GM3 involved in regulating cancer related genes in melanoma.
Gangliosides, GSLs, are expressed in the outer leaflet of the plasma membrane of animal cells and involved in a variety of functions, including serving as antigens, receptors for bacterial toxins, mediators of cell adhesion, and mediators and modulators of signal transduction. Moreover, the accumulated lines of evidence have shown that gangliosides play pivotal roles in cancer metastasis. As the first and simplest member in the metabolic series of the ganglioside family, GM3 is a metabolic precursor of more complex natural gangliosides, which in turn determines their contents and biological functions in cells and tissues. GM3 has been demonstrated to be involved in regulation of various processes including cell proliferation, differentiation, apoptosis, embryogenesis and oncogenesis, etc. However, it is difficult to understand the defined functional concepts of GM3 in cancer metastasis because GM3 indirectly exert their effects via regulating target genes.
Target genes indicated in cellular transformation and tumor progression have been divided into two categories: proto-oncogenes and tumor suppressor genes. In cancer, it can thus be speculated that an altered balance of tumor suppressor genes towards proto-oncogenes may contribute to tumor transformation. Alterations in proto-oncogenes and tumor suppressor genes are largely dependent on point mutation, amplification or translocation. Consequently, the normal control mechanisms that constrain the expression of genes are undermined, and thus the oncogene is continually expressed, resulting in tumor transformation . Similarly, genetic transformation has been linked to melanoma genesis and progression. These genes encompass many signaling pathways, including the RTK, PI3-K, Rb, p53, Wnt, and NF-κB pathways . Further evidence implicated the downstream oncogenes of these pathways in melanoma ranges from Ras, B-Raf, Kit, Mitf, Cyclin D1, CDK4 to HDM2 , . Although genetic discoveries related to melanoma transformation have been accelerated greatly in recent years, the involvement of GM3 in melanoma metastasis via these proto-oncogenes or tumor suppressor genes has not yet been clearly established.
The present chapter was aimed to give insights into the mechanisms that GM3 regulates melanoma metastasis via its target genes. 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 , . 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 , . 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 , . 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
2. Materials and methods
2.1. Cell lines and culture
Murine melanoma B16 cells were kindly provided by Dr. Kiyoshi Furukawa of Nagaoka University of Technology, Japan. The cells were maintained in medium containing Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Invitrogen Corporation, NY, USA) supplemented with 10% fetal bovine serum (TBD; Tianjin Hao Yang Biological Company, Tianjin, China), 100 U/ml penicillin, and 100 μg/ml streptomycin and incubated in a humidified (37°C, 5% CO2 and 95% air) incubator (Sanyo, Toyota, Japan). The cells were usually grown in a 60-mm culture dish (BD Falcon, CA, USA) and passaged once they reached 75% confluence. To observe the effects of pharmacological inhibitors on genes expression, cells were treated with pharmacological inhibitors for 24 h before analyzing genes expression by RT-PCR.
2.2. Chemicals and antibodies
LY294002 and LY303511 were purchased from Sigma-Aldrich (St. Louis, MD, USA). The Rneasy mini kit to extract total RNA was obtained from Qiagen (Hilden, Germany). The RT-PCR kit was from Takara Biotechnology Corporation (Dalian, China). All other reagents were from Invitrogen (Carlsbad, CA, USA), unless otherwise specified.
