Actinin-4 Research Articles

CRISPR/Cas9-Mediated Suppression of the Focal Segmental Glomerulosclerosis Protein Actinin-4 (ACTN4) in Thick Ascending Limbs (TALs) Increases Sodium Chloride Reabsorption by Regulating NKCC2 Trafficking

CRISPR/Cas9-Mediated Suppression of the Focal Segmental Glomerulosclerosis Protein Actinin-4 (ACTN4) in Thick Ascending Limbs (TALs) Increases Sodium Chloride Reabsorption by Regulating NKCC2 Trafficking

Benefits from Adjuvant Chemotherapy in Patients with Resected Non-Small Cell Lung Cancer: Possibility of Stratification by Gene Amplification of ACTN4 According to Evaluation of Metastatic Ability.

Simple SummaryThe establishment of biomarkers that can identify individuals at high risk of early recurrence after surgery will be an important issue in decision-making for perioperative therapy. In this review, we describe potential biomarkers for predicting the likelihood of recurrence in patients who undergo surgery for stage I NSCLC. ACTN4 is a possible biomarker for identifying patients at high risk of postoperative recurrence, and patients with gene amplification of ACTN4 might thus benefit the most from adjuvant chemotherapy.Surgical treatment is the best curative treatment option for patients with non-small cell lung cancer (NSCLC), but some patients have recurrence beyond the surgical margin even after receiving curative surgery. Therefore, therapies with anti-cancer agents also play an important role perioperatively. In this paper, we review the current status of adjuvant chemotherapy in NSCLC and describe promising perioperative therapies, including molecularly targeted therapies and immune checkpoint inhibitors. Previously reported biomarkers of adjuvant chemotherapy for NSCLC are discussed along with their limitations. Adjuvant chemotherapy after resective surgery was most effective in patients with metastatic lesions located just outside the surgical margin; in addition, these metastatic lesions were the most sensitive to adjuvant chemotherapy. Thus, the first step in predicting patients who have sensitivity to adjuvant therapies is to perform a qualified evaluation of metastatic ability using markers such as actinin-4 (ACTN4). In this review, we discuss the potential use of biomarkers in patient stratification for effective adjuvant chemotherapy and, in particular, the use of ACTN4 as a possible biomarker for NSCLC.

The FSGS protein Actinin‐4 (ACTN4) is expressed in Thick Ascending Limbs and interacts with NKCC2 and ALMS1 to regulate NKCC2 trafficking

NKCC2 is a Cl-dependent co-transporter that mediates all the NaCl absorption in the thick ascending limb (TAL). In humans, loss-of-function mutations in NKCC2 result in Bartter syndrome type 1, characterized by inability in concentrating urine, polyuria, and low blood pressure. Importantly, enhanced NKCC2 activity is associated with hypertension in humans and animal models. NKCC2 is present on the apical membrane and sub-apical space in the TAL and macula densa cells. We and others have shown that trafficking of NKCC2 to the apical membrane determines NKCC2-mediated NaCl absorption. These trafficking steps are controlled by signaling and protein-protein interactions. Very little is known about how NKCC2 endocytosis is controlled and only a handful of proteins that mediate NKCC2 internalization and trafficking are known. To identify new NKCC2 interacting partners we immunoprecipitated NKCC2 from TAL lysates using a C-terminus antibody and identified interacting proteins by mass spectrometry. We identified Actinin-4 (ACTN4), a protein studied in podocytes, that when mutated causes Focal segmental glomerulo-sclerosis (FSGS) in humans. We hypothesized that in addition to podocytes, ACTN4 is expressed along the nephron and is part of protein complex that binds apical NKCC2 and other proteins such as Alstrom Syndrome 1 (ALMS1) and promotes its endocytosis. To begin studying these mechanisms we used a targeted proteomics screen to identify new binding partners for ACTN4 in the TAL. We confirmed that glutathione-S-transferase (GST) ACTN4 (full length) pulled down NKCC2 and ALMS1 from TAL lysates identified by mass spectrometry (LC-MS) or Western blot. GST-ALMS1 also pulled down ACTN4, identified by LC-MS. Because there are no studies on the role of ACTN4 in NaCl reabsorption along the nephron, we studied its localization in the kidney. ACTN4 was abundant in glomeruli but also localized in cells along the nephron in a punctate vesicular pattern. ACTN4 was expressed in TALs and partially co-localized with NKCC2 the subapical space. To study if ACTN4 mediates NKCC2 trafficking, we generated adenoviruses expressing ACTN4 shRNA. In vivo transduction of the rat renal medulla decreased ACTN4 expression in TALs by 66±9% (p<0.04). Silencing ACTN4 in rat TALs increased the surface to total NKCC2 ratio by 49±16% (p<0.01) but did not affect total NKCC2, suggesting that ACTN4 regulates NKCC2 trafficking. To study the relationship of ACTN4 with ALMS1 and NKCC2, we generated nephron-specific ALMS1 knockout mice and measured surface NKCC2 and ACTN4. Deletion of ALMS1 increased surface to total NKCC2 ratio by 55±19% (p<0.05), but also decreased ACTN4 expression by 45±9% (p<0.05). We conclude that ACTN4 binds NKCC2 and ALMS1 and it is required for proper NKCC2 trafficking. It is likely that a large protein complex containing ACTN4 and ALMS1 mediates NKCC2 endocytosis to control renal salt reabsorption.

