Tumor Progression Locus Research Articles

Tpl2 exaggerated ischemic hepatic injury through induction of apoptosis: Downstream of caspase-9

Ischemia-reperfusion injury is a dynamic process that involves the two interrelated phases of local ischemic insult and inflammation-mediated reperfusion injury, and an important cause of liver damage occurring during surgical procedures and represents the main underlying cause of graft dysfunction post-transplantation. This study is a highlight into innate-adaptive immune crosstalk and cell activation cascades that lead to inflammation-mediated injury in livers stressed by ischemia-reperfusion. Tumor Progression Locus 2 (TPL2) is a serine-threonine kinase with essential functions in innate immune cells, where it transmits signals through Toll-like receptors family. To explore the role of Tpl2 in hepatic injury, liver ischemia/reperfusion injury was induced in wild type mice (Tpl2+/+) and in Tpl2-/- mice. They were sacrificed after 3-days post I/R. We determined TPL2 mRNA levels by quantitative PCR (qPCR), DNA and total RNA extraction from tissues was performed using the DNeasy and miRNeasy Kits, the extents of liver and function were studied. Tpl2 knockout mice produce low levels of proinflammatory cytokines expression, reduced release of LDH, ALT and less hepatic injury than wild type mice when exposed to I/R. Interestingly caspase-3 activation was significantly lower in Tpl2-/- mice. These results suggested that Tpl2 directly involved in apoptotic signal at the level of caspase-3 activation downstream of caspase-9, and that Tpl2 contributed to the exaggerated of ischemic liver injury through induction of apoptosis.

Tumor progression locus 2 (Tpl2) kinase as a novel therapeutic target for cancer: double-sided effects of Tpl2 on cancer.

Tumor progression locus 2 (Tpl2) is a mitogen-activated protein kinase (MAPK) kinase kinase (MAP3K) that conveys various intra- and extra-cellular stimuli to effector proteins of cells provoking adequate adoptive responses. Recent studies have elucidated that Tpl2 is an indispensable signal transducer as an MAP3K family member in diverse signaling pathways that regulate cell proliferation, survival, and death. Since tumorigenesis results from dysregulation of cellular proliferation, differentiation, and apoptosis, Tpl2 participates in many decisive molecular processes of tumor development and progression. Moreover, Tpl2 is closely associated with cytokine release of inflammatory cells, which has crucial effects on not only tumor cells but also tumor microenvironments. These critical roles of Tpl2 in human cancers make it an attractive anti-cancer therapeutic target. However, Tpl2 contradictorily works as a tumor suppressor in some cancers. The double-sided effects of Tpl2 originate from the specific upstream and downstream signaling environment of each tumor, since Tpl2 interacts with various signaling components. This review summarizes recent studies concerning the possible roles of Tpl2 in human cancers and considers its possibility as a therapeutic target, against which novel anti-cancer agents could be developed.

Suppression of mitochondrial biogenesis through toll-like receptor 4-dependent mitogen-activated protein kinase kinase/extracellular signal-regulated kinase signaling in endotoxin-induced acute kidney injury.

Although disruption of mitochondrial homeostasis and biogenesis (MB) is a widely accepted pathophysiologic feature of sepsis-induced acute kidney injury (AKI), the molecular mechanisms responsible for this phenomenon are unknown. In this study, we examined the signaling pathways responsible for the suppression of MB in a mouse model of lipopolysaccharide (LPS)-induced AKI. Downregulation of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), a master regulator of MB, was noted at the mRNA level at 3 hours and protein level at 18 hours in the renal cortex, and was associated with loss of renal function after LPS treatment. LPS-mediated suppression of PGC-1α led to reduced expression of downstream regulators of MB and electron transport chain proteins along with a reduction in renal cortical mitochondrial DNA content. Mechanistically, Toll-like receptor 4 (TLR4) knockout mice were protected from renal injury and disruption of MB after LPS exposure. Immunoblot analysis revealed activation of tumor progression locus 2/mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (TPL-2/MEK/ERK) signaling in the renal cortex by LPS. Pharmacologic inhibition of MEK/ERK signaling attenuated renal dysfunction and loss of PGC-1α, and was associated with a reduction in proinflammatory cytokine (e.g., tumor necrosis factor-α [TNF-α], interleukin-1β) expression at 3 hours after LPS exposure. Neutralization of TNF-α also blocked PGC-1α suppression, but not renal dysfunction, after LPS-induced AKI. Finally, systemic administration of recombinant tumor necrosis factor-α alone was sufficient to produce AKI and disrupt mitochondrial homeostasis. These findings indicate an important role for the TLR4/MEK/ERK pathway in both LPS-induced renal dysfunction and suppression of MB. TLR4/MEK/ERK/TNF-α signaling may represent a novel therapeutic target to prevent mitochondrial dysfunction and AKI produced by sepsis.

