Insulin-like growth factor-I induces CLU expression through Twist1 to promote prostate cancer growth

Accepted Manuscript Insulin-like growth factor-I induces CLU expression through Twist1 to promote prostate cancer growth Ario Takeuchi, Masaki Shiota, Eliana Beraldi, Daksh Thaper, Kiyoshi Takahara, Naokazu Ibuki, Michael Pollak, Michael E. Cox, Seiji Naito, Martin E. Gleave, Amina Zoubeidi PII: DOI: Reference:

S0303-7207(14)00016-1 http://dx.doi.org/10.1016/j.mce.2014.01.012 MCE 8752

To appear in:

Molecular and Cellular Endocrinology Molecular and Cellular Endocrinology

Received Date: Revised Date: Accepted Date:

19 June 2013 27 December 2013 14 January 2014

Please cite this article as: Takeuchi, A., Shiota, M., Beraldi, E., Thaper, D., Takahara, K., Ibuki, N., Pollak, M., Cox, M.E., Naito, S., Gleave, M.E., Zoubeidi, A., Insulin-like growth factor-I induces CLU expression through Twist1 to promote prostate cancer growth, Molecular and Cellular Endocrinology Molecular and Cellular Endocrinology (2014), doi: http://dx.doi.org/10.1016/j.mce.2014.01.012

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Insulin-like growth factor-I induces CLU expression through Twist1 to promote prostate cancer growth

Ario Takeuchi1, Masaki Shiota1, Eliana Beraldi1, Daksh Thaper1, Kiyoshi Takahara1, Naokazu Ibuki1, Michael Pollak2, Michael E. Cox1, Seiji Naito3, Martin E. Gleave1, and Amina Zoubeidi1

1

The Vancouver Prostate Centre and Department of Urologic Sciences, University of British

Columbia, Vancouver, British Columbia, Canada 2

Department of Medicine and Oncology, McGill University, Montreal, Quebec, Canada

3

Department of Urology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan

Short title: IGF-I induces CLU via Twist1 Keywords: clusterin; insulin-like growth factor-I; prostate cancer; STAT3; Twist1

Corresponding Author: Dr. Amina Zoubeidi The Vancouver Prostate Centre and Department of Urologic Sciences, University of British Columbia, 2660 Oak Street, Vancouver, British Columbia, Canada V6H 3Z6. Phone: +1-604-875-4818 Fax: +1-604-875-5654 E-mail: [email protected]

1

ABSTRACT Clusterin (CLU) is cytoprotective molecular chaperone that is highly expressed in castrate-resistant prostate cancer (CRPC). CRPC is also characterized by increased insulin-like growth factor (IGF)-I responsiveness which induces prostate cancer survival and CLU expression. However, how IGF-I induces CLU expression and whether CLU is required for IGF-mediated growth signaling remain unknown. Here we show that IGF-I induced CLU via STAT3-Twist1 signaling pathway. In response to IGF-I, STAT3 was phosphorylated, translocated to the nucleus and bound to the Twist1 promoter to activate Twist1 transcription. In turn, Twist1 bound to E-boxes on the CLU promoter and activated CLU transcription. Inversely, we demonstrated that knocking down Twist1 abrogated IGF-I induced CLU expression, indicating that Twist1 mediated IGF-I–induced CLU expression. When PTEN knockout mice were crossed with lit/lit mice, the resultant IGF-I deficiency suppressed Twist1 as well as CLU gene expression in mouse prostate glands. Moreover, both Twist1 and CLU knockdown suppressed prostate cancer growth accelerated by IGF-I, suggesting the relevance of this signaling not only in an in vitro, but also in an in vivo. Collectively, this study indicates that IGF-I induces CLU expression through sequential activation of STAT3 and Twist1, and suggests that this signaling cascade plays a critical role in prostate cancer pathogenesis.

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Introduction Prostate cancer is the most common solid malignant tumor among males in Western countries (Jemal et al., 2010). A series of epidemiological and biological studies demonstrate that the insulin-like growth factor (IGF) axis is a critical regulator of growth, survival, and metastatic potential in a variety of malignancies and is closely implicated in prostatic carcinogenesis and prostate cancer progression as well as resistance to castration therapy (Chan et al., 1998; Krueckl et al., 2004; Nickerson et al., 2001; Wolk et al., 1998). We have previously demonstrated that IGF-I promoted human prostate cancer cell growth and that increased IGF-I receptor (IGF-IR) expression and signaling are components of castrate resistant progression (Krueckl et al., 2004; Takahara et al., 2011). IGFs bind to the tyrosine kinase IGF-IR, which is a heterotetrameric type I receptor protein-tyrosine kinase composed of two ligand-binding α-subunits and two transmembrane β-subunits. The binding of ligand to IGF-IR induces auto-phosphorylation of the β-subunits of the receptor complex and further activation of the protein-tyrosine kinase activity (Hubbard et al., 1994; Weiss & Schlessinger 1998). Once activated, IGF-IR recruits and phosphorylates various downstream targets such as the insulin receptor substrate-1 and -2 which activate many signaling pathways, including Ras/Raf/mitogen-activated protein kinase (MAPK) and PI3K/Akt, as well as signal transducer and activator of transcription 3 (STAT3; Zong et al., 2000) resulting in cell growth and survival. Clusterin (CLU) is a stress-induced cytoprotective chaperone, and involved in many biological processes such as sperm maturation, tissue differentiation, tissue remodeling, membrane recycling, lipid transportation, cell proliferation and cell death. CLU has been shown expressed in many human cancers (Zhong et al., 2010). Increased levels of CLU have been reported in breast, colon, lung, bladder, prostate and other cancers (July et al., 2004; Kevans et al., 2009; Miyake et al., 2002; So et al., 2005; Steinberg et al., 1997). In prostate, CLU levels are low in benign prostate epithelial cells, but increase in prostate cancers with higher Gleason grade (Steinberg et al., 1997). Furthermore, CLU 3

expression increases as prostate cancers adapt to androgen-deprivation therapy (July et al., 2002). These data indicate that CLU is also implicated in prostate carcinogenesis and prostate cancer progression. Similarly, numerous evidences showed that a basic helix–loop–helix transcription factor Twist1 is also involved in pathogenesis of various cancers (Franco et al., 2011), including castration resistance in prostate cancer (Shiota et al., 2010). IGF-I axis induced CLU expression after irradiation via Src-MEK-ERK-EGR1 signaling in human breast cancer MCF-7 cells (Criswell et al., 2005). However, the mechanism and role of CLU induction by IGF-I in prostate cancer remain unrevealed. In this study, we set out to define links between IGF-I signaling and Twist1/CLU expression in prostate cancer, identifying STAT3 as a downstream effector of IGF-I.

