Prolactin-Induced Protein Is Required for Cell Cycle ... - Neoplasia

Volume 16 Number 4

April 2014

pp. 329–342.e14

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Prolactin-Induced Protein Is Required for Cell Cycle Progression in Breast Cancer1,2

Ali Naderi and Marion Vanneste Holden Comprehensive Cancer Center, Medical Education and Research Facility, University of Iowa, Iowa City, IA, USA

Abstract Prolactin-induced protein (PIP) is expressed in the majority of breast cancers and is used for the diagnostic evaluation of this disease as a characteristic biomarker; however, the molecular mechanisms of PIP function in breast cancer have remained largely unknown. In this study, we carried out a comprehensive investigation of PIP function using PIP silencing in a broad group of breast cancer cell lines, analysis of expression microarray data, proteomic analysis using mass spectrometry, and biomarker studies on breast tumors. We demonstrated that PIP is required for the progression through G1 phase, mitosis, and cytokinesis in luminal A, luminal B, and molecular apocrine breast cancer cells. In addition, PIP expression is associated with a transcriptional signature enriched with cell cycle genes and regulates key genes in this process including cyclin D1, cyclin B1, BUB1, and forkhead box M1 (FOXM1). It is notable that defects in mitotic transition and cytokinesis following PIP silencing are accompanied by an increase in aneuploidy of breast cancer cells. Importantly, we have identified novel PIP-binding partners in breast cancer and shown that PIP binds to β-tubulin and is necessary for microtubule polymerization. Furthermore, PIP interacts with actin-binding proteins including Arp2/3 and is needed for inside-out activation of integrin-β1 mediated through talin. This study suggests that PIP is required for cell cycle progression in breast cancer and provides a rationale for exploring PIP inhibition as a therapeutic approach in breast cancer that can potentially target microtubule polymerization. Neoplasia (2014) 16, 329–342.e14

Introduction Prolactin-induced protein (PIP) is widely expressed in breast cancer and has been used as a characteristic biomarker for the diagnostic evaluation of this disease [1]. Genomic studies have revealed that PIP is highly expressed in luminal A and molecular apocrine subtypes of breast cancer [2–4]. Molecular apocrine is a subtype of estrogen receptor (ER)–negative breast cancer that is characterized by the overexpression of steroid response genes such as androgen receptor (AR) and forkhead box A1 (FOXA1) [3,5,6]. Notably, a recent study has shown that PIP is one of the best biomarkers for the immunohistochemical identification of molecular apocrine tumors [7]. It is known that PIP expression is regulated by prolactin and androgen hormones [8]. In particular, AR engages in a transcriptional cooperation with prolactin-activated Stat5 and Runx2 to regulate PIP expression [8,9]. In addition, we have demonstrated that PIP is a cAMP responsive element binding protein 1 (CREB1) target gene that is induced by a positive feedback loop between AR and extracellular signal-regulated kinase (ERK) [10]. There is limited knowledge regarding the molecular function and binding partners of PIP in breast cancer. The available data indicate that PIP is a secreted protein with aspartyl protease activity that can degrade fibronectin and has the ability to bind and modulate CD4

receptor in T lymphocytes [11,12]. In addition, early studies using actin-sepharose columns have shown possible binding of PIP to actin in seminal fluid [13]; however, this finding has not been validated using more modern proteomic methods. Furthermore, the importance of PIP in cell proliferation has been demonstrated by the fact that purified PIP promotes growth of breast cancer cells and PIP expression is necessary for the proliferation of T-47D and MDA-MB-453 cell lines [9,10,14].

Address all correspondence to: Ali Naderi, MD, Holden Comprehensive Cancer Center, 3202 Medical Education and Research Facility, University of Iowa, 375 Newton Road, Iowa City, IA 52242. E-mail: [email protected] 1 Research reported in this publication was supported by the Holden Comprehensive Cancer Center at the University of Iowa and the National Cancer Institute of the National Institutes of Health under Award No. P30CA086862. Authors have no conflict of interests to disclose. 2 This article refers to supplementary materials, which are designated by Tables W1 to W9 and Figures W1 and W2 and are available online at www.neoplasia.com. Received 16 January 2014; Revised 6 March 2014; Accepted 24 March 2014 Copyright © 2014 Neoplasia Press, Inc. All rights reserved 1476-5586/14 http://dx.doi.org/10.1016/j.neo.2014.04.001

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Moreover, we have recently demonstrated that PIP mediates invasion of breast cancer cells in a process that partially depends on the degradation of fibronectin by this protein [10]. It is notable that the extracellular effects of PIP on fibronectin degradation are necessary for the outside-in activation of integrin-β1, which, in addition to the regulation of invasion, has a role in promoting cell proliferation [10]. Furthermore, it has been shown that coculture of PIP-silenced and naive T-47D cells does not reverse the growth inhibition induced by PIP silencing, which suggests a potential intracellular function for this protein [4]. Despite these findings, the underlying molecular mechanisms of PIP function in cell proliferation have remained largely unknown and require further studies. In this study, we investigated PIP function in breast cancer using small interfering RNA (siRNA) silencing in a broad group of breast cancer cell lines, analysis of expression microarray data, proteomic analysis by mass spectrometry (MS), and biomarker studies on primary breast tumors. We demonstrated that PIP is required for the progression through different phases of cell cycle and identified key molecular mechanisms and binding partners for this protein in breast cancer. Materials and Methods

Cell Culture Breast cancer cell lines MCF-7, T-47D, BT-474, HCC-202, HCC-1954, MDA-MB-453, SK-BR-3, MFM-223, and MDA-MB231 were obtained from American Type Culture Collection (Manassas, VA) and cultured as recommended by the provider.

RNA Interference PIP knockdown (KD) by siRNA silencing was performed as described before [15]. The following two siRNA-duplex oligos (Sigma-Aldrich, St Louis, MO) were applied: duplex 1—sense, 5′ CUCUACAAGGUGCAUUUAA and antisense, 5′UUAAAUGCACCUUGUAGAG; and duplex 2—sense, 5′CCUCUACAAGGUGCAUUUA and antisense, 5′UAAAUGCACCUUGUAGAGG. Transfections with siRNA Universal Negative Control No. 1 (SigmaAldrich) were used as controls. The effect of KD was assessed 72 hours after transfections. The average changes obtained for two duplexes are presented in manuscript.

Quantitative Real-Time Reverse Transcription–Polymerase Chain Reaction Quantitative real-time reverse transcription–polymerase chain reaction (qRT-PCR) to assess the expression levels of PIP (assay ID: Hs00160082_m1), cyclin D1 (Hs00765553_m1), cyclin E1 (Hs01026536_m1), cyclin B1 (Hs01565448_g1), forkhead box M1 (FOXM1) (Hs01073586_m1), TTK (Hs01009870_m1), BUB1 (Hs01557695_m1), and cell division cycle 20 ( CDC20) (Hs00426680_mH) was carried out using TaqMan Gene Expression Assays (Applied Biosystems, Grand Island, NY). Housekeeping gene ribosomal protein, large, P0 (RPLP0) was used as a control. Relative gene expression = gene expression in the KD group / average gene expression in the control group.

Western Blot Analysis Rabbit monoclonal PIP antibody (Novus Biologicals, Littleton, CO); rabbit antibodies for ERK1/2, phospho-ERK1/2 (Thr 202/ Tyr 204), c-Jun, phospho–c-Jun (Ser 63), Stat3, phospho-Stat3 (Try 705), Cdc2, phospho-Cdc2 (Tyr 15), focal adhesion kinase (FAK), and Talin-1 (Cell Signaling Technology, Danvers, MA); rabbit polyclonal integrin-β1

Neoplasia Vol. 16, No. 4, 2014

and mouse monoclonal Arp2/3 antibodies (Millipore, Billerica, MA); rabbit monoclonal phospho-FAK (Tyr 397) antibody (Life Technologies, Grand Island, NY); and mouse monoclonal β-tubulin antibody (SigmaAldrich) were applied at 1:1000 dilutions using 20 μg of each cell lysate. Rabbit α-tubulin antibody (Abcam, Cambridge, United Kingdom) was applied to assess loading. To extract protein from media, cell lines were cultured for 48 hours in serum-free media, followed by concentration using Amicon Ultra-15 (3 K) centrifugal filters (Millipore). A total of 100 μg from each concentrated sample was precipitated and used for immunoblot analysis.

Cell Proliferation Assay Cell proliferation assays were performed using Vybrant MTT Proliferation Assay Kit (Life Technologies) in eight replicates as previously published [10].

Cell Cycle Analysis Cell cycle analysis with propidium iodide was performed as described before [16]. Data analysis was carried using ModFit LT software (Verity Software House, Topsham, ME).

Immunohistochemistry Three sets of breast cancer tissue microarray (TMA) slides that are constituted of duplicate cores for a total of 210 malignant breast tumors (BRC1501-3) were obtained from Pantomics (Richmond, CA). Immunohistochemistry (IHC) staining was performed as described before [17]. Staining was carried out with rabbit PIP antibody at 1:100 dilution and mouse monoclonal antibodies (Dako, Carpinteria, CA) for AR (1:75 dilution), Ki-67 (1:100 dilution), and cytokeratin 5/6 (1:100 dilution). PIP score was defined as the percentage of PIP-positive cells (0-100) multiplied by the intensity of PIP cytoplasmic staining (1-3).

Immunofluorescence Immunofluorescence (IF) staining was performed as previously described [15,17], with mouse monoclonal β-actin and α-tubulin antibodies (Abcam) at 1:200 dilution, mitotic protein monoclonal 2 (MPM-2; Abcam) at 1:500 dilution, and rabbit polyclonal pericentrin antibody (Abcam) at 1:1000. Alexa 488 anti-mouse and Alexa 594 anti-rabbit (Life Technologies) were used as secondary antibodies. Quantification of pericentrin/nuclear ratio was performed on 100 nuclei, and experiments were carried out in duplicates. Percentage of multinucleated cells and percentage of cells stained with MPM-2 were assessed in PIP-KD and control experiments on 200 cells, and each experiment was carried out in four replicates.

Immunoprecipitation Immunoprecipitation (IP) of integrin-β1 using CHAPS buffer was performed as previously published [10]. To perform PIP-IP, T-47D cells were grown in 10-cm dishes to 60% confluency in full media and further cultured in serum-free media containing 100 nM dihydrotestosterone, (Thermo Fisher Scientific, Waltham, MA) for 48 hours. Conditioned media were then concentrated with Amicon Ultra-15 filters, and volume was adjusted to 500 μl with a nondenaturing lysis buffer containing 20 mM Tris-HCl (pH 8), 137 mM NaCl, 10% glycerol, 2 mM EDTA, and 1% NP-40. Cell lysate from each dish was extracted using 500 μl of lysis buffer. Subsequently, the extracted medium and lysate for each sample were mixed and subjected to IP as described [10]. PIP-IP was carried out using 2 μg of rabbit monoclonal PIP antibody, and control experiments followed the same process without a PIP antibody.



Proteomics Following PIP-IP, protein bands were separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie R-250 staining (Bio-Rad Laboratories, Hercules, CA).

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Control experiments included pulldown with Protein A-Sepharose beads (Life Technologies) without a PIP antibody. Processing of gels for MS and database search were carried out by the Proteomics Facility at the University of Iowa (Iowa City, IA). In summary, each PIP-IP and

Figure 1. PIP silencing in breast cancer cell lines and the effect of PIP expression on cell proliferation. (A and B) qRT-PCR demonstrates PIP-KD efficiencies with siRNA-duplex 1 (D1) and siRNA-duplex 2 (D2). PIP expression is relative to nontargeting siRNA (CTL). (C) Immunoblot analysis shows PIP protein following PIP-KD using cell extracts or (D) conditioned media. Fold changes (RR) in band density were measured relative to the control and represent the average change for two siRNA duplexes. (E and F) MTT assay measures cell proliferation following PIP-KD. The asterisk (*) is P value for each PIP-KD versus control (CTL).

