SFRP4 expression correlates with epithelial mesenchymal transitionlinked genes and poor overall survival in colon cancer patients

Landry E Nfonsam,Jana Jandova,Hunter C Jecius,Pamela N Omesiete,Valentine N Nfonsam

Abstract

Key words: Secreted frizzled-related protein 4;Epithelial mesenchymal transition;Colon cancer;Survival

INTRODUCTION

Colon cancer is an important contributor to worldwide cancer morbidity and mortality,affecting an estimated 1.2 million individuals and responsible for 0.6 million deaths every year[1].In the United States,colon cancer is among the most commonly diagnosed cancer with an estimated 97220 new cases expected by the end of 2018 and an overall life time risk of about 4.49% for men and 4.15% for women[2,3].Despite the decline in overall incidence and mortality of colon cancer over the past 30 years[4],there continues to be an alarming rise in the number of early-onset colon cancer cases in individuals younger than 50 years[5].Using the Surveillance Epidemiology and End Results Program Cancer Registries database,we previously demonstrated that this rise is most significant within the age group 40-44[6].About 70%-80% of early-onset colon cancer cases are sporadic,and these patients have aggressive features,are often diagnosed at a more advanced stage,and tend to have poor survival[5-11].

In 2016,we showed that the secreted frizzled-related protein 4 (SFRP4) gene was uniquely over-expressed in early-onset colon cancer patients[12].This is a protein coding gene and a member of theSFRPfamily.The cysteine-rich domain ofSFRP4is homologous to the putativeWNT-binding site of frizzled proteins,makingSFRP4,like otherSFRPs,a modulator ofWNTsignaling[13,14].As aWNTinhibitor,SFRP4prevents cell growth and proliferation and has been implicated in Pyle disease[15].SFRP4is expressed in mouse embryonic mesenchyme,cardiovascular system,and musculoskeletal system[16].Reports show thatSFRP4plays an essential role in ovarian cancer pathogenicity by inhibiting epithelial-mesenchymal transition (EMT) and cell migration,while promoting cell adhesion[13].

Although EMT is a typical process in embryonic development,heart valve formation,and other developmental processes,its role in promoting cancer metastasis is well documented[17,18].EMT provides a molecular environment that allows epithelial cells to lose polarity and cell-to-cell adhesion and gain migratory and invasive properties to become mesenchymal cells[17,18].The relationship betweenSFRP4and EMT-linked genes in colon cancer pathogenesis and the implication ofSFRP4overexpression in patient survival has not been investigated previously.In this study,we demonstrate a correlation betweenSFRP4gene expression and the expression of some key EMT-linked genes in colon cancer and show that the overexpression ofSFRP4associates with poor survival in colon cancer patients.

MATERIALS AND METHODS

Ethics statement

This study does not involve human subjects.It utilizes the publicly available deidentified colon adenocarcinoma (COAD) data set from The Cancer Genome Atlas(TCGA).

TCGA program

A landmark cancer genomics program molecularly characterized over 20000 primary cancer and matched normal samples spanning 33 cancer types.TCGA was formed in 2005 when the U.S.National Cancer and National Human Genome Research Institutes teamed together to support the launch of the project to map comprehensively various cancer genomic changes (https：//www.cancer.gov/about-nci/organization/ccg/research/structural-genomics/tcga/history/timeline).This joint effort brought together researchers from diverse disciplines and multiple institutions.Over the next dozen years,TCGA generated over 2.5 petabytes of genomic,epigenomic,transcriptomic,and proteomic data.The data,which has already led to improvements in our ability to diagnose,treat,and prevent cancer,will remain publicly available for anyone in the research community to use (https：// www.cancer.gov /aboutnci/organization/ccg/research/structural-genomics/tcga).

As of March 2019,the TCGA data portal contains 365463 pieces of genomic and clinical data for 45 projects;68 primary sites;33549 cases;22872 genes,and 3142246 mutations.The TCGA-COAD project contains 461 cases including two primary sites,colon and rectosigmoid junction,and the following disease types：Adenomas and adenocarcinomas,complex epithelial neoplasms;cystic,mucinous,and serous neoplasms,epithelial neoplasms,and no other specified carcinomas.

