Transcription factor EHF interacting with coactivator AJUBA aggravates malignancy and acts as a therapeutic target for gastroesophageal adenocarcinoma.

Transcriptional dysregulation of genes is a hallmark of tumors and can serve as targets for cancer drug development. However, it is extremely challenging to develop small-molecule inhibitors to target abnormally expressed transcription factors (TFs) except for the nuclear receptor family of TFs. Little is known about the interaction between TFs and transcription cofactors in gastroesophageal adenocarcinoma (GEA) or the therapeutic effects of targeting TF and transcription cofactor complexes. In this study, we found that ETS homologous factor (EHF) expression is promoted by a core transcriptional regulatory circuitry (CRC), specifically ELF3-KLF5-GATA6, and interference with its expression suppressed the malignant biological behavior of GEA cells. Importantly, we identified Ajuba LIM protein (AJUBA) as a new coactivator of EHF that cooperatively orchestrates transcriptional network activity in GEA. Furthermore, we identified KRAS signaling as a common pathway downstream of EHF and AJUBA. Applicably, dual targeting of EHF and AJUBA by lipid nanoparticles cooperatively attenuated the malignant biological behaviors of GEA in vitro and in vivo. In conclusion, EHF is upregulated by the CRC and promotes GEA malignancy by interacting with AJUBA through the KRAS pathway. Targeting of both EHF and its coactivator AJUBA through lipid nanoparticles is a novel potential therapeutic strategy.

The crescent-like Golgi ribbon is shaped by the Ajuba/PRMT5/Aurora-A complex-modified HURP.

BACKGROUND: Golgi apparatus (GA) is assembled as a crescent-like ribbon in mammalian cells under immunofluorescence microscope without knowing the shaping mechanisms. It is estimated that roughly 1/5 of the genes encoding kinases or phosphatases in human genome participate in the assembly of Golgi ribbon, reflecting protein modifications play major roles in building Golgi ribbon. METHODS: To explore how Golgi ribbon is shaped as a crescent-like structure under the guidance of protein modifications, we identified a protein complex containing the scaffold proteins Ajuba, two known GA regulators including the protein kinase Aurora-A and the protein arginine methyltransferase PRMT5, and the common substrate of Aurora-A and PRMT5, HURP. Mutual modifications and activation of PRMT5 and Aurora-A in the complex leads to methylation and in turn phosphorylation of HURP, thereby producing HURP p725. The HURP p725 localizes to GA vicinity and its distribution pattern looks like GA morphology. Correlation study of the HURP p725 statuses and GA structure, site-directed mutagenesis and knockdown-rescue experiments were employed to identify the modified HURP as a key regulator assembling GA as a crescent ribbon. RESULTS: The cells containing no or extended distribution of HURP p725 have dispersed GA membranes or longer GA. Knockdown of HURP fragmentized GA and HURP wild type could, while its phosphorylation deficiency mutant 725A could not, restore crescent Golgi ribbon in HURP depleted cells, collectively indicating a crescent GA-constructing activity of HURP p725. HURP p725 is transported, by GA membrane-associated ARF1, Dynein and its cargo adaptor Golgin-160, to cell center where HURP p725 forms crescent fibers, binds and stabilizes Golgi assembly factors (GAFs) including TRIP11, GRASP65 and GM130, thereby dictating the formation of crescent Golgi ribbon at nuclear periphery. CONCLUSIONS: The Ajuba/PRMT5/Aurora-A complex integrates the signals of protein methylation and phosphorylation to HURP, and the HURP p725 organizes GA by stabilizing and recruiting GAFs to its crescent-like structure, therefore shaping GA as a crescent ribbon. Therefore, the HURP p725 fiber serves a template to construct GA according to its shape. Video Abstract.

Case Report: Identification of a novel NTRK3-AJUBA fusion co-existing with ETV6-NTRK3 fusion in papillary thyroid carcinoma.

