Clin Transl Oncol DOI 10.1007/s12094-014-1253-z

RESEARCH ARTICLE

CMTM5 is reduced in prostate cancer and inhibits cancer cell growth in vitro and in vivo Y. Xiao • Y. Yuan • Y. Zhang • J. Li • Z. Liu • X. Zhang • Z. Sheng • T. Xu • X. Wang

Received: 1 August 2014 / Accepted: 27 October 2014 Ó Federacio´n de Sociedades Espan˜olas de Oncologı´a (FESEO) 2014

Abstract Purpose A novel tumor suppressor CKLF-like MARVEL transmembrane domain-containing member 5 (CMTM5) is reduced or undetectable in many kinds of cancers and inhibits tumor cells’ malignant features. To explore its role in prostate cancer (PCa), we detected its expression patterns in prostate tissues and PCa cells, and determined its anti-proliferation functions in PCa cells in vitro and in vivo. Methods The expression of CMTM5 in prostate tissue microarray, specimens and cell lines was evaluated by immunohistochemistry and Western blot, respectively. After being transfected with CMTM5 adenovirus or vector, the proliferation and migration of DU145 cells were detected by MTT assay and transwell assay, respectively. Furthermore, the effects of CMTM5 on tumor growth were performed in nude mice xenograft in vivo. Results We found CMTM5 was reduced in PCa tissues and cells compared with BPH tissues, and its expression in

Y. Xiao and Y. Yuan contributed equally to this work. Y. Xiao  Y. Yuan  J. Li  Z. Liu  X. Zhang  Z. Sheng  T. Xu (&)  X. Wang Department of Urology, Peking University People’s Hospital, Beijing 100044, China e-mail: [email protected] Y. Xiao Department of Urology, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325000, China Y. Yuan  Y. Zhang (&) Department of Urology, The Second Clinical Medical College of Jinan University, Shenzhen People’s Hospital, Shenzhen 518020, China e-mail: [email protected]

PCa tissues was related to the Gleason score. Moreover, after being transfected with adenovirus, ectopic expression of CMTM5-v1 in DU145 cells led to significant inhibition of cell proliferation and migration compared with the control, which may be attributed to decreased Akt activity. Finally, restoration of CMTM5 significantly suppressed tumor growth in vivo. Conclusions These results indicate that CMTM5 is downregulated in PCa and exhibit tumor suppressor activities in androgen-independent PCa cells. Loss of CMTM5 protein may be contributed to the development of PCa and it is a potential therapeutic target for castration-resistant prostate cancer. Keywords CMTM5  Prostate cancer  TM4SF  Tumor suppressor gene  Gene therapy

Introduction Prostate cancer(PCa) is the most common malignancy and the second leading cause of cancer-related death among males in the USA, and a major cause of morbidity and mortality in the Western world [1].There is no specific therapy for advanced PCa other than androgen deprivation therapy (ADT) for androgen-sensitive tumors [2]. However, most patients inevitably relapse and develop an incurable castration-resistant prostate cancer (CRPC) a few years later, which was formerly referred to as androgenindependent or hormone-refractory PCa and results in death within a few months [3, 4]. Therefore, a greater understanding of biological and physiological mechanisms that are involved in regulating prostate tumor growth and metastasis will help to discover new targets for CRPC and bring new hopes for those patients suffering from it.

