ATO Increases ROS Production and Apoptosis of Cells by Enhancing Calpain-Mediated Degradation of the Cancer Survival Protein TG2.

Transglutaminase 2 (TG2) is a critical cancer cell survival factor that activates several signalling pathways to foster drug resistance, cancer stem cell survival, metastasis, inflammation, epithelial-mesenchymal transition, and angiogenesis. All-trans retinoic acid (ATRA) and chemotherapy have been the standard treatments for acute promyelocytic leukaemia (APL), but clinical studies have shown that arsenic trioxide (ATO), alone or in combination with ATRA, can improve outcomes. ATO exerts cytotoxic effects in a variety of ways by inducing oxidative stress, genotoxicity, altered signal transduction, and/or epigenetic modification. In the present study, we showed that ATO increased ROS production and apoptosis ratios in ATRA-differentiated NB4 leukaemia cells, and that these responses were enhanced when TG2 was deleted. The combined ATRA + ATO treatment also increased the amount of nuclear factor erythroid 2-related factor 2 (NRF2) transcription factor, an adaptive regulator of the cellular oxidative stress response, and calpain proteolytic activity, resulting in TG2 degradation and the reduced survival of WT leukaemia cells. We further showed that the induced TG2 protein expression was degraded in the MCF-7 epithelial cell line and primary peripheral blood mononuclear cells upon ATO treatment, thereby sensitising these cell types to apoptotic signals.

Stretch-Induced Down-Regulation of HCN2 Suppresses Contractile Activity.

Although hyperpolarization-activated and cyclic nucleotide-gated 2 channels (HCN2) are expressed in multiple cell types in the gut, the role of HCN2 in intestinal motility is poorly understood. HCN2 is down-regulated in intestinal smooth muscle in a rodent model of ileus. Thus, the purpose of this study was to determine the effects of HCN inhibition on intestinal motility. HCN inhibition with ZD7288 or zatebradine significantly suppressed both spontaneous and agonist-induced contractile activity in the small intestine in a dose-dependent and tetrodotoxin-independent manner. HCN inhibition significantly suppressed intestinal tone but not contractile amplitude. The calcium sensitivity of contractile activity was significantly suppressed by HCN inhibition. Inflammatory mediators did not affect the suppression of intestinal contractile activity by HCN inhibition but increased stretch of the intestinal tissue partially attenuated the effects of HCN inhibition on agonist-induced intestinal contractile activity. HCN2 protein and mRNA levels in intestinal smooth muscle tissue were significantly down-regulated by increased mechanical stretch compared to unstretched tissue. Increased cyclical stretch down-regulated HCN2 protein and mRNA levels in primary human intestinal smooth muscle cells and macrophages. Overall, our results suggest that decreased HCN2 expression induced by mechanical signals, such as intestinal wall distension or edema development, may contribute to the development of ileus.

Mechanotransduction at the Plasma Membrane-Cytoskeleton Interface.

Mechanical cues are crucial for survival, adaptation, and normal homeostasis in virtually every cell type. The transduction of mechanical messages into intracellular biochemical messages is termed mechanotransduction. While significant advances in biochemical signaling have been made in the last few decades, the role of mechanotransduction in physiological and pathological processes has been largely overlooked until recently. In this review, the role of interactions between the cytoskeleton and cell-cell/cell-matrix adhesions in transducing mechanical signals is discussed. In addition, mechanosensors that reside in the cell membrane and the transduction of mechanical signals to the nucleus are discussed. Finally, we describe two examples in which mechanotransduction plays a significant role in normal physiology and disease development. The first example is the role of mechanotransduction in the proliferation and metastasis of cancerous cells. In this system, the role of mechanotransduction in cellular processes, including proliferation, differentiation, and motility, is described. In the second example, the role of mechanotransduction in a mechanically active organ, the gastrointestinal tract, is described. In the gut, mechanotransduction contributes to normal physiology and the development of motility disorders.

Bexarotene - a novel modulator of AURKA and the primary cilium in VHL-deficient cells.

