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Cancer Immunology, Immunotherapy : CII logoLink to Cancer Immunology, Immunotherapy : CII
. 2012 Aug 5;62(1):183–190. doi: 10.1007/s00262-012-1318-1

A knockdown of Maml1 that results in melanoma cell senescence promotes an innate and adaptive immune response

Shijun Kang 1, Jianmin Xie 1, Jingxia Miao 1, Rong Li 1, Wangjun Liao 1, Rongcheng Luo 1,
PMCID: PMC11029605  PMID: 22864395

Abstract

Maml1 is emerging as a coactivator of many signaling pathways, including the Notch and Wnt pathways. Targeting Maml1 in melanoma cells efficiently knocks down the downstream transcriptional repressors Hey1 and Hes1, resulting in melanoma cell senescence, cellular differentiation, and increased melanin production. Significantly, higher IFNβ and chemokine gene transcripts have been observed, together with increased STAT1 and decreased STAT3 and NF-κB signaling activities. Although decreased cell proliferation contributes to slower tumor growth in vivo, the depletion of NK and CD8+ T cells in an shMaml1-B16 tumor carrier mouse leads to more rapid tumor growth than that observed in control shC002-B16 tumors. This result demonstrates that the knockdown of Maml1 transcription and function contributes to increased immune surveillance. The knockdown of Maml1 transcription in the human melanoma cell line M537 also results in senescence, IFNβ upregulation, increased chemokine gene expression, and greater NK and CD8+ T cell migration in a transwell system. This study demonstrated that targeting Maml1-induced tumor cell senescence and differentiation may alter the tumor microenvironment and cytokine and chemokine profiles and may also promote innate and adaptive immune cell infiltration and function.

Keywords: shMaml1, Cellular senescence, Innate and adaptive immune cells, IFNβ and chemokines

Introduction

The developmental proteins such as Notch, Wnt, and Hedgehog are key regulators of cell fate, proliferation, and differentiation. Their signaling pathways are related and are frequently activated in neoplasms from cancer stem cells that are capable of self-renewal, which often leads to the development of tumor resistance to therapy, tumor recurrence, and metastasis [14]. The Notch receptor proteins are highly expressed in melanoma cells and are constitutively cleaved to release the intracellular domain (NICD). Mastermind-like protein-1 (Maml1) directly binds to the most conserved ankyrin repeat domain of the four Notch receptors (NICD1–4), and Maml1 also interacts with CSL to form a stable DNA-binding ternary complex, thereby leading to the transcriptional activation of Hes1 and Hey1 [48]. A recent study discovered that Maml1 also participates in the Wnt/β-catenin signaling pathway independently of the Notch signaling pathway. Maml1 is recruited by β-catenin to the cyclin D1 and c-Myc promoters to activate tumor cell survival [9]. In neural precursor cells, Maml1, β-catenin, and NICD form a complex within the promoter region of the Hes1 gene, inducing its expression and simultaneously promoting cellular proliferation and inhibiting differentiation [10]. These studies support a new role for Maml1 as an essential component of Notch and Wnt/β-catenin signaling-mediated tumorigenesis and suggest that Maml1 could be an alternative target for both the Notch and Wnt/β-catenin signaling pathways.

Hes1 encodes a basic helix-loop-helix transcription factor that functions as a classical effector of the Notch signaling pathway [48]. However, recent reports indicated that, in stem-like cells, Hedgehog elicited a strong Hes1 response independent of canonical Notch signaling [1114]. Elsewhere, Schreck’s group found that targeting the Notch pathway using a g-secretase inhibitor increases Hedgehog activity, and the inhibition of both Notch and Hedgehog dramatically decreases the growth of glioblastoma and melanoma cell lines and low-passage neurospheres derived from primary human tumors [15]. The Hes1 signaling pathway is activated in several types of tumor cells. Activation of this signaling pathway allows these tumor cells to evade differentiation; therefore, irreversible senescence contributes to tumorigenesis [16, 17]. Maml1 is an essential coactivator that facilitates Notch-induced Hes1 and Hey1 transcription [7, 18]. Recent studies have indicated that Maml1 directly regulates NF-κB signaling and cell survival [19]. All of these data support our hypothesis that targeting Maml1 may regulate the Notch, Wnt, and Hedgehog signaling pathways simultaneously and may serve as a novel anticancer strategy by inhibiting cell proliferation and promoting cell differentiation and irreversible senescence [1921].