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CKLF-like MAL and related proteins for vesicle trafficking and membrane link (MARVEL) transmembrane domain-containing family (CMTM) is a novel family of proteins consisting of nine members, chemokine-like factor (CKLF) and CMTM1–CMTM8. The members of CMTM link to chemokines and the transmembrane 4 superfamily (TM4SF), having important roles in immune and male reproductive systems as well as disease pathogenesis including tumorigenesis [5–13]. CMTM5 is structurally similar to the TM4SF [5], which includes typical tetraspanin and MARVEL-domain-containing proteins and involves in multiple cancers [14–18]. We are also interested in CMTM5 located at 14q11.2, an important locus associated with the pathogenesis of multiple carcinomas [19–21]. Considering the chromosomal location and the protein structure, CMTM5 may also be a potential tumor suppressor gene (TSG) involved in tumorigenesis. A previous study indicated that CMTM5-v1, the major expressed mRNA splicing variant of CMTM5, contained 156 amino acids and was broadly expressed in human fetal and adult tissues, but frequently epigenetically silenced by promoter methylation in cells derived from various cancers [22]. CMTM5 may exert tumor-suppressive activities on cervical, pancreatic and ovarian carcinomas, leukemia and the PCa cell line PC3 [22–27]. In the present study, we detected CMTM5 expression in benign prostate hyperplasia (BPH) and PCa tissues and cell lines, and identified the effects of CMTM5 restoration on DU145 cells proliferation and migration. Also, we examined the functions of CMTM5 in nude mice xenograft model in vivo.

Materials and methods Chemicals, reagents and antibodies A polyclonal antibody against CMTM5 of human origin (epitope mapping at the C-terminus, capable of detecting CMTM5 isoforms 1–5 of human origin), CMTM5-v1 adenovirus (ad-CMTM5) and empty vector were given as gifts by Prof. Wenling Han (Peking University Center for Human Disease Genomics, Beijing, China). Antibody specific for Akt, p-Akt(Ser473),CyclinD1, b-actin and GAPDH were from Cell Signaling Technologies (Danvers, MA, USA). Tissue microarray was purchased from Chaoying Biotechnology Co. (Xi’an, China). Other BPH tissues were obtained from patients undergoing transurethral resection of the prostate at the Peking University People’s Hospital with the patients’ consent and institutional ethics approval.

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Immunohistochemistry Immunohistochemistry was performed using the Envision TM ABC Kit in accordance with the manufacturer’s protocols. After counterstaining with hematoxylin, the sections were dehydrated and mounted. A previously established immunohistochemical scoring system for prostate carcinomas was used as the Ref. [28]. The percentage of positive cells was scored as 1, \25 %; 2, 25 % *50 %; 3, [50 %. The staining intensity was also scored: 0, absence of signal; 1, low-intensity signal (light brown); 2, moderate-intensity signal (brown); and 3, high-intensity signal (dark brown). The frequency and intensity scores were added to obtain the final score for each case, which was evaluated by two experienced observers, independently and without clinical data. The overall results of CMTM5 staining were assigned as ‘‘low expression’’ when the sum was 1–3, and ‘‘high expression’’ when 3–6 (except 3). Tissue samples were estimated in a consecutive analysis to ensure maximal internal consistency. All evaluations were conducted using a LEICA DM4000B/M microscope. Cell culture and infection Three human metastatic androgen-independent cell lines, DU145, PC3 and 22Rv1 (still weakly stimulated by dihydroxytestosterone), were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) or our collaborators. LNCaP was purchased from the Cell Resource Center of Peking Union Medical College (Beijing, China). Cell culture medium and reagents were from Invitrogen (Carlsbad, CA,USA). Cells were maintained in RPMI1640, 10 % FBS, penicillin (100 U/ml) and streptomycin (100 lg/ml) in a humidified atmosphere of 5 % CO2 air. Cells were infected with ad-CMTM5 or vector-containing adenovirus as described [22]. The efficiency of infection was monitored by ad-pEGFP. Cells with [75 % infection efficiency were used for further experiments. Cell proliferation assay Cells infected with ad-CMTM5 or vector were plated in 96-well plates at a density of 2,000 cells per well and then cultured at 37 °C. Cell proliferation was analyzed using the MTT Cell Proliferation Assay Kit (invitrogen, USA). 20 ll MTT reagent was added to each well and incubated for 4 h. The number of viable cells was calculated by absorbance measurements at 570 nm. Wound healing assay Cell mobility was assessed using a scratch wound assay. Adenovirus-infected cells were cultured in a 24-well plate

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until confluent. The cell layer was carefully wounded using sterile tips and washed twice with fresh medium. After incubation for 0 and 24 h, the cells were photographed at low magnification (409).

milk and incubated with primary antibodies in blocking solution overnight at 4 °C. Horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology) were used for detection of proteins by chemiluminescence (Thermo Fisher Scientific).

