Inhibitor of DNA Binding 4 (ID4)
Transcription factors containing a basic helix-loop-helix (bHLH) motif regulate expression of tissue-specific genes in a number of mammalian and insect systems, playing a key role in the differentiation processes. These proteins contain an HLH domain, which mediates homo- and heterodimerization, plus an adjacent DNA-binding region rich in basic amino acids. The bHLH proteins bind to a DNA sequence known as E-box (CANNTG).
Dominant-negative HLH proteins encoded by Id-related genes, such as Id4, also contain the HLH-dimerization domain but lack the DNA-binding basic domain. Consequently, Id proteins inhibit binding to DNA and transcriptional transactivation by heterodimerization with bHLH proteins (Lasorella et al. 2014). In mammals there are four known Id gene family members, known as Id1, Id2, Id3, and Id4. The identity between the HLH regions of Id proteins is very high, while the remaining regions of the proteins are not conserved. The N- and C-terminal fragments of Id proteins do not adopt a helical conformation, with the exception of Id4 fragment 27–64. This helix propensity is dictated by the presence of a poly-alanine tract between residues 39 and 57. This tract is consistent with similar tracts which are preferentially found in many transcription factors and generally act as a flexible spacer element located between the functional domains of a protein and therefore essential to protein conformation, protein–protein interactions, and/or DNA binding. It can be hypothesized that Id4 might exert unique functions through this structural feature (Patel et al. 2015). Additional structural and functional features may be uniquely imparted to Id4 protein by a proline-rich region present in the C-terminus. As proline is a strong promoter of intrinsic disorder, the proline-rich region of Id4 may lack a defined 3-D structure; this favors protein–protein interactions by presenting a larger interaction surface allowing multiple binding partners.
Despite the high similarity in the HLH domain, the Id proteins bind different targets with different affinities; for example, Id2 is the only Id family member that recognizes the retinoblastoma protein.
Typically, Id proteins are highly expressed during embryogenesis and expressed at lower levels in mature tissues, with the exception of some stem cells and many cancers (reviewed in Lasorella et al. 2014). Id proteins were described initially as inhibitors of differentiation and more recently as regulators of cell cycle progression, senescence, apoptosis, and tumorigenesis. Concerning the ability of Id proteins to control the developmental processes, Id4 in particular was shown to be involved in the differentiation of neurons, adipocytes and osteoblasts, and in the nervous system and mammary gland development.
Deregulated Id4 expression is also frequent in tumors. Id4 promoter hyper-methylation is a common event in a variety of tumors, including leukemia, prostate cancer, carcinomas of the gastrointestinal tract, and breast cancer, suggesting that it may play a role as a tumor suppressor (Umetani et al. 2005, Patel et al. 2015).
On the contrary, Id4 was found upregulated in glioblastoma multiforme (GBM) (Kuzontkoski et al. 2010) and upregulated/amplified in basal-like subtype of breast cancer (BLBC) and ovarian carcinoma (reviewed in Baker et al. 2016).
Id4 Regulates Neural Progenitor Proliferation/Differentiation and Maintains the Stem Cells Compartment in Glioblastoma Multiforme
It is well documented that the expression of each Id gene occurs in many regions of the developing nervous system in a complex and dynamic manner. Id4 expression is essentially restricted to the developing nervous system, whereas expression of Id1–3 is much more widespread during mouse embryogenesis (Jen et al. 1997). Early in neurogenesis, Id4 expression is prominent in the ventricular zone (VZ) of specific regions of the central nervous system (CNS), including the developing forebrain. Later, Id4 expression is apparent in the cortical plate of the telencephalon and the subventricular zone (SVZ) of the basal ganglia (Jen et al. 1997). Id4 expression is also observed in the postnatal and adult brains (Andres-Barquin et al. 1999).
Studies on knock-out mice revealed that Id4 is required for normal brain size and regulates neural stem cells proliferation and differentiation. In particular, Id4 regulates lateral expansion of the proliferative zone in the developing cortex and hippocampus. Since Id4 is required for the normal G1/S transition in early cortical progenitors, the absence of its expression compromises the proliferation of stem cells in the ventricular zone (Yun et al. 2004).
