Association of High Expression of Mitochondrial Fission Regulator 2 with Poor Survival of Patients with Esophageal Squamous Cell Carcinoma

doi:10.21203/rs.3.rs-617078/v1

Download PDF

Research Article

Association of High Expression of Mitochondrial Fission Regulator 2 with Poor Survival of Patients with Esophageal Squamous Cell Carcinoma

https://doi.org/10.21203/rs.3.rs-617078/v1

This work is licensed under a CC BY 4.0 License

Journal Publication

published 29 Dec, 2021

Read the published version in Journal of Cancer Prevention →

Version 1

posted

You are reading this latest preprint version

Background: Mitochondrial fission regulator 2 (MTFR2) is associated with mitochondrial fission, while few studies has assessed the associations between MTFR2 expression and clinical characteristics and prognosis of esophageal squamous cell carcinoma (ESCC).

Methods: We compared the expression of MTFR2 in 6 ESCC tumors and relative normal tissues by immunochemistry (IHC). To assess the effect of MTFR2 expression on clinicopathologic characteristics and survival, totally 115 paraffin embedded ESCC tissue samples were assessed by IHC staining. Furthermore, the associations between clinicopathological properties and MTFR2 expression in patients with ESCC was examined. The survival analysis was performed using the Cox regression models.

Results: We found that MTFR2 expression was significantly increased in ESCC tumors compared with normal esophageal epithelial cells (NEECs). IHC analysis of 115 paraffin embedded ESCC tumor specimens of the patients showed that the expression of MTFR2 was significantly associated with clinical stage (P <0.001), tumor classification (P <0.001), histological grade (P<0.001), and other clinicopathological characteristics. Both univariate and multivariate analyses showed that MTFR2 expression was significantly associated with the survival of ESCC patients.

Conclusion: The expression of MTFR2 is significantly associated with clinicopathologic characteristics and prognosis of ESCC. Thus, MTFR2 expression could serve as a potentially important prognostic biomarker and clinical target for patients with ESCC.

Surgery

Oncology

MTFR2

ESCC

IHC

prognosis.

Esophageal cancer is one of the most common malignant tumors in the upper digestive tract of the chest(1, 2). Esophageal squamous cell carcinoma (ESCC) is one of most common types of esophageal cancer(3, 4). Approximately 300,000 people die of esophageal cancer annually worldwide, with more than half in China⁵. Moreover, the mortality of esophageal cancer is ranked fourth in China(5). Recurrence and metastasis of ESCC are still the main causes for death and treatment failure, and have crucial impact on the prognosis of ESCC(6, 7). Although considerable progress has been made in diagnosis and treatment, the relevant predictors for early diagnosis and prognosis remain lacking. Therefore, identification of molecular biomarkers will promote the development of new diagnostic and prognostic predictors for early diagnosis and personalized treatment of patients with ESCC.

Mitochondrial fission regulator 2 (MTFR2), also known as similarity 54 family member A (FAM54A), belongs to the MTFR1/FAM54 family(8, 9). It has two subtypes and is produced by selective shearing. MTFR2 plays important roles in mitochondrial division and aerobic respiration(10), while few studies have assessed the effects of MTFR2 expression on clinicopathological characteristics and prognosis of ESCC. Currently, some studies have shown that MTFR2 is overexpressed in glioblastoma (GBM)(9), while the role of MTFR2 expression in ESCC remains unclear.

In this study, we analyzed the expression of MTFR2 in six paired ESCC and normal specimens and 115 paraffin embedded ESCC tumor samples by immunohistochemistry (IHC). We evaluated the associations of MTFR2 expression with clinicopathological characteristics of ESCC patients, and further explored the effect of expression of MTFR2 in ESCC on clinical outcome.

Study patients and tissue specimens

The study was approved by the Yantai Yuhuangding Hospital ethics committee and obtained consent from each patient before study. Besides the 6-pairs of ESCC patients, additional 115 paraffin-embedded tumor specimens from patients, who were pathologically diagnosed with ESCC from 2005 to 2010, were enrolled for the study at Yantai Yuhuangding Hospital. Detailed clinical information of the patients is shown in Table 1. Written informed consents were given to all patients and this study was approved by the IRB committee of Yantai Yuhuangding Hospital.

