Molecular Biology and Nanomedicine

© The Author(s) 2021. / Viewed: / Downloaded:140 / Cited:0 / DOI:10.37813/j.mbn.2707-4692.014

Role of TGF-β/Smad Signaling Pathway-Related Proteins in Clinicopathological Features and Prognosis and Survival of Patients with Nasopharyngeal Carcinoma





Article

Role of TGF-β/Smad Signaling Pathway-Related Proteinsin Clinicopathological Features and Prognosis and Survival of Patients withNasopharyngeal Carcinoma

Yanwei Rao 1, RuiYang 2, Huimin Tang 3,*, Zhanshu Ma 4,*

1     Department of Critical Care Medicine, Jilin ProvincialPeople’s Hospital, Changchun130021, China; ywrao@hotmail.com

2     Department of Vascular Surgery, the Fifth Hospital ofWuhan, Wuhan430074, China;YR1975@126.com

3     Department of Radiotherapy, NingboMedical Centre (Li Huili Hospital), Ningbo 315040, China

4     Radiotherapy Department, the Firstaffiliated Hospital of Chifeng university, Chifeng 024000, China

*Correspondence to:271758051@qq.com (Tang H); 6917088@qq.com (Ma Z)

Received: 18 November 2021; Accepted: 28February 2021; Published:10March 2021

Abstract: Nasopharyngeal carcinoma(NPC)is the most common canceroriginating in the nasopharynx with high incidence of nasopharyngeal carcinoma. This study aimsto identify possible prognostic factors of related proteins in the TGF-β/Smadsignaling pathway in NPC.The expression of TGF-β1, TGF-βRI, TGF-βRII, TGF-β2, Smad4, Smad7 and RUNX3 inNPC tissues and nasopharyngitis tissues was detected. Besides, the association ofTGF-β/Smad signaling pathway-related protein expressions and prognosis ofNPC was analyzed.Initially,the NPC tissues showed higher expression of TGF-β1 and Smad7 and lowerexpression of Smad4, TGF-βRII,TGF-β2 and RUNX3. Meanwhile, the nonkeratinizing differentiated squamous cellcarcinoma showed higher positive expression of TGF-βRI,patients in stage III-IV presentedhigher positive expression of TGF-β1 and Smad7, and patients with lymph nodemetastasis showed higher positive expression of TGF-β1 and Smad7. Furthermore,TGF-β1, TGF-β2 and Smad4 were independent factors for the prognosis of NPC. Our study suggests that NPC presents withup-regulated expression of positive expression rates of TGF-β1 and Smad7 anddown-regulated TGF-β2,TGF-βRⅡ, Smad4 and RUNX3, and additionally, TGF-β1, TGF-β2 and Smad4 areindependent factorsfor the prognosis of NPC, which could be regarded as the novel target for thetreatment of NPC.

Keywords: nasopharyngealcarcinoma; TGF-β; Smad; TGF-β/Smad signaling pathway; clinicopathological features; prognosis

1.Introduction

Nasopharyngealcarcinoma (NPC) ranks the 11th common cancer with high incidence in China, withthe rate of approximately 2.8/100,000 among men and about 1.9/100,000 amongwomen[1]. NPC has obvious geographicaldistribution characteristics, mostly occurred in the southern area of China[2]. Generally, NPC is caused by the riskfactors of Epstein-Barr virus (EBV), genetic predisposition, smoking andalcohol[3]. Radiotherapy is the basic clinicaltreatment for patients with early NPC, and intensive modulated radiationtherapy (IMRT) involved with a variety of systemic therapy has demonstrated asignificant effect in recent years[4].Although the local control rate ofIMRT for NPC can reach up to 90%, the prognosis of NPC reduced considerably[5].With further study of the regulationmechanism of NPC, a growing number of signaling pathways, such as the epidermal growth factorreceptor (EFGR) signaling pathway[6]and oncoprotein 18/stathmin signalingpathway[7]were disclosed, which would affectthe development process of NPC and play a key regulatory role in NPC.Therefore, the signaling pathway for the regulation of NPC caused extensiveconcern.

