Proteomic Analysis Identifies Tenascin-C Expression Is Upregulated in Uterine Fibroids

Abstract

Uterine leiomyomas (fibroids) are the most common gynecological tumors, which are enriched in the extracellular matrix (ECM). Fibroids are leading cause of abnormal uterine bleeding and hysterectomy. One of the major questions yet to be answered is the overproduction of specific ECM components in human uterine fibroids, particularly in relation to mutations in the driver gene mediator complex subunit 12 (MED12). Surgical specimens from 14 patients with uterine leiomyoma having fibroids and corresponding adjacent normal myometrium (ANM) were utilized to analyze genetic and proteomic expression patterns in the tissue samples. MED12 mutations in the fibroids were screened by Sanger sequencing. iTRAQ was used to label the peptides in small-, medium-, and large-sized fibroid samples of annotated MED12 mutation from the same patient. The mixtures of the peptides were fractionated by hydrophilic interaction liquid chromatography (HILIC) and analyzed by liquid chromatography–tandem mass spectrometry (LC-MS/MS) to identify the differential expression proteins. Using isobaric tagged-based quantitative mass spectrometry on 3 selected patients, ECM-related protein tenascin-C (TNC) was observed significantly upregulated (>1.5-fold) with a confidence corresponding to false discovery rate (FDR) <1% in small-, medium-, and large-sized fibroid samples regardless of MED12 mutation status. The TNC was validated on additional patient samples using Western blotting (WB) and immunohistochemistry (IHC) and confirmed significant overexpression of this protein in fibroids compared to matched ANM. Proteomic analyses have identified the increased ECM protein expression, TNC, as a hallmark of uterine fibroids regardless of MED12 mutations. Further functional studies focusing on the upregulated ECM proteins in leiomyogenesis will lead to the identification of novel ECM drug targets for fibroid treatment.

Keywords

ECM fibroids uterus infertility tumor

Introduction

Uterine leiomyomas or fibroids are benign smooth muscle tumors that are most commonly encountered in the uterus of approximately 70% of reproductive-aged women.^1
–3 Fibroids are often associated with severe symptoms that negatively impact the quality of patient's life.⁴ Many of these women experience symptoms such as abnormal uterine bleeding, pelvic discomfort, infertility, and adverse pregnancy outcomes.^5,6 Fibroids are the leading indication for hysterectomy in the United States and are responsible for substantial health-care cost up to US$34.4 billion dollars each year in treating this disease.⁷ The current treatments are limited due to the lack of understanding of the cellular mechanism that drives fibroid growth. While ovarian hormones estrogen and progesterone were reported to play a key role in fibroid growth,⁸ the exact causes of uterine fibroids are not completely known.

Recent comprehensive genome analyses have identified at least 4 major uterine fibroid subtypes with genetic alterations, namely, mediator complex subunit 12 (MED12) mutation, high mobility group AT-hook 2 (HMGA2) overexpression, biallelic fumarate hydratase (FH) inactivation, and collagen type IV α 5 (COL4A5) and collagen type IV α 6 (COL4A6) deletion.^9,10 The most common subtype is MED12 mutation in uterine fibroid, present in approximately 70% of all cases.¹¹ MED12 is a subunit of the transcriptional Mediator complex that regulates the interaction of RNA polymerase II with various transcriptional factors.^12,13 In our recent genetic screening of MED12 status of fibroids in the Australian population, we have identified 3% and 60% of uterine fibroids harbor-specific mutations in MED12 exon 1 and exon 2, respectively, of the 65 uterine fibroids studied.¹⁴ Almost all identified mutations were mainly missense mutations restricted to a codon encoding glycine 44 in exon 2, which is the most common hotspot for MED12 mutation.

