Tumor Biol. (2014) 35:12083–12090 DOI 10.1007/s13277-014-2509-5

RESEARCH ARTICLE

CD109 is a potential target for triple-negative breast cancer Ji Tao & Hongbin Li & Qingwei Li & Yu Yang

Received: 27 July 2014 / Accepted: 14 August 2014 / Published online: 23 August 2014 # International Society of Oncology and BioMarkers (ISOBM) 2014

Abstract The aim of this study is to explore the expression of CD109 in breast cancer stem cells and the relationship between CD109 protein and clinicopathological characteristics of breast cancer. CD44+/CD24− tumor cells (CSCs) were selected by flow cytometry. The protein expression of CD109 was analyzed by immunohistochemistry staining, and the relationship between CD109 and clinicopathological parameters of breast cancer was determined. CD109 positively regulated the proliferation of breast CSCs in vitro, and CD109 protein expression was significantly higher in triple-negative breast cancer (TNBC) compared to non-TNBC (63.78 vs. 3.71 %, P=0.001). Moreover, CD109 protein expression was related to the histological grade of breast cancer (P=0.015), whereas age (P=0.731), tumor size (P= 0.995), clinical stage (P=0.644), and lymph node metastasis (P=0.924) were not. In the logistic regression model, histological grade (P=0.001) and molecular type (P=0.001) were significantly related to CD109 expression. The patients with high expression of CD109 protein had significantly poorer postoperative disease-specific survival than those with no or low expression of CD109 protein (P=0.001). In the Cox regression, CD109 was an independent prognostic factor (P=0.001). CD109 is highly expressed in TNBC and is a potential biomarker for the initiation, progression, and differentiation of breast cancer tumors. J. Tao : Q. Li Department of Gastrointestinal Medical Oncology, The Affiliated Tumor Hospital of Harbin Medical University, No. 150 Haping Road, Harbin 150040, Heilongjiang Province, China H. Li Department of Breast and Lymphoma Medical Oncology, The Affiliated Tumor Hospital of Harbin Medical University, No. 150 Haping Road, Harbin 150040, Heilongjiang Province, China Y. Yang (*) Department of Oncology, The 2nd Hospital Affiliated to Harbin Medical University, Harbin 150086, China e-mail: [email protected]

Keywords Breast cancer . Stem cell . TNBC . Survival . CD109

Introduction Breast cancer remains a huge threat to women’s health [1]. Furthermore, patients with triple-negative breast cancer (TNBC)—negative expression of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 (HER2)—are insensitive to hormonal therapy and HER2targeted agents, so TNBC is often incurable and a leading cause of mortality [2, 3]. Unfortunately, there are currently no specific therapies to control the recurrence and metastasis of TNBC [4], and the mechanisms underlying the metastasis and chemotherapy resistance of TNBC are not fully understood [5]. Breast cancer stem cells are a small group of tumor cells with the capacity to self-renew, a strong ability to form solid breast tumors, and the ability to differentiate into a relatively quiescent primitive group of cancer cells that are considered the underlying factor of tumor recurrence and the main reason that breast cancers resist therapies [6]. Following a better understanding of cancer stem cell theory, stem cell-related genes in malignant tumors have gained more academic attention. Recently, some studies have suggested that high expression of CD109 antigen regulates the phenotype of cancer stem-like cells/cancer-initiating cells (CSCs/CICs) in the novel epithelioid sarcoma cell line ESX and might be a CSCs/CIC marker in epithelioid sarcoma [7]. The studies indicated that CD109 may be a promising prognostic biomarker and a molecular target of cancer therapy for sarcomas [7]. In another study, Cuppini et al. reported that CD109 is highly expressed in circulating endothelial and progenitor cells in recurrent glioblastomas [8]. CD109, a glycosylphosphatidylinositol-

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anchored protein, is a member of the α2 macroglobulin/C3, C4, C5 family of thioester-containing proteins, and high expression of the CD109 gene was detected in approximately half of examined lung, esophageal, and cervical squamous cell carcinomas [9]. Currently, studies addressing the function and specific mechanism of CD109 in the biological behavior of breast CSCs and TNBC are rare. Moreover, the relationship between CD109 protein expression and the clinicopathological features of breast cancer is not clear [10]. In this study, we selected breast CSCs and investigated the expression of the stem cell-related gene CD109 in order to lay a foundation for the management of TNBC.

