Anillin Expression Research Articles

Abstract 390: Expression of the actin-binding protein Anillin is a prognostic biomarker for primary ER positive breast cancer.

Abstract Identification of novel prognostic and predictive factors is important for individualizing prognosis and treatment for cancer patients. The present study aimed at examining the potential prognostic value of Anillin (ANLN) expression in breast cancer. ANLN, an actin-binding protein required for cytokinesis, was discovered using the Human Protein Atlas as being differentially expressed in breast cancer and, thus, a potential prognostic biomarker. The purpose of the Human Protein Atlas project (www.proteinatlas.org) is to generate validated antibodies towards the entire human proteome and to use these antibodies for protein profiling in normal tissues and cancer cells. The freely available Human Protein Atlas provides an opportunity to use antibody-based proteomics as a high-throughput strategy to discover novel cancer biomarkers of potential clinical relevance. ANLN has been shown to be up-regulated in several human tumor types including lung, renal and pancreatic tumors. For the purpose of the current study, tissue microarrays with tumor samples from three independent breast cancer cohorts were analyzed by immunohistochemistry (IHC). SiRNA mediated ANLN knockdown of breast cancer cell lines followed by western blot or IHC on cytospin was done for antibody validation. Cell viability assay and cell cycle assay was used to study the function of ANLN knocked breast cancer cells. The majority of breast tumors displayed moderate to strong nuclear positivity in slightly varying fractions, generally below 25%. High nuclear fraction of ANLN was associated with large tumor size, high histological grade, high proliferation and estrogen receptor (ER) negativity. Furthermore, high nuclear expression of ANLN was significantly associated with poor prognosis for patients with ER positive breast tumors in all three cohorts. SiRNA mediated ANLN knockdown was found to induce low cell viability for ER positive breast cancer cells but not for ER negative breast cancer cells. In addition, cell cycle studies showed that ANLN knockdown yielded a shift to G2/M phase, in both ER positive and ER negative breast cancer cells. In conclusion, our results strongly suggest that ANLN can be used as a prognostic biomarker for ER positive breast cancer patients. Citation Format: Kristina Magnusson, Gabriela Gremel, Fredrik Pontén, Karin Jirström. Expression of the actin-binding protein Anillin is a prognostic biomarker for primary ER positive breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 390. doi:10.1158/1538-7445.AM2013-390

Systematic antibody generation and validation via tissue microarray technology leading to identification of a novel protein prognostic panel in breast cancer.

BackgroundAlthough omic-based discovery approaches can provide powerful tools for biomarker identification, several reservations have been raised regarding the clinical applicability of gene expression studies, such as their prohibitive cost. However, the limited availability of antibodies is a key barrier to the development of a lower cost alternative, namely a discrete collection of immunohistochemistry (IHC)-based biomarkers. The aim of this study was to use a systematic approach to generate and screen affinity-purified, mono-specific antibodies targeting progression-related biomarkers, with a view towards developing a clinically applicable IHC-based prognostic biomarker panel for breast cancer.MethodsWe examined both in-house and publicly available breast cancer DNA microarray datasets relating to invasion and metastasis, thus identifying a cohort of candidate progression-associated biomarkers. Of these, 18 antibodies were released for extended analysis. Validated antibodies were screened against a tissue microarray (TMA) constructed from a cohort of consecutive breast cancer cases (n = 512) to test the immunohistochemical surrogate signature.ResultsAntibody screening revealed 3 candidate prognostic markers: the cell cycle regulator, Anillin (ANLN); the mitogen-activated protein kinase, PDZ-Binding Kinase (PBK); and the estrogen response gene, PDZ-Domain Containing 1 (PDZK1). Increased expression of ANLN and PBK was associated with poor prognosis, whilst increased expression of PDZK1 was associated with good prognosis. A 3-marker signature comprised of high PBK, high ANLN and low PDZK1 expression was associated with decreased recurrence-free survival (p < 0.001) and breast cancer-specific survival (BCSS) (p < 0.001). This novel signature was associated with high tumour grade (p < 0.001), positive nodal status (p = 0.029), ER-negativity (p = 0.006), Her2-positivity (p = 0.036) and high Ki67 status (p < 0.001). However, multivariate Cox regression demonstrated that the signature was not a significant predictor of BCSS (HR = 6.38; 95% CI = 0.79-51.26, p = 0.082).ConclusionsWe have developed a comprehensive biomarker pathway that extends from discovery through to validation on a TMA platform. This proof-of-concept study has resulted in the identification of a novel 3-protein prognostic panel. Additional biochemical markers, interrogated using this high-throughput platform, may further augment the prognostic accuracy of this panel to a point that may allow implementation into routine clinical practice.

