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Background: Metaplastic breast cancer (MpBC) is a therapeutically chemoresistant, aggressive, and heterogeneous breast cancer variant accounting for Citation Format: Tejaswini P Reddy, Bijan Mahboubi, Roberto R. Rosato, Liliana Guzman-Rojas, Wei Qian, Jianying Zhou, Baek Kim, Stacy Moulder, Helen Piwnica-Worms, Jenny C. Chang. Combined PI3K and NOS inhibition enhances efficacy of taxane-based chemotherapy in metaplastic breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-17-04.

Figure S1. Expression of ZNF326 a in human breast cancer tissues. The images show a representative scoring intensities following immunostaining with anti-ZNF326 antibody (Range of low, moderate and high). Figure S2. RT-PCR analysis of the candidate cancer genes downregulated by shRNA-pools in the (A) HCC70 and (B) MDA-MB-231 breast cancer cell lines. Figure S3. Stable ZNF326 knockdown in (A) HCC70, (B) HCC1569 and (C) MDA-MB-231 TNBC cell lines using two independent shRNAs was confirmed by qRT-PCR. (D) Ectopic expression of ZNF326 in MDA-MB-231 cells transduced with lentivirus particles expressing ZNF326 cDNA and empty vector (control) respectively. ZNF326 mRNA levels were quantified by qRT-PCR. Figure S4. Western blot analysis of mammosphere cell lysates of HCC70 (A) and MDA-MB-231 (B) with stable shRNA (left panel) and ectopic overexpressing ZNF326 (right panel). Figure S5. Gene expression-based outcome for breast cancer online (GOBO) was used to obtain the expression analysis of (A) DACH1 and (B) GATA3 in different breast cancer subtypes.

Although genomic sequencing has provided a better understating of the genetic landmarks in triple-negative breast cancer (TNBC), functional validation of candidate cancer genes (CCG) remains unsolved. In this study, we used a transposon mutagenesis strategy based on a two-step sleeping beauty (SB) forward genetic screen to identify and validate new tumor suppressors (TS) in this disease. We generated 120 siRNAs targeting 40 SB-identified candidate breast cancer TS genes and used them to downregulate expression of these genes in four human TNBC cell lines. Among CCG, whose SB-mediated genetic mutation resulted in increased cellular proliferation in all cell lines tested, the genes ADNP, AP2B1, TOMM70A, and ZNF326 showed TS activity in tumor xenograft studies. Subsequent studies showed that ZNF326 regulated expression of multiple epithelial–mesenchymal transition and cancer stem cell (CSC) pathway genes. It also modulated expression of TS genes involved in the regulation of migration and cellular invasion and was a direct transcriptional activator of genes that regulate CSC self-renewal. ZNF326 expression associated with TNBC patient survival, with ZNF326 protein levels showing a marked reduction in TNBC. Our validation of several new TS genes in TNBC demonstrate the utility of two-step forward genetic screens in mice and offer an invaluable tool to identify novel candidate therapeutic pathways and targets. Cancer Res; 77(15); 4089–101. ©2017 AACR.

Table S1. siRNA oligos of the candidate cancer genes. Table S2. List of lentivirus shRNA clones. Table S3. Primer sets used in the ChIP assay. Table S4. List of TaqMan probes. Table S5. Candidate cancer genes were screen in four TNBC cell lines using siRNA oligos.

