Phospho-DIGE Identified Phosphoproteins Involved in Pathways Related to Tumour Growth in Endometrial Cancer

Endometrial cancer (EC) is the most common gynecologic malignancy of the endometrium. This study focuses on EC and normal endometrium phosphoproteome to identify differentially phosphorylated proteins involved in tumorigenic signalling pathways which induce cancer growth. We obtained tissue samples from 8 types I EC at tumour stage 1 and 8 normal endometria. We analyzed the phosphoproteome by two-dimensional differential gel electrophoresis (2D-DIGE), combined with immobilized metal affinity chromatography (IMAC) and mass spectrometry for protein and phosphopeptide identification. Quantities of 34 phosphoproteins enriched by the IMAC approach were significantly different in the EC compared to the endometrium. Validation using Western blotting analysis on 13 patients with type I EC at tumour stage 1 and 13 endometria samples confirmed the altered abundance of HBB, CKB, LDHB, and HSPB1. Three EC samples were used for in-depth identification of phosphoproteins by LC-MS/MS analysis. Bioinformatic analysis revealed several tumorigenic signalling pathways. Our study highlights the involvement of the phosphoproteome in EC tumour growth. Further studies are needed to understand the role of phosphorylation in EC. Our data shed light on mechanisms that still need to be ascertained but could open the path to a new class of drugs that could hinder EC growth.


Introduction
Endometrial cancer (EC) is one of the most common gynecologic malignancies, affecting 3% of women [1]. Annually 10-20 per 100,000 women develop this type of cancer, and its incidence and mortality continuously increase [2]. ECs arise primarily from the endometrium's glandular tissue, the uterus's inner part. Clinically, two types of EC can be distinguished: estrogen-dependent type I and estrogen-independent type II [3]. Type I ECs represent around 90% of all ECs, frequently display endometrioid cell morphology, low grade (I or II) and are associated with a good prognosis. Conversely, type II is rarer, characterized by serous clear cells, undifferentiated carcinomas or carcinosarcomas, and shows poor prognosis [4]. Most ECs occur as sporadic tumours, being health and lifestyle conditions including obesity, metabolic syndrome, diabetes, polycystic ovary syndrome and high estrogen levels relevant to EC-predisposing factors [5][6][7][8][9][10].
Mechanisms underlying the carcinogenesis of EC are poorly known. In the last 20 years, several regulatory processes involved in ECs pathomechanism have been identified, being the disruptions of Wnt/β-catenin [11], Akt/PI3K/mTOR [12][13][14], MAPK/ERK [15] and VEGF/VEGFR [16] pathways, together with mutations in TP53-binding protein 1 (TP53) and Cyclin-dependent kinase inhibitor 2A (P16), the most common found in ECs or EC cell lines [17,18]. In particular, type I frequently carries a mutation in phosphatidylinositol 3-kinase (PI3K), Catenin beta-1 (CTNNB1), GTPase KRas (KRAS), and POLE (encoding for one domain of polymerase-ε) genes. In contrast, most type II tumours carry gain of function mutations in the TP53 gene and, at lower extent, mutation on (F-box/WD repeatcontaining protein 7) FBXW7, (AT-rich interactive domain-containing protein) ARID1A or (Receptor tyrosine-protein kinase erbB-2) ERBB2 gene amplification [4,19]. Genetic mutations and epigenetic changes lead to the deregulation of signal transduction pathways causing changes in standard cellular mechanisms and promoting tumour development and aggressiveness [20]. Phosphorylation/dephosphorylation events mediate cellular transduction signalling due to specific kinases and phosphatases. Indeed, protein phosphorylation is an essential control mechanism of cellular processes, including proliferation, cell death, metabolism, and intracellular trafficking [21]. Derangement of protein phosphorylation, including hyperactivation, malfunction or overexpression of kinases, is found in cancer.
Therefore, a more profound knowledge of the role of these signalling transduction pathways and the cross-talk between them is necessary to understand how ECs originate and identify new targeted therapy drugs. Mass spectrometry is a compelling technology for identifying cancer biomarkers [22,23] and understanding tumour pathophysiology mechanisms [24]. Phosphoproteomics has critical relevance for comprehending the molecular mechanisms involved in cancer, and the identification of phospho-proteome in EC might open a new therapeutic window for its treatment. Yongchao Dou et al. conducted a phospho-protein study on many patients giving information about proteomic markers involved in pathways leading to tumour development [19]. A similar successful approach led to identifying specific, actionable targets and the introduction of particular drugs for several types of cancers in clinics. [25,26]. Similarly, the identification of aberrant signalling downstream of (Epidermal growth factor receptor) EGFR mutation and (Receptor tyrosineprotein kinase erbB-2) Her2 amplification opened new therapeutic windows in lung, breast and colon cancer [27,28].
Indeed, in this work, we performed an analysis of phosphoproteome by using phosphor 2D-DIGE and MS in patients at an early stage of type I EC, with particular attention to the importance of phosphorylation of proteins involved in crucial signalling pathways, to identify processes that contribute to the onset and progression of EC.

