Correlation between VEGFR-2 receptor kinase domain-containing receptor (KDR) mRNA and angiotensin II receptor type 1 (AT1-R) mRNA in endometrial cancer

Cytokine 61 (2013) 639–644

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Correlation between VEGFR-2 receptor kinase domain-containing receptor (KDR) mRNA and angiotensin II receptor type 1 (AT1-R) mRNA in endometrial cancer _ Agnieszka W. Piastowska-Ciesielska a,⇑, Elzbieta Płuciennik b, Katarzyna Wójcik-Krowiranda c, c b Andrzej Bien´kiewicz , Magdalena Nowakowska , Karolina Pospiech b, Andrzej K. Bednarek b, Kamila Domin´ska a, Tomasz Oche˛dalski a a

Department of Comparative Endocrinology, Medical University of Lodz, Poland Department of Molecular Carcinogenesis, Medical University of Lodz, Poland c Clinical Division of Gynecological Oncology, Medical University of Lodz, Poland b

a r t i c l e

i n f o

Article history: Received 21 September 2012 Received in revised form 19 November 2012 Accepted 22 November 2012 Available online 28 December 2012 Keywords: Endometrial adenocarcinoma VEGFR-2/kinase-insert-domain containing receptor Angiotensin receptor type 1

a b s t r a c t Purpose: Angiogenesis, a multistep process that results in new blood vessel formation from preexisting vasculature is essential for both the growth of solid tumour and for metastasis. Stimulation of vascular endothelial growth factor receptor (VEGFR), a transmembrane glycoprotein, results in mitogenesis. Within this family of receptors, VEGFR 2/kinase-insert-domain containing receptor appears to be principally upregulated during tumorigenesis. The aim of this study was to determine the expression of VEGFR-2/kinase-insert-domain containing receptor (KDR) and its correlation with angiotensin receptor type 1 (AT1-R) and clinical factors in endometrial carcinoma. Methods: The expression of KDR and AT1-R was studied in endometrial carcinoma and normal endometrium by Real-time RT-PCR and Western blot analysis in 136 samples. The expression profile was correlated with the clinicopathological characteristics of endometrial adenocarcinoma. Results: We noted a significant correlation between the expression of KDR and AT1-R in tumour grade G1, G2 and G3 (Rs = 0.50; p = 0.002, Rs = 0.69; p = 0.0001, Rs = 0.52; p = 0.005, respectively). In stage I and stage II carcinoma, a significant correlation was also found between the expression of KDR and AT1-R (Rs = 0.70, p = 0.0001, Rs = 0.67; p = 0.001, respectively). Moreover significant correlation was observed between both KDR and AT1-R in tissue with different myometrial invasion (Rs = 0.54, p = 0.0001, Rs = 0.68; p = 0.0001; respectively for tumours with invasion into the inner half and invasion into the outer half). Conclusions: Basing on received correlation between AT1-R and KDR expression and previous results we speculate that angiotensin through AT1-R modulates KDR expression and thus have influence on local VEGF level. However, further studies are required to clarify the biological interaction between KDR, AT1-R and other hormonal regulators in endometrial carcinoma. Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction Endometrial carcinoma is a common gynaecological malignancy, although the mechanism of progression remains unclear. Development of endometrial cancer is caused by multimodal factors. Genetic mutations, hormonal and apoptotic dysregulations as well as additional factors play an important role in this tumour formation [1]. Endometrial carcinoma is the most frequent

