The role of a novel secretory peptidoglycan recognition protein with antibacterial ability from the Chinese Oak Silkworm Antheraea pernyi in humoral immunity.

Peptidoglycan recognition proteins (PGRPs) are a family of pattern recognition receptors that play a critical role in the immune response of invertebrates and vertebrates. Herein, the short ApPGRP-D gene was cloned from the model lepidopteran Antheraea pernyi. Quantitative PCR (qPCR) confirmed that ApPGRP-D is an immune-related protein and that the expression of ApPGRP-D can be induced by microorganisms. ApPGRP-D is a broad-spectrum pattern recognition protein that activates the prophenoloxidase cascade activation system and promotes the agglutination of microbial cells. Likely due to its amidase activity, ApPGRP-D can inhibit the growth of E. coli and S. aureus. In addition, we demonstrated for the first time that zinc ions, as important metal coenzymes, could promote multiple functions of ApPGRP-D but not its amidase activity.

A novel l-histidine based ionic liquid (LHIL) as an efficient corrosion inhibitor for mild steel.

A novel l-histidine based ionic liquid (LHIL) was developed and successfully synthesized. Its structure was confirmed by Fourier-transform infrared spectroscopy, UV-vis spectroscopy, X-ray photoelectron spectroscopy, 1H-NMR and high-resolution mass spectrometry. The outstanding corrosion inhibition effect of the LHIL on mild steel in 1 M hydrochloric acid was thoroughly evaluated by Tafel plots, electrochemical impedance spectroscopy, and localized electrochemical strategies. The results revealed that the corrosion of mild steel was effectively suppressed by the adsorption of LHIL on its surface, and the best inhibition efficiency reached 98.8%. The adsorption behavior of LHIL on steel obeyed the Langmuir adsorption isotherm, which involved both chemisorption and physisorption. Theoretical calculations indicated the strong chemisorption of LHIL on steel, as proved by the low energy gap (ΔE = 0.0522 eV) and high binding energy (E binding = 303.47 kcal mol-1), which clearly confirmed the effectiveness of LHIL for steel protection.

Anti-corrosion and self-healing coatings with polyaniline/epoxy copolymer-urea-formaldehyde microcapsules for rusty steel sheets.

Polyaniline (PANI)/Epoxy copolymer as a core material was synthesized via a chemical oxidation method. Various analytical techniques, including scanning electron microscope, Fourier transform infrared spectroscopy, energy dispersive spectroscopy, thermogravimetry, and electrochemical impedance spectroscopy, were used to characterize the morphology, compositions, and self-healing and anticorrosion properties of the prepared microcapsules and coatings. The prepared PANI/Epoxy copolymer showed the best electrochemical corrosion resistance when the ratio of PANI to epoxy was 0.05: 1 (wt.:wt.). For the mass fraction of the core (PANI/Epoxy copolymer) of 60.84 ± 0.06 wt%, the mean particle diameter of the prepared microcapsules was 4.20 ± 0.92 µm. The coatings with 15 wt% microcapsules possessed excellent self-healing performance and corrosion resistance. The low-frequency impedance modulus at 0.01 Hz of scratched coatings immersed in the NaCl solution for 24 h was 5.27 × 106 Ω·cm2. Scratched self-repairing coating samples were able to resist corrosion for 384 h; thus, the microcapsules can be used to significantly extend the service life of the coatings. Microcapsule-containing PANI/Epoxy copolymers are expected to find use in anticorrosion coating systems, where the coatings could be applied directly on rusty steel surfaces.

A novel hexamerin with an unexpected contribution to the prophenoloxidase activation system of the Chinese oak silkworm, Antheraea pernyi.

