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Regulation of cell survival by the HIP-55 signaling network† Chengzhi Yang,‡a Zenggang Li,‡b Zhi Shi,c Kangmin He,a Aiju Tian,a Jimin Wu,a Youyi Zhanga and Zijian Li*a HIP-55 (hematopoietic progenitor kinase 1 [HPK1]-interacting protein of 55 kDa) is the mammalian homologue of the yeast Abp1p. It contains a C-terminal Src homology 3 domain and an N-terminal actin depolymerization factor (ADF-H/C) domain. HIP-55 appears to be critical for organ development and immune response and is important for the regulation of the actin cytoskeleton through its interactions with F-actin and various cytoskeletal and cell signaling proteins. However, the function of HIP-55 in tumors remains unknown. Here, we found that HIP-55 is up-regulated or down-regulated in several types of tumor tissues in patients. Of these, lung cancer tissues had the highest expression of HIP-55. To gain full insight into the function of HIP-55 in lung cancer, microarray assay was performed using Affymetrix U133 Plus 2.0 expression arrays in both HIP-55 knockdown and scramble control A549 cells. The ingenuity pathway analysis tool was utilized to construct biological networks and analyze functions that might be associated with HIP-55. Functional analysis strongly suggested that HIP-55 may

Received 4th December 2013, Accepted 9th March 2014

be involved in cancer cell survival and cell death, which was then confirmed by further experimentation.

DOI: 10.1039/c3mb70552h

apoptosis of A549 cells treated with the anticancer agent etoposide. Our data suggested that HIP-55

Experimental results showed that downregulation of HIP-55 decreased the viability and increased the may be a newly discovered regulatory node in the growth signaling network and a new target for

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therapeutic interventions in proliferative disorders.

Introduction HIP-55 is short for the [HPK1]-interacting protein of 55 kDa (also known as mAbp1 or SH3P7). It is a multi-domain adaptor protein with an actin-binding domain at its N-terminus and an SH3 domain at its C-terminus. HIP-55 acts as an adaptor protein in many cellular processes such as cell signaling and endocytosis.1,2 HIP-55 functions as an actin-binding adaptor protein binding to F-actin but is not involved in actin polymerization, capping or bundling.1 Its association with dynamin suggests that it may connect dynamin to the actin cytoskeleton during the endocytic function. By interacting with dynamin, a a

Institute of Vascular Medicine, Peking University Third Hospital, Key Laboratory of Cardiovascular Molecular Biology and Regulatory Peptides, Ministry of Health, Key Laboratory of Molecular Cardiovascular Sciences, Ministry of Education and Beijing Key Laboratory of Cardiovasicular Receptors Research, Beijing 100191, China. E-mail: [email protected]; Fax: +86-10-82265519; Tel: +86-10-82265519 b Department of Pharmacology, Rollin Research Center, Emory University, Atlanta, GA, 30322, USA c Department of Cell Biology & Institute of Biomedicine, College of Life Science and Technology, Jinan University, Guangzhou, Guangdong 510632, China † Electronic supplementary information (ESI) available. See DOI: 10.1039/ c3mb70552h ‡ These authors contributed equally.

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GTPase involved in endocytosis, HIP-55 could functionally link the actin cytoskeleton to dynamin during endocytosis.3 HIP-55 is a key novel component of the immunological synapse that modulates T cell activation by connecting the actin cytoskeleton to TCRs for gene activation and endocytic processes.4 HIP-55 knockout T cells displayed defective T-cell proliferation, decreased cytokine production, and decreased up-regulation of the activation markers induced by TCR stimulation.4 HIP-55 interacts with HPK1 and regulates HPK1 and c-Jun N-terminal kinase (JNK), which are two important lymphocyte signaling molecules.5 In addition to binding to HPK1, HIP-55 might negatively regulate TCR signaling through down-regulation of TCR expression.6 HIP-55 can also interact with ZAP-70, a critical protein-tyrosine kinase in TCR signaling as this interaction was induced by TCR signaling.7 Thus, these results demonstrate the importance of HIP-55 as an adaptor protein in the TCR signaling and immune system function. Recent expression profiling studies pointed to a possible association of HIP-55 with tumor. HIP-55 is highly expressed in patients with squamous cervical cancer and in patients with pancreatic cancer lymph node metastasis.8,9 HIP-55 may also be a potential molecular marker in head and neck cancer.10 These findings suggest that HIP-55 could play a role in tumor development. To study the functions of HIP-55 in tumor, the expression of HIP-55 was first assayed in patient’s tumor

