- Research article
- Open Access
- Open Peer Review
The leukemia inhibitory factor (LIF) and p21 mediate the TGFβ tumor suppressive effects in human cutaneous melanoma
- Laure Humbert†1,
- Mostafa Ghozlan†1,
- Lucie Canaff1,
- Jun Tian1 and
- Jean-Jacques Lebrun1, 2Email author
© Humbert et al.; licensee BioMed Central. 2015
- Received: 12 December 2014
- Accepted: 6 March 2015
- Published: 29 March 2015
Cutaneous melanoma is the most lethal skin cancer and its incidence in developed countries has dramatically increased over the past decades. Localized tumors are easily treated by surgery, but advanced melanomas lack efficient treatment and are associated with very poor outcomes. Thus, understanding the processes underlying melanoma development and progression is critical. The Transforming Growth Factor beta (TGFβ) acts as a potent tumor suppressor in human melanoma, by inhibiting cell growth and preventing cellular migration and invasion.
In this study, we aimed at elucidating the molecular mechanisms underlying TGFβ-mediated tumor suppression. Human cutaneous melanoma cell lines, derived from different patients, were used to assess for cell cycle analysis, apoptosis/caspase activity and cell migration. Techniques involved immunoblotting, immunohistochemistry, real time PCR and luciferase reporter assays.
We found the leukemia inhibitory factor (LIF) to be strongly up-regulated by TGFβ in melanoma cells, defining LIF as a novel TGFβ downstream target gene in cutaneous melanoma. Interestingly, we also showed that TGFβ-mediated LIF expression is required for TGFβ-induced cell cycle arrest and caspase-mediated apoptosis, as well as for TGFβ-mediated inhibition of cell migration. Moreover, we found that TGFβ-mediated LIF expression leads to activation of transcription of the cell cycle inhibitor p21 in a STAT3-dependent manner, and further showed that p21 is required for TGFβ/LIF-mediated cell cycle arrest and TGFβ-induced gene activation of several pro-apoptotic genes.
Together, our results define the LIF/p21 signaling cascade as a novel tumor suppressive-like pathway in melanoma, acting downstream of TGFβ to regulate cell cycle arrest and cell death, further highlight new potential therapeutic strategies for the treatment of cutaneous melanoma.
- TGFβ/Smad signaling
- Tumor suppression
- Cell cycle arrest
- Cell migration
Skin cancer is the most common type of cancer worldwide, with an annual occurrence of almost 3 million cases. Cutaneous melanoma is one of the most aggressive and lethal human tumor, accounting for 75-80% of skin cancer-related deaths . Melanoma incidence has dramatically increased over the past decades and it is now the most common cause of cancer deaths among young people between the age of 20-35 . Melanomas have been classified into four clinical grades on the basis of their histology and prognosis. Grade IV melanomas are highly metastatic and refractory to conventional chemotherapeutic and biological reagents. Most patients have localized disease at the time of the diagnosis and are cured by surgical excision of the primary tumor, but melanomas can be highly malignant, and can metastasize to various organs including skin, lung, liver, brain and bone . The fifteen-year survival for stage I melanoma is 85% whereas it is only 5% for stage IV melanoma. . Melanoma display multifactorial etiology, yet its genetic and immunological background have not been elucidated. Thus, understanding the molecular and signaling mechanisms underlying melanoma formation and progression is a prerequisite for the development of more efficient treatments. At the molecular level, several signaling pathways have been implicated in the control of melanoma tumor formation, including the Ras-Raf-Mek-Erk cascade, which often exhibits activating mutations in cutaneous malignant melanoma . Other signaling pathways potentially implicated are PI3K/AKT, Wnt, NF-κB, Jnk/c-Jun, JAK/STAT and TGFβ . Contrary to frequent BRAF mutations which occur at a frequency of 50-80% , no genetic alterations of TGFβ signaling molecules have been identified in melanomas that could explain their resistance .
TGFβ signaling is initiated by the type II receptor (TβRII), a constitutively auto-phosphorylated serine/threonine kinase, which upon ligand binding recruits and transphosphorylates the type I receptor (TβRI), thereby activating its kinase activity . Activated TβRI then phosphorylates mediators known as receptor-regulated Smads (R-Smads), Smad2 and 3, and allows subsequent heterotrimerization with a common partner, Smad4 [7,8]. The Smad heterotrimer translocates to the nucleus where it can bind DNA and regulate transcription, along with transcription factors, co-activators or co-repressors .