NTRK fusions are validated oncogenic drivers of various adult and pediatric tumor types, including thyroid cancer, and serve as a therapeutic target. Recently, tropomyosin receptor kinase (TRK) inhibitors, such as entrectinib and larotrectinib, display promising therapeutic efficacy in NTRK-positive solid tumors. Although some NTRK fusion partners have been identified in thyroid cancer, the spectrum of NTRK fusion is not fully characterized. In this study, a dual NTRK3 fusion was identified by targeted RNA-Seq in a 47-year-old female patient with papillary thyroid carcinoma. The patient harbors a novel in-frame fusion between NTRK3 exon 13 and AJUBA exon 2, co-existing with a known in-frame fusion between ETV6 exon 4 and NTRK3 exon 14. The dual NTRK3 fusion was validated by Sanger sequencing and fluorescence in situ hybridization (FISH) but lack TRK protein expression as defined by pan-TRK immunohistochemistry (IHC). We supposed the pan-TRK IHC result to be falsely negative. In conclusion, we present the first case of a novel NTRK3-AJUBA fusion co-existing with a known ETV6-NTRK3 fusion in thyroid cancer. These findings extend the spectrum of translocation partners in NTRK3 fusion, and the effect of dual NTRK3 fusion on TRK inhibitor therapy and prognosis needs long-term follow-up.

LIM domain-containing protein Ajuba inhibits chemotherapy-induced apoptosis by negatively regulating p53 stability in colorectal cancer cells.

LIM protein-domain containing protein Ajuba (encoded by AJUBA) functions as a scaffold protein to regulate protein-protein interactions, signalling transduction and genes transcription. AJUBA expression is higher in colorectal cancer (CRC) tissues than normal tissues, but its specific molecular function in CRC progression is still not very clear. Here, we found that, in CRC cancer cell lines, overexpression of AJUBA decreased p53 levels, whereas knock-down of AJUBA significantly increased p53 levels. Although the presence of Ajuba did not influence p53 transcription, it formed a complex with p53 and MDM2 to promote the degradation of p53. AJUBA overexpression reduced the sensitivity of cancer cells to chemotherapeutic drugs and vice versa. In addition, chemotherapeutic drugs significantly induced AJUBA expression, which was largely dependent on the presence of p53. Therefore, Ajuba formed a negative feedback loop to regulate p53 expression and activity. In conclusion, as a novel p53-negative regulator, Ajuba inhibits the apoptosis of CRC cells induced by chemotherapeutic drugs and it may be a new therapeutic target for CRC treatment.

MiRNA-21 promotes differentiation of bone marrow mesenchymal stem cells into cardiomyocyte-like cells by regulating the Ajuba/Isl1 axis pathway.

Introduction: In this study, we aimed to investigate the role of miRNA-21 and the regulating pathway that promotes the differentiation of bone marrow mesenchymal stem cells (BMSCs). Methods: We used miR-21-OE, miR-21-KD, ajuba-OE and ajuba-KD plasmids to infect BMSCs. The expression of miRNA-21, ajuba, Isl1 and cTnI was detected by RT-qPCR, WB and immunofluorescence staining in groups. Results: MiRNA-21 over-expression increased the expression of Isl1, and vice versa. Ajuba over-expression decreased the expression of Isl1, and vice versa. Ajuba negatively regulated the differentiation of BMSCs into cardiomyocyte-like cells. Conclusions: MiRNA-21 could regulate differentiation of bone marrow mesenchymal stem cells (BMSCs) to cardiomyocyte-like cells through the ajuba/Isl1 axis pathway.

The LIM Protein AJUBA is a Potential Oncogenic Target and Prognostic Marker in Human Cancer via Pan-Cancer Analysis.

