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Ceramide targets xIAP and cIAP1 to sensitize metastatic colon and breast cancer cells to apoptosis induction to suppress tumor progression
- Amy V Paschall†1,
- Mary A Zimmerman†1,
- Christina M Torres1,
- Dafeng Yang1,
- May R Chen1,
- Xia Li1,
- Erhard Bieberich2,
- Aiping Bai3,
- Jacek Bielawski3,
- Alicja Bielawska3 and
- Kebin Liu1, 4Email author
© Paschall et al.; licensee BioMed Central Ltd. 2014
Received: 19 September 2013
Accepted: 9 January 2014
Published: 15 January 2014
Ceramide is a bioeffector that mediates various cellular processes, including apoptosis. However, the mechanism underlying ceramide function in apoptosis is apparently cell type-dependent and is not well-understood. We aimed at identifying molecular targets of ceramide in metastatic human colon and breast cancer cells, and determining the efficacy of ceramide analog in suppression of colon and breast cancer metastasis.
The activity of and mechanism underlying ceramide as a cytotoxic agent, and as a sensitizer for Fas-mediated apoptosis was analyzed in human cell lines established from primary or metastatic colon and breast cancers. The efficacy of ceramide analog LCL85 in suppression of metastasis was examined in preclinical mouse tumor models.
Exposure of human colon carcinoma cells to ceramide analog LCL85 results in apoptosis in a dose-dependent manner. Interestingly, a sublethal dose of LCL85 increased C16 ceramide content and overcame tumor cell resistance to Fas-mediated apoptosis. Subsequently, treatment of tumor cells with exogenous C16 ceramide resulted in increased tumor cell sensitivity to Fas-mediated apoptosis. LCL85 resembles Smac mimetic BV6 in sensitization of colon carcinoma cells to Fas-mediated apoptosis by inducing proteasomal degradation of cIAP1 and xIAP proteins. LCL85 also decreased xIAP1 and cIAP1 protein levels and sensitized metastatic human breast cancer cells to Fas-mediated apoptosis. Silencing xIAP and cIAP1 with specific siRNAs significantly increased the metastatic human colon carcinoma cell sensitivity to Fas-mediated apoptosis, suggesting that IAP proteins mediate apoptosis resistance in metastatic human colon carcinoma cells and ceramide induces IAP protein degradation to sensitize the tumor cells to apoptosis induction. Consistent with its apoptosis sensitization activity, subtoxic doses of LCL85 suppressed colon carcinoma cell metastatic potential in an experimental lung metastasis mouse model, as well as breast cancer growth and spontaneous lung metastasis in an orthotopic breast cancer mouse model.
We have identified xIAP and cIAP1 as molecular targets of ceramide and determined that ceramide analog LCL85 is an effective sensitizer in overcoming resistance of human cell lines established from metastatic colon and breast cancers to apoptosis induction to suppress metastasis in vivo.
Fas (also termed CD95 and TNFRSF6) is a member of the TNF death receptor superfamily. Despite other “non-apoptotic” cellular responses emanating from its signaling, the major and best known function of Fas is apoptosis. Fas is expressed on tumor cell surface, and its physiological ligand, FasL, is expressed on activated T cells and NK cells. Compelling experimental data from both human cancer patients and mouse tumor models indicate that the Fas-mediated apoptosis pathway plays a key role in suppression of cancer development and in host cancer immunosurveillance [1–3]. Furthermore, human cancer genomics data indicate that Fas is not significantly focally amplified across a dataset of 3131 tumors, but is significantly focally deleted across the entire dataset of these 3131 tumors, including human colorectal cancer (http://www.broadinstitute.org/tumorscape/pages/portalHome.jsf). These data thus strongly suggest that Fas functions as a tumor suppressor.
To avoid apoptosis, tumor cells tend to down-regulate Fas expression or alter the expression of key mediators of the Fas-mediated apoptosis signaling pathway to advance the disease [4–6]. This is well-supported by the phenomenon that resistance to apoptosis, including Fas-mediated apoptosis, is a hallmark in human cancers [5, 7], particularly in metastatic human colorectal cancer [2, 3, 8] and breast cancer . Therefore, therapeutic intervention of tumor cell resistance to Fas-mediated apoptosis potentially represents an effective approach to render tumor cell sensitivity to FasL+ cytotoxic T lymphocytes (CTL) of the host immunosurveillance system or to CTL-based adoptive cancer immunotherapy to suppress tumor progression [1, 5, 10].
