Tumor Biol. DOI 10.1007/s13277-013-1588-z

RESEARCH ARTICLE

Expression levels of serine/glycine metabolism-related proteins in triple negative breast cancer tissues Songmi Noh & Do Hee Kim & Woo Hee Jung & Ja Seung Koo

Received: 24 September 2013 / Accepted: 19 December 2013 # International Society of Oncology and BioMarkers (ISOBM) 2014

Abstract To evaluate the expression levels of serine/glycine metabolism-related proteins (PHGDH, PSAT, PSPH, SHMT, and GLDC) in six different subtypes of triple negative breast cancer (TNBC) patients and gain insight into their implications. Formalin-fixed, paraffin-embedded tissues from 129 TNBC patients were assembled into tissue microarrays. Immunohistochemical staining was performed for serine/glycine metabolism-related proteins (PHGDH, PSAT, PSPH, SHMT, and GLDC) and surrogate immunohistochemical markers (CK5/6, EGFR, claudin 3, claudin 4, claudin 7, E-cadherin, STAT1, interleukin-8 [IL-8], AR, and GGT-1) for identifying the molecular subtype of TNBC. TNBC subtype classifications included the following: basal-like (CK5/6-positive and/ or EGFR-positive), molecular apocrine (AR-positive and/or GGT-1-positive), claudin-low (claudin 3-, claudin 4-, claudin 7-negative and/or E-cadherin-negative), immune-related (IL8-negative and stromal STAT1-positive), mixed (features from two or more of the four subtypes), and null (no features from any of the four subtypes). Tissues from basal marker-positive patients showed increased expression levels of tumoral PHGDH compared with those from basal marker-negative patients (p=0.029); lack of stromal SHMT1 expression was significantly correlated with T stage (p=0.016). Multivariate Cox analysis revealed that a lack of stromal SHMT1

Electronic supplementary material The online version of this article (doi:10.1007/s13277-013-1588-z) contains supplementary material, which is available to authorized users. S. Noh Department of Pathology, CHA Gangnam Medical Center, CHA University, Seoul, South Korea S. Noh : D. H. Kim : W. H. Jung : J. S. Koo (*) Department of Pathology, Yonsei University College of Medicine, Severance Hospital, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752, South Korea e-mail: [email protected]

expression was an independent prognostic factor for predicting a shorter disease-free survival period (hazard ratio 4.002, 95 % confidence interval [CI] 1.077–14.83, p=0.038); furthermore, a lack of tumoral PHGDH expression was predictive of a shorter overall survival rate (hazard ratio 3.053, 95 % CI 1.002–9.305, p=0.050). In conclusion, the most abundantly expressed serine/glycine metabolism-related protein in basal-like TNBC tissues was tumoral PHGDH, and expression levels of stromal SHMT1 and tumoral PHGDH were inversely correlated with clinical prognostic factors. Also, this study is the first to assess serine/glycine relationships at the protein level in regards to clinical outcomes. Key word Glycine . Serine . Metabolism . Triple negative

Introduction The "Warburg effect" describes the phenomenon by which cancer cells of nearly all types preferentially carry out aerobic glycolysis at a high rate, even when oxygen is available, rather than a comparatively low rate of glycolysis followed by the tricarboxylic acid (TCA) cycle and mitochondrial oxidative phosphorylation, as occurs in most normal cells [1]. Since glycolytic rates are higher in cancer cells than in their normal tissues of origin, glycolytic intermediates are increased in tumor cells. Recent reports have suggested a connection between the metabolism of glycolytic intermediates and tumorigenesis, and the serine/glycine metabolic pathway is one of the major pathways involved in this link [2–5]. The serine/glycine metabolic pathway makes several contributions to glycolysis (Fig. 1). First, phosphoglycerate dehydrogenase (PHGDH) oxidizes the glycolytic intermediate 3phosphoglycerate (3PG) into 3-phosphohydroxypyruvate (pPYR); second, phosphoserine transaminase (PSAT) transaminates pPYR into phosphoserine (pSER). Finally,

Tumor Biol.

Fig. 1 Relationships between glycolysis, serine metabolism, and glycine metabolism