Proteomic Study
We used phosphor-DIGE coupled with MS of 8 EC and 8 CTRL for phosphoproteomics analysis. Proteomweaver software detected more than 2000 spots in both types of samples ( Figure 1).

Western Blotting
For the validation of the phosphoproteomic data, we selected three up-regulated phosphoproteins with fold change ≥ 4.5 (HBB, HPB1, LDHB) and one downregulated protein (CKB) with fold change ≤ 0.28 ( Figure 2) ( Figure S1). We used western blotting to validate the abundance of the phosphoproteins in 13 ECs vs. 13 CTRLs. A higher abundance was significant (Mann-Whitney sum-rank test p < 0.05) for HBB (p = 0.045) and HSPB1 (p = 0.0096), while LDHB showed an increase but not statistically noteworthy (p = 0.75). A decreased abundance was meaningful for CKB (p = 0.0019).

Figure 2.
Western blot analysis was utilized to confirm the alteration of phosphorylation of proteins HBB, HSPB1, LDHB and CKB in normal endometrium (C) and endometrial cancer (EC). The intensity of immunostained bands was normalized against the total protein intensities measured from the same blot stained with Red Ponceau. Results are displayed as a histogram (p < 0.05), and each bar represents mean ± standard deviation.

Bioinformatic Analysis
To understand the functional impact of differentially phosphorylated protein between ECs versus normal endometrium, we next performed bioinformatic enrichment using the gProfiler classification tool to categorize proteins based on their molecular function, biological processes, and cellular component ( Figure 3). In terms of molecular function, proteins were ranked into intramolecular oxidoreductase activity, protein folding chaperone, protein dimerization activity, protein homodimerization activity, unfolded protein binding, protein-disulfide reductase activity, disulfide oxidoreductase activity, and haemoglobin binding. Regarding the biological processes, proteins were categorized into protein folding, cell death regulation, cell death, protein metabolic process regulation, protein refolding, catalytic activity negative regulation, catalytic activity regulation, and mesenchyme migration. Considering the cellular compartment, proteins were organized into extracellular exosome, extracellular vesicle, extracellular organelle, extracellular membrane-bounded organelle, vesicle, extracellular space, extracellular region, and cytosol. Regarding the pathway, Reactome's tools indicate that these proteins are primarily involved in scavenging heme from plasma, Neutrophil degranulation, RNA Polymerase II Transcription Termination and AUF1 (hnRNP D0) binds and destabilizes mRNA.
IPA analysis (Figure 4) showed that identified proteins are required in top networks corresponding to (1)

