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Signal transducer and activator of transcription 5 (STAT5) promotes cell survival and instigates breast tumor formation, and in the normal breast it also drives alveolar differentiation and lactogenesis. However, whether STAT5 drives a differentiated phenotype in breast tumorigenesis and therefore impacts cancer spread and metastasis is unclear. We found in two genetically engineered mouse models of breast cancer that constitutively activated Stat5a(Stat5aca) caused precancerous mammary epithelial cells to become lactogenic and evolve into tumors with diminished potential to metastasize. We also showed that STAT5acareduced the migratory and invasive ability of human breast cancer cell lines in vitro. Furthermore, we demonstrated that STAT5acaoverexpression in human breast cancer cells lowered their metastatic burden in xenografted mice. Moreover, RPPA, Western blotting, and studies of ChIPseq data identified several EMT drivers regulated by STAT5. In addition, bioinformatic studies detected a correlation between STAT5 activity and better prognosis of breast cancer patients. Together, we conclude that STAT5 activation during mammary tumorigenesis specifies a tumor phenotype of lactogenic differentiation, suppresses EMT, and diminishes potential for subsequent metastasis.

Estrogen receptor alpha gene (ESR1) mutations occur frequently in ER-positive metastatic breast cancer, and confer clinical resistance to aromatase inhibitors. Expression of the ESR1Y537S mutation induced an epithelial–mesenchymal transition (EMT) with cells exhibiting enhanced migration and invasion potential in vitro. When small subpopulations of Y537S ESR1mutant cells were injected along with WT parental cells, tumor growth was enhanced with mutant cells becoming the predominant population in distant metastases. Y537S mutant primary xenograft tumors were resistant to the antiestrogen tamoxifen (Tam) as well as to estradiol (E2) withdrawal. Y537S ESR1mutant primary tumors metastasized efficiently in the absence of E2; however, Tam treatment significantly inhibited metastasis to distant sites. We identified a nine-gene expression signature, which predicted clinical outcomes of ER-positive breast cancer patients, as well as breast cancer metastasis to the lung. Androgen receptor (AR) protein levels were increased in mutant models, and the AR agonist dihydrotestosterone significantly inhibited estrogen-regulated gene expression, EMT, and distant metastasis in vivo, suggesting that AR may play a role in distant metastatic progression of ESR1mutant tumors.

IMPORTANCE: Birth defects affect approximately 1 in 33 children. Some birth defects are known to be strongly associated with childhood cancer (eg, trisomy 21 and acute leukemia). However, comprehensive evaluations of childhood cancer risk in those with birth defects have been limited in previous studies by insufficient sample sizes. OBJECTIVES: To identify specific birth defect–childhood cancer (BD-CC) associations and characterize cancer risk in children by increasing number of nonchromosomal birth defects. DESIGN, SETTING, AND PARTICIPANTS: This multistate, population-based registry linkage study pooled statewide data on births, birth defects, and cancer from Texas, Arkansas, Michigan, and North Carolina on 10 181 074 children born from January 1, 1992, to December 31, 2013. Children were followed up to 18 years of age for a diagnosis of cancer. Data were retrieved between September 26, 2016, and September 21, 2017, and data analysis was performed from September 2, 2017, to March 21, 2019. EXPOSURES: Birth defects diagnoses (chromosomal anomalies and nonchromosomal birth defects) recorded by statewide, population-based birth defects registries. MAIN OUTCOMES AND MEASURES: Cancer diagnosis before age 18 years, as recorded in state cancer registries. Cox regression models were used to generate hazard ratios (HRs) and 95% CIs to evaluate BD-CC associations and the association between number of nonchromosomal defects and cancer risk. RESULTS: Compared with children without any birth defects, children with chromosomal anomalies were 11.6 (95% CI, 10.4-12.9) times more likely to be diagnosed with cancer, whereas children with nonchromosomal birth defects were 2.5 (95% CI, 2.4-2.6) times more likely to be diagnosed with cancer before 18 years of age. An increasing number of nonchromosomal birth defects was associated with a corresponding increase in the risk of cancer. Children with 4 or more major birth defects were 5.9 (95% CI, 5.3-6.4) times more likely to be diagnosed with cancer compared with those without a birth defect. In the analysis of 72 specific BD-CC patterns, 40 HRs were statistically significant (adjusted P < .05) after accounting for multiple comparisons. Cancers most frequently associated with nonchromosomal defects were hepatoblastoma and neuroblastoma. CONCLUSIONS AND RELEVANCE: Several significant and novel associations were observed between specific birth defects and cancers. Among children with nonchromosomal birth defects, the number of major birth defects diagnosed was significantly and directly associated with cancer risk. These findings could inform clinical treatment for children with birth defects and may elucidate mechanisms that lead to these complex outcomes.

