Knockdown of DLK4 inhibits non-small cell lung cancer tumor growth by downregulating CKS2.

Non-small cell lung cancer (NSCLC) accounts for 85% of all lung cancer cases and is considered as the most common type of cancer. DLX4 was originally identified as a ß-globin gene suppressor in red blood cells, which plays critical roles in several types of cancers. However, the role and related mechanism of DLX4 in NSCLC are still unclear. The study aimed to uncover the expression of DLX4 in human NSCLC cells and tissues, reveal its possible role in NSCLC, and investigate the underlying mechanisms. Immunoblot and TCGA database were used to detect the expression of DLX4 in human NSCLC cells and tissues. CCK-8, colony formation, and FCM assays were conducted to detect the effects of DLX4 on the viability and cell cycle of NCI-H2170 and A549 cells. Immunoblot assays were further performed to investigate the possible mechanism underlying DLX4 affecting the growth of NSCLC. We revealed that knockdown of DLX4 inhibited NSCLC cell proliferation. We further revealed that DLX4 knockdown induced the NSCLC cell cycle arrest. Our results further showed that downregulation of DLX4 suppressed YB-1 expression, which further suppressed CKS2 expression, thereby suppressing tumor growth of NSCLC. In conclusion, DLX4 has the potential to serve as a promising drug for NSCLC treatment.

Analyzes In Silico Indicate the lncRNAs MIR31HG and LINC00939 as Possible Epigenetic Inhibitors of the Osteogenic Differentiation in PDLCs.

Chromatin conformation, DNA methylation pattern, transcriptional profile, and non-coding RNAs (ncRNAs) interactions constitute an epigenetic pattern that influences the cellular phenotypic commitment and impacts the clinical outcomes in regenerative therapies. Here, we investigated the epigenetic landscape of the SP7 transcriptor factor (SP7) and Distal-Less Homeobox 4 (DLX4) osteoblastic transcription factors (TFs), in human periodontal ligament mesenchymal cells (PDLCs) with low (l-PDLCs) and high (h-PDLCs) osteogenic potential. Chromatin accessibility (ATAC-seq), genome DNA methylation (Methylome), and RNA sequencing (RNA-seq) assays were performed in l- and h-PDLCs, cultured at 10 days in non-induced (DMEM) and osteogenic (OM) medium in vitro. Data were processed in HOMER, Genome Studio, and edgeR programs, and metadata was analyzed by online bioinformatics tools and in R and Python environments. ATAC-seq analyses showed the TFs genomic regions are more accessible in l-PDLCs than in h-PDLCs. In Methylome analyses, the TFs presented similar average methylation intensities (AMIs), without differently methylated probes (DMPs) between l- and h-PDLCs; in addition, there were no differences in the expression profiles of TFs signaling pathways. Interestingly, we identified the long non-coding RNAs (lncRNAs), MIR31HG and LINC00939, as upregulated in l-PDLCs, in both DMEM and OM. In the following analysis, the web-based prediction tool LncRRIsearch predicted RNA:RNA base-pairing interactions between SP7, DLX4, MIR31HG, and LINC00939 transcripts. The machine learning program TriplexFPP predicted DNA:RNA triplex-forming potential for the SP7 DNA site and for one of the LINC00939 transcripts (ENST00000502479). PCR data confirmed the upregulation of MIR31HG and LINC00939 transcripts in l-PDLCs (× h-PDLCs) in both DMEM and OM (p < 0.05); conversely, SP7 and DLX4 were downregulated, confirming those results observed in the RNA-Seq analysis. Together, these results indicate the lncRNAs MIR31HG and LINC00939 as possible epigenetic inhibitors of the osteogenic differentiation in PDLCs by (post)transcriptional and translational repression of the SP7 and DLX4 TFs.

Corrigendum: Transcription factors BARX1 and DLX4 contribute to progression of clear cell renal cell carcinoma via promoting proliferation and epithelial-mesenchymal transition.

[This corrects the article DOI: 10.3389/fmolb.2021.626328.].

DNA methylation-mediated differential expression of DLX4 isoforms has opposing roles in leukemogenesis.