Discussion
PTMs (post-translational modifications) are biochemical events that occur after translation and can affect the proteins' conformation, activity, and location in the cell [29]. There are more than 300 types of PTMs, and the most important of these, phosphorylation, is a modification related to carcinogenesis and cancer development, regulating numerous cellular processing, including cell growth, proliferation, death, metabolism, and signal transduction [30]. Mechanisms underlying the carcinogenesis of EC are poorly known. Therefore, a more profound knowledge of EC phosphoproteomics might have critical relevance for understanding molecular mechanisms and signalling transduction pathways underlying ECs development.
For this reason, we decided to conduct a phosphoproteomics study employing IMAC enrichment, 2D-DIGE and MS analysis identifying 38 phosphoproteins differently abundant in the EC tissue compared with the endometrial tissue. In addition, utilizing western blotting analysis, we confirmed in a new cohort of samples the significantly altered abundance of 3 phosphoproteins-HBB, CKB, HSPB1-in the EC vs. normal endometrial tissue.
HSPB1 is a small heat shock protein involved in environmental stress response that, upon activation, translocates from the cytoplasm to the nucleus and functions as a molecular chaperone. In response to several signals, it is phosphorylated by different kinases, including p90Rsk (ribosomal s6 kinase), PKC (Protein kinase C), PKD (Protein kinase D), PKG (Protein kinase G) and MAPKAP (MAP kinase-activated protein) kinases 2 and 3 [31]. The exact mechanism by which HSPB1 regulates cancer growth is still unknown [32]; however, this small protein is involved in several tumours and takes part in several distinctive cancer features, including resistance to stress response and actin organization [33]. Interestingly, data reported in the literature indicate that the phosphorylation and dephosphorylation state of HSPB1 controls the apoptosis of cancer cells [34,35]. Phosphorylation of HSPB1 in S15 and S82 is the most up-regulated event in treatment with chemotherapy in pancreatic cancer, raising the tumour resistance. Inhibition of this protein leads to increased apoptosis by chemotherapy [36].
LDHB, although not significantly increased, is a glycolytic enzyme that converts pyruvate and lactate [37]. This enzyme regulates autophagy and lysosomal activity leading to mainly lactate rather than glucose [38]. LDHB is phosphorylated primarily by the Aurora-A kinase enzyme. [39]. Aurora-A phosphorylation on Ser 162 induces the augmented glycolytic activity of LDHB, leading to tumour growth [40]. The two phosphorylated sites, S85 and S90, reported in this study were identified in non-small cell lung cancer [41]. CKB encodes for an enzyme that catalyzes the transfer reaction of phosphate between ATP and creatine phosphate, controlling energy homeostasis [42]. Its role in cancer, and especially in ECs, is obscure. Xu-Hui Li and colleagues have provided new evidence on the association between the expression of this gene and the survival and progression of ovarian cancer cells [43]. Phosphorylation of T35 is involved in different types of breast cancer, including HER2-positive breast cancer, luminal A and B breast cancer [44].
Considering HBB enrichment, its function is of great interest. Recently Yu Zheng et al. performing a single-cell RNA-Seq study on different types of tumours, found that HBB might induce a cytoprotective effect against the metastasis process [45]. Another study highlighted the cytoprotective role of the protein from oxidative stress in cervical cancer [46]. HBB protein phosphorylation in S10, S45, and T124 has been identified in breast cancer cells [41], while the phosphorylation in T5 has been detected in hepatocellular carcinoma [47] and pancreatic ductal adenocarcinoma [48].
Next, we performed bioinformatic analysis to understand the functional impact of differentially phosphorylated proteins between ECs compared to normal endometrium. Subsequently, we performed bioinformatic analysis to understand the functional impact of differentially phosphorylated proteins between ECs compared to normal endometrium. Our Reactome analysis revealed the possible involvement of the Neutrophil degranulation pathway in the early stage of EC. Neutrophils are the first cell responding to stimuli in infection and inflammation. They interact with cancer cells, allowing them to bind to the endothelium and promoting tumour advancement. Neutrophils can induce tumour progression through different mechanisms such as matrix remodelling, angiogenesis stimulation, and immunosuppression. An inflammatory response mediated by neutrophils can induce the activation of dormant cancer cells and a reactivation of the tumour. Their degranulation leads to the release of soluble granules with membrane proteins into cell surfaces, making possible extravasation of circulating tumour cells and their metastasization [49].
A limitation of this study is the limited number of phosphoproteins. A global LC/MS-MS phosphoprotein study would provide more information on possible pathological mechanisms of EC. Another limitation of this study is the need for more functional studies, which are required to ascertain the involvement of this phosphoprotein in EC development. We are organizing research in established/PDX endometrial cancer cells, transplanting them in immunocompromised mice to study the mechanisms of these key proteins.
In conclusion, proteomics highlighted the involvement of phosphoproteins in the early stages of EC. Bioinformatics tools have revealed several pathways related to oxidative stress, tumour development, and phosphoproteins involved in cell viability, cancer infiltration, and protein folding. Other studies are needed to understand the involvement of phosphorylation in the early stages of tumour growth.

