and TFF3 mRNAs Are Higher in Blood from Breast …downloads.hindawi.com/journals/jo/2018/4793498.pdfJournalofOncology Value TFF 1 5.00 4.00 3.00 2.00 1.00 0.00 group non metastatic

Research ArticleTFF1 and TFF3mRNAs Are Higher in Blood from BreastCancer Patients with Metastatic Disease than Those without

Marwa H Elnagdy1 Omar Farouk 2 Amal K Seleem1 and Hoda A Nada1

1Department of Medical Biochemistry Faculty of Medicine Mansoura University Egypt2Department of Surgical Oncology Faculty of Medicine Mansoura University Egypt

Correspondence should be addressed to Omar Farouk dr omarfaroukyahoocom

Received 26 November 2017 Revised 3 March 2018 Accepted 11 April 2018 Published 7 June 2018

Academic Editor P Neven

Copyright copy 2018 Marwa H Elnagdy et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Introduction Breast cancer metastasis occurs when tumor cells dissociate from the primary tumor and migrate to distant organsthrough the peripheral bloodstream or lymphatic drainage Circulating tumor cells (CTCs) originate from primary sites ormetastases and circulate in the patientsrsquo bloodstream Molecular assays for the detection and molecular characterization of CTCscan serve as a liquid biopsy and can represent an alternative to invasive biopsies as a source of tumor tissue in themetastatic patientsPatients and Methods We analyzed the presence of CTCs in the peripheral blood of 50 breast cancer patients by quantitative real-time reverse transcriptase polymerase chain reaction (RT-qPCR) to detect trefoil factor family (TFF) 1 and 3 genes Results Wefound significant difference in the level of both TFF1 and TFF3mRNA in the blood of nonmetastatic versusmetastatic breast cancerpatients (p= 0001 and p= 0038 respectively)TFF1mRNAwas detected at higher levels in 346ofmetastatic breast cancer patientsas compared to 0 of nonmetastatic (p= 0002) As regards TFF3 mRNA it was detected at higher levels in 462 of metastaticbreast cancer patients as compared to 4 of nonmetastatic (p= 0026) Moreover we found that the high level of both TFF1 andTFF3mRNA was related to estrogen status of the patients The detection of high level of TFF1mRNA in CTCs was associated withbonemetastases (778) while that ofTFF3was related to lymph node involvement (75) and lungmetastases (688)ConclusionThe combinedmeasurement of both TFF1 and TFF3mRNA level for differentiation of metastatic from nonmetastatic breast cancergave 5769 sensitivity and 833 specificity

1 Introduction

Breast cancer is the most common malignancy and secondcancer causing death in women Breast cancer screeninghelps to reduce mortality by early diagnosis of cancer andmetastases [1]

Metastasis is multiple steps process It starts with the localinvasion of surrounding host tissue by cells originating fromthe primary tumor These cells then intravasate into blood orlymphatic vessels where they disseminate to distant organsIn the target organ tumor cells adhere to capillary beds ofthis organThen they extravasate into the organ parenchymawhere they proliferate and start angiogenesis [2]

Circulating tumor cells (CTCs) are tumor cells thatoriginate from primary sites or metastases and circulate inthe patientsrsquo bloodstream Early detection of breast cancermetastasis cannot be done by repetitive metastatic biopsies

So the analysis of CTCs can provide an alternative liquidbiopsy as easy and noninvasive method for diagnosis ofmetastasis [3]

Molecular assays for the detection and molecular charac-terization of CTCs are based on the isolation of total RNAfrom viable CTCs and subsequent qRT-PCR amplificationof CTCs markers This provides valuable information on theexpression of cancer specific genes or mutations of onco-genes and tumor suppressor genes or epigenetic silencing oftumor suppressor genes and metastasis suppressorsThis waspreviously done by the study of mRNA expression of a panelof marker genes on CTCs [4]

CTCs occur in blood at a very low concentration incomparison to millions of blood cells (nearly 1 CTC per105ndash108 white blood cells) especially in patients with early-stage disease So CTCs should be firstly isolated and enriched

HindawiJournal of OncologyVolume 2018 Article ID 4793498 8 pageshttpsdoiorg10115520184793498

2 Journal of Oncology

before the characterization of genetic make-up using qRT-PCR [5]

Trefoil factor family (TFF) peptides are a family of growthfactor-like peptides which was first proposed byThim in 1988[6] They are characterized by the presence of unique three-loop structure stabilized by intrachain disulfide bonds in 1205721-5 2-4 3-6 configuration between six conserved cysteineresidues

There are three TFF peptides TFF1 TFF2 and TFF3Their genes are clustered on chromosome 21q223 [7] Theexpression of TFF genes has been detected in all tissuescontaining mucus-secreting cells eg in gastrointestinaltract ocular tissues salivary glands respiratory tissuesprostate seminal plasma cervical secretions and milkTheircolocalization with mucus helps to form a stable gel-likemucus layer that plays a role in protection and healing of themucosa from harmful agents mechanical stress viruses andharmful pathogens [8]

