Large-scale pan-cancer analysis reveals broad prognostic association between TGF-ß ligands, not Hedgehog, and GLI1/2 expression in tumors.

GLI1 expression is broadly accepted as a marker of Hedgehog pathway activation in tumors. Efficacy of Hedgehog inhibitors is essentially limited to tumors bearing activating mutations of the pathway. GLI2, a critical Hedgehog effector, is necessary for GLI1 expression and is a direct transcriptional target of TGF-ß/SMAD signaling. We examined the expression correlations of GLI1/2 with TGFB and HH genes in 152 distinct transcriptome datasets totaling over 23,500 patients and representing 37 types of neoplasms. Their prognostic value was measured in over 15,000 clinically annotated tumor samples from 26 tumor types. In most tumor types, GLI1 and GLI2 follow a similar pattern of expression and are equally correlated with HH and TGFB genes. However, GLI1/2 broadly share prognostic value with TGFB genes and a mesenchymal/EMT signature, not with HH genes. Our results provide a likely explanation for the frequent failure of anti-Hedgehog therapies in tumors, as they suggest a key role for TGF-ß, not Hedgehog, ligands, in tumors with elevated GLI1/2-expression.

GLI1/GLI2 functional interplay is required to control Hedgehog/GLI targets gene expression.

The Hedgehog-regulated transcription factors GLI1 and GLI2 play overlapping roles in development and disease; however, the mechanisms underlying their interplay remain elusive. We report for the first time that GLI1 and GLI2 physically and functionally interact in cancer cells. GLI1 and GLI2 were shown to co-immunoprecipitate in PANC1 pancreatic cancer cells and RMS13 rhabdomyosarcoma cells. Mapping analysis demonstrated that the zinc finger domains of both proteins are required for their heteromerization. RNAi knockdown of either GLI1 or GLI2 inhibited expression of many well-characterized GLI target genes (BCL2, MYCN, PTCH2, IL7 and CCND1) in PANC1 cells, whereas PTCH1 expression was only inhibited by GLI1 depletion. qPCR screening of a large set of putative canonical and non-canonical Hedgehog/GLI targets identified further genes (e.g. E2F1, BMP1, CDK2) strongly down-regulated by GLI1 and/or GLI2 depletion in PANC1 cells, and demonstrated that ANO1, AQP1 and SOCS1 are up-regulated by knockdown of either GLI1 or GLI2. Chromatin immunoprecipitation showed that GLI1 and GLI2 occupied the same regions at the BCL2, MYCN and CCND1 promoters. Furthermore, depletion of GLI1 inhibited GLI2 occupancy at these promoters, suggesting that GLI1/GLI2 interaction is required for the recruitment of GLI2 to these sites. Together, these findings indicate that GLI1 and GLI2 co-ordinately regulate the transcription of some genes, and provide mechanistic insight into the roles of GLI proteins in carcinogenesis.

Transcriptional repression of the tyrosinase-related protein 2 gene by transforming growth factor-ß and the Kruppel-like transcription factor GLI2.

BACKGROUND: Tyrosinase-Related Protein 2 (TRP2) is an enzyme involved in melanogenesis, that also exerts proliferative, anti-apoptotic and immunogenic functions in melanoma cells. TRP2 transcription is regulated by the melanocytic master transcription factor MITF. GLI2, a transcription factor that acts downstream of Hedgehog signaling, is also a direct transcriptional target of the TGF-ß/SMAD pathway that contributes to melanoma progression and exerts transcriptional antagonistic activities against MITF. OBJECTIVES: To characterize the molecular events responsible for TGF-ß and GLI2 repression of TRP2 expression. METHODS: In silico promoter analysis, transient cell transfection experiments with 5'-end TRP2 promoter deletion constructs, chromatin immuno-precipitation, and site-directed promoter mutagenesis were used to dissect the molecular mechanisms of TRP2 gene regulation by TGF-ß and GLI2. RESULTS: We demonstrate that TGF-ß and GLI2-specific TRP2 repression involves direct mechanisms that occur in addition to MITF downregulation by TGF-ß and GLI2. We identify two functional GLI2 binding sites within the TRP2 promoter that are critical for TGF-ß and GLI2 responsiveness, one of them overlapping a CREB binding site. GLI2 and CREB competing for the same cis-element is associated with opposite transcriptional outcome. CONCLUSION: Our results further refine the understanding of how TGF-ß and GLI2 control the phenotypic plasticity of melanoma cells. In particular, we identify critical GLI2-binding cis-elements within the TRP2 promoter region that allow for its transcriptional repression independently from MITF concomitant downregulation.