During the last decade, sphingolipids have emerged as bioeffectors that mediate various cellular processes, including proliferation and apoptosis of cancer cells [11–14]. Sphingolipid deregulation, namely the balance between ceramide and sphingosine 1-phosphate, has been implied as a key factor in tumor pathogenesis and apoptosis resistance [13, 15]. Although it has been demonstrated that de novo-generated ceramides may confer certain types of tumor cells with resistance to apoptosis , ceramide, the central molecule of the sphingolipid metabolism pathway, generally promotes apoptosis [17–19]. The role of ceramide in Fas-mediated apoptosis has also been well-documented . Ceramide enables Fas receptor to cluster to increase Fas-mediated apoptosis , and modulate Fas receptor activation [22, 23]. Ceramide has also been shown to regulate apoptosis through modulating key molecules of the Fas-mediated apoptosis pathways [22, 24–26]. Elevation of acid ceramidase, the enzyme that converts ceramide to sphingosine and subsequently sphingosine 1-phosphate, has been frequently observed in apoptosis-resistant cancer cells, including metastatic colon carcinoma cells [17, 27, 28]. These observations thus suggest that targeting ceramide metabolism to increase ceramide accumulation might be an effective approach to overcome cancer cell resistance to Fas-mediated apoptosis. In this study, we demonstrated that aromatic ceramide analog LCL85 effectively overcomes metastatic human colon and breast cancer cell resistance to Fas-mediated apoptosis at least partially through inducing proteasomal degradation of cIAP1 and xIAP in vitro. More significantly, we demonstrated that LCL85 effectively suppresses colon and breast cancer metastasis in vivo. Our data determined that LCL85 is potentially an effective apoptosis sensitizer that warrants further development as an adjunct agent to increase the efficacy of FasL+ CTL-based cancer immunotherapy.
BALB/c mice were obtained from National Cancer Institute (Frederick, MD). All studies are approved by the Georgia Regents University Institutional Animal Care and Use Committee (Protocol# 2011–0365).
All human cell lines established from primary and metastatic colon and breast cancer tissues (referred to as primary and metastatic human colon and breast cancer cell lines), and mouse breast cancer cell line 4 T1 were obtained from American Type Culture Collection (ATCC) (Manassas, VA). ATCC characterizes these cells by morphology, immunology, DNA fingerprint, and cytogenetics. Murine Colon26 cells were kindly provided by Dr. William E. Carson, III (Ohio State University, Columbus, OH).
BV6 was kindly provided by Genentech. Ceramide analogs B13 and LCL85 were synthesized by Lipidomics Shared Resource at Medical University of South Carolina . FasL (Mega-Fas Ligand®) was provided by Drs. Steven Butcher and Lars Damstrup (Topotarget A/S, Denmark). C16-ceramide was obtained from Santa Cruz Biotech, and was dissolved in dodecane:ethanol (2:98, v/v; 0.05% final concentration) as described . MG-132 and Z-VAD-FMK were obtained from Enzo Life Sciences (Farmingdale, NY).
Western blotting analysis
Western blotting analysis was performed as previously described . Anti-cIAP1 was obtained from R&D System (Minneapolis, MN). Anti-Bax and cIAP2 antibodies were obtained from Santa Cruz Biotech (Santa Cruz, CA). Anti-Bak and xIAP antibodies were obtained from Cell Signaling Biotech (Danvers, MA), anti-Bcl-2, and Bcl-xL antibodies were obtained from BD Biosciences (San Diego, CA), and anti-β-actin was obtained from Sigma (St Louis, MO).
Cell viability assays
Cell viability assay was carried out as previously described  using the MTT cell proliferation assay kit (ATCC, Manassas, VA).
Cells were treated with BV6, LCL85, or C16 ceramide for 1 h, followed by incubation with FasL for approximately 24 h. Apoptosis analysis was as previously described . Briefly, cells were then collected and incubated with propidium iodide (PI) and Annexin V (Biolegend), and analyzed by flow cytometry. The percentage of apoptosis was calculated by the formula: % apoptosis = % PI and AnnexinV double positive cells with FasL - % PI and Annexin V double positive cells without FasL.
Measurement of endogenous ceramide level
Cellular levels of endogenous ceramides were measured by Lipidomics Shared Resource, MUSC, using high-performance liquid chromatography-mass spectrometry approach (LC-MS/MS) as previously described. Ceramide levels were normalized to the total cellular protein contents.
Cell surface protein analysis
Tumor cells were stained with anti-Fas (BD biosciences), anti-FasL (BD biosciences), or anti-CD8 (Biolegend, San Diego, CA) mAbs. Isotype-matched control IgG (Biolegend) was used as a negative control. The stained cells were analyzed by flow cytometry. For FasL protein analysis, mouse lungs were digested in collagenase solution to make a single cell suspension. The cell suspension was stained with PE-conjugated FasL (BD Biosciences) or FITC-conjugated CD8 mAb, or both mAbs and analyzed by flow cytometry.