Previously, we used siMaml1 to inhibit the transcriptional activation of Hes1/Hey1 in melanoma cells, leading to the upregulation of multiple cytokines, including CCL2 [7]. However, researchers found that due to the low siRNA concentrations caused by cell division and intracellular siRNA turnover, the duration of siRNA silencing is short [22]. To analyze the effects of Maml1 on cell differentiation and senescence, the Maml1 gene must be silenced for a prolonged period of time. To accomplish this goal, we transfected several short-hairpin Maml1 (shMaml1) lentiviral expression plasmids to achieve sustained gene silencing, and we established a stable shMaml1-transfected B16 melanoma cell line (shMaml1-B16). We found that there were only a fraction of shMaml1-B16 cells undergoing apoptosis similar to the control plasmid shC002-B16-transfected cells or the parent B16 cells. However, we observed more shMaml1-B16 cells with increased senescence-associated-β-galactosidase (SA-β-Gal) staining and increased cell differentiation based on increased melanin production. These cells secrete several immune-stimulating cytokines and chemokines in vitro. In syngeneic mice, shMaml1-B16 cells formed tumors more slowly than control cells. Flow cytometry showed increased infiltration of immune cells, such as DC, NK, and CD8+ T cells, in these tumors.

Materials and methods

Establishment of shMaml1-B16 and shC002-B16 cells

MISSION shRNA clones (clone ID sh138: TRCN0000115138 targeting NM_175334.2-1187s1c1; clone ID sh139: TRCN0000115139 targeting NM_175334.2-1019s1c1; clone ID sh140: TRCN0000115140 targeting NM_175334.2-2544s1c1; clone ID shmix: mixture of TRCN0000115138, TRCN0000115139, and TRCN0000115140; human clone ID sh353: TRCN0000003353 targeting NM_014757.x-585s1c1; and clone ID sh356: TRCN0000003356 targeting NM_014757.x-1907s1c1) are sequence-verified, lentiviral plasmids (pLKO.1-puro) purchased from Sigma-Aldrich. B16F10 cells were transfected, and stable shMaml1-B16 cells were selected under optimized conditions using puromycin resistance (1 μg/ml) according the manufacturer’s protocol. Briefly, the siGENOME SMARTpool (10 μl) was diluted in 90 μl of serum-free DMEM containing transfection reagents, incubated at room temperature for 15 min, and added to a 2 × 105 cells/ml cell culture at a range of concentrations. Transfections were conducted twice on consecutive days, and the cells were allowed to continue to grow for an additional 2 days at 37 °C in the presence of 5 % CO2 before the puromycin selection. The nontarget, control lentiviral plasmid shC002 validated the specific knockdown of Maml1 by MISSION shMaml1.

Detection of SA-β-gal activity using confocal microscopy

SA-β-gal activity was measured at pH 6.0 for B16 cells and pH 5.5 for mouse tissue, as described previously. Briefly, the adherent cells were fixed with 0.5 % glutaraldehyde in PBS for 15 min, washed with PBS containing 1 mM MgCl2, and stained for 5–6 h in PBS containing 1 mM MgCl2, 1 mg/ml X-Gal (Roche, Indianapolis, IN, USA), 5 mM potassium ferricyanide, and 5 mM potassium ferrocyanide (Sigma-Aldrich, St. Louis, MO, USA). The images were photographed at several magnifications using a Nikon camera.