Loss of the gene von Hippel-Lindau (VHL) is associated with loss of primary cilia and is causally linked to elevated levels of Aurora kinase A (AURKA). We developed an image-based high-throughput screening (HTS) assay using a dual-labeling image analysis strategy that identifies both the cilium and the basal body. By using this strategy, we screened small-molecule compounds for the targeted rescue of cilia defects associated with VHL deficiency with high accuracy and reproducibility. Bexarotene was identified and validated as a positive regulator of the primary cilium. Importantly, the inability of an alternative retinoid X receptor (RXR) agonist to rescue ciliogenesis, in contrast to bexarotene, suggested that multiple bexarotene-driven mechanisms were responsible for the rescue. We found that bexarotene decreased AURKA expression in VHL-deficient cells, thereby restoring the ability of these cells to ciliate in the absence of VHL Finally, bexarotene treatment reduced the propensity of subcutaneous lesions to develop into tumors in a mouse xenograft model of renal cell carcinoma (RCC), with a concomitant decrease in activated AURKA, highlighting the potential of bexarotene treatment as an intervention strategy in the clinic to manage renal cystogenesis associated with VHL deficiency and elevated AURKA expression.

Transglutaminase 2 programs differentiating acute promyelocytic leukemia cells in all-trans retinoic acid treatment to inflammatory stage through NF-κB activation.

Differentiation syndrome (DS) is a life-threatening complication arising during retinoid treatment of acute promyelocytic leukemia (APL). Administration of all-trans retinoic acid leads to significant changes in gene expression, among the most induced of which is transglutaminase 2, which is not normally expressed in neutrophil granulocytes. To evaluate the pathophysiological function of transglutaminase 2 in the context of immunological function and disease outcomes, such as excessive superoxide anion, cytokine, and chemokine production in differentiated NB4 cells, we used an NB4 transglutaminase knock-out cell line and a transglutaminase inhibitor, NC9, which inhibits both transamidase- and guanosine triphosphate-binding activities, to clarify the contribution of transglutaminase to the development of potentially lethal DS during all-trans retinoic acid treatment of APL. We found that such treatment not only enhanced cell-surface expression of CD11b and CD11c but also induced high-affinity states; atypical transglutaminase 2 expression in NB4 cells activated the nuclear factor kappa (κ)-light-chain-enhancer of the activated B-cell pathway, driving pathogenic processes with an inflammatory cascade through the expression of numerous cytokines, including tumor necrosis factor alpha (TNF-α), interleukin 1 beta (IL-1ß), and monocyte chemoattractant protein 1. NC9 decreased the amount of transglutaminase 2, p65/RelA, and p50 in differentiated NB4 cells and their nuclei, leading to attenuated inflammatory cytokine synthesis. NC9 significantly inhibits transglutaminase 2 nuclear translocation but accelerates its proteasomal breakdown. This study demonstrates that transglutaminase 2 expression induced by all-trans retinoic acid treatment reprograms inflammatory signaling networks governed by nuclear factor κ-light-chain-enhancer of activated B-cell activation, resulting in overexpression of TNF-α and IL-1ß in differentiating APL cells, suggesting that atypically expressed transglutaminase 2 is a promising target for leukemia treatment.

Atypical antipsychotics induce both proinflammatory and adipogenic gene expression in human adipocytes in vitro.

