P27kip1 Pathway in Suppression of Cancer Cell Proliferation by RECK

Oncogene (2012) 31, 4128–4138 & 2012 Macmillan Publishers Limited All rights reserved 0950-9232/12 www.nature.com/onc ORIGINAL ARTICLE Involvement of the SKP2–p27KIP1 pathway in suppression of cancer cell proliferation by RECK

Y Yoshida1, K Ninomiya1, H Hamada2 and M Noda1

1Department of Molecular Oncology and Global COE Program, Kyoto University Graduate School of Medicine, Sakyo-ku, Kyoto, Japan and 2Laboratory of Oncology, Department of Life Sciences, Tokyo University of Pharmacy and Life Sciences, Tokyo, Japan

The membrane-anchored matrix metalloproteinase-regu- complex include three CDK inhibitors: p21 (CDKN1A, lator RECK is often downregulated in cancers; in some Cip1 and WAF1), p27 (also known as CDKN1B and cases, a significant correlation between the level of KIP1) and p57 (CDKN1C, KIP2 and BWS) (Frescas and residual RECK in resected tumors and patient survival Pagano, 2008). SKP2 upregulation therefore accelerates has been noted. Furthermore, restoration of RECK p27 degradation, leading to deregulated CDK activity and expression in certain cancer-derived cell lines results in cell-cycle progression as found in cancer cells. reduced tumorigenicity. Here we report that acute RECK RECK was first isolated as a transformation-suppressor expression in colon carcinoma cells results in cell cycle- gene by cDNA expression cloning (Noda et al., 1989; arrest accompanied by downregulation of a ubiquitin Takahashi et al., 1998) and found to encode a membrane- ligase component, S-phase kinase-associated protein 2 anchored regulator of several extracellular metalloprotei- (SKP2), and upregulation of its substrate, p27KIP1. Our nases, including MMP2, MMP7, MMP9, MT1-MMP, data indicate that RECK-induced growth suppression is at ADAM10 and CD13 (Takahashi et al., 1998; Oh et al., least partially dependent on p27, and that RECK and type 2001; Miki et al., 2007; Muraguchi et al., 2007; Omura I collagen share similar effects on the SKP2–p27 et al., 2009). These proteases have been implicated in tissue pathway. Importantly, in patients with lung, colorectal remodeling and cleavage of various extracellular molecules and bladder cancers, the RECK/SKP2 ratio is high in under normal and pathological conditions (Sternlicht and normal tissues and lower in the cancer tissues. These Werb, 2001). RECK mRNA is detectable widely in normal findings reveal a novel molecular pathway linking cell- human organs but is undetectable in a number of cell lines cycle progression to RECK downregulation, extracellular derived from human tumors (Takahashi et al., 1998; matrix degradation and SKP2 upregulation, and suggest Loayza-Puch et al., 2010). Downregulation of RECK is that treatment regimens that induce RECK expression found in a wide variety of tumors, and, in some cases, a could be promising cancer therapies. significant correlation between the level of residual RECK Oncogene (2012) 31, 4128–4138; doi:10.1038/onc.2011.570; in resected tumors and patient survival has been noted published online 12 December 2011 (Noda and Takahashi, 2007). In xenograft models, restored RECK expression in cancer cells results in Keywords: RECK; cell cycle; extracellular matrix; reduction in tumor angiogenesis, invasion, metastasis SKP2; p27; collagen (Takahashi et al., 1998; Oh et al., 2001) and, depending on the cell line employed, reduced tumorigenicity (Loayza- Puch et al., 2010). Thus, RECK downregulation is likely to be an important step towards, rather than a result of, Introduction carcinogenesis. Reck-deficient mice die around embryonic day 10.5 Protein phosphorylation (through the activity of cyclin- with reduced tissue integrity (accompanied by dramatic dependent kinases (CDKs)) and protein degradation reduction in collagen fibrils), arrested vascular develop- (through the ubiquitin–proteasome pathway) have major ment and precocious neuronal differentiation (Oh et al., roles in cell-cycle regulation. Recent evidence indicates the 2001; Muraguchi et al., 2007; Chandana et al., 2010). importance of S-phase kinase-associated protein 2 (SKP2) Embryo fibroblasts derived from these mice show upregulation and consequent p27KIP1 downregulation in unstable focal adhesions, increased speed of migration various cancers (Zhu, 2010). SKP2 is a component of a and reduced directional persistence during migration, SKP1-CUL1-F-box-protein (SCF)-type ubiquitin ligase and the phenotype can be partially suppressed on complex (termed SCFSKP2); known substrates of this fibronectin-coated dishes (Morioka et al., 2009). These studies, taken together with the effects RECK has on tumorigenesis, indicate that RECK functions via its Correspondence: Dr M Noda, Department of Molecular Oncology, effects on cell–cell and cell–extracellular matrix interac- Kyoto University Graduate School of Medicine, Yoshida-Konoe-cho, tions. The effect of RECK on intracellular signaling Sakyo-ku, Kyoto 606-8501, Japan. E-mail: [email protected] pathways, however, remains largely unexplored. In Received 26 June 2011; revised 29 October 2011; accepted 5 November particular, previous studies used stable manipulations 2011; published online 12 December 2011 of RECK gene activity (for example, stable transfections RECK regulates the SKP2–p27 pathway Y Yoshida et al 4129 and global gene knockouts) to explore the functions of expression profiles of the cells infected with Ad-Z or Ad- RECK (Noda et al., 2003; Noda and Takahashi, 2007), RECK using Affymetrix Human Genome U133Plus 2.0. but if RECK has negative effects on essential signaling Arrays (Affymetrix). Using the FunNet algorithm, pathways, such as those that affect cell proliferation