Deep Quantitative Proteomics Reveals Extensive Metabolic

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Deep quantitative proteomics reveals extensive metabolic reprogramming and cancer-like changes of ectopic endometriotic stromal cells Sergo Kasvandik, Külli Samuel, Maire Peters, Margus Eimre, Nadežda Peet, Anne Mari Roost, Lee Padrik, Kalju Paju, Lauri Peil, and Andres Salumets J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.5b00965 • Publication Date (Web): 14 Dec 2015 Downloaded from http://pubs.acs.org on December 15, 2015

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Journal of Proteome Research

Deep

quantitative

proteomics

reveals

extensive

metabolic

reprogramming and cancer-like changes of ectopic endometriotic stromal cells

Sergo Kasvandika,b,c, *, Külli Samuelb, Maire Petersb,c, *, Margus Eimred, Nadežda Peetd, Anne Mari Roostb,c, Lee Padrike, Kalju Pajud, Lauri Peila, Andres Salumetsb,c

a

Proteomics core facility, Institute of Technology, University of Tartu, Nooruse 1,

Tartu, Estonia b

Competence Centre on Health Technologies, Tiigi 61b, Tartu, Estonia

c

Tartu University Women’s Clinic, L. Puusepa 8, Tartu, Estonia

d

Chair of Pathological Physiology, Institute of Bio- and Translational Medicine,

University of Tartu, Ravila 19, Tartu, Estonia e

Tartu University Hospital, Women’s Clinic, L. Puusepa 8, Tartu, Estonia

*

Corresponding authors:

Sergo Kasvandik, (Phone: +372 737 4864. E-mail: [email protected]) or Maire Peters, (Phone: +372 731 8712. E-mail: [email protected])

Abstract Endometriosis is a prevalent health condition in women of reproductive age characterized by ectopic growth of endometrial-like tissue in the extrauterine 1

ACS Paragon Plus Environment


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environment. Thorough understanding of the molecular mechanisms underlying the disease is still incomplete. We dissected eutopic and ectopic endometrial primary stromal cell proteomes to a depth of nearly 6,900 proteins using quantitative massspectrometry with a spike-in SILAC standard. Acquired data revealed metabolic reprogramming of ectopic stromal cells with extensive upregulation of glycolysis and down-regulation of oxidative respiration, a wide-spread metabolic phenotype known as the Warburg effect and previously described in many cancers. These changes in metabolism are additionally accompanied by attenuated aerobic respiration of ectopic endometrial stromal cells as measured by live-cell oximetry and by altered mRNA levels of respective enzyme complexes. Our results additionally highlight other molecular changes of ectopic endometriotic stromal cells indicating reduced apoptotic potential, increased cellular invasiveness and adhesiveness, and altered immune function. Altogether, these comprehensive proteomics data refine the current understanding of endometriosis pathogenesis and present new avenues for therapies.

Keywords Endometrial stromal cells, endometriosis, endometrium, metabolism, peritoneal endometriosis, quantitative proteomics, SILAC, Warburg effect.

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Introduction Endometriosis is a common painful illness present in 10-15% of the female population of reproductive age and associated with chronic pelvic pain and infertility in over quarter of endometriosis patients. The disease is characterized by extrauterine growth of endometrial-like tissue, mostly in the peritoneal cavity, ovaries and other abdominal organs. Clinical diagnostics is complicated due to the need for laparoscopic surgery and lack of sufficiently specific blood biomarkers1. There is no specific cure available for endometriosis and the treatment mostly focuses on relieving of the symptoms. The molecular features of endometriosis are complex and still incompletely understood, although numerous proteins acting in multiple cellular processes have been speculated to be involved in the disease pathogenesis. These proteins mostly fall into the categories of regulating the cellular proliferation and apoptosis, adhesion and invasion, angiogenesis and immune functions2. The reciprocal epithelial to mesenchymal transitions have also been shown to be involved in the pathogenesis of pelvic endometriosis3, similar to those processes that determine invasive growth and the possibility of metastasis in cancer. Moreover, hypoxia and hypoxic injury have also been noted for ectopic endometriotic tissue and its molecular marker (HIF1A) has been reported to be elevated by several researchers4,5, suggesting that hypoxia accompanied metabolic alterations may additionally be present in endometriotic tissue. Endometriotic lesions have been proposed to originate from a single colony of progenitor endometrial stem cells reaching the ectopic sites via retrograde menstruation or less likely through lymphovascular dissemination, and subsequent 3



