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Targeting the HOX/PBX dimer in breast cancer
Abstract
The HOX genes are a family of closely related transcription factors that help to define the identity of cells and tissues during embryonic development and which are also frequently deregulated in a number of malignancies, including breast cancer. While relatively little is known about the roles that individual HOX genes play in cancer, it is however clear that these roles can be both contradictory, with some members acting as oncogenes and some as tumor suppressors, and also redundant, with several genes essentially having the same function. Here, we have attempted to address this complexity using the HXR9 peptide to target the interaction between HOX proteins and PBX, a second transcription factor that serves as a common co-factor for many HOX proteins. We show that HXR9 causes apoptosis in a number of breast cancer-derived cell lines and that sensitivity to HXR9 is directly related to the averaged expression of HOX genes HOXB1 through to HOXB9, providing a potential biomarker to predict the sensitivity of breast tumors to HXR9 or its derivatives. Measuring the expression of HOX genes HOXB1-HOXB9 in primary tumors revealed that a subset of tumors show highly elevated expression indicating that these might be potentially very sensitive to killing by HXR9. Furthermore, we show that while HXR9 blocks the oncogenic activity of HOX genes, it does not affect the known tumor-suppressor properties of a subset of HOX genes in breast cancer.
... Morgan and colleagues inhibited HOX-PBX interaction with short peptides mimicking the hexapeptide (referred to as HRX9), and showed that HRX9 inhibits tumour growth in many cancerous cell types [125][126][127][128][129]. In breast cancer, cell lines with high HOXB1-9 expression are most sensitive to HXR9. ...
... Morgan and colleagues inhibited HOX-PBX interaction with short peptides mimicking the hexapeptide (referred to as HRX9), and showed that HRX9 inhibits tumour growth in many cancerous cell types [125][126][127][128][129]. In breast cancer, cell lines with high HOXB1-9 expression are most sensitive to HXR9. In vivo, mice xenografted with the TNBC MDA-MB-231 cell line (that expresses HOXB1-9) and injected with HXR9 showed tumour growth retardation and longer survival [125]. This supports that the HOX-PBX interaction is crucial to the oncogenic activities of multiple HOX proteins, in a large repertoire of cancers, including solid or haematological malignancies [129]. ...
... HOXB1 is a direct target of HOXA1 and PBX during hindbrain segmentation and rhombomere 4 identity establishment [49,50]. It is over-expressed in some breast tumours, and HOXB1-PBX interaction blocking using the peptide HRX9 decreased tumour growth [125]. The EPHA2 ephrin receptor gene is regulated by HOXA1 and PBX1 in establishing rhombomere boundaries during hindbrain segmentation [223], and a recognized inducer of cell proliferation during mammary gland development [224]. ...
More than 25 years ago, the first literature records mentioned HOXA1 expression in human breast cancer. A few years later, HOXA1 was confirmed as a proper oncogene in mammary tissue. In the following two decades, molecular data about the mode of action of the HOXA1 protein, the factors contributing to activate and maintain HOXA1 gene expression and the identity of its target genes have accumulated and provide a wider view on the association of this transcription factor to breast oncogenesis. Large-scale transcriptomic data gathered from wide cohorts of patients further allowed refining the relationship between breast cancer type and HOXA1 expression. Several recent reports have reviewed the connection between cancer hallmarks and the biology of HOX genes in general. Here we take HOXA1 as a paradigm and propose an extensive overview of the molecular data centered on this oncoprotein, from what its expression modulators, to the interactors contributing to its oncogenic activities, and to the pathways and genes it controls. The data converge to an intricate picture that answers questions on the multi-modality of its oncogene activities, point towards better understanding of breast cancer aetiology and thereby provides an appraisal for treatment opportunities.
... Morgan et al. have found HXR9 peptides specifically target the interaction between HOX and PBX (78,168). HXR9 inhibits HOX function by preventing its binding to PBX, which leads to apoptosis in multiple mouse breast cancer derived cell lines (168). ...
... Morgan et al. have found HXR9 peptides specifically target the interaction between HOX and PBX (78,168). HXR9 inhibits HOX function by preventing its binding to PBX, which leads to apoptosis in multiple mouse breast cancer derived cell lines (168). In addition, the interaction between HOX and HXR9 has been shown to cause apoptosis in numerous cancers, including melanoma, mesothelioma, myeloma, renal cancer, prostate cancer, lung cancer, ovarian cancer, pancreatic cancer, squamous cell cancer of the head and neck, and oral cancer (58,78,95,98,168). ...
... HXR9 inhibits HOX function by preventing its binding to PBX, which leads to apoptosis in multiple mouse breast cancer derived cell lines (168). In addition, the interaction between HOX and HXR9 has been shown to cause apoptosis in numerous cancers, including melanoma, mesothelioma, myeloma, renal cancer, prostate cancer, lung cancer, ovarian cancer, pancreatic cancer, squamous cell cancer of the head and neck, and oral cancer (58,78,95,98,168). Kaspar and Reichert have already used HOX/HXR9-based treatments in cancer treatments, and Morgan and others are exploring a variant of HXR9 for intratumoral injection in clinical trials (168). ...
The homeobox (HOX) genes encoding an evolutionarily highly conserved family of homeodomain-containing transcriptional factors are essential for embryogenesis and tumorigenesis. HOX genes are involved in cell identity determination during early embryonic development and postnatal processes. The deregulation of HOX genes is closely associated with numerous human malignancies, highlighting the indispensable involvement in mortal cancer development. Since most HOX genes behave as oncogenes or tumor suppressors in human cancer, a better comprehension of their upstream regulators and downstream targets contributes to elucidating the function of HOX genes in cancer development. In addition, targeting HOX genes may imply therapeutic potential. Recently, novel therapies such as monoclonal antibodies targeting tyrosine receptor kinases, small molecular chemical inhibitors, and small interfering RNA strategies, are difficult to implement for targeting transcriptional factors on account of the dual function and pleiotropic nature of HOX genes-related molecular networks. This paper summarizes the current state of knowledge on the roles of HOX genes in human cancer and emphasizes the emerging importance of HOX genes as potential therapeutic targets to overcome the limitations of present cancer therapy.
... HXR9 was first shown to be cytotoxic to melanoma cell lines and primary melanoma cells and registered a reduction of B16F10 murine melanoma tumours growth in an orthotropic model [42]. Other experimental studies reported that HXR9 was able to inhibit the growth of several tumour types in mouse xenograft models, including non-small cell lung [43], breast [44], ovarian [44], and prostate cancer [45], and mesothelioma [46], melanoma [47], and meningioma [48]. ...
... HXR9 was first shown to be cytotoxic to melanoma cell lines and primary melanoma cells and registered a reduction of B16F10 murine melanoma tumours growth in an orthotropic model [42]. Other experimental studies reported that HXR9 was able to inhibit the growth of several tumour types in mouse xenograft models, including non-small cell lung [43], breast [44], ovarian [44], and prostate cancer [45], and mesothelioma [46], melanoma [47], and meningioma [48]. ...
... However, the mechanism associated with HOX-PBX inhibition and the resulting cell death through employing the HTL001 peptide is still to be fully elucidated, although generally in most solid tumours cell death is mediated by apoptosis [42,[44][45][46]50]. ...
In the last decades, HOX proteins have been extensively studied due to their pivotal role in transcriptional events. HOX proteins execute their activity by exploiting a cooperative binding to PBX proteins and DNA. Therefore, an increase or decrease in HOX activity has been associated with both solid and haematological cancer diseases. Thus, inhibiting HOX-PBX interaction represents a potential strategy to prevent these malignancies, as demonstrated by the patented peptide HTL001 that is being studied in clinical trials. In this work, a computational study is described to identify novel potential peptides designed by employing a database of non-natural amino acids. For this purpose, residue scanning of the HOX minimal active sequence was performed to select the mutations to be further processed. According to these results, the peptides were point-mutated and used for Molecular Dynamics (MD) simulations in complex with PBX1 protein and DNA to evaluate complex binding stability. MM-GBSA calculations of the resulting MD trajectories were exploited to guide the selection of the most promising mutations that were exploited to generate twelve combinatorial peptides. Finally, the latter peptides in complex with PBX1 protein and DNA were exploited to run MD simulations and the ΔGbinding average values of the complexes were calculated. Thus, the analysis of the results highlighted eleven combinatorial peptides that will be considered for further assays.
... This peptide mimics the 'hexapeptide' sequence in HOX proteins and penetrates cells efficiently. [23][24][25][26] In the present study, we detected whether HXR9 could block HOX/PBX interaction in ESCC cells through coimmunoprecipitation and fluorescence colocalization assay. As a control, a second peptide CXR9 was used which lacks a functional hexapeptide portion. ...
... The functional redundancy of HOX is derived from the sequence similarity of the homeodomain in HOX protein and interaction with defined cofactors which stabilize HOX and modulate specificity of target DNA binding. HXR9, an inhibitor of the interaction between HOX and cofactor PBX, targets a large subset of HOX proteins (members of paralogue groups 1-9), which was previously shown to cause apoptosis and inhibit tumor survival in a variety of tumor types, [23][24][25][26] implying that the HOX/ PBX interaction is a potential target for therapy. In the present study, we also showed that treating ESCC cells with HXR9 caused apoptosis and inhibited cell proliferation and tumor growth in all of the lines tested. ...
... In the present study, we also showed that treating ESCC cells with HXR9 caused apoptosis and inhibited cell proliferation and tumor growth in all of the lines tested. It has been reported that a rapid increase in c-fos expression as a response to HOX/PBX inhibition was activated to induce apoptosis, [23][24][25]33 which was also confirmed in our study. In addition, we found that PI3K-AKT and JAK-STAT pathway repression may be a response to antiproliferation and proapoptosis. ...
Homeobox genes are known to be classic examples of the intimate relationship between embryogenesis and tumorigenesis, which are a family of transcriptional factors involved in determining cell identity during early development, and also dysregulated in many malignancies. Previously, HOXB7, HOXC6 and HOXC8 were found abnormally up‐regulated in Esophageal squamous cell carcinoma (ESCC) tissues compared with normal mucosa and seen as poor prognostic predictors for ESCC patients, and were shown to promote cell proliferation and anti‐apoptosis in ESCC cells. These three HOX members have a high level of functional redundancy, making it difficult to target single HOX gene. This study is to explore whether ESCC cells are sensitive to HXR9 disrupting the interaction between multiple HOX proteins and their co‐factor PBX, which is required for HOX functions. ESCC cell lines(KYSE70,KYSE150,KYSE450) were treated with HXR9 or CXR9, Co‐immunoprecipitation and Immunofluorescent co‐localization were performed to observe HOX/PBX dimer formation. To further investigate whether HXR9 disrupts the HOX pro‐oncogenic function, CCK8 assay and colony formation assay were performed, apoptosis were assessed by flow cytometry, and tumor growth in vivo was investigated in xenograft model. RNA‐seq was used to study the transcriptome of HXR9‐treated cells. The results showed that HXR9 blocked HOX/PBX interaction, leading to subsequent transcription alteration of their potential target genes, which are involved in JAK‐STAT activation and apoptosis inducement. Meanwhile, HXR9 presented anti‐tumor phenotype, such as inhibiting cell proliferation, inducing cell apoptosis and retarding tumor growth significantly. Therefore, it suggested that targeting HOX/PBX may be a novel effective treatment for ESCC.
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... Recent studies have highlighted the therapeutic potential of inhibiting the interaction between HOX proteins and a common group of co-factors, the PBX proteins, using the synthetic peptide HXR9 [8]. The consistent phenotypic effect reported on inhibition of HOX-PBX interactions is induction of apoptosis that has been reported in a number of malignant cell types [9][10][11][12][13]. This has been associated with upregulation of c-Fos protein, which, whilst most often regarded as a proto-oncogene, may in some circumstances be pro-apoptotic [14]. ...
... The discovery that many HOX genes (particularly paralogous groups 1-8) require co-factor binding for stable interaction with DNA opens the potential for interfering with this interaction pharmaceutically using the HXR9 peptide. The effects of HXR9 have been demonstrated in a number of solid malignancies in vitro and in vivo [9,11,12,17]. In this paper, we demonstrate the sensitivity of oral cancer cells to this agent, similar to that reported for other cancers, but in addition, we demonstrate similar effects on cells derived from potentially malignant oral lesions and the lack of effect on normal oral keratinocytes. ...
... In this paper, we demonstrate the sensitivity of oral cancer cells to this agent, similar to that reported for other cancers, but in addition, we demonstrate similar effects on cells derived from potentially malignant oral lesions and the lack of effect on normal oral keratinocytes. This measure of selectivity was not seen on comparison of breast cancer cell lines with MCF10A cells where the sensitivity of three of the five cell lines tested was no different from the non-malignant MCF10A cells [11]. The data also suggests that HOX-PBX inhibition may be an effective strategy to prevent the development of OSCC due to its selective toxicity in PMOL cells. ...
Background:
High HOX gene expression has been described in many cancers, including oral squamous cell carcinoma and the functional roles of these genes are gradually being understood. The pattern of overexpression suggests that inhibition may be useful therapeutically. Inhibition of HOX protein binding to PBX cofactors by the use of synthetic peptides, such as HXR9, results in apoptosis in multiple cancers.
Methods:
Activity of the HOX-PBX inhibiting peptide HXR9 was tested in immortalised normal oral (NOK), potentially-malignant (PMOL) and squamous cell carcinoma (OSCC) cells, compared to the inactive peptide CXR9. Cytotoxicity was assessed by LDH assay. Expression of PBX1/2 and c-Fos was assessed by qPCR and western blotting. Apoptosis was assessed by Annexin-V assay.
Results:
PMOL and OSCC cells expressed PBX1/2. HOX-PBX inhibition by HXR9 caused death of PMOL and OSCC cells, but not NOKs. HXR9 treatment resulted in apoptosis and increased expression of c-Fos in some cells, whereas CXR9 did not. A correlation was observed between HOX expression and resistance to HXR9.
Conclusion:
Inhibition of HOX-PBX interactions causes selective apoptosis of OSCC/PMOL, indicating selective toxicity that may be useful clinically.
... Examples include HOXA4, the expression of which can block the spread of ovarian cancer cells [31], HOXA5 that has been shown to stabilize the P53 protein in breast cancer cells [32] and promote an epithelial phenotype [33], and HOXC8 which is inversely related to progression in ovarian cancer [31]. Intriguingly it seems that the regulation of HOX tumour suppressor targets may involve HOX binding to the promoter or enhancer region without PBX, as the identified binding sites are often for HOX only [33,34], or the HOX protein might have an additional function independent of that as a transcription factor [35]. ...
... HXR9 was originally shown to be cytotoxic to melanoma cell lines and primary melanoma cells and was reported to reduce the growth of B16F10 murine melanoma tumours in an orthotropic model [25]. Subsequently, HXR9 was shown to inhibit the growth of a range of tumour types in mouse xenograft models, including non-small cell lung [70], breast [34], ovarian [71], and prostate cancer [72], and mesothelioma [42], melanoma [73], and meningioma [74] ( Table 1). ...
... The mechanism by which HOX/PBX inhibition causes cell death remains to be fully elucidated (Figure 1). In most solid tumours cell death is mediated by apoptosis [25,34,42,71,72], although in a number of cancers, including some types of renal cancer, necrosis instead plays an important role [75]. However, both these events seem to be activated, at least in part, by a rapid increase IT, intratumoral; IP, intraperitoneal; IV, intravenous; NSCLC, non-small cell lung cancer; "tumour response", actual response of tumour to treatment; "sensitivity", prediction of tumour sensitivity to treatment in cFos expression, as preventing this using gene knock down strategies can achieve a partial rescue from HXR9mediated cell killing [25]. ...
