Genetic Variations in Inflammatory Response Genes and Their Association with the Risk of Prostate Cancer

Prostate cancer is a common cancer in men. Genetic variations in inflammatory response genes can potentially influence the risk of prostate cancer. We aimed to examine the association between PPARG Pro12Ala, NFKB1 -94 ins/del, NFKBIA -826C/T, COX-1 (50C>T), and COX-2 (-1195G>A) polymorphisms on prostate cancer risk. The genotypes of the polymorphisms were ascertained in 543 prostate cancer patients and 753 controls through PCR-RFLP and the risk association was evaluated statistically using logistic regression analysis. The NFKB1 -94 polymorphism was shown to decrease prostate cancer risk in both heterozygous and homozygous comparison models (odds ratios of 0.74 (95% CI = 0.58–0.96) (P = 0.02) and 0.57 (95% CI = 0.42–0.78) (P < 0.01), resp.). An opposite finding was observed for COX-2 (-1195) polymorphism (odds ratios of 1.58 (95% CI = 1.15–2.18) (P < 0.01) for heterozygous comparison model and 2.08 (95% CI = 1.48–2.92) (P < 0.01) for homozygous comparison model). No association was observed for other polymorphisms. In conclusion, NFKB1 -94 ins/del and COX-2 (-1195G>A) polymorphisms may be, respectively, associated with decreased and increased prostate cancer risk in the Chinese population.

Introduction
Prostate cancer is a form of cancer in which malignant cells develop in the prostate gland of the male reproductive system. Prostate cancer is the second leading type of cancer in men globally, with an age-standardized incidence rate of 31.1 per 100,000 men [1]. In 2012 alone, a total of 1,111,689 cases of prostate cancers were diagnosed, and approximately 307,471 deaths were attended [1]. In comparison, the estimated numbers of incidence and mortality of the cancer were 679,000 and 221,000, respectively, in 2002 [2]. Thus, a marked increase in the incidence and mortality of prostate cancer has been observed during the past decade, and development of an effective screening strategy for identifying individuals at risk of prostate cancer is greatly needed. This is especially true for the Chinese population, in which the mortality-toincidence rate ratio (MR/IR) has been consistently higher in comparison to the average MR/IR of Asian countries and Western populations [3,4].
Established etiological factors of prostate cancer include age, ethnicity, diet, and environmental carcinogen exposure [5]. The interactions among the above elements are believed to play significant roles in contributing to prostate carcinogenesis, although the precise mechanisms of this have not been fully understood [5]. In addition, in recent years, chronic inflammation of the prostate and, importantly, genetic profile of an individual have been shown to play an equally important role in oncogenesis of the prostate [6,7]. Epidemiological and molecular evidences implied that dysregulation of inflammatory response genes may promote the development and progression of approximately 20% of prostate malignancies by inducing DNA damage, cell proliferation enhancement, apoptosis inhibition, and angiogenesis simulation [8].
The most common cause of inflammatory response gene dysregulations is mutations in high penetrance genes [9]. Despite the fact that such mutations can strongly increase the risk of prostate cancer, they are only present in a small frequency in the general population [9]. In comparison, genetic variations in the form of single nucleotide polymorphisms (SNPs) in low penetrance genes can increase the risk of cancer modestly, but they are more prevalent in the general population and, thus, their overall impact could be substantial [10]. SNPs may alter the expression and functionality of the protein products [11], and many studies have showed a relationship between inherited variations in inflammatory response genes and the risk of prostate carcinogenesis [12][13][14][15][16]. However, the influence of several common SNPs in the inflammatory response genes, either individually or in combinations, on prostate cancer risk has been understudied in the Chinese population. This includes the PPARG Pro12Ala polymorphism, NFKB1 -94 ins/del polymorphism, NFKBIA -826C/T polymorphism, COX-1 (50C>T) polymorphism, and COX-2 (-1195G>A) polymorphism. The present study aimed to investigate the association of the above polymorphisms with the risk of prostate cancer in the Chinese population.

Study Subjects.
The study was approved by the Medical Ethics Committee of the Xuanwu Hospital, Beijing, China. All subjects were recruited from Xuanwu Hospital, Beijing, China, between October 2010 and August 2014. A total of 543 males who were diagnosed by qualified physicians as prostate cancer patients based on clinical, pathological, and laboratory tests were recruited into the study. To ensure consistency, only patients with high grade prostate cancer (Gleason score > 7) who had a PSA level of >10 ng/mL were included in the study. At the same period of time, a total of 753 healthy controls were enrolled into this study. Eligibility criteria for control included no individual history of cancer and no known genetic syndromes associated with prostate cancer and a PSA level of <2 ng/mL. Blood samples were collected from the subjects who meet the criteria mentioned above after obtaining written informed consent.

SNP Genotyping.
Genomic DNA extraction was done by using EasyPure Blood Genomic DNA Kit (TransGen Biotech, Beijing, China) after the blood samples were taken from peripheral blood of all subjects. The DNA was used for genotyping of the five SNPs by using the PCR-RFLP method. All PCR were performed in a 25 L reaction mixture containing standard PCR buffer, 1.5 mM MgCl 2 , 0.25 mM dNTP, 1 unit Taq polymerase, and 0.4 M of each primer. For all polymorphisms, approximately 10% of the samples were chosen at random and sequenced to confirm the genotypes from the amplified PCR product. The laboratory personnel performing the genotyping were blinded to the identity, the case-control status, and source of the samples.

PPARG Pro12Ala
Polymorphism. The PCR primers used for amplification of the region containing the PPARG Pro12Ala polymorphism were 5 -GCC AAT TCA AGC CCA GTC-3 and 5 -GAT ATG TTT GCA GAC AGT GTA TCA GTG AAG GAA TCG CTT TCC-3 . The annealing temperature of the reaction was 58 ∘ C. The PCR amplification produced a fragment of 270 bp in size, which was then digested using BstUI restriction enzyme. The fragment was cleaved into a 227 bp and a 43 bp fragment for the Ala/Ala genotype while the Pro/Pro genotype was uncut. Apart from that, heterozygous genotype (Pro/Ala) was detected by the presence of all the above bands in agarose gel.

NFKB1 -94 ins/del Polymorphism.
The PCR for amplification of the region containing the NFKB1 -94 ins/del polymorphism was performed by using 5 -TGG GCA CAA GTC GTT TAT GA-3 and 5 -CTG GAG CCG GTA GGG AAG-3 primers, which were annealed to the genomic DNA at 61 ∘ C. PflMI restriction enzyme was subsequently used to digest the amplified PCR product of 281 bp in size. For ins/ins genotype, the absence of restriction site resulted in the undigested fragment of 281 bp after the reaction. On the other hand, a 240 bp and a 45 bp fragment were obtained for the del/del genotype, while a 281 bp, a 240 bp, and a 45 bp fragment were found in the ins/del genotype.

NFKBIA -826C/T Polymorphism.
The NFKBIA -826C/T polymorphism was genotyped by first amplifying the region of interest by using the following PCR primers: 5 -GGT CCT TAA GGT CCA ATC G-3 and 5 -GTT GTG GAT ACC TTG CAC TA-3 at an annealing temperature of 59.5 ∘ C. This produced an amplicon of 200 bp in size. Restriction enzyme digestion was performed with BfaI restriction enzyme. The TT genotype was uncut by the enzyme and remained 200 bp, but the CC genotype was cleaved into 180 bp and 20 bp bands. Heterozygotes showed all three bands on agarose gel.

COX-1 (50C>T) Polymorphism.
The PCR primers used were 5 -GGT GCC CGG TGG GGA ATT TTC-3 and 5 -GAG GGG AAA GGA GGG GGT TG-3. The annealing temperature used was 60 ∘ C. The PCR reaction generated a product of 245 bp. Then, the fragment was digested by using SmuI restriction enzyme. The TT genotype was not cut by the enzyme, hence remaining as a 245 bp band on agarose gels. The CC genotyped, on the other hand, was cleaved into two fragments of 120 bp and 125 bp in size. Heterozygotes showed the presence of all the above bands in agarose gel.

COX-2 (-1195G>A) Polymorphism.
The PCR primers used for amplification of the region containing the COX-2 (1195G>A) polymorphism were 5 -CCC TGA GCA CTA CCC ATG AT-3 and 5 -GCCTTCATAGGAGATACTGG-3 . The annealing temperature of the reaction was 62 ∘ C. The PCR amplification gave a product of 273 bp in size, which was then digested using PvuII restriction enzyme. The AA genotype was undigested, while the PCR product was cleaved into a 220 bp and a 53 bp fragment for the GG genotype. Apart from that, heterozygous genotype (AG) was detected by the presence of all the above bands on the agarose gel.

Statistical Analysis.
Statistical analyses were carried out using the SPSS software package 18.0 (SPSS Inc., Chicago, IL). Mean age for cases and controls was evaluated by using Student's -test. On the other hand, chi square test ( 2 test) was used to determine the significance difference in the smoking status and the distribution of genotypes of the cases and controls. Odds ratios (ORs) and the corresponding 95% confidence intervals (CI) calculated by using logistic regression analysis were used to analyze the association between the polymorphic genotypes and prostate risk, based on heterozygous and homozygous comparison models, with the wild type genotype served as the reference. Odds ratios (ORs) of >1.00 indicated a positive risk association and vice versa. Interaction between the polymorphisms and smoking status, as well as between polymorphisms in related genes (NFKB1 and NFKBIA; COX-2 and COX-1), was tested by using the same logistic regression model as above, by using SPSS software. values of <0.05 were significant. For analyses involving gene-gene and gene-environment interaction, Bonferroni correction was applied for multiple testing, in which case the statistical significance was assumed at < 0.025. Table 1 shows the difference between cases and controls in terms of age and smoking status. The ages of cases ranged from 48 to 87 years, and those of controls ranged from 46 to 85 years. The mean age was 69.90 ± 8.43 for cases while the mean age was 69.38 ± 8.76 for controls. No significant difference was observed between the mean ages of both groups ( = 0.29). For both cases and controls, the frequency of never smokers was slightly higher than ever smokers. In cases, 247 (45.49%) were ever smokers and 296 (54.51%) were never smokers, while, in controls, 329 (43.69%) were ever smokers and 424 (56.31%) were never smokers. The difference in the distribution of ever and never smokers among cases and controls was not significant ( = 0.52).

Distribution of Genotypes.
The distribution of genotypes of the five polymorphisms in cases and controls is shown in Table 2. For the NFKB1 -94 ins/del polymorphism, the frequency of ins/ins, ins/del, and del/del genotypes was 198, 246, and 99 in cases and 212, 355, and 186 in controls. Significant difference was observed between cases and controls in the distribution of genotypes ( 2 = 13.12, < 0.01). Apart from that, another polymorphism which showed significant difference between cases and controls in the genotypic distribution was the COX-2 (-1195G>A) polymorphism. Among the cases, 71, 269, and 203 subjects had GG, GA, and AA genotypes, respectively. On the other hand, the distribution of the genotypes among the controls was 158, 378, and 217, respectively ( 2 = 18.34, < 0.01).
For all polymorphisms, the genotype distribution did not deviate significantly from Hardy Weinberg equilibrium, both among cases and among controls ( > 0.05). Table 3 shows the association between the five polymorphisms and prostate cancer risk in the population studied. For NFKB1 -94 ins/del polymorphism, a significantly decreased prostate cancer risk was observed in the heterozygous comparison model (ins/del versus ins/ins) and homozygous comparison model (del/del versus ins/ins). In heterozygous comparison model, the odds ratio was 0.74 (95% CI = 0.58-0.96), with a value of 0.02. On the other hand, in homozygous comparison model, the odds ratio was 0.57 (95% CI = 0.42-0.78, < 0.01).