Hexamerin was originally identified as a storage protein but later confirmed to be involved in many physiological processes. In the present study, we cloned and characterized a novel hexamerin complementary DNA sequence from the Chinese oak silkworm, Antheraea pernyi (Ap-hexamerin), which shows high homology with reported insect methionine-rich hexamerins. The tissue distribution and time course of expression demonstrated that Ap-hexamerin was predominantly synthesized in the fat body and the expression level was significantly increased in response to the microbial challenge, suggesting the relevance of Ap-hexamerin to immune responses. In further immune functional studies, Ap-hexamerin was confirmed to take part in the upregulation of prophenoloxidase (PPO) activation in A. pernyi haemolymph triggered by pathogen-associated molecular patterns (PAMPs). Additional molecular interaction analysis revealed that Ap-hexamerin is capable of binding the PAMPs used in the phenoloxidase assay, suggesting hexamerin in A. pernyi may positively regulate haemolymph PPO activation, acting as a pattern recognition protein.

Prevalence of somatic and germline mutations of Fumarate hydratase in uterine leiomyomas from young patients.

AIMS: Hereditary leiomyomatosis and renal cell cancer (HLRCC) syndrome is caused by germline mutations in the Fumarate hydratase (FH) gene. In young women, the syndrome often presents with symptomatic uterine leiomyomas, leading to myomectomy or hysterectomy. In this study, we aimed to investigate the incidence and mutational profiles of FH-negative leiomyomas from young patients, thus allowing for early identification and triage of syndromic patients for surveillance. METHODS AND RESULTS: We evaluated 153 cases of uterine leiomyomas from women aged up to 30 years for loss of FH expression by tissue microarray (TMA)-based immunohistochemical staining. Mutational analysis of tumours with loss of FH was carried out by polymerase chain reaction (PCR) amplification of 10 exons within the FH gene and subsequent Sanger sequencing. The status of promoter methylation was assessed by bisulphite sequencing. Loss of FH protein expression was detected in seven (4.6%) of 153 tested uterine leiomyomas from young patients. All FH-negative leiomyomas displayed staghorn vasculature and fibrillary/neurophil-like cytoplasm. We found that six (86%) of seven FH-negative tumours detected by immunohistochemistry harboured FH mutations, 50% of which contained germline mutations. In particular, the germline mutational rate in FH gene was 2.0% (three of 153 cases). Bisulphite sequencing analysis failed to detect promoter methylation in any of the seven tumours. CONCLUSION: Our study showed a relatively high rate of FH germline mutation in FH-negative uterine leiomyomas from patients aged up to 30 years. While genetic mutations confer protein expression loss, epigenetic regulation of the FH gene appears to be unrelated to this phenotype.

Clinicopathologic Features and Genetic Alterations of a Primary Osteosarcoma of the Uterine Corpus.

Primary osteosarcoma (OS) of the uterus is distinctly rare. We report a case of primary uterine OS with pulmonary metastasis in a 74-yr-old woman. Histopathologic features of the uterine tumor were in keeping with a pure chondroblastic OS composed of neoplastic cells with osteoblastic/chondroblastic differentiation and neoplastic bone formation. Despite treatment with Doxorubicin and Olaratumab and later with palliative radiation therapy, the patient died 7 mo after hysterectomy due to multiple distant metastases. A targeted next-generation sequencing assay based on a 637-gene panel was performed to analyze genetic alterations in this highly aggressive tumor, but no somatic mutations that are amenable to targeted therapy were detected. Rather, a 51-nucleotide deletion mutation including partial exon 2 of mediator complex subunit 12 (MED12), a gene commonly mutated in leiomyoma, breast fibroadenoma and phyllodes tumor, was identified. Given the MED12 mutation in this uterine OS, we propose possible mechanisms that account for the origin and development of this tumor.

An Epithelioid Smooth Muscle Neoplasm Mimicking a Signet Ring Cell Carcinoma in the Ovary.

