Contribution of Adrenal Glands to Intratumor Androgens and Growth of Castration-Resistant Prostate Cancer.

PURPOSE: Tumor androgens in castration-resistant prostate cancer (CRPC) reflect de novo intratumoral synthesis or adrenal androgens. We used C.B.-17 SCID mice in which we observed adrenal CYP17A activity to isolate the impact of adrenal steroids on CRPC tumors in vivo. EXPERIMENTAL DESIGN: We evaluated tumor growth and androgens in LuCaP35CR and LuCaP96CR xenografts in response to adrenalectomy (ADX). We assessed protein expression of key steroidogenic enzymes in 185 CRPC metastases from 42 patients. RESULTS: Adrenal glands of intact and castrated mice expressed CYP17A. Serum DHEA, androstenedione (AED), and testosterone (T) in castrated mice became undetectable after ADX (all P < 0.05). ADX prolonged median survival (days) in both CRPC models (33 vs. 179; 25 vs. 301) and suppressed tumor steroids versus castration alone (T 0.64 pg/mg vs. 0.03 pg/mg; DHT 2.3 pg/mg vs. 0.23 pg/mg; and T 0.81 pg/mg vs. 0.03 pg/mg, DHT 1.3 pg/mg vs. 0.04 pg/mg; all P ≤ 0.001). A subset of tumors recurred with increased steroid levels, and/or induction of androgen receptor (AR), truncated AR variants, and glucocorticoid receptor (GR). Metastases from 19 of 35 patients with AR positive tumors concurrently expressed enzymes for adrenal androgen utilization and nine expressed enzymes for de novo steroidogenesis (HSD3B1, CYP17A, AKR1C3, and HSD17B3). CONCLUSIONS: Mice are appropriate for evaluating adrenal impact of steroidogenesis inhibitors. A subset of ADX-resistant CRPC tumors demonstrate de novo androgen synthesis. Tumor growth and androgens were suppressed more strongly by surgical ADX than prior studies using abiraterone, suggesting reduction in adrenally-derived androgens beyond that achieved by abiraterone may have clinical benefit. Proof-of-concept studies with agents capable of achieving true "nonsurgical ADX" are warranted.

Inhibition of GLO1 in Glioblastoma Multiforme Increases DNA-AGEs, Stimulates RAGE Expression, and Inhibits Brain Tumor Growth in Orthotopic Mouse Models.

Cancers that exhibit the Warburg effect may elevate expression of glyoxylase 1 (GLO1) to detoxify the toxic glycolytic byproduct methylglyoxal (MG) and inhibit the formation of pro-apoptotic advanced glycation endproducts (AGEs). Inhibition of GLO1 in cancers that up-regulate glycolysis has been proposed as a therapeutic targeting strategy, but this approach has not been evaluated for glioblastoma multiforme (GBM), the most aggressive and difficult to treat malignancy of the brain. Elevated GLO1 expression in GBM was established in patient tumors and cell lines using bioinformatics tools and biochemical approaches. GLO1 inhibition in GBM cell lines and in an orthotopic xenograft GBM mouse model was examined using both small molecule and short hairpin RNA (shRNA) approaches. Inhibition of GLO1 with S-(p-bromobenzyl) glutathione dicyclopentyl ester (p-BrBzGSH(Cp)2) increased levels of the DNA-AGE N²-1-(carboxyethyl)-2'-deoxyguanosine (CEdG), a surrogate biomarker for nuclear MG exposure; substantially elevated expression of the immunoglobulin-like receptor for AGEs (RAGE); and induced apoptosis in GBM cell lines. Targeting GLO1 with shRNA similarly increased CEdG levels and RAGE expression, and was cytotoxic to glioma cells. Mice bearing orthotopic GBM xenografts treated systemically with p-BrBzGSH(Cp)2 exhibited tumor regression without significant off-target effects suggesting that GLO1 inhibition may have value in the therapeutic management of these drug-resistant tumors.

Testicular vs adrenal sources of hydroxy-androgens in prostate cancer.

