ABI1 Gene Research Articles

ABI1-based expression signature predicts breast cancer metastasis and survival.

Despite the current standard of care, breast cancer remains one of the leading causes of mortality in women worldwide, thus emphasizing the need for better predictive and therapeutic targets. ABI1 is associated with poor survival and an aggressive breast cancer phenotype, although its role in tumorigenesis, metastasis, and the disease outcome remains to be elucidated. Here, we define the ABI1‐based seven‐gene prognostic signature that predicts survival of metastatic breast cancer patients; ABI1 is an essential component of the signature. Genetic disruption of Abi1 in primary breast cancer tumors of PyMT mice led to significant reduction of the number and size of lung metastases in a gene dose‐dependent manner. The disruption of Abi1 resulted in deregulation of the WAVE complex at the mRNA and protein levels in mouse tumors. In conclusion, ABI1 is a prognostic metastatic biomarker in breast cancer. We demonstrate, for the first time, that lung metastasis is associated with an Abi1 gene dose and specific gene expression aberrations in primary breast cancer tumors. These results indicate that targeting ABI1 may provide a therapeutic advantage in breast cancer patients.

Abstract 2470: Defining the reciprocal regulation of Abi1 and the androgen receptor in prostate cancer

Abstract Background: Prostate cancer (PCa) is a slowly progressing disease that affects nearly 50% of males over the age of 60. Current therapies for patients diagnosed with advanced PCa include Androgen Deprivation Therapy (ADT) and anti-androgen receptor (Anti-AR) treatment. PCa treatment primarily targets the androgen receptor (AR), a ligand-activated transcription factor, which is a key driver of PCa tumor growth. Our previous research has demonstrated that ABI1, acts as a tumor suppressor in PCa. ABI1 is a scaffold protein and an integral member of the WAVE Regulatory Complex (WRC), a nucleation promoting factor. UCSC database indicates there is an AR binding site within the ABI1 gene, suggesting AR plays a role in the transcriptional regulation of ABI1. In this study, we aim to characterize the ABI1-dependent AR regulation in PCa. Method: We generated an ABI1 KO cell line model in LNCaP cells using CRISPR-Cas9. The efficiency of CRISPR-Cas9 was evaluated by Western Blotting and genomic alterations confirmed by DNA sequencing. In addition, we have generated ABI1 Isoform-specific rescue cell lines in our LNCaP ABI1 KO cell line with a binding mutation in the SH3 domain (ABI1-W485N) and an SH3 domain deleted (Abi1-ΔSH3) ABI1 protein. To understand AR and ABI1 dependent pathways we performed qPCR, co-immunoprecipitation, Western Blotting, ChIP, in vitro fluorescence spectra assay, immunofluorescence, proximity ligation assays. Results: In vitro binding assays indicate the interaction of AR, NTD poly-proline region, with the ABI1 SH3 domain. Expression of ABI1- W485N in our ABI1 CRISPR KO cell line showed decrease binding, while ABI1-ΔSH3 showed no binding to AR in co-IP assays compared to our control. Consequently, we saw decreased AR nuclear localization compared to our ABI1-WT control. Furthermore, decreased nuclear localization in our ABI1-W485N mutant was associated with decreased mRNA expression of hallmark AR target genes, Prostate-Specific-Antigen (KLK3), and TMPRSS2. ABI1 is an AR responsive gene and was confirmed with ChIP assays in an AR overexpression cell line. Further, the stimulation of AR transcriptional activity increased cell-cell adhesion in an ABI1 dependent system. Conclusions: Our preliminary studies demonstrate that AR and ABI1 have a negative feedback pathway. The loss of ABI1 resulted in a decrease of nuclear AR and a subsequent decrease in mRNA expression of AR target genes. AR can also modulate ABI1 expression on a transcriptional level as needed for this pathway to function. Future studies will investigate if anti-AR treatments could lead to the dysregulation of ABI1 and promote EMT through STAT3 activation until AR transcriptional regulation on ABI1 is restored. These findings will allow for novel insights into the mechanisms underlying AR and ABI1 relationship in neoplastic progression. Citation Format: Baylee A. Porter, Alaji Bah, Alfonso Urbanucci, Fan Zhang, Sonia Kung, Ladan Fazli, Martin Gleave, Gennady Bratslavsky, Leszek Kotula. Defining the reciprocal regulation of Abi1 and the androgen receptor in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2470.

RNA Splicing of the Abi1 Gene by MBNL1 contributes to macrophage-like phenotype modulation of vascular smooth muscle cell during atherogenesis.

