A short peptide protects from age-onset proteotoxicity.

Aberrant protein aggregation jeopardizes cellular functionality and underlies the development of a myriad of late-onset maladies including Alzheimer's disease (AD) and Huntington's disease (HD). Accordingly, molecules that mitigate the toxicity of hazardous protein aggregates are of great interest as potential future therapeutics. Here we asked whether a small peptide, composed of five amino acids (5MER peptide) that was derived from the human pro-inflammatory CD44 protein, could protect model nematodes from the toxicity of aggregative proteins that underlie the development of neurodegenerative disorders in humans. We found that the 5MER peptide mitigates the toxicity that stems from both; the AD-causing Aß peptide and a stretch of poly-glutamine that is accountable for the development of several disorders including HD, while minimally affecting lifespan. This protection was dependent on the activity of aging-regulating transcription factors and associated with enhanced Aß and polyQ35-YFP aggregation. A transcriptomic analysis unveiled that the peptide modifies signaling pathways, thereby modulating the expression of various genes, including these, which are known as protein homeostasis (proteostasis) regulators such as txt-13 and modifiers of proteasome activity. The knockdown of txt-13 protects worms from proteotoxicity to the same extent as the 5MER peptide, suggesting that the peptide activates the transcellular chaperone signaling to promote proteostasis. Together, our results propose that the 5MER peptide should be considered as a component of future therapeutic cocktails for the treatment of neurodegenerative maladies.

Distinct designer diamines promote mitophagy, and thereby enhance healthspan in C. elegans and protect human cells against oxidative damage.

Impaired mitophagy is a primary pathogenic event underlying diverse aging-associated diseases such as Alzheimer and Parkinson diseases and sarcopenia. Therefore, augmentation of mitophagy, the process by which defective mitochondria are removed, then replaced by new ones, is an emerging strategy for preventing the evolvement of multiple morbidities in the elderly population. Based on the scaffold of spermidine (Spd), a known mitophagy-promoting agent, we designed and tested a family of structurally related compounds. A prototypic member, 1,8-diaminooctane (VL-004), exceeds Spd in its ability to induce mitophagy and protect against oxidative stress. VL-004 activity is mediated by canonical aging genes and promotes lifespan and healthspan in C. elegans. Moreover, it enhances mitophagy and protects against oxidative injury in rodent and human cells. Initial structural characterization suggests simple rules for the design of compounds with improved bioactivity, opening the way for a new generation of agents with a potential to promote healthy aging.

Desiccation-induced changes in recombination rate and crossover interference in Drosophila melanogaster: evidence for fitness-dependent plasticity.

Meiotic recombination is evolutionarily ambiguous, as being associated with both benefits and costs to its bearers, with the resultant dependent on a variety of conditions. While existing theoretical models explain the emergence and maintenance of recombination, some of its essential features remain underexplored. Here we focus on one such feature, recombination plasticity, and test whether recombination response to stress is fitness-dependent. We compare desiccation stress effects on recombination rate and crossover interference in chromosome 3 between desiccation-sensitive and desiccation-tolerant Drosophila lines. We show that relative to desiccation-tolerant genotypes, desiccation-sensitive genotypes exhibit a significant segment-specific increase in single- and double-crossover frequencies across the pericentromeric region of chromosome 3. Significant changes (relaxation) in crossover interference were found for the interval pairs flanking the centromere and extending to the left arm of the chromosome. These results indicate that desiccation is a recombinogenic factor and that desiccation-induced changes in both recombination rate and crossover interference are fitness-dependent, with a tendency of less fitted individuals to produce more variable progeny. Such dependence may play an important role in the regulation of genetic variation in populations experiencing environmental challenges.

Sunlight assisted direct amide formation via a charge-transfer complex.

We report on the use of charge-transfer complexes between amines and carbon tetrachloride, as a novel way to activate the amine for photochemical reactions. This principle is demonstrated in a mild, transition metal free, visible light assisted, dealkylative amide formation from feedstock carboxylic acids and amines. The low absorption coefficient of the complex allows deep light penetration and thus scale up to a gram scale.

Experimental evolution of recombination and crossover interference in Drosophila caused by directional selection for stress-related traits.

