Regulation of MicroRNAs in Inflammation-Associated Colorectal Cancer: A Mechanistic Approach.

The development of colorectal cancer (CRC) is a multistage process. The inflammation of the colon as in inflammatory bowel disease (IBD) such as ulcerative colitis (UC) or Crohn's disease (CD) is often regarded as the initial trigger for the development of inflammation-associated CRC. Many cytokines such as tumor necrosis factor alpha (TNF-α) and interleukins (ILs) are known to exert proinflammatory actions, and inflammation initiates or promotes tumorigenesis of various cancers, including CRC, through differential regulation of microRNAs (miRNAs/miRs). miRNAs can be oncogenic miRNAs (oncomiRs) or anti-oncomiRs/tumor suppressor miRNAs, and they play key roles during colorectal carcinogenesis. However, the functions and molecular mechanisms of regulation of miRNAs involved in inflammation-associated CRC are still anecdotal and largely unknown. Consolidating the published results and offering perspective solutions to circumvent CRC, the current review is focused on the role of miRNAs and their regulation in the development of CRC. We have also discussed the model systems adapted by researchers to delineate the role of miRNAs in inflammation-associated CRC.

Role of Inflammation in the Development of Colorectal Cancer.

Chronic inflammation can lead to the development of many diseases, including cancer. Inflammatory bowel disease (IBD) that includes both ulcerative colitis (UC) and Crohnmp's disease (CD) are risk factors for the development of colorectal cancer (CRC). Many cytokines produced primarily by the gut immune cells either during or in response to localized inflammation in the colon and rectum are known to stimulate the complex interactions between the different cell types in the gut environment resulting in acute inflammation. Subsequently, chronic inflammation, together with genetic and epigenetic changes, have been shown to lead to the development and progression of CRC. Various cell types present in the colon, such as enterocytes, Paneth cells, goblet cells, and macrophages, express receptors for inflammatory cytokines and respond to tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1ß), IL-6, and other cytokines. Among the several cytokines produced, TNF-α and IL-1ß are the key pro-inflammatory molecules that play critical roles in the development of CRC. The current review is intended to consolidate the published findings to focus on the role of pro-inflammatory cytokines, namely TNF-α and IL-1ß, on inflammation (and the altered immune response) in the gut, to better understand the development of CRC in IBD, using various experimental model systems, preclinical and clinical studies. Moreover, this review also highlights the current therapeutic strategies available (monotherapy and combination therapy) to alleviate the symptoms or treat inflammation-associated CRC by using monoclonal antibodies or aptamers to block pro-inflammatory molecules, inhibitors of tyrosine kinases in the inflammatory signaling cascade, competitive inhibitors of pro-inflammatory molecules, and the nucleic acid drugs like small activating RNAs (saRNAs) or microRNA (miRNA) mimics to activate tumor suppressor or repress oncogene/pro-inflammatory cytokine gene expression.

The role of the redox/miR-6855-3p/PRDX5A axis in reversing SLUG-mediated BRCA2 silencing in breast cancer cells.