Risk Association: Overall.
For COX-2 (-1195G>A) polymorphism, a significantly increased prostate cancer risk was observed in the heterozygous comparison model (GA versus GG) and homozygous comparison model (AA versus GG). In heterozygous comparison model, the odds ratio was 1.58 (95% CI = 1.15-2.18), with a value of 0.01. On the other hand, in homozygous

Risk Association: Interaction of Polymorphisms with
Smoking Status. The interaction between smoking status and the polymorphisms in modulating prostate cancer risk was also formally investigated, and the results are shown in Table 4. Significant interaction with smoking status was observed only for the COX-2 (-1195G>A) polymorphism ( < 0.01), in which a significant risk association was observed between the AA genotype and ever smokers (OR = 1.76, 95% CI = 1.28-2.41, < 0.01).

Risk Association: Gene-Gene
Interactions. The effect of interactions between polymorphisms in related genes (NFKB1 and NFKBIA; COX-2 and COX-1) on the risk of prostate cancer was also examined ( Table 5). Both NFKB1 and NFKBIA combination and COX-2 and COX-1 combination showed statistically significant interactions ( = 0.03 and = 0.02, resp.). For the interactions between COX-2 -1195G>A and COX-1 (50C>T) polymorphisms, interaction between COX-2 (-1195AA) * COX-1 (50CT) genotypes conferred a risk increment with OR = 1.83 (95% CI = 1.06-3.17, = 0.03). However, this was deemed not statistically significant after application of Bonferroni correction, where < 0.025 was considered as significant. For the interactions between NFKB1 -94 ins/del and NFKBIA -826C/T polymorphisms, statistically significant prostate cancer risk association was not observed for any polymorphic combination investigated. No other genotypic interactions yielded a statistically significant association.

Discussion
In this study, we investigated and established the association of five inflammation-related genetic polymorphisms, namely, PPARG Pro12Ala, NFKB1 -94 ins/del, NFKBIA -826C/T, COX-1 (50C>T), and COX-2 (-1195G>A) polymorphisms, with prostate cancer risk in a Chinese population. These polymorphisms have been widely investigated for their associations with other cancers or in other populations, but report is scarce or unavailable for prostate cancer in the Chinese population. This novelty of data represents one of the strengths of the present study. Additionally, our study involved a considerably large number of subjects, which   There were two polymorphisms involved in each testing; hence, significance is assumed at < 0.025 after application of Bonferroni correction.
increases the reliability of the results obtained. Apart from that, our study analyzed the combined effect of polymorphisms in a similar pathway (i.e., NFKB1 and NFKBIA; COX-2 and COX-1), an approach which has been neglected in many previous reports. PPARG gene encodes for peroxisome proliferatoractivated receptor-(PPAR-), which is a ligand-activated transcription factor that is pleiotropic in nature. PPARis best known for its function in the regulation of adipogenesis and fatty acid metabolism, but it appears that the protein also plays a role in several carcinogenic processes, including apoptosis, cell cycle control, and, notably, inflammation [17]. The Pro12Ala polymorphism has been shown previously to affect the binding affinity of PPARtowards its target molecules [18]. Therefore, we postulated that this polymorphism could influence the risk of prostate cancer. However, we observed a lack of association between the PPARG Pro12Ala polymorphism and prostate cancer risk in our population. We propose that this observation could be due to the fact that although different genotypes of the polymorphism have different binding affinity to its target molecules, the difference is subtle and does not influence the ability of PPAR-in activating the transcription of its target molecules. Our result was in agreement with a study from Finland [19] and in partial agreement with another study from USA [20], which showed that the polymorphism was 6 BioMed Research International associated with an increased prostate cancer risk only among individuals with high body mass index.
On the other hand, NFKB1 and NFKBIA encode for NF-B1 and its inhibitor, I B , respectively. Both NF-B1 and I B are part of the NF-B pathway, which plays an important role in inflammation and other carcinogenic processes. The -94 ins/del polymorphism of NFKB1 and -826C/T polymorphism of NFKBIA have been shown to affect the transcriptional activity of the respective genes, as the polymorphisms were located in the promoter region of the genes [10,12]. We observed that the NFKB1 -94 ins/del polymorphism was associated with decreased risk of prostate cancer in both heterozygous and homozygous comparison models, suggesting that the variant del allele may be linked to a reduced inflammatory status among its carriers and, therefore, a reduced risk. This risk reduction was in agreement with an in vitro assay which showed that the del allele decreased the transcriptional activity of NFKB1, thereby truncating its role in promoting inflammation, which in turn led to the decreased prostate cancer risk [21]. Our finding was also in agreement with two other previous studies on prostate cancers, namely, Zhang et al. [22] in China and Kopp et al. [13] in Denmark. For NFKBIA -826C/T polymorphism, no significant association with prostate cancer risk was observed. Despite many previous studies reported on the association (or lack of association) between the NFKBIA polymorphism and risk of various cancers [10,[23][24][25], we believe that this is the first work which examined its association with prostate cancer risk.
Cyclooxygenases are enzymes which catalyze the conversion of arachidonic acid to prostaglandins, which promote and regulate the process of inflammation. There are two isoforms of cyclooxygenases, namely, the constitutively expressed cyclooxygenase-1 and inducibly expressed cyclooxygenase-2, which are encoded by COX-1 and COX-2 genes, respectively. The COX-1 (50C>T) polymorphism is located within the coding region of the gene and can potentially influence the functional importance of the enzyme produced [26]. On the other hand, the promoter COX-2 (-1195G>A) polymorphism can alter the transcriptional activity of the gene [27]. Our result showed the absence of prostate cancer risk association for the COX-1 (50C>T) polymorphism. This indicates that, despite its functional importance, the polymorphism is not sufficiently strong to result in significant risk alteration among its carriers. We also showed the presence of an association between COX-2 (-1195G>A) polymorphism and increased prostate cancer risk in both heterozygous and homozygous comparison models. This suggests that the variant A allele of the COX-2 polymorphism could contribute to an increased prostate cancer risk among its carriers. The observation that the A allele increases prostate cancer risk can be explained by the fact that the A allele creates a c-Myb transcription factor binding site in the gene, resulting in an increased transcription of COX-2 [27]. This in turn leads to the greater promotion of inflammation, which mediates the process of carcinogenesis. Our study is the first to evaluate the association between COX-1 (50C>T) polymorphism and prostate cancer risk, whereas several previous reports were available for the association between COX-2 (-1195G>A) polymorphism and prostate cancer risk. Our study results were in agreement with Sugie et al. [28] in a Japanese population, but Wu et al. (Taiwan) [29], Cheng et al. (USA) [30], and Kopp et al. (Denmark) [13] showed the absence of risk association between COX-2 (-1195G>A) polymorphism and prostate cancer risk.
In summary, we have demonstrated the presence of a positive prostate cancer risk association for COX-2 (-1195G>A) polymorphism, a negative risk association for NFKB1 -94 ins/del polymorphism, and absence of association for PPARG Pro12Ala polymorphism, NFKBIA -826C/T polymorphism, and COX-1 (50C>T) polymorphism. There are several limitations in this study. First, although the overall sample size was large, the sample size became small when analysis was performed separately between smokers and nonsmokers and when the genotypes were combined. In addition, we only included five polymorphisms which were considered more well established based on our literature review. Therefore, a more extensive examination for a greater number of polymorphisms in a larger sample size is needed and recommended for future work for reconfirmation of our findings.


extfieldsofstudy: Biology Medicine

s2fieldsofstudy: Biology Medicine

text:
Restin suppressed epithelial-mesenchymal transition and tumor metastasis in breast cancer cells through upregulating mir-200a/b expression via association with p73

Background Restin belongs to MAGE superfamily and is known as MAGE H1. Restin was firstly cloned from HL-60 cells treated with all-trans retinoic acid (ATRA). Previous studies showed a pro-apoptotic role of Restin in several cell lines. However, little information is available on its expression patterns and functions in vivo. Our study was performed to detect if Restin plays a role in breast cancer cells in vitro and in vivo. Methods and results Real-time PCR and western blot were conducted to detect Restin expression in multiple breast cancer cell lines and Restin level was negatively related with cell motility. Restin overexpression and knockdown stable cell lines were established by transducing lentivirus into MCF-7 and MDA-MB-231 cells. Cell morphology, wound closure assay, transwell migration and invasion assays were performed to detect if Restin inhibited EMT. Our data showed that Restin overexpressed cells exhibited classical epithelial cell morphology, and Restin overexpression resulted in activation of epithelial markers and suppression of mesenchymal markers, and inhibition of cell migration and invasion. Tumor xenograft model was used to characterize the biological functions of Restin in vivo. We found that Restin overexpression led to reduced lung metastasis. Real-time PCR, western blot, luciferase assay and ChIP assay were performed to identify the potential targets of Restin and the underlying molecular mechanisms. Among several master regulators of EMT, only ZEB1/2 levels were dramatically inhibited by Restin. Unexpectedly, Restin indirectly regulated ZEB1/2 expression at post-transcriptional level. We further identified mir-200a/b, well-characterized mediators controlling ZEB1/2 expression, were transcriptionally activated by Restin and the regulation was dependent on the p53 binding site in mir-200b/a/429 promoter. Further mechanical studies demonstrated Restin interacted with p73, one of p53 family members, which contributed to Restin-mediated activation of mir-200a/b and suppression of ZEB1/2. Conclusions Taken together, our results suggest that Restin inhibits EMT and tumor metastasis by controlling the expression of the tumor metastasis suppressor mir-200a/b via association with p73. Our findings not only establish a mechanistic link between Restin, EMT and tumor metastasis, but also provide strong evidence supporting the notion that MAGE Group II proteins may exert a tumor suppressive effect in vivo. Electronic supplementary material The online version of this article (doi:10.1186/s12943-015-0370-9) contains supplementary material, which is available to authorized users.

Background
The melanoma-associated antigen (MAGE) is one of the well-characterized members of the CTA family that contains at least 60 closely related proteins [1], including MAGE-A, B, C, D, E, F, G, H, L2, Necidin, I and J. According to the protein expression patterns and functions, the MAGE family has been divided into groups I and II [2,3]. Group I proteins, including MAGEs A, B and C, are expressed in many types of tumor tissues, but are not expressed in normal tissues [1]. Moreover, their expression is closely correlated with aggressive clinical course, the acquisition of resistance to chemotherapy, the occurrence, and poor clinical outcomes [1,3,4]. Contrast to group I proteins, Group II proteins, such as Necdin and Mage-D1, are universally expressed in all normal tissues but rarely in tumor tissues [5]. They are more likely associated with cell growth inhibition, cell cycle arrest, apoptosis, or cell differentiation [6]. For example, Necdin has been shown to be down-regulated in both carcinoma cell lines and primary tumors [7][8][9], suggesting Necdin is a potent tumor growth suppressor. Ectopic overexpression of Necdin in a mouse tumor cell line is reported to attenuate tumorigenicity and metastasis in vivo [10]. In addition, MAGE-D1 inhibited cell proliferation, migration and invasion of multiple human cancer cells [11]. Although Group II proteins emerge as novel tumor suppressor candidates in a wide range of human cancers, their roles in cancers remain poorly defined.
Restin belongs to MAGE Group II proteins and is known as MAGE H1 [12]. Restin was firstly cloned from the differentiated HL-60 cells induced by all-trans retinoic acid (ATRA) [13], an apoptosis and differentiation inducer. Bioinformatics analysis showed that Restin shared 49% homology with Necdin [14] and both of them were basic proteins. Further analysis found that Restin, Necdin and Mage-D1 had an alkaline conservative region, which is lowly expressed in tumor tissues [14]. Above data indicated that, similar to Necdin and Mage-D1, Restin belongs to Group II proteins. Bioinformatics data from GEO profiles show that Restin is rarely expressed in a variety of cancer cells, while its expression level is pretty high in normal cells. Restin was identified as one of pro-apoptotic genes that determined the response of multiple tumor cells to CD95-mediated apoptosis [15]. Fu HY et al. found that Restin overexpression in Hela cells promoted apoptosis [16]. Denis Selimovic et al. disclosed that Restin overexpression induced apoptosis of melanoma cells via interacting with p75 neurotrophin receptor (p75NTR), leading to the disruption of both NF-ƘB and extracellular signal-regulated kinase (ERK) pathways [12]. Thus, Restin may function as a tumor suppressor, which is similar to Necdin and Mage-D1. Nevertheless, little information is available on its expression patterns and functions, particularly its roles in tumorigenesis in vitro and in vivo.
The epithelial-mesenchymal transition (EMT) and the reverse process, termed the mesenchymal-epithelial transition (MET), play critical roles in embryogenesis, wound healing, tissue fibrosis, and carcinoma progression [17][18][19][20]. EMT is known to be a central mechanism responsible for invasiveness and metastasis of a variety of cancers [19][20][21]. During tumor development, epithelial cells undergo dynamic cytoskeletal rearrangement, and lose cell adhesion and epithelial components while acquiring mesenchymal and migratory phenotypes [20,22]. Therefore, targeting EMT may serve as an efficient strategy for the treatment of malignant and metastatic tumors.
Our present study, for the first time, demonstrated that Restin remarkably suppressed breast cancer metastasis through inhibiting EMT by controlling the expression and function of the tumor metastasis suppressor mir-200a/b via association with p73 in breast cancer cells.