Patients
During 2021 and 2022, a total of 26 patients (13 women suffering from EC and 13 non-EC controls) were recruited at the Institute for Maternal and Child Health-IRCCS "Burlo Garofolo" (Trieste, Italy). All EC patients had a type I EC at stage I. All procedures complied with the Declaration of Helsinki and were approved by the Institute's Technical and Scientific Committee. All patients signed informed consent forms. In Supplementary Table S1, we describe the clinical and pathological characteristics of the patients. The median age of patients was 70 years (IQR 59-74, Min = 59, Max = 74). The median age of controls was 44 years (IQR 42-47, Min = 32, Max = 51). The endometrial tissue samples were derived from ordinary leiomyomas in the selection of the controls. We excluded patients and controls with Human immunodeficiency virus (HIV), Hepatitis B virus (HBV), Hepatitis C virus (HCV), leiomyoma and adenomyosis.

Phosphoprotein and Phosphopeptide Isolation
The phosphoprotein isolation from EC and healthy endometrium tissue samples was performed by Phosphoprotein Enrichment Kit (Pierce, Appleton, USA) [50]. About 5 mg of tissue from the EC or the healthy endometrium was homogenized in lysis buffer (1% NP-40, 50 mM Tris-HCl (pH 8.0), NaCl 150 mM) with Phosphatase Inhibitor Cocktail Set II 1× (Millipore, Burlington, VT, USA) and 2 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM benzamidine. Protein concentration was determined by Bradford assay. According to the manufacturer's instructions, 3mg of tissue lysate was used for the phosphoprotein purification by immobilized metal affinity chromatography (IMAC).
3 EC was used to isolate phosphopeptides. Tissues were lysed, and proteins were digested with EasyPep Maxi MS Sample Prep Kit (Thermo Fisher, Boston, MA, USA). After protein digestion, peptides were transferred in dioxide (TiO 2 ) spin tips (Thermo Fisher) for phosphopeptide enrichment, according to the manufacturer's instructions.

Western Blotting
Western blotting analysis confirmed the altered abundance of 4 phosphoproteins, namely HBB, HPB1, LDHB and CKB, using the IMAC-enriched proteins derived from 13 ECs and 13 healthy samples, as previously described [52]. The phosphorylation status of these proteins was directly verified by LC-MS/MS (as described below) or by retrieving the information from the study by Yongchao Dou et al. [19]. For this analysis, 20 µg of phosphoproteins were loaded on 4-20% precast gel and then transferred to a nitrocellulose membrane. Once the proteins were transferred, the membrane was blocked by treatment with 5% defatted milk in TBS-tween 20 and incubated overnight at 4 • C with 1:500 diluted primary rabbit polyclonal antibody against HBB, with 1:700 diluted primary rabbit polyclonal antibody against HPB1, with 1:1000 diluted primary rabbit polyclonal antibody against LDHB, 1:1000 diluted primary rabbit polyclonal antibody against CKB. After primary antibody incubation, the membranes were washed with TBS-Tween 0.05% and incubated with HRP-conjugated anti-rabbit IgG and anti-mouse IgG (1:3000, Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). SuperSignal West Pico Chemiluminescent (Thermo Fisher Scientific Inc., Ottawa, ON, Canada) was used for protein band signal visualization. The intensities of the Immunostained bands were normalized with the total protein intensities measured by staining the membranes from the same blot with Red Ponceau solution (Sigma-Aldrich, St. Louis, MO, USA).