How Bad Is the Hedgehog? GLI-Dependent, Hedgehog-Independent Cancers on the Importance of Biomarkers for Proper Patients Selection.

Hedgehog (HH) signaling plays an important role both during embryonic development and adult life. It is involved in the regulation of cell differentiation, cell proliferation and tissue polarity, as well as in the maintenance of stem cells, tissue repair, and regeneration (Briscoe and Therond, 2013; Jiang and Hui, 2008). Three ligands, Indian, Sonic, and Desert HH, can activate this pathway. Binding of HH ligands to their receptor, PTCH1 (Figure 1) lift its inhibition on SMO, resulting in activation and nuclear translocation of GLI transcription factors (Javelaud et al., 2012). The vertebrate GLI gene family is composed of three distinct genes GLI1, GLI2, and GLI3, encoding Krüppel-like transcription factors. GLI proteins exhibit distinct regulations, biochemical properties, and target genes. GLI3 acts as the main repressor of the pathway in the absence of HH ligands, whereas, in their presence, GLI2 is the main HH effector that drives the expression of GLI1 (Briscoe and Therond, 2013).

Cell density sensing alters TGF-ß signaling in a cell-type-specific manner, independent from Hippo pathway activation.

Cell-cell contacts inhibit cell growth and proliferation in part by activating the Hippo pathway that drives the phosphorylation and nuclear exclusion of the transcriptional coactivators YAP and TAZ. Cell density and Hippo signaling have also been reported to block transforming growth factor ß (TGF-ß) responses, based on the ability of phospho-YAP/TAZ to sequester TGF-ß-activated SMAD complexes in the cytoplasm. Herein, we provide evidence that epithelial cell polarization interferes with TGF-ß signaling well upstream and independent of cytoplasmic YAP/TAZ. Rather, polarized basolateral presentation of TGF-ß receptors I and II deprives apically delivered TGF-ß of access to its receptors. Basolateral ligand delivery nonetheless remains entirely effective to induce TGF-ß responses. These data demonstrate that cell-type-specific inhibition of TGF-ß signaling by cell density is restricted to polarized epithelial cells and reflects the polarized distribution of TGF-ß receptors, which thus affects SMAD activation irrespective of Hippo pathway activation.

GLI2 cooperates with ZEB1 for transcriptional repression of CDH1 expression in human melanoma cells.

In melanoma cells, high expression of the transcription factor GLI2 is associated with increased invasive potential and loss of E-cadherin expression, an event reminiscent of the epithelial-to-mesenchymal transition (EMT). Herein, we provide evidence that GLI2 represses E-cadherin gene (CDH1) expression in melanoma cells via distinct mechanisms, enhancing transcription of the EMT-activator ZEB1 and cooperative repression of CDH1 gene transcription via direct binding of both GLI2 and ZEB1 to two closely positioned Kruppel-like factor-binding sites within the CDH1 promoter. GLI2 silencing rescued CDH1 expression except in melanoma cell lines in which the CDH1 promoter was hypermethylated. Proximity ligation assays identified GLI2-ZEB1 complexes in melanoma cell nuclei, proportional to endogenous GLI2 and ZEB1 expression, and whose accumulation was enhanced by the classical EMT inducer TGF-ß. These data identify GLI2 as a critical modulator of the cadherin switch in melanoma, a molecular process that is critical for metastatic spread of the disease.

Insights into the Transforming Growth Factor-ß Signaling Pathway in Cutaneous Melanoma.

Transforming growth factor-ß (TGF-ß) is a pleiotropic growth factor with broad tissue distribution that plays critical roles during embryonic development, normal tissue homeostasis, and cancer. While its cytostatic activity on normal epithelial cells initially defined TGF-ß signaling as a tumor suppressor pathway, there is ample evidence indicating that TGF-ß is a potent pro-tumorigenic agent, acting via autocrine and paracrine mechanisms to promote peri-tumoral angiogenesis, together with tumor cell migration, immune escape, and dissemination to metastatic sites. This review summarizes the current knowledge on the implication of TGF-ß signaling in melanoma.

