SNP of Aromatase Predict Long-term Survival and Aromatase Inhibitor Toxicity in Patients with Early Breast Cancer: A Biomarker Analysis of the GIM4 and GIM5 Trials.

PURPOSE: In estrogen receptor-positive (ER+) breast cancer, single-nucleotide polymorphisms (SNP) in the aromatase gene might affect aromatase inhibitors (AI) metabolism and efficacy. Here, we assessed the impact of SNP on prognosis and toxicity of patients receiving adjuvant letrozole. EXPERIMENTAL DESIGN: We enrolled 886 postmenopausal patients in the study. They were treated with letrozole for 2 to 5 years after taking tamoxifen for 2 to 6 years, continuing until they completed 5 to 10 years of therapy. Germline DNA was genotyped for SNP rs4646, rs10046, rs749292, and rs727479. Log-rank test and Cox model were used for disease-free survival (DFS) and overall survival (OS). Cumulative incidence (CI) of breast cancer metastasis was assessed through competing risk analysis, with contralateral breast cancer, second malignancies and non-breast cancer death as competing events. CI of skeletal and cardiovascular events were assessed using DFS events as competing events. Subdistribution HR (sHR) with 95% confidence intervals were calculated through Fine-Gray method. RESULTS: No SNP was associated with DFS. Variants rs10046 [sHR 2.03, (1.04-2.94)], rs749292 [sHR 2.11, (1.12-3.94)], and rs727479 [sHR 2.62, (1.17-5.83)] were associated with breast cancer metastasis. Three groups were identified on the basis of the number of these variants (0, 1, >1). Variant-based groups were associated with breast cancer metastasis (10-year CI 2.5%, 7.6%, 10.7%, P = 0.035) and OS (10-year estimates 96.5%, 93.0%, 89.6%, P = 0.030). Co-occurrence of rs10046 and rs749292 was negatively associated with 10-year CI of skeletal events (3.2% vs. 10%, P = 0.033). A similar association emerged between rs727479 and cardiovascular events (0.3% vs. 2.1%, P = 0.026). CONCLUSIONS: SNP of aromatase gene predict risk of metastasis and AI-related toxicity in ER+ early breast cancer, opening an opportunity for better treatment individualization.

The Role of Cyclin-Dependent Kinases (CDK) 4/6 in the Ovarian Tissue and the Possible Effects of Their Exogenous Inhibition.

The combination of cyclin-dependent kinase (CDK) 4/6 inhibitors with endocrine therapy is the standard treatment for patients with HR+/HER2- advanced breast cancer. Recently, this combination has also entered the early setting as an adjuvant treatment in patients with HR+/HER2- disease at a high risk of disease recurrence following (neo)adjuvant chemotherapy. Despite their current use in clinical practice, limited data on the potential gonadotoxicity of CDK4/6 inhibitors are available. Hence, fully informed treatment decision making by premenopausal patients concerned about the potential development of premature ovarian insufficiency and infertility with the proposed therapy remains difficult. The cell cycle progression of granulosa and cumulus cells is a critical process for ovarian function, especially for ensuring proper follicular growth and acquiring competence. Due to the pharmacological properties of CDK4/6 inhibitors, there could be a potentially negative impact on ovarian function and fertility in women of reproductive age. This review aims to summarize the role of the cyclin D-CDK4 and CDK6 complexes in the ovary and the potential impact of CDK4/6 inhibition on its physiological processes.

Anthracycline, taxane, and trastuzumab-based neoadjuvant chemotherapy in HER2-positive early breast cancer: phase II trial.