Data set

A total of 286 of 462 “Primary Tumor” samples from TCGA-COAD cohort was used in this study for gene expression and Kaplan-Meier survival analyses[19].Table 1 summarizes the gender,age,and tumor stage of samples.

Gene expression

SFRP4gene expression relative to the EMT genes,Fibronectin (FN1),Vimentin (VIM),Zinc Finger E-Box Binding Homeobox 1 (ZEB1),ZEB2,Twist Family BHLH Transcription Factor 1 (TWIST1),TWIST2,Snail Family Transcriptional Repressor 1(SNAI1),SNAI2,N-cadherin (CDH2),Claudin 4 (CLDN4),CLDN7,Tight Junction Protein 3 (TJP3),Mucin 1 (MUC1),and E-Cadherin (CDH1),was performed using the TCGA-COAD dataset as previously described[20].Pearson's and Spearman's rank coefficients and two-dimensional correlation scatter plots were generated using the University of California Santa Cruz Cancer Browser Interface (https：//xena browser.net)[21].

Survival analysis

The TCGA-COAD “Primary tumor” dataset was used to perform and visualize Kaplan-Meier overall survival analysis using the University of California Santa Cruz Xena Browser for cancer genomics (https：//xenabrowser.net/datapages/?Cohort-TCGA%20Colon%20Cancer%20(COAD))[22,23].Pvalues were obtained using the Kaplan-Meier Estimator.

RESULTS

Co-expression of SFRP4 with EMT genes

The role ofSFRP4in epithelial-mesenchymal transition was investigated in colon cancer by comparing its expression to that of some key EMT-linked genes using 286 colon cancer patients.We observed co-expression and a positive correlation by Pearson's and Spearman's rank coefficient analysis ofSFRP4with the EMT markersCDH2,FN1,VIM,ZEB1,ZEB2,TWIST1,TWIST2,SNAI1,SNAI2,Periostin (POSTN),Matrix Metallopeptidase 2 (MMP2),MMP9,MMP7,and Collagen 1 (COL1A1) (Figure 1 and Figure 2).A higher expression ofSFRP4and the EMT-linked markers wasobserved in samples that were more mesenchymal than epithelial.A negative correlation was observed betweenSFRP4andCLDN4,CLDN7,TJP3,MUC1,andCDH1(Figure 1 and Figure 3).Based on the Pearson's and Spearman's rank coefficients,SFRP4shows the strongest correlation withCDH2followed byMMP2,and the least correlation withCLDN7(Figure 2 and Figure 3).These data suggest a role forSFRP4in the EMT molecular pathway.

Table1 Gender,age and tumor stage of The Cancer Genome Atlas-colon adenocarcinoma dataset used for analysis

Colon cancer patients over-expressing SFRP4 have poor overall survival

We analyzed the effect ofSFRP4overexpression on colon cancer patient survival.Our data showed that colon cancer patients with higherSFRP4expression (≥ 8.661) had poor overall survival compared to patients with lowerSFRP4expression (＜ 8.661)(Figure 4A).A similar trend was observed when patient cohort was divided into three groups.Patients with intermediate levels ofSFRP4expression (7.467-10.05) showed survival curve that was between that of patients with higher (≥ 10.05) and lower (＜7.467) expression (Figure 4B).These data demonstrate thatSFRP4has an impact on colon cancer patient survival.

DISCUSSION

Very little is known about the role ofSFRP4in cancer pathogenesis,let alone its mechanism of action.Most of the speculations onSFRP4mechanism appear to focus on its role as aWNTsignaling modulator,through which it suppresses epithelial mesenchymal transition,a mechanism that is implicated in cancer metastasis and invasion[13,17,18].In ovarian cancer,SFRP4regulates metastasis by inhibiting EMTlinked genes,cell proliferation,and migration,while promoting apoptosis and upregulating cell adhesion molecules such asCDH1[13,24].A Study by Muleyet al[25]showed thatSFRP4can restrict ovarian serous cystadenocarcinoma growth in mice by interfering with endothelial cell function,suggestingSFRP4as a potent angiogenesis inhibitor.While these observations indicate thatSFRP4represses cancer pathogenesis,this is in contrast to our previous observation ofSFRP4upregulation in early-onset colon cancer[12]or its strong correlation with epithelial mesenchymal-linked genes observed in this study.