BACKGROUND: Population genetics predicts that tight linkage between new and/or pre-existing beneficial and deleterious alleles should decrease the efficiency of natural selection in finite populations. By decoupling beneficial and deleterious alleles and facilitating the combination of beneficial alleles, recombination accelerates the formation of high-fitness genotypes. This may impose indirect selection for increased recombination. Despite the progress in theoretical understanding, interplay between recombination and selection remains a controversial issue in evolutionary biology. Even less satisfactory is the situation with crossover interference, which is a deviation of double-crossover frequency in a pair of adjacent intervals from the product of recombination rates in the two intervals expected on the assumption of crossover independence. Here, we report substantial changes in recombination and interference in three long-term directional selection experiments with Drosophila melanogaster: for desiccation (~50 generations), hypoxia, and hyperoxia tolerance (>200 generations each). RESULTS: For all three experiments, we found a high interval-specific increase of recombination frequencies in selection lines (up to 40-50% per interval) compared to the control lines. We also discovered a profound effect of selection on interference as expressed by an increased frequency of double crossovers in selection lines. Our results show that changes in interference are not necessarily coupled with increased recombination. CONCLUSIONS: Our results support the theoretical predictions that adaptation to a new environment can promote evolution toward higher recombination. Moreover, this is the first evidence of selection for different recombination-unrelated traits potentially leading, not only to evolution toward increased crossover rates, but also to changes in crossover interference, one of the fundamental features of recombination.

Lengths of Orthologous Prokaryotic Proteins Are Affected by Evolutionary Factors.

Proteins of the same functional family (for example, kinases) may have significantly different lengths. It is an open question whether such variation in length is random or it appears as a response to some unknown evolutionary driving factors. The main purpose of this paper is to demonstrate existence of factors affecting prokaryotic gene lengths. We believe that the ranking of genomes according to lengths of their genes, followed by the calculation of coefficients of association between genome rank and genome property, is a reasonable approach in revealing such evolutionary driving factors. As we demonstrated earlier, our chosen approach, Bubble-sort, combines stability, accuracy, and computational efficiency as compared to other ranking methods. Application of Bubble Sort to the set of 1390 prokaryotic genomes confirmed that genes of Archaeal species are generally shorter than Bacterial ones. We observed that gene lengths are affected by various factors: within each domain, different phyla have preferences for short or long genes; thermophiles tend to have shorter genes than the soil-dwellers; halophiles tend to have longer genes. We also found that species with overrepresentation of cytosines and guanines in the third position of the codon (GC3 content) tend to have longer genes than species with low GC3 content.

Regulation of human protease-activated receptor 1 (hPar1) gene expression in breast cancer by estrogen.

A pivotal role is attributed to the estrogen-receptor (ER) pathway in mediating the effect of estrogen in breast cancer progression. Yet the precise mechanisms of cancer development by estrogen remain poorly understood. Advancing tumor categorization a step forward, and identifying cellular gene fingerprints to accompany histopathological assessment may provide targets for therapy as well as vehicles for evaluating the response to treatment. We report here that in breast carcinoma, estrogen may induce tumor development by eliciting protease-activated receptor-1 (PAR(1)) gene expression. Induction of PAR(1) was shown by electrophoretic mobility shift assay, luciferase reporter gene driven by the hPar(1) promoter, and chromatin-immunoprecipitation analyses. Functional estrogen regulation of hPar1 in breast cancer was demonstrated by an endothelial tube-forming network. Notably, tissue-microarray analyses from an established cohort of women diagnosed with invasive breast carcinoma exhibited a significantly shorter disease-free (P=0.006) and overall (P=0.02) survival of patients that were positive for ER and PAR(1), compared to ER-positive but PAR(1)-negative patients. We propose that estrogen transcriptionally regulates hPar(1), culminating in an aggressive gene imprint in breast cancer. While ER(+) patients are traditionally treated with hormone therapy, the presence of PAR(1) identifies a group of patients that requires additional treatment, such as anti-PAR(1) biological vehicles or chemotherapy.

PAR1 plays a role in epithelial malignancies: transcriptional regulation and novel signaling pathway.