Western blot Tumor xenograft model in nude mice Forty-eight hours after infection, cells were washed and lysed in 19 cell lysis buffer (Cell Signaling Technology Inc., Beverly, MA).Total cellular protein (40 lg) was separated on SDS-PAGE and transferred to nitrocellulose membrane(Millipore). Membranes were blocked in Tris– HCl buffered saline (TBS) solution with 5 % nonfat dry

All animal experimental protocols were approved by Peking University Institutional Animal Care and Use Committee. Male BALB/c nu/nu nude mice (6 weeks) were obtained from Peking University Experimental Animal Center. Briefly, PC3 cells (5 9 106) were injected

Fig. 1 Expression patterns of CMTM5 in prostate tissues and cells. A Representative images from immunohistochemical staining of CMTM5 protein in BPH (a, b) and PCa tissues (c, d). a, c High expression; b, d low expression. B CMTM5 expression in BPH tissues and PCa cells

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subcutaneously into the right dorsal flank of the nude mice. Once palpable tumors were formed, tumor growth was monitored daily using a linear caliper, and the volume (V) was estimated using the formula V = (L 9 W2)/2, where L was the length and W was the width of the tumor. When tumors reached an average volume of 50–100 mm3, mice were randomly divided into two groups for injection with ad-CMTM5 or vector (2.5 9 106 pfu each). Mice were killed 3 weeks after the virus injection, and tumors were excised, measured, weighed and photographed. Specimens were fixed in formalin and embedded in paraffin for IHC staining. Statistical analysis Statistical analysis was performed by Statistical Package of the Social Sciences (SPSS) software 17.0 (SPSS Inc., Chicago, IL, USA). Chi square test was used to analyze the relation between CMTM5 expression and different clinicopathological characteristics. Student’s t test was used to analyze the results from at least three independent experiments and presented as mean ± SEM. P \ 0.05 was considered as significant difference.

performed to detect CMTM5 expression of four PCa cell lines and BPH tissues. As shown in Fig. 1B, CMTM5 was expressed in BPH tissues, but was undetectable in all of the four cell lines. Restoration of CMTM5-v1 inhibits proliferation and migration of DU145 cells in vitro The above expression results of CMTM5 in PCa and effects of it on the phenotype of PC3 cells mentioned by the former study [22] indicate that CMTM5 is a potential TSG. To further determine whether CMTM5 exerts tumorsuppressive ability in PCa cells, we analyzed the effects of CMTM5 on the proliferation of DU145 cells. MTT assay revealed that ectopic CMTM5-v1 expression induced timedependent inhibition on tumor cell growth (Fig. 2a). Since other MARVEL domain-containing proteins, such as MAL, are methylated in carcinoma cell lines and suppress the motility, invasion and tumorigenicity of tumor cells, we also assessed the effects of CMTM5-v1 on the

Table 1 CMTM5 expression in prostate tissues and correlation between CMTM5 and clinicopathologic features in PCa patients Classification

Number

Results CMTM5 expression is downregulated in PCa tissues and cell lines A tissue microarray included 75 spots of different human prostate tissue samples (64 PCa and 11 BPH tissues) and 25 other BPH surgical specimens were used. In BPH tissues, CMTM5 expression was mainly detected in the membrane of the prostatic epithelia cells. In PCa tissues with CMTM5 high expression, it was mainly located in the cytoplasm of cancer cells (Fig. 1A). It indicated that 75 % (27/36) of BPH tissues had high CMTM5 expression. However, CMTM5 was decreased markedly in 64.1 % (41/64) of PCa tissues (Table 1). There was a significant difference in the ratio of high expression of CMTM5 between PCa (23/64, 35.9 %) and BPH tissues (27/36, 75.0 %) (P \ 0.001).The expression of CMTM5 had no correlation with the age, clinical stage and metastasis status. Interestingly, when compared with the expression levels of CMTM5 in PCa tissues with different Gleason scores, we found that there was a significant difference (P = 0.003) between the well and moderately differentiated tumors (GS B 7) and the poorly differentiated ones (GS C 8). The percentages of high expression cases were 47.7 versus 10.0 %, which hinted that CMTM5 expression might be correlated with the malignant grade of PCa. To further confirm this interesting finding, Western blot was

