TM4SF1 promotes esophageal squamous cell carcinoma metastasis by interacting with integrin α6

doi:10.1038/s41419-022-05067-2

. 2022 Jul 14;13(7):609.

doi: 10.1038/s41419-022-05067-2.

TM4SF1 promotes esophageal squamous cell carcinoma metastasis by interacting with integrin α6

Sicong Hou^#¹, Xin Hao^#^{2

3}, Jiajia Li^#², Siwei Weng⁴, Jiaxin Wang³, Tiantian Zhao³, Wenqian Li³, Xiaolin Hu³, Bing Deng², Jianguo Gu⁵, Qinglei Hang^{6

7}

Affiliations

¹ Department of Gastroenterology, The Affiliated Hospital of Yangzhou University, Yangzhou University, 225009, Yangzhou, Jiangsu, China. shou@yzu.edu.cn.
² Department of Gastroenterology, The Affiliated Hospital of Yangzhou University, Yangzhou University, 225009, Yangzhou, Jiangsu, China.
³ Department of Clinical Medicine, Medical College, Yangzhou University, 225001, Yangzhou, Jiangsu, China.
⁴ Department of stomatology, Clinical Traditional Chinese Medicine College of Yangzhou University, 225000, Yangzhou, Jiangsu, China.
⁵ Division of Regulatory Glycobiology, Institute of Molecular Biomembrane and Glycobiology, Tohoku Medical and Pharmaceutical University, Sendai, Miyagi, 981-8558, Japan. jgu@tohoku-mpu.ac.jp.
⁶ Division of Regulatory Glycobiology, Institute of Molecular Biomembrane and Glycobiology, Tohoku Medical and Pharmaceutical University, Sendai, Miyagi, 981-8558, Japan. hql219@hotmail.com.
⁷ Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, 77030, USA. hql219@hotmail.com.

^# Contributed equally.

PMID: 35835740
PMCID: PMC9283456
DOI: 10.1038/s41419-022-05067-2

TM4SF1 promotes esophageal squamous cell carcinoma metastasis by interacting with integrin α6

Sicong Hou et al. Cell Death Dis. 2022.

. 2022 Jul 14;13(7):609.

doi: 10.1038/s41419-022-05067-2.

Authors

Sicong Hou^#¹, Xin Hao^#^{2

3}, Jiajia Li^#², Siwei Weng⁴, Jiaxin Wang³, Tiantian Zhao³, Wenqian Li³, Xiaolin Hu³, Bing Deng², Jianguo Gu⁵, Qinglei Hang^{6

7}

Affiliations

¹ Department of Gastroenterology, The Affiliated Hospital of Yangzhou University, Yangzhou University, 225009, Yangzhou, Jiangsu, China. shou@yzu.edu.cn.
² Department of Gastroenterology, The Affiliated Hospital of Yangzhou University, Yangzhou University, 225009, Yangzhou, Jiangsu, China.
³ Department of Clinical Medicine, Medical College, Yangzhou University, 225001, Yangzhou, Jiangsu, China.
⁴ Department of stomatology, Clinical Traditional Chinese Medicine College of Yangzhou University, 225000, Yangzhou, Jiangsu, China.
⁵ Division of Regulatory Glycobiology, Institute of Molecular Biomembrane and Glycobiology, Tohoku Medical and Pharmaceutical University, Sendai, Miyagi, 981-8558, Japan. jgu@tohoku-mpu.ac.jp.
⁶ Division of Regulatory Glycobiology, Institute of Molecular Biomembrane and Glycobiology, Tohoku Medical and Pharmaceutical University, Sendai, Miyagi, 981-8558, Japan. hql219@hotmail.com.
⁷ Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, 77030, USA. hql219@hotmail.com.

^# Contributed equally.