OBJECTIVE: Neoadjuvant chemotherapy has become the preferred treatment in HER2-positive early breast cancer. Several trials investigated the neoadjuvant efficacy of dual HER2 blockade with anthracycline-free chemotherapy, whereas few data are available on single-agent trastuzumab and anthracycline-based regimens, which represent the standard of care in the adjuvant setting. This phase II, single-arm trial assessed anthracycline-based chemotherapy and trastuzumab as neoadjuvant treatment for high-risk HER2-positive breast cancer. METHODS: Forty-three patients with stage II-III HER2-positive breast cancer were treated with 4 courses of neoadjuvant 5-fluorouracil 600 mg/m2, epirubicin 90 mg/m2, cyclophosphamide 600 mg/m2 (FEC ×4) every 21 days, followed by 12 courses of weekly paclitaxel 80 mg/m2 and trastuzumab 2 mg/Kg IV (loading dose 4 mg/kg). RESULTS: Pathologic complete response (pCR) was observed in 22 (51%) of 43 patients. After a median follow-up of 6 years, the 5-year disease-free survival and overall survival were 85.8% (95% confidence interval 75.9%-97%) and 89.6% (80.4%-99.8%), respectively. A temporary decrease in left ventricular ejection fraction was observed in two patients. No cardiac death or congestive heart failure occurred. One patient died due to febrile neutropenia. CONCLUSIONS: FEC ×4 followed by paclitaxel and trastuzumab was associated with high pCR rates and favorable long-term outcomes. However, this regimen was associated with relevant hematologic toxicity.

Fraisinib: a calixpyrrole derivative reducing A549 cell-derived NSCLC tumor in vivo acts as a ligand of the glycine-tRNA synthase, a new molecular target in oncology.

Background and purpose: Lung cancer is the leading cause of death in both men and women, constituting a major public health problem worldwide. Non-small-cell lung cancer accounts for 85%-90% of all lung cancers. We propose a compound that successfully fights tumor growth in vivo by targeting the enzyme GARS1. Experimental approach: We present an in-depth investigation of the mechanism through which Fraisinib [meso-(p-acetamidophenyl)-calix(4)pyrrole] affects the human lung adenocarcinoma A549 cell line. In a xenografted model of non-small-cell lung cancer, Fraisinib was found to reduce tumor mass volume without affecting the vital parameters or body weight of mice. Through a computational approach, we uncovered that glycyl-tRNA synthetase is its molecular target. Differential proteomics analysis further confirmed that pathways regulated by Fraisinib are consistent with glycyl-tRNA synthetase inhibition. Key results: Fraisinib displays a strong anti-tumoral potential coupled with limited toxicity in mice. Glycyl-tRNA synthetase has been identified and validated as a protein target of this compound. By inhibiting GARS1, Fraisinib modulates different key biological processes involved in tumoral growth, aggressiveness, and invasiveness. Conclusion and implications: The overall results indicate that Fraisinib is a powerful inhibitor of non-small-cell lung cancer growth by exerting its action on the enzyme GARS1 while displaying marginal toxicity in animal models. Together with the proven ability of this compound to cross the blood-brain barrier, we can assess that Fraisinib can kill two birds with one stone: targeting the primary tumor and its metastases "in one shot." Taken together, we suggest that inhibiting GARS1 expression and/or GARS1 enzymatic activity may be innovative molecular targets for cancer treatment.

Targeting PIK3CA Actionable Mutations in the Circulome: A Proof of Concept in Metastatic Breast Cancer.

The study of circulating cancer-derived components (circulome) is considered the new frontier of liquid biopsy. Despite the recognized role of circulome biomarkers, their comparative molecular profiling is not yet routine. In advanced breast cancer (BC), approximately 40% of hormone-receptor-positive, HER2-negative BC cases harbor druggable PIK3CA mutations suitable for combined alpelisib/fulvestrant treatment. This pilot study investigates PIK3CA mutations in circulating tumor DNA (ctDNA), tumor cells (CTCs), and extracellular vesicles (EVs) with the aim of determining which information on molecular targetable profiling could be recollected in each of them. The in-depth molecular analysis of four BC patients demonstrated, as a proof-of-concept study, that it is possible to retrieve mutational information in the three components. Patient-specific PIK3CA mutations were found in both tissue and ctDNA and in 3/4 cases, as well as in CTCs, in the classical population (large-sized CD45-/EpCAM+/- cells), and/or in the "non-conventional" sub-population (smaller-sized CD44+/EpCAM-/CD45- cells). Consistent mutational profiles of EVs with CTCs suggest that they may have been released by CTCs. This preliminary evidence on the molecular content of the different circulating biomaterials suggests their possible function as a mirror of the intrinsic heterogeneity of BC. Moreover, this study demonstrates, through mutational assessment, the tumor origin of the different CTC sub-populations sustaining the translational value of the circulome for a more comprehensive picture of the disease.