Serum Actinin-4 Levels as a Potential Diagnostic and Prognostic Marker in Cervical Cancer.

Purpose The present study was aimed at determining the serum levels of actinin-4 (ACTN4) in cervical cancer (CC) and investigating the diagnostic and prognostic value of serum ACTN4 in CC. Materials and Methods We included 93 CC patients, 52 cervical intraepithelial neoplasia (CIN) patients, and 70 healthy women. Serum ACTN4 levels were assessed using an ELISA method. A receiver operating characteristic (ROC) curve was performed to evaluate the diagnostic value of serum ACTN4. The survival curves were used to display the overall survival distributions. Results Serum ACTN4 levels in CC patients were 48.39 ± 13.98 pg/mL which is significantly higher than those in CIN patients (32.72 ± 9.44 pg/mL; P < 0.001) and those in healthy controls (30.84 ± 8.08 pg/mL; P < 0.001). The ROC analysis demonstrated that the area under the curve (AUC) of ACTN4 was 0.852 (95%CI = 0.796–0.908), with sensitivity of 76.3% and specificity of 87.7%. Serum ACTN4 levels were associated with the FIGO stage, lymph node metastasis, and lymphovascular space invasion of CC (all P < 0.05). The survival curve suggested that high serum ACTN4 levels were related to poor prognosis. Conclusion Our findings suggest that serum ACTN4 levels may be valuable diagnostic and prognostic biomarkers for CC.

Prognostic impact of ACTN4 gene copy number alteration in hormone receptor-positive, HER2-negative, node-negative invasive breast carcinoma

BackgroundMost patients with hormone receptor (HR)-positive, human epidermal growth factor receptor type 2 (HER2)-negative breast cancer can be cured by surgery and endocrine therapy, but a significant proportion suffer recurrences. Actinin-4 is associated with cancer invasion and metastasis, and its genetic alteration may be used for breast cancer prognostication.MethodsThe copy number of the actinin-4 (ACTN4) gene was determined by fluorescence in situ hybridisation (FISH) in two independent cohorts totalling 597 patients (336 from Japan and 261 from the USA) with HR-positive, HER2-negative, node-negative breast cancer.ResultsIn the Japanese cohort, multivariate analysis revealed that a copy number increase (CNI) of ACTN4 was an independent factor associated with high risks of recurrence (P = 0.01; hazard ratio (HR), 2.95) and breast cancer death (P = 0.014; HR, 4.27). The prognostic significance of ACTN4 CNI was validated in the US cohort, where it was the sole prognostic factor significantly associated with high risks of recurrence (P = 0.04; HR, 2.73) and death (P = 0.016; HR, 4.01).ConclusionsCopy number analysis of a single gene, ACTN4, can identify early-stage luminal breast cancer patients with a distinct outcome. Such high-risk patients may benefit from adjuvant chemotherapy.