Trypsin Digestion and MS Analysis
For protein spot digestion, a 2-DE gel (200 µg of loaded phosphoproteins) was run and stained with SYPRO Ruby and Coomassie colloidal Blue for protein visualization. Protein spots were selected, digested, and analyzed by mass spectrometry by Ura et al. [53]. Protein spots were washed four times with 50 mM NH 4 HCO 3 and acetonitrile (ACN; Sigma-Aldrich, St. Louis, MO, USA) and dried under a vacuum in a SpeedVac system. Three microliters of 12.5 ng/µL sequencing grade modified trypsin (Promega, Madison, WI, USA) in 50 mM NH 4 HCO 3 was used to digest spots. Digestion was conducted overnight at 37 o C. Peptide extraction was performed with three changes extraction by 50% ACN/0.1% formic acid (FA; Fluka, Ammerbuch, Germany).
LC-MS/MS analyzed four microliters of each peptide mixture with an LTQ-Orbitrap XL mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) coupled to a nano-HPLC Ultimate 3000 (Dionex-Thermo Fisher Scientific). Peptides were separated at 250 nL/min in a 10 cm pico-frit column (75 µm ID, 15 µm Tip; (CoAnn Technologies 350 Hills St, Richland, WA 99354, United States) packed in-house with C18 material (Aeris Peptide 3.6 µm XB-C18, (Phenomenex, Torrance, California, United States) utilizing a linear gradient of ACN/0.1% formic acid from 3% to 40% in 20 min. Data were acquired with a Data Dependent Acquisition (DDA) approach: a full scan in the 300-1700 m/z range was performed at high resolution (60,000) on the Orbitrap. The ten most intense ions in the linear ion trap were selected for CID fragmentation and MS/MS data acquisition in low resolution (Top10 method). Raw data were analyzed with Proteome Discoverer 1.4 (Thermo Fisher Scientific) connected to a Mascot Search Engine (version 2.2.4, Matrix Science, London, UK). Spectra were searched against the human section of the Uniprot database (version 20200901) using the following parameters: enzyme specificity was set on trypsin with one missed cleavage allowed, and the precursor and fragment ions tolerance were 10 ppm and 0.6 Da, respectively. Carbamidomethylation of cys and oxidation of met residues were set as fixed and variable modifications, respectively. The Percolator algorithm assessed the False Discovery Rate (FDR) at protein and peptide levels. We considered those proteins identified with at least three unique peptides and FDR < 0.01 as positive hits. The precursor area detection node of Proteome Discoverer was used to estimate the abundance of each protein in the cases in which multiple proteins were identified within the same spot.
TiO 2 enriched samples were dried under vacuum and suspended in 50 µL of 3%ACN/0.1% formic acid. 5 µL of each sample was analyzed as described above but using a linear gradient of ACN/0.1% formic acid from 3% to 40% in 40 min. to identify phosphopeptides. The same sample was analyzed three times using the same chromatographic conditions but different instrumental methods: in particular, after the first acquisition with a Top10 DDA method, a second acquisition was performed using a static excluding list containing all peptides successfully identified in the first analysis. The third analysis was performed with a Top3 multi-stage activation method (MSA), described in Venerando et al. [54]. Data analysis was performed as reported above, adding phosphorylation of Ser and Thr residues as variable modifications.

Bioinformatic Analysis
Proteins identified by MS were enriched using the g-Profiler tool for their involvement in the biological processes, molecular function, and protein class. Ingenuity Pathway Analysis (IPA) generated bio-functions with p < 0.01. We only considered associations for the filter summary with high confidence or that had been observed experimentally.

Statistical Analysis
Differences were considered significant when spots showed a fold change ± 1.5 and satisfied the Mann-Whitney sum-rank test (p < 0.05). All analyses were conducted with Stata/IC 16.1 for Windows (StataCorp LP, College Station, TX, USA). Funding: This research was funded by the Italian Ministry of Health, grant number 5x1000 SD 18\19.

Institutional Review Board Statement:
The study was conducted according to the guidelines of the Declaration of Helsinki and was by the Institutional Review Board of IRCCS Burlo Garofolo and the Regional Ethics Committee (protocol code 5 × 1000 SD 18\19 approved in 2019 and CEUR-2020-Os-030).

Informed Consent Statement:
Informed consent was obtained from all subjects involved in the study.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author. The data are not publicly available due to ethical reasons.