Tumor Progression Locus 2-dependent Oxidative Burst Drives Phosphorylation of Extracellular Signal-regulated Kinase during TLR3 and 9 Signaling

Signal transduction via NFκB and MAP kinase cascades is a universal response initiated upon pathogen recognition by Toll-like receptors (TLRs). How activation of these divergent signaling pathways is integrated to dictate distinct immune responses to diverse pathogens is still incompletely understood. Herein, contrary to current perception, we demonstrate that a signaling pathway defined by the inhibitor of κB kinase β (IKKβ), MAP3 kinase tumor progression locus 2 (Tpl2/MAP3K8), and MAP kinase ERK is differentially activated by TLRs. TLRs 2, 4, and 7 directly activate this inflammatory axis, inducing immediate ERK phosphorylation and early TNFα secretion. In addition to TLR adaptor proteins, IKKβ-Tpl2-ERK activation by TLR4 is regulated by the TLR4 co-receptor CD14 and the tyrosine kinase Syk. Signals from TLRs 3 and 9 do not initiate early activation of IKKβ-Tpl2-ERK pathway but instead induce delayed, NADPH-oxidase-dependent ERK phosphorylation and TNFα secretion via autocrine reactive oxygen species signaling. Unexpectedly, Tpl2 is an essential regulator of ROS production during TLR signaling. Overall, our study reveals distinct mechanisms activating a common inflammatory signaling cascade and delineates differences in MyD88-dependent signaling between endosomal TLRs 7 and 9. These findings further confirm the importance of Tpl2 in innate host defense mechanisms and also enhance our understanding of how the immune system tailors pathogen-specific gene expression patterns.

Intestinal myofibroblast-specific Tpl2-Cox-2-PGE2 pathway links innate sensing to epithelial homeostasis.

Tumor progression locus-2 (Tpl2) kinase is a major inflammatory mediator in immune cell types recently found to be genetically associated with inflammatory bowel diseases (IBDs). Here we show that Tpl2 may exert a dominant homeostatic rather than inflammatory function in the intestine mediated specifically by subepithelial intestinal myofibroblasts (IMFs). Mice with complete or IMF-specific Tpl2 ablation are highly susceptible to epithelial injury-induced colitis showing impaired compensatory proliferation in crypts and extensive ulcerations without significant changes in inflammatory responses. Following epithelial injury, IMFs sense innate or inflammatory signals and activate, via Tpl2, the cyclooxygenase-2 (Cox-2)-prostaglandin E2 (PGE2) pathway, which we show here to be essential for the epithelial homeostatic response. Exogenous PGE2 administration rescues mice with complete or IMF-specific Tpl2 ablation from defects in crypt function and susceptibility to colitis. We also show that Tpl2 expression is decreased in IMFs isolated from the inflamed ileum of IBD patients indicating that Tpl2 function in IMFs may be highly relevant to human disease. The IMF-mediated mechanism we propose also involves the IBD-associated genes IL1R1, MAPK1, and the PGE2 receptor-encoding PTGER4. Our results establish a previously unidentified myofibroblast-specific innate pathway that regulates intestinal homeostasis and may underlie IBD susceptibility in humans.

Tpl2 induces castration resistant prostate cancer progression and metastasis.

