Carcinogenesis AdvanceCarcinogenesis Access published November 15, 2013

Repression of cathepsin E expression increases the risk of mammary carcinogenesis and links to poor prognosis in breast cancer

Manuscript ID: Manuscript Type: Date Submitted by the Author:

CARCIN-2013-00846.R2

Original Manuscript

28-Oct-2013

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Complete List of Authors:

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Yamamoto, Kenji; Kyushu University, Graduate School of Pharmaceutical Sciences Kawakubo, Tomoyo; Kyushu University, Graduate School of Pharmaceutical Sciences Yasukochi, Atsushi; Kyushu University, Graduate School of Pharmaceutical Sciences Toyama, Tatsuya; Nagoya City University, Graduate School of Medical Sciences Takahashi, Satoru; Nagoya City University, Graduate School of Medical Sciences Okamoto, Kuniaki; Nagasaki University, Graduate School of Biomedical Sciences Tsukuba, Takayuki; Nagasaki University, Graduate School of Biomedical Sciences Nakamura, Seiji; Kyushu University, Graduate School of Pharmaceutical Sciences Ozaki, Yasuhiko; Nagoya City University, Graduate School of Medical Sciences Nishigaki, Koichi; Saitama University, Faculty of Engineering Yamashita, Hiroko; Nagoya City University, Graduate School of Medical Sciences

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Keywords:

biomarker, breast cancer, carcinogenesis, cathepsin E, Wnt5a

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RESEARCH ARTICLE: 2nd Revised Ms (CARCIN-2013-00846.R1)

Repression of cathepsin E expression increases the risk of mammary carcinogenesis and links to poor prognosis in breast cancer

Kuniaki Okamoto5, Takayuki Tsukuba5, Seiji Nakamura2, Yasuhiko Ozaki 6, Koichi Nishigaki7, Hiroko Yamashita3,8, and Kenji Yamamoto1*

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Pe Proteolysis Research Laboratory, Graduate School of Pharmaceutical Sciences and

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Department of Oral and Maxillofacial Surgery, Graduate School of Dental Science,

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Kyushu University, Fukuoka 812-8582, Japan,

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Department of Oncology,

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Immunology and Surgery, 4Department of Experimental Pathology and Tumor

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Biology, and 6Department of Obstetrics and Gynecology, Graduate School of Medical Sciences, Nagoya City University, Nagoya 467-8601, Japan, 5Department of Dental Pharmacology, Graduated School of Biomedical Sciences, Nagasaki University, Nagasaki 852-8588, Japan, and 7Department of Functional Materials Sciences, Faculty of Engineering, Saitama University, Saitama 338-8587, Japan. 8

Present address: Breast and Endocrine, Surgery, Hokkaido University Hospital,

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Tomoyo Kawakubo1, Atsushi Yasukochi1,2, Tatsuya Toyama 3, Satoru Takahashi4,

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Kita-ku, Sapporo 060-8648, Japan

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To whom correspondence should be addressed: Proteolysis Research Laboratory,

Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka Japan.

Tel/Fax:

+81-92-642-6768;

[email protected]

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Running title: Cathepsin E in mammary gland

Email:

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812-8582,

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Abstract Despite advances in detection and treatment for breast cancer (BC), recurrence and death rates remains unacceptably high. Therefore, more convenient diagnostic and prognostic methods still required to optimize treatments among the patients. Here

novel prognostic marker for BC. Correlation analysis between the serum levels of CatE expression and clinicopathological parameters revealed that the activity levels,

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but not the protein levels, were negatively associated with the stages and

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progression of BC. Univariate and multivariate analyses demonstrated that the

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serum CatE activity was significantly correlated with favorable prognostic outcomes of the patients. The functional link of CatE expression to BC progression

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was further corroborated by in vivo and in vitro studies with mice exhibiting

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different CatE expression. Multiparous CatE-/- mice spontaneously developed mammary tumors concomitant with morphological transformation and altered growth characteristics of the mammary glands. These alterations were associated in part with the induction of epithelial-mesenchymal transition and the activation of β-catenin-dependent pathway in mammary cells. Loss of CatE strongly induced the translocation and accumulation of Wnt5a in the nuclei, thereby leading to the

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we report the clinical significance of the serum cathepsin E (CatE) activity as a

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aberrant trafficking, maturation and secretion of Wnt5a and the impaired signaling. The interaction of CatE and Wnt5a was verified by proximity ligation assay and by knockdown or restoration of CatE expression in the mammary cells. Consequently, our data demonstrate that CatE contributes to normal growth and development of

Summary

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Here we show that reduction of the serum level of CatE activity is associated with

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poor prognosis in BC patients. This association was further corroborated by in vivo

expression.

