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Fas double-negative feedback loop regulates human colon carcinoma cells sensitivity to Fas-related apoptosis
Biochemical and Biophysical Research Communications 408 (2011) 494–499
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
A let-7/Fas double-negative feedback loop regulates human colon carcinoma cells sensitivity to Fas-related apoptosis Li Geng a,1, Bin Zhu b,1, Bing-Hua Dai a,1, Cheng-Jun Sui a, Feng Xu a, Tong Kan a, Wei-Feng Shen a, Jia-Mei Yang a,⇑ a b
The Department of Special Treatment, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, No. 225, Changhai Road, Shanghai 200438, China The Second Department of Billiary Surgery, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, No. 225, Changhai Road, Shanghai 200438, China
a r t i c l e
i n f o
Article history: Received 7 April 2011 Available online 21 April 2011 Keywords: Fas Let-7 microRNA Interferon-c Apoptosis
a b s t r a c t Interferon-c (IFN-c) is considered essential for the regulation of anti-tumor reactions as it sensitizes Fas-related apoptosis in HT29 cells, but the mechanism is unclear. In the current study, our data demonstrated that IFN-c stimulation and Fas activation suppressed Dicer processing and let-7 microRNA biogenesis, while let-7 microRNA strongly inhibited Fas expression by directly targeting Fas mRNA. Accordingly, our results indicate that Fas and let-7 microRNAs form a double-negative feedback loop in IFN-c and Fas induced apoptosis in colon carcinoma cell line HT29, which may be an important synergistic mechanism in anti-tumor immune response. We also found that a let-7 microRNA inhibitor increased Fas expression and sensitized cells to Fas-related apoptosis, which may have future implications in colon carcinoma therapy. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction Apoptosis occurs frequently in epithelial cells located in the gastrointestinal tract, significantly contributing to epithelial cell turnover and the lymphocyte-mediated antitumor response [1]. Studies have demonstrated that epithelial cell apoptosis is regulated by the cell surface receptor Fas. Fas is a member of the tumor necrosis factor receptor family of death receptors and can induce apoptosis in sensitive cells by binding to their specific death ligands, including Fas ligand (FasL), tumor necrosis factor-a (TNF-a) and Fas-specific monoclonal antibody CH11 (mAb CH11) [2,3]. Fas is highly expressed in normal human colon epithelial cells, but its expression becomes progressively decreased during the transition from normal epithelium to adenocarcinoma in approximately 50% of observed cases [4–6]. It is also well documented that most colon carcinoma cells show impaired responses to Fas-related apoptosis. Consequently, from an immunotherapy standpoint, the extent of responsiveness of malignant cells to Fas signals may profoundly influence the overall efficacy of any anti-tumor lymphocyte-mediated response, highlighting the need to explore strategies to sensitize tumor cells to Fas-mediated apoptosis. Interferon-c (IFN-c), an important member of the interferon family that regulates antiviral, antiproliferative, and immunomodulatory responses, has been reported to be involved in the regula⇑ Corresponding author. Fax: +86 021 65562400. 1
E-mail address: [email protected] (J.-M. Yang). These authors contributed equally to this work.
0006-291X/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2011.04.074
tion of apoptotic processes. This involvement includes the sensitization of various target cells to Fas-mediated death, such as ovarian cancer cells [7], renal cell carcinoma cells [8] and prostate carcinoma cells [9]. For colon carcinoma, IFN-c can sensitize human primary colon carcinoma cells to Fas-mediated apoptosis by enhancing Fas expression [10,11]. Previous studies have demonstrated that many genes are involved in the process of IFN-c-mediated sensitization, including IFN consensus sequence-binding protein and caspase-1 [12]. The underlying mechanism behind how these genes contribute to IFN-c induction remains unclear; therefore, investigating this molecular mechanism warrants further study. MicroRNAs are a class of non-coding RNAs that post-transcriptionally regulate protein expression. MiRNA processing is initiated by nuclear RNase III Drosha and completed by cytoplasmic RNase III Dicer [13]. Drosha in a complex with DGCR8/Pasha cleaves a long primary transcript (pri-miRNA) liberating a precursor microRNA (pre-miRNA) with characteristic hairpin structure [14,15]. After its nuclear export, the pre-miRNA is further processed by Dicer into mature miRNA ranging in size from 18–24 nucleotides [16,17]. Recent studies highlighted the importance of microRNAs in cancer cell apoptosis. MicroRNAs miR-21 [18] and miR-17-92 cluster [19] were demonstrated to possess anti-apoptotic function in glioblastoma cells and lung cancer cells, while miR-29 [20] and miR-34 [21] exhibited pro-apoptotic functions by sensitizing cells to apoptosis. Based on previous studies, we hypothesized that certain microRNAs are also involved in the IFN-c sensitization of human colon carcinoma cells to Fas-mediated apoptosis. Here, we demon-
L. Geng et al. / Biochemical and Biophysical Research Communications 408 (2011) 494–499
strated that Fas expression was post transcriptionally regulated by mciroRNA let-7, while activated Fas influenced Dicer processing of precursor let-7 (pre-let-7). Our results indicate a double-negative feed back mechanism underlying the regulation of Fas protein expression in colon carcinoma cell apoptosis. 2. Materials and methods
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TG-30 and 50 -AAG CTT CAT TAG GCC ATT AAG ATG AGC ACC-30 . A pMiR-Report construct containing the Fas 30 -UTR with three point mutations in the seed sequence was synthesized with a QuikChange site-directed mutagenesis kit (Stratagene, Agilent Technologies, Palo Alto, CA). These reporters were transfected into HEK293 cells using lipofectamine 2000 (Invitrogen, Carlsbad, CA), and transfection efficiency was detected by co-transfection with the Renilla luciferase vector pRL-CMV (Promega, Madison, WI).
2.1. Mice and cell lines 2.5. Measurement of apoptotic cell death Female athymic nude mice (at 4–6 weeks of age) were purchased from the Transgenic Animal Research Center, Second Military Medical University. The mice were maintained and used in accordance with the institutional guidelines for animal care. The human embryo kidney epithelial cell line HEK293, the human colon adenocarcinoma cell line HT29 and HCT116, the human hepatocellular carcinoma cell lines HepG2 and Huh7 were all obtained from the American Type Culture Collection (ATCC, Manassas, VA). HEK293 cells were maintained in DMEM medium containing 10% FBS (Invitrogen, Carlsbad, CA). HT29 and HCT116 cells were maintained in McCoy’s 5A medium with L-glutamine (Invitrogen, Carlsbad, CA) containing 10% FBS. HepG2 and Huh7 cells were maintained in MEM medium with L-glutamine containing 10% FBS. 2.2. Reagents and antibodys Human rIFN-c was purchased from R&D Systems (Minneapolis, MN). Anti-Fas activating monoclonal antibody clone CH11 (mAb CH11) was purchased from Millipore Corporate (Billerica, MA). MicroRNA minics and GMR-miR™ microRNA inhibitors were purchased from Genepharma (Shanghai, China). 2.3. RNA preparation, reverse transcription and quantitative real time PCR Total RNA was prepared from cultured cells using TriZol Reagent (Invitrogen, Carlsbad, CA) according to manufacturer’s protocol. For mature microRNA assays: total RNA (100 ng) was used for cDNA preparation using microRNA specific stem-loop primers (Applied Biosystems (AB), Austin, TX). cDNA was then subjected to Taqman Quantitative Real Time PCR using microRNA specific Taqman primers and PCR master mix (AB, Austin, TX) using StepOne Plus Real time PCR system (AB, Austin, TX). U6RNB was used as endogenous control to calculate fold change using 2-DDCt method with StepOne Software V2.1 (AB, Austin, TX). For mRNA, the primary transcript of let-7a-1 (pri-let-7a-1) and pre-let-7a-1 assays: 1 lg of total RNA was subjected to DNase treatment (Sigma, St. Louis, MO) followed by cDNA preparation using Oligo dT primer (Fermentas, Glen Burnie, Maryland), dNTPs and MMLV-Reverse transcriptase (Fermentas, Glen Burnie, Maryland). The primers used for PCR were: for Fas: 50 -TTC CCA TCC TCC TGA CCAC-30 and 50 -CTC GTA AAC CGC TTC CCTC-30 ; actin-B: 50 -AGT TGC GTT ACA CCC TTT CTTG-30 and 50 -GCT GTC ACC TTC ACC GTT CC-30 ; pri-let-7a-1: 50 -GAT TCC TTT TCA CCA TTC ACC CTG GAT GTT-30 and 50 -TTT CTA TCA GAC CGC CTG GAT GCA GAC TTT-30 ; pre-let-7a-1: 50 -TGG GAT GAG GTA GTA GGT TC-30 and 50 -TAG GAA AGA CAG TAG ATT GTA TA-30 .