Materials and methods Patients and tissue specimens A total of 1,032 patients with histologically confirmed breast cancer and who underwent radical operations at Liaoning Cancer Hospital and Institute between January 2001 and January 2008 were enrolled for immunohistochemical staining and prognostic analysis. In addition, 82 cases were selected for Western blot analysis and 12 cases were chosen for CSC selection analysis from January 2012 to August 2013. The inclusion criteria were as follows: (a) curative operations were performed, (b) resected specimens were pathologically examined, (c) more than 15 lymph nodes were pathologically examined after operation, and (d) a complete medical record was available. The study protocol was approved by the Ethics Committee of Dalian Medical University. Experimental materials CD24-PE, CD44-FITC, CD2-FITC, CD3-APC, CD10-PE, CD16-FITC, CD18-APC, CD31-PE, CD326-FITC (EpCAM), and CD109-PE were obtained from BD Pharmingen (BD Co., USA). Ultra-low adherent plates, sterile cell scrapers, MammoCult® Basal Medium, MammoCult® Proliferation Supplement, Hanks’ balanced salt solution (HBSS), hydrocortisone, and heparin were purchased from STEMCELL Technologies Inc., Canada. Anti-CD109 antibody was obtained from Santa Cruz Biotechnology, Inc. The flow cytometer was obtained from BD Pharmingen. A modified Stem Cell RT2 ProfilerTM PCR Array and the ABI PRISM 7700 system (Applied Biosystems) were obtained from SABiosciences. Mammosphere generation test Complete MammoCult™ Medium (Human) was prepared by adding 50 mL thawed MammoCult™ Proliferation

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Supplements (Human) to 450 mL MammoCult™ Basal Medium (Human). Single cells were plated on ultra-low attachment plates (Corning, Acton, MA, USA) at a density of 20,000 viable cells/mL in Complete MammoCult™ Medium. The number of spheres for each well was evaluated after 5 days of culture. The numbers of mammospheres (>50 μm) in individual wells were counted in a blinded manner [11]. Tumor-forming ability of CD44+/CD24− tumor cells Human mammary tissue (1×1×1 cm per specimen) was transported from the operating room to the laboratory on ice in sterile specimen cups in Roswell Park Memorial Institute (RPMI) 1640 medium within 15 min. The tissue was transferred to sterile glass petri dishes, minced to 1× 1×1 mm with scalpels, washed three times with phosphate buffered saline (PBS), and then transferred to tissue dissociation flasks. Collagenase III was added to the minced tissue in the dissociation flasks and allowed to incubate at 37 °C for 3–4 h. Pipetting (to mix) with a 10-mL pipette was done every 15–20 min. At the end of the incubation, cells were filtered through a 45-μm nylon mesh and washed twice with PBS. Trypan blue stain was used to remove dead cells and count the cells on the cell plate. Cells to be injected were then suspended in RPMI 1640/Matrigel mix (1:1 volume) and injected into the appropriate area of the mammary fat pad of NOD/scid mice. Cell staining for flow cytometry Cells were counted and then transferred to a 5-mL tube, washed twice with HBSS with 2 % heat-inactivated calf serum (HICS; 5 min at 1,000 rpm), and then resuspended in 100 μL HBSS per 10 6 cells with 2 % HICS. Then, 5 μL Sandoglobulin solution (1 mg/mL) was added and incubated on ice for 10 min, after which the sample was washed twice with HBSS/2 % HICS and resuspended in 100 μL HBSS/2 % HICS per 106 cells. Antibodies (appropriate dilution per antibody) were then added and incubated for 20 min on ice and then washed twice with HBSS/2 % HICS. When needed, a secondary antibody addition was conducted by resuspending in 100 μL HBSS/2 % HICS per 106 cells, then adding 1–4 μL secondary antibody (depending on the secondary antibody and its concentration), followed by a 20-min incubation. When a streptavidin step was used, cells were resuspended in 100 μL HBSS/2 % HICS per 106 cells, and then 1 μL streptavidin, conjugated with the indicated fluorescent dye, was added, followed by a 20-min incubation. The cells were washed twice with HBSS/2 % HICS and resuspended in 0.5 mL HBSS/2 % HICS per 10 6 cells containing 7aminoactinomycin D (7AAD, 1 μg/mL final concentration).