Circulating miRNAs in Breast Cancer Diagnosis and Prognosis.

Great improvement has been made in the diagnosis and therapy of breast cancer patients. However, the identification of biomarkers for early diagnosis, prognosis, therapy assessment and monitoring, including drug resistance and the early detection of micro-metastases, is still lacking. Recently, circulating microRNAs (miRNAs), circulating freely in the blood stream or entrapped in extracellular vesicles (EVs), have been shown to have a potential diagnostic, prognostic or predictive power. In this review, recent findings are summarized, both at a preclinical and clinical level, related to miRNA applicability in the context of breast cancer. Different aspects, including clinical and technical challenges, are discussed, describing the potentialities of miRNA use in breast cancer. Even though more methodological standardized studies conducted in larger and selected patient cohorts are needed to support the effective clinical utility of miRNA as biomarkers, they could represent novel and accessible tools to be transferred into clinical practice.

Clinical outcomes of patients with breast cancer relapsing after (neo)adjuvant trastuzumab and receiving trastuzumab rechallenge or lapatinib-based therapy: a multicentre retrospective cohort study.

BACKGROUND: In the prepertuzumab era, we evaluated the clinical outcomes of patients with human epidermal growth factor receptor 2 (HER2)-positive metastatic breast cancer who underwent first-line trastuzumab-based or lapatinib-based therapy according to prior exposure to (neo)adjuvant trastuzumab. MATERIALS AND METHODS: In this multicentre retrospective cohort study conducted in 14 Italian centres of the Gruppo Italiano Mammella, consecutive patients undergoing first-line trastuzumab or lapatinib-based therapy were included. Analyses were performed according to the type of first-line therapy for metastatic disease (trastuzumab or lapatinib). Dichotomous clinical outcomes were analysed using logistic regression and time-to-event outcomes using Cox proportional hazard models controlling for relevant demographic, clinicopathological and therapy characteristics. RESULTS: Out of 450 patients included in the study, 416 (92%) received trastuzumab and 34 (7.5%) lapatinib. As compared with the trastuzumab cohort, more patients in the lapatinib cohort had a trastuzumab-free interval <1 month (37% vs 13.9%; p=0.017) and brain metastasis as first site of relapse (38.2% vs 9.4%; p<0.001). Among the 128 patients who relapsed after prior (neo)adjuvant trastuzumab, 101 (78.9%) received first-line trastuzumab and 27 (21.1%) first-line lapatinib. The following outcomes were observed with first-line lapatinib or trastuzumab, respectively: overall response rate 45.5% vs 61.3% (p=0.184), clinical benefit rate 68.2% vs 72.5% (p=0.691), median progression-free survival (PFS) 11.4 vs 12.0 months (p=0.814) and median overall survival (OS) 34.7 vs 48.2 months (p=0.722). In patients with brain metastasis as first site of relapse, median PFS was 12.2 vs 9.9 months (p=0.093) and median OS 33.7 vs 28.5 months (p=0.280), respectively. CONCLUSIONS: In patients with HER2-positive breast cancer relapsing after prior (neo)adjuvant trastuzumab, first-line treatment with trastuzumab or lapatinib was not associated with a significant difference in the clinical outcomes. A non-significant trend favouring the use of lapatinib was observed in patients with brain metastasis as the first site of relapse.