Tumor cells were treated (or untreated) for 24 h with mAb CH11 (1 lg/ml) of either human rIFN-c (250 U/ml; PeproTech, Rocky Hill, NJ), microRNA mimics (50 nM), or inhibitors (50 nM). Surviving cells were counted using the Cell Counting Kit-8 (Dojindo, Japan). Apoptotic cell death was evaluated by either detecting caspase-3/7-like activity (DEVD-ase activity) using the ApoOne Kit (Promega, USA) or by flow cytometry using the Annexin V-fluorescein isothiocyanate/PI reagent Kit (Nanjing KeyGen Biotech Co., Ltd. China) in accordance with the manufacturers’ protocols. 2.6. Cell surface Fas analysis IFN-c- (250 U/ml) or mAb CH11-(1 lg/ml) treated and untreated tumor cells were incubated with a biotinylated anti-Fas mAb or an isotype-matched control (Biolegend, San Diego, CA). Cells were then washed, incubated with Streptavidin-PE (Biolegend, San Diego, CA), and analyzed by flow cytometry. 2.7. Western blotting analysis Proteins were resolved with 10% SDS–polyacrylamide gels and transferred to Immobilon-P transfer membrane (Millipore, Billerica, MA). Primary antibodies used were mouse anti-Dicer mAb (Abcam, Cambridge, MA). GAPDH (antibody from Abcam) was used as endogenous control. 2.8. Anti-Fas mAb tumor treatment HT29 tumors were established (n = 5 per treatment group) on female athymic nude mice at 4–6 weeks of age by an intradermal injection of 107 HT29 cells (in 50 lL of McCoy’s 5A medium) into the hind flanks (day 0). On days 4, 6, and 8, a dose of 20 lg of anti-Fas mAb CH11 was injected intratumorally with 20 lg of let-7 inhibitors, control RNA (in 100 ll PBS) or IFN-c (1000 U). Tumor sizes were measured twice weekly. Mice were euthanized when tumors exceeded 400 mm3 in size. 2.9. Statistical analysis Data are expressed as mean ± SD of experiments performed in triplicate. All figures depicting flow cytometry data represent at least three independent experiments. Statistical comparisons were performed using Student’s t-test two treatment groups. 3. Results
2.4. DNA constructs and Luciferase assay
3.1. Let-7 microRNAs and Fas are inversely expressed in various carcinoma cell lines
For luciferase reporter assays: we cloned the 30 -UTR (17662144) of Fas (NM_000043) from human genomic DNA and cloned in pMiR-Report vector (Ambion, Austin, TX) using the Sca I and Hind III (New England Biolabs (Beijing) Ltd, China) site. The following primers were used: 50 -GAG CTC TGA ACA GGC AGG CCA CTT
Considering the potential contribution of microRNAs in the regulation of Fas, an online search using TargetScan 5.1 was used to find microRNAs predicted to bind to CD95/Fas mRNA, including the let-7/mir-98 family. Playing an important role in posttranscriptional regulation, microRNAs regulate gene expression
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through decreased translation, increased degradation of the target mRNA, or both [22]. Accordingly, we sought to determine if let-7 microRNAs and Fas are negatively correlated in carcinoma cell lines. We subsequently analyzed the expression of Fas mRNA and three mature microRNAs from the let-7/mir-98 family: let-7a, 7d and 7f. Our results indicate that let-7 microRNAs and Fas mRNA are inversely expressed in several carcinoma cell lines, including type 1 (HepG2 and Huh7) and type 2 (HT29 and HCT116) cells [23] (Fig. 1A). This observation suggests the presence of potential interactions between let-7 microRNAs and Fas in these carcinoma cell lines.
trol. Co-transfection of HEK293 cells with luciferase (mock, without the Fas 30 UTR) and the let-7a, 7d or 7f mimic did not significantly alter expression of the reporter. The expression of luciferase, however, was strongly decreased when the plasmid containing the Fas 3’UTR was co-transfected with let-7a, 7d or 7f mimics (Fig. 1B). This suppression was also abrogated by transfection of a plasmid containing a three-base mutation in the microRNA binding site. This finding indicates that let-7 microRNAs directly inhibit expression of Fas by binding to a defined target sequence. 3.3. Let-7 expression reduces in IFN-c/anti-Fas mAb treated HT29 cells
3.2. Let-7 microRNAs act directly at the Fas 30 UTR To demonstrate a direct negative effect of let-7 microRNAs on Fas expression, a dual-luciferase reporter assay was used. A fragment containing the target site of let-7/mir-98 microRNAs from the Fas 30 UTR was inserted into the luciferase construct, and a plasmid containing mutated Fas 30 UTR was used as a negative con-
To study the involvement of let-7 microRNAs in IFN-c-induced Fas up-regulation, Fas and let-7 microRNAs expression was analyzed in HT29 colon carcinoma cells following IFN-c/mAb CH11 treatment. Our data demonstrate an obvious up-regulation of Fas expression after IFN-c treatment both at mRNA level (Fig. 2A) and cell surface protein level (Fig. 2B), and the magnitude of this increase was significantly greater in IFN-c/mAb CH11 co-stimulated HT29 cells when compared to IFN-c- or mAb CH11-treated cells. For the mature let-7 microRNA, we found that the expression of let-7a, 7d and 7f were decreased in HT29 cells following IFN-c treatment. Interestingly, we also found that combined stimulation by IFN-c/mAb CH11 induced a significantly stronger decrease in let-7 microRNA levels compared to IFN-c treatment alone, while exposure to CH11 alone was unable to reduce let-7 expression in HT29 cells (Fig. 2A). As the mAb CH11 is a known activator of the Fas receptor, our results indicate that IFN-c treatment combined with Fas activation may further induce Fas up-regulation while simultaneously inducing endogenous let-7 microRNAs down-regulation. This indicates a synergistic mechanism underlying the regulation of Fas protein expression following IFN-c/mAb CH11 combined stimulation. To further confirm which process of the let-7 biogenesisis was affected by Fas activation, we also examined the expression levels of pre- and pri-let-7a-1. We found that pri-let-7a-1 was not affected following IFN-c/mAb CH11 treatment comparing to IFN-c treatment alone (Fig. 2C). This indicated that activated Fas suppressed let-7 biogenesis without significantly affecting let-7 transcribing and Drosha processing. However, pre-let-7a-1 was significantly increased following IFN-c/mAb CH11 combined stimulation. We therefore suggest that activated Fas may suppress Dicer processing and result in accumulation of pre-let-7a-1. This hypothesis was further confirmed by the decreased expression level of Dicer protein in a western blotting analysis (Fig. 2D). We also found that the pri-let-7a-1 and pre-let-7a-1 was both reduced following IFN-c stimulation (Fig. 2C), which indicated that IFN-c affected let-7 expression mainly at the transcriptional level. 3.4. Let-7 suppression sensitizes Fas-induced apoptosis of HT29 cells
Fig. 1. Let-7 regulates Fas expression in cancer cell lines by directly binding with its 30 UTR.A: the expression levels of Fas mRNA and mature let-7a and 7d and 7f in HepG2, HT29, Huh7 and HCT116 cells were analyzed with reverse transcription real-time PCR. HEK293 cells were used as the reference cell line. The relative quantity (RQ) values or their Ln values of the real-time PCR data are provided as mean ± SD. B: luciferase activity in HEK293 cells transiently transfected with either the luciferase construct alone or cotransfected with mimics of let-7a, let-7d, let-7f or negative control RNA (NC). Luciferase vectors used were parental (Mock), or contained the Fas-derived 30 UTR insert (Fas 30 UTR) or the mutated insert (Mutant Fas). All experiments were repeated three times. ⁄⁄represents p < 0.001.