Transforminggrowth factor β (TGF-β) is first found to induce phenotypic transformation infibroblasts, and one study found that TGF-β exerts a key regulatory function ona variety of tumor cells and non-tumor cells[8]. TGF-β can inhibit the replication ofnormal epithelial cells and regulate epithelial cells for migration, differentiationand apoptosis; meanwhile, TGF-β can promote proliferation and growth of tumorcells[9]. The number of amino acids for Smadproteins is generally 400 to 500 with molecular weight of about 42 ~ 60 ku, and Smad proteinsare the substrate for the extracellular kinase for TGF-β receptor yetdiscovered. Among which, smad4 in Smads family is a newly discovered tumorsuppressor gene, and smad7 play a role in inhibition of TGF-β signal pathway[10]. A previous study showed that, someabnormalities in TGF-β signaling pathway, such as downstream abnormal signalingproteins Smads due to gene mutations or loss, were also proved to be animportant mechanism related to carcinogenesis[11]. Therefore, this article intends tofurther investigate therole of TGF-β/Smad signaling pathway in clinicopathological features andprognosis of patients with NPC.


2. Material and methods

2.1. Ethical Statement

This study wascarried out in conformity to medical ethical standards and was approved byEthics Committee of Jilin Provincial People’s Hospital. Written informedconsents were obtained from eligible patients.

2.2. Subjects

The archivedparaffin blocks of biopsy specimens from 142 patients with pathologicallydiagnosed NPC in Jilin Provincial People’s Hospital between March 2009 andMarch 2011 were collected. There were 112 males and 30 females, aged from 19 to78 (median age of 44.6) years old. Patients were enrolled into this study ifthey met the following criteria: 1, diagnosed with NPC through clinical symptoms,signs, computed tomography (CT) or magnetic resonance imaging (MRI), andhistopathology; 2, with complete imaging data; 3, aged between 18 to 70 yearsold; 4, Karnofsky score not less than 70 points. Correspondingly, thesepatients were excluded: 1, patients receiving chemotherapy as well asradiotherapy sensitizers and radiation protective agent; 2, patientsexperienced surgery or had received radiotherapy or chemotherapy; 3, patientshad a history of other tumors. Specimens were fixed in 10% formalin, embeddedin paraffin and cut into 4 μm serial sections. According to the Internationalhistological classification criteria of World Health Organization (WHO) [12], specimens were classified as follows: thekeratinizing squamous cell carcinoma (n = 20), non-differentiated keratinizingsquamous cell carcinoma (n = 29) and non-keratinizing undifferentiated squamouscell carcinoma (n = 93). All cases had not received radiotherapy orchemotherapy before surgery. The clinical staging of NPC was based on AmericanJoint Committee on Cancer (AJCC) staging system edited in 2002 [13] and the cases were classified as follows:stage I (n = 4), stage II (n = 30), stage III (n = 56) and stage IV (n = 52). A total of 54 cases with nasopharyngitisconfirmed by biopsy over the same period were randomly selected for controlgroup. Among which there were 40 males and 14 females, aged between 30 ~ 70years old, with mean age of (44.2 ± 9.1) years old and the median age of 48years old. There was no significant difference of gender and age between twogroups (both p > 0.05).

2.3. ImmunohistochemicalStaining

Immunohistochemicalstaining (streptavidin-peroxidase (SP) method) was applied to detect theexpression of TGF-β1, TGF-βRI,TGF-βRII, TGF-β2, Smad4, Smad7 andRUNX3 in nasopharyngitis tissues and NPC tissues. Antigen retrieval and SPstaining were performed under instructions of an immunohistochemistry kit(purchased from Fuzhou Maixin Biotech. Co., Ltd, Fuzhou, China). The known NPCslice was used as a positive control, with phosphate buffer saline (PBS) as anegative control instead of primary antibody.