A 53-yr-old woman who presented with elevated renal indices was discovered to have a 4.5 cm right renal mass and an incidental 9.7 cm left ovarian mass on imaging studies. She underwent a partial nephrectomy and bilateral salpingo-oophorectomy, revealing a chromophobe renal cell carcinoma and an unusual ovarian neoplasm with epithelioid cells displaying prominent signet ring cell-like morphology. Immunohistochemical analysis of the ovarian neoplasm demonstrated that the tumor cells were diffusely immunoreactive for smooth muscle markers and negative for all tested cytokeratins and epithelial membrane antigen. On the basis of these results, the tumor was interpreted as an unusual epithelioid smooth muscle neoplasm with extensive signet ring cell-like features. Along with primary ovarian signet ring stromal tumors and sclerosing stromal tumors, this example adds epithelioid smooth muscle neoplasms with unusual cytologic alterations to the list of uncommon nonepithelial tumors that can simulate metastatic signet ring cell carcinoma (Krukenberg tumor) in the ovary.

Loss of Nuclear Localized Parathyroid Hormone-Related Protein in Primary Breast Cancer Predicts Poor Clinical Outcome and Correlates with Suppressed Stat5 Signaling.

PURPOSE: Parathyroid hormone-related protein (PTHrP) is required for normal mammary gland development and biology. A PTHLH gene polymorphism is associated with breast cancer risk, and PTHrP promotes growth of osteolytic breast cancer bone metastases. Accordingly, current dogma holds that PTHrP is upregulated in malignant primary breast tumors, but solid evidence for this assumption is missing. EXPERIMENTAL DESIGN: We used quantitative IHC to measure PTHrP in normal and malignant breast epithelia, and correlated PTHrP levels in primary breast cancer with clinical outcome. RESULTS: PTHrP levels were markedly downregulated in malignant compared with normal breast epithelia. Moreover, low levels of nuclear localized PTHrP in cancer cells correlated with unfavorable clinical outcome in a test and a validation cohort of breast cancer treated at different institutions totaling nearly 800 cases. PTHrP mRNA levels in tumors of a third cohort of 737 patients corroborated this association, also after multivariable adjustment for standard clinicopathologic parameters. Breast cancer PTHrP levels correlated strongly with transcription factors Stat5a/b, which are established markers of favorable prognosis and key mediators of prolactin signaling. Prolactin stimulated PTHrP transcript and protein in breast cancer cell lines in vitro and in vivo, effects mediated by Stat5 through the P2 gene promoter, producing transcript AT6 encoding the PTHrP 1-173 isoform. Low levels of AT6, but not two alternative transcripts, correlated with poor clinical outcome. CONCLUSIONS: This study overturns the prevailing view that PTHrP is upregulated in primary breast cancers and identifies a direct prolactin-Stat5-PTHrP axis that is progressively lost in more aggressive tumors.

Myositis following spine radiosurgery for metastatic disease: a case series.

OBJECTIVE Spinal stereotactic body radiation therapy (SBRT) has emerged as an attractive method to deliver high doses of radiation to oligometastatic spinal tumors with radioresistant histology. Because SBRT is a palliative therapy, attention to potential radiation toxicities is paramount when counseling patients. The objective of this study was to report radiation-induced myositis after SBRT, a previously undescribed complication. METHODS A total of 667 patients received 891 spine SBRT treatments (either 24 Gy in 1 fraction or 27 Gy in 3 fractions) from 2011 to 2016 and underwent retrospective review. Eleven patients were identified as having radiographic evidence of myositis following SBRT. Clinical and pathologic results were collected, including receipt of anti-vascular endothelial growth factor (VEGF) therapy, radiation dose, equivalent dose in 2-Gy fractions (EQD2), biologically effective dose (BED), and volume of muscle treated. Treatment toxicities were classified according to the Common Terminology Criteria for Adverse Events (CTCAE; version 4.03). Univariate statistical analyses were performed to evaluate the relationships between radiation fractionation schedule and myositis and between anti-VEGF therapy and myositis. RESULTS The cumulative incidence of myositis was 1.9% at 1 year. The median of the mean dose administered to muscle with myositis was 17.5 Gy. The median EQD2 was 55.1 Gy, and the median BED was 82.7 Gy. The median time to the development of clinical symptoms was 1.4 months, while the median time to imaging evidence was 4.7 months. Two patients (18.2%) had CTCAE grade 3 complications. Single-fraction spine SBRT (HR 4.5, 95% CI 1.2-16.9; p = 0.027) was associated with increased risk of developing myositis whereas receipt of anti-VEGF therapy was not (HR 2.2, 95% CI 0.6-7.1; p = 0.2). CONCLUSIONS Radiation myositis following spinal radiosurgery is a rare but important complication. Single-fraction treatment schedules may be associated with increased risk of myositis but should be validated in a larger series.