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control IP lane was sliced to 16 separate bands and analyzed by MS. Ingel digestion and sample processing were carried out as described before [18]. Database searching was performed by Mascot search engine (Matrix Science, Boston, MA), Spectrum Mill MS Proteomics Workbench (Agilent Technologies, Santa Clara, CA), and X! Tandem [The Global Proteome Machine Organization (The GPM), thegpm. org; version CYCLONE (2010.12.01.1)]. Scaffold (Proteome Software Inc, Portland, OR) was used to validate MS/MS-based peptide and protein identifications. Protein identifications were accepted at greater than 99.0% probability with at least four identified peptides [19]. Two replicates of PIP-IP and control experiments were analyzed by MS, and only hits that were present in both PIP-IP replicates and absent in the controls were accepted for further analysis.

Tubulin Polymerization Assay Quantitation of polymerized and soluble tubulin was carried out as described before [20]. Immunoblot analysis was performed using mouse monoclonal β-tubulin antibody and the band intensities of polymerized and soluble β-tubulin in each PIP-KD experiment were normalized to that of control siRNA.

Bioinformatics and Statistical Analysis Analysis of Gene Expression Data. Gene expression for 52 breast cancer cell lines was extracted from published microarray data by Neve et al. [21]. PIP transcriptional signature included genes that showed Pearson correlation coefficients (CCs) ≥ 0.5 with PIP expression (P b .001). Pearson CC analysis, proximity matrix, and clustering algorithms were performed using IBM SPSS Statistics 20 (Armonk, NY). Hierarchical


clustering of the PIP signature was carried out using centroid linkage method, and intervals were measured by CC values. Functional annotation of the PIP signature based on Gene Ontology was performed using The Database for Annotation, Visualization and Integrated Discovery (DAVID) Bioinformatics Resources (National Institute of Allergy and Infectious Diseases, Bethesda, MD) [22,23]. Analysis of Proteomics Data. Functional classification of PIPbinding partners was carried out using DAVID Bioinformatics Resources. The following parameters were used for the analysis: κ similarity overlap = 4, similarity threshold = 0.35, and multiple linkage threshold = 0.50. Enrichment score was obtained for each functional cluster. Canonical pathways associated with PIP-binding partners were derived using Ingenuity Pathway Analysis (Ingenuity Systems, Redwood City, CA). Statistical Analysis. Biostatistics was carried out using IBM SPSS Statistics 20. Student's t test and paired sample t test were applied for calculating the statistical significance. All error bars depict ± 2 SEM. Results

PIP Expression Is Necessary for Cell Proliferation We first characterized PIP expression in nine breast cancer cell lines from different molecular subtypes (Table W1). These included luminal A lines MCF-7 and T-47D, luminal B line BT-474, ERnegative luminal lines MDA-MB-453, HCC-202, MFM-223, and SK-BR-3, and ER-negative basal lines HCC-1954 and MDA-MB231. PIP expression using qRT-PCR was high in HCC-202, T-47D, MDA-MB-453, and HCC-1954 cells, intermediate in BT-474,

Figure 2. PIP expression in breast tumors. (A and B) PIP expression using IHC in breast tumors. Magnifications are at 10X. (C) PIP expression scores using IHC in molecular subtypes of breast cancer is shown. *P b .01 is for ER −/AR − versus other groups. (D) PIP-IHC scores in ER-negative tumors stratified on the basis of AR and CK5/6 status are shown. *P b .01 is for AR − tumors versus other groups.



MFM-223, and SK-BR-3 cell lines, and very low to undetectable in MCF-7 and MDA-MB-231 cells (Table W1 and Figure W1A). Notably, PIP-KD resulted in ≥ 80% reduction in PIP transcripts across seven cell lines with measurable PIP (Figure 1, A and B). In addition, PIP-KD was validated at the protein level using cell extracts in cell lines with high PIP expression (Figure 1C). In cell lines with an intermediate PIP expression, due to low levels of cellular PIP (Figure W1B), conditioned media were used to measure secreted PIP levels (Figure 1D). Importantly, all seven cell lines with measurable PIP levels showed a marked reduction of this protein following PIP-KD by ≥ 80% (Figure 1, C and D). We next examined the effect of PIP silencing on the proliferation of breast cancer cells using MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay and observed a significant reduction in cell proliferation following PIP-KD in T-47D, BT-474, MFM-223, HCC-1954, HCC-202, and SK-BR-3 cell lines (P b .01-.03; Figure 1, E and F). These results are similar to the effect of PIP silencing on MDA-MB-453 cells [10] and suggest that PIP expression is necessary for cell proliferation across different molecular subtypes of breast cancer.

PIP Expression in Molecular Subtypes of Breast Cancer We next investigated PIP expression in primary breast tumors to identify the pattern of PIP expression in various molecular subtypes of breast cancer and to study the association of biomarkers with PIP expression. In this process, we carried out IHC in a TMA cohort of 210 primary breast tumors (Table W2). To classify the cohort into established molecular subtypes, ER-positive tumors were subdivided into luminal A and luminal B groups using ErbB2 and Ki-67 expression patterns [24]. On the basis of this classification, luminal B was defined as ER-positive tumors with either ErbB2 overexpression or a Ki-67 index ≥ 14% (Table W3). In addition, ER-negative tumors were subdivided on the basis of their AR status [25], and cytokeratin 5/6 (CK5/6) staining

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was employed as a marker of basal-type tumors. Furthermore, we obtained IHC scores for PIP on the basis of the percentage and intensity of staining for this protein in each tumor (Figure 2, A and B). Next, we assessed the association of PIP expression with biomarkers and molecular subtypes. We observed that AR-positive tumors have a significantly higher PIP expression compared to AR negatives (P b .01; Table W3). In addition, PIP was highly expressed in luminal A, luminal B, and ER −/AR + molecular subtypes (Figure 2C and Table W3). Furthermore, among ER-negative tumors, PIP expression was associated with AR-positive status and was unrelated to CK5/6 staining (Figure 2D). It is notable that PIP expression was not associated with either tumor size or grade in this cohort (P N .1). These findings suggest that PIP is widely expressed in luminal A, luminal B, and ER −/AR + (molecular apocrine) subtypes of breast cancer. Moreover, PIP expression is associated with AR and is present in both luminal and basal ER-negative tumors.

PIP Transcriptional Signature Is Enriched with Cell Cycle Genes To investigate the functional role of PIP in breast cancer, we carried out a nonbiased genomic approach to study the transcriptional signature of this gene using a microarray data set of 52 breast cancer cell lines [21]. To identify PIP coregulated genes, we first calculated the Pearson CC for each gene expression in the data set with that of PIP and then obtained the list of genes that had Pearson CC values ≥ 0.5 (P b .001) with PIP expression. This PIP transcriptional signature is composed of 136 genes that had a highly coregulated expression pattern with PIP (Tables W4 and W5). We next performed hierarchical clustering analysis of PIP transcriptional signature and observed two main gene clusters in this signature on the basis of the direction of CC values with PIP expression (Figure 3A and Table W6).

Figure 3. PIP transcriptional signature. (A) Hierarchical clustering analysis of PIP transcriptional signature was performed using centroid linkage method, and intervals were measured by Pearson CCs. Functional annotations for gene clusters are demonstrated. FE, fold enrichment. (B) Functional annotation of PIP transcriptional signature based on Gene Ontology is presented. FEs and P values are shown.

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We subsequently conducted functional annotation of the PIP signature. Notably, this signature was highly enriched with cell cycle genes and in particular those related to mitotic transition and spindle checkpoint [Figure 3B, fold enrichment (FE) values = 11-56; P b .001]. These findings suggest a strong transcriptional coregulation between PIP and cell cycle–related genes that is most pronounced in relation to mitotic transition.

PIP Expression Is Required for G1-S Progression In view of the fact that PIP transcriptional signature is highly saturated with cell cycle genes and PIP expression is necessary for cell proliferation in breast cancer, we investigated the role of PIP in cell cycle progression using flow cytometry analysis on PIP-silenced cells. We observed that T-47D and MDA-MB-453 cells underwent a

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profound G1 arrest following PIP-KD manifested by a 10% to 20% increase in G0-G1 cell population compared to the controls with a corresponding decrease in the percentage of cells in S phase (P b .01; Figure 4, A and B). Furthermore, this G1 arrest was associated with a marked decrease in cyclin D1 and cyclin E1 expression following PIPKD (P b .01; Figure 4, C and D). Moreover, we examined the effect of PIP-KD on the level of ERK phosphorylation that is a required step for the transcriptional activation of cyclin D1 [26]. In addition, we have previously demonstrated a reduction in ERK phosphorylation in MDA-MB453 cells following PIP silencing [10]. Notably, phospho-/total ERK was markedly reduced by 0.14-fold following PIP-KD in T-47D cells (Figure 4E), which represents a similar pattern to that observed in MDA-MB-453 [10]. We also examined the effect of PIP silencing on

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Figure 4. The effect of PIP expression on G1 phase. (A) Cell cycle histograms following PIP-KD in T-47D are shown. (B) The percentage of cells in different phases of cell cycle following PIP-KD is shown. P b .01 is for ΔG0 -1 between PIP-KD and CTL groups. (C) Cyclin D1 and (D) cyclin E1 expression using qRT-PCR for PIP-KD relative to control is shown. (E) Immunoblot analysis measures the ratio of phospho (Ph)ERK to total (T) ERK, Ph–c-Jun to T–c-Jun, and Ph-Stat3 to T-Stat3 following PIP-KD. Fold changes were assessed relative to control. The average changes obtained for two duplexes are presented.



phosphorylation of c-Jun and STAT3 that are also involved in G1/S transition; however, we did not find any significant changes in the level of these proteins (Figure 4E). These data suggest that PIP silencing results in a profound G1 arrest in T-47D and MDA-MB453 cells associated with a reduction in the levels of cyclin D1, cyclin E1, and ERK phosphorylation.

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Cell cycle studies in MFM-223, SK-BR-3, HCC-1954, HCC-202, and BT-474 cell lines following PIP silencing revealed that these lines undergo a G2/M arrest manifested by a significant increase in G2/M phase and a marked increase in the percentage of aneuploidy by approximately 15% to 30% (P b .01; Figure 5, A–C). In addition,

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Figure 5. The effect of PIP expression on G2/M and aneuploidy. (A) The percentage of cells in different phases of cell cycle following PIPKD is shown. P values are for ΔG0 -1 and ΔG2/M between PIP-KD and control groups. (B) The change in percentage of aneuploidy between PIP-KD and control (CT) experiments is presented. (C) Cell cycle histograms following PIP-KD in BT-474 cell line are shown. (D) Cyclin D1 expression using qRT-PCR for PIP-KD relative to control (CTL) is shown. *P b .01 is for PIP-KD versus CTL groups. (E) T- and Ph-Cdc2 protein levels by immunoblot analysis for PIP-KD relative (RR) to control are shown. (F) Cyclin B1 expression for PIP-KD as explained in D is shown. The average changes obtained for two duplexes are presented.

