The regulation of PBXs and their emerging role in cancer

Abstract Pre‐B‐cell leukaemia transcription factor (PBX) proteins are a subfamily of evolutionarily conserved, atypical homeodomain transcription factors that belong to the superfamily of three amino acid loop extension (TALE) homeodomain proteins. Members of the PBX family play crucial roles in regulating multiple pathophysiological processes, such as the development of organs, congenital cardiac defects and carcinogenesis. The dysregulation of PBXs has been shown to be closely associated with many diseases, particularly cancer. However, the detailed mechanisms of PBX dysregulation in cancer progression are still inconclusive. In this review, we summarize the recent advances in the structures, functions and regulatory mechanisms of PBXs, and discuss their underlying mechanisms in cancer progression. We also highlight the great potential of PBXs as biomarkers for the early diagnosis and prognostic evaluation of cancer as well as their therapeutic applications. The information reviewed here may expand researchers’ understanding of PBXs and could strengthen the clinical implication of PBXs in cancer treatment.


| INTRODUC TI ON
Cancer is one of the most common diseases and the second leading cause of death in all countries worldwide. 1 According to data from global cancer statistics in 2020, new cancer cases were estimated to be over 19.3 million and the total number of cancer-related deaths more than 10.0 million worldwide. 2 The incidence and mortality of cancer are both rapidly growing, becoming a major barrier to increasing life expectancy. Although some important progress has been made in cancer treatment, it is still a major public health problem worldwide due to the lack of effective therapeutics. 3 Clinically, a large number of cancer patients are diagnosed at an advanced stage, with a poor prognosis. [4][5][6] This is mainly caused by difficulties involved in observing the early symptoms of cancer, deficiencies in effective prognostic evaluation methods and limited knowledge regarding the carcinogenesis mechanism. Therefore, it is important to elucidate the underlying mechanism of carcinogenesis, screen novel therapeutic targets of cancer and identify valuable biomarkers for the diagnosis and prognostic assessment.
Pre-B-cell leukaemia transcription factor (PBX) proteins are evolutionarily conserved, atypical homeodomain transcription factors that belong to the superfamily of three amino acid loop extension (TALE) homeodomain proteins. 7 Currently, four members of the PBX family, including PBX1, PBX2, PBX3 and PBX4, have been identified in the human genome. PBX proteins were initially identified as essential Hox cofactors playing crucial roles in early embryonic development and organogenesis 8 In recent years, an increasing number of studies have suggested that the dysregulation of PBXs is closely associated with many diseases, particularly cancer. [9][10][11] The high expression of PBXs has been observed in multiple solid tumours, such as prostate cancer (PCa), non-small-cell lung carcinoma (NSCLC) and glioma. [12][13][14] The aberrant expression and/or activity of PBXs contributes to cancer progression by regulating many aspects of cancer cell behaviour, including proliferation, apoptosis, cell cycle, epithelial-to-mesenchymal transition (EMT), invasion, metastasis and stemness. [15][16][17] These characteristics endow PBXs with great potential as therapeutic targets, and biomarkers for early diagnosis and prognostic evaluation in the clinical treatment of cancer patients.
Although PBXs have been shown to play crucial roles in various types of cancer, their detailed mechanisms and clinical implications in cancer progression are still unclear.
In this review, we summarize the recent findings in the structures, functions, and regulatory mechanisms of PBXs and focus on their underlying mechanisms in cancer progression. We also highlight the great potential of PBXs as biomarkers for the early diagnosis and prognostic evaluation of cancer and their therapeutic application.
Finally, we explore the future research directions of targeting PBXs for cancer therapy.