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transplantation and growth of the lesions by cellular migration and invasion 6. These molecular features are comparable to those of cancer cells, albeit not presenting the malignancy per se. Interestingly, endometriosis patients have been found to possess mildly elevated risk for various cancers, like ovarian and breast cancer, and melanoma, although, reduced risk for cervical cancer7. In general, these associations suggest that endometriosis patients may have derailments in certain cellular functions that govern eutopicity and proliferation and/or defective elimination of cells by the immune system. Nevertheless, the cellular safeguards that prevent malignant properties are still intact. To date, majority of endometriosis proteomic studies have utilized twodimensional

gel

electrophoresis

combined

with

matrix-assisted

laser

desorption/ionization (MALDI) MS to study different biofluids and endometrial tissue8. These studies have produced a list of differentially expressed proteins that appear to explain the various aspects of endometriotic cells, although consistency of findings for

the

eutopic

chromatography

endometrium tandem

has

not

been

mass-spectrometry

highly

(LC-MS/MS)

overlapping8. based

Liquid

quantitative

proteomics has only seldom been applied to endometriosis samples, which limits the attained proteome coverage of the previous studies. New developments in highresolution MS now enable deeper investigation of mammalian cells9 and can, therefore, expand the boundaries of the current knowledge on endometriosis. Endometrium and endometriotic lesions are composed of different types of cells with various phenotypes and functional properties. Endometrial stroma cells, which are the most prevalent population of cells in endometrium and in endometriotic lesions2, are very dynamic, growing and differentiating thorough the menstrual cycle 4


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and pregnancy. Furthermore, the resistance of endometriotic stromal cells to apoptosis is believed to support the growth and the spreading of the lesions in endometriosis10. In the current study we compared the proteomes of eutopic endometrial stromal cells (euESC) of endometriosis patients and fertile controls with the ectopic endometriotic stromal cells (ecESC) from lesions. For these purposes we used highthroughput, label-based quantitative mass spectrometry, which helped us to capture the shifts in molecular and cellular proteomes accompanying the development of endometriosis.

Experimental procedures Ethics statement and sample collection The study was approved by the Research Ethics Committee of the University of Tartu and informed written consent was obtained from the participants. Altogether, six (n=6) healthy controls and thirteen (n=13) endometriosis patients were enrolled for the study. Endometrial tissue samples and peritoneal endometriotic lesions were collected from endometriosis patients undergoing laparoscopy at the Tartu University Hospital Women’s Clinic. The diagnosis was histologically confirmed and disease severity was determined according to the American Society for Reproductive Medicine revised classification system11. The EM biopsies of controls were collected under local anaesthesia. The control group consisted of self-reported healthy and fertile women, having at least one child and recruited via public advertisement. A questionnaire was administered to all participants in order to obtain thorough 5



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information regarding the general and reproductive health characteristics, including menstrual cycle anamnesis, use of medications, presence of systemic diseases and other health conditions. All biopsy samples were collected using an EM suction catheter (Pipelle, Laboratoire CCD). All study participants were in reproductive age, had received no hormonal medication during previous three months before the recruitment and had regular menstrual cycle (28±5 days). Self-reported menstrual cycle day was used to estimate cycle phase and all biopsies were collected in the secretory phase. The demographic and clinical data of the study participants are summarized in Table S1.

Endometrial stromal cell isolation Endometrial tissue and lesions were immediately placed in an ice-cold 1:1 mixture of Dulbecco’s Modified Eagle’s Medium (DMEM) and Ham’s F-12 for transport to the laboratory. The tissue was washed in 7 ml of fresh medium to remove any debris or excess blood cells. The biopsies were dissociated in 5 ml DMEM medium without phenol red containing 0.5% collagenase (Sigma-Aldrich) in shaking incubator rotating at 110 rpm at 37°C until the biopsies were digested but not longer than 1 h. The dispersed cells were filtered through a 50 µm nylon mesh to remove undigested tissue pieces. Stromal cell isolation was carried out as previously described12. Briefly, the cells were resuspended in 10 ml of culture medium in a 15 ml tube. Sealed tubes were placed in an upright position for 10 min to sediment epithelial glands. The top 8 ml of medium containing stromal cells was collected and the epithelial cells on the bottom were discarded. The sedimentation process was repeated three times. Final purification of stromal cells was achieved by selective

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adherence of stromal cells to culture dishes for 30 min at 37°C in 5% CO2. Nonadhering epithelial cells were discarded by washing twice in 5 ml of culture medium.

Cell culture and stable isotope labelling by amino acids in cell culture Isolated ESCs were cultured for 2-5 passages in DMEM/F12 supplemented with 10% fetal bovine serum (FBS) with penicillin/streptomycin/amphotericin B (100 U/ml, 100 µg/ml, 0.25 µg/ml, respectively) at 37°C and under 5% CO2. For subsequent comparative analyses passage number of samples across groups was matched. Confluent cells were dissociated with 0.25% trypsin-EDTA (Gibco) and collected by centrifugation at 200 g for 6 min, followed by two washes with 2 ml PBS each. Cell pellets were kept at -80oC until further analysis. For the production of the stable isotope labelling by amino acids in cell culture (SILAC) standard, secretory phase ESCs from a healthy endometrium and Ishikawa cancer cells were separately cultured for 6 passages in DMEM supplemented with 0.266 mM heavy (13C615N2) lysine (Lys8) and 0.133 mM heavy (13C615N4) arginine (Arg10) (Cambridge Isotope Laboratories) and 10% dialysed FBS (Thermo Fisher Scientific). 200 mg/L light proline was also added to the labelling medium to suppress potential arginine to proline inter-conversion13. Ishikawa cancer cells were included as a standard to provide more optimal quantification of proteins important in proliferative capacity.