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MATERIAL AND METHODS Cell culture and transfection The human prostate cancer cell line, PC-3, was purchased from the American Type Culture Collection (ATCC authentication by isoenzymes analysis) and maintained in Dulbecco’s Modified Eagle’s Medium (DMEM; Thermo Scientific, Waltham, MA, USA) supplemented with 5% fetal-bovine serum (FBS). The human prostate cancer cell line LNCaP was kindly provided by Dr. Leland W.K. Chung (Cedars-Sinai Medical Center, Los Angeles, CA, USA), tested and authenticated by whole-genome and whole-transcriptome sequencing on Illumina Genome Analyzer IIx platform in 2009. LNCaP cells were maintained in RPMI 1640 (Thermo Scientific) supplemented with 5% FBS. Antibodies and reagents Antibodies against Myc (sc-815), CLU (sc-6419) and Twist1 (sc-81417) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-phosphorylated STAT3Tyr705 (p-STAT3Tyr705, #9131), anti-phosphorylated STAT3Ser727 (p-STAT3Ser727, #9134) and anti-STAT3 (#9139) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-Lamin B1 and anti-β-actin antibodies were purchased from Abcam (Cambridge, MA, USA) and Sigma (St Louis, MO, USA), respectively. Human recombinant IGF-I was obtained from Fitzgerald (Acton, MA, USA). Plasmids and siRNAs Twist1-Myc-Flag plasmid expressing C-terminally Myc-Flag-tagged Twist1 protein and the corresponding mock plasmid (Myc-Flag plasmid) were purchased from OriGene (Rockville, MD, USA). The Twist1 reporter plasmid (Twist–Luc) was kindly provided from Dr. Wang LH (Mount Sinai School of Medicine, New York, NY, USA; Cheng et al., 2008). CLU reporter plasmids (CLU–Luc –1,998/+254, –1,998/–702, –1,116/–702, and –707/+254) containing various lengths of the promoter and first exon of the wild-type human CLU gene were constructed as described previously (Shiota et al., 2011). The following double-stranded 25-bp siRNA oligonucleotides were commercially generated 5

(Invitrogen, Carlsbad, CA, USA): 5’-CUUCCUCGCUGUUGCUCAGGCUGUC-3’ for Twist1 siRNA #1; 5’-UUGAGGGUCUGAAUCUUGCUCAGCU-3’ for Twist1 siRNA #2. The sequence of siRNA

corresponding

to

the

human

CLU

initiation

site

in

exon

II

was

5’-GCAGCAGAGUCUUCAUCAU-3’ (Dharmacon Research Inc.). Stealth™ RNAi Negative Control Medium GC Duplex #2 (Invitrogen) was used as a control siRNA. Cells were transfected with the indicated siRNA or the indicated plasmid as previously described (Zoubeidi et al., 2010a; Zoubeidi et al., 2010b). Quantitative reverse transcription (RT)-PCR RNA extraction and RT-PCR were performed as described previously (Lamoureux et al., 2011). Real time monitoring of PCR amplification of cDNA was performed using the following primer pairs and probes, Twist1 (Hs00361186_m1), CLU (Hs00156548_m1) and GAPDH (Hs03929097_g1) (Applied Biosystems) on ABI PRISM 7900 HT Sequence Detection System (Applied Biosystems) with TaqMan Gene Expression Master Mix (Applied Biosystems). Target gene expression was normalized to GAPDH levels in respective samples as an internal control. The results are representative of at least three independent experiments. Western blot analysis Whole-cell extracts were obtained by lysis of cells in an appropriate volume of ice-cold RIPA buffer composed of 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.5% sodium deoxycholate, 1% Nonidet P-40, 0.1% sodium dodecyl sulfate (SDS) containing 1 mM Na3VO4, 1 mM NaF, 1 mM phenylmethylsulfonyl fluoride and protease inhibitor cocktail tablets (Complete, Roche Applied Science, Indianapolis, IN, USA). Nuclear and cytoplasmic extracts were obtained using CelLyticTM NuCLEARTM Extraction Kit (Sigma) according to manufacturer’s protocol. Cellular extracts were clarified by centrifugation at 13,000 x g for 10 min and protein concentrations of the extracts determined by a BCA protein assay kit (Thermo Scientific). Thirty micrograms of the extracts were boiled for 5 min in SDS sample buffer, separated by SDS-PAGE, and transferred onto a polyvinylidene difluoride (PVDF) membrane. Membranes were probed with dilutions of primary 6