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and in vitro studies using syngeneic multiparous mice showing different CatE

Keywords: biomarker/ breast cancer/ carcinogenesis/ cathepsin E/ Wnt5a

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mammary glands through proper trafficking and secretion of Wnt5a.

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INTRODUCTION Breast cancer (BC) is one of the most occurring cancers in women worldwide. Despite advances in diagnosis and treatment for BC, the prognosis and survival for most patients have not dramatically changed. Histological analyses of surgical

metastasis status, but seem not to be cost- and labor-effective. Therefore, more convenient diagnostic and prognostic methods for identifying a particular subgroup

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of patients with potentially poor prognosis are required to improve the overall

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outcome of BC. The circulating blood holds great promise as a reservoir of disease

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information and serves to provide a large variety of disease markers. We have thus been searching for new serum prognostic markers for BC.

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CatE is an intracellular aspartic proteinase, which is predominantly expressed in

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certain cell types including the immune system cells and the rapidly regenerating cells (1). The intracellular localization of CatE varies with different cell types (1). The strategic expression and localization suggest that CatE is associated with specific functions of each cell type. A role of CatE in immune responses against tumor cells has especially received special attention, because host defense against cancer cells is an important factor determining tumor evolution. It is also interesting

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specimens from BC patients are widely used to evaluate the tumor invasion and

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to note that, CatE displays a potent anticancer activity through manifold functions (1) and regulates the expression of proteins related to signaling, development, differentiation, and proliferation in mouse mammary gland (2). Loss of CatE was also shown to induce precancerous conditions in this tissue (2). Meanwhile, recent

cancers, although its clinical significance is controversial (3-7). To our knowledge, endogenous CatE inhibitors have not so far been identified in mammals. On the

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other hand, it has previously been reported that there are two CatE genes in normal

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and cancer cells in a different proportion (8) or two isozymic forms of CatE derived

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from the same gene (9, 10) and the abnormal molecular size enzyme in the pancreatic juice of the patient with ductal adenocarcinoma (11). These findings

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strongly suggest that some of CatE proteins produced in cancer cells are

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non-functional. Given that most of the previous studies were performed by immunological analyses such as immunohistochemistry, tissue microarray and ELISA or by gene expression profiling analyses such as quantitative RT-PCR or in situ hybridization to assess the total CatE expression levels, it seems likely that the conflicting reports seem to arise from differences in expression levels of some non-functional CatE between cancer cell types.

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studies suggest the clinical utility of CatE as a potential biomarker in certain human

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In this study, we show that reduction of the serum level of CatE activity is associated with poor prognosis in BC patients. The association of CatE expression with mammary carcinogenesis was further supported in in vivo and in vitro studies using four different genotypes of syngeneic multiparous mice showing different

risk of mammary carcinogenesis and links to poor prognosis in BC.

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MATERIALS AND METHODS

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Patients and samples

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The study included 293 consecutive Japanese women with BC, newly diagnosed at Nagoya City University Hospital between July 1999 and July 2008, and 107 control

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Japanese women who visited our hospital because of abnormalities found on breast

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screening or breast pain, and were confirmed without the disease. In some experiments, nine Japanese healthy women were chosen as another control group. The study protocol was approved by the institutional review boards and confirmed with the guidelines of the 1996 Declaration of Helsinki. All breast cancer patients except for those with Stage IV disease underwent surgical treatment (mastectomy or lumpectomy). Patients received adequate endocrine or chemotherapy for adjuvant or

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CatE expression. This study shows that repression of CatE expression increases the

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metastatic diseases. Mice All animals were maintained according to the guidelines of the Japanese Pharmacological Society in a specific pathogen–free facility at the Kyushu

approved by the Animal Research Committee of the Graduate School of Pharmaceutical Sciences, Kyushu University. WT, CatETG, CatE+/- and CatE–/– mice

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on the C57Bl/6 genetic background were used as described previously (2, 12, 13).