PMID: 35835740
PMCID: PMC9283456
DOI: 10.1038/s41419-022-05067-2

Abstract

Transmembrane-4 L-six family member-1 (TM4SF1) is a member of the L6 family and functions as a signal transducer to regulate tumor cell behaviors. However, the function and mechanism of TM4SF1 in esophageal squamous cell carcinoma (ESCC) metastasis remains unclear. Here, we find that TM4SF1 expression is increased and positively correlated with clinical TNM stage, N classification, differentiation, tumor size, and poor prognosis in ESCC patients. Interestingly, we demonstrate that TM4SF1 promotes ESCC cell adhesion, spreading, migration, and invasion, but not cell proliferation, in a laminin-dependent manner by interacting with integrin α6. Mechanistically, the TM4SF1/integrin α6/FAK axis signal pathway mediates cell migration under laminin-coating condition. Inhibiting FAK or knocking down TM4SF1 can attenuate TM4SF1-mediated cell migration and lung metastasis. Clinically, the TM4SF1/integrin α6/FAK axis positively correlates with ESCC. Altogether, these findings reveal a new mechanism of TM4SF1 in promoting ESCC metastasis via binding to integrin α6 and suggest that the cross-talk between TM4SF1 and integrin α6 may serve as a therapeutic target for ESCC.

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Conflict of interest statement

The authors declare no competing interests.

Figures

**Fig. 1. High TM4SF1 expression is significantly associated with poor prognosis.**
A The violinplot of TM4SF1 gene expression in EC tissue (T, n = 418) when compared with the normal esophageal tissue (N, n = 161), the data was generated via TNMplot. B The mRNA levels of *TM4SF1* in ESCC tissues and paired esophageal tissues were determined by qPCR. Graphic representation of the fold increases of mRNA in ESCC tissues (T) compared to paired esophageal tissues (N). The quantitative data were statistically analyzed as means ± s.d. (n = 27; ***P < 0.001, by paired t test). C Lysates from 8 paired ESCC samples (T) and adjacent normal tissues (N) were immunoblotted by anti-TM4SF1 antibody, GAPDH was used as a loading control. D Representative photos of TM4SF1 expression in ESCC tissues and non-cancerous tissues using TMA sections. IHC stainings of TM4SF1 in the non-cancerous specimen (I, II, and III), highly differentiated ESCC specimen (IV, V, and VI), moderately differentiated ESCC specimen (VII, VIII, and IX), and poorly differentiated ESCC specimen (X, XI, and XII). Original magnifications are ×40 in I, IV, VII, and X. Original magnifications are ×100 in II, V, VIII, and XI. Original magnifications are ×200 in III, VI, IX, and XII. Scale bar, 200 μm. E Kaplan–Meier analysis of overall survival in a cohort of ESCC patients (n = 109). The cumulative survival rate in patients with TM4SF1 highly-expressed (green line) is significantly lower than that in lowly-expressed ones (blue line). Statistical significance was determined by a log-rank test, P = 0.004.

**Fig. 2. The effects of TM4SF1 on cell proliferation, migration, and invasion in related ESCC cells.**
A Lysates from indicated ESCC cells were immunoblotted by anti-TM4SF1 antibody, β-actin was used as a loading control (upper panel). Quantitative data was determined by a two-tailed unpaired t test from 3 independent experiments (lower panel), Error bars are means ± s.d. B, D Lysates from control (Con) and TM4SF1 stably overexpressed (TM4SF1-OE) Eca109 cells (B), and Con, TM4SF1-knockdown (KD) KYSE-410 cells, and KD cells transduced with TM4SF1 (D) were immunoblotted by anti-TM4SF1 antibody, β-actin was used as a loading control (upper panel). Indicated cells were starved with serum-free RPMI 1640 for 24 h and then released with RPMI 1640 containing 10% FBS; the numbers of live cells were counted at the indicated time points. Cell numbers were normalized to those at 0 h. Statistical significance was determined by a two-tailed unpaired t test. Error bars are means ± s.d. (lower panel; n = 3; *n.s*., not statistically significant). C, E The migration and invasion abilities of indicated Eca109 (C) and KYSE-410 (E) cells were analyzed by transwell assay. The representative images were recorded by phase-contrast microscopy. The quantitative number of cells was obtained from 3 independent experiments. The P values were determined by two-tail unpaired t test (n.s., not statistically significant; **P < 0.01; ***P < 0.001 by two-tail unpaired t test). Error bars are means ± s.d. Scale bar, 250 µm.