Purpose : The LIM (Lin-11, Isl1, MEC-3) domain protein AJUBA is involved in multiple biological functions, and its aberrant expression is related to the occurrence and progression of various cancers. However, there are no analytical studies on AJUBA in pan-cancer. Methods: We performed a comprehensive pan-cancer analysis and explored the potential oncogenic roles of AJUBA, including gene expression, genetic mutation, protein phosphorylation, clinical diagnostic biomarker, prognosis, and AJUBA-related immune infiltration based on The Cancer Genome Atlas and Genotype-Tissue Expression databases. Results: The results revealed that the expression of AJUBA highly correlated with poor clinical outcomes in patients with different types of cancer. Meanwhile, AJUBA expression was positively correlated with cancer-associated fibroblasts in many human cancers, such as breast invasive carcinoma, colon adenocarcinoma, brain lower-grade glioma, lung adenocarcinoma (LUAD), and ovarian serous cystadenocarcinoma (OV). Gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses showed that AJUBA is mainly involved in protein serine/threonine kinase activity, cell-cell junction, covalent chromatin modification, and Hippo signaling pathway. Conclusion: The pan-cancer study reveals the oncogenic roles of AJUBA and provides a comprehensive understanding of the molecular biological genetic information of AJUBA in various tumors.

LIM protein Ajuba promotes liver cell proliferation through its involvement in DNA replication and DNA damage control.

The LIM-domain protein Ajuba is associated with cell proliferation, a fundamental process of tissue regeneration and cancer. We report that in the liver, Ajuba expression is increased during regeneration and in tumour cells and tissues. Knockout of Ajuba using CRISPR/Cas9 is embryonic lethal in mice. shRNA targeting of Ajuba reduces cell proliferation, delays cell entry into S-phase, reduces cell survival and tumour growth in vivo and increases expression of the DNA damage marker γH2AX. Ajuba binding partners include proteins involved in DNA replication and damage, such as SKP2, MCM2, MCM7 and RPA70. Taken together, our data support that Ajuba promotes liver cell proliferation associated with development, regeneration and tumour growth and is involved in DNA replication and damage repair.

hnRNPL-activated circANKRD42 back-splicing and circANKRD42-mediated crosstalk of mechanical stiffness and biochemical signal in lung fibrosis.

Increasing circular RNAs (circRNAs) are involved in the progression of idiopathic pulmonary fibrosis (IPF). However, circRNA biogenesis and circRNA-mediated crosstalk between mechanical stiffness and biochemical signals in IPF remain obscure. In this study, a novel circRNA-ankyrin repeat domain 42 (ANKRD42) from peripheral blood of patients with IPF, which participated in pulmonary fibrosis through the close communication of mechanical stiffness and biochemical signals, was identified. Mechanistic studies revealed that the heterogeneous nuclear ribonucleoprotein L (hnRNP L) activated the circANKRD42 reverse splicing biogenesis. The biogenetic circANKRD42 sponged miR-324-5p to promote the AJUBA expression, which blocked the binding between phosphorylated yes-associated protein 1 (YAP1) and large tumor suppressor kinase 1/2 (LATS1/2), leading to increased YAP1 entering the nucleus. circANKRD42 also sponged miR-136-5p to promote the YAP1 translation. Accumulating YAP1 in nucleus bound to TEAD, which initiated the transcription of genes related to mechanical stiffness. Finally, the therapeutic effect of circANKRD42 was evaluated in mice and the association between circANKRD42 and clinicopathological features was analyzed in IPF patients. Our findings supported that circANKRD42 is a promising biomarker and a potential therapeutic target related to cytoskeleton tension for IPF treatment.

Ajuba functions as a co-activator of C/EBPß to induce expression of PPARγ and C/EBPα during adipogenesis.

Adipogenesis is regulated by a complicated network of transcription factors among which PPARγ and C/EBP family members are the major regulators. During adipogenesis, C/EBPß is induced early and then transactivates PPARγ and C/EBPα, which cooperatively induce genes whose expressions give rise to the mature adipocyte phenotype. Identifying the factors that influence the expression and activity of C/EBPß should provide additional insight into the mechanisms regulating adipogenesis. Here, we demonstrate that depletion of Ajuba in 3T3-L1 cells significantly decreases mRNA and protein levels of PPARγ and C/EBPα and impairs adipocyte differentiation, while overexpression increases expression of these genes and promotes adipocyte differentiation. Moreover, restoration of C/EBPα or PPARγ expression in Ajuba-deficient 3T3-L1 cells improves the impaired lipid accumulation. Mechanistically, Ajuba interacts with C/EBPß and recruits CBP to facilitate the binding of C/EBPß to the promoter of PPARγ and C/EBPα, resulting in increased H3 histone acetylation and target gene expression. Collectively, these data indicate that Ajuba functions as a co-activator of C/EBPß, and may be an important therapeutic target for combating obesity-related diseases.

miR-433-3p Targets AJUBA to Inhibit Malignant Progression of Glioma.