Metaplastic breast cancer (MpBC) is a rare and highly chemoresistant breast cancer subtype, with a median survival of 8 months after metastatic disease, and no standard therapeutic options. MpBCs are enriched for epithelial-to-mesenchymal transition (EMT)/cancer stem cell (CSC) markers, produce high levels of nitric oxide (NO), and have a hyperactive phosphoinositide 3-kinase (PI3K) signaling pathway. NO activates multiple oncogenic pathways, such as PI3K and transforming growth factor beta (TGFβ), a regulator of EMT. Therefore, we hypothesized that pan-NOS inhibitor NG-monomethyl-l-arginine (L-NMMA) could augment the efficacy of α-specific PI3K inhibitor alpelisib in MpBC in vitro and in vivo models. Immunostaining analysis revealed that MpBC PDX tumors had elevated co-expression of iNOS and pAkt (60% vs 23%, p=0.04) relative to triple-negative breast cancer (TNBC) PDX tumors. MpBC PDX tumors had higher RPL39 A14V (66% vs 4.7%, p< 0.00) and PIK3CA hotspot mutation rates (50% vs 19.1%, p=0.31) than TNBC PDX tumors. L-NMMA was synergistic with alpelisib in MpBC cell lines with PIK3CA/PIK3R1 mutations and antagonistic in PIK3CA-wild type and PTEN-deleted models. In vivo evaluation using MpBC PDX tumors found that L-NMMA augmented the efficacy of alpelisib in reducing tumor volume in PIK3CA-mutated MpBC PDX models. Transcriptomic analysis found gene sets associated with EMT reversal, such as the formation of cornified envelope (NES = 2.04 Nom p These studies paralleled a phase 1b/2 clinical trial with L-NMMA+taxane chemotherapy in a cohort of anthracycline-refractory MpBC patients (NCT02834403). The clinical benefit rate was 40% (6/15), overall response rate was 20% (3/15), and one patient achieved a pathologic complete response. Relative to baseline tumors, the responder end-of-treatment tumors had undergone reversal of EMT, with decreased expression of iNOS and ALDH1. We find that combining L-NMMA and alpelisib is a novel therapeutic strategy to treat MpBC, and combination therapy is being tested in a first multicenter phase 2 study for patients with MpBC. Citation Format: Tejaswini P. Reddy, Akshjot Puri, Liliana Guzman-Rojas, Christoforos Thomas, Wei Qian, Jianying Zhou, Hong Zhao, Xiaoxian Li, Bijan Mahboubi, Adrian Oo, Cho Young-Jae, Baek Kim, Jose Thaiparambil, Maria Florencia Chervo, Roberto Rosato, Camila Ayerbe, Noah Giese, Stacy Moulder, Helen Piwnica-Worms, Funda Meric-Bernstam, Jenny C. Chang. NOS inhibition reverses epithelial-to-mesenchymal transition and synergizes with alpelisib in metaplastic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3447.

Metaplastic breast cancer (MpBC) is a rare and highly aggressive subset accounting for Citation Format: Maria Florencia Chervo, Chiara Mancino, Federica Giordano, Tejaswini P. Reddy, Wei Qian, Jianying Zhou, Liliana Guzman-Rojas, Roberto R. Rosato, Francesca Taraballi, Jenny C. Chang. Leukosome-based siRNA strategy for targeting ribosomal protein L39 (RPL39) in metaplastic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2709.

Purpose: Metaplastic breast cancer (MpBC) is a highly chemoresistant and aggressive breast cancer variant with limited therapeutic options. Most MpBCs harbor a triple-negative breast cancer (TNBC) phenotype, yet have a worse prognosis compared to TNBC. We and others discovered two key molecular alterations in MpBC associated with poor overall survival: 1) hyperactivation of the phosphoinositide 3-kinase (PI3K) signaling pathway and 2) enhanced inducible nitric oxide synthase (iNOS) signaling. Nitric oxide produced from iNOS is able to activate various oncogenic pathways, such as PI3K signaling. In MpBC, both PI3K and NOS signaling pathways may synergistically work to enhance chemoresistance. We propose to evaluate whether pan-NOS inhibitor NG-monomethyl-l-arginine (L-NMMA) augments the efficacy of alpha isoform-specific PI3K inhibitor alpelisib in MpBC in vitro and in vivo models. Methods: MpBC cell lines (Hs578T, BT549, SUM159) and MpBC Patient-Derived Xenograft (PDX) models were used in this study. Cell viability was determined with SRB Assay and the combination index was evaluated using Chou-Talalay Method. Flow cytometry was conducted to evaluate cellular apoptosis and cell cycle distribution analysis. In vivo efficacy of NOS and PI3K inhibition was assessed using MpBC PDX models. Transcriptional analysis of PDX tumors treated with vehicle control, single-agent (L-NMMA or alpelisib), or combination therapy (L-NMMA+alpelisib) was conducted to reveal top enriched pathways. Immunoblotting and immunofluorescence analyses of treated MpBC cell lines and PDX tissues were performed. Results: Combination of L-NMMA and alpelisib synergistically reduced cellular proliferation (CI Conclusion: Combined NOS and PI3K inhibition is a novel therapeutic strategy that may improve the efficacy of chemotherapy in patients with MpBC. Citation Format: Tejaswini P. Reddy, Akshjot Puri, Liliana Guzman-Rojas, Bijan Mahboubi, Wei Qian, Jianying Zhou, Baek Kim, Stacy Moulder, Helen Piwnica-Worms, Roberto Rosato, Jenny C. Chang. NOS inhibition augments PI3K inhibitor-induced DNA damage and mesenchymal-to-epithelial transition in metaplastic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2658.

