Tribbles in disease: Signaling pathways important for cellular function and neoplastic transformation

Abstract

The tribbles family of genes encodes pseudokinase proteins that are highly conserved in evolution. Instead of direct phosphorylation of target proteins, tribbles act as adaptors in signaling pathways for important cellular processes. These include mitogen-activated protein kinase kinase (MAPKK), CCAAT/enhancer-binding protein (C/EBP), activating transcription factor 4 (ATF4) and C/EBP-homologous protein (CHOP). Trib1 and Trib2 have been identified as myeloid oncogenes, and both may be involved in human leukemia. Tribbles proteins are also involved in a series of non-neoplastic disorders including metabolic and neurological diseases. The RAS/mitogen-activated protein kinase (MAPK) pathway molecules (in particular MAPK/ERK kinase 1 (MEK1) and C/EBP transcription factors) include tribbles-binding proteins that are involved in leukemogenesis, and the role of Trib1 as a linker between MAPK signaling and C/EBP degradation is proposed. Although the molecular function of tribbles is still under investigation, the research on tribbles in cellular processes, homeostasis of organisms and human diseases will provide valuable information for therapy of cancer as well as non-neoplastic disorders. (Cancer Sci 2011; 102: 1115–1122)

Intracellular signal transduction is important for regulating processes such as cell proliferation, survival and differentiation, and its dysregulated signaling is one of the most important hallmarks of cancer. Both gain-of-function and loss-of-function mutations within the receptor tyrosine kinase (RTK)/RAS/mitogen-activated protein kinase (MAPK) pathway have been identified in various human cancers.⁽¹⁾ In myeloid malignancies most mutations are found in RTK such as ABL1, PDGFR, FLT3 and c-KIT, resulting in ligand-independent activation. Mutations of downstream messengers or regulators including JAK2, SHP2, RAS and NF1 are less frequent but also induce significant dysfunction of the pathway (Fig. 1). As a result of mutations within the pathway, downstream molecules such as ERK, ELK, jun/fos and cyclin-dependent kinases (CDK) become constitutively activated, affecting proliferative activity and increasing survival potency of preneoplastic/neoplastic cells.

Figure 1.  The receptor tyrosine kinase (RTK)/RAS/mitogen-activated protein kinase (MAPK) pathway and Trib1. The molecules that are found mutated in human acute myeloid leukemia (AML) are indicated in red.

Dysregulation of transcriptional control is another important mechanism in carcinogenesis. Many transcription factors that control differentiation and/or lineage selection in both embryogenesis and adult tissues are mutated in human cancers. For example, CCAAT/enhancer-binding protein family proteins play key roles in differentiation of hematopoietic tissue, liver, breast and adipose tissue.⁽²⁾ Among these, C/EBPα is required for the development of granulocyte and it acts as an anti-proliferative molecule and a tumor suppressor in human leukemias.⁽³⁾ C/EBPα suppresses cell proliferation by stimulating p21, and by inhibiting CDK activity and E2F-mediated transcription. In human AML patients, loss-of-function mutations of C/EBPα are frequently observed.

The tribbles family of proteins are adaptors that link the MAPK pathway and C/EBP transcriptional regulation. This review describes the functions of tribbles and their regulation of important physiological conditions, and focuses on their role in cancer development and progression.

The tribbles family

The tribbles gene was first identified in Drosophila as a regulator of string/cdc25 in morphogenesis.⁽⁴⁾String activity is associated with cellular mitosis in early development of Drosophila except for a ventral furrow where mitosis is delayed compared with string RNA expression. Grosshans and Wieschaus⁽⁴⁾ predicted that negative regulators for string might be present and identified two genes, fruhstart and tribbles. They showed that overexpression of tribbles inhibited mitosis in Drosophila embryos. Mata et al.⁽⁵⁾ independently showed that tribbles induced degradation of string via the proteasome pathway. In addition, they demonstrated that overexpression of tribbles in imaginal disc cells blocked the cell cycle at G2, resulting in abnormal morphology of wings. Another group reached similar conclusions that tribbles acted as a mitotic inhibitor that blocked string at the post-translational level.⁽⁶⁾ In addition, it was proposed that snail and swift act as upstream factors of tribbles in mitotic regulation.^(4,7)