antibodies followed by incubation with horseradish peroxidase-conjugated secondary antibodies. After extensive washing, proteins were visualized by a chemiluminescent detection system (GE Healthcare, Buckinghamshire, UK). Luciferase reporter assay Prostate cancer cells were transfected with 0.5 μg of the indicated reporter plasmid, expression plasmid, siRNA, and 0.05 μg of pRL-TK as an internal control. 24 hours post transfection, media was changed to serum-free media followed by IGF-I treatment. The luciferase activities were measured using a Dual-Luciferase Reporter Assay System (Promega) and a microplate luminometer (EG&G Berthold). The Firefly luciferase activities were corrected by the corresponding Renilla luciferase activities and protein concentration. The results are representative of at least three independent experiments. Chromatin immunoprecipitation assay (ChIP assay) LNCaP cells were seeded and stimulated with 100 ng/ml IGF-I followed by paraformaldehyde cross-linking and micrococcal nuclease digestion to achieve a DNA smear of 200–1000 bp. ChIP assay on the indicated genes was performed using SimpleChIPTM Enzymatic Chromatin IP Kit (Agarose Beads) according to the manufacturer’s protocol (Cell Signaling Technology). Quantitative RT-PCR assay was performed using ABI PRISM 7900 HT Sequence Detection System with 2 μL of 20 μL DNA extraction, the primer pairs below and RT2 Real-TimeTM SYBR Green/Rox PCR master mix (Qiagen, Valencia, CA, USA). The results are representative of at least three independent experiments. The primer pairs for Twist1 promoter were Fw; 5’-TGCCTTTCCCATGGACTGGG-3’ and Rv; 5’-GAGTTCCAAAGGCCAAACCG-3’ as described previously (Cheng et al. 2008). The primer pairs for CLU –02 (GPH025704(–)02A) targeting around –1,403 bp, CLU –01 (GPH025704(–) 01A) targeting around –417 bp, CLU +07 (GPH025704(+)07A) targeting around +6,603 bp, and RPL30 gene (exon 3) were described previously (Shiota et al., 2011). Production and characterization of Ghrhr(lit/lit)/Cre/PTEN(fl/fl) The production of Ghrhr(lit/lit)/Cre/PTEN(fl/fl) was previously described (Takahara et al., 7

2013). Briefly, we crossed pbARR2-Cre, PTEN (fl/fl) mice (Wang et al., 2003) with GHRHR (lit/lit) mice (Yang et al., 1996) to produce lit/lit and lit/+ PTEN-/- mice. Mice serums and prostates were harvested in accordance with the guidelines of the Canadian Council on Animal Care and with appropriate institutional certification between 15 and 20 weeks of age. Nineteen mice were collected for each cohort after genotyping to clarify lit heterozygosity (lit/+) and homozygosity (lit/lit), Cre recombinase and PTEN fl/fl status from tail clip DNA. Serum IGF-I levels were measured using ELISA from (R&D Systems, Minneapolis, MN, USA). Murine Twist1 (Mm00442036_m1) and murine CLU (Mm00442773_m1) transcript levels were measured using RNA from mice prostates by quantitative RT-PCR normalized to murine GAPDH (Mm99999915_g1) transcript levels. Cell growth assay LNCaP cells (2.5 x 104) transfected with 10 nM of the indicated siRNA were plated in 24-well plates. The following day, media were changed into media supplemented with 1% serum from lit/lit or lit/+ mice with or without 100 ng/mL IGF-I. After incubation for 96 h, cell growth was measured using the crystal violet assay as described previously (Shiota et al., 2011). The results are representative of at least 3 independent experiments. Immunohistochemistry Prostate tissues from PTEN knockout lit/lit mice and PTEN knockout lit/+ mice at 15 and 20 weeks of age were obtained in accordance with the guidelines of the Canadian Council on Animal Care and with appropriate institutional certification. Immunohistochemical staining was conducted as previously described (Zoubeidi et al., 2010b) using the Ventana Discover XT TM autostainer (Ventana Medical System, Tuscan, AZ, USA) with enzyme labeled biotin streptavidin system and solvent resistant DAB Map kit by antibodies against CLU (Santa Cruz Biotechnology), IGF-IR (Sigma), p-STAT3Tyr705 (Cell Signaling Technology), Twist1 (Sigma). Statistical analysis All data were assessed using the Student’s t-test. Levels of statistical significance were set at P < 0.05. 8

RESULTS IGF-I induces both Twist1 and CLU expression in prostate cancer cells It is known that IGF-I activates Twist1 in NIH-3T3 fibroblasts (Dupont et al., 2001) and CLU expression in breast cancer cell lines (Criswell et al., 2005). Since Twist1 is a transcription factor, we intended to determine whether IGF-I induced CLU expression via Twist1 in human prostate cancer cells. First, we examined the appropriate concentration inducing Twist1 and CLU expression in LNCaP and PC-3 cells. Then, 100 ng/mL IGF-I most induced Twist1 as well as CLU expression both in LNCaP and PC-3 cells (data not shown). Then, we chose 100 ng/ml IGF-I for treatment thereafter. Next, LNCaP and PC-3 cell lines were treated with IGF-I in a time dependent manner and both Twist1 and CLU expression were evaluated at mRNA and protein levels. We found that IGF-I increased Twist1 as well as CLU expression at transcript level (Fig. 1A) and protein level (Fig. 1B) in LNCaP and PC-3 cells. Twist1 binds to CLU promoter region and regulates CLU expression The finding above prompted us to examine the functional link between Twist1 transcription factor and CLU. As shown in Fig. 2A, Twist1 knockdown using 2 different Twist1-specific siRNAs reduced basal CLU expression at both transcript and protein levels in LNCaP cells. Inversely, Twist1 over-expression up-regulated CLU mRNA and protein expression (Fig. 2B). Since Twist1 is known as an E-box (5’-CANNTG-3’) binding transcription factor (Li et al., 1995), we investigated whether Twist1 directly regulates CLU transcription. We first searched for putative E-box binding sites in the CLU promoter region between –2,000 bp and +500 bp from transcription start site (TSS) and found that are 10 E-boxes in CLU promoter as shown in Fig. 2C. We next analyzed CLU promoter activity using different truncated regions (Fig. 2C). We found that CLU promoter activity was highest between –1,998 bp and –702 bp in LNCaP cells which contains 7 E-boxes (Fig. 2D) as a potential Twist1-binding sites, similarly to the result using PC-3 cells (Shiota et al., 2012). To further evaluate if Twist1 can regulates CLU promoter activity, Twist1 was overexpressed and CLU transcriptional activity was analyzed using different CLU promoter 10