The HOX and PBX gene families encode transcription factors that have key roles in establishing the identity of cells and tissues in early development. Over the last 20 years it has become apparent that they are also dysregulated in a wide range of solid and haematological malignancies and have a predominantly pro-oncogenic function. A key mode of transcriptional regulation by HOX and PBX proteins is through their interaction as a heterodimer or larger complex that enhances their binding affinity and specificity for DNA, and there is growing evidence that this interaction is a potential therapeutic target in malignancies that include prostate, breast, renal, ovarian and lung cancer, melanoma, myeloma, and acute myeloid leukaemia. This review summarizes the roles of HOX and PBX genes in cancer and assesses the therapeutic potential of HOX/PBX dimer inhibition, including the availability of biomarkers for its application in precision medicine.
... Consistent with this, HOXB1 seems to have a TSG function in primary BC and was part of a set of HOX genes 25 downregulated in BC and AML (293)(294)(295). Likewise, HOXB1 expression was minimal in BC cell lines MCF-10A, MCF-7, and MDA-MB-231 cells (representing primary and metastatic BC), mirroring BC cell line qPCR results herein, but HOXB1-HOXB9 were upregulated in a small subset of primary BC tumour samples (296). ...
... However, only the flanks of the CpGI (covering the promoter/TSS and first exon) became demethylated, and its overall expression remained low. The role of this largely developmental gene in BC and PC is poorly known, but literature suggests upregulation in a subset of BC samples (296), with significant increase in expression in distant metastases (soft tissue and possibly bone) relative to primary tumours (HCMDB data; Weigman 2012). But in the majority of BC primary tumours HOXB1 is hypermethylated and non-expressed (293)(294)(295). ...
Breast cancer (BC) is globally the second most diagnosed cancer, and it is the commonest cancer of women. In Australia approximately 20,000 BC diagnoses and 3000 BC related deaths occurred in 2021 alone, in terms of cancer deaths second only to melanoma. BC has a strong propensity to metastasize to the bone, and while 5-year survival rates are >90% if BC is caught early as a primary tumour, survival rapidly worsens to less than 14% in those with skeletal metastases. Thus, novel means of counteracting this metastatic progression are greatly needed. Epigenetic changes, chemical modifications around the DNA including DNA methylation, histone modifications, and ncRNAs, help retain the plasticity of metastatic tumour cells and may play a greater role in 'driving' metastatic progression than acquired DNA mutations. Thus, if epigenetic changes drive metastatic progression, it follows that a subset of those epigenetic drivers or ‘epi-drivers’ may guide subsets of metastases such as skeletal metastases. Importantly, epigenetic modifications and mechanisms are potentially reversible, and are providing effective therapeutic targets in selected cancers, with more promising treatments on the horizon. Therefore, this study aimed to characterize and identify epi-drivers of BC bone metastasis, firstly by clarifying the epigenetic regulation of an integrin gene (ITGA2) known to have a role in BC bone metastasis, and secondly by identifying additional and novel epi-drivers in matched BC bone metastases and primary tumour samples.
Integrin ITGA2, which encodes a subunit of the α2β1 integrin collagen receptor, has known functions in bone metastasis, invasion, proliferation, and angiogenesis, and is subject to multiple layers of epigenetic regulation. In BC as well as prostate cancer (PC), which shares a strong propensity for bone metastasis, ITGA2 initially acts as a tumour suppressor gene (TSG) downregulated to promote initial spread and soft tissue metastasis. Secondary upregulation of ITGA2 likely induces bone metastasis, α2β1 engaging the abundant collagen in the bone matrix and guiding invasive pathways. Epigenetic changes likely contribute to ITGA2 regulation in BC bone metastatic progression. The ITGA2 promoter contains a large CpG island (CpGI), and herein it was revealed that in BC, methylation of this CpGI had limited ability to regulate the promoter and it was generally unmethylated in tumour samples and cell lines regardless of expression changes. This is in clear contrast to PC wherein methylation tightly controlled ITGA2 expression. Likewise, up-regulatory histone modifications were relatively uniform in BC cell lines, with classical repressive modifications absent. Instead, a selection of poorly understood histone acetylation modifications possibly regulated ITGA2 in BC along with transcription factors. Several novel transcription factor recognition motifs were identified in the ITGA2 promoter for NF-κB, FOXO3, FOXL2, EGR3, and β-catenin, all known ITGA2 up-regulators. Notably, NF-κB is upregulated by RANKL-RANK signalling, a bone remodelling pathway hijacked by BC cells to promote bone metastasis.
Herein noncoding RNAs were found to regulate ITGA2 in BC. A novel lncRNA, I2ALR, was found to be transcribed in antisense from a promoter nearby to that of ITGA2. Over-expression and knockdown studies revealed that I2ALR negatively regulated ITGA2, and modulated expression of ITGA2 pathways. I2ALR likely downregulated ITGA2 by forming a complex with the ITGA2 mRNA, supported by in-silico analyses. I2ALR was further found to be poly-adenylated via sequencing experiments, and in public datasets its expression correlated with improved survival in several cancer types, including BC. Contrastingly in PC, I2ALR apparently had little role in ITGA2 regulation and had no correlation to survival. Another noncoding RNA, micro-RNA-373-3p, negatively regulated by the oestrogen receptor (ER-α), was also shown herein to regulate ITGA2 expression. MiR-373-3p was proposed to be responsible, in part, for the initial downregulation of ITGA2 in BC progression and soft tissue metastasis, with the loss of ER-α in bone metastases possibly inducing miR-373-3p loss for ITGA2 upregulation. Both I2ALR and miR373 may offer novel therapeutic targets in BC. Overall, this suggests that distinct ITGA2 regulatory mechanisms operate in BC relative to PC.
It was likely that epigenetic changes to genes beyond that of ITGA2 alone were responsible for mediating BC bone metastasis. Patient paired primary tumour and metastases have recently proved to be an effective way of identifying novel epigenetic and transcriptional changes that may drive metastatic progression. Therefore, the identification of other novel epi-drivers of BC bone metastases was conducted with EPIC methylation array and RNA-seq on matched BC primary tumour and bone metastasis samples. Bone metastases greatly resembled their matched primary tumours in methylation patterns, and those differentially methylated sites shared across all bone metastases were largely demethylated, indicating continuation in the progression of global hypomethylation and site specific hypermethylation that characterizes primary cancer tumours in bone metastasis. As would be expected from widespread hypomethylation, RNA-seq indicated that most genes differentially expressed in bone metastases relative to primary tumours were upregulated. Many potential epi-drivers were identified and select genes were validated by qPCR, bisulfite sequencing, and examination of publicly available data. HOXB1, a developmental gene with low expression in most tissues was found to have a hypermethylated CpG island in primary tumours, with the 5′ and 3′ flanks of this significantly demethylated in bone metastases, and expression significantly increased in bone metastases. In public datasets HOXB1 was found to be upregulated with increasing BC grade. WNT5A and FLI1 were also validated as putative epi-drivers, both displayed significantly decreased 5′ promoter methylation and increased expression in bone metastases relative to primary tumours. WNT5A and FLI1 have known roles in BC progression and metastasis and (along with HOXB1) are likely epi-drivers of BC bone metastasis. Another putative epi-driver associated genes was the bone metastasis associated RANKL (of RANK-RANKL signaling and ITGA2 regulation), which was de-methylated and up-regulated in BC bone metastases.
Overall, these data suggest that a large range of both well characterized and novel BC associated pathways are likely epigenetically dysregulated to promote BC bone metastatic progression. These pathways and their constituent genes, including ITGA2, I2ALR, HOXB1, FLI1, WNT5A, and RANKL, may offer novel points of therapeutic intervention in BC bone metastasis, either through countering epigenetic changes or by targeting transcripts and proteins.
(Thesis submitted June 2022, final version submitted Feb 2023)
... Modulation of PBX1 activity by direct targeting or by targeting its downstream mediators might represent a novel therapeutic approach that likely hits MPN stem cells rather than the bulk of the disease. Small molecules targeting PBX1 have been developed and tested in cancer models (Morgan et al., 2012;Platais et al., 2018;Shen et al., 2018Shen et al., , 2019, and selective toxicity for neoplastic cells has been demonstrated in vitro (Liu et al., 2019); moreover, their therapeutic potential has been proposed for several types of tumors (Morgan et al., 2012). However, the high number of genes DE in JP LKS compared with WT, and the fact that some JP mice died likely of ineffective hematopoiesis, suggest some caution before considering PBX1 inhibition in patients. ...
... Modulation of PBX1 activity by direct targeting or by targeting its downstream mediators might represent a novel therapeutic approach that likely hits MPN stem cells rather than the bulk of the disease. Small molecules targeting PBX1 have been developed and tested in cancer models (Morgan et al., 2012;Platais et al., 2018;Shen et al., 2018Shen et al., , 2019, and selective toxicity for neoplastic cells has been demonstrated in vitro (Liu et al., 2019); moreover, their therapeutic potential has been proposed for several types of tumors (Morgan et al., 2012). However, the high number of genes DE in JP LKS compared with WT, and the fact that some JP mice died likely of ineffective hematopoiesis, suggest some caution before considering PBX1 inhibition in patients. ...
PBX1 regulates the balance between self-renewal and differentiation of hematopoietic stem cells and maintains proto-oncogenic transcriptional pathways in early progenitors. Its increased expression was found in myeloproliferative neoplasm (MPN) patients bearing the JAK2V617F mutation. To investigate if PBX1 contributes to MPN, and to explore its potential as therapeutic target, we generated the JP mouse strain, in which the human JAK2 mutation is induced in the absence of PBX1. Typical MPN features, such as thrombocythemia and granulocytosis, did not develop without PBX1, while erythrocytosis, initially displayed by JP mice, gradually resolved over time; splenic myeloid metaplasia and in vitro cytokine independent growth were absent upon PBX1 inactivation. The aberrant transcriptome in stem/progenitor cells from the MPN model was reverted by the absence of PBX1, demonstrating that PBX1 controls part of the molecular pathways deregulated by the JAK2V617F mutation. Modulation of the PBX1-driven transcriptional program might represent a novel therapeutic approach.
... The HOX genes are a family of proteins involved in cell and tissue development in the embryo and then subsequently play various roles in regulating proliferation, renewal and migration of adult cells [1]. In addition, they are frequently overexpressed in a range of solid tumours including those of the breast, prostate, lung, and kidneys, as well as numerous liquid cancers [2][3][4][5][6]. The normal, wildtype HOX genes are also significantly overexpressed in many types of leukemia, including acute myeloid leukemia (AML) [7]. ...
... mRNA expression levels were calculated using Livak's PCR calculation method [12] and then grouped into decades of age from 20's to 80's, as previously reported in [2]. ...
HOXA9 is a well characterised gene for its role in normal haemopoiesis and leukaemias where overexpression of HOXA9 in haemopoietic cells contributes to development of acute myeloid leukaemia (AML). This upregulation of HOXA9 may be sufficient in its own right to cause the disease. Little is known, however, about HOXA9 expression in healthy adults. Here, we demonstrated that HOXA9 expression increases dramatically with age in the peripheral blood mononuclear cells of healthy adults, with a significant proportion of over-55 year old individuals showing very high expression. This elevated expression of HOXA9 was not caused by chronic inflammation and HOXA9 was expressed highly in myeloid cells positive for AML cell markers as well as in cells positive for extracellular activation markers. This increasing incidence of elevated HOXA9 expression in older age is a potentially important observation as age is a well-established risk factor for the development of AML.
... 15 HOXB1 is also an important factor in early vertebrate development, and HOXB1 dysregulation may result in congenital heart defects. 16 Although numerous studies have reported crucial biological effects of HOXB1 in cancer development, 17,18 there has been no analysis of the role of HOXB1 in lung cancer. ...
... The overexpression of hsa-let-7g may promote lung cancer development by directly decreasing the expression level of HOXB1. HOXB1 is a vital regulator in cancer development, 17,18 and this is the first study to suggest a role of HOXB1 in lung cancer. Previous studies have shown key roles of miRNAs in both physiological and pathological processes. ...
Purpose:
The goal of this study was to explore the effects of hsa-let-7g on cell proliferation and apoptosis, and elucidate its role in lung cancer development.
Materials and methods:
The expression levels of has-let-7g and HOXB1 in tissues and cells were measured by qRT-PCR. An inhibitor of hsa-let-7g or one targeting a control messenger RNA were transfected into A549 and H1944 lung cancer cells, and the effects of hsa-let-7g dysregulation on cell viability and apoptosis were analyzed using CCK-8 and apoptosis detection assays. HOXB1 was confirmed as the target gene of hsa-let-7g, based on luciferase reporter assay results. The relationship between hsa-let-7g and HOXB1 was confirmed by co-transfection of inhibitors of hsa-let-7g and HOXB1 followed by Western blot, CCK-8, and apoptosis detection assays.
Results:
We observed high expression of hsa-let-7g in lung cancer tissues compared to the corresponding normal tissues, and generally higher expression of hsa-let-7g in patients with advanced tumor classification. The results of CCK-8 and apoptosis detection experiments showed that the inhibition of hsa-let-7g significantly inhibited proliferation of A549 and H1944 cells, but also promoted apoptosis. HOXB1 is a specific target of hsa-let-7g, and downregulation of HOXB1 in lung cancer cells reversed the suppressive effects caused by knocking down hsa-let-7g.
Conclusion:
These data collectively suggest that the expression of hsa-let-7g inhibits lung cancer cells apoptosis and promotes proliferation by down-regulating HOXB1. The results from this study demonstrate the potential of hsa-let-7g/HOXB1 axis as a therapeutic target for the treatment of lung cancer.
... Treatment. HXR9, an 18-amino acid peptide, competently inhibits the hexapeptide motif of HOX proteins and prevents HOX-PBX binding [19]. HOXB9n and HOXB9v expressing MCF7 cells were treated with HXR9 or a control peptide, CXR9 (Figures 6(a) and 6(b)). ...
... Additionally, HOX-PBX interactions involve a short HOX protein motif, the hexapeptide, located upstream of the homeodomain [33]. HXR9 is an 18-amino acid peptide and suppresses tumor proliferation by inhibiting HOX-PBX binding [19]. In our proliferation assay, HOXB9v expressing cell lines were sensitive to the HOX-PBX inhibitor HXR9, even though HOXB9v lacks the hexapeptide. ...
First identified as a developmental gene, HOXB9 is also known to be involved in tumor biological processes, and its aberrant expression correlates with poor prognosis of various cancers. In this study, we isolated a homeodomain-less, novel HOXB9 variant (HOXB9v) from human breast cancer cell line-derived mRNA. We confirmed that the novel variant was produced from variationless HOXB9 genomic DNA. RT-PCR of mRNA isolated from clinical samples and reanalysis of publicly available RNA-seq data proved that the new transcript is frequently expressed in human breast cancer. Exogenous HOXB9v expression significantly enhanced the proliferation of breast cancer cells, and gene ontology analysis indicated that apoptotic signaling was suppressed in these cells. Considering that HOXB9v lacks key domains of homeobox proteins, its behavior could be completely different from that of the previously described variationless HOXB9. Because none of the previous studies on HOXB9 have considered the presence of HOXB9v, further research analyzing the two transcripts individually is warranted to re-evaluate the true role of HOXB9 in cancer.
... HXR9 also caused apoptosis in the ovarian cancer cell line SK-OV3 and inhibited tumor growth of SK-OV3 xenografts [34]. Moreover, HXR9 induced apoptosis in breast cancer cell lines, correlating with their higher expression levels of HOX genes, and HXR9 injected intratumorally retarded in vivo tumor growth in MDA-MB-231 TNBC xenografts [37]. Furthermore, HXR9 had apoptotic activity in prostate cancer cells and inhibited in vivo tumor growth of LNCaP xenografts [38]. ...
... Another study noted lack of toxicity in mice regarding blood counts and liver histology changes [32]. However, no toxicity reports were made for mice bearing xenografts from ovarian [34], mesothelioma [39], prostate cancer [38], meningioma [186*], melanoma [187*], or breast cancer [37]. ...