Validation of tumor protein marker quantification by two independent automated immunofluorescence image analysis platforms.

Protein marker levels in formalin-fixed, paraffin-embedded tissue sections traditionally have been assayed by chromogenic immunohistochemistry and evaluated visually by pathologists. Pathologist scoring of chromogen staining intensity is subjective and generates low-resolution ordinal or nominal data rather than continuous data. Emerging digital pathology platforms now allow quantification of chromogen or fluorescence signals by computer-assisted image analysis, providing continuous immunohistochemistry values. Fluorescence immunohistochemistry offers greater dynamic signal range than chromogen immunohistochemistry, and combined with image analysis holds the promise of enhanced sensitivity and analytic resolution, and consequently more robust quantification. However, commercial fluorescence scanners and image analysis software differ in features and capabilities, and claims of objective quantitative immunohistochemistry are difficult to validate as pathologist scoring is subjective and there is no accepted gold standard. Here we provide the first side-by-side validation of two technologically distinct commercial fluorescence immunohistochemistry analysis platforms. We document highly consistent results by (1) concordance analysis of fluorescence immunohistochemistry values and (2) agreement in outcome predictions both for objective, data-driven cutpoint dichotomization with Kaplan-Meier analyses or employment of continuous marker values to compute receiver-operating curves. The two platforms examined rely on distinct fluorescence immunohistochemistry imaging hardware, microscopy vs line scanning, and functionally distinct image analysis software. Fluorescence immunohistochemistry values for nuclear-localized and tyrosine-phosphorylated Stat5a/b computed by each platform on a cohort of 323 breast cancer cases revealed high concordance after linear calibration, a finding confirmed on an independent 382 case cohort, with concordance correlation coefficients >0.98. Data-driven optimal cutpoints for outcome prediction by either platform were reciprocally applicable to the data derived by the alternate platform, identifying patients with low Nuc-pYStat5 at ~3.5-fold increased risk of disease progression. Our analyses identified two highly concordant fluorescence immunohistochemistry platforms that may serve as benchmarks for testing of other platforms, and low interoperator variability supports the implementation of objective tumor marker quantification in pathology laboratories.

Intracystic Papillary Carcinoma of the Breast in a Male Patient.

We report a case of intracystic papillary carcinoma of the right breast in a 59-year old man presenting with bloody nipple discharge for 1 week prior to presentation. Mammography, ultrasonography, and core needle aspiration were consistent with intracystic papillary carcinoma. The patient underwent right simple mastectomy. Pathology was also consistent with low grade intracystic papillary carcinoma. The 21-gene assay revealed a recurrence score of 0, corresponding to a 3% risk of distant recurrence at 10 years. A patient did not receive chemotherapy or post-mastectomy radiotherapy. The patient was placed on tamoxifen and has been free of disease to date.

The paracrine hormone for the GUCY2C tumor suppressor, guanylin, is universally lost in colorectal cancer.