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tissues. HIP-55 was found to be upregulated in twelve kinds of patient’s tumor tissues. These data raise the possibility that overexpression of HIP-55 may contribute to tumorigenesis. Because the highest expression of HIP-55 was found in lung cancer tissues, HIP-55 knockdown (KD) A549 lung cancer cells were established and a microarray assay was used to analyze the potential regulatory function of HIP-55. To better understand the biological significance of the obtained microarray data, we utilized the ingenuity pathway analysis tool to construct biological networks and analyze the function and pathways that might associate with HIP-55. Functional analysis suggested that HIP-55 may be involved in cancer cell survival and cell death, which was then confirmed by further experimentation. The results showed that HIP-55 enhanced the viability and decreased the apoptosis of A549 cells treated with etoposide (Eposin, Etopophos, Vepesid, and VP-16). Our study suggests a possible role of HIP-55 in promoting lung cancer cell survival and, importantly, identifies a novel target for anticancer therapeutic intervention.

Material and methods Cell culture The human non-small cell lung cancer (NSCLC) cell line A549 was maintained in DMEM with 10% fetal bovine serum and 100 units of penicillin–streptomycin at 37 1C under 5% CO2 atmosphere in a humidified incubator. Generation of HIP-55 KD stable cell lines Recombinant retroviruses carrying HIP-55 shRNA were prepared using pSilencer 5.1 (Ambion). The sequence for HIP-55 was designed as AACAGTGAACGTAGAGAATTG. To generate HIP-55-silenced stable cell lines, infected cells were selected in the presence of puromycin (1.25 mg ml1). Drug-resistant clones were collected, pooled, and expanded. The HIP-55 level was verified for each experiment. Western blotting Proteins were detected using West-Pico or West-Dura enhanced chemiluminescent detection reagents (Pierce) and a Kodak imaging system or films. Global gene expression analysis Total RNA was extracted using Trizol reagent and then was amplified, labeled and purified by using a GeneChip 3 0 IVT Express Kit (Cat#901229, Affymetrix, Santa Clara, CA, USA) following the manufacturer’s instructions to obtain biotin labeled cRNA. Ten micrograms of biotin-labelled cRNA (antisense RNA) was hybridized with a GeneChip Human U133 plus2.0. Array (Affymetrix). After hybridization and washing, the intensity of the fluorescence of the array chips was scanned using a Affymetrix GeneChips Scanner 3000. Raw data were normalized using the MAS 5.0 algorithm, Gene Spring Software 11.0 (Agilent technologies).