Drug development for Alzheimer’s disease has endeavored to lower amyloid β (Aβ) by either blocking production or promoting clearance. The benefit of combining these approaches has been examined in mouse models and shown to improve pathological measures of disease over single treatment; however, the impact on cellular and cognitive functions affected by Aβ has not been tested. We used a controllable APP transgenic mouse model to test whether combining genetic suppression of Aβ production with passive anti-Aβ immunization improved functional outcomes over either treatment alone. Compared with behavior before treatment, arresting further Aβ production (but not passive immunization) was sufficient to stop further decline in spatial learning, working memory, and associative memory, whereas combination treatment reversed each of these impairments. Cognitive improvement coincided with resolution of neuritic dystrophy, restoration of synaptic density surrounding deposits, and reduction of hyperactive mammalian target of rapamycin signaling. Computational modeling corroborated by in vivo microdialysis pointed to the reduction of soluble/exchangeable Aβ as the primary driver of cognitive recovery.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer diagnosed in more than 200,000 women each year and is recalcitrant to targeted therapies. Although TNBCs harbor multiple hyperactive receptor tyrosine kinases (RTKs), RTK inhibitors have been largely ineffective in TNBC patients thus far. We developed a broadly effective therapeutic strategy for TNBC that is based on combined inhibition of receptors that share the negative regulator PTPN12. Previously, we and others identified the tyrosine phosphatase PTPN12 as a tumor suppressor that is frequently inactivated in TNBC. PTPN12 restrains several RTKs, suggesting that PTPN12 deficiency leads to aberrant activation of multiple RTKs and a co-dependency on these receptors. This in turn leads to the therapeutic hypothesis that PTPN12-deficient TNBCs may be responsive to combined RTK inhibition. However, the repertoire of RTKs that are restrained by PTPN12 in human cells has not been systematically explored. By methodically identifying the suite of RTK substrates (MET, PDGFRβ, EGFR, and others) inhibited by PTPN12, we rationalized a combination RTK-inhibitor therapy that induced potent tumor regression across heterogeneous models of TNBC. Orthogonal approaches revealed that PTPN12 was recruited to and inhibited these receptors after ligand stimulation, thereby serving as a feedback mechanism to limit receptor signaling. Cancer-associated mutation of PTPN12 or reduced PTPN12 protein levels diminished this feedback mechanism, leading to aberrant activity of these receptors. Restoring PTPN12 protein levels restrained signaling from RTKs, including PDGFRβ and MET, and impaired TNBC survival. In contrast with single agents, combined inhibitors targeting the PDGFRβ and MET receptors induced the apoptosis in TNBC cells in vitro and in vivo. This therapeutic strategy resulted in tumor regressions in chemo-refractory patient-derived TNBC models. Notably, response correlated with PTPN12 deficiency, suggesting that impaired receptor feedback may establish a combined addiction to these proto-oncogenic receptors. Taken together, our data provide a rationale for combining RTK inhibitors in TNBC and other malignancies that lack receptor-activating mutations.

IMPORTANCE: Whole-exome sequencing (WES) has the potential to reveal tumor and germline mutations of clinical relevance, but the diagnostic yield for pediatric patients with solid tumors is unknown. OBJECTIVE: To characterize the diagnostic yield of combined tumor and germline WES for children with solid tumors. DESIGN: Unselected children with newly diagnosed and previously untreated central nervous system (CNS) and non-CNS solid tumors were prospectively enrolled in the BASIC3 study at a large academic children’s hospital during a 23-month period from August 2012 through June 2014. Blood and tumor samples underwent WES in a certified clinical laboratory with genetic results categorized on the basis of perceived clinical relevance and entered in the electronic health record. MAIN OUTCOMES AND MEASURES: Clinical categorization of somatic mutations; frequencies of deleterious germline mutations related to patient phenotype and incidental medically-actionable mutations. RESULTS: Of the first 150 participants (80 boys and 70 girls, mean age, 7.4 years), tumor samples adequate for WES were available from 121 patients (81%). Somatic mutations of established clinical utility (category I) were reported in 4 (3%) of 121 patients, with mutations of potential utility (category II) detected in an additional 29 (24%) of 121 patients. CTNNB1 was the gene most frequently mutated, with recurrent mutations in KIT, TSC2, and MAPK pathway genes (BRAF, KRAS, and NRAS) also identified. Mutations in consensus cancer genes (category III) were found in an additional 24 (20%) of 121 tumors. Fewer than half of somatic mutations identified were in genes known to be recurrently mutated in the tumor type tested. Diagnostic germline findings related to patient phenotype were discovered in 15 (10%) of 150 cases: 13 pathogenic or likely pathogenic dominant mutations in adult and pediatric cancer susceptibility genes (including 2 each in TP53, VHL, and BRCA1), 1 recessive liver disorder with hepatocellular carcinoma (TJP2), and 1 renal diagnosis (CLCN5). Incidental findings were reported in 8 (5%) of 150 patients. Most patients harbored germline uncertain variants in cancer genes (98%), pharmacogenetic variants (89%), and recessive carrier mutations (85%). CONCLUSIONS AND RELEVANCE: Tumor and germline WES revealed mutations in a broad spectrum of genes previously implicated in both adult and pediatric cancers. Combined reporting of tumor and germline WES identified diagnostic and/or potentially actionable findings in nearly 40% of newly diagnosed pediatric patients with solid tumors.