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Flow cytometry Flow cytometry was performed on FACSVantage. Cells were routinely sorted twice, and the cells were reanalyzed for purity, which typically was >95 %. Lineage+cells were firstly eliminated by anti-CD2, anti-CD3 anti-CD10, anti-CD16, anti-CD18, anti-CD31, and anti-CD326 during flow cytometry. Dead cells were eliminated by using the viability dye 7AAD. Second, CD44+/CD24− tumor cells were selected using CD44 and CD24 antibodies. Finally, CD 109+ CSCs were selected using CD109 antibody.

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Sections were then counterstained in Gill’s hematoxylin and dehydrated in ascending grades of methanol, before clearing in xylene and mounting under a coverslip. As a negative control, staining was also performed without primary antibodies. The reactivity of the anti-CD109 antibody was determined by the staining pattern of the tumor cell membrane and graded as follows: 0 (no staining), 1 (partial staining of the membrane), 2 (mild to moderate circumferential staining of the membrane), and 3 (strong circumferential staining of the membrane). If the score was 2 or 3 in more than 10 % of the tumor cells, it was considered to be positive.

Western blot analysis Statistical analysis The CSCs were separated from the clinical samples, and their total proteins were extracted using a total protein extraction kit (ProMab, Richmond, USA), followed by centrifugation. After quantification of protein concentrations using a BCA assay (Santa Cruz Biotech), the individual cell lysates (30 μg/lane) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride membranes. The membranes were blocked with 5 % fat-free dry milk in Tris buffered saline with Tween 20 and incubated with rabbit anti-CD109 (1:500) or rabbit anti-β-actin (1:5,000; Abcam) at 4 °C overnight. After the membranes had been washed, the bound antibodies were detected with horseradish peroxidase-conjugated anti-rabbit IgG at room temperature for 1 h and visualized using enhanced chemiluminescence (Santa Cruz Biotechnology). Purified mouse, rabbit, and goat IgG were used as the respective negative controls. The relative levels of targeting proteins to the control β-actin were determined using the ImmuNe software. Immunohistochemistry The procedure was similar to that previously reported, with minor modifications [6]. Briefly, breast tumor tissues were cut at a thickness of 4 μm using a cryostat. The sections were mounted on microscope slides, air dried, and then fixed in a mixture of 50 % acetone and 50 % methanol. The sections were then de-waxed with xylene, gradually hydrated with gradient alcohol, and washed with PBS. Sections were incubated for 60 min with the rabbit polyclonal CD109 antibody (1:500 dilution, Santa Cruz Biotechnology, Inc.). Following washing with PBS, sections were incubated for 30 min in the secondary biotinylated antibody (Multilink Swine anti-goat/ mouse/rabbit immunoglobulin; Dako Inc.). Following washing, avidin-biotin complex (1:1,000 dilution, Vector Laboratories Ltd) was applied to the sections for 30–60 min at room temperature. The immunoreactive products were visualized by catalysis of 3,3-diaminobenzidine by horseradish peroxidase in the presence of H2O2, following extensive washings.