Analysis of gene expression using RT-qPCR

The cells and tumors were homogenized in 1 ml Trizol (Invitrogen, Grand Island, NY, USA), and total RNA was precipitated from the aqueous phase using isopropyl alcohol after performing a chloroform separation. After DNase I treatment, cDNA was synthesized from 1 μg of total RNA using MLV-Reverse Transcriptase according to the manufacturer’s directions. Real-time PCR (RT-qPCR) was performed using an ABI PRISM® 7300 Sequence Detection System with a Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA). Changes in gene expression were calculated using comparative Ct values with β-actin as an endogenous gene control. The ΔCT was calculated, and the relative expression of each cytokine and chemokine was compared. Prevalidated primers and probes specific for Maml1 (Mm00614627_m1), Hey1 (Mm00468865_m1), Hes1 (Mm01342805_m1), CCL2 (Mm00441242_m1), CCL5 (Mm01302427_m1), CXCL9 (Mm00434946_m1), CXCL10 (Mm00445235_m1), CXCL11 (Mm00444662_m1), CCL22 (Mm00436439_m1), and β-actin (Mm00525061_m1) were purchased from Applied Biosystems.

Immunoblotting and signaling inhibitors

Preparation of cytosolic extracts was carried out using a Nuclear Extract Kit (Active Motif, CA, USA) according to the manufacturer’s recommendations. The concentrations of proteins in the nuclear and cytosolic fractions were measured using a microplate version of the Lowry assay (Micro BCA Protein Assay Reagent Kit, Pierce, IL, USA) with bovine serum albumin (BSA) standards. The lysates were separated using SDS-PAGE on a 10 % gel and then transferred to a nitrocellulose membrane. The membrane was blocked with blocking buffer (5 % nonfat dry milk in TBST (20 mM Tris–HCl, pH 7.5, and 0.5 % Tween 20)) for 1 h at room temperature and then incubated overnight with one of the following primary antibodies diluted in the blocking buffer: anti-phospho-STAT1, anti-phospho-STAT3, anti-phospho-IκBα (1:500; Cell Signaling Technologies, Beverly, MA, USA), rabbit anti-STAT1, anti-STAT3, anti-NFκB p65, or anti-β-actin (1:500; Santa Cruz Biotechnologies, Santa Cruz, CA, USA). An HRP-conjugated, anti-rabbit secondary antibody (Amersham, Arlington Heights, IL, USA) was used at a 1:10,000 dilution for 1 h at room temperature. The blots were visualized using an Amersham enhanced chemiluminescence (ECL) kit. A highly selective inhibitor of IκB kinase (BMS-345541), the STAT1 inhibitor fludarabine (Sigma-Aldrich, St. Louis, MO, USA), and the cell-permeable peptide STAT3 inhibitor LLL12 (BioVision, Mountain view, CA, USA) were applied to the cell cultures when necessary.

Tumor inoculation and tumor-infiltrating cell treatment and analysis

All mice were bred and maintained in a pathogen-free facility at the Medical Center. The mice were handled in accordance with the animal experimental guidelines set by the Institute of Animal Care and Use Committee (IACUC) for all experiments. C57BL/6 wild-type mice (6–7 weeks old) were purchased from the Laboratory Animal Center of Southern Medical University (Guangzhou, China). The mice were subcutaneously injected with shMaml1-B16, shC002-B16, and B16 cells (1 × 106) on the leg. Tumor size was measured on three orthogonal axes (a, b, and c) every 3–4 days, and tumor volumes were calculated as abc/2. The tumors were fixed in 10 % neutral formalin and embedded, and serial sections were histologically stained with H&E and were subjected to immunohistochemistry to visualize CD11b myeloid, F4/80 macrophage, and CD8+ T cells. CD8+ T cells or NK cells were depleted by injecting 100 μg/kg of anti-CD8 (clone 2.43.1) or anti-NK1.1 (clone PK136, BD Pharmingen, CA) antibodies, respectively, administered on days 14 and 21. Depletion was confirmed by flow cytometry analysis of peripheral blood using BD Pharmingen fluorescent-labeled anti-CD8α-FITC and anti-NK-PerCP antibodies [23].

Statistical analysis

One-way ANOVA and Dunnett’s posttest were performed using GraphPad InStat version 3.05 to compare the average volumes of tumors in different experimental groups. Two-tailed Student’s t-tests were performed to compare the means of the different treatment groups. p < 0.05 was accepted as significant.