In molecular cancer therapeutics only 10% of known cancer gene products are targetable with current pharmacological agents. Major oncogenic drivers, such as MYC and KRAS proteins are frequently highly overexpressed or mutated in multiple human malignancies. However, despite their key role in oncogenesis, these proteins are hard to target with traditional small molecule drugs due to their large, featureless protein interfaces and lack of deep pockets. In addition, they are inaccessible to large biologicals, which are unable to cross cell membranes. Designer interference peptides (iPeps) represent emerging pharmacological agents created to block selective interactions between protein partners that are difficult to target with conventional small molecule chemicals or with large biologicals. iPeps have demonstrated successful inhibition of multiple oncogenic drivers with some now entering clinical settings. However, the clinical translation of iPeps has been hampered by certain intrinsic limitations including intracellular localization, targeting tissue specificity and pharmacological potency. Herein, we outline recent advances for the selective inhibition of major cancer oncoproteins via iPep approaches and discuss the development of multimodal peptides to overcome limitations of the first generations of iPeps. Since many protein–protein interfaces are cell-type specific, this approach opens the door to novel programmable, precision medicine tools in cancer research and treatment for selective manipulation and reprogramming of the cancer cell oncoproteome.
... Extensive researches have demonstrated that HXR9 causes apoptosis and inhibits tumor survival in a variety of tumor types, including ESCC. [23][24][25][26][27][28] To validate the hypothesis that HXR9 enhances the efficacy of cisplatin in ESCC cells, we performed a CCK-8 assay and clonal formation assay to evaluate the cell viability after exposure to cisplatin, or HXR9, or the combination of cisplatin with HXR9 and to determine the effect of these treatments on tumor growth in ESCC mouse xenograft models. The results showed that a combination of HXR9 and cisplatin synergistically inhibited cell viability (Fig 7a, b). ...
... In addition, previous studies have indicated that there is a rapid accumulation of HXR9 in the first few hours and it then remains stable beyond 24 hours and local delivery of HXR9 into tumors in mice has not resulted in a local inflammatory response. 24,26 Therefore, intratumor delivery of HXR9 may be a feasible approach. Moreover, it was found that disruption of HOXB7/PBX by HXR9 caused transcription alteration of genes such as MPL, IL-15, IL-23A and IL-24, which encode cytokines involved in regulating the activation of JAK-STAT signaling pathway or induction of apoptosis in our previous study. ...
Background:
DNA damage repair is an important mechanism of platinum resistance. HOXB7 is one member of HOX family genes, which are essential developmental regulators and frequently dysregulated in cancer. Recently, its relevance in chemotherapy resistance and DNA damage repair has also been addressed. However, little is known regarding the association between HOXB7 and chemotherapy resistance in esophageal squamous cell carcinoma (ESCC).
Methods:
The association between HOXB7 expression detected by immunohistochemisty and tumor regression grade (TRG) and long-term survival was analyzed in 143 ESCC patients who underwent neoadjuvant chemotherapy. CCK8 assay was used to examine the effect of cisplatin in a panel of four ESCC cell lines. A stable cell strain with HOXB7 knockdown of KYSE150 and KYSE450 was established to explore the effect on cisplatin sensitivity. The interaction of HOXB7 with Ku70, Ku80 and DNA-PKcs was determined by GST-pull down, coimmunoprecipitation and immunofluorescent colocalization. Finally, we investigated whether disrupting HOXB7 function by a synthetic peptide HXR9 blocking the formation of HOXB7/PBX could enhance cisplatin sensitivity in vitro and in vivo.
Results:
High expression of HOXB7 was associated with cisplatin resistance and worse chemotherapy efficacy. HOXB7 knockdown reinforced cisplatin sensitivity. It was identified that HOXB7 interacts with Ku70, Ku80 and DNA-PKcs. HOXB7 knockdown was related to the downregulation of Ku70, Ku80 and DNA-PKcs as well as arrested cell cycle in S phase. HOXB7 inhibition by HXR9 had a synergistic effect to improve cisplatin sensitivity.
Conclusion:
HOXB7 may be a biomarker for the prediction of chemoresistance of ESCC and serves as a promising therapeutic target.
... Disrupting this partnership could, therefore, constitute a promising strategy for therapeutic approaches. Accordingly, an HX-mimicking peptide has been described to induce apoptosis in several cancer cell lines, including breast cancer cells (Morgan et al., 2012). Interestingly, it was also noticed that MCF7 cells were relatively insensitive to the HX-mimicking peptide when compared to other breast cancer-derived cell lines (Morgan et al., 2012). ...
... Accordingly, an HX-mimicking peptide has been described to induce apoptosis in several cancer cell lines, including breast cancer cells (Morgan et al., 2012). Interestingly, it was also noticed that MCF7 cells were relatively insensitive to the HX-mimicking peptide when compared to other breast cancer-derived cell lines (Morgan et al., 2012). Our work establishes that the majority of HOX proteins use additional TALE interaction interfaces that could act in a redundant, independent, or antagonistic way with the HX motif. ...
HOX proteins achieve numerous functions by interacting with the TALE class PBX and MEIS cofactors. In contrast to this established partnership in development and disease, how HOX proteins could interact with PBX and MEIS remains unclear. Here, we present a systematic analysis of HOX/PBX/MEIS interaction properties, scanning all paralog groups with human and mouse HOX proteins in vitro and in live cells. We demonstrate that a previously characterized HOX protein motif known to be critical for HOX-PBX interactions becomes dispensable in the presence of MEIS in all except the two most anterior paralog groups. We further identify paralog-specific TALE-binding sites that are used in a highly context-dependent manner. One of these binding sites is involved in the proliferative activity of HOXA7 in breast cancer cells. Together these findings reveal an extraordinary level of interaction flexibility between HOX proteins and their major class of developmental cofactors.
... PBX1, itself, is a product of a proto-oncogene, which suggests a role in tumorigenesis, metastasis, and chemoresistance in various cancers. Indeed, a previous study showed that increased expression of PBX1 promotes proliferation of melanoma and breast cancer cells [5][6][7][8]. These proliferative effects were dependent on the PBX1/HOX interaction. ...
... Although previous studies have reported that PBX1 can promote melanoma, breast cancer and lung cancer [5][6][7][8], the role that this protein plays in GC is unclear. ...
Pre-B-cell leukemia homeobox 1 (PBX1) was originally identified as a proto-oncogene in human leukemia. Although this protein has been shown to contribute to cellular development and tumorigenesis, the role of PBX1 in gastric carcinoma (GC) remains unclear. In this study, we observed increased expression of PBX1 in GC tissues compared with adjacent normal tissues. This increase in PBX1 expression levels negatively correlated with HOXB9 mRNA expression and was also associated with malignancy and metastasis. PBX1 promoted proliferation and metastasis of GC cells both in vitro and in vivo.These phenomena were also accompanied by epithelial-to-mesenchymal transition (EMT). Additionally, we observed that PBX1 promotes the expression of tumor growth and angiogenic factors. A structural model of the PBX1-HOX complex revealed that hydrophobic binding between PBX1 and the hexapeptide motif might be required for EMT induction. This analysis also demonstrated that the Phe252 residue in the first helix of the TALE homeodomain is involved in the latter hydrophobic binding reaction. In vitro data from PBX1 mutants suggest that PBX1 cannot promote tumorigenesis of GC cells via EMT induction when Phe252 residues lose hydrophobicity. It is likely that the presence of this residue is essential in facilitating hydrophobic binding with the hexapeptide motif. These findings suggest that PBX1 may be a potential target for GC treatment and this study provides a platform to elucidate the molecular mechanisms that underpin the role of PBX1 in GC tumorigenesis.
... HXR9, an 18 amino acid peptide inhibitor of this interaction, has been shown to reduce cell proliferation, tumor growth, and induce apoptosis, originally in melanoma cell lines [199]. HXR9 has been shown to be effective in a range of cancer cell types, including esophageal squamous cell carcinoma [200], glioblastoma [201], prostate cancer [202], oral squamous cell carcinoma [203], malignant mesothelioma [204], breast cancer [205], and ovarian cancer [206]. Furthermore, there has been some research on the differentiation inducer retinoic acid (RA) and the expression of HOX genes. ...
The HOX gene family encodes for regulatory transcription factors that play a crucial role in embryogenesis and differentiation of adult cells. This highly conserved family of genes consists of thirty-nine genes in humans that are located in four clusters, A–D, on different chromosomes. While early studies on the HOX gene family have been focused on embryonic development and its related disorders, research has shifted to examine aberrant expression of HOX genes and the subsequent implication in cancer prediction and progression. Due to their role of encoding master regulatory transcription factors, the abnormal expression of HOX genes has been shown to affect all stages of tumorigenesis and metastasis. This review highlights the novel role of the HOX family’s clinical relevance as both prognostic and diagnostic biomarkers in hematological and solid tumors.
... Other groups have investigated other miniproteins in TNBC. This is the case of KJ-Pyr-9 against MYC in the TNBC cell line MDA-MB-231 [58], an inhibitory peptide against HOX [59] in MDA-MB-231 cells or Omomyc against MYC in MDA-MB-231 cells and patient-derived TNBC xenografts [60]. So far, Omomyc is the only miniprotein-based inhibitor that has reached the clinical setting [61]. ...
Triple-negative breast cancer (TNBC) represents roughly one-sixth of all breast cancer patients, but accounts for 30–40% of breast cancer deaths. Due to the lack of typical biomarkers exploited clinically for breast cancer, it remains very difficult to treat. Moreover, its intrinsic high heterogeneity and proneness to become resistant to the drugs administered makes the treatment management very challenging for oncologists. Herein, we outline the different therapies used currently for TNBC and list the ongoing clinical trials to provide an overview of the most recent TNBC therapeutic landscape. In addition, we highlight the emerging therapies in the preclinical stage that hold the most promise, such as epigenetic modulators, CRISPR, miniproteins, radioconjugates, cancer vaccines, and PROTACs. Moreover, we navigate through the existing limitations and challenges which hamper the development of new and more effective treatments for TNBC. Lastly, we point to emerging new directions that may revolutionize future therapy for TNBC.
... The current strategy is to develop peptides targeting the interaction between HOX members and their cooperators [51]. HXR9, one of the most frequently used peptides to disrupt the HOX/PBX dimers competitively, effectively inhibits the growth of multiple tumors in vitro and in vivo [52][53][54]. However, the efficacies and outcomes of HXR9 and its derivatives encounter challenges for clinical applications [55]. ...
Homeobox genes include HOX and non-HOX genes. HOX proteins play fundamental roles during ontogenesis by interacting with other non-HOX gene-encoded partners and performing transcriptional functions, whereas aberrant activation of HOX family members drives tumorigenesis. In this study, gastric cancer (GC) expression microarray data indicated that HOXB9 is a prominent upregulated HOX member in GC samples significantly associated with clinical outcomes and advanced TNM stages. However, the functional role of HOXB9 in GC remains contradictory in previous reports, and the regulatory mechanisms are elusive. By in silico and experimental analyses, we found that HOXB9 was upregulated by a vital cell cycle-related transcription factor, E2F1. Depleting HOXB9 causes G1-phase cell cycle arrest by downregulating CDK6 and a subset of cell cycle-related genes. Meanwhile, HOXB9 contributes to cell division and maintains the cytoskeleton in GC cells. We verified that HOXB9 interacts with PBX2 to form a heterodimer, which transcriptionally upregulates CDK6. Knocking down CDK6 can phenocopy the tumor-suppressive effects caused by HOXB9 depletion. Blocking HOXB9 can enhance the anti-tumor effect of CDK6 inhibitors. In conclusion, we elucidate the oncogenic role of HOXB9 in GC and reveal CDK6 as its potent downstream effector. The E2F1-HOXB9/PBX2-CDK6 axis represents a novel mechanism driving gastric carcinogenesis and conveys prognostic and therapeutic implications. © 2023 The Pathological Society of Great Britain and Ireland.
... A small and unique peptide HXR9 was generated to destroy the interaction between HOX and PBX protein, which selectively damages leukemic cells [59]. In addition to treating AML, researches on HXR9 used in solid tumor such as melanoma, ovarian cancer [60], breast cancer [61] were also emerged. Take an example about this interaction abrogated by HXR9, the small HXR9 peptide hinders the binding ability of HOXA9 to PBX, resulting in an inhibition of meningioma growth [62]. ...
HOXA9 functioning as a transcription factor is one of the members of HOX gene family, which governs multiple cellular activities by facilitating cellular signal transduction. In addition to be a driver in AML which has been widely studied, the role of HOXA9 in solid tumor progression has also received increasing attention in recent years, where the aberrant expression of HOXA9 is closely associated with the prognosis of patient. This review details the signaling pathways, binding partners, post-transcriptional regulation of HOXA9, and possible inhibitors of HOXA9 in solid tumors, which provides a reference basis for further study on the role of HOXA9 in solid tumors.
... One potential strategy to inhibit HOX function is via the use of HXR9 peptides which prevents HOX binding to PBX, a transcription co-activator common to many HOX proteins. HXR9 causes apoptosis in multiple breast cancer cell lines (Morgan et al., 2012). Overcoming the functional redundancies among the different HOX family members is one of the main challenges for HOX-based therapeutic strategies (Feng et al., 2021). ...
Cancer is characterized as a reversion of a differentiated cell to a primitive cell state that recapitulates, in many aspects, features of embryonic cells. This review explores the current knowledge of developmental mechanisms that are essential for embryonic mouse mammary gland development, with a particular focus on genes and signaling pathway components that are essential for the induction, morphogenesis, and lineage specification of the mammary gland. The roles of these same genes and signaling pathways in mammary gland or breast tumorigenesis and metastasis are then summarized. Strikingly, key embryonic developmental pathways are often reactivated or dysregulated during tumorigenesis and metastasis in processes such as aberrant proliferation, epithelial-to-mesenchymal transition (EMT), and stem cell potency which affects cellular lineage hierarchy. These observations are in line with findings from recent studies using lineage tracing as well as bulk- and single-cell transcriptomics that have uncovered features of embryonic cells in cancer and metastasis through the identification of cell types, cell states and characterisation of their dynamic changes. Given the many overlapping features and similarities of the molecular signatures of normal development and cancer, embryonic molecular signatures could be useful prognostic markers for cancer. In this way, the study of embryonic development will continue to complement the understanding of the mechanisms of cancer and aid in the discovery of novel therapeutic targets and strategies.
... However, the majority of reports indicate that HOX genes have a pro-oncogenic role, including functions that support tumor growth and invasion such as angiogenesis, metastasis, and immune evasion [23] . At the cellular level, a generalized role for many HOX proteins in cancer appears to be to prevent apoptosis by inhibiting cFos [24][25][26][27] and dual specificity protease 1 (DUSP1) expression [26,28,29] . DUSP1 is a tumour suppressor gene [30] , and whilst cFos is generally considered to be a protoncogene, cFos protein can also induce apoptosis through the activation of the cell death ligand, FAS1 [31][32][33][34][35] . ...
It is now well established that the tumor microenvironment plays an essential role in the survival, growth, invasion, and spread of cancer through the regulation of angiogenesis and localized immune responses. This review examines the role of the HOX genes, which encode a family of homeodomain-containing transcription factors, in the interaction between prostate tumors and their microenvironment. Previous studies have established that HOX genes have an important function in prostate cancer cell survival in vitro and in vivo, but there is also evidence that HOX proteins regulate the expression of genes in the cancer cell that influence the tumor microenvironment, and that cells in the microenvironment likewise express HOX genes that confer a tumor-supportive function. Here we provide an overview of these studies that, taken together, indicate that the HOX genes help mediate cross talk between prostate tumors and their microenvironment.