Protease-activated receptor 1 (PAR(1)) is the first and prototype member of an established PAR family comprising four members. The role of PAR(1) in tumor biology has been established, and is characterized by a consistent direct correlation between overexpression of its levels and epithelial tumor aggressiveness. We have found that high expression of the human Par(1) (hPar(1)) gene in epithelial tumors is controlled largely at the transcriptional level. This led us to assign Egr-1, a transcription activator, as an inducer of hPar(1), and p53, a tumor suppressor gene, as an inhibitor, both acting to achieve fine tuning of hPar(1) in prostate carcinoma. High PAR(1) levels maintain prosurvival signals in tumor cells while silencing or ablation of the gene induce apoptosis. Studies of our hPar(1) transgenic mice, which overexpress hPar(1) in the mammary glands, revealed a novel PAR(1)-induced ß-catenin stabilization function. The components connecting PAR(1) to ß-catenin stabilization have been determined, assigning at first G(α)(13) as a selective immediate component. The PAR(1)-G(α) (13) axis recruits disheveled (DVL), an upstream signaling partner of the canonical Wnt signaling pathway. Silencing of DVL by siRNA-DVL potently abrogates PAR(1)-induced ß-catenin stabilization, demonstrating its critical role in the process. We, thus, propose that transcriptional regulation of hPar(1) gene over expression in epithelia malignancies initiates a novel signaling pathway, directly connecting to ß-catenin stabilization, a core event in both tumorigenesis and developmental processes.

Par genes: molecular probes to pathological assessment in breast cancer progression.

Taking the issue of tumor categorization a step forward and establish molecular imprints to accompany histopathological assessment is a challenging task. This is important since often patients with similar clinical and pathological tumors may respond differently to a given treatment. Protease-activated receptor-(1) (PAR(1)), a G protein-coupled receptor (GPCR), is the first member of the mammalian PAR family consisting of four genes. PAR(1) and PAR(2) play a central role in breast cancer. The release of N-terminal peptides during activation and the exposure of a cryptic internal ligand in PARs, endow these receptors with the opportunity to serve as a "mirror-image" index reflecting the level of cell surface PAR(1&2)-in body fluids. It is possible to use the levels of PAR-released peptide in patients and accordingly determine the choice of treatment. We have both identified PAR(1) C-tail as a scaffold site for the immobilization of signaling partners, and the critical minimal binding site. This binding region may be used for future therapeutic modalities in breast cancer, since abrogation of the binding inhibits PAR(1) induced breast cancer. Altogether, both PAR(1) and PAR(2) may serve as molecular probes for breast cancer diagnosis and valuable targets for therapy.

Etk/Bmx regulates proteinase-activated-receptor1 (PAR1) in breast cancer invasion: signaling partners, hierarchy and physiological significance.

BACKGROUND: While protease-activated-receptor 1 (PAR(1)) plays a central role in tumor progression, little is known about the cell signaling involved. METHODOLOGY/PRINCIPAL FINDINGS: We show here the impact of PAR(1) cellular activities using both an orthotopic mouse mammary xenograft and a colorectal-liver metastasis model in vivo, with biochemical analyses in vitro. Large and highly vascularized tumors were generated by cells over-expressing wt hPar1, Y397Z hPar1, with persistent signaling, or Y381A hPar1 mutant constructs. In contrast, cells over-expressing the truncated form of hPar1, which lacks the cytoplasmic tail, developed small or no tumors, similar to cells expressing empty vector or control untreated cells. Antibody array membranes revealed essential hPar1 partners including Etk/Bmx and Shc. PAR(1) activation induces Etk/Bmx and Shc binding to the receptor C-tail to form a complex. Y/A mutations in the PAR(1) C-tail did not prevent Shc-PAR(1) association, but enhanced the number of liver metastases compared with the already increased metastases obtained with wt hPar1. We found that Etk/Bmx first binds via the PH domain to a region of seven residues, located between C378-S384 in PAR(1) C-tail, enabling subsequent Shc association. Importantly, expression of the hPar1-7A mutant form (substituted A, residues 378-384), which is incapable of binding Etk/Bmx, resulted in inhibition of invasion through Matrigel-coated membranes. Similarly, knocking down Etk/Bmx inhibited PAR(1)-induced MDA-MB-435 cell migration. In addition, intact spheroid morphogenesis of MCF10A cells is markedly disrupted by the ectopic expression of wt hPar1. In contrast, the forced expression of the hPar1-7A mutant results in normal ball-shaped spheroids. Thus, by preventing binding of Etk/Bmx to PAR(1) -C-tail, hPar1 oncogenic properties are abrogated. CONCLUSIONS/SIGNIFICANCE: This is the first demonstration that a cytoplasmic portion of the PAR(1) C-tail functions as a scaffold site. We identify here essential signaling partners, determine the hierarchy of binding and provide a platform for therapeutic vehicles via definition of the critical PAR(1)-associating region in the breast cancer signaling niche.