In recent years, significant progress has been made in our understanding of the defining features of fibroids.^3,15

–18 The size and growth of fibroids involve proliferation of fibroid cells and increased accumulation of extracellular matrix (ECM). Additionally, uterine fibroids exhibited 50% greater ECM than adjacent normal myometrium tissue (ANM).^15,19
–21 The ECM is thought to play a key role in fibroid growth and structural organization, and the influence of the ECM on fibroids is generally considered to be affected by the abnormal composition, deposition, and function of specific ECM components.²² Several pathways have been implicated in promoting fibroid development, including the wingless-related integration site (WNT)/β-catenin pathway, transforming growth factor-β (TGF-β), prolactin, insulin-like growth factor (IGF1), and nuclear factor erythroid 2-related factor 2 (NRF2) signaling.^{10,18,19,23
–25}

An important finding is that several of these complex signaling pathways involved between fibroid cells and ECM are interconnected to one another and have the potential to converge into a common final pathway.²⁶ For example, the ECM stiffness transmitted mechanical signals to intracellular signaling via transmembrane integrin receptors.²⁶ Simultaneously, TGFs produced by the cells are released into the extracellular space, leading to massive deposition of collagen and therefore affect ECM stiffness.²⁶ This represents a dual signaling pathway occurring at the same time in uterine fibroid in relation to ECM stiffness.

In this study, using the combination of genetic and proteomic analyses and immunohistochemistry (IHC) validation of clinically annotated fibroids and corresponding ANM, we investigated the expression pattern of tenascin-C (TNC) in small-, medium-, and large-sized fibroid samples of MED12 mutation-positive and mutation-negative uterine fibroids. The TNC is a 300-kDa, multimodular ECM glycoprotein that confers the ability to interact with diverse binding partners such as other ECM proteins and cell surface receptors.²⁷ It is a key molecule in tissue remodeling and is highly expressed under pathological conditions in heart failure, atherosclerosis, and cancer.²⁸

Materials and Methods

Collection of Human Fibroid and ANM Tissues

The acquisition and research use of surgical specimens were approved by the Institutional Human Research Ethics Committee at the University of Newcastle. Fibroid and ANM samples were obtained from patients undergoing hysterectomy with an average age of 47 years with prior written informed consent. All bar one patients used in this study were white Caucasian presented with symptomatic fibroids. Patients received no hormonal treatments in the prior 6 months. We classified the various size fibroids in patients as small (<2 cm diameter), medium (2.1-4 cm diameter), and large (>4 cm diameter) fibroids. The collected tissues were processed as described by us.²³

Genomic DNA Extraction and Sanger Sequencing for MED12 Mutation

Small pieces of approximately 25 mg of fresh frozen fibroid or ANM tissues were used for genomic DNA extraction. Isolation of genomic DNA was performed using the QIAamp DNA Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. Following DNA amplification of MED12 exon 1 and exon 2, respectively, in fibroid and ANM samples, the purified PCR products were sequenced using the Sanger method as described previously.²⁹ Sequence chromatographs were analyzed for somatic mutations in exon 1 and exon 2 of MED12 using the Mutation Surveyor software (Softgenetics, State College, Pennsylvania).

Protein Sample Preparation and iTRAQ 4-plex Labeling and Processing

Freshly frozen fibroid or ANM tissue was weighted (75-100 mg) and cut into smaller pieces. Then, lysis buffer (0.1 mol/L sodium carbonate, 10 mmol/L sodium orthovanadate) containing protease inhibitor cocktail (Sigma, St. Louis, MO, USA) and phosphatase inhibitors PhosStop (Roche, Indianapolis, IN, USA) was added, and the sample preparation was homogenized using the beadbug homogenizer (Benchmark Scientific, Edison, NJ, USA). The homogenates were reduced, alkylated, and digested with trypsin as described.²³ Trypsin was inactivated by acidifying the samples to pH 2 using 10% trifluoroacetic acid. Peptides were desalted with C18-SD 4 mm/1 mL Extraction Disk Cartridge (EmporeTM, Bellefonte, PA, USA) before iTRAQ 4-plex (AB SCIEX, Framingham, MA, USA) labeling. 4-plex labeling of 3 fibroids (small, medium, and large) and 1 ANM tissue sample from each of the 3 patients were processed according to the manufacturer’s protocol. Labeled peptides were fractionated by hydrophilic interaction liquid chromatography as described¹⁴ before subjected to liquid chromatography–tandem mass spectrometry (LC-MS/MS).