All data were analyzed with SPSS (Version 13.0, Chicago, IL, USA). Relationships between tumor markers and other parameters were studied using the chi-square test, Fisher’s exact test, or independent t tests. Disease-specific survival was analyzed using the Kaplan–Meier method. The log-rank test was used to analyze survival differences. Multivariate analysis was performed using the Cox proportional hazards model with forward stepwise variable selection. P<0.05 was considered statistically significant.

Results CD109+ CSCs have stronger tumorigenicity Our previous studies have shown the tumorigenicity of CSCs and their ability to form mammospheres [6]. CD109 is expressed by CSCs, which are crucial for the development and progression of cancers [12]. To understand the regulation of CD109 on the proliferation and migration of breast CSCs, we characterized the expression of CD109 in CSCs and found that the percentage of CD109+ CSCs (70.42±21.51 %) was significantly higher than that of CD109− CSCs (29.68± 14.79 %) in 12 patients (P<0.05) (Fig. 1a). Furthermore, we found that CD109− CSCs cultured for 5 days did not form typical mammospheres. However, CD109+ breast CSCs cultured under the same conditions for 5 days did form many typical mammospheres of >60 μm (Fig. 1b), suggesting that CD109 positively regulated the proliferation of breast CSCs in vitro (P=0.01) (Fig. 1c). Next, we tested the effect of modulating CD109 expression on the tumorigenicity of breast CSCs in vivo. We found that implantation of 103 CD109+ breast CSCs resulted in solid tumor formation in four out of five NOD/scid mice, while implantation with 104 CD109− CSCs induced solid tumors in two out of five NOD/scid mice (Fig. 1d), suggesting that the tumorigenicity CD109+ breast CSCs was stronger than that of

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Fig. 1 The tumorigenicity of CD109+ cancer stem-like cells (CSCs). a Flow cytometry analysis of CD109+ CSCs in specimens. b CD109+ CSCs formed typical mammospheres while CD109− CSCs did not. c The ability of CD109+ CSCs and control groups to form mammospheres.

Single cells were plated on ultra-low attachment plates at a density of 20,000 viable cells/mL, and the number of spheres in each well was evaluated after 5 days of culture. d The rate of xenograft tumors in NOD/ scid mice

CD109− CSCs in our experimental system. Collectively, the rapid formation of typical mammospheres in vitro and solid tumors in vivo clearly indicated that the CD109+ breast CSCs had stronger tumorigenicity, which may contribute to the progression and metastasis of TNBC.

P=0.001) (Fig. 3, Table 1). Moreover, we observed that CD109 protein expression was associated with the histological grade of breast cancer (P=0.015), but not associated with age (P=0.731), tumor size (P=0.995), clinical stage (P= 0.924), or lymph node metastasis (P=0.924). In the logistic regression model, histological type (P=0.001) and molecular type (P=0.001) were significantly associated with CD109 expression (Tables 1 and 2).

Expression of CD109 in breast cancer and its association with clinicopathological characteristics CD109 showed significantly different expression between TNBC than non-TNBC patients at the protein level (P= 0.001) (Fig. 2). CD109 protein had significantly higher expression in TNBC than non-TNBC patients (63.78 vs. 3.71 %,

Association between CD109 protein expression and chemotherapeutic resistance We further studied the association between CD109 protein expression and chemotherapeutic sensitivity in 116 neoadjuvant chemotherapy breast cancers. CD109 protein was expressed in 0, 16.7, 33.3, and 50.0 % of patients with complete response, partial response, stable disease, and progressive disease (P=0.001), respectively (Table 3). Prognostic analysis

Fig. 2 Western blot analysis of the level of CD109 expression. CD109 showed higher expression in TNBC (nos. 1–3) than in non-TNBC (nos. 4–6) (P<0.01)