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there was a moderate degree of G1 arrest following PIP-KD in four of these cell lines (P b .01; Figure 5A). In contrast, T-47D and MDAMB-453 cells did not have an increase in aneuploidy following PIP silencing (Figure 5, A and B). These data suggest that PIP expression is required for progression through both G1 and G2/M phases of the cell cycle and the occurrence of G2/M arrest following PIP silencing is accompanied by an increase in aneuploidy. To identify a molecular basis for the observed cell cycle findings, we assessed the effect of PIP silencing on the expression of key genes involved in G1, G2/M checkpoint, and mitotic transition. We observed that cyclin D1 expression, a key regulator of G1, was significantly reduced in MFM-223, HCC1954, HCC202, and BT474 cells following PIP-KD (P b .01; Figure 5D). We next examined the effect of PIP silencing on Cdc2 (Cdk1) and cyclin B1 as main regulators of G2/M checkpoint [27]. Notably, there was a proportionate reduction in total and phospho-Cdc2 protein levels following PIP-KD in HCC-1954 and HCC-202 cell lines that suggests a decrease in the expression of this protein following PIP silencing (Figure 5E). Furthermore, cyclin B1 expression was markedly reduced by approximately 50% to 80% in all five lines (P b .01; Figure 5F). In view of the fact that functional annotation showed a strong correlation between PIP and mitotic transition (Figure 3), we also studied the effect of PIP silencing on some of the key mitotic genes in PIP signature. In this respect, we examined FOXM1, TTK, BUB1, and CDC20, which have a critical role in mitotic transition [28–30]. Expression levels of these genes were assessed following PIP-KD in BT-474, HCC-1954, MMF-223, SK-BR-3, HCC-202, and MDAMB-453 cell lines. Importantly, there was a significant reduction in FOXM1, TTK, BUB1, and CDC20 expression following PIP-KD in these cells that supports a functional role for PIP in the regulation of mitotic transition (P b .01; Figure 6, A–D). We next investigated whether the observed G2/M accumulation following PIP silencing is a result of an arrest in G2 or M phase of the cell cycle. This was studied using IF staining with the mitotic marker MPM-2 that stains mitotic cells after G2 phase [31]. IF for MPM-2 was carried out in MFM-223, BT-474, and HCC-1954 cell lines, and the percentage of nuclei stained with MPM-2 was assessed for PIP-KD and control experiments (Figure 6, E and F). We observed a significant increase in MPM-2 staining by two- to three-fold in PIP-KD cells compared to the control, suggesting an arrest in mitotic transition following PIP silencing (P b .01; Figure 6, E and F). Overall, these findings indicate that PIP expression is required for mitotic transition in breast cancer and the effect of PIP silencing on cell cycle corresponds to the transcriptional changes in key cell cycle genes.


an essential step in cytokinesis, was disrupted in some dividing PIP-KD cells (Figure W2A). We next assessed the formation of microtubules and pericentrin to nuclear ratio following PIP silencing in these cells, because supernumerary percentrins during cell division are associated with cytokinesis defect and multinucleation [33]. Notably, pericentrin/ nuclear ratios significantly increased following PIP-KD in all three cell lines by 1.4- to 2.2-fold compared to controls, suggesting that there are supernumerary percentrins (P b .03; Figures 7, B and C, and W2C). In addition, α-tubulin staining revealed that PIP silencing leads to multipolar spindle formation during cell division and an absence of distinct microtubules in multinuclear cells (Figures 7B and W2C). Moreover, we observed that there is a 20% to 60% increase in the number of multinucleated cells following PIP silencing in MFM-223, SK-BR-3, BT-474, HCC-1954, and HCC-202 cell lines (P b .01; Figure 7D). However, T-47D and MDA-MB-453 cells did not demonstrate a significant increase in the number of multinucleated cells after PIP-KD. Importantly, these findings are in agreement with the occurrence of aneuploidy among these cell lines. Overall, our data suggest that PIP silencing disrupts the organization of actin microfilaments and microtubules and leads to an increase in the number of pericentrins and multinucleated cells that are all indicators of a cytokinesis defect.

PIP Is Required for Inside-Out Activation of Integrin-β1 Signaling It is known that dysregulation of integrin-β1 signaling results in cell cycle defects in G1 progression and cytokinesis [34]. Therefore, we investigated the effect of PIP silencing on FAK phosphorylation (Tyr 397 ), which is a key downstream mediator of integrin-β1 signaling and integrin-β1 binding to talin-1 that is a required step for inside-out activation of integrins [35,36]. We observed that the level of phospho-FAK was generally low in breast cancer cell lines and was only detectable in HCC-1954 and HCC-202 cells. In addition, PIP-KD resulted in the reduction of phospho-/total FAK ratios by approximately 50-70% in these two lines, which was partly related to a relative increase in the total FAK levels following PIP-KD (Figure 7E). Furthermore, we found a baseline interaction between integrin-β1 and talin-1 in T-47D and MFM-223 cell lines using IP with integrin-β1 and immunoblot analysis with talin-1 (Figure 7F). Importantly, integrin-β1 binding to talin-1 was abrogated in T-47D cells and reduced by 0.5-fold in MFM-233 following PIP silencing (Figure 7E). These data suggest that PIP expression is necessary for inside-out activation and signaling effects of integrin-β1.

PIP Silencing Results in a Cytokinesis Defect We subsequently studied the effect of PIP expression on cytokinesis. It is established that microtubules and actin organization are essential for this process [32]. Therefore, we first examined the effect of PIP silencing on actin microfilaments using IF in BT-474, HCC-1954, and MFM-223 cell lines. We observed that actin organization was markedly disrupted after PIP-KD, resulting in abnormally shaped and large filopodia protrusions and irregular lamellipodia that were formed in multinucleated cells (Figures 7A and W2, A and B). In addition, as opposed to the control cells that demonstrated polarity in actin organization as evidenced by the orientation of filopodia and retraction fibers, there was a loss of polarity in actin filaments following PIP silencing (Figure 7A). Furthermore, formation of cleavage furrow,

Identification of PIP-Binding Partners To identify protein-binding partners for endogenous PIP, we carried out MS. These experiments were performed in T-47D cell line, which has a high level of endogenous PIP expression that was further induced using dihydrotestosterone. Because PIP is a secreted protein, IP was performed using a combination of cell extracts and conditioned media. The result of PIP pulldown was first validated using immunoblot analysis with PIP antibody (Figure 8A). PIP-IP bands were then separated using SDS-PAGE and Coomassie staining, followed by MS analysis (Figure 8B). We identified a total of 156 protein-binding partners for PIP that were reproducible between two replicate experiments (Table W7).



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Figure 6. The effect of PIP expression on mitosis. (A–D) The effects of PIP silencing on mitotic transition genes are presented. Expression of FOXM1, TTK, BUB1, and CDC20 are measured using qRT-PCR for PIP-KD relative to control. P values are for PIP-KD versus CTL groups in each cell line. The asterisk (*) denotes P b .01 in BUB1 experiments. The average changes obtained for two duplexes are presented. (E and F) IF with the mitotic marker MPM-2. IF staining for MPM-2/Alexa 488 was carried for PIP-KD and control siRNA experiments in MFM-223, BT-474, and HCC-1954 cell lines. Percentage of MPM-2 staining was calculated in 200 nuclei for each experiment, and the average changes obtained for two duplexes are presented. 4',6-diamidino-2-phenylindole (DAPI) DAPI staining was used to assess the nuclei. *P value is for PIP-KD versus control groups.

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Figure 7. The effect of PIP on cytokinesis and integrin signaling. (A) IF staining for β-actin/Alexa488 following PIP-KD in BT-474 cells is shown. Control and PIP-KD cells are shown during cell division (top panels), and a multinucleated cell following PIP-KD is shown in bottom panel. White arrow, filopodia (fil); yellow arrow, lamellipodia (lam); orange arrow, retraction fibers (rf); and magenta arrow, direction of actin polarity. (B) IF staining for α-tubulin (Tub) and pericentrin following PIP-KD in MFM-223 cells is shown. (C) Pericentrin to nuclear ratios following PIPKD are presented. DAPI staining was used to assess the nuclei. *P value is for PIP-KD versus control groups. (D) Change in percentage of multinucleated cells between PIP-KD and control cell lines is shown. IF following β-actin and DAPI staining was used to assess multinucleated cells. *P value is for PIP-KD versus control groups. (E) Immunoblot analysis measures the ratio of Ph-FAK (Tyr 397) to T-FAK following PIP-KD in cell lines. Fold changes were assessed relative to control. Experiments were carried out in four replicates using two PIP-siRNA duplexes or control siRNA, and mean changes (± SEM) were shown. (F) IP assesses integrin-β1 (ITG-β1) binding to talin-1 following PIP-KD. IP and immunoblot analysis were carried out with ITG-β1 and talin-1 antibodies, respectively. Membrane was stripped, and immunoblot analysis for ITG-β1 was used to assess loading. ITG-β1 immunoblot for input control is shown. Fold changes were assessed relative to control. Experiments were carried out in four replicates using two PIP-siRNA duplexes or control siRNA, and mean change (±SEM) is shown for each cell line.



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Figure 8. Identification of PIP-binding partners. (A) IP and immunoblot analysis (IB) with PIP antibody. The low molecular weight band may represent a PIP fragment product. (B) Coomassie staining of SDS-PAGE for PIP-IP and control pulldowns is shown. (C) Functional classification of PIP-binding partners is shown. Enrichment score and P value are shown. (D) IP with PIP antibody and IB with β-tubulin, PIP, and Arp2/3 antibodies in T-47D cells are shown. A nonspecific rabbit IgG was used for control IP. Membrane was stripped, and IB for PIP was used to assess loading. PIP immunoblot for input control is shown. (E) Microtubule polymerization assay measures polymerized and soluble tubulin fractions following PIP-KD. The amount of each fraction following PIP-KD was normalized to that of control, and the relative ratio of Pol/Sol fractions was obtained for each cell line. The average changes obtained for two duplexes are presented.

Functional classification of these binding partners using bioinformatics revealed six highly significant clusters (Figure 8C and Table W8). These functional clusters included translational elongation, ribonuclear protein, nucleosome and chromatin assembly, regulation of actin and cytoskeleton, clathrin-coated pit, and small guanosine diphosphate (GDP)-binding protein. In addition, we studied the functional association of PIP-binding partners with the canonical pathways using Ingenuity Pathway Analysis. The top identified pathways included eukaryotic Initiation Factor 2 (eIF2) and eIF4 signaling, followed by remodeling of epithelial adherens junctions, clathrinmediated endocytosis, regulation of actin-based motility by Rho, and integrin signaling (Table W9).

We further validated two of the identified PIP-binding partners that have key functions in cell cycle (Table W7). Notably, we identified β-tubulin as one of the top PIP-binding partners, which has a well-established role in mitosis [37]. In addition, Arp2/3 protein is another PIP-binding partner with a significant role in cytokinesis and promoting talin binding to integrins [38,39]. To validate these interactions, we carried out PIP-IP and performed immunoblot analysis with β-tubulin and Arp2/3 antibodies on pulldowns. Immunoblot analysis with PIP antibody was used to confirm a successful PIP pulldown (Figure 8D). Notably, we observed a strong interaction between PIP and β-tubulin and detected a specific interaction between PIP and Arp2/3 protein in PIP pulldown

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231, which do not have AR expression. This association between AR and PIP expression is also present among primary breast tumors and can be explained by the fact that AR is a transcriptional regulator of PIP [8–10]. Overall, the pattern of PIP association with molecular subtypes and biomarkers are similar between breast cancer cell lines and breast tumors. The effect of PIP on proliferation is explained by the fact that PIP expression is required for cell cycle progression in breast cancer cells and PIP silencing leads to defects in G1, mitosis, and cytokinesis. In addition, we show that PIP interacts with β-tubulin and is necessary for tubulin polymerization. This interaction is particularly significant because microtubules are known to have a critical role in mitotic transition, spindle assembly, and cytokinesis [37,40]. Moreover, proteomic data provide further evidence for the importance of PIP interaction with β-tubulin in the functional characterization of PIPbinding partners. In fact, a major portion of PIP interactions that contribute to the observed functional classification are known “Tubulin-Binding Proteins” (Figure 8C and Table W7). These include RNA-binding proteins, proteins involved in translation such as eIF4, heat shock proteins, clathrin, and 14-3-3 protein family [41– 43]. It is notable that 14-3-3 protein is a key regulator of cell cycle and clathrin is required for mitotic spindle function and endocytosis [42,43]. In addition, mRNA localization to microtubules contributes to the translation of genes involved in cell division such as cyclin B1 [44]. Therefore, PIP regulation of microtubular polymerization and its interaction with other tubulin-binding proteins would have a profound effect on cell cycle (Figure 9). Another major group of PIP interactions involves actin-binding proteins. Among these, Arp2/3 has a critical role in actin polymerization, and along with vinculin and talin, it provides a physical link between actin cytoskeleton and the integrin scaffold, which is needed to transform talin binding to integrin-β1 from a lowto high-affinity state [38]. Therefore, PIP interaction with Arp2/3 can

(Figure 8D). Taken together, our proteomic studies identified novel PIP-binding proteins.