Progression to metastatic castration resistant prostate cancer (CRPC) is the major lethal pathway of prostate cancer (PC). Herein, we demonstrated that tumor progression locus 2 (Tpl2) kinase is the fundamental molecule provoking progression and metastasis of CRPC. Tpl2 upregulates CXCR4 and focal adhesion kinase (FAK) to activate CXCL12/CXCR4 and FAK/Akt signalling pathway. Consequently, epithelial-mesenchymal transition (EMT) and stemness of androgen depletion independent (ADI) PC cells are induced, which is dependent on the kinase activity of Tpl2. In vitro, proliferation, clonogenicity, migration, invasion and chemoresistance of ADI PC cells were enhanced by Tpl2. In vivo, Tpl2 overexpression and downregulation showed significant stimulatory and inhibitory effects on tumorigenic and metastatic potential of ADI PC cells, respectively. Moreover, the prognostic effects of Tpl2 and expressional correlation between Tpl2 and EMT-related molecules/CXCR4 were validated in clinical PC databases. Since Tpl2 exerts metastatic progression promoting activities in CRPC, Tpl2 could serve as a novel therapeutic target for metastatic CRPC.

Physical and functional interaction of the TPL2 kinase with nucleophosmin

Tumor Progression Locus 2 (TPL2) is widely recognized as a cytoplasmic mitogen-activated protein 3 kinase with a prominent role in the regulation of inflammatory and oncogenic signal transduction. Herein we report that TPL2 may also operate in the nucleus as a physical and functional partner of nucleophosmin (NPM/B23), a major nucleolar phosphoprotein with diverse cellular activities linked to malignancy. We demonstrate that TPL2 mediates the phosphorylation of a fraction of NPM at threonine 199, an event required for its proteasomal degradation and maintenance of steady-state NPM levels. Upon exposure to ultraviolet C, Tpl2 is required for the translocation of de-phosphorylated NPM from the nucleolus to the nucleoplasm. NPM is an endogenous inhibitor of HDM2:p53 interaction and knockdown of TPL2 was found to result in reduced binding of NPM to HDM2, with concomitant defects in p53 accumulation following genotoxic or ribosomal stress. These findings expand our understanding of the function of TPL2 as a negative regulator of carcinogenesis by defining a nuclear role for this kinase in the topological sequestration of NPM, linking p53 signaling to the generation of threonine 199-phosphorylated NPM.

Genetic and pharmacologic inhibition of Tpl2 kinase is protective in a mouse model of ventilator-induced lung injury.

BackgroundMechanical stress induced by injurious ventilation leads to pro-inflammatory cytokine production and lung injury. The extracellular-signal-regulated-kinase, ERK1/2, participates in the signaling pathways activated upon mechanical stress in the lungs to promote the inflammatory response. Tumor progression locus 2 (Tpl2) is a MAP3kinase that activates ERK1/2 upon cytokine or TLR signaling, to induce pro-inflammatory cytokine production. The role of Tpl2 in lung inflammation, and specifically in the one caused by mechanical stress has not been investigated. The aim of the study was to examine if genetic or pharmacologic inhibition of Tpl2 could ameliorate ventilator-induced lung injury.MethodsAdult male wild-type and Tpl2-deficient mice were ventilated with normal or high tidal volume for 4 h. Additional wild-type mice were treated with a Tpl2 inhibitor either before or 30 min after initiation of high tidal ventilation. Non-ventilated mice of both genotypes served as controls. The development of lung injury was evaluated by measuring lung mechanics, arterial blood gases, concentrations of proteins, IL-6, and MIP-2 in bronchoalveolar lavage fluid (BALF) and by lung histology. Data were compared by Kruskal-Wallis non-parametric test and significance was defined as p < 0.05.ResultsMechanical ventilation with normal tidal volume induced a mild increase of IL-6 in BALF in both strains. High tidal volume ventilation induced lung injury in wild-type mice, characterized by decreased lung compliance, increased concentrations of proteins, IL-6 and MIP-2 in BALF, and inflammatory cell infiltration on histology. All indices of lung injury were ameliorated in Tpl2-deficient mice. Wild-type mice treated with the Tpl2 inhibitor, either prior of after the initiation of high tidal volume ventilation were protected from the development of lung injury, as indicated by preserved lung compliance and lower BALF concentrations of proteins and IL-6, than similarly ventilated, untreated wild-type mice.ConclusionsGenetic and pharmacologic inhibition of Tpl2 is protective in a mouse model of ventilator-induced lung injury, ameliorating both high-permeability pulmonary edema and lung inflammation.