Our previous findings have shown that increased inducible nitric oxide synthase (iNOS) expression is a poor prognostic indicator and associated with worse overall survival in TNBC patients. Growing evidence has also suggested that hyperactivation of the phosphoinositide-3-kinase (PI3K) survival signaling pathway is one of the most common oncogenic aberrations in TNBC. Nitric oxide (NO) is a unique molecule in its ability to target multiple oncogenic pathways in a spatial and temporal manner, such as PI3K, extracellular signal-regulated kinase (ERK), β-catenin pathway, transforming growth factor beta (TGFβ) signaling, and hypoxia-inducible factor (HIF). This study investigates the role of NO in PI3K-Akt pathway activation in human TNBC cells, in conditions with extracellular NO donor exposure and increased intracellular iNOS (NOS2) expression. TNBC cell lines BT549, HCC1937, Hs578T, SUM159, MDA-MB-231, and BT-20 were exposed to the NO donor DETA NONOate at increasing concentrations (0-500 µM) for 12 hours. Significant elevation in phosphorylation of Akt (Ser473 and Thr308) in response to increase NO donor concentration was found only in cell lines with wild-type PTEN and PIK3R1, such as SUM159, MDA-MB-231, and BT20. To evaluate whether NO-induced activation of Akt was PI3K-dependent, SUM159 (wild-type PTEN) and HCC1937 (PTEN mutant) cells were also pre-treated with PI3K inhibitor alpelisib before NO exposure. PI3K inhibition was only able to eliminate basal phospho-Akt and prevent NO-induced Akt activation in the SUM159 cell line. Furthermore, compared to vehicle control treated cells, we found that NO donor-induced activation enhanced phosphorylation in 15/18 Akt signaling proteins in SUM159 cells (PTEN intact) and 3/18 Akt signaling proteins in HCC1937 cells (PTEN loss). In Hs578T cells in which NOS2 was either overexpressed or knocked down via lentiviral transduction, we found that enhanced expression of NOS2 was associated with increased p-Akt expression and knock-down of NOS2 led to reduced p-Akt. This result suggests that iNOS, as opposed to other NOS isoforms, is more associated with modulating PI3K signaling in TNBC.We also analyzed TNBC data from The Cancer Genome Atlas and found that patients with positive iNOS mRNA expression had significantly higher phospho-Akt (Ser473 and Thr308) [p≤0.0001] expression compared to patients with no iNOS expression. When we stratified this patient cohort based on both iNOS and PTEN expression, we only found significantly higher phospho-Akt (Ser473 and Thr308) [p≤0.01] expression in patients with positive iNOS and PTEN expression, supporting the hypothesis that NO may impair PTEN’s function as a PI3K antagonist. Ongoing studies are directed to investigate mechanisms of iNOS-PTEN physical interactions, iNOS-dependent post-translational modifications to PTEN, and whether iNOS and PTEN are dual prognostic markers in TNBC patients. Citation Format: Tejaswini P. Reddy, Liliana Guzman-Rojas, Roberto R. Rosato, Wei Qian, Hong Zhao, Jenny C. Chang. Inducible nitric oxide synthase activates PI3K/Akt signaling via PTEN S-nitrosylation in triple-negative breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD3-01.