To investigate the potential implications of let-7 microRNAs in influencing antitumor therapeutic strategies, we used a let-7 microRNA inhibitor cocktail consisting of let-7a, let-7d and let-7f. Initially, we examined cell survival in the presence of let-7 inhibitor and other control agents following treatment with mAb CH11. We found that HT29 cell survival significantly decreased following combined treatment with a let-7 inhibitor and mAb CH11 for 24 h, as compared to cells treated with the negative control RNA/mAb CH11 combined treatment. Cell survival results demonstrate that the let-7 inhibitor/mAb CH11 and IFN-c/mAb CH11 combined treatments affected cells similarly (Fig. 3A). To further investigate apoptotic levels of HT29 cells, we assessed caspase-3/7 activity (Fig. 3B) and annexin A5 staining analysis (Fig. 3C). Following transfection with the let-7 inhibitor, we observed a significant in-
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Fig. 2. Fas activation suppressed let-7 biogenesis in IFN-c/Fas mAb-induced apoptosis. A: let-7a, 7d and 7f mature microRNAs and Fas mRNA expression levels in HT29 cells were analyzed following a 2 h treatment with IFN-c, Fas activating mAb CH11 or IFN-c/CH11. B: cell surface expression of Fas in HT29 cells after IFN-c, CH11 or IFN-c/CH11 treatment was determined by flow cytometry. The positive cell counts are provided. C: pri- and pre-let-7a-1 expression levels in HT29 cells were analyzed with reverse transcription real-time PCR following a 2 h treatment with IFN-c, Fas activating mAb CH11 or IFN-c/CH11. D: western blotting analysis of Dicer expression in all the treated groups. GAPDH was used as endogenous control. All experiments were repeated three times. For real-time PCR data, relative quantity (RQ) values or their Ln values are shown as mean ± SD. UN: untreated cells. ⁄⁄represents p < 0.001.
crease in mAb CH11-induced HT29 cell apoptosis as compared to the negative control RNA transfected cells. These results indicate that the let-7 inhibitor sensitizes HT29 cells to Fas-induced apoptosis in vitro. 3.5. Let-7 suppression facilitates tumor clearance in vivo Following the in vitro studies, we further investigated the effect of let-7 inhibitors on the regulation of CH11-induced HT29 cell apoptosis in vivo. HT29 tumors were established in immune-compromised mice, after a two-week treatment of mAb CH11 combined with either the let-7 inhibitor or IFN-c, HT29 tumor size was significantly reduced as compared to the negative control RNA/mAb CH11-treated tumors (Fig. 3D). These data indicate that let-7 inhibitors may function to increase the sensitivity of HT29 cells to anti-Fas mAb treatment in vivo, a finding that could ultimately influence the clinical approach to tumor therapy. 4. Discussion Increasing evidence suggests that microRNAs play a role in the regulation of the intrinsic and extrinsic apoptotic pathways, which have been extensively reviewed by Garofalo et al. [24]. In human colon carcinoma, gradual down-regulation of Fas expression has been observed throughout the course of tumor formation and progression [25]. It remains unknown, however, if microRNAs contribute to the regulation of Fas expression in colon carcinoma. Recent reports have also suggested that let-7 expression could influence the differentiation state of tumor cells and their sensitivity to CD95L and chemotherapeutic drugs [23]. Another most recent report also confirmed that let-7d, g and miR-98 regulated Fas-related apoptosis in T cells [26]. Our data further support these concepts by suggesting that microRNAs of the let-7 family regulate endoge-
nous Fas expression in HT29 cells, a line of moderately well-differentiated, non-transformed cells derived from a colon carcinoma. Decreased expression of Fas with concomitant enhancement of let-7 expression may partially explain the down-regulation of Fas in colon carcinoma. Down-regulation of microRNA let-7 has been observed in various cancer types, including head and neck, lung, colon and ovarian [23,27,28]. However, the regulatory mechanisms remain unknown. Regulation of miRNA biogenesis can be achieved at either transcriptional or posttranscriptional level. In our current study, we found that activated Fas can suppress Dicer processing and result in accumulation of pre-let-7a-1, indicating that let-7a may be post transcriptionally controlled following Fas activation. Recent studies have provided evidence that Dicer is a target for caspases during apoptosis [29]. Caspase-3 was further identified as the major caspase responsible for the cleavage of Dicer-1 [30]. In this regard, we suggest that caspase activation may greatly contribute to the Fas-mediated Dicer suppression and the posttranscriptional inhibition of let-7 maturation. Fas suppresses let-7, while let-7 downregulates Fas. This indicates a double-negative feedback mechanism underlying the regulation of Fas protein expression following IFN-c/mAb CH11 combined stimulation. A schematic view of this hypothesis is depicted in Fig. 4. Based on our data, we suggest that this mechanism may play an important role in Fas-related apoptosis, as it results in rapid up-regulation of membrane Fas protein following increased Fas up-regulation and activation. It is also possible that this mechanism may be involved in lymphocyte-mediated cell injury, specifically in the context of the anti-tumor immune response. As a key proinflammatory cytokine produced during immune reactions, IFN-c mediates numerous biologic activities. These include activities that are essential for the antitumor effects described in therapeutic paradigms and for the ability of Fas ligands to induce cytotoxicity [31]. Previous studies have demon-
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Fig. 3. Let-7 inhibitors sensitize HT29 cells to Fas-related apoptosis in vitro. HT29 cells were transfected with let-7 inhibitors (let-7 inh, a cocktail containing let-7a, let-7d and let-7f inhibitors) or negative control RNA (NC). Cells were treated with CH11 6 h following transfection. IFN-c was used as a positive control. A: cell survival was assessed using CCK8 after an additional 24 h and the survival percentages are expressed as the means ± SD. B: caspase-3/7-like (DEVDase) activity was measured 24 h following cell treatment. C: apoptotic cell rates were assessed using flow cytometry after an additional 12 h. D: human HT29 tumors were established on nude mice at day 0. Intratumoral injections of let-7 inhibitor, antibodies and IFN-c were performed on days 4, 6, and 8 (n = 5 per treatment group). Tumor size was measured two times a week. Tumor size is plotted for individual animals; each line represents the tumor size for a single animal. All the experiments were repeated three times, and data are expressed as the means ± SD. Statistical analysis was performed using the Student’s t-test, and the marker ⁄⁄represents p < 0.001.
Fig. 4. Fas activation regulates its own expression by causing the down-regulation of let-7 microRNAs. The proposed mechanism of IFN-c/Fas activator-induced HT29 cell apoptosis is depicted. In the HT29 cells lacking stimulation, high expression levels of let-7 microRNAs inhibit endogenous Fas expression by both the suppression of translation and the degradation of mRNA. Subsequent to IFN-c/Fas activator stimulation, membrane Fas is activated, affecting the Dicer processing and resulting in the downregulation of mature let-7 microRNAs. Membrane Fas levels are then further increased, resulting in the amplification of Fas-related apoptotic signals and the induction of apoptosis in HT29 cells. Upward and downward pointing black arrows represent up- and down-regulation, respectively.