|Positive||Caveolin-1||1.378||0.321||0.146||(1)7, (4)7, (5)7|
|E-cadherein||1.875||0.695||0.721||(1)10, (3)11, (5)12, (6)13|
|Rho B||2.247||0.427||0.318||(5)20, (6)20, (8)20|
|Tnf α||2.188||0.349||0.292||(4)23, (5)23|
|Negative||Integrin β5||0.783||1.465||1.754||(1)26, 27, (2)28, (3)29, (4)30|
|TGF-β1||0.571||2.124||3.309||(1)32, (4)33, (5)33|
|Src||0.639||1.347||1.925||(1)36, (3)36, (4)37, (5)36|
2.3. RNA extraction and RT-PCR
RNA extraction and analysis of amplified DNA were described in our previous work , , , [38-40]. The primers used in this study were designed with primer 3 software and syntehsized by Shanghai Genebase Biotechnology Corporation (Shanghai, China). Primer sequences used for the PCR in this study were as follows: Plau, Sense 5’-GCCCACAGA CCTGATGCTAT and Antisense 5’-TAGAGCCTTCTGGCCACACT; Plaur, Sense 5’-AGGTGGTGACAAGAGGCTGT and Antisense 5’-AGCTCTGGTCCAAAGAGGTG; gelsolin, Sense 5’-TCCAACAAGGTGCCAGTGGA and Antisense 5’- CAGCACAAAGGCATCGTTGG; Caveolin-1, Sense 5’-CTACAAGCCCAACAACAAGGC and Antisense 5’- AGGAAGCTCTTGATGCACGGT; Ly-GDI and Eef sequences are provided in our previous publications , . The mRNA levels of the genes under consideration, using Eef mRNA as an internal control, were determined by RT-PCR semiquantitatively, as described previously [4, 5, 9, 38-40]. Candidate mRNA values are expressed as a ratio of candicate mRNA to Eef mRNA and are usually expressed as unity for control experiments.
2.4. siRNA and cDNA constructs
Target sequences were designed and synthesized as previously described [4, 5, 9, 38-40]. Effective siRNA sequence targeting Ly-GDI can be found in our previous publications [5, 40]. For ectopic expression of Caveolin-1 or Ly-GDI, total RNA was isolated from mouse FBJ-S1 cells. After the first-strand cDNA was synthesized, a Caveolin-1 or Ly-GDI transcript was amplified using the following sense and antisense primers: Caveolin-1, Sense 5’-GCTAGCATGTCTGGGGGCAAATACGT and Antisense 5’-GGATCCTCATATCTCTTTCTGCGTGC; Ly-GDI, 5’- GCTAGCATGACGGAGAAGGATGCACAGCCA and Antisense 5’- GGATCCTCATTCTGTCCAATCCTTCT. The coding sequence of Caveolin-1 or Ly-GDI was inserted between NheI and BamHI digestion sites for sense expression in a pITES-puro3 expression vector with puromycin resistance (Clonetech, USA). The plasmids were prepared and confirmed by sequencing analysis.
In RNA interference experiments, B16 cells were transfected as previously described [4, 5, 9, 38-40]. In brief, B16 cells were transfected with Ly-GDI siRNA for 3 days and the stably transfected cells were further selected by G418 treatment. In control experiments, cells were transfected with scramble siRNA and also selected with G418. For ectopic expression experiments, B16 cells were transfected with Caveolin-1 or Ly-GDI cDNA contructs before analysing the mRNA expression of corresponding genes after 3 days. Control cells were transfected with empty vector.
3. Involvement of GM3 targeted genes in mediating melanoma metastasis
Accumulating evidence using thin-layer chromatography techniques has shown that the gangliosides GM3 and GD3 are predominantly expressed in melanoma cells and are present in relatively higher concentrations than the more complicated gangliosides GT1, GD1 and GM1 in adult brain tissue . In line with this report, GM3 has been detected in both human and murine melanoma cells by MAb M2590 , . Although GM3 has been identified several decades ago, the progress on the mechanism of GM3 in mediating melanoma metastasis is slow. Prior works have shown that GM3 facilitates melanoma B16 cells to metastasize in C57BL/6 mice , , but the mechanism remains unknown. In view of the complicated steps of melanoma metastasis and multiple biological functions of GM3, it is necessary to address the targets molecules through which GM3 exerts its functions on melanoma metastasis.