Results

ShMaml1-B16 cells demonstrated greater cell differentiation and senescence

Stable clones of the shMaml1-B16 cell line (shM138, shM140, shMmix) were established. Compared to the nontarget plasmid shC002-B16, the successful targeting of Maml1-promoted cell differentiation was demonstrated by increased melanin production. ShMaml1-B16 cells exhibited a large, flat morphology indicative of senescence, which was confirmed by enhanced SA-β-gal staining (Fig. 1a). A protein analysis of the B16 cells demonstrated the constitutive cleavage and high levels of expression of Notch 1 and Notch 4 but showed only low levels of expression of the Wnt signaling protein β-catenin, suggesting a dominant role for Notch signaling in B16 melanoma cells (we could not detect Hedgehog signaling protein, data not shown) (Fig. 1b). Total RNA was isolated from stable shMaml1-B16, shC002-B16, and parental B16 tumor cells, and RT-qPCR showed that the transcriptional repressors Hey1 and Hes1 abolished downstream Notch signaling (Fig. 1c). ShMaml1-B16 cells proliferated less than shC002-B16 or parental B16 tumor cells, but the difference in apoptotic rates among all cell lines was not significant in vitro (Fig. 1d).

Fig. 1.

Fig. 1

Establishment and characterization of a stable shMaml1-B16 cell line. a ShMaml1-B16 cells exhibited more intracellular melanin production and SA-β-gal staining than control cells. b Western blot of Notch and Wnt signaling proteins in B16 cells. c Successful knockdown of the transcriptional repressors Hey1 and Hes1, as well as Maml1, in shMaml1-B16 cells. d A lower proliferation rate but similar apoptosis rates are shown in shMaml1-B16 cells relative to shC002-B16 and parental WT B16 cells

Senescent shMaml1-B16 cells exhibited upregulated IFNβ and chemokine gene transcripts with increased STAT1 activity

Compared to shC002-B16 or parent B16 tumor cells, shMaml1-B16 cells showed greater expression of senescence-related cell cycle regulatory genes, including the cyclin-dependent kinase inhibitors p16 INK4A, p21 Cip1/Waf1, p27 Kip1, and p57 Kip2 (Fig. 2a). The expression levels of the IFNβ, CCL2, CCL5, and CCL18 transcripts were also increased in shMaml1-B16 cells relative to the shC002-B16 or parent B16 tumor cells (Fig. 2b).

Fig. 2.

Fig. 2

Changes in cytokine and chemokine expression in senescent shMaml1-B16 cells. a Upregulation of the cyclin-dependent kinase inhibitors p16 INK4A, p21 Cip1/Waf1, p27 Kip1, and p57 Kip2 in shMaml1-B16 cells compared to shC002-B16 or parent B16 tumor cells. b Upregulation of IFNβ, CCL2, CCL5, and CCL18 transcripts in shMaml1-B16 cells. c Levels of total and phosphorylated STAT1 were increased in shMaml1-B16 cells, but the phosphorylation of STAT3 and IκBα was inhibited. d Inhibition of STAT1 with fludarabine blocked the expression of CCL2, CCL5, and IFNβ in shMaml1-B16 cells, *p < 0.01

STATs and NF-κB have been reported to play essential roles in the synergistic transcriptional activation of multiple chemokines [2325]. We analyzed the expression and activation of these signaling pathways in shMaml1-B16, shC002-B16, and parental B16 cells. Western blot analysis showed an increase in both the total and phosphorylated levels of STAT1 in the shMaml1-B16 cells, but the phosphorylation of STAT3 and IκBα was inhibited (Fig. 2c). In the same cell line, inhibition of STAT1 with fludarabine blocked the expression of CCL2, CCL5, and IFNβ; however, inhibition of STAT3 using LLL12 or inhibition of IκBα kinase using BMS-345541 did not affect the expression of CCL2, CCL5, and IFNβ (Fig. 2d).