Tumor Progression Locus 2 (Tpl2) Kinase Promotes Chemokine Receptor Expression and Macrophage Migration during Acute Inflammation

In autoimmune diseases, the accumulation of activated leukocytes correlates with inflammation and disease progression, and, therefore, the disruption of leukocyte trafficking is an active area of research. The serine/threonine protein kinase Tpl2 (MAP3K8) regulates leukocyte inflammatory responses and is also being investigated for therapeutic inhibition during autoimmunity. Here we addressed the contribution of Tpl2 to the regulation of macrophage chemokine receptor expression and migration in vivo using a mouse model of Tpl2 ablation. LPS stimulation of bone marrow-derived macrophages induced early CCR1 chemokine receptor expression but repressed CCR2 and CCR5 expression. Notably, early induction of CCR1 expression by LPS was dependent upon a signaling pathway involving Tpl2, PI3K, and ERK. On the contrary, Tpl2 was required to maintain the basal expression of CCR2 and CCR5 as well as to stabilize CCR5 mRNA expression. Consistent with impairments in chemokine receptor expression, tpl2(-/-) macrophages were defective in trafficking to the peritoneal cavity following thioglycollate-induced inflammation. Overall, this study demonstrates a Tpl2-dependent mechanism for macrophage expression of select chemokine receptors and provides further insight into how Tpl2 inhibition may be used therapeutically to disrupt inflammatory networks in vivo.

Regulation of experimental autoimmune encephalomyelitis by TPL-2 kinase.

Tumor progression locus 2 (TPL-2) expression is required for efficient polarization of naive T cells to Th1 effector cells in vitro, as well as for Th1-mediated immune responses. In the present study, we investigated the potential role of TPL-2 in Th17 cells. TPL-2 was found to be dispensable for Th17 cell differentiation in vitro, and for the initial priming of Th17 cells in experimental autoimmune encephalomyelitis (EAE), a Th17 cell-mediated disease model for multiple sclerosis. Nevertheless, TPL-2-deficient mice were protected from EAE, which correlated with reduced immune cell infiltration, demyelination, and axonal damage in the CNS. Adoptive transfer experiments demonstrated that there was no T cell-intrinsic function for TPL-2 in EAE, and that TPL-2 signaling was not required in radiation-sensitive hematopoietic cells. Rather, TPL-2 signaling in radiation-resistant stromal cells promoted the effector phase of the disease. Importantly, using a newly generated mouse strain expressing a kinase-inactive form of TPL-2, we demonstrated that stimulation of EAE was dependent on the catalytic activity of TPL-2 and not its adaptor function to stabilize the associated ubiquitin-binding protein ABIN-2. Our data therefore raise the possibility that small molecule inhibitors of TPL-2 may be beneficial in multiple sclerosis therapy.

The biology of A20-binding inhibitors of NF-kappaB activation (ABINs).