A number of epidemiological studies have implicated calcium (Ca2+) signaling as a major factor in obesity that contributes to aberrant systems metabolism. Somewhat paradoxically, obesity correlates with decreased circulating Ca2+levels, leading to increased release of intracellular Ca2+stores from the endoplasmic reticulum. These findings suggest that insulin resistance associated with the obese state is linked to activation of canonical Ca2+signaling pathways. Mechanistically, increased intracellular Ca2+binds calmodulin (CaM) to activate a set of Ca2+/CaM-dependent protein kinases. In this research resource, we explore the metabolic functions and implications of Ca2+/CaM-dependent protein kinase kinase 2 (CaMKK2) as a metabolic effector of Ca2+/CaM action. We reveal the importance of CaMKK2 for gating insulin release from pancreatic β-cells while concomitantly influencing the sensitivity of insulin-responsive tissues. To provide a better understanding of the metabolic impact of CaMKK2loss, we performed targeted metabolomic analyses of key metabolic byproducts of glucose, fatty acid, and amino acid metabolism in mice null for CaMKK2. We quantified amino acids and acyl carnitines in 3 insulin-sensitive tissues (liver, skeletal muscle, plasma) isolated from CaMKK2−/−mice and their wild-type littermates under conditions of dietary stress (low-fat diet, normal chow, high-fat diet, and fasting), thereby unveiling unique metabolic functions of CaMKK2. Our findings highlight CaMKK2 as a molecular rheostat for insulin action and emphasize the importance of Ca2+/CaM/CaMKK2 in regulation of whole-body metabolism. These findings reveal that CaMKK2 may be an attractive therapeutic target for combatting comorbidities associated with perturbed insulin signaling.

Basal-like breast cancers (BLBCs) are aggressive, and their drivers are unclear. We have found that wild-type N-RASis overexpressed in BLBCs but not in other breast cancer subtypes. Repressing N-RASinhibits transformation and tumor growth, whereas overexpression enhances these processes even in preinvasive BLBC cells. We identified N-Ras-responsive genes, most of which encode chemokines; e.g., IL8. Expression levels of these chemokines and N-RASin tumors correlate with outcome. N-Ras, but not K-Ras, induces IL-8 by binding and activating the cytoplasmic pool of JAK2; IL-8 then acts on both the cancer cells and stromal fibroblasts. Thus, BLBC progression is promoted by increasing activities of wild-type N-Ras, which mediates autocrine/paracrine signaling that can influence both cancer and stroma cells.

Defining the molecular networks that drive breast cancer has led to therapeutic interventions and improved patient survival. However, the aggressive triple-negative breast cancer subtype (TNBC) remains recalcitrant to targeted therapies because its molecular etiology is poorly defined. In this study, we used a forward genetic screen to discover an oncogenic network driving human TNBC. SCYL1, TEX14, and PLK1 (“STP axis”) cooperatively trigger degradation of the REST tumor suppressor protein, a frequent event in human TNBC. The STP axis induces REST degradation by phosphorylating a conserved REST phospho-degron and bridging REST interaction with the ubiquitin-ligase βTRCP. Inhibition of the STP axis leads to increased REST protein levels and impairs TNBC transformation, tumor progression, and metastasis. Expression of the STP axis correlates with low REST protein levels in human TNBCs and poor clinical outcome for TNBC patients. Our findings demonstrate that the STP-REST axis is a molecular driver of human TNBC.

In breast cancer, a subset of tumor‐initiating cells (TIC) or “cancer stem cells” are thought to be responsible for tumor maintenance, treatment resistance, and disease recurrence. While current breast cancer stem cell markers (e.g., CD44high/CD24low/neg, ALDH positive) have allowed enrichment for such cells, they are not universally expressed and may actually identify distinct TIC subpopulations in the same tumor. Thus, additional markers of functional stem cells are needed. The STAT3 pathway is a critical regulator of the function of normal stem cells, and evidence is accumulating for its important role in breast cancer stem cells. However, due to the lack of a method for separating live cells based on their level of STAT3 activity, it remains unknown whether STAT3 functions in the cancer stem cells themselves, or in surrounding niche cells, or in both. To approach this question, we constructed a series of lentiviral fluorescent (enhanced green fluorescent protein, EGFP) reporters that enabled flow cytometric enrichment of cells differing in STAT3‐mediated transcriptional activity, as well as in vivo/in situ localization of STAT3 responsive cells. Using in vivo claudin‐low cell line xenograft models of human breast cancer, we found that STAT3 signaling reporter activity (EGFP+) is associated with a subpopulation of cancer cells enriched for mammosphere‐forming efficiency, as well as TIC function in limiting dilution transplantation assays compared to negative or unsorted populations. Our results support STAT3 signaling activity as another functional marker for human breast cancer stem cells thus making it an attractive therapeutic target for stem‐cell‐directed therapy in some breast cancer subtypes. StemCells2014;32:2571–2582