Neoadjuvant androgen deprivation therapy (NADT) is one strategy for the treatment of early-stage prostate cancer; however, the long-term outcomes of NADT with radical prostatectomy including biochemical failure-free survival are not promising. One proposed mechanism is incomplete androgen ablation. In this study, we aimed to evaluate the efficiency of serum hydroxy-androgen suppression in patients with localized high-risk prostate cancer under NADT (leuprolide acetate plus abiraterone acetate and prednisone) and interrogate the primary sources of circulating hydroxy-androgens using our recently described stable isotope dilution liquid chromatography mass spectrometric method. For the first time, three androgen diols including 5-androstene-3ß,17ß-diol (5-adiol), 5α-androstane-3α,17ß-diol (3α-adiol), 5α-androstane-3ß,17ß-diol (3ß-adiol), the glucuronide or sulfate conjugate of 5-adiol and 3α-adiol were measured and observed to be dramatically reduced after NADT. By comparing patients that took leuprolide acetate alone vs leuprolide acetate plus abiraterone acetate and prednisone, we were able to distinguish the primary sources of these androgens and their conjugates as being of either testicular or adrenal in origin. We find that testosterone, 5α-dihydrotestosterone (DHT), 3α-adiol and 3ß-adiol were predominately of testicular origin. By contrast, dehydroepiandrosterone (DHEA), epi-androsterone (epi-AST) and their conjugates, 5-adiol sulfate and glucuronide were predominately of adrenal origin. Our findings also show that NADT failed to completely suppress DHEA-sulfate levels and that two unappreciated sources of intratumoral androgens that were not suppressed by leuprolide acetate alone were 5-adiol-sulfate and epi-AST-sulfate of adrenal origin.

Association of Tissue Abiraterone Levels and SLCO Genotype with Intraprostatic Steroids and Pathologic Response in Men with High-Risk Localized Prostate Cancer.

Purpose: Germline variation in solute carrier organic anion (SLCO) genes influences cellular steroid uptake and is associated with prostate cancer outcomes. We hypothesized that, due to its steroidal structure, the CYP17A inhibitor abiraterone may undergo transport by SLCO-encoded transporters and that SLCO gene variation may influence intracellular abiraterone levels and outcomes.Experimental Design: Steroid and abiraterone levels were measured in serum and tissue from 58 men with localized prostate cancer in a clinical trial of LHRH agonist plus abiraterone acetate plus prednisone for 24 weeks prior to prostatectomy. Germline DNA was genotyped for 13 SNPs in six SLCO genes.Results: Abiraterone levels spanned a broad range (serum median 28 ng/mL, 108 nmol/L; tissue median 77 ng/mL, 271 nmol/L) and were correlated (r = 0.355, P = 0.001). Levels correlated positively with steroids upstream of CYP17A (pregnenolone, progesterone), and inversely with steroids downstream of CYP17A (DHEA, AED, testosterone). Serum PSA and tumor volumes were higher in men with undetectable versus detectable tissue abiraterone at prostatectomy (median 0.10 vs. 0.03 ng/dL, P = 0.02; 1.28 vs. 0.44 cc, P = 0.09, respectively). SNPs in SLCO2B1 associated with significant differences in tissue abiraterone (rs1789693, P = 0.0008; rs12422149, P = 0.03) and higher rates of minimal residual disease (tumor volume < 0.5 cc; rs1789693, 67% vs. 27%, P = 0.009; rs1077858, 46% vs. 0%, P = 0.03). LNCaP cells expressing SLCO2B1 showed two- to fourfold higher abiraterone levels compared with vector controls (P < 0.05).Conclusions: Intraprostatic abiraterone levels and genetic variation in SLCO genes are associated with pathologic responses in high-risk localized prostate cancer. Variation in SLCO genes may serve as predictors of response to abiraterone treatment. Clin Cancer Res; 23(16); 4592-601. ©2017 AACR.

Simultaneous quantitation of nine hydroxy-androgens and their conjugates in human serum by stable isotope dilution liquid chromatography electrospray ionization tandem mass spectrometry.