TFF peptides are involved in mucosal maintenance andrepair throughmotogenic and antiapoptotic propertiesTheyalso function as scatter factors proinvasive and angiogenicagents So they are important for digestive processes inthe normal gastrointestinal tract where they act uniquely astumor suppressors In other organs including the breast theiroverexpression leads to cancer development and metastasis[9]

2 Patients and Methods

21 Blood Collection and Patients Peripheral blood wascollected into EDTA-tubes from 50 breast cancer femalepatients They were classified into two groups 24 cases ofnonmetastatic breast cancer and 26 cases of metastatic breastcancer They were treated in the Oncology Center MansouraUniversity Hospital Thirteen cases were premenopausalTwenty-eight of the 50 patients had positive estrogen receptorin the primary tumor and 28 had positive progesteronereceptor The metastases were located in bone in 13 casesand in lung in 10 cases Thirty-eight cases had lymphnode involvement Blood was taken from 14 healthy femalevolunteers A written informed consent was taken beforesample withdrawal The study was performed in accordancewith the ethical standards laid down in Mansoura Faculty ofMedicine

Peripheral blood mononuclear cells (PBMC) and CTCswere isolated from each blood sample by centrifugationthrough a Ficoll density gradient (Biocoll Separating Solu-tion) with density 1077 gml [10] It was purchased fromBiochrom-Gmb cat no L 6113 Germany Then total RNAwas then extracted using miRNeasy mini kit (Qiagen cat no217004 Germany) [11] RNA was quantified by spectrometry[12]

22 Reverse Transcription of Extracted RNA to Produce cDNA[13] One 120583g (1000 ng) of RNA was reverse-transcribedusing Maxima First Strand cDNA Synthesis Kit provided byThermo Scientific USA cat no K1641 The volume of RNAtaken was calculated for each sample separately according

to RNA concentration measured by nanodrop The reactionwas done by adding the following to calculated volumeof RNA 4 120583l 5X reaction mix (containing the remainingreaction components reaction buffer dNTPs oligo (dT)and random hexamer primers) and 2 120583l maxima enzymemix (containing maxima reverse transcriptase and ThermoScientific Ribolock RNase inhibitor) and the reaction wascompleted to 20 120583l by nuclease-free water Thus each 1 120583lof the reaction contains 50 ng of RNA The tubes wereincubated for 10 minutes at 25∘C followed by 15 minutes at50∘C The reaction was terminated by heating at 85∘C for 5minutes

PCR was done using the 2x PCR master-mix solution (i-Taq) provided by iNtRONBiotechnology to check for Tm andproduct length [14] It was done in a total reaction volume of20 120583l using 10 120583l PCR reaction mixture (1X) 16 120583l templateDNA (80 ng) 08 120583l of 10 120583M forward primer (400 nM)08 120583l of 10 120583M reverse primer (400nM) and 68 120583l distilledwater

Gene-specific primerswere purchased from Invitrogen byThermo Fisher Scientific Primer sets for the PCR amplifica-tion genes were selected after testing the sequence of the threegenes from NCBI database [15] Then these sequences weresubmitted in Primer3 tool and checked for product lengthmelting temperature GC ratio self-complementarity and 31015840complementarity

The following assays targeting specific mRNAs wereincluded in the study Homo sapiens TFF1 mRNA (for-ward primer 51015840- CCC-AGT-GTG-CAA-ATA-AGG-GC-31015840and reverse primer 51015840- GCT-CTG-GGA-CTA-ATC-ACC-GT - 31015840) Homo sapiens TFF3 mRNA (forward primer 51015840-TTT-TCT-GTC-CCT-TTG-CTC-CCndash 31015840 and reverse primer51015840- CCA-CGA-CGC-AGC-AGA-AAT-AA -31015840) and Homosapiens ACTBmRNA (forward primer 51015840- GTG-GCC-GAG-GAC-TTT-GAT-TG ndash31015840 and reverse primer 51015840-GTG-GGG-TGG-CTT-TTA-GGA-TG ndash31015840)

23 Quantitative PCRAnalysis [16] Real-time PCRwas donefor quantification of TFF1 and TFF3 gene using SensiFASTSYBR Lo-ROX (purchased from Bioline London UK cat-alog number BIO-94005) For each reaction the followingwas used 10 120583l of SensiFAST SYBR Lo-ROX (1X) 08 120583l of 10120583M forward primer (400nM) 08 120583l of 10 120583M reverse primer(400nM) and 16 120583l of template (80 ng) and each reactionwas completed to reach a total volume of 20 120583l by nucleasesfree water (68 120583l)

Initial denaturation was done by heating for 1 min at 95∘Cfollowed by 40 cycles of denaturation at 95∘C for 5 secondsand annealing extension at 60∘C for 30 seconds in 7500 Fastamp 7500 Real-Time PCR System (Applied Biosystem ThemoFisher Scientific Life Technologies Corporation USA)Melt-ing curve analysis was done after amplification to confirmthe specificity of the product and to exclude the presence ofprimerndashdimers

The relative gene expression analysis was done by DeltaDelta cycle threshold (DDCT)method and the average DCTof the healthy volunteers for each target gene was used asthe calibrator sample [17] The amount of target normalized