| Structures of PBXs
The PBX family consists of PBX1, PBX2, PBX3 and PBX4, which share similar conserved structures ( Figure 1). PBX1-3 is about 430 amino acid residues long, while PBX4 is shorter, missing 78 amino acid residues in the N-terminal domain and a 30-residue stretch in the C-terminal domain. PBXs contain a 60-residue-long homeodomain (HD) that mediates their binding to DNA or other proteins. 18 For instance, HPIP competitively bound to the HD domain of PBX1 to inhibit the formation of PBX1-HOX complexes. 19 Besides, the HD and C-terminal of PBX family members mediated their interaction with MyoD and bHLH proteins, thereby inducing muscle differentiation. 20,21 In another study, PBX1 was found to directly interact with PDX1 via its HD and C-terminal domain to form a heterodimer, which bound to the promoter of somatostatin gene to activate its transcription. 22 Furthermore, the HD and C-terminal of PBX3 mediated its binding to RNX to form a DNA-binding complex, thereby enhancing the transcription of downstream target genes. 23 In addition, the HD and C-terminal of PBX proteins could also mediate their interaction with Hox/HOM-C proteins to play a role in transcriptional programmes. 24,25 PBX family members also contain two nuclear localization signals (NLSs) in the HD region and two distinct nuclear export sequence (NES), 26 which mediates their subcellular distribution by modulating the balance between the import and export pathways. In addition, PBX proteins include two highly conserved protein-protein interaction domains, including PBC-A and PBC-B. 27 The PBC-A domain of PBX members could mediate their interaction with PREP/MEIS proteins to form transcriptional regulation complexes, which play a role during development. 28 PBC-A domain also mediated the interaction of PBX1 with CRM1, which is required for the nuclear export of PBX1, whereas PBC-B domain in PBX1 could be specifically phosphorylated by PKA, leading to the accumulation of PBX1 in nucleus. 29 Moreover, the region of amino acids 75-230 (including partial PBC-A domain and complete PBC-B domain) in PBX4 was found to mediate the interaction of PBX4 with MEIS and HDAC1, resulting in the reduction of HDAC-mediated transcriptional inhibition. 30 In addition, NMHCB competitively interacted with PBC-B region of PBX1, resulting in the cytoplasmic accumulation of PBX1. 31 F I G U R E 1 Structure diagram of PBX proteins. PBX proteins share similar conserved structure. PBC-A and PBC-B are their family conserved domains, which mediate the interaction of PBXs with other proteins, including HDAC1, PKA, CRM1, NMHCB and PREP/MEIS. HD is primarily responsible for direct interaction between PBXs and DNA, which also mediates their interaction with PDX1, RNX, HPIP, MyoD/bHLH and Hox/HOM-C proteins. The proteins interacting with PBXs are exhibited above or under the lines at the corresponding regions. PBX, pre-B-cell leukaemia transcription factor; NLS: nuclear localization signals; NES: nuclear export sequence; PBC, protein interaction domain; HD, homeodomain; PKA: protein kinase A; HDAC1, histone deacetylase 1; PDX1, pancreatic and duodenal homeobox 1; RNX, radical nephrectomy, MyoD, Myogenic determination gene number; bHLH, basic helix-loop-helix protein; CRM1, chromosome region maintenance 1; NMHCA, non-muscle heavy chain myosin lla; PbxIP1, PBX1-interacting Protein; OCT-1, octamer transcription factor 1; FOXC1, Forkhead box C1; PREP, prolyl endopetidase; MEIS, myeloid ecotropic viral insertion site; HOM-C, homeotic gene complex

| Functions of PBXs
The PBX proteins are key transcription factors of multiple gene regulatory networks and regulate embryonic development, especially the anteroposterior patterning of the main body axis and the limbs by inducing the transcription of target genes involved in cell proliferation, apoptosis and differentiation via interacting with a subset of Hox proteins. 7 PBXs also play a fundamental and pleiotropic role in the organogenesis of multiple organs, including the heart, lung, thymus, pancreas and spleen. 32 Moreover, PBXs are strongly associated with cytoskeleton assembly and/or regulation. 31 In addition, PBXs have been shown to play crucial roles in the processes of haematopoiesis and embryonic stem cell (ESC) pluripotency. For instance, PBX1 is an essential factor for maintaining haematopoietic stem cells and progenitor expansion. 33 PBXs in the splenic niche also have non-cell autonomous functions that contribute to the modulation of haematopoiesis partly by targeting KitL/SCF and Cxcl12/SDF-1. 34 Furthermore, PBX1 was found to be involved in ESC pluripotency by regulating Fgf8 expression in CCE ES cells. 35 The complicated functions of PBXs indicate that their dysregulation can lead to various diseases, particularly cancer. Indeed, the aberrant expression of PBXs has been observed in many types of cancer. [12][13][14] Thus, further studies are required to elucidate the pathological mechanisms and clinical applications of PBXs in cancer.