Senescent shMaml1-B16 cells showed slower growth and increased immune cell infiltration in vivo

C57BL/6 mice were inoculated with ShMaml1-B16, shC002-B16, or parental B16 tumor cells (1 × 106). Tumor growth was observed, and the mean tumor volumes were calculated. The ShMaml1-B16 tumors were smaller than both the shC002-B16 and parental B16 tumors at day 18 (p = 0.015, p = 0.013) (Fig. 3a). RT-qPCR of tumor lysates showed a higher expression of IFNγ, IFNβ, CCL2, CCL5, CXCL9, CXCL10, and CXCL11 in the shMaml1-B16 tumors, thereby demonstrating that these cells are in an immunostimulatory microenvironment (Fig. 3b). Flow cytometry showed a higher frequency of CD4+ T, CD8+ T, and NK cells in the shMaml1-B16 tumors compared to the frequency in the shC002-B16 or parental B16 tumor cells (Fig. 3c). Depletion of NK (p = 0.026) and CD8+ T (p = 0.021) cells in the shMaml1-B16 tumor carrier mice led to a tumor growth rate that resembled the shC002-B16 tumor growth rate. These data demonstrate that a knockdown of Maml1 transcription and function contributes to slowed tumor growth not only by lowering the relative tumor cell proliferation rate but also through an increased infiltration of innate and adaptive immune cells (Fig. 3d). CD4 depletion (p = 0.144) did not change the growth rate of the shMaml1-B16 tumors. Depletion of NK (p = 0.175) or CD8+ T (p = 0.217) cells in the control shC002-B16 tumor carriers did not significantly change the tumor growth rate.

Fig. 3.

Fig. 3

Inhibition of senescent shMaml1-B16 tumor growth and increased immune cell infiltration. a Growth curves of shMaml1-B16, shC002-B16, or parental B16 tumor cells in syngeneic C57BL/6 mice. b RT-qPCR of immune cytokine and chemokine expression in shMaml1-B16 tumor lysates compared to shC002-B16 or parental WT B16 tumor cells. c Flow cytometry of tumor-infiltrating cells in shMaml1-B16 tumors. d Depletion of NK and CD8+ T cells accelerated shMaml1-B16 tumor growth

Induction of cellular senescence in human melanoma cells transfected with shMaml1

Stable clones of the shMaml1-M537 cell line (shM353, shM356) were established. Compared to the nontarget plasmid shC002-M537, the successful targeting of Maml1-promoted cell senescence was indicated by the large, flat morphology of the cells and enhanced SA-β-gal staining (Fig. 4a). Total RNA was isolated from stable shMaml1-M537, shC002-M537, and parental M537 tumor cells, and RT-qPCR confirmed that the Hey1/Hes1 and Maml1 transcriptional repressors were knocked down (data not shown). The cyclin-dependent kinase inhibitors p16 INK4A, p21 Cip1/Waf1, and p57 Kip2 were upregulated compared to the shC002-M537 or parent M537 tumor cells (Fig 4b, upper panel). Additionally, the expression of the IFNβ, CCL2, CCL3, CCL5, and CXCL9 transcripts in the shMaml1-M537 cells was increased compared to the expression of these transcripts in the shC002-M537 or parental M537 tumor cells (Fig 4b, lower panel). Finally, in a transwell system, the shMaml1-M537 cells attracted more NK (p < 0.05) and CD8+ T (p < 0.01) cells compared to the shC002-M537 or parental M537 tumor cells (Fig 4c).

Fig. 4.

Fig. 4

Senescent shMaml1-M537 cells expressed multiple cytokines and are more chemoattractive to NK and CD8+ T cells. a ShMaml1-M537 exhibited more SA-β-gal staining cells than control cells. b Upregulation of the cyclin-dependent kinase inhibitors p16 INK4A, p21 Cip1/Waf1, p27 Kip1, and p57 Kip2 in shMaml1-M537 cells compared to shC002-M537 or parent M537 tumor cells (upper panel). Upregulation of IFNβ, CCL2, CCL5, and CXCL9 transcripts in shMaml1-M537 cells (lower panel). c Migration assay of NK and CD8+ T cells in a transwell system that contains shC002-M537, shC002-M537, or parental M537 cells

Discussion

In this study, we show that a knockdown of Maml1 in melanoma cells results in tumor cell senescence and differentiation, leading to an upregulation of multiple immune cytokines and chemokines in the tumor microenvironment. This investigation further demonstrated that disrupting Maml1–Hes1-induced transcription repression improved immune surveillance and eventually contributed to the elimination of senescent tumor cells [23]. Wnt signaling results in the repression of p21 Cip1/Waf1, whereas Notch signaling represses p27 Kip1 and p57 Kip2 in part through its target gene Hes1 [26, 27]. We knocked down Maml1, a coactivator known to participate in both Notch- and Wnt-activated Hes1 transcription, in B16 melanoma cells. The knockdown effectively repressed the cyclin-dependent kinase inhibitors p16 INK4A, p21 Cip1/Waf1, p27 Kip1, and p57 Kip2, resulting in decreased cell proliferation and senescence [14, 17, 27]. Maml1 is emerging as a coactivator for many other pathways, but this report is the first to show that the inhibition of Maml1–Hes1 signaling induces cellular senescence and immune surveillance.