Id4 is expressed in oligodendrocyte precursor cells and may control the timing of oligodendrocyte differentiation. Enforced expression of Id4 in vitro stimulates proliferation and blocks differentiation of oligodendrocyte precursor cells (Bedford et al. 2005). Id4 was recently found in neural progenitor cells to directly interact with bHLH OLIG1 and OLIG2, two crucial transcription factors responsible for oligodendroglial differentiation. Id4 also mediates the inhibitory effects of bone morphogenetic protein-4 (BMP-4) on oligodendroglial differentiation that leads to astrocytic differentiation (Yun et al. 2004).
The molecular mechanism by which Id4 expression is downregulated during oligodendrocyte differentiation has remained unknown. Accumulating evidence, however, suggests that Id4 expression is repressed by DNA methylation at neighboring CpG islands. Interestingly, it was found that PRMT5, a type-II protein arginine methyltranferase, is required for maintaining the methylation status of CpG islands of Id2 and Id4, leading to gene silencing during glial cell differentiation (Huang et al. 2011). In addition, a transcriptional factor, RP58, was very recently reported to negatively regulate all four Id genes (Id1–Id4) in developing cerebral cortex. Consistently, Rp58 knockout (KO) mice demonstrated enhanced astrogenesis accompanied with an excess of neural stem cells (NSCs). Rp58 KO phenotypes were rescued by the knockdown of all Id genes in mutant cortical progenitors but not by the knockdown of each single Id gene. These findings establish RP58 as a novel key regulator that controls the self-renewal and differentiation of NSCs and restriction of astrogenesis by repressing all Id genes during corticogenesis (Hirai et al. 2012).
Id4 was found overexpressed in glioblastoma multiforme (GBM) when compared to normal brain tissue. In GBM it shows a robust expression with heterogenous staining pattern within the same tumor tissue. In particular, Id4 is preferentially expressed in cells of astrocytic lineage in oligodendroglioma and oligoastrocytoma tumors (Liang et al. 2005). Interestingly, the analysis of Id4 protein expression in human GBM specimens evidenced that the majority of Id4-positive cells resides near the vasculature, a location postulated to be the niche for brain tumor stem cells (Jeon et al. 2008).
Enforced Id4 expression can drive malignant transformation of primary murine Ink4a/ARF−/− astrocytes via deregulation of cell cycle and differentiation control. Id4 indeed increases the levels of both cyclin E (that leads to a hyperproliferative state) and Jagged1 to drive astrocytes into a neural stem-like cell state. These findings highlight the role of Id4 in controlling the “stemness” of neural cells during development of the central nervous system (Jeon et al. 2008). Further studies showed also that Id4 expression contributes to the chemoresistance of glioma stem cells. In particular, Id4 suppresses the expression of microRNA-9*, leading to the derepression of SOX2, a crucial player in cancer stem cells. SOX2 upregulation contributes, on one hand, to the maintenance of cancer stem cells compartment, and on the other hand, to glioma chemoresistance, through the transcriptional induction of drug resistance genes, such as those encoding the ATP-binding cassette transporters ABCC3/ABCC6 (Jeon et al. 2011).
Moreover, human glioblastoma xenografts overexpressing Id4 are characterized by higher tumor sizes compared to controls. This is not due to a higher proliferation rate of Id4-overexpressing cell clones but instead to the better vascularization of the derived xenograft tumors compared to control xenografts. Superficial blood vessels in xenografts expressing high Id4 levels are larger and more numerous than vessels in control tumors. The Id4-dependent mediator responsible for the enhanced angiogenesis is matrix GLA protein (MGP), a member of Vitamin-K-dependent family of proteins, which includes prothrombin (Kuzontkoski et al. 2010).
Unexpectedly, Id4 has been recently shown to reduce the invasive potential of GBM cells. This relies on the ability of Id4 to interact with and inhibit bHLH transcription factor Twist1, resulting in decreased levels of MMP2 production (Rahme and Israel 2015).
Id4 Controls Mammary Gland Development and Cancer
Increasing evidence supports a central role for Id4 in regulating mammary cell proliferation and lineage commitment. Comparison between the transcriptional signatures of the main cell populations of the mammary gland (e.g., luminal progenitors, committed/ mature luminal and basal cells) revealed that ID4 was one of the highest differentially expressed genes specific to basal cells of both human and murine origin (reviewed in Baker et al. 2016). Loss-of-function studies also showed that Id4-positive cells repopulated the adult mammary gland at a greater frequency than Id4-negative cells demonstrating the intrinsic stem cell activity of the Id4-positive basal cells (reviewed in Baker et al. 2016).