| Molecular mechanisms of PBXs regulation
Multiple mechanisms are involved in regulating PBXs expression and activity at different layers, such as transcription, post-transcription and post-translation ( Figure 2). Here, we exhibit several modes of PBXs regulation with a primary focus on the mechanisms of PBXs regulation in cancers.

| Regulation of PBXs at the transcription level
Retinoic acid (RA) is the most active form of vitamin A, and acts as a ligand for nuclear RA receptors (RARs) to regulate embryonic development, differentiation, and growth. It has been reported that the expression of PBXs was significantly upregulated by RA at the transcription level during endodermal and neuronal differentiation of P19 cells. 36

| Post-transcriptional regulation of PBXs by non-coding RNAs
Non-coding RNAs (ncRNAs) are essential regulatory factors that are involved in all pathological and physiological processes by regulating gene expression and chromatin structure. [40][41][42] According to their size and biogenesis, ncRNAs are divided into different classes, including microRNAs (miRNAs), circular RNAs (circRNAs) and long ncRNAs (lncRNAs). 6,43,44 In recent years, a large number of studies have shown that ncRNAs contribute to the post-transcriptional regulation of PBXs (Table 1).
MicroRNAs are short single-stranded ncRNA molecules that play critical roles in post-transcriptional gene regulation by repressing translation and/or promoting mRNA degradation via targeting the 3′-untranslated region (3′UTR) of messenger RNAs (mRNAs). 45,46 PBXs are direct targets of miRNAs in multiple malignant tumours. For instance, Muller et al. revealed that miR-198 significantly downregulated the expression of PBX1 by directly targeting the 3′UTR of its mRNA, resulting in reduced tumour growth, metastasis, and the increased survival of PCa patients. 47 PBX1 was also found to be a target of miR-31-3p in head and neck squamous cell carcinoma (HNSCC). 48 Xu et al. found that PBX2 was a downstream target of miR-1915-3p. MiR-1915-3 directly bound to the 3'UTR of PBX2 mRNA, subsequently downregulating its expression in lung cancer cells. 49 Moreover, PBX2 was identified as a target of miRNA let-7c in acute myeloid leukaemia (AML) cells. 50 PBX3 was also reported to be a direct target of let-7c, which could then bind to the 3'UTR of PBX3 and inhibit its expression in colorectal cancer. 51 In addition, PBX3 could also be regulated by a series of miRNAs, such as miR-144, miR-129-5p, miR-4458 and miR-320a, in multiple types of cancer. [52][53][54][55][56] All of these miRNAs have been shown to directly target the 3′UTR of PBX3.

| IMPLI C ATI ON OF PBX S IN C AN CER PROG RE SS I ON
The dysregulation of PBXs is closely associated with many diseases, particularly cancer. [9][10][11] It has been reported that the aberrant expression and activity of PBXs contribute to cancer progression by regulating gene regulatory networks involved in distinct cellular processes, including cell proliferation, apoptosis, cell cycle, EMT, invasion and metastasis as well as the stemness of cancer cells [15][16][17] ( Table 2). Thus, a comprehensive understanding of PBXs in cancer occurrence and development will provide novel insights for early diagnosis, prognostic evaluation and the design of treatment strategies for cancer patients.

| Roles of PBXs in regulating cancer-related signalling pathways
An increasing amount of evidence has demonstrated that PBXs play crucial roles in cancer progression by modulating cancer-related sig- and ERK1/2, leading to proliferation inhibition and apoptosis promotion in glioma. 79 In addition, in hepatocellular carcinoma (HCC) cells, PBX3 was found to mediate the effect of miR-302A on cell proliferation and apoptosis by targeting the expression of p-p38, p-ERK1/2 and p-JNK. 80 The PI3K/AKT signalling pathway is a classical carcinogenic pathway and contributes to many aspects of cancer progression, such as cell proliferation, metastasis and drug resistance. 81 The knockdown of PBX3 in cervical cancer cells was found to inhibit cell proliferation in vitro and tumour size and weight in vivo via the inactivation of the PI3K/AKT signalling pathway via downregulating p-AKT expression. 82 In addition, PBX3 was found to promote the invasion and metastasis of GC cells by upregulating p-AKT expression. 83 These findings suggest that PBXs might be core regulators in the signalling pathway networks during cancer progression.