The FSGS protein Actinin‐4 (ACTN4) is expressed in Thick Ascending Limbs and interacts with NKCC2 and ALMS1 to regulate NKCC2 trafficking.

Thick ascending limb (TAL) of the loop of Henle reabsorbs 30% NaCl via the apical Na/K/2Cl cotransporter (NKCC2). Maintenance of steady‐state surface NKCC2 levels is results from a balance between endocytosis, exocytosis and recycling. These trafficking steps are controlled by signaling and protein‐protein interactions. Very little is known about how NKCC2 endocytosis is controlled and only a handful of proteins that mediate NKCC2 internalization and trafficking are known. To identify new NKCC2 interacting partners we immunoprecipitated NKCC2 from TAL lysates using a C‐terminus antibody and identified interacting proteins by mass spectrometry. We identified Actinin‐4 (ACTN4), a protein studied in podocytes, that when mutated causes Focal segmental glomerulo‐sclerosis (FSGS) in humans. We hypothesized that in addition to podocytes, ACTN4 is expressed along the nephron and is part of protein complex that binds apical NKCC2 and other proteins such as Alstrom Syndrome 1 (ALMS1) and promotes its endocytosis. To begin studying these mechanisms we used a targeted proteomics screen to identify new binding partners for ACTN4 in the TAL. We confirmed that glutathione‐S‐transferase (GST) ACTN4 (full length) pulled down NKCC2 and ALMS1 from TAL lysates identified by mass spectrometry (LC‐MS) or Western blot. GST‐ALMS1 also pulled down ACTN4, identified by LC‐MS. Because there are no studies on the role of ACTN4 in NaCl reabsorption along the nephron, we studied its localization in the kidney. ACTN4 was abundant in glomeruli but also localized in cells along the nephron in a punctate vesicular pattern. ACTN4 was expressed in TALs and partially co‐localized with NKCC2 the subapical space. To study if ACTN4 mediates NKCC2 trafficking, we generated adenoviruses expressing ACTN4 shRNA. In vivo transduction of the rat renal medulla decreased ACTN4 expression in TALs by 66±9% (p&lt;0.04). Silencing ACTN4 in rat TALs increased the surface to total NKCC2 ratio by 49±16% (p&lt;0.01) but did not affect total NKCC2, suggesting that ACTN4 regulates NKCC2 trafficking. To study the relationship of ACTN4 with ALMS1 and NKCC2, we generated nephron‐specific ALMS1 knockout mice and measured surface NKCC2 and ACTN4. Deletion of ALMS1 increased surface to total NKCC2 ratio by 55±19% (p&lt;0.05), but also decreased ACTN4 expression by 45±9% (p&lt;0.05). We conclude that ACTN4 binds NKCC2 and ALMS1 and it is required for proper NKCC2 trafficking. It is likely that a large protein complex containing ACTN4 and ALMS1 mediates NKCC2 endocytosis to control renal salt reabsorption.Support or Funding InformationNIH

The role of actinin-4 (ACTN4) in exosomes as a potential novel therapeutic target in castration-resistant prostate cancer