Ly-GDI, a Rho GTPase dissociation inhibitor beta, is also known as RhoGDI2, Arhgdib or D4-GDI. It belongs to a family of RhoGDIs 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, GAP, and the effector targets . Among GDIs, Ly-GDI differs substantially at the structural level from the other two GDIs and is regarded as an invasive and metastatic suppressor gene in human bladder cancer cells , . In line with these findings, Ota
As a first step, we evaluated the effect of endogenous GM3 on Ly-GDI mRNA expression. Our data reveal that Ly-GDI mRNA expression is always proportional to endogenous GM3 contents (Table. 1), which suggests possible roles of GM3 in Ly-GDI regulation. To further confirm the notion that GM3 is responsible for Ly-GDI regulation, GM3 was exogenously added in the culture medium of GM3-depleted cells, such as B11 clone and CAH-3 cells, as well as B16 parental cells. Our data reveal that exogenous GM3 significantly bound to biological membranes, which resulted in upregulation of Ly-GDI expression in three cell lines . Reciprocally, GM3 depletion was carried out by incubating cells with D-PDMP, which in turn suppresses Ly-GDI expression. Collectively, our data demonstrated that GM3 contents play pivotal role in regulating Ly-GDI expression .
To keep the discussion focused, we next aimed to characterize the signaling pathway of GM3 in regulating Ly-GDI expression. In light of the possible role of PI3-K pathway in GM3 signaling cascade , , we examined the effects of PI3-K inhibitor, LY294002, on Ly-GDI mRNA expression in the absence or presence of GM3. Treatment of B16 and CSSH-1 cells with LY294002 not only suppressed Ly-GDI mRNA expression, but also reversed the effects of GM3 on increasing Ly-GDI mRNA expression. Similarly, this pharmacological intervention was also effective in suppressing Akt phosphorylation at Ser 473 and Thr 308 without altering total Akt levels. To further exclude the possible non-specific effects of PI3-K inhibitors, we transfected B16 cells with Akt1 or Akt2 siRNA. Our results revealed that Akt1 or Akt2 knock down decreases Ly-GDI expression at mRNA level and the effects of suppression are more profound in Akt1/2 doubly silenced cells. It is noteworthy that GM3 are not able to upregulate Ly-GDI expression in Akt1/2 double knock down cells. Collectively, our results demonstrate that GM3 regulate Ly-GDI expression via PI3-K/Akt pathway .
Although we had found that PI3-K/Akt pathway plays pivotal role in regulating Ly-GDI expression, the question is easily raised whether GM3 directly activates PI3-K pathway or not. In view of the important role of Pdpk1 and mTOR complexes in PI3-K signaling cascade , we determined their expression in B11 cells. Our data revealed that GM3 knock down concurrently reduces Pdpk1 and Raptor expression, whereas induces Rictor expression. Taking the advantage of siRNA technique, we further found that Pdpk1 and Raptor, but not Rictor knock down abolished GM3 effects on Ly-GDI induction via blocking Akt phosphorylation at Thr 308 . From these observations, we demonstrate that GM3 induces Ly-GDI expression via PI3-K, Pdpk1, AktThr308 and mTOR/Raptor pathway in melanoma B16 cells (Fig. 1). More importantly, the experiments were also carried out to determine if Ly-GDI is the key molecule in mediating melanoma B16 cells anchorage-independent growth. Our data demonstrate that Ly-GDI knock down significantly increased the proliferating ability of B16 cells in soft agar or serum free medium . It is noteworthy that expression of GM3 is gradually increased during the progression of melanoma malignancy. For instance, GM3 was not detected in normal or naevi skin, but 60% of primary and 75% of metastatic melanoma expressed GM3 . Our data along with previously published results  suggest that relatively lower levels of GM3 and Ly-GDI at the early stage of melanoma facilitate melanoma cells to undergo invasive proliferation in vigorous environment. These observations also provide insights into the molecular basis of GM3 on augmenting melanoma invasive proliferation at the early stage of pathology.