strated that IFN-c treatment up-regulates cell surface Fas expression at both the RNA and protein levels across multiple cancer cell types. IFN-c, however, has multiple effects in vivo and treatment
with a high dose of this cytokine may not be acceptable for clinical applications. In this study, we demonstrated that the let-7 inhibitor increased the sensitivity of HT29 cells to Fas-related
L. Geng et al. / Biochemical and Biophysical Research Communications 408 (2011) 494–499
apoptosis. This observation indicates that let-7 suppression may be exploited as a novel target in the treatment of human colon carcinoma. It is also possible that the combined administration of let-7 inhibitors and IFN-c may reduce the dosage of IFN-c required for clinical efficacy. This combination may be beneficial in reducing adverse effects, and it remains a focus of future clinical investigations in our laboratory. Conflict of interest All of the authors declare that there is no competing financial interest in relation to the work described in this manuscript. References [1] M.O. Hengartner, The biochemistry of apoptosis, Nature 407 (2000) 770–776. [2] M.E. Peter, R.C. Budd, J. Desbarats, S.M. Hedrick, A.O. Hueber, M.K. Newell, L.B. Owen, R.M. Pope, J. Tschopp, H. Wajant, D. Wallach, R.H. Wiltrout, M. Zornig, D.H. Lynch, The CD95 receptor: apoptosis revisited, Cell 129 (2007) 447–450. [3] B.C. Barnhart, P. Legembre, E. Pietras, C. Bubici, G. Franzoso, M.E. Peter, CD95 ligand induces motility and invasiveness of apoptosis-resistant tumor cells, EMBO J. 23 (2004) 3175–3185. [4] J. Strater, I. Wellisch, S. Riedl, H. Walczak, K. Koretz, A. Tandara, P.H. Krammer, P. Moller, CD95 (APO-1/Fas)-mediated apoptosis in colon epithelial cells: a possible role in ulcerative colitis, Gastroenterology 113 (1997) 160–167. [5] S. Nagata, Fas ligand-induced apoptosis, Annu. Rev. Genet. 33 (1999) 29–55. [6] M. Merger, J.L. Viney, R. Borojevic, D. Steele-Norwood, P. Zhou, D.A. Clark, R. Riddell, R. Maric, E.R. Podack, K. Croitoru, Defining the roles of perforin Fas/ FasL, and tumour necrosis factor alpha in T cell induced mucosal damage in the mouse intestine, Gut 51 (2002) 155–163. [7] E.J. Kim, J.M. Lee, S.E. Namkoong, S.J. Um, J.S. Park, Interferon regulatory factor1 mediates interferon-gamma-induced apoptosis in ovarian carcinoma cells, J. Cell Biochem. 85 (2002) 369–380. [8] Y. Tomita, V. Bilim, N. Hara, T. Kasahara, K. Takahashi, Role of IRF-1 and caspase-7 in IFN-gamma enhancement of Fas-mediated apoptosis in ACHN renal cell carcinoma cells, Int. J. Cancer 104 (2003) 400–408. [9] W.A. Selleck, S.E. Canfield, W.A. Hassen, M. Meseck, A.I. Kuzmin, R.C. Eisensmith, S.H. Chen, S.J. Hall, IFN-gamma sensitization of prostate cancer cells to Fas-mediated death: a gene therapy approach, Mol. Ther. 7 (2003) 185– 192. [10] F. Leithauser, J. Dhein, G. Mechtersheimer, K. Koretz, S. Bruderlein, C. Henne, A. Schmidt, K.M. Debatin, P.H. Krammer, P. Moller, Constitutive and induced expression of APO-1, a new member of the nerve growth factor/tumor necrosis factor receptor superfamily, in normal and neoplastic cells, Lab. Invest. 69 (1993) 415–429. [11] U. von Reyher, J. Strater, W. Kittstein, M. Gschwendt, P.H. Krammer, P. Moller, Colon carcinoma cells use different mechanisms to escape CD95-mediated apoptosis, Cancer Res. 58 (1998) 526–534. [12] K. Liu, S.I. Abrams, Coordinate regulation of IFN consensus sequence-binding protein and caspase-1 in the sensitization of human colon carcinoma cells to Fas-mediated apoptosis by IFN-gamma, J. Immunol. 170 (2003) 6329–6337.
499
[13] V.N. Kim, MicroRNA biogenesis: coordinated cropping and dicing, Nat. Rev. Mol. Cell Biol. 6 (2005) 376–385. [14] A.M. Denli, B.B. Tops, R.H. Plasterk, R.F. Ketting, G.J. Hannon, Processing of primary microRNAs by the Microprocessor complex, Nature 432 (2004) 231– 235. [15] Y. Lee, C. Ahn, J. Han, H. Choi, J. Kim, J. Yim, J. Lee, P. Provost, O. Radmark, S. Kim, V.N. Kim, The nuclear RNase III Drosha initiates microRNA processing, Nature 425 (2003) 415–419. [16] E. Bernstein, A.A. Caudy, S.M. Hammond, G.J. Hannon, Role for a bidentate ribonuclease in the initiation step of RNA interference, Nature 409 (2001) 363– 366. [17] S.W. Knight, B.L. Bass, A role for the RNase III enzyme DCR-1 in RNA interference and germ line development in Caenorhabditis elegans, Science 293 (2001) 2269–2271. [18] J.A. Chan, A.M. Krichevsky, K.S. Kosik, MicroRNA-21 is an antiapoptotic factor in human glioblastoma cells, Cancer Res. 65 (2005) 6029–6033. [19] H. Matsubara, T. Takeuchi, E. Nishikawa, K. Yanagisawa, Y. Hayashita, H. Ebi, H. Yamada, M. Suzuki, M. Nagino, Y. Nimura, H. Osada, T. Takahashi, Apoptosis induction by antisense oligonucleotides against miR-17-5p and miR-20a in lung cancers overexpressing miR-17-92, Oncogene 26 (2007) 6099–6105. [20] J.L. Mott, S. Kobayashi, S.F. Bronk, G.J. Gores, Mir-29 regulates Mcl-1 protein expression and apoptosis, Oncogene 26 (2007) 6133–6140. [21] G.T. Bommer, I. Gerin, Y. Feng, A.J. Kaczorowski, R. Kuick, R.E. Love, Y. Zhai, T.J. Giordano, Z.S. Qin, B.B. Moore, O.A. MacDougald, K.R. Cho, E.R. Fearon, p53Mediated activation of miRNA34 candidate tumor-suppressor genes, Curr. Biol. 17 (2007) 1298–1307. [22] M.A. Valencia-Sanchez, J. Liu, G.J. Hannon, R. Parker, Control of translation and mRNA degradation by miRNAs and siRNAs, Genes Dev. 20 (2006) 515–524. [23] S. Shell, S.M. Park, A.R. Radjabi, R. Schickel, E.O. Kistner, D.A. Jewell, C. Feig, E. Lengyel, M.E. Peter, Let-7 expression defines two differentiation stages of cancer, Proc. Natl. Acad. Sci. USA 104 (2007) 11400–11405. [24] M. Garofalo, G.L. Condorelli, C.M. Croce, G. Condorelli, MicroRNAs as regulators of death receptors signaling, Cell Death Differ. 17 (2010) 200–208. [25] P. Moller, K. Koretz, F. Leithauser, S. Bruderlein, C. Henne, A. Quentmeier, P.H. Krammer, Expression of APO-1 (CD95), a member of the NGF/TNF receptor superfamily, in normal and neoplastic colon epithelium, Int. J. Cancer 57 (1994) 371–377. [26] S. Wang, Y. Tang, H. Cui, X. Zhao, X. Luo, W. Pan, X. Huang, N. Shen, Let-7/miR98 regulate Fas and Fas-mediated apoptosis, Genes Immun. 12 (2011) 149–154. [27] Y. Akao, Y. Nakagawa, T. Naoe, Let-7 microRNA functions as a potential growth suppressor in human colon cancer cells, Biol. Pharm Bull. 29 (2006) 903–906. [28] S.M. Johnson, H. Grosshans, J. Shingara, M. Byrom, R. Jarvis, A. Cheng, E. Labourier, K.L. Reinert, D. Brown, F.J. Slack, RAS is regulated by the let-7 microRNA family, Cell 120 (2005) 635–647. [29] A.A. Matskevich, K. Moelling, Stimuli-dependent cleavage of dicer during apoptosis, Biochem. J. 412 (2008) 527–534. [30] M.M. Ghodgaonkar, R.G. Shah, F. Kandan-Kulangara, E.B. Affar, H.H. Qi, E. Wiemer, G.M. Shah, Abrogation of DNA vector-based RNAi during apoptosis in mammalian cells due to caspase-mediated cleavage and inactivation of Dicer1, Cell Death Differ. 16 (2009) 858–868. [31] K. Schroder, P.J. Hertzog, T. Ravasi, D.A. Hume, Interferon-gamma: an overview of signals, mechanisms and functions, J. Leukoc. Biol. 75 (2004) 163–189.