Maml1 was shown to cause the phosphorylation of IκBα and affect IκBα stability. Maml1 also acts as a coactivator of the NF-κB subunit p65 in NF-κB-dependent transcription. These findings indicate that Maml1 is a novel modulator of NF-κB signaling and has a role in the regulation of cell survival [28, 29]. The activation or suppression of Notch1 and Hes1 has been correlated with the phosphorylation and activation of STAT3, and targeting the STAT3/p63/Notch axis induces several types of tumor cell differentiation [3032]. Additionally, interactions between activated STAT3 and constitutively active NF-κB have been studied in tumors by Yu’s group, and STAT3 was shown to be a target for enhancing anti-tumor immunity [3336]. Here, we show that targeting the Notch/Wnt coactivator Maml1 results in melanoma cell differentiation and senescence that not only inhibits the activation of both STAT3 and NF-κB but also stimulates the activity of STAT1. Consequently, the expression of immune cytokines and chemokines is significantly increased, resulting in increased NK and CD8+ T cell infiltration and the establishment of an immunostimulatory microenvironment [36, 37].

Tumor cell differentiation has been associated with the activation of cellular senescence [38]. Treatments that induce senescence and tumor cell differentiation may need to be accompanied by chemotherapeutic drugs or radiation. Treatment-induced senescence was shown to be one of the key determinants of tumor response to therapy both in vitro and in vivo. Clinical and preclinical studies have shown that the expression of different biological classes of senescence-associated genes in tumor cells has significant prognostic implications [3941]. Although senescent cells do not proliferate, they remain metabolically active and secrete proteins with either tumor-suppressing or tumor-promoting activities [42, 43]. A role has been reported for the innate immune system in eliminating senescent cells from tumors upon p53 reexpression, and CD4+ T cells have been shown to mediate anti-tumor effects through Myc-induced cellular senescence [44, 45]. We have shown that mouse shMaml1-B16 cells and human shMaml1-M537 cells undergoing senescence expressed multiple senescence-associated immune cytokines and chemokines, both in culture and in tumor microenvironments. Consequently, more NK and CD8+ T cell infiltrations occurred, demonstrating that the disruption of Maml1- and Notch-induced transcription repression may be a possible strategy for cancer treatment. Maml1 may participate in the Notch and Wnt signaling pathways during normal tissue development and cell differentiation; the knockdown of Maml1 blocked the transcriptional repression of Hes1 and affected the activation of STAT1, STAT3, and NF-κB. This specific knockdown of Maml1 in tumor cells has tremendous therapeutic potential; therefore, further studies of the involvement of Maml1 in the Notch and Wnt pathways are necessary.

Acknowledgments

This work was supported by the Province of Guangdong Science Grant (10251051501000008).

Conflict of interest

The authors declare that they have no conflict of interest.

Abbreviations

Maml1

Mastermind-like protein-1

siMaml1

Small interfering Maml1 RNA

Hes1

Hairy/enhancer-of-split 1

Hey1

Hairy/enhancer-of-split related with YRPW motif 1

CCL2

MCP-1, monocyte chemoattractant-1

CCL3

Macrophage inflammatory protein-1α (MIP-1α)

CCL5

Regulated upon activation normal T cell expressed and secreted (RANTES)

CCL18

Macrophage inflammatory protein-4 (MIP-4, PARC)

CXCL9

Monokine induced by gamma interferon (Mig)

CXCL10

Interferon gamma-induced protein-10 (IP-10)

CXCL11

Interferon-inducible T cell alpha chemoattractant (I-TAC, IP-9)

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