Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2021 Oct;22(4):1102.
doi: 10.3892/etm.2021.10535. Epub 2021 Aug 2.

Sema4C modulates the migration of primary tumor-associated lymphatic endothelial cells via an ERK-mediated pathway

Jin Peng  1 , Xijiang Liu  2 , Chengcheng Li  1 , Min Gao  1 , Hongyan Wang  1
Affiliations

Sema4C modulates the migration of primary tumor-associated lymphatic endothelial cells via an ERK-mediated pathway

Jin Peng et al. Exp Ther Med. 2021 Oct.

Abstract

Although lymphatic endothelial cells (LECs) serve a positive role in tumor lymphatic metastasis, the regulation of LECs undergoing migration similar to that of tumor cells remains poorly understood. A previous study revealed that semaphorin 4C (Sema4C) could be a marker of LECs in cervical cancer. Thus, the present study aimed to understand the mechanism via which Sema4C could promote the development of tumor-associated characteristics in LECs in cervical cancer. Primary tumor-associated LECs (TLECs) were distinguished from cervical cancer by flow cytometry. The promigratory ability was assessed using the Transwell assay. Lentivirus infection was used to alter the expression of Sema4C in TLECs. Confocal laser scanning was used to determine the infection efficiency of lentivirus infection. Sema4C/ERK/E-cadherin pathway was measured by reverse transcription-quantitative PCR and western blotting. The co-localization of Sema4C and the lymphatic marker lymphatic vessel endothelial hyaluronan receptor 1 was verified. Primary tumor-associated LECs (TLECs) were isolated from a mouse xenograft cervical tumor model. It was revealed that overexpressing Sema4C stimulated the migratory ability of TLECs, downregulated E-cadherin expression and stimulated ERK phosphorylation, whereas knocking down Sema4C had the opposite effects. The treatment of PD98059 (ERK inhibitor) blocked the pro-migratory ability of TLECs, which indicated a dependence on the ERK signaling pathway. It was identified that the Sema4C/ERK/E-cadherin pathway may be critical for the migration of TLECs, which may promote lymph node metastasis. Therefore, Sema4C could be a promising target for the treatment of cervical cancer with lymphatic metastasis.

Keywords: E-cadherin; extracellular signal-regulated kinase; semaphorin 4C; tumor-associated lymphatic endothelial cells.