Bladder cancer is the fifth most frequent tumor in men and ninth in women in the United States. Due to a high likelihood of recurrence, effective chemoprevention is a significant unmet need. Estrogen receptors (ERs), primarily ERβ, are expressed in normal urothelium and urothelial carcinoma, and blocking ER function with selective ER modulators such as tamoxifen inhibits bladder cancer cell proliferation in vitro. Herein, the chemoprotective potential of tamoxifen was evaluated in female mice exposed to the bladder-specific carcinogen, N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN). Carcinogen treatment resulted in a 76% tumor incidence and increased mean bladder weights in comparison to controls. In contrast, mice receiving tamoxifen concurrent (8–20 weeks) or concurrent and subsequent (8–32 weeks) to BBN administration had no change in bladder weight and only 10% to 14% incidence of tumors. Non-muscle-invasive disease was present in animals treated with tamoxifen before (5–8 weeks) or after (20–32 weeks) BBN exposure, while incidence of muscle-invasive bladder carcinoma was reduced. ERβ was present in all mice and thus is a potential mediator of the tamoxifen chemoprotective effect. Surprisingly, ERα expression, which was detected in 74% of the mice exposed to BBN alone but not in any controlmice, was correlated with tumor incidence, indicating a possible role for this receptor in carcinogen-induced urothelial tumorigenesis. Thus, these data argue that both ERα and ERβ play a role in modulating carcinogen-induced bladder tumorigenesis. Administration of tamoxifen should be tested as a chemopreventive strategy for patients at high risk for bladder cancer recurrence.

Purpose: Despite growing concerns toward maintaining participants' privacy, individual investigators collecting tissue and other biological specimens for genomic analysis are encouraged to obtain informed consent for broad data sharing. Our purpose was to assess the effect on research enrollment and data sharing decisions of three different consent types (traditional, binary, or tiered) with varying levels of control and choices regarding data sharing.Methods: A single-blinded, randomized controlled trial was conducted with 323 eligible adult participants being recruited into one of six genome studies at Baylor College of Medicine in Houston, Texas, between January 2008 and August 2009. Participants were randomly assigned to one of three experimental consent documents (traditional, n = 110; binary, n = 103; and tiered, n = 110). Debriefing in follow-up visits provided participants a detailed review of all consent types and the chance to change data sharing choices or decline genome study participation.Results: Before debriefing, 83.9% of participants chose public data release. After debriefing, 53.1% chose public data release, 33.1% chose restricted (controlled access database) release, and 13.7% opted out of data sharing. Only one participant declined genome study participation due to data sharing concerns.Conclusion: Our findings indicate that most participants are willing to publicly release their genomic data; however, a significant portion prefers restricted release. These results suggest discordance between existing data sharing policies and participants' judgments and desires.

Normal neutrophil development requires G‐CSF signaling, which includes activation of Stat3. Studies of G‐CSF‐mediated Stat3 signaling in cell culture and transgenic mice have yielded conflicting data regarding the role of Stat3 in myelopoiesis. The specific functions of Stat3 remain unclear, in part, because two isoforms, Stat3α and Stat3β, are expressed in myeloid cells. To understand the contribution of each Stat3 isoform to myelopoiesis, we conditionally overexpressed Stat3α or Stat3β in the murine myeloid cell line 32Dcl3 (32D) and examined the consequences of overexpression on cell survival and differentiation. 32D cells induced to overexpress Stat3α, but not Stat3β, generated a markedly higher number of neutrophils in response to G‐CSF. This effect was a result of decreased apoptosis but not of increased proliferation. Comparison of gene expression profiles of G‐CSF‐stimulated, Stat3α‐overexpressing 32D cells with those of cells with normal Stat3α expression revealed novel Stat3 gene targets, which may contribute to neutrophil expansion and improved survival, most notably Slc28a2, a purine nucleoside transporter, which is critical for maintenance of intracellular nucleotide levels and prevention of apoptosis, and Gpr65, an acid‐sensing, G protein‐coupled receptor with pro‐oncogenic and antiapoptotic functions.

Similar to the bone marrow, the mammary gland contains a distinct population of Hoechst‐effluxing side population cells, mammary gland side population cells (MG‐SPs). To better characterize MG‐SPs, their microarray gene profiles were compared to the remaining cells, which retain Hoechst dye (mammary gland non‐side population cells [MG‐NSPs]). For analysis, Gene Ontology (GO) that describes genes in terms of biological processes and Ontology Traverser (OT) that performs enrichment analysis were used. OT showed that MG‐SP‐specific genes were enriched in the GO categories of cell cycle regulation and checkpoints, multidrug‐resistant transporters, organogenesis, and vasculogenesis. The MG‐NSP‐upregulated genes were enriched in the GO category of cellular organization and biogenesis, which includes basal epithelial markers, p63, smooth muscle actin, myosin, α6 integrin, cytokeratin (CK) 14, and luminal markers CK8 and CD24. Additional studies showed that a higher percentage of MG‐SPs exist in the G1 phase of the cell cycle compared with the MG‐NSPs. G1 cell cycle block of MG‐SPs may be explained by higher expression of cell cycle‐negative regulatory genes such as transforming growth factor‐β2, insulin‐like growth factor binding protein‐5, P18INK4C, and wingless‐5a (Wnt‐5a). Accordingly, a smaller percentage of MG‐SPs expressed nuclear β‐catenin, possibly as a consequence of the higher expression of Wnt‐5a. In conclusion, microarray gene profiling suggests that MG‐SPs are a lineage‐deficient mammary gland subpopulation expressing key genes involved in cell cycle regulation, development, and angiogenesis.