Accelerated carcinogenesis following liver regeneration is associated with chronic inflammation-induced double-strand DNA breaks.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer mortality worldwide and is considered to be the outcome of chronic liver inflammation. Currently, the main treatment for HCC is surgical resection. However, survival rates are suboptimal partially because of tumor recurrence in the remaining liver. Our aim was to understand the molecular mechanisms linking liver regeneration under chronic inflammation to hepatic tumorigenesis. Mdr2-KO mice, a model of inflammation-associated cancer, underwent partial hepatectomy (PHx), which led to enhanced hepatocarcinogenesis. Moreover, liver regeneration in these mice was severely attenuated. We demonstrate the activation of the DNA damage-response machinery and increased genomic instability during early liver inflammatory stages resulting in hepatocyte apoptosis, cell-cycle arrest, and senescence and suggest their involvement in tumor growth acceleration subsequent to PHx. We propose that under the regenerative proliferative stress induced by liver resection, the genomic unstable hepatocytes generated during chronic inflammation escape senescence and apoptosis and reenter the cell cycle, triggering the enhanced tumorigenesis. Thus, we clarify the immediate and long-term contributions of the DNA damage response to HCC development and recurrence.

Tamoxifen induces heparanase expression in estrogen receptor-positive breast cancer.

PURPOSE: Mammalian heparanase degrades heparan sulfate, the main polysaccharide of the basement membrane. Heparanase is an important determinant in cancer progression, acting via the breakdown of extracellular barriers for invasion, as well as release of heparan sulfate-bound angiogenic and growth-promoting factors. The present study was undertaken to elucidate molecular mechanisms responsible for heparanase overexpression in breast cancer. EXPERIMENTAL DESIGN: To characterize heparanase regulation by estrogen and tamoxifen and its clinical relevance for breast tumorigenesis, we applied immunohistochemical analysis of tissue microarray combined with chromatin immunoprecipitation assay, reverse transcription-PCR, and Western blot analysis. RESULTS: A highly significant correlation (P<0.0001) between estrogen receptor (ER) positivity and heparanase overexpression was found in breast cancer. Binding of ER to heparanase promoter accompanied estrogen-induced increase in heparanase expression by breast carcinoma cells. Surprisingly, heparanase transcription was also stimulated by tamoxifen, conferring a proliferation advantage to breast carcinoma cells grown on a naturally produced extracellular matrix. Heparanase overexpression was invariably detected in ER-positive second primary breast tumors, developed in patients receiving tamoxifen for the initial breast carcinoma. The molecular mechanism of the estrogenlike effect of tamoxifen on heparanase expression involves recruitment of transcription coactivator AIB1 to the heparanase promoter. CONCLUSIONS: Heparanase induction by ligand-bound ER represents an important pathway in breast tumorigenesis and may be responsible, at least in part, for the failure of tamoxifen therapy in some patients. Our study provides new insights on breast cancer progression and endocrine therapy resistance, offering future strategies for delaying or reversing this process.

Mammary gland tissue targeted overexpression of human protease-activated receptor 1 reveals a novel link to beta-catenin stabilization.