**Fig. 3. TM4SF1 promotes laminin-mediated cell adhesion, migration, and spreading in ESCC cells.**
A Adhesion ability of Con and TM4SF1-OE Eca109 cells upon various ECM proteins. Cell suspensions were planted on the different ECM (laminin, collagen, fibronectin, and fibrinogen)-coated plates for 40 min at 37 °C. BSA was used as a control. Attached cells were stained and checked by colorimetric detection. The quantitative data were determined by a two-tailed unpaired t test. Error bars are means ± s.d. (n = 3; ***P < 0.001; *P < 0.05 by two-tail unpaired t test). B Indicated Eca109 cells were cultured on the laminin-coated dishes, starved with serum-free RPMI 1640 for 24 h and then released with RPMI 1640 containing 10% FBS, the numbers of live cells were counted at the indicated times. Cell numbers were normalized to those at 0 h. Statistical significance was determined by a two-tailed unpaired t test. Error bars are means ± s.d. (n = 3; n.s., not statistically significant). C, D The migration ability of indicated Eca109 (C) and KYSE-410 (D) cells was determined by transwell assay on laminin-coated condition. Representative photos were taken and then the migrated cells were counted. Statistical significance was determined by a two-tailed unpaired t test. Error bars are means ± s.d. (n = 3; ***P < 0.001; **P < 0.01). E Indicated Eca109 cells were detached, suspended in serum-free RPMI 1640 for 1 h, and then plated on the laminin-precoated (10 µg/ml) dishes. After incubation for 40 min, spread cells were fixed with PFA, and the representative photos were taken (left panel). The percentages of adherent cells were determined by a two-tailed unpaired t test. Error bars are means ± s.d. (right panel; n = 3; ***P < 0.001). Scale bar, 50 µm.

**Fig. 4. Laminin promotes the interaction between TM4SF1 and integrin α6 in Eca109 cells.**
A Con- and TM4SF1 overexpressed- Eca109 cell lysates (under laminin-coating condition) were immunoblotted by anti-p-FAK, anti-FAK, anti-p-PI3K p85 (Try458), anti- PI3K p85, anti-pAKT, and anti-AKT antibodies. B List of TM4SF1-associated cell membrane proteins in Eca109 cells under laminin-coating condition, identified by mass spectrometric analysis. C Co-IP of endogenous TM4SF1 with endogenous integrin α6, β4, and β1 in cell lysate. TM4SF1 was immunoprecipitated from Eca109 cells, which were cultured on the dishes with or without laminin-coating, and immunoblotted with antibodies against integrin α6, β4, β1, and TM4SF1. The input was immunoblotted with antibodies against integrin α6, β4, β1, p-FAK, FAK, TM4SF1, and β-actin. D Co-IP of endogenous TM4SF1 with endogenous integrin α6, β4, and β1 in ESCC patient tissues. TM4SF1 was immunoprecipitated from homogenized ESCC patient tissues lysate and immunoblotted with antibodies against integrin α6, β4, β1, and TM4SF1.

