|Note|| RhoBTB2 is one of the three members of the RhoBTB family in vertebrates. The RhoBTB family was identified during the study of the genes encoding Rho-related proteins in the lower eukaryote Dictyostelium discoideum (Rivero et al., 2002). All three RhoBTB proteins may be implicated in tumorigenesis.|
| || Architecture of RhoBTB proteins. The figure shows the three human (Hs) RhoBTB subfamily members as well as the Drosophila (Dm) and Dictyostelium (Dd) orthologues. The simplified phylogenetic tree on the left illustrates the relationship among the proteins (overall percentage similarity between branches). The different domains are indicated with colours.|
|Description|| RhoBTB2 is 727 amino acids long. All RhoBTB proteins share the same domain architecture: a GTPase domain is followed by a proline-rich region, a tandem of two BTB domains and a C-terminal region (Figure 2) |
The GTPase domain is Rho-related and contains a Rho insert that is longer than usual, two insertions and one deletion, as well as a few deviations from the GTPase consensus of most Rho GTPases. Chang et al. (2006) reported the inability of this domain to bind GTP. However the construct used in that study lacked one of the GTPase motifs, G5. Subsequent work with the full-length protein and a more complete GTPase domain showed that this domain is actually able to bind GTP (Manjarrez et al., 2014).
The proline-rich region links the GTPase to the first BTB domain. This region could act as a SH3 domain-binding site.
The BTB domain (broad complex, tramtract and bric-a-brac) is an evolutionary conserved protein-protein interaction domain that participates in homomeric and heteromeric associations with other BTB domains. The BTB domain was also identified as a component of multimeric cullin3-dependent ubiquitin ligase complexes. The first BTB domain is bipartite, being interrupted by an insertion of unknown function. The BTB domains of RhoBTB allow the formation of homodimers and of heterodimers with other proteins of the RhoBTB family (Berthold et al., 2008).
The C-terminus is a region conserved in all members of the RhoBTB subfamily. It predictably folds as 4 consecutive alpha-helices and one beta-strand and may constitute a RING finger domain (Manjarrez et al., 2014). Many RING finger domains function as ubiquitin ligases. RhoBTB2 does not bear a CAAX motif that is typical for classical Rho GTPases and serves for localization of the protein to membranes.
|Expression|| RHOBTB2 is weakly expressed, with relatively higher levels in neural and cardiac tissues. It is also expressed in fetal tissues (Ramos et al., 2002; Nagase et al., 1998). During mouse embryogenesis high and specific expression has been observed in the central and peripheral nervous system and comparatively weaker in the gut, but the mRNA becomes undetectable at embryonic day 18.5 (St-Pierre et al., 2004). One study has addressed the expression of RHOBTB2 during mammogenesis in the mouse and found that transcripts are expressed at low but constant levels. Attempts to study the spatial pattern of expression in the mammary gland using in situ hybridization were inconclusive because of undetectable mRNA levels (St-Pierre et al., 2004). |
RhoBTB2 levels increase upon initiation of prophase and decrease at telophase. RhoBTB2 levels also increase during drug-induced apoptosis. Both effects depend on the E2F1 transcription factor (Freeman et al., 2007).
RHOBTB2 is a TP53 candidate target gene (Garritano et al., 2013).
Expression of RHOBTB2 has been found decreased in breast, lung, bladder and stomach cancers and in osteosarcomas (Hamaguchi et al., 2002; Knowles et al., 2005; Cho et al., 2008; Shi et al., 2008; Dong et al., 2012; Han et al., 2013; Jin et al., 2013) as well as in cell lines derived from breast, lung and bladder tumors and head and neck squamous cell carcinomas (Hamaguchi et al., 2002; Knowles et al., 2005; McKinnon et al., 2008) Loss of RHOBTB2 expression has been found to correlate with promoter methylation in breast and bladder cancers (Shi et al., 2008; Mehri Hajikhan et al., 2012; Han et al., 2013).