constructs. As shown in Fig. 2E, Twist1 increased luciferase activity of CLU–Luc –1,998/–702, but not CLU–Luc –1,116/–702. These findings suggested that the cis-element of CLU promoter region containing –1,998/–1,116 bp was activated by Twist1. Inversely, Twist1 knockdown reduced CLU promoter transcriptional activity (Fig. 2F). To confirm Twist1 binding to CLU promoter region, we performed ChIP assay against CLU gene in LNCaP cells. The results showed that Twist1 bound to CLU promoter regions around –1,400 bp from TSS represented by CLU –02, while they did not bind to the CLU gene regions around +6,600 bp from TSS and RPL30 gene (Fig. 2G). IGF-I induces CLU expression via Twist1 To further evaluate whether Twist1 expression is required for IGF-I–induced CLU expression, Twist1 was silenced and levels of Twist1 and CLU were evaluated in the absence or presence of IGF-I. Twist1-specific siRNA successfully down-regulated both basal and IGF-I–induced Twist1 expression (Fig. 3A, left) as well as CLU mRNA at basal level. Interestingly, we found that Twist1 knockdown abrogated IGF-I–induced CLU expression at mRNA levels (Fig. 3A, right) and protein levels (Fig. 3B) in both LNCaP and PC-3 cells. These data suggest that Twist1 is involved in basal CLU expression, as well as required for IGF-I–induced CLU expression. In addition, luciferase reporter assay using CLU reporter plasmid also revealed that Twist1 knockdown ameliorated CLU induction by IGF-I (Fig. 3C). IGF-I activates STAT3 transcription factor, resulting in Twist1 up-regulation To investigate the mechanism of Twist1/CLU induction by IGF-I, we focused on STAT3 transcription factor because it was reported that STAT3 transcriptionally regulated Twist1 expression (Cheng et al., 2008). We next analyzed the effect of IGF-I on STAT3 phosphorylation. Our data showed that IGF-I induces STAT3 phosphorylation only on Tyr705 but not on Ser727, resulting in an activation of STAT3 (Fig. 4A). This result was supported by the finding that IGF-I facilitated STAT3 translocation into nucleus (Fig. 4B). As a result, IGF-I stimulated STAT3 binding to Twist1-promotor region, resulting in activation of Twist1 transcription (Fig. 4C), thereby augmented Twist1 binding to CLU-promoter region (Fig. 4D). Consistently, reporter assay using Twist1 reporter plasmid showed 11

an increased transcription of Twist1 gene by IGF-I (Fig. 4E). These data show that IGF-I promoted STAT3 activation and STAT3 is at least in part needed to mediate the IGF-I–induced Twist1 expression and subsequent CLU expression. IGF-I/Twist1/CLU signaling plays a critical role in mice prostate cancer proliferation To investigate the biologic relevance of the above findings, we assessed Twist1 and CLU expression in prostate tissues from PTEN knockout mice (Wang et al., 2003) crossed with lit/lit mice, which harbor growth-hormone-releasing hormone receptor (GHRHR) mutation abolishing GHRHR function (Wang et al., 2003). Lack of growth-hormone-releasing hormone signaling in lit/lit mice results in marked reduction of serum growth hormone, which in turn leads to reducing serum IGF-I level (Fig. 5A), known to correlate with IGF-I level in prostate (Wang et al., 2008). Consistently with the preceding in vitro data, Twist1 and CLU expression were lower in prostate tissues, harvested between 15 and 20 weeks of age, from PTEN knockout lit/lit mice compared with those of PTEN knockout lit/+ mice (Fig. 5B). As well, immunohistochemistry against prostate tissues from PTEN knockout lit/lit mice and PTEN knockout lit/+ mice suggested that prominent decreased levels of IGF-IR and Twist1 in lit/lit mice (Fig. 5C). Previously we showed that prostate cancer grew less rapidly in lit/lit mice compared with lit/+ mice in an in vitro as well as an in vivo (Takahara et al., 2011). Subsequently, we examined whether cell proliferation induced by IGF-I is affected by Twist1 or CLU silencing. As we previously reported, LNCaP cell growth was promoted by IGF-I, while this growth promotion was almost completely abolished by either Twist1 or CLU knockdown (Fig. 6A), suggesting that both Twist1 and CLU are important downstream mediators of IGF-I–induced prostate cancer growth.

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DISCUSSION In this study, we identified a novel mechanism by which IGF-I regulates CLU expression in prostate cancer cells via the STAT3-Twist1 pathway. We went on to demonstrate that once IGF-I activates STAT3, STAT3 translocates to the nucleus, binds to the Twist1 promoter, resulting in Twist1 up-regulation. Twist1 subsequently binds to E-boxes on CLU promoter and enhance CLU expression, thereby creating a feed-forward loop which leads to increase of cell proliferation in prostate cancer (Fig. 6B). Our data showed that IGF-I induced STAT3 phosphorylation in prostate cancer cells, confirming previous reports in human fibroblasts (HEK293T cells; Zong et al., 2000) and suggesting that the role of IGF-I on STAT3 activation was conserved across different cell lines. Once phosphorylated, STAT3 translocated to the nucleus and regulated Twist1 expression in breast cancer cells (Cheng et al., 2008). STAT3 activation positively correlated with Twist1 expression in breast cancer tissues (Cheng et al., 2008). Similarly, epidermal-growth factor (EGF)–induced Twist1 transcription was reported to be mediated by STAT3 in several cancer cells (Lo et al., 2007). Moreover, in hepatocellular carcinoma, activated STAT3 and Twist1 expressions were positively correlated (Zhang et al., 2012). Thus, the connection between STAT3 and Twist1 has been established in various cancers including prostate cancer (Cheng et al., 2008; Cho et al., 2013; Hsu et al., 2012; Teng et al., 2013). Additionally, in this study, STAT3 phosphorylation at Tyr705 was induced by IGF-I concurret with nuclear translocation, which is consistent with the previous report (Wen et al., 1995). Moreover, we found that STAT3 bound to Twist1 promoter region in prostate cancer, leading to Twist1 gene expression, which was increased by IGF-I treatment. Twist1, a basic helix–loop–helix transcription factor, has been described as a proto-oncogene (Hamamori et al., 1997; Quertermous et al., 1994) that promoted breast cancer metastasis (Yang et al., 2004). Similar to CLU, Twist1 was also up-regulated in various malignant tumors, including prostate cancer (Wallerand et al., 2010; Wang et al., 2004). Moreover, we have recently shown that Twist1 was involved in prostate cancer growth (Shiota et al., 2008) as well as resistance to castration through 13