Effect of dose-dense adjuvant chemotherapy in hormone receptor positive/HER2-negative early breast cancer patients according to immunohistochemically defined luminal subtype: an exploratory analysis of the GIM2 trial.

BACKGROUND: Luminal A-like and luminal B-like subtypes have different sensitivity to (neo)adjuvant chemotherapy, but their role in predicting dose-dense (DD) efficacy in the high-risk setting is unknown. In this exploratory analysis of the Gruppo Italiano Mammella 2 (GIM2) trial, we investigated DD efficacy according to luminal-like subtypes. METHODS: Patients with node-positive early breast cancer were randomised to receive either DD or standard-interval (SI) anthracycline-based chemotherapy followed by paclitaxel. In our analysis, luminal A-like cohort was identified as having a Ki67 < 20% and a progesterone receptor (PgR) ≥ 20%; luminal B-like cohort as having a Ki67 ≥ 20% and/or a PgR < 20%. RESULTS: Out of 2003 patients enrolled in the GIM2 trial, 412 had luminal A-like and 638 luminal B-like breast cancer. After a median follow-up of 7.9 years, disease-free survival (DFS) was 80.8% (95% confidence interval [CI] 76.4-84.5) and 70.5% (66.5-74.2) in luminal A-like and luminal B-like cohorts; overall survival (OS) was 91.6% (88.2-94.1) and 85.1% (81.7-87.9), respectively. We found no significant interaction between treatment and luminal subtype (interaction p = 0.603 and 0.535 for DFS and OS, respectively). When DD efficacy was investigated separately in each cohort, luminal-B like cohort appeared to benefit more from the DD schedule both in terms of DFS (unadjusted hazard ratio [HR] 0.72 [95% CI 0.54-0.96]) and OS (unadjusted HR 0.61 [95% CI 0.40-0.94]), compared with the luminal A-like cohort (unadjusted HR for DFS 0.89 [95% CI 0.59-1.33]; unadjusted HR for OS 0.83 [95% CI 0.45-1.54]). CONCLUSIONS: No significant interaction between luminal-like subtype and treatment was observed. Patients in the luminal B-like cohort seemed to benefit more from DD schedule.

Optimization of a WGA-Free Molecular Tagging-Based NGS Protocol for CTCs Mutational Profiling.

Molecular characterization of Circulating Tumor Cells (CTCs) is still challenging, despite attempts to minimize the drawbacks of Whole Genome Amplification (WGA). In this paper, we propose a Next-Generation Sequencing (NGS) optimized protocol based on molecular tagging technology, in order to detect CTCs mutations while skipping the WGA step. MDA-MB-231 and MCF-7 cell lines, as well as leukocytes, were sorted into pools (2-5 cells) using a DEPArray™ system and were employed to set up the overall NGS procedure. A substantial reduction of reagent volume for the preparation of libraries was performed, in order to fit the limited DNA templates directly derived from cell lysates. Known variants in TP53, KRAS, and PIK3CA genes were detected in almost all the cell line pools (35/37 pools, 94.6%). No additional alterations, other than those which were expected, were found in all tested pools and no mutations were detected in leukocytes. The translational value of the optimized NGS workflow is confirmed by sequencing CTCs pools isolated from eight breast cancer patients and through the successful detection of variants. In conclusion, this study shows that the proposed NGS molecular tagging approach is technically feasible and, compared to traditional NGS approaches, has the advantage of filtering out the artifacts generated during library amplification, allowing for the reliable detection of mutations and, thus, making it highly promising for clinical use.

Gonadotropin Releasing Hormone Agonists Have an Anti-apoptotic Effect on Cumulus Cells.

