biomedicines Article Increased Expressions of Matrix Metalloproteinases (MMPs) in Prostate Cancer Tissues of Men with Type 2 Diabetes Andras Franko 1,2,3 , Lucia Berti 2,3 , Jörg Hennenlotter 4 , Steffen Rausch 4, Marcus O. Scharpf 5, Martin Hrabe˘ de Angelis 3,6, Arnulf Stenzl 4 , Andreas Peter 2,3,7, Andreas L. Birkenfeld 1,2,3, Stefan Z. Lutz 1,8, Hans-Ulrich Häring 1,2,3 and Martin Heni 1,2,3,7,* 1 Department of Internal Medicine IV, Division of Diabetology, Endocrinology and Nephrology, University Hospital Tübingen, 72076 Tübingen, Germany; [email protected] (A.F.); [email protected] (A.L.B.); [email protected] (H.-U.H.) 2 Institute for Diabetes Research and Metabolic Diseases of the Helmholtz Centre Munich at the University of Tübingen, 72076 Tübingen, Germany; [email protected] 3 German Center for Diabetes Research (DZD), 85764 Neuherberg, Germany 4 Department of Urology, University Hospital Tübingen, 72076 Tübingen, Germany; [email protected] (J.H.); steff[email protected] (S.R.); [email protected] (A.S.) 5 Institute of Pathology, University Hospital Tübingen, 72076 Tübingen, Germany; [email protected] 6 Institute of Experimental Genetics, Helmholtz Zentrum München, German Research Center for Environmental Health, 85764 Neuherberg, Germany; [email protected] 7 Institute for Clinical Chemistry and Pathobiochemistry, Department for Diagnostic Laboratory Medicine, University Hospital Tübingen, 72076 Tübingen, Germany; [email protected] 8 Clinic for Geriatric and Orthopedic Rehabilitation Bad Sebastiansweiler, 72116 Mössingen, Germany; [email protected] * Correspondence: [email protected]; Tel.: +49-7071-29-82714 Received: 3 October 2020; Accepted: 12 November 2020; Published: 16 November 2020 Abstract: Type 2 diabetes (T2D) is associated with worse prognosis of prostate cancer (PCa). The molecular mechanisms behind this association are still not fully understood. The aim of this study was to identify key factors, which contribute to the more aggressive PCa phenotype in patients with concurrent T2D. Therefore, we investigated benign and PCa tissue of PCa patients with and without diabetes using real time qPCR. Compared to patients without diabetes, patients with T2D showed a decreased E-cadherin/N-cadherin (CDH1/CDH2) ratio in prostate tissue, indicating a switch of epithelial-mesenchymal transition (EMT), which is a pivotal process in carcinogenesis. In addition, the gene expression levels of matrix metalloproteinases (MMPs) and CC chemokine ligands (CCLs) were higher in prostate samples of T2D patients. Next, prostate adenocarcinoma PC3 cells were treated with increasing glucose concentrations to replicate hyperglycemia in vitro. In these cells, high glucose induced expressions of MMPs and CCLs, which showed significant positive associations with the proliferation marker proliferating cell nuclear antigen (PCNA). These results indicate that in prostate tissue of men with T2D, hyperglycemia may induce EMT, increase MMP and CCL gene expressions, which in turn activate invasion and inflammatory processes accelerating the progression of PCa. Keywords: prostate cancer; diabetes; epithelial-mesenchymal transition; matrix metalloproteinase; CC chemokine ligand Biomedicines 2020, 8, 507; doi:10.3390/biomedicines8110507 www.mdpi.com/journal/biomedicines Biomedicines 2020, 8, 507 2 of 13 1. Introduction The association between prostate cancer (PCa) and type 2 diabetes (T2D) is complex [1]. Many studies suggest that patients with T2D may have a reduced risk for developing PCa [2,3], however the underlying molecular mechanisms are not fully understood. One possible explanation is proposed by the reduced PCa detection in patients with diabetes due to lower prostate-specific antigen (PSA) levels compared to patients without diabetes [4]. Nevertheless, when patients with T2D are diagnosed with PCa, they are characterized by activated carcinogenic pathways [5] and suffer from more aggressive PCa [6,7] with a markedly poorer prognosis compared to patients without T2D [8,9]. Both high insulin and glucose levels are postulated to drive PCa carcinogenesis in patients with T2D [10], however the exact underlying molecular mechanisms for the accelerated PCa progression in case of coexistent T2D is not determined yet. In this study, we aimed to identify key pathways and their regulators including epithelial-mesenchymal transition (EMT), inflammation, and invasion, which could accelerate PCa progression in patients with T2D. One of the crucial carcinogenic processes in PCa is EMT, which is defined as a switch from epithelial characteristics to mesenchymal features [11]. EMT is characterized by decreased expression of epithelial E-cadherin (encoded by the CDH1 gene) and increased level of mesenchymal N-cadherin (encoded by the CDH2 gene), which consequently leads to a reduced CDH1/CDH2 ratio. Several studies showed that EMT promotes metastasis in PCa [12]. In PCa, CC chemokine ligands (CCLs) are central regulators for inflammatory pathways [13,14]. The chemokine CCL2 was described to induce proliferation and migration of PCa cells [15]. Angiogenesis and invasion are two major carcinogenic processes, which are both controlled by matrix metalloproteinases (MMPs) [16,17]. Increased production of MMP7 and MMP9 was associated with malignant progression of PCa [18]. To study the effect of diabetes on the progression of PCa, we examined benign prostate (BEN) and PCa tissues of PCa patients with and without T2D. Furthermore, to replicate hyperglycemia in vitro, PC3 prostate adenocarcinoma cells were treated with increasing glucose concentrations. The expressions of candidate genes involved in EMT (CDH1 and CDH2), inflammatory pathways (CCL2 and CCL5) and invasion (MMP7, MMP9 and MMP14), were measured using real time qPCR both in human prostate tissues and PC3 cells. 2. Experimental Section 2.1. Human Samples Newly diagnosed PCa patients without prior anti-cancer therapy were recruited prior to radical prostatectomy. Tissue sampling was performed by an experienced uropathologist (M.S.) and all prostate cancers were classified as adenocarcinoma. PCa as well as benign tissues were immediately snap-frozen in liquid nitrogen and stored at 80 C. For histological confirmation, hematoxylin and − ◦ eosin staining was performed on paraffinized samples. Patient cohorts were age- and BMI-matched and n = 17–17 benign and n = 11–11 tumor tissue samples from patients with and without type 2 diabetes were included (Figure1), as recently described [ 5]. Most of the patients with T2D received anti-diabetic medication (Table1). To analyze PCa at similar tumor stages, all PCa samples had a Gleason score of 7a and 7b. Informed written consent was obtained from all participants, and the Ethics Committee of the University of Tübingen (575/2018BO2) approved the protocol in accordance with the Declaration of Helsinki. Total RNA was isolated using Allprep RNA/DNA/protein kit (Qiagen, Hilden, Germany) in accordance with the manufacturer’s description. Biomedicines 2020, 8, 507 3 of 13 Biomedicines 2020, 8, x FOR PEER REVIEW 3 of 13 Figure 1. Benign prostate (BEN) (n = 17–17) and prostate cancer (PCa) (n = 11–11) tissues of PCa patientsFigure 1. with Benign (T2D) prostate and patients (BEN) without (n = 17–17) (noT2D) and typeprosta 2 diabeteste cancer were (PCa) studied. (n = 11–11) Age ( Atissues) and BMIof PCa (B) datapatients are shownwith (T2D) as Tukey and patients box plots. without Statistical (noT2D) significance type 2 diabetes was calculated were studied. using Mann–WhitneyAge (A) and BMI tests (B) anddata consideredare shown asas pTukey< 0.05. box No plots. statistical Statistical significant significance differences was calculated were found. using Mann–Whitney tests and considered as p < 0.05. No statistical significant differences were found. Table 1. Most patients with T2D were treated with antidiabetic medications. Numbers (nr) denote the numberTable 1. ofMost patients, patients who with received T2D thewere respective treated with treatment. antidiabetic medications. Numbers (nr) denote the number of patients,T2D who received the respectiveT2D Patients treatment. with T2D Patients with Therapy T2D T2DBEN Patients Samples with T2D PatientsPCa with Samples InsulinTherapy BEN Samples 2 PCa Samples 1 MetforminInsulin 102 1 6 RepaglinidMetformin 10 2 6 1 GlimepirideRepaglinid 2 0 1 1 Acerbose 1 0 Glimepiride 0 1 Sitagliptin 0 1 Acerbose 1 0 Diet modification only 6 4 Total nrSitagliptin 170 1 11 Diet modification only 6 4 Total nr 17 11 2.2. Cell Culture 2.2. CellThe Culture human prostate adenocarcinoma cell line PC3 was purchased from CLS-Cell line services GmbHThe (Eppelheim, human prostate Germany). adenocarcinoma Cells were maintained cell line PC in3 lowwas glucose purchased (5.5 mM)from DMEMCLS-Cell (Gibco line /Thermoservices FisherGmbH Scientific,(Eppelheim, Karlsruhe, Germany). Germany) Cells supplemented were maintained with 5%in fetallow bovineglucose serum (5.5 (FBS) mM) (Bio&Sell, DMEM Feucht,(Gibco/Thermo Germany) Fisher [19 ].Scientific, One day Karlsruhe, before treatment, Germany) medium supplemented was changed with 5% to fetal DMEM bovine with serum 0.2% FBS.(FBS) Following (Bio&Sell, theFeucht, addition Germany) of medium [19]. One containing day before 5.5, treatment, 11.25 or 17.5medium mM was glucose, changed the cellsto DMEM were incubatedwith 0.2% FBS. for