androgen receptor (Shiota et al., 2010). Collectively, Twist1 plays a key role in the development and progression of prostate cancer similar to that ascribed to CLU. Like Twist1 (Dupont et al., 2001), CLU was also known to be induced by IGF-I (Criswell et al., 2005). In this study, Twist1 knockdown decreased basal CLU transcript and protein, as well as IGF-I–induced CLU expression, indicating that IGF-I–induced CLU expression was mediated by Twist1. Furthermore, we found that Twist1 regulated CLU transcription by reporter assay and ChIP assay. These findings link Twist1 regulation of CLU expression, under IGF-I stimulation, as a potential pathway that promotes prostate cancer growth. The pbARR2-Cre, PTEN (fl/fl) mouse model, which lead to de novo formation of prostate tumors, is one model that mimicks human prostate cancer from initiation to local invasion and metastasis (Trotman et al., 2003; Wang et al., 2003). Prostate-specific loss of PTEN expression resulted in invasive carcinoma with lymphovascular invasion within 12 weeks, which progressed to lung metastasis (Wang et al., 2003). Deletion or mutation of the tumor suppressor PTEN gene has been implicated in many human cancers and has been seen in up to 30% of primary prostate cancers and >64% of prostate metastases, making PTEN an important candidate gene for prostate cancer development and progression (Majumder & Sellers 2005; Suzuki et al., 1998). Furthermore, CLU expression was elevated in PTEN knockout mice (Wang et al., 2003). To define links between IGF-I signaling and CLU in prostate cancer growth, we crossed pbARR2-Cre, PTEN (fl/fl) mice with GHRHR (lit/lit) mice. It has been known that in lit/lit mice, several proto-oncogenic pathways including MAPK and PI3K/Akt were down-regulated (Takahara et al., 2011). In addition, in PTEN knockout lit/lit mice model, Twist1 as well as CLU expression was reduced in IGF-1–deficient lit/lit mice, which supported our in vitro data that IGF-I induced Twist1 and CLU expression in LNCaP and PC-3 cells. Furthermore, this study suggested that Twist1 as well as CLU plays key roles in IGF-1–induced prostate cancer cell proliferation. Since IGF-I has been a well-known promoter of prostate cancer growth, these data identified Twist1 and CLU as important mediators of IGF-I–stimulated prostate pathogenesis in this model. 14

In summary, we identified a novel Twist1/CLU pathway stimulated by IGF-I involving STAT3 phosphorylation, and then CLU. Therefore, signaling from IGF-I to CLU provided a molecular mechanism that might explain at least in part the influence of IGF-I on prostate cancer.

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Declaration of interest There are no conflicts of interest.

Funding This study was supported by the Terry Fox New Frontiers Program, the Canadian Institutes of Health Research, the Prostate Cancer Foundation USA.

Acknowledgements We are grateful to Dr. Lu-Hai Wang (Mount Sinai School of Medicine, New York, NY, USA) providing the Twist–Luc reporter plasmid. We thank Howard Tearle and Ladan Fazli1 for their excellent technical assistance.

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Cancer Res 3 1707–1711 Suzuki H, Freije D, Nusskern DR, Okami K, Cairns P, Sidransky D, Isaacs WB & Bova GS 1998 Interfocal heterogeneity of PTEN/MMAC1 gene alterations in multiple metastatic prostate cancer tissues. Cancer Res 58 204–209 Takahara K, Tearle H, Ghaffari M, Gleave ME, Pollak M & Cox ME 2011 Human prostate cancer xenografts in lit/lit mice exhibit reduced growth and androgen-independent progression. Prostate 71 525–537 Takahara K, Ibuki N, Ghaffari M, Tearle H, Ong CJ, Azuma H, Gleave ME, Pollak M & Cox ME 2013 The influence of growth hormone/insulin-like growth factor deficiency on prostatic dysplasia in pbARR2-Cre, PTEN knockout mice. Prostate Cancer Prostatic Dis 16 239–247 Teng J, Wang X, Xu Z & Tang N 2013 HBx-dependent activation of Twist mediates STAT3 control of epithelium-mesenchymal transition of liver cells. J Cell Biochem 114 1097–1104 Trotman LC, Niki M, Dotan ZA, Koutcher JA, Di Cristofano A, Xiao A, Khoo AS, Roy-Burman P, Greenberg NM, Van Dyke T, Cordon-Cardo C & Pandolfi PP 2003 Pten dose dictates cancer progression in the prostate. PLoS Biol 1 E59 Wallerand H, Robert G, Pasticier G, Ravaud A, Ballanger P, Reiter RE & Ferrière JM 2010 The epithelial-mesenchymal transition-inducing factor TWIST is an attractive target in advanced and/or metastatic bladder and prostate cancers. Urol Oncol 28 473–479 Wang S, Gao J, Lei Q, Rozengurt N, Pritchard C, Jiao J, Thomas GV, Li G, Roy-Burman P, Nelson PS, Liu X & Wu H 2003 Prostate-specific deletion of the murine Pten tumor suppressor gene leads to metastatic prostate cancer. Cancer Cell 4 209–221 Wang X, Ling MT, Guan XY, Tsao SW, Cheung HW, Lee DT & Wong YC 2004 Identification of a novel function of TWIST, a bHLH protein, in the development of acquired taxol resistance in human cancer cells. Oncogene 23 474–482 Wang Z, Luque RM, Kineman RD, Ray VH, Christov KT, Lantvit DD, Shirai T, Hedayat S, Unterman TG, Bosland MC, Prins GS & Swanson SM 2008 Disruption of growth hormone signaling retards 20

