Mitochondrial UCP4 and bcl-2 expression in imprints of breast carcinomas: Relationship with DNA ploidy and classical prognostic factors

Pathology – Research and Practice 207 (2011) 377–382

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Original article

Mitochondrial UCP4 and bcl-2 expression in imprints of breast carcinomas: Relationship with DNA ploidy and classical prognostic factors Maria Gonidi, Anna-Maria Athanassiadou, Efstratios Patsouris, Angelos Tsipis, Stavros Dimopoulos, Vassiliki Kyriakidou, George Chelidonis, Pauline Athanassiadou ∗ 1st Pathology Department and Cytology Unit, National & Kapodistrian University of Athens, Medical School, 75 Mikras Asias, 11527 Athens, Greece

a r t i c l e

i n f o

Article history: Received 5 November 2010 Received in revised form 26 February 2011 Accepted 21 March 2011 Keywords: UCP4 bcl-2 DNA ploidy Breast carcinomas

a b s t r a c t Mitochondria are the bioenergetic and metabolic centers of cells and play an important role in the regulation of cell death. The mitochondrial apoptosis pathway is controlled by the bcl-2 protein family. Overexpression of mitochondrial uncoupling protein 4 (UCP4) can promote proliferation and inhibit apoptosis and differentiation. Imprint smears obtained from 124 tumors were studied immunocytochemically, and results were correlated with prognostic markers. There were 112 ductal and 12 lobular carcinomas. The positivity of UCP4 was correlated with lymph node metastases (p = 0.005), positive ER and PR expression (p < 0.0001 for both), as well as positivity for p53 (p < 0.0001) and Ki-67 (p < 0.0001). Decreased expression of bcl-2 correlated with increased expression of UCP4 (p = 0.001). Regarding DNA ploidy, UCP4 positivity was correlated with aneuploid tumors (p = 0.002). Negative expression of bcl-2 was correlated with poorly differentiated carcinomas (p < 0.0001), as well as with positive expression of p53 (p < 0.0001) and Ki-67 (p < 0.0001). Logistic regression revealed that ploidy and p53 expression had an impact on UCP4. These findings encourage future investigations regarding the potential role of UCPs not only into mechanisms underlying breast cancer, but also as a novel candidate to the design and development of more effective therapeutic strategies. © 2011 Published by Elsevier GmbH.

1. Introduction Apoptosis is a conserved mechanism present in multicellular organisms which are dependent on an ordered removal of damaged or unwanted cells both during their development and in the adult life [1,2]. Apoptosis can be triggered by a large variety of different stimuli. However, only two major intracellular apoptosis signaling pathways have been identified, the death receptor pathway and the mitochondrial pathway [1,3]. Mitochondria are the bioenergetic and metabolic centers of cells and play an important role in the regulation of cell death. During apoptosis, mitochondria suffer specific damages that result in loss of their function. The complex role of mitochondria, in cell apoptosis, focuses on several mitochondrial proteins which are able to activate cellular apoptotic programs directly [1,2,4]. The mitochondrial apoptosis pathway is controlled by the bcl-2 protein family. This family contains members with either pro- or anti-apoptotic activity. Antiapoptotic members, like bcl-2 and bcl-XL, contain four homology BH domains, whereas proapoptotic members, such as Bax and Bak, have three BH domains [5].

∗ Corresponding author. Tel.: +30 2107462171; fax: +30 2107462171. E-mail address: [email protected] (P. Athanassiadou). 0344-0338/$ – see front matter © 2011 Published by Elsevier GmbH. doi:10.1016/j.prp.2011.03.007