Nano-LC/MS/MS sample preparation 7



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For proteomics analyses we used primary cultures of stromal cells from endometrium of healthy controls (n=6), stromal cells from endometrium of endometriosis patients (n=5) and stromal cells from peritoneal lesions (n=5). Cell pellets were lysed and prepared essentially as described by Erde et al14 with minor modifications. Briefly, the cells were lysed in 4% SDS, 100 mM Tris-HCl pH 7.5, 10 mM DTT with incubation at 95oC for 5 min, followed by probe sonication (20x 1 s pulses). After protein concentration measurements in 8 M urea, 100 mM Tris-HCl pH 7.5 using tryptophan fluorescence (excitation/emission wavelengths of 295/350 nm, respectively) the samples were spiked 1:1 with the SILAC standard. The standard consisted of 2:1 mixture of healthy secretory phase ESCs and Ishikawa cancer cells labelled with heavy lysine and arginine. The mixtures were on-filter (30 kDa molecular weight cut-off; Millipore) digested with 1:50 proteomics grade dimethylated porcine trypsin (Sigma-Aldrich) and in the presence of 1.0% sodium deoxycholate. After phase transfer removal of the detergent with ethyl acetate the samples were fractionated into six fractions using strong cation exchange (SCX) and C18 StageTips15. Peptides were eluted, dried and reconstituted in 0.5% TFA.

Nano-LC/MS/MS analysis SCX peptide fractions were separated on Ultimate 3000 RSLCnano system (Dionex) using a C18 cartridge trap-column (Dionex) in backflush configuration and an in-house packed (3 µm C18 particles, Dr Maisch) analytical 50 cm x 75 µm emitter-column (New Objective). Peptides were eluted at 200 nl/min with a 8-40% B 240 min gradient (buffer A: 0.1% formic acid, buffer B: 80% acetonitrile + 0.1% formic acid) to a Q Exactive (Thermo Fisher Scientific) tandem mass spectrometer operating 8


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with a top-15 strategy and a cycle time of 1.2 s. Briefly, one 350-1400 m/z MS scan at a resolution of R=70000 was followed by higher-energy collisional dissociation fragmentation (normalized collision energy of 27) of up to 15 most intense ions (charge states +2 to +6) at R=17500. MS and MS/MS ion target values were set to 3e6 and 5e4, respectively, fragmented ions were dynamically excluded for 80 s.

Mass-spectrometric raw data processing Raw data were identified and quantified with MaxQuant 1.4.0.8 software package16. Labelling state (multiplicity) was set to 2 and Lys8 and Arg10 were defined as the heavy amino acids. Methionine oxidation, asparagine/glutamine deamidation and protein N-terminal acetylation were set as variable modifications, while cysteine carbamidomethylation was set as a fixed modification. Search was performed with the MaxQuant Andromeda search engine against UniProtKB (www.uniprot.org) human reference proteome database (September 1, 2014) using the trypsin digestion rule (cleavage after lysine and arginine without proline restriction). First and main search MS mass tolerances were ≤20 and ≤4.5 ppm, respectively. MS/MS tolerance was ≤20 ppm. Only protein identifications with minimally one (n≥1) peptide of at least seven (n≥7) amino acids long were accepted and transfer of identifications between runs was enabled. For protein quantification minimally two ratio (n≥2) measurements were required with at least three (n≥3) points across a chromatographic peak. Signal integration of missing label channels (requantification) was allowed. Peptide-spectrum match and protein false discovery rate was kept below 1% (FDR≤1%) using a target-decoy approach. All other parameters were set to default. The mass spectrometry raw files along with MaxQuant 9



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identification and quantification output (txt folder) have been deposited to the ProteomeXchange Consortium17 via the PRIDE partner repository with the dataset identifier PXD002845.

Reviewer account details: Username: [email protected] Password: efBupa9q

Statistical and enrichment analysis of proteomics data Statistical analysis was performed using MaxQuant Perseus module and Microsoft Excel’s Real Statistics Resource Pack. To account for mixing errors, protein H/L ratios were normalized by shifting median log H/L ratio to 0. After inverting and log transformation of the ratio data, only proteins with at least three quantitative measurements per sample group were retained. Histograms were plotted for measured ratios and checked for conformity with the normal distribution. To detect significantly changed proteins, one-way analysis of variance (one-way ANOVA) was conducted along with multiple testing correction using the Benjamini & Hochberg’s FDR procedure below 0.05 (FDR≤5%) level. Tukey-Kramer’s method was applied for post-hoc comparisons and significance was reported for proteins with a post-hoc comparison p-value of

Deep Quantitative Proteomics Reveals Extensive Metabolic

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