Mammalian homologues of tribbles have been identified as C5FW in dog, NIPK in rat or C8FW in humans.^(8–10) Currently, the mammalian tribbles family consists of three genes, Trib1/C8FW/SKIP1, Trib2/C5FW/SKIP2/SINK and Trib3/NIPK/SKIP3. The tribbles family of proteins is characterized by their central pseudokinase domain that is well conserved during evolution (Fig. 2A). The amino acid sequences of tribbles are highly conserved between human and mouse (amino acid identities: Trib1, 97.5%; Trib2, 99.2%; Trib3, 81.2%) (Fig. 2B), and high degrees of similarity are also noted within the human family of sequences (TRIB1/TRIB2, 71.3%; TRIB1/TRIB3, 53.3%; TRIB2/TRIB3, 53.7%). The pseudokinase domain of each tribbles consists of 12 subdomains that belong to the protein kinase superfamily.⁽¹¹⁾ While the amino acid sequences of those subdomains in each kinase family are mostly diverse, there are conserved amino acids/motifs that are required for enzymatic function. Moreover, the predicted 3-D structure of tribbles is consistent with that of a protein kinase (SWISS-MODEL, http://swissmodel.expasy.org/).⁽¹²⁾ However, several consensus sequences for protein phosphorylation are missing. The complete lack of GXGX₂GXV in subdomain I, the loss of histidine and asparagine in HRDLKX₂N in VIB, and lack of the DFG triplet in VII are important variations specific to tribbles (Fig. 2B). Given these sequence characteristics, it is believed that none of the tribbles possess kinase activity and experimental data are consistent with that interpretation.⁽¹²⁾

Figure 2.  The structure of tribbles proteins. (A) Tribbles consists of N-terminal, pseudokinase and C-terminal domains. The number of amino acids for each domain in Drosophila and mammals are indicated. (B) Amino acid sequences of the pseudokinase domain in human and murine tribbles. The conserved amino acid sequences that are characteristic to the kinase superfamily are indicated in red. (C) Three unique motifs: catalytic loop, MEK1 binding motif and COP1 binding motif.

The N-terminal regions are barely conserved in Drosophila and the three mammalian tribbles. A putative nuclear localization signal, [K/R]₂X₂[D/E]X[D/E], has been proposed by Hegedus et al.⁽¹²⁾ and it is well conserved in three mammalian tribbles, although the possibility remains to be confirmed experimentally. Two important motifs have been identified within the tribbles C-terminal domain. A hexapeptide motif [D/E]QXVP[D/E] was identified in Trib3 as an E3 ubiquitin ligase COP1 binding site (Fig. 2C),⁽¹³⁾ and all three members contain the motif. This motif is important for a core function of tribbles as proteasome-mediated degradation of the C/EBP family of proteins is achieved using the motif as described at the next chapter. Another important motif in the C-terminus is an MEK binding site that is conserved by all members (Fig. 2C).⁽¹⁴⁾ Proteins such as C/EBP transcription factors and retinoic acid receptors interact with tribbles at the pseudokinase domain (Yokoyama T and Nakamura T, unpublished data, 2011).⁽¹⁵⁾

Tribbles function as signaling modulators and mediators

Soon after discovery of tribbles in Drosophila, slbo, the Drosophila homologue of C/EBP, was found to be regulated by tribbles.⁽¹⁶⁾slbo is required for migration of border cells during Drosophila development, and protein expression of slbo is positively regulated by the ubiquitin hydrolase Ubp64 and negatively regulated by tribbles, strongly suggesting its role in ubiquitination. In mammalian cells tribbles homologues also negatively regulate the C/EBP family of transcription factors. Keeshan et al.⁽¹⁷⁾ observed that overexpression of Trib2 reduced C/EBPα protein in a proteasome-dependent manner, resulting in myeloid differentiation. We have shown that Trib1 accelerates degradation of C/EBPα in AML cells and this action is MEK-dependent.⁽¹⁴⁾ Moreover, Trib2 increases degradation of C/EBPβ in 3T3-L1 preadipocytes, suppressing adipocytic differentiation.⁽¹⁸⁾ Trib2 and Trib3 are rapidly downregulated during adipocytic differentiation of 3T3-L1 cells, and Trib2 (but not Trib3) enhances degradation of C/EBPβ. Interestingly, both Trib2 and Trib3 could inhibit adipocytic differentiation, and Trib3 suppresses AKT phosphorylation instead of downregulating C/EBPβ. Yamamoto et al.⁽¹⁹⁾ further observed interaction between C/EBPβ and Trib1, and upregulation of C/EBPβ in Trib1 knockout mice. These data suggest that both Trib1 and Trib2 could induce proteasomal degradation of C/EBP α and β.

