 genes were differentially expressed in GRM1-silenced MDA-MB-231 cells, with some of these falling into four major canonical pathways associated with acute inflammation, specifically leukocyte migration/chemotaxis. Upregulation of three of these genes (CXCL1, IL6, IL8) and their corresponding protein was confirmed by qPCR analysis and ELISA in GRM1-manipulated TNBC cells. Upregulation of these cytokines enhanced endothelial adhesion and transmigration of neutrophils in co-culture assays and in 4T1 mouse tumors. Our results suggest mGluR1 may serve as a novel endogenous regulator of inflammation in TNBC.

Racial Differences in 21-Gene Recurrence Scores Among Patients With Hormone Receptor-Positive, Node-Negative Breast Cancer.

Purpose The 21-gene recurrence score (RS) breast cancer assay is clinically used to quantify risk of 10-year distant recurrence by category (low, < 18; intermediate, 18 to 30; high, ≥ 31) for treatment management among women diagnosed with hormone receptor-positive, human epidermal growth factor receptor 2-negative, lymph node-negative breast cancer. Although non-Hispanic black (NHB) women have worse prognosis compared with non-Hispanic white (NHW) women, the equivalency of 21-gene RS across racial groups remains unknown. Patients and Methods Using the Metropolitan Detroit Cancer Surveillance System, we identified women who were diagnosed with hormone receptor-positive, human epidermal growth factor receptor 2-negative, lymph node-negative invasive breast cancer between 2010 and 2014. Multinomial logistic regression was used to quantify racial differences in 21-gene RS category. Results We identified 2,216 women (1,824 NHW and 392 NHB) with invasive breast cancer who met clinical guidelines for and underwent 21-gene RS testing. The mean RS was significantly higher in NHBs compared with NHWs (19.3 v 17.0, respectively; P = .0003), where NHBs were more likely to present with high-risk tumors compared with NHWs (14.8% v 8.3%, respectively; P = .0004). These differences were limited to patients younger than 65 years at diagnosis, among whom NHBs had significantly higher RS compared with NHWs (20 to 49 years: 23.6 v 17.3, respectively; P < .001 and 50 to 64 years: 19.6 v 17.4, respectively; P = .023). NHBs remained more likely to have high-risk tumors compared with NHWs after adjusting for age, clinical stage, tumor grade, and histology (odds ratio [OR], 1.75; 95% CI, 1.18 to 2.59). Conclusion NHBs who met clinical criteria for 21-gene RS testing had tumors with higher estimated risks of distant recurrence compared with NHWs. Further study is needed to elucidate whether differences in recurrence are observed for these women, which would have clinical implications for 21-gene RS calibration and treatment recommendations in NHB patients.

The homeobox gene Gax inhibits angiogenesis through inhibition of nuclear factor-kappaB-dependent endothelial cell gene expression.

The growth and metastasis of tumors are heavily dependent on angiogenesis, but much of the transcriptional regulation of vascular endothelial cell gene expression responsible for angiogenesis remains to be elucidated. The homeobox gene Gax is expressed in vascular endothelial cells and inhibits proliferation and tube formation in vitro. We hypothesized that Gax is a negative transcriptional regulator of the endothelial cell angiogenic phenotype and studied its regulation and activity in vascular endothelial cells. Several proangiogenic factors caused a rapid down-regulation of Gax mRNA in human vascular endothelial cells, as did conditioned media from breast cancer cell lines. In addition, Gax expression using a replication-deficient adenoviral vector inhibited human umbilical vein endothelial cell migration toward proangiogenic factors in vitro and inhibited angiogenesis in vivo in Matrigel plugs. To identify putative downstream targets of Gax, we examined changes in global gene expression in endothelial cells due to Gax activity. Gax expression resulted in changes in global gene expression consistent with a quiescent, nonangiogenic phenotype, with increased expression of cyclin kinase inhibitors and decreased expression of genes implicated in endothelial cell activation and angiogenesis. Further analysis revealed that Gax down-regulated numerous nuclear factor-kappaB (NF-kappaB) target genes and decreased the binding of NF-kappaB to its target sequence in electrophoretic mobility shift assays. To our knowledge, Gax is the first homeobox gene described that inhibits NF-kappaB activity in vascular endothelial cells. Because NF-kappaB has been implicated in endothelial cell activation and angiogenesis, the down-regulation of NF-kappaB-dependent genes by Gax suggests one potential mechanism by which Gax inhibits the angiogenic phenotype.

Prolonged treatment with angiostatin reduces metastatic burden during radiation therapy.

Ionizing radiation (IR) and concomitant angiostatin (AS) produce greater than additive local antitumor effects. We examined whether prolonged AS treatment added to IR reduces proliferation of lung metastases from LLC primary tumors. Flank tumors were treated with 40 Gy with or without AS (25 mg/kg/day). IR plus a 14-day course of AS improved local tumor control and blocked the increase in lung weights observed in the group receiving IR plus a 2-day course of AS group. Animals treated with prolonged AS exhibited no increase in lung weight and no macrometastases. These findings suggest that long-term treatment with antiangiogenic compounds may be effective in preventing metastases from IR-treated tumors as well as increasing the local antitumor effects of radiotherapy.