PIP Is Necessary for Microtubule Polymerization

To assess whether PIP binding to β-tubulin has a regulatory effect on microtubule polymerization, we measured the effect of PIP silencing on the polymerized and soluble fractions of tubulin in T-47D and HCC1954 cell lines. Immunoblot analysis was carried out using a β-tubulin antibody to measure polymerized and soluble tubulin fractions. The amount of each fraction following PIP silencing was normalized to that of siRNA control, and the relative ratio of polymerized to soluble (Pol/Sol) fractions was obtained for each cell line. We observed that Pol/Sol tubulin ratio was markedly reduced following PIP-KD to 0.31- and 0.06-fold of controls in T-47D and HCC-1954 cell lines, respectively (Figure 8E). These findings suggest that PIP is required for tubulin polymerization in breast cancer cells. Discussion PIP is widely expressed in breast cancer and is used as a characteristic biomarker in this disease; however, the molecular functions of PIP have remained largely unknown. Therefore, we carried out a comprehensive study to identify the underlying mechanisms for PIP function in breast cancer. In this process, we employed seven breast cancer cell lines that encompass luminal A, luminal B, and molecular apocrine subtypes. It is notable that these molecular subtypes also correspond to the pattern of PIP expression among primary breast tumors. Previous studies have shown a positive regulatory role for PIP in cell proliferation [9,10]. In this study, we demonstrated that PIP expression is necessary for the proliferation of all breast cancer cell lines that have a detectable level of PIP. Although PIP expression varied among different cell lines, the impact of PIP on cell proliferation was similar across these cells. Moreover, the only two cell lines with undetectable levels of PIP were MCF-7 and MDA-MBFn PIP

Fn-f

ITG

Arp2/3

FAK

β-tubulin

vinculin

Talin

PIP Histones

PIP FOXM1

Figure 9. A schematic model for PIP regulation of cell cycle. The proposed mechanisms by which extracellular and intracellular PIP can regulate cell cycle are depicted. Fn, fibronectin; Fn-f, fibronectin fragments; ITG, integrin-β1. Red arrows indicate positive regulation. Cell membrane has been depicted by a circular line. Arp2/3, β-tubulin, and histones interact with PIP based on our study.



explain the marked reduction in talin binding to integrin-β1, which is a required step for inside-out activation of integrins, and a decrease in FAK phosphorylation following PIP silencing. It is notable that outside-in signaling of integrin-β1 binding to integrin-linked kinase 1 (ILK1) is also regulated by PIP in a process that partially depends on fibronectin fragmentation (Figure 9), [10]. In addition to Arp2/3, some of the other PIP-binding partners including gelsolin, cofilin 1, F-actin–capping protein, α-actinin, and small GTP-binding proteins have key roles in actin organization, formation of focal adhesions, and cytokinesis [39,45]. In fact, our study provides functional evidence for the importance of these interactions as shown by a defect in cytokinesis following PIP silencing that is associated with abnormal actin organization (Figures 7 and W2). Importantly, some of the PIPbinding partners such as Rho-GTPase are known to coordinate cytokinesis and cell polarity [46]. Overall, our findings indicate that a key feature of PIP function is the regulation of cytoskeleton (Figure 9). Our data suggest that PIP expression is necessary for both G1/S and mitotic progression in breast cancer cells. However, the impact of PIP on each phase of cell cycle varies among breast cancer lines. In this respect, we observed two main patterns for the effect of PIP silencing on cell cycle. In one group constituted of T-47D and MDA-MB-453 cell lines, there was a severe degree of G1 arrest that was not associated with mitotic arrest or aneuploidy. Importantly, this pattern was associated with a profound reduction in cyclin D1 expression following PIP silencing. In comparison, the remaining cell lines developed a moderate degree of G1 arrest accompanied by mitotic arrest and a marked increase in aneuploidy (Figure 5, A and B). Furthermore, our genomics data on PIP transcriptional signature and a marked reduction in key mitotic transition genes such as cyclin B1, BUB1, and CDC20 following PIP down-regulation suggest the importance of PIP expression in mitosis. It is notable that the emergence of aneuploidy can be a consequence of defects in both mitotic checkpoint and cytokinesis [47]. In particular, dysregulation of mitotic and spindle checkpoint genes such as BUB1 and FOXM1 have been associated with aneuploidy [48,49]. Importantly, this increase in aneuploidy can further contribute to a reduction in cell proliferation [50]. We observed that PIP expression is necessary for the transcription of multiple genes involved in cell cycle progression. Some of these effects can be explained by PIP regulation of the upstream signaling pathways. For example, cyclin D1 is a target of integrin-β1 mediated through the activation of ERK [34]. Therefore, the effect of PIP on cyclin D1 can be explained by the fact that G1 arrest in T-47D cells is accompanied by a marked reduction in ERK phosphorylation associated with an abrogation of talin binding to integrin-β1. In addition, PIP transcriptional signature shows a robust pattern of coregulation between PIP and mitotic transition genes. This finding along with a profound decrease in the expression of mitotic genes observed following PIP silencing suggest that PIP is required to maintain a balance in the expression of key genes involved in mitotic transition. This is especially important because both overexpression and down-regulation of some of the mitotic genes such as BUB1 can lead to abnormal mitosis and aneuploidy [48,51]. Notably, FOXM1 is known to be a transcription factor for multiple genes involved in cell cycle progression including cyclin B1, BUB1, CDC20, and polo-like kinase (PLK) [28]. Therefore, PIP regulation of FOXM1 expression can explain many of the transcriptional changes observed following PIP silencing. In addition, soluble tubulin has been shown

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to interact with histones and regulate transcription [52]. In view of the fact that PIP interacts with histones (Table W7) and also regulates soluble tubulin levels, the possibility of a transcriptional role for PIP deserves further studies (Figure 9). In summary, we propose that PIP has a versatile function in breast cancer resulting from a diverse range of both intracellular and extracellular binding partners (Figure 9). As a consequence of these functional interactions, PIP can regulate key cellular processes including outside-in and inside-out activation of integrin-β1, transcription of key cell cycle genes such as FOXM1, and cytoskeletal organization including microtubule polymerization. The net effect of these molecular functions is the fact that PIP can profoundly influence cell cycle progression in breast cancer cells (Figure 9). Importantly, our findings provide a rationale for the tantalizing possibility of exploring PIP as a therapeutic target in breast cancer. References [1] Clark JW, Snell L, Shiu RP, Orr FW, Maitre N, Vary CP, Cole DJ, and Watson PH (1999). The potential role for prolactin-inducible protein (PIP) as a marker of human breast cancer micrometastasis. Br J Cancer 81, 1002–1008. [2] Teschendorff AE, Naderi A, Barbosa-Morais NL, Pinder SE, Ellis IO, Aparicio S, Brenton JD, and Caldas C (2006). A consensus prognostic gene expression classifier for ER positive breast cancer. Genome Biol 7, R101. [3] Doane AS, Danso M, Lal P, Donaton M, Zhang L, Hudis C, and Gerald WL (2006). An estrogen receptor-negative breast cancer subset characterized by a hormonally regulated transcriptional program and response to androgen. Oncogene 25, 3994–4008. [4] Baniwal SK, Chimge NO, Jordan VC, Tripathy D, and Frenkel B (2013). Prolactin-induced protein (PIP) regulates proliferation of luminal A type breast cancer cells in an estrogen-independent manner. PLoS One 8, e62361. [5] Farmer P, Bonnefoi H, Becette V, Tubiana-Hulin M, Fumoleau P, Larsimont D, Macgrogan G, Bergh J, Cameron D, and Goldstein D, et al (2005). Identification of molecular apocrine breast tumours by microarray analysis. Oncogene 24, 4660–4671. [6] Naderi A and Hughes-Davies L (2008). A functionally significant cross-talk between androgen receptor and ErbB2 pathways in estrogen receptor negative breast cancer. Neoplasia 10, 542–548. [7] Lehmann-Che J, Hamy AS, Porcher R, Barritault M, Bouhidel F, Habuellelah H, Leman-Detours S, de Roquancourt A, Cahen-Doidy L, and Bourstyn E, et al (2013). Molecular apocrine breast cancers are aggressive estrogen receptor negative tumors overexpressing either HER2 or GCDFP15. Breast Cancer Res 15, R37. [8] Carsol JL, Gingras S, and Simard J (2002). Synergistic action of prolactin (PRL) and androgen on PRL-inducible protein gene expression in human breast cancer cells: a unique model for functional cooperation between signal transducer and activator of transcription-5 and androgen receptor. Mol Endocrinol 16, 1696–1710. [9] Baniwal SK, Little GH, Chimge NO, and Frenkel B (2012). Runx2 controls a feed-forward loop between androgen and prolactin-induced protein (PIP) in stimulating T47D cell proliferation. J Cell Physiol 227, 2276–2282. [10] Naderi A and Meyer M (2012). Prolactin-induced protein mediates cell invasion and regulates integrin signaling in estrogen receptor-negative breast cancer. Breast Cancer Res 14, R111. [11] Caputo E, Manco G, Mandrich L, and Guardiola J (2000). A novel aspartyl proteinase from apocrine epithelia and breast tumors. J Biol Chem 275, 7935–7941. [12] Gaubin M, Autiero M, Basmaciogullari S, Métivier D, Mis hal Z, Culerrier R, Oudin A, Guardiola J, and Piatier-Tonneau D (1999). Potent inhibition of CD4/TCR-mediated T cell apoptosis by a CD4-binding glycoprotein secreted from breast tumor and seminal vesicle cells. J Immunol 162, 2631–2638. [13] Schaller J, Akiyama K, Kimura H, Hess D, Affolter M, and Rickli EE (1991). Primary structure of a new actin-binding protein from human seminal plasma. Eur J Biochem 196, 743–750. [14] Cassoni P, Sapino A, Haagensen DE, Naldoni C, and Bussolati G (1995). Mitogenic effect of the 15-kDa gross cystic disease fluid protein (GCDFP-15) on breast-cancer cell lines and on immortal mammary cells. Int J Cancer 60, 216–220.