Figure1 SFRP4 co-expresses with epithelial mesenchymal transition-linked genes in colon cancer.

Our previous report on the overexpression ofSFRP4in early-onset colon cancer informed us of the potential role ofSFRP4in colon cancer particularly with respect to age of onset[12].Our data showed thatSFRP4co-expresses and correlates positively with the EMT-linked genesCDH2,FN1,VIM,SNAI1,SNAI2,ZEB1,ZEB2,TWIST1,POSTN,COL1A1,MMP2,MMP7,andMMP9,many of which promote epithelial mesenchymal transition by repressing proteins that maintain tissue integrity such asCDH1[26,27].Some of the hallmarks of EMT are cell separation and invasion,characterized by loss of cell-cell adhesion and gain of migratory and invasive properties[18].Given the role ofSFRP4as an EMT inhibitor,overexpression in earlyonset colon cancer might be a response mechanism to serve as a check on EMT in the wake of increase expression of EMT genes.

However,overexpression ofSFRP4may not always correlate with EMT suppression.There is accumulating evidence thatSFRPfamily proteins may also have oncogenic potential in certain cancer types[28,29].While the binding and sequestering ofWNTligand bySFRPs inhibitWNTsignaling[30-33],binding may also allowSFRPs to act as carriers forWNTproteins in a diffusion gradient,thus expanding the range and activity ofWNTsignaling.This has been reported inXenopuswhereSFRP3andCrescentwere shown potentially to activate canonicalWNTsignaling by promoting the diffusion ofWNT8andWNT11[34].There have also been reports on the dimerization of the N-terminal cysteine-rich domain ofSFRPs and Frizzled proteins,hence suggestingSFRPs direct activation ofWNTsignaling by mimicking theWNTligand[35-37].Additionally,it has been suggested that by antagonizing each other,theSFRPsindirectly activateWNTsignaling,as seen in rat renal organogenesis whereSFRP2was reported to inhibitSFRP1activities[38].Also,studies have shown thatWNTregulation could be concentration dependent with lowSFRPconcentrations activatingWNTsignaling and high concentrations inhibiting it[39].

Consistent with the concept ofSFRPsfunctioning as oncogenes,SFRP1has been reported to be over-expressed in basal-like breast cancers and implicated in brainspecific metastases,whileSFRP2induces mammary tumor formation in canines by blocking apoptosis[40,41].BothSFRP1andSFRP2promote cellular invasion,proliferation,andin vivotumor growth in renal cancer[42,43],andSFRP1in gastric cancer.SFRP2enhancesWNT3Asignaling in HEK293 and HSG cells[44]and activatesWNTsignaling through direct interaction withFZ4/7in the mouse[45].Interestingly,overexpression ofSFRP4has also been observed in stomach cancer where it associates with poor patient outcomes and interacts strongly with EMT-linked genes in gliomas[29].Taken together,these observations suggest that besides being aWNTsignaling inhibitor,theSFRPfamily members,includingSFRP4,also have oncogenic potentials,and their mechanism of action might be context dependent or cancer type dependent.

Consistent with the important roleMMP2plays in the breakdown of the extracellular matrix and the EMT process,particularly in cell invasion and metastasis,SFRP4expression was strongly correlated withMMP2.In addition toCDH1,SFRP4was negatively correlated with many other cell adhesion molecules.Given the role these genes play as gatekeepers of EMT and metastasis,it follows thatSFRP4is upregulated with EMT following downregulation of cell-adhesion molecules.Overexpression ofSFRP4is also consistent with overexpression of other EMT regulators we previously reported in early-onset colon cancer,such as cartilage oligomeric matrix protein,(COMP) which catalyzes collagen fibrillation to maintain the extracellular matrix[20,46].

Figure2 Epithelial mesenchymal transition-linked genes with positive correlation with SFRP4 expression.