Mass Spectrometry and Data Analysis

Peptides were analyzed by LC-MS/MS using a nanoflow liquid chromatography (Thermo Dionex, Ultimate 3000 RSLCnano; Thermo Scientific, Waltham, MA, USA) and Q-Exactive Plus Orbitrap mass spectrometer (Thermo Scientific, Waltham, MA, USA) with a nano-electrospray emitter at the sampling region as described.¹⁴ The raw MS data were processed with the Proteome Discoverer software package (version2.0.0.802; Thermo Scientific). Proteins and peptides were identified by searching against the UniProt Human reference proteome database (downloaded October 21, 2016, with a total of 48 140 entries). The following search parameters were used: mass tolerances in MS and MS/MS modes were 10 and 20 ppm, respectively; trypsin was designated as the digestion enzyme, and up to 2 missed cleavages were allowed; S-carbamidomethylation of cysteine residues; oxidation on methionine; phosphorylation on serine, threonine, and tyrosine; and acetylation and methylation of lysine and deamidation of asparagine and glutamine were set as variable modifications. The false discovery rate (FDR) was set to 1% for positive identification of proteins, peptides, and phosphorylation sites. Data were analyzed using Gitools 2.3.1 software to generate heat maps for TNC expression between patients. Proteins represented by at least 2 unique peptides were included in the analysis. We set a threshold for a protein to be upregulated if the level of expression was 1.5-fold higher than the corresponding ANM sample and downregulated if it was 0.66-fold lower than the corresponding ANM sample. A q-value must be below 0.05 to identify significant changes in expression.

Western Blotting

Fibroid or ANM tissue extracts were solubilized using protein lysis buffer. The proteins were then separated by size using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; 10% Mini-PROTEAN TGX Gels; Bio-Rad, Hercules, California, USA) and transferred to nitrocellulose membrane (Amersham Protran 0.45 µmol/L NC). The membranes were incubated with primary antibodies followed by secondary antibody (Jackson ImmunoResearch Labs, West Grove, Pennsylvania). The immunoreactive proteins were detected using LAS-3000 Imager (Fujifilm, Tokyo, Japan). The primary antibodies were rabbit anti-TNC (1:1000; Abcam) and mouse anti-β-actin (1:4000; Developmental Studies Hybridoma Bank, Iowa). The digital images of the Western blotting bands were quantified by Image J plugin software (National Institutes of Health, USA).

Immunohistochemistry

Immunohistochemistry was performed as described by us.³⁰ Briefly, fresh tissues were washed 3 times with phosphate-buffered saline (PBS) for 15 minutes per wash and fixed overnight at 4°C in 10% buffered formalin. Postfixation, tissues were embedded in paraffin and 5-μm sections were prepared. Tissue sections were deparaffinized, quenched to eliminate internal peroxidase activity, and incubated with TNC antibody (1:2000; HPA01306; Sigma) overnight at 4°C. Following 3 washes in phosphate-buffered saline with 0.1% Tween-20, tissue sections were incubated for 1 hour in biotinylated secondary antibody, followed by incubation in horseradish peroxidase (HRP) streptavidin (1:250; Jackson ImmunoResearch Labs). Tissue sections were exposed to diaminobenzidine (Sigma) to develop antibody signal. Nuclei were counterstained using hematoxylin. Images were scanned using Aperio AT2 slide scanner (Leica Biosystems, Victoria, Australia). Quantification of TNC staining was performed using a Halo image analysis platform (Indica Labs, New Mexico). As TNC is an extracellular protein, the proportion and intensity scores were individually quantified and were compared with normal adjacent tissue from the same patient.