BACKGROUND: Although colorectal cancer is a disease characterized by sequential accumulation of mutations in epithelial cells, mechanisms leading to genomic vulnerability contributing to tumor initiation remain undefined. GUCY2C has emerged as an intestine-specific tumor suppressor controlling epithelial homeostasis through circuits canonically disrupted in cancer. Surprisingly, the GUCY2C tumor suppressor is universally overexpressed by human colorectal cancer cells. This apparent paradox likely reflects silencing of GUCY2C through loss of its paracrine hormone guanylin. Here, we quantified expression of guanylin mRNA and protein in tumors and normal epithelia from patients with colorectal cancer. METHODS: Guanylin mRNA was quantified in tumors and normal adjacent epithelia from 281 patients by the reverse transcriptase-polymerase chain reaction. Separately, the guanylin protein was quantified by immunohistochemistry in 54 colorectal tumors and 30 specimens of normal intestinal epithelium. RESULTS: Guanylin mRNA in colorectum varied more than a 100-fold across the population. Guanylin mRNA was reduced 100- to 1,000-fold in >85% of tumors compared with matched normal adjacent mucosa (P < 0.001). Loss of guanylin mRNA was greatest in tumors from patients <50 years old (P < 0.005) and with the highest expression in normal adjacent mucosa (Spearman correlation coefficient = 0.61; P < 0.001). In a separate validation cohort, guanylin protein was detected in all 30 normal colorectal mucosa specimens, but in none of 54 colorectal tumors. CONCLUSIONS: Colorectal cancer may initiate as a disease of paracrine hormone insufficiency through loss of guanylin expression, silencing the GUCY2C tumor suppressor and disrupting homeostatic mechanisms regulating colorectal epithelia cells. IMPACT: Intestinal tumorigenesis may be prevented by oral GUCY2C hormone replacement therapy.

Prolactin-Stat5 signaling in breast cancer is potently disrupted by acidosis within the tumor microenvironment.

INTRODUCTION: Emerging evidence in estrogen receptor-positive breast cancer supports the notion that prolactin-Stat5 signaling promotes survival and maintenance of differentiated luminal cells, and loss of nuclear tyrosine phosphorylated Stat5 (Nuc-pYStat5) in clinical breast cancer is associated with increased risk of antiestrogen therapy failure. However, the molecular mechanisms underlying loss of Nuc-pYStat5 in breast cancer remain poorly defined. METHODS: We investigated whether moderate extracellular acidosis of pH 6.5 to 6.9 frequently observed in breast cancer inhibits prolactin-Stat5 signaling, using in vitro and in vivo experimental approaches combined with quantitative immunofluorescence protein analyses to interrogate archival breast cancer specimens. RESULTS: Moderate acidosis at pH 6.8 potently disrupted signaling by receptors for prolactin but not epidermal growth factor, oncostatin M, IGF1, FGF or growth hormone. In breast cancer specimens there was mutually exclusive expression of Nuc-pYStat5 and GLUT1, a glucose transporter upregulated in glycolysis-dependent carcinoma cells and an indirect marker of lactacidosis. Mutually exclusive expression of GLUT1 and Nuc-pYStat5 occurred globally or regionally within tumors, consistent with global or regional acidosis. All prolactin-induced signals and transcripts were suppressed by acidosis, and the acidosis effect was rapid and immediately reversible, supporting a mechanism of acidosis disruption of prolactin binding to receptor. T47D breast cancer xenotransplants in mice displayed variable acidosis (pH 6.5 to 6.9) and tumor regions with elevated GLUT1 displayed resistance to exogenous prolactin despite unaltered levels of prolactin receptors and Stat5. CONCLUSIONS: Moderate extracellular acidosis effectively blocks prolactin signaling in breast cancer. We propose that acidosis-induced prolactin resistance represents a previously unrecognized mechanism by which breast cancer cells may escape homeostatic control.

Global profiling of prolactin-modulated transcripts in breast cancer in vivo.