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A let-7/Fas double-negative feedback loop regulates human colon carcinoma cells sensitivity to Fas-related apoptosis Li Geng a,1, Bin Zhu b,1, Bing-Hua Dai a,1, Cheng-Jun Sui a, Feng Xu a, Tong Kan a, Wei-Feng Shen a, Jia-Mei Yang a,⇑ a b
The Department of Special Treatment, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, No. 225, Changhai Road, Shanghai 200438, China The Second Department of Billiary Surgery, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, No. 225, Changhai Road, Shanghai 200438, China
a r t i c l e
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Article history: Received 7 April 2011 Available online 21 April 2011 Keywords: Fas Let-7 microRNA Interferon-c Apoptosis
a b s t r a c t Interferon-c (IFN-c) is considered essential for the regulation of anti-tumor reactions as it sensitizes Fas-related apoptosis in HT29 cells, but the mechanism is unclear. In the current study, our data demonstrated that IFN-c stimulation and Fas activation suppressed Dicer processing and let-7 microRNA biogenesis, while let-7 microRNA strongly inhibited Fas expression by directly targeting Fas mRNA. Accordingly, our results indicate that Fas and let-7 microRNAs form a double-negative feedback loop in IFN-c and Fas induced apoptosis in colon carcinoma cell line HT29, which may be an important synergistic mechanism in anti-tumor immune response. We also found that a let-7 microRNA inhibitor increased Fas expression and sensitized cells to Fas-related apoptosis, which may have future implications in colon carcinoma therapy. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction Apoptosis occurs frequently in epithelial cells located in the gastrointestinal tract, significantly contributing to epithelial cell turnover and the lymphocyte-mediated antitumor response [1]. Studies have demonstrated that epithelial cell apoptosis is regulated by the cell surface receptor Fas. Fas is a member of the tumor necrosis factor receptor family of death receptors and can induce apoptosis in sensitive cells by binding to their specific death ligands, including Fas ligand (FasL), tumor necrosis factor-a (TNF-a) and Fas-specific monoclonal antibody CH11 (mAb CH11) [2,3]. Fas is highly expressed in normal human colon epithelial cells, but its expression becomes progressively decreased during the transition from normal epithelium to adenocarcinoma in approximately 50% of observed cases [4–6]. It is also well documented that most colon carcinoma cells show impaired responses to Fas-related apoptosis. Consequently, from an immunotherapy standpoint, the extent of responsiveness of malignant cells to Fas signals may profoundly influence the overall efficacy of any anti-tumor lymphocyte-mediated response, highlighting the need to explore strategies to sensitize tumor cells to Fas-mediated apoptosis. Interferon-c (IFN-c), an important member of the interferon family that regulates antiviral, antiproliferative, and immunomodulatory responses, has been reported to be involved in the regula⇑ Corresponding author. Fax: +86 021 65562400. 1
E-mail address: [email protected] (J.-M. Yang). These authors contributed equally to this work.
0006-291X/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2011.04.074
tion of apoptotic processes. This involvement includes the sensitization of various target cells to Fas-mediated death, such as ovarian cancer cells [7], renal cell carcinoma cells [8] and prostate carcinoma cells [9]. For colon carcinoma, IFN-c can sensitize human primary colon carcinoma cells to Fas-mediated apoptosis by enhancing Fas expression [10,11]. Previous studies have demonstrated that many genes are involved in the process of IFN-c-mediated sensitization, including IFN consensus sequence-binding protein and caspase-1 [12]. The underlying mechanism behind how these genes contribute to IFN-c induction remains unclear; therefore, investigating this molecular mechanism warrants further study. MicroRNAs are a class of non-coding RNAs that post-transcriptionally regulate protein expression. MiRNA processing is initiated by nuclear RNase III Drosha and completed by cytoplasmic RNase III Dicer [13]. Drosha in a complex with DGCR8/Pasha cleaves a long primary transcript (pri-miRNA) liberating a precursor microRNA (pre-miRNA) with characteristic hairpin structure [14,15]. After its nuclear export, the pre-miRNA is further processed by Dicer into mature miRNA ranging in size from 18–24 nucleotides [16,17]. Recent studies highlighted the importance of microRNAs in cancer cell apoptosis. MicroRNAs miR-21 [18] and miR-17-92 cluster [19] were demonstrated to possess anti-apoptotic function in glioblastoma cells and lung cancer cells, while miR-29 [20] and miR-34 [21] exhibited pro-apoptotic functions by sensitizing cells to apoptosis. Based on previous studies, we hypothesized that certain microRNAs are also involved in the IFN-c sensitization of human colon carcinoma cells to Fas-mediated apoptosis. Here, we demon-
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strated that Fas expression was post transcriptionally regulated by mciroRNA let-7, while activated Fas influenced Dicer processing of precursor let-7 (pre-let-7). Our results indicate a double-negative feed back mechanism underlying the regulation of Fas protein expression in colon carcinoma cell apoptosis. 2. Materials and methods
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TG-30 and 50 -AAG CTT CAT TAG GCC ATT AAG ATG AGC ACC-30 . A pMiR-Report construct containing the Fas 30 -UTR with three point mutations in the seed sequence was synthesized with a QuikChange site-directed mutagenesis kit (Stratagene, Agilent Technologies, Palo Alto, CA). These reporters were transfected into HEK293 cells using lipofectamine 2000 (Invitrogen, Carlsbad, CA), and transfection efficiency was detected by co-transfection with the Renilla luciferase vector pRL-CMV (Promega, Madison, WI).
2.1. Mice and cell lines 2.5. Measurement of apoptotic cell death Female athymic nude mice (at 4–6 weeks of age) were purchased from the Transgenic Animal Research Center, Second Military Medical University. The mice were maintained and used in accordance with the institutional guidelines for animal care. The human embryo kidney epithelial cell line HEK293, the human colon adenocarcinoma cell line HT29 and HCT116, the human hepatocellular carcinoma cell lines HepG2 and Huh7 were all obtained from the American Type Culture Collection (ATCC, Manassas, VA). HEK293 cells were maintained in DMEM medium containing 10% FBS (Invitrogen, Carlsbad, CA). HT29 and HCT116 cells were maintained in McCoy’s 5A medium with L-glutamine (Invitrogen, Carlsbad, CA) containing 10% FBS. HepG2 and Huh7 cells were maintained in MEM medium with L-glutamine containing 10% FBS. 2.2. Reagents and antibodys Human rIFN-c was purchased from R&D Systems (Minneapolis, MN). Anti-Fas activating monoclonal antibody clone CH11 (mAb CH11) was purchased from Millipore Corporate (Billerica, MA). MicroRNA minics and GMR-miR™ microRNA inhibitors were purchased from Genepharma (Shanghai, China). 2.3. RNA preparation, reverse transcription and quantitative real time PCR Total RNA was prepared from cultured cells using TriZol Reagent (Invitrogen, Carlsbad, CA) according to manufacturer’s protocol. For mature microRNA assays: total RNA (100 ng) was used for cDNA preparation using microRNA specific stem-loop primers (Applied Biosystems (AB), Austin, TX). cDNA was then subjected to Taqman Quantitative Real Time PCR using microRNA specific Taqman primers and PCR master mix (AB, Austin, TX) using StepOne Plus Real time PCR system (AB, Austin, TX). U6RNB was used as endogenous control to calculate fold change using 2-DDCt method with StepOne Software V2.1 (AB, Austin, TX). For mRNA, the primary transcript of let-7a-1 (pri-let-7a-1) and pre-let-7a-1 assays: 1 lg of total RNA was subjected to DNase treatment (Sigma, St. Louis, MO) followed by cDNA preparation using Oligo dT primer (Fermentas, Glen Burnie, Maryland), dNTPs and MMLV-Reverse transcriptase (Fermentas, Glen Burnie, Maryland). The primers used for PCR were: for Fas: 50 -TTC CCA TCC TCC TGA CCAC-30 and 50 -CTC GTA AAC CGC TTC CCTC-30 ; actin-B: 50 -AGT TGC GTT ACA CCC TTT CTTG-30 and 50 -GCT GTC ACC TTC ACC GTT CC-30 ; pri-let-7a-1: 50 -GAT TCC TTT TCA CCA TTC ACC CTG GAT GTT-30 and 50 -TTT CTA TCA GAC CGC CTG GAT GCA GAC TTT-30 ; pre-let-7a-1: 50 -TGG GAT GAG GTA GTA GGT TC-30 and 50 -TAG GAA AGA CAG TAG ATT GTA TA-30 .