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Supplementary References [59] [53] Lehmann BD, Bauer JA, Chen X, Sanders ME, Chakravarthy AB, Shyr Y, and Pietenpol JA (2011). Identification of human triple-negative breast cancer subtypes and preclinical models for selection of targeted therapies. J Clin Invest 121, 2750–2767. [54] Magklara A, Brown TJ, and Diamandis EP (2002). Characterization of androgen receptor and nuclear receptor co-regulator expression in human breast cancer cell lines exhibiting differential regulation of kallikreins 2 and 3. Int J Cancer 100, 507–514. [55] Heiser LM, Sadanandam A, Kuo WL, Benz SC, Goldstein TC, Ng S, Gibb WJ, Wang NJ, Ziyad S, and Tong F, et al (2012). Subtype and pathway specific responses to anticancer compounds in breast cancer. Proc Natl Acad Sci U S A 109, 2724–2729. [56] Naderi A and Liu J (2010). Inhibition of androgen receptor and Cdc25A phosphatase as a combination targeted therapy in molecular apocrine breast cancer. Cancer Lett 298, 74–87. [57] Naderi A and Hughes-Davies L (2008). A functionally significant cross-talk between androgen receptor and ErbB2 pathways in estrogen receptor negative breast cancer. Neoplasia 10, 542–548. [58] Doane AS, Danso M, Lal P, Donaton M, Zhang L, Hudis C, and Gerald WL (2006). An estrogen receptor-negative breast cancer subset characterized by a

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hormonally regulated transcriptional program and response to androgen. Oncogene 25, 3994–4008. Neve RM, Chin K, Fridlyand J, Yeh J, Baehner FL, Fevr T, Clark L, Bayani N, Coppe JP, and Tong F, et al (2006). A collection of breast cancer cell lines for the study of functionally distinct cancer subtypes. Cancer Cell 10, 515–527. Subik K, Lee JF, Baxter L, Strzepek T, Costello D, Crowley P, Xing L, Hung MC, Bonfiglio T, and Hicks DG, et al (2010). The expression patterns of ER, PR, HER2, CK5/6, EGFR, Ki-67 and AR by immunohistochemical analysis in breast cancer cell lines. Breast Cancer 4, 35–41. Ginestier C, Adélaïde J, Gonçalvès A, Repellini L, Sircoulomb F, Letessier A, Finetti P, Geneix J, Charafe-Jauffret E, and Bertucci F, et al (2007). ERBB2 phosphorylation and trastuzumab sensitivity of breast cancer cell lines. Oncogene 26, 7163–7169. Lombardi DP (2004). The Tetraspanin Metastasis Suppressor Gene, KAI1/CD82, and the Proto-Oncogene, Her-2/neu, as Molecular Determinants of Metastasis in Breast Cancer Patients. Defense Technical Information Center Annual Summary Report, contract number DAMD17-03-1-0559, 1-29 (http://handle.dtic.mil/100.2/ADA433099). Agarwal R, Gonzalez-Angulo AM, Myhre S, Carey M, Lee JS, Overgaard J, Alsner J, Stemke-Hale K, Lluch A, and Neve RM, et al (2009). Integrative analysis of cyclin protein levels identifies cyclin b1 as a classifier and predictor of outcomes in breast cancer. Clin Cancer Res 15, 3654–3662.

Figure W1. PIP expression in breast cancer cell lines. (A) Relative PIP expression in breast cancer cell lines to that of HCC202 cells presented in a logarithmic scale (base 2). (B) PIP protein expression in BT-474 and MFM-223 cell lines following PIP-knockdown (KD) using cell lysate samples. CTL: cells transfected with the control-siRNA. RR: relative ratio to control.

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Figure W2. PIP effect on cytokinesis. (A-B) Immunofluorescence (IF) staining for β-Actin in HCC-1954 and MFM-223 cell lines. White arrow: filopodia (fil), blue arrow: cleavage furrow (cf). (A) Cleavage furrow formation is present in dividing control cells (CT) and is absent in dividing cells with PIP-knockdown (KD). Multinucleated cells are shown following PIP-KD in HCC-1954 (A) and MFM-223 cells (B). IF was carried out with β-Actin antibody and Alexa488 was used as secondary antibody. (C) IF staining for α-Tub/Pericentrin. IF was carried out with, α-Tubulin and Pericentrin antibodies in BT-474 cell line. Alexa488 and Alexa 594 antibodies were used as secondaries. There is an absence of distinct microtubules and presence of supernumerary pericentrins in PIP-KD cells. Magnifications are shown for each panel.



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Table W1. Characteristics of Breast Cancer Cell Lines Used in the Functional Study of PIP. Cell Line

ER

ErbB2

AR

Subtype

PIP −ΔCT

MCF-7 T-47D BT-474 HCC-202 HCC-1954 MDA-MB-453 SK-BR-3 MFM-223 MDA-MB-231

POS POS POS NEG NEG NEG NEG NEG NEG

NEG NEG POS POS POS POS POS NEG NEG

NEG POS POS POS POS POS NEG POS NEG

Luminal A Luminal A Luminal B Luminal Basal A Luminal Luminal Luminal Basal B

− 17.5 (±0.08) − 4.9 (±0.04) − 12.2 (±0.08) − 1.1 (±0.13) − 7.3 (±0.02) − 6.2 (±0.06) − 11.1 (±0.08) − 11.8 (±0.03) − 20 (±0) *

Cell subtype and AR status were obtained from Lehmann BD et al. (2011) [53], Magklara A et al.(2002) [54], Heiser LM et al. (2012) [55], Naderi A et al. (2010) [56], Naderi A et al. (2008) [57], Doane AS et al. (2006) [58], and Neve RM et al. (2006) [59]. ErbB2 status was obtained from Subik K et al. (2010) [60], Ginestier C et al. (2007) [61], Lombardi DP et al. (2004) [62], and Agarwal R et al. (2009) [63]. POS, positive; NEG, negative. PIP − ΔCT is −Δ cycle threshold value (±SEM) for PIP expression using qRT-PCR. * PIP expression was not detectable after 40 cycles of qRT-PCR.

Table W2. Characteristics of the TMA Cohort. Feature

Status

Percentage

Histology

IDC * Others ** Negative Positive 0-1 2-3 0-1 N2

85% 15% 51% 49% 52% 48% 68% 32%

ER ErbB2 p53

Percentage is calculated in a total of 210 primary breast tumors. * IDC, invasive ductal carcinoma. ** Others: Ductal carcinoma in situ, Paget disease, invasive lobular carcinoma, invasive micropapillary carcinoma, invasive tubulo-lobular carcinoma, invasive papillary carcinoma, lobular carcinoma in situ, invasive carcinoma with apocrine features, invasive tubular carcinoma, intraductal carcinoma, and tubular mixed carcinoma.

Table W3. Association of PIP Expression with Molecular Features in Breast Cancer Cohort. Biomarkers Marker Status PIP (SEM) P value

ErbB2 b2 112 (8) N.1

2-3 126 (9)

ER Neg 111 (9) N.1

Pos 126 (9)

AR Neg 80 (11) b .01 *

Pos 132 (7)

p53 Neg 119 (7) N.1

Pos 118 (11)

Molecular Subtypes Subtype Frequency PIP (SEM) P value

Luminal A 28% 116 (12) P b .01 **

Luminal B 21% 136 (12) P b .01 **

ER−/AR+ 31% 141 (11) P b .01 **

ER−/AR − 20% (Basal: 10%) 62 (12)

Mean scores for PIP expression are demonstrated in 210 primary breast tumors. Luminal B, ER+ with Ki-67 index ≥ 14% or ErbB2 staining score of 3; AR +, ≥ 10% nuclear staining; Basal, CK5/6 +; SEM, standard error of mean. * P value for AR-negative (Neg) versus AR-positive (Pos) tumors. ** P values versus ER−/AR− group.

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Table W4. Proximity Matrix for PIP Coregulated Genes. Each Gene has Pearson CC ≥0.5 with PIP Expression at a Significant Level of P b .001.




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342.e5

Table W4. (continued)

(continued on next page)

342.e6



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(continued on next page)

342.e8



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Table W5. Transcriptional Signature of PIP Coregulated Genes. Gene

CC

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342.e9

Table W5. (continued ) Table W5. (continued)

Pb

Function *

Gene

CC

Pb

Function *

Fatty acid pathway Hydrolysis Lysosomal Cell migration Mitochondrial Steroid receptor Cell migration Survival and migration G-protein receptor Ca2 + function Apoptosis Cytoskeletal

VIPR1 WSB1 XBP1 ZDHHC3 PATZ1 BUB1 CCNA2 CCNB2 CDC20 CDC5L CENPE CTPS DDX18 HJURP DNMT1 E2_EPF E2F3 FBXO5 FAM64A PRR11 QTRTD1 NCAPG2 SPDL1 IARS FASTKD1 FOXM1 GLS_C HCAP_G HNRPD_E HNRPH1 DLGAP5 NUP205 EHBP1 KIF4A MAD2L1 METAP1 MSH2 NCL NUP54 OSBPL11 PDCD5 PLK PLS3 PMS1 PMSCL1 PSMD1 RAB6KIFL RHEB2 RUVBL2 SFRS10 SFRS7 SIL ORNT1 SMC4L1 SNRPD1 STK12 (Aurora B) TSN TTK UBA2 UBE2C UPF3B USP1 XPO1 ZRF1

0.5 0.551 0.505 0.538 0.516 −0.607 −0.584 −0.574 −0.588 −0.531 −0.565 −0.531 −0.54 −0.662 −0.563 −0.52 −0.504 −0.566 −0.521 −0.599 −0.503 −0.589 −0.536 −0.504 −0.512 −0.54 −0.52 −0.557 −0.562 −0.613 −0.548 −0.54 −0.542 −0.515 −0.561 −0.515 −0.507 −0.559 −0.578 −0.559 −0.64 −0.535 −0.616 −0.514 −0.578 −0.522 −0.522 −0.518 −0.521 −0.533 −0.539 −0.59 −0.53 −0.578 −0.584 −0.5 −0.512 −0.639 −0.581 −0.541 −0.539 −0.555 −0.563 −0.562

.001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001

G-protein receptor Proteasome Transcription Cell surface stability Transcription Mitosis Cell cycle Mitosis Mitosis Cell cycle Mitosis Cell growth Cell growth and division Centrosome function Epigenetics

ACOX1 ACY1 AGA AGR2 ALDH6A1 AR ARHGEF16 ARHH ARRB1 ATP2A3 BIK ANKRA2 CACFD1 CPD CRAT SGSM3 LRP10 DUSP4

0.518 0.504 0.525 0.592 0.515 0.59 0.5 0.572 0.547 0.554 0.545 0.527 0.522 0.606 0.51 0.702 0.603 0.51

.001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001

EGFL3 ERRB3

0.506 0.501

.001 .001

FKSG28 SRD5A3 TMEM132A TCTN1 RHBDF1 FMO4 FRAT1 FZD4 GABRA3 HMIC HNF3A HOXC10 HPIP ICA1 ITPR1 KHNYN TRIL KIF13B KMO LDB1 LDB3 LFG NFATC4 P24B P2RX4 P2RY6 PAPSS2 PCK2 PDEF PISD PRKAG1 PRKCH PRSS8 PXMP4 RAB5B SEMA3F SERF2 SERHL SH3GLB2 NHE2 SPRY1 SPTLC2 SSBP2 SUOX TJP3 TM7SF1 TM7SF2 TM9SF1 TMEM8 TSPAN1 TST ULK1

0.583 0.572 0.563 0.523 0.523 0.502 0.503 0.569 0.513 0.504 0.518 0.501 0.564 0.517 0.545 0.562 0.587 0.632 0.52 0.532 0.598 0.501 0.513 0.561 0.547 0.504 0.521 0.501 0.521 0.565 0.514 0.641 0.535 0.518 0.552 0.511 0.513 0.518 0.512 0.517 0.521 0.645 0.502 0.595 0.52 0.505 0.646 0.503 0.506 0.552 0.506 0.527

.001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001 .001

Peptidase Mitochondrial neurofibromatosis 2 (NF-2) signaling Lipid metabolism mitogen-activated protein kinase (MAPK) signaling epidermal growth factor receptor (EGFR) signaling Proliferation Androgen metabolism Cell death Hedgehog signaling EGFR signaling Metabolism Wnt signaling Wnt signaling Neurotransmitter Metabolism Steroid response Transcription factor estrogen receptor-alpha (ESR) signaling Secretory function Ca2 + signaling Cytokine secretion Cytoskeletal Metabolism Transcription Cytoskeletal Apoptosis Transcription Protein trafficking Ion channel G-protein receptor Metabolism Metabolism Transcription Mitochondrial Metabolism Protein kinase Sserine protease Protein transport Cell motility Serine hydrolase Channel protein Fibroblast growth factor signaling Metabolism Genome stability Mitochondrial Cytoskeletal mitosis Sterol metabolism Autophagy Adhesion Growth and motility Mitochondrial Autophagy (continued on next page)

Cell cycle Mitosis Mitosis RNA synthesis Mitosis Mitosis RNA synthesis Cell cycle Metabolism Mitosis RNA function RNA function Mitosis Nuclear transport Cytoskeletal actin Cytokinesis Mitosis Cell cycle Genome stability Transcription Nuclear transport Lipid metabolism Apoptosis Cell cycle Actin binding Genome stability RNA function Proteasome Cytokinesis Ras-GTPase DNA repair Splicing factor Splicing factor Mitosis Mitochondrial Mitosis RNA function Mitosis and cytokinesis Chromosomal function Mitosis Protein modification Mitosis RNA function DNA repair Protein export Transcription

List of genes that have Pearson CCs ≥ 0.5 with PIP expression at a significance level of P b .001. Raw data for gene expression values were extracted from Affymetrix microarray data set published by Neve RM et al. 2006. * Proposed gene Function is derived from GeneCards (www.genecards.org).