We calculated hazard rates for recurrence in patients with primary breast cancer (stage I, II; no adjuvant therapy). Previous publications have indicated a peak in hazard rates for recurrence (or death) at approximately 2–3 years after diagnosis of primary breast cancer. However, there have been conflicting reports concerning the presence of a second peak at 5–7 years after diagnosis. In this study, we estimated hazard functions by the Nelson–Aalen method and fit by cubic–linear and cubic–cubic–linear models to test for the presence of one or two peaks, respectively. We identified two peaks in hazard of recurrence, one at 2 years and another at 5 years. The 5-year peak, though statistically significant, represents very small differences in patient outcome. This additional peak may be an artifact of interval censoring due to a tendency to follow-up patients at specific bench-mark time points.

Trastuzumab/chemotherapy combinations have already shown superior results in metastatic breast cancer patients. The purpose of this study is to determine the clinical efficacy of neoadjuvant trastuzumab and docetaxel in women with locally advanced breast cancer, with or without metastatic disease. Treatment-naive women with HER2-overexpressing locally advanced breast cancer, with or without metastatic disease, were included. Patients received trastuzumab 4 mg/kg loading dose intravenously then 2 mg/kg weekly. On day 22, docetaxel 100 mg/m2every 3 weeks for 4 cycles was added to weekly trastuzumab. Patients then underwent surgery and subsequent 4 cycles of AC (doxorubicin/cyclophosphamide; 60/600 mg/m2) without trastuzumab. Weekly trastuzumab was resumed 1 month after completion of AC and continued for a year. Preliminary results from the first 22 patients with median follow-up of 15.5 months (range, 2-38 months) are reported. Of these, 9 patients (40.9%) had inflammatory breast cancer, and 6 patients (27.3%) had stage IV breast cancer. Seventeen of 22 patients (77.3%) had objective clinical response, with a clinical complete response in 9 patients (40.9%). Two patients (9.1%) had decline in cardiac function and 7 patients (31.8%) experienced neutropenia, with 2 deaths (9.1%) from neutropenic sepsis. Eight patients (36.4%) have relapsed, 3 with local skin recurrence (13.6%) and 5 with distant recurrence, of whom 1 had liver metastasis (4.5%) and 4 had brain metastasis (18.2%). Combined neoadjuvant trastuzumab and docetaxel induced high clinical response rates for HER2- overexpressing breast cancer, in particular for inflammatory breast cancer. A high rate of brain metastasis was noted, particularly in patients with baseline metastatic disease.

Control of anthrax toxin and capsule synthesis, the two major virulence factors of Bacillus anthracis, has been associated with two regulatory genes, atxA and acpA, located on virulence plasmids pXO1 and pXO2, respectively. We used transcriptional profiling to determine whether atxA and/or acpA control genes other than those already described and to investigate functional similarities of the regulators. Transcription was assessed in a pXO1(+) pXO2(+) parent strain and in isogenic mutants in which one or both regulatory genes were deleted. We determined that in addition to the toxin and capsule genes, atxA controls expression of numerous other genes on both plasmids and the chromosome. Generally, plasmid-encoded genes were more highly regulated than chromosomal genes, and both positive and negative effects were observed. Certain atxA-regulated genes were affected synergistically in an atxA acpA mutant. Yet overall, acpA appears to be a minor regulator with fewer targets than atxA. In contrast to previous reports of acpA function in attenuated strains, acpA had a minimal influence on capsule gene transcription and capsule synthesis in a genetically complete strain. Surprisingly, acpA expression was positively affected by atxA, although atxA-activated capsule gene transcription is not acpA dependent. The newly discovered atxA-regulated targets include genes predicted to encode secreted proteins and proteins with roles in transcriptional regulation and signaling. Regulation of chromosomal genes by atxA is particularly intriguing, given that many of the target genes have homologues in other Bacillus species that lack atxA homologues. Given the global effect of atxA on gene expression in B. anthracis, previous assumptions regarding reduced virulence of strains harboring single plasmids must be reassessed and the potential roles of newly identified atxA-regulated genes should be investigated.