Castration resistant prostate cancer (CRPC), the fatal form of prostate cancer, remains androgen dependent despite castrate levels of circulating testosterone (T) and 5α-dihydrotestosterone (DHT). To investigate mechanisms by which the tumor can synthesize its own androgens and develop resistance to abiraterone acetate and enzalutamide, methods to measure a complete androgen profile are imperative. Here, we report the development and validation of a stable isotope dilution liquid chromatography electrospray ionization tandem mass spectrometric (SID-LC-ESI-MS/MS) method to quantify nine human hydroxy-androgens as picolinates, simultaneously with requisite specificity and sensitivity. In the established method, the fragmentation patterns of all nine hydroxy-androgen picolinates were identified, and [13C3]-5α-androstane-3α, 17ß-diol and [13C3]-5α-androstane-3ß, 17ß-diol used as internal standards were synthesized enzymatically. Intra-day and inter-day precision and accuracy corresponds to the U.S. Food and Drug Administration Criteria for Bioanalytical Method Validation. The lower limit of quantitation (LLOQ) of nine hydroxy-androgens is 1.0pg to 2.5pg on column. Diols which have been infrequently measured: 5-androstene-3ß, 17ß-diol and 5α-androstane-3α, 17ß-diol can be determined in serum at values as low as 1.0pg on column. The method also permits the quantitation of conjugated hydroxy-androgens following enzymatic digestion. While direct detection of steroid conjugates by electrospray-ionization tandem mass spectrometry has advantages the detection of unconjugated and conjugated steroids would require separate methods for each set of analytes. Our method was applied to pooled serum from male and female donors to provide reference values for both unconjugated and conjugated hydroxy-androgens. This method will allow us to interrogate the involvement of the conversion of 5-androstene-3ß, 17ß-diol to T, the backdoor pathway involving the conversion of 5α-androstane-3α, 17ß-diol to DHT and the inactivation of DHT to 5α-androstane-3ß, 17ß-diol in advanced prostate cancer.

Discovery of (R)-2-(6-Methoxynaphthalen-2-yl)butanoic Acid as a Potent and Selective Aldo-keto Reductase 1C3 Inhibitor.

Type 5 17ß-hydroxysteroid dehydrogenase, aldo-keto reductase 1C3 (AKR1C3) converts Δ(4)-androstene-3,17-dione and 5α-androstane-3,17-dione to testosterone (T) and 5α-dihydrotestosterone, respectively, in castration resistant prostate cancer (CRPC). In CRPC, AKR1C3 is implicated in drug resistance, and enzalutamide drug resistance can be surmounted by indomethacin a potent inhibitor of AKR1C3. We examined a series of naproxen analogues and find that (R)-2-(6-methoxynaphthalen-2-yl)butanoic acid (in which the methyl group of R-naproxen was replaced by an ethyl group) acts as a potent AKR1C3 inhibitor that displays selectivity for AKR1C3 over other AKR1C enzymes. This compound was devoid of inhibitory activity on COX isozymes and blocked AKR1C3 mediated production of T and induction of PSA in LNCaP-AKR1C3 cells as a model of a CRPC cell line. R-Profens are substrate selective COX-2 inhibitors and block the oxygenation of endocannabinoids and in the context of advanced prostate cancer R-profens could inhibit intratumoral androgen synthesis and act as analgesics for metastatic disease.

Current advances in intratumoral androgen metabolism in castration-resistant prostate cancer.

PURPOSE OF REVIEW: Androgen deprivation therapy is a cornerstone in the treatment of advanced prostate cancer and has extended the lives of countless patients. Unfortunately, many of these patients eventually succumb to metastatic castration-resistant prostate cancer (mCRPC). The efficacy of abiraterone acetate (AA, Zytiga) and enzalutamide (Enza, Xtandi) in the mCRPC setting prove that these tumors remain androgen-driven. We review recent studies that have shown that intratumoral androgen biosynthesis plays a significant role in the ever-evolving mCRPC tumor and we discuss the therapeutic implications of these findings. RECENT FINDINGS: A novel abiraterone metabolite, 17-(pyridin-3-yl)androsta-4,16-dien-3-one (D4A), possesses robust antitumor activity in rodent models via the inhibition of androgen biosynthetic enzymes and antagonism of the androgen receptor. The TMPRSS2 : ERG fusion drives aldo-keto reductase 1C3 (AKR1C3) expression and activity to facilitate androgen biosynthesis and activate the androgen receptor in prostate cancer. Intracrine androgen formation and AKR1C3 expression and activity have been found to confer resistance to enzalutamide. SUMMARY: These studies highlight the significant role that intratumoral androgen biosynthesis plays in the mCRPC tumor. The therapeutic implications include the inhibition of AKR1C3 in tumors that become resistant to current drugs such as abiraterone acetate or Enza and the potential administration of D4A as an mCRPC therapeutic.