The determinationof results: brown particles occurred inside the nucleus and (or) the cytoplasmfor Smad4 and TGF-ΒRI as positive, brown colorationoccurred for TGF-β1, TGF-βRII,TGF-β2, Smad7, and RUNX3 inside membrane and (or) cytoplasm as positive.Staining results were determined based on each slice with five high-powerfields, where positive cells were calculated within 500 cells, marked (-) fornumber of positive cells < 20% and (+) for ≥ 20%. The results determined bytwo independent pathologists, or decided by senior pathologist in case ofdispute.

2.4. Reverse TranscriptionQuantitative Polymerase Chain Reaction (RT-qPCR)

Tissues orperipheral blood cells in 1 mL of Trizol homogenizing tube, were placed on ice,with ethylene diamine tetraacetic acid (EDTA) used as anticoagulant. Total RNAisolation was performed with RNA extraction tool according to the instructionsof reagent (Shanghai Huashun Biotechnology Company, Shanghai, China). RNA wasreversely transcribed at 42°Cfor60 min using 20 units of avian myeloblastosis virus (AMV) reverse transcriptase(BBI), 0.5 ng oligosaccharides (dT) 12–18 primer, 0.5 mM nucleotides and 20 unitsof ribonuclease (20 μL). The sample was heated for 10 min and then the reactionwas terminated when the temperature was up to 70°C. After reversetranscription, the reaction system (20 μL) consisted of 1 × PCR reactionbuffer, 0.2 mM nucleotides, and 0.5 μM primers(Table 1)and 1 unit of DNA polymerase. The reactionconditions for TGF-βRII,Smad4 and Smad7 were as follows: at 95°C for 30 s, cooling to 55°C for another30 s, and finally warmed to 72°C for45 s. The reaction cycle for TGF-βRII was set to 30, and for Smad4 and Smad7 were 28. The reactionconditions for TGF-β1 and RUNX3 were as follows: pre-denaturation at 95°Cfor 30 s, 45 cycles of cooling at 95°C foranother 5 s, and finally warmed to 60°C for 30 s. The reaction conditions for TGF-β2and TGF-βRI wereas follows: pre-denaturationat 95°C for 3 min, 45 cycles ofdenaturation at 94°Cfor 50 s,annealing at 60°Cfor 50 s, andextension at 72°Cfor 50 s. Finally,all the proteins was extended at 72°C for 10 min. PCR products were visualized by2% agarose gel electrophoresis containing ethidium bromide as fluorescent dye.


Table 1. Theprimer sequences for RT-qPCR.

Gene

Primer sequence (5'-3')

TGF-β1

F: TGG CGA TAC CTC AGC AAC

R: CTC GTG GAT CCA CTT CCA  G

TGF-βRI

F: ACC TTC TGA TCC ATC CCT T

R: CCC AAA GCT GTC AGC CTAG

TGF-βRII

F:AGC AACTGC AGCATC ACCTC

RTGA TGTCTG AGAAGA TGTCC

TGF-β2

F AAA ATG CAC TAC TGT GTG C

R: CTG CAT TTG CAA GAC TTT AC

Smad4

F:GCA TCG ACA GAGACA TACAG

R:CAA CAG TAA CAA TAG GGCAG

Smad7

F:ACC GCAGCA GTTACC CCATCT T

R:GGC TACCGG CTG TTG AA

RUNX3

F: TTA CGA GGG GCG GTC GTA CGC  GGG

R AAA ACG ACC GAC GCG AAC GCC TCC

β-actin

F:ACC ACA GTC CAT GCC ATC AC

R:TCC ACC ACC CTGTTG CTG TA

Notes: RT-qPCR, reverse transcription quantitativepolymerase chain reaction; TGF-β, transforming growth factor β; F, forward; R, reverse.

2.5. Follow-up

Follow-up wasstarted when the treatment completed. The follow-up, performed every 3 monthsin the first two years of follow-up, included checking, mail and telephoneinterview. By February 31, 2016, no patients were lost to follow-up. Records ofsurvival condition of each patient were recorded during the follow-up period.All patients had completed clinical data and five-year follow-up records.