INTRODUCTION: Glioma is the most aggressive and malignant type of tumors among primary intracranial tumors. miR-433-3p has been verified to be correlated with the formation and progression of many types of cancers. METHODS: In this study, the effects of miR-433-3p and AJUBA on the proliferation, migration, and invasion of glioma and the molecular mechanisms were investigated. We analyzed bioinformatics databases and conducted cell biology experiments to determine that compared with adjacent tissue and normal cells, the expression level of miR-433-3p in glioma tissue and cells was lower, while the expression level of AJUBA was higher. Overexpressing miR-433-3p could significantly inhibit the proliferation, migration, and invasion of glioma cells and promote cell apoptosis. RESULTS: In addition, after overexpressing miR-433-3p and AJUBA, it was found that overexpressing AJUBA could attenuate the inhibitory effect of overexpressing miR-433-3p on the proliferation, migration, and invasion of glioma cells, which suggested that miR-433-3p regulated the biological function of glioma by downregulating AJUBA expression. CONCLUSION: These results proved that miR-433-3p could target to inhibit the expression of AJUBA, thus inhibiting the biological function and malignant progression of glioma.

Evidence for AJUBA-catenin-CDH4-linked differentiation resistance of mesenchymal stem cells implies tumorigenesis and progression of head and neck squamous cell carcinoma: a single-cell transcriptome approach.

An increasing number of reports indicate that mesenchymal stem cells (MSCs) play an essential role in promoting tumorigenesis and progression of head and neck squamous cell carcinoma (HNSCC). However, the molecular mechanisms underlying this process remain unclear. Using the MSC model system, this study analyzes the molecular pathway by which differentiation resistant MSCs promote HNSCC. MSCs were cultured in osteogenic differentiation media and harvested on days 12 and 19. Cells were stained for cell differentiation analysis using Alizarin Red. The osteogenesis-resistant MSCs (OR-MSCs) and MSC-differentiation-derived osteoblasts (D-OSTBs) were identified and subjected to the single-cell transcriptome analysis. Gene-specific analyses of these two sub-populations were performed for the patterns of differential expression. A total of 1 780 differentially expressed genes were determined to distinguish OR-MSCs significantly from D-OSTB. Notably, AJUBA, ß-catenin, and CDH4 expression levels were upregulated considerably within the OR-MSCs compared to D-OSTBs. To confirm their clinical relevance, a survey of a clinical cohort revealed a high correlation among the expression levels of AJUBA, ß-catenin and CDH4. The results shed new light that OR-MSCs participate in the development of HNSCC via a pathway mediated by AJUBA, ß-catenin, CDH4, and CTNNB1, thereby implying that MSC-based therapy is a promising therapeutic approach in the management of HNSCC.

Ajuba transactivates N-cadherin expression in colorectal cancer cells through interaction with Twist.

Ajuba is a multiple LIM domain-containing protein and functions as a transcriptional coregulator to modulate many gene expressions in various cellular processes. Here, we describe that the LIM domain of Ajuba interacts with Twist, and the Twist box is a pivotal motif for the interaction. Biologically, Ajuba enhances transcription of target gene N-cadherin as an obligate coactivator of Twist. The enhancement is achieved by binding to the E-box element within N-cadherin promoter as revealed by luciferase reporter and chromatin immunoprecipitation assays. Mechanistic investigation demonstrates that Ajuba recruits CBP and Twist to form a ternary complex at the Twist target promoter region and concomitantly enhances histone acetylation at these sites. These findings identify that Twist is a new interacting protein of Ajuba and Ajuba/Twist/CBP ternary complex may be a potential treatment strategy for Twist-related tumour metastasis.