RNAi-based silencing of gene expression in tumor cells was done as previously described . Briefly, SW620 cells were transiently transfected with scramble siRNA (Dharmacon), and human xIAP- and cIAP1-specific siRNAs (Santa Cruz Biotech), respectively, using Lipofectamine 2000 (Invitrogen) for approximately 24 h. Cells were then harvested. Part of the cells were used for RT-PCR analysis of xIAP and cIAP expression. Another part of the cells were cultured in the absence or presence of FasL for approximately 24 h and then analyzed for apoptosis.
Liver toxicity analysis
LCL85 was injected to BALB/c mice (5 mg/kg body weight) i.v. Peripheral blood was collected from mice 3 days later using Multivette 600 Z-gel tubes (SARSTEDT). Serum was separated by centrifugation and measured for complete liver enzyme profile at Georgia Laboratory Animal Diagnostic Service (Athens, GA).
Colon cancer experimental lung metastasis
Colon 26 cells (1.5×105 cells/mouse) were injected to BALB/c mice iv. LCL85 (0, 1 and 5 mg/kg body weight) was injected iv to tumor-bearing mice at days 3, 6, 9 and 12 after tumor injection. Mice were sacrificed at day 14 and analyzed for lung metastasis as previously described .
Breast cancer spontaneous lung metastasis
4 T1 cells (1×104 cells/mouse) were injected to the mammary fat pad. LCL85 (2.5 mg/kg body weight) was injected to the tumor-bearing mice at days 7, 10, 13, and 16 after tumor injection. Mice were sacrificed 29 days after tumor injection, and analyzed for primary tumor growth and lung metastasis. To determine the efficacy of LCL85 on metastasis, 4 T1 cells (1×104 cells/mouse) were injected to the mammary fat pad. Primary tumors were surgically removed 16 days later. Mice were treated with LCL85 (2.5 mg/kg body weight) at days 10, 13, and 16 after surgery. Mice were sacrificed and analyzed for lung metastasis 19 days after surgery.
Where indicated, data were represented as the mean ± SD. Statistical analysis was performed using two-sided t test, with p-values < 0.05 considered statistically significant.
Ceramide analog effectively sensitizes metastatic human colon and breast cancer cell apoptosis resistance
Next, we used SW620 and LS411N cells to determine whether the above observed tumor cell growth inhibition is due to apoptosis. SW620 and LS411N cells were cultured in the presence of LCL85 and FasL, and analyzed for apoptosis. Staining cells with Annexin V and PI revealed that LCL85 induces apoptosis of SW620 and LS411N cells in a dose-dependent manner. However, LCL85 alone at low doses only induced a small degree of apoptosis (Figure 2C). In contrast, a sublethal dose of LCL85 dramatically increased SW620 and LS411N cell sensitivity to FasL-induced apoptosis (Figure 2C & D).
LCL85 increases cellular C16 ceramide level to sensitize colon carcinoma cells to apoptosis
xIAP and cIAP1 are molecular targets of LCL85
BV6 targets IAP proteins to induce apoptosis
We then analyzed the effects of LCL85 on IAP proteins in metastatic human colon carcinoma cells. SW620 cells were treated with LCL85 and analyzed for IAP protein levels at various time points. Among the 3 IAP proteins, xIAP protein levels dramatically decreased 12 h after LCL85 treatment. cIAP1 protein was also decreased, albeit at a smaller degree. cIAP2 protein level was not significantly changed by LCL85 treatment (Figure 7B). To determine whether LCL85 also decreases xIAP protein levels in metastatic human breast cancer cells, MDA-MB-231 cells were treated with LCL85, and analyzed for xIAP and cIAP protein levels. It is clear that LCL85 decreases xIAP and cIAP1 protein levels in a dose-dependent manner (Figure 7C). Next, SW620 cells were cultured in the presence of a sublethal dose of BV6 (5 μM) and FasL, and analyzed for apoptosis. It is clear that BV6 dramatically increased SW620 cell sensitivity to FasL-induced apoptosis (Figure 7D). Our results thus revealed that LCL85 targets xIAP and cIAP1 to sensitize metastatic human colon carcinoma cells to Fas-mediated apoptosis.