Several models have been proposed to explain how bcl-2 blocks cell death: (a) by sequestering the proforms of caspases by indirect interaction, (b) by forming ionic channels, and (c) by inhibiting apoptosis-associated release of cytochrome c from the mitochondria, which leads to the activation of caspase 3 [6]. A variety of key events in apoptosis focus on mitochondria, including the release of caspase activators (such as cytochrome c), changes in electron transport, loss of mitochondrial transmembrane potential, altered cellular oxidation–reduction, and participation of pro- and antiapoptotic Bcl-2 family proteins [7,8]. Mitochondria are recognized as regulators of cell death via apoptosis and necrosis, and as modifier events via mitochondrial DNA (mt DNA) alterations providing a possible proliferative advantage to the tumor cells, in addition to their essential role in cell survival [9]. The mitochondrial uncoupling proteins (UCPs) form a distinct subfamily of the mitochondrial anion carrier protein (MACP) gene family. Although their phenotypes are known in detail only for brown adipose tissue-specific UCP1 and some plant UCPs, it is a general consensus that mammalian ubiquitous UCP2 and skeletal muscle-specific UCP3 might participate in the regulation of body weight (with dysfunctions leading to obesity), in adaptive thermogenic processes including fever, and in the reduction of the excessive formation of reactive oxygen species (ROS) [10].

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Recently revealed brain-specific UCP4 has been suggested to participate in apoptosis in the brain. This speculation could also be extended for another brain-specific UCP, termed BMCP1 or UCP5 (which appeared to be the most distant member in the UCP family). UCP4 maps to human chromosome 6p11.2–q12. Consistent with its potential role as an uncoupling protein, UCP4 is localized to the mitochondria, and its ectopic expression in mammalian cells reduces mitochondrial membrane potential. These findings suggest that UCP4 may be involved in thermoregulatory heat production and metabolism in the brain [11]. Overexpression of UCP4 can promote proliferation and inhibit apoptosis and differentiation of preadipocytes. The underlying mechanism whereby the expression of UCP4 promotes bioenergetics adaption and cell survival was recently elucidated by the role of extracellular signal-regulated kinases (ERKs). Activation of ERKs is necessary and sufficient for the increased dependency on glucose utilization by neural cells expressing the brain uncoupling protein UCP4. The switch from oxidative toward glycolytic metabolism reduces the production of toxic reactive oxygen species (ROS) and increases cellular resistance to toxicity induced by 3-nitropropionic acid, promoting cell survival [12]. The aim of this study was to investigate in imprints of resected breast carcinomas the relationship between DNA ploidy and UCP4 and bcl-2 expression with different clinicopathological factors. 2. Materials and methods The study population included 124 patients (aged 43–83) who had undergone surgery for breast cancer. Imprint smears were obtained immediately after tumor removal at the operation theater. From each specimen, smears were prepared and stained according to the Papanicolaou and Giemsa procedure to confirm the presence of cancer cells from the tumor site. After air drying, additional cytological smears were fixed with 5% buffer formalin for 20 min and stored until used by an immunocytochemical procedure. All histopathological diagnoses were performed using sections from samples that were used for the imprints. 2.1. Immunocytochemistry Imprint smears were obtained from each patient by touching the cut surface of the freshly resected tumor tissues. We prefer to use imprint smears instead of paraffin-embedded tissue sections and cells (cell blocks), as the latter present a lot of difficulties with regard to immunoreactivity. Depending on the thickness of the section, there are always numbers of sliced or overlapped cells, thus leading either to false low or to false high immunoreactivity, respectively [13–15]. Furthermore, tissue fixation and, to a lesser degree, tissue processing are potential causes of variation in the reproducibility of immunohistochemical staining [16]. Besides that, in cytological preparations, the cells are whole, a larger surface of the tumor is sampled, and tissue is preserved for subsequent pathological and molecular analyses [15,17–19]. It is now firmly established that a wide variety of markers can be applied to cytological preparations and that immunocytochemistry correlates well with immunohistochemistry [20–24]. Immunostaining was performed by the Avidin–Biotin Complex immunoperoxidase method with the use of antiUCP4 (Menarini, Italy) at a dilution of 1:200, antiKi-67 (Menarini, Italy) at a dilution of 1:50, antip53 clone DO7 (Menarini, Italy) at a dilution of 1:50, and antibcl-2 (Menarini, Italy) at a dilution of 1:50.Smears were incubated for 45 min with a normal rabbit serum diluted 1:40 in PBS. Then, the smears were rinsed in three changes of PBS for 5 min each and incubated overnight with primary antibodies. After washing in PBS, the smears were incubated with rabbit anti-

Fig. 1. Breast adenocarcinoma cells with positive cytoplasmic reaction for bcl-2 (500×).