prostate carcinogenesis in the Probasin/TAg rat. Endocrinology 149 1366–1376 Weiss A & Schlessinger J 1998 Switching signals on or off by receptor dimerization. Cell 94 277–280 Wen Z, Zhong Z & Darnell JE Jr 1995 Maximal activation of transcription factor by Stat1 and Stat3 requires both tyrosine and serine phosphorylation. Cell 82 241–250 Wolk A, Mantzoros CS, Andersson SO, Bergström R, Signorello LB, Lagiou P, Adami HO & richopoulos D 1998 Insulin-like growth factor 1 and prostate cancer risk: a population-based, case-control study. J Natl Cancer Inst 90 911–915 Yang J, Mani SA, Donaher JL, Ramaswamy S, Itzykson RA, Come C, Savagner P, Gitelman I, Richardson A & Weinberg RA 2004 Twist, a master regulator of morphogenesis, plays an essential role in tumor metastasis. Cell 117 927–939 Yang XF, Beamer WG, Huynh H & Pollak M 1996 Reduced growth of human breast cancer xenografts in hosts homozygous for the lit mutation. Cancer Res 56 1509–1511 Zhang CH, Xu GL, Jia WD, Li JS, Ma JL, Ren WH, Ge YS, Yu JH, Liu WB & Wang W 2012 Activation of STAT3 signal pathway correlates with Twist and E-cadherin expression in hepatocellular carcinoma and their clinical significance. J Surg Res 174 120–129 Zhong B, Sallman DA, Gilvary DL, Pernazza D, Sahakian E, Fritz D, Cheng JQ, Trougakos I, Wei S & Djeu JY 2010 Induction of clusterin by AKT--role in cytoprotection against docetaxel in prostate tumor cells. Mol Cancer Ther 9 1831–1841 Zong CS, Chan J, Levy DE, Horvath C, Sadowski HB & Wang LH 2000 Mechanism of STAT3 activation by insulin-like growth factor I receptor. J Biol Chem 275 15099–15105 Zoubeidi A, Ettinger S, Beraldi E, Hadaschik B, Zardan A, Klomp LW, Nelson CC, Rennie PS & Gleave ME 2010a Clusterin facilitates COMMD1 and I-κB degradation to enhance NF-κB activity in prostate cancer cells. Mol Cancer Res 8 119–130 Zoubeidi A, Zardan A, Wiedmann RM, Locke J, Beraldi E, Fazli L & Gleave ME 2010b Hsp27 promotes insulin-like growth factor-I survival signaling in prostate cancer via p90Rsk-dependent phosphorylation and inactivation of BAD. Cancer Res 70 2307–2317 21

FIGURE LEGENDS

Figure 1. IGF-I increases Twist1 expression followed by CLU up-regulation. (A) LNCaP (left) and PC-3 (right) cells were treated with 100 ng/mL IGF-I. After the indicated duration, quantitative RT-PCR was performed using the primer pairs and probes for Twist1, CLU and GAPDH. Each transcript level from non-treated cells was set as 1. Boxes, mean; bars, ±s.d. *P < 0.05 (compared with no treatment). (B) LNCaP (left) and PC-3 (right) cells were treated with 100 ng/mL IGF-I. After the indicated duration, whole-cell extracts were analyzed by SDS–PAGE and western blot analysis with specific antibodies.

Figure 2. Twist1 binds to CLU promoter region and regulates CLU expression. (A) LNCaP cells were transfected with 40 nM of the indicated siRNA. At 72 h after transfection, quantitative RT-PCR was performed using the primers and probes for Twist1, CLU and GAPDH. Each transcript level from cells transfected with control siRNA was set as 1. Boxes, mean; bars, ±s.d. *P < 0.05 (compared with control siRNA). Whole-cell extracts were analyzed by SDS–PAGE and western blot analysis with specific antibodies. (B) LNCaP cells were transfected with 1.0 μg/mL of the indicated expression plasmid. At 72 h after transfection, quantitative RT-PCR was performed using the primers and probes for CLU and GAPDH. Each transcript level from mock-transfected cells was set as 1. Boxes, mean; bars, ±s.d. *P < 0.05 (compared with mock). Whole-cell extracts were analyzed by SDS–PAGE and western blot analysis with specific antibodies. (C) Schematic representation of the promoter region and 5’ end of the CLU gene. Black box, E-boxes (5’-CANNTG-3’); gray box, CLE; white box, AP-1–binding site. CLU–Luc plasmids (–1,998/+254, –1,998/–702, –1,116/–702, and –707/+254) used in (d), (e) and (f) are shown. (D) LNCaP cells were cotransfected with 0.5 μg/mL of the various CLU–Luc plasmids and 0.05 μg/mL of pRL-TK. The luciferase activity of CLU–Luc –1,998/+254 was set as 1. Boxes, mean; bars, ±s.d. *P < 0.05 (compared with CLU-Luc –1,998/+254). (E) LNCaP cells were cotransfected with 0.5 μg/mL of the various CLU–Luc plasmids, 0.5 μg/mL of Myc-Flag or 23