Journal of Oncology 3

Table 1 The median value of TFF1 and TFF3 between nonmetastatic and metastatic group

Nonmetastaticgroup(n=24)

Metastatic group(n=26) P valuelowast

TFF1lowastMedian(min-max) 016 (003-07) 047 (003-43) 0001

TFF3lowastMedian (min-max) 035 (001-097) 0585 (012-106) 0038

Significant P lt 005 lowastMannndashWhitney to show the difference in the median value of TFF1 and TFF3mRNA between nonmetastatic and metastatic groups

Figure 1 Box-whisker plot illustrates median (min-max) of TFF1 and TFF3mRNA level among the studied groups

to an endogenous reference and relative to a calibrator wascalculated The fold change is obtained by 2ndash119863119863119862119879 Thismethod assigns a value of 07 to the calibrator sample and allother quantities are expressed as an n-fold difference relativeto the calibrator

3 Statistical Analysis

Data was analyzed using SPSS 21 Parametric data wereexpressed in mean plusmn standard deviation Nonparametricdata were expressed in median minimum and maximumNormality of data was first tested by one sample K-S testMannndashWhitney was used to show the difference in themedian value of TFF1 and TFF3 mRNA between non-metastatic and metastatic groups Fisherrsquos exact test wasused to show number and percentage of their high andlow expression between studied groups P value lt 005 wasconsidered as statistically significant

4 Results

In our study all cases were femalesThey were cross-matchedwith each other as regards ageThemean age of cases was 575plusmn 76 years for nonmetastatic group and 560 plusmn 123 years formetastatic

There was significant difference in the median value ofTFF1 and TFF3 mRNA level between nonmetastatic andmetastatic groups (Table 1 Figure 1) The value of 07units was considered as cut-off value for both TFF1 andTFF3 mRNA level above which samples were considered tohave higher concentration and below which samples wereconsidered to have low concentration (Table 2 Figure 2)For both TFF1 and TFF3 this cut-off value was calculatedby the median of the fold change of the calibrator samplescalculated by 2ndash119863119863119862119879method According to this value 346ofmetastatic breast cancer patients showed high level of TFF1mRNA as compared to 0 of nonmetastatic Regarding TFF3mRNA level 462 of metastatic breast cancer patients hadhigher level as compared to 4 of nonmetastatic (Table 3Figure 3) Cases with high TFF1 mRNA were analyzed inrelation to the estrogen status of patients to show that 889were premenopausal (Table 4) 667 had ER positive pri-mary tumor and 444 had PR positive primary tumor Theanalysis of patients with high TFF3 mRNA concentrationsrevealed that 688 and 563 had ER and PR positiveprimary tumor respectively (Table 4)

The high level of TFF1 mRNA was related to the site ofmetastasis to show that 778 of cases had bone metastasiswhile 75 of cases with high TFF3 mRNA level showedlymph node involvement and 688 showed lung metastasis(Table 4)


Table 2The performance analysis of TFF1 and TFF3mRNA within metastatic and nonmetastatic groupsconsidered the value of 07 units ascut-off for both TFF1 mRNA and TFF3mRNA above which samples were considered to have higher concentration and below which sampleswere considered to have low concentration

Variables AUC Cut-off Sensitivity(95CI)

Specificity(95CI)

PPV(95CI)

NPV(95CI)

TFF1 0784 gt 07 3462(172-557)

1000(858-1000)

1000(664-1000)

585(421-737)

TFF3 0671 gt 07 4615(266-666)

8333(626-953)

750(476-927)

588(407-754)

CombinedTFF1ampTFF3 0705 ------- 5769

(369-766)833

(626-953)789

(544-939)645

(454-808)AUC area under the curve PPV positive predictive value NPV negative predictive value and CI confidence interval

Figure 2 ROC curve illustrates performance analysis of TFF1 and TFF3mRNA level within metastatic and nonmetastatic groups

Table 3 The number and percentage of high and low levels of TFF1and TFF3 mRNA between non metastatic and metastatic groups(regarding cut-off 070)

Nonmetastatic group(n=24)No ()

Metastatic group(n=26)No()

p-value

TFF1lowastHigh level 0 (00) 9 (346) 0002Low level 24 (1000) 17 (654)TFF3lowastHigh level 4 (167) 12 (462) 0026Low level 20(833) 14(538)Significant P lt 005 lowastFisherrsquos exact test to show number and percentage oftheir high and low expression between studied groups

5 Discussion

In this study we measured the level of both TFF1 andTFF3 mRNA in nonmetastatic and metastatic breast cancerpatientsThis was done bymeasuringmRNA in RNA isolatedfrom blood by density gradient centrifugation

We found significant difference in the concentration ofTFF1 mRNA in the blood of nonmetastatic patients versusmetastatic (p=0001) The median was 016 (003-07) innonmetastatic as compared to 047 (003-43) in metastaticpatients The median value of 07 units for TFF1 mRNAdetected in cells isolated from peripheral blood of fourteenhealthy women was defined as the threshold below whichsamples were considered to have lower TFF1mRNA concen-trations and above which samples were considered to havehigher concentrations This was used as the cut-off valueto differentiate nonmetastatic from metastatic patients Thisvalue gives us 3462 sensitivity and 100 specificity