Protease-activated receptor 1 (PAR1) is emerging with distinct assignments in tumor biology. We show that tissue targeted overexpression of hPar1 in mice mammary glands results in precocious hyperplasia, characterized by a dense network of ductal side branching and accelerated proliferation. These glands exhibit increased levels of wnt-4 and wnt-7b and a striking beta-catenin stabilization. Nuclear localization of beta-catenin is observed in hPar1 transgenic mouse tissue sections but not in the wild-type, age-matched counterparts. PAR1 induces beta-catenin nuclear localization also in established epithelial tumor cell lines of intact beta-catenin system (transformed on the background of mismatch repair system; RKO cells). We propose hereby that PAR1-mediated beta-catenin stabilization is taking place primarily via the increase of Wnt expression. Enforced expression of a specific Wnt antagonist family member, secreted frizzled receptor protein 5 (SFRP5), efficiently inhibited PAR1-induced beta-catenin stabilization. Likewise, application of either SFRP2 or SFRP5 on epithelial tumor cells completely abrogated PAR1-induced beta-catenin nuclear accumulation. This takes place most likely via inhibition of Wnt signaling at the level of cell surface (forming a neutralizing complex of "Receptors-SFRP-Wnt"). Furthermore, depletion of hPar1 by small interfering RNA (siRNA) vectors markedly inhibited PAR1-induced Wnt-4. The striking stabilization of beta-catenin, inhibited by SFRPs on one hand and Wnt-4 silencing by hPar1 siRNA on the other hand, points to a novel role of hPar1 in Wnt-mediated beta-catenin stabilization. This link between PAR1 and beta-catenin may bear substantial implications both in developmental and tumor progression processes.

Heparanase promotes growth, angiogenesis and survival of primary breast tumors.

Despite great strides toward diagnosis and therapy, breast cancer remains a most threatening disease in its incidence, morbidity and mortality; therefore, additional knowledge regarding the molecular mechanisms contributing to breast cancer progression, as well as new targets for drug discovery are highly needed. Heparanase is the predominant enzyme involved in cleavage of heparan sulfate, the main polysaccharide component of the extracellular matrix. Experimental and clinical data indicate that heparanase plays important roles in cancer metastasis and angiogenesis. In breast carcinoma patients, heparanase expression correlates with the metastatic potential of the tumor. The present study was undertaken to investigate the role of heparanase in local growth and angiogenesis of primary breast tumors. MCF-7 breast carcinoma cells were stable transfected with the human heparanase (H-hpa) cDNA, or empty vector (mock), and injected into the mammary pad of nude mice. MRI was applied to monitor progression of tumor growth and angiogenesis. We demonstrate that tumors produced by cells overexpressing heparanase grew faster and were 7-fold larger than tumors produced by mock transfected cells. This enhanced growth was accompanied by increased tumor vascularization and a higher degree of vessel maturation. Histological examination ascribed the differences in tumor growth to heparanase-stimulated cell proliferation and survival. In-vitro experiments reinforced heparanase role as a survival factor under stress conditions. Moreover, H-hpa tumor cells infiltrate into the adjacent stroma, promoting formation of highly vascularized fibrous bands. Our results emphasize the significance and clarify the involvement of heparanase in primary breast cancer progression by generating a supportive microenvironment that promotes tumor growth, angiogenesis and survival.

Identification of a novel functional androgen response element within hPar1 promoter: implications to prostate cancer progression.

Human protease-activated receptor-1 (hPar1) plays a role in malignant and physiological invasion processes. We have identified a functional androgen response element (ARE) located in the hPar1 promoter upstream of the transcription start site at -1791 to -1777. Dihydrotestosterone treatment of the prostate cancer cell line LNCaP increased endogenous hPar1 mRNA levels, consistent with the threefold increase in promoter activity of hPar1-luciferase reporter construct. Specific binding of the hPar1-derived ARE to LNCaP nuclear extracts was demonstrated by electrophoretic mobility shift assay. This binding was abrogated by antiandrogen receptor (anti-AR) antibodies or excess cold oligonucleotide but not by a mutated oligonucleotide. Moreover, using chromatin immunoprecipitation assays, we confirm the in vivo interaction between the AR and ARE domain of the hPar1 promoter. In parallel, we show that hormone ablation therapy markedly reduces the otherwise high hPar1 expression levels in prostate cancer biopsy specimens. We suggest that the hPar1 gene is regulated transcriptionally by androgens, representing one of several target genes effectively reduced during hormone ablation therapy. A major limitation of hormonal deprivation is that it causes only a temporary remission, and the cancer eventually reappears in a more malignant, androgen-independent form. hPar1 is also overexpressed in CL1 cells, an aggressively metastasizing, hormone-independent subclone of LNCaP, and in PC3 prostate adenocarcinoma lacking AR in a mechanism yet to be fully elucidated. These data may imply that hPar1 expression correlates with prostate cancer progression in androgen-dependent and -independent phases and therefore, provides an instrumental, therapeutic target for treatment in prostate cancer.