The rate of distant metastasis was significantly higher among patients exhibiting CD109 expression compared with the rate

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Fig. 3 The CD109 expression in 1,032 surgical tissue samples from patients with breast cancer was characterized by immunohistochemistry. a Negative anti-CD109 staining in the surrounding non-tumor areas. b Negative or slight anti-CD109 staining in the non-TNBC tumors. c High anti-CD109 staining in the TNBC tumors (magnification ×400)

Table 1 The relationship between CD109 expression and the clinicopathological factors (n=1,032) Varies

Number

Age <35 years 156 >35 years 876 Tumor size T1 192 T2 684 T3 156 Differentiation grade I 132 II 459 III 441 Clinical stage DCIS 117 IDC 915 Lymph node metastasis pN0 420 pN1 356 pN2 pN3 Molecular type TNBC Non-TNBC

215 41

CD109+ (n (%))

X2 value 0.118

0.731

0.011

0.995

8.428

0.015

0.214

0.644

25 (16.03) 131 (14.95) 29 (15.10) 103 (15.06) 24 (15.38) 9 (6.82) 72 (15.69) 75 (17.01)

Discussion

16 (13.68) 140 (15.30) 0.478

0.924

61 (14.52) 53 (14.89)

CD109 has been identified as a co-receptor for transforming growth factor (TGF)-β and a negative regulator of TGF-β Table 2 Logistic regression analysis of the factors related to CD109 expression

35 (16.28) 7 (17.07) 446.468

196 836

P value

among CD109-negative patients. Of the 156 patients with CD109 protein expression, 96 (61.5 %) developed 5-year postoperative distant metastasis, whereas only 161 (18.4 %) of patients without CD109 protein expression developed 5year postoperative distant metastasis (P=0.001). Survival analysis showed the patients with high expression of CD109 protein had significantly poorer postoperative disease-specific survival than those with no or low expression (P=0.001) (Fig. 4). In the Cox regression, tumor size (P=0.017), histological grade (P=0.001), clinical stage (P=0.012), lymph node metastasis (P=0.015), molecular type (P=0.004), and CD109 (P=0.001) were identified as independent prognostic factors (Table 4).

125 (63.78) 31 (3.71)

DCIS ductal carcinoma in situ, IDC invasive ductal carcinoma

Characteristics

OR

95 % confidence interval for OR

P value

Histological type Molecular type Constant

1.773 1.536 0.127

1.458–2.158 1.246–1.893

0.001 0.001

0.001

12088 Table 3 Correlations between CD109 protein expression and chemotherapeutic resistance in breast cancer (n=116;)

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Organs metastasis

Number

CD109+ (n (%))

CD109− (n (%))

X2 value

P value

Complete response Partial response Stable disease Progressive disease

8 56 30 22

0 (0) 3 (16.67) 6 (33.33) 9 (50.00)

8 (8.16) 53 (54.08) 24 (24.49) 11 (11.22)

19.318

0.001

Fig. 4 Kaplan–Meier curves of overall survival by factors that were found to be independent prognostic factors in Cox regression analysis: CD109 status (P=0.001) (a), tumor size (P=0.001) (b), histological type

(P=0.01) (c), clinical stage (P=0.001) (d), lymph node metastasis (P= 0.015) (e), and molecular type (P=0.001) (f)

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Table 4 Cox model regression analysis of the breast cancer prognostic factors Varies

OR

95 % confidence interval for OR

P value

Age Tumor size Histological type Clinical stage Lymph node metastasis Molecular type CD 109