Restin expression level was negatively related with tumor cell mobility
Previous study showed that Restin overexpression induced apoptosis of melanoma cells [12], indicating Restin may be associated with cell malignancy. Thus, we first tested if Restin overexpression inhibited cell proliferation in two breast cancer cell lines. Contrast to melanoma cells, Restin overexpression failed to inhibit cell proliferation determined by 3 [H] incorporation assay ( Figure 1A). Next, we detected if Restin was related with cell mobility, another tumor cell malignant feature. To test this, Restin expression level was determined in nonmetastatic, low-metastatic and high-metastatic cells. As expected, Restin mRNA level was pretty high in two non-metastatic cells, MCF-10A and HMEC, while it was moderately decreased in low-metastatic MCF-7 cells ( Figure 1B). However, Restin was barely detectable in high-metastatic MDA-MB-231 and MDA-MB-451 cells ( Figure 1B). Restin protein levels showed the similar trend ( Figure 1C). TGF-β is a strong inducer of cell mobility in epithelial cells [23]. As expected, Restin expression level was gradually decreased upon TGF-β treatment in MCF-7 cells ( Figure 1D). Above data indicated that Restin expression level is negatively related with tumor cell mobility.

Restin prevented EMT in breast cancer cells
EMT is a crucial program for the invasion and metastasis of epithelial tumors that involves loss of cell-cell adhesion and increase of cell mobility [19][20][21]. To examine if Restin plays a role in EMT process, cell morphology and EMT markers were detected. Restin overexpression and knockdown stable cell lines were generated in MDA-MB-231 and MCF-7 cells and Restin expression levels were confirmed by western blot (Additional file 1: Figure S1). The Control MDA-MB-231 cells retained their elongated spindle-like morphology with sharp borders ( Figure 2A). However, Restin overexpressed MDA-MB-231 cells displayed a cobblestone-like epithelial phenotype, grew as a monolayer and formed islands of grouped cells with tight cell-cell contact (Figure 2A). In addition to morphological changes, the expression levels of critical genes for EMT process were determined. E-cadherin plays a central role in maintaining epithelial cell-cell adhesion and polarity [24]. Real-time PCR showed a remarkable increase in E-cadherin expression in Restin overexpressed cells ( Figure 2B). The concomitant increase in ZO-1 expression was observed either. In contrast, Restin overexpresssion resulted in a strong repression on the expression of mesenchamal markers, including N-cadherin, Vimentin and Fibronectin, in MDA-MB-231 cells ( Figure 2B). Similar results were confirmed by western blot analysis ( Figure 2C). Thus, morphological alteration from a fibroblastic to an epithelial phenotype and an increase of E-cadherin level upon Restin overexpression strongly indicate that Restin inhibits EMT process. Furthermore, we determined if knockdown of Restin promoted EMT. To this end, cell morphology and EMT markers were tested upon Restin knockdown in low-mobility MCF-7 cells. Although knockdown of Restin failed to change cell morphology (data not shown), these cells apparently exhibited a spindle-like morphology upon TGF-β induction ( Figure 2D). Consistently, E-cadherin and ZO-1 expression were significantly inhibited, whereas Vimentin and Fibronectin expression were moderately enhanced in Restin knockdown cells as compared to si-Control group ( Figure 2E). Our data suggest that Restin inhibits EMT in breast cancer cells.

Restin suppressed migration and invasiveness of breast cancer cells
Fibroblast cells that have undergone MET are characterized by their decreased motility and loss of invasiveness. Therefore, we detected whether Restin overexpression resulted in decreased motility of MDA-MB-231 and MDA-MB-451 cells determined by wound closure assay, transwell migration and invasion assays. As shown in Figure 3A, the percentage of wound closure for Control MDA-MB-231 cells  Figure 3B). Next, we performed transwell matrigel invasion assay to assess whether Restin inhibited cell invasiveness. As shown in Figure 3C, Restin overexpression caused a reduction of invasive cells by more than 41.65% and 37.61% in MDA-MB-231 and MDA-MB-451 cells compared with their Control cells. We also found that Restin overexpressed cells displayed significantly less adherence to fibronectin compared with Control group ( Figure 3D). These results suggest that Restin suppresses cell migration and invasiveness of metastatic breast cancer cells.

Restin inhibited lung metastasis in vivo
In order to evaluate if Restin-mediated inhibition of EMT indeed plays a role in vivo, tumor growth and lung metastasis were monitored in NOD-SCID mice implanted with Control and Restin overexpressed MDA-MB-231 cells.
The tumor growth rate showed no statistic difference between Control and Restin overexpression groups ( Figure 4A, p > 0.05). Consistently, the tumor size was similar in the two groups ( Figure 4B). Interestingly, the tumors formed by Control MDA-MB-231 cells gave rise to 13.32 ± 1.659 metastatic nodules per lung ( Figure 4C). However, Restin overexpression resulted in a 2.7-fold decrease of lung metastases ( Figures 4C and D). Our in vivo data indicate that the morphological changes caused by Restin overexpression is closely related to decreased lung metastasis.

Restin downregulated ZEB1/2 expression at post-transcriptional level
Downregulation of E-cadherin is one of the critical markers of EMT in human breast cancers [25]. FOXC2, Twist, ZEB1, ZEB2, Slug and Snail are identified transcription factors that repress E-cadherin transcription and overexpression of each gene induces EMT and promotes malignant cell invasion and dissemination [21,24]. Considering the positive regulation of Restin on E-cadherin, we hypothesize that Restin may control the expression of those transcription factors. We found that the expression levels of ZEB1 and ZEB2 were significantly inhibited by 76.4% and 70.0%, respectively, upon Restin overexpression ( Figure 5A). However, a slight reduction in Twist, Snail and Slug mRNA levels was observed ( Figure 5A). Consistently, ZEB1 and ZEB2 protein levels were apparently inhibited by Restin overexpression ( Figure 5B). However, no apparent difference was observed in Twist and Slug protein levels. On the contrary, Restin knockdown resulted in a dramatic increase in ZEB1 and ZEB2 levels, while only a marginal effect on Twist and Slug expression ( Figure 5C, lane 3 versus lane 1). Consistent with TGF-βmediated negative regulation on Restin, TGF-β treatment further enhanced ZEB1 and ZEB2 expression levels ( Figure 5C, lane 4 versus lane 2).
Next, we determined if ZEB1/2 was involved in Restin-mediated inhibition of EMT. E-cadherin, Vimentin and Fibronectin levels were tested upon ZEB1 knockdown (Additional file 1: Figure S2). Control MCF-7 cells transfected with ZEB1 siRNAs showed moderately enhanced E-cadherin and decreased Vimentin and Fibronectin expression ( Figure 5D, lane 2 relative to lane 1), which is consistent with ZEB1 function in promoting EMT.
Restin knockdown led to reduction of E-cadherin and increase of Vimentin and Fibronectin ( Figure 5D, lane 3 relative to lane 1). Additional knockdown of ZEB1 effectively reversed the expression of these proteins, and their levels were comparable to that in Control cells ( Figure 5D, lane 4 relative to lane 2). Collectively, these data indicate that ZEB1 is a crucial mediator of Restininduced phenotypes.
Previous studies showed that Restin was localized in nucleus and it interacted with several transcription factors [26,27], indicating it may regulate ZEB1 expression at the transcriptional level. To test this, ZEB1 promoter activity was detected by luciferase reporter assay. We found that Restin overexpression failed to activate the transcription of ZEB1 ( Figure 5E). Since Restin suppressed ZEB1 and ZEB2 mRNA levels, it is possible that Restin may regulate ZEB1 expression at the posttranscriptional level. As expected, Restin overexpression strongly repressed the luciferase activities driven by ZEB1 3'UTR in a dose-dependent manner ( Figure 5F). Furthermore, an enhanced luciferase activity was observed upon Restin knockdown (Additional file 1: Figure S3).

mir-200a/b was responsible for Restin-mediated downregulation of ZEB1
Accumulating evidences demonstrate that mir-200 family is a powerful microRNA family controlling ZEB1/2 expression [23,28]. To determine if mir-200 members are involved in Restin-mediated downregulation of ZEB1/2, mir-200a, mir-200b, mir-429, mir-200c and mir-141 levels were determined upon Restin overexpression and knockdown. As shown in Figure 6A, overexpression of Restin significantly enhanced mir-200a and mir-200b expression by 2.71-and 2.07-fold, while their levels were greatly inhibited by Restin knockdown (Figure 6B), hinting that Restin may regulate the transcription of mir-200b/a/429. However, the expression levels of mir-200c and mir-141, which are located in another genomic cluster, were not altered by Restin overexpression (Additional file 1: Figure S4). Our hypothesis was further confirmed by luciferase reporter assay showing that Restin significantly increased the luciferase activity driven by mir-200b/a/429 promoter but not mir-200c/141 promoter ( Figure 6C and Additional file 1: Figure S5). Next we determined if mir-200b/a is indeed responsible for Restin-mediated downregulation of ZEB1 and ZEB2. To this end, a mutant ZEB1 3'UTR with deletion of mir-200b binding sites was constructed. Consistent with above data, Restin overexpression decreased the luciferase activities driven by ZEB1 3'UTR, while failed to reduce the luciferase activities driven by mutant ZEB1 3'UTR ( Figure 6D).