BACKGROUND: Ovaries are sensitive to chemotherapy, which may lead to early depletion of primordial follicle reserve. One strategy for gonadal function preservation is temporary ovarian suppression with Gonadotropin Releasing Hormone agonists (GnRHa) during chemotherapy. To date, GnRHa protective mechanism of action remains not fully elucidated. METHODS: We collected 260 immature cumulus cell-oocyte complexes (COC) from 111 women < 38 years old, with a normal ovarian reserve. The COC were randomly assigned to the following groups: a) control; culture with the addition of b) GnRHa; c) cyclophosphamide; d) cyclophosphamide plus GnRHa. After in vitro treatments, RNA and proteins were extracted from oocytes and cumulus cells (CC), separately. Potential effects of drugs were evaluated on GnRH receptors, apoptosis pathways, ceramide pathway, and glutathione synthesis by quantitative PCR and, whenever possible, by Western blot. RESULTS: Cyclophosphamide triggered activation of the extrinsic pathway of apoptosis mediated by BAX in CC. The co-administration of GnRHa inhibited the apoptosis pathway in CC. According to our model, the GnRHa does not directly act on oocytes, which do not express GnRH receptors. Moreover, glutathione synthesis was decreased after GnRHa treatment both in CC and oocytes. CONCLUSION: Our data suggest that the protective mechanisms induced by GnRHa is mediated by an anti-apoptotic effect on CC.

Increases in Tumor N-Glycan Polylactosamines Associated with Advanced HER2-Positive and Triple-Negative Breast Cancer Tissues.

PURPOSE: Using a recently developed matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) method, human breast cancer formalin-fixed paraffin-embedded (FFPE) tissue sections and tissue microarrays (TMA) are evaluated for N-linked glycan distribution in the tumor microenvironment. EXPERIMENTAL DESIGN: Tissue sections representing multiple human epidermal growth factor receptor 2 (HER2) receptor-positive and triple-negative breast cancers (TNBC) in both TMA and FFPE slide format are processed for high resolution N-glycan MALDI-IMS. An additional FFPE tissue cohort of primary and metastatic breast tumors from the same donors are also evaluated. RESULTS: The cumulative N-glycan MALDI-IMS analysis of breast cancer FFPE tissues and TMAs indicate the distribution of specific glycan structural classes to stromal, necrotic, and tumor regions. A series of high-mannose, branched and fucosylated glycans are detected predominantly within tumor regions. Additionally, a series of polylactosamine glycans are detected in advanced HER2+, TNBC, and metastatic breast cancer tissues. Comparison of tumor N-glycan species detected in paired primary and metastatic tissues indicate minimal changes between the two conditions. CONCLUSIONS AND CLINICAL RELEVANCE: The prevalence of tumor-associated polylactosamine glycans in primary and metastatic breast cancer tissues indicates new mechanistic insights into the development and progression of breast cancers. The presence of these glycans could be targeted for therapeutic strategies and further evaluation as potential prognostic biomarkers.

Purificación del fibrinógeno gamma A/gamma prima (үA/ү') por cromatografía líquida rápida de proteínas / Purification of fibrinogen gamma A/gamma prima (үA/ү') by fast protein liquid chromatography / Purificação do fibrinogênio gamma A/gamma prima (үA/ү') por cromatografía liquida rápida de proteínas

Resumen Una fracción del fibrinógeno circulante contiene una variante de la cadena ү que se origina por empalme alternativo del ARNm, denominada ү' cuya concentración en plasma se ha relacionado con un incremento en el riesgo de padecer enfermedades cardiovasculares. Por tanto, el objetivo de este trabajo fue diseñar un método de purificación del fibrinógeno үA/ү' más eficiente en relación a los descritos en la literatura, a partir de plasma humano. Se purificó el fibrinógeno үA/ү' a partir del fibrinógeno total obtenido por precipitación con β-alanina, mediante la separación por cromatografía líquida rápida de proteínas. Se confirmó la presencia de fibrinógeno үA/ү' mediante Western blot; su concentración fue determinada por ELISA. El método mostró ventajas en comparación con los métodos clásicos de separación, por ejemplo, que cantidades menores de muestra pudieron ser fraccionadas cuantitativamente en componentes puros en menor tiempo (30 min). Por tanto, se puede concluir que la técnica utilizada para la purificación de las variantes del fibrinógeno, correspondiente al Fg үA/үA y Fg үA/ү', es un método de separación eficiente que permite purificar el Fg үA/ү' libre de contaminantes principales, como lo confirma la inmunoelectroforesis.