Regulation of heparanase gene expression by estrogen in breast cancer.

Numerous epidemiological studies clearly suggest that estrogen is one of the main driving forces in breast tumorigenesis, but precise mechanisms of cancer promotion by estrogen remain poorly understood. Classically, tumorigenic effects of estrogen have been attributed to its ability to directly promote the proliferation of breast cancer cells. In addition to abnormal proliferation, interactions between tumor cells and surrounding stromal components (e.g., enzymatic remodeling and degradation of extracellular matrix) are critical for cancer progression, angiogenesis, and metastasis. We now report that in breast carcinomas, estrogen may promote these pathological tumor-stromal interactions through up-regulation of heparanase gene expression. Heparanase is an endoglycosidase degrading heparan sulfate, of the basement membrane and extracellular matrix. This cleavage affects tumor-stromal interaction, neovascularization, local invasion, and metastatic spread. However, little is known about transcriptional regulation of the heparanase gene. We identified four putative estrogen response elements in the heparanase promoter region and found that transcription of a luciferase reporter gene driven by the heparanase promoter was significantly increased in estrogen-receptor positive MCF-7 human breast carcinoma cells after estrogen treatment. Estrogen-induced heparanase mRNA transcription in estrogen receptor-positive, but not in estrogen receptor-negative, breast cancer cells, confirmed the promoter study data. The estrogen effects on heparanase mRNA expression levels were abolished in the presence of the pure antiestrogen ICI 182,780, indicating that the classic estrogen receptor pathway is involved in transcriptional activation of heparanase. In vivo, exposure to estrogen augmented levels of heparanase protein in MCF-7 cells embedded in Matrigel plugs and correlated with increased plug vascularization. Collectively, our data suggest a new molecular pathway through which estrogen, independent of its proliferative effect, may induce heparanase overexpression and, thus, promote tumor-stromal interactions, critical for breast carcinoma development and progression.

Heparanase mediates cell adhesion independent of its enzymatic activity.

Heparanase is an endo-beta-D-glucuronidase that cleaves heparan sulfate and is implicated in diverse physiological and pathological processes. In this study we report on a novel direct involvement of heparanase in cell adhesion. We demonstrate that expression of heparanase in nonadherent lymphoma cells induces early stages of cell adhesion, provided that the enzyme is expressed on the cell surface. Heparanase-mediated cell adhesion to extracellular matrix (ECM) results in integrin-dependent cell spreading, tyrosine phosphorylation of paxillin, and reorganization of the actin cytoskeleton. The surface-bound enzyme also augments cell invasion through a reconstituted basement membrane. Cell adhesion was augmented by cell surface heparanase regardless of whether the cells were transfected with active or point mutated inactive enzyme, indicating that heparanase functions as an adhesion molecule independent of its endoglycosidase activity. The combined feature of heparanase as an ECM-degrading enzyme and a cell adhesion molecule emphasizes its significance in processes involving cell adhesion, migration, and invasion, including embryonic development, neovascularization, and cancer metastasis.

Human protease-activated receptor 1 expression in malignant epithelia: a role in invasiveness.

While protease-activated receptors (PARs) play a traditional role in vascular biology, they emerge with surprisingly new assignments in tumor biology. PAR1 expression correlates with the invasion properties of breast carcinoma, whereas human PAR1 antisense reduces their ability to migrate through Matrigel. Part of the molecular mechanism of PAR1 invasion involves the formation of focal contact complexes on PAR1 activation. PAR1 induces angiogenesis in animal models in vivo and exhibits an oncogenic phenotype of enhanced ductal complexity when overexpressed in mouse mammary glands.