Tumor cells were treated (or untreated) for 24 h with mAb CH11 (1 lg/ml) of either human rIFN-c (250 U/ml; PeproTech, Rocky Hill, NJ), microRNA mimics (50 nM), or inhibitors (50 nM). Surviving cells were counted using the Cell Counting Kit-8 (Dojindo, Japan). Apoptotic cell death was evaluated by either detecting caspase-3/7-like activity (DEVD-ase activity) using the ApoOne Kit (Promega, USA) or by flow cytometry using the Annexin V-fluorescein isothiocyanate/PI reagent Kit (Nanjing KeyGen Biotech Co., Ltd. China) in accordance with the manufacturers’ protocols. 2.6. Cell surface Fas analysis IFN-c- (250 U/ml) or mAb CH11-(1 lg/ml) treated and untreated tumor cells were incubated with a biotinylated anti-Fas mAb or an isotype-matched control (Biolegend, San Diego, CA). Cells were then washed, incubated with Streptavidin-PE (Biolegend, San Diego, CA), and analyzed by flow cytometry. 2.7. Western blotting analysis Proteins were resolved with 10% SDS–polyacrylamide gels and transferred to Immobilon-P transfer membrane (Millipore, Billerica, MA). Primary antibodies used were mouse anti-Dicer mAb (Abcam, Cambridge, MA). GAPDH (antibody from Abcam) was used as endogenous control. 2.8. Anti-Fas mAb tumor treatment HT29 tumors were established (n = 5 per treatment group) on female athymic nude mice at 4–6 weeks of age by an intradermal injection of 107 HT29 cells (in 50 lL of McCoy’s 5A medium) into the hind flanks (day 0). On days 4, 6, and 8, a dose of 20 lg of anti-Fas mAb CH11 was injected intratumorally with 20 lg of let-7 inhibitors, control RNA (in 100 ll PBS) or IFN-c (1000 U). Tumor sizes were measured twice weekly. Mice were euthanized when tumors exceeded 400 mm3 in size. 2.9. Statistical analysis Data are expressed as mean ± SD of experiments performed in triplicate. All figures depicting flow cytometry data represent at least three independent experiments. Statistical comparisons were performed using Student’s t-test two treatment groups. 3. Results
2.4. DNA constructs and Luciferase assay
3.1. Let-7 microRNAs and Fas are inversely expressed in various carcinoma cell lines
For luciferase reporter assays: we cloned the 30 -UTR (17662144) of Fas (NM_000043) from human genomic DNA and cloned in pMiR-Report vector (Ambion, Austin, TX) using the Sca I and Hind III (New England Biolabs (Beijing) Ltd, China) site. The following primers were used: 50 -GAG CTC TGA ACA GGC AGG CCA CTT
Considering the potential contribution of microRNAs in the regulation of Fas, an online search using TargetScan 5.1 was used to find microRNAs predicted to bind to CD95/Fas mRNA, including the let-7/mir-98 family. Playing an important role in posttranscriptional regulation, microRNAs regulate gene expression
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through decreased translation, increased degradation of the target mRNA, or both [22]. Accordingly, we sought to determine if let-7 microRNAs and Fas are negatively correlated in carcinoma cell lines. We subsequently analyzed the expression of Fas mRNA and three mature microRNAs from the let-7/mir-98 family: let-7a, 7d and 7f. Our results indicate that let-7 microRNAs and Fas mRNA are inversely expressed in several carcinoma cell lines, including type 1 (HepG2 and Huh7) and type 2 (HT29 and HCT116) cells [23] (Fig. 1A). This observation suggests the presence of potential interactions between let-7 microRNAs and Fas in these carcinoma cell lines.
trol. Co-transfection of HEK293 cells with luciferase (mock, without the Fas 30 UTR) and the let-7a, 7d or 7f mimic did not significantly alter expression of the reporter. The expression of luciferase, however, was strongly decreased when the plasmid containing the Fas 3’UTR was co-transfected with let-7a, 7d or 7f mimics (Fig. 1B). This suppression was also abrogated by transfection of a plasmid containing a three-base mutation in the microRNA binding site. This finding indicates that let-7 microRNAs directly inhibit expression of Fas by binding to a defined target sequence. 3.3. Let-7 expression reduces in IFN-c/anti-Fas mAb treated HT29 cells
3.2. Let-7 microRNAs act directly at the Fas 30 UTR To demonstrate a direct negative effect of let-7 microRNAs on Fas expression, a dual-luciferase reporter assay was used. A fragment containing the target site of let-7/mir-98 microRNAs from the Fas 30 UTR was inserted into the luciferase construct, and a plasmid containing mutated Fas 30 UTR was used as a negative con-
To study the involvement of let-7 microRNAs in IFN-c-induced Fas up-regulation, Fas and let-7 microRNAs expression was analyzed in HT29 colon carcinoma cells following IFN-c/mAb CH11 treatment. Our data demonstrate an obvious up-regulation of Fas expression after IFN-c treatment both at mRNA level (Fig. 2A) and cell surface protein level (Fig. 2B), and the magnitude of this increase was significantly greater in IFN-c/mAb CH11 co-stimulated HT29 cells when compared to IFN-c- or mAb CH11-treated cells. For the mature let-7 microRNA, we found that the expression of let-7a, 7d and 7f were decreased in HT29 cells following IFN-c treatment. Interestingly, we also found that combined stimulation by IFN-c/mAb CH11 induced a significantly stronger decrease in let-7 microRNA levels compared to IFN-c treatment alone, while exposure to CH11 alone was unable to reduce let-7 expression in HT29 cells (Fig. 2A). As the mAb CH11 is a known activator of the Fas receptor, our results indicate that IFN-c treatment combined with Fas activation may further induce Fas up-regulation while simultaneously inducing endogenous let-7 microRNAs down-regulation. This indicates a synergistic mechanism underlying the regulation of Fas protein expression following IFN-c/mAb CH11 combined stimulation. To further confirm which process of the let-7 biogenesisis was affected by Fas activation, we also examined the expression levels of pre- and pri-let-7a-1. We found that pri-let-7a-1 was not affected following IFN-c/mAb CH11 treatment comparing to IFN-c treatment alone (Fig. 2C). This indicated that activated Fas suppressed let-7 biogenesis without significantly affecting let-7 transcribing and Drosha processing. However, pre-let-7a-1 was significantly increased following IFN-c/mAb CH11 combined stimulation. We therefore suggest that activated Fas may suppress Dicer processing and result in accumulation of pre-let-7a-1. This hypothesis was further confirmed by the decreased expression level of Dicer protein in a western blotting analysis (Fig. 2D). We also found that the pri-let-7a-1 and pre-let-7a-1 was both reduced following IFN-c stimulation (Fig. 2C), which indicated that IFN-c affected let-7 expression mainly at the transcriptional level. 3.4. Let-7 suppression sensitizes Fas-induced apoptosis of HT29 cells
Fig. 1. Let-7 regulates Fas expression in cancer cell lines by directly binding with its 30 UTR.A: the expression levels of Fas mRNA and mature let-7a and 7d and 7f in HepG2, HT29, Huh7 and HCT116 cells were analyzed with reverse transcription real-time PCR. HEK293 cells were used as the reference cell line. The relative quantity (RQ) values or their Ln values of the real-time PCR data are provided as mean ± SD. B: luciferase activity in HEK293 cells transiently transfected with either the luciferase construct alone or cotransfected with mimics of let-7a, let-7d, let-7f or negative control RNA (NC). Luciferase vectors used were parental (Mock), or contained the Fas-derived 30 UTR insert (Fas 30 UTR) or the mutated insert (Mutant Fas). All experiments were repeated three times. ⁄⁄represents p < 0.001.