or however, we were unable to determine which pathways viability, stable manipulation of RECK would result in affected by RECK led to cell-cycle arrest (Table 1b). the selection of populations with resistance to, or In an attempt to find individual genes that were both functional compensations for, RECK activity. There- altered by RECK and relevant to the observed fore, we took advantage of the high efficiency and bioactivity, we filtered the data using gene ontology synchrony of adenovirus-mediated gene transfer to terms such as cell proliferation (GO:0008283), cell cycle allow us to study the effects of acute RECK expression (GO:0007049) and G1/S transition of the mitotic in cancer cells. Our data indicate that RECK can cell cycle (GO:0000082). Through this approach, we suppress cell proliferation and induce cellular senescence found a modest (B0.5-fold) downregulation of SKP2 when acutely expressed in cancer cells, and that at least a (Figure 2a), which raised the interesting possibility that part of these effects can be attributed to downregulation the major substrates of SKP2 (for example, the CDK- of SKP2 and consequent upregulation of p27. These inhibitors p21, p27 and p57) may serve as effectors of findings have revealed an important link between the RECK to induce cell-cycle arrest. inhibition of extracellular proteolysis by RECK and the Our microarray data indicated that p27 mRNA is inhibition of intracellular proteolysis by the ubiquitin– detectable but not much affected by RECK expression in proteasome system. They also suggest that RECK SW620 cells, and that the levels of p21 and p57 mRNAs reactivation may be a promising new strategy for are low in these cells (Figure 2a). SKP2 downregulation manipulating the SKP2–p27 pathway in cancer cells. after RECK expression and the little effects of RECK on p27 expression were also confirmed by RT–PCR (Figure 2b, lanes 1–3). At the protein level, however, Results SKP2 downregulation was accompanied by a significant upregulation of p27 (Figure 2c, lane 3), which is Biological effects of acute RECK expression in malignant cells consistent with the widely accepted model that SKP2 To study the effects of acute RECK expression in cancer promotes p27 degradation. cells, we generated an adenoviral vector containing a We also examined the levels of SKP2 and p27 in human RECK cDNA (Ad-RECK). When this virus was another human tumor cell line, H1080 fibrosarcoma infected into a v-K-ras-transformed NIH3T3 subline, DT (Rasheed et al., 1974), stably transfected with a control (Noda et al.,1983),thecells,asexpected,showedincreased plasmid vector (V) or the vector expressing RECK (R) RECK expression (Figure 1a) and increased spreading (Figure 2d). The cells expressing RECK grew more (Figure 1b). We determined the gene expression profiles of slowly than the control cells (Figure 2e), and SKP2 DT cells infected with the control virus (Ad-Z) or Ad- downregulation (both at the mRNA and protein levels) RECK using Affymetrix Mouse Genome 430 2.0 Arrays and p27 upregulation (at the protein level) were also (Affymetrix, Santa Clara, CA, USA). Through pathway observed (Figure 2b, lane 5; Figure 2d, lane 2). Small analysis using the FunNet algorithm (Prifti et al., 2008), interfering RNA (siRNA)-mediated knockdown of we detected significant upregulation of two pathways and RECK also resulted in SKP2 upregulation and p27 downregulation of nine pathways after RECK expression downregulation (Figure 2f, lane 4), indicating an active (Table 1a). These changes largely implicate RECK in role for RECK in regulating the levels of SKP2 and p27 regulation of cellular metabolism and proliferation. in this system. Hence, in two tumor-derived cell lines We also examined the effects of Ad-RECK in SW620, (SW620 and HT1080), RECK expression was found to a cell line derived form a metastatic lesion of colon induce SKP2 downregulation and p27 upregulation. adenocarcinoma (Leibovitz et al., 1976). At 48 h after Does a similar link between RECK and the SKP2– infection, the cells infected by Ad-RECK were signifi- p27 pathway exist in nonmalignant cells? We addressed cantly fewer in number (Figure 1c, panels 5 and 6) as this question using two systems. First, in the mouse compared with the controls (Figure 1c, panels 1–4). fibroblast cell line NIH3T3, the level of endogenous Cell-cycle analysis indicated that after RECK expres- Reck is known to heavily depend on cell density (Hatta sion, the cells in the G1 phase of the cycle increased, et al., 2009). Here we found that the level of Reck at whereas the cells in the S phase decreased, and the sub- different cell densities was inversely correlated with the G1 populations stayed low (Figure 1d, panels 3 and 4), level of SKP2 and positively correlated with the level of indicating the occurrence of cell-cycle arrest rather than p27 (Figures 2g and h). Second, in a mouse embryo apoptosis. Moreover, the Ad-RECK-infected cells showed fibroblast (MEF) line derived from a tamoxifen- elevated b-galactosidase activity at pH 6 (Figure 1e, inducible conditional Reck-knockout mouse, inactiva- panel 3), suggesting the induction of cellular senescence tion of endogenous Reck gene by treatment with 4- (Dimri et al., 1995). hydroxytamoxifen (OHT) resulted in SKP2 upregulation and p27 downregulation (Figure 2i). These findings RECK induces SKP2 downregulation and p27 not only demonstrate the correlation among the levels of upregulation in SW620 cells Reck, SKP2 and p27 in nonmalignant cells but also To address the question of how RECK induces cellular indicate the active role for Reck in the regulation of senescence in SW620 cells, we determined the gene SKP2 and p27 expression.