The systematic identification of genetic events driving cellular transformation and tumor progression in the absence of a highly recurrent oncogenic driver mutation is a challenge in cutaneous oncology. In cutaneous squamous cell carcinoma (cuSCC), the high UV-induced mutational burden poses a hurdle to achieve a complete molecular landscape of this disease. Here, we utilized the Sleeping Beauty transposon mutagenesis system to statistically define drivers of keratinocyte transformation and cuSCC progression in vivo in the absence of UV-IR, and identified both known tumor suppressor genes and novel oncogenic drivers of cuSCC. Functional analysis confirms an oncogenic role for the ZMIZ genes, and tumor suppressive roles for KMT2C, CREBBP and NCOA2, in the initiation or progression of human cuSCC. Taken together, our in vivo screen demonstrates an extremely heterogeneous genetic landscape of cuSCC initiation and progression, which can be harnessed to better understand skin oncogenic etiology and prioritize therapeutic candidates., Author summary Non-melanoma skin cancers, the most common cancers in the US, are caused by UV skin exposure. Nearly 1 million cases of cutaneous squamous cell carcinoma (cuSCC) are diagnosed in the US each year. While most cuSCCs are highly treatable, more than twice as many individuals die from this disease as from melanoma. The high burden of UV-induced DNA damage in human skin poses a challenge for identifying initiating and cooperating mutations that promote cuSCC development and for defining potential therapeutic targets. Here, we describe a genetic screen in mice using a DNA transposon system to mutagenize the genome of keratinocytes and drive squamous cell carcinoma in the absence of UV. By sequencing where the transposons selectively integrated in the genomes of normal skin, skin with pre-cancerous lesions and skin with fully developed cuSCCs from our mouse model, we were able to identify frequently mutated genes likely important for this disease. Our analysis also defined cooperation between sets of genes not previously appreciated in cuSCC. Our mouse model and ensuing data provide a framework for understanding the genetics of cuSCC and for defining the molecular changes that may lead to the future therapies for patients.

A central challenge in oncology is how to kill tumors containing heterogeneous cell populations defined by different combinations of mutated genes. Identifying these mutated genes and understanding how they cooperate requires single-cell analysis, but current single-cell analytic methods, such as PCR-based strategies or whole-exome sequencing, are biased, lack sequencing depth or are cost prohibitive. Transposon-based mutagenesis allows the identification of early cancer drivers, but current sequencing methods have limitations that prevent single-cell analysis. We report a liquid-phase, capture-based sequencing and bioinformatics pipeline, Sleeping Beauty (SB) capture hybridization sequencing (SBCapSeq), that facilitates sequencing of transposon insertion sites from single tumor cells in a SB mouse model of myeloid leukemia (ML). SBCapSeq analysis of just 26 cells from one tumor revealed the tumor’s major clonal subpopulations, enabled detection of clonal insertion events not detected by other sequencing methods and led to the identification of dominant subclones, each containing a unique pair of interacting gene drivers along with three to six cooperating cancer genes with SB-driven expression changes.

Cytoskeletal-associated proteins play an active role in coordinating the adhesion and migration machinery in cancer progression. To identify functional protein networks and potential inhibitors, we screened an internalizing phage (iPhage) display library in tumor cells, and selected LGRFYAASG as a cytosol-targeting peptide. By affinity purification and mass spectrometry, intracellular annexin A2 was identified as the corresponding binding protein. Consistently, annexin A2 and a cell-internalizing, penetratin-fused version of the selected peptide (LGRFYAASG-pen) co-localized and specifically accumulated in the cytoplasm at the cell edges and cell-cell contacts. Functionally, tumor cells incubated with LGRFYAASG-pen showed disruption of filamentous actin, focal adhesions and caveolae-mediated membrane trafficking, resulting in impaired cell adhesion and migration in vitro. These effects were paralleled by a decrease in the phosphorylation of both focal adhesion kinase (Fak) and protein kinase B (Akt). Likewise, tumor cells pretreated with LGRFYAASG-pen exhibited an impaired capacity to colonize the lungs in vivo in several mouse models. Together, our findings demonstrate an unrecognized functional link between intracellular annexin A2 and tumor cell adhesion, migration and in vivo grafting. Moreover, this work uncovers a new peptide motif that binds to and inhibits intracellular annexin A2 as a candidate therapeutic lead for potential translation into clinical applications.