Twist1–Myc-Flag expression plasmid and 0.05 μg/mL of pRL-TK. The luciferase activity of CLU–Luc with mock plasmid was set as 1. Boxes, mean; bars, ±s.d. *P < 0.05 (compared with mock). (F) LNCaP cells were cotransfected with 0.5 μg/mL of the CLU–Luc –1,998/+254 plasmid, 20 nM of the indicated siRNA and 0.05 μg/mL of pRL-TK. The luciferase activity of CLU–Luc –1,998/+254 alone was set as 1. Boxes, mean; bars, ±s.d. *P < 0.05 (compared with control siRNA). (G) ChIP assays were conducted on nuclear extracts from LNCaP cells using the indicated antibodies. The quantitative RT-PCR was carried out using immunoprecipitated DNAs, soluble chromatin, and specific primer pairs for the CLU and RPL30 genes: CLU –02 targeting around –1,403 bp from TSS, CLU –01 targeting around –417 bp from TSS, CLU +07 targeting around +6,603 bp from TSS and RPL30 as negative control. The results of immunoprecipitated samples were corrected for the results of the corresponding soluble chromatin samples. Boxes, mean; bars, ±s.d. *P < 0.05 (compared with IgG).

Figure 3. IGF-I induces CLU expression via Twist1. (A) LNCaP (left) and PC-3 (right) cells were transfected with 40 nM of the indicated siRNA and incubated for 48 h, and then cells were treated with 100 ng/mL IGF-I. After 1.5 h (Twist1) or 12 h (CLU), quantitative RT-PCR was performed using the primer pairs and probes for Twist1, CLU and GAPDH. Each transcript level from non-treated cells was set as 1. Boxes, mean; bars, ±s.d. *P < 0.05 (compared with no treatment). (B) LNCaP (left) and PC-3 (right) cells were transfected with 40 nM of the indicated siRNA and incubated for 48 h, and then cells were treated with 100 ng/mL IGF-I. After the indicated duration, whole-cell extracts were analyzed by SDS–PAGE and western blot analysis with specific antibodies. (C) LNCaP cells were cotransfected with 0.5 μg/mL of the CLU–Luc –1,998/+254 plasmid, 20 nM of the indicated siRNA and 0.05 μg/mL of pRL-TK, incubated for 24 h, and then cells were and treated with IGF-I. After 24 h, the luciferase activity of CLU–Luc –1,998/+254 transfected with control siRNA without IGF-I was set as 1. Boxes, mean; bars, ±s.d. *P < 0.05 (compared with no treatment).

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Figure 4. IGF-I activates STAT3, resulting in Twist1 up-regulation. (A) LNCaP cells were treated with 100 ng/mL IGF-I. After the indicated duration, whole-cell extracts were analyzed by SDS–PAGE and western blot analysis with specific antibodies. (B) LNCaP cells were treated with 100 ng/mL IGF-I. After 30 min, cells were harvested and fractioned into nuclear and cytoplasmic extracts, and then extracts were analyzed by SDS–PAGE and western blot analysis with specific antibodies. (C) ChIP assays were performed on nuclear extracts from LNCaP cells treated with 100 ng/mL IGF-I for 30 min using mouse IgG or anti-STAT3 antibody. The quantitative RT-PCR was performed using immunoprecipitated DNAs, soluble chromatin and specific primer pairs for the Twist1-promoter region. Results of immunoprecipitated samples were corrected for the results of the corresponding soluble chromatin samples. Boxes, mean; bars, ±s.d. *P < 0.05 (compared with no treatment). **P < 0.05 (compared with treatment with IGF-I). (D) ChIP assays were performed on nuclear extracts from LNCaP cells treated with 100 ng/mL IGF-I for 6 h using mouse IgG or anti-Twist1 antibody. The quantitative RT-PCR was performed using immunoprecipitated DNAs, soluble chromatin and specific primer pairs for the CLU-promoter region. Results of immunoprecipitated samples were corrected for the results of the corresponding soluble chromatin samples. Boxes, mean; bars, ±s.d. *P < 0.05 (compared with no treatment). (E) LNCaP cells were cotransfected with 0.5 μg/mL of Twist–Luc plasmid and 0.05 μg/mL of pRL-TK, incubated for 36 h (Twist1), and then cells were treated with 100 ng/mL IGF-I. After 4 h (Twist1), the luciferase activity of Twist–Luc without IGF-I was set as 1. Boxes, mean; bars, ±s.d. *P < 0.05 (compared with no treatment.

Figure 5. IGF-I/Twist1/CLU signaling in IGF-I-deficient mice. (A) Serum IGF-I concentration was measured from PTEN knockout lit/+ mice and PTEN knockout lit/lit mice. Boxes, mean; bars, ±s.e.m. *P < 0.05 (compared with lit/+). (B) Quantitative RT-PCR was performed using the primer pairs and probes for murine Twist1, murine CLU and murine GAPDH. Each transcript level from lit/+ mice was set as 1. Boxes, mean; bars, ±s.e.m. *P < 0.05 (compared with lit/+). (C) Immunohistochemistry 25

against the indicated antibodies in prostate tissues from PTEN knockout lit/+ mice and PTEN knockout lit/lit mice was shown.

Figure 6. IGF-I promotes prostate cancer cell growth in IGF-I-deficient serum through Twist1 and CLU. (A) LNCaP cells transfected with 10 nM of the indicated siRNA were cultured in 1% serum from lit/lit or lit/+ mice (left), or 1% serum from lit/lit mice with or without 100 ng/mL IGF-I (right). After 96 h, growth of cells transfected with control siRNA and cultured in 1% serum from lit/lit mice was set as 1. Boxes, mean; bars, ±s.d. *P < 0.05 (compared with lit/lit or IGF-I–). **P < 0.05 (compared with control siRNA). (B) Schematic representation of signaling pathway from IGF-I to CLU in this study.

26

Figure 1 Takeuchi et al.