⇑ Corresponding author. Address: Department of Comparative Endocrinology, Faculty of Biomedical Sciences and Postgraduate Training, Medical University of Lodz, Zeligowskiego 7/9, Lodz 90-752, Poland. Tel./fax: +48 42 677 93 18. E-mail address: [email protected] (A.W. Piastowska-Ciesielska). 1043-4666/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.cyto.2012.11.017

gynaecological cancer worldwide. In Poland, endometrial carcinoma takes second place after breast cancer in women and is considered the most common genital cancer [2]. Angiogenesis is one of the main physiological processes responsible for neovascularisation, growth, and metastasis of many different tumours [3–5]. The course of this process is regulated by a dynamic balance between angiogenic stimuli and angiogenic inhibitors that are produced in the target tissues and at distant sites [4]. Vascular endothelial growth factor (VEGF) is the predominant stimulator of angiogenesis with a specific mitogenic action on cells and aggressive tumour behaviour. It acts on specific tyrosine kinase receptors, VEGFR-1/Flt-1, VEGFR-2/Flt-1/KDR (KDR) and VEGFR-3/ Flt-4 [6,7]. However, KDR is the major VEGF receptor in human malignancies [8]. Binding of VEGF to KDR on cells results in receptor

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phosphorylation and triggering a cascade of events leading to endothelial cell proliferation, migration, apoptosis inhibition and maturation of vascular structures [9]. Angiotensin II (AngII) is a peptide with a direct, potent vasopressive effect on the peripheral vasculature, and plays a pivotal role in electrolyte and circulatory homeostasis. In addition, AngII may act as a mitotic factor by inducing or regulating gene expression in cell cycle progression [1,10,11]. The AngII type 1 receptor (AT1-R) is also frequently expressed in various human tumours, and AT1-R blockade effectively reduced tumour progression and metastasis in vivo [2]. AT1-R is known to induce angiogenesis, cellular proliferation, and inflammatory responses, in addition to promoting antiapoptotic activity, and several experiments have provided evidence that AGTR1 blockers, such as losartan, can function as anticancer treatments [12–16]. In the presented study, we revealed a possible correlation between the expression of KDR and AT1-R and clinicopathological characteristics of primary endometrial adenocarcinoma. We determined the above mentioned molecular factors both at the mRNA (real-time RT-PCR) and protein levels (Immunobloting). 2. Materials and methods 2.1. Patients and endometrial tissue specimens One hundred thirty six of endometrial carcinoma surgically treated from 2008 to 2011 at the Clinical Division of Gynecological Oncology, Medical University of Lodz, was included in this study. Twenty eight control samples were obtained from patients operated due to gynaecological diseases other than of endometrial origin. Clinicopathological information was obtained from patient charts. None of the patients had received radiotherapy or chemotherapy prior to surgery. The clinical stage of the disease was defined according to the FIGO criteria. Histological grade was based on the degree of glandular differentiation, and tumours were graded as: G1 (percentage of solid growth in the tumour mass up to 5%); G2 (percentage of solid growth between 6% and 50%); G3 (percentage of solid growth above 50%). The depth of myometrial invasion was defined as the percentage of the myometrium invaded by the tumour. The expression of KDR and AT1-R was analysed in 136 cases of endometrial carcinoma. Patient ages ranged from 31 to 83 years, with a mean age of 62 years. In the control group of patients (28 cases) age ranged from 33 to 68 with a mean age of 51 years. There were 40 cases classified as G1 (patient age range 36–85; average 61 years), 73 cases of G2 (patient age range 31–83; average 62 years), 23 cases classified as G3 (patient age range 36–79; average 64 years). The FIGO classifications was as follows, 82 samples were identified as I (patient age range 35–83; average 62 years), 19 as II (patient age range 43–83; average 62 years), 22 as III/IV (patient age range 51–79; average 66 years), in 13 cases FIGO classifications was not denoted. In 60 cases invasion of the myometrium exceeded upper half (patient age range 35–83; average 63 years) in 67 was under upper half (patient age range 31–83; average 60 years). In 26 cases lymph node metastasis were detected (patient age range 36–83; average 65 years). Distributions of histological grade and stage are listed in Table 1. Samples of specimens were stained with Hematoxylin and Eosin (H&E) for light microscopic study and evaluated to confirm the tumour stage and histological type (Fig. 1). The study was approved by the Ethics Committee at Medical University of Lodz. Experiments involving human subjects were conducted according to the Declaration of Helsinki. 2.2. RNA extraction, cDNA synthesis and Real-time RT-PCR analysis Tumour samples were stored at 80 °C in RNAlater (Life Technology, Corporation), and RNA was isolated using the TRIzol