mouse biotinylated immunoglobulins diluted in 1:200 followed by the ABC/HPR. Visualization was achieved by a final incubation in 3,3 -diaminobenzide tetrahydrochloride. The smears were counterstained with Mayer’s hematoxylin. Smears of known positive reactivity for all markers were incubated as positive controls, and negative controls were stained by omitting the primary antibodies incubation. Results were interpreted by two independent cytologists. In cases where staining was heterogeneous, the examined fields included those with the highest and lowest percentage of stained cells. The immunostaining for each protein was determined as positive or negative by a cut off value determined as follows: p53, UCP4, and bcl-2 staining was interpreted as positive when >10% of the tumor cells showed distinct nuclear or cytoplasmic staining; Ki67 was regarded as positive when >25% showed distinct nuclear staining (Figs. 1 and 2, for bcl-2 and UCP4, respectively). Histological classification was performed according to World Health Organization criteria [25], and pathological staging was performed according to the American Joint Committee on Cancer system [26]. Additional information was recorded concerning the patients’ menopausal status (ER/PR) as determined immunohistochemically and HER-2 expression. 2.2. Image analysis cytometry One imprint smear was stained using the Thionin Feulgen procedure in order to be used for image analysis. DNA measurements were performed with Image Pro Plus Software (␯ 5.1, Media Cybernetics Inc., Maryland, USA). A light microscope (Nikon Eclipse 80i, Nikon Corp., Tokyo, Japan) at a mag-

Fig. 2. Breast adenocarcinoma cells with positive cytoplasmic reaction for UCP4 (500×).

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Table 1 Clinicopathological characteristics of the patients. Variables

Number

Histological type Ductal Lobular Histological grade I II III Tumor size <2 cm >2 cm Lymph node status 0 1–3 >4 Menopausal status Premenopausal Postmenopausal ER status (−) (+) PR status (−) (+) HER-2 (−) (+)

Percentage (%)

112 12

90.3 9.7

12 52 60

9.7 41.9 48.4

66 58

53.2 46.8

24 46 54

19.3 37.1 43.6

32 92

25.8 74.2

40 84

32.3 67.7

62 62

50 50

74 50

59.7 40.3

bcl-2 expression with factors such as grade, lymph node metastasis, tumor size, ER, PR, and HER-2 status. Fig. 3. DNA histogram revealing a diploid distribution with peak at 2c.

3. Results nification of 400× was used in order to identify the heterogeneity of the nucleus and to measure accurately the optical density. A Nikon color camera (Nikon DS-2MW, Nikon Corp., Tokyo, Japan) with microscope adapter was plugged to the microscope for image capture. From each slide, at least 4 optical fields with the most cellular areas (hot spot) were selected and captured as images. All images were performed with the following procedure: (i) image calibration based on stain and optical density, (ii) image calibration based on microscope lens, with micrometric scale, in measurements unit microns, (iii) control cells detection and counting (at least 600 lymphocytes), (iv) tumor cells detection and counting (at least 300 cells), and (v) measurements of parameters as area, aspect ratio, diameter, roundness, and cell stain integrity (for both control and tumor cells). Data were automatically exported to Microsoft Excel spreadsheets, and DNA index and ploidy histograms were produced. The procedure was performed for all nuclei, and the overall mean represents DNA content or DNA Index (DI). The mean IOD of control cells serves as the diploid standard (2c) and reference for DI calculation for targeted cells. DNA histograms are also generated. DNA ploidy classification was based on DNA index and histograms according to the 4th updated ESACP consensus report on diagnostic DNA image analysis. A tumor was classified as diploid if DNA index ranged from 0.9 to 1.1 and the relevant DNA histogram revealed only one peak at 2c (Fig. 3), and as aneuploid if either of the previous two criteria was absent.