The DHEA-sulfate depot following P450c17 inhibition supports the case for AKR1C3 inhibition in high risk localized and advanced castration resistant prostate cancer.

Prostate cancer is the second leading cause of cancer death in the United States. Treatment of localized high-risk disease and de novo metastatic disease frequently leads to relapse. These metastatic castration resistant prostate cancers (mCRPC) claim a high mortality rate, despite the extended survival afforded by the growing armamentarium of androgen deprivation, radiation and immunotherapies. Here, we review two studies of neoadjuvant treatment of high-risk localized prostate cancer prior to prostatectomy, the total androgen pathway suppression (TAPS) trial and the neoadjuvant abiraterone acetate (AA) trial. These two trials assessed the efficacy of the non-specific P450c17 inhibitor, ketoconazole and the specific P450c17 inhibitor, AA, to inhibit tissue and serum androgen levels. Furthermore, a novel and validated stable isotope dilution liquid chromatography electrospray ionization selected reaction monitoring mass spectrometry assay was used to accurately quantify adrenal and gonadal androgens in circulation during the course of these trials. The adrenal androgens, Δ(4)-androstene-3,17-dione, dehydroepiandrosterone and dehydroepiandrosterone sulfate were significantly reduced in the patients receiving ketoconazole or AA compared to those who did not. However, in both trials, a significant amount of DHEA-S (∼20 µg/dL) persists and thus may serve as a depot for intratumoral conversion to the potent androgen receptor ligands, testosterone (T) and 5α-dihydrotestosterone (DHT). The final step in conversion of Δ(4)-androstene-3,17-dione and 5α-androstanedione to T and DHT, respectively, is catalyzed by AKR1C3. We therefore present the case that in the context of the DHEA-S depot, P450c17 and AKR1C3 inhibition may be an effective combinatorial treatment strategy.

Intense androgen-deprivation therapy with abiraterone acetate plus leuprolide acetate in patients with localized high-risk prostate cancer: results of a randomized phase II neoadjuvant study.

PURPOSE: Cure rates for localized high-risk prostate cancers (PCa) and some intermediate-risk PCa are frequently suboptimal with local therapy. Outcomes are improved by concomitant androgen-deprivation therapy (ADT) with radiation therapy, but not by concomitant ADT with surgery. Luteinizing hormone-releasing hormone agonist (LHRHa; leuprolide acetate) does not reduce serum androgens as effectively as abiraterone acetate (AA), a prodrug of abiraterone, a CYP17 inhibitor that lowers serum testosterone (< 1 ng/dL) and improves survival in metastatic PCa. The possibility that greater androgen suppression in patients with localized high-risk PCa will result in improved clinical outcomes makes paramount the reassessment of neoadjuvant ADT with more robust androgen suppression. PATIENTS AND METHODS: A neoadjuvant randomized phase II trial of LHRHa with AA was conducted in patients with localized high-risk PCa (N = 58). For the first 12 weeks, patients were randomly assigned to LHRHa versus LHRHa plus AA. After a research prostate biopsy, all patients received 12 additional weeks of LHRHa plus AA followed by prostatectomy. RESULTS: The levels of intraprostatic androgens from 12-week prostate biopsies, including the primary end point (dihydrotestosterone/testosterone), were significantly lower (dehydroepiandrosterone, Δ(4)-androstene-3,17-dione, dihydrotestosterone, all P < .001; testosterone, P < .05) with LHRHa plus AA compared with LHRHa alone. Prostatectomy pathologic staging demonstrated a low incidence of complete responses and minimal residual disease, with residual T3- or lymph node-positive disease in the majority. CONCLUSION: LHRHa plus AA treatment suppresses tissue androgens more effectively than LHRHa alone. Intensive intratumoral androgen suppression with LHRHa plus AA before prostatectomy for localized high-risk PCa may reduce tumor burden.

Targeted androgen pathway suppression in localized prostate cancer: a pilot study.