Restin activated mir-200b/a/429 transcription in a p73-dependent manner
To investigate the molecular mechanisms by which Restin upregulated mir-200b/a transcription, a series of mir-200b/ a/429 promoter truncations were constructed ( Figure 7A) and their luciferase activities were determined. As shown in Figure 7B, Restin dramatically enhanced the luciferase activities driven by WT mir-200b/a/429 promoter (P1,-1574/+120). However, Restin failed to activate the mir-200b/a/429 promoter activities with deletion of p53 binding sites (-1412/-1408) in P2 truncation (-1000/+120) and another two shorter truncations (P3 and P4). Above data suggest that p53 binding site is critical for Restin-mediated activation of mir-200b/a/429 transcription. To further demonstrate this, we introduced a luciferase reporter construct in which the p53 binding site had been mutated ( Figure 7C, bottom panel). As expected, the luciferase activities driven by WT mir-200b/a/429 promoter was increased by 1.69fold upon Restin overexpression, whereas Restin was unable to upregulate the activities of mir-200b/a/429 promoter with mutations in p53 binding site ( Figure 7C). These data indicate that Restin directly or indirectly activates the transcription of mir-200b/a/429 through p53 binding site on its promoter. The p53 family comprises three genes that encode for p53, p63 and p73 [29]. To identify which factor is involved in Restin-activated mir-200b/a/429 transcription, the expression levels of three proteins were first detected in several breast cancer cell lines. It is well-documented that MCF-7 cells contain wild-type p53, MDA-MB-231 cells carry mutant p53 with greater levels, and MDA-MB-157 cells expressed no p53 [30]. As shown in Figure 7D, p53 was moderately expressed in MCF-7 cells, and was undetectable in MDA-MB-157 cells, whereas a high level of mutant p53 protein was observed in MDA-MB-231 cells. All cell lines were negative for p63 (data not shown), whereas contained detectable and comparable p73 levels ( Figure 7D). It has been demonstrated that MDA-MB-231 cells have mutant p53 due to an arginine to lysine mutation at position 280 and the mutant p53 does not retain the tumor suppressive ability of wild-type p53 [30]. We compared the luciferase activities driven by mir-200b/a/429 promoter in above three cell lines and found that Restin activated the luciferase activities in a comparable manner whatever the expression level and function of p53 in those cells (Additional file 1: Figure S6). Therefore, we postulate that p53 may not participate in Restin-mediated transcriptional activation of mir-200b/a/429. Considering the undetectable level of p63, we hypothesize that p73 may play a role in Restin-mediated upregulation of mir-200b/a. To test this, reciprocal co-immunoprecipitation was performed to detect the interaction between p73 and Restin. HERK-293 cells were transiently transfected with Flag-tagged Restin and His-tagged p73 plasmids. Upon immunoprecipitation of p73 using an anti-His antibody, Restin was coimmunoprecipitated ( Figure 7E, upper panel). Likewise, in the immunoprecipitate of Flag-Restin, p73 protein was recovered ( Figure 7E, lower panel). Endogenous interaction of p73 and Restin was also examined in MCF-7 cells. Anti-Restin antibody could co-immunoprecipitate endogenous p73 (Additional file 1: Figure S7). As expected, endogenous interaction between p53 and Restin was not found in MCF-7 cells determined by co-immunoprecipitation assay (Additional file 1: Figure S8).
Next we investigated if Restin upregulated mir-200b/a transcription in a p73-dependent manner. siRNAs mediated p73 knockdown was confirmed by western blot (Additional file 1: Figure S9

Discussion
MAGE group II proteins function as putative tumor suppressors in a variety of cancers [5,6]. However, its clinical effects on tumor development and the underlying mechanisms are poorly characterized. Restin is a novel member of MAGE group II proteins [12]. Selimovic   p73) are involved in above process. p53 suppresses EMT by transactivating the expression of mir-200 family members in primary hepatocellular carcinomas (HCCs) and 9 HCC cell lines [34]. Simlar to p53, p73 and p63 can directly associate with the mir-200b/a/429 promoter and activate mir-200 transcription in ovarian carcinoma cells [35]. Although MDA-MB-231 cells or MDA-MB-157 cells possess mutant p53 or no p53 expression, Restin still activated mir-200b/a/429 transcription, indicating p53 protein may not participate in above process and Restin may not associate with p53. Our hypothesis was proven by our data showing that no endogenous interaction between Restin and p53 was found. By performing Co-IP experiment, we found that Restin can closely interact with p73. Moreover, p73 knockdown diminished Restin-mediated regulation on mir-200a/b and ZEB1/2. Thus, our observations provide new insights into the role of p53 family members in mediating Restin's function in tumorigenicity.
Based on the chromosomal locations, the mir-200 family can be divided into two clusters: the mir-200b/a/ 429 cluster containing mir-200a, mir-200b and mir-429, and the mir-200c/141 cluster, which contains mir-200c and mir-141 [32]. Although both clusters contained putative p53 binding sites, Restin drove mir-200b/a/429 transcription rather than that of mir-200c/141 via association with p73. Interestingly, a recent report found that in mammary epithelial cells, p53 serves as a transcriptional activator of mir-200c/141, but not of the mir-200b/a/429 [36]. We speculate that the transcription of mir-200 clusters by p53 family members may be dependent on the expression level and functions of p53 family members, the promoter methylation status of mir-200 promoters or other transcriptional co-factors, which need to be further elucidated.
p53 is involved in tumor initiation as well as tumor progression [37][38][39]. However, deletions or mutations of p53 are frequently found in cancers [39][40][41][42][43]. p63 and p73 are rarely mutated in a large number of tumors examined to date [42]. Therefore, in the absence of p53, these proteins may be involved in the control of the ZEB/mir-200 equilibrium and EMT-MET plasticity. Our data showed that Restin interacted with p73 rather than p53 and upregulated mir-200b/a expression even in p53 mutant cells. Our results shed light on the transcriptional regulation of Restin on mir-200s, which has potential therapeutic value in the development of approaches aimed at modifying mir-200s expression for the treatment of diverse forms of human cancers, especially those cancers with p53 mutation.
Restin was identified from HL-60 cells induced by alltrans-retinoic acid (ATRA). Restin expression level was undetectable in HL-60 cells, whereas its level was sharply increased upon ATRA treatment [13,14]. Consistently, we observed that Restin expression was pretty high in normal breast epithelial cells compared to breast cancer cells. Its  expression in low-metastatic cells was higher than that in high-metastatic cells, hinting that Restin expression level is negatively related to tumor cell malignancy. Our data was supported by current evidence showing that Necdin, another member of MAGE group II proteins, was expressed at a low level in MDA-MB-468 and MDA-MB-231 cells, but exhibited much higher levle in low-metastatic MCF-7 cells [8]. Contrast to Restin and Necdin, MAGE-A and C, belong to MAGE group I proteins, exerted an oncogenic role in breast cancer and exhibited a positive correlation with tumor malignancy [1,3,4]. Considering the undetectable expression of Restin in high-metastatic MDA-MB-231 cells and the dramatic inhibition of tumor metastasis by Restin overexpression, we hypothesize that Restin may function as a tumor-suppressor in breast cancers. Our ongoing studies are being performed to detect Restin expression in normal breast tissues, para-carcinoma tissues and carcinoma tissues and to statistically analyze the association of its expression level with tumor malignant grade, tumor metastasis, relapse, and clinical prognosis, which will strongly support our hypothesis.

Conclusions
Taken together, our study, for the first time, demonstrated that Restin strongly inhibited lung metastasis in vivo, which strongly suggests that Restin may serve as a potent tumor suppressor in breast cancers although our study is still in a preliminary stage. Our current data not only shed a light on the function of Restin in tumor development in light of its inhibition on EMT process but also facilitate other researchers to extend or verify its function in other type of cancers.

Materials and methods
Cell culture and reagents MCF-7, MDA-MB-157, MDA-MB-231 and MDA-MB-451 breast cancer cells were grown in Dulbecco's modified Eagle's medium (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C in a humidified atmosphere of 5% CO2. MCF-10A, a spontaneously immortalized human breast epithelial cell line, was maintained in DMEM/F12 medium (Invitrogen Life Technologies, Carlsbad, CA). Human mammary epithelial cells (HMEC) were cultured in mammary epithelial cell complete medium (Invitrogen). All cell lines were obtained from the ATCC. p73 and ZEB1 siRNAs (Stealth™ siRNAs) were synthesized from Invitrogen and were dissolved in DEPC-treated H 2 O at a concentration of 20 pmol/μl as a stock.

Constructs
The following plasmids were bought from Addgene

Transfection and luciferase reporter assay
Cells were seeded onto 24-well plates and transfected with 800 ng of luciferase vectors (ZEB promoter, mir-200 promoter, ZEB 3'UTR (WT and mutant)) per well using Lipofectamine 2000 Transfection Reagent (Invitrogen). 50 ng of pBind plasmids were cotransfected to normalize for transfection efficiency. 24 h later, cells were lysed with PLB lysing buffer. The luciferase activities were measured with the dual-luciferase reagent assay kit (Promega, Madison, WI) according to the manufacturer's instructions using the TD-20/20 Luminometer (Turner Designs).
All reporter assays are shown as relative luciferase activities (averaged ratios of Firefly luciferase: Renilla FSE) and are representative of three experiments.

Quantitative real-time PCR
RNAs were collected using Trizol Reagent (Invitrogen). Reverse transcription was carried out using 2 μg RNA in a 20 μl reaction volume using the Superscript III Reverse Transcription Kit (Invitrogen). Real-time PCR was performed with the SYBR® Premix Ex Taq TM (Takara Biotechnology, Dalian, China) in 20 μl reactions using ABI PRISM ® 7500 Real-Time PCR System (Applied Biosystems, Grand Island, NY). GAPDH was used as an internal control. Primer sequences are listed in the Additional file 1: Table S1. For mir-200 detection, RNAs were reverse transcribed by miScript Reverse Transcription Kit (Qiagen, Valencia, CA). qPCR of mir-200a and 200b was performed using miScript SYBR Green PCR Kits (Qiagen). mir-200 expression was normalized with U6.

Western blot
MDA-MB-231 cells were harvested and lysed in a RIPA buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate, and 50 mM NaF. One tablet of protease inhibitor mixture (Complete Mini, Roche Applied Science) was added just prior to use. Then 30 μg protein lysates were separated by 12% SDS-PAGE and transferred onto nitrocellulose. After blocking in a 5% non-fat dried milk solution in washing buffer containing 10 mmol/l Tris (pH 7.5), 50 mmol/l NaCl, and 0.02% Tween 20 (TBST), membranes were incubated overnight at 4°C with primary antibodies. After washed three times with TBST, membranes were incubated for 1 h with horseradish peroxidase-coupled secondary antibodies at room temperature. Signals were detected with the ECL kit (Pierce, Rockford, IL).

Antibodies
The following antibodies were used: rabbit polyclonal anti-Slug

Co-immunoprecipitation assay
Human 293 T cells were seeded onto the 10-cm plates and cotransfected with Flag-tagged Restin and His-tagged p73 constructs using Lipofectamine 2000 Transfection Reagent (Invitrogen). 2 days after transfection, cells were lysed in precooled RIPA buffer for 30 min on ice, followed by 15 min of centrifugation. Cell lysates were precleaned with protein G-Sepharose beads (GE Healthcare) for 30 min at 4°C. After centrifugation at 14,000 rpm at 4°C for 15 min, cleaned lysates were incubated with either anti-Flag or anti-His antibody on a rocking platform for 4 h at 4°C. Protein complexes were immunoprecipitated by incubation with protein G-Sepharose beads for 30 min at 4°C and then washed three times with lysis buffer. Immunoprecipitated proteins were eluted with 50 μl of Laemmli loading buffer and separated by 10% SDS-PAGE. Protein expression was evaluated by Western blot analysis with either His or Flag monoclonal antibodies.

Wound closure assay
MDA-MB-231 cells were seeded onto 6-cm cell culture dishes at a density of 5 × 10 5 . A wound was incised in the central area of the confluent culture by a 10 μl pipette tip. 12 h after scratch, phase-contrast pictures were taken by a Nikon microscope using a 10x phase contrast objective.
Cell migration and invasion assays 2 × 10 4 MDA-MB-231 or MDA-MB-451 cells resuspended in 200 μl serum-free medium were seeded in the upper chamber with serum-containing medium in the lower chamber of 24-well transwell plates (BD Biosciences, San Jose, CA). After 6 h, the experiment was terminated by wiping the cells from the wells with a cotton swab and fixed and stained with 0.05% crystal violet for 2 h. Matrigel invasion assays were done using the BD BioCoat Matrigel Invasion Chamber (BD Biosciences). The procedures and the analyses were the same as those for the transwell migration assay except for the presence of the Matrigel.
Cell adhesion assay 96-well cell culture plates were pretreated with 20 μg/ml fibronectin at 37°C for 2 h and blocked with 0.5% BSA solution for 1 h. MDA-MB-231 or MDA-MB-451 cells were plated onto 96-well cell culture plates at a density of 1×10 4 cells/well. Cells were allowed to adhere for 30 min, after which the media were removed and nonadhered cells were rinsed away. Adhered cells were fixed with 4% paraformaldehyde, stained with crystal violet for 10 min and dissolved in 2% SDS. The absorbance was measured at 550 nm with an ELISA reader (Bio-Tek, Winooski, VT).