Oncogene RECK regulates the SKP2–p27 pathway Y Yoshida et al 4130 DT Ad-Z Ad-RECK 12

Ad-RECK Ad-Z

RECK

GAPDH

1 2

SW620 Cell cycle analysis 12 1 200 160 mock 120 mock 80 Cell count 40 0 34 2 200 160 Ad-Z 120

Ad-Z 80 Cell count 40 0 3 200 56 160 Ad-RECK 120 80 Cell count

Ad-RECK 40 0 0 200 400 600 800 1000 Fluorescent intensity 4 X-gal stain Summary 80 mock Ad-Z Ad-RECK m 60 Z R 40

20 Proportion (%)

12 3 0 sub-G1 G1 S G2M Figure 1 Effects of acute RECK expression in DT and SW620 cells. (a) Immunoblot detection of RECK (upper panel) in DT cells at 48 h after infection with Ad-Z (lane 1) or Ad-RECK (lane 2). GAPDH (lower panel) was detected as the loading control. (b) Morphology of DT cells infected with Ad-Z (panel 1) or Ad-RECK (panel 2). Scale bar: 50 mm. (c) Morphology of SW620 cells at 48 h after mock treatment (panels 1 and 2) or after infection with Ad-Z (panels 3 and 4) or Ad-RECK (panels 5 and 6). Scale bar: 200 mm (panels 1, 3 and 5), 50 mm (panels 2, 4 and 6). See Figure 2c (top panel) for RECK protein expression. (d) Cell-cycle analysis of SW620 cells at 48 h after mock treatment (panel 1) or after infection with Ad-Z (panel 2) or Ad-RECK (panel 3). Proportion of the cells in each phase was determined by ﬂow cytometry of propidium iodide-stained cells as shown in panels 1–3, and the data are summarized in panel 4. (e) Senescence-associated b-galactosidase activity in the SW620 cells at 48 h after mock treatment (panel 1) or after infection with Ad-Z (panel 2) or Ad-RECK (panel 3). Scale bar: 50 mm.

The role of p27 in RECK-mediated growth suppression in of the control (mock-infected or Ad-Z-infected cells) SW620 cells (Figure 3b, panels 1–3 and Figure 3c, bars 1–3). To assess the role of p27 in RECK-mediated growth The si-p27 efﬁciently depleted p27 (Figure 3a, lane 3), arrest, we treated SW620 cells for 24 h with the control alleviated growth suppression to about 80% of the siRNA (si-C) or an siRNA-targeting p27 (si-p27) and then control (Figure 3b, panel 9; Figure 3c, bar 9) and infected with either Ad-Z or Ad-RECK and incubated for attenuated the senescence-associated b-galactosidase additional 72 h (Figure 3). Under these conditions, activity in Ad-RECK-infected cells (Figure 3d, panel 3). Ad-RECK alone had an effect to suppress the cell number These results suggest that p27 has an active role in RECK- (probably via cell-cycle arrest, Figure 1d) to about 40% induced growth suppression and senescence.

Oncogene RECK regulates the SKP2–p27 pathway Y Yoshida et al 4131 Table 1 KEGG pathways affected by acutely expressed RECK in DT and SW620 cells Rank ID Term List hits List total Population hits Population total P-value a. DT Upregulated 1 51 Fructose and mannose metabolism 3 31 37 6223 7.8EÀ04 2 520 Amino sugar and nucleotide sugar metabolism 3 31 44 6223 1.3EÀ03 Downregulated 1 1100 Metabolic pathways 134 516 1253 6223 4.7EÀ04 2 3010 Ribosome 40 516 132 6223 1.1EÀ13 3 4110 Cell cycle 28 516 138 6223 6.2EÀ06 4 4120 Ubiquitin-mediated proteolysis 26 516 151 6223 2.4EÀ04 5 4722 Neurotrophin signaling pathway 25 516 142 6223 2.2EÀ04 6 240 Pyrimidine metabolism 23 516 104 6223 9.6EÀ06 7 4666 Fc gamma R-mediated phagocytosis 20 516 105 6223 3.2EÀ04 8 3420 Nucleotide excision repair 11 516 45 6223 8.6EÀ04 9 3030 DNA replication 11 516 36 6223 1.0EÀ04 b. SW620 Upregulated 1 970 Aminoacyl-tRNA biosynthesis 5 37 41 5187 8.7EÀ06 2 260 Glycine, serine and threonine metabolism 3 37 30 5187 1.2EÀ03 Downregulated 1 100 Steroid biosynthesis 6 109 17 5187 7.7EÀ07