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Table W6. Dendrogram Using Centroid Linkage.

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Table W7. Identified PIP-Binding Proteins (Protein Threshold at 99% and Peptide Threshold at 0.1% false discovery rate [FDR]). No 1

1.1 1.2 2 3 4

4.1 5 6 7 7.1 8 8.1 9 10 10.1 11 12

12.1 12.2 13 14 15 16 17 18 19 20 21 22 22.1 22.2 23 24 25

Accession No. Cluster of tubulin β chain; Organism Species (OS) = Homo sapiens; Gene Name (GN) = TUBB; PE = 1; Splice Variant (SV) = 2 (TBB5_HUMAN) Tubulin β chain; OS = H sapiens; GN = TUBB; PE = 1; SV = 2 Tubulin β-4B chain; OS = H sapiens; GN = TUBB4B; Protein Existence (PE) = 1; SV = 1 Heat shock 70-kDa protein 1A/1B; OS = H sapiens; GN = HSPA1A; PE = 1; SV = 5 Unconventional myosin-Id; OS = H sapiens; GN = MYO1D; PE = 1; SV = 2 Cluster of heat shock protein HSP 90-β; OS = H sapiens; GN = HSP90AB1; PE = 1; SV = 4 (HS90B_HUMAN) Heat shock protein HSP 90-β; OS = H sapiens; GN = HSP90AB1; PE = 1; SV = 4 Heterogeneous nuclear ribonucleoproteins A2/B1; OS = H sapiens; GN = HNRNPA2B1; PE = 1; SV = 2 Neuroblast differentiation-associated protein AHNAK; OS = H sapiens; GN = AHNAK; PE = 1; SV = 2 Cluster of prelamin-A/C; OS = H sapiens; GN = LMNA; PE = 1; SV = 1 (LMNA_HUMAN) Prelamin-A/C; OS = H sapiens; GN = LMNA; PE = 1; SV = 1 Cluster of 40S ribosomal protein S3a; OS = H sapiens; GN = RPS3A; PE = 1; SV = 2 (RS3A_HUMAN) 40S ribosomal protein S3a; OS = H sapiens; GN = RPS3A; PE = 1; SV = 2 40S ribosomal protein S3; OS = H sapiens; GN = RPS3; PE = 1; SV = 2 Cluster of α-enolase; OS = H sapiens; GN = ENO1; PE = 1; SV = 2 (ENOA_HUMAN) α-enolase; OS = H sapiens; GN = ENO1; PE = 1; SV = 2 60S ribosomal protein L10a; OS = H sapiens; GN = RPL10A; PE = 1; SV = 2 Cluster of serine/arginine-rich splicing factor 6; OS = H sapiens; GN = SRSF6; PE = 1; SV = 2 (SRSF6_HUMAN) Serine/arginine-rich splicing factor 6; OS = H sapiens; GN = SRSF6; PE = 1; SV = 2 Serine/arginine-rich splicing factor 4; OS = H sapiens; GN = SRSF4; PE = 1; SV = 2 60S ribosomal protein L8; OS = H sapiens; GN = RPL8; PE = 1; SV = 2 40S ribosomal protein S6; OS = H sapiens; GN = RPS6; PE = 1; SV = 1 Pyruvate kinase PKM; OS = H sapiens; GN = PKM; PE = 1; SV = 4 40S ribosomal protein S18; OS = H sapiens; GN = RPS18; PE = 1; SV = 3 Ras GTPase-activating protein-binding protein 1; OS = H sapiens; GN = G3BP1; PE = 1; SV = 1 UPF0568 protein C14orf166; OS = H sapiens; GN = C14orf166; PE = 1; SV = 1 Heterogeneous nuclear ribonucleoprotein A1; OS = H sapiens; GN = HNRNPA1; PE = 1; SV = 5 Heat shock protein β-1; OS = H sapiens; GN = HSPB1; PE = 1; SV = 2 60S ribosomal protein L10; OS = H sapiens; GN = RPL10; PE = 1; SV = 4 Cluster of histone H1.3; OS = H sapiens; GN = HIST1H1D; PE = 1; SV = 2 (H13_HUMAN) Histone H1.3; OS = H sapiens; GN = HIST1H1D; PE = 1; SV = 2 Histone H1.2; OS = H sapiens; GN = HIST1H1C; PE = 1; SV = 2 60S acidic ribosomal protein P0; OS = H sapiens; GN = RPLP0; PE = 1; SV = 1 tRNA-splicing ligase RtcB homolog; OS = H sapiens; GN = C22orf28; PE = 1; SV = 1 Serine/arginine-rich splicing factor 9; OS = H sapiens; GN = SRSF9; PE = 1; SV = 1

TBB5_HUMAN [2]

TBB5_HUMAN TBB4B_HUMAN HSP71_HUMAN MYO1D_HUMAN HS90B_HUMAN

HS90B_HUMAN ROA2_HUMAN AHNK_HUMAN LMNA_HUMAN LMNA_HUMAN RS3A_HUMAN RS3A_HUMAN RS3_HUMAN ENOA_HUMAN ENOA_HUMAN RL10A_HUMAN SRSF6_HUMAN [4]

SRSF6_HUMAN SRSF4_HUMAN (+2) RL8_HUMAN RS6_HUMAN KPYM_HUMAN RS18_HUMAN G3BP1_HUMAN CN166_HUMAN ROA1_HUMAN HSPB1_HUMAN RL10_HUMAN H13_HUMAN [2] H13_HUMAN H12_HUMAN RLA0_HUMAN (+1) RTCB_HUMAN SRSF9_HUMAN



Table W7. (continued) (continued ) No 26 27 28 29 29.1 30 30.1 30.2 31 32

32.1 33 34 34 35 36 37 38 39 39.1 40 41 42 43 44 45 46 47 48 49

49.1 49.2 50 51 52 53 54 55

40S ribosomal protein S11; OS = H sapiens; GN = RPS11; PE = 1; SV = 3 Clathrin heavy chain 1; OS = H sapiens; GN = CLTC; PE = 1; SV = 5 40S ribosomal protein S16; OS = H sapiens; GN = RPS16; PE = 1; SV = 2 Cluster of annexin A2; OS = H sapiens; GN = ANXA2; PE = 1; SV = 2 (ANXA2_HUMAN) Annexin A2; OS = H sapiens; GN = ANXA2; PE = 1; SV = 2 Cluster of Q5VU59_HUMAN Q5VU59_HUMAN Tropomyosin α-3 chain; OS = H sapiens; GN = TPM3; PE = 1; SV = 2 ADP/ATP translocase 2; OS = H sapiens; GN = SLC25A5; PE = 1; SV = 7 Cluster of eukaryotic translation initiation factor 4 γ 1; OS = H sapiens; GN = EIF4G1; PE = 1; SV = 4 (IF4G1_HUMAN) Eukaryotic translation initiation factor 4 γ 1; OS = H sapiens; GN = EIF4G1; PE = 1; SV = 4 60S ribosomal protein L19; OS = H sapiens; GN = RPL19; PE = 1; SV = 1 Cluster of dystonin; OS = H sapiens; GN = DST; PE = 1; SV = 4 (DYST_HUMAN) Cluster of dystonin; OS = H sapiens; GN = DST; PE = 1; SV = 4 (DYST_HUMAN) EH domain-containing protein 1; OS = H sapiens; GN = EHD1; PE = 1; SV = 2 Peptidyl-prolyl cis-trans isomerase A; OS = H sapiens; GN = PPIA; PE = 1; SV = 2 GTP-binding nuclear protein Ran; OS = H sapiens; GN = RAN; PE = 1; SV = 3 60S ribosomal protein L23a; OS = H sapiens; GN = RPL23A; PE = 1; SV = 1 Cluster of AP-2 complex subunit α-1; OS = H sapiens; GN = AP2A1; PE = 1; SV = 3 (AP2A1_HUMAN) AP-2 complex subunit α-1; OS = H sapiens; GN = AP2A1; PE = 1; SV = 3 Elongation factor 2; OS = H sapiens; GN = EEF2; PE = 1; SV = 4 60S ribosomal protein L13a; OS = H sapiens; GN = RPL13A; PE = 1; SV = 2 40S ribosomal protein S25; OS = H sapiens; GN = RPS25; PE = 1; SV = 1 EF-hand domain-containing protein D1; OS = H sapiens; GN = EFHD1; PE = 1; SV = 1 60S ribosomal protein L26; OS = H sapiens; GN = RPL26; PE = 1; SV = 1 AP-2 complex subunit μ; OS = H sapiens; GN = AP2M1; PE = 1; SV = 2 Ataxin-2-like protein; OS = H sapiens; GN = ATXN2L; PE = 1; SV = 2 60S ribosomal protein L15; OS = H sapiens; GN = RPL15; PE = 1; SV = 2 60S ribosomal protein L24; OS = H sapiens; GN = RPL24; PE = 1; SV = 1 Cluster of thyroid hormone receptor–associated protein 3; OS = H sapiens; GN = THRAP3; PE = 1; SV = 2 (TR150_HUMAN) Thyroid hormone receptor–associated protein 3; OS = H sapiens; GN = THRAP3; PE = 1; SV = 2 THRAP3 protein (fragment); OS = H sapiens; GN = THRAP3; PE = 2; SV = 1 Gelsolin; OS = H sapiens; GN = GSN; PE = 1; SV = 1 Nucleophosmin; OS = H sapiens; GN = NPM1; PE = 1; SV = 2 Histone H4; OS = H sapiens; GN = HIST1H4A; PE = 1; SV = 2 Cofilin 1; OS = H sapiens; GN = CFL1; PE = 1; SV = 3 Glutathione S-transferase μ 3; OS = H sapiens; GN = GSTM3; PE = 1; SV = 3 Prolactin-inducible protein; OS = H sapiens; GN = PIP; PE = 1; SV = 1

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Table W7. (continued) (continued ) Accession No.