The family of A20-Binding Inhibitors of NF-kappaB (ABINs) consists of three proteins, ABIN-1, ABIN-2 and ABIN-3, which were originally identified as A20-binding proteins and inhibitors of cytokines and Lipopolysaccharide (LPS) induced NF-kappaB activation. ABIN family members have limited sequence homology in a number of short regions that mediate A20-binding, ubiquitin-binding, and NF-kappaB inhibition. The functional role of A20 binding to ABINs remains unclear, although an adaptor function has been suggested. ABIN-1 and ABIN-3 expression is upregulated when cells are triggered by NF-kappaB-activating stimuli, suggesting a role for these ABINs in a negative feedback regulation of NF-kappaB signaling. Additional ABIN functions have been reported such as inhibition of TNF-induced hepatocyte apoptosis, regulation of HIV-1 replication for ABIN-1, and Tumor Progression Locus 2 (TPL-2)-mediated Extracellular signal-Regulated Kinase (ERK) activation for ABIN-2. In mice, ABIN-1 overexpression reduces allergic airway inflammation and TNF-mediated liver injury, ABIN-2 overexpression delays liver regeneration, and ABIN-3 overexpression partially protects against LPS-induced acute liver failure. Analysis of mice deficient in ABIN-1 or ABIN-2 demonstrates the important immune regulatory function of ABINs. Future studies should clarify the functional implication of the A20-ABIN interaction in supporting ABINs' mechanisms of action.

Implication of the Tpl2 Kinase in Inflammatory Changes and Insulin Resistance Induced by the Interaction Between Adipocytes and Macrophages

Adipose tissue inflammation is associated with the development of insulin resistance. In obese adipose tissue, lipopolysaccharides (LPSs) and saturated fatty acids trigger inflammatory factors that mediate a paracrine loop between adipocytes and macrophages. However, the inflammatory signaling proteins underlying this cross talk remain to be identified. The mitogen-activated protein kinase kinase kinase tumor progression locus 2 (Tpl2) is activated by inflammatory stimuli, including LPS, and its expression is up-regulated in obese adipose tissue, but its role in the interaction between adipocytes and macrophages remains ill-defined. To assess the implication of Tpl2 in the cross talk between these 2 cell types, we used coculture system and conditioned medium (CM) from macrophages. Pharmacological inhibition of Tpl2 in the coculture markedly reduced lipolysis and cytokine production and prevented the decrease in adipocyte insulin signaling. Tpl2 knockdown in cocultured adipocytes reduced lipolysis but had a weak effect on cytokine production and did not prevent the alteration of insulin signaling. By contrast, Tpl2 silencing in cocultured macrophages resulted in a marked inhibition of cytokine production and prevented the alteration of adipocyte insulin signaling. Further, when Tpl2 was inhibited in LPS-activated macrophages, the produced CM did not alter adipocyte insulin signaling and did not induce an inflammatory response in adipocytes. By contrast, Tpl2 silencing in adipocytes did not prevent the deleterious effects of a CM from LPS-activated macrophages. Together, these data establish that Tpl2, mainly in macrophages, is involved in the cross talk between adipocytes and macrophages that promotes inflammatory changes and alteration of insulin signaling in adipocytes.

The role of tumor progression locus 2 protein kinase in glial inflammatory response

Tumor progression locus 2 (Tpl2)/cancer Osaka thyroid kinase is a newer member of MAP3K family that is now known for its essential role in tumor necrosis factor-aplha (TNFα) expression in macrophages, but its pro-inflammatory signaling, if any, in glia is unknown. When cultures of murine microglia and astrocytes were exposed to lipopolysaccharide, there was a rapid activation (i.e., phosphorylation) of Tpl2 in parallel to the activation of down-stream effector MAPKs, that is, extracellular signal regulated kinase (ERK), p38 MAPK and C-Jun N-terminal kinase (JNK). Pre-incubation of the cultures with a Tpl2 inhibitor selectively suppressed the activation of the primary down-stream target, that is, ERK relative to p38 MAPK and JNK. That Tpl2 activation was functionally involved in glial inflammatory response was indicated by a reduced release of the cytokines, i.e. TNFα and the expression of inducible nitric oxide synthase in the presence of the kinase inhibitor. Furthermore, over-expression of a wild-type Tpl2 construct in C-6 glia resulted in an enhanced transcriptional activation of inducible nitric oxide synthase, while transfection with a dominant negative form of Tpl-2 had the opposite effect. The findings assign an important pro-inflammatory signaling function for Tpl2 pathway in glial cells.