Possible mechanisms by which RECK downregulates studies. Our findings in vitro predicted that direct SKP2 inverse correlations between the levels of SKP2 and How does RECK downregulate SKP2? RECK, a RECK might be found in individual clinical samples if membrane-anchored glycoprotein, is unlikely to regu- RECK regulates SKP2 in vivo. To test this possibility, late gene expression directly; RECK is more likely to we searched the GEO profile database (NCBI) for regulate proteolysis of some molecules in the vicinity of relatively large sets of data on human tumor samples the cell surface, thereby affecting intracellular signaling and found that RECK was downregulated and SKP2 and gene expression. Several lines of evidence implicate upregulated in two of the common human tumors, lung RECK in the protection of extracellular matrix compo- and colorectal, as well as urinary bladder tumor. First, nents through inhibition of extracellular proteases (see among 32 colorectal adenomas and corresponding Introduction). We therefore tested the effects of two normal mucosas (Sabates-Bellver et al., 2007), high- major extracellular matrix components, type I collagen RECK/low-SKP2 profiles in all normal mucosa and and fibronectin on SKP2 expression in SW620 cells low-RECK/high-SKP2 profiles in all but one adenoma (Figures 4a and b). Interestingly, SKP2 expression was samples were found (Figure 5a). Second, among 60 strongly suppressed when the cells were plated on type I urinary bladder tissues (Dyrskjot et al., 2004), high- collagen (Figure 4a, lane 2) but not on fibronectin RECK/low-SKP2 profiles were found mainly in normal (Figure 4b, lane 2). In fact, collagenolytic activity of urothelium, normal mucosa and carcinoma in situ, SW620 cells was greatly diminished after Ad-RECK whereas low-RECK/high-SKP2 profiles were found in infection, as visualized by in situ zymography using DQ more malignant tumors: superficial transitional cell type I collagen (Figure 4c). We also found that the effect carcinoma and muscle invasive carcinoma (Figure 5b). of type I collagen to downregulate SKP2 could be Third, among 108 lung tissues (Landi et al., 2008) suppressed by the protein kinase C inhibitor (Figure 5c), high-RECK/low-SKP2 profiles were found GF109203X (Toullec et al., 1991) (Figure 4e) and the in normal tissues (right half) and low-RECK/high-SKP2 Rho-family GTPase inhibitor toxin B (Just et al., 1995) profiles were found in tumor tissues (left half) with only (Figure 4f), but not by the Src inhibitor PP2 (Hanke a few exceptions. These data demonstrate that a direct et al., 1996) (Figure 4d). Protein kinase C and Rho- inverse correlation between the levels of RECK and family GTPases are known to be involved in integrin SKP2 is found in lung, colon and bladder tissues and signaling (Caswell et al., 2009). Importantly, the SKP2 that a low-RECK/high-SKP2 ratio shows strong asso- downregulation induced by RECK was also sensitive to ciation with malignancy in these tissues. GF109203X and toxin B (Figures 4g and h), suggesting that type I collagen and RECK share a common signaling pathway (for example, integrin signaling) in Discussion downregulating SKP2. In this study, we demonstrated that RECK expression The levels of RECK and SKP2 in human tissues suppresses cell proliferation of three malignant cell lines: SKP2 upregulation (Zhu, 2010) and RECK down- DT, SW620 and HT1080. RECK is downregulated by regulation (Noda and Takahashi, 2007) in cancer cells multiple signaling pathways at multiple levels (Takaha- have been documented, albeit separately, in many shi et al., 1998; Sasahara et al., 1999; Hatta et al., 2009;

Oncogene RECK regulates the SKP2–p27 pathway Y Yoshida et al 4132 abSW620 SW620 HT1080 c SW620 2500 Ad-Z Ad-RECK mZ RVR mZ R 2000 RECK 140 bp RECK 1500 SKP2 164 bp SKP2 1000 500 p27 169 bp p27

GeneChip signal 0 HPRT1 132 bp GAPDH SKP2 p21 p27 p57 12345 123

deHT1080 HT1080 growth curve fRECK / HT1080

VR ) v - si-C si-R

5 300 RECK 250 RECK 200 Vector SKP2 150 SKP2 p27 100 p27 50 RECK GAPDH GAPDH Cell number (10 0 01234567 12 1234 Day

ghiNIH3T3 Reckfl/- MEF NIH3T3 Density: low high OHT: -+ Density: low high Reck Reck Skp2 Skp2 p27 p27 Gapdh Gapdh