Table 1 Clinicopathologic characteristics of patients. Parameter

(%)

Grade 1 2 3

29.4 53.7 16.9

FIGO I II III/ IV n/a

60.3 14.0 16.1 9.6

Myometrial invasion Inner half Outer half n/a Lymph node metastasis Negative Positive

49.3 44.1 6.6 80.9 19.1

reagent (Life Technology, Corporation). cDNA synthesis was performed using 10 lg of total RNA at a total volume of 100 ll with ImProm RT-II reverse transcriptase (Promega, Poland) as described previously [2]. Reverse transcriptions were performed under the following conditions: 5 min incubation at 25 °C and 60 min at 42 °C, and heating at 70 °C for 15 min. The synthesised cDNA was diluted with sterile deionised water to 150 ll, and 2 ll of cDNA were used in the PCR reaction. Real-time RT-PCR was performed with a Light Cycler 480 II (Roche, Poland) as described previously [2]. We analysed the relative expression of genes (KDR, AT1-R). Their expression level was normalised to the mean expression of three reference genes (RPS17, RPLPO, H3F3A) [17,18]. The detection temperature was set above the non-specific/primer-dimer melting temperature. The following primer pairs were used: KDR sense primer 50 -CACCACTCAAACGCTGACATGTA-30 and antisense primer 50 -AAGA GTGCGCCAACGAGC-30 ; AT1-R sense primer 50 -ATTCGACC CAGGTGATCAAA-30 and antisense primer 50 -ATTTGGAAACAGCTTGGTGG30 ; H3F3A sense primer 50 -AGG ACT TTA AAA GAT CTG CGC TTC CAG AG-30 ; and antisense primer 50 -ACC AGA TAG GCC TCA CTT GCC TCC TGC-30 ; RPLPO sense primer: 50 -ACG GAT TAC ACC TTC CCA CTT GCT AAA AGG TC-30 ; and antisense primer: 50 -AGC CAC AAA GGC AGA TGG ATC AGC CAA G-30 ; RPS17 sense primer: 50 AAG CGC GTG TGC GAG GAG ATC G-30 ; and antisense primer 50 TCG CTT CAT CAG ATG CGT GAC ATA ACC TG-30 . The Universal Human Reference RNA (composed of 10 cell lines) was used as a calibrator for each reaction (STratagene). 2.3. Immunoblotting Total protein from samples was extracted as described previously [2,19] Protein quantity was measured with the use of QubitÒ Protein Assay Kit (Life Technologies, Corporation) according to the manufacturer’s protocol. Equal amounts (30 lg) of samples lysates were separated using SDS–PAGE and transferred to a PVDF membrane (Sigma–Aldrich, Poland). The membrane was blocked in TBST (20 mM Tris–HCL, 500 mM NaCl, 0.05%, Tween-20, pH 7.5) containing 5% dry milk. The membrane was probed with the following antibodies: KDR (sc-101820, Santa Cruz Biotechnology Inc.,) and AT1-R (sc-1173, Santa Cruz Biotechnology Inc.). The probes were then labelled with Alkaline Phosphatase-conjugated anti-mouse or antirabbit antibody (Sigma–Aldrich, Poland). The colour reaction was induced using NovexÒ AP Chromogenic Substrate (BCIP/NBT) (Life Technology, Corporation), bands were visualised on membranes. A densitometric analysis of protein levels was performed with ImageJ

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Fig. 1. Haematoxylin and eosin (HE) stain of human endometrial carcinoma. (A) Section of grade 1 (G1), (A – magnification  200). (B) Section of grade 2 (G2), (B – magnification  200). (C) Section of grade 3 (G3), (C – magnification  200).

