Considering the Downregulation of Tpm1.6 and Tpm1.7 in Squamous Cell Carcinoma of Esophagus As a Potent Biomarker

Research Article

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Considering the downregulation of Tpm1.6 and Tpm1.7 in squamous cell carcinoma of esophagus as a potent biomarker

Maryam Zare*,1, Faranak Hadi2 & Mohammad Reza Alivand3 1Department of Biology, Faculty of Sciences, Payame Noor University, Tehran, Iran 2Department of Biology, Faculty of Sciences, Lorestan University, Khorramabad, Iran 3Department of Medical Genetics, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran *Author for correspondence: [email protected]

Aim: Squamous cell carcinoma of esophagus (SCCE) is an aggressive disease with a poor prognosis. Tropomyosins attach to actin microfilaments, providing its stability. Nonmuscle cells express Tpm isoforms such as Tpm1.6 and Tpm1.7 which are involved in cytoskeleton functional properties regulation. Materials & methods: The expression of Tpm1.6 and Tpm1.7 was analyzed in SCCE tissues and its association with clinicopathological parameters and survival of patients was assessed. Results: Tpm1.6 and Tpm1.7, besides TPM1 mRNA decreased considerably in SCCE tissues relative to normal esophageal tissues (p < 0.001). TPM1 downregulation level was significantly associated with the degree of tumor differentiation (p = 0.017). Conclusion: Tpm1.6 and Tpm1.7 suppression play a crucial role in esophagus tumorigenesis and could be associated with SCCE poor prognosis.

First draft submitted: 16 February 2018; Accepted for publication: 14 May 2018; Published online: 27 September 2018

Keywords: squamous cell carcinoma of esophagus • tropomyosin • biomarker • clinicopathological • differentiation • downregulation • histological grade • survival

Esophageal cancer is a highly lethal malignancy, which ranks as the eighth most common cancer and the sixth most widespread cause of cancer-related deaths globally. More than 450,000 new cases are diagnosed each year worldwide [1–4]. Adenocarcinoma of esophagus and squamous cell carcinoma of esophagus (SCCE), as the most frequent form throughout the world, are major histological types of esophageal cancer. The incidence of SCCE is highly variable according to region, the most prevalent being south eastern and central Asia which accounts for 79% of the total worldwide SCCE cases [5,6]. Iran is located in the Asian esophageal cancer belt and has the greatest prevalence rate [2,5,7]. The absence of a serous membrane on the outer surface of the esophagus promotes the speedy progression of SCCE to nearby tissues and metastasis to local lymph nodes and other organs. Despite improvement in multimodality therapy, the prognosis of SCCE remains poor and the overall 5-year survival rate is still low, given that more than 85% of SCCE patients die within 2 years of diagnosis [8,9]. Therefore, comprehensive understanding of basic alterations in SCCE is essential to make possible advances in its diagnosis and new treatment strategies. Although studies have indicated the involvement of both genetic and epigenetic alterations in SCCE carcinogenesis [10–17], the precise mechanism of SCCE is unknown and requires further investigation. The actin cytoskeleton plays an essential role in the regulation of multiple cellular functions and maintenance of a normal cell phenotype. Disorganization of the actin cytoskeleton is commonly associated with neoplastic transformation [18,19]. The expression of cytoskeleton-related proteins such as gelsolin, vinculin, caldesmon and myosin light chain alters during transformation, leading to profound changes in the organization of the cytoskeleton [20–23]. Tropomyosins (Tpm) are a family of rod-like, coiled-coil dimer proteins that bind to actin microﬁlaments, providing structural stability and modulating cytoskeleton functions [19,24–26]. In mammals, Tpms are expressed from four genes (α, β, γ and δ or TPM1, TPM2, TPM3 and TPM4, respectively) that produce more than 40 isoforms through different promoters and the process of alternative splicing which are expressed variably in different tissues. The family is separated into high molecular weight (HMW; 284–285 amino acids [a.a.] and ∼38 kD) and low