BackgroundVascular smooth muscle cells (VSMC) switch to macrophage‐like cells after cholesterol loading, and this change may play an important role in atherogenesis. Muscleblind‐like splicing regulator 1 (MBNL1) is a well‐known splicing factor that has been implicated in many cellular processes. However, the role of MBNL1 in VSMC macrophage‐like transdifferentiation is largely unknown. In this study, we aim to characterize the role of MBNL1‐induced gene splicing during atherogenesis.MethodsThe expression of MBNL1 and Abelson interactor 1 (Abi1) splice variants (Abi1‐e10 and Abi1‐Δe10) was compared between artery tissues from healthy donors and atherosclerosis patients. Regulatory mechanisms of MBNL1‐induced Abi1 gene splicing were studied, and the signal pathways mediated by Abi1 splice variants were investigated in VSMC.ResultsLoss of MBNL1 was found in the macrophage‐like VSMC (VSMC‐M) in artery wall from atherosclerosis patients. In vitro and in vivo evidence confirmed that Abi1 is one of the MBNL1 target genes. Loss of MBNL1 significantly induces the Abi1‐Δe10 isoform expression. Compared to the known actin organization activities of the Abi1 gene, we discovered a novel action of Abi1‐Δe10, whereby Abi1‐Δe10 activates Rac1 independent of upstream stimulation and triggers the Rac1‐NOX1‐ROS pathway, which results in increased expression of transcription factor Kruppel‐like factor 4 (KLF4). While Abi1‐Δe10 inhibits contractile VSMC biomarkers expression and cell contraction, it stimulates VSMC proliferation, migration and macrophage‐like transdifferentiation.ConclusionLoss‐of‐function of MBNL1 activates VSMC‐M transdifferentiation to promote atherogenesis through regulating Abi1 RNA splicing.

Abi1 loss drives prostate tumorigenesis through activation of EMT and non-canonical WNT signaling

BackgroundProstate cancer development involves various mechanisms, which are poorly understood but pointing to epithelial mesenchymal transition (EMT) as the key mechanism in progression to metastatic disease. ABI1, a member of WAVE complex and actin cytoskeleton regulator and adaptor protein, acts as tumor suppressor in prostate cancer but the role of ABI1 in EMT is not clear.MethodsTo investigate the molecular mechanism by which loss of ABI1 contributes to tumor progression, we disrupted the ABI1 gene in the benign prostate epithelial RWPE-1 cell line and determined its phenotype. Levels of ABI1 expression in prostate organoid tumor cell lines was evaluated by Western blotting and RNA sequencing. ABI1 expression and its association with prostate tumor grade was evaluated in a TMA cohort of 505 patients and metastatic cell lines.ResultsLow ABI1 expression is associated with biochemical recurrence, metastasis and death (p = 0.038). Moreover, ABI1 expression was significantly decreased in Gleason pattern 5 vs. pattern 4 (p = 0.0025) and 3 (p = 0.0012), indicating an association between low ABI1 expression and highly invasive prostate tumors. Disruption of ABI1 gene in RWPE-1 cell line resulted in gain of an invasive phenotype, which was characterized by a loss of cell-cell adhesion markers and increased migratory ability of RWPE-1 spheroids. Through RNA sequencing and protein expression analysis, we discovered that ABI1 loss leads to activation of non-canonical WNT signaling and EMT pathways, which are rescued by re-expression of ABI1. Furthermore, an increase in STAT3 phosphorylation upon ABI1 inactivation and the evidence of a high-affinity interaction between the FYN SH2 domain and ABI1 pY421 support a model in which ABI1 acts as a gatekeeper of non-canonical WNT-EMT pathway activation downstream of the FZD2 receptor.ConclusionsABI1 controls prostate tumor progression and epithelial plasticity through regulation of EMT-WNT pathway. Here we discovered that ABI1 inhibits EMT through suppressing FYN-STAT3 activation downstream from non-canonical WNT signaling thus providing a novel mechanism of prostate tumor suppression.

Abstract 5307: Androgen Receptor promotes localization of E-cadherin to cell-cell junctions through Abi1 signaling

Abstract According to American Cancer Society, prostate cancer is a slow progressing disease that affects nearly 50 percent of males over the age of 60. Every year, 165,000 new cases are diagnosed and 30,000 deaths are reported. Current therapies such as Androgen Depravation Therapy (ADT) and Anti-Androgen treatments such as Enzalutamide increase quality of life and extend survival but do not provide a definitive treatment and are ineffective in highly metastatic prostate cancer. Castrate Resistant Prostate Cancer (CRPC) is an untreatable highly metastatic form of prostate cancer and once diagnosed the survival rate is 1-2 years. Hence, the understanding of molecular mechanisms of CRPC is of critical importance. We previously linked prostate cancer to Abi1 gene. Abl interactor 1 (Abi1) is a scaffolding protein and an integral member of the WAVE Regulatory Complex (WRC). WRC is actin nucleation promoting factor that acts upstream of Arp2/3 to enhance the speed of actin polymerization and is critical for various cellular functions including cell-cell junction formation. Our previous research using a prostate cancer mouse models shows that loss of Abi1 expression leads to down-regulation of cellular adhesion proteins such as E-cadherin. Studies using our CRISPR KO RWPE cell line indicate that loss of Abi1 disrupts cell-cell junction formation via decreased levels of E-cadherin. Analysis of patient data demonstrated loss of Abi1 in a subset of CRPC patients. Castrate resistant tumor growth is androgen independent therefore anti-androgen therapy is ineffective. In search of a link between the androgen signaling and the role of Abi1 in prostate cancer we found that Abi1 gene contains androgen receptor (AR) binding sites. Consistent with the idea that Abi1 is an AR responsive gene, expression levels of Abi1 are increased following AR stimulation with the synthetic androgen, R1881, of the hormone sensitive cell line LNCaP. We also observed increased levels of E-cadherin at the cell-cell junctions. In contrast, cells lacking Abi1 do not form proper cell junctions neither respond to AR stimulation in the same way as wild type cells. We propose that Abi1 is a critical part of androgen signaling by promoting cell-cell junctions and tissue integrity. Supported by NCI R01 CA161018 (LK). Citation Format: Baylee Porter, Disharee Nath, Susan A. Krum, Gennady Bratslavsky, Vladimir A. Kuznetsov, Leszek Kotula. Androgen Receptor promotes localization of E-cadherin to cell-cell junctions through Abi1 signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5307.