| The effect of PBXs on cancer proliferation and apoptosis
The ability to maintain proliferation and escape apoptosis is the most

PBX3
Overexpression of PBX3 promoted the invasion and metastasis of GC cell by facilitating EMT process, possibly via the AKT signalling pathway. 83 NSCLC PBX1 PBX1 inhibited the proliferation of NSCLC cells and increased the phosphorylation of histone H3. Knockdown of PBX1 promoted the proliferation of NSCLC cells. 129

PBX2
The expression of PBX2 was associated with prognosis in NSCLC. Knockdown of PBX2 decreased VCM expression in NSCLC cells. 13 PCa PBX1 Overexpression of PBX1 promoted cell proliferation and enhanced the resistance of PCa cells to doxorubicin and cisplatin. USP9x was a PBX1-specific deubiquitinase and, which promoted the degradation of PBX1 by increasing its Lys-48-linked polyubiquitination. 12

PBX3
Androgen decreased the expression of PBX2 by upregulating Let-7d in PCa cells.

AML PBX2
The expression of PBX2 was regulated by let-7c and might contribute to the AML phenotype.

PBX3
High expression of PBX3 was associated with poor prognosis in AML patients. PBX3 knockdown improved the survival of leukemic mice and reduced the leukaemia burden via decreasing the capacity of LSCs and promoting LSC apoptosis. 105 Glioma PBX3 Knockdown of PBX3 suppressed proliferation and induced apoptosis in glioma cells, while PBX3 overexpression significantly facilitated glioma cell proliferation. The effect of PBX3 on proliferation depended on its regulation on cell cycle progression. 14

HCC PBX3
PBX3 mediated the effect of miR-302a on proliferation and apoptosis in HCC cells. 80 Melanoma PBX1 PBX1 was a target of PLZF. Knockdown of PBX1 by PLZF inhibited melanoma cell growth by reducing the expression of HoxB7 target genes, including angiopoietin-2 and MMP-9.

PBX2
PBX2 formed a dimer with HOXB7, and the dimer decreased the expression of c-FOS by upregulated miR-221 and miR-222 in melanoma cells. 118

PBX3
PBX3 was a target of miR-495. Knockdown of PBX3 inhibited the proliferation, migration and invasion of melanoma cells. 92 LC PBX3 PBX3 was closely associated with the stemness of hepatoma cells, and its degradation was in ubiquitin-proteasome system-independent manner. 104 OC PBX1 High PBX1 expression was associated with shorter survival in post-chemotherapy OC patients. Overexpression of PBX1 promoted CSC-like phenotypes and enhanced the resistance of OC cells to platinum by upregulating STAT3 via binding to its promoter. 76

PBX3
PBX3 silencing inhibited the progression of OC cells. PBX3 mediated the effect of HCG11/miR-144-3p on OC progression. 60 The regulation of PBXs on proliferation and apoptosis may be partially due to their positive modulation of cell cycle progression.
It has been reported that PBX1 knockdown in ccRCC cells inhibited cell viability and proliferation by blocking cell cycle progression.
Mechanistically, silencing PBX1 downregulated Cyclin D1 expression via decreasing the p-STAT3 level. 74  cells. 63 Collectively, these data confirm that PBXs can act as oncoproteins to promote cell proliferation and/or inhibit apoptosis in different types of cancer, at least partially due to their regulation on cell cycle progression, but the detailed mechanisms are still not fully understood. Nonetheless, elucidating their underlying mechanism in cancer proliferation and apoptosis would help to precisely utilize PBXs-based therapeutics in particular type of cancer.

| PBXs and EMT in cancer
Epithelial-to-mesenchymal transition is an essential biological process for maintaining the physiological functions of cells and its aberrant activation of EMT endows cancer cells with enhanced invasive characteristics and increased drug resistance. Cells undergoing EMT exhibit decreased epithelial marker (e.g. E-cadherin) and increased mesenchymal marker (e.g. vimentin and N-cadherin) levels. 1 PBXs have been shown to be crucial regulators of EMT during cancer