In the present study the level ofTFF1mRNAwas differentsignificantly between the 2 groups (p=0002) It was high in346 of metastatic breast cancer patients as compared to0 of nonmetastatic This is in agreement with the resultsdescribed by Lasa et al [4] which showed that TFF1 ispositively expressed in the blood of 17 of metastatic breastcancer patients

As regards TFF3 we found significant difference inthe level of mRNA in the blood of nonmetastatic versusmetastatic patients (p=0038) The median of TFF3 was 035(001-097) in nonmetastatic as compared to 0585 (012-106)in metastatic group The median value of 07 units for TFF3

Journal of Oncology 5Va

lue T

FF 1

500

400

300

200

100

000

group

met

asta

tic

non

met

asta

tic

Valu

e TFF

3

1200

1000

800

600

400

200

000

group

met

asta

tic

non

met

asta

tic

Figure 3 Dot plot chart illustrates distribution of high TFF1 andTFF3mRNA levels within studied groups

mRNA detected in cells isolated from peripheral blood offourteen healthy women was defined as the threshold belowwhich samples were considered to have lower TFF3 mRNAconcentrations and above which samples were consideredto have higher concentrations This value gives us 4615sensitivity and 8333 specificity

In our study there was significant difference in the levelof TFF3 mRNA between the 2 groups (p=0026) It was highin 462 of metastatic breast cancer patients as compared to4 of nonmetastatic This is in agreement with the results ofstudy done by Livak and Schmittgen which showed that TFF3is positively expressed in the blood of 20ofmetastatic breastcancer patients [17]

The higher levels of TFF1 and TFF3 mRNA in CTCs ofsome metastatic breast cancer patients confirmed that theyact as tumor progression factors This might be explained bythe role described by Chaiyarit et al as signal transducers todecrease apoptosis increase tumor cell motility and increaseangiogenesis [18]

On the other hand the low level ofTFF1 andTFF3mRNAin the remaining metastatic breast cancer patients may beconsistent with the results of Buache et al [19] who proposedthat the TFF1 and TFF3 play a role during mammary glandmorphogenesis ontogenesis and remodeling So loss ofthese functions can lead to cancer progression [20]

Among the 9 cases of metastatic breast cancer (MBC)that have high TFF1 mRNA level we found that 8 caseswere premenopausal (889) This may confirm the resultsof the retrospective study of Markicevic et al [21] whichshowed that levels of TFF1 expression were significantlyhigher in breast tissue samples in premenopausal patientsthan in postmenopausal (p=002) The results of Ishibashi etal [22] showed that serum TFF1 in breast cancer patientswho were immunohistologically positive for TFF1 was signif-icantly higher than patients who were immunohistologicallynegative for TFF1 (P= 0017) Also Bohn et al [23] noticed intheir study that there was no significant difference betweenMBC and primary breast cancer (PBC) groups (P gt 005) asregards expression rates of TFF1

Within the 9 cases of MBC that have high TFF1 mRNAlevel we found 6 cases with ER + primary tumor (667)This is in accordance with the results of Haakensen et al[24] and Markicevic et al [22] who showed that TFF1 wasdifferentially expressed according to serum estradiol levelsand it was higher in patients with ER+ breast cancer

The previous results were in agreement with the studydone by Prest et al which concluded the presence of pro-moter containing an estrogen-response element (ERE) thatregulates the expression of the TFF1 The induction of TFF1is a primary response to estrogen and it is mediated by thebinding of the estrogen receptor complex to a 13-bp near-palindromic estrogen-response element GG TC AC GG TGGC C located 400 bases upstream of the TFF1 transcriptionstart site [25]

As regards TFF1 mRNA concentration in serum samplesof PR + breast cancer patients we found that only 4 cases withhigh TFF1 were PR+ (444) These results were differentfrom the results of Markicevic et al [21] who showed thatTFF1 was higher in patients with PR + breast cancer Theyexplained this by the fact that progesterone is estrogen-regulated protein and its level is related to estrogen levelCrosier et al [26] proved that there was a relationshipbetween the expression of the estrogen receptor progesteronereceptor and TFF1 in breast cancer In our study the smallsample size may be the cause of this difference

As regards the level of TFF3 mRNA in CTCs in ER +breast cancer tissue we found 688 (11 of 16) of patients withhigh TFF3 level had ER + primary tumor This may confirmthe results of Ahmed et al [27] who stated that TFF3 proteinexpression is associated with estrogen receptor expressionThey found that TFF3 expression was not detected in most ofthe tumors that do not express the estrogen receptor Therewas a strong positive correlation between estrogen receptorand TFF3 protein expression in tumor tissue They foundalso that the levels of TFF3 expression in the primary tumorwere associated strongly with its level in the correspondingmetastatic cells and the level was increased as the tumor cellsmoved along the metastatic cascade from the primary tumor


Table 4 The number and percentage of high TFF1 and TFF3 mRNA level in ER and PR positive and negative breast cancer patients Thenumber and percentage of high TFF1 mRNA level in pre- and postmenopausal breast cancer patients in cases with and without bonemetastasis and the number and percentage of high TFF3 mRNA level in patients with and without lymph node involvement and lungmetastasis