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Assay

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The assay of CatE activity in sera was performed using KYS-1 as a substrate by a slight modification of the method of Yasuda et al. (14). Reaction mixtures contained

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80 µl of 50 mM sodium acetate buffer, pH 4.0, 10 µl of 200 µM substrate solution,

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and 10 µl of sample solution containing 1-5 µl of sera and 0.1% Triton X-100. After 10-min incubation at 40 °C, the reaction was terminated by adding 2 ml of 10 mM Tris-HCl buffer, pH 8.6. ELISA Two different monoclonal antibodies against human CatE were custom-made at Genostaff Co. (Tokyo, Japan). The first monoclonal antibody was added to 96-well

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University Station for Collaborative Research. All animal experiments were

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plates and incubated at 4 °C overnight. After washing with Tris-buffered saline (TBS), pH 7.4, containing 0.05% Tween-20 (TBS-T), the plates were incubated with 1% Blocking One (nacalai tesque Inc., Tokyo, Japan)/TBS-T at 37 °C for 1 h. After washing, serum (15 µl) was added to the plate and incubated for 2 h at room

antibody (2 µg/ml in 1% Blocking One/TBS-T) were added to each plate and incubated for 1 h at room temperature. After washing, 100µl of avidin-conjugated

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peroxidase (2 µg/ml in 1% Blocking One/TBS-T) was added to the plates and

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incubated for 30 min at room temperature. Then the plates were washed and

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incubated with 100 µl of TMB for 20 min at room temperature in dark. The reaction was terminated by adding 100 µl of 1 N hydrochloride.

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Immunochemical analyses

Immunohistochemical analysis was performed on mouse mammary tissue sections that had been formalin-fixed and paraffin-embedded, essentially according to the method described previously (13). For immunoblotting, various antibodies including purified polyclonal antibodies to human CatE (15), human CatE propeptide (16), and rat CatE (16) were used. Polyclonal anti-Wnt5a antibodies raised in rabbits against the synthetic peptide corresponding to the sequence

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temperature. After washing, 100 µl of the biotin-labeled secondary monoclonal

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between amino acids 190 and 240 of human Wnt5a were obtained from Abcam Plc (Cambridge, UK). All other antibodies used were purchased from various commercial sources. Primary culture of mammary cells

essentially according to the method described previously (17). Briefly, mammary glands were digested for 6 h at 37 ºC in EpiCult-B medium (StemCell Technologies

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Inc., Tukwila, WA) containing 5% fatal bovine serum, 300 U/ml collagenase, 100

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U/ml hyaluronidase, 10 ng/ml recombinant human epidermal growth factor, 10

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ng/ml recombinant human basic fibroblast growth factor, and 0.0004% heparin. After lysis of the red blood cells, the resultant organoid pellet was subjected to

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sequential dissociation by gentle pipetting in TrypLE Express (GIBCO) for 2 min

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and in 5mg/ml Dispase®II (Godo Shusei Co., Ltd., Chiba, Japan) plus 1mg/ml DNaseI for 1 min. The cell suspension was then filtered through 40-micron cell strainer and suspended in Epicult-B defined medium. Mammosphere assay Mammosphere formation was performed in 24-well plates coated with Matrigel (Growth Factor Reduced Matrigel, BD Bioscience), essentially according to the

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Primary mammary cells were prepared from 8-12-week-old virgin female mice,

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method described previously (18). In vitro formation assay of capillary-like structure was performed according to the method described previously (19). Quantitative RT-PCR analysis Quantitative RT-PCR was performed essentially according to the method as

Wisp1 (68 bp), and β-catenin (68 bp). Primers used to amplify the transcripts are as follows: Gapgh; (forward) TGT CCG TCG TGG ATC TGA C, (reverse) CCT GCT