Abstract 4602: Abi1 promotes breast cancer progression by regulating cell-matrix adhesion

Abstract Breast cancer is one of the most common diagnosed cancers in the United States. There will be approximately 266,120 new breast cancer cases in the U.S. and over 40,000 patients are predicted to die from the malignant progression of their breast cancer tumors in 2018. Traditional therapeutic methods of the invasive breast cancer include surgery accompanied by chemotherapy. However, following the initial therapy patients often relapse and eventually die from tumor metastasis. Abl interactor 1, or Abi1, is an adaptor protein which regulates actin polymerization through participation in WAVE (Wiscott-Aldrich syndrome protein family verprolin homologous) complex. The WAVE regulatory complex (also called WRC), regulates actin based cellular behavior, such as cellular adhesion and cell migration, by activating Arp2/3 complex and promoting actin polymerization. Previous studies indicated the direct interaction between integrin α4 and Abi1, which suggests a potential role of Abi1 in regulating cell-matrix adhesion signaling by involved in the inside-out integrin signaling pathways. In order to test the above hypothesis, we developed two genetic engineered Abi1 knocked-out breast cell lines from either HER2+ breast cancer cell line SKBR3 or normal breast epithelial cell line MCF10A by using CRISPR-Cas9 technique. After confirming inactivation of Abi1 gene by Western blotting, DNA and RNA sequencing, cells were harvested and analyzed for integrins levels. Western blotting results indicated dysregulation of multiple integrins in the Abi1 knock-out cell lines. The results from subsequent immunofluorescence experiments of the Abi1 knock-out cell lines also suggested changes in cell-matrix adhesion signaling using staining for scaffold protein paxillin and phosphorylated Focal-Adhesion-Kinase (FAK) as readouts. Our findings suggest that Abi1 might functions as a key regulator in breast cancer tumor progression by playing an important role in regulating signal pathways in cell-matrix adhesion. Supported by the Carol Baldwin Foundation of CNY, and in part by NIH-NCI grant No. R01 CA161018. Citation Format: Xiang Li, Angelina Regua, Gennady Bratslavsky, Vladimir A. Kuznetsov, Leszek Kotula. Abi1 promotes breast cancer progression by regulating cell-matrix adhesion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4602.

Abi1 loss drives prostate tumorigenesis through activation of EMT and noncanonical WNT signaling.

280 Background: Prostate cancer is characterized by heterogeneity of mechanisms which are poorly understood but pointing to epithelial plasticity as the key mechanism in progression to metastatic disease. ABI1, a member of WAVE complex and actin cytoskeleton regulator and adaptor protein, is proposed to act as tumor suppressor in prostate cancer, but the mechanism of tumor progression due to Abi1 loss is not clear. Methods: To address Abi1’s role in prostate cancer we used CRISPR-based gene editing and retroviral expression to manipulate Abi1 levels in prostate cancer cell lines. Levels of Abi1 expression in prostate organoid tumor cell lines were evaluated by Western blotting and/or RNA sequencing. Association of Abi1 loss with tumor grade was evaluated by immunohistochemistry. Results: Abi1 expression is downregulated in tumor organoid cell lines from metastatic bone and lymph node biopsies. Moreover, low Abi1 expression is associated with high-grade prostate tumors (GG3 or higher, p < 0.001). Disruption of Abi1 gene in a benign prostate epithelial cell line RWPE-1 resulted in a gain of invasive phenotype, which is characterized by loss of cell-cell adhesion markers and increased migratory ability of RWPE-1 Abi1 KO spheroids. Through RNA sequencing and protein expression analysis we discovered that Abi1 loss leads to activation of non-canonical WNT signaling and EMT pathways, which are rescued by re-expression of Abi1. Furthermore, increase in STAT3 phosphorylation upon Abi1 inactivation and evidence for high affinity interaction of FYN-SH2 domain with Abi1 pY421 support the model that Abi1 acts as a gatekeeper of the non-canonical WNT-EMT pathway activation downstream from FZD2 receptor. The gene expression profile of Abi1-EMT-WNT pathway overlaps with the reported gene signature of high-risk prostate tumors. Conclusions: Abi1 contributes to prostate cancer progression and epithelial plasticity through regulation of EMT-WNT pathways. Understanding of Abi1’s role may provide more mechanistic understanding of prostate cancer tumor progression.