High TFF1 mRNA level (n=9) High TFF3 mRNA level (n=16)No No

ERPositive 6 667 11 688Negative 3 333 5 313PRPositive 4 444 9 563Negative 5 556 7 438Lymph node involvementPresent 12 750Absent 4 250Lung metastasisPresent 11 688Absent 5 313Menopause statusPremenopausal 8 889Postmenopausal 1 111Bone metastasisPresent 7 778Absent 2 222

Among the 16 cases that had high concentration of TFF3mRNA we found 9 cases with positive PR expression inprimary tumor (563) This is in accordance with Ahmedet al [27] who had foundstrong positive correlation betweenprogesterone receptor and TFF3 protein expression in tumortissue

The relation between the concentration of TFF3 mRNAand estrogen status is explained by the results of May andWestley who stated that TFF3 is an estrogen-responsive geneand its expression level is positively correlated with ER statusin breast cancerPandey et al [28] found that the coexpressionof TFF3 and ER positivity in breast cancer increased tumorinvasion and metastatic seeding

We should focus on the fact that ER TFF1 and TFF3 areexpressed in normal breast epithelial cells and are importantfor the normal physiology of the breast epithelium [29]All of them are under the control of estrogen The switchfrom a beneficial to a malignant behavior may result fromthe loss of tissue architecture and matrix remodeling whichposits the epithelial cells near fibroblasts muscle nerve andendothelial cells in an invasive tumor in contrast to thecompartmentalization provided by the myoepithelial cellsin normal mammary gland Besides breakdown in tissuearchitecture the inversion of cell polarity facilitates directsecretion of TFF peptides into the ectopic location of thetumor stroma where it will exert its biological effects [27]

In our study of the 9 cases ofMBC patients that have highlevel of TFF1mRNA there were 7 cases with bone metastasis(778) This is in agreement with the study of Wang et al[30] who found that 433 of patients with bone metastasis

exhibited a high expression level of TFF1 Similarly Smid etal [31] considered TFF1 most differentially expressed gene (Plt 0015) in breast cancer metastasis to bone

The fact that TFF1 may contribute to tumor metastasis tobone is proved by its high expression in MBC Emami et alexplained this by the ability of TFF1 to dimerizewith cysteine-rich molecules as cysteine-rich intestinal protein 1 (CRIP1)which also may be overexpressed in breast cancer and mightbe an interacting partner for TFF1 [32] The presence of ERpositive primary tumors has the strongest association withmetastasis to the bone [31]

In the present study out of the 16 cases that had highTFF3mRNA concentration we found 12 cases (75) had lymphnode metastases This confirmed the observation of Ahmedet al [27] that there was significant correlation between theoverexpressed TFF3 in breast cancer and the lymph nodemetastases They also found that the expression of TFF3 washigher in malignant cells that have metastasized away fromthan in those within the primary tumor

In our study we found 11 cases of high TFF3mRNA levelhad lung metastasis (688)This is consistent with the studyof Pandey et al [28] who made forced expression of TFF3 inmice and examined their ability to form metastatic nodulesThey found 4 of 6 mice with expressed TFF3 developed lungnodulesThey explained this by the increase in hypoxanthine-guanine phosphoribosyl transferase (hHPRT) activity inlung of mice with ER+ mammary carcinoma cell lines thatdeveloped increased expressed TFF3

Finally we analyzed the results of the combined analysisof TFF1 and TFF3 in the blood of breast cancer patientsThey


gave 5769 sensitivity and 833 specificity This means thatif both genes are overexpressed in blood of breast cancerpatients the risk of development of metastasis will be 5769

The use of TFF1 and TFF3 as markers in the CTCsof breast cancer patients is hopeful Their upregulation isassociated with breast cancer tumorigenesis Their detectionin CTCs of breast cancer patients confirms that the CTCsoriginated from the breast and maintains the properties ofbreast cancer cells [33]

The high concentration of TFF1 and TFF3 mRNAs inwomen with ER + primary tumors is exciting This mayraise the possibility that the measurement of TFF1 and TFF3mRNAs in cells isolated from peripheral blood can help theprediction of endocrine response degree and duration ofresponse The use of antiestrogens can antagonize estrogenmediated induction of TFF1 and TFF3mRNA expression

Conflicts of Interest

The authors declare that they have no conflicts of interest

References

[1] R L Siegel K D Miller and A Jemal ldquoCancer statisticsrdquo ACancer Journal for Clinicians pp 67ndash77 2017

[2] J E Talmadge and I J Fidler ldquoAACR centennial series thebiology of cancer metastasis historical perspectiverdquo CancerResearch vol 70 no 14 pp 5649ndash5669 2010

[3] A Van De Stolpe K Pantel S Sleijfer L W Terstappen andJ M J Den Toonder ldquoCirculating tumor cell isolation anddiagnostics Toward routine clinical userdquo Cancer Research vol71 no 18 pp 5955ndash5960 2011