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Low expression, n (%)

High expression, n (%)

P

\0.001*

Type BPH

36

9 (25.0)

27 (75.0)

PCa

64

41 (64.1)

23 (35.9)

Age (years) \70

32

21 (65.6)

11 (34.4)

C70

32

20 (62.5)

12 (37.5)

B7

44

23 (52.3)

21 (47.7)

C8

20

18 (90.0)

2 (10.0)

0.794

Gleason score 0.004*

Primary tumor T2

34

24 (70.6)

10 (29.4)

T3–T4

30

17 (56.7)

13 (43.3)

0.247

28 36

17 (60.7) 24 (66.7)

11 (39.3) 12 (33.3)

0.622

?

28

19 (67.9)

9 (32.1)

0.577

-

36

22 (61.1)

14 (38.9)

Clinical stage II III–IV Metastasis

Lymph node metastasis ?

23

14 (60.9)

9 (39.1)

-

41

27 (65.9)

14 (34.1)

0.690

Distant metastasis ?

24

18 (75.0)

6 (25.0)

-

40

23 (57.5)

17 (42.5)

* P \ 0.05

0.158

Clin Transl Oncol Fig. 2 Ectopic expression of CMTM5-v1-inhibited cell proliferation and migration of androgen-independent DU145 cells. a CMTM5-v1 inhibited the proliferation of DU145 cells in a time-dependent manner. The relative growth OD values assessed by MTT are shown above. Data represent the mean ± SEM of OD570 of at least three independent experiments. (*P \ 0.05). b CMTM5-v1 slowed down cell migration of DU145 as assessed by wound healing assay. Photos were taken after the scratch at 0 and 24 h. c Protein expressions of CMTM5, Akt, pAkt and cyclin D1 were detected by Western blot

migration of DU145 cells. Wound healing assay was conducted with the ad-CMTM5-infected cells and the vector control cells. The results showed that CMTM5-v1expressing cells had decreased migration ability compared with the control (Fig. 2b). To elucidate the molecular mechanisms underlying the tumor cell growth and migration inhibition by CMTM5, several proteins which may participate in the process were assessed by Western blot. There were significant decreased protein levels of phosphorylated Akt and its downstream cyclin D1in CMTM5-v1-transfected cells compared with the control (Fig. 2c), consistent with the inhibition of biologic behaviors. These results suggest that CMTM5 may repress PCa cells through regulating PI3 K-Akt signaling, which is one of the most important pathways in tumor progression. Restoration of CMTM5-v1 inhibits xenograft tumor growth in vivo To confirm the in vivo tumor-suppressive ability of CMTM5, tumor formation in nude mice was induced by injecting PC3 cells (Fig. 3a).Within 3 weeks after adenovirus injection, the solid tumor of the ad-CMTM5 group

had a much slower growth curve than the control group (Fig. 3b). In addition, the weight of tumors caused by CMTM5-v1-expressing cells was significantly smaller than the control (P \ 0.05; Fig. 3c). These results indicated that tumor growth was completely suppressed in the animals injected with ad-CMTM5.

Discussion CMTM5 is a newly discovered gene belonging to the human CKLF-like MARVEL transmembrane domaincontaining family. There was frequent CpG methylation of CMTM5 promoter in carcinoma cell lines [22], leading to its loss of expression. Ectopic expression of CMTM5-v1 can inhibit cell colony formation, viability and migratory ability in several tumor cell lines including PC3. However, the role of CMTM5 in the development of PCa has not been reported yet. Here, we tried to elaborate it by investigating the expression, function and preliminary mechanisms of the potential TSG. In the present study, we found that the expression of CMTM5 was reduced in most of the PCa tissues (64.1 %). More importantly, CMTM5 expression had no correlation