BACKGROUND: We have previously shown that the zinc finger transcription repressor SNAI2 (SLUG) represses tumor suppressor BRCA2-expression in non-dividing cells by binding to the E2-box upstream of the transcription start site. However, it is unclear how proliferating breast cancer (BC) cells that has higher oxidation state, overcome this repression. In this study, we provide insight into the mechanism of de-silencing of BRCA2 gene expression by PRDX5A, which is the longest member of the peroxiredoxin5 family, in proliferating breast cancer cells. METHODS: We used cell synchronization and DNA affinity pulldown to analyze PRDX5A binding to the BRCA2 silencer. We used oxidative stress and microRNA (miRNA) treatments to study nuclear localization of PRDX5A and its impact on BRCA2-expression. We validated our findings using mutational, reporter assay, and immunofluorescence analyses. RESULTS: Under oxidative stress, proliferating BC cells express PRDX5 isoform A (PRDX5A). In the nucleus, PRDX5A binds to the BRCA2 silencer near the E2-box, displacing SLUG and enhancing BRCA2-expression. Nuclear PRDX5A is translated from the second AUG codon in frame to the first AUG codon in the PRDX5A transcript that retains all exons. Mutation of the first AUG increases nuclear localization of PRDX5A in MDA-MB-231 cells, but mutation of the second AUG decreases it. Increased mitronic hsa-miRNA-6855-3p levels under oxidative stress renders translation from the second AUG preferable. Mutational analysis using reporter assay uncovered a miR-6855-3p binding site between the first and second AUG codon in the PRDX5A transcript. miR-6855-3p mimic increases accumulation of nuclear PRDX5A and inhibits reporter gene translation. CONCLUSION: Oxidative stress increases miR-6855-3p expression and binding to the inter-AUG sequence of the PRDX5A transcript, promoting translation of nuclear PRDX5A. Nuclear PRDX5A relieves SLUG-mediated BRCA2 silencing, resulting in increased BRCA2-expression.

Carbon monoxide sensitizes cisplatin-resistant ovarian cancer cell lines toward cisplatin via attenuation of levels of glutathione and nuclear metallothionein.

Cisplatin resistance remains a major impediment to effective treatment of ovarian cancer. Despite initial platinum responsiveness, thiol-containing peptides and proteins, glutathione (GSH) and metallothionein (MT), bind and inactivate cisplatin in cancer cells. Indeed, high levels of GSH and MT in ovarian cancers impart cisplatin resistance and are predictive of poor prognosis. Cystathionine ß-synthase (CBS), an enzyme involved in sulfur metabolism, is overexpressed in ovarian cancer tissues and is itself associated with cisplatin resistance. Treatment with exogenous carbon monoxide (CO), a known inhibitor of CBS, may mitigate cisplatin resistance in ovarian cancer cells by attenuation of GSH and MT levels. Using a photo-activated CO-releasing molecule (photoCORM), [Mn(CO)3(phen)(PTA)]CF3SO3 (phen = 1,10-phenanthroline, PTA = 1,3,5-triza-7-phosphaadamantane) we assessed the ability of CO to sensitize established cisplatin-resistant ovarian cancer cell lines to cisplatin. Cisplatin-resistant cells, treated with both cisplatin and CO, exhibited significantly lower cell viability and increased poly (ADP-ribose) polymerase (PARP) cleavage versus those treated with cisplatin alone. These cisplatin-resistant cell lines overexpressed CBS and had increased steady state levels of GSH and expression of nuclear MT. Both CO treatment and lentiviral-mediated silencing of CBS attenuated GSH and nuclear MT expression in cisplatin resistant cells. We have demonstrated that CO, delivered from a photoCORM, sensitizes established cisplatin-resistant cell lines to cisplatin. Furthermore, we have presented strong evidence that the effects of CO in circumventing chemotherapeutic drug resistance is at least in part mediated by the inactivation of endogenous CBS.

The Role of Epithelial to Mesenchymal Transition in Human Amniotic Membrane Rupture.

Context: Biochemical weakening of the amnion is a major factor preceding preterm premature rupture of membranes (PPROMs), leading to preterm birth. Activation of matrix metalloproteinases (MMPs) is known to play a key role in collagen degradation of the amnion; however, epithelial to mesenchymal transition (EMT) that is also induced by MMP activation has not been investigated as a mechanism for amnion weakening. Objective: To measure amniotic EMT associated with vaginal delivery (VD) compared with unlabored cesarean sections (CSs), and to assess changes in amniotic mechanical strength with pharmacologic inhibitors and inducers of EMT, thus testing the hypothesis that EMT is a key biochemical event that promotes amniotic rupture. Findings: (1) Amnions taken from VD contained a significantly increased number of mesenchymal cells relative to epithelial cells compared with unlabored CS by fluorescence-activated cell sorting analysis (60% vs 10%); (2) tumor necrosis factor (TNF)-α stimulation of amniotic epithelial cells increased expression of the mesenchymal marker vimentin after 2 days; (3) EMT inhibitor, etodolac, significantly increased the time and mechanical pressure required to rupture the amnion; and (4) TNF-α and another pharmacologic EMT inducer, ethacridine, decreased the time and mechanical pressure required for amnion rupture, further confirming that the mesenchymal phenotype significantly weakens the amnion. Conclusions: This work demonstrated amniotic cell EMT was associated with labor and EMT decreased the tensile strength of the amnion. These findings suggest a role for EMT in the pathophysiology of PPROM and may provide a basis for development of therapies to prevent preterm labor.