Loss of Abelson Interactor-1 Is Linked to Inflammatory Hematopoiesis and Accelerated Aging of Hematopoietic System

Background: Hematopoietic stem cells (HSC) ensure homeostasis and lifelong maintenance of hematopoietic system, but with age, they gradually lose quiescence, self-renewal potential, and system restoration capacity. HSC aging results in a differentiation shift towards myeloid lineage, anemia, thrombocytosis, decrease in T and B cells, imbalance in macrophage function, and increased osteoclast activity. Mechanisms involved in HSC aging include increased mTOR activity and ROS production, impaired autophagy, epigenetic reprograming, and cumulative DNA damage. Intriguingly, cellular and molecular similarities between aging and inflammation have led to a novel concept of “inflammation-associated aging of hematopoiesis”. Understanding the molecular mechanisms responsible for this process may impact strategies targeting age-related diseases, including neoplasms. However, to date only few primary animal models of inflammation have shown bone marrow failure, so new animal models need to be established to provide mechanistic insight into the long-term implications of chronic inflammation on the hematopoietic system.We have previously shown that bone marrow-specific deletion of an adapter protein Abelson interactor-1 (Abi1) leads to a myeloproliferative neoplasm (MPN)-like disease in 35-56-week-old mice, mechanistically associated with increased activity of Src Family Kinases (SFKs), STAT3 and NF-κB. At both transcript and protein levels, Abi-1 is also significantly reduced in HSCs and granulocytes from patients with primary myelofibrosis (PMF), and Abi-1-deficient HSC in human PMF show increased SFK-STAT3-NF-κB signaling (Chorzalska, ASH 2017).Methods: Myeloid/lymphoid, stem/progenitor populations profiling by FACS, bone marrow transplantation assays, transcriptomics and proteomics analyses as well pro-inflammatory cytokine profiling and histopathology analyses were performed on the transgenic Abi-1KO mice carrying bone marrow-selective knockout at 4 weeks post-recombination, upon confirming both inducible inactivation of the Abi1flox allele and loss of Abi-1 protein in the marrow (Fig.1A, B).Results: To better understand initial systemic events that lead to the development of MPN-like disease in aged Abi-1KO mice we have now characterized early changes within the hematopoietic system associated with loss of Abi-1. Blood count analysis indicated leukocytosis, anemia and thrombocytosis, and an increase in the fraction of myeloid (CD11b+/Gr-1+) as well as macrophage/monocyte (F4/80+) cells at the expense of lymphoid (B220+) cells in Abi-1KO relative to Abi-1WT mice (Fig.1C). Previously reported 2.6-fold increase in Abi-1KO LT-HSCs (Chorzalska, ASH 2017) was now shown to be associated with 30% increase in number of LT-HSCs is in the S/G2/M phases of the cell cycle relative to Abi-1WT LT-HSCs (Fig. 1D). Lethally irradiated recipient C57BL/6 wild-type mice transplanted with bone marrow cells from Abi-1KO relative to Abi-1WT mice (in the absence of competitor cells) showed progressive loss of chimerism in primary and secondary recipients (Fig. E). Genome-wide gene expression analysis of Abi-1WT vs. Abi-1KO LSK cells showed significant overexpression of genes regulated by or involved in regulation of the NF-κB pathway (Fig. 1F). Plasma cytokine levels showed 2-fold increase in IL-1B, IL-12, IL-17, IL-23, IL-27, and MCP-1 and nearly 10-fold increase in INFγ (Fig. 1G). Label-free, intensity-based quantitative proteomic analysis of bone marrow from 20-week-old Abi-1KO and Abi-1WT mice showed abundance of peptides derived from Mac-1, myeloperoxidase, STAT1, STAT3, and SFKs - Hck and Fgr, confirming not only activation of SFKs and STAT3 signaling, but also increase in proteins associated with myeloid lineages (Fig. 1H). Loss of bone density (Fig. 1I) and a significant decrease in thymus size (Fig. 1J) were observed in Abi-1KO mice relative to Abi-1WT mice.Conclusions: In sum, phenotypic analyses performed 4-10-weeks post Abi1 gene inactivation indicate changes consistent with accelerated aging of hematopoietic system that are mechanistically linked to inflammatory SFK-STAT3-NF-κB signaling. To our knowledge this is the first animal model linking accelerated inflammation-driven aging of hematopoietic system to development of an MPN in aged mice. [Display omitted] DisclosuresOlszewski:Genentech: Research Funding; TG Therapeutics: Research Funding; Spectrum Pharmaceuticals: Consultancy, Research Funding. Reagan:Pfizer: Research Funding; Alexion: Honoraria; Takeda Oncology: Research Funding.