Figure 2 Downregulation of SKP2 after RECK expression in two human tumor cell lines. (a) Normalized GeneChip data for the major transcripts of SKP2, p21 (CDKN1A), p27 (CDKN1B) and p57 (CDKN1C) expressed in SW620 cells at 48 h after infection with Ad-Z (gray bar) or Ad-RECK (black bar). (b) Detection of RECK, SKP2 and p27 transcripts by semi-quantitative RT–PCR. Total RNA from SW620 cells at 48 h after mock treatment (m) or infection with Ad-Z (Z) or Ad-RECK (R) (lanes 1–3) and total RNA from HT1080 cells stably transfected with a control vector (V) or the vector expressing RECK (R) (lanes 4 and 5) were analyzed. HPRT1 was used as a loading control. (c) Immunoblot detection of the RECK, SKP2 and p27 proteins in whole-cell lysates of SW620 at 48 h after mock treatment (lane 1) or after infection with Ad-Z (lane 2) or Ad-RECK (lane 3). GAPDH was used as a loading control. (d) Immunoblot detection of RECK, SKP2 and p27 proteins in whole-cell lysates of HT1080 cells stably transfected with a control vector (lane 1) or the vector expressing RECK (lane 2). (e) Growth curves of HT1080 cells stably transfected with a control vector (open squares) or a vector expressing RECK (ﬁlled squares). Bar represents mean±s.d. (n ¼ 3). (f) Effects of RECK knockdown on the levels of SKP2 and p27. RECK-transfected HT1080 cells plated on the previous day were incubated in growth medium (lane 2) or treated with vehicle (lane 1), a control siRNA (lane 3) or an siRNA targeting RECK (lane 4) for 48 h. Whole-cell lysates were subjected to immunoblot assay. Experiments were repeated three times with similar results. (g) Morphology of NIH3T3 cells plated at two different cell densities (3 Â 104 (low) and 2.5 Â 105 (high) per well in six-well plate) and incubated for 48 h. Scale bar: 50 mm. (h) Effects of cell density on the levels of Reck, Skp2 and p27 proteins in NIH3T3 cells. Lysates of the cells shown in (g) were subjected to immunoblot assay. GAPDH was used as a loading control. (i) Effects of conditional Reck-knockout on the levels of Skp2 and p27 in MEFs. R1B2-6 cells incubated in the absence or presence of 250 nM 4-hydroxytamoxifen for 96 h and then in GM for 48 h were subjected to immunoblot assay.

Loayza-Puch et al., 2010). Inhibitory signaling by of the hepatocellular carcinoma line SMMC-7721 (Fu negative regulators of cell division, like RECK, would et al., 2007) are in agreement with this model. Not have to be overcome prior to the cell division, which surprisingly, not all cancer cells respond in the same occurs during processes such as development, wound manner to b1 integrin. In the hepatocellular carcinoma line healing and tissue maintenance, and this may explain HepG2, for example, b1 integrin signaling enhances SKP2 why RECK is downregulated by several signaling expression (Zhang et al.,2003).Hence,detailedmolecular pathways. mechanisms underlying how cancer cells can evade Our data are consistent with the model that RECK RECK-induced SKP2 downregulation need to be clarified inhibits cell division by protecting pericellular extra- in future studies. cellular matrix (for example, collagens), thereby enhancing Skp2-deficient mice are reported to be smaller than integrin signaling that leads to SKP2 downregulation and normal, and their embryo fibroblasts (MEFs) grow consequent p27 upregulation (Figure 6). The previous more slowly than wild-type MEFs (Nakayama et al., findings that Rac1 downregulates SKP2 in rat vascular 2000). These phenotypes are alleviated in Skp2/p27 smooth muscle cells (Bond et al., 2008) and that b1 double mutant mice (Nakayama et al., 2004), suggesting integrin downregulates SKP2 and suppresses proliferation that p27 overexpression is the cause of these phenotypes

Oncogene RECK regulates the SKP2–p27 pathway Y Yoshida et al 4133 vehicle si-Control si-p27

SW620 v si-C si-p27 RECK mock

SKP2 1 4 7

p27

GAPDH Ad-Z 123 2 5 8

mock Ad-Z Ad-RECK Ad-RECK 100 3 6 9

75 * vehicle si-Control si-p27 50 123

0 Relative cell number (%) 123 456 789 Ad-RECK v si-C si-p27

Figure 3 Involvement of p27 in Ad-RECK-induced growth arrest and cellular senescence. (a) Effects of p27 siRNA on the levels of RECK, SKP2 and p27 in SW620 cells after infection with Ad-RECK. SW620 cells pretreated for 24 h with vehicle (lane 1), a control siRNA (lane 2) or an siRNA targeting p27 (lane 3) were infected with Ad-RECK and incubated for 72 h. Whole-cell lysates were subjected to immunoblot assay. (b) Effects of p27 siRNA on the growth of SW620 cells after infection with Ad-Z or Ad-RECK. SW620 cells pretreated for 24 h with either vehicle (panels 1–3), the control siRNA (panels 4–6) or the p27 siRNA (panels 7–9) were left uninfected (mock; panels 1, 4 and 7) or infected with either Ad-Z (panels 2, 5 and 8) or Ad-RECK (panels 3, 6 and 9) and incubated for 72 h. Scale bar: 100 mm. (c) Effects of p27 siRNA on Ad-RECK-induced reduction in cell number. Cells treated as described in b were counted. The data are presented as the ratios to the vehicle-treated controls. Bar represents mean±s.e.m. (n ¼ 3). (d) Effects of p27 siRNA on Ad-RECK-induced activation of senescence-associated b-galactosidase. Cells treated as described in a were subjected to X-gal staining at pH 6. Scale bar: 50 mm.

