Luiz F. Zerbini

Genomic Counter-Stress Changes Induced by the Relaxation Response

Background Mind-body practices that elicit the relaxation response (RR) have been used worldwide for millennia to prevent and treat disease. The RR is characterized by decreased oxygen consumption, increased exhaled nitric oxide, and reduced psychological distress. It is believed to be the counterpart of the stress response that exhibits a distinct pattern of physiology and transcriptional profile. We hypothesized that RR elicitation results in characteristic gene expression changes that can be used to measure physiological responses elicited by the RR in an unbiased fashion. Methods/Principal Findings We assessed whole blood transcriptional profiles in 19 healthy, long-term practitioners of daily RR practice (group M), 19 healthy controls (group N1), and 20 N1 individuals who completed 8 weeks of RR training (group N2). 2209 genes were differentially expressed in group M relative to group N1 (p<0.05) and 1561 genes in group N2 compared to group N1 (p<0.05). Importantly, 433 (p<10−10) of 2209 and 1561 differentially expressed genes were shared among long-term (M) and short-term practitioners (N2). Gene ontology and gene set enrichment analyses revealed significant alterations in cellular metabolism, oxidative phosphorylation, generation of reactive oxygen species and response to oxidative stress in long-term and short-term practitioners of daily RR practice that may counteract cellular damage related to chronic psychological stress. A significant number of genes and pathways were confirmed in an independent validation set containing 5 N1 controls, 5 N2 short-term and 6 M long-term practitioners. Conclusions/Significance This study provides the first compelling evidence that the RR elicits specific gene expression changes in short-term and long-term practitioners. Our results suggest consistent and constitutive changes in gene expression resulting from RR may relate to long term physiological effects. Our study may stimulate new investigations into applying transcriptional profiling for accurately measuring RR and stress related responses in multiple disease settings.

Targeting Transcription Factors for Cancer Gene Therapy

A high proportion of oncogenes and tumor suppressor genes encode transcription factors. Deregulated expression or activation and inactivation of transcription factors as well as mutations and translocations play critical roles in tumorigenesis. Furthermore, the majority of oncogenic signaling pathways converge on sets of transcription factors that ultimately control gene expression patterns resulting in tumor formation and progression as well as metastasis. Under normal physiological conditions whole sets of genes with similar functions are regulated by highly specific, tightly regulated upstream transcriptional regulators, whereas in cancer aberrant activation of these transcription factors leads to deregulated expression of multiple gene sets associated with tumor development and progression. The activity of these transcription factors can be modulated by multiple mechanisms including posttranslational modifications. Activation or inactivation of transcription factors promote cancer development, cell survival and proliferation and induce tumor angiogenesis. Since many of these transcription factors are inactive under normal physiological conditions and their expression and activities are tightly regulated, these transcription factors represent highly desirable and logical points of therapeutical interference in cancer development and progression. Three major families of transcription factors have emerged as important players in human cancer and are validated targets in drug discovery for cancer therapy: 1) the NF-kappaB and AP-1 families of transcription factors, 2) the STAT family members and 3) the steroids receptors. This review aims to elucidate the divergent molecular mechanisms involved in the deregulated activation of transcription factor signaling in malignant transformation, although additional transcription factor families such as the Ets factors, ATF family members, basic helix-loop-helix transcription factors etc. are additional critical transcriptional regulators in human cancer. We explore new approaches to specifically inhibit these transcription factors in cancer in order to validate them as a drug targets. Efforts to develop novel viral vectors for therapeutic applications are also discussed.

GADD45 proteins: central players in tumorigenesis

The Growth Arrest and DNA Damage-inducible 45 (GADD45) proteins have been implicated in regulation of many cellular functions including DNA repair, cell cycle control, senescence and genotoxic stress. However, the pro-apoptotic activities have also positioned GADD45 as an essential player in oncogenesis. Emerging functional evidence implies that GADD45 proteins serve as tumor suppressors in response to diverse stimuli, connecting multiple cell signaling modules. Defects in the GADD45 pathway can be related to the initiation and progression of malignancies. Moreover, induction of GADD45 expression is an essential step for mediating anti-cancer activity of multiple chemotherapeutic drugs and the absence of GADD45 might abrogate their effects in cancer cells. In this review, we present a comprehensive discussion of the functions of GADD45 proteins, linking their regulation to effectors of cell cycle arrest, DNA repair and apoptosis. The ramifications regarding their roles as essential and central players in tumor growth suppression are also examined. We also extensively review recent literature to clarify how different chemotherapeutic drugs induce GADD45 gene expression and how its up-regulation and interaction with different molecular partners may benefit cancer chemotherapy and facilitate novel drug discovery.