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molecular weight (245–248 a.a. and ∼32 kD) groups [18,19]. Multiple isoforms of Tpm are expressed in nonmuscle cells, including Tpm2.1sm/cy (Tpm1.2), Tpm1.6cy (Tpm1.6) and Tpm1.7cy (Tpm1.7) [27]. The prominent function of Tpms in muscle contraction is clearly defined, while they possess more complex functions related to the organization and dynamics of the cytoskeleton in nonmuscle cells [19,24,25]. Major isoforms of Tpm, including Tpm1.6, are downregulated in transformed cells and are likely associated with transformed phenotypic characteristics [18,28,29]. Moreover, reduced expression of Tpm has been found in epithelial cell-originated human cancers such as breast, bladder, colon, prostate and renal cell carcinoma (RCC) and oral squamous cell carcinoma [28,30–34]. It has also been shown that Tpm2.1 possesses tumor suppressor activity, since its re-expression in transformed cells could reverse several aspects of tumorigenic phenotypes, such as anchorage- independent growth and resistance to anoikis [18,35]. We have previously reported the downregulation of HMW Tpms in the SCCE cell line and further identified the possible underlying mechanism of its suppression [13]. Considering the importance of Tpm in tumorigenicity, the association of Tpm alteration in human cancers with the clinicopathological characters of patients has rarely been analyzed [33,34]. In the present study, the expression pattern of TPM1 gene and its alternative spliced products of Tpm1.6 and Tpm1.7 were evaluated in freshly collected SCCE tumor and normal esophageal tissues and its association with prognosis and survival rates of patients have been assessed for the first time. The findings reveal significant downregulation of Tpm1.6 and Tpm1.7 in SCCE. Moreover, decreased expression of these isoforms is shown to correlate with suppression of TPM1 mRNA, indicating that tropomyosin downregulation occurs essentially at the transcription level in esophageal cancer.

Materials & methods Tissue samples Fresh tumor tissue samples along with adjacent normal tissue samples of esophagus were obtained from SCCE patients following esophagectomy. Total samples of 49 patients (26 males and 23 females) were included in this study. All samples were collected immediately after surgery, snap frozen in liquid nitrogen and transferred to the laboratory. The samples were stored at -80◦C until use. All samples were conﬁrmed for origination from the esophagus and the status of differentiation was identiﬁed by pathological examination. The age of the patients at the time of diagnosis ranged from 47 to 88 years with a mean of 62 years. The survival rate of patients was monitored for 5 years. This study was approved by the ethical committee of Imam Khomeini Hospital and the tumor bank of Tehran University of Medical Sciences. No patients were given radiotherapy or chemical therapy before surgery.

Western blotting To obtain the total proteins, the frozen normal and tumor samples were powdered in liquid nitrogen and lysed in Tris-Triton buffer containing 10 mM Tris (pH 7.4), 100 mM NaCl, 1 mM EDTA, 1% triton X-100, 10% glycerol, 0.1% SDS, 0.5% deoxycholate and a cocktail of protease inhibitors (2 μg/ml aprotinin, 5 μg/ml leupeptin, 2 μg/ml pepstatin A, 1 mM phenylmethane sulfonyl fluoride (PMSF [Sigma-Aldrich, MO, USA]). The lysates were centrifuged at 4◦C and the supernatant fractions were measured for protein concentration based on the Bradford method using bovine serum albumin (BSA) as the standard. Equal quantities of proteins were separated on 12.5% SDS-polyacrylamide gel and transferred to nitrocellulose membrane blocked in 5% skim milk for 1 h at room temperature. The blocked membranes were then incubated with the primary antibody of mouse anti-tropomyosin TM311 (Sigma-Aldrich) for 1 h at room temperature, washed three-times, incubated with the secondary antibody of anti-mouse horseradish peroxidase conjugated antibody (Sigma-Aldrich) for 1 h at room temperature. After being washed again for three-times, the signals were visualized with enhanced chemiluminescence (ECL Plus; Amersham), and exposed to Fuji x-ray film in a dark room. The membranes were stripped and reprobed with anti-β-actin antibody (Sigma-Aldrich) for verification of equal sample loading.