PURPOSE: Ligand-mediated activation of the androgen receptor (AR) is critical for prostate cancer (PCa) survival and proliferation. The failure to completely ablate tissue androgens may limit suppression of PCa growth. We evaluated combinations of CYP17A and 5-α-reductase inhibitors for reducing prostate androgen levels, AR signaling, and PCa volumes. PATIENTS AND METHODS: Thirty-five men with intermediate/high-risk clinically localized PCa were randomly assigned to goserelin combined with dutasteride (ZD), bicalutamide and dutasteride (ZBD), or bicalutamide, dutasteride, and ketoconazole (ZBDK) for 3 months before prostatectomy. Controls included patients receiving combined androgen blockade with luteinizing hormone-releasing hormone agonist and bicalutamide. The primary outcome measure was tissue dihydrotestosterone (DHT) concentration. RESULTS: Prostate DHT levels were substantially lower in all experimental arms (0.02 to 0.04 ng/g v 0.92 ng/g in controls; P < .001). The ZBDK group demonstrated the greatest percentage decline in serum testosterone, androsterone, and dehydroepiandrosterone sulfate (P < .05 for all). Staining for AR and the androgen-regulated genes prostate-specific antigen and TMPRSS2 was strongly suppressed in benign glands and moderately in malignant glands (P < .05 for all). Two patients had pathologic complete response, and nine had ≤ 0.2 cm(3) of residual tumor (defined as a near-complete response), with the largest numbers of complete and near-complete responses in the ZBDK group. CONCLUSION: Addition of androgen synthesis inhibitors lowers prostate androgens below that achieved with standard therapy, but significant AR signaling remains. Tissue-based analysis of steroids and AR signaling is critical to informing the search for optimal local and systemic control of high-risk prostate cancer.

Development, validation and application of a stable isotope dilution liquid chromatography electrospray ionization/selected reaction monitoring/mass spectrometry (SID-LC/ESI/SRM/MS) method for quantification of keto-androgens in human serum.

Prostate cancer is the most frequently diagnosed form of cancer in males in the United States. The disease is androgen driven and the use of orchiectomy or chemical castration, known as androgen deprivation therapy (ADT) has been employed for the treatment of advanced prostate cancer for over 70 years. Agents such as GnRH agonists and non-steroidal androgen receptor antagonists are routinely used in the clinic, but eventually relapse occurs due to the emergence of castration-resistant prostate cancer. With the appreciation that androgen signaling still persists in these patients and the development of new therapies such as abiraterone and enzalutamide that further suppresses androgen synthesis or signaling, there is a renewed need for sensitive and specific methods to quantify androgen precursor and metabolite levels to assess drug efficacy. We describe the development, validation and application of a stable isotope dilution liquid chromatography electrospray ionization selected reaction monitoring mass spectrometry (SID-LC/ESI/SRM/MS) method for quantification of serum keto-androgens and their sulfate and glucuronide conjugates using Girard-T oxime derivatives. The method is robust down to 0.2-4pg on column, depending on the androgen metabolite quantified, and can also quantify dehydroepiandrosterone sulfate (DHEA-S) in only 1µL of serum. The clinical utility of this method was demonstrated by analyzing serum androgens from patients enrolled in a clinical trial assessing combinations of pharmacological agents to maximally suppress gonadal and adrenal androgens (Targeted Androgen Pathway Suppression, TAPS clinical trial). The method was validated by correlating the results obtained with a hydroxylamine derivatization procedure coupled with tandem mass spectrometry using selected reaction monitoring that was conducted in an independent laboratory.

Enhanced radiation response in radioresistant MCF-7 cells by targeting peroxiredoxin II.

In our previous study, we identified that a protein target, peroxiredoxin II (PrxII), is overexpressed in radioresistant MCF+FIR3 breast-cancer cells and found that its expression and function is associated with breast-cancer radiation sensitivity or resistance. Small interference RNA (siRNA) targeting PrxII gene expression was able to sensitize MCF+FIR3 radioresistant breast-cancer cells to ionizing radiation. The major focus of this work was to investigate how the radiation response of MCF+FIR3 radioresistant cells was affected by the siRNA that inhibits PrxII gene expression. Our results, presented here, show that silencing PrxII gene expression increased cellular toxicity by altering cellular thiol status, inhibiting Ca(2+) efflux from the cells, and perturbing the intracellular Ca(2+) homeostasis. By combining radiotherapy and siRNA technology, we hope to develop new therapeutic strategies that may have potential to enhance the efficacy of chemotherapeutic agents due to this technology's property of targeting to specific cancer-related genes.