Statistical analyses
Statistical significance of the studies was analyzed by Student's t test. Differences with P values of <0.05 are considered significant.

Additional file
Additional file 1: Table S1. Primers used for quantitative real-time PCR. Figure S1. Western blot was performed to confirm Restin expression levels in Restin overexpressed MDA-MB-231 cells and Restin knockdown MCF-7 cells. Figure S2. Western blot was performed to detect ZEB1 expression levels in cells transfected with negative control and ZEB1 siRNAs. Figure S3. ZEB1 3'UTR activity was determined in HEK293 cells by luciferase reporter assay upon Restin knockdown. HEK293 cells were seeded onto 24-well plates and transfected with ZEB1 3'UTR plasmids and different dose of Restin knockdown lentivirus (si-Restin). Figure S4. mir-200c and mir-141 levels were determined in Control and Restin overexpressed MDA-MB-231 cells by real-time PCR. Figure S5. mir-200c/ 141 promoter activity was determined by luciferase reporter assay. Figure S6. mir-200b/a/429 promoter activities were measured by luciferase reporter assay in multiple cell lines. Figure S7. Co-immunoprecipitation assay was performed to detect the endogenous interaction between Restin and p73. (Upper panel) MCF-7 cell extracts were immunoprecipitated with mouse IgG or anti-p73 antibody and then blotted with anti-Restin antibody. (Lower panel) Cell extracts were immunoprecipitated with mouse IgG or anti-Restin antibody and then blotted with anti-p73 antibody. Input, total cell lysates. Figure S8. Co-immunoprecipitation assay was performed to detect the endogenous interaction between Restin and p53. MCF-7 cell extracts were immunoprecipitated with mouse IgG or anti-p53 antibody and then blotted with anti-Restin (upper panel) and anti-MDM2 (C-18) antibodies (lower panel). MDM2 p90 is a positive control. Input, total cell lysates. Figure S9. Western blot was performed to detect p73 expression levels in cells transfected with negative control and p73 siRNAs.


Co-regulation of mitochondrial respiration by proline dehydrogenase/oxidase and succinate

Proline dehydrogenase/oxidase (PRODH/POX) is a mitochondrial protein critical to multiple stress pathways. Because of the roles of PRODH/POX in signaling, and its shared localization to the mitochondrial inner membrane with the electron transport chain (ETC), we investigated whether there was a direct relationship between PRODH/POX and regulation of the ETC. We found that PRODH/POX binds directly to CoQ1 and that CoQ1-dependent PRODH/POX activity required functional Complex III and Complex IV. PRODH/POX supported respiration in living cells during nutrient stress; however, expression of PRODH/POX resulted in an overall decrease in respiratory fitness. Effects on respiratory fitness were inhibited by DHP and NAC, indicating that these effects were mediated by PRODH/POX-dependent reactive oxygen species (ROS) generation. PRODH/POX expression resulted in a dose-dependent down-regulation of Complexes I–IV of the ETC, and this effect was also mitigated by the addition of DHP and NAC. We found that succinate was an uncompetitive inhibitor of PRODH/POX activity, inhibited ROS generation by PRODH/POX, and alleviated PRODH/POX effects on respiratory fitness. The findings demonstrate novel cross-talk between proline and succinate respiration in vivo and provide mechanistic insights into observations from previous animal studies. Our results suggest a potential regulatory loop between PRODH/POX and succinate in regulation of mitochondrial respiration.

Introduction
Proline dehydrogenase (PRODH), a.k.a. proline oxidase (POX), is a mitochondrial inner membrane protein. Oxidation of proline to pyrroline-5-carboxylate (P5C) mediates the proline cycle that shuttles NADP(H)/NADP(+) redox equivalents between mitochondria and cytosol and forms a metabolic interlock with the pentose phosphate pathway (Hagedorn and Phang 1983;Phang 1985). Conversion of P5C to glutamine or ornithine also connects the proline cycle to the TCA and urea cycles, respectively.
PRODH/POX is a mediator of genotoxic, inflammatory, and metabolic stress signaling. Originally identified in a screen of p53-induced genes (Polyak et al. 1997;Campbell et al. 1997), induction of PRODH/POX by p53 was subsequently shown to result in mitochondrial-mediated apoptosis through generation of reactive oxygen species (ROS) in the form of mitochondrial superoxide (Donald et al. 2001;Hu et al. 2007;Liu et al. 2005). During hypoxia, tumor cells respond with either PRODH/POX-mediated ATP generation under conditions of low glucose, or pro-survival, ROS-mediated autophagy induction when glucose levels were normal (Liu et al. 2012). Activation of PRODH/POX by PPAR-γ results in PRODH/POX-dependent superoxide production that induces beclin-1 gene expression and activates protective autophagy versus the toxic effects of oxidized low-density lipoproteins (Zabirnyk et al. 2010). In adipocytes, PRODH/POX activation of PPAR-α through ROS-dependent and FOXO1-mediated induction prevents cell death and inflammation (Lettieri Barbato et al. 2014). In RKO colorectal cancer cells, induction of nutrient stress by either glucose withdrawal or treatment of cells with rapamycin results in increased AMP-activated protein kinase (AMPK)-dependent PRODH/POX catalytic activity, PRODH/POX-dependent cellular ATP generation, and activation of the pentose phosphate pathway (Pandhare et al. 2009). In C. elegans, AMPK-mediated upregulation of proline catabolism results in generation of ROS that promote an adaptive endogenous stress defense and increased lifespan (Zarse et al. 2012). Treatment of embryonic stem cells with proline induces their differentiation into either a primitive, ectoderm-type cell (Washington et al. 2010) or a novel, reversible epiblast stem cell-like state (Casalino et al. 2011). Thus, depending on cellular and environmental context, PRODH/POX can mediate programmed cell death, promote cell survival, or induce differentiation.
The mitochondrial respiratory electron transport chain (ETC) consists of a series of four protein complexes, known as Complexes I-IV, that pass electrons from the electron donors NADH and FADH through a series of redox reactions, creating an electrochemical proton gradient that is coupled with oxidative phosphorylation and the synthesis of ATP by ATP synthase (Chaban et al. 2014). Complex I, aka NADH dehydrogenase, passes electrons from NADH to reduce ubiquinone to ubiquinol. Complex II, aka succinate dehydrogenase (SDH), couples the oxidation of succinate to fumarate in the tricarboxylic acid (TCA) cycle with the reduction of ubiquinone to ubiquinol in the ETC (Hagerhall 1997). Electrons are transferred from this ubiquinol pool to Complex III, coenzyme Q-cytochrome C reductase, which passes the electrons to the hemeprotein cytochrome C. The final step is the transfer of electrons to Complex IV, cytochrome C oxidase, which passes the electrons to molecular oxygen to form water and drive the proton gradient that powers ATP synthase.
The SDH holoenzyme consists of four subunits, known as SDHA-D. SDHA contains the succinate binding site and flavin-adenine dinucleotide (FAD) redox center; SDHB contains three iron-sulfur centers required for electron transfer to ubiquinone, and SDHC and SDHD are two hydrophilic subunits responsible for anchoring the enzyme in the mitochondrial inner membrane. The ubiquinonebinding site of the holoenzyme is in a pocket formed by SDHB, SDHC, and SDHD at the surface of the matrix face of the mitochondrial inner membrane (Lancaster 2002;Horsefield et al. 2004;Sun et al. 2005). Mutations in SDHB, SDHC, and SDHD have been found in hereditary paraganglioma (Baysal et al. 2000;Niemann and Muller 2000;Astuti et al. 2001), gastrointestinal stromal tumors (GISTs) (Pasini et al. 2008), thyroid (Ricketts et al. 2010;Zantour et al. 2004), and renal tumors (Ricketts et al. 2008;Vanharanta et al. 2004). There is increasing evidence that mutations in SDH can lead to aberrant ROS generation through errors introduced in electron transport, and that this can result in tumorigenesis. ROS can be generated at the SDHA subunit during impaired electron transport (Yankovskaya et al. 2003). SDHC inactivation mutation in C. elegans and 3T3 mouse fibroblasts and electron leakage, oxidative stress, apoptosis and increased transformation and tumor growth (Adachi et al. 1998;Ishii et al. 2005).
Because of the pleiotropic role of PRODH/POX in cellular energetics and signaling, and its shared localization with the ETC on the inner membrane of the mitochondria, we sought to determine whether there was a direct relationship between PRODH/POX and regulation of the ETC. Using a PRODH/POX-expressing DLD colorectal cancer cell model and mouse mitochondria, we demonstrate that PRODH/POX passes electrons directly to Coenzyme Q1 (CoQ1), and that acute proline treatment in PRODH/ POX-expressing cells resulted in Complex I-and Complex II-independent oxidative respiration during nutrient stress conditions. In contrast, exposure of cells to PRODH/ POX and proline resulted in a significant time and dependent decrease in total oxidative respiration due to PRODH/ POX-dependent ROS production. PRODH/POX had dosedependent effect on the protein levels of individual subunits of Complexes I-IV of the ETC, which was reversed with the PRODH/POX inhibitor DHP and the antioxidant l-NAC. We show here that succinate inhibits PRODH/ POX through uncompetitive inhibition, and treatment of cells with succinate inhibits production of PRODH/POXdependent ROS, mitigates inhibition of respiration by PRODH/POX, and restores protein levels of ETC complexes in PRODH/POX-treated cells. These results suggest that PRODH/POX acts as a regulator of cellular respiration and that PRODH/POX activity is functionally linked to levels of succinate, potentially linking them as metabolic regulators.

Mouse liver mitochondria isolation
Mouse liver mitochondria were prepared in the method of Chappell and Hansford (Birnie 1972). In brief, freshly harvested livers were minced and washed 3× in ice-cold sucrose buffer (0.25 M sucrose, 3.4 mM tris-HCl, 1 mM EDTA, pH 7.4). Liver tissue was homogenized by 5 passes in a Dounce homogenizer. Homogenates were then centrifuged once at 478×g for 10 min, and the supernatant centrifuged at 10,000×g for 7 min. The pellet was washed with 25 ml ice-cold sucrose buffer and centrifuged 4 times at 10,000×g for 7 min, then resuspended in 3 ml ice-cold sucrose buffer. Protein concentration was determined using a BCA kit following the manufacturer's instructions (Life Technologies).

Measurement of PRODH/POX catalytic activity
PRODH/POX activity was measured as previously described, with minor modification (Pandhare et al. 2009). After treatment, cells were washed with cold PBS and harvested by trypsinization. Cells were resuspended in cold sucrose buffer [0.25 M sucrose, 3.5 mM Tris, and 1 mM EDTA (pH 7.4)] containing 1× protease inhibitor cocktail 1 and 2 (Sigma) and then sonicated for 20 s at a setting of 20 % (Branson Sonifier 450; Branson Ultrasonics Corp., Danbury, CT). Total protein was determined using the BCA protein assay (Pierce). A 1 ml reaction mixture containing 0.1 M KPO 4 , pH 7.2, 0.12 mg/ml o-aminobenzaldehyde (OAB), 0.012 mg/ml cytochrome C, 5 mM proline, and cell extract containing 50-100 μg protein was incubated for 20-60 min at 37 °C. The reaction was terminated by addition of 20 μl of OAB (10 mg/ml in 6 N HCl). The samples were centrifuged and the absorbance of the OAB-P5C complex was measured at 440 nm. A standard calibration curve was generated using purified P5C.