Real-time RT-PCR Total RNA was extracted according to manufacturer’s instructions using TriPure Isolation Reagent (Roche, Mannheim, Germany) and the precipitated RNA was dissolved in 20-μl Diethyl Pyrocarbonate (DEPC)-treated water. To remove any genomic DNA contamination, the isolated RNA was treated with RNase-free DNase I (Fer- mentas, INC, MD, USA). cDNA was synthesized using RevertAid First Strand cDNA Synthesis Kit, according to manufacturer’s protocol (Fermentas). Following cDNA synthesis, real-time PCR was performed using LightCycler 4 (Roche, Basel, Switzerland) and SYBER Green method in a total volume of 20 μl containing 0.5 pmol/μlof each primer, 4 μl of SYBER Green master mixture (Roche, Mannheim, Germany), 10 μl of PCR grade water

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Normal Normal Tumor Tumor Tumor TumorNormal Tumor

Tpm2.1 Tpm1.6 Tpm2.1 Tpm1.7 Tpm1.6 Tpm1.7

β -actin β-actin

Figure 1. Expression of Tpm1.6 and Tpm1.7 in normal and tumor tissue of squamous cell carcinoma of esophagus patients. Western blot was used to evaluate the expression level of Tpm1.6 and Tpm1.7 proteins in tumor and adjacent normal samples of the esophagus using TM311 monoclonal antibody which recognizes Tpm1.6, Tpm1.7 and Tpm2.1 isoforms. (A & B) Tpm1.6 and Tpm1.7 are expressed in normal tissue, but are remarkably downregulated in tumor tissue of squamous cell carcinoma of esophagus. The membrane was reprobed with anti-β-actin monoclonal antibody for loading control. and 2 μl of cDNA. All reactions were done in duplicate. The amplification was carried out under the following conditions: 5 min at 95◦C for pre-incubation, followed by 40 cycles of quantification as follows: 10 s at 95◦C, 10 s at 58◦Cand10sat72◦C, ending with one cycle of melting curve step as follows: 40 s at 65◦C ramping to 95◦Cat a ramp rate of 0.1◦C/s. For internal control, glyceraldehyde-3-phosphate dehydrogenase (GAPDH)wasused.The primer sequence applied for the desired genes were as follows: TPM1 forward; 5-GCT GGT TGA GGA AGA GTT GG-3, TPM1 reverse; 5-CTC TCA TCT GCT GCC TTC TC-3 and GAPDH forward: 5-ACA GTC AGCCGCATCTTC-3, GAPDH reverse: 5-CTC CGA CCT TCA CCT TCC-3. Relative expression of the desired mRNA was quantified using iCycler iQ software (Bio-Rad Laboratories, CA, USA).

Statistical analysis Statistical analysis was performed using SPSS (version 23) software. The values are presented as mean ± standard error of the mean. The Pearson chi-square test was used to analyze the associations between TPM1 and the clinicopathological characteristics. Fisher’s exact test was used when the number of group members was less than ﬁve. The Kaplan–Meier estimate and log-rank test (Mantel–Cox) were applied to evaluate the survival rate. p-value < 0.05 was considered as statistically signiﬁcant.

Results Tpm1.6 & Tpm1.7 expression in tumor & normal tissues of esophagus The expression pattern of Tpm1.6 and Tpm1.7 in SCCE was determined by western blot analysis by the use of TM311 monoclonal antibody that could recognize Tpm2.1, Tpm1.6 and Tpm1.7 isoforms. The results of western blot analysis showed a high level of expression of the Tpm1.6 and Tpm1.7 isoforms in the normal esophageal tissues, while their expression was notably lower level or were not observable in the tumor tissues (Figure 1).

Expression of TPM1 mRNA in tumor & normal tissues of esophagus The expression level of TPM1 mRNA in SCCE and adjacent normal esophageal tissues from each patient were analyzed by real time RT-PCR. The results showed that the tumor tissues exhibited signiﬁcantly lower levels of TPM1 mRNA expression compared with the adjacent normal tissues (n = 49; p < 0.001). According to the real time RT-PCR results, the tumor tissues exhibited severe TPM1 downregulation which ranged from 76 to 94% when compared with the normal tissues (Figure 2).

