Giulia Federici

TAZ is required for metastatic activity and chemoresistance of breast cancer stem cells.

Metastatic growth in breast cancer (BC) has been proposed as an exclusive property of cancer stem cells (CSCs). However, formal proof of their identity as cells of origin of recurrences at distant sites and the molecular events that may contribute to tumor cell dissemination and metastasis development are yet to be elucidated. In this study, we analyzed a set of patient-derived breast cancer stem cell (BCSC) lines. We found that in vitro BCSCs exhibit a higher chemoresistance and migratory potential when compared with differentiated, nontumorigenic, breast cancer cells (dBCCs). By developing an in vivo metastatic model simulating the disease of patients with early BC, we observed that BCSCs is the only cell population endowed with metastatic potential. Gene-expression profile studies comparing metastagenic and non-metastagenic cells identified TAZ, a transducer of the Hippo pathway and biomechanical cues, as a central mediator of BCSCs metastatic ability involved in their chemoresistance and tumorigenic potential. Overexpression of TAZ in low-expressing dBCCs induced cell transformation and conferred tumorigenicity and migratory activity. Conversely, loss of TAZ in BCSCs severely impaired metastatic colonization and chemoresistance. In clinical data from 99 BC patients, high expression levels of TAZ were associated with shorter disease-free survival in multivariate analysis, thus indicating that TAZ may represent a novel independent negative prognostic factor. Overall, this study designates TAZ as a novel biomarker and a possible therapeutic target for BC.

Systems Analysis of the NCI-60 Cancer Cell Lines by Alignment of Protein Pathway Activation Modules with “-OMIC” Data Fields and Therapeutic Response Signatures

The NCI-60 cell line set is likely the most molecularly profiled set of human tumor cell lines in the world. However, a critical missing component of previous analyses has been the inability to place the massive amounts of “-omic” data in the context of functional protein signaling networks, which often contain many of the drug targets for new targeted therapeutics. We used reverse-phase protein array (RPPA) analysis to measure the activation/phosphorylation state of 135 proteins, with a total analysis of nearly 200 key protein isoforms involved in cell proliferation, survival, migration, adhesion, etc., in all 60 cell lines. We aggregated the signaling data into biochemical modules of interconnected kinase substrates for 6 key cancer signaling pathways: AKT, mTOR, EGF receptor (EGFR), insulin-like growth factor-1 receptor (IGF-1R), integrin, and apoptosis signaling. The net activation state of these protein network modules was correlated to available individual protein, phosphoprotein, mutational, metabolomic, miRNA, transcriptional, and drug sensitivity data. Pathway activation mapping identified reproducible and distinct signaling cohorts that transcended organ-type distinctions. Direct correlations with the protein network modules involved largely protein phosphorylation data but we also identified direct correlations of signaling networks with metabolites, miRNA, and DNA data. The integration of protein activation measurements into biochemically interconnected modules provided a novel means to align the functional protein architecture with multiple “-omic” data sets and therapeutic response correlations. This approach may provide a deeper understanding of how cellular biochemistry defines therapeutic response. Such “-omic” portraits could inform rational anticancer agent screenings and drive personalized therapeutic approaches. Mol Cancer Res; 11(6); 676–85. ©2013 AACR.

Proliferation State and Polo‐Like Kinase1 Dependence of Tumorigenic Colon Cancer Cells

Tumor‐initiating cells are responsible for tumor maintenance and relapse in solid and hematologic cancers. Although tumor‐initiating cells were initially believed to be mainly quiescent, rapidly proliferating tumorigenic cells were found in breast cancer. In colon cancer, the proliferative activity of the tumorigenic population has not been defined, although it represents an essential parameter for the development of more effective therapeutic strategies. Here, we show that tumorigenic colon cancer cells can be found in a rapidly proliferating state in vitro and in vivo, both in human tumors and mouse xenografts. Inhibitors of polo‐like kinase1 (Plk1), a mitotic kinase essential for cell proliferation, demonstrated maximal efficiency over other targeted compounds and chemotherapeutic agents in inducing death of colon cancer‐initiating cells in vitro. In vivo, Plk1 inhibitors killed CD133+ colon cancer cells leading to complete growth arrest of colon cancer stem cell‐derived xenografts, whereas chemotherapeutic agents only slowed tumor progression. While chemotherapy treatment increased CD133+ cell proliferation, treatment with Plk1 inhibitors eliminated all proliferating tumor‐initiating cells. Quiescent CD133+ cells that survived the treatment with Plk1 inhibitors could be killed by subsequent Plk1 inhibition when they exited from quiescence. Altogether, these results provide a new insight into the proliferative status of colon tumor‐initiating cells both in basal conditions and in response to therapy and indicate Plk1 inhibitors as potentially useful in the treatment of colorectal cancer. Stem Cells2012;30:1819–1830

Concise Review: Stem Cell-Derived Erythrocytes as Upcoming Players in Blood Transfusion