in Skp2-deficient mice. Interestingly, some oncogenic It is presently unclear why the siRNA-mediated manipulations (for example, transfection of mutant Ras knockdown of p27 could rescue the proliferation of plus E1A or deletion of the tumor suppressor gene Pten Ad-RECK-infected cells only partially (Figure 3c, bar or Arf) induce cellular senescence in Skp2-deficient 9). One possibility is that RECK suppresses cell MEFs (Lin et al., 2010). This is consistent with our proliferation through multiple mechanisms, and this finding that RECK (which downregulates SKP2) could be at the levels of SKP2 targets as well as RECK induces cellular senescence in SW620, a cell line carrying targets. mutations in KRAS and p53. The transcriptome data in the public database p27-deficient mice, on the other hand, are reported to (Dyrskjot et al., 2004; Sabates-Bellver et al., 2007; show gigantism and pituitary tumors (Frescas and Pagano, Landi et al., 2008) (Figure 5) strongly support the 2008).Ifp27isamajoreffectorofRECK,Reck-deficient clinical relevance of our findings. As SKP2 is a mice and p27-deficient mice would be expected to show promising target of cancer therapy (Frescas and Pagano, some overlapping phenotypes. Global Reck-deficient mice, 2008; Zhu, 2010), the newly discovered link between however, die too early (Oh et al., 2001; Muraguchi et al., RECK and SKP2 may open a new avenue to the 2007) and provide no direct answers to this question; the therapeutic manipulations of this important pathway. embryonic lethality of Reck-deficient mice probably We recently reported on an assay system for screening reflects the multiplicity of RECK targets/effectors that chemicals that activate Reck transcription (Murai et al., are essential for embryogenesis. Phenotypes of hypo- 2010). One possible approach would be to use such morphic and/or conditional Reck-knockout mice may chemicals to induce RECK expression and consequent provide more useful information in this regard. SKP2 downregulation in cancer cells.

Oncogene RECK regulates the SKP2–p27 pathway Y Yoshida et al 4134 123

non Col RECK

SKP2 mock

p27

GAPDH 12 Ad-Z

non FN SKP2

GAPDH

1 2 Ad-RECK

PP2 (µM) GF109203X (µM) Toxin B (pg/ml) 0 1.5 4.5 0 0.5 1.5 4.5 SKP2 0 10 non n Col n Col GAPDH SKP2 SKP2 Col GAPDH GAPDH

GF109203X (µM) Toxin B (pg/ml) 0 4.5 0 50 mZR mZR mZR mZR

SKP2

GAPDH

Figure 4 Effects of type I collagen on SKP2 expression. (a) Effects of type I collagen on SKP2 and p27 expression. SW620 cells plated on a regular dish (lane 1) or a dish coated with type I collagen (lane 2) were incubated for 48 h, and their lysates were subjected to immunoblot assay to estimate the levels of the indicated proteins. (b) Effects of fibronectin (FN) on SKP2 expression. SW620 cells plated on a regular dish (lane 1) or a dish coated with FN (lane 2) were incubated for 48 h, and their lysates were subjected to immunoblot assay to estimate the levels of SKP2 and the loading control GAPDH. (c) Collagenolytic activity of SW620 cells left uninfected (mock) or infected with Ad-Z or Ad-RECK as visualized by in situ zymography using a quenched fluorescent substrate, DQ type I collagen. Typical fluorescence/phase-contrast images of a relatively large cluster (row 1), small cluster (row 2) and single cell (row 3) are shown. Scale bar: 20 mm. (d–h) Effects of signaling inhibitors on collagen-mediated SKP2 downregulation in SW620 cells. Cells plated on regular (non) or collagen-coated (Col) dishes were treated for 48 h with either PP2 (a Src inhibitor, d), GF109203X (a PKC inhibitor, e) or Toxin B (a Rho/Rac/Cdc42 inhibitor, f) at the indicated doses and subjected to immunoblot assay. (g and h) Effects of signaling inhibitors on Ad-RECK-mediated SKP2 downregulation in SW620 cells. Cells were mock-treated (m) or infected with Ad-Z (Z) or Ad-RECK (R), treated for 48 h with GF109203X (g) or Toxin B (h) at the indicated dose and subjected to immunoblot assay.