Super-enhancer-driven AJUBA is activated by TCF4 and involved in epithelial-mesenchymal transition in the progression of Hepatocellular Carcinoma.

Background and Aims: Aberrant transcriptional programs are highly regulated processes that play important roles in the development and progression of hepatocellular carcinoma (HCC). Emerging evidence suggests that super-enhancers (SEs) often drive critical oncogene expression. However, SE-associated genes in HCC pathogenesis are still poorly understood. Methods: We performed integrative ChIP-seq and Hi-C analyses of HCC cells and identified ajuba LIM protein (AJUBA) as a SE-associated gene. We evaluated AJUBA expression in HCC using immunohistochemistry, immunoblotting, and qRT-PCR. ChIP and luciferase reporter assays were performed to demonstrate that transcription factor 4 (TCF4) bound to AJUBA-associated SEs. We then assessed the role of AJUBA in HCC using both in vitro and in vivo assays. Epithelial-mesenchymal transition (EMT) was examined using immunofluorescence and immunoblotting assays. Furthermore, we used immunoprecipitation and BiFC assays to explore the underlying mechanisms. Results: We identified AJUBA as a SE-associated oncogene in HCC regulated by TCF4. High AJUBA expression was related to an aggressive phenotype and unfavorable outcome in HCC patients. AJUBA knockdown significantly reduced cell migration and invasion capacities both in vitro and in vivo. Furthermore, AJUBA overexpression in HCC recruited tumor necrosis factor associated factor 6 (TRAF6), enhancing the phosphorylation of Akt and increasing Akt activity toward GSK-3ß, thus promoting EMT. Conclusions: Our results provide functional and mechanistic links between the SE-associated gene AJUBA and tumor EMT in aggressive HCC.

The LIM Protein Ajuba Augments Tumor Metastasis in Colon Cancer.

Colorectal cancer, along with its high potential for recurrence and metastasis, is a major health burden. Uncovering proteins and pathways required for tumor cell growth is necessary for the development of novel targeted therapies. Ajuba is a member of the LIM domain family of proteins whose expression is positively associated with numerous cancers. Our data shows that Ajuba is highly expressed in human colon cancer tissue and cell lines. Publicly available data from The Cancer Genome Atlas shows a negative correlation between survival and Ajuba expression in patients with colon cancer. To investigate its function, we transduced SW480 human colon cancer cells, with lentiviral constructs to knockdown or overexpress Ajuba protein. The transcriptome of the modified cell lines was analyzed by RNA sequencing. Among the pathways enriched in the differentially expressed genes, were cell proliferation, migration and differentiation. We confirmed our sequencing data with biological assays; cells depleted of Ajuba were less proliferative, more sensitive to irradiation, migrated less and were less efficient in colony formation. In addition, loss of Ajuba expression decreased the tumor burden in a murine model of colorectal metastasis to the liver. Taken together, our data supports that Ajuba promotes colon cancer growth, migration and metastasis and therefore is a potential candidate for targeted therapy.

Hsa_circ_0128846 promotes tumorigenesis of colorectal cancer by sponging hsa-miR-1184 and releasing AJUBA and inactivating Hippo/YAP signalling.