Prostate cancer is the second leading cause of cancer death in men in the United States. Several novel therapeutic agents have been developed for castration-resistant prostate cancer (CRPC), but the prognosis for patients with CRPC remains poor. The identification of novel therapeutic targets for CRPC is an urgent issue. Exosomes are small vesicles secreted by a variety of cells, and exosomes derived from cancer cells have been reported to circulate in the patient’s bodily fluids, promoting metastasis and invasion. We aimed to identify novel therapeutic targets for CRPC by proteomic analysis of serum exosomes. Exosomes were isolated by ultracentrifugation of sera from 36 men with metastatic prostate cancer: untreated (n = 8), well-controlled with primary androgen deprivation therapy (ADT) (n = 8), and CRPC (n = 20). We identified 823 proteins in the serum exosomes. Six proteins were increased in CRPC patients compared with untreated patients. In contrast, only ACTN4 was increased in the CRPC patients compared to the ADT patients. We focused on ACTN4 as a candidate for targeted therapeutics. ACTN4 was highly expressed in the prostate cancer cell line DU145 as well as exosomes from this line. RNA interference-mediated downregulation of ACTN4 significantly attenuated cell proliferation and invasion in DU145 cells. ACTN4 could be a potential therapeutic target for CRPC.

Prognostic significance of gene amplification of ACTN4 in stage I and II oral tongue cancer

Despite complete resection of the early stage of oral tongue cancer by partial glossectomy, late cervical lymph node metastasis is frequently observed. Gene amplification of ACTN4 (protein name: actinin-4) is closely associated with the metastatic potential of various cancers. This retrospective study was performed to demonstrate the potential usefulness of ACTN4 gene amplification as a prognostic biomarker in patients with stage I/II oral tongue cancer. Fifty-four patients with stage I/II oral tongue cancer were enrolled retrospectively, in accordance with the reporting recommendations for tumour marker prognostic studies (REMARK) guidelines. The copy number of ACTN4 and the protein expression of actinin-4 were evaluated by fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC), respectively. The overall survival time of patients with gene amplification of ACTN4 was significantly shorter than that of patients without gene amplification (P=0.0010, log-rank test). Gene amplification of ACTN4 was a significant independent risk factor for death in patients with stage I/II oral tongue cancer (hazard ratio 6.08, 95% confidence interval 1.66–22.27). Gene amplification of ACTN4 is a potential prognostic biomarker for overall survival in oral tongue cancer.

α Actinin 4 (ACTN4) Regulates Glucocorticoid Receptor-mediated Transactivation and Transrepression in Podocytes

Glucocorticoids are a general class of steroids that possess renoprotective activity in glomeruli through their interaction with the glucocorticoid receptor. However, the mechanisms by which glucocorticoids ameliorate proteinuria and glomerular disease are not well understood. In this study, we demonstrated that α actinin 4 (ACTN4), an actin-cross-linking protein known to coordinate cytoskeletal organization, interacts with the glucocorticoid receptor (GR) in the nucleus of human podocytes (HPCs), a key cell type in the glomerulus critical for kidney filtration function. The GR-ACTN4 complex enhances glucocorticoid response element (GRE)-driven reporter activity. Stable knockdown of ACTN4 by shRNA in HPCs significantly reduces dexamethasone-mediated induction of GR target genes and GRE-driven reporter activity without disrupting dexamethasone-induced nuclear translocation of GR. Synonymous mutations or protein expression losses in ACTN4 are associated with kidney diseases, including focal segmental glomerulosclerosis, characterized by proteinuria and podocyte injury. We found that focal segmental glomerulosclerosis-linked ACTN4 mutants lose their ability to bind liganded GR and support GRE-mediated transcriptional activity. Mechanistically, GR and ACTN4 interact in the nucleus of HPCs. Furthermore, disruption of the LXXLL nuclear receptor-interacting motif present in ACTN4 results in reduced GR interaction and dexamethasone-mediated transactivation of a GRE reporter while still maintaining its actin-binding activity. In contrast, an ACTN4 isoform, ACTN4 (Iso), that loses its actin-binding domain is still capable of potentiating a GRE reporter. Dexamethasone induces the recruitment of ACTN4 and GR to putative GREs in dexamethasone-transactivated promoters, SERPINE1, ANGPLT4, CCL20, and SAA1 as well as the NF-κB (p65) binding sites on GR-transrepressed promoters such as IL-1β, IL-6, and IL-8 Taken together, our data establish ACTN4 as a transcriptional co-regulator that modulates both dexamethasone-transactivated and -transrepressed genes in podocytes.