Table 1

Clinicopathological characteristics of patients and MTFR2 expression in ESCC
Characteristics	Number of cases (%)
Sex
Female	6 (5.22)
Male	109 (94.78)
Age(years)
>60	56 (48.70)
≤60	59 (51.30)
Clinical stage
I/II	79 (68.70)
III/IV	36 (31.30)
T stage
1	20 (17.39)
2	36 (31.30)
3	40 (34.78)
4	19 (16.52)
N stage
0	76 (66.09)
1	33 (28.70)
2	6 (5.22)
M stage
0	110 (95.65)
1	5 (4.35)
Vital status
Alive	53 (46.09)
Dead	62 (53.91)
Histological differentiation
High	22(19.13)
Moderate	67 (58.26)
Low	26 (22.61)
Expression of MTFR2
Low	77 (67.00)
High	38 (33.00)

IHC

MTFR2 expression was performed in 115 paraffin embedded ESCC specimens and 6 pairs of ESCC and normal tissue samples by IHC, as previously described(11). In IHC, we used rabbit monoclonal antibody as target anti-MTFR2 antibody (1:200). Samples without any antibody were used as the corresponding blank control(11). In qualitative analysis, we selected 6 samples, which contained cancer and adjacent normal tissues, for comparison(12). Two independent pathologists who did not know the relevant clinical information evaluated the results of IHC staining of tumor Sect. (13). The positive staining score of cancer cells was divided into the following five grades: 0 (0), 1 (< 10%), 2 (11–50%), 3 (51–75%), and 4 (> 75%). The staining intensity was graded as follows: 0(negative = no staining), 1 (weak = light yellow), 2 (moderate = yellow brown), and 3 (strong = brown). The staining index (SI) was defined as the staining intensity score × the proportion of positive staining tumor cells (ranging from 0 to 12). Simultaneously, we defined SI ≥ 6 as high expression and SI < 6 as low expression.

Statistical analysis

The relationships between MTFR2 expression level and sex, age, TNM stage and clinical stage were analyzed using the Spearman correlation method. The effect of MTFR2 expression on clinicopathological characteristics was assessed using the test or Fisher’s exact test. For survival analysis, the effect of clinicopathological characteristics and MTFR2 expression on prognosis of ESCC were examined by the Kaplan-Meier analysis and log rank test, and further evaluated by univariate and multivariate Cox regression analyses. All statistical analyses were performed using SPSS software, version 20 (SPSS Inc., Chicago, IL, USA). Statistical significance was set at P < 0.05.

MTFR2 is highly expressed in ESCC tissues

After IHC in 115 specimens, we found that MTFR2 was highly expressed in ESCC tissues. To validate the expression of MTFR2 in ESCC, we further analyzed the expression of MTFR2 in 6 pairs of ESCC tumor and adjacent normal tissues. Compared to that in normal tissues, the expression of MTFR2 in ESCC tissues was significantly overregulated as shown in Fig. 1. Therefore, these results indicated that MTFR2 was overexpressed in ESCC tumors. In addition, to evaluate the association between MTFR2 expression and clinicopathological characteristics of patients with ESCC, we analyzed such associations in 115 ESCC patients. These patients included 25 patients with stage I, 53 patients with stage II, 32 patients with stage III, and 5 patients with stage IV, respectively. Out IHC staining showed that 38 cases (33.00%) had high MTFR2 protein expression and 77 cases (67.00%) had low or no expression (Table 1).