Nucleoside triphosphate diphosphohydrolase-2 (NTPDase2/CD39L1) is the dominant ectonucleotidase expressed by rat astrocytes

Inflammatory and degenerative pathophysiological processes within the CNS are important causes of human disease. Astrocytes appear to modulate these reactions and are a major source of inflammatory mediators, e.g. extracellular adenine nucleotides, in nervous tissues. Actions following extracellular nucleotides binding to type 2 purinergic receptors are regulated by ectonucleotidases, including members of the CD39/ecto-nucleoside triphosphate diphosphohydrolase family. The ectonucleotidases of astrocytes expressed by rat brain rapidly convert extracellular ATP to ADP, ultimately to AMP. RT-PCR, immunocytochemistry as well as Western blotting analysis demonstrated expression of multiple ecto-nucleoside triphosphate diphosphohydrolase family members at both the mRNA and protein level. By quantitative real-time PCR, we identified Entpd2 (CD39L1) as the dominant Entpd gene expressed by rat hippocampal, cortical and cerebellar astrocytes. These data in combination with the elevated ecto-ATPase activity observed in these brain regions, suggest that NTPDase2, an ecto-enzyme that preferentially hydrolyzes ATP, is the major ecto-nucleoside triphosphate diphosphohydrolase expressed by rat astrocytes. NTPDase2 may modulate inflammatory reactions within the CNS and could represent a useful therapeutic target in human disease.

A novel role for GADD45beta as a mediator of MMP-13 gene expression during chondrocyte terminal differentiation

The growth arrest and DNA damage-inducible 45β (GADD45β) gene product has been implicated in the stress response, cell cycle arrest, and apoptosis. Here we demonstrated the unexpected expression of GADD45β in the embryonic growth plate and uncovered its novel role as an essential mediator of matrix metalloproteinase-13 (MMP-13) expression during terminal chondrocyte differentiation. We identified GADD45β as a prominent early response gene induced by bone morphogenetic protein-2 (BMP-2) through a Smad1/Runx2-dependent pathway. Because this pathway is involved in skeletal development, we examined mouse embryonic growth plates, and we observed expression of Gadd45β mRNA coincident with Runx2 protein in pre-hypertrophic chondrocytes, whereas GADD45β protein was localized prominently in the nucleus in late stage hypertrophic chondrocytes where Mmp-13 mRNA was expressed. In Gadd45β-/- mouse embryos, defective mineralization and decreased bone growth accompanied deficient Mmp-13 and Col10a1 gene expression in the hypertrophic zone. Transduction of small interfering RNA-GADD45β in epiphyseal chondrocytes in vitro blocked terminal differentiation and the associated expression of Mmp-13 and Col10a1 mRNA in vitro. Finally, GADD45β stimulated MMP-13 promoter activity in chondrocytes through the JNK-mediated phosphorylation of JunD, partnered with Fra2, in synergy with Runx2. These observations indicated that GADD45β plays an essential role during chondrocyte terminal differentiation.

Differential expression of GADD45β in normal and osteoarthritic cartilage: Potential role in homeostasis of articular chondrocytes

OBJECTIVE Our previous study suggested that growth arrest and DNA damage-inducible protein 45beta (GADD45beta) prolonged the survival of hypertrophic chondrocytes in the developing mouse embryo. This study was undertaken, therefore, to investigate whether GADD45beta plays a role in adult articular cartilage. METHODS Gene expression profiles of cartilage from patients with late-stage osteoarthritis (OA) were compared with those from patients with early OA and normal controls in 2 separate microarray analyses. Histologic features of cartilage were graded using the Mankin scale, and GADD45beta was localized by immunohistochemistry. Human chondrocytes were transduced with small interfering RNA (siRNA)-GADD45beta or GADD45beta-FLAG. GADD45beta and COL2A1 messenger RNA (mRNA) levels were analyzed by real-time reverse transcriptase-polymerase chain reaction, and promoter activities were analyzed by transient transfection. Cell death was detected by Hoechst 33342 staining of condensed chromatin. RESULTS GADD45beta was expressed at higher levels in cartilage from normal donors and patients with early OA than in cartilage from patients with late-stage OA. All chondrocyte nuclei in normal cartilage immunostained for GADD45beta. In early OA cartilage, GADD45beta was distributed variably in chondrocyte clusters, in middle and deep zone cells, and in osteophytes. In contrast, COL2A1, other collagen genes, and factors associated with skeletal development were up-regulated in late OA, compared with early OA or normal cartilage. In overexpression and knockdown experiments, GADD45beta down-regulated COL2A1 mRNA and promoter activity. NF-kappaB overexpression increased GADD45beta promoter activity, and siRNA-GADD45beta decreased cell survival per se and enhanced tumor necrosis factor alpha-induced cell death in human articular chondrocytes. CONCLUSION These observations suggest that GADD45beta might play an important role in regulating chondrocyte homeostasis by modulating collagen gene expression and promoting cell survival in normal adult cartilage and in early OA.