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Fig. 3 Restoration of CMTM5-v-inhibited tumor growth in vivo. a Representative images of the xenograft tumors and immunohistochemical staining of CMTM5 in tumor tissues after ad-CMTM5 or vector injection. b, c CMTM5-v1 effectively suppressed tumor

growth in nude mice. The average tumor volume and weight of the two groups are expressed as mean ± SEM in 5–6 inoculated sites for each group (*P \ 0.05)

with patients’ clinical stage and metastasis status in these 64 cases, but there was a significant difference of its expression between the two groups of patients with different Gleason scores, supporting that CMTM5 may participate in the progression of PCa. CMTM5 protein in PCa cells was also undetectable, but re-expressed after infection of ad-CMTM5. Similar to other tumor cell lines, exogenous CMTM5-v1 expression in silenced cancer cells dramatically suppressed not only tumor cell proliferation, but also their migration abilities through attenuating Akt activity. Moreover, introducing the CMTM5-v1 gene into cancer xenograft significantly slowed down tumor growth. Our findings demonstrate that CMTM5 possesses tumor suppressor functions in PCa, and its epigenetic silencing is probably involved in tumor pathogenesis. As we know, the two cell lines we selected for the study are androgen independent, and the new finding of this study may provide some novel idea for treatment with CRPC. Studies on CMTM5 or other MARVEL domain-containing proteins will uncover their roles and mechanisms in cancer and may also promote the application in CRPC therapy. Though the detailed molecular mechanisms of CMTM5 functioning in PCa are poorly characterized, we speculate that it may be attributed to the predicted MARVEL domain. MARVEL, a common feature of physins,

gyrins, occludin, MAL and CMTM families, is a membrane-associating domain and may be a part of the machinery of membrane apposition events [29], such as transport vesicle biogenesis, neurotransmitter secretion and polarized membrane trafficking. CMTM8, another member of the CMTM family, is the first identified protein with a MARVEL domain that can regulate epidermal growth factor (EGFR) signaling by accelerating the membrane receptor internalization and subsequent degradation in tumor cells [10]. In addition, CMTM7 also inhibits cancer cell growth and represses oncogenic EGFR signaling through promoting EGFR internalization and further suppressing the Akt signaling pathway [30].Thus, we suppose that the mechanisms of CMTM5 acting on PCa cells might be also involved in membrane protein sorting and trafficking, which supports the value of assessing whether CMTM5 can regulate the EGFR or vascular endothelial growth factor receptor (VEGFR) signaling, both wellacknowledged targets of molecular-targeted therapy in other cancers, but does not work in CRPC. Further evaluation of this hypothesis will provide additional information on the sensitivity of molecular-targeted therapy. Since the CMTM family was just recently discovered in 2003, it is a novel family of proteins, with very limited

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studies conducted thus far for their molecular functions. The primary results of our study provided evidences that CMTM5 was a potent TSG and with putative suppressive functions in androgen-independent PCa cells in vitro and in vivo. A better understanding of the tumor-suppressive role of CMTM5 will significantly improve our knowledge of the roles of the CMTM family of proteins in carcinogenesis, and might lead to application of a new PCa therapeutic target for those ADT-resistant tumors.

Conclusions CMTM5 is significantly down-regulated or undetectable in PCa tissues and cell lines. Restoration of CMTM5-v1 inhibits PCa cell proliferation and migration in vitro and tumor growth in vivo. Thus, CMTM5 acts as a tumor suppressor in PCa and targeting it may constitute a potential therapeutic strategy for CRPC. Acknowledgments This work was supported by the National Natural Science Foundation of China (NSFC) 81072099. We thank Drs. Wenling Han for her generous offer of adenovirus vector and antibody. We also thank Weidong Yu, Yidong Niu and Henan Li for some technical support, and Jianqiang Dong and the Department of Pathology, Peking University People’s Hospital for support for IHC and staining evaluation. Conflict of interest

The authors declare no conflict of interest.

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CMTM5 is reduced in prostate cancer and inhibits cancer cell growth in vitro and in vivo.

A novel tumor suppressor CKLF-like MARVEL transmembrane domain-containing member 5 (CMTM5) is reduced or undetectable in many kinds of cancers and inh...
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