Although genomic sequencing has provided a better understating of the genetic landmarks in triple-negative breast cancer (TNBC), functional validation of candidate cancer genes (CCG) remains unsolved. In this study, we used a transposon mutagenesis strategy based on a two-step sleeping beauty (SB) forward genetic screen to identify and validate new tumor suppressors (TS) in this disease. We generated 120 siRNAs targeting 40 SB-identified candidate breast cancer TS genes and used them to downregulate expression of these genes in four human TNBC cell lines. Among CCG, whose SB-mediated genetic mutation resulted in increased cellular proliferation in all cell lines tested, the genes ADNP, AP2B1, TOMM70A, and ZNF326 showed TS activity in tumor xenograft studies. Subsequent studies showed that ZNF326 regulated expression of multiple epithelial–mesenchymal transition and cancer stem cell (CSC) pathway genes. It also modulated expression of TS genes involved in the regulation of migration and cellular invasion and was a direct transcriptional activator of genes that regulate CSC self-renewal. ZNF326 expression associated with TNBC patient survival, with ZNF326 protein levels showing a marked reduction in TNBC. Our validation of several new TS genes in TNBC demonstrate the utility of two-step forward genetic screens in mice and offer an invaluable tool to identify novel candidate therapeutic pathways and targets. Cancer Res; 77(15); 4089–101. ©2017 AACR.

Techniques that are largely used for protein interaction studies and the discovery of intracellular receptors, such as affinity-capture complex purification and the yeast two-hybrid system, may produce inaccurate data sets owing to protein insolubility, transient or weak protein interactions or irrelevant intracellular context. A versatile tool for overcoming these limitations, as well as for potentially creating vaccines and engineering peptides and antibodies as targeted diagnostic and therapeutic agents, is the phage-display technique. We have recently developed a new technology for screening internalizing phage (iPhage) vectors and libraries using a ligand/receptor-independent mechanism to penetrate eukaryotic cells. iPhage particles provide a unique discovery platform for combinatorial intracellular targeting of organelle ligands along with their corresponding receptors and for fingerprinting functional protein domains in living cells. Here we explain the design, cloning, construction and production of iPhage-based vectors and libraries, along with basic ligand-receptor identification and validation methodologies for organelle receptors. An iPhage library screening can be performed in ∼8 weeks.

SignificanceTriple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype. Despite extensive cancer genome-sequencing efforts, there is still an incomplete understanding of the genetic networks driving TNBC. Here, we usedSleeping Beautytransposon mutagenesis to identify genes that cooperate with mutantPtenin the induction of TNBC. We identified 12 candidate TNBC trunk drivers and a larger number of progression genes. Subsequent functional validation studies identified eight human TNBC tumor suppressor genes, including the GATA-like transcriptional repressorTRPS1, which was shown to inhibit lung metastasis by deregulating the expression of multiple serpin and epithelial-to-mesenchymal transition (EMT) pathway genes. Our study provides a better understanding of the genetic forces driving TNBC and discovered genes with clinical importance in TNBC.

Processes that promote cancer progression such as angiogenesis require a functional interplay between malignant and nonmalignant cells in the tumor microenvironment. The metalloprotease aminopeptidase N (APN; CD13) is often overexpressed in tumor cells and has been implicated in angiogenesis and cancer progression. Our previous studies of APN-null mice revealed impaired neoangiogenesis in model systems without cancer cells and suggested the hypothesis that APN expressed by nonmalignant cells might promote tumor growth. We tested this hypothesis by comparing the effects of APN deficiency in allografted malignant (tumor) and nonmalignant (host) cells on tumor growth and metastasis in APN-null mice. In two independent tumor graft models, APN activity in both the tumors and the host cells cooperate to promote tumor vascularization and growth. Loss of APN expression by the host and/or the malignant cells also impaired lung metastasis in experimental mouse models. Thus, cooperation in APN expression by both cancer cells and nonmalignant stromal cells within the tumor microenvironment promotes angiogenesis, tumor growth, and metastasis.

Mammalian cell membranes provide an interface between the intracellular and extracellular compartments. It is currently thought that cytoplasmic signaling adapter proteins play no functional role within the extracellular tumor environment. Here, by selecting combinatorial random peptide libraries in tumor-bearing mice, we uncovered a direct, specific, and functional interaction between CRKL, an adapter protein [with Src homology 2 (SH2)- and SH3-containing domains], and the plexin-semaphorin-integrin domain of β 1 integrin in the extracellular milieu. Through assays in vitro, in cellulo, and in vivo, we show that this unconventional and as yet unrecognized protein–protein interaction between a regulatory integrin domain (rather than a ligand-binding one) and an intracellular adapter (acting outside of the cells) triggers an alternative integrin-mediated cascade for cell growth and survival. Based on these data, here we propose that a secreted form of the SH3/SH2 adaptor protein CRKL may act as a growth-promoting factor driving tumorigenesis and may lead to the development of cancer therapeutics targeting secreted CRKL.