Actin organization and dynamics: novel regulatory mechanisms from the biophysical to the tissue level

Properly regulated actin organization and dynamics are crucial for cellular processes such as cell migration, cell division, and maintenance of cell–cell junctions. Speakers in the “Actin Organization and Dynamics” Minisymposium at the ASCB 2012 meeting highlighted new and interesting mechanisms that underlie the regulation of actin dynamics. The session was broad in scope, ranging from in vitro biochemical and biophysical studies with purified components to cellular- and tissue-level studies. There was an emphasis on severing mechanisms and mechanics, as well as the complexities of regulatory pathways in cells. Hyeran Kang (Yale University), a postdoc with Enrique M. De La Cruz, presented work on how cations affect filament mechanics and severing via the regulatory protein, cofilin. Kang identified, using structural bioinformatics, two potential filament-specific cation-binding sites and classified them as “polymerization” and “stiffness” sites based on the effects that mutations at the sites have on salt-dependent assembly and bending mechanics, respectively. Cofilin enhances actin filament bending and twisting compliance, and it is hypothesized that local mechanical discontinuities in partially decorated filaments promote severing at boundaries of bare and cofilin-decorated segments. Using native and engineered yeast actin mutants, Kang showed that dissociation of stiffness site cations by human cofilin drives changes in filament bending mechanics and that this is required for severing. Pinar Gurel (Geisel School of Medicine at Dartmouth), a graduate student with Harry Higgs, described research investigating the severing activity of INF2, a formin linked to two human genetic diseases: Charcot-Marie-Tooth disease and focal segmental glomerulosclerosis. Using a combination of classic biochemical experiments and total internal reflection fluorescence (TIRF) microscopy, Gurel showed that, in addition to the normal formin binding site at the barbed end, INF2 binds the sides of actin filaments stoichiometrically (one INF2 per subunit) and with submicromolar affinity. INF2-catalyzed filament severing occurs within INF2-decorated regions of partially and fully decorated filaments. This property, along with INF2’s ability to bind ADP-Pi filaments, distinguishes INF2-mediated filament severing from that of cofilin. Dennis Breitsprecher (Brandeis University), a postdoc with Bruce Goode, presented recent work demonstrating a new role for Srv2/cyclase-associated protein. Breitsprecher observed, using dual-color TIRF microscopy, that the N-terminal half of Srv2 (N-Srv2) increases the severing activity of cofilin without appreciably affecting cofilin binding to filaments. Electron microscopy and single-particle analysis revealed that N-Srv2 self-associates into hexameric ring structures. N-terminal truncations weaken N-Srv2 oligomerization, compromise enhanced severing activity, and yield actin organization and cell polarity defects in cells. Peter Bieling (University of California, San Francisco), a postdoc with Dyche Mullins and Dan Fletcher, evaluated how reconstituted dendritic actin network growth responds to external loads. Dendritic actin networks assembled in vitro from micron-sized patterns of surface-immobilized nucleation-promoting factor were challenged with force applied via an atomic force microscope (AFM) cantilever, and actin assembly was monitored using multicolor TIRF microscopy and AFM. Bieling demonstrated that the dendritic actin networks dynamically adapt to external forces; the actin network growth velocity decreases with load, while actin density in the force-generating region of the network increases with load. Barbed-end capping is force-dependent and slows with increasing load. Colleen G. Bilancia (University of North Carolina, Chapel Hill), a postdoc with Mark Peifer, presented work on how Enabled (Ena) and Diaphanous (Dia) coordinate to regulate actin filament barbed-end polymerization. Overexpressing active Dia in Drosophila D16 cells promotes long, stable filopodial protrusions, while overexpressing Ena leads to dynamic, fan-like protrusions. Coexpression of Dia and Ena promotes protrusions distinct from those produced with expression of either regulator independently. Bilancia noted that filopodia retract when active Dia and Ena colocalize, suggesting that Ena negatively regulates Dia, possibly through direct binding interactions. Indeed, Bilancia showed that the Ena EVH1 domain directly binds the Dia FH1 domain, and the EVH1 domain inhibits Dia-mediated in vitro pyrene actin assembly and filopodia formation in cells. Ann Miller (University of Michigan) described her lab's finding that the cytokinesis regulator anillin plays a novel role in regulating epithelial cell–cell junctions. Using Xenopus embryos as a model system, Miller showed that a population of anillin localizes at cell–cell junctions in both dividing and nondividing cells. Further, anillin functionally regulates junctional integrity. Both tight junctions and adherens junctions are disrupted when anillin is knocked down. Anillin binds actin filaments and the small GTPase RhoA, both of which regulate cell–cell junction structure and function. Miller's group tested the effects of anillin expression on RhoA activation and found that anillin knockdown results in increased spontaneous flares of active RhoA that are prominent at cell–cell junctions in both dividing cells and nondividing regions of the epithelium.