2.6. Statistical Analysis

SPSS 21.0 softwarepackage (IBM Corp., Armonk, New York, USA) was used for statistical analysis ofexperimental data. The χ2 test and Fisher's exact probability in 2 × 2 tableswere used for the differences of TGF-β/Smad signaling pathway-related proteinsexpression. Spearman rank correlation analysis was used for correlationanalysis with p < 0.05 indicated a significant difference. Forunivariate analysis, Kaplan-Meier survival curve was used to analyze therelationship between TGF-β/Smad signaling pathway-related proteins expressionand prognosis of NPC, with log-rank test for the difference of survival curves.COX proportional hazard model was also used for multivariate survival analysis.

3. Results

3.1. The Positive ProteinExpression of TGF-β1 and Smad7 was Increased while that of Smad4, TGF-βRII,TGF-β2 and RUNX3 was Decreased in NPC tissues

Immunohistochemicalstaining was conducted to examine the protein expression of TGF-β/Smadsignaling pathway-related proteins in tissues. The results showed that, TGF-βRI expressed mainly in the cytoplasm and(or) nucleus, TGF-βRII expressed mainly in themembrane and (or) cytoplasm, Smad4 protein expressed in the cytoplasm and (or)within the nucleus, TGF-β2, Smad7 and RUNX3 expressed in the cytoplasm withpositive signals localized in the cytoplasm. The difference of positiveexpression rates of TGF-βRIbetween NPC and nasopharyngitis tissues were notstatistically significant (p > 0.05). However, the positiveexpression rates of TGF-β1 and Smad7 in the NPC tissues were higher than thatin the nasopharyngitis tissues, and the positive expression rate of Smad4,TGF-βRII, TGF-β2 and RUNX3 in the NPCtissues were significantly lower than those in the nasopharyngitis tissues (allp < 0.05)(Table 2 and Figure 1). The results above revealed that higherpositive protein expression of TGF-β1, Smad7 and lower that of Smad4, TGF-βRII, TGF-β2 and RUNX3 were found in NPCtissues.


Figure 1. NPC tissues show higher positiveprotein expression of TGF-β1 and Smad7 and lower Smad4, TGF-βRII, TGF-β2 and RUNX3 (SP × 400). Notes: Panel A, positive expression of TGF-β1; Panel B, positiveexpression of Smad7; Panel C, negative expression of TGF-β2; Panel D,negative expression of RUNX3; Panel E, negative expression of TGF-βRI;Panel F, negative expression of TGF-βRII; Panel G, negativeexpression of Smad4. NPC, nasopharyngeal carcinoma; TGF-β, transforming growth factor β.

Table 2. The protein expression of TGF-β/Smadsignaling pathway-related proteins between NPC tissues and nasopharyngitistissues.

Factor

Result

NPC tissues (n = 142)

Nasopharyngitis tissues (n = 54)

χ2

p

TGF-β1

Positive

90

12

26.550

<0.001

Negative

52

42

26.550

2.526

<0.001

0.112

TGF-βR

Positive

101

32

Negative

41

22

2.526

4.020

0.112

0.045

TGF-βR

Positive

92

43

Negative

50

11

18.08

0.045

0.001

TGF-β2

Positive

100

50

10.710

Negative

42

4

18.08

0.001

0.022

Smad4

Positive

98

46

5.249

Negative

44

8

18.08

<0.05

Smad7

Positive

54

6

13.340

<0.001

Negative

88

48

18.08

<0.05

RUNX3

Positive

80

49

20.580

<0.001

Negative

62

5

18.08

<0.05

Notes: TGF-β, transforming growthfactor β; NPC, nasopharyngeal carcinoma.