By using a germ-line knockout mouse model, Dong and coworkers showed that during the normal mammary gland development Id4 expression is required for ductal expansion and branching morphogenesis as well as cell proliferation induced by estrogen and/or progesterone (reviewed in Baker et al. 2016). p38 MAPK is activated in Id4-null mammary cells, and this activation is required for the reduced proliferation and increased apoptosis observed in Id4-ablated mammary glands. Therefore, Id4 promotes mammary gland development by suppressing p38 MAPK activity (reviewed in Baker et al. 2016).
In a more recent study based on the use of a cre-inducible mouse model of Id4 deletion (reviewed in Baker et al. 2016), a delay in ductal morphogenesis was observed, confirming previous evidence. Interestingly, a striking increase in the expression of ERα (Esr1), PR, and FoxA1 was observed in both the basal and luminal cellular subsets of Id4-deficient mammary glands (reviewed in Baker et al. 2016). Id4 was found to be part of the transcriptional complex that could potentially repress ERα and Foxa1 promoters. The lack of Id4 DNA binding domain in Id4 suggests that it most likely regulates transcription as part of a larger protein complex. Of note, in the same study an impairment of the enzymatic cascade responsible for estrogen biosynthesis in the ovary was also observed in Id4-deficient females. Thus, compromised ovarian function and decreased circulating estrogen likely contribute to the mammary ductal defects evident in Id4-deficient mice.
A role for Id4 in mammary and ovarian tumorigenesis has been also proposed. In particular, an overexpression and amplification of Id4 has been reported in basal-like breast cancer (BLBC) and in ovarian cancer, and the complex regulatory network involving Id4, ERα, and BRCA1 in these tumors still remains to be fully elucidated. BRCA1 and ERα mRNA expression have been shown to correlate in sporadic breast cancers (Roldán et al. 2006), while Id4 is negatively correlated to both BRCA1 and ERα in sporadic basal-like breast cancer (reviewed in Baker et al. 2016). Beger and coworkers (2001) identified Id4 as an upstream negative regulator of the BRCA1 promoter in luminal breast cancer cell lines. The inhibitory effect of Id4 on BRCA1 has been subsequently confirmed in ERα-negative breast cancer cell lines (reviewed in Baker et al. 2016).
In clinical breast cancer, Id4 expression is exclusive to ERα-negative subtypes of breast cancer (reviewed in Baker et al. 2016). This evidence has led researchers to suggest that Id4 loss may be important in the development of ERα-dependent breast cancers (where Id4 hypermethylation has been reported), and conversely, Id4 may suppress BRCA1 and ERα in Id4-positive BLBC (reviewed in Baker et al. 2016), resulting in poor survival outcomes (reviewed in Baker et al. 2016). Accordingly, Id4 depletion in a basal-like MMTV-Wnt-1 mammary tumor model results in reexpression of ERα and activation of the ERα signaling network, indicated by the expression of FOXA1 (reviewed in Baker et al. 2016).
Altogether these data suggest that Id4 may be exerting similar inhibitory effects on BRCA1 and ERα and conversely that BRCA1 and ERα may demonstrate redundancy in inhibiting Id4 (reviewed in Baker et al. 2016).
Regulation of Id4 expression in breast cancer is a field of intensive study. Recently, it has been shown that Id4 expression is also regulated by miRNAs. In particular, Id4 3′-UTR was found to be targeted by microRNA-335 in breast cancer cells, resulting in BRCA1 induction and decrease in genomic instability. Accordingly, Id4 and miR-335 expression are inversely correlated in breast cancer specimens (reviewed in Baker et al. 2016). In addition, ID4 may be repressed by miR-342 (breast cancer cell line MDA-MB-231) (reviewed in Baker et al. 2016), leading also in this case to BRCA1 induction, and by senescence-associated miR485-5p (Napolitano et al. 2014).