ABSTRACTControl of anthrax toxin and capsule synthesis, the two major virulence factors of Bacillus anthracis, has been associated with two regulatory genes, atxAand acpA, located on virulence plasmids pXO1 and pXO2, respectively. We used transcriptional profiling to determine whether atxAand/or acpAcontrol genes other than those already described and to investigate functional similarities of the regulators. Transcription was assessed in a pXO1+pXO2+parent strain and in isogenic mutants in which one or both regulatory genes were deleted. We determined that in addition to the toxin and capsule genes, atxAcontrols expression of numerous other genes on both plasmids and the chromosome. Generally, plasmid-encoded genes were more highly regulated than chromosomal genes, and both positive and negative effects were observed. Certain atxA-regulated genes were affected synergistically in an atxA acpAmutant. Yet overall, acpAappears to be a minor regulator with fewer targets than atxA. In contrast to previous reports of acpAfunction in attenuated strains, acpAhad a minimal influence on capsule gene transcription and capsule synthesis in a genetically complete strain. Surprisingly, acpAexpression was positively affected by atxA,although atxA-activated capsule gene transcription is not acpAdependent. The newly discovered atxA-regulated targets include genes predicted to encode secreted proteins and proteins with roles in transcriptional regulation and signaling. Regulation of chromosomal genes by atxAis particularly intriguing, given that many of the target genes have homologues in other Bacillusspecies that lack atxAhomologues. Given the global effect of atxAon gene expression in B. anthracis, previous assumptions regarding reduced virulence of strains harboring single plasmids must be reassessed and the potential roles of newly identified atxA-regulated genes should be investigated.

Proliferating cells within the terminal end buds of the virgin female rat mammary gland are the most susceptible to chemical carcinogen induced tumorigenesis. We hypothesized that selective ablation of proliferating cells in the mammary gland would reduce mammary tumor incidence upon carcinogen challenge. Selective ablation of proliferating cells was achieved by intraductal injections of Adv-RSV-tk and gancyclovir administration. Despite efficient viral transduction of the thymidine kinase protein and the apparent elimination of >90% of the prolif-erating cells, the rats exhibited a higher incidence of MNU induced mammary tumors arising with shorter latency as compared to control animals. Several possible explanations of the puzzling relationship between elimination of cycling cells and increased tumor incidence are discussed and alternative strategies for the prevention of breast cancer are proposed.

Reproductive history is a consistent risk factor for human breast cancer. Epidemiological studies have repeatedly demonstrated that early age of first pregnancy is a strong protective factor against breast cancer and provides a physiologically operative model to achieve a practical mode of prevention. In rodents, the effects of full‐term pregnancy can be mimicked by a three‐week exposure to low doses of estrogen and progesterone. Neither hormone alone is sufficient to induce protection. The cellular and molecular mechanisms that underlie hormone‐induced refractoriness are largely unresolved. Our recent studies have demonstrated that an early cellular response that is altered in hormone‐treated mammary cells is the initial proliferative burst induced by the chemical carcinogen methylnitrosourea. The decrease in proliferation is also accompanied by a decrease in the ability of estrogen receptor‐positive cells to proliferate. RNA expression of several mammary cell‐cycle‐related genes is not altered in hormone‐treated mice; however, immunohistochemical assays demonstrate that the protein level and nuclear compartmentalization of the p53 tumor suppressor gene are markedly upregulated as a consequence of hormone treatment. These results support the hypothesis that hormone stimulation, at a critical period in mammary development, results in cells with persistent changes in the intracellular regulatory loops governing proliferation and response to DNA damage. A corollary to this hypothesis is that the genes affected by estrogen and progesterone are independent of alveolar differentiation‐specific genes. Suppressive subtractive hybridization‐PCR methods have identified several genes that are differentially expressed as a consequence of prior estrogen and progesterone treatment. Future experiments are aimed at determining the mechanisms of hormone‐induced upregulation of p53 protein expression as part of the overall goal of identifying and functionally characterizing the genes responsible for the refractory phenotype.

The small heat shock protein hsp27 is associated with aggressive tumor behavior in certain subsets of breast cancer patients. Previously we demonstrated that hsp27 overexpression in breast cancer cells increased in vitro and in vivo invasiveness, suggesting that hsp27 influences the metastatic process. To investigate this role for hsp27, we have utilized MDA-MB-231 breast cancer cells that overexpress hsp27 and cDNA expression array technology. We demonstrate that hsp27 overexpression up-regulates MMP-9 expression and activity and down-regulates Yes expression. Furthermore, our results suggest that Yes may be involved in regulating MMP-9 expression, as well as in vitro invasion. Reconstitution of Yes expression by transfection into hsp27-overexpressing cells decreased MMP-9 expression, and increased in vitro invasiveness, abrogating the phenotype conferred by hsp27 overexpression. Therefore, our results provide a new potential mechanism by which hsp27 affects the metastatic cascade--through regulation of MMP-9 and Yes expression.