... One approach to overcoming this is to target multiple HOX proteins through their interaction with PBX using an inhibitory peptide (HXR9) that acts as a competitive antagonist by mimicking the conserved hexapeptide region in HOX paralogue group proteins 1 to 10 that mediates PBX binding. 164 HXR9 has been shown to cause apoptosis in a range of cancers both in vitro and in vivo, including prostate, 8 breast, 9 lung, 10 renal, 11 ovarian, 165 oral 12 and oesophageal cancer, 166 as well as mesothelioma, 14 myeloma, 167 melanoma 168,169 and acute myeloid leukaemia. 170 A potential difficulty with this approach though is the apparent tumour suppressor function of some ...
The HOX genes are a highly conserved group of transcription factors that have key roles in early development, but which are also highly expressed in most cancers. Many studies have found strong associative relationships between the expression of individual HOX genes in tumours and clinical parameters including survival. For the majority of HOX genes, high tumour expression levels seem to be associated with a worse outcome for patients, and in some cases, this has been shown to result from the activation of pro‐oncogenic genes and pathways. However, there are also many studies that indicate a tumour suppressor role for some HOX genes, sometimes with conclusions that contradict earlier work. In this review, we have attempted to clarify the role of HOX genes in cancer by focusing on their downstream targets as identified in studies that provide experimental evidence for their activation or repression. On this basis, the majority of HOX genes would appear to have a pro‐oncogenic function, with the notable exception of HOXD10, which acts exclusively as a tumour suppressor. HOX proteins regulate a wide range of target genes involved in metastasis, cell death, proliferation and angiogenesis, and activate key cell signalling pathways. Furthermore, for some functionally related targets, this regulation is achieved by a relatively small subgroup of HOX genes.
... The role of the whole MEIS protein family (MEIS1, MEIS2, and MEIS3) in solid cancers has been recently reviewed by Girgin and colleagues [105]. MEIS1 overexpression in several solid cancers has an important oncogenic function, for example, in breast [106,107], colorectal [108], human esophageal squamous cell [109,110], neuroblastoma [111], ovarian [112], and prostate cancers [113], where its upregulation has been associated to cancer etiology, progression, and increased invasiveness. ...
Recently MEIS1 emerged as a major determinant of the MLL-r leukemic phenotype. The latest and most efficient drugs effectively decrease the levels of MEIS1 in cancer cells. Together with an overview of the latest drugs developed to target MEIS1 in MLL-r leukemia, we review, in detail, the role of MEIS1 in embryonic and adult hematopoiesis and suggest how a more profound knowledge of MEIS1 biochemistry can be used to design potent and effective drugs against MLL-r leukemia. In addition, we present data showing that the interaction between MEIS1 and PBX1 can be blocked efficiently and might represent a new avenue in anti-MLL-r and anti-leukemic therapy.
... Recurrent chordoma derived cell lines were significantly more susceptible towards HOX/PBXinhibition than primary cell lines. In line with this, a higher increase of CFOS expression following HXR9 treatment was detected in recurrent chordoma cell lines [57]. ...
Background
Tumor recurrence is one of the major challenges in clinical management of chordoma. Despite R0-resection, approximately 50% of chordomas recur within ten years after initial surgery. The underlying molecular processes are poorly understood resulting in the lack of associated therapeutic options. This is not least due to the absence of appropriate cell culture models of this orphan disease.
Methods
The intra-personal progression model cell lines U-CH11 and U-CH11R were compared using array comparative genomic hybridization, expression arrays, RNA-seq, and immunocytochemistry. Cell line origin was confirmed by short tandem repeat analysis. Inter-personal cell culture models ( n = 6) were examined to validate whether the new model is representative. Cell viability after HOX/PBX complex inhibition with small peptides was determined by MTS assays.
Results
Using whole genome microarray analyses, striking differences in gene expression between primary and recurrent chordomas were identified. These expression differences were confirmed in the world’s first intra-personal model of chordoma relapse consisting of cell lines established from a primary (U-CH11) and the corresponding recurrent tumor (U-CH11R). Array comparative genomic hybridization and RNA-sequencing analyses revealed profound genetic similarities between both cell lines pointing to transcriptomic reprogramming as a key mechanism of chordoma progression. Network analysis of the recurrence specific genes highlighted HOX/PBX signaling as a common dysregulated event. Hence, HOX/PBX complexes were used as so far unknown therapeutic targets in recurrent chordomas. Treating chordoma cell lines with the complex formation inhibiting peptide HXR9 induced cFOS mediated apoptosis in all chordoma cell lines tested. This effect was significantly stronger in cell lines established from chordoma relapses.
Conclusion
Clearly differing gene expression patterns and vulnerabilities to HOX/PBX complex inhibition in highly therapy resistant chordoma relapses were identified using the first intra-personal loco-regional and further inter-personal chordoma progression models. For the first time, HOX/PBX interference was used to induce cell death in chordoma and might serve as the basic concept of an upcoming targeted therapy for chordomas of all progression stages.
... Cancer cells' sensitivity to this peptide is highly correlated to their HOX expression profile, albeit the subset of HOX genes which act as oncosuppressors is not affected [34]. This peptide has shown to be effective in oesophageal and oral squamous cell carcinomas [36,37], melanoma [35,38], ovarian cancer (OC) [39], breast cancer [40], meningioma [41], prostate cancer [42], and leukaemia [43]. The use of RNA interference mechanisms [44,45] and the control of HOX methylation status [46] are additional tools that can control HOX expression with therapeutic function. ...
Simple Summary
Cancer is the second leading cause of death worldwide, right after cardiovascular diseases, and the invasion and metastatization correspond to the foremost cause of cancer-related deaths. Here, we reviewed the state of the art regarding the importance of HOX transcription factors in these last steps of cancer progression and described five of their complex mechanisms of regulation, including the miRs and lncRNAs interference. This information highlights the importance of HOX in the suppression and induction of disease advancement and point out the potential of HOX products as therapeutic targets for diverse cancer types.
Abstract
Invasion and metastasis correspond to the foremost cause of cancer-related death, and the molecular networks behind these two processes are extremely complex and dependent on the intra- and extracellular conditions along with the prime of the premetastatic niche. Currently, several studies suggest an association between the levels of HOX genes expression and cancer cell invasion and metastasis, which favour the formation of novel tumour masses. The deregulation of HOX genes by HMGA2/TET1 signalling and the regulatory effect of noncoding RNAs generated by the HOX loci can also promote invasion and metastasis, interfering with the expression of HOX genes or other genes relevant to these processes. In this review, we present five molecular mechanisms of HOX deregulation by which the HOX clusters products may affect invasion and metastatic processes in solid tumours.
... A peptide, for instance, designed to disrupt the HOX/PBX/DNA interaction reduced the proliferation of T3M4 PC cells, K562 leukemia cells, and MJT1 melanoma cells (Aulisa et al., 2009). Two small peptides, HXR9 and CXR9, were shown to induce apoptosis in NSCLC cells, breast, ovarian, prostate, and meningioma cells by disrupting the interaction of HOX and PBX (Plowright et al., 2009;Morgan et al., 2010;Morgan et al., 2012;Ando et al., 2014). In neuroblastoma, stable PBX1-MEIS1 interaction provides an overexpression of Phox2b (Di Zanni et al., 2015). ...
MEIS proteins are historically associated with tumorigenesis, metastasis, and invasion in cancer. MEIS and associated PBX-HOX proteins may act as tumor suppressors or oncogenes in different cellular settings. Their expressions tend to be misregulated in various cancers. Bioinformatic analyses have suggested their upregulation in leukemia/lymphoma, thymoma, pancreas, glioma, and glioblastoma, and downregulation in cervical, uterine, rectum, and colon cancers. However, every cancer type includes, at least, a subtype with high MEIS expression. In addition, studies have highlighted that MEIS proteins and associated factors may function as diagnostic or therapeutic biomarkers for various diseases. Herein, MEIS proteins and associated factors in tumorigenesis are discussed with recent discoveries in addition to how they could be modulated by noncoding RNAs or newly developed small-molecule MEIS inhibitors.
... Previous studies about the analysis of HOX gene expression in breast cancer in tumor cell lines have found that these transcriptional factors play a key role in the pathogenesis of this neoplasia [61][62][63]64 . ...
Breast cancer represents the most commonly cancer in women, second only to skin cancer. The percentage of mortality risk for breast cancer is 1 in 37 women (2.7%), which makes breast cancer represent the second cause of cancer death in women 1. Usually, the 61% of the pathological cases were diagnosed when the tumor was identified in the breast tissue, while a third of the cases were diagnosed when the disease was in advanced or metastases condition 1. Recently, new researches based on previously published work in systemic chemotherapy and endocrine therapy field, have reduced the incidence rates. However, drug resistance, stem-like capability or stemness, still represent a reducing factor of the successful treatment. The patients with breast cancer recurrences, always show drug resistance. Therefore, new research will play a key role in order to better understand the molecular mechanisms that controls neoplastic transformation and resistance to the therapy 1. The identification of new molecular targets and understanding their role in the progression and development of breast cancer metastases will open up new possibilities for the development of active molecules against breast cancer. The quinonic nucleus is common to many natural and synthetic products associated with anticancer and antibacterial activities 2 , these compounds are typically DNA-intercalating agents because of the ability of their large, planar polycycles to bind strongly between the base pairs through hydrogen bonds and π-stacking interactions 3,4. They usually have side chains or sugar substitutes and basic nitrogens, which upon protonation further strengthen the DNA binding. Examples of quinone derivatives with antitumor activity include mitoxantrone, doxorubicin, mitomycin, streptonigrin and actinomycin D (AMD) 5-7. All known quinonic DNA intercalators have the potential to disrupt the normal function of DNA, leading to cell death. This DNA damage can be caused either by the parent form or by its metabolic conversion to electrophilic or radical species 8. In a recent paper, we described a potent anticancer synthetic iminoquinone, the 5H-pyrido[3,2-a]phenoxazin-5-one (PPH), able to inhibit a large number of lymphoblastoid and solid-tumor-derived cells at submicromolar concentrations. Interestingly, this compound also exhibits high inhibitory activity on the proliferation of wild-type and multidrug-resistant KB cells 9. In fact, "multidrug resistance" (MDR), a phenomenon due to the prolonged use of chemotherapy agents, currently constitutes the major problem in cancer treatment. From these studies, we concluded that the excellent cytotoxic activity of PPH results from the intercalation at the middle 5'-GC-3' base pairs of the octamer [d(GAAGCTTC)]2 10 (Figure 1). Breast cancer in women is the second most commonly cancer, after skin cancer. The percentage of mortality risk for breast cancer is 1 in 37 women (2.7%), which makes breast cancer represent the second cause of cancer death in women. Recently, new research based on previously published work in systemic chemotherapy and endocrine therapy field, have improved the incidence rates. The quinonic nucleus is common to many natural and synthetic products associated with anticancer and antibacterial activities, these compounds are typically DNA-intercalating agents. The Class I Homeobox genes (HOX in human and hox in mouse) control embryonic development and specific determination of positional identity anteroposterior axis of the human body. The HOX genes, are 39 transcription factors related to morphological, physiological disease. It has been demonstrated that any deregulation into the network is able to induce neoplastic transformation. Particularly, HOXC locus collaborating with lncRNA HOTAIR play a key role in breast cancer. In this study, our group evaluated the chemical and metabolic stability of new anticancer molecule 5H-pyro[3,2-a] phenoxazin-5-one (PPH). In a recent paper, we have already demonstrated that a new and potent anticancer synthetic iminoquinone, the 5H-pyrido[3,2-a]phenoxazin-5-one (PPH), is able to inhibit a large number of lymphoblastoid and solid-tumor-derived cells at submicromolar concentrations. Based on our previous research, we decided to analyze the cytotoxic effect and capability of PPH to control the lncRNA HOTAIR and HOXC locus gene expression in human breast cancer cells MCF-7, in order to demonstrate its role like potential new breast cancer antitumor drug. Figure 1: 5H-pirido[3,2-a] fenossazin-5-one (PPH)
... To date, the only effective inhibitors of HOX-PBX binding are the HXR9 peptide and its derivatives, originally derived from the HOXA9-PBX1 interacting peptide 66 . HXR9 was shown to inhibit the growth of a range of tumours in mouse xenograft models, including non-small cell lung, breast, ovarian, and prostate cancer, mesothelioma, melanoma, and meningioma [66][67][68][69][70][71][72] . HXR9 is supposed to act by preventing the binding of HOX-PBX complexes to the DNA. ...
Both onco-suppressor PREP1 and the oncogene MEIS1 bind to PBX1. This interaction stabilizes the two proteins and allows their translocation into the nucleus and thus their transcriptional activity. Here, we have combined cross-linking mass-spectrometry and systematic mutagenesis to detail the binding geometry of the PBX1-PREP1 (and PBX1-MEIS1) complexes, under native in vivo conditions. The data confirm the existence of two distinct interaction sites within the PBC domain of PBX1 and unravel differences among the highly similar binding sites of MEIS1 and PREP1. The HR2 domain has a fundamental role in binding the PBC-B domain of PBX1 in both PREP1 and MEIS1. The HR1 domain of MEIS1, however, seem to play a less stringent role in PBX1 interaction with respect to that of PREP1. This difference is also reflected by the different binding affinity of the two proteins to PBX1. Although partial, this analysis provides for the first time some ideas on the tertiary structure of the complexes not available before. Moreover, the extensive mutagenic analysis of PREP1 identifies the role of individual hydrophobic HR1 and HR2 residues, both in vitro and in vivo.
... 203 In vivo, HXR9 inhibited tumor growth in multiple xenograft studies. 201,202,[204][205][206] Thus far, no weight loss, unusual blood counts, or liver histology has been noted, suggesting minimal toxicity of the peptide. 202 A second generation of this drug, HTL001, is presently beginning clinical trials. ...
During animal development, HOX transcription factors determine the fate of developing tissues to generate diverse organs and appendages. The power of these proteins is striking: mis-expressing a HOX protein causes homeotic transformation of one body part into another. During development, HOX proteins interpret their cellular context through protein interactions, alternative splicing, and post-translational modifications to regulate cell proliferation, cell death, cell migration, cell differentiation, and angiogenesis. Although mutation and/or mis-expression of HOX proteins during development can be lethal, changes in HOX proteins that do not pattern vital organs can result in survivable malformations. In adults, mutation and/or mis-expression of HOX proteins disrupts their gene regulatory networks, deregulating cell behaviors and leading to arthritis and cancer. On the molecular level, HOX proteins are composed of DNA binding homeodomain, and large regions of unstructured, or intrinsically disordered, protein sequence. The primary roles of HOX proteins in arthritis and cancer suggest that mutations associated with these diseases in both the structured and disordered regions of HOX proteins can have substantial functional effects. These insights lead to new questions critical for understanding and manipulating HOX function in physiological and pathological conditions.
... In breast cancer cells, the higher the expression of HOXB1 through to HOXB9, the greater the cytotoxic effect. Surprisingly, HXR9 blocked the oncogenic activity of HOX genes, but it did not affect the tumor-suppressor or differentiation properties of some of the HOX genes tested [129][130][131][132]. Another approach to suppress HOX transcription factors that was quite effective in suppressing early-stage mammary tumors in cancer-prone transgenic C3(1)-SV40TAg mice was to use lipidoid nanoparticles of HOXA1 siRNA by local mammary intraductal delivery [133]. ...
Knowledge of the role of HOX proteins in cancer has been steadily accumulating in the last 25 years. They are encoded by 39 HOX genes arranged in 4 distinct clusters, and have unique and redundant function in all types of cancers. Many HOX genes behave as oncogenic transcriptional factors regulating multiple pathways that are critical to malignant progression in a variety of tumors. Some HOX proteins have dual roles that are tumor-site specific, displaying both oncogenic and tumor suppressor function. The focus of this review is on how HOX proteins contribute to growth or suppression of metastasis. The review will cover HOX protein function in the critical aspects of epithelial-mesenchymal transition, in cancer stem cell sustenance and in therapy resistance, manifested as distant metastasis. The emerging role of adiposity in both initiation and progression of metastasis is described. Defining the role of HOX genes in the metastatic process has identified candidates for targeted cancer therapies that may combat the metastatic process. We will discuss potential therapeutic opportunities, particularly in pathways influenced by HOX proteins.