Blood transfusions have become indispensable to treat the anemia associated with a variety of medical conditions ranging from genetic disorders and cancer to extensive surgical procedures. In developed countries, the blood supply is generally adequate. However, the projected decline in blood donor availability due to population ageing and the difficulty in finding rare blood types for alloimmunized patients indicate a need for alternative red blood cell (RBC) transfusion products. Increasing knowledge of processes that govern erythropoiesis has been translated into efficient procedures to produce RBC ex vivo using primary hematopoietic stem cells, embryonic stem cells, or induced pluripotent stem cells. Although in vitro‐generated RBCs have recently entered clinical evaluation, several issues related to ex vivo RBC production are still under intense scrutiny: among those are the identification of stem cell sources more suitable for ex vivo RBC generation, the translation of RBC culture methods into clinical grade production processes, and the development of protocols to achieve maximal RBC quality, quantity, and maturation. Data on size, hemoglobin, and blood group antigen expression and phosphoproteomic profiling obtained on erythroid cells expanded ex vivo from a limited number of donors are presented as examples of the type of measurements that should be performed as part of the quality control to assess the suitability of these cells for transfusion. New technologies for ex vivo erythroid cell generation will hopefully provide alternative transfusion products to meet present and future clinical requirements. Stem Cells2012;30:1587–1596

Erythropoietin activates cell survival pathways in breast cancer stem-like cells to protect them from chemotherapy.

Recombinant erythropoietin (EPO) analogs [erythropoiesis-stimulating agents (ESA)] are clinically used to treat anemia in patients with cancer receiving chemotherapy. After clinical trials reporting increased adverse events and/or reduced survival in ESA-treated patients, concerns have been raised about the potential role of ESAs in promoting tumor progression, possibly through tumor cell stimulation. However, evidence is lacking on the ability of EPO to directly affect cancer stem-like cells, which are thought to be responsible for tumor progression and relapse. We found that breast cancer stem-like cells (BCSC) isolated from patient tumors express the EPO receptor and respond to EPO treatment with increased proliferation and self-renewal. Importantly, EPO stimulation increased BCSC resistance to chemotherapeutic agents and activated cellular pathways responsible for survival and drug resistance. Specifically, the Akt and ERK pathways were activated in BCSC at early time points following EPO treatment, whereas Bcl-xL levels increased at later times. In vivo, EPO administration counteracted the effects of chemotherapeutic agents on BCSC-derived orthotopic tumor xenografts and promoted metastatic progression both in the presence and in the absence of chemotherapy treatment. Altogether, these results indicate that EPO acts directly on BCSC by activating specific survival pathways, resulting in BCSC protection from chemotherapy and enhanced tumor progression.

Dexamethasone targeted directly to macrophages induces macrophage niches that promote erythroid expansion

Cultures of human CD34pos cells stimulated with erythroid growth factors plus dexamethasone, a model for stress erythropoiesis, generate numerous erythroid cells plus a few macrophages (approx. 3%; 3:1 positive and negative for CD169). Interactions occurring between erythroblasts and macrophages in these cultures and the biological effects associated with these interactions were documented by live phase-contrast videomicroscopy. Macrophages expressed high motility interacting with hundreds/thousands of erythroblasts per hour. CD169pos macrophages established multiple rapid ‘loose’ interactions with proerythroblasts leading to formation of transient erythroblastic island-like structures. By contrast, CD169neg macrophages established ‘tight’ interactions with mature erythroblasts and phagocytosed these cells. ‘Loose’ interactions of CD169pos macrophages were associated with proerythroblast cytokinesis (the M phase of the cell cycle) suggesting that these interactions may promote proerythroblast duplication. This hypothesis was tested by experiments that showed that as few as 103 macrophages significantly increased levels of 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide incorporation frequency in S/G2/M and cytokinesis expressed by proerythroblasts over 24 h of culture. These effects were observed also when macrophages were co-cultured with dexamethasone directly conjugated to a macrophage-specific CD163 antibody. In conclusion, in addition to promoting proerythroblast proliferation directly, dexamethasone stimulates expansion of these cells indirectly by stimulating maturation and cytokinesis supporting activity of macrophages.

Preparation and Use of Reverse Protein Microarrays

Reverse‐phase protein array (RPPA) is a multiplex, high‐throughput proteomic technique for profiling the activation status of signal transduction pathways involved in cancer survival and progression, potentially allowing for identification of new biomarkers and drug targets. On RPPA, the entire patient proteome is immobilized on a spot and single proteins can be quantified across a set of samples, spotted on the same array, with high specificity and sensitivity. Array immunostaining and signal amplification systems are used to generate a signal proportional to the concentration of the analyte. Dedicated scanners and software are used to detect spots, measure intensity, subtract background, normalize signal, and generate a numeric value as output. The generated output file is then analyzed using several different bioinformatic and biostatistical tools. In this unit, the RPPA procedure is described in depth, from sample handling and preparation to data analysis, with particular emphasis on tissue sample analysis. Curr. Protoc. Protein Sci. 75:27.7.1‐27.7.29.