To investigate the potential implications of let-7 microRNAs in influencing antitumor therapeutic strategies, we used a let-7 microRNA inhibitor cocktail consisting of let-7a, let-7d and let-7f. Initially, we examined cell survival in the presence of let-7 inhibitor and other control agents following treatment with mAb CH11. We found that HT29 cell survival significantly decreased following combined treatment with a let-7 inhibitor and mAb CH11 for 24 h, as compared to cells treated with the negative control RNA/mAb CH11 combined treatment. Cell survival results demonstrate that the let-7 inhibitor/mAb CH11 and IFN-c/mAb CH11 combined treatments affected cells similarly (Fig. 3A). To further investigate apoptotic levels of HT29 cells, we assessed caspase-3/7 activity (Fig. 3B) and annexin A5 staining analysis (Fig. 3C). Following transfection with the let-7 inhibitor, we observed a significant in-
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Fig. 2. Fas activation suppressed let-7 biogenesis in IFN-c/Fas mAb-induced apoptosis. A: let-7a, 7d and 7f mature microRNAs and Fas mRNA expression levels in HT29 cells were analyzed following a 2 h treatment with IFN-c, Fas activating mAb CH11 or IFN-c/CH11. B: cell surface expression of Fas in HT29 cells after IFN-c, CH11 or IFN-c/CH11 treatment was determined by flow cytometry. The positive cell counts are provided. C: pri- and pre-let-7a-1 expression levels in HT29 cells were analyzed with reverse transcription real-time PCR following a 2 h treatment with IFN-c, Fas activating mAb CH11 or IFN-c/CH11. D: western blotting analysis of Dicer expression in all the treated groups. GAPDH was used as endogenous control. All experiments were repeated three times. For real-time PCR data, relative quantity (RQ) values or their Ln values are shown as mean ± SD. UN: untreated cells. ⁄⁄represents p < 0.001.
crease in mAb CH11-induced HT29 cell apoptosis as compared to the negative control RNA transfected cells. These results indicate that the let-7 inhibitor sensitizes HT29 cells to Fas-induced apoptosis in vitro. 3.5. Let-7 suppression facilitates tumor clearance in vivo Following the in vitro studies, we further investigated the effect of let-7 inhibitors on the regulation of CH11-induced HT29 cell apoptosis in vivo. HT29 tumors were established in immune-compromised mice, after a two-week treatment of mAb CH11 combined with either the let-7 inhibitor or IFN-c, HT29 tumor size was significantly reduced as compared to the negative control RNA/mAb CH11-treated tumors (Fig. 3D). These data indicate that let-7 inhibitors may function to increase the sensitivity of HT29 cells to anti-Fas mAb treatment in vivo, a finding that could ultimately influence the clinical approach to tumor therapy. 4. Discussion Increasing evidence suggests that microRNAs play a role in the regulation of the intrinsic and extrinsic apoptotic pathways, which have been extensively reviewed by Garofalo et al. [24]. In human colon carcinoma, gradual down-regulation of Fas expression has been observed throughout the course of tumor formation and progression [25]. It remains unknown, however, if microRNAs contribute to the regulation of Fas expression in colon carcinoma. Recent reports have also suggested that let-7 expression could influence the differentiation state of tumor cells and their sensitivity to CD95L and chemotherapeutic drugs [23]. Another most recent report also confirmed that let-7d, g and miR-98 regulated Fas-related apoptosis in T cells [26]. Our data further support these concepts by suggesting that microRNAs of the let-7 family regulate endoge-
nous Fas expression in HT29 cells, a line of moderately well-differentiated, non-transformed cells derived from a colon carcinoma. Decreased expression of Fas with concomitant enhancement of let-7 expression may partially explain the down-regulation of Fas in colon carcinoma. Down-regulation of microRNA let-7 has been observed in various cancer types, including head and neck, lung, colon and ovarian [23,27,28]. However, the regulatory mechanisms remain unknown. Regulation of miRNA biogenesis can be achieved at either transcriptional or posttranscriptional level. In our current study, we found that activated Fas can suppress Dicer processing and result in accumulation of pre-let-7a-1, indicating that let-7a may be post transcriptionally controlled following Fas activation. Recent studies have provided evidence that Dicer is a target for caspases during apoptosis [29]. Caspase-3 was further identified as the major caspase responsible for the cleavage of Dicer-1 [30]. In this regard, we suggest that caspase activation may greatly contribute to the Fas-mediated Dicer suppression and the posttranscriptional inhibition of let-7 maturation. Fas suppresses let-7, while let-7 downregulates Fas. This indicates a double-negative feedback mechanism underlying the regulation of Fas protein expression following IFN-c/mAb CH11 combined stimulation. A schematic view of this hypothesis is depicted in Fig. 4. Based on our data, we suggest that this mechanism may play an important role in Fas-related apoptosis, as it results in rapid up-regulation of membrane Fas protein following increased Fas up-regulation and activation. It is also possible that this mechanism may be involved in lymphocyte-mediated cell injury, specifically in the context of the anti-tumor immune response. As a key proinflammatory cytokine produced during immune reactions, IFN-c mediates numerous biologic activities. These include activities that are essential for the antitumor effects described in therapeutic paradigms and for the ability of Fas ligands to induce cytotoxicity [31]. Previous studies have demon-
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Fig. 3. Let-7 inhibitors sensitize HT29 cells to Fas-related apoptosis in vitro. HT29 cells were transfected with let-7 inhibitors (let-7 inh, a cocktail containing let-7a, let-7d and let-7f inhibitors) or negative control RNA (NC). Cells were treated with CH11 6 h following transfection. IFN-c was used as a positive control. A: cell survival was assessed using CCK8 after an additional 24 h and the survival percentages are expressed as the means ± SD. B: caspase-3/7-like (DEVDase) activity was measured 24 h following cell treatment. C: apoptotic cell rates were assessed using flow cytometry after an additional 12 h. D: human HT29 tumors were established on nude mice at day 0. Intratumoral injections of let-7 inhibitor, antibodies and IFN-c were performed on days 4, 6, and 8 (n = 5 per treatment group). Tumor size was measured two times a week. Tumor size is plotted for individual animals; each line represents the tumor size for a single animal. All the experiments were repeated three times, and data are expressed as the means ± SD. Statistical analysis was performed using the Student’s t-test, and the marker ⁄⁄represents p < 0.001.
Fig. 4. Fas activation regulates its own expression by causing the down-regulation of let-7 microRNAs. The proposed mechanism of IFN-c/Fas activator-induced HT29 cell apoptosis is depicted. In the HT29 cells lacking stimulation, high expression levels of let-7 microRNAs inhibit endogenous Fas expression by both the suppression of translation and the degradation of mRNA. Subsequent to IFN-c/Fas activator stimulation, membrane Fas is activated, affecting the Dicer processing and resulting in the downregulation of mature let-7 microRNAs. Membrane Fas levels are then further increased, resulting in the amplification of Fas-related apoptotic signals and the induction of apoptosis in HT29 cells. Upward and downward pointing black arrows represent up- and down-regulation, respectively.