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Biological functions and network analysis The biological network data were generated through the use of the Ingenuity Pathway Analysis (IPA) software (www.ingenuity. com), a web-delivered application that evaluates biological networks. A data set of proteins from A549 cells was uploaded into IPA. Each protein identifier was mapped to its corresponding protein object in the Ingenuity Pathways Knowledge Base (IPKB). These proteins were then used as the starting point for generating biological networks based upon the identities of the focus proteins and interactions with genes–proteins that were reported in the literature. IPA calculated a significance score for each network. The score was generated using a p-value calculation and was displayed as the negative log of that p-value, indicating the likelihood that the assembly of a set of focus proteins in a network could be explained by random chance alone. A score of 2 indicates that there is a 1 in 100 chance that the focus proteins are together in a network due to random chance. Therefore, networks with scores of 2 or higher have at least 99% confidence of not being generated by random chance alone. Biological functions or canonical pathways were then calculated and assigned to each network by using findings that had been extracted from the scientific literature and stored in the IPKB. The biological function assigned to each network was ranked according to the significance of that biological function to the network. Cell viability assay A549 cells were incubated on 96-well plates (4  103 cells per well) with etoposide at the indicated concentrations for 24 h. The cell viability was then analyzed using a CCK-8 assay (DOJINDO LABORATORISE, Japan) and measured using a microplate reader at an absorbance of 450 nm. The cell viability was calculated as % of control using the following formula: number of treated cells/number of control cells  100%. Cell apoptosis assay For apoptosis analysis, cells were harvested and washed with PBS, then stained using 7-AAD and Annexin5-PE. Samples were analyzed using Guava flow cytometer systems (Millipore). Immunohistochemistry (IHC) analysis Slides (AccuMax A701 VI) were stained using an anti-HIP55 antibody (BD126146, 1 : 300). The staining was performed manually using a VECTASTAIN Kit (Vector Laboratories, Inc. CA, USA) in the following manner. Slides were deparaffinized using xylene and graded ethyl alcohol and then rinsed in water. Antigen retrieval was performed by boiling the slides in 0.01 M citrate buffer in a microwave oven at maximum power for 6 minutes and at half-maximum power for 10 minutes, followed by a 30 minute cooling at room temperature and rinsed in water. The slides were then subjected to treatment with 0.05% Triton-X100 in PBS (PBS-T) for 5 minutes and washed with PBS, followed by sequential treatment with the following reagents in a humidified chamber after quenching endogenous peroxides with 3% H2O2 in MeOH: blocking serum with avidin for 20 minutes at room temperature, anti-HIP-55

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antibody overnight at 40 1C, secondary antibody for 20 minutes at room temperature, hydrogen peroxidase for 15 minutes at room temperature, and peroxidase substrate solution for 20 minutes at room temperature. Wash buffer (PBS-T) steps were included between each step. The stained slides were then counterstained with hematoxylin and coverslipped. Statistical analysis Student’s t-test was used to compare individual data points among each group. The significance was determined at p o 0.05.

Results HIP-55 was up-regulated in cancer patient’s specimens To date, it is still unknown whether HIP-55 plays a role in tumor development. To study the functions of HIP-55 in tumor development, the expression of HIP-55 was first assayed in tumor and normal tissues. HIP-55 was found by immunohistochemistry staining to be up-regulated in six types of tumor tissues (including brain, skin, esophagus, lung, thyroid and cervix) and down-regulated in breast tumor tissues when compared with the corresponding normal tissues from the same patient. The highest expression was identified in lung cancer patient’s specimens (Fig. 1). These data raise the possibility that overexpression of HIP-55 may play a role in tumor, especially in lung tumor. The establishment of a stable HIP-55 knock-down A549 cell line To investigate the function of HIP-55 in lung cancer, we used RNAi to reduce cellular levels. The stable HIP-55 stable knockdown A549 cell line was established expressing distinct shRNA

Fig. 2 Expression of HIP-55 in A549 scramble (SCR) cells and HIP-55 knockdown (KD) cells. (A) A549 scramble cells and HIP-55 KD cell lines were grown in the presence of serum and lysed for Western blot analysis. Expression of HIP-55 was determined by the HIP-55 antibody from BD Biosciences (BD: 612614). Actin was used as a loading control. (B) Quantification of HIP-55 expression fold change was analyzed as mean  SD of three independent Western blot experiments. (C) A549 scramble cell and HIP-55 KD cell lines were grown in the presence of serum and immunofluorescence analysis was performed using HIP-55 antibody from Santa Cruz (sc-79975). (D) Quantification of HIP-55 expression fold change was analyzed as mean  SD of three independent immunofluorescence experiments.

that targets the sequence in the 3 0 -noncoding region of HIP-55 mRNA. Compared with A549 cells harboring a scramble control shRNA (SCR), knock down cells showed significantly diminished HIP-55 protein expression (Fig. 2A). The effectiveness of knock down was about 90% (Fig. 2B). Similar results were obtained

Fig. 1 Expression of HIP-55 in a tissue array is shown. Immunohistochemistry (IHC) was performed on a tissue array with different paired normal and tumor tissue samples (AccuMax) using HIP55 antibody (BD). (A) Magnification: 4 and 10. (B) Summary of HIP55 expression in different tumor tissues based on the tissue array IHC results.

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from HIP-55 immunohistochemistry detection by laser scanning confocal microscopy (Fig. 2C and D).