... In this regard, the key regulators in the control of cell proliferation, are cell cycle and apoptosis. Previous studies reported that HOXB9 participates in the process of cell proliferation (Nagel et al. 2007), apoptosis (Morgan et al. 2012) and cell cycle, and in various cancers Hence, we hypothesized that HOXB9 might also play an important role in regulating cell proliferation, apoptosis and cell cycle in prostate cancer. Subsequently, we performed CCK-8 assays to detect differences in cell proliferation between negative control groups and experimental groups. ...
The Homeobox B9 (HOXB9) is a homeodomain-containing transcription factor that participates in the progression of various malignancies. Nevertheless, the functional role of HOXB9 in prostate cancer cells is largely unknown. Hence, we aimed to address the effect of HOXB9 on the progression of prostate cancer cells. Small interfering RNA (siRNA) against HOXB9 was used to downregulate HOXB9 expression in PC3 and DU145 cells. Western blotting was performed to detect the expression levels of HOXB9 and other related proteins. Cell proliferation was tested by the Cell Counting Kit-8 (CCK-8) and cell cycle and apoptosis were investigated by flow cytometry. Angiogenesis was examined using tube formation assays The Transwell assays were carried out to assess the migratory and invasive capacities of cells. Here, we found that HOXB9 knockdown significantly reduced cell proliferation via inducing cell cycle arrest at G1 phase. This treatment also reduced angiogenesis, migration and invasion abilities of PC3 and DU145 cells in vitro. We also found that HOXB9 knockdown inhibits the activation of the PI3K/AKT signaling pathway in prostate cancer cells. In conclusion, our findings revealed that HOXB9 promotes prostate cancer progression and might be a novel and effective therapeutic target for human prostate cancer.
... These results support the idea of targeting the HOX transcription complex in the targeted therapy of NPM1 mutated AML. In some solid cancers, including lung [60], breast [61], prostate [62], melanoma [63], and AML cell lines [64], HXR9 is a potent cell penetrating peptide inhibitor targeting HOX proteins by inhibiting the interaction with PBX cofactors. Alharbi et al. evaluated the mechanism of HXR9 induced cell death and found that HXR9 promotes apoptosis and necroptosis and its cytotoxicity can be enhanced by inhibiting protein kinase C (PKC) in AML cell lines [65]. ...
Acute myeloid leukemia (AML) is a clonal disorder of hematopoietic progenitor cells and the most common malignant myeloid disorder in adults. Several gene mutations such as in NPM1 (nucleophosmin 1) are involved in the pathogenesis and progression of AML. The aim of this study was to identify genes whose expression is associated with driver mutations and survival outcome. Genotype data (somatic mutations) and gene expression data including RNA-seq, microarray, and qPCR data were used for the analysis. Multiple datasets were utilized as training sets (GSE6891, TCGA, and GSE1159). A new clinical sample cohort (Semmelweis set) was established for in vitro validation. Wilcoxon analysis was used to identify genes with expression alterations between the mutant and wild type samples. Cox regression analysis was performed to examine the association between gene expression and survival outcome. Data analysis was performed in the R statistical environment. Eighty-five genes were identified with significantly altered expression when comparing NPM1 mutant and wild type patient groups in the GSE6891 set. Additional training sets were used as a filter to condense the six most significant genes associated with NPM1 mutations. Then, the expression changes of these six genes were confirmed in the Semmelweis set: HOXA5 (P = 3.06E-12, FC = 8.3), HOXA10 (P = 2.44E-09, FC = 3.3), HOXB5 (P = 1.86E-13, FC = 37), MEIS1 (P = 9.82E-10, FC = 4.4), PBX3 (P = 1.03E-13, FC = 5.4) and ITM2A (P = 0.004, FC = 0.4). Cox regression analysis showed that higher expression of these genes - with the exception of ITM2A - was associated with worse overall survival. Higher expression of the HOX genes was identified in tumors harboring NPM1 gene mutations by computationally linking genotype and gene expression. In vitro validation of these genes supports their potential therapeutic application in AML.
... Such interaction shuts down the expression of various genes involved in apoptosis [8,237,238]. Disruption of HOX/PBX interaction impacts cell proliferation and restores cell death in various solid tumor cell models [238], including melanoma [236,239], renal cell carcinoma [240], ovarian cancer (such as the SK-OV3 cell model or primary samples [241,242]), breast cancer [243], prostate cancer [244], asbestos-associated mesothelioma [245], and oral and esophageal squamous cell carcinoma [246,247]. HXR9 was also evaluated in AMLs based on the relevance of HOXA9 and PBX3 interaction in AML [29, 75,248,249], but other expressed HOX/PBX3 interfaces may also be disrupted by HXR9 in AML. ...
HOXA9 (Homeobox A9) is a homeotic transcription factor known for more than two decades to be associated with leukemia. The expression of HOXA9 homeoprotein is associated with anterior–posterior patterning during embryonic development, and its expression is then abolished in most adult cells, with the exception of hematopoietic progenitor cells. The oncogenic function of HOXA9 was first assessed in human acute myeloid leukemia (AML), particularly in the mixed-phenotype associated lineage leukemia (MPAL) subtype. HOXA9 expression in AML is associated with aggressiveness and a poor prognosis. Since then, HOXA9 has been involved in other hematopoietic malignancies and an increasing number of solid tumors. Despite this, HOXA9 was for a long time not targeted to treat cancer, mainly since, as a transcription factor, it belongs to a class of protein long considered to be an “undruggable” target; however, things have now evolved. The aim of the present review is to focus on the different aspects of HOXA9 targeting that could be achieved through multiple ways: (1) indirectly, through the inhibition of its expression, a strategy acting principally at the epigenetic level; or (2) directly, through the inhibition of its transcription factor function by acting at either the protein/protein interaction or the protein/DNA interaction interfaces.
... Along the same line, over-expression of HOXA9 is found in glioblastoma multiforme (GBM) and ovarian cancer (Cheng et al., 2005;Costa et al., 2010;Gaspar et al., 2010). In contrast, HOXA9 plays a tumor-suppressing role in lung and breast; it is downregulated in these corresponding cancers and can block cancer progression when ectopically expressed (Makiyama et al., 2005;Morgan et al., 2012;Svingen and Tonissen, 2003). ...
HOXA9 is reported as one of the most commonly altered genes in different solid tumours and leukemia in human. Mapping HOXA9-related protein-protein interactions is therefore important for the understanding of the molecular cues underlying HOXA9-mediated tumorigenesis and cancer progression. In a first part, we analyzed the domain(s) of interaction of PBX1 with HOXA9 in order to understand the interaction mechanism between these two canonical partners. Then, we enlarged our study by searching new candidate cofactors of HoxA9. Using computational prediction methods, based on 54 microarray experiments from GEO, including 49 breast cancer samples belonging to 7 breast cancer subtypes, and 5 non-cancerous control breast tissues, we identified 22 transcription factors (TF) as putative HOXA9 cofactors. These cofactors were compared to cofactors identified using a new high-throughput BiFC screening, previously used to sort candidate HoxA9 cofactors in MDA-MB231 breast cancer cells. Finally, we improved the BiFC screening method by designing a new Gateway destination vector for the human cDNA library.
... The cooperative role of HOX and TALE in cancer is best established during leukemogenesis, where it has been demonstrated that PBX3 and MEIS1 are important cofactors for the transformation/immortalization activity of HOX proteins (in particular HOXA9) in hematopoietic stem cells (Li et al., 2013;Rozovskaia et al., 2001). Such cooperative role has rarely been shown in solid cancers (see for example (Fernandez et al., 2008)) and is principally deduced from indirect studies using a HOX-PBC interaction inhibitory peptide in cancer cell lines (see for example (Morgan et al., 2012). Moreover, the role of PBC, MEIS and PREP was analyzed with one member of each subfamily in most cancer studies (PBX1, MEIS1 and PREP1), which asks for the role of the other TALE members (PBX2-4, MEIS2-3 and PREP2). ...
HOX and TALE genes encode homeodomain (HD)-containing transcription factors that act in concert in different tissues to coordinate cell fates and morphogenesis throughout embryonic development. These two evolutionary conserved families contain several members that form different types of protein complexes on DNA. Mutations affecting the expression of HOX or TALE genes have been reported in a number of cancers, but whether and how the two gene families could be perturbed together has never been explored systematically. As a consequence, the putative collaborative role between HOX and TALE members for promoting or inhibiting oncogenesis remains to be established in most cancer contexts. Here, we address this issue by considering HOX and TALE expression profiling in normal and cancer adult tissues, using normalized RNA-sequencing expression data deriving from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) research projects. Information was extracted from 28 cancer types originating from 21 different tissues, constituting a unique comparative analysis of HOX and TALE expression profiles between normal and cancer contexts in human. We present the general and specific rules that could be deduced from this large-scale comparative analysis. Overall this work provides a precious annotated support to better understand the role of specific HOX/TALE combinatorial codes in human cancers.
... HOX are highly conserved transcription factors playing crucial roles in development, and several HOX genes are associated with cancer (7)(8)(9). Many previous studies have demonstrated abnormal HOX expression in breast cancer tissues and culture cells, and furthermore, their roles in tumorigenesis and metastasis of breast cancer (10)(11)(12)(13)(14). In addition, many HOX genes, such as HOXB5, HOXB7, HOXB13, HOXC10, HOXC11, and non-coding RNAs in HOX clusters are associated with endocrine resistance to breast cancer via repression of ER expression or activation of receptor tyrosine kinase pathways (15)(16)(17)(18)(19). ...
Tamoxifen (TAM) is commonly used to treat estrogen receptor (ER)-positive breast cancer. Despite the remarkable benefits, resistance to TAM presents a serious therapeutic challenge. Since several HOX transcription factors have been proposed as strong candidates in the development of resistance to TAM therapy in breast cancer, we generated an in vitro model of acquired TAM resistance using ER-positive MCF7 breast cancer cells (MCF7-TAMR), and analyzed the expression pattern and epigenetic states of HOX genes. HOXB cluster genes were uniquely up-regulated in MCF7-TAMR cells. Survival analysis of in slico data showed the correlation of high expression of HOXB genes with poor response to TAM in ER-positive breast cancer patients treated with TAM. Gain- and loss-of-function experiments showed that the overexpression of multi HOXB genes in MCF7 renders cancer cells more resistant to TAM, whereas the knockdown restores TAM sensitivity. Furthermore, activation of HOXB genes in MCF7-TAMR was associated with histone modifications, particularly the gain of H3K9ac. These findings imply that the activation of HOXB genes mediate the development of TAM resistance, and represent a target for development of new strategies to prevent or reverse TAM resistance.
... An alternative strategy to targeting HOX proteins is to inhibit their interaction with the PBX co-factor, which can be achieved using a short, cell-penetrating peptide (HXR9) that mimics the conserved hexapeptide in HOX proteins responsible for PBX binding [4]. HXR9 has been shown to induce apoptosis in a range of solid cancers, both in vitro and in vivo, including those of the lung [5], breast [6] and prostate [7], and melanoma [8]. It has also been shown to be cytotoxic for malignant B cells [9], and a number of AML cell lines [10]. ...
The HOX genes encode a family of transcription factors that have key roles in both development and malignancy. Disrupting the interaction between HOX proteins and their binding partner, PBX, has been shown to cause apoptotic cell death in a range of solid tumors. However, despite HOX proteins playing a particularly significant role in acute myeloid leukemia (AML), the relationship between HOX gene expression and patient survival has not been evaluated (with the exception of HOXA9), and the mechanism by which HOX/PBX inhibition induces cell death in this malignancy is not well understood. In this study, we show that the expression of HOXA5, HOXB2, HOXB4, HOXB9, and HOXC9, but not HOXA9, in primary AML samples is significantly related to survival. Furthermore, the previously described inhibitor of HOX/PBX dimerization, HXR9, is cytotoxic to both AML-derived cell lines and primary AML cells from patients. The mechanism of cell death is not dependent on apoptosis but instead involves a regulated form of necrosis referred to as necroptosis. HXR9-induced necroptosis is enhanced by inhibitors of protein kinase C (PKC) signaling, and HXR9 combined with the PKC inhibitor Ro31 causes a significantly greater reduction in tumor growth compared to either reagent alone.
... However, the only effective HOX/PBX binding inhibitors are the HXR9 peptide and its derivatives. In a study on breast cancer, the sensitivity of breast cancer cell to killing by HXR9 was shown to be strongly related to the expression of HOXB1 through to HOXB9 [49]. However, it has not been reported in other cancers. ...
The purpose of this study was to investigate the HOX gene expression profile in laryngeal squamous cell carcinoma (LSCC) and assess whether some genes are associated with the clinicopathological features and prognosis in LSCC patients. The HOX gene levels were tested by microarray and validated by qRT-PCR in paired cancerous and adjacent noncancerous LSCC tissue samples. The microarray testing data of 39 HOX genes revealed 15 HOX genes that were at least 2-fold upregulated and 2 that were downregulated. After qRT-PCR evaluation, the three most upregulated genes (HOXB9, HOXB13, and HOXD13) were selected for tissue microarray (TMA) analysis. The correlations between the HOXB9, HOXB13, and HOXD13 expression levels and both clinicopathological features and prognosis were analyzed. Three HOX gene expression levels were markedly increased in LSCC tissues compared with adjacent noncancerous tissues ( P<0.001 ). HOXB9 was found to correlate with histological grade ( P<0.01 ) and prognosis ( P<0.01 ) in LSCC. In conclusion, this study revealed that HOXB9, HOXB13, and HOXD13 were upregulated and may play important roles in LSCC. Moreover, HOXB9 may serve as a novel marker of poor prognosis and a potential therapeutic target in LSCC patients.
... 6,7 HXR9 peptide which targets the interaction between HOX proteins and PBX1 induced apoptosis in breast cancer-derived cell lines. 22 PREP1 and MEIS1 competition for PBX1 binding regulates protein stability and tumorigenesis through the Notch signaling pathway. 23,24 Recently, PREP1 was identified to be a novel epithelial-to-mesenchymal transition (EMT) inducer and pro-metastatic transcription factor in human non-small cell lung cancer cells. ...
Risk biomarkers for estrogen receptor (ER) negative breast cancer have clear value for breast cancer prevention. We previously reported a set of lipid metabolism (LiMe) genes with high expression in the contralateral unaffected breasts (CUBs) of ER-negative cancer cases. We now further examine LiMe gene expression in both tumor and CUB, and investigate the role of Pre-B-cell leukemia homeobox-1 (PBX1) as a candidate common transcription factor for LiMe gene expression. mRNA was extracted from laser-capture microdissected epithelium from tumor and CUB of 84 subjects (28 ER-positive cases, 28 ER-negative cases, 28 healthy controls). Gene expression was quantitated by qRT-PCR. Logistic regression models were generated to predict ER status of the contralateral cancer. Protein expression of HMGCS2 and PBX1 was measured using immunohistochemistry. The effect of PBX1 on LiMe gene expression was examined by overexpressing PBX1 in MCF10A cells with or without ER, and by suppressing PBX1 in MDA-MB-453 cells. The expression of DHRS2, HMGCS2, UGT2B7, UGT2B11, ALOX15B, HPGD, UGT2B28 and GLYATL1 was significantly higher in ER-negative versus ER-positive CUBs, and predicted ER status of the tumor in test and validation sets. In contrast, LiMe gene expression was significantly lower in ER-negative than ER-positive tumors. PBX1 overexpression in MCF10A cells up-regulated most LiMe genes, but not in MCF10A cells overexpressing ER. Suppressing PBX1 in MDA-MB-453 cells resulted in decrease of LiMe gene expression. Four binding sites of PBX1 and cofactor were identified in three lipid metabolism genes using ChIP-qPCR. These data suggest a novel role for PBX1 in the regulation of lipid metabolism genes in benign breast, which may contribute to ER-negative tumorigenesis. This article is protected by copyright. All rights reserved.