Overlapping activities of TGF-ß and Hedgehog signaling in cancer: therapeutic targets for cancer treatment.

Recent advances in the field of cancer therapeutics come from the development of drugs that specifically recognize validated oncogenic or pro-metastatic targets. The latter may be mutated proteins with altered function, such as kinases that become constitutively active, or critical components of growth factor signaling pathways, whose deregulation leads to aberrant malignant cell proliferation and dissemination to metastatic sites. We herein focus on the description of the overlapping activities of two important developmental pathways often exacerbated in cancer, namely Transforming Growth Factor-ß (TGF-ß) and Hedgehog (HH) signaling, with a special emphasis on the unifying oncogenic role played by GLI1/2 transcription factors. The latter are the main effectors of the canonical HH pathway, yet are direct target genes of TGF-ß/SMAD signal transduction. While tumor-suppressor in healthy and pre-malignant tissues, TGF-ß is often expressed at high levels in tumors and contributes to tumor growth, escape from immune surveillance, invasion and metastasis. HH signaling regulates cell proliferation, differentiation and apoptosis, and aberrant HH signaling is found in a variety of cancers. We discuss the current knowledge on HH and TGF-ß implication in cancer including cancer stem cell biology, as well as the current state, both successes and failures, of targeted therapeutics aimed at blocking either of these pathways in the pre-clinical and clinical settings.

Halofuginone inhibits the establishment and progression of melanoma bone metastases.

TGF-ß derived from bone fuels melanoma bone metastases by inducing tumor secretion of prometastatic factors that act on bone cells to change the skeletal microenvironment. Halofuginone is a plant alkaloid derivative that blocks TGF-ß signaling with antiangiogenic and antiproliferative properties. Here, we show for the first time that halofuginone therapy decreases development and progression of bone metastasis caused by melanoma cells through the inhibition of TGF-ß signaling. Halofuginone treatment of human melanoma cells inhibited cell proliferation, phosphorylation of SMAD proteins in response to TGF-ß, and TGF-ß-induced SMAD-driven transcription. In addition, halofuginone reduced expression of TGF-ß target genes that enhance bone metastases, including PTHrP, CTGF, CXCR4, and IL11. Also, cell apoptosis was increased in response to halofuginone. In nude mice inoculated with 1205 Lu melanoma cells, a preventive protocol with halofuginone inhibited bone metastasis. The beneficial effects of halofuginone treatment were comparable with those observed with other anti-TGF-ß strategies, including systemic administration of SD208, a small-molecule inhibitor of TGF-ß receptor I kinase, or forced overexpression of Smad7, a negative regulator of TGF-ß signaling. Furthermore, mice with established bone metastases treated with halofuginone had significantly less osteolysis than mice receiving placebo assessed by radiography. Thus, halofuginone is also effective in reducing the progression of melanoma bone metastases. Moreover, halofuginone treatment reduced melanoma metastasis to the brain, showing the potential of this novel treatment against cancer metastasis.

Crosstalk between TGF-ß and hedgehog signaling in cancer.

Hedgehog (HH) and TGF-ß signals control various aspects of embryonic development and cancer progression. While their canonical signal transduction cascades have been well characterized, there is increasing evidence that these pathways are able to exert overlapping activities that challenge efficient therapeutic targeting. We herein review the current knowledge on HH signaling and summarize the recent findings on the crosstalks between the HH and TGF-ß pathways in cancer.

Expression of microphthalmia-associated transcription factor (MITF), which is critical for melanoma progression, is inhibited by both transcription factor GLI2 and transforming growth factor-ß.

The melanocyte-specific transcription factor M-MITF is involved in numerous aspects of melanoblast lineage biology including pigmentation, survival, and migration. It plays complex roles at all stages of melanoma progression and metastasis. We established previously that GLI2, a Kruppel-like transcription factor that acts downstream of Hedgehog signaling, is a direct transcriptional target of the TGF-ß/SMAD pathway and contributes to melanoma progression, exerting antagonistic activities against M-MITF to control melanoma cell invasiveness. Herein, we dissected the molecular mechanisms underlying both TGF-ß and GLI2-driven M-MITF gene repression. Using transient cell transfection experiments with M-MITF promoter constructs, chromatin immunoprecipitation, site-directed mutagenesis, and electrophoretic mobility shift assays, we identified a GLI2 binding site within the -334/-296 region of the M-MITF promoter, critical for GLI2-driven transcriptional repression. This region is, however, not needed for inhibition of M-MITF promoter activity by TGF-ß. We determined that TGF-ß rapidly repressed protein kinase A activity, thus reducing both phospho-cAMP-response element-binding protein (CREB) levels and CREB-dependent transcription of the M-MITF promoter. Increased GLI2 binding to its cognate cis-element, associated with reduced CREB-dependent transcription, allowed maximal inhibition of the M-MITF promoter via two distinct mechanisms.