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Gene expression changes and functional analysis associated with HIP-55 in A549 cells To identify tumor gene expression associated with the acquired HIP-55 protein, we performed mRNA microarray analysis to compare the control and HIP-55-knockdown A549 cells using Affymetrix U133 Plus 2.0 expression arrays. According to the statistical criteria described in the methods, we observed significant changes in the expression of genes involved in HIP-55-knockdown A549 cells (Table S1, ESI†). We next sought to identify the major bio-functional categories associated with HIP-55 deficiency. High-level functions were calculated by Ingenuity Pathway Analysis (IPA) (Fig. 3). The top three scores of diseases and disorders were cancer, immunological disease and inflammatory response and the top three scores of molecular and cellular functions were cellular growth and proliferation, cellular development and cellular movement. Together, these data suggest that HIP-55 could play an important role in the growth and proliferation and HIP-55 dysfunction could be involved in tumorigenesis. Data network analysis and visualization In order to further explore how the gene expression changes in HIP-55-knockdown A549 cells were related, we used the IPA program to analyze data from different genes. The IPA program analyzes a large genomic data set to find the most significant networks and canonical pathways relevant to the data set based on a calculated probability score. As described in the methods, networks with scores of 2 or higher have at least 99% confidence of not being generated by random chance alone.

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Therefore, based on the computed scores, the top two direct networks with scores of 44 were found to be significant in the database (Fig. 4A and B). In addition, high-level functions were calculated and assigned to each direct network if the significance of the association between the network and the biological function had p o 0.01. The functions displayed in Fig. 4C represent the top 3 high-level functions from the local analysis, which provides an overview of the biological functions associated with a given network ((1), network1; (2), network2). The analysis showed that HIP-55 regulates two major functional networks that control cell death, suggesting a critical role of HIP-55 in tumor survival. HIP-55 promoted tumor survival by inhibiting etoposide induced apoptosis Biological network and functional analysis suggested that HIP-55 could play an important role in tumor survival. To validate the microarray findings, we assessed the effects of HIP-55 knockdown on A549 cell viability when exposed to etoposide, an anticancer (‘‘antineoplastic’’ or ‘‘cytotoxic’’) chemotherapy commonly used in clinics. The results show that loss of function of HIP-55 significantly decreased the viability of A549 cells (Fig. 5A). One critical anti-tumor effect of etoposide is to induce tumor apoptosis. To test the effect of HIP-55 on apoptosis induced by etoposide, flow cytometry was used to measure cell apoptosis. The results show that knockdown of HIP-55 resulted in a significant increase of cell apoptosis (Fig. 5B and C). In order to further confirm these results, cleavage of caspase-3 (an apoptotic marker) was tested. The cleavage of caspase-3 was significantly higher in HIP-55 knockdown cells than the scramble cells (Fig. 5D and E). These results suggest a critical role of HIP55 in the inhibition of apoptosis induced by etoposide.

Fig. 3 Functional analysis of down-regulation of HIP-55 in A549 cells. In the top panel, top ten high-level functions associated with down-regulation of HIP-55 are displayed in a bar graph format as shown by IPA. In the bottom panel, details of diseases and disorders/molecular and cellular functional categories also appear in a list format.

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Fig. 4 Functional network analysis of the differentially expressed genes associated with down-regulation of HIP-55. (A and B) The score of the top two networkswas 44 (networks 1 and 2). Nodes represent genes, with their shape indicating the functional class of the gene, and multiple edges indicating the biological relationships between the nodes. (C) High-level functions are analyzed for the network.

