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.

An important aspect of Tpl2 activation is its interaction with upstream components of the nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) pathway, such as the inhibitor of κ B (IκB) kinase β (IKKβ/IKK2) [27][28][29]. In the steady state, the entire pool of Tpl2 is held in complex with ABIN2 and p105. This interaction stabilizes Tpl2 but also prevents Tpl2 and NF-κB from activating their downstream signaling cascades by inhibiting the kinase activity of Tpl2 and the proteolysis of NF-κB precursor protein p105 [28,30,31]. Upon activation of Tpl2 by various stimuli, IKK phosphorylates p105/NF-κB1 at Ser927 and Ser932, which flags p105 for proteasomal degradation and releases Tpl2 from the complex [32,33]. The newly liberated Tpl2 phosphorylates its substrates but is unstable to be targeted for proteasome-mediated degradation, thus restricting prolonged activation of Tpl2 and its downstream signaling pathways [31,34]. Furthermore, dissociated p105/NF-κB1 is proteolysed into p50, which dimerizes with other NF-κB family members, translocates into the nucleus, and regulates the transcription of over 400 genes [6,30].
There are two Tpl2 isoforms; 58 kDa (Tpl2 long) and 52 kDa (Tpl2 short) [6]. Three ATGs at the 5' end of Tpl2 mRNA give rise to 467-, 464-, and 438-a.a. proteins. The 467-and 464-a.a. proteins are detected as a 58-kDa band (p58), whereas the 438-a.a. protein is detected as a 52-kDa (p52) band in western blot analysis [14]. Although the p58 and p52 isoforms are expressed at similar levels in most tissue types, the 58-kDa isoform is predominant when Tpl2 is overexpressed in 293 cells. A recent study has shown that phosphorylation at Thr-290 plays an obligatory role in Tpl2 activation by external signals [31,35-37]. Both p52 and p58 isoforms of Tpl2 form a stable complex with p105 in the resting state. However, the p58 isoform is preferentially phosphorylated at Thr-290 by stimulation, which is more efficient when p58 is binding to p52 [34]. Furthermore, the p58 isoform is released from p105 preferentially upon stimulation compared with the p52 isoform [34]. The released p58 is active but undergoes rapid proteasome-mediated degradation, which is dependent on the phosphorylation at Thr-290 [34].

Tpl2 Kinase-Mediated Downstream Signal Transduction
There are four major MAPKs in mammals; extracellular signal-regulated kinases (ERKs), extracellular signal-regulated kinase 5 (ERK5), c-Jun N-terminal kinases (JNKs), and p38 MAPKs ( Figure 1) [3,4]. In general, growth factors, cellular stresses, and either growth factors or stresses are considered as the main activator of ERK1/2, JNK/p38-MAPKs, and ERK5 cascade, respectively. However, accumulated evidence has suggested cross talks between various components of the pathways. These multiple interactions between the different MAPK cascades serve to provoke more diverse and precise cellular responses to various intra-and extra-cellular stimuli [5,38].
The innate immune response, the first line of defense against infections, is orchestrated by macrophages, dendritic cells, natural killer cells, and neutrophils. The crucial initial step to provoke the innate immune response is the detection of pathogen-associated molecules by TLRs. TLRs activate the Tpl2-MEK-ERK pathway in macrophages, which regulates not only the production of cytokines but also the cellular responses to TNF, IL-1β, and CD40 ligand [60][61][62][63][64]. Production of prostaglandin E2 (PGE2) and its regulatory enzyme, COX-2, in monocytes are also regulated by the Tpl2-ERK-mitogen-and stress-activated protein kinase-1 (MSK1) pathway [65,66]. Furthermore, Tpl2 is required for the optimal induction of p38 MAPK in the stimulation of bone marrow-derived dendritic cells by LPS or CpG [43,67]. This suggests that Tpl2 transduces a broad inflammatory signal in innate immune cells.
In the adaptive immune system, Tpl2 is expressed in B and T lymphocytes [14,22,50]. The CD40-Tpl2-ERK signaling pathway mediates immunoglobulin isotype switching in B cells [14], insensitivity to LPS-mediated B cell apoptosis [59], and IL-12-mediated helper T cell differentiation [22,38]. For example, in a TNF-driven Crohn's-like inflammatory bowel disease mouse model, the absence of Tpl2 reduced numbers of memory CD4 + and peripheral CD8 + lymphocytes and ameliorated the onset and progression of the disease [68].