Benzo[a]pyrene‐7,8‐diol‐9,10‐epoxide inhibits gap junction intercellular communication via phosphorylation of tumor progression locus 2 in WB‐F344 rat liver epithelial cells

Benzo[a]pyrene-7,8-diol-9,10-epoxide (B[a]PDE), a major metabolite of benzo[a]pyrene, has been reported to function as a human carcinogen. However, the molecular mechanism of how B[a]PDE regulates signaling pathways during tumor promotion remains unclear. In this study, we investigated the effects of B[a]PDE on the regulation of gap junction intercellular communication (GJIC), one of the major carcinogenic processes, and its main regulatory signaling pathways using WB-F344 rat liver epithelial (WB-F344 RLE) cells. Treatment of benzo[a]pyrene or B[a]PDE resulted in GJIC inhibition, and B[a]PDE was more active at lower concentrations than benzo[a]pyrene in the suppression of GJIC. This suggests that B[a]PDE is a stronger GJIC inhibitor. B[a]PDE at 1 µM reversibly inhibited GJIC in WB-F344 RLE cells, which was attributable to hyperphosphorylation of connexin43 (Cx43) via phosphorylation of mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK) and extracellular signal-regulated kinase (ERK). We found that B[a]PDE induced phosphorylation of tumor progression locus 2 (Tpl2), a direct upstream regulator of MEK. Tpl2 inhibitor recovered B[a]PDE-induced GJIC inhibition and attenuated B[a]PDE-induced MEK/ERK phosphorylation in WB-F344 RLE cells. Collectively, our results suggest that B[a]PDE suppresses GJIC by activating Tpl2 and subsequently the MEK/ERK pathway and Cx43 phosphorylation in WB-F344 RLE cells. These results outline the potential importance of Tpl2 as a novel therapeutic target for B[a]PDE-induced GJIC inhibition during cancer promotion.

Tpl2 Kinase Impacts Tumor Growth and Metastasis of Clear Cell Renal Cell Carcinoma

Due to the innate high metastatic ability of renal cell carcinoma (RCC), many patients with RCC experience local or systemic relapses after surgical resection. A deeper understanding of the molecular pathogenesis underlying advanced RCC is essential for novel innovative therapeutics. Tumor progression locus 2 (Tpl2), upregulated in various tumor types, has been reported to be associated with oncogenesis and metastatic progression via activation of the MAPK signaling pathway. Herein, the relevance of Tpl2 in tumor growth and metastasis of RCC is explored. Inspection of The Cancer Genome Atlas (TCGA) indicated that Tpl2 overexpression was significantly related to the presence of metastases and poor outcome in clear cell RCC (ccRCC), which is the most aggressive subtype of RCC. Moreover, expression of Tpl2 and CXCR4 showed a positive correlation in ccRCC patients. Depletion of Tpl2 by RNAi or activity by a Tpl2 kinase inhibitor in human ccRCC cells remarkably suppressed MAPK pathways and impaired in vitro cell proliferation, clonogenicity, anoikis resistance, migration, and invasion capabilities. Similarly, orthotopic xenograft growth and lung metastasis were significantly inhibited by Tpl2 silencing. Furthermore, Tpl2 knockdown reduced CXCL12-directed chemotaxis and chemoinvasion accompanied with impaired downstream signaling, indicating potential involvement of Tpl2 in CXCR4-mediated metastasis. Taken together, these data indicate that Tpl2 kinase is associated with and contributes to disease progression of ccRCC. Tpl2 kinase activity has prognostic and therapeutic targeting potential in aggressive clear cell renal cell carcinoma.

Tpl2 knockout keratinocytes have increased biomarkers for invasion and metastasis