BACKGROUND: Previously, we reported the expression of DLX4 isoforms (BP1 and DLX7) in myeloid leukemia, but the functional role of DLX4 isoforms remains poorly understood. In the work described herein, we further determined the underlying role of DLX4 isoforms in chronic myeloid leukemia (CML) leukemogenesis. METHODS: The expression and methylation of DLX4 isoforms were detected by real-time quantitative PCR (RT-qPCR) and real-time quantitative methylation-specific PCR (RT-qMSP) in patients with CML. The functional role of DLX4 isoforms was determined in vitro and in vivo. The molecular mechanism of DLX4 isoforms in leukemogenesis was identified based on chromatin immunoprecipitation with high-throughput sequencing (ChIP-Seq)/assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-Seq) and RNA sequencing (RNA-Seq). RESULTS: BP1 expression was increased in patients with CML with unmethylated promoter, but DLX7 expression was decreased with hypermethylated promoter. Functionally, overexpression of BP1 increased the proliferation rate of K562 cells with S/G2 promotion, whereas DLX7 overexpression reduced the proliferation rate of K562 cells with G1 arrest. Moreover, K562 cells with BP1 overexpression increased the tumorigenicity in NCG mice, whereas K562 cells with DLX7 overexpression decreased the tumorigenicity. Mechanistically, a total of 91 genes including 79 messenger RNAs (mRNAs) and 12 long noncoding RNAs (lncRNAs) were discovered by ChIP-Seq and RNA-Seq as direct downstream targets of BP1. Among the downstream genes, knockdown of RREB1 and SGMS1-AS1 partially revived the proliferation caused by BP1 overexpression in K562 cells. Similarly, using ATAC-Seq and RNA-Seq, a total of 282 genes including 151 mRNA and 131 lncRNAs were identified as direct downstream targets of DLX7. Knockdown of downstream genes PTPRB and NEAT1 partially revived the proliferation caused by DLX7 overexpression in K562 cells. Finally, we also identified and validated a SGMS1-AS1/miR-181d-5p/SRPK2 competing endogenous RNA (ceRNA) network caused by BP1 overexpression in K562 cells. CONCLUSIONS: The current findings reveal that DNA methylation-mediated differential expression of DLX4 isoforms BP1 and DLX7 plays opposite functions in leukemogenesis. BP1 plays an oncogenic role in leukemia development, whereas DLX7 acts as a tumor suppressor gene. These results suggest DLX4 as a therapeutic target for antileukemia therapy.

Evaluating the Expression of Candidate Homeobox Genes and Their Role in Local-Site Inflammation in Mucosal Tissue Obtained from Children with Non-Syndromic Cleft Lip and Palate.

Craniofacial development including palatogenesis is a complex process which requires an orchestrated and spatiotemporal expression of various genes and factors for proper embryogenesis and organogenesis. One such group of genes essential for craniofacial development is the homeobox genes, transcriptional factors that are commonly associated with congenital abnormalities. Amongst these genes, DLX4, HOXB3, and MSX2 have been recently shown to be involved in the etiology of non-syndromic cleft lip and palate. Hence, we investigated the gene and protein expression of these genes in normal and cleft affected mucosal tissue obtained from 22 children, along with analyzing their role in promoting local-site inflammation using NF-κB. Additionally, we investigated the role of PTX3, which plays a critical role in tissue remodeling and wound repair. We found a residual gene and protein expression of DLX4 in cleft mucosa, although no differences in gene expression levels of HOXB3 and MSX2 were noted. However, a significant increase in protein expression for these genes was noted in the cleft mucosa (p < 0.05), indicating increased cellular proliferation. This was coupled with a significant increase in NF-κB protein expression in cleft mucosa (p < 0.05), highlighting the role of these genes in promotion of pro-inflammatory environment. Finally, no differences in gene expression of PTX3 were noted.

Transcription Factors BARX1 and DLX4 Contribute to Progression of Clear Cell Renal Cell Carcinoma via Promoting Proliferation and Epithelial-Mesenchymal Transition.