1.34s software (Wayne Rasband, National Institutes of Health, USA. http://rsb.info.nih.gov/ij/). The results were normalised for glyceraldehyde-3-phosphate dehydrogenase (GAPDH, sc-59540, Santa Cruz Biotechnology Inc.). Values of protein expression are given in arbitrary units (AU). 2.4. Statistical analysis Graph Pad Prism 5 was used for statistical analysis and statistical significance was accepted at 0.05. All data were expressed as means ± SEM. The unpaired two-tailed t-test was used to compare groups with continuous variable data. One way ANOVA non parametric analysis (Kruskal–Wallis test) was also performed to test difference among groups of continuous variables. The correlations between the expression levels of KDR, AT1-R and clinical factors were performed using a non-parametric Spearman Rank Correlation test. Roche algorithm was used to calculate the relative gene expression levels [20]. 3. Results 3.1. Expression of KDR KDR mRNA was detected within all endometrial samples (normal and tumour) using Real-time RT-PCR. Real-time RT-PCR results were confirmed in protein expression by immunoblotting. The lowest KDR mRNA expression was detected in tissues classified as G3 (40.85 ± 7.14) but this decrease was statistically insignificant in comparison with G1 and G2 (48.74 ± 8.81, 45.22 ± 3.13 respectively). A similar tendency was observed on the protein level of KDR G3 versus G1 (8.28 ± 5.3, 13.99 ± 1.4 p < 0.05) (Fig. 2). In the FIGO stages the highest KDR mRNA was detected in stage I (47.73 ± 3.27) and the lowest in stage II (28.43 ± 6.22). The protein analysis confirmed the mRNA level, and showed statistically significant differences between stage II and stage I (1.09 ± 0.60, 7.07 ± 1.0 p < 0.05) (Fig. 2). There was the tendency between KDR mRNA expression and diverse invasion of the myometrium i.e. it was higher in tumours with invasion into the outer of the myometrium than in tumours with invasion into the inner half (21.14 ± 3.69; 19.91 ± 2.96). KDR mRNA and protein level was also analysed in the normal endometrium (148.40 ± 3.66; 32.45 ± 3.66, respectively). 3.2 Expression of AT1-R Similarly to our previous results in case of AT1-R the lowest level of mRNA was noted in G3 (3.97 ± 0.63), and this difference was statistically significant in comparison with G1 (17.77 ± 2.35 p < 0.05). Moreover in this group of patients statistically significant difference was also observed between G1 and G2 (17.77 ± 2.35; 5.87 ± 1.19 p < 0.05). In protein analysis the statistically significant difference between G1/G2 and G1/G3 was confirmed (5.53 ± 1.18,

2.30 ± 0.43 and 5.53 ± 1.18, 0.57 ± 0.18 p < 0.05 respectively) (Fig. 3). In the FIGO stages lowest AT1-R mRNA was in stage III/ IV (4.53 ± 0.68) and significant differences were noted between stages I and II when compare to III/IV (9.76 ± 2.16; 9.73 ± 2.43 versus 4.53 ± 0.68 p < 0.05). This observation was also received in protein analysis: lowest AT1-R protein was in stage III (1.05 ± 0.10). Significant difference was noted between stage III/ IV compare to stages I and II (1.05 ± 0.10; 9.76 ± 2.16, 9.73 ± 2.43 p < 0.05) (Fig. 3). Similarly to KDR there was no statistically significant difference between AT1-R expression and tumours with diverse invasion of the myometrium, but AT1-R mRNA expression was higher in tumours with invasion into the inner half of the myometrium than in tumours with invasion into the outer half (10.13 ± 2.06; 8.84 ± 1.63). AT1-R mRNA was also expressed in the normal endometrium. 3.3. Expression of KDR in endometrial cancer tissues correlates with expression of AT1-R In G1, G2 and G3, a significant correlation was noted between the levels of KDR and AT1-R (Rs = 0.50; p = 0.002, Rs = 0.69; p = 0.0001, Rs = 0.52; p = 0.005, respectively). We also found a significant correlation between the expression levels of KDR and AT1-R in stage I and II (Rs = 0.70, p = 0.0001, Rs = 0.67; p = 0.001, respectively). As of importance, significant correlation was observed between both KDR and AT1-R in tissue with different myometrial invasion (Rs = 0.54, p = 0.0001, Rs = 0.68; p = 0.0001; respectively for tumours with invasion into the inner half and invasion into the outer half). 4. Discussion Our previous study performed on endometrial cancer samples revealed a correlation between the expression of angiotensin receptors (AT1-R, AT2-R), ERa and VEGF on both mRNA and protein levels and the clinicopathological characteristics of this tumour type. Moreover our analysis also showed that in an early stage of differentiation of endometrial cancer there is a correlation between AT1-R and VEGF [2]. These results lead us to investigate the influence of AT1-R expression on VEGF downstream pathway, i.e. the expression of KDR and its correlation with clinical factors. As a result, we observed that the relative expression of the investigated genes correlated with tumour differentiation and the clinical stage of the carcinoma. VEGF is an important angiogenic factor in gynecological malignancies. Beyond its stimulatory activity on endothelial cell proliferation and vascular maturation, growing evidence suggests that VEGF produced by cancer cells has also an autocrine role by stimulating cancer cell growth and inhibiting apoptosis through binding to its own VEGF membrane receptors [6]. Three high-affinity cognate endothelial receptors for VEGF have been identified: VEGFR-1/Flt-1, VEGFR-2/Flk-1/KDR, and VEGFR-3/