By interacting with C/EBP-homologous protein (CHOP) (C/EBPζ), C/EBPβ is bound to the promoter region of Trib3 and upregulates its transcription,⁽²⁰⁾ suggesting there might be a regulatory loop between the tribbles family of proteins and C/EBP transcription factors.⁽²¹⁾ Ohoka et al.⁽²²⁾ showed that Trib3 interacts with CHOP but does not promote its degradation. However, the same group later identified the CHOP N-terminal domain that is required for its ubiquitination as well as p300 binding.⁽²³⁾ They found Trib3 interacts with the same region and competes with p300, suggesting that Trib3 might play some role in turnover and transcriptional activation of CHOP.

ATF4 is a downstream target of PERK kinase activation that is closely linked to the endoplasmic reticulum (ER) stress reaction.⁽²⁴⁾Trib3 is induced by hypoxic stress and its overexpression may suppress stress signals through promoting proteolysis of ATF4.⁽²⁵⁾ The functional role of Trib3 in ER stress signaling was further delineated when it was shown that Trib3 induction by ethanol was blocked by ER stress inhibitors such as 4-phenyl butyric acid and taurine-ursodeoxycholic acid.⁽²⁶⁾ Trib3 and ATF4 are isolated as a protein complex and they bind to the cyclic AMP responsive element, which represses transcriptional induction of CHOP.^(27,28)

It was proposed that degradation of target proteins by tribbles is mediated by the E3 ubiquitin ligase COP1.⁽¹³⁾ Trib3 promotes ubiquitination of acetyl-coenzyme A carboxylase (ACC) in adipose tissue. Trib3 interacts with both ACC and COP1 using distinct domains, promoting COP1-dependent ubiquitination of ACC. The COP1 binding consensus sequence [D/E]QXVP[D/E] has been well conserved among the three tribbles during evolution,⁽¹²⁾ suggesting that COP1 binding by the tribbles family of proteins is essential for their targets in protein degradation.

Kiss-Toth et al.⁽²⁹⁾ clarified that Trib1-3 interact with mitogen-activated protein kinase kinase (MAPKK) including MEK1, MKK4 or MKK7. They showed that phosphorylation of ERK is enhanced by Trib1 overexpression. This was confirmed in bone marrow (BM) cells and myeloid leukemia cells with Trib1 upregulation.^(14,30) We identified a distinct motif ILXHPW[F/L] at the C-terminus as a MEK1 interaction domain, and all three tribbles could interact with MEK1.⁽¹⁴⁾ Besides, overexpression of Trib1 inhibited MEKK1-induced stress kinase signaling.⁽³¹⁾ This was further supported by data showing that Trib1 interacts with MKK4 and blocks AP-1 activation downstream of JNK in smooth muscle cells.⁽³²⁾ It is therefore proposed that interaction between tribbles and MEK/MAPK signaling molecules is cell-type dependent and there may be differences in affinity between each tribbles and MAPKK.^(31,33,34) Pujol et al.⁽³⁵⁾ identified NIPI-3, a Caenorhabditis elegans homolog of Trib1, as an upstream molecule of MAPKK/SEK-1. NIPI-3 is required for nlp-29 induction in response to infection and wounding and the function of NIPI-3 is mediated by MAPK signaling.

Tribbles interaction and involvement was also reported in AKT, bone morphogenic protein (BMP), peroxisome proliferator-activated receptor (PPAR) or nuclear factor (NF)-κB signaling, and the results are summarized in Table 1.