[4] A Lasa A Garcia C Alonso et al ldquoMolecular detection ofperipheral blood breast cancermRNA transcripts as a surrogatebiomarker for circulating tumor cellsrdquo PLoS ONE vol 8 no 9Article ID e74079 2013

[5] L E Lowes and A L Allan ldquoRecent advances in the molecularcharacterization of circulating tumor cellsrdquo Cancers vol 6 no1 pp 595ndash624 2014

[6] L Thim ldquoA surprising sequence homologyrdquo Biochemical Jour-nal vol 253 no 1 p 309 1988

[7] S Kjellev ldquoThe trefoil factor family - Small peptides withmultiple functionalitiesrdquo Cellular and Molecular Life Sciencesvol 66 no 8 pp 1350ndash1369 2009

[8] M H Samson EM Vestergaard NMilman S S Poulsen andENexo ldquoCirculating serum trefoil factors increase dramaticallyduring pregnancyrdquo Scandinavian Journal of Clinical and Labo-ratory Investigation vol 68 pp 369ndash374 2008

[9] F E May ldquoThe potential of trefoil proteins as biomarkers inhuman cancerrdquo Biomarkers in Medicine vol 6 no 3 pp 301ndash304 2012

[10] D English and B R Andersen ldquoSingle step separation ofred blood cells granulocytes and mononuclear leukocytes ondiscontinuous density gradients of Ficoll Hypaquerdquo Journal ofImmunological Methods vol 5 no 3 pp 249ndash252 1974

[11] D E Macfarlane and C E Dahle ldquoIsolating RNA from wholebloodndashthe dawn of RNA-based diagnosisrdquoNature vol 362 no6416 pp 186ndash188 1993

[12] J Sambrook E F Fritschi and T ManiatisMolecular cloning alaboratory manual Cold Spring Harbor Laboratory Press NewYork NY USA 1989

[13] WM Freeman S J Walker and K E Vrana ldquoQuantitative RT-PCR pitfalls and potentialrdquo Biotechniques vol 26 no 1 pp 112ndash122 1999

[14] R K Saiki D H Gelfand S Stoffel et al ldquoPrimer-directedenzymatic amplification of DNA with a thermostable DNApolymeraserdquo Science vol 239 no 4839 pp 487ndash491 1988

[15] J Ye G Coulouris I Zaretskaya I Cutcutache S Rozen andT Madden ldquoPrimer-BLAST a tool to design target-specificprimers for polymerase chain reactionrdquo BMC Bioinformaticsvol 13 article 134 2012

[16] R Higuchi C Fockler G Dollinger and R Watson ldquoKineticPCR analysis real-time monitoring of DNA amplificationreactionsrdquo Nature Biotechnology vol 11 no 9 pp 1026ndash10301993

[17] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the 2-ΔΔCT methodrdquoMethods vol 25 no 4 pp 402ndash408 2001

[18] P Chaiyarit A Utrawichian C Leelayuwat et al ldquoInvestigationof trefoil factor expression in saliva and oral mucosal tissuesof patients with oral squamous cell carcinomardquo Clinical OralInvestigations vol 16 no 6 pp 1549ndash1556 2012

[19] E Buache N Etique F Alpy et al ldquoDeficiency in trefoil factor1 (TFF1) increases tumorigenicity of human breast cancer cellsand mammary tumor development in TFF1-knockout micerdquoOncogene vol 30 no 29 pp 3261ndash3273 2011

[20] D J Andrew and A J Ewald ldquoMorphogenesis of epithelialtubes Insights into tube formation elongation and elabora-tionrdquo Developmental Biology vol 341 no 1 pp 34ndash55 2010

[21] M Markicevic R Dzodic M Buta et al ldquoTrefoil factor 1 inearly breast carcinoma a potential indicator of clinical outcomeduring the first 3 years of follow-uprdquo International Journal ofMedical Sciences vol 11 no 7 pp 663ndash673 2014

[22] Y IshibashiHOhtsuM Ikemura et al ldquoSerumTFF1 andTFF3but not TFF2 are higher in women with breast cancer than inwomen without breast cancerrdquo Scientific Reports vol 7 no 12017

[23] O L Bohn I Nasir andA Brufsky ldquoBiomarker profile in breastcarcinomas presenting with bone metastasisrdquo InternationalJournal of Clinical and Experimental Pathology vol 3 no 2 pp139ndash146 2009

[24] V D Haakensen T Bjoslashro T Luders et al ldquoSerum estradiollevels associated with specific gene expression patterns innormal breast tissue and in breast carcinomasrdquo BMC Cancervol 11 article no 332 2011

[25] S J Prest F E B May and B R Westley ldquoThe estrogen-regulated protein TFF1 stimulates migration of human breastcancer cellsrdquo The FASEB Journal vol 16 no 6 pp 592ndash5942002

[26] M Crosier D Scott R G Wilson C D Griffiths F E Mayand B R Westley ldquoHigh expression of the trefoil protein TFF1in interval breast cancersrdquo American Journal of Pathology vol159 pp 215ndash221 2001

[27] A R H Ahmed A B Griffiths M T Tilby B R Westley andF E B May ldquoTFF3 is a normal breast epithelial protein and isassociated with differentiated phenotype in early breast cancerbut predisposes to invasion andmetastasis in advanced diseaserdquoThe American Journal of Pathology vol 180 no 3 pp 904ndash9162012