|Localisation|| The localisation of the endogenous RhoBTB2 protein has not been investigated extensively. In cells ectopically expressing RhoBTB2 the protein tends to form aggregates in the cytoplasm (Aspenström et al., 2004 ; Berthold et al., 2008). When expressed at moderate levels it displays a vesicular pattern, frequently in the proximity of microtubules (Chang et al., 2006 ; Berthold et al., 2008).|
|Function|| RHOBTB2 was initially described as a gene homozygously deleted in breast cancer samples and was proposed as a candidate tumor supressor gene (Hamaguchi et al., 2002). The mechanisms by which RhoBTB2 exerts this and other roles remain speculative. When expressed in cancer cell lines, RhoBTB2 inhibits proliferation, induces apoptosis and inhibits cell migration and invasion (Mao et al., 2011; Jin et al., 2013). |
Following functions have been proposed for RhoBTB2:
1. RhoBTB2 as adaptor of cullin3-dependent ubiquitin ligases. The first BTB domain binds to the N-terminal region of Cullin 3, but not other cullins. RhoBTB2 is itself a substrate for the Cullin 3-based ubiquitin ligase complex (Wilkins et al., 2004). RhoBTB proteins appear to exist in an inactive state through an intramolecular interaction of the BTB domain region with the GTPase domain (Berthold et al., 2008). This model has been refined recently to show that the HSP90AA1 (Hsp90) chaperone machinery unlocks RhoBTB, enabling GTP binding and interaction with Cullin 3 and the COPS8 (COP9) signalosome. COP9 deneddylates Cullin 3 and stabilises the complex (Manjarrez et al. 2014).
RhoBTB2, like RhoBTB1 and RhoBTB3, interacts with LLRC41 (leucine rich repeat containing 41, MUF1). MUF1 is a nuclear protein and carries a BC-box that functions as a linker in multicomponent Cullin 5-dependent ubiquitin ligase complexes (Schenková et al., 2012). MUF1 may be a substrate for RhoBTB-Cullin 3 ubiquitin ligase complexes. The function of MUF1 is unknown, but it is suspected to be involved in the DNA damage response.
2. RhoBTB2, cell growth and apoptosis. Overexpression of RhoBTB2 in the breast cancer cell line T-47D (a cell line that lacks RHOBTB2 transcripts) effectively suppressed cell growth in vitro (Hamaguchi et al., 2002). Overexpression of RhoBTB2 in osteosarcoma cells significantly arrested cells at G1 and resulted in apoptosis (Jin et al., 2013). In the thyroid carcinoma cell line SW579 treatment with recombinant RhoBTB2 for 24 hours inhibited proliferation and provoked an increase of the apoptotic ratio through the mitochondrial apoptotic signalling pathway (Wang et al., 2015), but it's not clear how the exogenously added protein exerts those actions.
It was shown that overexpression of RhoBTB2 leads to a short-term increase in cell cycle progression and proliferation, but long-term expression has a negative effect on proliferation (Freeman et al., 2007). The growth arrest effect has been explained by the downregulation of CCND1 (cyclin D1). Cyclin D1 is upstream of cyclin E, and the overexpression of any of both prevented the growth arrest effect of RhoBTB2 (Yoshihara et al., 2007). The effect on cyclin D1 is probably post-transcriptional, but only partially dependent on proteasomal degradation (Collado et al., 2007). RHOBTB2 has been identified as a target of the E2F1 transcription factor. RhoBTB2 levels also increase during drug-induced apoptosis in an E2F1-dependent manner, and the downregulation of RHOBTB2 delays the onset of apoptosis (Freeman et al., 2007).
3. RhoBTB2 and chemokine expression. Downregulation of RhoBTB2 by RNA interference in primary lung epithelial cells causes a decrease in CXCL14 mRNA expression. The same effect was observed in keratinocytes and is apparently independent of Cullin 3-mediated protein degradation (McKinnon et al., 2008).