Increased sensitivity of African American triple negative breast cancer cells to nitric oxide-induced mitochondria-mediated apoptosis.

BACKGROUND: Breast cancer is a complex heterogeneous disease where many distinct subtypes are found. Younger African American (AA) women often present themselves with aggressive form of breast cancer with unique biology which is very difficult to treat. Better understanding the biology of AA breast tumors could lead to development of effective treatment strategies. Our previous studies indicate that AA but not Caucasian (CA) triple negative (TN) breast cancer cells were sensitive to nitrosative stress-induced cell death. In this study, we elucidate possible mechanisms that contribute to nitric oxide (NO)-induced apoptosis in AA TN breast cancer cells. METHODS: Breast cancer cells were treated with various concentrations of long-acting NO donor, DETA-NONOate and cell viability was determined by trypan blue exclusion assay. Apoptosis was determined by TUNEL and caspase 3 activity as well as changes in mitochondrial membrane potential. Caspase 3 and Bax cleavage, levels of Cu/Zn superoxide dismutase (SOD) and Mn SOD was assessed by immunoblot analysis. Inhibition of Bax cleavage by Calpain inhibitor, and levels of reactive oxygen species (ROS) as well as SOD activity was measured in NO-induced apoptosis. In vitro and in vivo effect of NO treatment on mammary cancer stem cells (MCSCs) was assessed. RESULTS AND DISCUSSION: NO induced mitocondria-mediated apoptosis in all AA but not in CA TN breast cancer cells. We found significant TUNEL-positive cells, cleavage of Bax and caspase-3 activation as well as depolarization mitochondrial membrane potential only in AA TN breast cancer cells exposed to NO. Inhibition of Bax cleavage and quenching of ROS partially inhibited NO-induced apoptosis in AA TN cells. Increase in ROS coincided with reduction in SOD activity in AA TN breast cancer cells. Furthermore, NO treatment of AA TN breast cancer cells dramatically reduced aldehyde dehydrogenase1 (ALDH1) expressing MCSCs and xenograft formation but not in breast cancer cells from CA origin. CONCLUSIONS: Ethnic differences in breast tumors dictate a need for tailoring treatment options more suited to the unique biology of the disease.

γ-Aminobutyric Acid Is Synthesized and Released by the Endothelium: Potential Implications.

RATIONALE: Gamma aminobutyric acid (GABA), a neurotransmitter of the central nervous system, is found in the systemic circulation of humans at a concentration between 0.5 and 3 µmol/L. However, the potential source of circulating GABA and its significance on the vascular system remains unknown. We hypothesized that endothelial cells (ECs) may synthesize and release GABA to modulate some functions in the EC and after its release into the circulation. OBJECTIVE: To assess whether GABA is synthesized and released by the EC and its potential functions. METHODS AND RESULTS: Utilizing the human umbilical vein ECs and aortic ECs, we demonstrated for the first time that ECs synthesize and release GABA from [1-(14)C]glutamate. Localization of GABA and the presence of the GABA-synthesizing enzyme, glutamic acid decarboxylase in EC were confirmed by immunostaining and immunoblot analysis, respectively. The presence of GABA was further confirmed by immunohistochemistry in the EC lining the human coronary vessel. EC-derived GABA regulated the key mechanisms of ATP synthesis, fatty acid, and pyruvate oxidation in EC. GABA protected EC by inhibiting the reactive oxygen species generation and prevented monocyte adhesion by attenuating vascular cell adhesion molecule -1 and monocyte chemoattractant protein-1 expressions. GABA had no relaxing effect on rat aortic rings. GABA exhibited a dose-dependent fall in blood pressure. However, the fall in BP was abolished after pretreatment with pentolinium. CONCLUSIONS: Our findings indicate novel potential functions of endothelium-derived GABA.