CRC PBX3
Overexpression of PBX3 promoted the migration and invasion of CRC cells by activating the MAPK signalling pathway. 78 LSCC PBX3 PBX3 promoted the malignant progression by enhancing EMT progress in LSCC. PBX3 mediated the effect of circCORO1C/let-7c-5p on the malignant progression of LSCC. 69 Acute erythroleukaemia PBX2 PBX2 was downregulated in acute erythroleukaemia by hsa-let-7c-5. 121 Ewing sarcomas PBX2 Knockdown of GPR64 decreased the expression of PBX2 in Ewing sarcomas cells. 131 PBX3 PBX3 was associated with bad prognosis in Ewing sarcomas 132 HNSCC PBX1 Low expression of PBX1 was closely associated with poor prognosis in HNSCC. PBX1 was a target of miR-31-3p. 48 Lung cancer PBX2 PBX2 could mediate the negative regulation of miR-1915-3p on apoptosis in lung cancer. 49 PBX3 PBX3 overexpression facilitated the proliferation and metastasis of lung cancer cells.
Silencing of PBX3 exhibited an opposite effect. PBX3 was a target of miR-144. 120 ccRCC PBX1 High expression of PBX1 was associated with poor OS in ccRCC. PBX1 knockdown suppressed ccRCC cell viability, proliferation and cell cycle progression by inactivating the JAK2/STAT3 signalling pathway. 74 Oesophageal cancer PBX1 PBX1 acted as pioneer transcription factor to mediate the binding of FoxC1 to ZEB2 promoter in oesophageal cancer cells. 88 Neuroblastoma PBX1 Overexpression of PBX1 inhibited proliferation and anchorage-independent growth, and promoted RA-dependent and -independent differentiation in neuroblastoma. Knockdown of PBX1 produced an aggressive growth phenotype and RA resistance. 110 Hodgkin lymphoma PBX1 PBX1 was upregulated in Hodgkin lymphoma and affected the differentiation of Hodgkin lymphoma cells by activating NFIB and TLX2. 133 Embryonal carcinoma PBX1 PBX2 PBX3 The expression of PBX1, PBX2, and PBX3 was upregulated during endodermal and neuronal differentiation of embryonal carcinoma P19 cells in a RAR-dependent subtype-unspecific manner following RA treatment. 36 Cervical cancer PBX2 rs2856437 at PBX2 was closely associated with invasive squamous cervical cancer 134

PBX3
Upregulation of PBX3 promoted the proliferation of cervical cancer cells by activating the AKT signalling pathway. High expression of PBX3 was associated with poor prognosis in cervical cancer.  pathway. 78 In addition, PBX3 was found to mediate the effects of several miRNAs, such as miR-144, miR-495 and miR-98, on invasion and metastasis in cancer. 53,92,93 The molecular mechanisms involved in invasion and metastasis are very complicated and have not been fully understood; however, these findings strongly suggest that PBXs are effective targets for preventing cancer invasion and metastasis as well as cancer progression.

| PBXs in tumour angiogenesis
Angiogenesis is the process where new capillaries grow from preexisting blood vessels, which is crucial for the growth and metastasis of a large number of solid tumours. 94

Cancer stem cells (CSCs) play crucial roles in cancer progression
and are recognized as one of the main causes of drug resistance, metastasis, and recurrence in cancer. [97][98][99] They are also important targets for cancer therapy. 100

| Regulation effect of PBXs on chemotherapy sensitivity in cancer
The development of drug resistance is considered to be one of the main causes of chemotherapy failure, cancer recurrence and finally patient death. 106 In-depth investigation into the regulatory mecha-  Accumulating evidence revealed that the aberrant expression patterns of PBXs endow them with great potential as biomarkers for the diagnosis, prognosis and treatment of cancer (Table 3) 110 These data confirmed that high PBX1 expression is an independently and favourable prognostic biomarker for NB patients. Moreover, PBX1 expression is associated with the sensitivity or resistance of NB cells to retinoid and can be used as a biomarker to predict the response of NB cells to retinoid differentiation therapy. 111 In another study, Qiu et

CO N FLI C T O F I NTE R E S T
The authors declare that they have no competing interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data sharing not applicable to this article as no data sets were generated or analysed during the current study.