Possible utility of actinin-4 as a predictive biomarker of the efficacy of postoperative adjuvant chemotherapy for completely resected early stage lung adenocarcinoma.

e20003Background: We previously identified gene amplification and protein overexpression of actinin-4 (ACTN4) associated with prognosis in the early stage of lung adenocarcinoma. However, predictiv...

Traumatically injured astrocytes release a proteomic signature modulated by STAT3-dependent cell survival.

Molecular markers associated with CNS injury are of diagnostic interest. Mechanical trauma generates cellular deformation associated with membrane permeability with unknown molecular consequences. We used an in vitro model of stretch-injury and proteomic analyses to determine protein changes in murine astrocytes and their surrounding fluids. Abrupt pressure-pulse stretching resulted in the rapid release of 59 astrocytic proteins with profiles reflecting cell injury and cell death, i.e., mechanoporation and cell lysis. This acute trauma-release proteome was overrepresented with metabolic proteins compared with the uninjured cellular proteome, bearing relevance for post-traumatic metabolic depression. Astrocyte-specific deletion of signal transducer and activator of transcription 3 (STAT3-CKO) resulted in reduced stretch-injury tolerance, elevated necrosis and increased protein release. Consistent with more lysed cells, more protein complexes, nuclear and transport proteins were released from STAT3-CKO versus nontransgenic astrocytes. STAT3-CKO astrocytes had reduced basal expression of GFAP, lactate dehydrogenase B (LDHB), aldolase C (ALDOC), and astrocytic phosphoprotein 15 (PEA15), and elevated levels of tropomyosin (TPM4) and α actinin 4 (ACTN4). Stretching caused STAT3-dependent cellular depletion of PEA15 and GFAP, and its filament disassembly in subpopulations of injured astrocytes. PEA15 and ALDOC signals were low in injured astrocytes acutely after mouse spinal cord crush injury and were robustly expressed in reactive astrocytes 1 day postinjury. In contrast, α crystallin (CRYAB) was present in acutely injured astrocytes, and absent from uninjured and reactive astrocytes, demonstrating novel marker differences among postinjury astrocytes. These findings reveal a proteomic signature of traumatically-injured astrocytes reflecting STAT3-dependent cellular survival with potential diagnostic value.

The biological role of actinin-4 (ACTN4) in malignant phenotypes of cancer.

Invasion and metastasis are malignant phenotypes in cancer that lead to patient death. Cell motility is involved in these processes. In 1998, we identified overexpression of the actin-bundling protein actinin-4 in several types of cancer. Protein expression of actinin-4 is closely associated with the invasive phenotypes of cancers. Actinin-4 is predominantly expressed in the cellular protrusions that stimulate the invasive phenotype in cancer cells and is essential for formation of cellular protrusions such as filopodia and lamellipodia. ACTN4 (gene name encoding actinin-4 protein) is located on human chromosome 19q. ACTN4 amplification is frequently observed in patients with carcinomas of the pancreas, ovary, lung, and salivary gland, and patients with ACTN4 amplifications have worse outcomes than patients without amplification. In addition, nuclear distribution of actinin-4 is frequently observed in small cell lung, breast, and ovarian cancer. Actinin-4, when expressed in cancer cell nuclei, functions as a transcriptional co-activator. In this review, we summarize recent developments regarding the biological roles of actinin-4 in cancer invasion.