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Fig. 2. Expression analysis of the VEGFR-2/kinase-insert-domain containing receptor (KDR) in endometrial carcinoma. The relative expression of KDR was normalised to mean expression of reference genes. The relative protein level of KDR was normalised to GAPDH. (A) Relative expression of total KDR mRNA in different grades (grade 1 – G1, grade 2 – G2, grade 3 – G3). (B) Data presents mean level of KDR protein in different grades (grade 1 – G1, grade 2 – G2, grade 3 – G3). Asterisk denotes significance difference between protein levels of G1 and G2. (C) Relative expression of total KDR mRNA in different stages (stage I – SI, stage II – SII, stage III/IV – SIII/IV). (D) Mean level of KDR protein in different stages (stage I – SI, stage II – SII, stage III/IV – SIII/IV). Asterisk denotes significance difference between proteins levels of S I and S II. B0 – D’. Representative immunoblots with antibodies against human KDR presented immunopositive bands.

Flt-4 [21]. VEGFR-1/Flt-1 and VEGFR-2/Flk-1/KDR (KDR) are cell surface receptor tyrosine kinases (RTKs) [21]. KDR is exclusively expressed in endothelial cells and appears to play a pivotal role in endothelial cell differentiation and vasculogenesis, moreover various studies have provided evidence for the role of KDR in tumor vascularisation, growth, and metastasis [7,21]. Moreover, An et al. in their reverse transcriptase and real-time PCR analysis of tissue samples obtained from the patients with lung cancer, and normal lung specimens, showed that the KDR mRNA expression and protein level were significantly higher in normal tissue [22]. We have also observed higher KDR mRNA expression in normal tissue (148.40 ± 3.66) then in endometrial adenocarcinoma (G1-32.84%; G2-30.22%; G3-27.85%). Moreover,

immunoblotting assay recognised the active form of the KDR in both type of samples (32.45 ± 3.66 normal; G1-43.11%; G231.18%; G3-25.53%). Higher KDR expression in normal endometrial tissue can be explained varied vascularisation in this type of tissue during menstrual cycle. The highest expression of VEGFR-2/Flt-1/KDR is noticed in the proliferative and the early or early-mid secretory phases [23]. Some authors suggested role of estradiol in upregulation this type of receptor through VEGF mechanism [24]. Angiotensin II (Ang II), which is a major effector peptide of the renin-angiotensin system (RAS), is well known to be an important factor in hypertension [25]. A local autocrine or paracrine RAS exists in a number of tissues [3,10,26,27]. It has been reported that