Table 1.   Tribbles interacting proteins

Protein	Tribbles	Mode of interaction	Biological significance	Reference
ATF5, CSNK2B, FN1, GRN, HLA-B, LTBP4, MCM3AP, LTBP4, MCM3AP, MYO18A, HRSP12, PSMA3, RBM4, RPSA, COP9, TEF1g, GIT1, GDF9 and TAT are potent Trib3 interaction partners of unknown significance.^(^52^) BMP, bone morphogenic protein; C/EBP, CCAAT/enhancer-binding protein; CHOP, C/EBP-homologous protein; ER, endoplasmic reticulum; IL, interleukin; PPAR, peroxisome proliferator-activated receptor; TGF, transforming growth factor; TNF, tumor necrosis factor; TRAIL, TNF-related apoptosis-inducing ligand.
COP1	Trib1-3	Interaction by the C-terminal [D/E]QXVP[D/E] motif	Degradation of target proteins	13,41,42
String (cdc25)	Tribbles (drosophila)	Proteasome-mediated degradation	Inhibition of mitosis and cell cycle at G2 in embryo	4,7
slbo (c/ebp)	Tribbles (drosophila)	Proteasome-mediated degradation	Regulation of cell migration during wing formation	16
C/EBPα	Trib1 and Trib2	Proteasome-mediated degradation	Myeloid leukemia induction	14,17,41
C/EBPβ	Trib1 and Trib2	Proteasome-mediated degradation	Inhibition of adipocytic differentiation; modification of toll-like receptor signaling	18,19
ATF4	Trib3	Proteasome-mediated degradation	Suppression of stress signal upon hypoxia	25,27
CHOP	Trib3	Interaction by CHOP N-terminus	Induction of apoptosis upon ER stress	22,23
ACC	Trib3	Proteasome-mediated degradation	Suppression of adipogenesis	13
MEK1	Trib1-3	Interaction by the C-terminal ILXHPW[F/L] motif	Enhancement of ERK phosphorylation and myeloid leukemia induction	14,29,34
MKK4	Trib1 and Trib3	Interaction by the C-terminus	Suppression of vascular smooth muscle cell migration	14,29
MKK7	Trib2 and Trib3	Interaction by kinase-like domain	Suppression of IL8 production in monocytes by inhibiting JNK and ERK phosphorylation	29
AKT1/2	Trib2 and Trib3	Inhibition of AKT kinase activity	Suppression of adipocytic differentiation; disruption of insulin signaling in adipose tissue	55
MLK3	Trib3	Interaction by Trib3 N-terminus	Induction of pancreatic β cell death	56
BMPRII	Trib3	Interaction by the tail domain of BMPRII in the absence of BMP stimuli	Promotion of osteogenic differentiation by degrading Smurf1	36
Smurf1	Trib3	Interaction by Trib3 pseudokinase domain	Induction of Smurf1 degradation	36
PPARγ	Trib3	Interaction by Trib3 pseudokinase domain	Inhibition of adipogenic differentiation by suppression of PPARγ transcriptional activities	58
p65/RelA	Trib2	Inhibition of p65 phosphorylation	Apoptotic induction upon TNF and TRAIL stimuli	37
SIAH1	Trib3	Degradation of Trib3 by SIAH1	Inhibition of TGFβ signaling	52
CtIP	Trib3	Interaction by each C-terminal domain	Unknown	38

Although it remains unknown where endogenous tribbles is located, overexpression experiments have shown predominant nuclear localization of three mammalian Trib proteins with some exception.^{(8,14,31,32,36,37)} Nuclear localization of tribbles is not exclusive, therefore tribbles proteins could associate with their partners both in the nucleus and cytoplasm. Nuclear co-localization and direct interaction between Trib1 and RARα has also been reported.⁽¹⁵⁾ Interestingly, Trib3 is recruited to nuclear dot-like structures under low ultraviolet exposure, and Trib3 and CtIP are co-localized in that structure, suggesting Trib3 may be involved in the DNA repair machinery.⁽³⁸⁾

Tribbles in cancer

Trib1 and Trib2 are leukemia disease genes. Trib2 was first identified as a downregulated gene upon γ-secretase inhibitor treatment of Notch-dependent mouse T-ALL cell lines.⁽¹⁷⁾ Retrovirus-mediated gene transfer of Trib2 into mouse BM cells induced monocytic differentiation, inhibited granulocytic differentiation and accelerated self-renewal activity. Transplantation of Trib2-expressing BM cells into lethally irradiated mice resulted in AML development at 100% penetrance. Subsequently, Trib1 was identified as a collaborator of Hoxa9 and Meis1 in myeloid leukemogenesis.⁽³⁰⁾ Cooperative genes for Hoxa9/Meis1 were identified as common targets of retroviral integration when retrovirus was used as an insertional mutagen,⁽³⁹⁾ and Trib1 was identified as the most frequent common integration site of the Hoxa9/Meis1 retrovirus in AML. Trib1 is by itself a transforming gene for myeloid cells but also significantly accelerates progression of Hoxa9/Meis1 AML. Specific genetic interaction between Trib1 and Hoxa9/Meis1 was further confirmed by identifying Trib1-retroviruses in Trib1-AML at Hoxa7, Hoxa9 or Meis1 loci. Consistent with the results, Keeshan et al.⁽⁴⁰⁾ confirmed that Trib2 also cooperates with Hoxa9.