Predictive Significance of Actinin-4 (ACTN4) Gene Expression in Early-Stage Non-Small Cell Lung Cancer (NSCLC): Early-Stage Non-Small Cell Lung Cancer

Predictive Significance of Actinin-4 (ACTN4) Gene Expression in Early-Stage Non-Small Cell Lung Cancer (NSCLC): Early-Stage Non-Small Cell Lung Cancer

Distinct outcome of stage I lung adenocarcinoma with ACTN4 cell motility gene amplification

BackgroundEven if detected at an early stage, a substantial number of lung cancers relapse after curative surgery. However, no method for distinguishing such tumors has yet been established. Patients and methodsThe copy number of the actinin-4 (ACTN4) gene was determined by fluorescence in situ hybridization on tissue microarrays comprising 543 surgically resected adenocarcinomas of the lung. ResultsAmplification (an increase in the copy number by ≥2.0 fold) of the ACTN4 gene was detected in two of seven lung adenocarcinoma cell lines and 79 (15%) of 543 cases of pathological stage I–IV lung adenocarcinoma. Multivariate analysis revealed that ACTN4 gene amplification was the most significant independent factor associated with an extremely high risk of death (hazard ratio, 6.78; P = 9.48 × 10-5, Cox regression analysis) among 290 patients with stage I lung adenocarcinoma. The prognostic significance of ACTN gene amplification was further validated in three independent cohorts totaling 1033 patients. ConclusionsAmplification of the ACTN4 gene defines a small but substantial subset of patients with stage I lung adenocarcinoma showing a distinct outcome. Such patients require intensive medical attention and might benefit from postoperative adjuvant chemotherapy.

1186P - Gene Amplification of ACTN4 in Lung Cancer: A Novel Prognostic Indicator for Stage I Adenocarcinoma of The Lung

ABSTRACT Background Even if detected at an early stage, a substantial number of lung cancers relapse after surgery. Patients with such tumors are likely to benefit from adjuvant therapy, but methods for identifying such patients have yet to be established. Methods We retrospectively analyzed multiple cohorts totaling 1744 patients who underwent resection of lung adenocarcinoma. Expression of actinin-4 protein in tumors was evaluated immunohistochemically, and copy numbers of the actinin-4 (ACTN4) gene were determined by fluorescence in situ hybridization. Results Amplification of the ACTN4 gene correlated significantly with smoking history (P = 0.02), pathological stage (P = 0.002), and histological differentiation (P Conclusions Amplification of the actinin-4 gene defines a subset of stage I lung adenocarcinoma with distinctly poor outcomes. Disclosure All authors have declared no conflicts of interest.

Abstract 695: Significant loss of nuclear expression of Actinin-4 in metastatic breast carcinoma and lymph nodes: A novel biomarker for metastatic breast carcinoma

Abstract Background: Actinin-4 is an actin cross-linked protein. The expression and subcellular distribution of actinin-4 (ACTN4) has been associated with multiple roles in tumorigenicity and cancer metastasis. In the present study, we developed a novel antibody against human actinin-4 and investigated the clinical utility of actinin-4 as a biomarker in metastatic breast cancer. Methods and findings: We generated a novel monoclonal antibody (AB-Actinin-4) using hybridoma technology and native ACTN4 protein as an immunogen. The antibody specifically recognized both native and denatured human Actinin-4 protein and displayed high affinity as assessed by ELISA, immunohistochemistry assay, western blot and flow cytometry. Using this antibody and ELISA as well as immunohistochemical techniques, we examined plasma levels and tissue expression of actinin-4 in healthy controls’ and breast cancer patients’ samples. 50 plasma samples from healthy controls and breast cancer patients were analyzed for the presence of soluble actinin-4. ELISA results showed significantly decreased actinin-4 levels in plasma samples from patients with breast carcinomas compared to healthy control plasma. A total of 156 cases, including 70 normal, 36 non-metastatic breast carcinoma, 50 breast carcinoma tissues with regional metastasis, and 50 matched metastatic lymph nodes were examined. The immunoreactivity of actinin-4 was located mainly in nucleus and cytoplasm of cells. To assess the difference between groups, the staining intensity and intracellular distribution were compared and the intensity in nucleus and cytoplasm was separately scored as 0, 1+, 2+ and 3+. Grades 0 and 1+ were defined as negative and 2+ and 3+ as positive. Significant differences between the groups were found in nuclear staining. 92% of normal and 89% of non-metastatic breast carcinoma samples displayed positive nuclear staining. In contrast, only 29% of metastatic lymph nodes and 51% of metastatic breast cancer tissues showed strong nuclear staining. Conclusion: Actinin-4 demonstrated significantly decreased expression in plasma from breast cancer patients. Metastasis leads to significant loss of the nuclear expression of Actinin-4 in breast cancer tissues, in comparison to normal and non-metastatic breast carcinoma tissues. Our data suggests that Actinin-4 might have significant impact in breast cancer metastasis. Actinin-4 may serve as a reliable biomarker for the diagnosis and prognosis of metastatic breast cancer as well as could be a therapeutic target. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 695. doi:1538-7445.AM2012-695