Cystathionine: A novel oncometabolite in human breast cancer.

In this study, we have identified cystathionine (CTH), a sulfur containing metabolite, to be selectively enriched in human breast cancer (HBC) tissues (∼50-100 pmoles/mg protein) compared with undetectable levels in normal breast tissues. The accumulation of CTH, specifically in HBC, was attributed to the overexpression of cystathionine beta synthase (CBS), its synthesizing enzyme, and the undetectable levels of its downstream metabolizing enzyme, cystathionine gamma lyase (CGL). Interestingly both CBS and CGL could not be detected in normal breast tissues. We further observed that CTH protected HBC cells against excess reactive oxygen species (ROS) and chemotherapeutic drug-induced apoptosis. Moreover, CTH promoted both mitochondrial and endoplasmic reticulum homeostasis in HBC cells. As both the mitochondria and the endoplasmic reticulum are key organelles regulating the onset of apoptosis, we reasoned that endogenous CTH could be contributing towards increasing the apoptotic threshold in HBC cells. An increased apoptotic threshold is a hallmark of all cancer types, including HBC, and is primarily responsible for drug resistance. Hence this study unravels one of the possible pathways that may contribute towards drug resistance in HBC.

Role of cystathionine ß-synthase in human breast Cancer.

Cystathionine ß-synthase (CBS) is an enzyme in the transulfuration pathway that can catalyze the condensation of homocysteine (Hcy) and cysteine (Cys) to hydrogen sulfide (H2S) and cystathionine (CTH). CBS-derived H2S is important in angiogenesis and drug resistance in colon and ovarian cancers, respectively. However, the mechanisms by which cancer cell-derived H2S is utilized by cancer cells as a protective agent against host-derived activated macrophages are not yet investigated. This study investigated the mechanistic role of CBS-derived H2S in the protection of human breast cancer (HBC) cells against activated macrophages. HBC patient-derived tissue arrays and immunoblot analysis of HBC cells exhibited significantly increased levels of CBS when compared with their normal counterparts. This was associated with increased levels of H2S and CTH. Silencing of CBS in HBC cells caused a significant decrease in the levels of H2S and CTH but did not affect the growth of these cells per se, in in vitro cultures. However CBS-silenced cells exhibited significantly reduced growth in the presence of activated macrophages and in xenograft models. This was associated with an increase in the steady state levels of reactive aldehyde-derived protein adducts. Exogenous addition of H2S countered the effects of CBS silencing in the presence of macrophages. Conversely overexpression of CBS in human breast epithelial (HBE) cells (which do not naturally express CBS) protected them from activated macrophages, which were otherwise susceptible to the latter.

Acid gradient across plasma membrane can drive phosphate bond synthesis in cancer cells: acidic tumor milieu as a potential energy source.