Association between MTFR2 expression clinicopathologic characteristics of ESCC

As shown in Table 2, we found that there was no significant difference in MTFR2 expression for age and sex (P > 0.05), while the significant differences in MTFR2 expression were found for clinical stage (P < 0.001), pathological grade (P = 0.029), T/N stage (P < 0.001) and M classification (P = 0.040) (Table 2). These results suggested that MTFR2 expression was significantly associated with advanced tumors. Additionally, we also analyzed the correlations between MTFR2 expression and clinicopathologic characteristics. As shown in Table 3, there was statistically significant correlations between MTFR2 expression and clinical stage (r = 0.460, P༜0.001) (Fig. 2), T stage (r = 0.318, P = 0.001), N stage (r = 0.373, P༜0.001), M stage (r = 0.213, P = 0.040), and histological differentiation (r = 0.192, P = 0.038). Therefore, MTFR2 was significantly related to overall clinical stage, TNM stage, and poor pathological differentiation of ESCC, supporting a role of MTFR2 in cancer progression of ESCC.

Table 2

The association of MTFR2 expression with clinicopathological characteristics of ESCC patients
Characteristics	No. of cases	MTFR2 expression		test P	Fisher’s exact test P
Characteristics	No. of cases	Low (%)	High (%)	test P	Fisher’s exact test P
Sex				0.351	0.267
Female	6	5 (83.33)	1 (16.77)
Male	109	72 (66.06)	37 (33.94)
Age(years)				0.213	0.097
≤60	56	40 (71.43)	16 (28.57)
>60	59	37 (62.71)	22 (37.29)
Clinical stage				༜0.001	༜0.001
I/II	79	65 (82.29)	14 (17.71)
III/IV	36	12 (33.33)	24 (66.67)
T stage				༜0.001	༜0.001
1	20	17 (85.00)	3 (15.00)
2	36	27 (75.00)	9 (25.00)
3	40	27 (67.50)	13 (32.50)
4	19	6 (31.58)	13 (68.42)
N stage				༜0.001	༜0.001
0	76	60 (78.95)	16 (21.05)
1	33	16 (48.48)	17 (51.52)
2	6	1 (16.67)	5 (83.33)
M stage				0.040	0.037
0	110	76 (69.09)	34 (30.91)
1	5	1 (20.00)	4 (80.00)
Histological differentiation				0.029	0.015
High	22	17 (77.27)	5 (22.73)
Moderate	67	47 (70.15)	20 (29.85)
Low	26	13 (50.00)	13 (50.00)

Table 3

Spearman correlation analysis between MTFR2 expression and clinicopathologic characteristics
Variables	MTFR2 expression level Spearman’s correlation	P
Clinical stage	0.460	༜0.001
T stage	0.318	0.001
N stage	0.373	༜0.001
M stage	0.213	0.040
Vital status	0.427	༜0.001
Histological differentiation	0.192	0.038

MTFR2 expression is associated with survival of ESCC

In the patients with low MTFR2 expression, the rate of cumulative 5-year survival was 61.0% (95% CI, 60.6%, 75.9%), while that was only 15.8% (95% CI, 15.2%, 28.1%) in patients with high MTFR2 expression, indicating that the 5-year survival rate in the low MTFR2 expression group was significantly higher than in high MTFR2 group

Furthermore, we found that the survival time of ESCC patients was negatively correlated with MTFR2 expression (r = -0.580, P༜0.001), and the patients with high MTFR2 expression had worse survival than those with low expression (log-rank: P༜0.001, Fig. 3A). Our stratified analysis also showed the survival differences in several subgroups. There were significant differences in survival between MTFR2 expression in patients with early disease stage (stage I /II) (log-rank, P ༜ 0.001, Fig. 3B) and those with advanced disease (stage III/IV) (P༜0.001, Fig. 3C). Similarly, there were significant survival differences between the MTFR2 expression in patients with T1-2, T3-4, positive lymph nodes, negative lymph nodes, non-distant metastasis and distant metastasis groups (All P༜0.001, Fig. 4), respectively. These findings in the subgroups further indicated that high expression of MTFR2 had shorter survival than low expression of MTFR2, supporting the role of MTFR2 expression could be potential prognostic biomarkers for ESCC.