Schizophrenia requires lifelong treatment, potentially causing systemic changes in metabolic homeostasis. In the clinical setting, antipsychotic treatment may differentially lead to weight gain among individual patients, although the molecular determinants of such adverse effects are currently unknown. In this study, we investigated changes in the expression levels of critical regulatory genes of adipogenesis, lipid metabolism and proinflammatory genes during the differentiation of primary human adipose-derived stem cells (ADSCs). These cells were isolated from patients with body mass indices <25 and treated with the second-generation antipsychotics olanzapine, ziprasidone, clozapine, quetiapine, aripiprazole and risperidone and the first-generation antipsychotic haloperidol. We found that antipsychotics exhibited a marked effect on key genes involved in the regulation of cell cycle, signal transduction, transcription factors, nuclear receptors, differentiation markers and metabolic enzymes. In particular, we observed an induction of the transcription factor NF-KB1 and NF-KB1 target genes in adipocytes in response to these drugs, including the proinflammatory cytokines TNF-α, IL-1ß, IL-8 and MCP-1. In addition, enhanced secretion of both IL8 and MCP-1 was observed in the supernatant of these cell cultures. In addition to their remarkable stimulatory effects on proinflammatory gene transcription, three of the most frequently prescribed antipsychotic drugs, clozapine, quetiapine and aripiprazole, also induced the expression of essential adipocyte differentiation genes and the adipocyte hormones leptin and adiponectin, suggesting that both glucose and fat metabolism may be affected by these drugs. These data further suggest that antipsychotic treatments in patients alter the gene expression patterns in adipocytes in a coordinated fashion and priming them for a low-level inflammatory state.

Transglutaminase 2 associated with PI3K and PTEN in a membrane-bound signalosome platform blunts cell death.

Atypically expressed transglutaminase 2 (TG2) has been identified as a poor prognostic factor in a variety of cancers. In this study, we evaluated the contribution of TG2 to the prolonged cell survival of differentiated acute promyelocytic leukaemia (APL) cells in response to the standard treatment with combined retinoic acid (ATRA) and arsenic trioxide (ATO). We report that one advantage of ATRA + ATO treatment compared to ATRA alone diminishes the amount of activated and non-activated CD11b/CD18 and CD11c/CD18 cell surface integrin receptors. These changes suppress ATRA-induced TG2 docking on the cytosolic part of CD18 ß2-integrin subunits and reduce cell survival. In addition, TG2 overexpresses and hyperactivates the phosphatidylinositol-3-kinase (PI3K), phospho-AKT S473, and phospho-mTOR S2481 signalling axis. mTORC2 acts as a functional switch between cell survival and death by promoting the full activation of AKT. We show that TG2 presumably triggers the formation of a signalosome platform, hyperactivates downstream mTORC2-AKT signalling, which in turn phosphorylates and inhibits the activity of FOXO3, a key pro-apoptotic transcription factor. In contrast, the absence of TG2 restores basic phospho-mTOR S2481, phospho-AKT S473, PI3K, and PTEN expression and activity, thereby sensitising APL cells to ATO-induced cell death. We conclude, that atypically expressed TG2 may serve as a hub, facilitating signal transduction via signalosome formation by the CD18 subunit with both PI3K hyperactivation and PTEN inactivation through the PI3K-PTEN cycle in ATRA-treated APL cells.

Cancer-preventive rexinoid modulates neutral lipid contents of mammary epithelial cells through a peroxisome proliferator-activated receptor γ-dependent mechanism.

Synthetic rexinoids effectively suppress both estrogen receptor-positive and estrogen receptor-negative mammary tumors in animal models, which makes them prime candidates for a novel class of cancer-preventive agents. When used in combination with chemotherapy for non-small-cell lung cancer, the rexinoid bexarotene was most effective for patients who developed hypertriglyceridemia as a side effect. Although serum triglycerides originate from the liver, the effect of bexarotene on lipogenesis in breast epithelial cells is not known. Gene expression studies with normal mammary epithelial cells indicated that rexinoids modulate lipid metabolism, particularly enzymes involved in triglyceride synthesis. High-content analysis revealed dose-dependent accumulation of neutral lipids within adipocyte differentiation-related protein-associated cytoplasmic lipid droplets after long-term bexarotene treatment. Bexarotene also induced mRNA and protein levels for peroxisome proliferator-activated receptor (PPAR) γ, whereas selective knockdown of PPARγ attenuated the induction of both lipid droplets and adipocyte differentiation-related protein. Pharmacological activation of PPARγ, but not PPARα or retinoic acid receptors, effectively induced lipid accumulation. Furthermore, the combination of the PPARγ agonist rosiglitazone with bexarotene synergistically suppressed the growth of human mammary epithelial cells and revealed a strong, nonlinear, inverse correlation of cell growth with lipid droplet accumulation in the cell population. These findings indicate that rexinoids activate a lipogenic program in mammary epithelial cells through a retinoid X receptor/PPARγ-mediated mechanism. It is noteworthy that combining low doses of bexarotene with the PPARγ agonist rosiglitazone provides effective growth suppression of mammary epithelial cells, potentially dissociating systemic adverse effects associated with standard bexarotene treatment from the antiproliferative effects on mammary epithelium.