TNF-α is a multifunctional cytokine, which is synthesized as a 26kDa (233 amino acids) membrane-bound propeptide (pro-TNF-α) and is secreted upon cleavage by TNF-α converting enzyme . Studies on the effects of TNF-α using experimental models of invasion and metastasis have shown that it can often act as a tumor promoting factor . More specifically for melanoma, TNF-α has been reported to upregulate the expression of integrin subunits, which in turn enhance the interaction of human melanoma cells with ECM substrates , . The more recent study from this laboratory showed that TNF-α induces integrin expression, cell attachment and invasion via fibronectin in human melanoma cells . In light of these prior works, our recent data demonstrate that TNF-α located downstream of Ly-GDI to mediate melanoma metastasis (Fig. 4) , , . To verify that TNF-α follows the same GM3 signaling cascade as Ly-GDI, we carried out the same experiments as above Ly-GDI. In line with GM3 regulating Ly-GDI signaling pathway , our results revealed that GM3 regulates TNF-α mRNA and protein expression via PI3-K, Akt and mTOR pathway , , suggesting that TNF-α is indeed the downstream target of Ly-GDI through which mediate biological functions of GM3 and Ly-GDI in melanoma metastasis. Once TNF-α was found to be a link in the chain of GM3 signaling, we focused on its biological effects on melanoma metastasis. The results demonstrate that TNF-α is able to enhance melanoma migration via inducing MMP-9 expression and activity , which will be further discussed in “MMP-9 and MMP-2” part (Fig. 1). On the other hand, Venessa
3.3. MMP-9 and MMP-2
As above discussion, TNF-α is able to enhance melanoma migration via inducing MMP-9 expression and activity , we next aimed to elucidate its mechanism. In order to determine the role of MMP-9 in cell migration, we examined the effects of an MMP-9 agonist and antagonist on cell migration, as stimulation of MMP-9 expression by TNF-α has been reported in several studies , . We therefore used TNF-α as a positive control against the MMP-9 inhibitor GM6001. RT-PCR results demonstrate that TNF-α markedly induces MMP-9 expression and activity, which is reversed by GM6001 treatment . Furthermore, cell migration tested by transwell experiments showed that the numbers of cells migrating were consistent with MMP-9 expression . This finding is in accordance with previously published data showing that TNF-α increases human melanoma cell invasion and migration
In addition to MMP-9, it is noteworthy that MMP-2 is also induced by endogenous GM3 (Table. 1). Although we are still not figure out the mechanism that GM3 positively regulates MMP-2 expression in melanoma B16 cells, MMP-2 has been implicated to be associated with progression of the melanoma . In more detail, although all skin and nodal metastasis were negative for MMP-2, higher MMP-2 concentrations were observed in patients with metastatic disease (stage IV) than in those with primary melanoma (stage I) or in controls . In addition, Liu
3.4. Plau and Plaur
During the course of MMP-9 and MMP-2 investigations, experimental evidence had led to the recognition of Plau and Plaur , . Indeed, Plau and Plaur are highly expressed in advanced stages of primary and metastatic melanoma progression . In addition, Bianchini
Gelsolin is a representative actin-regulatory protein with an 82kDa mass and is present in most vertebrate tissues. Gelsolin controls the length of actin polymers
Caveolin-1 is a 22-24 KDa protein originally identified as a structural component of caveolae, specialized invagination of the plasma membrane. These caveolae represent compartments in which key signaling transduction molecules are concentrated to provide an efficient system for cellular cross talk. However, relatively little information is available concerning the role of Caveolin-1 in melanomas. Early studies demonstrate that increased Caveolin-1 expression was associated with enhanced malignancy in a non-cutaneous, retinal melanoma . An even more recent study identified exosomes in the plasma of melanoma patients with high levels of Caveolin-1.In this particular case, exosomes are associated with malignant tumor progression as a small vesicle secreted by both normal and tumoral cells . These prior works demonstrate that Caveolin-1 are relevant to function in melanoma metastasis.
Additional studies support the notion that presence of Caveolin-1 helps melanoma metastasis. Felicetti et al. proposed that Caveolin-1 expression is associated with increased metastatic potential in different human melanoma cell lines. Specifically, Caveolin-1 expression increased cell proliferation, anchorage-independent growth, migration and invasion in WM983A melanoma cell line. Alternatively, Caveolin-1 down-regulation in metastatic Caveolin-1 overexpressing melanomas reduces their proliferation, as well as their tumorigenicity . Consistent with prior works , we further found that Caveolin-1 was regulated by endogenous GM3 (Table. 1). More importantly, our recent data revealed that Caveolin-1 is able to regulate TNF-α (Fig. 4), which in turn mediates melanoma migration or invasion through MMP-9 as discussed above . Our data along with previous reports  further implicated the important role of GM3-enriched membrane subdomain, especially Caveolae, in melanoma metastasis.