Finally, we performed both univariate and multivariate Cox regression analyses to assess the associations between MTFR2 expression and survival of patients with ESCC. Our results showed that the expression of MTFR2 was a significantly independent prognostic predictor for ESCC (aHR, 3.04, 95%CI, 1.75–5.30; P༜0.001; Table 4).

Table 4

Univariate and multivariable analysis on association between MTFR2 expression and survival in patients with ESCC
Variables	Univariate analysis				Multivariate analysis
Variables	No. of patients	P	cHR^†	95% CI	P	aHR^*	95% CI
MTFR2 expression		༜ 0.001			༜ 0.001
Low	77		1.00			1.00
High	38		3.12	1.66–5.27		3.04	1.75–5.30
^†cHR: crude hazard ratio
^*aHR: adjusted hazard ratio, HR was adjusted by age, sex, clinical stage, T/N/M stage, and Histological differentiation

In this study, MTFR2 expression were relatively higher in tumor lesions than that in normal tissues. MTFR2 was highly expressed in ESCC tumor specimens, and it was significantly correlated with clinical stage, T, N, M classification, histological differentiation, and metastasis of ESCC. The expression of MTFR2 increased with the progression of ESCC, indicating that high expression of MTFR2 could be related to the progression of ESCC. Our current findings demonstrated that MTFR2 was an independent prognostic factor for prognosis of patients with ESCC, and the patients with high MTFR2 expression had worse survival than those with corresponding low expression. Therefore, it is likely that MTFR2 expression may have significant clinical values as a new prognostic predictor and one of potential novel targets for future targeted treatment of ESCC.

As a mitochondrion fission factor, MTFR2 is related to mitochondrion fission, participates in aerobic respiration and has antioxidant effects(8, 14). While MTFR2 may play a key role in the induction of intrinsic apoptosis(15, 16). Compared with normal cells, mitochondrion induces tumor-related programming and accelerates growth of tumor cells and blood vessels(17, 18). Some studies have shown that MTFR2 may transcriptionally regulate dual specificity protein kinase TTK(9). MTFR2 is one of the key genes related to TTK, which can be regulated by activating the promoter of TTK(19, 20). Furthermore, it is involved in regulation of proliferation of glioma stem cells (GSCs) and thus may affect the prognosis of glioblastoma(9, 17). Most recently, MTFR2 has been reported to promote the proliferation, migration, and invasion of oral squamous carcinoma(21), and MTFR2-dependent regulation of TTK was involved in maintaining GSCs in glioblastoma and might serve as a potential novel druggable target for glioblastoma. It might regulate the expression of TTK for participation in up-regulation and expression of GSCs in glioblastoma. In addition to cell proliferation, TTK was also involved in centrosome duplication, DNA damage response, and organ development(22). Another study showed that reduced TTK levels could result in abnormal mitoses, activate apoptosis and reduce survival of breast cancer(23).

TTK depletion might seriously damage the viability and ability to form colonies of TNBC cell lines(24). Thus, TTK might be an independent predictor of prognosis, and it appears biologically plausible that MTFR2 might play important roles in activation of regulation, promotion of expression of TTK in ESCC tumors, and occurrence and development of ESCC. However, the exact mechanisms underlying such associations require further exploration.

Hassan et al.(10) showed that the expression of MTFR2 was related to poor prognosis, suggesting that MTFR2 might be involved in the progression and invasion of several cancers(25, 26). Such findings may highlight the potential role of MTFR2 in ESCC as future new targets for treatment.

Although our study found that MTFR2 was overexpressed in ESCC, and was significantly associated with advanced disease and prognosis, which was consistent with the research on glioma⁹, our research has some limitations. First, we only analyzed the expression levels of cell protein, while we did not specifically analyze other aspects of genetic alterations. Second, our sample size is relatively small, and all study patients are from a single hospital, and our findings could be due to by chance. Moreover, the selection bias may also exist. Nevertheless, the current preliminary findings from this small study might serve as a basis for future hypothesis generation and testing as well as validation in large well-designed prospective studies such consortia, multi-center study. Finally, more studies are needed to explore the exact molecular mechanisms underlying the associations.