The role of TGFβ in cancer is complex and ranges from cell growth inhibition to regulation of cell migration and invasion [6,9,10]. In several types of cancer, such as breast cancer, TGFβ exerts a dual role: while it acts as a potent cell cycle inhibitor and a pro-apoptotic factor in normal and premalignant states, these tumor suppressive effects are lost in more advanced tumors and replaced by tumor promoting effects leading to metastasis [6,9-11]. In melanocytic systems, the role of TGFβ is different. While TGFβ acts as a potent tumor suppressor in normal melanocytes through the regulation of the plasminogen activation system, it also inhibits cell migration and cell invasion in melanoma of various stages [12,13]. Regarding cell growth inhibition, it has been reported that normal melanocytes in culture are sensitive to the growth-inhibitory effects of TGFβ, whereas melanoma cell lines demonstrate various degrees of resistance to these effects [14,15]. However, TGFβ is perfectly capable of inducing Smad signaling and Smad-dependent transcription in melanomas, suggesting that desensitization to the anti-proliferative activity of TGFβ is highly specific to cell cycle progression [12,16]. Also, several studies have shown an increased expression and secretion of the TGFβ isoforms in melanoma cell lines compared to normal melanocytes, suggesting that TGFβ signaling is still active in these cells [14,17-20]. While it seems that TGFβ acts as a potent tumor suppressor in melanocytic systems, the TGFβ tumor suppressive mechanisms have not been thoroughly investigated in melanoma .
Previous work from our lab showed that TGFβ inhibits human cutaneous melanoma cell migration and invasion through regulation of the plasminogen activator system . We found by analysis of the transcriptome of two human melanoma cell lines, WM793B (Vertical growth phase melanoma, VPG, Stage I) and WM278 (VPG, Stage II), that one particular gene, the leukemia inhibitory factor (LIF), appeared to be strongly upregulated by TGFβ. Two previous studies have reported the induction of LIF mRNA and/or protein by TGFβ in Schwann cells  and glioblastoma  and shown this upregulation to be Smad-dependant by binding to a Smad binding element in LIF promoter. LIF is a member of the interleukin 6 (IL-6) family of cytokines, which includes IL-11, IL-27 and Oncostatin M (OSM) [24-26]. LIF signals through LIF receptor (LIF-R) which shares the gp130 subunit with other members of its class and which activates the JAK-STAT pathway [24-26]. LIF is expressed at the embryo stage and in many adult cell types and has been shown to be crucial for blastocyst implantation, maintenance of hematopoietic stem cells, differentiation, cell growth, inflammation, cachexia in animals, mammary gland involution after lactation, neurogenesis, and tissue regeneration [25,26]. Its role in cell growth is unclear as it was shown to both positively and negatively regulate proliferation [24-26], suggesting that these effects may be tissue-specific. Promotor studies and CHIP assays have shown that members of the LIF family such as Oncostatin M and IL-6 upregulate p21 expression through the Jak/STAT pathway, but this has not yet been investigated in melanoma cells [27,28].
TGFβ has been shown to regulate the expression of p21, to suppress the expression of genes important for cell cycle progression and to induce the expression of genes important for senescence [9,11]. However, the role of p21 in apoptosis is paradoxical and more investigations are needed [29-35]. Several studies have reported that p21 was detected in primary melanomas and metastatic lesions, while p21 levels were low or undetectable in melanocytic nevi. p21 might play an important role in melanoma progression, but the mechanisms are unknown.
In the present study, we aimed to understand the molecular mechanisms underlying TGFβ growth inhibition and apoptosis in human melanoma cells.
Recombinant human TGFβ and LIF were purchased from Peprotech (Dollard des Ormeaux, Quebec, Canada). Tissue culture medium RPMI1640 was from Hyclone (Logan, UT, US). FBS, antibiotics (penicillin/streptomycin) and Lipofectamine 2000 were from Life Science (Grand Island, NY, USA). Antibodies against LIF, p15, p21, c-myc, p-Stat3 (Tyr705), Stat3 and β-tubulin were from Santa Cruz Biotechnologies (Santa Cruz, CA, USA). Scrambled, p21 and LIF siRNAs were from Sigma (Oakville, ON, Canada). D-luciferin and Lumi-light plus were from Roche Diagnostics (Laval, Qc, Canada). MMLV reverse transcriptase and random primers were from Life Science (Grand Island, NY, USA).