Mutagenic potential of DNA glycation: miscoding by (R)- and (S)-N2-(1-carboxyethyl)-2'-deoxyguanosine.

Elevated circulating glucose resulting from complications of obesity and metabolic disease can result in the accumulation of advanced glycation end products (AGEs) of proteins, lipids, and DNA. The formation of DNA-AGEs assumes particular importance as these adducts may contribute to genetic instability and elevated cancer risk associated with metabolic disease. The principal DNA-AGE, N(2)-(1-carboxyethyl)-2'-deoxyguanosine (CEdG), is formed as a mixture of R and S isomers at both the polymer and monomer levels. In order to examine the miscoding potential of this adduct, oligonucleotides substituted with (R)- and (S)-CEdG and the corresponding triphosphates (R)- and (S)-CEdGTP were synthesized, and base-pairing preferences for each stereoisomer were examined using steady-state kinetic approaches. Purine dNTPs were preferentially incorporated opposite template CEdG when either the Klenow (Kf(-)) or Thermus aquaticus (Taq) polymerases were used. The Kf(-) polymerase preferentially incorporated dGTP, whereas Taq demonstrated a bias for dATP. Kf(-) incorporated purines opposite the R isomer with greater efficiency, but Taq favored the S isomer. Incorporation of (R)- and (S)-CEdGTP only occurred opposite dC and was catalyzed by Kf(-) with equal efficiencies. Primer extension from a 3'-terminal CEdG was observed only for the R isomer. These data suggest CEdG is the likely adduct responsible for the observed pattern of G transversions induced by exposure to elevated glucose or its alpha-oxoaldehyde decomposition product methylglyoxal. The results imply that CEdG within template DNA and the corresponding triphosphate possess different syn/anti conformations during replication which influence base-pairing preferences. The implications for CEdG-induced mutagenesis in vivo are discussed.

Advanced glycation end products of DNA: quantification of N2-(1-Carboxyethyl)-2'-deoxyguanosine in biological samples by liquid chromatography electrospray ionization tandem mass spectrometry.

Methylglyoxal (MG) and related alpha-oxoaldehydes react with proteins, lipids, and DNA to give rise to covalent adducts known as advanced glycation end products (AGEs). Elevated levels of AGEs have been implicated in the pathological complications of diabetes, uremia, Alzheimer's disease, and possibly cancer. There is therefore widespread interest in developing sensitive methods for the in vivo measurement of AGEs as prognostic biomarkers and for treatment monitoring. The two diastereomeric MG-DNA adducts of N(2)-(1-carboxyethyl)-2'-deoxyguanosine (CEdG) are the primary glycation products formed in DNA; however, accurate assessment of their distribution in vivo has not been possible since there is no readily available quantitative method for CEdG determination in biological samples. To address these issues, we have developed a sensitive and quantitative liquid chromatography electrospray ionization tandem mass spectrometry assay using the stable isotope dilution method with an (15)N(5)-CEdG standard. Methods for CEdG determination in urine or tissue extracted DNA are described. Changes in urinary CEdG in diabetic rats in response to oral administration of the AGE inhibitor LR-90 are used to demonstrate the potential utility of the method for treatment monitoring. Both stereoisomeric CEdG adducts were detected in a human breast tumor and normal adjacent tissue at levels of 3-12 adducts/10(7) dG, suggesting that this lesion may be widely distributed in vivo. Strategies for dealing with artifactual adduct formation due to oxoaldehyde generation during DNA isolation and enzymatic workup procedures are described.

The role of peroxiredoxin II in radiation-resistant MCF-7 breast cancer cells.