Aminopeptidase N (APN, CD13; EC 3.4.11.2) is a transmembrane metalloprotease with several functions, depending on the cell type and tissue environment. In tumor vasculature, APN is overexpressed in the endothelium and promotes angiogenesis. However, there have been no reports ofin vivoinactivation of the APN gene to validate these findings. Here we evaluated, by targeted disruption of the APN gene, whether APN participates in blood vessel formation and function under normal conditions. Surprisingly, APN-null mice developed with no gross or histological abnormalities. Standard neurological, cardiovascular, metabolic, locomotor, and hematological studies revealed no alterations. Nonetheless, in oxygen-induced retinopathy experiments, APN-deficient mice had a marked and dose-dependent deficiency of the expected retinal neovascularization. Moreover, gelfoams embedded with growth factors failed to induce functional blood vessel formation in APN-null mice. These findings establish that APN-null mice develop normally without physiological alterations and can undergo physiological angiogenesis but show a severely impaired angiogenic response under pathological conditions. Finally, in addition to vascular biology research, APN-null mice may be useful reagents in other medical fields such as malignant, cardiovascular, immunological, or infectious diseases.

We previously showed that the human AKNA gene encodes an AT-hook transcription factor that regulates the expression of costimulatory cell surface molecules on lymphocytes. However, AKNA cDNA probes hybridize with multiple transcripts, suggesting either the existence of other homologous genes or a complex regulation operating on a single gene. Here we report evidence for the latter, as we find that AKNA is encoded by a single gene that spans a 61-kb locus of 24 exons on the fragile FRA9E region of human chromosome 9q32. This gene gives rise to at least nine distinct transcripts, most of which are expressed in a tissue-specific manner in lymphoid organs. Many of the AKNA transcripts originate from alternative splicing; others appear to derive from differential polyadenylation and promoter usage. The alternative AKNA transcripts are predicted to encode overlapping protein isoforms, some of which (p70 and p100) are readily detectable using a polyclonal anti-AKNA antisera that we generated. We also find that AKNA PEST-dependent cleavage into p50 polypeptides is targeted to mature B cells and appears to be required for CD40 upregulation. The unusual capacity of the AKNA gene to generate multiple transcripts and proteins may reflect its functional diversity, and it may also provide a fail-safe mechanism that preserves AKNA expression.

LILIANA GUZMAN-ROJAS, JENNIFER C. SIMS-MOURTADA, ROBERTO RANGEL &HECTOR MARTINEZ-VALDEZ Department of Immunology, The University of TexasMD Anderson Cancer Center, Houston, Texas, USASUMMARYWithin germinal centres, B lymphocytes are destined to die by apoptosis via Fas signalling, unlessthey are positively rescued by antigen and by signals initiated by CD40–CD154 interactions. Thus,while the germinal centre microenvironment can become a virtual graveyard for most B lympho-cytes that fail to bind antigen with high affinity, it concomitantly provides the necessary stimuli forthe survival of cells that successfully accomplish affinity maturation. Such dichotomy in thephysiology of germinal centre reaction that results in survival of the functional B-cell repertoire andthe elimination of abnormal cells, dictates the fate towards B-cell homeostasis or disease.Consequently, the death and survival-signalling arms within germinal centres predominantlyreside on the timely and controlled expression of Fas and its ligand (FasL), and CD40 andCD154, respectively. In keeping with this notion, lymphoproliferation or deficient immunity aredocumented landmarks of inactivation of either the Fas/FasL or CD40/CD154 signalling pathways.The present review considers two different scenarios in the control of B-cell survival and deathwithin germinalcentres. Thefirst is anidealistic scenario, inwhicha discriminatory andco-ordinatesignalling initiated by the CD40/CD154 and Fas/FasL pairs, respectively, leads the rescue of thefunctional B-cell repertoire and the elimination of the abnormal phenotype. The second is a gloomyscenario in which both the lack and the hyperexpression of either receptor/ligand pairs, are seen asequally deleterious.INTRODUCTIONJust like a good education in a prestigious university campus,the acquisition of B-cell memory from nai¨ve cells takes placewithin a highly specialized microenvironment, namely thegerminal centres (GC) of the secondary lymphoid organs.The screening process for recruitment of GC candidates isinitiated by the induction of immunoglobulin (Ig)M