TPM1 mRNA expression & clinicopathological characteristics of SCCE patients As mentioned, real time RT-PCR was used to evaluate the expression level of TPM1 mRNA in SCCE patients. The tumor and normal samples from 49 patients (including 26 males and 23 females) were used in this study. The ages of the patients at the time of diagnosis were 47–88 years with a mean of 62 years, which was used as the cutoff for dividing the patients into two groups (<62; n = 22 and >62; n = 27). The downregulation level of TPM1 mRNA in the tumor tissue ranged from 76 to 94%. The patients were divided into a very high TPM1 downregulation

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120

100

60 Normal

40 Tumor * * Relative TPM1 expression 20 * * *

0 12345 Tissue samples of SCCE patients

120

100

Relative TPM1 expression *

0 Normal Tumor

Figure 2. Expression of TPM1 mRNA in tumor and normal tissue of squamous cell carcinoma of esophagus patients. Real time RT-PCR was used to analyze the expression of TPM1 mRNA in tumor and adjacent normal samples of the esophagus. (A) TPM1 mRNA is shown to be severely downregulated in squamous cell carcinoma of esophagus tumor tissue in comparison with adjacent normal tissue (the results of five patients; p < 0.001). (B) Mean downregulation of TPM1 in 49 tumor samples compared with normal samples (*p < 0.001). SCCE: Squamous cell carcinoma of esophagus.

group(n=29)andahighTPM1 downregulation group (n = 20) according to the mean of downregulation levels (85%). The correlation between TPM1 expression and clinicopathological characteristics was assessed by statistical analysis (Table 1). The results show that there was no signiﬁcant association between TPM1 downregulation and gender (p > 0.05) or age (p > 0.05) of patients. While, the level of TPM1 reduction is signiﬁcantly correlated with the histological grade of the tumor (p = 0.017) and higher downregulation of TPM1 mRNA was observed in the poorly differentiated samples. In other words, SCCE patients with poorly differentiated tumors had a higher probability of TPM1 downregulation than patients with well or moderately differentiated tumors.

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Table 1. Analysis of relationship between TPM1 mRNA downregulation and clinicopathological characteristics of squamous cell carcinoma of esophagus patients. Characteristics TPM1 downregulation level X2 p-value High (<85%) Very high (≥85%) Age: 1.39 0.238 – Ͻ62 years 11 (50%) 11 (50%) – Ͼ62 years 9 (33.3%) 18 (66.7%) Sex: 0.05 0.821 – Male 11 (42.3%) 15 (57.7%) – Female 9 (39.1%) 14 (60.9%) Histological grade: 8.09 0.017 – Poor 7 (24.1%) 22 (75.9%) – Moderate 5 (62.5%) 3 (37.5%) – Well 8 (66.7%) 4 (33.3%)

TPM1 expression & 5-year survival rate in SCCE patients The patients in this study were followed for ﬁve or more years after surgery. During follow-up, 13 patients (13/49; 26.5%) died. Ten of these patients (10/29; 34.5%) exhibited very high downregulation (≥85%) and three (3/20; 15%) exhibited high downregulation (<85%). The results showed that 5-year survival for the high downregulation group was 85% and for the very high downregulation group was 65.5%. In other words, patients with very high TPM1 downregulation had a decreased chance of survival for 5 years in comparison with patients with high TPM1 downregulation. Moreover, the mean survival time in high downregulation group (<85%) was 56.75 months and for the very high downregulation group was 47.96 months. Therefore, expression of TPM1 seems to be a favorable factor for patient survival. Despite the lower survival rate and shorter survival time for the very high downregulation group, there was no signiﬁcant correlation between the survival rates of patients and TPM1 expression level (p > 0.05) (Figure 3).