3.2. NPC Tissues Show HighermRNA Expression of TGF-β1 and Smad7, and Lower that of Smad4, TGF-βRII, TGF-β2and RUNX3

Next, RT-qPCR wasconducted to examine the mRNA expression of TGF-β/Smad signalingpathway-related proteins in tissues. The mRNA expression of TGF-β1 and Smad7 inthe NPC tissues was significantly higher than those in the nasopharyngitistissues (p < 0.01). The mRNA expression of TGF-βRI between the NPC and nasopharyngitistissues was not statistically significant (p > 0.05). The mRNAexpression of TGF-βRII, TGF-β2, Smad4 and RUNX3 inthe NPC tissues was lower than that in the nasopharyngitis tissues (all p< 0.05) (Figure 2). Therefore, the higher mRNA expression of TGF-β1and Smad7 and lower that of Smad4, TGF-βRII, TGF-β2 and RUNX3 were found in the NPCtissues.






Figure 2.NPC tissues show higher mRNA expression ofTGF-β1 and Smad7, and lower Smad4, TGF-βRII, TGF-β2 and RUNX3. Note: NPC, nasopharyngeal carcinoma; TGF-β, transforming growthfactor β.


3.3. NPC is Relevant to theHistological Classification, the Clinical Staging and Lymph Node Metastasis

Subsequently,results of examining the relationship between TGF-β/Smad signaling pathway andclinicopathological features of NPC showed that no significant difference wasfound in the positive expression rates of TGF-β1, TGF-βRI, TGF-βRII, TGF-β2, Smad4, Smad7 and RUNX3 in NPCpatients with different gender and age (all p > 0.05). The positiveexpression rate of TGF-βRI innon-keratinizing differentiated squamous cell carcinoma was significantly lowerthan that in non-keratinizing undifferentiated squamous cell carcinoma (p< 0.05). The positive expression rates of TGF-β1 and Smad7 in patients in stage III-IV were significantlyhigher than patients in stage I-II (both p < 0.05). The positiveexpression rate of Smad4 in patients without lymph node metastasis wassignificantly higher than those with lymph node metastasis (p <0.05). The positive expression rates of TGF-β1 and Smad7 in patients with lymphnode metastasis were higher than those without lymph node metastasis (all p< 0.05) (Table 3). All these results suggested that NPC wasrelated to the histological classification, the clinical staging and lymph nodemetastasis.

3.4. The Prognostic Survival isRelated to TGF-β1, TGF-β2, Smad4 and Smad7






Follow-up was performedto observe the association of TGF-β/Smad expression and prognosis of NPCpatients. The total five-year survival rate of patients was 47.8%. There weresignificant differences for the five-year survival rate in positive cases ofTGF-β1, TGF-β2, Smad4 and Smad7 when compared with those negative cases (all p< 0.05). However, no significant difference was found for the five-yearsurvival rate between the positive cases of TGFβRI, TGFβRIIand RUNX3 and those negative cases (all p > 0.05)(Figure 3). Therefore, TGF-β1, TGF-β2, Smad4 and Smad7were related to the prognosis of patients in NPC.


Figure 3. The prognostic survival of NPCis related to TGF-β1, TGF-β2, Smad4 and Smad7. Notes: Panel A, curve for TGF-β1; Panel B, curve for TGF-βRI; Panel C, curvefor TGF-βRII; Panel D, curve for TGF-β2; Panel E, curve forSmad4; Panel F, curve for Smad7; Panel G, curve for RUNX3. NPC, nasopharyngeal carcinoma; TGF-β, transforming growthfactor β.


Table3. Therelationship between TGF-β/Smad expressions and clinicopathological features inNPC tissues.