The HOX and PBX genes encode transcription factors that have key roles in development and cancer, both independently and as a heterodimer within a complex of proteins that recognizes specific sequences in DNA and can both activate and repress transcription of target genes. Due to functional redundancy amongst HOX proteins, knock down or knock out studies of individual genes often do not result in an altered phenotype. An alternative approach is to target the interaction between HOX and PBX proteins, which is dependent on a conserved hexapeptide region within HOX. To this end, several peptides have been developed based on the hexapeptide sequence which act as competitive antagonists of HOX/PBX binding, including HXR9 and HTL001. Here, we review the methodology that has been used in these studies, including peptide syntheses, cell culture, assays, and mouse models.
HOX genes constitute a family of evolutionarily conserved transcription factors that play pivotal roles in embryonic development, tissue patterning, and cell differentiation. These genes are essential for the precise spatial and temporal control of body axis formation in vertebrates. In addition to their developmental functions, HOX genes have garnered significant attention for their involvement in various diseases, including cancer. Deregulation of HOX gene expression has been observed in numerous malignancies, where they can influence tumorigenesis, progression, and therapeutic responses. This review provides an overview of the diverse roles of HOX genes in development, disease, and potential therapeutic targets, highlighting their significance in understanding biological processes and their potential clinical implications.
HOXC6 plays an essential part of the carcinogenesis of solid tumors, but its functional relevance within the immune contexture in patients with colorectal cancer (CRC) is still uncertain. We intended to investigate the predictive value of HOXC6 expression for survival outcomes and its correlation with immune contexture in CRC patients by utilizing the Cancer Genome Atlas database (n = 619). Validation was performed in cohorts from Zhongshan Hospital (n = 200) and Shanghai Cancer Center (n = 300). Immunohistochemical (IHC) staining was utilized to compare the levels of immunocytes infiltrating the tumor between the groups with high and low expression of HOXC6. Elevated levels of HOXC6 expression in CRC tissues were linked to malignant progression and poor prognosis. HOXC6 as a risk factor for survival of CRC patients was confirmed. Receiver operating characteristic analysis confirmed its diagnostic value, and a reliable prognostic nomogram was constructed. KEGG analysis and GSEA showed that HOXC6 participated in immune regulation, and its expression was tightly linked to the abundance of infiltrating immunocytes. HOXC6 was upregulated in patients diagnosed with CRC within the two cohorts, and high HOXC6 levels were correlated with a worse prognosis. The high-HOXC6 expression group showed increased infiltration of Treg cells, CD68⁺ macrophages, CD66b⁺ neutrophils, and CD8⁺ T-cells and elevated levels of PD-L1 and PD-1, but decreased levels of granzyme B and perforin. These findings suggest that HOXC6 abundance in patients with CRC determines a poor prognosis, promotes an immunoevasive environment, and directs CD8⁺ T-cell dysfunction. HOXC6 is expected to become a prospective biomarker for the outcome of CRC.
The roles of HOX transcription factors as oncogenes and tumor suppressor genes, and the NF-KB pathway in chronic inflammation, both leading to cancer are well-established. HOX transcription factors are members of an evolutionarily conserved family of proteins required for anteroposterior body axis patterning during embryonic development, and are often dysregulated in cancer. The NF-KB pathway aids inflammation and immunity but it is also important during embryonic development. It is frequently activated in both solid and hematological malignancies. NF-KB and HOX proteins can influence each other through mutual transcriptional regulation, protein-protein interactions, and regulation of upstream and downstream interactors. These interactions have important implications both in homeostasis and in disease. In this review, we summarize the role of HOX proteins in regulating inflammation in homeostasis and disease- with a particular emphasis on cancer. We also describe the relationship between HOX genes and the NF-KB pathway, and discuss potential therapeutic strategies.
Le cancer du rein représente 3% des cancers de l’adulte. Il s’agit d’une pathologie tumorale hétérogène insidieuse dont le pronostic reste généralement bon lorsque le cancer est découvert précocement. Malheureusement, la survie à 5 ans ne dépasse pas les 10% lorsque la découverte se fait au stade métastatique. La prise en charge des patients repose sur la chirurgie et sur l’utilisation d’une thérapie systémique aux stades avancés ou métastatiques. Bien que ces dernières années, le cancer du rein ait fait l’objet d’une révolution thérapeutique avec l’arrivée de thérapies ciblées anti-angiogéniques et d’immunomodulateurs, les taux de réponse ne dépassent pas les 30%. Il y a donc un intérêt tout particulier à identifier de nouvelles cibles thérapeutiques dans ce cancer pour espérer de meilleurs traitements.Pour cela, nous travaillons sur le rôle du facteur de transcription HOXA9 et sur son inhibition par de petites molécules, capables d’entrer en compétition avec sa liaison à l’ADN, d’inhiber son activité transcriptionnelle et, par conséquent, les programmes oncogéniques qu’il régule. Deux molécules inhibitrices ont été sélectionnées.Dans un premier temps, nous avons utilisé une base de données publique et montréqu’HOXA9 était surexprimé dans le sous-type papillaire et que son expression était corrélée à la survie des patients dans le sous-type à cellules claires.Puis, à l’aide de deux lignées cellulaires humaines représentant les deux principaux types histologiques de carcinome à cellules rénales, nous avons montré qu’elles exprimaientfortement HOXA9 et évalué ses effets biologiques par invalidation par ARN interférence.Les effets biologiques observés ont été comparés à ceux obtenus par le traitement avecles molécules inhibitrices. Nous avons ainsi pu montrer que l’invalidation d’HOXA9entraînait la mort des cellules et impactait la migration cellulaire. De plus, le traitementpar les molécules inhibitrices impacte également la survie des cellules et leur migration.Pour identifier les gènes régulés indirectement ou directement par HOXA9, une analyse transcriptomique après invalidation a identifié qu’HOXA9 régule des gènes impliqués dans la voie du TNFα, et notamment certains gènes déjà décrits dans le carcinome rénal tels que BMP2. BMP2 étant connu pour réguler la sous-population de cellules souches cancéreuses (CSC), nous avons évalué le rôle d’HOXA9 dans cette population et montréqu’HOXA9 est surexprimé lors de la formation de tumorosphères, une caractéristique des CSC et que son invalidation abolit totalement la formation de ces sphères. Pour confirmer ces résultats, des cellules invalidées pour HOXA9 ont été implantées orthotopiquement à des souris immunodéficientes et montrent qu’HOXA9 est nécessaire à la tumorigénicité.Enfin, pour évaluer le rôle d’HOXA9 dans les métastases du cancer rénal et leur ciblage potentiel, nous avons généré un modèle métastatique pulmonaire et montré que l’invalidation d’HOXA9 est associée à une diminution de la formation de métastases pulmonaires du carcinome rénal.
Background
Sinonasal papilloma has a tendency toward local destruction, recurrence, and malignant transformation. This study aimed to unravel mechanisms in the malignant transformation of sinonasal papillomas using RNA‐seq.
Methods
The cohort consisted of 37 consecutive patients; tumor histology included a continuum spectrum (sinonasal papillomas/dysplastic/carcinomas‐in‐situ/invasive squamous cell carcinomas). These were microdissected and RNA was subjected to whole‐transcriptome shotgun sequencing.
Results
RNA‐seq and pathway analysis showed that the highest expressed genes/potential drivers were development‐ and differentiation‐related genes. The protein expression of six highly upregulated genes (HOXA9, EN1, DUX4, CA9, CD1a, and CK5/6) validated the RNA‐seq results. HOXA9 and CA9 were found to be expressed in most of the carcinoma samples but were largely negative in papillomas; all of the CA9‐negative carcinomas were recurrent.
Conclusions
We conclude that sinonasal carcinomas arising from papillomas are mainly defined by overexpressed developmental/homeobox genes, which provide the potential for transformation/plasticity, along with differentiation and proliferation behavior of neoplastic cells. Our results support HOXA9 and CA9 as biomarkers for carcinomas, with CA9 emerging as a predictive marker of recurrence.
Background
HOXB9 is a homeobox transcription factor which plays an important role in carcinoma development. This protein has been shown to inhibit cancer cell proliferation. However, the mechanisms that underpin HOXB9-mediated inhibition of cellular proliferation remain to be elucidated.
Methods
In this study, two gastric cancer cell lines, SGC7901 and MKN45, were transfected with plasmids pLVX-HOXB9 and shHOXB9. These transfections resulted in the over-expression of the HOXB9 gene in the SGC7901/HOXB9 cells and knockdown of the HOXB9 gene in the MKN45/shHOXB9 cells.
Results
Over-expression of the HOXB9 gene in the SGC7901/HOXB9 cells caused an increase in the apoptotic rate and a concomitant reduction in metastatic ability compared with the knocked-down MKN45/shHOXB9 cells. Moreover, a reduction in the expression of the phosphorylated-Akt protein was observed in the SGC7901/HOXB9 cells, while an increase in expression of the same protein was observed in the MKN45/shHOXB9 cells. We also observed that HOXB9 mediated a reduction in both NF-κB and N-cadherin and Snail protein expression. Conversely, HOXB9 caused an increase in the expression of E-cadherin.
Conclusions
In summary, this study reports that HOXB9 can suppress both phosphorylated-Akt expression and NF-κB activity. The latter phenomenon affects Snail protein expression and the inhibition of gastric carcinoma proliferation.
Background:
Increased transcription of the human endogenous retrovirus group HERV-K (HML-2) is often seen during disease. Although the mechanism of its tissue-specific activation is unclear, research shows that LTR CpG hypomethylation alone is not sufficient to induce its promoter activity and that the transcriptional milieu of a malignant cell contributes, at least partly, to differential HML-2 expression.
Results:
We analyzed the relationship between LTR sequence variation and promoter expression patterns in human breast cancer cell lines, finding them to be positively correlated. In particular, two proviruses (3q12.3 and 11p15.4) displayed increased activity in almost all tumorigenic cell lines sampled. Using a transcription factor binding site prediction algorithm, we identified two unique binding sites in each 5' LTR that appeared to be associated with inducing promoter activity during neoplasia. Genomic analysis of the homologous proviruses in several non-human primates indicated post-integration genetic drift in two transcription factor binding sites, away from the ancestral sequence and towards the active form. Based on the sequences of 2504 individuals from the 1000 Genomes Project, the active form of the 11p15.4 site was found to be polymorphic within the human population, with an allele frequency of 51%, whereas the activating mutation in the 3q12.3 provirus was fixed in humans but not present in the orthologous provirus in chimpanzees or gorillas.
Conclusions:
These data suggest that stage-specific transcription factors at least partly contribute to LTR promoter activity during transformation and that, in some cases, transcription factor binding site polymorphisms may be responsible for the differential HML-2 expression often seen between individuals.
HOX genes encode an evolutionarily conserved set of transcription factors that control how the phenotype of an organism becomes organized during development based on its genetic makeup. For example, in bilaterian-type animals, HOX genes are organized in gene clusters that encode anatomic segment identity, that is, whether the embryo will form with bilateral symmetry with a head (anterior), tail (posterior), back (dorsal), and belly (ventral). Although HOX genes are known to regulate stem cell (SC) differentiation and HOX genes are dysregulated in cancer, the mechanisms by which dysregulation of HOX genes in SCs causes cancer development is not fully understood. Therefore, the purpose of this manuscript was (i) to review the role of HOX genes in SC differentiation, particularly in embryonic, adult tissue-specific, and induced pluripotent SC, and (ii) to investigate how dysregulated HOX genes in SCs are responsible for the development of colorectal cancer (CRC) and acute myeloid leukemia (AML). We analyzed HOX gene expression in CRC and AML using information from The Cancer Genome Atlas study. Finally, we reviewed the literature on HOX genes and related therapeutics that might help us understand ways to develop SC-specific therapies that target aberrant HOX gene expression that contributes to cancer development.
Transcription factors regulate gene expression in cells and control cellular development, function, and death. Dysregulation of transcription factors is often associated with disease, including cancer. As such, transcription factors are attractive targets for design of therapeutics against disease. Transcription factors function using protein-protein and protein-DNA interactions that occur over relatively large surface areas: this lack of a small and defined “ligand binding site” has proven to be challenging to target with small molecules. Peptide therapeutics, therefore, provide an alternate approach toward design of inhibitory agents. Transcription factors are conveniently modular by design: just the small domain that is responsible for the transcription factor's DNA binding or a protein-protein interaction or another function, can serve as the basis for novel peptide therapeutics. In this review, examples of peptides that directly interfere with transcription factors will be discussed.
Hox homeodomain proteins are developmental regulators that determine body plan in a variety of organisms. A majority of the vertebrate Hox proteins bind DNA as heterodimers with the Pbx1 homeodomain protein. We report here the 2.35 Å structure of a ternary complex containing a human HoxB1–Pbx1 heterodimer bound to DNA. Heterodimer contacts are mediated by the hexapeptide of HoxB1, which binds in a pocket in the Pbx1 protein formed in part by a three–amino acid insertion in the Pbx1 homeodomain. The Pbx1 DNA-binding domain is larger than the canonical homeodomain, containing an additional α helix that appears to contribute to binding of the HoxB1 hexapeptide and to stable binding of Pbx1 to DNA. The structure suggests a model for modulation of Hox DNA binding activity by Pbx1 and related proteins.
Multiple factors including long-term treatment with tamoxifen are involved in the development of selective estrogen receptor (ER) modulator resistance in ERα-positive breast cancer. Many underlying molecular events that confer resistance are known but a unifying theme is yet to be revealed. In this report, we provide evidence that HOXB7 overexpression renders MCF-7 cells resistant to tamoxifen via cross-talk between receptor tyrosine kinases and ERα signaling. HOXB7 is an ERα-responsive gene. Extended treatment of MCF-7 cells with tamoxifen resulted in progressively increasing levels of HOXB7 expression, along with EGFR and EGFR ligands. Up-regulation of EGFR occurs through direct binding of HOXB7 to the EGFR promoter, enhancing transcriptional activity. Finally, higher expression levels of HOXB7 in the tumor significantly correlated with poorer disease-free survival in ERα-positive patients with breast cancer on adjuvant tamoxifen monotherapy. These studies suggest that HOXB7 acts as a key regulator, orchestrating a major group of target molecules in the oncogenic hierarchy. Functional antagonism of HOXB7 could circumvent tamoxifen resistance.
The HOX genes encode a family of transcription factors that are dysregulated in several malignancies and have been implicated in oncogenesis and cancer cell survival. Disruption of HOX protein function using the peptide HXR9 has shown anti-tumor effects against melanoma, lung cancer and renal cancer. In this report, we evaluated the expression of all 39 HOX genes in a panel of six malignant B-cell lines, including multiple myeloma cells and found different levels of expression of HOX family members suggesting that they also have a role in malignant B-cell survival. We show that disrupting HOX function using the peptide HXR9 induces significant cytotoxicity in the entire panel of cell lines. Importantly, we found that the cytotoxic effects of HXR9 can be enhanced by combining it with ch128.1Av, an antibody-avidin fusion protein specific for the human transferrin receptor 1 (CD71). Iron starvation induced by the fusion protein contributes to the enhanced effect and involves, at least in part, the induction of a caspase-independent pathway. These results show the relevance of HOX proteins in malignant B-cell survival and suggest that our therapeutic strategy may be effective in the treatment of incurable B-cell malignancies such as multiple myeloma.