2.3. Statistical analysis The statistical analysis was performed using PASW Statistics 18 (SPSS Inc., Chicago, IL, USA). The significance level was set at 0.05. Pearson’s 2 test (with continuity correction for 2×2 tables) and logistic regression were used to compare DNA ploidy, UCP4, and

The clinicopathological characteristics for the 124 breast tumors studied are given in Table 1. There were 112 ductal and 12 lobular carcinomas. Thirty-two women were premenopausal and 92 postmenopausal. Twelve had histological grade I, 52 grade II, and 60 grade III. As for tumor size, 66 were smaller than 2 cm and 58 greater than 2 cm. Tumors were classified into three groups according to the number of positive lymph nodes. Twenty-four patients had no lymph node metastasis, 46 patients had one to three positive lymph nodes, and 54 patients had four or more positive lymph nodes. Eighty-four (67.7%) of the 124 tumors were ER-positive, and 62 (50.0%) were PR-positive. HER-2 positivity was 40.3% as determined immunohistochemically. Table 2 shows the distribution of UCP4, p53, bcl-2, Ki-67 staining pattern, and DNA ploidy in all cases of breast carcinomas. UCP4 was expressed in 72.6%, p53 in 71.0%, Ki-67 in 53.2%, and bcl-2 in

Table 2 Distribution of UCP4, p53, Ki-67, and bcl-2 staining expression, as well as DNA ploidy in 124 tumors of breast carcinoma. Immunostaining UCP4 − + p53 − + Ki-67 − + bcl-2 − + DNA ploidy Diploid Aneuplioid

Frequency (number)

Expression (%)

34 90

27.4 72.6

36 88

29.0 71.0

58 66

46.8 53.2

76 48

61.3 38.7

44 80

35.5 64.5

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Table 3 Results of 2 analysis between UCP4 and bcl-2 staining patterns and several clinicopathological parameters in 124 patients with breast carcinoma. Variable

Total

Staining pattern UCP4

Grade I/II III Tumor size <2 cm >2 cm Lymph nodes status − + ER status − + PR status − + HER-2 status − + p53 − + Ki-67 − + DNA ploidy Aneuploid Diploid

Bcl-2

− (%)

+ (%)

p-Value

− (%)

+ (%)

p-Value

26(40.6) 8(13.3)

38(59.4) 52(86.7)

0.001

26(40.6) 50(83.3)

38(59.4) 10(16.7)

<0.0001

22(33.3) 12(20.7)

44(66.7) 46(79.3)

0.170

34(51.5) 42(72.4)

32(48.5) 16(27.6)

0.028

24(40.0) 10(15.6)

36(60.0) 54(84.4)

0.005

36(60.0) 40(62.5)

24(40.0) 24(37.5)

0.919

20(50.0) 14(16.7)

20(50.0) 70(83.3)

<0.0001

24(60.0) 52(61.9)

16(40.0) 32(38.1)

0.995

26(41.9) 8(12.9)

36(58.1) 54(87.1)

0.001

42(67.7) 34(54.8)

20(32.3) 28(45.2)

0.197

18(24.3) 16(32.0)

56(75.7) 34(68.0)

0.463

48(64.9) 28(56.0)

26(35.1) 22(44.0)

0.420

22(61.1) 12(13.6)

14(38.9) 76(86.4)

<0.0001

12(33.3) 64(72.7)

24(66.7) 24(27.3)

<0.0001

28(48.3) 6(9.1)

30(51.7) 60(90.9)

<0.0001

22(37.9) 54(81.8)

36(62.1) 12(18.2)

<0.0001

14(17.5) 20(45.5)

66(82.5) 24(54.5)

0.002

54(67.5) 22(50.0)

26(32.5) 22(50.0)