In conclusion, in the current study, we found that high expression of MTFR2 is overexpressed in and correlated with ESCC progression. In addition, MTFR2 expression was significantly associated with survival of patients with ESCC, particularly in aggressive tumors. Thus, our current findings might support that MTFR2 could be an independent biomarker for prognosis and serve as a potential new therapeutic target for ESCC.

MTFR2, Mitochondrial fission regulator 2; ESCC, esophageal squamous cell carcinoma; IHC, immunochemistry; NEECs, normal esophageal epithelial cells, GSCs, glioma stem cells.

Ethics approval and consent to participate

All procedures performed in studies involving human participants were in accordance with the ethical standards of the Ethics Committee of the Yantai Yuhuangding Hospital Affiliated Hospital of Qingdao University and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Consent for publication

Not applicable.

Availability of data and materials

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests

The authors declare that they have no competing interests.

Funding

This article is one of the results of the Key Research and Development Plan of Yantai City(2018SFGY110).

Authors' contributions

HL, XZ, WZ, WL, and JM carried out the majority of the experiment, data analysis, and wrote the manuscript. They were all helped by CL,YZ, RZ, and YS. All authors reviewed and approved the final manuscript.

Acknowledgements

Not applicable.

Torre LA, Bray F, Siegel RL, Ferlay J, Lortet-Tieulent J, Jemal A. Global cancer statistics, 2012. Cancer J Clin. 2015;65(2):87–108.
Huang Z, Zhong L. Research progress on radiotherapy of esophageal carcinoma. Chinese Journal of Clinical Oncology. 2016.
Ren QG, Yang SL, Hu JL, Li PD, Chen YS, Wang QS. Evaluation of HO-1 expression, cellular ROS production, cellular proliferation and cellular apoptosis in human esophageal squamous cell carcinoma tumors and cell lines. Oncol Rep. 2016;35(4):2270–6.
Hsieh MS, Yang PW, Wong LF, Lee JM. The AXL receptor tyrosine kinase is associated with adverse prognosis and distant metastasis in esophageal squamous cell carcinoma. Oncotarget. 2016;7(24):36956–70.
Lin Y, Totsuka Y, He Y, Kikuchi S, Qiao Y, Ueda J, et al. Epidemiology of esophageal cancer in Japan and China. Journal of epidemiology. 2013;23(4):233–42.
Kleinberg L, Forastiere AA. Chemoradiation in the management of esophageal cancer. Journal of clinical oncology: official journal of the American Society of Clinical Oncology. 2007;25(26):4110–7.
Han X, Lu N, Pan Y, Xu J. Nimotuzumab Combined with Chemotherapy is a Promising Treatment for Locally Advanced and Metastatic Esophageal Cancer. Medical science monitor: international medical journal of experimental clinical research. 2017;23:412–8.
Monticone M, Panfoli I, Ravera S, Puglisi R, Jiang MM, Morello R, et al. The nuclear genes Mtfr1 and Dufd1 regulate mitochondrial dynamic and cellular respiration. Journal of cellular physiology. 2010;225(3):767–76.
Wang J, Xie Y, Bai X, Wang N, Yu H, Deng Z, et al. Targeting dual specificity protein kinase TTK attenuates tumorigenesis of glioblastoma. Oncotarget. 2018;9(3):3081–8.
Dastsooz H, Cereda M, Donna D, Oliviero S. A Comprehensive Bioinformatics Analysis of UBE2C in Cancers. International journal of molecular sciences. 2019;20(9).
Li J, Gong LY, Song LB, Jiang LL, Liu LP, Wu J, et al. Oncoprotein Bmi-1 renders apoptotic resistance to glioma cells through activation of the IKK-nuclear factor-kappaB Pathway. Am J Pathol. 2010;176(2):699–709.