Actinin-4 gene amplification in ovarian cancer: a candidate oncogene associated with poor patient prognosis and tumor chemoresistance

Actinin-4, an isoform of non-muscular α-actinin, enhances cell motility by bundling the actin cytoskeleton. We previously reported a prognostic implication of high immunohistochemical expression of actinin-4 protein in ovarian cancers. Chromosomal gain or amplification of the 19q12–q13 region has been reported in ovarian cancer. We hypothesized that the actinin-4 (ACTN4) gene might be a target of the 19q12–q13 amplicon and play an essential role of ovarian cancer progression. In total, 136 advanced-stage ovarian cancers were investigated for the copy number of the ACTN4 gene on chromosome 19q13, using fluorescence in situ hybridization, and the correlation of the ACTN4 copy number with actinin-4 protein immunoreactivity and major clinicopathological factors was investigated. A higher copy number (≧4 copies) of the ACTN4 gene was detected in 29 (21%) cases and was highly associated with the intensity of actinin-4 immunoreactivity (P<0.0001), a high histological tumor grade (P=0.030), a clear-cell adenocarcinoma histology (P=0.012), resistance to first-line chemotherapies (P=0.028), and poor patient outcome (P=0.0011). Univariate analyses using the Cox regression model showed that a higher ACTN4 gene copy number was able to predict patient outcome more accurately than high actinin-4 immunoreactivity (relative risk: 2.48 vs 1.55). Multivariate analysis showed that a higher copy number of the ACTN4 gene and the degree of residual disease were independent prognostic factors for overall patient survival. The actinin-4 gene may be a target of the 19q amplicon, acting as a candidate oncogene, and serve as a predictor of poor outcome and tumor chemoresistance in patients with advanced-stage ovarian cancers.

α-Actinin-4 Is Selectively Required for Insulin-induced GLUT4 Translocation

Insulin induces GLUT4 translocation to the muscle cell surface. Using differential amino acid labeling and mass spectrometry, we observed insulin-dependent co-precipitation of actinin-4 (ACTN4) with GLUT4 (Foster, L. J., Rudich, A., Talior, I., Patel, N., Huang, X., Furtado, L. M., Bilan, P. J., Mann, M., and Klip, A. (2006) J. Proteome Res. 5, 64-75). ACTN4 links F-actin to membrane proteins, and actin dynamics are essential for GLUT4 translocation. We hypothesized that ACTN4 may contribute to insulin-regulated GLUT4 traffic. In L6 muscle cells insulin, but not platelet-derived growth factor, increased co-precipitation of ACTN4 with GLUT4. Small interfering RNA-mediated ACTN4 knockdown abolished the gain in surface-exposed GLUT4 elicited by insulin but not by platelet-derived growth factor, membrane depolarization, or mitochondrial uncoupling. In contrast, knockdown of alpha-actinin-1 (ACTN1) did not prevent GLUT4 translocation by insulin. GLUT4 colocalized with ACTN4 along the insulin-induced cortical actin mesh and ACTN4 knockdown prevented GLUT4-actin colocalization without impeding actin remodeling or Akt phosphorylation, maintaining GLUT4 in a tight perinuclear location. We propose that ACTN4 contributes to GLUT4 traffic, likely by tethering GLUT4 vesicles to the cortical actin cytoskeleton.