No

RS11_HUMAN (+1)

56

CLH1_HUMAN 56.1 RS16_HUMAN (+1) 57 ANXA2_HUMAN [4] 57.1 ANXA2_HUMAN (+3) 57.2 Q5VU59_HUMAN [2] Q5VU59_HUMAN TPM3_HUMAN

58 59

ADT2_HUMAN 60 IF4G1_HUMAN

IF4G1_HUMAN

61

RL19_HUMAN

62

DYST_HUMAN

63

DYST_HUMAN

64

EHD1_HUMAN

65

PPIA_HUMAN (+4)

66

RAN_HUMAN 66.1 RL23A_HUMAN 66.2 AP2A1_HUMAN 67 AP2A1_HUMAN EF2_HUMAN

67.1

RL13A_HUMAN

68

RS25_HUMAN

69

EFHD1_HUMAN

70

RL26_HUMAN

71

AP2M1_HUMAN (+2)

72

ATX2L_HUMAN

73

RL15_HUMAN (+2)

74

RL24_HUMAN (+2)

75

TR150_HUMAN [5]

76 77

TR150_HUMAN 78 Q05D20_HUMAN (+3) GELS_HUMAN NPM_HUMAN

78.1 79

H4_HUMAN 80 COF1_HUMAN 81 GSTM3_HUMAN PIP_HUMAN (continued on next page)

81.1

Accession No. Cluster of F-actin–capping protein subunit β; OS = H sapiens; GN = CAPZB; PE = 1; SV = 4 (CAPZB_HUMAN) F-actin–capping protein subunit β; OS = H sapiens; GN = CAPZB; PE = 1; SV = 4 Cluster of peroxiredoxin-1; OS = H sapiens; GN = PRDX1; PE = 1; SV = 1 (PRDX1_HUMAN) Peroxiredoxin-1; OS = H sapiens; GN = PRDX1; PE = 1; SV = 1 Peroxiredoxin-2; OS = H sapiens; GN = PRDX2; PE = 1; SV = 5 Fructose-bisphosphate aldolase A; OS = H sapiens; GN = ALDOA; PE = 1; SV = 2 40S ribosomal protein S15a; OS = H sapiens; GN = RPS15A; PE = 1; SV = 2 KH domain-containing, RNA-binding, signal transduction-associated protein 1; OS = H sapiens; GN = KHDRBS1; PE = 1; SV = 1 Leucine-rich repeat-containing protein 59; OS = H sapiens; GN = LRRC59; PE = 1; SV = 1 60S ribosomal protein L12; OS = H sapiens; GN = RPL12; PE = 1; SV = 1 Tropomodulin-3; OS = H sapiens; GN = TMOD3; PE = 1; SV = 1 Heterogeneous nuclear ribonucleoprotein H; OS = H sapiens; GN = HNRNPH1; PE = 1; SV = 4 60S ribosomal protein L21; OS = H sapiens; GN = RPL21; PE = 1; SV = 2 Cluster of ADP-ribosylation factor 1; OS = H sapiens; GN = ARF1; PE = 1; SV = 2 (ARF1_HUMAN) ADP-ribosylation factor 1; OS = H sapiens; GN = ARF1; PE = 1; SV = 2 ADP-ribosylation factor 4; OS = H sapiens; GN = ARF4; PE = 1; SV = 3 Cluster of eukaryotic initiation factor 4A-I; OS = H sapiens; GN = EIF4A1; PE = 1; SV = 1 (IF4A1_HUMAN) Eukaryotic initiation factor 4A-I; OS = H sapiens; GN = EIF4A1; PE = 1; SV = 1 60S ribosomal protein L14; OS = H sapiens; GN = RPL14; PE = 1; SV = 4 ATP synthase subunit α, mitochondrial; OS = H sapiens; GN = ATP5A1; PE = 1; SV = 1 40S ribosomal protein S7; OS = H sapiens; GN = RPS7; PE = 1; SV = 1 Histone H2A type 1-B/E; OS = H sapiens; GN = HIST1H2AB; PE = 1; SV = 2 AP-2 complex subunit β; OS = H sapiens; GN = AP2B1; PE = 1; SV = 1 ELAV-like protein 1; OS = H sapiens; GN = ELAVL1; PE = 1; SV = 2 Peroxiredoxin-6; OS = H sapiens; GN = PRDX6; PE = 1; SV = 3 60S ribosomal protein L11; OS = H sapiens; GN = RPL11; PE = 1; SV = 2 78-kDa glucose-regulated protein; OS = H sapiens; GN = HSPA5; PE = 1; SV = 2 40S ribosomal protein S17-like; OS = H sapiens; GN = RPS17L; PE = 1; SV = 1 Cluster of F-actin–capping protein subunit α-1; OS = H sapiens; GN = CAPZA1; PE = 1; SV = 3 (CAZA1_HUMAN) F-actin–capping protein subunit α-1; OS = H sapiens; GN = CAPZA1; PE = 1; SV = 3 Phosphoglycerate mutase 1; OS = H sapiens; GN = PGAM1; PE = 1; SV = 2 Phosphoglycerate kinase 1; OS = H sapiens; GN = PGK1; PE = 1; SV = 3 Cluster of stress-70 protein, mitochondrial; OS = H sapiens; GN = HSPA9; PE = 1; SV = 2 (GRP75_HUMAN) Stress-70 protein, mitochondrial; OS = H sapiens; GN = HSPA9; PE = 1; SV = 2

CAPZB_HUMAN

CAPZB_HUMAN PRDX1_HUMAN [2] PRDX1_HUMAN PRDX2_HUMAN ALDOA_HUMAN RS15A_HUMAN KHDR1_HUMAN

LRC59_HUMAN RL12_HUMAN TMOD3_HUMAN HNRH1_HUMAN (+2) RL21_HUMAN (+1) ARF1_HUMAN [4]

ARF1_HUMAN (+1) ARF4_HUMAN (+1) IF4A1_HUMAN

IF4A1_HUMAN RL14_HUMAN ATPA_HUMAN RS7_HUMAN H2A1B_HUMAN (+14) AP2B1_HUMAN ELAV1_HUMAN PRDX6_HUMAN RL11_HUMAN (+2) GRP78_HUMAN RS17L_HUMAN (+1) CAZA1_HUMAN

CAZA1_HUMAN PGAM1_HUMAN PGK1_HUMAN GRP75_HUMAN

GRP75_HUMAN (continued on next page)

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Table W7. (continued) No 82

82.1 82.2 83 84 85 86 87 88 88.1 88.2 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114

Cluster of transformer-2 protein homolog β; OS = H sapiens; GN = TRA2B; PE = 1; SV = 1 (TRA2B_HUMAN) Transformer-2 protein homolog β; OS = H sapiens; GN = TRA2B; PE = 1; SV = 1 Transformer-2 protein homolog α; OS = H sapiens; GN = TRA2A; PE = 1; SV = 1 Heterogeneous nuclear ribonucleoprotein M; OS = H sapiens; GN = HNRNPM; PE = 1; SV = 3 Heterogeneous nuclear ribonucleoprotein A3; OS = H sapiens; GN = HNRNPA3; PE = 1; SV = 2 40S ribosomal protein S10; OS = H sapiens; GN = RPS10; PE = 1; SV = 1 60S ribosomal protein L18a; OS = H sapiens; GN = RPL18A; PE = 1; SV = 2 Malate dehydrogenase, mitochondrial; OS = H sapiens; GN = MDH2; PE = 1; SV = 3 Cluster of 14–3–3 protein β/α; OS = H sapiens; GN = YWHAB; PE = 1; SV = 3 (1433B_HUMAN) 14-3-3 protein β/α; OS = H sapiens; GN = YWHAB; PE = 1; SV = 3 14-3-3 protein ζ/δ; OS = H sapiens; GN = YWHAZ; PE = 1; SV = 1 Ras GTPase-activating protein-binding protein 2; OS = H sapiens; GN = G3BP2; PE = 1; SV = 2 Ras-related protein Rab-1B; OS = H sapiens; GN = RAB1B; PE = 1; SV = 1 Catenin α-1; OS = H sapiens; GN = CTNNA1; PE = 1; SV = 1 RNA-binding protein EWS; OS = H sapiens; GN = EWSR1; PE = 1; SV = 1 60S ribosomal protein L3; OS = H sapiens; GN = RPL3; PE = 1; SV = 2 Cathepsin D; OS = H sapiens; GN = CTSD; PE = 1; SV = 1 Chloride intracellular channel protein 1; OS = H sapiens; GN = CLIC1; PE = 1; SV = 4 Fatty acid synthase; OS = H sapiens; GN = FASN; PE = 1; SV = 3 Guanine nucleotide-binding protein subunit β-2–like 1; OS = H sapiens; GN =; GNB2L1; PE = 1; SV = 3 Α-actinin-4; OS = H sapiens; GN = ACTN4; PE = 1; SV = 2 Splicing factor, proline- and glutamine-rich; OS = H sapiens; GN = SFPQ; PE = 1; SV = 2 ARF6 protein; OS = H sapiens; GN = ARF6; PE = 2; SV = 1 40S ribosomal protein S5; OS = H sapiens; GN = RPS5; PE = 1; SV = 4 Barrier-to-autointegration factor; OS = H sapiens; GN = BANF1; PE = 1; SV = 1 60S ribosomal protein L28; OS = H sapiens; GN = RPL28; PE = 1; SV = 3 Ribosomal protein S19 (Fragment); OS = H sapiens; PE = 2; SV = 1 Histone H1.5; OS = H sapiens; GN = HIST1H1B; PE = 1; SV = 3 Single-stranded DNA-binding protein, mitochondrial; OS = H sapiens; GN = SSBP1; PE = 1; SV = 1 Transferrin receptor protein 1; OS = H sapiens; GN = TFRC; PE = 1; SV = 2 40S ribosomal protein S14; OS = H sapiens; GN = RPS14; PE = 1; SV = 3 60S ribosomal protein L22; OS = H sapiens; GN = RPL22; PE = 1; SV = 2 60S ribosomal protein L31; OS = H sapiens; GN = RPL31; PE = 1; SV = 1 60S ribosomal protein L35; OS = H sapiens; GN = RPL35; PE = 1; SV = 2 THO complex subunit 4; OS = H sapiens; GN = ALYREF; PE = 1; SV = 3 Serine/arginine repetitive matrix protein 2; OS = H sapiens; GN = SRRM2; PE = 1; SV = 2 Peptidyl-prolyl cis-trans isomerase FKBP4; OS = H sapiens; GN = FKBP4; PE = 1; SV = 3


Table W7. (continued) Accession No.