Skin cancer is the most common form of cancer in the USA, with an estimated two million cases diagnosed annually. Tumor progression locus 2 (Tpl2), also known as MAP3K8, is a serine/threonine protein kinase in the mitogen-activated protein kinase signal transduction cascade. Tpl2 was identified by our laboratory as having a tumor suppressor function in skin carcinogenesis, with the absence of this gene contributing to heightened inflammation and increased skin carcinogenesis. In this study, we used gene expression profiling to compare expression levels between Tpl2 (+/+) and Tpl2 (-) (/-) keratinocytes. We identified over 2000 genes as being differentially expressed between genotypes. Functional annotation analysis identified cancer, cell growth/proliferation, cell death, cell development, cell movement and cell signaling as the top biological processes to be differentially regulated between genotypes. Further microarray analysis identified several candidate genes, including Mmp1b, Mmp2, Mmp9 and Mmp13, involved in migration and invasion to be upregulated in Tpl2 (-) (/-) keratinocytes. Moreover, Tpl2 (-/-) keratinocytes had a significant downregulation in the matrix metalloproteinase (MMP) inhibitor Timp3. Real-time PCR validated the upregulation of the MMPs in Tpl2 (-/-) keratinocytes and zymography confirmed that MMP2 and MMP9 activity was higher in conditioned media from Tpl2 (-/-) keratinocytes. Immunohistochemistry confirmed higher MMP9 staining in 12-O-tetradecanoylphorbol-13-acetate-treated skin from Tpl2 (-/-) mice and grafted tumors formed from v-ras(Ha) retrovirus-infected Tpl2 (-/-) keratinocytes. Additionally, Tpl2 (-/-) keratinocytes had significantly higher invasion, malignant conversion rates and increased endothelial cell tube formation when compared with Tpl2 (+/+) keratinocytes. In summary, our studies reveal that keratinocytes from Tpl2 (-/-) mice demonstrate a higher potential to be invasive and metastatic.

Tpl2 Inhibitors Thwart Endothelial Cell Function in Angiogenesis and Peritoneal Dissemination

Angiogenesis is critical in the development of cancer, which involves several angiogenic factors in its peritoneal dissemination. The role of protein tumor progression locus 2 (Tpl2) in angiogenic factor-related endothelial cell angiogenesis is still unclear. To understand the precise mechanism(s) of Tpl2 inhibition in endothelial cells, this study investigated the role of Tpl2 in mediating angiogenic signals using in vitro, in vivo, and ex vivo models. Results showed that inhibition of Tpl2 inhibitor significantly reduced peritoneal dissemination in a mouse model by positron emission tomography/computed tomography imaging. Simultaneously, inhibiting Tpl2 blocked angiogenesis in tumor nodules and prevented angiogenic factor-induced proliferating cell nuclear antigen (PCNA) in endothelial cells. Vascular endothelial growth factor (VEGF) or chemokine (C-X-C motif) ligand 1 (CXCL1) increased Tpl2 kinase activity and phosphorylation in a dose- and time-dependent manner. Furthermore, Tpl2 inhibition or ablation by siRNA prevented the angiogenic signal-induced tube formation in Matrigel plug assay or aortic ring assay. Inhibiting Tpl2 also prevented the angiogenic factor-induced chemotactic motility and migration of endothelial cells. Tpl2 inhibition by CXCL1 or epidermal growth factor in endothelial cells was associated with inactivation of CCAAT/enhancer binding protein β, nuclear factor κ light-chain enhancer of activated B cells, and activating protein 1 and suppression of VEGF expression. Thus, Tpl2 inhibitors thwart Tpl2-regulated VEGF by inactivating transcription factors involved in angiogenic factor-triggered endothelial cell angiogenesis. These results suggest that the therapeutic inhibition of Tpl2 may extend beyond cancer and include the treatment of other diseases involving pathologic angiogenesis.

Honokiol thwarts gastric tumor growth and peritoneal dissemination by inhibiting Tpl2 in an orthotopic model