Abstract A fraction of the circulating fibrinogen contains a variant of the y chain that is originated by mRNA alternative splicing denominated ү' whose concentration in plasma has been related to an increase in the risk of cardiovascular diseases. Thus, the objective of this work was to design a more efficient үA/ү' fibrinogen purification method in relation to those described in the literature from human plasma. The үA/ү' fibrinogen was purified from the total fibrinogen obtained by precipitation with β-alanine, using separation by fast protein liquid chromatography. Fibrinogen үA/ү', was confirmed by Western blot and its concentration was determined by ELISA. The method showed advantages compared to classical separation methods, for example, smaller amounts of sample could be fractionated quantitatively into pure components in less time (30 min). Therefore, it can be concluded that the technique used for the purification of fibrinogen variants, corresponding to Fg үA/үA and Fg үA/ү', is an efficient separation method that allows purifying the Fg үA/ү' free of main contaminants, as confirmed by immunoelectrophoresis.

Resumo Uma fracção do fibrinogênio circulante contém uma variante da cadeia y que origina-se por junção alternativa do ARNm, chamada ү' cuja concentração em plasma está asociada com um incremento no resgo de sofrer doenças cardiovasculares. Asim, o objetivo em este trabalho foi desenhar um método de purificação do fibrinogênio үA/ү' mais eficiente em relação aos que estão descritos na literatura, a partir do plasma humano. Purificou-se o fibrinogênio үA/ү' a partir do fibrinogênio total conseguido pela precipitação com β-alanina, através da separação por cromatografia líquida rápida de proteínas. Se confirmou por Western blot o fibrinogênio үA/ү'. A sua concentração foi determinada por ELISA. O método apresenta vantagens em comparação com os métodos clássicos de separação, por exemplo, quantidades menores de mostra podem ser fraccionadas quantitativamente em componentes puros, em tempos mais curtos (30 min). Entao, pode-se concluir que a técnica utilizada, para a purificação das variantes do fibrinogênio correspondente ao Fg үA/үA e Fg үA/ү', é um método de separação eficiente que permite purificar o Fg үA/ү' livre de contaminantes principais, como o confirma a imunoeletroforese.

Analysis of in vitro ADCC and clinical response to trastuzumab: possible relevance of FcγRIIIA/FcγRIIA gene polymorphisms and HER-2 expression levels on breast cancer cell lines.

BACKGROUND: Trastuzumab is a humanized monoclonal antibody (mAb) currently used for the treatment of breast cancer (BC) patients with HER-2 overexpressing tumor subtype. Previous data reported the involvement of FcγRIIIA/IIA gene polymorphisms and/or antibody-dependent cellular cytotoxicity (ADCC) in the therapeutic efficacy of trastuzumab, although results on these issues are still controversial. This study was aimed to evaluate in vitro the functional relationships among FcγRIIIA/IIA polymorphisms, ADCC intensity and HER-2 expression on tumor target cells and to correlate them with response to trastuzumab. PATIENTS AND METHODS: Twenty-five patients with HER-2 overexpressing BC, receiving trastuzumab in a neoadjuvant (NEO) or metastatic (MTS) setting, were genotyped for the FcγRIIIA 158V>F and FcγRIIA 131H>R polymorphisms by a newly developed pyrosequencing assay and by multiplex Tetra-primer-ARMS PCR, respectively. Trastuzumab-mediated ADCC of patients' peripheral blood mononuclear cells (PBMCs) was evaluated prior to therapy and measured by (51)Chromium release using as targets three human BC cell lines showing different levels of reactivity with trastuzumab. RESULTS: We found that the FcγRIIIA 158F and/or the FcγRIIA 131R variants, commonly reported as unfavorable in BC, may actually behave as ADCC favorable genotypes, in both the NEO (P ranging from 0.009 to 0.039 and from 0.007 to 0.047, respectively) and MTS (P ranging from 0.009 to 0.032 and P = 0.034, respectively) patients. The ADCC intensity was affected by different levels of trastuzumab reactivity with BC target cells. In this context, the MCF-7 cell line, showing the lowest reactivity with trastuzumab, resulted the most suitable cell line for evaluating ADCC and response to trastuzumab. Indeed, we found a statistically significant correlation between an increased frequency of patients showing ADCC of MCF-7 and complete response to trastuzumab in the NEO setting (P = 0.006). CONCLUSIONS: Although this study was performed in a limited number of patients, it would indicate a correlation of FcγR gene polymorphisms to the ADCC extent in combination with the HER-2 expression levels on tumor target cells in BC patients. However, to confirm our findings further experimental evidences obtained from a larger cohort of BC patients are mandatory.

