Slit2 and Robo1 induce opposing effects on metastasis of hepatocellular carcinoma Sk-hep-1 cells

Abstract

The neural guidance molecular, Slit2, and its cognate receptor, Robo1, play critical roles in the development of the nervous system, nevertheless, their functions are not limited to this system. Numerous studies have shown decreased Slit2 expression in a wide variety of cancers, highlighting its potential as a tumor suppressor. However, the Slit2/Robo1 signaling axis was reported to induce either suppressive or stimulatory effects on tumor growth and metastasis, depending on cellular context. There is a paucity of information on the effects of the Slit2/Robo1 signaling axis on the growth and metastasis of human hepatocellular carcinoma (HCC). Large-scale data mining of the Oncomine database has revealed heterogeneous expression of Slit2 in HCC. We screened the Sk-hep-1, a cell line showing a relatively high level of Slit2, and low level of Robo1 expression. After Slit2 knockdown and Robo1 overexpression in these cells, we found Slit2 and Robo1 exerted opposing effects on tumor growth and metastasis both in in vitro and in vivo models. Slit2 knockdown and Robo1 overexpression in Sk-hep-1 cells promoted tumor growth and metastasis, suggesting a negative and positive role for Slit2 and Robo1, respectively, in tumor progression. Robo1 overexpression upregulated matrix metalloproteinase (MMP)2, -9 and membrane-type1 MMP (MT1-MMP) expression, stimulated MMP2, but not MMP9 activation, and downregulated expression of TIMP1 and 2. The PI3K/Akt signaling pathway is of importance in regulating MMP2 expression in Sk-hep-1 cells, since Robo1 overexpression stimulated phosphorylation of Akt while the PI3K inhibitor LY294002, significantly inhibited the upregulation of MMP2 and also the enhanced cell invasion induced by Robo1 overexpression. We postulate that Robo1 promotes tumor invasion partly by the upregulation of MMP2 after activation of PI3K/Akt signaling pathway. Notably, Slit2 knockdown caused the upregulation of Robo1 expression both at the mRNA and protein levels. Thus, the stimulatory effects of Slit2 knockdown on tumor progression can be ascribed, at least in part, to the upregulation of Robo1 and its positive role in tumor progression.

The Role of the Slit/Robo Signaling Pathway

“…We and Kong et al showed that in lung cancer, Slit2/Robo1 inhibited lung cancer cell migration via inhibiting the Myob/RhoA signaling pathway 50. Meanwhile, Yuan, M et al showed high expression of slit2 and down-regulation of Robo1 markedly enhanced migration of hepatic cancer cell 104. The authors demonstrated that Robo1 overexpression upregulated matrix metalloproteinase (MMP)-2, MMP-9, and membrane-type1 MMP (MT1-MMP) expression, thereby promoting tumor metastasis 104.…”

“…Meanwhile, Yuan, M et al showed high expression of slit2 and down-regulation of Robo1 markedly enhanced migration of hepatic cancer cell 104. The authors demonstrated that Robo1 overexpression upregulated matrix metalloproteinase (MMP)-2, MMP-9, and membrane-type1 MMP (MT1-MMP) expression, thereby promoting tumor metastasis 104. In malignant melanoma, MMP2 and MMP14 was showed to be downregulated by Slit3/Robo2 and thereby inhibiting cancer cell migration 105.…”

Identification and validation of an immune-related prognostic signature for hepatocellular carcinoma

“…ROBO1 is overexpressed in HCC samples [38]. Overexpression of ROBO1 could promote HCC cell growth and metastasis both in vitro and in vivo [39]. STC2 is upregulated in HCC, thereby facilitating HCC proliferation and survival as well as mediating chemotherapeutic resistance [40][41][42].…”

Slit2-Robo1 signaling enhances nasopharyngeal carcinoma metastasis via PI3K/AKT/β-catenin pathway

“…Slit2/Robo1 signaling has been associated with Akt phosphorylation and upregulation of β-catenin activity [25,26]. Yuan et al found Slit2/Robo1 promotes tumor invasion partly by upregulation of MMP2 after activation of PI3K/Akt signaling pathway in human hepatocellular carcinoma cell line [25].…”

“…Slit2/Robo1 signaling has been associated with Akt phosphorylation and upregulation of β-catenin activity [25,26]. Yuan et al found Slit2/Robo1 promotes tumor invasion partly by upregulation of MMP2 after activation of PI3K/Akt signaling pathway in human hepatocellular carcinoma cell line [25]. PI3K/Akt/ β-catenin signaling is highly activated in NPC [27] Activation of the PI3K/Akt signaling pathway results in aberrant cell proliferation, tumorigenesis and metastatic competence [28][29][30].…”