Abstract 1088: Defining bone metastatic potential of Abi1 CRISPR prostate cancer cell lines

Abstract Prostate cancer is the second most common cancer in American men, and it is the third leading cause of cancer-related deaths in American men. Distant stage and metastatic disease is often treated with androgen-deprivation therapy (ADT); however, this treatment ultimately fails, leading to castration-resistant prostate cancer (CRPC). This further emphasizes the need for better therapeutic targets. Our work focuses on understanding the role of the Abi1 gene in prostate cancer tumor progression. Our current in vitro data demonstrate an invasive phenotype with our CRISPR-mediated Abi1 knockout (KO) cell lines. In total, using CRISPR-mediated technology we have generated four Abi1 gene KO cell lines, PC-3, DU145, LnCaP, and RWPE-1. Importantly, the androgen-dependent cell line, LNCaP, demonstrated upregulation of Abi1 to the androgen receptor stimulation and the opposite response to the receptor inhibition upon treatment with enzalutamide. These data indicated that low Abi1 level could be induced by ADT therapies and are consistent with our previous observation of low Abi1/WAVE complex in prostate tumor samples. To learn more about this mechanism, we set out to investigate the role of Abi1 in castrated mice. The overall goal of the experiment is to compare the ability of cells lacking the Abi1 gene to engraft in bone under androgen deprivation condition and compare it to the control cells and examine any change in the engraftment phenotype. It is hoped that this research will lead to better understanding of the Abi1/WAVE complex role in CRPC. Supported by NCI R01 CA16101. Citation Format: Alexander Nappi, Dawn Post, Disharee Das, Ishita Joshi, Megan Oest, Gennady Bratslavsky, Leszek Kotula. Defining bone metastatic potential of Abi1 CRISPR prostate cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1088.

Involvement of genes encoding ABI1 protein phosphatases in the response of Brassica napus L. to drought stress

In this report we characterized the ArabidopsisABI1 gene orthologue and Brassica napus gene paralogues encoding protein phosphatase 2C (PP2C, group A), which is known to be a negative regulator of the ABA signaling pathway. Six homologous B. napus sequences were identified and characterized as putative PP2C group A members. To gain insight into the conservation of ABI1 function in Brassicaceae, and understand better its regulatory effects in the drought stress response, we generated transgenic B. napus plants overexpressing A. thalianaABI1. Transgenic plants subjected to drought showed a decrease in relative water content, photosynthetic pigments content and expression level of RAB18- and RD19A-drought-responsive marker genes relative to WT plants. We present the characterization of the drought response of B. napus with the participation of ABI1-like paralogues. The expression pattern of two evolutionarily distant paralogues, BnaA01.ABI1.a and BnaC07.ABI1.b in B. napus and their promoter activity in A. thaliana showed differences in the induction of the paralogues under dehydration stress. Comparative sequence analysis of both BnaABI1 promoters showed variation in positions of cis-acting elements that are especially important for ABA- and stress-inducible expression. Together, these data reveal that subfunctionalization following gene duplication may be important in the maintenance and functional divergence of the BnaABI1 paralogues. Our results provide a framework for a better understanding of (1) the role of ABI1 as a hub protein regulator of the drought response, and (2) the differential involvement of the duplicated BnaABI1 genes in the response of B. napus to dehydration-related stresses.Electronic supplementary materialThe online version of this article (doi:10.1007/s11103-015-0334-x) contains supplementary material, which is available to authorized users.

Abstract A51: Disruption of Abi1/Hssh3bp1 expression induces prostatic intraepithelial neoplasia in the conditional Abi1/Hssh3bp1 KO mice

Abstract Prostate cancer is one of the leading causes of cancer-related deaths in the United States and a leading diagnosed non-skin cancer in American men. Genetic mutations underlying prostate cancer tumorigenesis include alterations of tumor suppressor genes. We previously proposed that ABI1/hSSH3BP1 acts as a tumor suppressor gene (Macoska, et al. 2001 Neoplasia 3, 99-104). Here, we tested this hypothesis by searching for ABI1 gene mutations in primary prostate tumors from patients, and by analyzing the consequences of prostate-specific disruption of the mouse Abi1/Hssh3bp1 orthologue. We sequenced the ABI1/hSSH3BP1 gene and identified mutations in 15 out of 44 prostate tumors. Moreover, complementation growth-, proliferation- and xenograft- assays using the LNCaP cell line, which contains the loss-of-function Abi1 mutation, and stably expressed wt ABI gene, were consistent with the tumor suppressor hypothesis. To test the hypothesis further, we disrupted the gene in the mouse prostate by breeding the Abi1 floxed strain with the probasin promoter-driven Cre recombinase strain. Histopathological evaluation of mice indicated development of prostatic intraepithelial neoplasia (PIN) in Abi1/Hssh3bp1 KO mouse as early as the 8th month, but no progression beyond PIN was observed in mice as old as 12 months. Observed decreased levels of E-cadherin, β-catenin, and WAVE2 in mouse prostate suggest abnormal cellular adhesion as the mechanism underlying PIN development due to Abi1 disruption. In summary, this study provides further evidence for the hypothesis that Abi1 down-regulation plays a role in the development of prostate cancer and warrants further investigations of Abi1's role in prostate cancer tumorigenesis. Citation Format: Xiaoling Xiong, Leszek Kotula, Anna Chorzalska, Patrycja Dubielecka, Julie White, Yogindra Vedvyas, Cyrus V. Hedvat, Adriana Haimovitz-Friedman, Jason A. Koutcher, Janet A. Sawicki. Disruption of Abi1/Hssh3bp1 expression induces prostatic intraepithelial neoplasia in the conditional Abi1/Hssh3bp1 KO mice [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr A51.