BACKGROUND: Prolactin (PRL) is essential for normal mammary gland development. PRL promotes mammary tumor formation in rodents and elevated serum prolactin is associated with increased risk of estrogen-receptor positive breast cancer in women. On the other hand, PRL may also exert pro-differentiation effects and act to suppress invasive features of established breast cancer. Previously published limited global transcript profiling analyses of prolactin-regulated gene expression in human breast cancer cells have exclusively been performed in vitro. The present study aimed to shed new light on how PRL modulates estrogen receptor (ER)-positive breast cancer through global transcript profiling of a human breast cancer xenograft model in vivo. METHODS: The prolactin-responsive human T47D breast cancer cell line was xenotransplanted into nude mice and global transcript profiling was carried out following treatment with or without human PRL for 48 h. A subset of PRL-modulated transcripts was further validated using qRT-PCR and immunohistochemistry. RESULTS: The in vivo analyses identified 130 PRL-modulated transcripts, 75 upregulated and 55 downregulated, based on fold change >1.6 and P-value <0.05. From this initial panel of transcripts, a subset of 18 transcripts with established breast cancer-relevance were selected and validated by qRT-PCR. Some but not all of the transcripts were also PRL-modulated in vitro. The selected PRL-modulated transcripts were tested for dependence on Stat5, Jak1 or Jak2 activation, and for co-regulation by 17ß-estradiol (E2). The protein encoded by one of the PRL-regulated transcripts, PTHrP, was examined in a panel of 92 human breast cancers and found by in situ quantitative immunofluorescence analysis to be highly positively correlated with nuclear localized and tyrosine phosphorylated Stat5. Gene Ontology analysis revealed that PRL-upregulated genes were enriched in pathways involved in differentiation. Finally, a gene signature based on PRL-upregulated genes was associated with prolonged relapse-free and metastasis-free survival in breast cancer patients. CONCLUSIONS: This global analysis identified and validated a panel of PRL-modulated transcripts in an ER-positive human breast cancer xenotransplant model, which may have value as markers of relapse-free and metastasis-free survival. Gene products identified in the present study may facilitate ongoing deciphering of the pleiotropic effects of PRL on human breast cancer.

STAT5A/B gene locus undergoes amplification during human prostate cancer progression.

The molecular mechanisms underlying progression of prostate cancer (PCa) to castrate-resistant (CR) and metastatic disease are poorly understood. Our previous mechanistic work shows that inhibition of transcription factor Stat5 by multiple alternative methods induces extensive rapid apoptotic death of Stat5-positive PCa cells in vitro and inhibits PCa xenograft tumor growth in nude mice. Furthermore, STAT5A/B induces invasive behavior of PCa cells in vitro and in vivo, suggesting involvement of STAT5A/B in PCa progression. Nuclear STAT5A/B protein levels are increased in high-grade PCas, CR PCas, and distant metastases, and high nuclear STAT5A/B expression predicts early disease recurrence and PCa-specific death in clinical PCas. Based on these findings, STAT5A/B represents a therapeutic target protein for advanced PCa. The mechanisms underlying increased Stat5 protein levels in PCa are unclear. Herein, we demonstrate amplification at the STAT5A/B gene locus in a significant fraction of clinical PCa specimens. STAT5A/B gene amplification was more frequently found in PCas of high histologic grades and in CR distant metastases. Quantitative in situ analysis revealed that STAT5A/B gene amplification was associated with increased STAT5A/B protein expression in PCa. Functional studies showed that increased STAT5A/B copy numbers conferred growth advantage in PCa cells in vitro and as xenograft tumors in vivo. The work presented herein provides the first evidence of somatic STAT5A/B gene amplification in clinical PCas.

Dormant cancer cells contribute to residual disease in a model of reversible pancreatic cancer.