Materials and methods pCAcc plasmid, to obtain pCAhRECK. The 5.2-kb Cla1 fragment (RECK expression cassette) from pCAhRECK was Cell lines and recombinant adenoviruses inserted into the ClaI site of the pAx3 cosmid vector to obtain NIH3T3, DT, SW620, HT1080 and HT1080 transfectants pAx3hRECK. Replication-defective Ad-RECK virus was were maintained in growth medium as described previously generated by transfecting pAx3hRECK into HEK-293 cells (Oh et al., 2001; Hatta et al., 2009). The 3.2-kb Mlu1–Msc1 following the protocols described previously (Miyake et al., fragment of human RECK cDNA was inserted between the 1996). The control virus expressing the bacterial lacZ gene has corresponding sites of pCAmm, a modiﬁed version of the been described elsewhere (Yoshida and Hamada, 1997). The

Oncogene RECK regulates the SKP2–p27 pathway Y Yoshida et al 4135 Colorectal adenoma (GDS2947) 3 P = 6 x 10 -17 RECK SKP2 1 2 0 1 Reck – SKP2 -1

Relative GeneChip Count 0 Patients 1 - 32 Patients 1 - 32 Normal

Normal mucosa Adenoma Adenoma

Urinary bladder (GDS1479) P = 1 x 10 -4 6 RECK SKP2 1

4 0

2 Reck – SKP2 -1

0 Relative GeneChip Count Normal STCC-CISSTCC MI CIS Normal

urothelium Carcinoma mucosa STCC / MI Normal / CIS

Lung adenocarcinoma (GDS3257) 2 P = 1.8 x 10 -20 RECK SKP2 0.2

1 0

Reck – SKP2 -0.2

Relative GeneChip Count 0 Tumor Stage : I II IIIIVI II III I IIIII IV I II III IV IIIIIII II III IV Normal Gender : FFMFMMFFFFMMFM FMMF MMMF Never smoker Former Smoker Current Smoker Never smoker Former Smoker Current Smoker Tumor tissues Normal tissues Figure 5 The levels of RECK and SKP2 in human tissues. (a) Colorectal adenomas and normal mucosas from 32 patients (GEO accession no. GDS2947) (Sabates-Bellver et al., 2007). The data for RECK (205407_at) and SKP2 (203625_x_at) were obtained from the GEO database (NCBI), and the ratios to the mean value of all samples (32 adenomas and 32 normal mucosas) were calculated. (b) Carcinoma in situ (CIS) lesions of the urinary bladder (GDS1479) (Dyrskjot et al., 2004). The probes and methods are the same as (a). STCC-CIS, superﬁcial transitional cell carcinoma with surrounding CIS lesion; STCC, superﬁcial transitional cell carcinoma without surrounding CIS lesion; MI, muscle invasive carcinoma. (c) Cigarette smoking effect on lung adenocarcinoma (GDS3257) (Landi et al., 2008). The probes and methods are the same as (a). F, female; M, male.

MEF line (R1B2-6) was derived from a 13.5-day mouse embryo additional 48 h before RNA preparation. HT1080 transfec- (genotype: ReckR1B/R1BD;CAG-CreER) using the cold trypsin tants (5 Â 104/60-mm dish) were harvested 48 h after plating. method (Freshney, 1987), followed by extensive passaging at Total RNA was extracted from these cells using the RNeasy the splitting ratio of 1:3 in aMEM supplemented with 10% Kit (Qiagen, Hilden, Germany) was used to generate feral bovine serum on collagen-coated dishes. Generation of biotinylated cRNA, and the cRNA was hybridized to the mice with ReckR1B allele (containing two loxP sites ﬂanking Mouse Genome 430 2.0 Array (for DT) or Human Genome Reck exon 1) will be described elsewhere (Yamamoto et al., U133Plus 2.0 Array (for SW620 and HT1080) (Affymetrix) submitted for publication). The CreER transgenic mouse following the manufacturer’s instructions (URL:http:// (STOCK Tg(CAG-cre/Esr1)5Amc/J) was obtained from the www.affymetrix.com/index.affx). The hybridization intensity Jackson Laboratory (Bar Habor, ME, USA). was determined with Affymetrix GeneChip Scanner 3000 and normalized against controls. The lists of transcripts whose Gene expression proﬁling signals were increased (ratio of normalized values >2) or DT cells (5 Â 104/60-mm dish) or SW620 cells (2 Â 105/60-mm decreased (ratio of normalized values o 0.5) in the RECK- dish) were infected (multiplicity of infection (MOI) ¼ 600) with expressing counterparts (and positive at least in one of the two Ad-Z or Ad-RECK at 16 h after plating and incubated for an samples) were used to detect affected pathways using the

Oncogene RECK regulates the SKP2–p27 pathway Y Yoshida et al 4136 pore, Billerica, MA, USA) and HRP-conjugated anti-mouse Collagen IgG-F(ab0)2 monoclonal antibody (Cell Signaling Technology) followed by recording with LAS-3000. For mouse proteins, the same antibodies were used except for Skp2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA). (Integrin) RECK