Argiropoulos et al.⁽⁴¹⁾ identified Trib2 as a downstream target gene of Meis1 in Meis1/NUP98-HOXD13 leukemia cells and they speculated that Trib2 replaces Meis1 function in leukemogenesis. However, this is an interesting issue given the functional similarity between Trib1 and Trib2, and specific genetic interaction between Trib1 and Meis1; further study is needed to define the relationship between tribbles and homeodomain proteins in leukemogenesis.

The molecular mechanism of tribbles-induced leukemogenesis is an important issue for understanding tribbles function as well as the biological nature of leukemia. First, phosphorylation of ERK1/2 is enhanced in Trib1-transfected HeLa cells and BaF3 cells as well as leukemia cells derived from Trib1-induced AML upon cytokine stimulation.⁽³⁰⁾ The MEK1 binding motif ILLHPWF in the C-terminal region of Trib1 was then identified (Figs 2C,3A). A Trib1 mutant lacking the motif was unable to enhance phosphorylation of ERK1/2 or prolong self-renewal of BM cells.⁽¹⁴⁾ More importantly, the mutant that lacked the ILLHPWF motif could neither induce AML nor accelerate Hoxa9/Meis1-induced AML. These results indicate that MEK interaction and enhancement of the MEK/ERK pathway is required for the leukemogenic activity of Trib1.

Figure 3.  The role of Trib1 in leukemogenesis. (A) Trib1 functions as an adaptor between the MEK/ERK pathway and C/EBPα. Trib1 interacts with MEK1 and enhances phosphorylation of ERK1/2, which promotes cell proliferation and suppresses apoptosis (1). In addition, Trib1 recruits COP1 to C/EBPα (2) and ERK phosphorylation is required for promotion of C/EBPα ubiquitination (3). C/EBPα is also downregulated by hnRNP E2, which requires ERK phosphorylation (4). Proteasome-mediated degradation and post-transcriptional suppression of C/EBPα results in myeloid differentiation (5). (B) Cooperative partners (indicated as blue circles) for Trib1 in leukemogenesis were identified by retroviral tagging. MLL chimeras and Hoxa9 are involved in human AML, Gata2 in DS-AMKL, and loss of Nf1 in juvenile chronic myeloid leukemia (JCML). Cooperation between Bcl11a and Nf1 mutations was reported.⁽⁵⁰⁾ C/EBP, CCAAT/enhancer-binding protein; DS-AMKL, down syndrome-related acute megakaryocytic leukemia; ERK, extracellular signal-regulated kinase; hnRNP, heterogeneous nuclear ribonucleoprotein; MEK1, MAPK/ERK kinase 1.

Second, downregulation of C/EBPα p42 (the full-length isoform) was observed in Trib2-induced AML as well as in 32D and U937 myeloid cells overexpressing Trib2. As discussed above, it was proposed that Trib2 induces downregulation of C/EBPα through proteasome-dependent degradation. An E3 ubiquitin ligase COP1 that is required for degradation of ACC by Trib3 also mediates C/EBPα degradation by Trib2.⁽⁴²⁾ The COP1 binding motif is required for both degradation of C/EBPα and leukemogenic activity of Trib2. All three tribbles members could interact with COP1 through the C-terminal DQXVP sequence (Figs 2C,3A); however, while Trib1 and Trib2 could induce degradation of C/EBPα, Trib3 could not.⁽⁴³⁾ These data suggest degradation of C/EBPα is causally related to the leukemogenic activity of Trib1 and Trib2. Importantly, the Trib1 mutant lacking the MEK1 binding motif also failed to degrade C/EBPα,⁽¹⁴⁾ suggesting degradation of C/EBPα by Trib1 is dependent on activation of the MEK1/ERK pathway, which was further confirmed by the finding that MEK inhibitor U0126 suppressed degradation of C/EBPα by Trib1.