RT-PCR analysis indicated that LCL85 does not alter the mRNA levels of IAP proteins in human colon carcinoma cells (Figure 7E). Proteasome inhibitor MG-132 blocked LCL85-induced xIAP degradation, whereas caspase inhibitor Z-VAD did not block LCL85-induced xIAP degradation (Figure 7F). Our data thus suggest that LCL85 mediates proteasome-dependent degradation of xIAP protein.
To validate the functions of xIAP and cIAP1 in Fas-mediated apoptosis in human colon carcinoma cells, SW620 cells were transfected with xIAP- and cIAP1-specific siRNAs, respectively (Figure 8A), and analyzed the tumor cell sensitivity to FasL-induced apoptosis (Figure 8B). Silencing xIAP or cIAP1 significantly increased the tumor cell to FasL-induced apoptosis (Figure 8C). Our data thus suggest that IAP proteins mediate apoptosis resistance in metastatic human colon carcinoma cells, and ceramide sensitizes the tumor cell to Fas-mediated apoptosis at least partially through inducing cIAP1 and xIAP degradation.
LCL85 also targets Bcl-xL
Ceramide analog and Smac mimetic additively sensitize metastatic human colon carcinoma cells to apoptosis induction
Sensitivity of mouse tumor cells to LCL85-sensitized and Fas-mediated apoptosis
Toxicity of LCL85
Mouse serum toxicity profiles of LCL85 *
Serum enzyme/protein level
LCL85 (mg/kg body weight)
Control (n = 2)
1 (n = 4)
5 (n = 3)
65 ± 7.1
67.5 ± 11.6
66.7 ± 5.1
20.5 ± 4.9
37.8 ± 10.2
57.3 ± 24.2
3.2 ± 0.1
3.1 ± 0.1
3.2 ± 0.2
205 ± 29.7
198.5 ± 48.7
176.7 ± 23
150.5 ± 0.7
152.3 ± 2.2
151 ± 3
5.2 ± 0.1
6.4 ± 1
6.9 ± 0.5
108.5 ± 0.7
111 ± 2.6
111.7 ± 2.9
16 ± 0
14.3 ± 2.2
15.7 ± 2.1
Anion Gap (mmol/L)
31 ± 0
33.5 ± 4.2
30.7 ± 2.5
10.3 ± 0.1
10.2 ± 0.6
9.9 ± 0.3
8.8 ± 3.7
9.2 ± 1.9
8.5 ± 0.5
74.5 ± 9.2
68.8 ± 12.4
86 ± 4.6
24.5 ± 3.5
29.3 ± 4
26.3 ± 1.5
Total Bilirubin (mg/dl)
0.1 ± 0
0.1 ± 0
0.1 ± 0
Total Protein (g/dl)
4.5 ± 0.1
4.5 ± 0.3
4.8 ± 0.2
LCL85 suppresses colon carcinoma metastatic potential in an experimental lung metastasis mouse model in vivo
LCL85 suppresses spontaneous breast cancer metastasis in vivo
Ceramide mediates apoptosis through multiple mechanisms. It has been reported that ceramide mediates Fas receptor clustering, capping and activation to promote Fas-mediated apoptosis [21–23]. Ceramide has also been shown to regulate Bcl-x alternative splicing to decrease Bcl-xL level , and mediates Bak, Bax and Bcl-2 functions in the intrinsic apoptosis pathway [39–43]. The effects of ceramide on these apoptosis mediators are apparently cell type- or cellular context-dependent since LCL85 only alters the expression level of Bcl-xL in human colon and breast cancer cells. Here, we identified xIAP and cIAP1 as targets of the ceramide signaling pathways in both metastatic human colon and breast cancer cells. We observed that LCL85 effectively decreased cIAP1 and xIAP protein levels in metastatic human colon and breast cancer cells. Consistent with the decreased xIAP1 and cIAP1 protein levels, metastatic human colon carcinoma cells exhibited increased sensitivity to FasL-induced apoptosis. Furthermore, treatment of metastatic human colon carcinoma cells with cIAP1 and xIAP-specific inhibitor BV6 also significantly increased tumor cell sensitivity to FasL-induced apoptosis . Therefore, our data suggest that xIAP1 and cIAP1 proteins are responsible, at least in part, for the apoptosis-resistant phenotype in metastatic human colon and breast cancers, and LCL85 overcomes metastatic human colon and breast cancer cell resistance to Fas-mediated apoptosis at least partially through inducing proteasomal degradation of xIAP and cIAP1 proteins.