Aggressive cancers exhibit an efficient conversion of high amounts of glucose to lactate accompanied by acid secretion, a phenomenon popularly known as the Warburg effect. The acidic microenvironment and the alkaline cytosol create a proton-gradient (acid gradient) across the plasma membrane that represents proton-motive energy. Increasing experimental data from physiological relevant models suggest that acid gradient stimulates tumor proliferation, and can also support its energy needs. However, direct biochemical evidence linking extracellular acid gradient to generation of intracellular ATP are missing. In this work, we demonstrate that cancer cells can synthesize significant amounts of phosphate-bonds from phosphate in response to acid gradient across plasma membrane. The noted phenomenon exists in absence of glycolysis and mitochondrial ATP synthesis, and is unique to cancer. Biochemical assays using viable cancer cells, and purified plasma membrane vesicles utilizing radioactive phosphate, confirmed phosphate-bond synthesis from free phosphate (Pi), and also localization of this activity to the plasma membrane. In addition to ATP, predominant formation of pyrophosphate (PPi) from Pi was also observed when plasma membrane vesicles from cancer cells were subjected to trans-membrane acid gradient. Cancer cytosols were found capable of converting PPi to ATP, and also stimulate ATP synthesis from Pi from the vesicles. Acid gradient created through glucose metabolism by cancer cells, as observed in tumors, also proved critical for phosphate-bond synthesis. In brief, these observations reveal a role of acidic tumor milieu as a potential energy source and may offer a novel therapeutic target.

Gestational diabetes, preeclampsia and cytokine release: similarities and differences in endothelial cell function.

Gestational diabetes, pre-eclampsia as well as intra-uterine infection during pregnancy affects the function of the endothelium both in the mother and the fetus leading to endothelial dysfunction. Gestational diabetes is also associated with an increased incidence of pre-eclampsia and it is likely that both the hyperglycemia as well as the release of cytokines especially TNFα during hyperglycemia may play an important role in the pathogenesis of endothelial dysfunction leading to preeclampsia. Similarly, some but not all studies have suggested that infection of the mother under certain circumstances can also lead to preeclampsia as women with either a bacterial or viral infection were at a higher risk of developing preeclampsia, compared to women without infection and infection also leads to a release in TNFα. Endothelial cells exposed to either high glucose or TNFα leads to an increase in the production of H2O2 and to a decrease in endothelial cell proliferation. The cellular and molecular mechanisms involved in this phenomenon are discussed.Gestational diabetes, pre-eclampsia as well as intra-uterine infection during pregnancy has profound effects on the fetus and long term effects on the neonate. All three conditions affect the function of the endothelium both in the mother and the fetus leading to endothelial dysfunction. Gestational diabetes is also associated with an increased incidence of pre-eclampsia and it is likely that both the hyperglycemia as well as the release of cytokines especially TNFα during hyperglycemia may play an important role in the pathogenesis of endothelial dysfunction leading to preeclampsia. It has also been suggested although not universally accepted that under certain circumstances maternal infection may also predispose to pre-eclampsia. Pre-eclampsia is also associated with the release of TNFα and endothelial dysfunction. However, the cellular and molecular mechanism(s) leading to the endothelial dysfunction by either hyperglycemia or by the cytokine TNFα appear to be different. In this chapter, we explore some of the similarities and differences leading to endothelial dysfunction by both hyperglycemia and by the inflammatory cytokine TNFα and the cellular and molecular mechanism(s) involved.

Proteomic identification of mitochondrial targets of arginase in human breast cancer.

We have previously reported arginase expression in human breast cancer cells and demonstrated that the inhibition of arginase by N(ω) hydroxy L-arginine (NOHA) in MDA-MB-468 cells induces apoptosis. However, arginase expression and its possible molecular targets in human breast tumor samples and potential clinical implications have not been fully elucidated. Here, we demonstrate arginase expression in human breast tumor samples, and several established breast cancer cell lines, in which NOHA treatment selectively inhibits cell proliferation. The over-expression of Bcl2 in MDA-MB-468 cells abolished NOHA-induced apoptosis, suggesting that the mitochondria may be the main site of NOHA's action. We, therefore, undertook a proteomics approach to identify key mitochondrial targets of arginase in MDA-MB-468 cells. We identified 54 non-mitochondrial and 13 mitochondrial proteins that were differentially expressed in control and NOHA treated groups. Mitochondrial serine hydroxymethyltransferase (mSHMT) was identified as one of the most promising targets of arginase. Both arginase II (Arg II) and mSHMT expressions were higher in human breast tumor tissues compared to the matched normal and there was a strong correlation between Arg II and mSHMT protein expression. MDA-MB-468 xenografts had significant upregulation of Arg II expression that preceded the induction of mSHMT expression. Small inhibitory RNA (siRNA)-mediated inhibition of Arg II in MDA-MB-468 and HCC-1806 cells led to significant inhibition of both the mSHMT gene and protein expression. As mSHMT is a key player in folate metabolism, our data provides a novel link between arginine and folate metabolism in human breast cancer, both of which are critical for tumor cell proliferation.