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TCA CCA CCT TCT TG, Sfrp1; (forward) GTG GTT CAA GAT GTG CTC CA,

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(reverse) TCA GAG CAG CCA ACA TGC, Wisp1; (forward) AAA GGG CAT GTG

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CAT TAT CC, (reverse) AAG CAT GCT GTA AGC TAG TTC TGA, β-catenin; (forward) GCA GCA GCA GTT TGT GGA, (reverse) TGT GGA GAG CTC CAG

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TAC ACC. Light Cycler Universal Probe Master (Roche Diagnostics GmbH)

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specific for each sequence was applied to number 80 (for Gapdh and Sfrp1), number 63 (for Wisp1), and number 25 (for β-catenin). All reactions were performed in triplicate for three dependent experiments. Gapdh was used as an endogenous control. Transfection Mouse CatE cDNA was subcloned from the pBluescript SK plasmid containing

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described previously (20). Amplicon sizes were Gapdh (75 bp), Sfrp1 (69 bp),

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full-length mouse CatE cDNA (21) into pcDNA4/HisMax©A vector. The construct was verified by DNA sequence analysis and restricting enzyme digestion analysis. Primary cultures of CatE-/- mammary cells were transiently transfected with pcDNA4/HisMax©A-mCatE or pcDNA4/HisMax©A-MOCK (empty vector) using

following the manufacturer’s protocol. For RNA interference, primary cultures of WT mammary cells were transiently transfected with control siRNA-A (sc-37007;

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Santa Curz Biotechnology, Inc., Santa Cruz, CA) or cathepsin E siRNA (sc-41474;

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Santa Curz Biotechnology, Inc.), using siRNA Transfection Reagent (Santa Curz

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Biotechnology, Inc.) according to the manufacturer’s protocol. After 72 h, the cells were washed with PBS three times and then harvested with ice-cold RIPA Buffer

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(nacalai tesque Inc., Kyoto, Japan) containing phosphatase inhibitor cocktail (Sigma

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Aldrich, Inc., Saint Louis) and protease inhibitors and then lysed and centrifuged at 10,000xg for 10 min. The supernatant was used as the cell extract. The cultured medium were also concentrated to a final volume of 500 µl by using Amicon Centriprep YM-3. PLA analysis PLA was performed using the Duolink® II Kit (Olink Bioscience, Uppsala, Sweden)

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X-tremeGENE HP DNA Transfection Reagent (Roche Diagnostics GmbH)

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according to the manufacturer’s instruction. This kit is based on the use of two unique and bi-functional probes called PLATM, each probe consisting of a secondary antibody attached to a unique synthetic oligonucleotide that acts as a reporter. After a 30 min fixation with 4% paraformaldehyde and blocking with PBS containing 5%

two primary antibodies depending on the experiment (single protein detection or detection of interacting proteins). After washing, the sections were incubated with

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the secondary oligonucleotide-linked antibodies (PLA probes) provided in the kit.

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After hybridization for 1 h at 37 °C, ligation for 30 min at 37 °C, and amplification

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for 100 min at 37 °C, the oligonucleotides bound to the antibodies were detected using a fluorescent probe (Detection Kit 563). The products were visualized with

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fluorescently labeled oligonucleotides and the sections counterstained using

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Duolink II Mounting Medium with 4’,6-diamidino-2-phenylindole. When two PLA probes were in close proximity to each other ( 50

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Age range (years)

0.19

5.42 ± 2.27 2.74 ± 0.17

0.039*

Number of positive

≥2 0

lymph nodes Grade

≥1 1 or 2

256

4.43 ± 2.74

5

1.75 ± 1.49

117

4.50 ± 2.71

144 148

4.28 ± 2.77 4.69 ± 2.65

86 133

3.71 ± 2.51 4.59 ± 2.60

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Repression of cathepsin E expression increases the risk of mammary carcinogenesis and links to poor prognosis in breast cancer.

Despite advances in detection and treatment for breast cancer (BC), recurrence and death rates remain unacceptably high. Therefore, more convenient di...
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