Glycogen synthase kinase 3 regulates cell death and survival signaling in tumor cells under redox stress.

Targeting tumor-specific metabolic adaptations is a promising anticancer strategy when tumor defense mechanisms are restrained. Here, we show that redox-modulating drugs including the retinoid N-(4-hydroxyphenyl)retinamide (4HPR), the synthetic triterpenoid bardoxolone (2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid methyl ester), arsenic trioxide (As2O3), and phenylethyl isothiocyanate (PEITC), while affecting tumor cell viability, induce sustained Ser9 phosphorylation of the multifunctional kinase glycogen synthase kinase 3ß (GSK3ß). The antioxidant N-acetylcysteine decreased GSK3ß phosphorylation and poly(ADP-ribose) polymerase cleavage induced by 4HPR, As2O3, and PEITC, implicating oxidative stress in these effects. GSK3ß phosphorylation was associated with up-regulation of antioxidant enzymes, in particular heme oxygenase-1 (HO-1), and transient elevation of intracellular glutathione (GSH) in cells surviving acute stress, before occurrence of irreversible damage and death. Genetic inactivation of GSK3ß or transfection with the non-phosphorylatable GSK3ß-S9A mutant inhibited HO-1 induction under redox stress, while tumor cells resistant to 4HPR exhibited increased GSK3ß phosphorylation, HO-1 expression, and GSH levels. The above-listed findings are consistent with a role for sustained GSK3ß phosphorylation in a signaling network activating antioxidant effector mechanisms during oxidoreductive stress. These data underlie the importance of combination regimens of antitumor redox drugs with inhibitors of survival signaling to improve control of tumor development and progression and overcome chemoresistance.

Trastuzumab quantification in serum: a new, rapid, robust ELISA assay based on a mimetic peptide that specifically recognizes trastuzumab.

Trastuzumab, a humanized monoclonal antibody directed against the epidermal growth factor receptor 2 (HER2), is a milestone in the treatment of HER2-overexpressing breast cancer patients. An enzyme-linked immunosorbent assay (ELISA) for trastuzumab has been developed for routine use in the laboratory to support clinical and pharmacokinetic studies to optimize therapy. The method relies on an antigen peptide linked to a 96-well plate via the streptavidin/biotin system. The peptide sequence mimics the extracellular portion of the HER2 receptor that is recognized by trastuzumab. The calibration range of the assay is 10 to 360 ng/mL per well, corresponding to a trastuzumab serum concentration from 5 to 180 µg/mL with a lower limit of quantification of 10 µg/mL. Validation results demonstrate that trastuzumab can be accurately and precisely quantified in human serum using this assay. The procedure was also tested in sera obtained from breast cancer patients to evaluate trastuzumab serum levels, confirming the applicability of method that could be a valid assay to use in daily laboratory practice.

Hydrodynamic characterization of recombinant human fibrinogen species.