Significance The progression of many solid tumors is associated with increased vascularization. We previously recognized involvement in tumor development and angiogenesis of tumor stromal cells expressing the CD13 protease aminopeptidase. The basic biological concept of participation of nontumor cells in the cancer stroma microenvironment is strengthened in the present study by our finding that a CD11b + CD13 + myeloid subset of bone marrow-derived cells affects pericyte biology and angiogenesis and thereby influences tumor growth and metastasis. Therapeutic implications of the identification of specific CD11 + CD13 + myeloid bone marrow-derived cells as participants in the mechanism of tumor angiogenesis merit further investigation.

Phage display screening allows the study of functional protein–protein interactions at the cell surface, but investigating intracellular organelles remains a challenge. Here we introduce internalizing-phage libraries to identify clones that enter mammalian cells through a receptor-independent mechanism and target-specific organelles as a tool to select ligand peptides and identify their intracellular receptors. We demonstrate that penetratin, an antennapedia-derived peptide, can be displayed on the phage envelope and mediate receptor-independent uptake of internalizing phage into cells. We also show that an internalizing-phage construct displaying an established mitochondria-specific localization signal targets mitochondria, and that an internalizing-phage random peptide library selects for peptide motifs that localize to different intracellular compartments. As a proof-of-concept, we demonstrate that one such peptide, if chemically fused to penetratin, is internalized receptor-independently, localizes to mitochondria, and promotes cell death. This combinatorial platform technology has potential applications in cell biology and drug development., Phage display screening can unravel protein–protein interactions, but its application has been mainly restricted to the cell surface. Here, a phage-based reagent is introduced that allows the targeting of combinatorial peptides to cell organelles, providing a tool for the discovery of intracellular ligand-receptors.

Hydrogels have become a promising research focus because of their potential for biomedical application. Here we explore the long-range, electrostatic interactions by following the effect of trans-acting (pH) and cis-acting factors (peptide mutation) on the formation of Au-phage hydrogels. These bioinorganic hydrogels can be generated from the bottom-up assembly of Au nanoparticles (Au NP) with either native or mutant bacteriophage (phage) through electrostatic interaction of the phage pVIII major capsid proteins (pVIII). The cis-acting factor consists of a peptide extension displayed on the pVIII that mutates the phage. Our results show that pH can dictate the direct-assembly and stability of Au-phage hydrogels in spite of the differences between the native and the mutant pVIII. The first step in characterizing the interactions of Au NP with phage was to generate a molecular model that identified the charge distribution and structure of the native and mutant pVIII. This model indicated that the mutant peptide extension carried a higher positive charge relative to the native pVIII at all pHs. Next, by monitoring the Au-phage interaction by means of optical microscopy, elastic light scattering, fractal dimension analysis as well as Uv-vis and surface plasmon resonance spectroscopy, we show that the positive charge of the mutant peptide extension favors the opposite charge affinity between the phage and Au NP as the pH is decreased. These results show the versatility of this assembly method, where the stability of these hydrogels can be achieved by either adjusting the pH or by changing the composition of the phage pVIII without the need of phage display libraries.

T-cell homing within germinal centres (GCs) is required for humoral B-cell responses. However, the mechanisms implicated in the recruitment of T cells into the GC are not completely understood. Here we show, by immunohistology, and Northern and Western blots, that in vivo human GC B lymphocytes can express CxC and CC chemokines. Moreover, B-cell subset-specific experiments reveal that interleukin (IL)-8 and regulated on activation, normal, T-cell expressed, and secreted (RANTES) are predominantly expressed by GC centroblast and centrocytes, suggesting that chemokine expression is essential at stages in which B-lymphocytes engage in active antigen-dependent interactions with T lymphocytes. In keeping with this hypothesis, we show that the T cells recruited into the GC correlatively express the receptors for IL-8 and RANTES. We propose that chemokine expression is a key B-cell function that facilitates T-lymphocyte recruitment into the GCs and supports cognate B-cell : T-cell encounters. Moreover, our data are consistent with the impaired homing of T cells to secondary lymphoid organs in mice that are either deficient in CC and CxC chemokines or their receptors.