Expression and Gene Amplification of Actinin-4 in Invasive Ductal Carcinoma of the Pancreas

An invasive growth pattern is one of the hallmarks of pancreatic ductal carcinoma. Actinin-4 is an actin-binding protein associated with enhanced cell motility, invasive growth, and lymph node metastasis. Actinin-4 might play an important role in the development and progression of pancreatic cancer. The expression of actinin-4 was examined immunohistochemically in 173 cases of invasive pancreatic ductal carcinoma. The copy number of the actinin-4 (ACTN4) gene was calculated by fluorescence in situ hybridization. The expression of actinin-4 was stably knocked down by short hairpin RNA, and tumorigenicity was evaluated by orthotopic implantation into mice with severe combined immunodeficiency. The expression level of actinin-4 was increased in 109 (63.0%) of 173 cases of pancreatic cancer. Kaplan-Meier survival curves revealed that patients with increased expression of actinin-4 had a significantly poorer outcome (P=0.00001, log-rank test). Multivariate analysis by the Cox proportional hazard model showed that high expression of actinin-4 was the most significant independent negative predictor of survival (hazard ratio, 2.33; P=0.000009). Amplification (defined as more than four copies per interphase nucleus) of the ACTN4 gene was detected in 11 (37.9%) of 29 cases showing increased expression of actinin-4. Knockdown of actinin-4 expression inhibited the destructive growth of cancer cells in the pancreatic parenchyma. Recurrent amplification of chromosome 19q13.1-2 has been reported in pancreatic cancer, but the exact target gene has not been identified. Actinin-4 contributes to the invasive growth of pancreatic ductal carcinoma, and ACTN4 is one of the candidate oncogenes in this chromosome locus.

Ruffling with Rab5 and RN-tre

Lanzetti et al. investigated receptor tyrosine kinase (RTK)-mediated regulation of the actin cytoskeleton and discovered that Rab5, a small guanosine triphosphatase (GTPase) previously implicated in intracellular trafficking, is critical to circular ruffling (an RTK-dependent process that involves cytoskeletal remodeling). Expression of a Rab5 dominant-negative mutant in mouse embryo fibroblasts (MEFs) inhibited platelet-derived growth factor (PDGF)-mediated production of circular ruffles, without affecting edge ruffles (which depend on Ras), as did overexpression of the Rab5-specific GTPase-activating protein (GAP) RN-tre. Experiments in which MEFs were transfected with wild-type or mutant forms of Rab5, Ras, Rac, or phosphatidylinositol 3-kinase (PI3K) alone or in combination suggested that independent signals from Rab5, PI3K, and Rac were all required for circular ruffling. Mutational analysis indicated that suppression of circular ruffling (and the related process of macropinocytosis) by RN-tre did not depend solely on its GAP activity but also on a region in its C terminus: Only a mutant that lacked both GAP activity and this region failed to inhibit PDGF-dependent circular ruffling. The authors used mass spectrometry to show that actinin-4 (ACTN4), a nonmuscle isoform of α-actinin, interacted with this region of RN-tre. Endogenous RN-tre and ACTN4 exhibited PDGF-dependent coimmunoprecipitation, and PDGF-dependent ruffling was impaired in cells in which RN-tre or ACTN4 expression was inhibited through RNA interference. RN-tre interacted with F-actin through regions distinct from the region that bound ACTN4. Thus, Rab5 mediates RTK-dependent cytoskeletal signals involved in circular ruffling and RN-tre acts as a Rab5 effector--possibly by connecting Rab5, ACTN4 and F-actin--as well as a Rab5 GAP. L. Lanzetti, A. Palamidessi, L. Areces, G. Scita, P. P. Di Fiore, Rab5 is a signalling GTPase involved in actin remodelling by receptor tyrosine kinases. Nature 429 , 309-314 (2004). [Online Journal]