The initiation and progression of pancreatic ductal adenocarcinoma (PDAC) is governed by a series of genetic and epigenetic changes, but it is still unknown whether these alterations are required for the maintenance of primary and metastatic PDAC. We show here that the c-Myc oncogene is upregulated throughout the entire process of neoplastic progression in human PDAC and in genetically engineered mice that express mutant Kras. To experimentally address whether c-Myc is essential for the growth and survival of cancer cells, we developed a novel mouse model that allows a temporally and spatially controlled expression of this oncogene in pancreatic progenitors and derived lineages of the exocrine pancreas. Unlike previous reports, upregulation of c-Myc was sufficient to induce the formation of adenocarcinomas after a short latency without additional genetic manipulation of cell survival pathways. Deficiency in Cdkn2a increased the rate of metastasis but had no effect on tumor latency or c-Myc-mediated cancer maintenance. Despite a macroscopically complete regression of primary, metastatic, and transplantable tumors following the ablation of c-Myc, some cancer cells remained dormant. A significant number of these residual neoplastic cells expressed cancer stem cell markers, and re-expression of exogenous c-Myc in these cells led to rapid cancer recurrence. Collectively, the results of this study suggest that c-Myc plays a significant role in the progression and maintenance of PDAC, but besides targeting this oncogene or its downstream effectors, additional therapeutic strategies are necessary to eradicate residual cancer cells to prevent disease recurrence.

Low levels of Stat5a protein in breast cancer are associated with tumor progression and unfavorable clinical outcomes.

INTRODUCTION: Signal transducer and activator of transcripton-5a (Stat5a) and its close homologue, Stat5b, mediate key physiological effects of prolactin and growth hormone in mammary glands. In breast cancer, loss of nuclear localized and tyrosine phosphorylated Stat5a/b is associated with poor prognosis and increased risk of antiestrogen therapy failure. Here we quantify for the first time levels of Stat5a and Stat5b over breast cancer progression, and explore their potential association with clinical outcome. METHODS: Stat5a and Stat5b protein levels were quantified in situ in breast-cancer progression material. Stat5a and Stat5b transcript levels in breast cancer were correlated with clinical outcome in 936 patients. Stat5a protein was further quantified in four archival cohorts totaling 686 patients with clinical outcome data by using multivariate models. RESULTS: Protein levels of Stat5a but not Stat5b were reduced in primary breast cancer and lymph node metastases compared with normal epithelia. Low tumor levels of Stat5a but not Stat5b mRNA were associated with poor prognosis. Experimentally, only limited overlap between Stat5a- and Stat5b-modulated genes was found. In two cohorts of therapy-naïve, node-negative breast cancer patients, low nuclear Stat5a protein levels were an independent marker of poor prognosis. Multivariate analysis of two cohorts treated with antiestrogen monotherapy revealed that low nuclear Stat5a levels were associated with a more than fourfold risk of unfavorable outcome. CONCLUSIONS: Loss of Stat5a represents a new independent marker of poor prognosis in node-negative breast cancer and may be a predictor of response to antiestrogen therapy if validated in randomized clinical trials.

Cyclin D3 compensates for the loss of cyclin D1 during ErbB2-induced mammary tumor initiation and progression.

Cyclin D1 regulates cell proliferation and is a candidate molecular target for breast cancer therapy. This study addresses whether Cyclin D1 is indispensable for ErbB2-associated mammary tumor initiation and progression using a breast cancer model in which this cell-cycle regulator can be genetically ablated prior to or after neoplastic transformation. Deficiency in Cyclin D1 delayed tumor onset but did not prevent the occurrence of mammary cancer in mice overexpressing wild-type ErbB2. The lack of Cyclin D1 was associated with a compensatory upregulation of Cyclin D3, which explains why the targeted downregulation of Cyclin D1 in established mammary tumors had no effect on cancer cell proliferation. Cyclin D1 and D3 are overexpressed in human breast cancer cell lines and primary invasive breast cancers, and Cyclin D3 frequently exceeded the expression of Cyclin D1 in ErbB2-positive cases. The simultaneous inhibition of both cyclins in mammary tumor cells reduced cancer cell proliferation in vitro and decreased the tumor burden in vivo. Collectively, the results of this study suggest that only the combined inhibition of Cyclin D1 and D3 might be a suitable strategy for breast cancer prevention and therapy.