4. RhoBTB2 and vesicle transport. Knockdown of endogenous RhoBTB2 hindered the ER to Golgi apparatus transport of a VSVG-GFP reporter and resulted in the altered distribution of the fusion protein. Ectopic RhoBTB2 distributes in a vesicular pattern occasionally adjacent to microtubules and an intact microtubule network seems to be required for the mobility of RhoBTB2 (Chang el al., 2006).
5. RhoBTB2 and the actin filament system. RhoBTB2 displays only a moderate influence on the morphology and actin organisation of porcine aortic endothelial cells upon ectopic expression. It does not interact with the GTPase-binding domain of WASP, PAK1 or RTKN (Rhotekin), which are well-known effectors of many typical Rho GTPases (Aspenström et al., 2004).
|Homology|| There are three RhoBTB proteins in vertebrates: RhoBTB1, RhoBTB2 and RhoBTB3 (Figure 2). RhoBTB2 is very similar to RhoBTB1, while RhoBTB3 displays very low similarity to these. Orthologues have been found in amoebae and in insects but they are absent in plants and fungi.|
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|Entity|| Breast cancer|
|Note|| RHOBTB2 was found homozygously deleted in 3.5% of 200 breast tumors. A mutation analysis revealed two somatic missense mutations (E5 G>A D299N and E9 C>A P647T) and one missense mutation (E5 A>C D368A). In the same study expression of RHOBTB2 appeared extinguished in about 42% of 19 breast cancer cell lines (Hamaguchi et al., 2002). |
A more extensive mutation analysis of 100 sporadic breast cancers revealed some polymorphisms as well as two somatic mutations in the promoter (-238G>A, -121C>T) and 5'UTR (+48G>A) of RHOBTB2. The analysis of 17 CC: TXT: familial breast tumours ID: 10062> negative for BRCA1/BRCA2mutations failed to reveal additional mutations in the coding region of RHOBTB2 (Ohadi et al., 2007).
No mutations were found in the promoter or exon 7 in 32 breast cancers of a Han Chinese population. This study revealed an intronic polymorphism common in this population; the variant IVS7 + 53C >G correlated with HER2 and p53 expression but not with age, tumor stage or estrogen or progesterone receptor expression (Fu et al., 2014.
Using semi-quantitative PCR RHOBTB2 mRNA was found absent (56% of 87 breast tumors vs. 9% of normal tissue) or significantly reduced. RHOBTB2 Promoter methylation was detected in over 33% of a large collection of breast tumor samples and was rare in normal tissue. Loss of RHOBTB2 expression correlated with promoter methylation. Moreover, RHOBTB2 promoter methylation associated with more advance tumor stages, p53 mutation and HER2-positive status (Han et al., 2013). A similar correlation between promoter methylation and RHOBTB2 downregulation has been reported in a sample of 50 paired breast cancer and normal tissues. In this study promoter methylation was found more frequently associated to progesterone receptor negative tumors (Tang et al., 2014). Significantly more frequent promoter methylation was also found in a collection of 50 breast tumor (34%) and blood samples (46%) compared to normal samples (20% or less) in another study (Mehri Hjikhan et al., 2012).
A frequent pattern of loss of RHOBTB2, CDH1 and TP53 together with gain of COX2 and MYC was found in a single cell in situ hybridization analysis of 13 samples simultaneously carrying ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC) of the breast. DCIS is the precursor lesion for invasive breast cancer. The study aimed at understanding the dynamics of genomic alterations in the progression from DCIS to IDC and included several oncogenes and tumor suppressor genes (Heselmayer-Haddad et al., 2012).
|Oncogenesis|| RHOBTB2 was proposed as a candidate tumor suppressor gene based on the fact that its re-expression in T-47D (a breast cancer cell line that lacks RHOBTB2 transcripts) caused growth inhibition, whereas expression of the mutant D299N did not have the same effect. Neither the D299N nor a D368A mutant found in some tumors presented abolished binding to Cullin 3 (Hamaguchi et al., 2002). Overexpression of RhoBTB2 in T-47D inhibits cell proliferation and colony formation while promoting apoptosis, but was found not to influence the invasion and migration ability of the cells in a Transwell system (Mao et al., 2011). |
The mutations found in the promoter and 5'UTR of RHOBTB2 in some breast tumors might affect regulation of gene expression (Ohadi et al., 2007). Lack of RHOBTB2 expression in T-47D is apparently due to RHOBTB2 promoter methylation (Han et al., 2013). Methylation of RHOBTB2 and other genes in peripheral blood is a potential epigenetic marker for predicting the risk of breast cancer development (Khakpour et al., 2015).