**Fig. 5. The Laminin-TM4SF1-integrin α6-FAK signaling axis mediates ESCC cell migration.**
A–C Immunoblotting of p-FAK, FAK, TM4SF1, integrin α6, and β-actin in (left panel) and migration ability of (right panel) TM4SF1-overexpressed, integrin α6-overexpressed, and TM4SF1/integrin α6 double overexpressed Eca109 cells (A), Con-, TM4SF1-knockdown-, related TM4SF1 rescued-, integrin α6- overexpressed, and TM4SF1-rescued/integrin α6- overexpressed KYSE-410 cells (B), and integrin α6-knockdown- and related α6 rescued- Con or TM4SF1 overexpressed Eca109 cells (C) under laminin coating condition (LE: long exposure; SE: short exposure). Representative photos were taken, and then the migrated cells were counted. Statistical significance was determined by a two-tailed unpaired t test. Error bars are means ± s.d. (right panel; n = 3; *P < 0.05; **P < 0.01; ***P < 0.001). D Immunoblotting of p-FAK, FAK, TM4SF1, integrin α6, and β-actin in (upper panel) and migration ability of (lower panel) TM4SF1-overexpressed, integrin α6-overexpressed, and TM4SF1/integrin α6 double overexpressed KYSE-410 cells pre-treated with or without VS-4718 (1 μM) for 24 h. Representative photos were taken and then the migrated cells were counted. Statistical significance was determined by a two-tailed unpaired t test. Error bars are means ± s.d. (right panel; n = 3; n.s., not statistically significant; *P < 0.05; ***P < 0.001).

**Fig. 6. TM4SF1/integrin α6/FAK signaling axis promotes ESCC metastasis.**
A, C TM4SF1 overexpressed Eca109 (A) and KYSE-510 (C) cells were treated with or without VS-4718 (1 μM) for 24 h, the migration ability was further determined by transwell assay on laminin-coating condition. The number of migrated cells were counted. Statistical significance was determined by a two-tailed unpaired t test. Error bars are means ± s.d. (n = 3; n.s., not statistically significant; **P < 0.01). B, D The representative images (left panels) and number (right panels) of lung metastatic nodules of BALB/c-nude mice with tail vein injection of Eca109-Con and Eca109-TM4SF1-OE cells (B) and KYSE-510 -Con and KYSE-510 -TM4SF1-OE cells (D) are presented. Scale bar, 250 µm. Mice were administered with or without VS-4718 (50 mg/kg) by oral gavage two times a day. The numbers of lung metastasis were statistically analyzed by a two-tailed unpaired t test. Error bars are means ± s.d. (n = 5; n.s., not statistically significant; *P < 0.05; **P < 0.01). E The representative images (left panel) and number (right panel) of lung metastatic nodules of BALB/c-nude mice with tail vein injection of Con-, TM4SF1-knockdown-, related TM4SF1 rescued-, integrin α6- overexpressed, and TM4SF1-rescued/integrin α6- overexpressed KYSE-410 cells are presented. Scale bar, 250 µm. The numbers of lung metastasis were statistically analyzed by a two-tailed unpaired t test. Error bars are means ± s.d. (n = 5; n.s., not statistically significant; ***P < 0.001; ****P < 0.0001).

**Fig. 7. TM4SF1-integrin α6-FAK signaling axis is positively related to ESCC.**
A Immunohistochemical stainings of TM4SF1, integrin α6, and p-FAK in representative ESCC tissue microarray (n = 109). Score = 0–3 (low) and score = 4–12 (high) indicate TM4SF1, integrin α6, and p-FAK levels in representative tumor tissues. Scale bar, 200 µm. B Correlation between TM4SF1, integrin α6, and p-FAK protein levels in patients with ESCC. The P value was calculated from a χ² test. (C-G) Kaplan–Meier analysis of overall survival in a cohort of ESCC patients (n = 109), stratified by the protein level of integrin α6 (C), p-FAK (D), low expression level of TM4SF1/integrin α6 /p-FAK and high expression level of TM4SF1/integrin α6/p-FAK (E), low expression level of TM4SF1/high expression level of integrin α6 and high expression level of TM4SF1/integrin α6 (F), or low expression level of TM4SF1/high expression level of p-FAK and high expression level of TM4SF1/p-FAK (G). Statistical significance was determined by a log-rank test, P < 0.001 (C), P = 0.003 (D), P = 0.008 (E), P = 0.005 (F), or P < 0.001 (G). H Working model for the role of TM4SF1 in regulating laminin-mediated signaling and metastasis.