INTRODUCTION: Fibrinogen is a key component of the blood coagulation system and plays important, diverse roles in several relevant pathologies such as thrombosis, hemorrhage, and cancer. It is a large glycoprotein whose three-dimensional molecular structure is not fully known. Furthermore, circulating fibrinogen is highly heterogeneous, mainly due to proteolytic degradation and alternative mRNA processing. Recombinant production of human fibrinogen allows investigating the impact on the three-dimensional structure of specific changes in the primary structure. METHODS: We performed analytical ultracentrifugation analyses of a full-length recombinant human fibrinogen, its counterpart purified from human plasma, and a recombinant human fibrinogen with both Aα chains truncated at amino acid 251, thus missing their last 359 amino acid residues. RESULTS: We have accurately determined the translational diffusion and sedimentation coefficients (Dt(20,w)(0), s(20,w)(0)) of all three species. This was confirmed by derived molecular weights within 1% for the full length species, and 5% for the truncated species, as assessed by comparison with SDS-PAGE/Western blot analyses and primary structure data. No significant differences in the values of Dt(20,w)(0) and s(20,w)(0) were found between the recombinant and purified full length human fibrinogens, while slightly lower and higher values, respectively, resulted for the recombinant truncated human fibrinogen compared to a previously characterized purified human fibrinogen fragment X obtained by plasmin digestion. CONCLUSIONS: Full-length recombinant fibrinogen is less polydisperse but hydrodynamically indistinguishable from its counterpart purified from human plasma. Recombinant Aα251-truncated human fibrinogen instead behaves differently from fragment X, suggesting a role for the Bß residues 1-52 in inter-molecular interactions. Overall, these new hydrodynamic data will constitute a reliable benchmark against which models of fibrinogen species could be compared.

Modeling of fibrin gels based on confocal microscopy and light-scattering data.

Fibrin gels are biological networks that play a fundamental role in blood coagulation and other patho/physiological processes, such as thrombosis and cancer. Electron and confocal microscopies show a collection of fibers that are relatively monodisperse in diameter, not uniformly distributed, and connected at nodal points with a branching order of ∼3-4. Although in the confocal images the hydrated fibers appear to be quite straight (mass fractal dimension D(m) = 1), for the overall system 1, joined at randomly distributed nodal points. The resulting 3D network strikingly resembles real fibrin gels and can be sketched as an assembly of densely packed fractal blobs, i.e., regions of size ξ, where the fiber concentration is higher than average. The blobs are placed at a distance ξ0 between their centers of mass so that they are overlapped by a factor η =ξ/ξ0 and have D(m) ∼1.2-1.6. The in silico gels' structure is quantitatively analyzed by its 3D spatial correlation function g(3D)(r) and corresponding power spectrum I(q) = FFT(3D[g3D(r)]), from which ρ, d, D(m), η, and ξ0 can be extracted. In particular, ξ0 provides an excellent estimate of the gel mesh size. The in silico gels' I(q) compares quite well with real gels' elastic light-scattering measurements. We then derived an analytical form factor for accurately fitting the scattering data, which allowed us to directly recover the gels' structural parameters.

The role of unstructured extensions in the rotational diffusion properties of a globular protein: the example of the titin i27 module.

The possibility of predicting the overall shape of a macromolecule in solution from its diffusional properties has gained increasing importance in the structural genomic era. Here we explore and quantify the influence that unstructured and flexible regions have on the motions of a globular protein, a situation that can occur from the presence of such regions in the natural sequence or from additional tags. I27, an immunoglobulin-like module from the muscle protein titin, whose structure and properties are well characterized, was selected for our studies. The backbone dynamics and the overall tumbling of three different constructs of I27 were investigated using (15)N NMR relaxation collected at two (15)N frequencies (60.8 and 81.1 MHz) and fluorescence depolarization spectroscopy after labeling of a reactive cysteine with an extrinsic fluorophore. Our data show that the presence of disordered tags clearly exerts a frictional drag that increases with the length of the tags, thus affecting the module tumbling in solution. We discuss the use and the limitations of current approaches to hydrodynamic calculations, especially when having to take into account local flexibility.