Dysregulation of transcription factors contributes to the carcinogenesis and progression of cancers. However, their roles in clear cell renal cell carcinoma remain largely unknown. This study aimed to evaluate the clinical significance of TFs and investigate their potential molecular mechanisms in ccRCC. Data were accessed from the cancer genome atlas kidney clear cell carcinoma cohort. Bioinformatics algorithm was used in copy number alterations mutations, and differentially expressed TFs' analysis. Univariate and multivariate Cox regression analyses were performed to identify clinically significant TFs and construct a six-TF prognostic panel. TFs' expression was validated in human tissues. Gene set enrichment analysis (GSEA) was utilized to find enriched cancer hallmark pathways. Functional experiments were conducted to verify the cancer-promoting effect of BARX homeobox 1 (BARX1) and distal-less homeobox 4 (DLX4) in ccRCC, and Western blot was performed to explore their downstream pathways. As for results, many CNAs and mutations were identified in transcription factor genes. TFs were differentially expressed in ccRCC. An applicable predictive panel of six-TF genes was constructed to predict the overall survival for ccRCC patients, and its diagnostic efficiency was evaluated by the area under the curve (AUC). BARX1 and DLX4 were associated with poor prognosis, and they could promote the proliferation and migration of ccRCC. In conclusion, the six-TF panel can be used as a prognostic biomarker for ccRCC patients. BARX1 and DLX4 play oncogenic roles in ccRCC via promoting proliferation and epithelial-mesenchymal transition. They have the potential to be novel therapeutic targets for ccRCC.

Homeodomain protein DLX4 facilitates nasopharyngeal carcinoma progression via up-regulation of YB-1.

Nasopharyngeal carcinoma (NPC) is a malignant tumor in nasopharynx tissues and lacks effective treatment strategies. Dysregulation of distal-less homeobox 4 (DLX4) participates in the development of tumors. Understanding the regulatory mechanism of DLX4 in NPC progression may address this issue. Here, we first identified an up-regulation of DLX4 in NPC cell lines compared to normal epithelial cells. Data from colony formation and transwell assays showed that knockdown of DLX4 inhibited cell proliferation and invasion of NPC, respectively. Moreover, DLX4 knockdown blocked the cell cycle of NPC at G1 phase, suggesting the antitumor effect of DLX4 knockdown on NPC. The downstream target of DLX4 was identified as Y-box binding protein 1 (YB-1), whose expression was increased by over-expression of DLX4, while decreased by knockdown of DLX4. The binding capacity between DLX4 and YB-1 was verified by chromatin immunoprecipitation (ChIP), and the result showed that DLX4 could not directly bind to the promoter of YB-1. Mechanically, YB-1 over-expression reversed the effects of DLX4 knockdown on cell proliferation, cell cycle arrest and cell invasion of NPC. In conclusion, our findings indicated that DLX4 promoted NPC progression via up-regulation of YB-1, which would shed light on therapeutic schedule in NPC.

Expression of nestin, transcription factor SOX3 and homeobox protein DLX-4 in the facial tissue of children with cleft lip and palate / Expresión de nestina, factor de transcripción SOX3 y proteína homeobox DLX-4 en el tejido facial de niños con fisura labiopalatina

The failure of fusion of nasal and maxillary processes results in cleft lip and palate (CLP), which is one of the most common birth defects. The morphopathogenesis of this pathology is multifactorial and still largely unclear. The aim of this study was to evaluate the presence of nestin, transcriptor factor SOX3 (Sox3) and homeobox protein DLX-4 (Dlx-4) in complete unilateral (CU) and complete bilateral (CB) CLP affected facial tissue. Oral mucosa tissue samples were obtained from 17 CUCLP and 13 CBCLP patients during surgical cleft correction and 6 unaffected control subjects. Obtained tissue sections were stained with hematoxylin and eosin and by immunohistochemistry for nestin, Sox3 and Dlx-4. The intensity of staining was graded semiquantitatively. Nestin-positive structures were detected in all CUCLP and CBCLP patients' tissue samples, varying from moderate number of nestin-positive structures to numerous. Sox3 immunoreactivity was more prominent in epithelial cells in both patient groups with frequently patchy distribution. Mainly moderate number of Dlx-4-positive cells was observed in most of tissue samples. Statistically significant moderate positive correlation was found between nestin and Sox3 factors in CUCLP patient group (Spearman's rank correlation coefficient = .517, P = .034). Increase of nestinpositive structures suggests its role in the regulation of the remodeling of tissue in both CUCLP and CBCLP affected tissue. Dominance of Sox3 positivity in cleft affected epithelium indicates its possible role in (compensatory) formation of defective oral epithelium of CUCLP and CBCLP patients. The reduced expression of Dlx-4 implicates its limited regulatory role on the craniofacial development in CUCLP and CBCLP affected tissue.