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References

1. Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al. Global Cancer Statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2021;71:209–49. doi: 10.3322/caac.21660. - DOI - PubMed
1. Huang J, Koulaouzidis A, Marlicz W, Lok V, Chu C, Ngai CH, et al. Global burden, risk factors, and trends of esophageal cancer: an analysis of cancer registries from 48 countries. Cancers (Basel) 2021;13:141. doi: 10.3390/cancers13010141. - DOI - PMC - PubMed
1. Yaseen IH, Monk PN, Partridge LJ. Tspan2: a tetraspanin protein involved in oligodendrogenesis and cancer metastasis. Biochem Soc Trans. 2017;45:465–75. doi: 10.1042/BST20160022. - DOI - PMC - PubMed
1. Jiang X, Zhang J, Huang Y. Tetraspanins in cell migration. Cell Adh Migr. 2015;9:406–15. doi: 10.1080/19336918.2015.1005465. - DOI - PMC - PubMed
1. Huang YK, Fan XG, Qiu F. TM4SF1 promotes proliferation, invasion, and metastasis in human liver cancer cells. Int J Mol Sci. 2016;17:661. doi: 10.3390/ijms17050661. - DOI - PMC - PubMed

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[1] Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al. Global Cancer Statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2021;71:209–49. doi: 10.3322/caac.21660. - DOI - PubMed

[2] Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al. Global Cancer Statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2021;71:209–49. doi: 10.3322/caac.21660. - DOI - PubMed

[3] Huang J, Koulaouzidis A, Marlicz W, Lok V, Chu C, Ngai CH, et al. Global burden, risk factors, and trends of esophageal cancer: an analysis of cancer registries from 48 countries. Cancers (Basel) 2021;13:141. doi: 10.3390/cancers13010141. - DOI - PMC - PubMed

[4] Huang J, Koulaouzidis A, Marlicz W, Lok V, Chu C, Ngai CH, et al. Global burden, risk factors, and trends of esophageal cancer: an analysis of cancer registries from 48 countries. Cancers (Basel) 2021;13:141. doi: 10.3390/cancers13010141. - DOI - PMC - PubMed

[5] Yaseen IH, Monk PN, Partridge LJ. Tspan2: a tetraspanin protein involved in oligodendrogenesis and cancer metastasis. Biochem Soc Trans. 2017;45:465–75. doi: 10.1042/BST20160022. - DOI - PMC - PubMed

[6] Yaseen IH, Monk PN, Partridge LJ. Tspan2: a tetraspanin protein involved in oligodendrogenesis and cancer metastasis. Biochem Soc Trans. 2017;45:465–75. doi: 10.1042/BST20160022. - DOI - PMC - PubMed

[7] Jiang X, Zhang J, Huang Y. Tetraspanins in cell migration. Cell Adh Migr. 2015;9:406–15. doi: 10.1080/19336918.2015.1005465. - DOI - PMC - PubMed

[8] Jiang X, Zhang J, Huang Y. Tetraspanins in cell migration. Cell Adh Migr. 2015;9:406–15. doi: 10.1080/19336918.2015.1005465. - DOI - PMC - PubMed

[9] Huang YK, Fan XG, Qiu F. TM4SF1 promotes proliferation, invasion, and metastasis in human liver cancer cells. Int J Mol Sci. 2016;17:661. doi: 10.3390/ijms17050661. - DOI - PMC - PubMed

[10] Huang YK, Fan XG, Qiu F. TM4SF1 promotes proliferation, invasion, and metastasis in human liver cancer cells. Int J Mol Sci. 2016;17:661. doi: 10.3390/ijms17050661. - DOI - PMC - PubMed

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TM4SF1 promotes esophageal squamous cell carcinoma metastasis by interacting with integrin α6

Affiliations

TM4SF1 promotes esophageal squamous cell carcinoma metastasis by interacting with integrin α6

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Abstract

Conflict of interest statement

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