Although several signaling pathways have been suggested to be involved in the cellular response to ionizing radiation, the molecular basis of tumor resistance to radiation remains elusive. We have developed a unique model system based upon the MCF-7 human breast cancer cell line that became resistant to radiation treatment (MCF+FIR30) after exposure to chronic ionizing radiation. By proteomics analysis, we found that peroxiredoxin II (PrxII), a member of a family of peroxidases, is up-regulated in the radiation-derived MCF+FIR3 cells but not in the MCF+FIS4 cells that are relatively sensitive to radiation. Both MCF+FIR3 and MCF+FIS4 cell lines are from MCF+FIR30 populations. Furthermore, the resistance to ionizing radiation can be partially reversed by silencing the expression of PrxII by PrxII/small interfering RNA treatment of MCF+FIR3 resistant cells, suggesting that PrxII is not the sole factor responsible for the resistant phenotype. The relevance of this mechanism was further confirmed by the increased radioresistance in PrxII-overexpressing MCF+FIS4 cells when compared with vector control cells. The up-regulation of the PrxII protein in radioresistant cancer cells suggested that human peroxiredoxin plays an important role in eliminating the generation of reactive oxygen species by ionizing radiation. The present finding, together with the observation that PrxII is also up-regulated in response to ionizing radiation in other cell systems, strengthens the hypothesis that the PrxII antioxidant protein is involved in the cellular response to ionizing radiation and functions to reduce the intracellular reactive oxygen species levels, resulting in increased resistance of breast cancer cells to ionizing radiation.

Co-activation of ERK, NF-kappaB, and GADD45beta in response to ionizing radiation.

NF-kappaB has been well documented to play a critical role in signaling cell stress reactions. The extracellular signal-regulated kinase (ERK) regulates cell proliferation and survival. GADD45beta is a primary cell cycle element responsive to NF-kappaB activation in anti-apoptotic responses. The present study provides evidence demonstrating that NK-kappaB, ERK and GADD45beta are co-activated by ionizing radiation (IR) in a pattern of mutually dependence to increase cell survival. Stress conditions generated in human breast cancer MCF-7 cells by the administration of a single exposure of 5 Gy IR resulted in the activation of ERK but not p38 or JNK, along with an enhancement of the NF-kappaB transactivation and GADD45beta expression. Overexpression of dominant negative Erk (DN-Erk) or pre-exposure to ERK inhibitor PD98059 inhibited NF-kappaB. Transfection of dominant negative mutant IkappaB that blocks NF-kappaB nuclear translocation, inhibited ERK activity and GADD45beta expression and increased cell radiosensitivity. Interaction of p65 and ERK was visualized in living MCF-7 cells by bimolecular fluorescence complementation analysis. Antisense inhibition of GADD45beta strikingly blocked IR-induced NF-kappaB and ERK but not p38 and JNK. Overall, these results demonstrate a possibility that NF-kappaB, ERK, and GADD45beta are able to coordinate in a loop-like signaling network to defend cells against the cytotoxicity induced by ionizing radiation.

Response of cyclin B1 to ionizing radiation: regulation by NF-kappaB and mitochondrial antioxidant enzyme MnSOD.

BACKGROUND: To understand the molecular response of tumor cells to therapeutic ionizing radiation (IR), we previously reported that human breast cancer cells derived following chronic exposure to fractionated ionizing radiation (MCF+FIR) showed a transient radioresistance. MCF+FIR cells also demonstrated increased activity of NF-kappaB, increased expression of the mitochondrial antioxidant enzyme (MnSOD), and increased expression of a cell cycle regulatory protein (Cyclin B1). The present studies were designed to determine the relationship of NF-kappaB, MnSOD and Cyclin B1 expression in cellular adaptive responses to ionizing radiation. MATERIALS AND METHODS: The first intron of the cyclin B1 gene with a putative NF-kappaB element was cloned into the pGL3 luciferase reporter (pGL3CB1EI1). PGL3CB1EI1 and control NF-kappaB luciferase activities were determined in MCF-7 and MCF+FIR cells treated with a single dose of radiation, over expression of the dominant negative mutant IkB (mIkB) or over expression of the SOD2 gene. RESULTS: MCF+FIR cells derived from fractionated IR demonstrated increased transactivation of the pGL3CB1EI1 and NF-kappaB controlled reporter activities, relative to the parental cell line. Transfection of dominant negative mutant IkB that inhibits NF-kappaB nuclear translocation, inhibited pGL3CB1EI1 and NF-kappaB activity, indicating the NF-kappaB dependence of pGL3CB1EI1 mediated transcription. In addition, over expression of the human SOD2 gene (MnSOD) inhibited NF-kappaB and pGL3CB1EI1 activity, indicating that superoxide or some species derived from superoxide may have participated in the up-regulation of reporter activity in response to chronic exposure to fractionated ionizing radiation. These results provide evidence suggesting that a signaling pathway involving NF-kappaB and Cyclin B1 may contribute to adaptive radioresistance induced by chronic exposure to fractionated IR and support the conclusion that MnSOD appears to be a negative regulator of this pathway.