Features

Case

TGF-β1

P

TGF-βRI

P

TGF-βRII

P

TGF-β2


P

Smad4

P

Smad7

P

RUNX3

P

+

-

+

-

+

-

+

-


+

-

+

-

+

-

Gender




0.995



0.544



0.808




0.955



0.754



0.802



0.229

Male

112

71

41


81

31


72

40


79

33



78

34


42

70


66

46


Female

30

19

11


20

10


20

10


21

9



20

10


12

18


14

16


Age  (years)




0.779



0.931



0.147




0.512



0.754



0.922



0.897

45

77

48

29


55

22


54

23


56

21



54

23


29

48


43

34


≥45

65

42

23


46

19


38

27


44

21



44

21


25

40


37

28


Pathological  type




0.052



0.028



0.580




0.978



0.505



0.415



0.698

NonkeratinizingSquamousCellCarcinoma

20

8

12


14

6


11

9


14

6



16

4


5

15


10

10


Nonkeratinizing  undifferentiated squamous cell carcinoma

29

21

8


15

14


20

9


20

9



19

10


11

18


18

11


Nonkeratinizing  differentiated squamous cell carcinoma

93

61

32


72

21


61

32


66

27



63

30


38

55


52

41


Skull  base encroachment




0.960



0.420



0.225




0.217



0.698



0.165



0.166

No

87

55

32


64

23


53

34


58

29



59

28


37

50


53

34


Yes

55

35

20


37

18


39

16


42

13



39

16


17

38


27

28


Clinical  stage




0.024



0.937



0.991




0.981



0.054



0.046



0.259

I+II

34

16

18


24

10


22

12


24

10



28

6


8

26


22

12


III+IV

108

74

34


77

31


70

38


76

32



70

38


46

62


58

50


Lymph  node metastasis




0.034



0.530



0.266




0.516



0.001



0.001



0.151

No

60

32

28


41

19


42

18


44

16



57

3


5

55


38

22


Yes

82

58

24


60

22


50

32


56

26



41

41


49

33


42

40


Notes:TGF-β,transforming growth factor β; NPC, nasopharyngeal carcinoma.


3.5. TGF-β1, TGF-β2 and Smad4are Independent Factors for the Prognosis of NPC

COXproportional hazard model was applied to multivariate survival analysis for theprognosis of NPC patients. The clinicopathological features, including TGF-β1,TGF-βRI, TGF-βRII, TGF-β2, Smad4, Smad7, RUNX3, clinicalstage and lymph node metastasis, were included into COX proportional hazardmodel. The results showed that TGF-β1, TGF-β2 and Smad4 were independentfactors for the prognosis of NPC (all p < 0.05)(Table 4).

Table4. Multivariatesurvival analysis for the prognosis of NPC patients.

Factor

B

SE

Wald

Sig.

Exp  (B)

95%  CI

TGF-β1

0.826

0.347

5.682

0.017

2.284

1.158–4.505

TGF-βRI

0.344

0.284

1.468

0.226

1.410

0.809–2.459

TGF-βRII

0.138

0.264

0.271

0.603

1.147

0.683–1.927

TGF-β2

−1.071

0.250

18.295

0.001

0.343

0.210–0.560

Smad4

−2.584

1.186

4.744

0.029

0.076

0.007–0.772

Smad7

−0.319

0.582

0.301

0.583

0.727

0.232–2.273

RUNX3

−0.187

0.242

0.595

0.440

0.830

0.516–1.334

Clinical  stage

0.371

0.272

1.860

0.173

1.449

0.850–2.470

Lymph  node metastasis

−0.120

0.335

0.127

0.721

0.887

0.460–1.711

Notes: TGF-β, transforming growthfactor β; NPC, nasopharyngeal carcinoma; B, regression coefficient; S.E,standard error; OR, odd ratio; CI, confidence interval.

4. Discussion

NPCis a serious threat to human health, and most NPC are poorly differentiated,which prone to easy invasion and metastasis [14]. Currently, IMRT is widely used in thetreatment for NPC, which significantly improve life of quality and survivalrate of NPC patients. However, researchers found that the relapse andmetastasis of NPC after treatment occurred in a certain proportion of patients[15]. Therefore, researchers try to reveal thepotential mechanism of NPC on the genetic level, in order to provide new ideasfor the treatment of NPC and improve the cure rate of NPC.