0.085

124

38.7% of the tumors studied. Forty-four (35.5%) of the tumors were diploid, and 80 (64.5%) were aneuploid. The results of the 2 analysis for the clinicopathological factors studied are shown in Table 3. Differentiation was associated with UCP4, as 86.7% of poorly differentiated carcinomas were also UCP4-positive (p = 0.001). Positive expression of UCP4 was associated with lymph node metastases (84.4% of tumors, p = 0.005), positive expression of ER (83.3% of tumors, p < 0.0001), PR (87.1% of tumors, p = 0.001), p53 (86.4% of tumors, p < 0.0001), and Ki-67 (90.9% of tumors, p < 0.0001). Finally, UPC4 expression was also correlated with DNA ploidy, as 82.5% of positive UCP4 tumors were also aneuploid (p = 0.002). Decreased expression of bcl-2 correlated with increased expression of UCP4 (2 = 13.343, p = 0.001). Specifically, 84.2% of tumors with negative bcl-2 expression had positive UCP4 expression. Bcl-2 negative expression was correlated with poorly differentiated carcinomas (83.3% of tumors, p < 0.0001) and positive expression of p53 (72.7% of tumors, p < 0.0001) and Ki-67 (81.8% of tumors p < 0.0001) (Table 3). Logistic regression results (Table 4), including all parameters studied, revealed that only DNA ploidy and p53 expression had an impact on UCP4 (UCP4-positive expression was associated with positive expression of p53 and aneuploid tumors). 4. Discussion The complex role of mitochondria in mammalian cell apoptosis came into focus when biochemical studies identified several mitochondrial proteins that are able to activate several apoptotic programs directly [27–30]. During apoptosis, mitochondrial membrane permeability (MMP) increases, and the release into the cytosol of pro-apoptotic factors (procaspases, caspase activators and caspase-independent factors, such as apoptosis-inducing factor(AIF)) leads to the apoptotic phenotype. Despite progress regarding the mitochondrial-mediated cell death, many questions remain to be answered.

Apart from this pivotal role of mitochondria during the execution phase of apoptosis, there is increasing evidence that reactive oxygen species (ROS) produced by the mitochondria can be involved in cell death [31]. Reactive oxygen species (ROS) are produced as by-products of cellular metabolic pathways and function as a critical second messenger in a variety of intracellular signaling pathways. A defect or deficiency in the anti-oxidant defense system and/or the excessive intracellular generation of ROS renders an oxidatively stressed cell [32]. Direct or indirect involvement of ROS in numerous diseases has been documented by several authors [33–36]. Increasing evidence points toward a central role of oxidative stress in the pathogenesis of E2-induced breast cancer [37]. Although estrogen has been shown to act as an antioxidant in several tissues, there is a strong controversy regarding their oxidative effect in estrogen-dependent tissues [38–40]. To explore further involvement of ROS in breast cancer cells, we tried to correlate UCP isoform-UCP4- and apoptotic and prognostic markers at the immunocytochemical level, as UCPs are good candidates for controlling mitochondrial ROS. From a physiological viewpoint, it is worth noting that mitochondrial H2 O2 can diffuse into the nucleus, where it acts as a mitogen. Excessive mitochondrial generation of H2 O2 can overwhelm the antioxidant defenses of the cytosol and cause DNA damage, which can lead to mutation and activation of protooncogenes [41]. Increased ROS production is therefore a possible mitochondrial mutator phenotype, leading to further DNA damage and genetic instability [42]. Uncoupling proteins (UCPs) as negative regulators of mitochondrial oxidant productions are expected to decrease ROS productions [43,44]. Kamga et al. [45] showed the effects of UCP4 on ROS homeostasis and on mitochondrial antioxidants by contrasting the effects of UCP4-silencing and overexpression. Their findings suggest that UCP4 may regulate ROS levels by modifying mitochondrial antioxidants, especially those that are important in reducing H2 O2 and preventing the formation of hydroxyl radicals. Consistent with the few previous studies, we found a positive immunoreaction of

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Table 4 Logistic regression results for UCP 4. Factors

Coefficients (B)

St. Error

Wald statistic

Degrees of freedom (df)

p-Value

EXP(B)