A light-activated NO donor attenuates anchorage independent growth of cancer cells: Important role of a cross talk between NO and other reactive oxygen species.

It is established that high concentrations of nitric oxide(1) (NO), as released from activated macrophages, induce apoptosis in breast cancer cells. In this study, we assessed the potential of a light-activated NO donor [(Me2bpb)Ru(NO)(Resf)], a recently reported apoptototic agent, in suppressing the anchorage independent growth potentials of an aggressive human breast cancer cell line. Our results demonstrated the down regulation of anchorage independent growth by light activated NO treatment in the aggressive human breast cancer cell line MDA-MB-231 and afforded insight into the associated mechanism(s). The investigation revealed an up-regulation of the bioactivity of catalase with an accompanied reduction in the endogenous levels of H2O2, a direct substrate of catalase and a recently identified endogenous growth modulator in breast cancer cells. An earlier publication reported that endogenous superoxide (O2(-)) in human breast cancer cells constitutively inhibits catalase bioactivity (at the level of its protein), resulting in increased H2O2 levels. Interestingly in this study, O2(-) was also found to be down- regulated following NO treatment providing a basis for the observed increase in catalase bioactivity. Cells silenced for the catalase gene exhibited compromised reduction in anchorage independent growth upon light activated NO treatment. Collectively this study detailed a mechanistic cross talk between exogenous NO and endogenous ROS in attenuating anchorage independent growth.

Endothelial cell function in utero-placental circulation physiology and pathophysiology.

Endothelial cells in the utero-placental circulation play an important physiological role in maintaining the fetoplacental vessels in a vasodilated state as these vessels are non-innervated. These endothelial cells produce both prostacyclin and nitric oxide which in addition to causing vasodilation also prevent platelet aggregation and adhesion of platelets to endothelial cells. Most investigators are of the opinion that energy metabolism of endothelial cells and ATP generation is mainly glycolytic. Glycolytic activity in endothelial cells is increased during proliferation to maintain ATP at normal levels by an increase in the expression of the glucose transporter. More recent studies have reported the existence of a functional F1F0 ATP synthase on the surface of HUVEC and it has been found to be enzymatically active in the synthesis of ATP. Additional studies utilizing very early passage HUVEC need to be carried out to ascertain the relative contribution of oxidative phosphorylation compared with the glycolytic pathway for ATP synthesis in normal pregnancy as well as in abnormal states like preeclampsia, diabetes, intrauterine injection as well as intrauterine growth restriction.

Induction of a feed forward pro-apoptotic mechanistic loop by nitric oxide in a human breast cancer model.

We have previously demonstrated that relatively high concentrations of NO [Nitric Oxide] as produced by activated macrophages induced apoptosis in the human breast cancer cell line, MDA-MB-468. More recently, we also demonstrated the importance of endogenous H2O2 in the regulation of growth in human breast cancer cells. In the present study we assessed the interplay between exogenously administered NO and the endogenously produced reactive oxygen species [ROS] in human breast cancer cells and evaluated the mechanism[s] in the induction of apoptosis. To this end we identified a novel mechanism by which NO down regulated endogenous hydrogen peroxide [H2O2] formation via the down-regulation of superoxide [O2 (.-)] and the activation of catalase. We further demonstrated the existence of a feed forward mechanistic loop involving protein phosphatase 2A [PP2A] and its downstream substrate FOXO1 in the induction of apoptosis and the synthesis of catalase. We utilized gene silencing of PP2A, FOXO1 and catalase to assess their relative importance and key roles in NO mediated apoptosis. This study provides the potential for a therapeutic approach in treating breast cancer by targeted delivery of NO where NO donors and activators of downstream players could initiate a self sustaining apoptotic cascade in breast cancer cells.