Discussion Lung cancer is a leading cause of cancer-related death worldwide, resulting in more than 1.3 million deaths per year.11,12 Although improvements in molecular diagnostics and targeted therapies were achieved in recent several decades, the average 5 year survival rate for lung cancer is still below 20%.13–15 New targets are eagerly needed for this disease. HIP-55 is an actin binding protein that plays a novel role in T-cell regulation. Our results suggested the potential ability of HIP55 to be a target for lung cancer therapeutics. Firstly HIP-55 was found to be highly upregulated in tumor tissues from different cancer patients while not in the adjacent normal tissues by an IHC-based survey using an anti-HIP55 specific antibody. These results indicate that many cancers highly express the HIP 55 protein, regardless of the tumor type. Further shRNA based RNAi experiments in the lung cancer adenocarcinoma cell line A549 revealed that down-regulation of HIP-55 significantly sensitizes this type of cells to the commonly used chemotherapy reagent etoposide. This is the first report of a functional relationship between HIP-55 expression and cancer. Microarray technology has been used to study different types of cancer16–19 and is a useful tool to profile gene expression patterns that can facilitate diagnosis, predict response to therapy, find new biomarkers and examine the development of drug resistance in cancer.20–23 Recent expression profiling studies from other groups show that HIP-55 is highly expressed in squamous cervical cancer patients and in pancreatic cancer

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with lymph node metastasis.8,9 Our results reveal that HIP-55 is up-regulated or down-regulated in several types of patient tumor tissues, including the brain, skin, esophagus, lung, thyroid, breast and cervix. These results suggest a broad role of HIP-55 across different kinds of cancer types. Since HIP-55 is a F-actin binding protein and F-actin was reported to play critical roles in tumorigenesis and metastasis, whether the effect of HIP-55 on tumor development is mediated through F-actin needs to be addressed further. Microarray data from HIP-55 knockdown cells indicate a close relationship of HIP-55 with immunological disease, which is consistent with the known function of HIP-55 in T-cell proliferation. We also found an even closer relationship of HIP-55 with cancer than immunological disease in this ingenuity pathway analysis. These results suggest that HIP-55 may play more important roles in cancer development than in immunological disease. We also found that HIP-55 expression correlates with inflammatory response, organismal injury and abnormalities and renal and urological diseases. However, these have a much weaker relationship when compared with cancer and immunological disease. We performed pathway analysis using these microarray data and found that HIP-55 was involved in multiple cell signaling pathways including cellular growth and proliferation, cellular development, cellular movement and lipid metabolism pathways. Some of these cell processes and signaling pathways are closely related to actin

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Fig. 5 Effect of HIP-55 on A549 cell survival treated with the chemotherapy reagent etoposide. A549 scramble (SCR) cells and HIP-55 knockdown (KD) cells were treated with etoposide. (A) Cell viability was assayed 24 h after treatment with different doses of etoposide by the CCK-8 assay. (B) Apoptotic cells were determined by flow cytometric analysis. (C) Quantification of apoptotic cells was determined by flow cytometric analysis. (D) Whole-cell lysates were harvested for Western blot analysis of cleaved caspase-3. (E) Quantification of cleaved caspase-3 standardized to actin.

functions or T-cell functions such as cellular movement or inflammatory response, respectively. These results are consistent with the known HIP-55 functions. We also found cell processes and signaling pathways such as organismal injury and abnormalities, cellular growth, proliferation and cellular development which are not directly related to the known HIP-55 function. These results suggest the existence of novel functions of HIP-55 that may drive the role of this protein in cancer development. In conclusion, we found dysregulation of HIP-55 to be associated with tumor survival and have analyzed changes in genes that occur following the down-regulation of HIP-55 in A549 cells. Global gene profiling and computational-specific regulatory network and functional analyses provide both global and specific information associated with HIP-55 function in lung cancer, which we verified experimentally. These results presented here may provide clues to elucidate the mechanism of HIP-55 in cancer and to discover novel diagnostic markers. Our study also provides the basis for the development of new therapeutic strategies for lung and other cancers.

Disclosure statement The authors have no conflict of interest.

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Acknowledgements We sincerely thank Dr Haian Fu from Emory University for scientific advice, experimental assistance, and providing reagents. This work was supported by grants from the National Natural Science Foundation of China (No. 81070078 and 81270157), the National Basic Research Program of China (Grant no. 2011CB503903) and Beijing Municipal Natural Science Foundation (7102158). This work was supported in part by Emory Global Health Institute.

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Regulation of cell survival by the HIP-55 signaling network.

HIP-55 (hematopoietic progenitor kinase 1 [HPK1]-interacting protein of 55 kDa) is the mammalian homologue of the yeast Abp1p. It contains a C-termina...
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