95% confidence interval for EXP(B) Lower

p53 Ploidy Constant

2.353 −1.450 0.127

0.495 0.492 0.400

22.620 8.674 0.101

UCP4 in the majority of our breast cancer cell cases studied (90 of 124 cases (72.6%)), as well as a statistically significant correlation between UCP4 and prognostic indicators, such as increased grade, presence of more than four positive lymph nodes, ER and PR-positive receptors, DNA ploidy, proliferative markers, and P53positive nuclear expression. Data on higher apoptotic activity in poorly differentiated tumors have been revealed, whereas in well-differentiated neoplasms, this activity is relatively low. The prognostic value of P53 and bcl-2 expression regarding treatment outcome in breast cancer patients has been extensively evaluated, but the results are inconclusive, although bcl-2, as an antiapoptotic protein, is commonly – but not always – associated with improved prognosis in breast cancer [46–48]. Dysregulation of a normal cell death mechanism plays an important role in the pathogenesis and progression of breast cancer, as well as in responses of tumors to therapeutic intervention. Sastre-Serra et al. [49] detected the presence of UCP1 and its homologues UCP2,3,4,5 by real time PCR and Western Blotting in breast cancer cell lines and found that all isoforms of uncoupling proteins were strongly expressed in ER-positive breast cancer cells compared to negative ones, while estrogen treatment decreased uncoupling protein. Based on these results, they suggest that estrogens, through an ER-dependent mechanism, may downregulate uncoupling proteins and increase oxidative stress in breast cancer, in contrast to other studies that have found that estrogen-induced increase in mitochondrial coupling is a reasonable mechanism to enhance ATP synthesis needed for cell proliferation and a harmful strategy leading to oxidative stress. Nevertheless, it is difficult to reconcile the pro-oxidant estrogen role. In our series, after immunocytochemical detection in breast cancer cells, we found stronger positive expression of UCP4 (83.3%) in the ER-positive and PR-positive (87.1%) cancer cells, compared with ER and PR negative expression, as expected. These results are in agreement with those described in a few previous reports [47–49]. Interestingly, our results of bcl-2 expression in association with overexpression of UCP4 and ER-positive breast cancer cells were inconclusive, as we failed to find any reasonable result of the study of these parameters. Although the results of these observations are rather unclear, it is conceivable that deregulation of bcl-2 may result in deregulation of apoptosis of breast cancer cells and in worsening prognosis. Overexpression of UCPs protects cells from oxidative stress while its absence may cause production of ROS. Derdák et al. [50] induced colon tumors with azoxymethane (AOM) in mice deficient for uncoupling protein-2 (UCP2). In their study, they showed that UCP2−/− mice develop more aberrant crypt foci and colon tumors than UCP2+/+, in association with changes indicative of increased ROS production, disrupted balance between intestinal epithelial cell proliferation and apoptosis, and increased expression of bcl-2. Findings regarding bcl-2 expression as an anti-apoptotic marker in breast tumor cell lines have been controversial. To achieve in this study a maximal effect of bcl-2 and UCP4 expression, at the immunocytological level, we correlated these two proteins and a panel of other parameters, as has been already mentioned. As expected, in high grade tumor cells (grade III) and in tumor cells

1 1 1

<0.0001 0.003 0.751

10.519 0.235 1.136

3.989 0.089

Upper 27.742 0.616

with a high proliferative rate (Ki-67-positive expression 53%) and strongly positive nuclear P53 expression (71%), we found a strong cytoplasmic expression for UCP4 protein and a statistically significant downregulation of bcl-2 expression. Furthermore, in our study, we found an association between aneuploid tumors and positive expression of UCP4, but in these cases bcl-2 expression is more probable to be negative. Although UCPs are involved in several important processes of the mammary gland biology, the role of UCPs and especially that of UCP4 in breast cancer has not yet been explored [51,52]. In agreement with the role of ROS and UCPs in apoptosis signaling, inhibition of apoptosis by anti-apoptotic bcl-2 is associated with a protection against ROS and/or shift of the cellular redox potential to a more reduced state. In addition, the fact that active forms of cell death in yeast and plants also involve ROS suggests the existence of an ancestral redox-sensitive death signaling pathway that has been independent of caspases and bcl-2 [53]. On the whole, the results of our study indicate a strong correlation between the immunocytochemical expression of UCP4 of breast cancer tumors and several clinicopathological parameters. Although we failed to find any statistically significant association between the apoptotic marker bcl-2 and the UCP4 expression in the ER-positive cases, our findings encourage future investigations regarding the potential role of uncoupling proteins not only into the mechanisms underlying breast cancer, but also as a novel candidate for the design and development of more effective therapeutic strategies. Despite a breathtaking rate of progress, many questions regarding the mitochondria-mediated cell death pathways remain to be answered. One of the most elusive is the biochemical mechanism for the release of the apoptotic proteins from the mitochondria and the relationship between the release of apoptogenic proteins and the loss of mitochondrial functions, such as uncoupling and defects in ATP/ADP exchange” [2].

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