SLC22A5/OCTN2 expression in breast cancer is induced by estrogen via a novel intronic estrogen-response element (ERE).

Estrogen signaling is a critical pathway that plays a key role in the pathogenesis of breast cancer. In a previous transcriptional profiling study, we identified a novel panel of estrogen-induced genes in breast cancer. One of these genes is solute carrier family 22 member 5 (SLC22A5), which encodes a polyspecific organic cation transporter (also called OCTN2). In this study, we found that estrogen stimulates SLC22A5 expression robustly in an estrogen receptor (ER)-dependent manner and that SLC22A5 expression is associated with ER status in breast cancer cell lines and tissue specimens. Although the SLC22A5 proximal promoter is not responsive to estrogen, a downstream intronic enhancer confers estrogen inducibility. This intronic enhancer contains a newly identified estrogen-responsive element (ERE) (GGTCA-CTG-TGACT) and other transcription factor binding sites, such as a half ERE and a nuclear receptor related 1 (NR4A2/Nurr1) site. Estrogen induction of the luciferase reporter was dependent upon both the ERE and the NR4A2 site within the intronic enhancer. Small interfering RNA against either ER or Nurr1 inhibited estrogen induction of SLC22A5 expression, and chromatin immunoprecipitation assays confirmed the recruitment of both ER and Nurr1 to this enhancer. In functional assays, knockdown of SLC22A5 inhibited L: -carnitine intake, resulted in lipid droplet accumulation, and suppressed the proliferation of breast cancer cells. These results demonstrate that SLC22A5 is an estrogen-dependent gene regulated via a newly identified intronic ERE. Since SLC22A5 is a critical regulator of carnitine homeostasis, lipid metabolism, and cell proliferation, SLC22A5 may serve as a potential therapeutic target for breast cancer in the future.

Redistribution of the SWI/SNF Complex Dictates Coordinated Transcriptional Control over Epithelial-Mesenchymal Transition of Normal Breast Cells through TGF-ß Signaling.

Therapeutic targets in cancer cells defective for the tumor suppressor ARID1A are fundamentals of synthetic lethal strategies. However, whether modulating ARID1A function in premalignant breast epithelial cells could be exploited to reduce carcinogenic potential remains to be elucidated. In search of chromatin-modulating mechanisms activated by anti-proliferative agents in normal breast epithelial (HME-hTert) cells, we identified a distinct pattern of genome-wide H3K27 histone acetylation marks characteristic for the combined treatment by the cancer preventive rexinoid bexarotene (Bex) and carvedilol (Carv). Among these marks, several enhancers functionally linked to TGF-ß signaling were enriched for ARID1A and Brg1, subunits within the SWI/SNF chromatin-remodeling complex. The recruitment of ARID1A and Brg1 was associated with the suppression of TGFBR2, KLF4, and FoxQ1, and the induction of BMP6, while the inverse pattern ensued upon the knock-down of ARID1A. Bex+Carv treatment resulted in fewer cells expressing N-cadherin and dictated a more epithelial phenotype. However, the silencing of ARID1A expression reversed the ability of Bex and Carv to limit epithelial-mesenchymal transition. The nuclear levels of SMAD4, a canonical mediator of TGF-ß action, were more effectively suppressed by the combination than by TGF-ß. In contrast, TGF-ß treatment exceeded the ability of Bex+Carv to lower nuclear FoxQ1 levels and induced markedly higher E-cadherin positivity, indicating a target-selective antagonism of Bex+Carv to TGF-ß action. In summary, the chromatin-wide redistribution of ARID1A by Bex and Carv treatment is instrumental in the suppression of genes mediating TGF-ß signaling, and, thus, the morphologic reprogramming of normal breast epithelial cells. The concerted engagement of functionally linked targets using low toxicity clinical agents represents an attractive new approach for cancer interception.

