GM3 suppresses anchorage‐independent growth via Rho GDP dissociation inhibitor beta in melanoma B16 cells

Abstract

Ly-GDI, Rho GTPase dissociation inhibitor beta, was found to be expressed parallel to the GM3 level in mouse B16 cells whose GM3 contents were modified by B4galt6 sense, B4galt6 antisense cDNA, or St3galt5 siRNA transfection. Ly-GDI expression was increased on GM3 addition to these cells and decreased with D-PDMP treatment, a glucosylceramide synthesis inhibitor. Suppression of GM3 or Ly-GDI by RNAi was concomitantly associated with an increase in anchorage-independent growth in soft agar. These results clearly indicate that GM3 suppresses anchorage-independent growth through Ly-GDI. GM3 signals regulating Ly-GDI expression was inhibited by LY294002, siRNA against Akt1 and Akt2 and rapamycin, showing that GM3 signals are transduced via the PI3K/Akt/ mTOR pathway. Either siRNA towards Rictor or Raptor suppressed Ly-GDI expression. The Raptor siRNA suppressed the effects of GM3 on Ly-GDI expression and Akt phosphorylation at Thr 308 , suggesting GM3 signals to be transduced to mTOR-Raptor and AktThr 308 , leading to Ly-GDI stimulation. siRNA targeting Pdpk1 reduced Akt phosphorylation at Thr 308 and rendered the cells insensitive to GM3 stimulation, indicating that Akt-Thr 308 plays a critical role in the pathway. The components aligned in this pathway showed similar effects on anchorage-independent growth as GM3 and Ly-GDI. Taken together, GM3 signals are transduced in B16 cells through PI3K, Pdpk1, Akt Thr308 and the mTOR/Raptor pathway, leading to enhanced expression of Ly-GDI mRNA, which in turn suppresses anchorage-independent growth in melanoma B16 cells. (Cancer Sci 2011; 102: 1476-1485 L y-GDI, a Rho GTPase dissociation inhibitor beta, is also known as RhoGDI2 or D4-GDI. It belongs to a family of Rho GDP dissociation inhibitors (RhoGDI) including RhoGDI1 and RhoGDI3. The family is named for its ability to inhibit the dissociation of bound GDP from its partner Rho GTPase, which regulates interactions with regulatory guanine nucleotide exchange factors, GTPase-activating proteins, and effector targets. (1) Rho GTPases, such as RhoA, Rac1 and CDC42, are important regulators of cellular migration and adhesion, (2,3) and regulate the dynamics of b-actin, resulting in stress fibers, lamellipodia, and filopodia formation. In contrast to other GDIs, Ly-GDI interacts poorly with RhoA, Rac and Cdc42 in vivo. (4) Although Ly-GDI differs substantially at the structural level from the other two GDIs, RhoGDI-1 and RhoGDI-3, these three GDI exhibit very similar biological regulatory activities on Rho family proteins. (5-7) Ly-GDI is regarded as an invasion and metastasis suppressor gene in human bladder cancer cells. (8,9) For example, the truncated Ly-GDI has been shown to promote metastasis of colon cancer. (10) However, Ly-GDI was reported to be highly expressed in human breast cancer cells and its involvement in tumor cell invasion has been suggested. (11) Thus, function of Ly-GDI is controversial, and furthermore, it remains unknown as to how Ly-GDI is regulated in cells and how its regulation is related to tumor be...

Unconventional myosin VIIA promotes melanoma progression

“…RhoGDI2 also promotes tumor growth, invasion and metastasis in ovarian carcinoma (Stevens et al, 2011) and in breast cancer (Gu et al, 2008;Zhang et al, 2009;Zhang and Zhang, 2006). It has been reported that RhoGDI2 suppresses anchorage-independent growth in melanoma B16 cells (Wang et al, 2011). In our study, we demonstrated that depletion of Myo7a inhibited melanoma cell motility by downregulating expression of RhoGDI2.…”

Changing the Nature of Melanoma Cells by Manipulation of Ganglioside Expression

“…These results give further support to the notion that GM3 reduction enhances invasive proliferating ability of B16 cells in rigorous condition. It is also the characteristic of tumor cells that the proliferation was deregulated and the cells can escape the rigorous environment (Wang et al, 2011).…”

Emerging GM3 Regulated Biomarkers in Malignant Melanoma

“…To keep the discussion focused, we will discuss the relationship between GM3 contents and their abilities to regulate proto-oncogenes or tumor suppressor genes, which in turn mediate melanoma metastasis. To screen GM3 target genes, we obtained cells (CSSH-1) that overexpressed B4galt6 cDNA and cells (CAH-3) that suppressed its expression, which in turn result in GM3 modulation [4], [5]. In the CSSH-1 cells, GM3 contents were doubled, but in the CAH-3 cells, GM3 expression was halved compared with vector transfectant control, SM-1 and CM-1, respectively [4], [5].…”

“…To screen GM3 target genes, we obtained cells (CSSH-1) that overexpressed B4galt6 cDNA and cells (CAH-3) that suppressed its expression, which in turn result in GM3 modulation [4], [5]. In the CSSH-1 cells, GM3 contents were doubled, but in the CAH-3 cells, GM3 expression was halved compared with vector transfectant control, SM-1 and CM-1, respectively [4], [5]. To further confirm the roles of GM3 in melanoma cells, St3gal5 silenced cells were established by transfecting B16 cells with St3gal5 siRNA and it was found that the introduction of St3gal5 siRNA to B16 cells resulted in GM3 depletion as compared with the scrambled siRNA transfectant control [4], [5].…”

“…In the CSSH-1 cells, GM3 contents were doubled, but in the CAH-3 cells, GM3 expression was halved compared with vector transfectant control, SM-1 and CM-1, respectively [4], [5]. To further confirm the roles of GM3 in melanoma cells, St3gal5 silenced cells were established by transfecting B16 cells with St3gal5 siRNA and it was found that the introduction of St3gal5 siRNA to B16 cells resulted in GM3 depletion as compared with the scrambled siRNA transfectant control [4], [5]. Moreover, we would elucidate the mechanism that GM3 regulate melanoma metastasis via the genes, such as Ly-GDI, TNF-α, MMP-9, MMP-2, Caveolin-1 and Plaur, etc.…”