Down-regulation of vitamin D receptor in mammospheres: implications for vitamin D resistance in breast cancer and potential for combination therapy.

Vitamin D signaling in mammary cancer stem cells (MCSCs), which are implicated in the initiation and progression of breast cancer, is poorly understood. In this study, we examined vitamin D signaling in mammospheres which are enriched in MCSCs from established breast cancer cell lines. Breast cancer cells positive for aldehyde dehydrogenase (ALDH(+)) had increased ability to form mammospheres compared to ALDH(-) cells. These mammospheres expressed MCSC-specific markers and generated transplantable xenografts in nude mice. Vitamin D receptor (VDR) was significantly down-regulated in mammospheres, as well as in ALDH(+) breast cancer cells. TN aggressive human breast tumors as well as transplantable xenografts obtained from SKBR3 expressed significantly lower levels of VDR but higher levels of CD44 expression. Snail was up-regulated in mammospheres isolated from breast cancer cells. Inhibition of VDR expression by siRNA led to a significant change in key EMT-specific transcription factors and increased the ability of these cells to form mammospheres. On the other hand, over-expression of VDR led to a down-regulation of Snail but increased expression of E-cad and significantly compromised the ability of cells to form mammospheres. Mammospheres were relatively insensitive to treatment with 1,25-dihydroxyvitamin D (1,25D), the active form of vitamin D, compared to more differentiated cancer cells grown in presence of serum. Treatment of H-Ras transformed HMLE(HRas) cells with DETA NONOate, a nitric oxide (NO)-donor led to induction of MAP-kinase phosphatase -1 (MKP-1) and dephosphorylation of ERK1/2 in the mammospheres. Combined treatment of these cells with 1,25D and a low-concentration of DETA NONOate led to a significant decrease in the overall size of mammospheres and reduced tumor volume in nude mice. Our findings therefore, suggest that combination therapy using 1,25D with drugs specifically targeting key survival pathways in MCSCs warrant testing in prospective clinical trial for treatment of aggressive breast cancer.

Mitochondrial-associated nitric oxide synthase activity inhibits cytochrome c oxidase: implications for breast cancer.

Nitric oxide (NO) is produced and nitric oxide synthase (NOS) activity is expressed in many types of tumor cells, but their precise role in tumor proliferation has not been clearly elucidated. Recently, it has been observed that patients with triple-negative breast tumors expressing NOS have a significantly worse prognosis compared to those that do not express any NOS. We observed that NOS activity was associated with the mitochondria in two breast cancer cell lines, ZR-75-30 and BT-474, compared with another NO-producing benign breast epithelial cell line, MCF-12F, in which no significant mitochondrial-associated NOS activity was detected. The rate of proliferation of the malignant cells expressing mitochondrial-associated NOS was decreased in the presence of an inhibitor of NO synthesis, but it had no effect on the normal breast epithelial cells, MCF-12F, which also expressed NOS, but not associated with mitochondria. The basal rate of proliferation was not affected by ODQ, an inhibitor of soluble guanylate cyclase, indicating that the effects of the endogenous NO produced by the malignant cell lines on proliferation are cGMP independent. Our results indicate that mitochondrial-associated NOS activity exhibited by the cancer cell lines ZR-75-30 and BT-474 inhibited cytochrome c oxidase, resulting in increased production of hydrogen peroxide (H2O2), which inhibited protein phosphatase 2A activity. This resulted in the maintenance of Akt and ERK1/2 in a phosphorylated state, leading to cell proliferation.

Maintenance of higher H2O2 levels, and its mechanism of action to induce growth in breast cancer cells: important roles of bioactive catalase and PP2A.