The expression of Id4, as well as that of Id2, can be induced by mutant p53 proteins (Fontemaggi et al. 2009, Fontemaggi et al. 2010). In breast cancer cell lines the transcriptional transactivation of Id4 promoter is exerted by the complex mutant p53/E2F-1/p300. Accordingly, Id4 protein expression is enriched in breast cancer tissues showing p53 overexpression (predicting the presence of p53 mutations) (reviewed in Baker et al. 2016). The net biological output of the transcriptional activation of Id4 gene by mutant p53 is the increase of the angiogenic potential of mutant p53-carrying tumor cells. The mutant p53/Id4 axis promotes endothelial cells proliferation and migration in vitro. In addition, the analysis of human breast cancer cases revealed that a higher microvessel density is present in the Id4-positive population than in Id4-negative one.
At the molecular level, Id4 protein binds to the mRNAs of proangiogenic factors like CXCL8 (IL8) and CXCL1 (GRO-alpha), containing AU-rich (ARE) elements in their 3′UTR, resulting in an increased stability and a higher rate of translation of these transcripts (Fontemaggi et al. 2009). Significantly, the most expressed cytokines in HER2 tumors, displaying the highest correlation between p53 and Id4 expressions among all breast cancer subtypes, are just CXCL8 and CXCL1.
Studies performed in 4 T1 invasive mouse mammary cancer cells also evidenced the ability of Id4 to promote features typical of the cancer stem cells, such as the tumor sphere-forming ability (Park et al. 2011). In addition, Id4 is involved in the induction of the expression of ABCC3, belonging to the family of ABC transporters.
ID4 Is Amplified and Oncogenic in Ovarian Cancer
As mentioned above, the chromosomal region containing ID4 (6p22) is amplified in 32% of high-grade serous ovarian cancers (TCGA 2011). Also, Id4 is overexpressed in most primary ovarian cancers and ovarian cancer cell lines, but not in normal ovary, fallopian tube (FT), and other tissues (Ren et al. 2012). A tumor-penetrating nanocomplex (TPN), comprised of siRNA complexed with a tandem tumor-penetrating and membrane-translocating peptide, which enables the specific delivery of siRNA deep into the tumor parenchyma, was employed in vivo to evaluate the role of Id4 as an oncogene in tumors, where Id4 expression is elevated, such as those of the ovary. Treatment of ovarian tumor-bearing mice with Id4-specific TPN suppressed growth of established tumors and significantly improved survival suggesting that targeting Id4 expression is a viable therapeutic strategy in cancers that overexpress Id4 (Ren et al. 2012).
ID4 Acts as a Tumor Suppressor in Prostate Cancer
Id4 acts as a tumor suppressor, for example, in the normal prostate where it is highly expressed; this expression decreases in prostate cancer in a stage-dependent manner as a result of ID4 promoter hypermethylation (reviewed in Patel et al. 2015). High-grade tumors are associated with the absence of Id4 expression (reviewed in Patel et al. 2015). In this context, Id4 exerts antiproliferative effects, in part, by increasing expression of the classical tumor suppressor genes p27 and p21 (reviewed in Patel et al. 2015). This work highlights the divergent and context-dependent roles of Id4 in different hormone-dependent cancers.
Id4 is a member of the inhibitor-of-DNA-binding family of HLH proteins that comprises four members (Id1–4). The Id group of proteins was reported to promote proliferation and inhibit differentiation in several cell types. Because they lack a DNA-binding domain at the N-terminus, Id proteins are generally thought to exert their function by forming heterodimers with bHLH proteins, preventing these other proteins from forming transcriptionally active homodimers or heterodimers with the ubiquitous E proteins on consensus E-boxes present on target promoters. The gene encoding Id4 is required for neuroprogenitor cell proliferation and proper differentiation, as well as for mammary and prostate development. The expression of Id proteins, which is very low in adult tissues, can be reactivated in human cancers, and deregulated Id signaling may promote multiple attributes of malignancy. Id4 may function as a tumor suppressor or as an oncogene in different tumoral contexts. Overexpression of Id4 has been reported in glioblastoma multiforme, where it promotes the maintenance of cancer cell stemness and neovascularization. Id4 is also emerging as a lineage commitment oncogene that is highly expressed and amplified in a subset of basal-like breast cancers and may represent a powerful therapeutic target for this subset of tumors.
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