Cutaneous melanoma cell lines WM793B and WM278 cell lines were isolated from the primary tumors of a 37-year-old male patient and a 62-year-old female patient and were kindly provided by Dr Louise Larose (McGill University, Montreal, Canada). The WM278 cell line harbors a V600E mutation in the BRAF gene, and a hemizygous deletion of PTEN. NRas and CDK4 are wild type. WM793B cells are positive for a V600E BRAF mutation and carry a W274X mutation as well as a hemizygous deletion of PTEN. This cell line also has a mutation K22Q of CDK4. NRas is wildtype.
Cells were cultured at 37°C in RPMI1640 medium supplemented with 10% FBS and antibiotics under a humidified atmosphere of 5% CO2.
Cell cycle analysis
Melanoma cells were plated in 24-well plates, serum starved overnight, and treated or not with TGF-β (200 pM) for 24 hours in a medium containing 2% FBS. Cells were washed in PBS and fixed in ethanol 70% for 2 hours. When ready for analysis, cells were resuspended in a solution containing 50 μg/ml propidium iodide, 50 μg/ml RNAse A and 0.1% Triton X-100. Cell cycle analysis was measured using an Accuri C6 flow cytometer (BD Biosciences, Mississauga, ON, Canada).
Quantitative real time PCR
Total RNAs were extracted with Trizol (Life Science, Grand Island, NY, USA) according to the manufacturer’s instructions. One μg of RNA was reverse transcribed using M-MLV reverse transcriptase and random primers. Amplification of cDNA was performed by quantitative real time PCR (qPCR) (Bio-Rad iQ Sybr Green supermix, Mississauga, ON, Canada and RotorGene Corbett, San Francisco, CA, USA). Human GAPDH was used as a housekeeping gene. The qPCR conditions were: 3 minutes 95°C, then 40 cycles of 10 seconds at 94°C, 10 seconds at 60°C and 20 seconds at 72°C.
Cells were lysed for 30 minutes at 4°C in RIPA buffer (50 mM Tris-HCL pH 7.4, 150 mM NaCl, 1% triton X-100, 1 mM EDTA, 1 mM EGTA, 1 mM DTT) supplemented with protease inhibitors (1 mM PMSF, 10 μg/ml leupeptin and aprotinin and 2 μg/ml of pepstatin A). For analysis of phosphorylation, 100 mM sodium fluoride, 10 mM sodium pyrophosphate and 100 mM sodium ortho-vanadate were added. For analysis of LIF expression, conditioned media were concentrated using Amicon® Ultra-15 Centrifugal Filter Unit with Ultracel-30 membrane (Billerica, MA, USA). Total lysates or concentrated media were immunoblotted by SDS-PAGE against specific antibodies. Immunoreactivity was revealed by chemiluminescence using Lumi-light PLUS (Roche, Mississauga, ON, Canada). Protein levels were quantified by densitometric analysis (ImageJ software, http://rsb.info.nih.gov/nih-image/).
Melanoma cells were plated in 96-well plates, starved overnight, then treated or not for 72 hours with TGFβ (200 pM) in medium supplemented with 2% FBS. Caspase 3/7 activity was measured by luminescence using Caspase 3/7 assay (Promega, Madison, WI, USA) according to the manufacturer’s instructions.
Promoter-reporter constructs transfection and luciferase assay
The p21-luc and p21inr-luc constructs were kindly provided by Dr Xiao Fan Wang. For transient transfection, WM278 or WM793B cells were plated in 6-well dishes in RPMI1640, 10% FBS (1-4 × 105 cells per well), and incubated overnight. The next day, cells were transfected with Lipofectamine reagent with 80nM siRNA. After 24 hours cells were transfected with 1 μg of promoter-reporter construct and 0.5 μg of Renilla luciferase construct per well. The following day, cells were serum-starved in RPMI overnight and cultured with or without 100 pmol TGF-β for 16 h. Cells were washed in PBS and lysed in 250 μl of passive lysis buffer (25 mM glycylglycine, 15 mM MgSO4, 4 mM EGTA, 1 mM DTT and 1% Triton X-100) on ice. Supernatants were collected by centrifugation (12,000 rpm, 20 minutes, 4°C). Luciferase activity was measured in a Fluostar Optima luminometer (BMG Labtech) using 45 μl of cell lysate and D-luciferin. Firefly luciferase activity was normalized to Renilla luciferase activity.