Several studies have shown that the MAPK pathway affects C/EBPα function. MAPK activation induced by the BCR-ABL fusion protein upregulates hnRNP E2 and downregulates miR-328.⁽⁴⁴⁾C/EBPα mRNA is one of the targets of hnRNP E2 transcriptional repression, and the translational activity of C/EBPα is restored when MAPK activity is blocked (Fig. 3A). Furthermore, using AML cell lines that possess FLT3 mutations showing constitutive activation of the MAPK pathway, phosphorylation of C/EBPα at serine 21 was induced and granulocytic differentiation was suppressed.⁽⁴⁵⁾ Conversely, inhibition of the MAPK pathway with a specific inhibitor PD98059 or overexpression of the C/EBPα S21A mutant induced granulocytic differentiation. These results support the role of MAPK signaling in C/EBPα function, and Trib1 and Trib2 could mediate that pathway as adaptors. Another important motif for C/EBPα degradation was observed as a conserved LRDLKLRK motif within the pseudokinase domain.⁽⁴²⁾

A potential oncogenic role of TRIB1 has been suggested in human AML. On chromosome 8q24 TRIB1 is located 1.5 Mb from c-MYC. It has been believed that the target of 8q24 amplification in human AML is c-MYC. However, Storlazzi et al. have shown that TRIB1 is overexpressed at least in some cases of AML with 8q24 amplification while c-MYC expression is not detected, and similar results were obtained in AML with ring chromosome 8.^(46,47) Nevertheless, upregulation of MYC is undoubtedly important in AML, suggesting cooperative roles between TRIB1 and MYC.⁽⁴⁸⁾

In search of genetic abnormalities that involve tribbles in human leukemia, we recently identified a somatic point mutation of TRIB1 in a case of Down syndrome (DS)-related acute megakaryocytic leukemia (AMKL) (Yokoyama T, Toki T, Aoki Y, Kanezaki R, Park M, Kanno Y, Takahara T, Yamazaki Y, Ito E, Hayashi Y and Nakamura T, unpublished data, 2011). The G : T point mutation was observed within the pseudokinase domain resulting in amino acid conversion from arginine to leucine (R107L). When the R107L mutant was expressed in mouse BM cells and transferred into lethally irradiated recipients, acceleration of AML development and further enhancement of MAPK phosphorylation was observed, suggesting that the mutation is a gain-of-function mutation. This case exhibited a loss of function mutation of GATA1 in its blastic phase.⁽⁴⁹⁾ Interestingly, the TRIB1 mutation preceded the GATA1 mutation at the hematopoietic stem cell level and emerged at a very early stage of leukemogenesis, and the TRIB1 mutation is necessary but not sufficient for leukemogenesis.

In DS-related leukemogenesis, GATA2 upregulation contributes to dysregulated megakaryocytic proliferation in the absence of GATA1.⁽⁵⁰⁾ In accordance with this result, we identified Gata2 as a common retroviral integration site in Trib1-induced AML (Table 2). The data strongly suggest cooperation between Trib1 and the Gata transcriptional network (Fig. 3B). Moreover, the same study identified Bcl11a as the second most frequent integration site (Table 2). Recently, Yin et al.⁽⁵¹⁾ showed that Bcl11a cooperates with Nf1 mutations. The data raised the possibility that Bcl11a is a cooperative partner of the MAPK pathway where both Nf1 and Trib1 are important components (Fig. 3B).

Table 2.   Common integration sites and candidate cooperative genes in Trib1-induced AML

Candidate gene	Chromosome locus	Incidence, % (n = 37)	Products	Known common site in RTCGD
RTCGD, Mouse Retrovirus Tagged Cancer Gene Database (http://rtcgd.abcc.ncifcrf.gov/).
Hoxa9, Hoxa7	6B3	35.1	Transcription factor	Yes
Bcl11a	11A3.2	24.3	Transcription factor	Yes
Sox4	13A3-A5	13.5	Transcription factor	Yes
Anp32b	4B2	8.1	Histone chaperone	Yes
Gata2	6D1	5.4	Transcription factor	No
AK044786	2A1	5.4	Unknown	No
1600014C10Rik	7B1	5.4	Unknown	No