Overexpression ofSFRP4in colon cancer patients resulted in poor survival that correlated with the survival analysis observed for colon cancer patients overexpressingCOMP[20].Given the implication ofWNTsignaling in many developmental and disease pathways[47-49]and the knowledge thatSFRP4inhibitsWNTsignaling,it is possible that early deaths might be due to secondary complications following overexpression ofSFRP4,which interferes with the normal functions of neighboring healthy cells.However,it is also possible that early deaths are due primarily to disease independent ofSFRP4,whose overexpression might only be a consequence of the disease.

Although highSFRPexpression in colon cancer was observed in this study,others have suggested thatSFPR4expression in tumor may be contributed by the stroma fraction.This was documented in studies by Vincentet al[29]where a strong correlation betweenSFRP4and the stroma was observed in 14 cancer types,including colorectal.Nevertheless,considering that the stroma constitutes an integral part of the tumor microenvironment,its expression ofSFRP4will undoubtedly constitute an important disease mechanism for potential targeted therapy.

In summary,we had previous shown thatSFRP4was over-expressed in early-onset colon cancer[12]and report hereSFRP4expression was correlated with EMT-linked genes.These findings highlightSFRP4as an important contributor to the pathogenesis of colon cancer,particularly early-onset colon cancer,and therefore as a potential biomarker for the early diagnosis of colon cancer.Considering thatSFRP4modulatesWNTsignaling during cell proliferation,migration,and EMT,which are all hallmarks of cancer pathogenesis.Overexpression ofSFRP4in early-onset colon cancer and co-expression with EMT-linked genes provide an important first step towards understanding its molecular mechanism in young patients,particularly in the context of cancer metastasis and cell invasion.

Figure3 Epithelial mesenchymal transition-linked genes with negative correlation SFRP4 express.

Figure4 Overall survival of colon cancer patients with low,intermediate or high SFRP4 expression.

ARTICLE HIGHLIGHTS

Research background

Colon cancer affects 1.2 million people around the world and causes about 0.6 million deaths annually.Although the overall incidence and mortality is decreasing,there has been an alarming increase in early-onset cases (＜ 50 years).These younger patients are often diagnosed at late stages with more aggressive tumors and tend to have poorer survival.

Research motivation

We previously showed that secreted frizzled-related protein 4 (SFRP4),speculated to play an essential role in cancer by inhibiting epithelial mesenchymal transition (EMT),is over-expressed in younger patients.

Research objectives

The aim of our study was to investigate whether there is a correlation between SFRP4 expression and EMT-linked genes in colon cancer and whether SFRP4 over-expression affects patient survival using a larger cohort of patients.

Research methods

Correlation betweenSFRP4expression and EMT-linked genes along with survival analysis were performed using the University of California Santa Cruz Cancer browser interface using publicly available The Cancer Genome Atlas-colon adenocarcinoma cohort of colon cancer cases.

Research results

SFRP4is co-expressed with EMT-linked markers,such asCDH2,FN1,VIM,TWIST1,TWIST2,SNAI1,SNAI2,ZEB1,ZEB2,POSTN,MMP2,MMP7,MMP9,andCOL1A1,in colon cancer patients.SFRP4expression negatively correlated with the EMT-linked suppressorsCLDN4,CLDN7,TJP3,MUC1,andCDH1.Mesenchymal-like samples showed higher expression ofSFRP4and EMT-linked markers relative to epithelial-like samples.This implicatesSFRP4in colon cancer EMT mechanism.Additionally,patients with colon tumors over-expressingSFRP4presented with significantly poorer overall survival.

Research conclusions

Results of this study suggest a potential role ofSFRP4in colon cancer aggressiveness,disease progression,and poorer patient survival.Although the role ofSFRPs includingSFRP4asWNTsignaling inhibitors is well documented in the literature,data presented in this manuscript suggest that theSFRPfamily proteins might also activateWNTsignaling and promote cell proliferation,therefore demonstrating its oncogenic potential and suggesting its mechanism of action might be context dependent.

Research perspectives

More extensive and detailed mechanisticin vitroandin vivostudies will have to be performed to confirm these initial observations in colon cancer patients.

ACKNOWLEDGEMENTS

We would like to thank the patients without whom this work would not be possible.