Systematic classification of melanoma cells by phenotype-specific gene expression mapping.

There is growing evidence that the metastatic spread of melanoma is driven not by a linear increase in tumorigenic aggressiveness, but rather by switching back and forth between two different phenotypes of metastatic potential. In vitro these phenotypes are respectively defined by the characteristics of strong proliferation/weak invasiveness and weak proliferation/strong invasiveness. Melanoma cell phenotype is tightly linked to gene expression. Taking advantage of this, we have developed a gene expression-based tool for predicting phenotype called Heuristic Online Phenotype Prediction. We demonstrate the predictive utility of this tool by comparing phenotype-specific signatures with measurements of characteristics of melanoma phenotype-specific biology in different melanoma cell lines and short-term cultures. We further show that 86% of 536 tested melanoma lines and short-term cultures are significantly associated with the phenotypes we describe. These findings reinforce the concept that a two-state system, as described by the phenotype switching model, underlies melanoma progression.

TGF-ß/SMAD/GLI2 signaling axis in cancer progression and metastasis.

The Hedgehog (HH) and TGF-ß signaling pathways represent essential regulators of cell proliferation and differentiation during embryogenesis. Pathway deregulation is a characteristic of various cancers. Recently, evidence for a convergence of these pathways at the level of the GLI2 transcription factor in the context of tumor initiation and progression to metastasis has emerged. This short review summarizes recent knowledge about GLI2 function and mechanisms of action downstream of TGF-ß in cancer.

GLI2 and M-MITF transcription factors control exclusive gene expression programs and inversely regulate invasion in human melanoma cells.

We recently identified GLI2, the most active of GLI transcription factors, as a direct TGF-ß/SMAD target, whose expression in melanoma cells is associated with increased invasiveness and metastatic capacity. In this work, we provide evidence that high GLI2 expression is inversely correlated with that of the melanocyte-specific transcription factor M-microphthalmia transcription factor (M-MITF) and associated transcriptional program. GLI2-expressing cell lines were characterized by the loss of M-MITF-dependent melanocytic differentiation markers and reduced pigmentation. The balance between M-MITF and GLI2 expression did not correlate with the presence or absence of BRAF-activating mutations, but rather was controlled by two distinct pathways: the TGF-ß pathway, which favors GLI2 expression, and the protein kinase A (PKA)/cAMP pathway, which pushes the balance toward high M-MITF expression. Furthermore, overexpression and knockdown experiments demonstrated that GLI2 and M-MITF reciprocally repress each other's expression and control melanoma cell invasion in an opposite manner. These findings thus identify GLI2 as a critical transcription factor antagonizing M-MITF function to promote melanoma cell phenotypic plasticity and invasive behavior.

Efficient TGF-ß/SMAD signaling in human melanoma cells associated with high c-SKI/SnoN expression.

BACKGROUND: SKI and SnoN proteins have been shown to inhibit TGF-ß signaling, acting both as transcriptional co-repressors in the cell nucleus, and as sequestrators of SMAD proteins in the cytoplasm. TGF-ß, on the other hand, induces rapid, proteasome-mediated, degradation of both proteins. How elevated SKI and SnoN protein levels co-exist with active autocrine TGF-ß signaling in cancer cells is yet to be understood. RESULTS: In this study, we found elevated SKI and SnoN protein levels in a panel of melanoma cell lines, as compared to normal melanocytes. There was no correlation between SKI protein content and the capacity of melanoma cells to invade Matrigel™, to form subcutaneous tumors, or to metastasize to bone after intracardiac inoculation into nude mice. Nor did we find a correlation between SKI expression and histopathological staging of human melanoma. TGF-ß induced a rapid and dose-dependent degradation of SKI protein, associated with SMAD3/4 specific transcriptional response and induction of pro-metastatic target genes, partially prevented by pharmacologic blockade of proteasome activity. SKI knockdown in 1205Lu melanoma cells did not alter their invasive capacity or transcriptional responses to TGF-ß, and did not allow p21 expression in response to TGF-ß or reveal any growth inhibitory activity of TGF-ß. CONCLUSIONS: Despite high expression in melanoma cells, the role of SKI in melanoma remains elusive: SKI does not efficiently interfere with the pro-oncogenic activities of TGF-ß, unless stabilized by proteasome blockade. Its highly labile nature makes it an unlikely target for therapeutic intervention.