Discussion SCCE is a deadly malignancy that occurs at high incidence in some regions, particularly in Iran, where it ranks as the second most common cancer and constitutes >90% of all esophageal cancers [7]. However, its molecular alterations have been rarely investigated in this region. To obtain more insight into molecular changes during esophageal tumorigenesis, the expression levels of Tpm1.6 and Tpm1.7 were assessed in tumor samples of SCCE relative to adjacent normal esophageal tissue. Tropomyosins bind to actin filaments and are involved in the assembly and stabilization of the cytoskeleton [18,25,28]. Tpm1.6 and Tpm1.7 are the products of the TPM1 gene located on chromosome 9p13.2-p13.1 [18,36]. Suppressed expression of TPM1 and TPM2 genes has been shown in several cancers and is involved in the re- arrangement of actin cytoskeleton architecture and transformed characteristics. Additionally, restoration of Tpms could alter tumorigenic phenotypes and suggest a role of tumor suppressor gene for Tpm1.6 [32,37]. The results of the present study showed that Tpm1.6 and Tpm1.7 proteins were markedly downregulated in SCCE tumor tissues, but were expressed at a considerably higher level in the adjacent normal tissues. Similar results were observed in our previous work on the SCCE cell line and primary cultures [13]. In support of this, downregulation of HMW Tpms has been documented in cancers of the breast [30,35,38], urinary bladder [28], colon [30,39,40], neuroblastoma [41],prostate[31] and esophageal adenocarcinoma [42]. Inversely, increased expression of Tpm2.1 in metastatic breast tumors [38], Tpm1.6 in pancreatic cancer [43] and the tropomyosin α 4 chain in SCCE has also been found [44]. In the current study, significant downregulation of TPM1 mRNA along with downregulation of Tpm1.6 and Tpm1.7 proteins in SCCE indicate transcriptional suppression of the TPM1 gene. Downregulation of TPM1 and upregulation of TPM2 has been reported by Zhao et al. in esophageal adenocarcinoma [42]. TPM2 expression also has decreased significantly in colorectal cancer [40]. A former study used RT-PCR and indicated the expression of TPM1 mRNA in primary, but not in metastatic, breast and colon carcinoma [30]. Considering the less sensitivity of RT-PCR for quantitation of gene expression, the application of real time RT-PCR could provide more accurate quantitative comparison of TPM1 expression.

future science group 10.2217/pme-2018-0015 Research Article Zare, Hadi & Alivand

Survival functions

1.0 High TPM1 downregulation (<85%)

0.8

Very high TPM1 downregulation (≥85%)

0.6 Cum survival 0.4

0.2 Down_reg_g <85 = >85 <85-censored 0.0 = >85-censored

0.00 10.00 20.00 30.00 40.00 50.00 60.00 Months

Figure 3. Overall survival rate of patients in high and very high TPM1 downregulation groups. Patients with very high downregulation of TPM1 (lower level of TPM1) show worse overall survival in comparison to those with high downregulation (higher level of TPM1).

Although downregulation of HMW Tpms has been more documented in cancers, a number of studies have shown the overexpression of low molecular weight isoforms such as TPM3 in SCCE that correlate with invasion and poor survival rates [45] and TPM1λ, an isoform of TPM1 gene, in the malignant breast cell lines associated with malignant transformation [46]. Increased expression of TPM1, TPM2, TPM3 and TPM4 has been additionally found in the sera of ovarian cancer patients [47]. Regarding the suppression of TPM1, the involvement of DNA methylation and histone deacetylation has been demonstrated in breast and colon cancer and fibrosarcoma [30,32,39]. We previously have identified the crucial role of promoter hypermethylation in TPM1 and TPM2 gene downregulation in SCCE [13]. TPM1 expression can also be regulated by miR-21 [48] whose upregulation has been shown in SCCE [49,50]. Tropomyosin involvement in actin cytoskeleton stability makes it logical that TPM1 suppression through miR-21, which is important in epithelial–mesenchymal transition [50,51], could promote the migration and invasion of tumor cells. Considering the severe downregulation of TPM1, the relationship between TPM1 expression and the clinicopathological characteristics of SCCE patients was additionally analyzed. Importantly, the results revealed that decreased TPM1 expression occurring in tumor samples had no significant correlation with the gender or age of