Although previous studies using mouse inflammatory disease models suggest that Tpl2 has predominantly pro-inflammatory functions, in some circumstances, Tpl2 inhibits pro-inflammatory cytokines and functions in an anti-inflammatory manner [38,42]. For example, Tpl2 deficiency resulted in over-production of pro-inflammatory cytokines [43] and reduced synthesis of an anti-inflammatory cytokine, IL-10 [69]. In a mouse model of ovalbumin (OVA)-induced bronchoalveolar inflammation, Tpl2 ablation led to increase in both OVA-specific and total IgE and shifted the balance of sytokine production toward the Th2 cytokines [44]. Those contradictory roles of Tpl2 in the immune system indicate that Tpl2 can have ambivalent effects on cancer development and progression in a cell-type-specific manner.

Tpl2 in the Development and Progression of Human Cancers
The MAPK signaling pathway including Tpl2 regulates the development and progression of cancers [70]. Since Tpl2 interacts with many upstream and downstream signaling components, the roles of Tpl2 in human cancers is complex (Figure 2). Either over-expression or reduced-expression of this gene can promote tumorigenesis depending on cancer types [6] ( Table 1). The complexity might originate from the specific extra-and intra-cellular signaling context of each human cancer.
Tpl2 conveys various oncogenic signals in a variety of human solid tumors. Tpl2 expression is up-regulated in approximately 40% of human breast cancer specimens [62,64]. Since Tpl2 gene amplification was detected in eight out of the Tpl2-over-expressing 14 breast cancer specimens, increased number of Tpl2 gene could be a possible mechanism for Tpl2 over-expression [64]. Tpl2 directly interacts with Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1) and induces the phosphorylation of Pin1 on Ser16, which results in cyclin D1 up-regulation and breast cancer development [78]. In addition, interleukin-22 (IL-22) phosphorylates Tpl2 through IL-22R1, activates the MEK-ERK, JNK, and STAT3 signaling pathways, and results in the epithelial-mesenchymal transition (EMT) through AP-1 in breast cancer [79].
There is considerable evidence on the role of Tpl2 in cancer metastasis. Proteinase-activated receptor 1 (PAR1) activated by several proteinases including thrombin and matrix metalloproteinase-1 (MMP-1) promotes cell transformation, tumor metastasis, and angiogenesis in a variety of cancers including prostate cancer and melanoma [101][102][103][104]. Tpl2 transduces the PAR1 signals to regulate the expression of MMPs and other secreted molecules both in fibroblasts and tumor cells [74]. Tpl2 is also required for PAR1 to engage a Rac1 and focal adhesion kinase (FAK)-dependent pathway, to activate ERK and JNK1, and to promote reorganization of the actin cytoskeleton and cell migration [74,83]. Pancreatic adenocarcinoma upregulated factor (PAUF), an endogenous ligand of TLR2 and TLR4, is overproduced in certain types of cancer including pancreatic cancers [105]. PAUF activatesTLR2-mediated TPL2-MEK-ERK signaling pathway to increase expression of pro-tumorigenic cytokines, but inhibits TLR-mediated NF-κB signaling, thereby facilitating tumor growth and escape from innate immune surveillance [80].
Peritoneal dissemination of cancer cells is closely associated with poor oncologic outcomes [65][66][67]106]. Angiogenic factors including vascular endothelial growth factor (VEGF) and chemokine (C-X-C motif) ligand 1 (CXCL1) are involved in this process [107][108][109][110]. VEGF and CXCL1 increase phosphorylation and kinase activity of Tpl2 in a dose-and time-dependent manner [81]. For example, in gastric cancer, Tpl2 inhibition significantly induced endoplasmic reticulum stress, inhibited EMT, and reduced peritoneal dissemination of cancer cells, which was accompanied by angiogenesis blockage [81,82]. These findings suggest that Tpl2 inhibitors could lead to the development of novel simultaneous anti-angiogenic and anti-metastatic treatment strategies.