Deletions of chromosome 18q are among the most common segmental aneusomies compatible with life. The estimated frequency is approximately 1/40,000 live births [Cody JD, Pierce JF, Brkanac Z, Plaetke R, Ghidoni PD, Kaye CI, Leach RJ. 1997. Am. J. Med. Genet. 69:280–286]. Most deletions are terminal encompassing as much as 36 Mb, but interstitial deletions have also been reported. We have evaluated 42 subjects with deletions of 18q at our institution. This is the largest number of individuals with this chromosome abnormality studied by one group of investigators. Here we report the physical findings in these individuals. We have compared our findings with those of previously reported cases and have found a significantly different incidence of several minor anomalies in our subjects. We also describe here several anomalies not previously reported in individuals with deletions of 18q, including short frenulum, short palpebral fissures, disproportionate short stature, overlap of second and third toes, and a prominent abdominal venous pattern. Characteristics found in subjects were analyzed for correlation with cytogenetic breakpoints. Several traits were found to correlate with the extent of the deletion. Large deletions were associated with significantly decreased head circumference and ear length as well as the presence of proximally placed and/or anomalous thumbs. Individuals with the smallest deletions were more likely to have metatarsus adductus. Although relatively few genotype/phenotype correlations were apparent, these data demonstrate that correlations with breakpoint are possible. This implies that more correlations will become evident when the more precise molecularly based genotyping is completed. These correlations will identify critical regions on the chromosome in which genes responsible for specific abnormal phenotypes are located. Am. J. Med. Genet. 85:455–462, 1999. © 1999 Wiley-Liss, Inc.

Summary Using sucrose gradient centrifugation, human breast cancer estrogen receptors appear in two molecular forms sedimenting at either 8S or 4S fractions. The sum of these two fractions has been valuable clinically in helping to predict response to hormonal therapies. It has been suggested that the 4S receptor may not be of predictive value and that the practice of using only dextran-coated charcoal methodology might thus overestimate the number of patients whose tumors might be hormonally dependent. The present study correlates clinical response to hormonal manipulations with 8S and 4S receptors as determined by sucrose density gradient centrifugation. Patients with only 4S positive receptors had a 56% response rate to hormonal manipulation compared with a 52% response rate in patients with positive 8S alone or with both 8S and 4S receptors. Although patient numbers are small in this and the other three published series addressing this question, our data do not confirm two previously published studies suggesting clinical importance of 8S/4S fractionation.

Despite the widespread usage of hydroxyurea in the treatment of both malignant and nonmalignant diseases and a recent expansion in the recognition of its potential therapeutic applications, there have been few detailed studies of hydroxyurea's pharmacokinetic (PK) behavior and oral bioavailability. Parenteral administration schedules have been evaluated because of concerns about the possibility for significant interindividual variability in the PK behavior and bioavailability of hydroxyurea after oral administration. In this PK and bioavailability study, 29 patients with advanced solid malignancies were randomized to treatment with 2,000 mg hydroxyurea administered either orally or as a 30-minute intravenous (IV) infusion accompanied by extensive plasma and urine sampling for PK studies. After 3 weeks of treatment with hydroxyurea (80 mg/kg orally every 3 days followed by a 1-week washout period), patients were crossed over to the alternate route of administration, at which time extensive PK studies were repeated. Three days later, patients continued treatment with 80 mg/kg hydroxyurea orally every 3 days for 3 weeks, followed by a 1-week rest period. Thereafter, 80 mg/kg hydroxyurea was administered orally every 3 days. Twenty-two of 29 patients had extensive plasma and urine sampling performed after treatment with both oral and IV hydroxyurea. Oral bioavailability (F) averaged 108%. Moreover, interindividual variability in F was low, as indicated by 19 of 22 individual F values within a narrow range of 85% to 127% and a modest coefficient of variation of 17%. The time in which maximum plasma concentrations (Cmax) were achieved averaged 1.22 hours with an average lag time of 0.22 hours after oral administration. Except for Cmax, which was 19.5% higher after IV drug administration, the PK profiles of oral and IV hydroxyurea were very similar. The plasma disposition of hydroxyurea was well described by a linear two-compartment model. The initial harmonic mean half-lives for oral and IV hydroxyurea were 1.78 and 0.63 hours, respectively, and the harmonic mean terminal half-lives were 3.32 and 3.39 hours, respectively. For IV hydroxyurea, systemic clearance averaged 76.16 mL/min/m2 and the mean volume of distribution at steady-state was 19.71 L/m2, whereas Cloral/F and Voral/F averaged 73.16 mL/min/m2 and 19.65 L/m2, respectively, after oral administration. The percentage of the administered dose of hydroxyurea that was excreted unchanged into the urine was nearly identical after oral and IV administration—36.84% and 35.82%, respectively. Additionally, the acute toxic effects of hydroxyurea after treatment on both routes were similar. Relationships between pertinent PK parameters and the principal toxicity, neutropenia, were sought, but no pharmacodynamic relationships were evident. From PK, bioavailability, and toxicologic standpoints, these results indicate that there are no clear advantages for administering hydroxyurea by the IV route except in situations when oral administration is not possible and/or in the case of severe gastrointestinal impairment.

Intra−abdominal operations are relatively high risk for pulmonary complications. Previous research has more intensely investigated cardiac operative risk, but recent work suggests that significant pulmonary complications may be more common than cardiac complications and associated with longer length of stay. This study identified risk indicators for pulmonary complications after elective abdominal operations.