Thestudy found that the expression of TGF-β1 and Smad7 have an increase tendencyin NPC patients. TGF-β1 is one of the three isoforms from TGF-β superfamily,mainly expressed in the immune system, and the cancer cells may overproduceTGF-β as well as induce TGF-β production of peripheral tumor inmicroenvironment, which indicated that TGF-β1 expression would be increased inNPC [16]. In addition, TGF-β1 would play a dual rolein tumor development process, which not only play a role in the inhibition oftumor cell proliferation, but also induce apoptosis of liver cells, bone marrowcells, and epithelial cells[17]. The study concentrating on the mechanismof TGF-β1 in colorectal cancer showed that, TGF-β1 produced by tumor cells cancause reduced permeability of antigen presenting cell, thereby increasing thedegree of malignancy of the tumor and significantly reducing patient survivalrate, which is consistent with the results of our study [18]. Smad7 is an inhibitory factor of TGF-βsignaling pathway, abnormal expression of which can lead to TGF-β signalingpathway disorder, resulting in a variety of diseases. For example, Smad7exhibit low expression in normal epithelial tissues, whereas in humanpancreatic cancer, Smad7 expression was significantly increased; all of whichis consistent with the results of this experiment[19]. The study found that both TGF-β1 and Smad7expression in NPC have shown up-regulation in tumor cells, which demonstratetheir synergistic promotion role in tumor development.

Thestudy also found that, RUNX3, TGF-β2, and Smad4 expressions in the NPC tissueswere lower than those in the nasopharyngitis tissues. Smad4 is one of theindependent prognostic factors of NPC, and low expression of Smad4 is indicatedof poor prognosis. TGF-βRII isone of the TGF-β1 receptors on the other hand. The researchers found the factthat the cancer cells become insensitive to TGF-β was related to reducedexpression or inactivation of TGF-βRII in tumor cell surface, and the deletion or inactivation of itsexpression can promote tumor proliferation, invasion and metastasis [20]. Tumor suppressor protein RUNX3 is downstreamtranscription factor, and RUNX3 protein expression coded by RUNX3 gene wassignificantly down-regulated in NPC, with a suggestion that RUNX3 may be aprotective factor in NPC; additionally, RUNX3 inactivation is a reflection ofweakened defense force as well, RUNX3 and TGF-β2 expressions in NPC werepositively correlated, which indicated that these two factors may have asynergistic effect on the progress of NPC as a protective factor[21]. Smad gene family located on chromosome18q21, and Smad proteins are essential regulatory factor in TGFβ signalingpathway [22]. Researchers who also detected lowexpression of Smad4 in colorectal cancer had proposed low Smad4 expressionwould inhibit tumor growth pathways by blocking TGF-β, so as to promote the developmentand progression of colorectal cancer; in addition, low expression of Smad4 wasindependent of tumor invasion and metastasis promoted by TGF-β, which alsosuggesting that Smad4 was an independent prognostic factor for NPC[23]. Furthermore, studies about the prognosticvalue of Smad4 expressions in colorectal cancer showed that cancer withmetastasis have lower expression of Smad4, and low expression of Smad4 was themain reason for the metastasis of colorectal cancer[24,25].

Insummary, the findings of this study suggest that TGF-β1 and Smad7 expressionwere up-regulated and the expressions of TGF-β2, TGF-βRII, Smad4 and RUNX3 were down-regulated inNPC. Besides, TGF-β1, TGF-β2 and Smad4 were independent factors in theprognosis of NPC. Thus, up-regulation of TGF-β2 and Smad4 expressions anddown-regulation of TGF-β1 expression could be an effective way for NPCtreatment. Yet without adequate experimental evidence, the experimental resultsfor the clinical significance remain to be confirmed.

Funding: None.

Conflictsof Interest: All authors declare no conflict of interest.

Copyright Statement

CC BY

©2021 the authors. This article is an open  access article licensed under the terms and conditions of the CREATIVE  COMMONS ATTRIBUTION (CC BY) LICENSE (http://creativecommons.org/licenses/by/4.0/).

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