Hsa_circ_0128846 was found to be the most significantly up-regulated circRNA in our bioinformatics analysis. However, the role of hsa_circ_0128846 in colorectal cancer has not been explored. We thus aim to explore the influence and mechanism of hsa_circ_0128846 in colorectal cancer by sponging its downstream miRNA target miR-1184. We collected 40 colorectal cancer patients' tumour tissues to analyse the expression of hsa_circ_0128846, miR-1184 and AJUBA using qRT-PCR and Western blot where needed. Then, we constructed stably transfected SW480 and HCT116 cells to study the influence of hsa_circ_0128846, miR-1184 and AJUBA on colorectal cancer cell phenotypes. To obtain reliable results, a plethora of experiments including RNA immunoprecipitation assay, flow cytometry, EdU incorporation assay, wound healing migration assay, transwell invasion assay and live imaging of nude mice xenograft assay were performed. The binding relationship between hsa_circ_0128846, miR-1184 and AJUBA mRNA in colorectal cancer was validated by reported gene assay. In colorectal cancer tissues, circ_0128846 and AJUBA were both significantly up-regulated, while miR-1184 was significantly down-regulated compared with healthy tissues. Meanwhile, hsa_circ_0128846 can absorb miR-1184 to promote the progression of CRC in vivo and SW480 and HCT116 cell phenotypes in vitro. The knockdown of AJUBA, a downstream target of miR-1184, reversed the effect of miR-1184 in CRC cells via enhancing the phosphorylation of the Hippo/YAP signalling pathway proteins MST1, LATS1 and YAP. This study revealed that hsa_circ_0128846 contributed to the development of CRC by decreasing the expression of miR-1184, thereby increasing AJUBA expression and inactivating Hippo/YAP signalling.

Knockout of Ajuba Attenuates the Growth and Migration of Hepatocellular Carcinoma Cells.

Ajuba has been found to be mutated or aberrantly regulated in several human cancers and plays important roles in cancer progression via different signaling pathways. However, little is known about the role of Ajuba in hepatocellular carcinoma (HCC). Here, we found an upregulation of Ajuba expression in HCC tissues compared with normal liver tissues, while a poor prognosis was observed in HCC patients with high Ajuba expression. Knockout of Ajuba in HCC cells inhibited cell growth in vitro and in vivo, suppressed cell migration, and enhanced the cell apoptosis under stress. Moreover, re-expression of Ajuba in Ajuba-deficient cells could restore the phenotype of Ajuba-deficient cells. In conclusion, these results indicate that Ajuba is upregulated in HCC and promotes cell growth and migration of HCC cells, suggesting that Ajuba could possibly be a new target for HCC diagnosis and treatment.

Ajuba: An emerging signal transducer in oncogenesis.

The LIM protein Ajuba contains an unstructured proline/glycine-rich preLIM region in the N terminus and conserved tandem LIM motifs in the C terminus. Additionally, Ajuba contains both nuclear export sequences (NES) and nuclear localization sequences (NLS), which enable Ajuba shuttle between the cytoplasm and the nucleus. Thus, Ajuba can act as a versatile scaffold participating in assembly of variety of protein complexes to execute multiple cellular functions including cell adhesion, motility, mitosis, survival, gene expression, microRNA processing and mechanical force sensing. Numerous studies have demonstrated that Ajuba plays important roles in oncogenesis and progression by regulating major signalling pathways such as Wnt, RAS/ERK, JAK/STAT and Hippo, and by acting as a co-regulator of key transcription factors such as Snail, Sp1 and nuclear hormone receptors. Clinically, Ajuba is highly expressed in various types of tumors and can be a marker for prognosis and diagnosis. In this review, we aim to summarize the up-to-date literatures on the signaling pathways mediated by Ajuba and its associated protein complexes in oncogenesis, and to discuss Ajuba as a potential target for new therapeutics to treat cancers.

Dual-strand tumor suppressor miR-193b-3p and -5p inhibit malignant phenotypes of lung cancer by suppressing their common targets.