Andl CD, Mizushima T, Nakagawa H, Oyama K, Harada H, Chruma K, et al. Epidermal growth factor receptor mediates increased cell proliferation, migration, and aggregation in esophageal keratinocytes in vitro and in vivo. J Biol Chem. 2003;278(3):1824–30.
Yu DL, Li HW, Wang Y, Li CQ, You D, Jiang L, et al. Acyl-CoA dehydrogenase long chain expression is associated with esophageal squamous cell carcinoma progression and poor prognosis. OncoTargets therapy. 2018;11:7643–53.
Rahman J, Rahman S. Mitochondrial medicine in the omics era. Lancet. 2018;391(10139):2560–74.
Tonachini L, Monticone M, Puri C, Tacchetti C, Pinton P, Rizzuto R, et al. Chondrocyte protein with a poly-proline region (CHPPR) is a novel mitochondrial protein and promotes mitochondrial fission. Journal of cellular physiology. 2004;201(3):470–82.
Monticone M, Tonachini L, Tavella S, Degan P, Biticchi R, Palombi F, et al. Impaired expression of genes coding for reactive oxygen species scavenging enzymes in testes of Mtfr1/Chppr-deficient mice. Reproduction. 2007;134(3):483–92.
Jelluma N, Brenkman AB, van den Broek NJ, Cruijsen CW, van Osch MH, Lens SM, et al. Mps1 phosphorylates Borealin to control Aurora B activity and chromosome alignment. Cell. 2008;132(2):233–46.
Sabharwal SS, Schumacker PT. Mitochondrial ROS in cancer: initiators, amplifiers or an Achilles' heel? Nature reviews Cancer. 2014;14(11):709–21.
Hudler P, Britovsek NK, Grazio SF, Komel R. Association between polymorphisms in segregation genes BUB1B and TTK and gastric cancer risk. Radiol Oncol. 2016;50(3):297–307.
Kaistha BP, Honstein T, Müller V, Bielak S, Sauer M, Kreider R, et al. Key role of dual specificity kinase TTK in proliferation and survival of pancreatic cancer cells. Br J Cancer. 2014;111(9):1780–7.
Wang W, Xiong M, Jiang L, Chen Z, Shao Y. MTFR2 Promotes the Proliferation, Migration, and Invasion of Oral Squamous Carcinoma by Switching OXPHOS to Glycolysis. Frontiers in oncology. 2020;10:858.
Liu X, Winey M. The MPS1 family of protein kinases. Annual review of biochemistry. 2012;81:561–85.
Daniel J, Coulter J, Woo JH, Wilsbach K, Gabrielson E. High levels of the Mps1 checkpoint protein are protective of aneuploidy in breast cancer cells. Proc Natl Acad Sci USA. 2011;108(13):5384–9.
Maire V, Baldeyron C, Richardson M, Tesson B, Vincent-Salomon A, Gravier E, et al. TTK/hMPS1 is an attractive therapeutic target for triple-negative breast cancer. PLoS One. 2013;8(5):e63712.
Nath S, Banerjee T, Sen D, Das T, Roychoudhury S. Spindle assembly checkpoint protein Cdc20 transcriptionally activates expression of ubiquitin carrier protein UbcH10. J Biol Chem. 2011;286(18):15666–77.
Perrotta I, Bruno L, Maltese L, Russo E, Donato A, Donato G. Immunohistochemical analysis of the ubiquitin-conjugating enzyme UbcH10 in lung cancer: a useful tool for diagnosis and therapy. The journal of histochemistry cytochemistry: official journal of the Histochemistry Society. 2012;60(5):359–65.

Download PDF

Journal Publication

published 29 Dec, 2021

Read the published version in Journal of Cancer Prevention →

Version 1

posted

You are reading this latest preprint version

Association of High Expression of Mitochondrial Fission Regulator 2 with Poor Survival of Patients with Esophageal Squamous Cell Carcinoma

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Materials And Methods

Study patients and tissue specimens

IHC

Statistical analysis

Results

MTFR2 is highly expressed in ESCC tissues

Association between MTFR2 expression clinicopathologic characteristics of ESCC

MTFR2 expression is associated with survival of ESCC

Discussion

Conclusions

Abbreviations

Declarations

References

Status:

Journal Publication

Version 1