phosphoserine phosphatase (PSPH) dephosphorylates pSER, thereby generating serine. In glycine metabolism, glycine is transformed into methylene-tetrahydrofolate by glycine decarboxylase (GLDC). Serine hydroxymethyltransferase (SHMT) catalyzes conversion of serine to glycine in a reversible manner and therefore connects serine metabolism with glycine metabolism. Recent studies have revealed increased expression of PHGDH in breast cancer and melanoma patients [3, 4] and GLDC overexpression in lung cancer cells [5]. Furthermore, serine/glycine metabolism-related proteins are believed to be associated with tumorigenesis. Triple negative breast cancer (TNBC), characterized by a lack of expression of the estrogen receptor (ER), the progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER-2) [6], is associated with poor prognosis because of the current lack of effective therapies and its associated heterogeneous tumors. TNBC has been classified into basal-like, molecular apocrine, immune-related, and claudin-low subtypes according to gene profiling studies [7]. Because these molecular subtypes exhibit different histological and clinical features, responses to therapies, and prognoses, they have also been proposed to possess distinct metabolic features. In support of this hypothesis, increased expression levels of the glycolysis-related proteins GLUT-1, CAIX and MCT4 have been reported in basal-like TNBC cells [8, 9]. These findings suggest that increased glycolytic activity, especially serine/glycine metabolism of glycolytic intermediates, is associated with basal-like TNBC. In this study, we investigated whether there were any relationships between the expression levels of serine/glycine metabolism-related

proteins (PHGDH, PSAT, PSPH, SHMT, and GLDC) and the prognoses of different molecular subtypes of TNBC. This question is important to answer, because few studies have investigated these relationships.

Materials and methods Patient selection A total of 129 patients who were diagnosed with TNBC and had never undergone either preoperative chemotherapy or hormone therapy were enrolled in this study. Enrollment occurred at Yonsei University College of Medicine in Seoul, South Korea, between 2000 and 2005. This study was approved by the Institutional Review Board of Yonsei University Severance Hospital. Here, TNBC is defined as any breast cancer lacking expression of ER, PR, and HER-2 as assessed by immunohistochemical (IHC) staining; furthermore, HER-2 expression must not be detected by fluorescent in situ hybridization (FISH). A cut-off value of 1 % or more positively stained nuclei was used to define tissues as positive for ER and PR [10]. HER2 staining was analyzed according to the American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) guidelines using the following categories: 0 = no immunostaining; 1+ = weak incomplete membranous staining, less than 10 % of tumor cells; 2+ = complete membranous staining, either uniform or weak in at least 10 % of tumor cells; and 3+ = uniform intense membranous staining in at least 30 % of tumor cells [11]. HER-2 immunostaining was considered

Tumor Biol.

positive when strong (3+) membranous staining was observed, whereas cases with 0 to 1+ were regarded as negative. Cases showing 2+ HER-2 expression were evaluated for HER-2 detection by FISH. All samples were independently reviewed by a breast pathologist (J.S. Koo). Histology diagnosis was confirmed with hemotoxylin and eosin (H&E)-stained slides, and histological grades were assessed using the Nottingham Grading System [12]. Clinicopathologic parameters evaluated in each breast cancer patient included patient’s age at initial diagnosis, lymph node metastasis, tumor recurrence, distant metastasis, and survival. Tissue microarrays On H&E-stained tumor slides, representative areas were selected and corresponding spots were marked on the surfaces of the paraffin blocks. Using biopsy needles, selected areas were punched out and 3-mm tissue cores were placed into 6×5 arrays of recipient blocks. Tissues of invasive tumors were extracted. More than two tissue cores were extracted for each patient to minimize extraction bias. For IHC staining, 5-μmthick sections from each tissue microarray block were prepared. Each tissue core was assigned to a unique tissue microarray location number linked to a database containing other clinicopathologic data. Table 1 Clones, dilutions, and sources of antibodies used

EGFR epidermal growth factor receptor, GGT gammaglutamyltransferase, STAT signal transducer and activator of transcription, IL interleukin, PHGDH phosphoglycerate dehydrogenase, PSAT phosphoserine transaminase, PSPH phosphoserine phosphatase, SHMT serine hydroxymethyltransferase, GLDC glycine decarboxylase

Antibody

Immunohistochemistry IHC staining was performed on formalin-fixed, paraffinembedded tissue with microarray preparation using antibodies as described in Table 1. Briefly, 5-μm-thick sections were obtained with a microtome, transferred onto adhesive slides, and dried at 62 °C for 30 min. After incubation with primary antibodies, immunodetection was performed with biotinylated anti-mouse immunoglobulin antibodies, followed by incubation with peroxidase-labeled streptavidin using a labeled streptavidin–biotin kit with 3,3′-diaminobenzidine chromogen as a substrate. The primary antibody incubation step was omitted in the negative control sample. Positive control tissue was used according to the manufacturer’s recommendations. Slides were counterstained with Harris Hematoxylin. Interpretation of immunohistochemical staining All IHC markers, including GLDC, PSAT, PSPH, PHGDH, and SHMT, were assessed by light microscopy. A three-step categorization process was performed to interpret IHC results for each marker. First, the proportion of stained cells was graded on a scale of 0 to 2 (0 = negative, 1 =

glycine metabolism-related proteins in triple negative breast cancer tissues.

To evaluate the expression levels of serine/glycine metabolism-related proteins (PHGDH, PSAT, PSPH, SHMT, and GLDC) in six different subtypes of tripl...
492KB Sizes 0 Downloads 0 Views