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. 2014 Apr;16(4):329-42.e1-14.
doi: 10.1016/j.neo.2014.04.001.

Prolactin-induced protein is required for cell cycle progression in breast cancer

Ali Naderi  1 , Marion Vanneste  2
Affiliations

Prolactin-induced protein is required for cell cycle progression in breast cancer

VSports - Ali Naderi et al. Neoplasia. 2014 Apr.

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. VSports手机版.

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Figures

Figure W1
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.
Figure W2
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.
Figure 1
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).
Figure 2
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 < .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 < .01 is for AR − tumors versus other groups.
Figure 3
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.
Figure 4
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 < .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.
Figure 5
Figure 5
The effect of PIP expression on G2/M and aneuploidy. (A) The percentage of cells in different phases of cell cycle following PIP-KD 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 < .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.
Figure 6
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 < .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.
Figure 7
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 PIP-KD 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 (Tyr397) 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.
Figure 8
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.
Figure 9
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.
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References

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Supplementary References

    1. Lehmann B.D., Bauer J.A., Chen X., Sanders M.E., Chakravarthy A.B., Shyr Y., Pietenpol J.A. Identification of human triple-negative breast cancer subtypes and preclinical models for selection of targeted therapies. J Clin Invest. 2011;121:2750–2767. - PMC - PubMed
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    1. Naderi A., Liu J. Inhibition of androgen receptor and Cdc25A phosphatase as a combination targeted therapy in molecular apocrine breast cancer. Cancer Lett. 2010;298:74–87. - PubMed
    1. Naderi A., Hughes-Davies L. A functionally significant cross-talk between androgen receptor and ErbB2 pathways in estrogen receptor negative breast cancer. Neoplasia. 2008;10:542–548. - PMC - PubMed

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