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the patients. Similar results have been found in RCC and oral squamous cell carcinoma [33,34]. Otherwise, the differentiation degree of tumors has been shown to be significantly associated with TPM1 expression level. Poorly differentiated tumors show higher downregulation of TPM1 compared with moderately and well-differentiated tumors, which indicate that TPM1 downregulation would be a major early event in esophageal cancer development, increasing according to tumor progression. This finding is inconsistent with the results of a recent study which found no significant correlation between TPM1 expression and the histological grade of tumors in OSCC. It was shown that patients with Stage I–II cancer had higher TPM1 expression compared with patients with lymph node metastasis. Moreover, low TPM1 levels were accompanied by a poorer overall survival and cancer-specific survival of patients [34].Wanget al. reported significant TPM1 downregulation in RCC tissues, both at the protein and mRNA level, that was associated with tumor size, smoking status, Fuhrman grade and prognosis [33]. Intense TPM1 downregulation also has been found in pancreatic cancer patients and drug-resistant pancreatic cancer cell lines and correlated with worse survival [52]. Whereas, elevated levels of tropomyosin 3 in hepatocellular carcinoma were observed to be associated with poor recurrence-free survival of patients [53].Yuet al. have also shown that overexpression of tropomyosin 3 in Stage III SCCE compared with Stage I illustrates its importance in invasion and metastasis [54]. As far as is known, the relationship between Tpm downregulation and the survival rate of patients has not been studied in SCCE. Although the current results showed no significant correlation between survival of patients with TPM1 downregulation level, but it clearly indicates that patients with very high downregulation of TPM1 have decreased odds for 5-year survival, which further emphasizes the significance of TPM1 gene in the poor prognosis for SCCE patients. However, the small sample size in this study and a lack of clinical records for other histopathological characteristics means that further analysis with larger and more complete samples of SCCE are required, particularly regarding moderately and well-differentiated tumors.

Conclusion The uniform results of TPM1 downregulation in the TE15 cell line of SCCE [13] and tumor tissue of patients from a high-incidence area indicates that, despite the different origins of the samples, the same molecular and genetic alterations are involved in the tumorigenesis of SCCE, at least in the population of high risk area for esophageal cancer. Because a higher downregulation level of TPM1 was observed in poorly differentiated samples, it could be considered to be a primary event that may affect the survival rate. Thus, TPM1 expression may serve as a potent biomarker for SCCE monitoring.

Future perspective Despite the high mortality rate of esophageal cancer, its molecular mechanism is rather unknown. Further investigation of esophageal cancer tumorigenesis could provide more effective prevention and treatment approaches. In this regard, the alteration of other Tpm isoforms as central components of the actin cytoskeleton could clarify their functional role in esophageal cancer. The monitoring of Tpm/TPM downregulation in the serum of patients could provide new diagnostic approach for early detection of esophageal cancer patients.

Executive Summary

• The expression pattern of Tpm1.6 and Tpm1.7 in addition to TPM1 gene was assessed in squamous cell carcinoma of esophagus (SCCE) tissue samples. • Tpm1.6 and Tpm1.7 were significantly downregulated in SCCE tumor tissues compared with the normal esophageal tissues. • TPM1 mRNA was considerably downregulated in SCCE tumor tissues. • The association between TPM1 downregulation and histological grade of tumors and survival of SCCE patients was assessed for the first time. • Higher downregulation of TPM1 was observed in the poorly differentiated tumor tissues of SCCE. • Downregulation of TPM1 appears to be an early and universal event in esophageal carcinogenesis which probably is associated with poor prognosis and may affect the survival rate of SCCE patients. • TPM1 could be considered as a putative tumor suppressor gene that plays an important role in SCCE tumorigenesis. • TPM1 could potentially be used as a candidate biomarker for esophageal cancer.

future science group 10.2217/pme-2018-0015 Research Article Zare, Hadi & Alivand

Authors’ contributions M Zare conceived and designed the study, performed the experiments, analyzed the data and participated in manuscript writing, F Hadi and MR Alivand collected the clinicopathological data, helped to revise the manuscript and performed the statistical analysis. All authors read and approved the final manuscript.

Financial & competing interests disclosure The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest inorfinan- cial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.

Ethical disclosure The authors state that they have followed the principles outlined in the Declaration of Helsinki for human experimental investi- gations. In addition, for application of human subjects, informed consent has been obtained from the participants involved. The study was approved by the ethical committee of Imam Khomeini Hospital and the tumor bank of Tehran University of Medical Sciences.

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future science group 10.2217/pme-2018-0015 Research Article Zare, Hadi & Alivand

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