siRNA transfection Validated siRNAs were obtained from Ambion (hRECK, #. 29149), Santa Cruz p27KIP1 (sc-29430) and Dharmacon (Chicago, IL, USA; control duplex, D-001210-03). The cells (2.5 Â 105/well) were seeded onto six-well plates with DMEM SKP2 supplemented with 5% fetal bovine serum without antibiotics. GF signals Sixteen hours after plating, siRNA was transfected using Lipofectamine RNAiMAX (Invitrogen) at a final RNA concentration of 100 nM. p27 Cell cycle analysis Cells infected with recombinant adenovirus at an MOI of 600 Cell cycle were incubated for 48 h, washed twice with ice-cold phosphate- buffered saline (PBS) (À), fixed in 1% methanol in PBS for progression 20 min on ice and washed twice with PBS (À). The cells were resuspended in 5 ml ice-cold 70% ethanol, kept at À20 1C for 4 h, washed twice with PBS (À) containing 0.1% Triton X-100 Figure 6 A model consistent with our findings. RECK protects and 5 mg/ml bovine serum albumin, resuspended in 1 ml PBS collagen from proteolytic degradation, and collagen downregulates (À) containing 20 mg/ml propidium iodide, 250 mg DNase-free SKP2 (possibly through integrin signaling), leading to decreased RNaseA and incubated at room temperature for 30 min in the p27-degradation and consequent suppression of proliferation. dark. The stained cells were analyzed using FACScan (Becton Downregulation of RECK gene expression by growth factor (GF) Dickinson, Franklin Lakes, NJ, USA) with the CELLQuest as well as oncogenic signals have been found in previous studies (Takahashi et al., 1998; Sasahara et al., 1999; Hatta et al., 2009). software. There may be other pathways by which RECK suppresses cancer cell proliferation. Detection of senescence-associated b-galactosidase Senescence-associated b-galactosidase activity was detected as online tool FunNet (Prifti et al., 2008). The lists of normalized previously described (Dimri et al., 1995; Serrano et al., 1997) values were also filtered using GO-term numbers to focus on with some modifications. Briefly, the cells were washed once certain categories of genes. Gene expression data on human with PBS (À) (pH 7.2), fixed with 0.5% glutaraldehyde tumor samples were downloaded from the GEO profile (PBS (À) (pH 7.2)), washed in PBS (À) (pH 6.0) containing database at NCBI (http://www.ncbi.nlm.nih.gov/sites/entrez? 1mM MgCl2 and incubated in X-gal solution (1 mg/ml db ¼ geo&term). X-gal (Nacalai, Kyoto, Japan), 0.12 mM K3Fe(CN)6, 0.12 mM K4Fe(CN)6 and 1 mM MgCl2 in PBS at pH 6.0) for 10 h at 37 1C. RT–PCR The cDNA was synthesized using total RNA (the same batches of samples used in the GeneChip assay), oligo(dT) In situ zymography primer (PerkinElmer, Waltham, MA, USA), and SuperScript SW620 cells (2 Â 104 cell /35-mm glass-base dish (Iwaki, Chiba, II (Invitrogen, Carlsbad, CA, USA). The PCR amplification of Japan)) were infected with adenovirus at MOI of 600. Sixteen cDNA fragments was performed with Taq PCR Core Kit hours after infection, the cells were treated with freshly (Qiagen), under conditions optimized for linearity of amplifi- neutralized Cellmatrix type I collagen (Nitta Gelatin, Osaka, cation, using the following primers (Takara Bio, Shiga, Japan): Japan) and FITC-labeled DQ type I collagen (Molecular RECK (HA073007), p27 (HA039060), HPRT1 (HA031580) Probes, Eugene, OR, USA) as described previously (Kondo and SKP2 (sense: 50-ACCTTTCTGGGTGTTCTGGA-30, et al., 2007). After additional 48-h incubation, FITC signals, antisense: 50-AAGATTCAGCTGGGTGATGG-30). The representing cleaved DQ type I collagen, were recorded using PCR products were separated by electrophoresis (2% agarose) the confocal laser microscope (Olympus, Tokyo, Japan). and quantified using LAS-3000 (Fujifilm, Tokyo, Japan). Treatment with inhibitors Immunoblot assay SW620 cells (1 Â 105) plated onto regular or type I collagen- Proteins in the cell lysates prepared, as described previously coated six-well plates (Iwaki) on the previous day were (Oh et al., 2001), were quantified using the DC protein assay exposed to an inhibitor for 48 h. PP2, LY294002, PD98059, kit (Bio-Rad, Hercules, CA, USA), separated by SDS–PAGE GF109203X and Toxin B were purchased from Calbiochem (10% acrylamide) and subjected to indirect immunoblot assay (Rockville, ML, USA). using the following antibodies as primary antibodies (antigen (source)): RECK (5B11D12) (Takahashi et al., 1998), p27 (BD Biosciences, Pharmingen, San Jose, CA, USA), SKP2 (Cell Signaling Technology, Danvers, MA, USA), and GAPDH Conflict of interest (6C5, Ambion, Carlsbad, CA, USA). Specific bands were visualized using the Enhanced Chemiluminescence kit (Milli- The authors declare no conflict of interest.

Oncogene RECK regulates the SKP2–p27 pathway Y Yoshida et al 4137 Acknowledgements of the manuscript, Hai-Ou Gu and Aiko Nishimoto for technical assistance and Aki Miyazaki for secretarial assistance. This work We thank Chiaki Takahashi for the RECK cDNA, Mako was supported by JSPS Grant-in-Aid for Creative Scientiﬁc Yamamoto for R1B2-6 cells, Hitoshi Kitayama and Tomoko ResearchandMEXTGrant-in-AidonPriorityAreas. Matsuzaki for discussions, David Alexander for critical reading

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