Tissue sections (5 μm) from a melanoma microarray slide (ME1004a, US Biomax) were stained for p-Smad3 and LIF at the Goodman Cancer Research Histology core laboratory (McGill University, Montreal, Canada). LIF staining was revealed using Bajoran purple chromogen and p-Smad3 was developed using 3,3′-Diaminobenzidine (DAB) staining. The staining was scored from 0 to 4, where 0 means no staining while 4 means a strong staining. Representative pictures were taken at 40X magnification.
Cells were plated on top of 24-well cell culture Transwell inserts (BD Biosciences, Mississauga, ON, Canada) and stimulated or not with TGFβ (200 pM) for 48 hours after an overnight serum starvation. The bottom chambers contained medium supplemented with 10% FBS as a chemoattractant. The migratory cells located on the filter of the bottom chamber were fixed for 10 minutes in paraformaldehyde and stained with 0.5% crystal violet. Images were taken using phase contrast light microscopy and migratory cells were counted using ImageJ software.
Ethics and consent
This study did not require any ethics statement or any written informed consent for participation from participants, as no participant, patient tissue samples but only cell lines in culture were used in the study.
TGFβ induces cell cycle arrest and apoptosis in human cutaneous melanoma cell lines
LIF upregulation by TGFβ is required for TGFβ-mediated cell cycle arrest and apoptosis
TGFβ exerts its tumor suppressive effects in melanoma through regulation of the cyclin-dependent kinase inhibitor p21
LIF is required for TGFβ-mediated p21 upregulation
LIF is required for the anti-metastatic effects of TGFβ in a p21-independent manner
Low response to TGFβ and low LIF expression correlate with melanoma aggressiveness
The role of p21 as a cell cycle inhibitor has been well characterized in other tissues and cell types [37,43,44]. Through its amino-terminal CDK-cyclin inhibitory domain, p21 binds to both the cyclin subunit and the CDK subunit of CDK-cyclin complexes, preventing them from binding to p107, p130, and Rb, which are involved in cell cycle progression. p21 also directly inhibits DNA synthesis by disrupting DNA-polymerase binding to DNA. Nonetheless, the exact function of p21 in regulating apoptosis remains unclear and even controversial, as both pro- and anti-apoptotic p21 activities have been previously reported [45,46]. Many studies are indicative of an anti-apoptotic role for p21 in different target tissues. For example, p21-deficient lymphomas with a p53 deficient background showed a higher apoptotic rate than p21-proficient lymphomas, indicating a protective role for p21 against apoptosis . Similarly, p21 depletion in human embryonic fibroblasts was reported to induce cell death in these cells . In hepatoma cells, p21 was found to bind caspase 3, thereby preventing caspase activation and Fas-induced apoptosis . In addition, p21 was also found to inhibit stress-induced apoptosis . Indeed, p21 prevents stress-induced apoptosis mediated by the JNK and p38 signaling pathways by binding to and inhibiting the activity of the MAP3K5 (ASK1; MEKK5) in human rhabdomyosarcoma cells  and by binding to JNK kinases, further preventing their activation by upstream kinases . On the other hand, multiple reports have also documented a pro-apoptotic role for p21. For instance, p21 overexpression in ovarian cancer cells was found to enhance susceptibility to cisplatin-induced apoptosis . Reports showed that p21 could also facilitate deoxycholic acid-induced apoptosis in primary mouse hepatocytes  and ceramide-induced apoptosis in human hepatoma cells . Similarly, thymocytes from mice carrying a p21 transgene targeted for restricted expression in the T cell lineage were found to be hypersensitive to radiation-induced programmed cell death . These studies suggest that p21 can also act as a cell death inducer even though the molecular mechanisms underlying these effects are not fully elucidated. Altogether, these studies highlight the fact that the role of p21 in regulating cell death is clearly context-dependent. Our study indicates that, in the context of human cutaneous melanoma, p21 acts as a potent pro-apoptotic factor. We also showed that p21 acts downstream of the TGFβ/LIF signaling cascade and that it promotes caspase-dependent cell death though induced-expression of pro-apoptotic molecules, such as Bax and Bim.