The Role of ARID1A in the Nonestrogenic Modulation of IGF-1 Signaling.

Gaining pharmacologic access to the potential of ARID1A, a tumor suppressor protein, to mediate transcriptional control over cancer gene expression is an unresolved challenge. Retinoid X receptor ligands are pleiotropic, incompletely understood tools that regulate breast epithelial cell proliferation and differentiation. We found that low-dose bexarotene (Bex) combined with the nonselective beta-blocker carvedilol (Carv) reduces proliferation of MCF10DCIS.com cells and markedly suppresses ARID1A levels. Similarly, Carv synergized with Bex in MCF-7 cells to suppress cell growth. Chromatin immunoprecipitation sequencing analysis revealed that under nonestrogenic conditions Bex + Carv alters the concerted genomic distribution of the chromatin remodeler ARID1A and acetylated histone H3K27, at sites related to insulin-like growth factor (IGF) signaling. Several distinct sites of ARID1A enrichment were identified in the IGF-1 receptor and IRS1 genes, associated with a suppression of both proteins. The knock-down of ARID1A increased IGF-1R levels, prevented IGF-1R and IRS1 suppression upon Bex + Carv, and stimulated proliferation. In vitro IGF-1 receptor neutralizing antibody suppressed cell growth, while elevated IGF-1R or IRS1 expression was associated with poor survival of patients with ER-negative breast cancer. Our study demonstrates direct impact of ARID1A redistribution on the expression and growth regulation of IGF-1-related genes, induced by repurposed clinical drugs under nonestrogenic conditions. IMPLICATIONS: This study underscores the possibility of the pharmacologic modulation of the ARID1A factor to downregulate protumorigenic IGF-1 activity in patients with postmenopausal breast cancer undergoing aromatase inhibitor treatment.

Chemoprevention of hormone receptor-negative breast cancer: new approaches needed.

Results from clinical trials have demonstrated that it is possible to prevent estrogen-responsive breast cancers by targeting the estrogen receptor with selective estrogen receptor modulators (SERMs) (tamoxifen, raloxifene, or lasofoxifene) or with aromatase inhibitors (AIs) (anastrozole, letrozole, or exemestene). Results from breast cancer treatment trials suggest that aromatase inhibitors may be even more effective in preventing breast cancer than SERMs. However, while SERMs and aromatase inhibitors do prevent the development of many ER-positive breast cancers, these drugs do not prevent ER-negative breast cancer. These results show that new approaches are needed for the prevention of this aggressive form of breast cancer. Our laboratory and clinical efforts have been focused on identifying critical molecular pathways in breast cells that can be targeted for the prevention of ER-negative breast cancer. Our preclinical studies have demonstrated that other nuclear receptors, such as RXR receptors, vitamin D receptors, as well as others are critical for the growth of ER-negative breast cells and for the transformation of these cells into ER-negative cancers. Other studies show that growth factor pathways including those activated by EGFR, Her2, and IGFR, which are activated in many ER-negative breast cancers, can be targeted for the prevention of ER-negative breast cancer in mice. Clinical studies have also shown that PARP inhibitors are effective for the treatment of breast cancers arising in BRCA-1 or -2 mutation carriers, suggesting that targeting PARP may also be useful for the prevention of breast cancers arising in these high-risk individuals. Most recently, we have demonstrated that ER-negative breast cancers can be subdivided into four distinct groups based on the kinases that they express. These groups include ER-negative/Her-2-positive groups (the MAPK and immunomodulatory groups) and ER-negative/Her2-negative groups (the S6K and the cell cycle checkpoint groups). These groups of ER-negative breast cancers can be targeted with kinase inhibitors specific for each subgroup. These preclinical studies have supported the development of several clinical trials testing targeted agents for the prevention of breast cancer. The results of a completed Phase II cancer prevention trial using the RXR ligand bexarotene in women at high risk of breast cancer will be reviewed, and the current status of an ongoing Phase II trial using the EGFR and Her2 kinase inhibitor lapatinib for the treatment of women with DCIS breast cancer will be presented. It is anticipated that in the future these molecularly targeted drugs will be combined with hormonal agents such as SERMs or aromatase inhibitors to prevent all forms of breast cancer.