Abl interactor 1 regulates Src-Id1-matrix metalloproteinase 9 axis and is required for invadopodia formation, extracellular matrix degradation and tumor growth of human breast cancer cells

Abl interactor 1 (Abi1) is a key regulator of actin polymerization/depolymerization. The involvement of Abi1 in the development of abnormal cytoskeletal functions of cancer cells has recently been reported. It remains unclear, however, how Abi1 exerts its effects in tumor cells and whether it contributes to tumor progression in vivo. We report here a novel function for Abi1 in the regulation of invadopodia formation and Src-inhibitor of differentiation protein 1 (Id1)-matrix metalloproteinase (MMP)-9 pathway in MDA-MB-231 human breast cancer cells. Abi1 is found in the invadopodia of MDA-MB-231 cells. Epigenetic silencing of the Abi1 gene by short hairpin RNA in MDA-MB-231 cells impaired the formation of invadopodia and resulted in downregulation of the Src activation and Id1/MMP-9 expression. The decreased invadopodia formation and MMP-9 expression correlate with a reduction in the ability of these cells to degrade extracellular matrix. Remarkably, the knockdown of Abi1 expression inhibited tumor cell proliferation and migration in vitro and slowed tumor growth in vivo. Taken together, these results indicate that the Abi1 signaling plays a critical role in breast cancer progression and suggest that this pathway may serve as a therapeutic target for the treatment of human breast cancer.

Expression of the Arabidopsis mutant ABI1 gene alters abscisic acid sensitivity, stomatal development, and growth morphology in gray poplars.

The consequences of altered abscisic acid (ABA) sensitivity in gray poplar (Populus x canescens [Ait.] Sm.) development were examined by ectopic expression of the Arabidopsis (Arabidopsis thaliana) mutant abi1 (for abscisic acid insensitive1) gene. The expression resulted in an ABA-insensitive phenotype revealed by a strong tendency of abi1 poplars to wilt, impaired responsiveness of their stomata to ABA, and an ABA-resistant bud outgrowth. These plants therefore required cultivation under very humid conditions to prevent drought stress symptoms. Morphological alterations became evident when comparing abi1 poplars with poplars expressing Arabidopsis nonmutant ABI1 or wild-type plants. abi1 poplars showed increased stomatal size, enhanced shoot growth, and retarded leaf and root development. The increased stomatal size and its reversion to the size of wild-type plants by exogenous ABA indicate a role for ABA in regulating stomatal development. Enhanced shoot growth and retarded leaf and root development support the hypothesis that ABA acts independently from drought stress as a negative regulator of growth in shoots and as a positive regulator of growth in leaves and roots. In shoots, we observed an interaction of ABA with ethylene: abi1 poplars exhibited elevated ethylene production, and the ethylene perception inhibitor Ag(+) antagonized the enhanced shoot growth. Thus, we provide evidence that ABA acts as negative regulator of shoot growth in nonstressed poplars by restricting ethylene production. Furthermore, we show that ABA has a role in regulating shoot branching by inhibiting lateral bud outgrowth.

Knockdown of Abl interactor 1 expression impairs invadopodia formation, tumor angiogenesis, and markedly slows tumor growth of breast cancer cells.

Abstract Abstract #2054 Background: Abl interactor 1 (Abi1) is a key regulator of actin polymerization/depolymerization. Previous studies have revealed that Abi1 interacts with Wiskott–Aldrich syndrome protein (Wasp) family proteins, and regulates the nucleation promoting activity of Wasp proteins. In addition to Wasp family proteins, Abi1 also binds to a variety of other signaling molecules, including those involved in the signal transductions of small GTP-binding protein Rac and PI3 kinase. The ability to interact with diverse signaling pathways places Abi1 at a central position in the signaling network that regulates cell motility and proliferation. The involvement of Abi1 in the development of abnormal cytoskeletal functions of cancer cells has recently been reported. It remains unclear, however, how Abi1 exerts its effects in tumor cells and whether it contributes to tumor progression in vivo.
 Material and Methods: The short hairpin RNA (shRNA)-mediated gene silencing was used, in conjunction with murine tumor xenograft models, to examine the effects of Abi1 knockdown on cytoskeleton remodeling, cell migration, invasion, as well as tumor growth and angiogenesis of MDA-MB231 breast cancer cells.
 Results: We report here a novel function for Abi1 in the regulation of invadopodia formation as well as in the tumor angiogenesis and invasion of breast cancer cells, possibly through the modulation of Src and matrix metalloproteinase 9 (MMP-9) activities. Abi1 co-localizes with invadopodia, the specialized adhesive/invasive structures believed to control the invasion of tumor cells, in MDA-MB231 breast cancer cells. Epigenetic silencing of the Abi1 gene by shRNA in MDA-MB231 cells impaired the formation of invadopodia and decreased the activity of c-Src and MMP-9. The decreased invadopodia formation and MMP-9 activity correlated with a reduction in the ability of these cells to degrade extracellular matrix (ECM). Remarkably, the knockdown of Abi1 expression inhibited tumor invasion and angiogenesis and markedly slowed tumor growth in xenograft mouse models. Taken together, these results indicate that the Abi1 signaling plays a critical role in breast cancer progression and suggest that this pathway may serve as a therapeutic target for the treatment of human breast cancer. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2054.