Expression of ErbB2 enhances radiation-induced NF-kappaB activation.

Her-2/neu (ErbB2) oncogene, the second member of the epidermal growth factor receptor (EGFR) family, encodes a transmembrane tyrosine kinase receptor in Her-2-positive tumors. Accumulating evidences demonstrate that signaling networks activated by EGFR and transcription factor NF-kappaB are associated with cell response to ionizing radiation (IR). The present study shows that overexpression of ErbB2 enhanced NF-kappaB activation induced by IR in human breast carcinoma MCF-7 cells transfected with ErbB2 genes (MCF-7/ErbB2). Stable transfection of dominant-negative mutant IkappaB (MCF-7/ErbB2/mIkappaB) or treatment with anti-ErbB2 antibody, Herceptin, inhibited NF-kappaB activation and radiosensitized MCF-7/ErbB2 cells. Consistent with NF-kappaB regulation, basal and IR-induced Akt, a kinase downstream of ErbB2, was activated in MCF-7/ErbB2 cells and inhibited by Herceptin. To identify specific genes affected by ErbB2-mediated NF-kappaB activation, a group of IR-responsive elements Cyclin B1, Cyclin D1, Bcl-2, Bcl/XL, BAD and BAX were evaluated. Basal levels of prosurvival elements Cyclin B1, Cyclin D1, Bcl-2 and Bcl/XL but not apoptotic BAD and BAX were upregulated in MCF-7/ErbB2 cells with striking enhancements in Bcl-2 and Bcl/XL. IR further induced Cyclin B1 and Cyclin D1 expression that was reduced by Herceptin. Bcl-2 kept a high steady level after Herceptin+IR treatment and, in contrast to control MCF-7/Vector cells, Bcl/XL was inhibited in MCF-7/ErbB2 cells by Herceptin+IR treatment. However, all four prosurvival proteins were downregulated by inhibition of NF-kappaB in MCF-7/ErbB2/mIkappaB cells. These results thus provide evidence suggesting that overexpression of ErbB2 is able to enhance NF-kappaB response to IR, and that a specific prosurvival network downstream of NF-kappaB is triggered by treatments using anti-ErbB2 antibody combined with radiation.

Manganese superoxide dismutase-mediated gene expression in radiation-induced adaptive responses.

Antioxidant enzymes are critical in oxidative stress responses. Radioresistant variants isolated from MCF-7 human carcinoma cells following fractionated ionizing radiation (MCF+FIR cells) or overexpression of manganese superoxide dismutase (MCF+SOD cells) demonstrated dose-modifying factors at 10% isosurvival of 1.8 and 2.3, respectively. MCF+FIR and MCF-7 cells (exposed to single-dose radiation) demonstrated 5- to 10-fold increases in MnSOD activity, mRNA, and immunoreactive protein. Radioresistance in MCF+FIR and MCF+SOD cells was reduced following expression of antisense MnSOD. DNA microarray analysis and immunoblotting identified p21, Myc, 14-3-3 zeta, cyclin A, cyclin B1, and GADD153 as genes constitutively overexpressed (2- to 10-fold) in both MCF+FIR and MCF+SOD cells. Radiation-induced expression of these six genes was suppressed in fibroblasts from Sod2 knockout mice (-/-) as well as in MCF+FIR and MCF+SOD cells expressing antisense MnSOD. Inhibiting NF-kappa B transcriptional activity in MCF+FIR cells, by using mutant I kappa B alpha, inhibited radioresistance as well as reducing steady-state levels of MnSOD, 14-3-3 zeta, GADD153, cyclin A, and cyclin B1 mRNA. In contrast, mutant I kappa B alpha was unable to inhibit radioresistance or reduce 14-3-3 zeta, GADD153, cyclin A, and cyclin B1 mRNAs in MCF+SOD cells, where MnSOD overexpression was independent of NF-kappa B. These results support the hypothesis that NF-kappa B is capable of regulating the expression of MnSOD, which in turn is capable of increasing the expression of genes that participate in radiation-induced adaptive responses.