Statistical Analysis

Data were analyzed and graphed with GraphPad Prism 6.0 (GraphPad Software, San Diego, California). Values were presented as mean ± standard error of the mean. Statistical significance was calculated using one-way analysis of variance and Wilcoxon matched pairs test. P values <.05 were considered statistically significant. For mass spectrometry data analysis, the Student t test value comparing protein expression differences between the ANM and the fibroid across patients was corrected to P values using the Benjamini-Hochberg method.³¹

Results

Proteome Analysis Reveals TNC, an ECM Protein, Overexpression in Genetically Annotated Uterine Fibroids

Multiple uterine fibroids and corresponding ANM tissues were collected from consented patients (n = 14) who had undergone surgery (hysterectomy) for the removal of fibroids as described previously²³ (Supplementary Figure 1). The different fibroid sizes in patients were categorized as small (<2 cm diameter), medium (2.1-4 cm diameter) and large (>4 cm diameter) fibroids (Supplementary Figure 1). Notably, we avoided the necrotic area of the fibroid as they lack cellular and tissue integrity. The suitability of the tissue collections was evaluated and confirmed by a pathologist. The ANM was obtained at least 2 cm from adjacent fibroid tissue. The ANM and fibroid tissues were immediately processed as described by us²³ before genetic screening and proteomic procedures. We performed proteomic analysis of the 3 fibroids (small, medium, and large) and 1 ANM tissue from each of the 3 patients using the 4-plex iTRAQ labeling approach to determine protein expression patterns in uterine fibroids (n = 12 tissue samples; 9 fibroids and 3 ANM).¹⁴ iTRAQ is advanced technique in labeling of complex samples³² and enabled cross-comparisons between various size fibroids and ANM samples in the same LC-MS run, thereby reducing experimental variation. Using the iTRAQ mass spectrometry approach, we found an overall total of 1061, 1081, and 1116 upregulated proteins in small, medium, and large fibroid samples of annotated MED12 mutation.¹⁴ The majority of upregulated proteins that were common across fibroid size range were of ECM origin.

In this study, we focused on one of the significant upregulated ECM proteins, TNC (Figure 1). We compared TNC expression of genetically annotated fibroids from each size category with ANM control tissue samples. Based on the MED12 mutation screening, we selected 3 of the 14 patients (n = 12 tissue samples; 9 fibroids and 3 ANM) harboring both MED12 mutation-positive and mutation-negative tumors and performed the proteomic analysis. This set of tumors (6 of 9) that were MED12 mutation positive included all 4 possible base substitutions affecting codon 44 (G44C, G44D, G44S, and G44V mutations). For the proteomic procedure, the control ANM tissue was labeled with iTRAQ 114, and 3 fibroids (small, medium, and large) were labeled with iTRAQ 115, 116, and 117, respectively, from a 4-plex kit, to allow accurate quantitation of proteins and peptides in the complex sample. For example, this approach detected the iTRAQ-modified form of peptide L126-R137 from TNC, which was upregulated across the fibroid size range (Figure 1C; Supplementary Table S1).

Analysis of small fibroids versus ANM control revealed that TNC in small fibroid was upregulated with an average of 3-fold higher than the ANM in all patients (Figures 1A and 2B; P = .04). In medium-sized fibroid versus ANM analysis, TNC was upregulated with an average of 2.6-fold higher expression in fibroids than ANM (Figure 1A and B; P = .0063). In addition, we detected overexpression of TNC in large fibroid, with a 2.64-fold increase expression than ANM (Figure 1A and B; P = .0003). Taken together, the overexpression of TNC across the fibroid size range strongly suggested that TNC is potentially involved in fibroid growth irrespective of MED12 mutations. The proteomic analyses presented here are based on a predefined criterion of q < .05 with relative expression levels at least >1.5-fold or <0.66-fold compared to ANM for upregulated and downregulated proteins, respectively; proteins represented by at least 2 unique iTRAQ-labeled peptides were included in the analyses. The P values were then calculated by Student t test comparing the TNC protein expression differences between ANM and fibroid across the 3 patients and adjusted for multiple testing by the Benjamini-Hochberg method³¹ (*P < .05 is considered statistically significance after the Benjamini-Hochberg correction for multiple testing).