Similarly, the role of LIF in cell growth regulation has not been clearly established. Evidence shows that LIF inhibits the differentiation of embryonic stem cells to maintain their pluripotentiality  and positively regulates the proliferation of germ cells, hematopoietic progenitors, megakaryocytes, myoblasts, and neural cells . Conversely, LIF inhibits proliferation and induces differentiation of leukemic myeloid cells , promotes differentiation of adipocytes , cardiac muscle cells , and cardiac stem cells . Exogenous LIF was reported to act as a growth factor for melanoblasts and melanocytes , yet another study showed that LIF did not exert any growth stimulatory effect in melanoma cells . Our results clearly indicate that LIF inhibits melanoma cell growth downstream of the TGFβ signaling pathway. We found that LIF mediates both TGFβ-induced G1 arrest and apoptosis in melanoma, indicating a tumor suppressor-like role for this cytokine. This is also consistent with previous reports indicating that LIF could induce G1 arrest in medullary cancer cells  and retinal microvascular endothelial cells . In terms of cell death, LIF has been reported to induce apoptosis in mammary epithelial cells , but was found to inhibit apoptosis in other cell types, such as olfactory sensory neurons  and myoblast cells [63,64]. Thus, similar to p21, LIF function as a regulator of apoptosis appears to be cell type- and tissue-specific.
Mounting evidence show that increased nuclear pSTAT3 expression in various solid tumors, such as lung , breast , head and neck , as well as thyroid [68,69], is correlated with either reduced tumor size, reduced aggressiveness or enhanced survival outcomes thus pointing towards a rather tumor-suppressive role of pSTAT3 in these cancers. Our results support these findings and show that that TGFβ-induced LIF expression through activation of STAT3 further leads to p21 gene transcription and TGFβ-mediated cell cycle arrest and apoptosis in melanoma.
Moreover, our study shows that in addition to its role in mediating TGFβ tumor suppressor effects, LIF also acts downstream of TGFβ to prevent tumor progression by inhibiting cell migration, in a p21-independent manner. This indicates that LIF is a major regulator of the TGFβ effects in cutaneous melanoma, not only relaying TGFβ-mediated cell cycle arrest and apoptosis but also TGFβ-mediated cell migration inhibition. Thus, our results define LIF signaling as a potent tumor suppressor and as a potential suppressor of metastasis in human melanoma. In fact, this is consistent with a recent study showing that LIF receptor (LIF-R) also acts as a metastasis suppressor in breast cancer . In that context, LIF-R acts downstream of the microRNA miR-9 but upstream of Hippo signaling . The authors further found that loss of LIF-R expression in non-metastatic breast cancer cells induced a metastatic behavior. Consistently, we show here that LIF itself also contributes to prevention of tumor metastasis in melanoma, by mediating the TGFβ inhibitory effects on cell migration.
Collectively, our results indicate that the TGFβ/LIF/p21 signaling axis plays a major role in controlling tumor formation and tumor progression in melanoma. Interestingly, a recent clinical study from Tas et al., examining 60 patients with a pathologically confirmed diagnosis of melanoma, revealed that chemotherapy-responsive melanoma patients have higher levels of serum TGFβ compared to chemotherapy-refractory patients . Moreover, melanoma patients with high levels of serum TGFβ also showed favorable overall survival compared to patients with lower levels . These findings are in agreement with our results showing that TGFβ, via LIF/STAT3 activation, leads to the suppression of the invasive phenotype in melanoma and highlight TGFβ as a favorable prognosis marker and protective growth factor against tumor metastasis in human melanoma.
The authors would like to thank Drs. A. Spatz and L. Larose for kindly providing the melanoma cell lines and Dr. X.F. Wang for providing the p21-luciferase reporter constructs. This work was supported by grants from the Canadian Institutes for Health Research (CIHR to JJL).
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