Tribbles involvement in non-hematopoietic malignancies.  Bowers et al.⁽²⁵⁾ found that TRIB3 is highly expressed in primary breast and colorectal cancer. In addition, expression of TRIB3 in human colon or prostate cancer cell lines was upregulated under hypoxic conditions, suggesting that Trib3 could be induced by HIF1α and might play a role in cellular survival in vivo. High levels of TRIB3 expression in lung, esophagus and colon cancer were also reported.⁽⁵²⁾ Another study indicated that a high level of TRIB3 expression was correlated with high metastatic activity and a poor prognosis of human colorectal cancer, and that TRIB3 knockdown suppressed growth of colorectal cancer cells.⁽⁵³⁾

In a series of human skin cancer, TRIB2 was found overexpressed in melanoma.⁽⁵⁴⁾ Interestingly, the expression levels of TRIB2 correlated with cytoplasmic localization of FOXO3a, and TRIB2 knockdown restored nuclear localization of FOXO3a. The TRIB2-mediated downregulation of FOXO3a increased cell proliferation, colony formation, wound healing and in vivo tumor growth, indicating the oncogenic nature of TRIB2 in certain non-hematopoietic neoplasms.

It remains to be determined which genetic pathways and target molecules of tribbles are involved in non-hematopoietic cancers. In this respect, FOXO family transcription factors are good candidates, although more comprehensive studies in other tumor types are needed. Moreover, given the fact that TRIB3 is induced in ER stress and hypoxic conditions, ATF4/CHOP and AKT pathways are important in the treatment for tribbles-expressing cancer.

Tribbles in non-neoplastic disorders

Metabolic disorders.  Trib3 is upregulated in the liver by fasting and its overexpression completely blocks insulin action through inhibition of AKT phosphorylation. These findings suggest that Trib3 promotes glucose output from the liver under fasting conditions and that its abnormal expression may contribute to insulin resistance and thereby promote hyperglycemia.⁽⁵⁵⁾ AKT inhibition by Trib3 is mediated by MLK3, a MAPK pathway-related kinase, and this inhibition induces BAX translocation resulting in apoptosis of pancreatic β cells.⁽⁵⁶⁾

The foregoing results suggest that Trib3 could be a potential target for the treatment of type 2 diabetes.⁽⁵⁷⁾ Knockdown of PGC1, a PPARγ coactivator, in the liver induced fasting hypoglycemia and decreased expression of Trib3.⁽⁵⁷⁾ Furthermore, physical interaction between Trib3 and PPARγ was identified by Takahashi et al.⁽⁵⁸⁾ They showed that Trib3 overexpression downregulated PPARγ target genes in 3T3-L1 adipocytic progenitors and suggested that Trib3 is a potent negative regulator of PPARγ in adipogenesis. Based on this hypothesis, mutations/single nucleotide polymorphisms (SNP) of Trib3 have been examined in human populations. Prudente et al.⁽⁵⁹⁾ reported that the Q84R SNP of Trib3 is significantly associated with insulin resistance-related cardiovascular risk in type 2 diabetes. They found insulin-induced AKT phosphorylation is more significantly reduced by the introduction of Trib3 R84 into HepG2 cells than by that of Q84. The same group subsequently exhibited Q84R, which is also associated with chronic kidney disease with type 2 diabetes.⁽⁶⁰⁾ R84 exhibited greater protein stability than Q84, resulting in increased binding affinity to ATF4.⁽⁶¹⁾ The Trib3/ATF4 complex competitively inhibits the activity of cAMP response element-binding protein (CREB) that is a key regulator of insulin exocytosis in pancreatic β cells, and R84 reduces proliferation and enhances apoptosis of β cells. Moreover, R84 expression in U937 monocytic cells increased cell adhesion to HUVEC cells, VCAM1 and ICAM1 expression, and phosphorylation of ERK1/2 and MEK in the steady state. With these results Formoso et al.⁽⁶²⁾ concluded that the TRIB3 R84 variant is associated with unbalanced insulin signaling and susceptibility to atherosclerosis.