[28] V Pandey Z-S Wu M Zhang et al ldquoTrefoil factor 3 promotesmetastatic seeding and predicts poor survival outcome ofpatients with mammary carcinomardquo Breast Cancer Researchvol 16 no 5 p 429 2014

[29] R PoulsomAMHanby E-N Lalani FHauserWHoffmannand G W H Stamp ldquoIntestinal trefoil factor (TFF 3) and pS2(TFF 1) but not spasmolytic polypeptide (TFF 2) mRNAs areco-expressed in normal hyperplastic and neoplastic humanbreast epitheliumrdquo The Journal of Pathology vol 183 no 1 pp30ndash38 1997

[30] H Wang J Molina J Jiang M Ferber S Pruthi and TJatkoe ldquoGene expressionmarkers in circulating tumor cellsmaypredict bone metastasis and response to hormonal treatment inbreast cancerrdquoMolecular and Clinical Oncology vol 1 no 6 pp1031ndash1038 2013

[31] M Smid Y Wang J G M Klijn et al ldquoGenes associated withbreast cancer metastatic to bonerdquo Journal of Clinical Oncologyvol 24 no 15 pp 2261ndash2267 2006

[32] S Emami N Le Floch E Bruyneel et al ldquoInduction ofscattering and cellular invasion by trefoil peptides in src- andRhoA-transformed kidney and colonic epithelial cellsrdquo TheFASEB Journal vol 15 no 2 pp 351ndash361 2001

[33] D A Smirnov D R Zweitzig B W Foulk et al ldquoGlobal geneexpression profiling of circulating tumor cellsrdquoCancer Researchvol 65 no 12 pp 4993ndash4997 2005

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before the characterization of genetic make-up using qRT-PCR [5]

Trefoil factor family (TFF) peptides are a family of growthfactor-like peptides which was first proposed byThim in 1988[6] They are characterized by the presence of unique three-loop structure stabilized by intrachain disulfide bonds in 1205721-5 2-4 3-6 configuration between six conserved cysteineresidues

There are three TFF peptides TFF1 TFF2 and TFF3Their genes are clustered on chromosome 21q223 [7] Theexpression of TFF genes has been detected in all tissuescontaining mucus-secreting cells eg in gastrointestinaltract ocular tissues salivary glands respiratory tissuesprostate seminal plasma cervical secretions and milkTheircolocalization with mucus helps to form a stable gel-likemucus layer that plays a role in protection and healing of themucosa from harmful agents mechanical stress viruses andharmful pathogens [8]

TFF peptides are involved in mucosal maintenance andrepair throughmotogenic and antiapoptotic propertiesTheyalso function as scatter factors proinvasive and angiogenicagents So they are important for digestive processes inthe normal gastrointestinal tract where they act uniquely astumor suppressors In other organs including the breast theiroverexpression leads to cancer development and metastasis[9]

2 Patients and Methods

21 Blood Collection and Patients Peripheral blood wascollected into EDTA-tubes from 50 breast cancer femalepatients They were classified into two groups 24 cases ofnonmetastatic breast cancer and 26 cases of metastatic breastcancer They were treated in the Oncology Center MansouraUniversity Hospital Thirteen cases were premenopausalTwenty-eight of the 50 patients had positive estrogen receptorin the primary tumor and 28 had positive progesteronereceptor The metastases were located in bone in 13 casesand in lung in 10 cases Thirty-eight cases had lymphnode involvement Blood was taken from 14 healthy femalevolunteers A written informed consent was taken beforesample withdrawal The study was performed in accordancewith the ethical standards laid down in Mansoura Faculty ofMedicine

Peripheral blood mononuclear cells (PBMC) and CTCswere isolated from each blood sample by centrifugationthrough a Ficoll density gradient (Biocoll Separating Solu-tion) with density 1077 gml [10] It was purchased fromBiochrom-Gmb cat no L 6113 Germany Then total RNAwas then extracted using miRNeasy mini kit (Qiagen cat no217004 Germany) [11] RNA was quantified by spectrometry[12]

22 Reverse Transcription of Extracted RNA to Produce cDNA[13] One 120583g (1000 ng) of RNA was reverse-transcribedusing Maxima First Strand cDNA Synthesis Kit provided byThermo Scientific USA cat no K1641 The volume of RNAtaken was calculated for each sample separately according

to RNA concentration measured by nanodrop The reactionwas done by adding the following to calculated volumeof RNA 4 120583l 5X reaction mix (containing the remainingreaction components reaction buffer dNTPs oligo (dT)and random hexamer primers) and 2 120583l maxima enzymemix (containing maxima reverse transcriptase and ThermoScientific Ribolock RNase inhibitor) and the reaction wascompleted to 20 120583l by nuclease-free water Thus each 1 120583lof the reaction contains 50 ng of RNA The tubes wereincubated for 10 minutes at 25∘C followed by 15 minutes at50∘C The reaction was terminated by heating at 85∘C for 5minutes