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Fig. 3. Expression analysis of the angiotensin receptor type 1 (AT1-R) in endometrial carcinoma. The relative expression of AT1-R was normalised to men expression of reference genes. AT1-R protein levels was analysed by western blotting. (A) Relative expression of total AT1-R mRNA in the different grades (grade 1 – G1, grade 2 – G2, grade 3 – G3). Asterisk denotes significance difference between mRNA levels of G1/G2 and G1/G3. (B) Data presents mean level of AT1-R protein in the different grades (grade 1 – G1, grade 2 – G2, grade 3 – G3). Asterisk denotes significance difference between mRNA levels of G1/G2 and G1/G3. (C) Relative expression of total AT1-R mRNA in the different stages (stage I – SI, stage II – SII, stage III/IV – SIII/IV). Asterisk denotes significance difference between mRNA levels of SI – SII and SI – SIII/IV. (D) Mean level of AT1-R mRNA in the different stages (stage I – SI, stage II – SII, stage III/IV – SIII/IV). Asterisk denotes significance difference between mRNA levels of SI – SII and SI – SIII/IV. B0 – D0 . Representative immunoblots with antibodies against human AT1-R presented immunopositive bands.

Ang II is involved in the development and invasion of some cancers, including breast, ovarian and pancreatic ones [28–30]. The existence of all RAS factors has been confirmed, and it is suggested that the most active RAS factors, Ang II and AT1-R, are actively involved in tumour biology in endometrial adenocarcinoma [1,3]. Imanishi et al. demonstrated that Ang II induced a significant increase of KDR mRNA in a dose- and time-dependent manner in human bone marrow-derived endothelial progenitor cells (EPCs). In addition, they showed that flow cytometric analysis revealed that Ang II up-regulated KDR protein expression in EPCs. This group postulated that both the up-regulation of KDR and the potentiation of VEGF mitogenic effects in Ang II-stimulated EPCs

could be abolished by the pretreatment of the AT1-specific receptor antagonist, suggesting that these effects occurred via the AT1 receptor [31]. This data are consistent with our observation that there is a significant correlation between AT-1R and KDR in the cells with change in the differentiation/proliferation balance. Similarly Akhavan et al. noted that angiotensin II significantly enhanced the expression of VEGF and this effect was abolished by the losartan in B16F10 melanoma cells. These authors postulated, that angiotensin II through activation of AT1-R can stimulate the expression of VEGF in melanoma cells [32]. As for endometrial cancer, Yokoyama et al. analysed correlation between the presence of VEGF-D and VEGFR-3 and

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clinicopathological factors and revealed that VEGFR-3 receptor, which is expressed in lymph vessels binds VEGF-C and VEGF-D [33]. These authors showed also that VEGF-D in carcinoma and stromal cells and VEGFR-3 in carcinoma and endothelial cells had no correlation with histological type, grading, ovarian metastasis, and age at surgery. However, they found significant correlation between increased levels of VEGF-D protein in carcinoma and stromal cells and VEGFR-3 protein in carcinoma cells with vessel space invasion and cervical invasion [33]. In the presented study we demonstrated that KDR and VEGF expression in endometrial cancer had no correlation with histological type and grading. However we revealed, that levels of KDR protein and mRNA in endometrial cancer significantly correlated with the AT1-R protein and mRNA level. We found positive correlation between KDR and AT1-R at various grades and stages of tumour differentiation.

[10]

[11]

[12]

[13]

[14]

[15]

5. Conclusions [16]

These results could together support the hypothesis indicating that the angiotensin through AT-1R modulates KDR expression and thus has influence on local VEGF level. Moreover, the obtained results confirmed a significant role of KDR and AT1-R in endometrial tumorigenesis and suggest an efficient paracrine loop of Angiotensin II acted via AT1-R on KDR and VEGF in endometrial cancer cells. However, further studies ought to be conducted.

[17]

[18]

[19]

Acknowledgements [20]

This work was supported by the Ministry of Science and Higher Education Grant N403293 536 and the Medical University of Lodz Grants 502-03/0-078-04/502-04-008 and 502-03/0-078-08/50204-008. We would like to thank Mr. Rafał Kacprzak and Mr. Daniel Kacprzak for their support.

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[23]

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