Recently, we further elucidated a novel function of Tpl2 kinase in the disease progression of genito-urinary cancers [94,95]. In clear cell renal cell carcinoma (ccRCC) with innate high metastatic ability, Tpl2 mRNA levels are significantly elevated compared with normal kidneys [6]. Its downstream signaling components, MKK and ERK, were also activated in human RCC cases [111,112]. In our study, we detected that up-regulation of Tpl2 is significantly associated with the presence of metastases and poor clinical outcomes in human ccRCCs [84]. Moreover, elevated Tpl2 activity enhanced tumorigenic and metastatic potential of ccRCC cells significantly in preclinical ccRCC models through activation of the MAPK signaling and cross talk with the CXCL12-CXCR4-directed chemotaxis and chemoinvasion [84].
The expression of TLR4 is closely associated with the severity of prostate cancer [113]. Tpl2 has an important role in the downstream MAPK signaling of TLRs under acute or chronic inflammatory conditions [14,50,55,58,59]. Chronic prostatic inflammation is a major inducing factor of prostate cancer, which supports the possible connection between the Tpl2 signaling pathway and the development of prostate cancer [114]. Importantly, Tpl2 was over-expressed in human castration resistant prostate cancer (CRPC) and Tpl2 drove CRPC growth through the MEK-ERK and NF-κB signaling pathway [85]. We further demonstrated that Tpl2 induces EMT and maintains stemness of CRPC cells, which increase proliferation, clonogenic, migration, and invasion abilities of CRPC cells significantly [86]. Although more detailed studies are necessary to elucidate the detailed mechanism, we found that FAK-Akt and CXCL12-CXCR4 axis may be alternative downstream signaling pathways activated by Tpl2 kinase in CRPC cells, through previous study [86].

Tpl2 as a Key Player in Inflammatory Cancer Microenvironment
Cancer-related inflammation aids proliferation and survival of malignant cells, stimulates angiogenesis and metastasis, inhibits adaptive immunity to tumor cells, and alters tumor-responses to anti-cancer therapies [115][116][117]. Activation of oncogenes or inactivation of tumor suppressor genes leads to alterations in nearby inflammatory cytokine network, which is of great importance in the processes of cancer-related inflammation [118]. Given the impact of Tpl2 on the innate and acquired immunity, Tpl2 could contribute to cancer progression and metastasis via tumor-associated inflammatory response.

Tpl2 as a Tumor-Suppressor Gene
In contrast with the strong evidence associated with the oncogenic roles of Tpl2, under certain conditions, Tpl2 may serve tumor suppressive roles. In one study, Tpl2 knockout (Tpl2 −/− ) mice, when crossed with mice with a T cell receptor transgene that provokes T cell lymphoma, showed a higher incidence of the tumor [75]. Moreover, the Tpl2 −/− mice had a significantly higher incidence of tumor initiation and faster malignant progression in a chemical-induction mouse skin cancer model [94,95]. Mechanistically, oncogenic effects of Tpl2 ablation were mediated by increased NF-κB activity, which ultimately induced both skin tumorigenesis and inflammation [95]. Comparative gene expression profiling between Tpl2 +/+ and Tpl2 −/− mice further demonstrated that MMP1b/2/9/13 that stimulate the migration and invasion of cancer cells are up-regulated in Tpl2 −/− keratinocytes [96].
Tpl2 may have a broader role in dictating the balance between cell survival and death. Tumor protein p53 has a crucial role in tumor suppression, in part by regulating apoptosis. In breast cancer cells, reduced Tpl2 expression was associated with resistance to TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis, a p53-independent process [97]. Tpl2 utilizes similar mechanisms in lung carcinogenesis and meets the requirement for a suppressor gene [98]. Low Tpl2 levels were correlated with reduced lung cancer patient survival and accelerated onset and multiplicity of urethane-induced lung tumors in mice. Tpl2 was found to antagonize oncogene-induced cell transformation and survival through JNK dependent up-regulation of nucleophosmin (NPM), which is required for the optimal p53 response to oncogenic or genotoxic stresses [98]. Collectively, these data indicate that Tpl2 is a positive regulator of the p53 pathway in human lung cancer.