4

Twist1

LNCaP

*

3.5

CLU

3 2.5 2

*

1.5

**

* *

1 0.5

IGF-I

0 0h

1.5 h

B

6h

12 h

24 h

Relative mRNA expression

Relative mRNA expression

A 3

6

12

*

2

CLU

*

2.5

*

*

IGF-I (h)

*

*

1 0.5 0 0h

IGF-I

1.5 h

6h

PC-3 24

* *

1.5

LNCaP 0

Twist1

PC-3

0

6

12

24

IGF-I (h)

Twist1

Twist1

CLU

CLU

ȕ-actin

ȕ-actin

12 h

24 h

Figure 2 Takeuchi et al.

B

LNCaP

Twist1

1.2

LNCaP

CLU

1 0.8 0.6

CLU

0.4

*

*

0.2

Twist1

0 Twist Twist1 Control Twist1 siRNA siRNA siRNA #2 #1

Relative mRNA expression

ȕ-actin

CLU

2 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0

LNCaP

IB;Myc Twist1-Myc-Flag

+1

CLE

AP-1

-702

-1998 CLU-Luc㻌 -1998/-702

Luciferase -702

-1116 CLU-Luc㻌 -1116/-702

Luciferase +254 Luciferase

-707

E

LNCaP

*

Relative luciferase activity

Relative luciferase activity

CLU-Luc㻌 -707/+254

2 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0

2.5 2

1 0.8 0.6 0.4

*

*

0.2 0 Control Twist1 Twist1 siRNA siRNA #1 siRNA #2

*

*

1 0.5 0 CLU-Luc CLU-Luc CLU-Luc CLU-Luc -1998/+254 -1998/-702 -1116/-702 -707/+254

1.2

G

LNCaP CLU-Luc

Myc-Flag Twist1-Myc-Flag

LNCaP

1.5

CLU-Luc CLU-Luc CLU-Luc CLU-Luc -1998/+254 -1998/-702 -1116/-702 -707/+254

1.2

E-box +254 Luciferase

CLU-Luc㻌 -1998/+254

F

+500

Clusterin -1998

D

ȕ-actin

Twist1-Myc-Flag

-2000

C

LNCaP

*

CLU

Myc-Flag

Immunoprecipitants/input

1.4

Relative luciferase activity

Relative mRNA expression

A

LNCaP

1

IgG Histone H3 Twist1

0.8 0.6

*

0.4 0.2 0 CLU -02 CLU -01 CLU +07

RPL30

Figure 3 Takeuchi et al.

Relative mRNA expression

LNCaP Twist1

3 2.5 2 1.5 1 0.5

0 IGF-I

+

㸫 Control siRNA Twist1 siRNA #1

2.5 Relative mRNA expression

*

2

PC-3 Twist1

*

1.5 1 0.5

0 IGF-I

+

㸫

B

Control siRNA Twist1 siRNA #1

1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 IGF-I

Relative mRNA expression

Control siRNA Twist1 siRNA #1

3.5

6

12 24

Control siRNA

Twist1 siRNA #1 0

+

PC-3

6

12 24

C Relative luciferase activity

0

PC-3 CLU

*

㸫

LNCaP Control siRNA

+

Control siRNA Twist1 siRNA #1

1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 IGF-I

LNCaP CLU

*

㸫

Relative mRNA expression

A

3.5

IGF-I (h)

0

6

0

6

12 24

IGF-I (h)

Twist1

Twist1

CLU

CLU

ȕ-actin

ȕ-actin

LNCaP CLU-Luc

Control siRNA Twist1 siRNA #1

*

3 2.5 2 1.5 1 0.5 0

IGF-I

12 24

Twist1 siRNA #1

㸫

+

Figure 4 Takeuchi et al.

A

LNCaP 15

30

60

B

IGF-I (min)

LNCaP –

p-STAT3Tyr705

+ C

IGF-I㸫 LNCaP IGF-I+ Twist1 promoter

IgG

*

STAT3

IGF-I C

STAT3

STAT3

Lamin B1

ȕ-actin

ȕ-actin

D 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0

N

p-STAT3Ser727

E 2.5

Immunoprecipitant/input

Immunoprecipitant/input

C

N

LNCaP CLU -02

2

2.5

*

1.5 1 0.5

0 – Ctrl IGF-I LNCaP

Relative luciferase activity

0

LNCaP Twist-Luc

*

2 1.5 1 0.5 0

+ LNCaP IGF (6h)

IGF-I

㸫

+

Figure 5 Takeuchi et al.

300

Serum IGF-I

250 200 150 100

*

50 0 lit/+

C

lit/lit mice

lit/+ mice

CLU

lit/lit

IGF-IR

B Relative mRNA expression

Serum IGF-I concentration (ng/mL)

A

1.6

Twist1

1.4

CLU

1.2 1 0.8 0.6

*

0.4 0.2 0 lit/+

p-STAT3Tyr705

lit/lit

Twist1

Figure 6 Takeuchi et al.

A 1.4

LNCaP

*

1.2 1

1.4

Media lit/lit lit/+

**

Relative cell growth

Relative cell growth

1.6

**

0.8 0.6 0.4 0.2

*

1.2 1

LNCaP

Media IGF-I㸫 IGF-I+

**

0.8

**

0.6 0.4 0.2

0

0 Control Twist1 siRNA siRNA #1

B

CLU siRNA

IGF-I

Control Twist1 siRNA siRNA #1

IGF-I

CLU siRNA

IGF-I

IGF-IR P

STAT3 Cancer cell growth Nucleus P

STAT3

Twist1 Twist1

CLU

Highlights



Insulin-like growth factor-I (IGF-I) transcriptionally increased Twist1 levels as well as clusterin (CLU) expression.



At transcriptional level, Twist1 regulated CLU expression and mediated CLU induction by IGF-I.



As upstream pathway of Twist1, STAT3 mediated Twist1/CLU induction by IGF-I.



Twist1 and CLU expressions were suggested to be lower in IGF-I–deficient mice.



Both Twist1 and CLU knockdown suppressed prostate cancer cell proliferation promoted by IGF-I.

27