0.952 1.665 1.520 1.176 1.707

0.708–1.281 1.094–2.533 1.179–1.959 1.036–1.335 1.101–2.648

0.746 0.017 0.001 0.012 0.015

1.535 1.148–2.052 2.267 1.779–2.889

0.004 0.001

signaling [13]. In addition, CD109 promotes localization of the TGF-β receptors into the caveolar compartment in the presence of ligand and facilitates TGF-β-receptor degradation [14]. Thus, CD109 regulates TGF-β receptor endocytosis and degradation to inhibit TGF-β signaling. Some studies have shown that human cells overexpressing CD109 exhibit enhanced proliferation compared with control cells, so this molecule is known to be involved in tumorigenesis [15, 16]. Recently, circulating endothelial and progenitor cells (CECs and CEPs, respectively) have been considered with increasing interest as predictive biomarkers [8]. CD109 is highly expressed in a subtype of CECs and CEPs, and a baseline count of CD109+ CECs higher than 41.1/mL (first quartile) is associated with increased progression-free survival and overall survival [8]. Therefore, CD109+ CECs are a potential predictive marker. In another study, Emori et al. evaluated the expression of CD109 protein in 80 clinical specimens of soft tissue sarcoma and found a strong correlation between CD109 protein expression and the prognosis [7]. The reported studies indicated that there is a significant correlation between CD109 and the prognosis of some malignant solid tumors and that CD109 may be a molecular target of cancer therapy for cancers, especially for CSCs [17, 18]. However, the relationship between CD109 protein expression and biological behaviors is still unclear, as is the association with the prognosis. Only one study, which had a small sample size and did not include prognosis or analysis of sensitivity to chemotherapy, has found that CD109 has high expression in TNBC [19]. Furthermore, studies addressing the expression of stem cell genes in breast cancer and the relationship between stem cell gene expression and clinicopathological characteristics and prognosis of TNBC are sparse. Nevertheless, studies of cancer stem cell-related genes may lead to a new therapeutic method for the treatment of TNBC. In this study, we sorted and identified breast CSCs and successfully selected CD109+ CSCs. CD109 showed higher expression in CSCs than in non-CSCs. Most TNBCs expressed CD109 protein whereas non-TNBCs did not. We

investigated the relationship between CD109 expression and the biological behavior of the breast cancer stem cell and the clinicopathological characteristics of breast cancer. CD109 protein was associated with both the differentiation grade and the molecular type of the breast cancer. Recently, Ozbay et al. found that CD109 protein expression is significantly related to the tumor grade of vulvar squamous cell carcinoma [12]. In our study, high CD109 protein expression was correlated with the chemotherapeutic resistance of TNBC in neoadjuvant chemotherapy. The patients with high expression of CD109 protein had a significantly higher rate of distant metastasis and poorer postoperative disease-specific survival than those with no or low expression of CD109 protein, and Cox regression showed that CD109 was an independent prognostic factor. This outcome suggests that CD109 is associated with breast cancer stem-like cells and that its expression may be implicated in self-renewal and tumorigenesis via activation of its downstream target genes. CD109 may play a role in breast cancer oncogenesis and may be a potential biomarker for the initiation, progression, and differentiation of TNBC.

Conclusion CD109 is highly expressed in TNBC and tumor stem cells and is a potential biomarker for the initiation, progression, and differentiation of breast cancer tumors. It would be a potential target for TNBC. However, the underlying genetic mechanism of CD109 as it is expressed in breast cancer stem cells remains unclear. Hence, the relationship between CD109 gene expression and the biological behavior of breast cancer stem cells needs further investigation. Acknowledgments This study was funded by the China National Natural Science Foundation (No. 81102029 and 81172047) and Liaoning National Natural Science Foundation (No. 2013021006). Conflicts interests None

References 1. Desantis C, Ma J, Bryan L, Jemal A. Breast cancer statistics, 2013. CA Cancer J Clin. 2014;64(1):52–62. 2. Wright MH, Calcagno AM, Salcido CD, et al. Brca1 breast tumors contain distinct CD44+/CD24- and CD133+ cells with cancer stem cell characteristics. Breast Cancer Res. 2008;10:R10. 3. Liu R, Wang X, Chen GY, et al. The prognostic role of a gene signature from tumorigenic breast-cancer cells. N Engl J Med. 2007;356:217–26. 4. Gao N, Xu H, Liu C, Xu H, Chen G, Wang X, Li Y, Wang Y. Nestin: predicting specific survival factors for breast cancer. Tumour Biol. 2014 5. Xu D, Xu H, Ren Y, Liu C, Wang X, Zhang H, et al. Cancer stem cellrelated gene periostin: a novel prognostic marker for breast cancer. PLoS One. 2012;7(10):e46670.