Abi1 gene silencing by short hairpin RNA impairs Bcr-Abl-induced cell adhesion and migration in vitro and leukemogenesis in vivo

Abl interactor (Abi) 1 was first identified as the downstream target of Abl tyrosine kinases and was found to be dysregulated in leukemic cells expressing oncogenic Bcr-Abl and v-Abl. Although the accumulating evidence supports a role of Abi1 in actin cytoskeleton remodeling and growth factor/receptor signaling, it is not clear how it contributes to Bcr-Abl-induced leukemogenesis. We show here that Abi1 gene silencing by short hairpin RNA attenuated the Bcr-Abl-induced abnormal actin remodeling, membrane-type 1 metalloproteinase clustering and inhibited cell adhesion and migration on fibronectin-coated surfaces. Although the knock down of Abi1 expression did not affect growth factor-independent growth of Bcr-Abl-transformed Ba/F3 cells in vitro, it impeded competitive expansion of these cells in non obese diabetic (NOD)/ severe combined immuno-deficiency (SCID) mice. Remarkably, the knock down of Abi1 expression in Bcr-Abl-transformed Ba/F3 cells impaired the leukemogenic potential of these cells in NOD/SCID mice. Abi1 contributes to Bcr-Abl-induced leukemogenesis in part through Src family kinases, as the knock down of Abi1 expression attenuates Bcr-Abl-stimulated activation of Lyn. Together, these data provide for the first time the direct evidence that supports a critical role of Abi1 pathway in the pathogenesis of Bcr-Abl-induced leukemia.

Interplay between JA, SA and ABA signalling during basal and induced resistance against Pseudomonas syringae and Alternaria brassicicola

We have examined the role of the callose synthase PMR4 in basal resistance and beta-aminobutyric acid-induced resistance (BABA-IR) of Arabidopsis thaliana against the hemi-biotrophic pathogen Pseudomonas syringae and the necrotrophic pathogen Alternaria brassicicola. Compared to wild-type plants, the pmr4-1 mutant displayed enhanced basal resistance against P. syringae, which correlated with constitutive expression of the PR-1 gene. Treating the pmr4-1 mutant with BABA boosted the already elevated levels of PR-1 gene expression, and further increased the level of resistance. Hence, BABA-IR against P. syringae does not require PMR4-derived callose. Conversely, pmr4-1 plants showed enhanced susceptibility to A. brassicicola, and failed to show BABA-IR. Wild-type plants showing BABA-IR against A. brassicicola produced increased levels of JA. The pmr4-1 mutant produced less JA upon A. brassicicola infection than the wild-type. Blocking SA accumulation in pmr4-1 restored basal resistance, but not BABA-IR against A. brassicicola. This suggests that the mutant's enhanced susceptibility to A. brassicicola is caused by SA-mediated suppression of JA, whereas the lack of BABA-IR is caused by its inability to produce callose. A. brassicicola infection suppressed ABA accumulation. Pre-treatment with BABA antagonized this ABA accumulation, and concurrently potentiated expression of the ABA-responsive ABI1 gene. Hence, BABA prevents pathogen-induced suppression of ABA accumulation, and sensitizes the tissue to ABA, causing augmented deposition of PMR4-derived callose.

Yeast complementation reveals a role for an Arabidopsis thaliana late embryogenesis abundant (LEA)‐like protein in oxidative stress tolerance

A functional cloning approach using the oxidant-sensitive yeast mutant, Deltayap1, was employed to identify plant genes involved in tolerance of oxidative stress. In this screen, we identified an Arabidopsis late embryogenesis-abundant (LEA)-like protein, AtLEA5, which increased the tolerance of Deltayap1 cells to the oxidants H(2)O(2), diamide, menadione and tert-butyl hydroperoxide. Unlike canonical LEAs, AtLEA5 is constitutively expressed in roots and reproductive organs but not in seeds. In leaves of short-day grown plants, AtLEA5 transcripts exhibited a diurnal pattern of regulation, where transcripts were repressed in the light and abundant in the dark. Expression of AtLEA5 in leaves was induced by oxidants, ABA and dehydration. Use of abi1-1 (ABA-insensitive) and aba1-1 (ABA-deficient) Arabidopsis mutants indicated that drought induction of AtLEA5 required ABA synthesis but was independent of the ABI1 gene product. Abscisic acid and H(2)O(2) induction of AtLEA5 was also independent of the OXI1 protein kinase. Constitutive overexpression of AtLEA5 resulted in increased root growth and shoot biomass, both in optimal conditions and under H(2)O(2) stress. However, in comparison with wild type, photosynthesis in overexpressing plants was more susceptible to drought. These features suggest that AtLEA5 has a unique function among LEA proteins in that it plays a specific role in protection against oxidative stress involving decreased photosynthesis. This protein functions as part of a complex network of defences that contribute to robustness of plants under stress by minimizing the negative effects of oxidation.