Honokiol is known to suppress the growth of cancer cells; however, to date, its antiperitoneal dissemination effects have not been studied in an orthotopic mouse model. In the present study, we evaluated the antiperitoneal dissemination potential of Honokiol in an orthotopic mouse model and assessed associations with tumor growth factor-β1 (TGFβ1) and cells stimulated by a carcinogen, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Our results demonstrate that tumor growth, peritoneal dissemination and peritoneum or organ metastasis of orthotopically implanted MKN45 cells were significantly decreased in Honokiol-treated mice and that endoplasmic reticulum (ER) stress was induced. Honokiol-treated tumors showed increased epithelial signatures such as E-cadherin, cytokeratin-18 and ER stress marker. In contrast, decreased expression of vimentin, Snail and tumor progression locus 2 (Tpl2) was also noted. TGFβ1 and MNNG-induced downregulation of E-cadherin and upregulation of Tpl2 were abrogated by Honokiol treatment. The effect of Tpl2 inhibition in cancer cells or endothelial cells was associated with inactivation of CCAAT/enhancer binding protein B, nuclear factor kappa-light-chain-enhancer of activated B cell and activator protein-1 and suppression of vascular endothelial growth factor. Inhibition of Tpl2 in gastric cancer cells by small interfering RNA or pharmacological inhibitor was found to effectively reduce growth ability and vessel density in vivo. Honokiol-induced reversal of epithelial-to-mesenchymal transition (EMT) and ER stress-induced apoptosis via Tp12 may involve the paralleling processes. Taken together, our results suggest that the therapeutic inhibition of Tpl2 by Honokiol thwarts both gastric tumor growth and peritoneal dissemination by inducing ER stress and inhibiting EMT.

Ebola virus VP35 induces high-level production of recombinant TPL-2–ABIN-2–NF-κB1 p105 complex in co-transfected HEK-293 cells

Activation of PKR (double-stranded-RNA-dependent protein kinase) by DNA plasmids decreases translation, and limits the amount of recombinant protein produced by transiently transfected HEK (human embryonic kidney)-293 cells. Co-expression with Ebola virus VP35 (virus protein 35), which blocked plasmid activation of PKR, substantially increased production of recombinant TPL-2 (tumour progression locus 2)–ABIN-2 [A20-binding inhibitor of NF-κB (nuclear factor κB) 2]–NF-κB1 p105 complex. VP35 also increased expression of other co-transfected proteins, suggesting that VP35 could be employed generally to boost recombinant protein production by HEK-293 cells.

Abstract 1422: Interleukin-17A activates tumor progression locus 2 to induce neoplastic cell transformation and breast carcinogenesis.

Abstract Interleukin-17 (IL-17) is a pro-inflammatory cytokine secreted by activated CD4+ memory T cells. Although the role of T cells in cancer promotion has been examined, little is known about the underlying molecular mechanisms of interleukin-17 A (IL-17A) in carcinogenesis. In the present study, we first found that IL-17A induces neoplastic transformation of JB6 Cl41 cells through activation of tumor progression locus 2 (TPL2). IL-17A activates MEKs-ERKs, JNKs-c-Jun, and STAT3 signaling pathways, which are inhibited by a TPL2 kinase inhibitor (TKI). Furthermore, IL-17A activates c-fos and c-jun promoter activity, resulting in increased activator protein-1 (AP-1) activity. When small interfering RNA (siRNA) of IL-17A receptor (IL-17R), IL-17A, and TPL2 were introduced into JB6 Cl41 cells, respectively, IL-17A-induced AP-1 activity was significantly decreased compared to control cells. Similarly, TPL2 inhibition suppressed AP-1 activity induced by IL-17A. The knockdown of IL-17R and TKI treatment in JB6 Cl41 cells resulted in decreased IL-17A-induced cell transformation. The in vivo chorioallantoic membrane (CAM) assay also showed that IL-17A increased tumor formation of JB6 Cl41 cells, whereas TKI inhibited the tumorigenesis promoted by IL-17A. Consistent with these observations, knockdown of IL-17A and/or inhibition of TPL2 attenuated tumorigenecity of human breast cancer MCF7 cells. Moreover, immunohistochemical staining showed that both expressions of IL-17A and TPL2 were highly upregulated in human breast cancers. Together, our findings point to a critical role for the IL-17A-induced TPL2 signaling pathway in supporting cancer-associated inflammation in the tumor microenvironment. Therapeutic approaches that target this pathway may therefore effectively inhibit carcinogenesis. Citation Format: Garam Kim, Hong Seok Choi. Interleukin-17A activates tumor progression locus 2 to induce neoplastic cell transformation and breast carcinogenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1422. doi:10.1158/1538-7445.AM2013-1422 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.