Hox genes, a highly conserved subgroup of the homeobox superfamily, have crucial roles in development, regulating numerous processes including apoptosis, receptor signalling, differentiation, motility and angiogenesis. Aberrations in Hox gene expression have been reported in abnormal development and malignancy, indicating that altered expression of Hox genes could be important for both oncogenesis and tumour suppression, depending on context. Therefore, Hox gene expression could be important in diagnosis and therapy.
Ovarian cancer still has a relatively poor prognosis due to the frequent occurrence of drug resistance, making the identification of new therapeutic targets an important goal. We have studied the role of HOX genes in the survival and proliferation of ovarian cancer cells. These are a family of homeodomain-containing transcription factors that determine cell and tissue identity in the early embryo, and have an anti-apoptotic role in a number of malignancies including lung and renal cancer.
We used QPCR to determine HOX gene expression in normal ovary and in the ovarian cancer cell lines SK-OV3 and OV-90. We used a short peptide, HXR9, to disrupt the formation of HOX/PBX dimers and alter transcriptional regulation by HOX proteins.
In this study we show that the ovarian cancer derived line SK-OV3, but not OV-90, exhibits highly dysregulated expression of members of the HOX gene family. Disrupting the interaction between HOX proteins and their co-factor PBX induces apoptosis in SK-OV3 cells and retards tumour growth in vivo.
HOX/PBX binding is a potential target in ovarian cancer.
Genomic integration of human papillomavirus (HPV) DNA accounts for more than 90% of cervical cancers. High-risk genital HPVs
encode E6 proteins that can interact with a cellular ubiquitin ligase E6-associated protein (E6AP) and target the tumor suppressor
p53 for ubiquitin-mediated proteolysis. Currently, how this critical event is regulated is largely unknown. Here we report
that activating transcription factor 3 (ATF3), a broad DNA damage sensor whose expression is frequently downregulated in cervical
cancer, interacted with E6 and prevented p53 from ubiquitination and degradation mediated by the viral protein. Consistent
with its role as a potent E6 antagonist, ATF3 expressed enforcedly in HPV-positive SiHa cells activated p53, leading to expression
of p53-target genes (e.g. p21 and PUMA), cell cycle arrest and apoptotic cell death. The leucine zipper domain of ATF3 appears indispensable for these
effects as an ATF3 mutant lacking this domain failed to interact with E6 and activate p53 in the cervical cancer cells. The
prevention of p53 degradation was unlikely caused by binding of ATF3 to the tumor suppressor, but rather was a consequence
of disruption of the E6-E6AP interaction by ATF3. These results indicate that ATF3 plays a key role in a mechanism defending
against HPV-induced carcinogenesis, and could serve as a novel therapeutic target for HPV-positive cancers.
The HOX genes are a family of homeodomain-containing transcription factors that determine the identity of cells and tissues during embryonic development. They are also known to behave as oncogenes in some haematological malignancies. In this study, we show that the expression of many of the HOX genes is highly elevated in primary non-small-cell lung cancers (NSCLCs) and in the derived cell lines A549 and H23. Furthermore, blocking the activity of HOX proteins by interfering with their binding to the PBX co-factor causes these cells to undergo apoptosis in vitro and reduces the growth of A549 tumours in vivo. These findings suggest that the interaction between HOX and PBX proteins is a potential therapeutic target in NSCLC.
The HOX genes are a family of homeodomain containing transcription factors that determine embryonic tissue identity and also have regulatory and oncogenic roles in adult cells. We quantified the expression of HOX genes in normal kidney tissue, primary tumors and derived cell lines, and examined their role in renal cancer cell survival.
Quantitative polymerase chain reaction was used to evaluate HOX gene expression in cells and tissues. HOX gene function was disrupted using a peptide that blocks the interaction between HOX proteins and their PBX cofactor. Apoptosis was assessed by annexin/propidium iodide staining and direct measurement of caspase activity.
Primary renal tumors and derived cell lines showed abnormal HOX gene expression. Furthermore, blocking HOX activity by targeting the interaction between HOX and its cofactor PBX caused apoptotic and necrotic cell death in the renal cancer cell lines CaKi-2 and 769-P, while sparing normal adult kidney cells.
Our findings suggest that the HOX/PBX dimer is a potential therapeutic target in renal cancer.
Hox genes encode homeodomain-containing transcriptional regulators that function during development to specify positional identity along embryonic axes. The homeodomain is composed of a flexible N-terminal arm and three alpha helices, and it differentially binds DNA. A number of homeodomains recognize sites containing a TAAT core motif. The product of the murine Hoxd-4 (Hox-4.2) gene functions in a positive autoregulatory fashion in P19 cells that is dependent on two TAAT motifs in the Hoxd-4 promoter. This effect is specific in that murine HOXA-1 (HOX-1.6) is unable to activate transcription through the Hoxd-4 autoregulatory element. Here we show that this is due to an inability of the HOXA-1 homeodomain to bind a HOXD-4 recognition site effectively. We have produced chimeras between HOXD-4 and HOXA-1 to map specific residues responsible for this functional difference. When positions 2 and 3 in the N-terminal arm of HOXA-1 were converted to HOXD-4 identity, both strong DNA binding and transcriptional activation were rescued. This substitution appears to confer an increased DNA-binding ability on the HOXA-1 homeodomain, since we were unable to detect a high-affinity recognition sequence for HOXA-1 in a randomized pool of DNA probes. The contribution of position 3 to DNA binding has been implicated by structural studies, but this is the first report of the importance of position 2 in regulating homeodomain-DNA interactions. Additionally, specific homeodomain residues that confer major differences in DNA binding and transcriptional activation between Hox gene products have not been previously determined. Identity at these two positions is generally conserved among paralogs but varies between Hox gene subfamilies. As a result, these residues may be important for the regulation of target gene expression by specific Hox products.
E2a-Pbx1 is a chimeric transcription factor oncoprotein produced by the t(1;19) translocation in human pre-B cell leukemia. Class I Hox proteins bind DNA cooperatively with both Pbx proteins and oncoprotein E2a-Pbx1, suggesting that leukemogenesis by E2a-Pbx1 and Hox proteins may alter transcription of cellular genes regulated by Pbx-Hox motifs. Likewise, in murine myeloid leukemia, transcriptional coactivation of Meis1 with HoxA7/A9 suggests that Meis1-HoxA7/9 heterodimers may evoke aberrant gene transcription. Here, we demonstrate that both Meis1 and its relative, pKnox1, dimerize with Pbx1 on the same TGATTGAC motif selected by dimers of Pbx proteins and unidentified partner(s) in nuclear extracts, including those from t(1;19) pre-B cells. Outside their homeodomains, Meis1 and pKnox1 were highly conserved only in two motifs required for cooperativity with Pbx1. Like the unidentified endogenous partner(s), both Meis1 and pKnox1 failed to dimerize significantly with E2a-Pbx1. The Meis1/pKnox1-interaction domain in Pbx1 resided predominantly in a conserved N-terminal Pbx domain deleted in E2a-Pbx1. Thus, the leukemic potential of E2a-Pbx1 may require abrogation of its interaction with members of the Meis and pKnox families of transcription factors, permitting selective targeting of genes regulated by Pbx-Hox complexes. In addition, because most motifs bound by Pbx-Meis1/pKnox1 were not bound by Pbx1-Hox complexes, the leukemic potential of Meis1 in myeloid leukemias may involve shifting Pbx proteins from promoters containing Pbx-Hox motifs to those containing Pbx-Meis motifs.
Several melanomas, carcinomas, glioblastomas and leukemias showed coordinated expression of HOXB7 and bFGF with exception of the SkBr3 mammary carcinoma that was negative for both. Transduction of HOXB7 gene into SkBr3 cells, induced bFGF expression, increased growth rate, independence from serum withdrawal and ability to form colonies in semisolid medium. ELISA assay showed that most of bFGF was associated to cell lysate when cells were cultured at 1% serum whereas in cells kept to 10% serum bFGF was detected both within cell lysate or secreted into cell supernatants. Antisense oligos to bFGF inhibited the growth of cells cultured in 1%, indicating that beside the possible activation of additional genes other than bFGF by HOXB7 transduction, only bFGF induction accounts for the observed results. Moreover, since inhibition of cell proliferation occurred in cells kept in 1% but not 10% serum, a bFGF intracrine loop appears operative in serum starved SkBr3/HOXB7 cells. Also, these results further indicate bFGF as target of HOXB7.
Expression of the p53 gene protects cells against malignant transformation1, 2. Whereas control of p53 degradation has been a subject of intense
scrutiny, little is known about the factors that regulate p53 synthesis1, 2. Here we show that p53 messenger RNA levels are low in a large
proportion of breast tumours. Seeking potential regulators of p53 transcription,
we found consensus HOX binding sites3, 4 in the p53 promoter5. Transient transfection of Hox/HOXA5 activated the p53 promoter.
Expression of HOXA5 in epithelial cancer cells expressing wild-type p53, but
not in isogenic variants lacking the p53 gene6, led to
apoptotic cell death. Moreover, breast cancer cell lines and patient tumours
display a coordinate loss of p53 and HOXA5 mRNA and protein expression. The
HOXA5 promoter region was methylated in 16 out of 20 p53-negative breast tumour
specimens. We conclude that loss of expression of p53 in human breast cancer
may be primarily due to lack of expression of HOXA5.
HOX genes are evolutionarily highly conserved. The HOX proteins which they encode are master regulators of embryonic development and continue to be expressed throughout postnatal life. The 39 human HOX genes are located in four clusters (A-D) on different chromosomes at 7p15, 17q21 [corrected] 12q13, and 2q31 respectively and are assumed to have arisen by duplication and divergence from a primordial homeobox gene. Disorders of limb formation, such as hand-foot-genital syndrome, have been traced to mutations in HOXA13 and HOXD13. Evolutionary conservation provides unlimited scope for experimental investigation of the functional control of the Hox gene network which is providing important insights into human disease. Chromosomal translocations involving the MLL gene, the human homologue of the Drosophila gene trithorax, create fusion genes which exhibit gain of function and are associated with aggressive leukaemias in both adults and children. To date 39 partner genes for MLL have been cloned from patients with leukaemia. Models based on specific translocations of MLL and individual HOX genes are now the subject of intense research aimed at understanding the molecular programs involved, and ultimately the design of chemotherapeutic agents for leukaemia. Investigation of the role of HOX genes in cancer has led to the concept that oncology may recapitulate ontology, a challenging postulate for experimentalists in view of the functional redundancy implicit in the HOX gene network.
Epithelial-mesenchymal transition (EMT) is increasingly recognized as a mechanism whereby cells in primary noninvasive tumors acquire properties essential for migration and invasion. Microarray analyses of microdissected epithelial cells from bone metastasis revealed a HOXB7 overexpression that was 3-fold higher than in primary breast carcinomas and 18-fold higher compared with normal breast. This led us to investigate the role of HOXB7 in neoplastic transformation of breast cells. Expression of HOXB7 in both MCF10A and Madin-Darby canine kidney (MDCK) epithelial cells resulted in the acquisition of both phenotypic and molecular attributes typical of EMT. Loss of epithelial proteins, claudin 1 and claudin 7, mislocalization of claudin 4 and E-cadherin, and the expression of mesenchymal proteins, vimentin and alpha-smooth muscle actin, were observed. MDCK cells expressing HOXB7 exhibited properties of migration and invasion. Unlike MDCK vector-transfected cells, MDCK-HOXB7 cells formed highly vascularized tumors in mice. MDCK-HOXB7 cells overexpressed basic fibroblast growth factor (bFGF), had more active forms of both Ras and RhoA proteins, and displayed higher levels of phosphorylation of p44 and p42 mitogen-activated protein kinase (MAPK; extracellular signal-regulated kinases 1 and 2). Effects initiated by HOXB7 were reversed by specific inhibitors of FGF receptor and the Ras-MAPK pathways. These data provide support for a function for HOXB7 in promoting tumor invasion through activation of Ras/Rho pathway by up-regulating bFGF, a known transcriptional target of HOXB7. Reversal of these effects by HOXB7-specific siRNA further suggested that these effects were mediated by HOXB7. Thus, HOXB7 overexpression caused EMT in epithelial cells, accompanied by acquisition of aggressive properties of tumorigenicity, migration, and invasion.
Homeobox genes encode transcription factors which function in body axis patterning in the developing embryo. Recent evidence suggests that the maintenance of specific HOX expression patterns is necessary for regulating the homeostasis of adult tissues as well. In this study, HOXB7 transformed human mammary epithelial cells, MCF10A, to grow in minimally supplemented medium, to form colonies in Matrigel, and display resistance to ionizing radiation. Searching for protein partners of HOXB7 that might contribute to resistance to ionizing radiation, we identified four HOXB7-binding proteins by GST pull-down/affinity chromatography and confirmed their interactions by coimmunoprecipitation in vivo. Interestingly, all four HOXB7-binding proteins shared functions as genomic caretakers and included members of the DNA-dependent protein kinase holoenzyme (Ku70, Ku80, DNA-PK(cs)) responsible for DNA double-strand break repair by nonhomologous end joining pathway and poly(ADP) ribose polymerase. Exogenous and endogenous expression of HOXB7 enhanced nonhomologous end joining and DNA repair functions in vitro and in vivo, which were reversed by silencing HOXB7. This is the first mechanistic study providing definitive evidence for the involvement of any HOX protein in DNA double-strand break repair.
The promise of retinoids as chemopreventive agents in breast cancer is based on the differentiation and apoptosis induced upon their binding to the retinoic acid (RA) receptor beta (RARbeta). We have previously shown that HOXA5 induces apoptosis in breast cancer cells. In this study, we investigated whether RA/RARbeta and HOXA5 actions intersect to induce apoptosis and differentiation in breast cancer cells. We found that HOXA5 expression can be induced by RA only in RARbeta-positive breast cancer cells. We have, for the first time, identified the RA response element in HOXA5, which was found to be located in the 3' end of the gene. Chromatin immunoprecipitation assays showed that RARbeta binds directly to this region in vivo. Overexpression of RARbeta strongly enhances RA responsiveness, and knocking down RARbeta expression abolishes RA-mediated induction of HOXA5 expression in breast cancer cells. In addition, there is coordinated loss of both HOXA5 and RARbeta expression during neoplastic transformation and progression in the breast epithelial cell model, MCF10A. Knockdown of HOXA5 expression partially abrogates retinoid-induced apoptosis and promotes cell survival upon RA treatment. These results strongly suggest that HOXA5 acts directly downstream of RARbeta and may contribute to retinoid-induced anticancer and chemopreventive effects.
Our previous studies have shown that HOXB7 mRNA is overexpressed in approximately 50% of invasive breast carcinomas and promotes tumor progression in breast cancer cells grown as xenografts in mice. In silico analysis of published microarray data showed that high levels of HOXB7 predict a poor outcome in HER-2-positive (P = 0.046), but not in HER-2-negative breast cancers (P = 0.94). To study the function of HOXB7 in vivo in the context of HER-2 overexpression, we generated mouse mammary tumor virus (MMTV)-Hoxb7 transgenic mice, and then crossed them with MMTV-HER-2/neu transgenic mice. In the mice carrying both Hoxb7 and HER-2/neu transgenes, Hoxb7 plays a dual role in mammary tumorigenesis. In double transgenic mice, overexpression of Hoxb7 delayed tumor onset and lowered tumor multiplicity. However, consistent with the clinical data, once the tumors appeared, their growth was faster and metastasis to the lungs occurred at a higher frequency. Our data show, for the first time, that deregulated expression of Hoxb7 in mammary tumor cells can significantly modulate HER-2/neu-oncogene induced tumorigenesis in vivo.