Oxygen consumption rate assays
XF24 cell culture plates were seeded with 5 × 10 5 cells and allowed to attach overnight. For PRODH/POX expression, cells were washed 3× with PBS and plated in DMEM containing the indicated amount of DOX and media treatment. One hour prior to the assay, cells were washed 2× with PBS and plated in Seahorse assay media containing 5 mM glucose and 2 mM glutamine, and adjusted to pH 7.4. Cells were incubated for 1 h at 37 °C in a no CO 2 incubator. Assay reagents (DMSO, oligomycin, FCCP, rotenone, and antimycin A) were loaded at a final concentration of 2.5 µM. Seahorse calibration, mitochondrial fitness tests, and oxygen consumption measurements were performed as per manufacturer's instructions (Seahorse Biotechnology, Massachusetts).

Measurement of reactive oxygen species
Cells were washed 3× with Dulbecco's PBS (DPBS), then placed in DPBS containing 5 µM of 5-(and-6)chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H 2 DCFDA; Life Technologies). Cells were exposed to the dye for 20 min in a 37 °C incubator. The fluorescence intensity was determined on an adherent cell laser cytometer (ACAS; Meridian Instruments, Inc. Okemos, MI) using 488 nm excitation and 560 nm fluorescence detection. Quantitation was based on total protein measured per well using the BCA protein quantitation kit as per manufacturer's protocol (Life Technologies).

Measurement of electron transfer
Catalytic activity of SDH and PRODH/POX was measured using 2,6-dichlorophenolindophenol (DCIP) in the method of Hatefi and Stiggall with modification (Hatefi and Stiggall 1978). 50-100 µg of mouse mitochondria was suspended in 1 ml assay buffer containing 2 mM KCN, 20 mM KPO 4 , 50 µM DCIP, 100 µM CoQ1, and either 10 mM methylsuccinate or proline. Reactions were incubated at 37 °C for 10 min. Changes in absorbance at 600 nm were calculated versus negative control and blank.

Crosslinking and co-immunoprecipitation
Mitochondrial proteins were cross-linked using dithiobi s(succinimidylpropionate) (DSP) according to manufacturer's instructions (Thermo Fisher Scientific; Waltham, MA). Briefly, 200 mg of mouse mitochondrial fractions was washed 3× in ice-cold PBS. Mitochondria were suspended in cold PBS containing 2.5 mM of DSP and incubated on ice for 2 h. Reaction was quenched by addition of 50 mM Tris-HCl. Immunoprecipitation of Complex II was performed using the monoclonal Complex II immunocapture antibody (Abcam, Cambridge, MA) following manufacturer's protocol. Mitochondria were solubilized by the addition of 1 % lauryl maltoside (LM) and 1× protease inhibitor cocktail 1 and 2 (Sigma Aldrich) and incubated on ice for 30 min. Mitochondria were centrifuged at 14,000×g at 4 °C for 10 min to pellet insoluble material. Lysates were incubated with either 10 µg of Complex II antibody, PRODH monoclonal antibody (A-11; Santa Cruz Biotechnology), or mouse IgG control (Abcam) on ice for 1.5 h. 50 µl of a 50 % slurry of TrueBlot Anti-Mouse IP Beads (Rockland Antibodies, Limerick, PA) was added and samples placed on a rotator overnight at 4 °C. Beads were washed 3× with cold PBS containing 0.1 % LM and suspended in 100 µl of Laemelli SDS-page buffer.

Western blotting
Cell lysates were prepared and quantified according to established methods. To each well of a 4-12 % or 12 % SDS-polyacrylamide gel, 15-30 μg total protein was applied, electrophoresed, and transferred to nitrocellulose membrane using an iBlot semi-dry transfer apparatus (Life Technologies). Membranes were blocked using Tris-buffered saline with 5 % nonfat milk (pH 7.6; Sigma).

Statistical methods
For analysis of mechanisms of inhibition of POX by SDH inhibitors and succinate, data in this study were evaluated using linear and nonlinear regression analysis, Lineweaver-Burk double-reciprocal plot analysis, and analysis of covariance (ANCOVA). Precise estimates of V max and K m were estimated using modern nonlinear regression (Bates and Watts 1988) methods. Intercepts (1/v0) and slopes (K m /V max ), which were back-calculated from the V max and K m estimates obtained from nonlinear regression analyses, were found to be virtually identical to intercept and slope estimates obtained from linear regression analyses using the double-reciprocal Lineweaver-Burk strategy. ANCOVA was used to determine statistical equivalence of intercept and slope estimates in connection with inferences regarding competitive or uncompetitive inhibition in the CoQ1, SUCC, and TTFA analyses (N AWaC2014; Alvord 2014; Venables et al. 2002). Statistical analyses were performed with the R Statistical Language and Environment (Team RC 2014).

Coenzyme Q1 is electron acceptor for PRODH/POX
It was recently shown that recombinant PRODH/POX isolated from S. Cerevisae bound directly to CoQ1 and that this was the mechanism by which PRODH/POX fed electrons from proline into the electron transport chain (Wanduragala et al. 2010). In addition, we have previously demonstrated that PRODH/POX catalysis of proline can be used to support ATP generation under conditions of acute nutrient stress (Pandhare et al. 2009).
To assess PRODH/POX utilization of CoQ1 as an electron acceptor in our DLD-POX cell model, we added increasing amounts of CoQ1 to DLD-POX lysates and monitored PRODH/POX catalytic activity. As shown in supports respiration independent of Complex I and II activity. a POX activity assay showing the increase in POX activity in the presence of increasing CoQ1. DLD-POX cells were grown in 0.2 ng/ml DOX to allow POX expression. Homogenized cell lysate containing 100 μg protein was incubated in assay buffer with 0, 25, 50, 100, 250, and 500 μM of CoQ1 at 37 °C for 20 min. Absorbance at 440 nm was measured to assess the amount of OAB-P5C complex formed. A P5C standard curve was used to calculate the P5C concentration. Data shown represent mean ± SEM. Compared to CoQ1 = 0, all values are significant to p < 0.001. b Double-reciprocal analysis of CoQ1-dependent POX activity. DLD-POX lysate with 100 µg protein was incubated with 1, 2, 5, or 10 mM of proline and 25, 50, 75, or 100 µM of CoQ1 in assay buffer at 37 °C for 20 min. Absorbance at 440 nm was used to measure the amount of OAB-P5Ccomplex formed and a P5C standard curve used to calculate the P5C concentration. c PRODH/POX activity in DLD-POX lysates and mouse mitochondria show similar dependence on ETC function. PRODH/POX-containing lysates (200 µg protein) or mouse mitochondria (50 µg protein) were incubated in assay buffer at 37 °C for 30 min. Sensitivity of PRODH/POX activity to ETC function was measured by the addition of 50 µM ROT, 2 mM TTFA, 5 µM of AA5, 100 µM of AMA, and 1 mM KCN. Data shown represent mean ± SEM. *p < 0.01 compared to its respective control. d PRODH/POX expression is required for proline to support respira-tion during acute nutrient stress. DLD-VEC control cells (VEC) and DLD-POX cells (POX) were grown in 0.2 ng/ml DOX for 48 h. Cells were incubated in glucose-free media alone (CON) or with the addition of 5 mM proline (+PRO) for 1 h prior to Seahorse XF24 analysis. OCR was measured and a respiratory profile was established by the addition of 2.5 µM DMSO, OLIGO, FCCP, and ROT/AMA at the indicated time points. *p < 0.01 compared to vector control. e Seahorse XF24 oxygen consumption analysis of DLD-POX control cells. Cells were grown in 20 ng/ml DOX to suppress PRODH/POX expression. 1 h prior to analysis, cells were incubated in assay media containing 5 mM glucose and either 0 (CON), 1, or 5 mM proline (PRO). OCR was measured at the indicated timepoints. ROT and AA5 were added at 2.5 uM to inhibit Complex I and Complex II activity, respectively. AMA was added at 2.5 μM to inhibit Complex III. Compared to control, *p < 0.01; + p < 0.02. f Experimental details as described for (e), except in this case, DLD-POX cells were grown in 0.2 ng/ ml DOX for 48 h prior to assay. Compared to control, + p < 0.001; *p < 0.01. The following abbreviations apply: AA5 atpenin A5, AMA antimycin A, CoQ1 coenzyme Q1, DMSO dimethyl sulfoxide, DOX doxycycline, FCCP carbonyl cyanide p-trifluoromethoxyphenylhydrazone, KCN potassium cyanide, OAB 2-aminobenzaldehyde, OCR oxygen consumption rate, OLIGO oligomycin, PRODH/POX proline oxidase, ROT rotenone, TTFA 2-thenoyltrifluoroacetone CoQ1 in a dose-dependent and saturable manner. These data indicate that PRODH/POX utilizes CoQ1 as an electron acceptor in the oxidation of proline to P5C.
To further characterize its interaction with CoQ1, we performed PRODH/POX activity assays using increasing concentrations of proline and CoQ1 to generate a Lineweaver-Burk plot (Fig. 1b). Under our experimental conditions, the V max and K m values were 9.72 ± 0.35 µM P5C min −1 and 0.42 ± 0.098 mM proline, respectively, using 25 µM of CoQ1. The V max and K m increased to 21.97 ± 0.47 µM P5C min −1 and 1.2 ± 0.09 mM proline, respectively, using 100 µM CoQ1. In a global analysis of covariance of the four data sets, there was no significant difference in the slopes at the α = 0.01 level of confidence, p = 0.011. This indicates that PRODH/POX binds directly to CoQ1 without the formation of a tertiary complex, consistent with recent reports (Wanduragala et al. 2010).
To determine whether PRODH/POX activity was dependent on ETC function, we compared PRODH/POX activity in DLD-POX cell lysates and mouse mitochondria in the presence of ETC inhibitors. We examined PRODH/ POX activity in the presence of CoQ1 alone or in combination with rotenone (ROT), an inhibitor of Complex I, 2-thenoyltrifluoroacetone (TTFA) or atpenin A5 (AA5), inhibitors of Complex II, antimycin A (AMA), an inhibitor of Complex III, or potassium cyanide (KCN), an inhibitor of Complex IV. In both isolated mitochondria and DLD-POX cell lysates, addition of ROT only modestly affected PRODH/POX activity, whereas addition of TTFA significantly reduced PRODH/POX activity (Fig. 1c). This was not the case with AA5, which had no effect on PRODH/ POX activity. When AMA or KCN was added to the reaction, PRODH/POX catalytic activity was dramatically reduced. Thus, in both DLD-POX cells and mouse mitochondria, the transfer of electrons from proline to CoQ1 by PRODH/POX was dependent on downstream electron transfer from CoQ1 to Complexes III and IV.
Acute expression of PRODH/POX during nutrient stress led to PRODH/POX-and proline-dependent ATP generation and cell survival (Pandhare et al. 2009). To determine whether PRODH/POX supported oxidative respiration during acute nutrient stress, we incubated either DLD-VEC cells, containing the vector construct, or DLD-POX cells, grown in 0.2 ng/ml doxycycline (DOX) to allow PRODH/ POX expression, for 48 h in proline-free media. We then placed the cells in glucose-free media with or without the addition of 5 mM proline for 1 h, and measured cellular oxygen consumption rate (OCR). Whereas the DLD-VEC cells did not respond to proline treatment, the DLD-POX cells were able to support a stable respiratory profile (Fig. 1d).
For additional evidence that PRODH/POX could support respiration solely through proline oxidation, we incubated induced and uninduced DLD-POX cells in proline-free media for 48 h, and then in media containing 0, 1, and 5 mM proline for 1 h, and measured cellular OCR. After measurement of basal respiration, ROT and AA5 were added to eliminate the contributions of Complex I and Complex II to respiration. As shown in Fig. 1e, f, addition of ROT and AA5 reduced respiration to approximately 30 % of normal basal respiration in cells in which PRODH/ POX expression was uninduced. The addition of 5 mM proline to uninduced control cells resulted in only a slight recovery of respiration (Fig. 1e). In contrast, addition of proline to PRODH/POX-expressing cells restored respiration at both 1 mM (approximately 50 % basal) and 5 mM (approximately 80 % basal; Fig. 1f). Addition of AMA inhibited respiration in all conditions shown in Fig. 1e, f, indicating that the observed oxygen consumption was dependent on Complex III. This data indicates that proline is a PRODH/POX-dependent respiratory substrate during acute nutrient stress and that it acts independently of Complex I and Complex II but requires functional Complex III.