Ectopic expression of RHOBTB2 in two human metastatic breast cancer cell lines, MDA-MB-231 and MDA-MB-435, inhibits cell migration and invasiveness through a mechanism that involves upregulation of the metastasis suppressor BRMS1 and decreased phosphorylation of EZR (ezrin) and Akt2 (Ling et al., 2010). Ezrin is a cytoskeleton and signaling molecule that regulates cell adhesion, migration and invasion. Akt2 is a kinase involved in invasiveness of breast cancer cells and is able to phosphorylate ezrin (Freeman and Cress 2010)
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|Entity|| Lung cancer|
|Note|| A mutation analysis revealed a missense mutation (E5 T>G Y284D) in a lung tumour cell line. In the same study expression of RHOBTB2 appeared extinguished in about 50% of 14 lung cancer cell lines (Hamaguchi et al., 2002). |
In an immunohistochemistry study of 172 tissue samples from different subtypes of lung adenocarcinomas frequent (70% of tumors) downregulation of RHOBTB2 was found that correlated with the degree of invasiveness (Dong et al, 2012).
|Oncogenesis|| The Y284D mutant protein presents abolished binding to Cullin 3 and has consequently a longer half-life than the wild type protein. The mutation resides in the dimerisation interface of the first BTB domain and could prevent proper folding (Wilkins et al., 2004).|
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|Entity|| Gastric cancer|
|Note|| In a study on primary gastric cancers loss of heterozygosity was found in 29% of 95 tumors. Sequence analysis identified several polymorphisms and one missense somatic mutation (E5 C>T R275W) of unknown effect (Cho et al., 2008).|
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|Entity|| Bladder cancer|
|Note|| A loss of heterozygosity (LOH) and mutation analysis on 54 tumour samples and 32 cell lines of bladder cancer revealed LOH in the target region in 42% of informative tumours and 38% of cell lines. Sequence analysis revealed numerous polymorphisms and one missense somatic mutation (E5 G>C E349D) of unknown effect. One polymorphism (E7 G>A G561S) may have some functional effect. In addition, expression of RHOBTB2 was found reduced by 2 to 20-fold in 9 of 12 cell lines with predicted LOH in the region of interest (Knowles et al. 2005). Significantly higher RHOBTB2 promoter methylation correlated with decreased expression compared to normal tissue was reported in a study of 75 bladder cancer samples (Shi et al., 2008).|
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|Entity|| Head and neck squamous cell carcinoma|
|Note|| Expression of RHOBTB2 was found reduced in four clonal keratinocyte cell lines derived from patients with HNSCC. This was accompanied by reduced expression of the chemokine CXCL14 (McKinnon et al., 2008).|
|Oncogenesis|| RhoBTB2 seems to be required for expression of the chemokine CXCL14 (McKinnon et al., 2008). CXCL14 controls leukocyte migration and angiogenesis and its expression is frequently lost in diverse epithelial tumours, including most HNSCCs.|
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|Note|| Expression of RhoBTB2 was analysed in 121 osteosarcoma specimens and was found decreased compared to normal regions of the specimens. Expression correlated with the primary location and local recurrence of the tumor (Jin et al., 2013).|
|Oncogenesis|| RhoBTB2 may affect the cell cycle. Overexpression of RhoBTB2 in osteosarcoma cells significantly arrested cells at G1 and resulted in apoptosis (Jin et al., 2013).|
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