Benefits of Combined All-Trans Retinoic Acid and Arsenic Trioxide Treatment of Acute Promyelocytic Leukemia Cells and Further Enhancement by Inhibition of Atypically Expressed Transglutaminase 2.

Randomized trials in acute promyelocytic leukemia patients have shown that treatment with a combination of all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) is superior in efficacy to monotherapy, with significantly decreased mortality. So far, there are little data available to explain the success of the ATRA and ATO combination treatment in molecular terms. We showed that ATRA- and ATO-treated cells had the same capacity for superoxide production, which was reduced by two-thirds in the combined treatment. Secreted inflammatory biomarkers (monocyte chemoattractant protein-1 [MCP-1], interleukin-1 beta [IL-1ß] and tumor necrosis factor-α [TNF-α]) were significantly decreased and were further reduced in a transglutaminase 2 (TG2) expression-dependent manner. The amount of secreted TNF-α in the supernatant of NB4 TG2 knockout cells was close to 50 times lower than in ATRA-treated differentiated wild-type NB4 cells. The irreversible inhibitor of TG2 NC9 not only decreased reactive oxygen species production 28-fold, but decreased the concentration of MCP-1, IL-1ß and TNF-α 8-, 15- and 61-fold, respectively in the combined ATRA + ATO-treated wild-type NB4 cell culture. We propose that atypical expression of TG2 leads to the generation of inflammation, which thereby serves as a potential target for the prevention of differentiation syndrome.

Translation of Effects of Retinoids and Rexinoids: Extraction and Quality Assessment of RNA from Formalin-Fixed Tissues.

Retinoids and rexinoids directly and selectively activate their nuclear receptors, resulting in changes in the transcript levels of their target genes. Consequently, quantitating mRNA levels transcribed from cognate target genes is the most accurate measure of retinoid action. These changes can serve as relevant endpoints in biomarker trials, as well as in vivo preclinical studies. In gene expression analyses of archival material such as formalin-fixed paraffin-embedded (FFPE) tissues, assessing the quality of the extracted RNA is essential for the validation of the studies. With next generation sequencing (NGS) becoming the method of choice for gene expression profiling, RNA quality has become a critical aspect of study feasibility. In this chapter, we describe a method to extract RNA and to assess the intactness of RNA samples extracted from paraffin-embedded tissues.

Akt phosphorylates and suppresses the transactivation of retinoic acid receptor alpha.

The transactivation of nuclear receptors is regulated by both ligand binding and phosphorylation. We previously showed that RARalpha (retinoic acid receptor alpha) phosphorylation by c-Jun N-terminal kinase contributes to retinoid resistance in a subset of NSCLC cells (non-small cell lung cancer cells), but the aetiology of this resistance in the remainder has not been fully elucidated [Srinivas, Juroske, Kalyankrishna, Cody, Price, Xu, Narayanan, Weigel and Kurie (2005) Mol. Cell. Biol. 25, 1054-1069]. In the present study, we report that Akt, which is constitutively activated in NSCLC cells, phosphorylates RARalpha and inhibits its transactivation. Biochemical and functional analyses showed that Akt interacts with RARalpha and phosphorylates the Ser96 residue of its DNA-binding domain. Mutation of Ser96 to alanine abrogated the suppressive effect of Akt. Overexpression of a dominant-negative form of Akt in an NSCLC cell line decreased RAR phosphorylation, increased RAR transactivation and enhanced the growth-inhibitory effects of an RAR ligand. The findings presented here show that Akt inhibits RAR transactivation and contributes to retinoid resistance in a subset of NSCLC cells.