TNC is One of the Significantly Upregulated ECM Protein During Early Stages of Uterine Leiomyogenesis

The TNC was one of the significantly upregulated ECM proteins observed in small fibroids, suggesting that it is upregulated at the earliest stages of uterine leiomyogenesis. Here, we selected 1 small or medium fibroid containing either MED12-positive or MED12-negative mutation and corresponding ANM tissue (total n = 24) from each of the 8 patients (Figure 2) and analyzed for TNC expression by Western blotting (WB; Figure 3A). The majority of the positive mutations included in the WB analysis were clustered around codon 44 (Figure 2), which is the most common hot spot mutations in MED12. The WB results revealed significantly higher levels of TNC in small fibroids harboring MED12-positive mutation compared to corresponding ANM in the 8 patients (Figure 3A; 8 ANM vs 8 fibroids with MED12-positive mutation; P = .0013). Although the higher level of TNC protein was also detected in fibroids without MED12 mutations, the difference did not reach the statistical significance (Figure 3A; 8 ANM vs 8 fibroids with MED12-negative mutation; P = .08). Next, we performed immunohistochemical analysis of TNC on fibroids and ANM using tissue microarrays. Small fibroids with MED12-positive mutations exhibited upregulation of TNC compared to ANM, supporting WB results (representative IHC images are shown in Figure 3B). Quantitative analysis of TNC expression using immunoproportion and immunointensity scores (Figure 3B, right panel) confirmed increased staining and therefore TNC upregulation in fibroids compared to ANM (209 cores in 22 patient; immunoproportion score: ANM vs fibroids with MED12-positive mutation, P = .0050; ANM vs fibroids with MED12-negative mutation, P = .0003; immunointensity score: ANM vs fibroids with MED12-positive mutation, P = .0028; ANM vs fibroids with MED12-negative mutation, P = .0014).

STRING Analysis Identified ECM Receptor and Focal Adhesion Interactions and PI3K-Akt Signaling

Based on our quantitative proteomic results, we mapped out the list of common ECM proteins (including collagen, type II α 1, collagen, type III α 1, collagen, type V α 1, collagen, type VII α 1, fibromodulin, periostin, alpha-1-antitrypsin, SPARC-related modular calcium-binding protein 2, SPARC-like 1, TGF-β-induced protein ig-h3, TNC, and versican core protein) that were upregulated across the fibroid size range (n = 9 fibroids) and used pathway analysis using STRING version 10.5 to identify the associated protein–protein interaction pathways. These analyses highlighted a number of important protein networks including ECM receptor and focal adhesion interactions and PI3K-Akt signaling (Figure 4). The involvement of these ECM protein networks in fibroids suggested that each one of these upregulated proteins is likely to interact with one another to promote and increase ECM deposition in fibroid.

Discussion

Uterine fibroids affect millions of women worldwide and are a major source of abnormal uterine bleeding that leads to hysterectomy or other surgical interventions.^1
–3,5,7 Despite their prevalence and enormous medical and economic impact, the exact underlying pathobiology of uterine fibroids is not clear. Consequently, nonsurgical or nonhormonal therapeutic options are still limited. Hysterectomy remains the definitive treatment for women with symptomatic uterine fibroid;³³ however; this surgical option is unsuitable for patients wishing to remain fertile. Therefore, there is an obvious need for an alternative therapy or drug treatments that would be useful to block leiomyoma growth without the unwanted side effects.