PubMed Disclaimer

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
TLECs isolated from a mouse xenograft tumor. (A) Localization of Sema4C was detected in mouse tumor tissue. In lymphatic vessels, Sema4C (green) was co-localized with LYVE1 (red). Nuclei were identified via DAPI staining (blue). Scale bars, 20 µm. (B) LYVE1 immunohistochemical analysis indicated that day 7 after U14 cell injection was the most suitable time for isolation of TLECs. Arrowheads demonstrate TLEC positive staining. Scale bars, 20 µm. (C) Tumor image indicated the tumor size at day 7 after injection, and quantification of the numbers of lymphatic vessels on day 5, 7 and 14 was performed. (D) Tumor tissues were dissociated into a cell suspension and then stained for LYVE1-phycoerythrin. LYVE1-positive cells were separated via flow cytometry. The control group was used non-specific IgG. (E) TLECs were cultured in EBM-2 medium for 5 and 10 days. Scale bars, 10 µm. (F) VEGFR3-positive TLECs were identified via immunohistochemistry. Scale bars, 100 µm. The experiments were repeated three times. **P<0.01. TLECs, tumor-associated lymphatic endothelial cells; LYVE1, lymphatic vessel endothelial hyaluronan receptor 1; Sema4C, semaphorin 4C; LMVD, lymphatic microvessel density; SSC, side-scattered light; con, control.
Figure 2
Figure 2
Role of Sema4C expression in the migratory ability of TLECs. (A) TLECs treated with lentiviral medium only, lentiviral control vector for Sema4C siRNA, lentiviral control vector for full-length Sema4C, Sema4C siRNA and full-length Sema4C. Scale bars, 10 µm. (B) TLECs with LV3-siRNA or LV5-Sema4C were generated, and Sema4C mRNA expression was measured via reverse transcription-quantitative PCR. (C) Migratory ability of cells was assessed using a Transwell assay. (D) Quantification of the number of cells at the bottom of the Transwell chamber. The experiments were repeated three times. *P<0.05, **P<0.01 LV3-siRNA group vs. LV3NC group; LV5-Sema4C group vs. LV5NC group. Sema4C, semaphorin 4C; siRNA, small interfering RNA; NC, negative control; TLECs, tumor-associated lymphatic endothelial cells.
Figure 3
Figure 3
Role of Sema4C expression on E-cadherin and ERK1/2 expression of tumor-associated lymphatic endothelial cells. (A) Assessment of the mRNA expression levels of Sema4C, E-cadherin and ERK1/2 using reverse transcription-quantitative PCR. (B) Protein expression levels of Sema4C, E-cadherin, p-ERK1/2 and total ERK1/2 were detected using western blot analysis. (C) Semiquantitative analysis of protein expression levels of Sema4C, E-cadherin, p-ERK1/2 and total ERK1/2. (D) Expression of p-ERK1/2 was normalized to total ERK1/2, and represented as fold change over the control group. The experiments were repeated three times. *P<0.05 LV5-Sema4C group vs. LV5 NC group, **P<0.01 LV3-siRNA group vs. LV3NC group. Sema4C, semaphorin 4C; p, phosphorylated; siRNA, small interfering RNA; NC, negative control.
Figure 4
Figure 4
ERK inhibitor reverses effects on the migratory ability of TLECs induced by Sema4C. (A) Migratory ability of TLECs was assessed using cells treated with lentiviral medium only (control group), LV3-siRNA, LV5-Sema4C, LV3-siRNA + PD98059 and LV5-Sema4C + PD98059. Scale bars, 100 µm. (B) Quantification of the number of cells from the bottom of the Transwell inserts was performed. The experiments were repeated three times. *P<0.05 vs. control group; ##P<0.01 vs. LV3-siRNA group. Sema4C, semaphorin 4C; siRNA, small interfering RNA; TLECs, tumor-associated lymphatic endothelial cells.
Figure 5
Figure 5
Sema4C regulates E-cadherin expression via the ERK pathway. mRNA expression levels of (A) Sema4C, (B) E-cadherin and (C) total ERK1/2 in TLECs treated with lentiviral medium only (control group), LV3-siRNA, LV5-Sema4C, LV3-siRNA + PD98059 and LV5-Sema4C + PD98059. (D) Protein expression levels of Sema4C, E-cadherin, p-ERK1/2 and total ERK1/2 in TLECs treated with lentiviral medium only, LV3-siRNA, LV5-Sema4C, LV3-siRNA + PD98059 and LV5-Sema4C + PD98059 detected using western blot analysis. Semiquantitative analysis of protein expression levels of (E) Sema4C, (F) E-cadherin, (G) p-ERK1/2 and (H) total ERK1/2. (I) Expression of p-ERK1/2 was normalized to total ERK1/2, and represented as fold change over the control group. The experiments were repeated three times. *P<0.05, **P<0.01 vs. control group; ##P<0.01 vs. LV3-siRNA group; &P<0.05 LV5-Sema4C group. Sema4C, semaphorin 4C; p, phosphorylated; siRNA, small interfering RNA; TLECs, tumor-associated lymphatic endothelial cells.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Frumovitz M. Sentinel lymph node biopsy for cervical cancer patients-what's it gonna take? Gynecol Oncol. 2017;144:3–4. doi: 10.1016/j.ygyno.2016.12.009. - DOI - PubMed
    1. Li H, Wu X, Cheng X. Advances in diagnosis and treatment of metastatic cervical cancer. J Gynecol Oncol. 2016;27(e43) doi: 10.3802/jgo.2016.27.e43. - DOI - PMC - PubMed
    1. Hoshino A, Lyden D. Metastasis: Lymphatic detours for cancer. Nature. 2017;546:609–610. doi: 10.1038/546609a. - DOI - PubMed
    1. Lucas ED, Tamburini BAJ. Lymph node lymphatic endothelial cell expansion and contraction and the programming of the immune response. Front Immunol. 2019;10(36) doi: 10.3389/fimmu.2019.00036. - DOI - PMC - PubMed
    1. Saito A. EMT and EndMT: Regulated in similar ways? J Biochem. 2013;153:493–495. doi: 10.1093/jb/mvt032. - DOI - PubMed

Grants and funding

Funding: The present study was supported by grants from the National Natural Science Foundation of China (grant nos. 81502238 and 81602286), the Department of Medical and Health Science Technology of Shandong Province (grant no. 2016w0345), the Department of Science Technology of Jinan City (grant no. 201705051), the China Postdoctoral Science Foundation (grant no. 2019T120594) and Natural Science Foundation General Project of Shandong Province (grant no. ZR2020MH230).