Over the past few decades, studies of Src and the SFKs have given new insights into the role of these proteins in regulating cell adhesion, invasiveness and motility in cancer cells and in tumor vasculature, rather than directly influencing cell proliferation . Src expression and activity are increased in melanoma cell lines and in melanoma tumors
Src can activate STAT3, STAT5 and other downstream targets in melanoma . The expression of STAT3 is highly expressed in both primary and metastatic melanoma in humans, although the expression level is variable . In addition, STAT3 is activated in human melanoma, but not in melanocytic or in benign melanocytic neoplasms . Moreover, blocking STAT3 signaling in mouse B16 melanoma cells resulted in the release of soluble factors capable of inducing apoptosis and cell-cycle arrest .
3.8. Rho B
Recent studies confirmed the role of the Rho proteins in cancer by showing their involvement in cell transforamtion, invasion, metastasis and angiogenesis. The major members of the Rho subfamily comprise the Rho A, Rho B and Rho C proteins. Rho B is quite different from Rho A and Rho C in many aspects, although it shares 87% homology . For example, Rho B has a tumor suppressive role, including inhibiting cell proliferation and inducing apoptosis in several human cancer cells, and inhibiting tumor growth in murine model, in contrast, activation of Rho A promotes cell malignant transformation, proliferation, invasion and metastasis, like other small GTPases such as Ras, Rac1 and Cdc42  Moreover, Rho B, unlike the constitutively expressed Rho A, is inducible by genotoxic stress, such as U.V. light, growth factors (TGF-β1) and chemotherapeutic drugs (cisplatin and 5-FU). In our experimental system, we found that mRNA expression of Rho B is suppressed by Ly-GDI knocking down . This observation partially implied that Rho B located downstream of Ly-GDI to mediate its inhibitory effects on melanoma invasive proliferation and would also exerts its effects at early stage of melanoma progression (Fig. 1).
3.9. Other genes
During the course of our investigation in melanoma metastasis, we also found the involvement of some pro-oncogenes or tumor suppressor genes, such as TGF-β1, N-Cam, integrin β5, PKN1 or E-cadherin
Metastasis, the spread of malignant tumor cells from a primary site to distant sites, is the most life-threatening complication of cancer and a major problem of cancer treatment , . The metastatic process consists of multiple steps: 1) invasive proliferation as benign tumor at the primary site 2) dissociation of tumor cell(s) from the primary site with a concomitant loss of cell-cell and cell-ECM adhesions; 3) tumor-cell adhesion to and subsequent local digestion of basement membrane; 4) retraction of endothelial cells and subsequent intravasation; 5) survival within the vasculature; 6) extravasation from vasculature at a distinct site and 7) growth in a “foreign” or ectopic organ environment , . In view of these prior theories, our data summarized here reinforce the notion that GM3 potentially plays a dual role in melanoma development, as has been described in our previously published works4-6, , . At early stage of melanoma metastasis, lower level of GM3 induces melanoma invasive proliferation via Src, Rho B and Ly-GDI
GSLs, sialylated glycosphingolipids; RTK, receptor tyrosine kinase; PI3-K, phosphatidylinositol-3-kinase; Rb, retinoblastoma; PKN1, protein kinase 1; MMP-9, matrix matelloproteinase-9; MMP-2, matrix metalloproteinase-2; Apaf1, apoptotic protease activating factor 1; TNF-α, tumor necrosis factor-α; Plau, urokinase-type plasminogen activator; Plaur, urokinase-type plasminogen activator receptor; TGF-β1, transforming growth factor-β1; TGFBR2, transforming growth factor, beta receptor 2; RhoGDI, RhoGDP dissociation inhibitors; GAP, GTPase-activating proteins; Pdpk1, 3-phosphoinositide dependent protein kinase-1; mTOR, mammalian target of rapamycin; Raptor, regulatory associated protein of mTOR; ECM, extracellular matrix; d-GM3, De-N-acetyl GM3; IFNγ, interferon γ; SFKs, Src-family kinases; STAT3, signal transducer and activator of transcription 3; STAT5, signal transducer and activator of transcription 5; JAK1, Janus kinase 1.