Transcriptomic and phospho-proteomic analyzes of erythroblasts expanded in vitro from normal donors and from patients with polycythemia vera.

Erythropoiesis is a tightly regulated process which becomes decoupled from its normal differentiation program in patients with polycythemia vera (PV). Somatic mutations in JAK2 are commonly associated with this myeloid proliferative disorder. To gain insight into the molecular events that are required for abnormally developing erythroid cells to escape dependence on normal growth signals, we performed in vitro expansion of mature erythroblasts (ERY) from seven normal healthy donors and from seven polycythemic patients in the presence of IL3, EPO, SCF for 10, 11, or 13 days. Normal ERYs required exposure to the glucocorticoid dexamethasone (Dex) for expansion, while PV‐derived ERYs expanded in the absence of dexamethasone. RNA expression profiling revealed enrichment of two known oncogenes, GPR56 and RAB4a, in PV‐derived ERYs along with reduced expression levels of transcription factor TAL1 (ANOVA FDR < 0.05). While both normal and polycythemic‐derived ERYs integrated signaling cascades for growth, they did so via different signaling pathways which are represented by their differential phospho‐profiles. Our results show that normal ERYs displayed greater levels of phosphorylation of EGFR, PDGFRβ, TGFβ, and cKit, while PV‐derived ERYs were characterized by increased phosphorylation of cytoplasmic kinases in the JAK/STAT, PI3K, and GATA1 pathways. Together these data suggest that PV erythroblast expansion and maturation may be maintained and enriched in the absence of dexamethasone through reduced TAL1 expression and by accessing additional signaling cascades. Members of this acquired repertoire may provide important insight into the pathogenesis of aberrant erythropoiesis in myeloproliferative neoplasms such as polycythemia vera. Am. J. Hematol. 88:723–729, 2013.

C-Met/miR-130b axis as novel mechanism and biomarker for castration resistance state acquisition

Although a significant subset of prostate tumors remain indolent during the entire life, the advanced forms are still one of the leading cause of cancer-related death. There are not reliable markers distinguishing indolent from aggressive forms. Here we highlighted a new molecular circuitry involving microRNA and coding genes promoting cancer progression and castration resistance. Our preclinical and clinical data demonstrated that c-Met activation increases miR-130b levels, inhibits androgen receptor expression, promotes cancer spreading and resistance to hormone ablation therapy. The relevance of these findings was confirmed on patients’ samples and by in silico analysis on an independent patient cohort from Taylor’s platform. Data suggest c-Met/miR-130b axis as a new prognostic marker for patients’ risk assessment and as an indicator of therapy resistance. Our results propose new biomarkers for therapy decision-making in all phases of the pathology. Data may help identify high-risk patients to be treated with adjuvant therapy together with alternative cure for castration-resistant forms while facilitating the identification of possible patients candidates for anti-Met therapy. In addition, we demonstrated that it is possible to evaluate Met/miR-130b axis expression in exosomes isolated from peripheral blood of surgery candidates and advanced patients offering a new non-invasive tool for active surveillance and therapy monitoring.

Preclinical rationale for TGF-β inhibition as a therapeutic target for the treatment of myelofibrosis

To assess the role of abnormal transforming growth factor-beta (TGF-β) signaling in the pathogenesis of primary myelofibrosis (PMF), the effects of the TGF-β receptor-1 kinase inhibitor SB431542 on exxa0vivo expansion of hematopoietic cells in cultures from patients with JAK2V617+-polycythemia vera (PV) or PMF (JAK2V617F+, CALRpQ365f+, or unknown) and from normal sources (adult blood, AB, or cord blood, CB) were compared. In cultures of normal sources, SB431542 significantly increased by 2.5-fold the number of progenitor cells generated by days 1-2 (CD34+) and 6 (colony-forming cells) (CB) and that of precursor cells, mostly immature erythroblasts, by days 14-17 (AB and CB). In cultures of JAK2V617F+-PV, SB431542 increased by twofold the numbers of progenitor cells by day 10 and had no effect on that of precursors cells by days 12-17 (∼fourfold increase in all cases). In contrast, SB431542 had no effect on the number of either progenitor or precursor cells in cultures of JAK2V617F+ and CALR pQ365fs+ PMF. These ontogenetic- and disease-specific effects were associated with variegation in the ability of SB431542 to induce CD34+ cells from AB (increased), CB (decreased), or PV and PMF (unaffected) into cycle and erythroblasts in proliferation (increased for AB and PV and unaffected for CB and PMF). Differences in expansion of erythroblasts from AB, CB, and PV were associated with differences in activation of TGF-β signaling (SHCY317, SMAD2S245/250/255, and SMAD1S/S/SMAD5S/S/SMAD8S/S) detectable in these cells by phosphoproteomic profiling. In conclusion, treatment with TGF-β receptor-1 kinase inhibitors may reactivate normal hematopoiesis in PMF patients, providing a proliferative advantage over the unresponsive malignant clone.