It has been well-documented that Smac mimetic BV6 specifically targets cIAP1 and cIAP2 proteins to induce apoptosis through activating the TNFα signaling pathway [36, 45]. However, it has also been shown that xIAP, rather than cIAP1 and cIAP2, is the critical target of BV6 in Fas-mediated apoptosis [44, 46]. Strikingly, we observed that LCL85 also sensitizes tumor cells to Fas-mediated apoptosis through inducing proteasomal degradation of xIAP. LCL85 treatment increased endogenous C16 ceramide level and exogenous C16 ceramide is effective in sensitizing the apoptotic resistant metastatic human colon carcinoma cells to Fas-mediated apoptosis. Therefore, it is possible that LCL85 sensitizes tumor cells to Fas-mediated apoptosis at least in part through inducing C16 ceramide accumulation, resulting in ceramide-mediated xIAP and cIAP1 proteasomal degradation. However, the molecular mechanisms underlying the crosstalk network between ceramide analog, C16 ceramide and IAP proteins remain to be elucidated.
Ceramide analog-mediated direct cytotoxicity often depends on administering a high dose of the agent . In this study, LCL85 exhibited potent anti-tumor cytotoxicity, suggesting that LCL85 is potentially an effective therapeutic agent in cancer therapy. However, LCL85 also exhibited toxicity in a dose-dependent manner. Therefore, LCL85 might also be toxic if used in high doses. Interestingly, we demonstrated that a sublethal dose of LCL85 is not cytotoxic but effectively sensitizes metastatic human colon carcinoma cells to FasL-induced apoptosis in vitro. This observation is significant since a sublethal dose of LCL85 might be safe and yet an effective sensitizer in FasL+ CTL-based cancer immunotherapy.
Tumor-reactive CTLs primarily use the perforin and Fas/FasL effector mechanisms to induce target tumor cell apoptosis. Immunosuppression of CTL activation and effector functions by immuno- suppressive cells is a major challenge in cancer immunotherapy. However, recent studies revealed that the immuno- suppressive Treg cells only selectively suppress the perforin pathway without inhibiting CTL activation and proliferation in vivo[48, 49], suggesting that Treg cells may not suppress the Fas/FasL effector mechanism of CTL in vivo. Indeed, our recent study showed that tumor-infiltrating CTLs in tumor-bearing mice and CTLs from human colon and breast cancer patients are FasL+. Therefore, the Fas/FasL effector mechanism might be functional in the immuno suppressive tumor microenvironment. However, metastatic human colon and breast cancer cells are often resistant to Fas-mediated apoptosis [8, 9]. Therefore, a therapeutic agent that can sensitize tumor cell Fas resistance may represent an effective enhancer of CTL-based cancer immunotherapy against metastatic colon and breast cancers. Our data suggest that LCL85 is potentially such an agent. Although LCL85 does not effectively sensitize Colon 26 cells to FasL-induced apoptosis, LCL85 is effective in suppressing Colon 26 cell metastatic potential in vivo, suggesting that other host factors, such as IFN-γ and TNFα secreted by T cells, might also act to sensitize the tumor cells to apoptosis in vivo, which requires further study.
We envision that a sublethal dose of LCL85 can be used as a sensitizer in cancer immunotherapy for metastatic colon and breast cancers. This idea is analogous to a “one-two punch” concept. First, cancer patients are treated with a non-cytotoxic dose of LCL85 to sensitize cancer cells to apoptosis. Once “sensitized”, patients are then treated with FasL+ CTLs-based immunotherapy to suppress cancer metastasis. Our in vivo tumor suppression studies showed that low doses (2.5-5 mg/kg body weight) of LCL85 exhibited potent tumor suppression activity in immune-competent mice in vivo (Figure 12). A previous study showed that lack of ceramide accumulation in target cells is a significant cause of resistance to cytotoxic T lymphocyte (CTL)-induced apoptosis . In this study, we observed that a large portion of the tumor-infiltrating CTLs are FasL+ (Figure 12C), and low doses of LCL85 effectively suppresses colon and breast tumor growth and metastasis in immune-competent mice (Figures 12 & 13). Our observations thus indicate that LCL85 might sensitize tumor cells to CTL-induced apoptosis through inducing ceramide accumulation in the tumor cells in vivo, which requires further investigation. Nevertheless, our data suggest that LCL85, although effective as a single agent in suppression of tumor development at high doses, might be more valuable if used at a sublethal dose as a sensitizer for enhancing the efficacy of FasL-based cancer therapy, particularly CTL-based cancer immunotherapy.
We thank Dr. Wayne J. Fairbrother for advice and critical reading of the manuscript. This work was supported by National Institutes of Health grants CA133085, CA168512-DRP1 (to KL), C06RR018823, P30CA138313, and P20RR017677 (to AB).
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