La falla en la fusión de los procesos nasal y maxilar son causante de la fisura labiopalatina (FLP), que es uno de los defectos congénitos más comunes. La morfopatogenia de esta patología es multifactorial y aún poco clara. El objetivo de este estudio fue evaluar la presencia de nestina, el factor transcriptor SOX3 (Sox3) y la proteína homeobox DLX-4 (Dlx-4) en todo el tejido facial afectado por FLP bilateral unilateral (FU) y bilateral completa (FB). Se obtuvieron muestras de tejido de mucosa oral de 17 pacientes FUFLP y 13 FBFLP durante la corrección quirúrgica de la fisura y de 6 sujetos de control no afectados. Las secciones de tejido obtenidas se tiñeron con hematoxilina y eosina y mediante inmunohistoquímica para nestina, Sox3 y Dlx-4. La intensidad de la tinción fue graduada semicuantitativamente. Se detectaron estructuras positivas para nestina en todas las muestras de tejido de pacientes FUFLP y FBFLP, variando desde un número moderado a numerosas estructuras positivas para nestina. La inmunorreactividad de Sox3 fue más prominente en las células epiteliales en ambos grupos de pacientes con distribución frecuentemente irregular. Se observó un número principalmente moderado de células Dlx-4-positivas en la mayoría de las muestras de tejido. Se encontró una correlación positiva moderada estadísticamente significativa entre los factores de nestina y Sox3 en el grupo de pacientes FUFLP (coeficiente de correlación de rangos de Spearman = 0,517, P = 0,034). El aumento de estructuras positivas para nestina sugiere su papel en la regulación de la remodelación de tejido, tanto en tejido afectado por FUFLP como FBFLP. La dominancia de la positividad de Sox3 en el epitelio afectado de la fisura, indica su posible papel en la formación (compensatoria) del epitelio oral defectuoso de pacientes FUFLP y FBFLP. La expresión reducida de Dlx-4 implica su función reguladora limitada en el desarrollo craneofacial en tejido afectado por FUFLP y FBFLP.

Homeoprotein DLX4 expression is increased in inflammatory breast cancer cases from an urban African-American population.

Protein expression of Distal-less homeobox 4 (DLX4) was analyzed in inflammatory breast cancer (IBC) cases from an African-American (AA) population to determine if a) DLX4 gene over expression exists in this cohort and b) if the overexpression is associated with breast cancer clinicopathological characteristics (ER, PR, HER2, triple-negative). Twenty-nine blocks of formalin-fixed paraffin-embedded (FFPE) tissue from well-characterized human IBC cases were used for immunohistochemical staining (IHC). IHC results were assigned an intensity and percentage score. Percentage scores were assigned as 0, 1, 2, 3, or 4 and intensity scores were assigned 0, 1+, 2+ or 3+. For the analysis of the IHC, a percentage score of 3 or 4 and an intensity score of 2+ or 3+ were categorized as high. Chi-square or Fisher's exact tests were used to compare the high and low groups. In this cohort, 89.7% (26 out of 29) of IBC cases showed high percentages of positive cells staining for the DLX4 protein, while 40.0% (12 out of 30) of normal breast tissue from reduction mammoplasty cases demonstrated DLX4 expression (p < 0.01). In IBC patients, 65.5% of cases showed a high level of staining intensity, compared to 20.0% of normal breast tissues (test, p = 0.001). Intensity to DLX4 was higher in the HER2 negative status (78.3%) than the HER2 positive status (16.7%) (test, p = 0.011). DLX4 expression is higher in the IBC cases in this study of an urban AA population than in normal breast tissue cases. HER2 negative status is positively associated with high intensity of DLX4.