Proteins containing AT hooks bind A/T-rich DNA through a nine-amino-acid motif and are thought to co-regulate transcription by modifying the architecture of DNA, thereby enhancing the accessibility of promoters to transcription factors. Here we describe AKNA, a human AT-hook protein that directly binds the A/T-rich regulatory elements of the promoters of CD40 and CD40 ligand (CD40L) and coordinately regulates their expression. Consistent with its function, AKNA is a nuclear protein that contains multiple PEST protein-cleavage motifs, which are common in regulatory proteins with high turnover rates. AKNA is mainly expressed by B and T lymphocytes, natural killer cells and dendritic cells. During B-lymphocyte differentiation, AKNA is mainly expressed by germinal centre B lymphocytes, a stage in which receptor and ligand interactions are crucial for B-lymphocyte maturation. Our findings show that an AT-hook molecule can coordinately regulate the expression of a key receptor and its ligand, and point towards a molecular mechanism that explains homotypic cell interactions.

We report the identification of a human cDNA encoding a 25 kDa protein of relevant evolutionary and lymphoid interest (PRELI). PRELI was cloned by screening a B lymphocyte-specific cDNA library with a probe generated by mRNA differential display. PRELI amino acid sequence is 85% similar to the avian px19 protein, expressed within the blood islands and in the liver during avian embryo development. PRELI and px19 contain tandem repeats (A/TAEKAK) of the late embryogenesis abundant (LEA) motif, characteristic of a group of survival molecules and originally thought to be present only in plant proteins. Interestingly, PRELI expression is high in the fetal liver, a major site for B cell lymphopoiesis, while the mRNA levels in other fetal tissues such as the brain, lung, and kidney are comparatively low. At the adult stage, PRELI expression is drastically reduced in the liver but exhibits high mRNA levels in the spleen, brain, lung and kidney tissues, suggesting that PRELI expression may be important for the development of vital and immunocompetent organs. Moreover, PRELI is also highly expressed in the adult lymph nodes and peripheral blood leukocytes, further stressing that at the adult stage, PRELI expression may be important during secondary immune responses. Consistent with this hypothesis, the expression of PRELI is predominant within germinal centers (GC), a stage in which B lymphocytes are under a stressful selection pressure. Taken together these data: (i) strongly support the notion that the conserved LEA motif represents a phylogenetic link between plants and animals, (ii) reveal a novel molecule whose expression may play a role in the maturation of distinct human tissues, and (iii) suggest that PRELI expression may be important for GC B lymphocytes.

El cáncer cervicouterino representa un grave problema de salud pública, debido a la asociación de la neoplasia con el virus del papiloma humano; actualmente se realizan estudios usando estrategias dirigidas a combatir este patógeno, mediante vacunas, que podrían ser de gran utilidad para el control de la progresión de la enfermedad. El estudio tanto de la inmunología humoral como celular ha servido para el desarrollo de vacunas. Así, la utilización de partículas virales sintéticas para el estudio de anticuerpos neutralizantes y el uso de proteínas tempranas virales, entre otras, para la inducción de inmunidad mediada por células, han sido la pauta para realizar estudios que dirijan la respuesta inmune para prevenir la infección celular tanto hacia células infectadas no transformadas como hacia células transformadas viralrnente con resultados favorables.

Interleukin-11 (IL-11) is a pleiotropic cytokine approved by the FDA against chemotherapy-induced thrombocytopenia. From a combinatorial selection in a cancer patient, we isolated an IL-11-like peptide mapping to domain I of the IL-11 (sequence CGRRAGGSC). Although this motif has ligand attributes, it is not within the previously characterized interacting sites. Here we design and validate in-tandem binding assays, site-directed mutagenesis and NMR spectroscopy to show (i) the peptide mimics a receptor-binding site within IL-11, (ii) the binding of CGRRAGGSC to the IL-11R alpha is functionally relevant, (iii) Arg4 and Ser8 are the key residues mediating the interaction, and (iv) the IL-11-like motif induces cell proliferation through STAT3 activation. These structural and functional results uncover an as yet unrecognized receptor-binding site in human IL-11. Given that IL-11R alpha has been proposed as a target in human cancer, our results provide clues for the rational design of targeted drugs.