Effect of PRODH/POX on respiratory fitness
Our data indicated that PRODH/POX and proline can support respiration during acute nutrient stress. We investigated the effects of PRODH/POX expression on overall respiratory fitness and capacity. We grew DLD-VEC control cells and DLD-POX cells for 3 days with 20, 0.2, 0.02 and 0 ng/ ml of DOX to allow for increasing levels of PRODH/POX expression. Manipulation of DOX concentration in DLD-VEC cells had no effect on respiration (Fig. 2a), but DLD-POX cells showed a decrease in both basal and maximal respiratory capacity that was directly proportional to DOX concentration (Fig. 2b).
We examined the effects on respiration resulting from increasing duration of PRODH/POX expression alone or with the addition of proline to the media. DLD-POX cells were grown in either 20 ng/ml DOX as a negative control or 0.2 ng/ml of DOX to allow PRODH/POX expression for 3 or 5 days in the presence or absence of 5 mM added proline. In control cells, proline alone had no effect on basal or maximal respiration, even after 5 days (Fig. 2c). In contrast, expression of PRODH/POX significantly decreased basal and maximal respiration at both 3 and 5 days, and proline addition exacerbated this effect (Fig. 2c). Thus, PRODH/POX-dependent inhibition of respiration can be modulated by the duration of PRODH/POX expression and the availability of proline.
PRODH/POX is a well-established superoxide generator, and the majority of effects of PRODH/POX on signaling are due to generation of ROS (Liu et al. 2006;D'Aniello et al. 2015;Pang and Curran 2014). We investigated whether effects of PRODH/POX on respiration were due to ROS production. DLD-POX cells were grown Oxygen consumption rate (OCR) was measured and a cellular respiratory profile was established by the addition of 2.5 µM DMSO, OLIGO, FCCP, and ROT/AMA at the indicated timepoints. b Increasing PRODH/ POX expression correlates with lower cellular respiration. DLD-POX cells were grown in the indicated amount of DOX and oxygen consumption rate was analyzed as in (a). Values with DOX = 0.02 and 0.0 were compared to DOX = 20; *p ≤ 0.01; + p < 0.02. c Prolonged PRODH/ POX expression and addition of proline correlates with greater decreases in respiration. DLD-POX cells were grown in 0.2 ng/ml DOX to allow PRODH/POX expression for 3 days (3D POX) or 5 days (5D POX) alone or in media supplemented with 5 mM proline (3D POX + PRO and 5D POX + PRO). Respiration by these cells was compared to DLD-POX cells that had been grown for 5 days in 20 ng/ml DOX to suppress PRODH/ POX expression, alone or in combination with 5 mM proline (5D CON and 5D CON + PRO, respectively). Oxygen consumption rate was measured and a respiratory profile established as described in (a). Values for 3D POX + PRO, 5D POX, and 5D POX + PRO were compared to 5D CON. + p ≤ 0.05; *p < 0.01. d Inhibition of PRODH/POX-mediated ROS decreases effects on respiration. DLD-POX cells were grown for 48 h in either 20 ng/ml DOX (CON), or 0.2 ng/ml DOX alone (POX) or in combination with 5 or 10 mM of N-acetyl-l-cysteine (NAC). Oxygen consumption rate was measured and a respiratory profile established as described in (a). Compared to control, *p < 0.001; Compared to POX, + p < 0.01. e PRODH/POX expression down-regulated ETC component proteins. DLD-POX cells were grown in the indicated ng/ml concentration of doxycycline (DOX) for 48 h, alone or in combination with 10 mM DHP or NAC. Whole cell lysates were harvested, and protein expression of subunits of Complex I (NDUFA10), Complex II (SDHA and SDHB), Complex III (CIII-R, Reiske Fe-S subunit), and Complex IV (COX IV) were analyzed by Western blotting. β-Actin was used as a protein loading control. The band intensities shown below each panel were quantified using Image Studio, normalized to β-actin control, and expressed as the level relative to untreated control (lane 1 20 ng/ml DOX). Values for DHP and NAC were compared to the mean ± STD of the ratios for 0.2 ng/mL DOX (lane 3 e and lane 2, Fig. 5a) combined with those for 0.02 ng/ml DOX (lane 4 e) Although the DOX concentration for DHP and NAC treatment was 0.02 ng/mL DOX, the values at 0.02 ng/ml DOX were consistently lower than those at 0.2 ng/ml DOX. Thus, the values used represent a higher distribution for the PRODH/POX-mediated effect on ETC proteins. DHP and NAC increased the values of the ETC proteins, i.e., decreased the effect of PRODH/POX. *Value greater than 2 standard deviations of aforementioned distribution, denoting 95 % confidence limits in 0.2 ng/ml DOX for 3 days with 5 mM proline alone or with the addition of 5 or 10 mM of the antioxidant N-acetyl-l-cysteine (NAC). Expression of PRODH/POX with proline showed a dramatic suppression of oxidative respiration, and NAC mitigated their inhibitory effects on and preserved both basal and maximal respiratory capacity (Fig. 2d). These results demonstrate that basal and maximal respiratory capacity are modulated by levels of PRODH/ POX induction and proline availability, and that this effect is mediated by ROS generation.
As shown in Fig. 2, PRODH/POX down-regulates cellular respiration through ROS production. We examined the effects of PRODH/POX-dependent ROS on ETC component proteins. To correlate changes in proteins to functional effects on respiration, we grew DLD-POX cells in 20, 2, 0.2, and 0.02 ng/ml of DOX and monitored ETC proteins at increasing levels of PRODH/POX expression (Fig. 2e,  lanes 1-4, respectively). In addition, cells were incubated in 0.02 ng/ml of DOX together with either the PRODH/ POX inhibitor dehydroproline (DHP) (Fig. 2e, lane 5) or NAC (Fig. 2e, lane 6). To assess statistical significance of DHP and NAC, we calculated mean ± standard deviation of the values for 0.2 and 0.02 ng/ml DOX from Figs. 2e and 5a. Increasing PRODH/POX expression decreased levels of the NDUFA10 subunit of Complex I, SDHA and SDHB, the Reiske subunit of cytochrome C reductase (Complex III), and subunit IV of cytochrome C oxidase (COX IV, Complex IV). Importantly, SDHA, SDHB and CIII-R were restored by either NAC or DHP or by both (Fig. 2e, lanes 5 and 6, respectively. See legend to Fig. 2e). Compared to the POX levels, the values for DHP and NAC indicated by an asterisk were outside of 2 standard deviations from the mean of the POX controls. Taken together, these results provide preliminary evidence that PRODH/POX modulates respiration through ROS-mediated down-regulation of ETC component proteins.

PRODH/POX is inhibited by Complex II inhibitors and succinate
We showed in Fig. 1c that TTFA inhibited PRODH/POX activity whereas AA5 did not. The crystal structure of SDH with bound TTFA or AA5 has been solved; both are competitive inhibitors of the ubiquitin-binding site, with AA5 binding at a site more deeply embedded in the enzyme's catalytic site (Sun et al. 2005;Miyadera et al. 2003;Horsefield et al. 2006). To compare the inhibitory potency of TTFA and AA5 for PRODH/POX versus SDH, we performed enzyme activity assays with either proline or succinate as the substrate. Carboxin, an additional competitive inhibitor of the ubiquitin-binding site of SDH (Ruprecht et al. 2009), was also tested. Both TTFA and carboxin inhibited SDH and PRODH/POX activity with similar efficacy, with TTFA inhibiting 60 % and 35 % and carboxin inhibiting 36 and 42 % of SDH and PRODH/POX activity, respectively. In contrast, AA5 was selective toward SDH, inhibiting its activity by 83 % whereas it only inhibited PRODH/POX activity by 9 % versus control (Fig. 3a).
To elucidate the mechanism by which TTFA inhibited PRODH/POX activity, we performed POX activity assays to construct a Lineweaver-Burk plot (Fig. 3b). V max was only modestly decreased, being 17.2 ± 0.4 µM P5C min −1 for control and 16.4 ± 0.3 µM P5C min −1 with the addition of 2 mM TTFA. In contrast, 2 mM TTFA increased K m from 0.65 ± 0.05 mM proline to 2.7 ± 0.35 mM proline. Statistical tests were performed for the equivalence of the y-intercepts for TTFA data pairs. All p values were greater than 0.05, indicating that the y-intercepts for TTFA data were statistically equivalent. Together, these results suggest that TTFA acts as a competitive inhibitor of PRODH/POX. We compared the mechanism by which succinate inhibits PRODH/POX activity. We again performed PRODH/ POX activity assays to construct a Lineweaver-Burk plot (Fig. 3c). Addition of 5 mM succinate decreased both V max and K m from 19.1 ± 0.4 to 8.8 ± 0.3 µM P5C min −1 and 1.1 ± 0.08 mM proline and 0.55 ± 0.09 mM proline, respectively, versus control. In a global analysis of covariance of the four experimental data sets, there was no significant difference in the slopes at the α = 0.05 level of confidence, p = 0.75. This indicates succinate inhibits PRODH/ POX activity through uncompetitive inhibition.
To examine whether PRODH/POX and SDH could be physically associated on the mitochondrial inner membrane, we performed cross-linking and co-immunoprecipitation assays. We incubated mouse mitochondria with the protein cross-linker DSP, dissolved the mitochondria with 1 % laurel maltoside and immunoprecipitated using an antibody versus either PRODH/POX or Complex II. We then immunoblotted for PRODH/POX and the A and B subunits of SDH. Immunoprecipitation of PRODH/POX resulted in co-immunoprecipitation of both SDHA and SDHB. In addition, PRODH/POX co-immunoprecipitated with Complex II when an anti-Complex II was used (Fig. 3d). These data indicate that PRODH/POX and Complex II co-localize on the surface of the mitochondrial matrix membrane within the length of the DSP spacer arm (12 angstroms).

Succinate inhibits PRODH/POX-dependent ROS generation
Our data suggested that PRODH/POX and proline control mitochondrial respiration through ROS-mediated downregulation of ETC proteins. In addition, our data showed that succinate inhibits catalytic activity of PRODH/POX. We were intrigued whether succinate would modulate the PRODH/POX effect on the ETC. DLD-POX cells were grown in 0.2 ng/ml of DOX to allow PRODH/POX expression, alone or in combination with 5, 10, or 20 mM of methyl-succinate. Cells were then treated with general ROS indicator 5-(and-6)-chloromethyl-2′, 7′-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H 2 DCFDA) to detect total cellular ROS. PRODH/POX expression resulted in a threefold increase in detectable ROS generation versus uninduced control; this ROS generation was inhibited by succinate in a dose-dependent manner (Fig. 4a).
To determine whether succinate inhibition of PRODH/ POX protected against the PRODH/POX-dependent downregulation of respiration, we grew DLD-VEC and DLD-POX cells in media containing proline alone or together with methyl-succinate. DLD-VEC control cells showed no change in basal and maximal respiration whether treated with proline, methyl-succinate, or a combination of both (Fig. 4b). In contrast, PRODH/POX-expressing cells showed a marked reduction of respiration in response to proline treatment that was inhibited by co-treatment with methyl-succinate (Fig. 4c). Taken together, this data indicates that succinate inhibits the effects of PRODH/POX and proline on respiration through the inhibition of PRODH/ POX-and proline-derived ROS.