BP1, a potential biomarker for breast cancer prognosis.

Homeobox genes are critical in tumor development. An isoform protein of DLX4 called BP1 is expressed in 80% of invasive ductal breast carcinomas. BP1 overexpression is implicated in an aggressive phenotype and poor prognosis. BP1 upregulation is associated with estrogen receptor negativity so those tumors do not respond to antiestrogens. Breast cancer is the second leading cause of death in women. BP1 could serve as both a novel prognostic biomarker for breast cancer and a therapeutic target. In this review, we address the role of BP1 protein in tumorigenesis of breast cancer and four other malignancies. A number of functions of BP1 in cancer are also discussed.

Homeobox transcription factor DLX4 is not necessary for skin development and homeostasis.

Dlx4 is a member of a family of homeobox genes with homology to Drosophila distal-less (dll) gene. We show that Dlx4 expression pattern partially overlaps with its cis-linked gene Dlx3 during mouse development as well as in neonatal and adult skin. In mice, Dlx4 is expressed in the branchial arches, embryonic limbs, digits, nose, hair follicle and in the basal and suprabasal layers of mouse interfollicular epidermis. We show that inactivation of Dlx4 in mice did not result in any overtly gross pathology. Skin development, homeostasis and response to TPA treatment were similar in mice with loss of Dlx4 compared to wild-type counterparts.

DLX4 hypermethylation is a prognostically adverse indicator in de novo acute myeloid leukemia.

Hypermethylation of distal-less homeobox 4 (DLX4) has been increasingly identified in several cancers. Our study was aimed to determine the role of DLX4 methylation in regulating DLX4 expression and further analyze its clinical significance in de novo acute myeloid leukemia (AML) patients. DLX4 methylation level was detected by real-time quantitative methylation-specific PCR and bisulfite sequencing PCR. Treatment with 5-aza-2'-deoxycytidine (5-aza-dC) was used for demethylation studies. Clinical significance of DLX4 methylation was obtained by the comparison between the patients with and without DLX4 methylation. DLX4 was significantly methylated in AML patients compared with controls (P < 0.001). DLX4 methylation was negatively associated with DLX7 (the shorter DLX4 isoform) (R = -0.202, P = 0.021) but not BP1 (the longer DLX4 isoform) (R = -0.049, P = 0.582) expression in AML patients. DLX7 and BP1 messenger RNA (mRNA) were significantly increased after 5-aza-dC treatment in leukemic cell lines THP1 and Kasumi-1. DLX4 methylated patients showed significantly higher frequency of U2AF1 mutation compared with DLX4 unmethylated patients (P = 0.043). Both all AML and non-M3 patients with DLX4 methylation presented significantly lower complete remission rate than those with DLX4 unmethylation (P = 0.001 and <0.001, respectively). DLX4 methylated cases had significantly shorter overall survival than DLX4 unmethylated cases among both all AML (P = 0.003), non-M3 AML (P = 0.001), and cytogenetically normal AML (P = 0.032). Multivariate analysis confirmed that DLX4 methylation was independent risk factor in both all AML and non-M3 patients. Our study indicates that DLX4 hypermethylation is negatively associated with DLX7 expression and predicts poor clinical outcome in de novo AML patients.

BP1 overexpression is associated with adverse prognosis in de novo acute myeloid leukemia.

To investigate DLX4 isoforms expression and their clinical significance in acute myeloid leukemia (AML). DLX4 transcript variant 1 (BP1) expression was significantly up-regulated in AML patients compared with normal controls. However, DLX4 transcript variant 2 (DLX7) was significantly down-regulated in AML patients. Both in the overall AML and the non-M3 AML cohorts, those patients with high BP1 expression (BP1(high)) showed significantly lower rates of complete remission than those with low BP1 expression (BP1(low)). BP1(high) cases had significantly shorter overall survival than BP1(low) cases in the overall AML cohort, non-M3 AML, and cytogenetically normal AML (CN-AML). Multivariate analysis confirmed the independent prognostic value of BP1 expression among both the overall AML cohort and non-M3 AML as well as CN-AML patients. However, we did not observe the impact of DLX7 expression on prognosis in AML patients. Our study reveals that BP1 overexpression serves as an independent risk factor in de novo AML patients.