Reduced PDEF Expression Increases Invasion and Expression of Mesenchymal Genes in Prostate Cancer Cells

The epithelium-specific Ets transcription factor, PDEF, plays a role in prostate and breast cancer, although its precise function has not been established. In prostate cancer, PDEF is involved in regulating prostate-specific antigen expression via interaction with the androgen receptor and NKX3.1, and down-regulation of PDEF by antiproliferative agents has been associated with reduced PDEF expression. We now report that reduced expression of PDEF leads to a morphologic change, increased migration and invasiveness in prostate cancer cells, reminiscent of transforming growth factor beta (TGFbeta) function and epithelial-to-mesenchymal transition. Indeed, inhibition of PDEF expression triggers a transcriptional program of genes involved in the TGFbeta pathway, migration, invasion, adhesion, and epithelial dedifferentiation. Our results establish PDEF as a critical regulator of genes involved in cell motility, invasion, and adhesion of prostate cancer cells.

The receptor tyrosine kinase Axl is an essential regulator of prostate cancer proliferation and tumor growth and represents a new therapeutic target

Deregulation of the receptor tyrosine kinase Axl has been implicated in the progression of several human cancers. However, the role of Axl in prostate cancer remains poorly understood, and the therapeutic efficacy of Axl targeting remains untested. In this report we identified Axl as a new therapeutic target for prostate cancer. Axl is consistently overexpressed in prostate cancer cell lines and human prostate tumors. Interestingly, the blockage of Axl gene expression strongly inhibits proliferation, migration, invasion and tumor growth. Furthermore, inhibition of Axl expression by small interfering RNA regulates a transcriptional program of genes involved in cell survival, strikingly all connected to the nuclear factor-κB pathway. Additionally, blockage of Axl expression leads to inhibition of Akt, IKKα and IκBα phosphorylation, increasing IκBα expression and stability. Furthermore, induction of Akt phosphorylation by insulin-like growth factor 1 in Axl knockdown cells restores Akt activity and proliferation. Taken together, our results establish an unambiguous role for Axl in prostate cancer tumorigenesis with implications for prostate cancer treatment.

The receptor tyrosine kinase Axl in cancer: Biological functions and therapeutic implications

The receptor tyrosine kinase Axl has been implicated in the malignancy of different types of cancer. Emerging evidence of Axl upregulation in numerous cancers, as well as reports demonstrating that its inhibition blocks tumor formation in animal models, highlight the importance of Axl as a new potential therapeutic target. Furthermore, recent data demonstrate that Axl plays a pivotal role in resistance to chemotherapeutic regimens. In this review we discuss the functions of Axl and its regulation and role in cancer development, resistance to therapy, and its importance as a potential drug target, focusing on acute myeloid leukemia, breast, prostate and non‐small cell lung cancers.

A novel pathway involving melanoma differentiation associated gene-7/interleukin-24 mediates nonsteroidal anti-inflammatory drug-induced apoptosis and growth arrest of cancer cells.

Numerous studies show that nonsteroidal anti-inflammatory drugs (NSAIDs) are effective in chemoprevention or treatment of cancer. Nevertheless, the mechanisms underlying these antineoplastic effects remain poorly understood. Here, we report that induction of the cancer-specific proapoptotic cytokine melanoma differentiation associated gene-7/interleukin-24 (MDA-7/IL-24) by several NSAIDs is an essential step for induction of apoptosis and G(2)-M growth arrest in cancer cells in vitro and inhibition of tumor growth in vivo. We also show that MDA-7/IL-24-dependent up-regulation of growth arrest and DNA damage inducible 45 alpha (GADD45alpha) and GADD45gamma gene expression is sufficient for cancer cell apoptosis via c-Jun NH(2)-terminal kinase (JNK) activation and growth arrest induction through inhibition of Cdc2-cyclin B checkpoint kinase. Knockdown of GADD45alpha and GADD45gamma transcription by small interfering RNA abrogates apoptosis and growth arrest induction by the NSAID treatment, blocks JNK activation, and restores Cdc2-cyclin B kinase activity. Our results establish MDA-7/IL-24 and GADD45alpha and GADD45gamma as critical mediators of apoptosis and growth arrest in response to NSAIDs in cancer cells.