Emerging studies suggest that microRNAs (miRNAs) play multiple roles in cancer malignancy, including proliferation and acquisition of metastatic potential. Differentially expressed miRNAs responsible for the malignancy of lung cancer were searched by miRNA microarray using a previously established brain metastatic lung cancer model. Twenty-five miRNAs were down-regulated in brain metastatic lung cancer cells. Among those, miR-193b-3p and -5p were chosen for further studies. Their function in metastatic potential and proliferation was examined using Transwell invasion, wound healing, and colony forming assays. The underlying mechanism of tumor-suppressor miR-193b-3p and -5p was explored using reverse transcriptase quantitative polymerase chain reaction (RT-qPCR), Western blot, Argonaute 2-RNA immunoprecipitation (Ago2-RIP), and reporter assays. Both strands of miR-193b were down-regulated in brain metastatic lung cancer cells and in tissues from lung cancer patients. Overexpression of miR-193b-3p and -5p inhibited invasive and migratory activities and diminished clonogenic ability. Conversely, inhibition of miR-193b-3p or -5p increased the metastatic potential and colony forming ability. Cyclin D1 (CCND1), Ajuba LIM Protein (AJUBA), and heart development protein with EGF like domains 1 (HEG1) were identified as common target genes of miR-193b-3p and -5p. A reporter assay and an Ago2-RIP experiment showed that both miRNAs directly bind to the 3' untranslated region (3'UTR) of the target mRNA. Knockdown of target gene reduced the proliferative and metastatic potential of primary and metastatic lung cancer cells. Our results demonstrate miR-193b is a dual-strand tumor suppressor and a novel therapeutic target for lung cancer.

The LIM protein Ajuba/SP1 complex forms a feed forward loop to induce SP1 target genes and promote pancreatic cancer cell proliferation.

BACKGROUND: The aim of this study is to explore the molecular mechanism of the LIM protein Ajuba and the transcription factor SP1 in the pathogenesis and progression of PDAC. Ajuba is a newly defined transcriptional co-regulator and plays important role in various cancer development, while SP1 is a classic transcription factor, and is closely related with a variety of gene expression and cancer development including PDAC. METHODS: The expression of Ajuba and SP1 in PDAC tissues was detected by immunohistochemistry (IHC), and the correlation between expression level and clinical prognosis of Ajuba and SP1 was extensively analyzed using online tools. The interaction between Ajuba and SP1 was examined by co-immunoprecipitation (co-IP) and GST-pulldown assays. Stable cell lines were established via lentiviral infection, and was examined by qRT-PCR and western blot assays. The effects of Ajuba/SP1 on PDAC cell proliferation were examined using CCK8 and colony formation assays. Luciferase reporter and chromatin immunoprecipitation (ChIP) assays were employed to examine the transcription activity. RESULTS: The expression level (protein and mRNA) of Ajuba and SP1 was elevated in PDAC tissues and was positively correlated; patients with high Ajuba and SP1 expression had a poor prognosis. Mechanistically, Ajuba binds to the C-terminus of SP1 and functions as a co-activator to enhance SP1 gene expression and promote cell proliferation; the promoter of Ajuba contains functional SP1 responsive elements and Ajuba itself is a target gene of SP1. CONCLUSION: Ajuba functions as a co-activator of SP1 to induce its target gene, and that Ajuba itself is a target genes of SP1. Ajuba/SP1 complex could form a feed forward loop to drive SP1 target gene transcription and promote cell proliferation of pancreatic cancer cells. Ajuba and SP1 might be biomarkers for PDAC diagnostics, prognosis and targets for new therapeutics.

Organization and function of tension-dependent complexes at adherens junctions.

Adherens junctions provide attachments between neighboring epithelial cells and a physical link to the cytoskeleton, which enables them to sense and transmit forces and to initiate biomechanical signaling. Examination of the Ajuba LIM protein Jub in Drosophila embryos revealed that it is recruited to adherens junctions in tissues experiencing high levels of myosin activity, and that the pattern of Jub recruitment varies depending upon how tension is organized. In cells with high junctional myosin, Jub is recruited to puncta near intercellular vertices, which are distinct from Ena-containing puncta, but can overlap Vinc-containing puncta. We identify roles for Jub in modulating tension and cellular organization, which are shared with the cytohesin Step, and the cytohesin adapter Sstn, and show that Jub and Sstn together recruit Step to adherens junctions under tension. Our observations establish Jub as a reporter of tension experienced at adherens junctions, and identify distinct types of tension-dependent and tension-independent junctional complexes. They also identify a role for Jub in mediating a feedback loop that modulates the distribution of tension and cellular organization in epithelia.