We assessed the catalase bioactivity and hydrogen peroxide (H2O2) production rate in human breast cancer (HBC) cell lines and compared these with normal human breast epithelial (HBE) cells. We observed that the bioactivity of catalase was decreased in HBC cells when compared with HBE cells. This was also accompanied by an increase in H2O2 steady-state levels in HBC cells. Silencing the catalase gene led to a further increase in the steady-state level of H2O2 which was also accompanied by an increase in growth rate of HBC cells. Catalase activity was up regulated on treatment with superoxide (O2⁻) scavengers such as pegylated SOD (PEG-SOD, indicating inhibition of catalase by the increased O2⁻ produced by HBC cells. Transfection of either catalase or glutathione peroxidase to HBC cells decreased intracellular H2O2 levels and led to apoptosis of these cells. The H2O2 produced by HBC cells inhibited PP2A activity accompanied by increased phosphorylation of Akt and ERK1/2. The importance of catalase bioactivity in breast cancer was further confirmed as its bioactivity was also decreased in human breast cancer tissues when compared to normal breast tissues. We conclude that inhibition of catalase bioactivity by O2⁻ leads to an increase in steady-state levels of H2O2 in HBC cells, which in turn inhibits PP2A activity, leading to phosphorylation of ERK 1/2 and Akt and resulting in HBC cell proliferation.

DICER1 loss and Alu RNA induce age-related macular degeneration via the NLRP3 inflammasome and MyD88.

Alu RNA accumulation due to DICER1 deficiency in the retinal pigmented epithelium (RPE) is implicated in geographic atrophy (GA), an advanced form of age-related macular degeneration that causes blindness in millions of individuals. The mechanism of Alu RNA-induced cytotoxicity is unknown. Here we show that DICER1 deficit or Alu RNA exposure activates the NLRP3 inflammasome and triggers TLR-independent MyD88 signaling via IL18 in the RPE. Genetic or pharmacological inhibition of inflammasome components (NLRP3, Pycard, Caspase-1), MyD88, or IL18 prevents RPE degeneration induced by DICER1 loss or Alu RNA exposure. These findings, coupled with our observation that human GA RPE contains elevated amounts of NLRP3, PYCARD, and IL18 and evidence of increased Caspase-1 and MyD88 activation, provide a rationale for targeting this pathway in GA. Our findings also reveal a function of the inflammasome outside the immune system and an immunomodulatory action of mobile elements.

High motility of triple-negative breast cancer cells is due to repression of plakoglobin gene by metastasis modulator protein SLUG.

One of highly pathogenic breast cancer cell types are the triple negative (negative in the expression of estrogen, progesterone, and ERBB2 receptors) breast cancer cells. These cells are highly motile and metastatic and are characterized by high levels of the metastasis regulator protein SLUG. Using isogenic breast cancer cell systems we have shown here that high motility of these cells is directly correlated with the levels of the SLUG in these cells. Because epithelial/mesenchymal cell motility is known to be negatively regulated by the catenin protein plakoglobin, we postulated that the transcriptional repressor protein SLUG increases the motility of the aggressive breast cancer cells through the knockdown of the transcription of the plakoglobin gene. We found that SLUG inhibits the expression of plakoglobin gene directly in these cells. Overexpression of SLUG in the SLUG-deficient cancer cells significantly decreased the levels of mRNA and protein of plakoglobin. On the contrary, knockdown of SLUG in SLUG-high cancer cells elevated the levels of plakoglobin. Blocking of SLUG function with a double-stranded DNA decoy that competes with the E2-box binding of SLUG also increased the levels of plakoglobin mRNA, protein, and promoter activity in the SLUG-high triple negative breast cancer cells. Overexpression of SLUG in the SLUG-deficient cells elevated the motility of these cells. Knockdown of plakoglobin in these low motility non-invasive breast cancer cells rearranged the actin filaments and increased the motility of these cells. Forced expression of plakoglobin in SLUG-high cells had the reverse effects on cellular motility. This study thus implicates SLUG-induced repression of plakoglobin as a motility determinant in highly disseminating breast cancer.