Succinate inhibition of PRODH/POX restores levels of ETC component proteins
Because succinate inhibited PRODH/POX-dependent ROS generation, we investigated whether succinate treatment could modulate PRODH/POX-mediated down-regulation of ETC proteins. DLD-POX cells were grown in either 20 ng/ml DOX or 0.2 ng/ml DOX to allow PRODH/POX expression alone or in combination with increasing concentrations of succinate. To link effects on ETC proteins with effects on ROS (Fig. 4a), we used increasing concentrations of succinate. Expression of PRODH/POX resulted in down-regulation of subunits of Complexes I-IV of the ETC (Fig. 5, lane 1 vs lane 2). Addition of increasing PRODH/POX activity is regulated by succinate and SDH inhibitors and co-localizes with SDH on the mitochondrial inner membrane. a PRODH/POX and SDH share similar sensitivity to SDH inhibitors except AA5. Mouse mitochondria (440 µg protein) were incubated in reaction buffer containing either 10 mM of succinate or proline alone (Control) or in the presence of 1 mM TTFA, 1 mM Carboxin, or 5 µM AA5 at 37 °C for 10 min. Absorbance was measured at 600 nm and results calculated as a percent of unreacted control dye. Data shown represent mean ± SEM (n = 3) of comparisons against their respective controls. *p < 0.001; + p < 0.05. b Doublereciprocal analysis of TTFA-dependent inhibition of PRODH/POX activity. DLD-POX lysate containing 100 µg protein was incubated with 1, 2, 5, or 10 mM of proline and 0, 0.5, 1 and 2 mM of TTFA in assay buffer at 37 °C for 20 min. Absorbance at 440 nm was used to measure the amount of OAB-P5C complex formed and a P5C standard curve used to calculate the P5C concentration. c Double-recipro-cal analysis of succinate-dependent inhibition of PRODH/POX activity. DLD-POX lysate containing 100 µg protein was incubated with 1, 2, 5, or 10 mM of proline and 0, 0.5, 1, and 5 mM of succinate in assay buffer at 37 °C for 20 min. Absorbance at 440 nm was used to measure the amount of OAB-P5Ccomplex formed and a P5C standard curve used to calculate the P5C concentration. d Co-immunoprecipitation of PRODH/POX and Complex II. 4 mg of mouse mitochondria was cross-linked with DSP, then solubilized and incubated with either PRODH/POX (POX IP) or Complex II antibody (CII IP). Control samples were incubated with an equal concentration of a non-specific mouse IgG (CON). Samples were incubated with beads coated with anti-mouse IgG overnight. Cross-linker was cleaved and proteins solubilized with SDS-PAGE buffer. Control and IP lysates were immunoblotted versus a 5 % input control (IN) using PRODH/ POX, SDHA, and SDHB antibodies. Data is representative of three separate experiments concentrations of succinate resulted in restoring protein levels for Complexes I-III (Fig. 5, lanes 3-6). Levels of the COX IV subunit of Complex IV were not restored by succinate treatment, but it was the only exception of all of the ETC subunits examined.
Since both the decrease in PRODH/POX-generated ROS and the mitigation of the PRODH/POX-mediated decrease in ETC protein levels appeared to be dependent on the concentration of succinate, we plotted the individual ETC proteins versus succinate and showed a general concentration dependence (Fig. 5b). This suggested that succinate-dependent inhibition of PRODH/ POX was a common mechanism. With this hypothesis, we compared the values for ETC proteins in combination. Furthermore, we included the values for ETC proteins at 0.2 ng/ml DOX from Fig. 2e. These values represent mean ± STD of ETC proteins. With SUC = 0, 0.635 ± 0.091, N = 10, SUC = 10 mM, mean = 0.782 ± 0.122, N = 5, different from SUC = 0 with p = 0.0203; SUC = 20 mM, values were 0.768 ± 0.156, N = 5, different from SUC = 0, with p = 0.0546. These values and statistical differences support our working hypothesis of a common mechanism underlying the changes in ETC proteins.

Discussion
In this study, we have examined the interaction of PRODH/ POX with Coenzyme Q1 and with the ETC. While PRODH/POX supports respiration in the short term, PRODH/POX expression results in a decrease of total cellular respiration through down-regulation of proteins of the ETC. Most interestingly, the effects of PRODH/POX on respiration and the ETC can be inhibited by succinate, resulting in a potential regulatory loop. c Succinate inhibits the decrease in respiration induced by PRODH/ POX and proline. DLD-POX cells were grown for 48 h in 0.2 ng/ ml of DOX to allow PRODH/POX expression. Oxygen consumption rate (OCR) was measured and a respiratory profile was established as described for (b). Comparison of 10 mM succinate to 5 mM PRO, + p < 0.01. Comparison of 5 mM POX to 5 MM POX + 10 mM SUC, *p < 0.05. Data represents mean ± standard error of the mean (n = 3) for all panels Our analysis of the relationship between PRODH/POX and CoQ1 suggested that POX can transfer proline-derived electrons directly to CoQ1 without the formation of a tertiary complex. In addition, selective inhibitors indicated that PRODH/POX did not require the activity of Complex I or Complex II to support respiration. This is consistent with studies in other diverse model systems of PRODH/POX activity, including Z. meas (corn) mitochondria, purified S. cerevisiae PRODH/POX, and PutA from E. coli (Elthon and Stewart 1982;Wanduragala et al. 2010;Moxley et al. 2011;Abrahamson et al. 1983). In addition, PRODH/POX dependence on a functional ETC in our DLD-POX expression system was almost identical to that of isolated mouse mitochondria. Thus, we believe our DLD expression system accurately reflects the relationship between PRODH/ POX and the ETC.
PRODH/POX was necessary and sufficient to support proline-dependent oxidative respiration during acute nutrient stress, consistent with previous publications showing its pro-survival role during nutrient stress (Pandhare et al. 2009;Liu et al. 2012). In addition, extended expression of PRODH/POX (48-96 h) resulted in the down-regulation of basal oxidative respiration and maximal respiratory capacity. This effect was dependent on the duration and intensity of POX expression as well as the addition of exogenous proline, and was inhibited by the co-treatment with NAC. Reduction of respiratory capacity correlated with reduction in levels of protein subunits of Complexes I-IV of the ETC, which was reversed by co-treatment of cells with either DHP or NAC. These effects are most readily explained as resulting from proline oxidation and ROS generation by PRODH/POX.
Inhibition of PRODH/POX by TTFA was through competitive inhibition. Both TTFA and carboxin have previously been shown to compete for the ubiquinone-binding site of SDH that is formed when the 4 SDH subunits combine to form the holoenzyme (Sun et al. 2005;Ruprecht et al. 2009). Based on this, one possibility we considered was that PRODH/POX required the ubiquitin-binding site of SDH for its activity. However, AA5, which has been shown to bind deeper in the ubiquinone-binding pocket of SDH (Miyadera et al. 2003;Horsefield et al. 2006), showed very little inhibition of PRODH/POX. In addition, the interaction of PRODH/POX with CoQ1 indicated direct binding to PRODH/POX. We think it more likely that the SDH inhibitors compete for the ubiquitin-binding site on PRODH/POX. The observation that TTFA and carboxin can inhibit PRODH/POX and SDH to a similar degree at the concentrations used may warrant caution in their use as SDH-selective inhibitors.
Although succinate inhibition of PRODH/POX has been investigated (Kowaloff et al. 1977;Norden and Matanganyidze 1974), to our knowledge this is the first study to examine the effects of this inhibition on PRODH/POXmediated ROS generation and mitochondrial metabolism. Our analysis showed that succinate inhibits POX through  (Fig. 4a). a Whole cell lysates were harvested, and protein expression of PRODH/POX and of subunits of Complex I (NDUFA10), Complex II (SDHA, SDHB), Complex III (CIII-R, Reiske Fe-S subunit), and Complex IV (COX IV) was analyzed by Western blotting. The band intensities shown below each panel were quantified using Image Studio, normalized to β-actin control, and expressed as the level relative to untreated control (lane 1 20 ng/ml DOX). b Since succinate decreased PRODH/POX-mediated ROS in a dose-dependent manner (Fig. 4a) and the levels of proteins in a suggested a succinate concentration effect, we constructed a combination plot of protein levels as the fraction of control against succinate concentrations (see text). For the value with treatment by POX (0.2 ng/ml DOX) without succinate, we combined the values from a with those obtained in Fig. 2e. We then compared the values statistically. The value with 10 mM succinate was different from that without succinate, *p = 0.020. At 20 mM succinate, the difference was borderline significant, + p = 0.055 uncompetitive inhibition. This mechanism makes it very unlikely that the observed effects are due to non-selective inhibition of PRODH/POX by succinate. Treatment with succinate inhibited PRODH/POX-dependent ROS production and prevented PRODH/POX-dependent ROS effects on oxidative respiration. Succinate also protected ETC component proteins from PRODH/POX ROS-mediated down-regulation with almost the same efficacy as DHP and NAC.
As an uncompetitive inhibitor, the affinity of succinate is for the enzyme-substrate complex of PRODH/POX and proline rather than for the enzyme binding site for proline. Additionally, our data show that PRODH/POX and SDH co-localize on the mitochondrial inner membrane, where local substrate concentrations would have the potential to affect the activities of both enzymes. Thus, in the presence of low levels of proline, higher levels of succinate could act to inhibit PRODH/POX activity and ROS generation. This may provide an additional level of regulation of PRODH/POX stress signaling until cellular levels of TCA cycle intermediates fall below a critical point. The specific role that SDH plays in the transmission of the PRODH/ POX-generated ROS signal remains to be elucidated, but a coordinated role with PRODH/POX would be consistent with the observation that SDH-mediated ROS generation was essential for a hypoxia-dependent stress response in mouse lung sections (Paddenberg et al. 2003(Paddenberg et al. , 2012 and that Complex II has been found to be a site of ROS generation resulting from proline oxidation (Goncalves et al. 2014).
The data presented here introduce a novel relationship between PRODH/POX, proline, and succinate and the regulation of respiration. Depending on cellular context, this relationship could provide an additional point of regulation between the identified inducers of PRODH/POX and regulation of cellular energy levels and routing of metabolites. More intriguing is the possibility that the proline found in extracellular collagen may serve both as a source of energy for tumors and tissues through proline catabolism, and also as a signaling and metabolic link between the extracellular environment during development and tumor formation. It is becoming increasingly apparent that dysregulation of metabolism and the resulting change in metabolite levels affect not only cell transformation (Ward et al. 2010;Lu et al. 2013), but influence tumor cell aggressiveness and metastatic potential, as has been shown recently in ovarian cancer and ovarian cancer stem cells (Vermeersch et al. 2014(Vermeersch et al. , 2015. The role of PRODH/POX as a direct mediator of ROS-dependent signaling and central mediator of metabolic intermediates will, no doubt, continue to play an interesting role in cellular development and transformation.

Summary
This work shows for the first time the regulation of respiratory fitness by PRODH/POX through downregulation of ETC proteins, and the inhibition of these effects by succinate. The existence of a regulatory loop between PRODH/ POX, the ETC, and succinate would link the various stress pathways that induce PRODH/POX with ETC regulation and TCA cycle metabolite flux. Changes in proline and succinate homeostasis may be a mechanism of epigenetic gene regulation during differentiation and tumorigenesis, and this possibility should be investigated further.