TGF-beta-RI kinase inhibitor SD-208 reduces the development and progression of melanoma bone metastases.

Melanoma often metastasizes to bone where it is exposed to high concentrations of TGF-ß. Constitutive Smad signaling occurs in human melanoma. Because TGF-ß promotes metastases to bone by several types of solid tumors including breast cancer, we hypothesized that pharmacologic blockade of the TGF-ß signaling pathway may interfere with the capacity of melanoma cells to metastasize to bone. In this study, we tested the effect of a small molecule inhibitor of TGF-ß receptor I kinase (TßRI), SD-208, on various parameters affecting the development and progression of melanoma, both in vitro and in a mouse model of human melanoma bone metastasis. In melanoma cell lines, SD-208 blocked TGF-ß induction of Smad3 phosphorylation, Smad3/4-specific transcription, Matrigel invasion and expression of the TGF-ß target genes PTHrP, IL-11, CTGF, and RUNX2. To assess effects of SD-208 on melanoma development and metastasis, nude mice were inoculated with 1205Lu melanoma cells into the left cardiac ventricle and drug was administered by oral gavage on prevention or treatment protocols. SD-208 (60 mg/kg/d), started 2 days before tumor inoculation prevented the development of osteolytic bone metastases compared with vehicle. In mice with established bone metastases, the size of osteolytic lesions was significantly reduced after 4 weeks treatment with SD-208 compared with vehicle-treated mice. Our results demonstrate that therapeutic targeting of TGF-ß may prevent the development of melanoma bone metastases and decrease the progression of established osteolytic lesions.

Smad7 restricts melanoma invasion by restoring N-cadherin expression and establishing heterotypic cell-cell interactions in vivo.

The list of transforming growth factor-beta (TGF-ß)-related proteins in non-canonical TGF-ß signaling is growing. Examples include receptor-Smads directing micro-RNA processing and inhibitory-Smads, e.g. Smad7, directing cell adhesion. Human skin grafts with fluorescently tagged melanoma cells revealed Smad7-expressing cells positioned themselves proximal to the dermal-epidermal junction and failed to form tumors, while control cells readily invaded and formed tumors within the dermis. Smad7 significantly inhibited ß-catenin T41/S45 phosphorylation associated with degradation and induced a 4.5-fold increase in full-length N-cadherin. Cell adhesion assays confirmed a strong interaction between Smad7-expressing cells and primary dermal fibroblasts mediated via N-cadherin, while control cells were incapable of such interaction. Immunofluorescent analysis of skin grafts indicated N-cadherin homotypic interaction at the surface of both Smad7 cells and primary dermal fibroblasts, in contrast to control melanoma cells. We propose that Smad7 suppresses ß-catenin degradation and promotes interaction with N-cadherin, stabilizing association with neighboring dermal fibroblasts, thus mitigating invasion.

GLI2-mediated melanoma invasion and metastasis.