Epigenetic inactivation of DLX4 is associated with disease progression in chronic myeloid leukemia.

Aberrant DNA methylation of various genes has been identified to be associated with disease progression in chronic myeloid leukemia (CML). Our study was intended to investigate DLX4 methylation pattern in different clinical stages of CML and further determine its role in regulating DLX4 expression. Real-time quantitative methylation-specific PCR and bisulfite sequencing PCR were applied to detect DLX4 methylation. 5-aza-2'-deoxycytidine (5-aza-dC) was used for demethylation studies. DLX4 was significantly hypermethylated in CML patients (P = 0.002) especially in blastic phase (BC) stage (P < 0.001) as compared with controls. Moreover, DLX4 methylation level in BC stage was significantly higher than in chronic phase (CP) stage (P < 0.001). DLX4 methylation density was significantly increased during the progression of CML among the tested two patients (P < 0.001). DLX4 hypermethylation occurred with the highest incidence in BC stage (83%), lower incidence in acute phase (AP) stage (43%), and the lowest incidence in CP stage (26%) (P = 0.001). Moreover, t(9; 22) with additional alteration cases had significantly higher frequency of DLX4 hypermethylation compared with the other cytogenetics (P = 0.010). Significantly negative correlation was observed between DLX4 methylation and DLX4-TV2 (the shorter DLX4 isoform) expression (R = -0.382, P = 0.001, n = 78) but not between DLX4 methylation and BP1 (the longer DLX4 isoform) expression (R = 0.134, P = 0.244, n = 78) in CML patients. Both DLX4-TV2 and BP1 mRNA were significantly increased after 5-aza-dC treatment in K562 cell line (P < 0.001). Our study indicated that hypermethylation of DLX4 correlated with disease progression of CML. Moreover, DLX4 expression was regulated by its methylation in CML.

Aberrant DNA methylation of DLX4 and SIM1 is a predictive marker for disease progression of uterine cervical low-grade squamous intraepithelial lesion.

BACKGROUND: A few uterine cervical low-grade squamous intraepithelial lesions (LSILs) are known to progress with high-risk human papillomavirus (hrHPV). METHODS: One hundred and thirteen patients were classified into four groups according to their cervical cytology, hrHPV infection, and follow up. Cytology samples were examined for aberrant DNA methylation of DLX4 and SIM1 genes and protein expressions. CaSki cells were treated with 5-Aza-2'-deoxycytidine (5-aza-dC). RESULTS: Group 1 was negative for intraepithelial lesions or malignancies. LSIL in group 2 showed a continuance of LSIL for longer than 365 days and LSIL in group 3 showed an upgrading to high-grade (H) SIL or higher (HSIL+) within 365 days of LSIL diagnosis. Group 4 was squamous cell carcinoma. All but group 1 were infected with hrHPV. Significant differences existed in the frequency of DNA methylation between groups 2 and 3 (p = 0.044), between groups 3 and 4 (p = 0.020) for DLX4, and between groups 1 and 3 (p = 0.0003), and groups 2 and 3 (p = 0.005) for the SIM1 gene. DLX4 protein expression was significantly reduced in the DLX4 methylation positive tissues (p = 0.008). The 5-aza-dC treatment restored DLX4 mRNA expressions of CaSki cells (p < 0.005). The LSIL cases with DNA methylation of the SIM1 gene, or both genes, progressed faster to HSIL+ than did the others (p = 0.033 and p = 0.045, respectively). CONCLUSION: Aberrant DNA methylation of the DLX4 and SIM1 genes should be a novel progression marker for uterine cervical LSIL with hrHPV infection.

Increased copy number of the DLX4 homeobox gene in breast axillary lymph node metastasis.