A striking feature of uterine fibroids is excessive deposition of ECM. The ECM components including collagen subtypes, proteoglycans, and fibronectin contributed to the majority of the volume in fibroid, further adding to its tumor bulk size, and stiffness of the fibroids, which would eventually lead to clinical symptoms such as abnormal uterine bleeding and discomfort.^15,18 One can rationalize that dissolving ECM will lead to the regression of these tumors. As a proof of concept, numerous studies have reported that the ECM-degrading enzyme collagenase is effective in vitro in suppressing the size of these tumors.^34,35 For the more precise strategy of targeting ECM in uterine fibroids, we need to define differential components of the ECM proteins in fibroid. We have shown that common set of upregulated proteins in small-, medium-, and large-sized fibroids harboring annotated MED12 mutation, respectively, was predominantly ECM proteins.¹⁴ This study has validated and confirmed one of the significant upregulated ECM proteins and, in particular, TNC as potential molecular marker for diagnosing uterine fibroids. Notably, our study is the first to explore the heterogeneity of fibroids within the same patient and provides strong evidence that any changes observed are attributable to the disease state and not a consequence of patient-to-patient differences. This is a major concern as other studies do not take into account inter- and intrapatient variation that may lead to incorrect conclusions that are not applicable to the wide range of fibroid types.

The frequent identification of MED12 mutations in uterine fibroids provides another genetic marker to monitor these nodules of smooth muscle origin.^11,36 Our previous genetic screening using Sanger sequencing provided clear differences when comparing the peaks between the fibroid mutated MED12 and the corresponding ANM. We learnt that multiple fibroids in a patient can display diverse genetic profiles, the majority of which affected glycine at codon 44 in exon 2 of MED12. Our genomic data are in line with other literatures showing that exon 2 of the MED12 gene is the most frequently reported genetic alteration in fibroids.

From the patient’s genetic information, our goal is to determine whether the MED12 mutation status and fibroid size modulate ECM protein expression patterns. Abundant ECM deposition is one of the key hallmarks of this disease, which contributes to fibroid expansion and bulk-type symptoms. Following our proteomic analyses using 4-plex iTRAQ labeling method, we observed an ECM-related protein TNC that was upregulated in all 3 patients, irrespective of MED12 mutation status and fibroid size. We validated the expression of TNC in fibroids and corresponding ANM by WB and IHC examination. Taken together (using proteomics, WB and IHC), our findings demonstrated a significant overexpression of TNC in fibroid compared to matched ANM.

The TNC is a large ECM glycoprotein of about 300-kDa with a multimodular structure that contains 4 distinct domains: an N-terminal assembly domain, followed by a series of epidermal growth factor-like repeats (EGF-L), a series of fibronectin type III-like repeats (FNIII), and a C-terminal fibrinogen-like globular (FBG) domain.^27,28,37 Each of these domains can interact with a great variety of ECM proteins including collagens, periostin, fibronectin, as well as various cell surface integrin receptors.^28,38 The consequence of this interaction between TNC and binding partner on cellular signaling still needs intensive study, and it is not clear whether these interactions are responsible for the many tumorigenic effects attributed to TNC. Since fibroid are made up of excessive ECM relative to the surrounding uterine smooth muscle,³ we can only speculate that these different subsets of ECM proteins are likely to interact with the distinct domains of TNC that leads to abundant ECM production in fibroid. Our STRING analysis also recognized the involvement of these protein networks in fibroids (Figure 4). Further studies will be required to target the binding sites on the multidomain structure of TNC to study the relationship between the interacting proteins and the TNC.

The TNC is reported to be transiently expressed during embryonic tissue development^39,40 and is absent or greatly reduced in most adult tissues.⁴¹ Furthermore, high levels of TNC are associated with a variety of cancers including bladder, bone, brain, breast, colon, kidney, larynx, liver, lung, lymphomas, mouth, ovaries, pancreas, prostate, skin, stomach, soft tissues, and uterus.^28,37 The TNC has been shown to play an important role in cell proliferation,^42,43 migration,⁴⁴ and tumor invasion.⁴⁵ Our study is the first to compare the expression levels of TNC in fibroids relative to ANM. In this regard, overexpression of TNC in uterine fibroid certainly represents the most valuable candidate for further functional and clinical investigations.