Cleavage Furrow Organization Requires PIP2-Mediated Recruitment of Anillin

Cell division is achieved by a plasma membrane furrow that must ingress between the segregating chromosomes during anaphase [1-3]. The force that drives furrow ingression is generated by the actomyosin cytoskeleton, which is linked to the membrane by an as yet undefined molecular mechanism. A key component of the membrane furrow is anillin. Upon targeting to the furrow through its pleckstrin homology (PH) domain, anillin acts as a scaffold linking the actomyosin and septin cytoskeletons to maintain furrow stability (reviewed in [4, 5]). We report that the PH domain of anillin interacts with phosphatidylinositol phosphate lipids (PIPs), including PI(4,5)P(2), which is enriched in the furrow. Reduction of cellular PI(4,5)P(2) or mutations in the PH domain of anillin that specifically disrupt the interaction with PI(4,5)P(2), interfere with the localization of anillin to the furrow. Reduced expression of anillin disrupts symmetric furrow ingression that can be restored by targeting ectopically expressed anillin to the furrow using an alternate PI(4,5)P(2) binding module, a condition where the septin cytoskeleton is not recruited to the plasma membrane. These data demonstrate that the anillin PH domain has two functions: targeting anillin to the furrow by binding to PI(4,5)P(2) to maintain furrow organization and recruiting septins to the furrow.

Anillin expression is a marker of favourable prognosis in patients with renal cell carcinoma

The clinical course of renal cell carcinoma (RCC) can be difficult to predict. In this study we evaluated the prognostic value of anillin and Ki-67 in predicting survival in RCC. Immunohistochemical analysis using anillin and Ki-67 antibodies was performed on tissue microarrays constructed from paraffin-embedded specimens from 152 patients with primary RCC. The mean follow-up time was 90 months. Levels of anillin and Ki-67 staining were correlated with clinical factors, pathological features and survival. Anillin expression in cytoplasmic and nuclear fractions of the RCC cell line 786-O was examined using Western blot analysis. Cytoplasmic anillin immunopositivity was detected in 121 (83%) tumours. Nuclear anillin expression was present in 40 (27%) tumours and increased Ki-67 activity in 98 (66%) tumours. A positive association was found between nuclear anillin and Ki-67 proliferation activity (p=0.005). The mean RCC-specific survival times for anillin immunopositive and immunonegative tumours were 158 (95% CI 143-173) and 109 (78-141) months, respectively, with p=0.03. Increased Ki-67 activity showed a tendency towards a poorer prognosis, although this was not statistically significant. In the Cox regression analysis for cytoplasmic anillin, nuclear anillin or Ki-67 rate, and age, gender, stage and nuclear grade, the only significant factor in RCC-specific survival was stage (p<0.001). Western blot analysis showed anillin expression in both nuclear and cytosolic fractions of the RCC cell line. To conclude, anillin expression can be observed both in the cytoplasm and nuclei in patients with RCC. Cytoplasmic anillin expression is a marker of favourable prognosis in RCC patients.

The Septin-Binding Protein Anillin Is Overexpressed in Diverse Human Tumors

Anillin is an actin-binding protein that can bind septins and is a component of the cytokinetic ring. We assessed the anillin expression in 7,579 human tissue samples and cell lines by DNA microarray analysis. Anillin is expressed ubiquitously but with variable levels of expression, being highest in the central nervous system. The median level of anillin mRNA expression was higher in tumors than normal tissues (median fold increase 2.58; 95% confidence intervals, 2.19-5.68, P < 0.0001) except in the central nervous system where anillin mRNA levels were lower in tumors. We developed a sensitive reverse transcription-PCR strategy to show that anillin mRNA is expressed in cell lines and in cDNA panels derived from fetal and adult tissues, thus validating the microarray data. We compared anillin with Ki67 mRNA expression and found a significant linear relationship between anillin and Ki67 mRNA expression (Spearmann r approximately 0.6, P < 0.0001). Anillin mRNA expression was analyzed during tumor progression in breast, ovarian, kidney, colorectal, hepatic, lung, endometrial, and pancreatic tumors and in all tissues there was progressive increase in anillin mRNA expression from normal to benign to malignant to metastatic disease. Finally, we used anti-anillin sera and found nuclear anillin immunoreactivity to be widespread in normal tissues, often not correlating with proliferative compartments. These data provide insight into the existence of nonproliferation-associated activities of anillin and roles in interphase nuclei. Thus, anillin is overexpressed in diverse common human tumors, but not simply as a consequence of being a proliferation marker. Anillin may have potential as a novel biomarker.