No

TRA2B_HUMAN [2]

115 116

TRA2B_HUMAN 117 TRA2A_HUMAN 118 HNRPM_HUMAN 119 ROA3_HUMAN 120 RS10_HUMAN 121 RL18A_HUMAN 122 MDHM_HUMAN (+5) 123 1433B_HUMAN [3] 124 1433B_HUMAN (+1) 125 1433Z_HUMAN 126 G3BP2_HUMAN (+1) RAB1B_HUMAN (+1)

126

CTNA1_HUMAN (+1) 127 EWS_HUMAN (+3) RL3_HUMAN (+6)

127.1

CATD_HUMAN

128

CLIC1_HUMAN

129

FAS_HUMAN

130

GBLP_HUMAN

131

ACTN4_HUMAN (+2) 132 SFPQ_HUMAN 133 Q6FH17_HUMAN 134 RS5_HUMAN (+4) 135 BAF_HUMAN 136 RL28_HUMAN (+1) 137 Q8WVX7_HUMAN 138 H15_HUMAN SSBP_HUMAN

138.1

TFR1_HUMAN (+1)

138.2

RS14_HUMAN

139

RL22_HUMAN (+3)

140

RL31_HUMAN 141 RL35_HUMAN THOC4_HUMAN (+1)

142

SRRM2_HUMAN

143

FKBP4_HUMAN

144

Accession No. Actin-related protein 2/3 complex subunit 3; OS = H sapiens; GN = ARPC3; PE = 1; SV = 3 40S ribosomal protein S26; OS = H sapiens; GN = RPS26; PE = 1; SV = 3 Transcription factor A, mitochondrial; OS = H sapiens; GN = TFAM; PE = 1; SV = 1 Polypyrimidine tract-binding protein 1; OS = H sapiens; GN = PTBP1; PE = 1; SV = 1 Adenine phosphoribosyltransferase; OS = H sapiens; GN = APRT; PE = 1; SV = 2 Protein FAM98A; OS = H sapiens; GN = FAM98A; PE = 1; SV = 1 Galectin-7; OS = H sapiens; GN = LGALS7; PE = 1; SV = 2 Triosephosphate isomerase; OS = H sapiens; GN = TPI1; PE = 1; SV = 3 Histone H2B type 1-A; OS = H sapiens; GN = HIST1H2BA; PE = 1; SV = 3 DNA repair protein XRCC1; OS = H sapiens; GN = XRCC1; PE = 1; SV = 2 L-lactate dehydrogenase A chain; OS = H sapiens; GN = LDHA; PE = 1; SV = 2 Cluster of serine/threonine-protein phosphatase PP1-α catalytic subunit; OS = H sapiens; GN = PPP1CA; PE = 1; SV = 1 (PP1A_HU Cluster of serine/threonine-protein phosphatase PP1-α catalytic subunit; OS = H sapiens; GN = PPP1CA; PE = 1; SV = 1 (PP1A_HU Cluster of chloride intracellular channel protein 3; OS = H sapiens; GN = CLIC3; PE = 1; SV = 2 (CLIC3_HUMAN) Chloride intracellular channel protein 3; OS = H sapiens; GN = CLIC3; PE = 1; SV = 2 14-3-3 protein ε; OS = H sapiens; GN = YWHAE; PE = 1; SV = 1 MUC1 isoform J14; OS = H sapiens; GN = MUC1; PE = 2; SV = 1 cDNA FLJ59433, highly similar to elongation factor 1-γ; OS = H sapiens; PE = 2; SV = 1 Non-POU domain-containing octamer-binding protein; OS = H sapiens; GN = NONO; PE = 1; SV = 4 Actin-related protein 2/3 complex subunit 4; OS = H sapiens; GN = ARPC4; PE = 1; SV = 3 60-kDa heat shock protein, mitochondrial; OS = H sapiens; GN = HSPD1; PE = 1; SV = 2 DNA ligase 3; OS = H sapiens; GN = LIG3; PE = 1; SV = 2 40S ribosomal protein S15; OS = H sapiens; GN = RPS15; PE = 1; SV = 2 Matrin-3; OS = H sapiens; GN = MATR3; PE = 1; SV = 2 Serine/arginine-rich splicing factor 10; OS = H sapiens; GN = SRSF10; PE = 1; SV = 1 Cluster of Ras-related protein Rab-10; OS = H sapiens; GN = RAB10; PE = 1; SV = 1 (RAB10_HUMAN) Ras-related protein Rab-10; OS = H sapiens; GN = RAB10; PE = 1; SV = 1 Ras-related protein Rab-13; OS = H sapiens; GN = RAB13; PE = 1; SV = 1 Actin-related protein 3; OS = H sapiens; GN = ACTR3; PE = 1; SV = 3 U1 small nuclear ribonucleoprotein A; OS = H sapiens; GN = SNRPA; PE = 1; SV = 3 Fragile X mental retardation syndrome–related protein 2; OS = H sapiens; GN = FXR2; PE = 1; SV = 2 Anterior gradient protein 2 homolog; OS = H sapiens; GN = AGR2; PE = 1; SV = 1 Clathrin interactor 1; OS = H sapiens; GN = CLINT1; PE = 1; SV = 1 Cell division control protein 42 homolog; OS = H sapiens; GN = CDC42; PE = 1; SV = 2

ARPC3_HUMAN (+2) RS26_HUMAN TFAM_HUMAN PTBP1_HUMAN APT_HUMAN FA98A_HUMAN (+4) LEG7_HUMAN TPIS_HUMAN H2B1A_HUMAN (+19) XRCC1_HUMAN (+3) LDHA_HUMAN (+1) PP1A_HUMAN

PP1A_HUMAN

CLIC3_HUMAN

CLIC3_HUMAN 1433E_HUMAN B6ECA3_HUMAN B4DUP0_HUMAN NONO_HUMAN (+1)

ARPC4_HUMAN (+1) CH60_HUMAN DNLI3_HUMAN RS15_HUMAN (+4) MATR3_HUMAN (+3) SRS10_HUMAN RAB10_HUMAN [3]

RAB10_HUMAN RAB13_HUMAN (+1) ARP3_HUMAN (+2) SNRPA_HUMAN (+6)

FXR2_HUMAN

AGR2_HUMAN EPN4_HUMAN CDC42_HUMAN



Naderi and Vanneste

342.e13

Table W8. Functional Classification of PIP Proteomics Data. Cluster 1

Enrichment Score = 33.10409316169969

P Value

UNIPROT_ID 823648 779090 801125 781935 783263 793235 772335 782492 778688 797877 808990 796446 810885 808508 803377 802424 796817 791016 816191 789968 799749 800698 784832 821689 796227 817032 785872 777984 773042 786531 788428 790706 786137 799716 814248 820040

Protein Name Ribosomal protein L23a pseudogene 63 Ribosomal protein L15 pseudogene 22 Ribosomal protein L8; ribosomal protein L8 pseudogene 2 Ribosomal protein S25 pseudogene 8; ribosomal protein S25 Ribosomal protein L12 pseudogene 2; ribosomal protein L12 Ribosomal protein L21 pseudogene 134; ribosomal protein L21 Ribosomal protein S26 pseudogene 38; ribosomal protein S26 Ribosomal protein L22 pseudogene 11; ribosomal protein L22 Ribosomal protein L18a pseudogene 6; ribosomal protein L18a Ribosomal protein S5 Ribosomal protein S10; ribosomal protein S10 pseudogene 4 Ribosomal protein L31 pseudogene 49; ribosomal protein L31 Ribosomal protein L11 Ribosomal protein S6 pseudogene 25; ribosomal protein S6 Ribosomal protein L35; ribosomal protein L35 pseudogene 1 Ribosomal protein L3; similar to 60S ribosomal protein L3 (L4) Ribosomal protein L10a pseudogene 6; ribosomal protein L10a Ribosomal protein L13a pseudogene 7; ribosomal protein L13a Ribosomal protein S3 pseudogene 3; ribosomal protein S3 Ribosomal protein S3A pseudogene 5; ribosomal protein S3a Ribosomal protein S19 pseudogene 3; ribosomal protein S19 Ribosomal protein L24; ribosomal protein L24 pseudogene 6 Ribosomal protein S15 pseudogene 5; ribosomal protein S15 Fragile X mental retardation, autosomal homolog 2 Ribosomal protein, large, P0 pseudogene 2; ribosomal protein Ribosomal protein S14 Ribosomal protein L26 pseudogene 33; ribosomal protein L26 Ribosomal protein L14 Ribosomal protein S7; ribosomal protein S7 pseudogene 11 Ribosomal protein L19; ribosomal protein L19 pseudogene 12 Ribosomal protein S15a pseudogene 17; ribosomal protein S15a Ribosomal protein S16 pseudogene 1 Ribosomal protein S11 pseudogene 5; ribosomal protein S11 Ribosomal protein L10; ribosomal protein L10 pseudogene 15 Ribosomal protein S18 pseudogene 12 Ribosomal protein L28

9.3E-76

Cluster 2

Enrichment Score: 13.296661105241336

P Value

UNIPROT_ID 814227 824519 816714 806558 786723 826437 810069 797048 825011 826656 825901 800520 776456 798354 776781 806156 791398 802485 816065

Protein Name Heterogeneous nuclear ribonucleoprotein A2/B1 Heterogeneous nuclear ribonucleoprotein A1-like 3 FUS-interacting protein (serine/arginine-rich) 1 THO complex 4 Matrin 3 Heterogeneous nuclear ribonucleoprotein H1 (H) Heterogeneous nuclear ribonucleoprotein A3 Transformer 2 β homolog (Drosophila) Splicing factor, arginine/serine-rich 4 Splicing factor proline/glutamine-rich Polypyrimidine tract binding protein 1 Serine/arginine repetitive matrix 2 Non-POU domain containing, octamer-binding Small nuclear ribonucleoprotein polypeptide A Transformer 2 α homolog (Drosophila) Splicing factor, arginine/serine-rich 6 Heterogeneous nuclear ribonucleoprotein M ELAV (embryonic lethal, abnormal vision, Drosophila)-like 1 Splicing factor, arginine/serine-rich 9

5.14E-32

Cluster 3


P Value

UNIPROT_ID 775719 784692 781820 784865 799393 783255

Protein Name Histone cluster Histone cluster Histone cluster Histone cluster Histone cluster Histone cluster

2.47E-12 1, H2ae; histone cluster 1, H2ab 1, H4l; histone cluster 1 1, H1d 1, H1c 1, H1b 1, H2ba (continued on next page)

342.e14


Naderi and Vanneste


Table W8. (continued) (continued ) Cluster 4


P Value

UNIPROT_ID 775114 819298 799685 773758

Protein Name Gelsolin (amyloidosis, Finnish type) Capping protein (actin filament) muscle Z-line, β Capping protein (actin filament) muscle Z-line, α 1 Similar to actin related protein 2/3 complex subunit 3

6.01E-08

Cluster 5


P Value

UNIPROT_ID 777280 797086 789199 783500

Protein Name Adaptor-related protein complex 2, β 1 subunit Adaptor-related protein complex 2, α 1 subunit Adaptor-related protein complex 2, mu 1 subunit Clathrin, heavy chain (Hc)

4.18E-09

Cluster 6


P Value

UNIPROT_ID 794683 824702 823808 819977 825257 803034

Protein Name ADP-ribosylation factor 4 RAB13, member RAS oncogene family; similar to hCG24991 RAB10, member RAS oncogene family RAB1B, member RAS oncogene family ADP-ribosylation factor 6 ADP-ribosylation factor 1

9.54E-11

Table W9. Canonical Pathways Associated with PIP-Binding Partners. Canonical Pathways

− Log (P Value)

Ratio to Total

Molecules

EIF2 Signaling

4.37E+01

1.94E-01

Regulation of eIF4 and p70S6K signaling

1.55E+01

1.03E-01

Remodeling of epithelial adherens junctions Clathrin-mediated endocytosis signaling Regulation of actin-based motility by Rho Integrin signaling

7.03E+00 5.46E+00 4.20E+00 3.60E+00

1.14E-01 5.05E-02 6.59E-02 3.85E-02

RPL11, RPL24, RPL22, RPS18, RPL14, RPL26, EIF4G1 RPS17/RPS17L, RPS11, RPS7, RPL35, RPS3A, RPL18A, RPL19 RPL12, RPL8, PPP1CA, RPS5, RPS3, RPS10, RPL31, RPL3, RPS19 RPL21, RPL23A, RPLP0, RPL10A, RPS6, RPL15, RPS16, RPS26 RPL28, RPL10, EIF4A1, RPS15, RPS15A, RPS25, RPL13A, RPS14 RPS18, RPS19, RPS17/RPS17L, EIF4G1, RPS11, RPS7, RPS6 RPS6, RPS3A, RPS16, RPS26, EIF4A1, RPS15, RPS15A, RPS25 RPS5, RPS3, RPS10, RPS14 ARF6, ACTR3, TUBB4B, CTNNA1, ARPC3, ACTN4, TUBB, ARPC4 AP2B1, AP2M1, AP2A1, ARF6, ACTR3, CLTC, TFRC, ARPC3, CDC42 ARPC4 ACTR3, CFL1, ARPC3, CDC42, GSN, ARPC4 ARF1, ARF6, ACTR3, ARF4, ARPC3, ACTN4, CDC42, ARPC4