BACKGROUND: The transforming growth factor-beta (TGF-beta) pathway, which has both tumor suppressor and pro-oncogenic activities, is often constitutively active in melanoma and is a marker of poor prognosis. Recently, we identified GLI2, a mediator of the hedgehog pathway, as a transcriptional target of TGF-beta signaling. METHODS: We used real-time reverse transcription-polymerase chain reaction (RT-PCR) and western blotting to determine GLI2 expression in human melanoma cell lines and subsequently classified them as GLI2high or as GLI2low according to their relative GLI2 mRNA and protein expression levels. GLI2 expression was reduced in a GLI2high cell line with lentiviral expression of short hairpin RNA targeting GLI2. We assessed the role of GLI2 in melanoma cell invasiveness in Matrigel assays. We measured secretion of matrix metalloproteinase (MMP)-2 and MMP-9 by gelatin zymography and expression of E-cadherin by western blotting and RT-PCR. The role of GLI2 in development of bone metastases was determined following intracardiac injection of melanoma cells in immunocompromised mice (n = 5-13). Human melanoma samples (n = 79) at various stages of disease progression were analyzed for GLI2 and E-cadherin expression by immunohistochemistry, in situ hybridization, or RT-PCR. All statistical tests were two-sided. RESULTS: Among melanoma cell lines, increased GLI2 expression was associated with loss of E-cadherin expression and with increased capacity to invade Matrigel and to form bone metastases in mice (mean osteolytic tumor area: GLI2high vs GLI2low, 2.81 vs 0.93 mm(2), difference = 1.88 mm(2), 95% confidence interval [CI] = 1.16 to 2.60, P < .001). Reduction of GLI2 expression in melanoma cells that had expressed high levels of GLI2 substantially inhibited both basal and TGF-beta-induced cell migration, invasion (mean number of Matrigel invading cells: shGLI2 vs shCtrl (control), 52.6 vs 100, difference = 47.4, 95% CI = 37.0 to 57.8, P = .024; for shGLI2 + TGF-beta vs shCtrl + TGF-beta, 31.0 vs 161.9, difference = -130.9, 95% CI = -96.2 to -165.5, P = .002), and MMP secretion in vitro and the development of experimental bone metastases in mice. Within human melanoma lesions, GLI2 expression was heterogeneous, associated with tumor regions in which E-cadherin was lost and increased in the most aggressive tumors. CONCLUSION: GLI2 was directly involved in driving melanoma invasion and metastasis in this preclinical study.

JNK supports survival in melanoma cells by controlling cell cycle arrest and apoptosis.

JNK1/2 proteins belong to the family of stress-activated protein kinases. They play a complex role in growth regulation, inducing either cell death or growth support. In this report, we provide evidence that, in human melanoma cells, JNK inhibition with the small molecule inhibitor SP600125 induces either predominantly a G2/M arrest or apoptosis depending on the cell line. In 1205Lu cells, JNK inhibition induced cell cycle arrest through p53-dependent induction of p21 Cip1/Waf1 expression, while in WM983B cells, induction of apoptosis by JNK inhibition was accompanied by p53, Bad and Bax induction, not p21 Cip1/Waf1. JNK inhibition with the small molecule inhibitor SP600125 slowed growth of all cell lines, although the effect was markedly greater in cells exhibiting high phospho- (P-)JNK1 levels. Specific gene knockdown of JNK1 by means of siRNA oligonucleotides inhibited cell growth only in melanoma cell lines exhibiting high P-JNK1 levels. siRNAs directed against JNK2 did not reduce cell growth in any of the cell lines tested. Together, our findings demonstrate that JNK, and in particular the JNK1 isoform, support the growth of melanoma cells, by controlling either cell cycle progression or apoptosis depending on the cellular context.

Transforming growth factor-beta suppresses the ability of Ski to inhibit tumor metastasis by inducing its degradation.

c-Ski is an important corepressor of transforming growth factor-beta (TGF-beta) signaling through its ability to bind to and repress the activity of the Smad proteins. It was initially identified as an oncogene that promotes anchorage-independent growth of chicken and quail embryo fibroblasts when overexpressed. Although increased Ski expression is detected in many human cancer cells, the roles of Ski in mammalian carcinogenesis have yet to be defined. Here, we report that reducing Ski expression in breast and lung cancer cells does not affect tumor growth but enhances tumor metastasis in vivo. Thus, in these cells, Ski plays an antitumorigenic role. We also showed that TGF-beta, a cytokine that is often highly expressed in metastatic tumors, induces Ski degradation through the ubiquitin-dependent proteasome in malignant human cancer cells. On TGF-beta treatment, the E3 ubiquitin ligase Arkadia mediates degradation of Ski in a Smad-dependent manner. Although Arkadia interacts with Ski in the absence of TGF-beta, binding of phosphorylated Smad2 or Smad3 to Ski is required to induce efficient degradation of Ski by Arkadia. Our results suggest that the ability of TGF-beta to induce degradation of Ski could be an additional mechanism contributing to its protumorigenic activity.