12090 6. Liu CG, Lu Y, Wang BB, Zhang YJ, Zhang RS, Lu Y, et al. Clinical implications of stem cell gene Oct-4 expression in breast cancer. Ann Surg. 2011;253(6):1165–71. 7. Emori M, Tsukahara T, Murase M, Kano M, Murata K, Takahashi A, et al. High expression of CD109 antigen regulates the phenotype of cancer stem-like cells/cancer-initiating cells in the novel epithelioid sarcoma cell line ESX and is related to poor prognosis of soft tissue sarcoma. PLoS One. 2013;8(12):e84187. 8. Cuppini L, Calleri A, Bruzzone MG, Prodi E, Anghileri E, Pellegatta S, et al. Prognostic value of CD109+ circulating endothelial cells in recurrent glioblastomas treated with bevacizumab and irinotecan. PLoS One. 2013;8(9):e74345. 9. COTZIAS GC, BORG DC, SELLECK B. Virtual absence of turnover in cadmium metabolism: Cd109 studies in the mouse. Am J Physiol. 1961;201:927–30. 10. Hockla A, Radisky DC, Radisky ES. Mesotrypsin promotes malignant growth of breast cancer cells through shedding of CD109. Breast Cancer Res Treat. 2010;124(1):27–38. 11. Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF. Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci U S A. 2003;100(7):3983–8. 12. Ozbay PÖ, Ekinci T, Yiǧit S, Yavuzcan A, Uysal S, Soylu F, et al. Investigation of prognostic significance of CD109 expression in women with vulvar squamous cell carcinoma. Oncol Targets Ther. 2013;6:621–7.

Tumor Biol. (2014) 35:12083–12090 13. Man XY, Finnson KW, Baron M, Philip A. CD109, a TGF-β coreceptor, attenuates extracellular matrix production in scleroderma skin fibroblasts. Arthritis Res Ther. 2012;14(3):R144. 14. Bizet AA, Tran-Khanh N, Saksena A, Liu K, Buschmann MD, Philip A. CD109-mediated degradation of TGF-β receptors and inhibition of TGF-β responses involve regulation of SMAD7 and Smurf2 localization and function. J Cell Biochem. 2012;113(1):238–46. 15. Wang Y, Inger M, Jiang H, Tenenbaum H, Glogauer M. CD109 plays a role in osteoclastogenesis. PLoS One. 2013;8(4): e61213. 16. Hagikura M, Murakumo Y, Hasegawa M, Jijiwa M, Hagiwara S, Mii S, et al. Correlation of pathological grade and tumor stage of urothelial carcinomas with CD109 expression. Pathol Int. 2010;60(11):735–43. 17. Ohshima Y, Yajima I, Kumasaka MY, Yanagishita T, Watanabe D, Takahashi M, et al. CD109 expression levels in malignant melanoma. J Dermatol Sci. 2010;57(2):140–2. 18. Hagiwara S, Murakumo Y, Sato T, Shigetomi T, Mitsudo K, Tohnai I, et al. Up-regulation of CD109 expression is associated with carcinogenesis of the squamous epithelium of the oral cavity. Cancer Sci. 2008;99(10):1916–23. 19. Hasegawa M, Moritani S, Murakumo Y, Sato T, Hagiwara S, Suzuki C, et al. CD109 expression in basal-like breast carcinoma. Pathol Int. 2008;58(5):288–94.