strated that IFN-c treatment up-regulates cell surface Fas expression at both the RNA and protein levels across multiple cancer cell types. IFN-c, however, has multiple effects in vivo and treatment
with a high dose of this cytokine may not be acceptable for clinical applications. In this study, we demonstrated that the let-7 inhibitor increased the sensitivity of HT29 cells to Fas-related
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apoptosis. This observation indicates that let-7 suppression may be exploited as a novel target in the treatment of human colon carcinoma. It is also possible that the combined administration of let-7 inhibitors and IFN-c may reduce the dosage of IFN-c required for clinical efficacy. This combination may be beneficial in reducing adverse effects, and it remains a focus of future clinical investigations in our laboratory. Conflict of interest All of the authors declare that there is no competing financial interest in relation to the work described in this manuscript. References [1] M.O. Hengartner, The biochemistry of apoptosis, Nature 407 (2000) 770–776. [2] M.E. Peter, R.C. Budd, J. Desbarats, S.M. Hedrick, A.O. Hueber, M.K. Newell, L.B. Owen, R.M. Pope, J. Tschopp, H. Wajant, D. Wallach, R.H. Wiltrout, M. Zornig, D.H. Lynch, The CD95 receptor: apoptosis revisited, Cell 129 (2007) 447–450. [3] B.C. Barnhart, P. Legembre, E. Pietras, C. Bubici, G. Franzoso, M.E. Peter, CD95 ligand induces motility and invasiveness of apoptosis-resistant tumor cells, EMBO J. 23 (2004) 3175–3185. [4] J. Strater, I. Wellisch, S. Riedl, H. Walczak, K. Koretz, A. Tandara, P.H. Krammer, P. Moller, CD95 (APO-1/Fas)-mediated apoptosis in colon epithelial cells: a possible role in ulcerative colitis, Gastroenterology 113 (1997) 160–167. [5] S. Nagata, Fas ligand-induced apoptosis, Annu. Rev. Genet. 33 (1999) 29–55. [6] M. Merger, J.L. Viney, R. Borojevic, D. Steele-Norwood, P. Zhou, D.A. Clark, R. Riddell, R. Maric, E.R. Podack, K. Croitoru, Defining the roles of perforin Fas/ FasL, and tumour necrosis factor alpha in T cell induced mucosal damage in the mouse intestine, Gut 51 (2002) 155–163. [7] E.J. Kim, J.M. Lee, S.E. Namkoong, S.J. Um, J.S. Park, Interferon regulatory factor1 mediates interferon-gamma-induced apoptosis in ovarian carcinoma cells, J. Cell Biochem. 85 (2002) 369–380. [8] Y. Tomita, V. Bilim, N. Hara, T. Kasahara, K. Takahashi, Role of IRF-1 and caspase-7 in IFN-gamma enhancement of Fas-mediated apoptosis in ACHN renal cell carcinoma cells, Int. J. Cancer 104 (2003) 400–408. [9] W.A. Selleck, S.E. Canfield, W.A. Hassen, M. Meseck, A.I. Kuzmin, R.C. Eisensmith, S.H. Chen, S.J. Hall, IFN-gamma sensitization of prostate cancer cells to Fas-mediated death: a gene therapy approach, Mol. Ther. 7 (2003) 185– 192. [10] F. Leithauser, J. Dhein, G. Mechtersheimer, K. Koretz, S. Bruderlein, C. Henne, A. Schmidt, K.M. Debatin, P.H. Krammer, P. Moller, Constitutive and induced expression of APO-1, a new member of the nerve growth factor/tumor necrosis factor receptor superfamily, in normal and neoplastic cells, Lab. Invest. 69 (1993) 415–429. [11] U. von Reyher, J. Strater, W. Kittstein, M. Gschwendt, P.H. Krammer, P. Moller, Colon carcinoma cells use different mechanisms to escape CD95-mediated apoptosis, Cancer Res. 58 (1998) 526–534. [12] K. Liu, S.I. Abrams, Coordinate regulation of IFN consensus sequence-binding protein and caspase-1 in the sensitization of human colon carcinoma cells to Fas-mediated apoptosis by IFN-gamma, J. Immunol. 170 (2003) 6329–6337.
499
[13] V.N. Kim, MicroRNA biogenesis: coordinated cropping and dicing, Nat. Rev. Mol. Cell Biol. 6 (2005) 376–385. [14] A.M. Denli, B.B. Tops, R.H. Plasterk, R.F. Ketting, G.J. Hannon, Processing of primary microRNAs by the Microprocessor complex, Nature 432 (2004) 231– 235. [15] Y. Lee, C. Ahn, J. Han, H. Choi, J. Kim, J. Yim, J. Lee, P. Provost, O. Radmark, S. Kim, V.N. Kim, The nuclear RNase III Drosha initiates microRNA processing, Nature 425 (2003) 415–419. [16] E. Bernstein, A.A. Caudy, S.M. Hammond, G.J. Hannon, Role for a bidentate ribonuclease in the initiation step of RNA interference, Nature 409 (2001) 363– 366. [17] S.W. Knight, B.L. Bass, A role for the RNase III enzyme DCR-1 in RNA interference and germ line development in Caenorhabditis elegans, Science 293 (2001) 2269–2271. [18] J.A. Chan, A.M. Krichevsky, K.S. Kosik, MicroRNA-21 is an antiapoptotic factor in human glioblastoma cells, Cancer Res. 65 (2005) 6029–6033. [19] H. Matsubara, T. Takeuchi, E. Nishikawa, K. Yanagisawa, Y. Hayashita, H. Ebi, H. Yamada, M. Suzuki, M. Nagino, Y. Nimura, H. Osada, T. Takahashi, Apoptosis induction by antisense oligonucleotides against miR-17-5p and miR-20a in lung cancers overexpressing miR-17-92, Oncogene 26 (2007) 6099–6105. [20] J.L. Mott, S. Kobayashi, S.F. Bronk, G.J. Gores, Mir-29 regulates Mcl-1 protein expression and apoptosis, Oncogene 26 (2007) 6133–6140. [21] G.T. Bommer, I. Gerin, Y. Feng, A.J. Kaczorowski, R. Kuick, R.E. Love, Y. Zhai, T.J. Giordano, Z.S. Qin, B.B. Moore, O.A. MacDougald, K.R. Cho, E.R. Fearon, p53Mediated activation of miRNA34 candidate tumor-suppressor genes, Curr. Biol. 17 (2007) 1298–1307. [22] M.A. Valencia-Sanchez, J. Liu, G.J. Hannon, R. Parker, Control of translation and mRNA degradation by miRNAs and siRNAs, Genes Dev. 20 (2006) 515–524. [23] S. Shell, S.M. Park, A.R. Radjabi, R. Schickel, E.O. Kistner, D.A. Jewell, C. Feig, E. Lengyel, M.E. Peter, Let-7 expression defines two differentiation stages of cancer, Proc. Natl. Acad. Sci. USA 104 (2007) 11400–11405. [24] M. Garofalo, G.L. Condorelli, C.M. Croce, G. Condorelli, MicroRNAs as regulators of death receptors signaling, Cell Death Differ. 17 (2010) 200–208. [25] P. Moller, K. Koretz, F. Leithauser, S. Bruderlein, C. Henne, A. Quentmeier, P.H. Krammer, Expression of APO-1 (CD95), a member of the NGF/TNF receptor superfamily, in normal and neoplastic colon epithelium, Int. J. Cancer 57 (1994) 371–377. [26] S. Wang, Y. Tang, H. Cui, X. Zhao, X. Luo, W. Pan, X. Huang, N. Shen, Let-7/miR98 regulate Fas and Fas-mediated apoptosis, Genes Immun. 12 (2011) 149–154. [27] Y. Akao, Y. Nakagawa, T. Naoe, Let-7 microRNA functions as a potential growth suppressor in human colon cancer cells, Biol. Pharm Bull. 29 (2006) 903–906. [28] S.M. Johnson, H. Grosshans, J. Shingara, M. Byrom, R. Jarvis, A. Cheng, E. Labourier, K.L. Reinert, D. Brown, F.J. Slack, RAS is regulated by the let-7 microRNA family, Cell 120 (2005) 635–647. [29] A.A. Matskevich, K. Moelling, Stimuli-dependent cleavage of dicer during apoptosis, Biochem. J. 412 (2008) 527–534. [30] M.M. Ghodgaonkar, R.G. Shah, F. Kandan-Kulangara, E.B. Affar, H.H. Qi, E. Wiemer, G.M. Shah, Abrogation of DNA vector-based RNAi during apoptosis in mammalian cells due to caspase-mediated cleavage and inactivation of Dicer1, Cell Death Differ. 16 (2009) 858–868. [31] K. Schroder, P.J. Hertzog, T. Ravasi, D.A. Hume, Interferon-gamma: an overview of signals, mechanisms and functions, J. Leukoc. Biol. 75 (2004) 163–189.