Identifying Cell Cycle Modulators That Selectively Target ARID1A Deficiency Using High-Throughput Image-Based Screening.

ARID1A, a component of the chromatin remodeling complex SWI/SNF, is an evolutionarily conserved complex that uses the energy of adenosine triphosphate hydrolysis to remodel chromatin structure and functions as a master regulator of gene transcription. Recent genomic studies have revealed that ARID1A is one of the most frequently mutated genes in human cancers. However, therapeutic approaches that selectively target ARID1A-mutant tumors are not yet clinically available. Our previous study showed that ARID1A facilitates chromatin response and cell cycle checkpoint activation after DNA damage. Therefore, an ARID1A deficiency may result in therapeutic vulnerabilities in cell cycle modulators. The goals of our study were to develop a novel screening approach, based on fluorescent ubiquitination-based cell cycle indicators (FUCCI), and to identify chemical agents that can selectively modulate the cell cycle transition in ARID1A-deficient cancer cells. Using this high-throughput assay, we screened 2643 compounds and identified six potential chemical modulators that can selectively modulate the cell cycle in ARID1A-deficient cells; these agents may be useful for developing new therapeutics for ARID1A-mutant tumors. In summary, our study demonstrates that FUCCI cell-based high-content screening is a powerful and effective approach for identifying cell cycle modulators and can be applied to multigenotypic screening for targeted cancer therapeutics.

Natural product ß-thujaplicin inhibits homologous recombination repair and sensitizes cancer cells to radiation therapy.

Investigation of natural products is an attractive strategy to identify novel compounds for cancer prevention and treatment. Numerous studies have shown the efficacy and safety of natural products, and they have been widely used as alternative treatments for a wide range of illnesses, including cancers. However, it remains unknown whether natural products affect homologous recombination (HR)-mediated DNA repair and whether these compounds can be used as sensitizers with minimal toxicity to improve patients' responses to radiation therapy, a mainstay of treatment for many human cancers. In this study, in order to systematically identify natural products with an inhibitory effect on HR repair, we developed a high-throughput image-based HR repair screening assay and screened a chemical library containing natural products. Among the most interesting of the candidate compounds identified from the screen was ß-thujaplicin, a bioactive compound isolated from the heart wood of plants in the Cupressaceae family, can significantly inhibit HR repair. We further demonstrated that ß-thujaplicin inhibits HR repair by reducing the recruitment of a key HR repair protein, Rad51, to DNA double-strand breaks. More importantly, our results showed that ß-thujaplicin can radiosensitize cancer cells. Additionally, ß-thujaplicin sensitizes cancer cells to PARP inhibitor in different cancer cell lines. Collectively, our findings for the first time identify natural compound ß-thujaplicin, which has a good biosafety profile, as a novel HR repair inhibitor with great potential to be translated into clinical applications as a sensitizer to DNA-damage-inducing treatment such as radiation and PARP inhibitor. In addition, our study provides proof of the principle that our robust high-throughput functional HR repair assay can be used for a large-scale screening system to identify novel natural products that regulate DNA repair and cellular responses to DNA damage-inducing treatments such as radiation therapy.

Retinoids and rexinoids in cancer prevention: from laboratory to clinic.