Signal transducer and activator of transcription-3 and breast cancer prognosis.

Signal transducer and activator of transcription-3 (Stat3) is frequently activated in breast cancer and multiple lines of evidence suggest that Stat3 promotes tumor progression. However, the prognostic value of Stat3 in human breast cancer remains controversial and associations range from favorable to unfavorable based on four outcome studies of 62, 102, 255 and 517 patients. Cellular Stat3 protein expression was measured in three studies whereas nuclear localized, tyrosine phosphorylated Stat3 (Nuc-pYStat3) was used as the readout in only one study. We therefore retrospectively analyzed the prognostic value of Nuc-pYStat3 in a larger material of 721 breast cancer specimens. Overall, patients whose tumors were positive for Nuc-pYStat3 tended to have improved survival, but the trend did not reach statistical significance (P=0.08). When specimens were stratified by tumor grade, patients with low grade but not high grade tumors that were positive for Nuc-pYStat3 had significantly prolonged overall survival in univariate analysis (P=0.014) but not in multivariate analyses. Unexpectedly, quantitative immunofluoresence detection revealed highest levels of Nuc-pYStat3 in normal breast epithelia and gradual loss of Nuc-pYStat3 during progression from DCIS, invasive ductal carcinoma, and lymph node metastases. Levels of Nuc-pYStat3 correlated positively with levels of Nuc-pYStat5, a favorable prognostic marker, in invasive ductal carcinomas. Furthermore, NucpYStat3 levels correlated strongly with protein levels of nuclear localized Stat5a (r=0.633, P<0.001) but not Stat5b. Our data does not support the notion that Nuc-pYStat3 is an independent marker of prognosis in breast cancer, although future studies may reveal prognostic utility within molecularly characterized subtypes of breast cancer.

Loss of nuclear localized and tyrosine phosphorylated Stat5 in breast cancer predicts poor clinical outcome and increased risk of antiestrogen therapy failure.

PURPOSE: To investigate nuclear localized and tyrosine phosphorylated Stat5 (Nuc-pYStat5) as a marker of prognosis in node-negative breast cancer and as a predictor of response to antiestrogen therapy. PATIENTS AND METHODS: Levels of Nuc-pYStat5 were analyzed in five archival cohorts of breast cancer by traditional diaminobenzidine-chromogen immunostaining and pathologist scoring of whole tissue sections or by immunofluorescence and automated quantitative analysis (AQUA) of tissue microarrays. RESULTS: Nuc-pYStat5 was an independent prognostic marker as measured by cancer-specific survival (CSS) in patients with node-negative breast cancer who did not receive systemic adjuvant therapy, when adjusted for common pathology parameters in multivariate analyses both by standard chromogen detection with pathologist scoring of whole tissue sections (cohort I; n = 233) and quantitative immunofluorescence of a tissue microarray (cohort II; n = 291). Two distinct monoclonal antibodies gave concordant results. A progression array (cohort III; n = 180) revealed frequent loss of Nuc-pYStat5 in invasive carcinoma compared to normal breast epithelia or ductal carcinoma in situ, and general loss of Nuc-pYStat5 in lymph node metastases. In cohort IV (n = 221), loss of Nuc-pYStat5 was associated with increased risk of antiestrogen therapy failure as measured by univariate CSS and time to recurrence (TTR). More sensitive AQUA quantification of Nuc-pYStat5 in antiestrogen-treated patients (cohort V; n = 97) identified by multivariate analysis patients with low Nuc-pYStat5 at elevated risk for therapy failure (CSS hazard ratio [HR], 21.55; 95% CI, 5.61 to 82.77; P < .001; TTR HR, 7.30; 95% CI, 2.34 to 22.78; P = .001). CONCLUSION Nuc-pYStat5 is an independent prognostic marker in node-negative breast cancer. If confirmed in prospective studies, Nuc-pYStat5 may become a useful predictive marker of response to adjuvant hormone therapy.