Sine Oculis Homeobox Homolog1Promotes Cell Proliferation And Metastasis Of Cervical Cancer

Objective:The malignant proliferation and metastasis are the fundamental traits of tumor cells. This study investigated how Sine oculis homeobox homolog1(SIX1) could promote proliferation of cervical cancer cells and tumor metastasis.Materials and Methods:Gene expression levels were measured by real-time RT-PCR, ELISA, immunoblotting or immunohistochemstry. Cell surface protein expression was measured by flow cytometry. Lymphangiogenesis was assessed by immunohistochemstry. Bioinformatic analysis of RNA-sequencing data from The Cancer Genome Atlas (TCGA) was performed using Bioconductor edgeR, Onto-Tools and Gene set enrichment analysis packages. The cells in different cell cycle phases were detected by flow cytometry. Tumor cell proliferation was measured by Cell Counting kit-8and soft agar assay in vitro. Transwell chamber was used to analysis the migration and invasion of tumor cells in vitro. The ability of cells adhesion to extracellular matrix (ECM) was determined by adhesion assay. The MMP-2and MMP-9in cultural supernatants were measured by gelatin zymography. The anoikis were detected by flow cytometry. The effects of SIX1on lymphangiogenesis in vitro were evaluated using migration assay and tube-formation assay of human lymphatic endothelial cells (HLECs). Orthotopic and ectopic xenograft model of cervical cancer were used to analysis lymphatic metastasis of tumor cells in vivo. Experimental metastasis model was used to analysis tumor cell arrest, extravasation and development of metastatic lesions in target organs. Metastasis was measured using bioluminescence and fluorescence imaging. Immunoprecipitation were used to detect the interaction between proteins. Chromatin immunoprecipitation were used to detect the binding of proteins to the VEGF-C promoter.Results:(1) The expression of SIX1was induced by E7oncoprotein of human papillomaviruses in cervical intraepithelial neoplasia and cervical cancer. The increase of SIX1expression resulted in the up-regulation of multiple genes related to the initiation of DNA replication, including the genes coding for the proteins in minichromosome maintenance complex (MCM2, MCM3, MCM6), DNA polymerase a-primase complex (POLA1, PRIM1, PRIM2), clamp loader (RFC3, RFC4, RFC5), DNA polymerase8complex (POLD3) and DNA polymerase s complex (POLE2). In line with this, the increase of SIX1expression accelerated Gl-to-S phase progression, and promoted the proliferation of cervical cancer cells and the growth of cervical cancer. Consistently, knock-down of SIX1could slow down Gl-to-S phase progression, and suppress tumor cell proliferation and tumor growth. Importantly, SIX1could more efficiently promote anchorage-independent cell growth.(2) Lymphangiogenesis and lymph-node metastasis in cervical cancer were closely correlated with higher expression of SIX1in tumor cells. By enhancing VEGF-C expression in tumor cells, SIX1could augment the promoting effect of tumor cells on the migration or tube-formation of lymphatic endothelial cells (LECs) in vitro and lymphangiogenesis in vivo. SIX1enhanced TGF-β-induced activation of SMAD2/3and coordinated with the SMAD pathway to modulate VEGF-C expression. Together, SIX1and TGF-β induced much higher expression of VEGF-C in tumor cells than each of them alone. Despite its effect promoting VEGF-C expression, TGF-β could inhibit lymphangiogenesis by directly inhibiting tube-formation by LECs. However, the increased production of VEGF-C not only directly promoted migration and tube-formation of LECs but also thwarted the inhibitory effect of TGF-β on LECs.(3) SIX1could up-regulate the expression of a5and β1, but not other integrin subunits that were identified to affect prognosis of various cancers. SIX1promoted the expression of α5β1to enhance the adhesion capacity of tumor cells in vitro and tumor cell arrest in circulation in vivo. By up-regulating α5β1expression, SIX1also augmented ECM-α5β1-mediated regulation of gene expression to enhance the metastatic potential of cervical cancer cells, including in the increased production of active MMP-2and MMP-9, up-regulation of anti-apoptotic genes (BCL-XL and BCL2) and down-regulation of pro-apoptotic genes (BIM and BAX). Blocking α5β1abrogated the regulatory effect of SIX1on gene expression, and also abolished the promotional effect of SIX1on invasive capability of tumor cells. Furthermore, knock-down of a5could abolish the promoting effect of SIX1on the development of metastatic lesions in both experimental and spontaneous metastasis model.Conclusions:SIX1functions as a master regulator in DNA replication and promotes proliferation of cervical cancer cells. SIX1promotes lymphangiogenesis in cervical cancer by coordinating with TGF-β1to increase VEGF-C expression. Moreover, SIX1up-regulates α5β1expression to promote metastatic capacity of cervical cancer cells. These findings indicate that the increase of SIX1expression plays an important role in tumorigenesis, progression and metastasis of cervical cancer. These results also suggest that SIX1might be considered as valuable marker for proliferative and metastatic potential of cervical cancer cells, or a therapeutic target in cervical cancer treatment.