Deng et al.⁽⁶³⁾ reported that expression of Trib1 and Trib2 was increased in vulnerable regions of human atherosclerotic plaques. Treatment of human macrophages with oxidized low-density lipoprotein (LDL) upregulated Trib2, and exogenous expression of Trib2 in macrophages reduced IL-10 expression. They concluded that Trib2 upregulation is a potential pro-inflammatory process and may contribute to the instability of atherosclerotic plaques. Using a similar approach, Eder et al.⁽³⁴⁾ showed that LPS induced IL-8 expression in the human macrophage cell line THP1. They also showed that LPS stimulation increased LDL uptake by macrophages but this phenomenon was Tribbles independent.⁽⁶⁴⁾ They found that increased LDL uptake was associated with JNK phosphorylation but none of the three Trib genes changed expression. Perhaps Trib1 and Trib2 are downstream of LPS-induced LDL uptake and JNK signaling. In fact, the same group screened signaling components that modulated the promoter activity of IL8,⁽³¹⁾ and identified Trib1 as one such molecule. Inflammatory cytokine responses were modulated in white adipose tissue in Trib1 mutant mice.⁽⁶⁵⁾ Moreover, increased plasma lipid levels and hepatic very low density lipoprotein (VLDL) production were observed in Trib1 mutant mice, suggesting that high Trib1 expression may be a risk factor for myocardial infarction.⁽⁶⁶⁾

Cardiovascular diseases.  Advanced glycation end products (AGE) are important in collagen deposition in diabetic cardiomyopathy, and Trib3 overexpression is accompanied with AGE.⁽⁶⁷⁾ Trib3’s induction of AGE is linked to activation of p38-MAPK, again indicating the important function of tribbles in RAS/MAPK signaling. Although the importance of Trib3 in glucose consumption as well as insulin signaling is supported by these studies, Trib3 knockout mice exhibited normal hepatic insulin signaling and glucose homeostasis.⁽⁶⁸⁾ Nevertheless, Trib3 expression is increased by ER stress in myocardial infarction, and overexpression of Trib3 increased pathological cardiac remodeling after infarction.⁽⁶⁹⁾

BMP signaling is abrogated in 60% of familial pulmonary hypertension (PAH) and 30% of idiopathic PAH cases. Phenamil treatment attenuates vascular remodeling of PAH, and Trib3 is a key molecule as Trib3 is induced by phenamil.^(70,71) The result indicates that Trib3 is a potential molecular target for the therapy of PAH.

Other disorders.  Possible roles of TRIB2 in human autoimmune disorders have been proposed. Zhang et al.⁽⁷²⁾ identified TRIB2 as an autoantigen in autoimmune uveitis. Using mRNA tagging, Cvetkovic-Lopes et al.⁽⁷³⁾ identified TRIB2 as an upregulated gene in Pabpc1-hypocretin transgenic mice and detected anti-TRIB2 immune activity in the sera of narcolepsy patients. The results suggest that narcolepsy might be an autoimmune disorder and showed the possible importance of TRIB2 as a diagnostic tool for the disease.

Possible involvement of TRIB3 in bone and cartilage disorders was also reported. TRIB3 expression was significantly increased in cells obtained from osteoarthritis cartilage.⁽⁷⁴⁾ As shown in other cell types, overexpression of TRIB3 inhibits AKT phosphorylation resulting in reduced chondrocyte survival and proteoglycan synthesis.

Future direction and conclusion

Multiple genetic and epigenetic mutations are required for pre-neoplastic cells to gain competitive growth advantages over their normal counterparts. The specific interaction of tribbles with their cooperative partners such as MLL-Hox/Meis axis, Bcl11a and GATA is important not only for understanding carcinogenic mechanisms but also for exploring novel cancer therapeutic approaches (Fig. 3B). Detection of Trib2 autoantibody in narcolepsy patients is also important, and it raises the possibility that tribbles proteins may be good candidates for cancer immunotherapy because the cancer-specific antigen may be expressed in tribbles-overexpressing leukemic, colon cancer or melanoma cells that overexpress tribbles.

None of the knockout mice for the three tribbles genes have shown significant phenotypic manifestation,^(68,75) suggesting that there might be functional redundancy and overlapping functions of the tribbles family genes, and therefore double or triple mutant mice should be tested to clarify their physiological roles.

Acknowledgments

This work was supported by a Grant-in-Aid for Scientific Research on Priority Areas “Integrative Research Toward the Conquest of Cancer” (T.N.) and for Young Scientists (T.Y.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

Disclosure Statement

The authors have no conflict of interest.