Early in the age of modern medicine the consequences of vitamin A deficiency drew attention to the fundamental link between retinoid-dependent homeostatic regulation and malignant hyperproliferative diseases. The term "retinoid" includes a handful of endogenous and a large group of synthetic derivatives of vitamin A. These multifunctional lipid-soluble compounds directly regulate target genes of specific biological functions and critical signaling pathways to orchestrate complex functions from vision to development, metabolism, and inflammation. Many of the retinoid activities on the cellular level have been well characterized and translated to the regulation of processes like differentiation and cell death, which play critical roles in the outcome of malignant transformation of tissues. In fact, retinoid-based differentiation therapy of acute promyelocytic leukemia was one of the first successful examples of molecularly targeted treatment strategies. The selectivity, high receptor binding affinity and the ability of retinoids to directly modulate gene expression programs present a distinct pharmacological opportunity for cancer treatment and prevention. However, to fully exploit their potential, the adverse effects of retinoids must be averted. In this review we provide an overview of the biology of retinoid (activated by nuclear retinoic acid receptors [RARs]) and rexinoid (engaged by nuclear retinoid X receptors [RXRs]) action concluded from a long line of preclinical studies, in relation to normal and transformed states of cells. We will also discuss the past and current uses of retinoids in the treatment of malignancies, the potential of rexinoids in the cancer prevention setting, both as single agents and in combinations.

The SOX11 transcription factor is a critical regulator of basal-like breast cancer growth, invasion, and basal-like gene expression.

Basal-like breast cancers (BLBCs) are aggressive breast cancers associated with poor survival. Defining the key drivers of BLBC growth will allow identification of molecules for targeted therapy. In this study, we performed a primary screen integrating multiple assays that compare transcription factor expression and activity in BLBC and non-BLBC at the RNA, DNA, and protein levels. This integrated screen identified 33 transcription factors that were elevated in BLBC in multiple assays comparing mRNA expression, DNA cis-element sequences, or protein DNA-binding activity. In a secondary screen to identify transcription factors critical for BLBC cell growth, 8 of the 33 candidate transcription factors (TFs) were found to be necessary for growth in at least two of three BLBC cell lines. Of these 8 transcription factors, SOX11 was the only transcription factor required for BLBC growth, but not for growth of non-BLBC cells. Our studies demonstrate that SOX11 is a critical regulator of multiple BLBC phenotypes, including growth, migration, invasion, and expression of signature BLBC genes. High SOX11 expression was also found to be an independent prognostic indicator of poor survival in women with breast cancer. These results identify SOX11 as a potential target for the treatment of BLBC, the most aggressive form of breast cancer.

Mechanical unloading increases caveolin expression in the failing human heart.

OBJECTIVE: Implantation of a left ventricular assist device (LVAD) in the failing human heart initiates structural and functional changes termed reverse remodeling. Mechanical unloading improves cardiac adrenergic responsiveness and lipid metabolism, processes regulated by caveolar function. We tested the hypothesis that mechanical unloading alters the expression of caveolins and these changes are linked to altered expression of markers of reverse remodeling. METHODS: Paired human myocardial samples were obtained from patients who received an LVAD as a bridge to cardiac transplantation. Transcript levels were measured using real-time Q-RT-PCR in RNA prepared from 34 pairs of formalin-fixed myocardial tissue blocks. Caveolin-1 and -3 protein levels were determined from frozen tissue (n=5) by Western blots. Caveolin-3 localization was demonstrated by immunohistochemistry. RESULTS: Caveolin-1 protein levels were upregulated in all LVAD-patients after mechanical unloading (P=0.002). Caveolin-1 mRNA was increased in 76% of the patients (n=34, P<0.001). Larger induction of caveolin-1 was associated with greater suppression of ANF. Caveolin-3 transcript levels increased in 82% of the cohort, along with a 2.5-fold induction of caveolin-2. Sarcolemmal caveolin-3 staining was increased after LVAD-support, although no change in total caveolin-3 protein was detected. The mRNA levels of the caveolin-associated CD36 also increased with unloading. Patients with ischemic cardiomyopathy showed greater induction of CD36 (P<0.05) than non-ischemic cases, as well as highly correlated changes in the expression of caveolin isoforms. CONCLUSION: Mechanical unloading induces the expression of caveolins and CD36. The induction of caveolin-1 and the reciprocal suppression of ANF suggest that the changes in the expression of both genes are linked to decreased hemodynamic load. Enhanced caveolin expression during mechanical unloading of failing human hearts may be a part of the reverse remodeling of lipid metabolism, nitric oxide production and adrenergic signaling.