DLX4 is a homeobox gene strongly implicated in breast tumor progression and invasion. Our main objective was to determine the DLX4 copy number status in sentinel lymph node (SLN) metastasis to assess its involvement in the initial stages of the axillary metastatic process. A total of 37 paired samples of SLN metastasis and primary breast tumors (PBT) were evaluated by fluorescence in situ hybridization, quantitative polymerase chain reaction and array comparative genomic hybridization assays. DLX4 increased copy number was observed in 21.6% of the PBT and 24.3% of the SLN metastasis; regression analysis demonstrated that the DLX4 alterations observed in the SLN metastasis were dependent on the ones in the PBT, indicating that they occur in the primary tumor cell populations and are maintained in the early axillary metastatic site. In addition, regression analysis demonstrated that DLX4 alterations (and other DLX and HOXB family members) occurred independently of the ones in the HER2/NEU gene, the main amplification driver on the 17q region. Additional studies evaluating DLX4 copy number in non-SLN axillary lymph nodes and/or distant breast cancer metastasis are necessary to determine if these alterations are carried on and maintained during more advanced stages of tumor progression and if could be used as a predictive marker for axillary involvement.

DLX4 upregulates TWIST and enhances tumor migration, invasion and metastasis.

The distal-less homeobox gene 4 (DLX4) is a member of the DLX family of homeobox genes. Although absent from most normal adult tissues, DLX4 is widely expressed in leukemia, lung, breast, ovarian and prostate cancers. However the molecular targets, mechanisms and pathways that mediate the role of DLX4 in tumor metastasis are poorly understood. In this study, we found that DLX4 induces cancer cells to undergo epithelial to mesenchymal transition (EMT) through TWIST. Overexpression of DLX4 increased expression of TWIST expression in cancer cell lines, resulting in increased migratory and invasive capacity. Likewise, knocking down expression of DLX4 decreased TWIST expression and the migration ability of cancer cell lines. DLX4 bound to regulatory regions of the TWIST gene. Both western blotting and immunohistochemistry staining showed that the expression of DLX4 and TWIST are correlated in most of breast tumors. Taken together, these data from both cell models and tumor tissues demonstrate that DLX4 not only upregulates TWIST expression but also induces EMT and tumor metastasis. Altogether, we propose a new pathway in which DLX4 drives expression of TWIST to promote EMT, cancer migration, invasion and metastasis.

BP1, an isoform of DLX4 homeoprotein, negatively regulates BRCA1 in sporadic breast cancer.

INTRODUCTION: Several lines of evidence point to an important role for BP1, an isoform of DLX4 homeobox gene, in breast carcinogenesis and progression. BRCA1 is a well-known player in the etiology of breast cancer. While familial breast cancer is often marked by BRCA1 mutation and subsequent loss of heterozygosity, sporadic breast cancers exhibit reduced expression of wild type BRCA1, and loss of BRCA1 expression may result in tumor development and progression. METHODS: The Cister algorithm and Genomatix program were used to identify potential BP1 binding sites in BRCA1 gene. Real-time PCR, Western blot and immunohistochemistry analysis were performed to verify the expression of BRCA1 and BP1 in cell lines and breast cancer tissues. Double-stranded siRNA transfection was carried out for silencing BP1 expression. ChIP and EMSA were used to confirm that BP1 specifically binds to BRCA1. RESULTS: A putative BP1 binding site was identified in the first intron of BRCA1, which was confirmed by chromatin immunoprecipiation and electrophoresis mobility shift assay. BP1 and BRCA1 expression were inversely correlated in breast cancer cell lines and tissues, suggesting that BP1 may suppress BRCA1 transcription through consensus sequence binding. CONCLUSIONS: BP1 homeoprotein represses BRCA1 expression through direct binding to its first intron, which is consistent with a previous study which identified a novel transcriptional repressor element located more than 500 base pairs into the first intron of BRCA1, suggesting that the first intron plays an important role in the negative regulation of BRCA1. Although further functional studies are necessary to confirm its repressor activity towards BRCA1, the elucidation of the role of BP1 in breast tumorigenesis holds great promise in establishing BP1 as a novel target for drug therapy.