The molecular mechanism of TNC is still unclear; however, a recent study revealed that TNC influences the cell signaling pathway to gain epithelial–mesenchymal transition, accompanied by decreased expression of E-cadherin and the rise in ECM proteins vimentin and several matrix metalloproteinases.⁴⁶ This event leads to increase in cell motility and metastasis.^47,48 Another study reported that breast cancer cells produce TNC as a metastatic niche component to successfully infiltrate and colonize the lungs.⁴⁹ The TNC activates Wnt and Notch signaling in tumor cells, which eventually supports the survival and fitness of the tumor cells.^49
–51 The TGF-β is known to regulate the transcription of at least some tenascin isoforms such as tenascin-W in breast cancer metastases ⁵²; however, less is known about the association between TGF-β and TNC in the development of uterine fibroid, particularly during fibroid expansion. Our previous proteomic analysis revealed that TGF-β was upregulated in the various size (small, medium, and large) fibroids when compared to the ANM.¹⁴ It is possible that TNC interacts with TGF-β signaling pathway to promote ECM formation and cell phenotypic changes in fibroid growth. Continuous in-depth research is nevertheless warranted to better understand the functional role of TNC and other ECM-related proteins in leiomyogenesis.

This study is the first to compare TNC protein expression patterns of fibroids versus ANM in humans. Furthermore, we employed genomic and proteomic analysis to study the relationship between exon 2 MED12 mutation status, fibroid size, and TNC expression. This analysis revealed that TNC was upregulated in all patients, despite variations in fibroid size and the MED12 gene. Furthermore, we validated the expression levels of TNC, as it was one of the significantly upregulated ECM proteins identified in small fibroids and these fibroids are indicative of early stages of leiomyogenesis. Since excessive amount of ECM is a distinct feature of fibroids, targeting upregulated ECM proteins is a rational approach for tackling this disease. It is essential to understand the mechanisms and mediators responsible for the overproduction of specific ECM components. This understanding will provide opportunity to develop intervening strategies that will inhibit the production of these ECM proteins, thereby, to eventually shrink the fibroid size and to improve tumor-related bleeding.

Supplemental Material

Supplementary_Figure_1 - Proteomic Analysis Identifies Tenascin-C Expression Is Upregulated in Uterine Fibroids

Supplementary_Figure_1 for Proteomic Analysis Identifies Tenascin-C Expression Is Upregulated in Uterine Fibroids by M. Fairuz B. Jamaluddin, Prathima B. Nagendra, Pravin Nahar, Christopher Oldmeadow, and Pradeep S. Tanwar in Reproductive Sciences

Supplemental Material

Supplementary_Table_1 - Proteomic Analysis Identifies Tenascin-C Expression Is Upregulated in Uterine Fibroids

Supplementary_Table_1 for Proteomic Analysis Identifies Tenascin-C Expression Is Upregulated in Uterine Fibroids by M. Fairuz B. Jamaluddin, Prathima B. Nagendra, Pravin Nahar, Christopher Oldmeadow, and Pradeep S. Tanwar in Reproductive Sciences

Footnotes

Acknowledgments

LC-MS/MS was achieved with thanks to Nathan Smith from the University of Newcastle Analytical and Biomolecular Research Facility.

Declaration of Conflicting Interests

The author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: Prathima B. Nagendra is a recipient of the University of Newcastle Postgraduate Research Fellowship.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: Work in the Tanwar lab was in part supported by funding from the National Health and Medical Research Council, the Australian Research Council, the Cancer Institute NSW, and the John Hunter Hospital Charitable Trust.

Supplemental Material

Supplementary material is available for this article online.

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