Tumor suppressor p53 is a transcription factor that induces growth arrest and/or apoptosis in response to cellular stress. In recent years, many genes have been identified as p53-regulated genes; however, no single target gene has been shown to be required for the apoptotic effect. Using microarray analysis, we have identified the transcription factor early growth response 2 (EGR2) as a target of the p53 family, specifically p53, p63 and p73. EGR2 expression was up-regulated by DNA damage-induced p53 activity, as well as by overexpression of p53 family genes. Furthermore, we identified a responsive element to p53, TAp63, and TAp73 within the EGR2 gene. This response element is highly conserved between human and rodents. We also found that overexpression of EGR2 induced apoptosis when combined with anticancer agents. Conversely, inactivation of EGR2 attenuated p53-mediated apoptosis. The results presented here suggest that EGR2 is a direct transcriptional target of p53 family that can in part mediate the p53-dependent apoptotic pathway.
Tumor suppressor p53 is a transcription factor that induces growth arrest and/or apoptosis in response to cellular stress. In recent years, many genes have been identified as p53-regulated genes; however, no single target gene has been shown to be required for the apoptotic effect. Using microarray analysis, we have identified the transcription factor early growth response 2 (EGR2) as a target of the p53 family, specifically p53, p63 and p73. EGR2 expression was up-regulated by DNA damage-induced p53 activity, as well as by overexpression of p53 family genes. Furthermore, we identified a responsive element to p53, TAp63, and TAp73 within the EGR2 gene. This response element is highly conserved between human and rodents. We also found that overexpression of EGR2 induced apoptosis when combined with anticancer agents. Conversely, inactivation of EGR2 attenuated p53-mediated apoptosis. The results presented here suggest that EGR2 is a direct transcriptional target of p53 family that can in part mediate the p53-dependent apoptotic pathway.
Hox genes encode transcription factors that play a central role in the specification of regional identities along the anterior to posterior body axis. In the developing mouse embryo, Hox genes from all four genomic clusters are involved in range of developmental processes, including the patterning of skeletal structures and the formation of several organs. However, the functional redundancy observed either between paralogous genes, or among neighboring genes from the same cluster, has hampered functional analyses, in particular when synergistic, cluster-specific functions are considered. Here, we report that mutant mice lacking the entire HoxA cluster in mesodermal lineages display the expected spectrum of postnatal respiratory, cardiac and urogenital defects, previously reported for single gene mutations. Likewise, mild phenotypes are observed in both appendicular and axial skeleton. However, a striking effect was uncovered in the hematopoietic system, much stronger than that seen for Hoxa9 inactivation alone, which involves stem cells (HSCs) as well as the erythroid lineage, indicating that several Hoxa genes are necessary for normal hematopoiesis to occur. Finally, the combined deletions of Hoxa and Hoxd genes reveal abnormalities in axial elongation as well as skin morphogenesis that are likely the results of defects in epithelial-mesenchymal interactions.
Hox genes encode transcription factors of crucial importance in the pattern formation of a large spectrum of species. Several studies have now proposed a role for these developmental genes in cancer biology. It has been suggested that HOXA5 possesses growth-suppressive properties through activation of p53 expression in human breast tissue. To assess the genetic cooperation that may exist between Hoxa5 and p53 in tumorigenesis, we generated Hoxa5/p53 compound mutant mice. The presence of Hoxa5 null alleles increased the susceptibility of p53(-/-) mice to develop tumors with a high prevalence for thymic lymphoma, suggesting that the loss of function of the two genes collaborate in tumor formation. To extend our analysis to mammary tumorigenesis, we performed Hoxa5/p53 whole mammary gland transplantations into wild-type hosts. In the p53(-/-) background, the presence of one Hoxa5 mutant allele had no impact on mammary tumor formation. In contrast, the complete loss of Hoxa5 function influenced the tumorigenic outcome of p53(+/-) mammary glands. However, the collaborative nature of this interaction did not depend on the transcriptional regulation of p53 by Hoxa5. Altogether, our data establish that Hoxa5 and p53 cooperate in mammary tumorigenesis in vivo.
The Secretariat, UKCCCR, The Medical Research Council, 20 Park Crescent, London W1N 4AL, United Kingdom
During 1973, 4 new epithelial tumor cell lines were isolated from pleural effusions from breast cancer patients. The authors describe 3 of these lines: MDA MB 134, with a mean chromosome number of 43; MDA MB 175, with a mean chromosome number of 49; and MDA MB 231, with a mean chromosome number between 65 and 69. The authors isolated the same cell type from 4 to 10 effusions from MDA MB 134 and from 6 of 8 effusions from MDA MB 175. Pleural effusions found as a source of breast tumor cells to be cultured and studied in vitro have the following advantages: large amounts of material and the possibility of obtaining sequential samples from the same patient; high viability of tumor cells; scarcity or absence of fibroblasts; and the possibility of separating the tumor cells from other 'contaminating' cell types by differences in their speed or degree of attachment to the flask. All lines from different patients differed, as seen grossly and microscopically. All lines from sequential pleural effusions from the same patient were apparently alike. No viruses or mycoplasmas were detected in any line.
Developmental fates along the anterior-posterior axes of animals are controlled by clustered homeotlc genes which In vertebrates
are called Hox genes. The gene clusters are similar and probably functionally homologous in animals as different as nematodes,
files, and mammals. A new set of names for Hox genes was recently agreed upon by many workers In the field. Remarkably, the
order of the Hox genes along the chromosome reflects where they are expressed along the body axis1. This simple principle is reflected in the new nomenclature system.
Hox homeodomain proteins are developmental regulators that determine body plan in a variety of organisms. A majority of the vertebrate Hox proteins bind DNA as heterodimers with the Pbx1 homeodomain protein. We report here the 2.35 A structure of a ternary complex containing a human HoxB1-Pbx1 heterodimer bound to DNA. Heterodimer contacts are mediated by the hexapeptide of HoxB1, which binds in a pocket in the Pbx1 protein formed in part by a three-amino acid insertion in the Pbx1 homeodomain. The Pbx1 DNA-binding domain is larger than the canonical homeodomain, containing an additional alpha helix that appears to contribute to binding of the HoxB1 hexapeptide and to stable binding of Pbx1 to DNA. The structure suggests a model for modulation of Hox DNA binding activity by Pbx1 and related proteins.
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We have shown that the 26 bp bx1 element from the regulatory region of Distal-less is capable of imposing control by the homeotic genes Ultrabithorax and abdominal-A on a general epidermal activator in Drosophila. This provides us with an assay to analyze the sequence requirements for specific repression by these Hox genes. Both the core Hox binding site, 5'-TAAT, and the adjacent EXD 5'-TGAT core site are required for repression by Ultrabithorax and abdominal-A. The Distal-less bx1 site thus fits with the model of Hox protein binding specificity based on the consensus PBX/HOX-family site TGATNNAT[g/t][g/a], where the key elements of binding specificity are proposed to lie in the two base pairs following the TGAT. A single base pair deletion in the bx1 sequence generates a site, bx1:A(-)mut, that on the consensus PBX/HOX model would be expected to be regulated by the Deformed Hox gene. We observed, however, that the bx1:A(-)mut site was regulated predominantly by Sex combs reduced, Ultrabithorax and abdominal-A. The analysis of this site indicates that the specificity of action of Hox proteins may depend not only on selective DNA binding but also on specific post-binding interactions.
Homeotic (Hox) genes regulate the identity of structures along the anterior-posterior axis of most animals. The low DNA-binding specificities of Hox proteins have raised the question of how these transcription factors selectively regulate target gene expression. The discovery that the Extradenticle (Exd)/Pbx and Homothorax (Hth)/Meis proteins act as cofactors for several Hox proteins has advanced the view that interactions with cofactors are critical to the target selectivity of Hox proteins. It is not clear, however, to what extent Hox proteins also regulate target genes in the absence of cofactors. In Drosophila melanogaster, the Hox protein Ultrabithorax (Ubx) promotes haltere development and suppresses wing development by selectively repressing many genes of the wing-patterning hierarchy, and this activity requires neither Exd nor Hth function. Here, we show that Ubx directly regulates a flight appendage-specific cis-regulatory element of the spalt (sal) gene. We find that multiple monomer Ubx-binding sites are required to completely repress this cis-element in the haltere, and that individual Ubx-binding sites are sufficient to mediate its partial repression. These results suggest that Hox proteins can directly regulate target genes in the absence of the cofactor Extradenticle. We propose that the regulation of some Hox target genes evolves via the accumulation of multiple Hox monomer binding sites. Furthermore, because the development and morphological diversity of the distal parts of most arthropod and vertebrate appendages involve Hox, but not Exd/Pbx or Hth/Meis proteins, this mode of target gene regulation appears to be important for distal appendage development and the evolution of appendage diversity.
Although epithelial ovarian cancers (EOCs) have been thought to arise from the simple epithelium lining the ovarian surface or inclusion cysts, the major subtypes of EOCs show morphologic features that resemble those of the müllerian duct-derived epithelia of the reproductive tract. We found that HOX genes, which normally regulate mullerian duct differentiation, are not expressed in normal ovarian surface epithelium (OSE), but are expressed in different EOC subtypes according to the pattern of mullerian-like differentiation of these cancers. Ectopic expression of Hoxa9 in tumorigenic mouse OSE cells gave rise to papillary tumors resembling serous EOCs. In contrast, Hoxa10 and Hoxa11 induced morphogenesis of endometrioid-like and mucinous-like EOCs, respectively. Hoxa7 showed no lineage specificity, but promoted the abilities of Hoxa9, Hoxa10 and Hoxa11 to induce differentiation along their respective pathways. Therefore, inappropriate activation of a molecular program that controls patterning of the reproductive tract could explain the morphologic heterogeneity of EOCs and their assumption of müllerian-like features.
Herein we focus on recent studies of knock out mice that demonstrate a function for the clustered homeobox (Hox) genes in normal hematopoiesis, on papers that point to their general involvement in human leukemia, and discuss the advances in the understanding of the mechanisms underlying their role in these processes.
Expression analysis and gain- or loss- of function studies have shown that Hox play an important role in the regulation of early stages of hematopoiesis, including the self-renewal of hematopoietic stem cells (HSCs)/early progenitors. In the area of leukemia, numerous models of murine leukemia have demonstrated a role for Hox in the pathobiology of the disease. Moreover, the identification of multiple Hox genes as partners of chromosomal translocations and the observed global deregulation of Hox genes and cofactors demonstrated by gene profiling of cells from leukemic patients, have unequivocally shown a major function for Hox genes and cofactors in a wide spectrum of human leukemia.
The identification of Hox genes as HSC regulators has been exploited to develop strategies to efficiently expand HSCs ex vivo, a key step to the success of therapies based on HSC transplantation and the understanding of mechanisms underlying HSC regulation. As leukemia is the result of deregulation of normal HSC development, the elucidation of the role of Hox in the pathobiology of the disease is helping to understand how HSCs self-renew and differentiate, and moreover, should facilitate the development of strategies for the management of leukemia.
The surprising variation in the number of Hox clusters and the genomic architecture within vertebrate lineages, especially within the ray-finned fish, reflects a history of duplications and subsequent lineage-specific gene loss. Recent research on the evolution of conserved non-coding sequences (CNS) in Hox clusters promises to reveal interesting results for functional and phenotypic diversification.
Hox genes encode homeodomain-containing transcription factors that pattern the body axes of animal embryos. It is well established that the exquisite DNA-binding specificity that allows different Hox proteins to specify distinct structures along the body axis is frequently dependent on interactions with other DNA-binding proteins which act as Hox cofactors. These include the PBC and MEIS classes of TALE (Three Amino acid Loop Extension) homeodomain proteins. The PBC class comprises fly Extradenticle (Exd) and vertebrate Pbx homeoproteins, whereas the MEIS class includes fly Homothorax (Hth) and vertebrate Meis and Prep homeoproteins. Exd was first implicated as a Hox cofactor based on mutant phenotypes in the fly. In vertebrates, PBC and MEIS homeobox proteins play important roles in development and disease. In this review, we describe the evidence that these functions reflect a requirement for Pbx and Meis/Prep proteins as Hox cofactors. However, there is mounting evidence that, like in the fly, Pbx and Meis/Prep proteins function more broadly, and we also discuss how "Hox cofactors" function as partners for other, non-Hox transcription factors during development. Conversely, we review the evidence that Hox proteins have functions that are independent of Pbx and Meis/Prep cofactors and discuss the possibility that other proteins may participate in the DNA-bound Hox complex, contributing to DNA-binding specificity in the absence of, or in addition to, Pbx and Meis/Prep.
RRAD, a small Ras-related GTPase, is highly expressed in human skeletal muscle, lung, and heart. Although loss of expression of RRAD in breast cancer cells has been reported and it may act as an oncogene, the mechanism of silencing is unknown.
We examined (1) mRNA expression of RRAD in lung and breast cancer cell lines using RT-PCR and (2) methylation status of lung and breast cancers.
Loss of RRAD expression was found in 14 of 20 (70%) NSCLC cell lines, 11 of 11 (100%) SCLC cell lines, and 8 of 10 (80%) breast cancer cell lines; expression was not affected in normal bronchial and mammary epithelial cells. Treatment of 23 expression-negative cell lines with a demethylating agent restored expression in all cases. We developed a methylation-specific assay from the analysis of bisulfite sequencing of the 5' region of RRAD in expression-negative and positive cell lines, which resulted in good concordance between methylation and expression. Primary lung and breast cancers showed hypermethylation in 89 of 214 (42%) and 39 of 63 (62%) cases, respectively. RRAD hypermethylation correlated with smoking history and poorer prognosis in lung adenocarcinomas.
We conclude that epigenetic silencing of RRAD is a frequent event in lung and breast cancers, and analysis of it may provide novel opportunities for prognosis and therapy of these cancers.
Vertebrae display distinct morphological features at different levels of the body axis. Links between collinear Hox gene activation and the progressive mode of body axis elongation have provided a fascinating blueprint of the mechanisms for establishing these morphological identities. In this review, we first discuss the regulation and possible role of collinear Hox gene activation during body formation and then highlight the direct role of Hox genes in controlling cellular movements during gastrulation, therefore contributing to body formation. Additional related research aspects, such as imaging of chromatin regulation, roles of micro RNAs and evolutional findings are also discussed.
Malignant melanoma is a cancer that arises from melanocyte cells in a complex but well-studied process, and which can only be successfully treated prior to metastasis as it is highly resistant to conventional therapies. A number of recent reports have indicated that members of the HOX family of homeodomain-containing transcription factors are deregulated in melanoma, and may actually be required to maintain proliferation. In this report, we describe the use of a novel, cell-permeable antagonist of the interaction between HOX proteins and PBX, a second homeodomain-containing transcription factor that modifies HOX activity. This antagonist can block the growth of murine B16 cells and trigger apoptosis both in vitro and in vivo when administered to mice with flank tumors.
Members of the epidermal growth factor receptor family (EGFR/ERBB1, ERBB2/HER2, ERBB3/HER3 and ERBB4/HER4) are key targets for inhibition in cancer therapy. Critical for activation is the formation of an asymmetric dimer by the intracellular kinase domains, in which the carboxy-terminal lobe (C lobe) of one kinase domain induces an active conformation in the other. The cytoplasmic protein MIG6 (mitogen-induced gene 6; also known as ERRFI1) interacts with and inhibits the kinase domains of EGFR and ERBB2 (refs 3-5). Crystal structures of complexes between the EGFR kinase domain and a fragment of MIG6 show that a approximately 25-residue epitope (segment 1) from MIG6 binds to the distal surface of the C lobe of the kinase domain. Biochemical and cell-based analyses confirm that this interaction contributes to EGFR inhibition by blocking the formation of the activating dimer interface. A longer MIG6 peptide that is extended C terminal to segment 1 has increased potency as an inhibitor of the activated EGFR kinase domain, while retaining a critical dependence on segment 1. We show that signalling by EGFR molecules that contain constitutively active kinase domains still requires formation of the asymmetric dimer, underscoring the importance of dimer interface blockage in MIG6-mediated inhibition.
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