Tpl2 ablation also promoted intestinal inflammation through down-regulation of IL-10 levels and regulatory T-cell numbers in the intestinal mucosa of Tpl2 −/− mice. The inflammation was responsible for the tumorigenesis of colitis-associated cancer [69]. In addition, cell-specific ablation of Tpl2 in intestinal myofibroblasts (IMFs) developed significantly increased numbers and sizes of colon cancers, which were associated with enhanced epithelial proliferation and decreased apoptosis [99]. Tpl2-deficient IMFs up-regulated HGF production and became less sensitive to the negative feedback by TGF-β3. These results indicate that Tpl2 normally suppresses colon epithelial tumorigenesis through the negative regulation of the HGF-c-Met pathway.
Finally, Tpl2 participates as an upstream of the MEK-ERK pathway in the positive regulation effects of IL-12 on the functions of human effector memory CD8 + cytotoxic T lymphocytes, which plays a major role in adaptive anti-tumor immune responses [100].
Given the important roles of Tpl2 in tumorigenesis, metastasis, and cancer-related neo-angiogenesis, Tpl2-targeting agents may also provide novel insights into potential anti-cancer therapeutics strategies in several cancers in which Tpl2 plays an important role as a tumor promoting gene [134]. Moreover, Tpl2 acts on both tumor cells and inflammatory tumor microenvironments through diverse signaling pathways including the MAPK cascades, which would offer multi-modal therapeutic mechanisms. Besides the MAPK pathway, TNFα-Tpl2 mediated pathways could be additional therapeutic targets for developing anti-tumor agents since TNFα-mediated COX2 expression plays an important role in inflammation and carcinogenesis. For example, luteolin (2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4-chromenone), which inhibits Tpl2 activity by direct binding in an ATP-competitive manner, primarily targeted Tpl2 involved in cancer-associated inflammation and exerted potent anti-tumor activities [145]. Luteolin attenuated TNFα-induced COX-2 expression by down-regulating the transactivation of NF-κB and AP-1 [145].
However, any therapeutic use of kinase inhibitors should consider the cost-benefit ratio carefully. Although small molecule inhibitors exist for the targeting of the Tpl2-MEK-ERK pathway, side effects could be a serious issue. Consistent with the diminished chemokine receptor expression levels in both resting and activated Tpl2 −/− macrophages, Tpl2 ablation resulted in impaired in vivo macrophage recruitment to tissue sites of inflammation [146]. Although these findings provided scientific bases of how Tpl2 inhibition could provide an alternative treatment for a variety of autoimmune diseases, the physiological effect of Tpl2 ablation could significantly impair the immunologic responses of cancer patients to a variety of infections as well as to mutated cancer antigens. Furthermore, HGF deregulation has been causally associated with tumorigenesis in breast, skin, stomach, prostate, skin, and lung [147,148]. Since fibroblast-specific HGF up-regulation by Tpl2 inhibition was reported in colorectal carcinogenesis [99], potentially enhanced carcinogenesis by activation of HGF-c-Met signaling pathways need to be exercised in the future clinical use of Tpl2 inhibitors in chronic inflammatory diseases [149]. Unfortunately, the role of Tpl2 in tumorigenesis is complex, as either over-expression or reduced-expression can promote tumor formation depending on the cancer type [6]. Therefore, Tpl2 inhibitors may paradoxically lead to the development of secondary malignancies by suppressing anti-tumorigenic mechanisms of Tpl2 kinase in some tissues including skin and lung [94,98].

Conclusions
Anti-cancer targeting agents have major advantages for clinical uses compared with conventional cytotoxic chemotherapeutic agents; cancer specific effects and less systemic toxicities. Since targeted therapeutic agents are developed against specific molecular targets, it is of crucial importance to elucidate optimal target candidates. Tpl2, a MAP3K, participates in a broad range of cancer-related signaling pathways and induces tumorigenesis and progression of many human cancers. Several Tpl2 inhibitors have been developed for immunological disorders; they could be tested for anti-cancer effects using various cancer in vitro and in vivo models preclinically. Given that Tpl2 signals are cell type-specific as well as stimulus-specific, Tpl2 could be a novel and attractive therapeutic target for many different malignancies with cancer-specific dependence toward Tpl2-mediated oncogenic pathways, such as breast, colorectal, gastric, prostate, and kidney cancer. However, due to the probability of secondary malignancies by Tpl2 targeting, clinical application of Tpl2 as a novel therapeutic target for advanced cancer patients needs to be further validated.