We have used an in vivo tumor model to evaluate the consequences of p53 tumor suppressor protein deficiency in a tissue-specific context. By breeding MMTV-rastransgenic mice, which are highly susceptible to the development of mammary and salivary tumors, with p53−/−mice, we generated three classes of animals which contained the MMTV-rastransgene but differed in their p53 functional status (ras/p53+/+, ras/p53+/−, or ras/p53−/−). ras/p53−/−mice developed tumors more rapidly than animals of the other two genotypes; however, the distribution of tumors was unexpectedly altered. Whereas the most frequently observed tumors in ras/p53+/+and ras/p53+/−mice were of mammary origin, ras/p53−/−mice developed primarily salivary tumors. In addition, the mammary and salivary tumors from ras/p53−/−mice consistently exhibited a number of unfavorable characteristics, including higher histologic grades, increased growth rates, and extensive genomic instability and heterogeneity, relative to tumors from ras/p53+/+mice. Interestingly, the increased growth rates of ras/p53−/−tumors appear to be due to impaired cell cycle regulation rather than decreased apoptosis, suggesting that p53-mediated tumor suppression can occur independent of its role in apoptosis.

Increasing evidence indicates that routine preoperative diagnostic spirometry (pulmonary function tests [PFTs]) before elective abdominal surgery does not predict individual risk of postoperative pulmonary complications and is overutilized. This economic evaluation estimates potential savings from reduced use of preoperative PFTs.

The acquired ability of tamoxifen to stimulate tumor growth has been suggested as one mechanism for the development of treatment failure in breast cancer. We have reported that tamoxifen-stimulated MCF-7 breast tumors in nude mice display reduced tamoxifen levels as compared with tamoxifen-inhibited tumors and an altered metabolite profile with isomerization oftrans-4-hydroxytamoxifen to a weak antiestrogen and the production of metabolite E, an estrogenic metabolite. To investigate further the importance of tamoxifen metabolism in this model, we quantified levels of tamoxifen and major metabolites in tamoxifen-stimulated as compared with tamoxifen-inhibited MCF-7 tumors growing in nude mice and employed tamoxifen analogs resistant to metabolism. Tamoxifen-stimulated tumors have a relative abundance ofcis-4-hydroxytamoxifen and metabolite E. However, in vivo treatment of mice carrying tamoxifen-stimulated tumors with fixed-ring nonisomerizable tamoxifen analogs or with nafoxidine, a nonsteroidal antiestrogen with a different structure, nonetheless resulted in tumor growth stimulation. Tumors were also stimulated by a deoxytamoxifen analog resistant to conversion to metabolite E. Growth of tamoxifen-stimulated tumors was inhibited by a pure steroidal antiestrogen, ICI 182, 780, suggesting the need for clinical trials of this drug in patients with tamoxifen resistance. Growth of tamoxifen-stimulated tumors was further stimulated by estrogen replenishment, and this estrogen stimulation could be blocked by tamoxifen indicating that tamoxifen has both agonist and antagonist properties in these tumors. This study suggests that tamoxifen-stimulated tumor growth in this model is not due to isomerization or metabolism of tamoxifen to less antiestrogenic or more estrogenic metabolities. The mechanisms by which tamoxifen acquires more potent in vivo agonist properties, resulting in tumor growth stimulation over time, remain to be defined.

OBJECTIVE:Internists frequently evaluate preoperative cardiopulmonary risk and comanage cardiac and pulmonary complications, but the comparative incidence and clinical importance of these complications are not clearly delineated. This study evaluated incidence and length of stay for both cardiac and pulmonary complications after elective laparotomy.

Heat shock proteins (hsp's) are induced in cells when exposed to different environmental stressful conditions. We have found that breast cancer cells sometimes express high levels of several hsp's, which may both augment the aggressiveness of these tumors and make them more resistant to treatment. We have shown that hsp70 is an ominous prognostic sign as detected by Western blot assays in node-negative breast tumors, and that hsp27 increases specific resistance to doxorubicin in breast cancer cell lines. These findings have direct clinical application, and suggest that modulating hsp expression may be a therapeutic target for reversal of hsp-associated detrimental cellular effects.

With the proliferation of potential prognostic factors for breast cancer, it is becoming increasingly more difficult for physicians and patients to integrate the information provided by these factors into a single accurate prediction of clinical outcome. Here we review Cox's proportional hazards model, recursive partitioning, correspondence analysis, and neural networks for their respective capabilities in analyzing censored survival data in the presence of multiple prognostic factors, and we present some clinical applications where these models have been used.

Summary Neural Network Analysis, a form of artificial intelligence, was successfully used to predict the clinical outcome of node-positive breast cancer patients. A Neural Network was trained to predict clinical outcome using prognostic information from 1008 patients. During training, the network received as input information tumor hormone receptor status, DNA index and S-phase determination by flow cytometry, tumor size, number of axillary lymph nodes involved with tumor, and age of the patient, as well as lengtl of clinical followup, relapse status, and time of relapse. The ability of the trained Network to determine relapse probability was then validated in a separate set of 960 patients. The Neural Network was as powerful as Cox Regression Modeling inidentifying breast cancer patients at high and low risk for relapse.

Gαi2mediates proliferation stimulated by Smoothened in the mammary gland.

Local hyperthermia with gold nanoshells sensitizes cancer stem cells to radiation treatment in mouse and human breast cancer preclinical models.