PCR was done using the 2x PCR master-mix solution (i-Taq) provided by iNtRONBiotechnology to check for Tm andproduct length [14] It was done in a total reaction volume of20 120583l using 10 120583l PCR reaction mixture (1X) 16 120583l templateDNA (80 ng) 08 120583l of 10 120583M forward primer (400 nM)08 120583l of 10 120583M reverse primer (400nM) and 68 120583l distilledwater

Gene-specific primerswere purchased from Invitrogen byThermo Fisher Scientific Primer sets for the PCR amplifica-tion genes were selected after testing the sequence of the threegenes from NCBI database [15] Then these sequences weresubmitted in Primer3 tool and checked for product lengthmelting temperature GC ratio self-complementarity and 31015840complementarity

The following assays targeting specific mRNAs wereincluded in the study Homo sapiens TFF1 mRNA (for-ward primer 51015840- CCC-AGT-GTG-CAA-ATA-AGG-GC-31015840and reverse primer 51015840- GCT-CTG-GGA-CTA-ATC-ACC-GT - 31015840) Homo sapiens TFF3 mRNA (forward primer 51015840-TTT-TCT-GTC-CCT-TTG-CTC-CCndash 31015840 and reverse primer51015840- CCA-CGA-CGC-AGC-AGA-AAT-AA -31015840) and Homosapiens ACTBmRNA (forward primer 51015840- GTG-GCC-GAG-GAC-TTT-GAT-TG ndash31015840 and reverse primer 51015840-GTG-GGG-TGG-CTT-TTA-GGA-TG ndash31015840)

23 Quantitative PCRAnalysis [16] Real-time PCRwas donefor quantification of TFF1 and TFF3 gene using SensiFASTSYBR Lo-ROX (purchased from Bioline London UK cat-alog number BIO-94005) For each reaction the followingwas used 10 120583l of SensiFAST SYBR Lo-ROX (1X) 08 120583l of 10120583M forward primer (400nM) 08 120583l of 10 120583M reverse primer(400nM) and 16 120583l of template (80 ng) and each reactionwas completed to reach a total volume of 20 120583l by nucleasesfree water (68 120583l)

Initial denaturation was done by heating for 1 min at 95∘Cfollowed by 40 cycles of denaturation at 95∘C for 5 secondsand annealing extension at 60∘C for 30 seconds in 7500 Fastamp 7500 Real-Time PCR System (Applied Biosystem ThemoFisher Scientific Life Technologies Corporation USA)Melt-ing curve analysis was done after amplification to confirmthe specificity of the product and to exclude the presence ofprimerndashdimers

The relative gene expression analysis was done by DeltaDelta cycle threshold (DDCT)method and the average DCTof the healthy volunteers for each target gene was used asthe calibrator sample [17] The amount of target normalized












4 Results







Specificity(95CI)

PPV(95CI)

NPV(95CI)

TFF1 0784 gt 07 3462(172-557)

1000(858-1000)

1000(664-1000)

585(421-737)

TFF3 0671 gt 07 4615(266-666)

8333(626-953)

750(476-927)

588(407-754)


(369-766)833

(626-953)789

(544-939)645






p-value


5 Discussion






lue T

FF 1

500

400

300

200

100

000

group

met

asta

tic

non

met

asta

tic

Valu

e TFF

3

1200

1000

800

600

400

200

000

group

met

asta

tic

non

met

asta

tic






























References







































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of






























4 Results







Specificity(95CI)

PPV(95CI)

NPV(95CI)

TFF1 0784 gt 07 3462(172-557)

1000(858-1000)

1000(664-1000)

585(421-737)

TFF3 0671 gt 07 4615(266-666)

8333(626-953)

750(476-927)

588(407-754)


(369-766)833

(626-953)789

(544-939)645






p-value


5 Discussion






lue T

FF 1

500

400

300

200

100

000

group

met

asta

tic

non

met

asta

tic

Valu

e TFF

3

1200

1000

800

600

400

200

000

group

met

asta

tic

non

met

asta

tic






























References







































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of






















Specificity(95CI)

PPV(95CI)

NPV(95CI)

TFF1 0784 gt 07 3462(172-557)

1000(858-1000)

1000(664-1000)

585(421-737)

TFF3 0671 gt 07 4615(266-666)

8333(626-953)

750(476-927)

588(407-754)


(369-766)833

(626-953)789

(544-939)645






p-value


5 Discussion






lue T

FF 1

500

400

300

200

100

000

group

met

asta

tic

non

met

asta

tic

Valu

e TFF

3

1200

1000

800

600

400

200

000

group

met

asta

tic

non

met

asta

tic






























References







































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















lue T

FF 1

500

400

300

200

100

000

group

met

asta

tic

non

met

asta

tic

Valu

e TFF

3

1200

1000

800

600

400

200

000

group

met

asta

tic

non

met

asta

tic






























References







































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of






































References







































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of

























References







































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of






























of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of























of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



















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and TFF3 mRNAs Are Higher in Blood from Breast …downloads.hindawi.com/journals/jo/2018/4793498.pdfJournalofOncology Value TFF 1 5.00 4.00 3.00 2.00 1.00 0.00 group non metastatic