Inhibition of v-Abl transformation in 3T3 cells overexpressing different forms of the Abelson interactor protein Abi-1

The abi-1 gene encodes a protein that binds and is phosphorylated by the Abelson protein tyrosine kinase. Constructs expressing a full-length abi-1 cDNA, and a smaller cDNA arising from an alternatively spliced form, were generated and tested for their effect on transformation of NIH3T3 cells by the Abelson murine leukemia virus. Overexpression of both forms of the protein strongly inhibited transformation by the wild-type P160 strain of the virus, but not by the non-interacting mutant P90A strain. The inhibition required the SH3 domain of Abi-1, suggesting that a direct interaction was required for the effect. Rare breakthrough P160 transformants of the Abi-1 overexpressing lines were found to have downregulated Abi-1 protein levels by a post-transcriptional mechanism.

ABI1 protein phosphatase 2C is a negative regulator of abscisic acid signaling.

The plant hormone abscisic acid (ABA) is a key regulator of seed maturation and germination and mediates adaptive responses to environmental stress. In Arabidopsis, the ABI1 gene encodes a member of the 2C class of protein serine/threonine phosphatases (PP2C), and the abi1-1 mutation markedly reduces ABA responsiveness in both seeds and vegetative tissues. However, this mutation is dominant and has been the only mutant allele available for the ABI1 gene. Hence, it remained unclear whether ABI1 contributes to ABA signaling, and in case ABI1 does regulate ABA responsiveness, whether it is a positive or negative regulator of ABA action. In this study, we isolated seven novel alleles of the ABI1 gene as intragenic revertants of the abi1-1 mutant. In contrast to the ABA-resistant abi1-1 mutant, these revertants were more sensitive than the wild type to the inhibition of seed germination and seedling root growth by applied ABA. They also displayed increases in seed dormancy and drought adaptive responses that are indicative of a higher responsiveness to endogenous ABA. The revertant alleles were recessive to the wild-type ABI1 allele in enhancing ABA sensitivity, indicating that this ABA-supersensitive phenotype results from a loss of function in ABI1. The seven suppressor mutations are missense mutations in conserved regions of the PP2C domain of ABI1, and each of the corresponding revertant alleles encodes an ABI1 protein that lacked any detectable PP2C activity in an in vitro enzymatic assay. These results indicate that a loss of ABI1 PP2C activity leads to an enhanced responsiveness to ABA. Thus, the wild-type ABI1 phosphatase is a negative regulator of ABA responses.

ABI-1, a Human Homolog to Mouse Abl-Interactor 1, Fuses theMLL Gene in Acute Myeloid Leukemia With t(10;11)(p11.2;q23)

Recurrent translocation t(10;11) has been reported to be associated with acute myeloid leukemia (AML). Recently, two types of chimeric transcripts, MLL-AF10 in t(10;11)(p12;q23) andCALM-AF10 in t(10;11)(p13;q14), were isolated. t(10;11) is strongly associated with complex translocations, including invins(10;11) and inv(11)t(10;11), because the direction of transcription of AF10 is telomere to centromere. We analyzed a patient of AML with t(10;11)(p11.2;q23) and identified ABI-1 on chromosome 10p11.2, a human homolog to mouse Abl-interactor 1 (Abi-1), fused with MLL. Whereas the ABI-1 gene bears no homology with the partner genes of MLL previously described, the ABI-1 protein exhibits sequence similarity to protein of homeotic genes, contains several polyproline stretches, and includes asrc homology 3 (SH3) domain at the C-terminus that is required for binding to Abl proteins in mouse Abi-1 protein. Recently, e3B1, an eps8 SH3 binding protein 1, was also isolated as a human homolog to mouse Abi-1. Three types of transcripts of ABI-1 gene were expressed in normal peripheral blood. Although e3B1 was considered to be a full-length ABI-1, the MLL-ABI-1fusion transcript in this patient was formed by an alternatively spliced ABI-1. Others have shown that mouse Abi-1 suppresses v-ABL transforming activity and that e3B1, full-length ABI-1, regulates cell growth. In-frame MLL-ABI-1 fusion transcripts combine the MLL AT-hook motifs and DNA methyltransferase homology region with the homeodomain homologous region, polyproline stretches, and SH3 domain of alternatively spliced transcript of ABI-1. Our results suggest that the ABI-1 gene plays a role in leukemogenesis by translocating to MLL. © 1998 by The American Society of Hematology.