- Email: [email protected]
Chemokine CXCL14 is a multistep tumor suppressor
Author’s Accepted Manuscript Chemokine CXCL14 is a multistep tumor suppressor Xiaoyan Yang, Chihiro Miyamoto, Tetsu Akasaka, Kazuhito Izukuri, Yojiro Maehata, Takeharu Ikoma, Shigeyuki Ozawa, Ryu-Ichiro Hata www.elsevier.com
PII: DOI: Reference:
S1349-0079(15)00108-5 http://dx.doi.org/10.1016/j.job.2015.08.003 JOB134
To appear in: Journal of Oral Biosciences Received date: 29 July 2015 Accepted date: 15 August 2015 Cite this article as: Xiaoyan Yang, Chihiro Miyamoto, Tetsu Akasaka, Kazuhito Izukuri, Yojiro Maehata, Takeharu Ikoma, Shigeyuki Ozawa and Ryu-Ichiro Hata, Chemokine CXCL14 is a multistep tumor suppressor, Journal of Oral Biosciences, http://dx.doi.org/10.1016/j.job.2015.08.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Chemokine CXCL14 is a multistep tumor suppressor Xiaoyan Yang
a,b
, Chihiro Miyamoto
a,b
, Tetsu Akasaka
a,b
a,b
, Kazuhito Izukuri
,
Yojiro Maehata a,b, Takeharu Ikoma c, Shigeyuki Ozawa c and Ryu-Ichiro Hataa,b,*
a
Oral Health Science Research Center, b Department of Oral Science, c Department of
Oral and Maxillofacial Surgery, Graduate School of Kanagawa Dental University, Yokosuka, 238-8580, Japan *Correspondence should be addressed to: Ryu-Ichiro Hata, Oral Health Science Research Center, Graduate School of Kanagawa Dental University, Yokosuka, 238-8580,
Japan,
Phone:
+81-46-822-9587;
Fax+81-46-822-9587;
E-mail:
[email protected]
Abstract Background The multistep nature of cancer that involves carcinogenesis, an increase in tumor size, and invasion and/or metastasis is well recognized. These respective steps depend on the
1
mutation of several genes, epigenetic changes in cells, and interactions between cancer cells and other cells in the microenvironment. To identify novel intercellular tumor suppressors, we screened for genes whose expression was down-regulated in oral cancer cells.
Highlight The chemokine, CXCL14, was down-regulated in oral carcinoma cells, and its up-regulation suppressed tumor cell growth in vivo, indicating that CXCL14 is a tumor growth suppressor. Moreover, to investigate whether CXCL14 suppresses tumors originating from the other cells in a paracrine or endocrine fashion, we generated CXCL14 transgenic (Tg) mice. The Tg mice exhibited a suppressed rate of carcinogenesis, decreased volume of transplanted tumors, and reduced pulmonary metastasis, as well as an increased survival rate following tumor cell injection, compared with wild-type mice. The CXCL14-expressing Tg mice showed no apparent abnormality when observed up to 2 years of age, and, interestingly, in a normal human population an individual was found to have a blood level of CXCL14 protein 10-fold
2
higher than the average but did not present any apparent abnormalities.
Conclusion These data indicate that CXCL14 expressed at high levels does not have severe side effects, and, thus, we propose CXCL14 as a promising molecular target for cancer suppression/prevention.
Key words: Chemokine; CXCL14; Tumor suppression; Multistep tumor suppressor; Molecular preventive medicine
3
1. Introduction: present problems of cancer therapy There has been a great deal of progress in the fight against cancer. However, cancer remains the leading cause of death worldwide. The major problems faced in cancer therapy are the side effects, including those of chemotherapy, as well as metastasis and the recurrence of drug-resistant cancers. For the past 30 years, dozens of oncogenes that stimulate cancer progression have been identified in cancer patients. Chemotherapy mostly targets the proteins encoded by these oncogenes, but such genes also play pivotal roles in normal development and homeostasis of our body; thus, anticancer drugs often suppress normal cell functions and induce side effects [1, 2]. There is, however, an additional side effect of chemotherapy: cancer cells that are exposed to one anticancer drug often develop a multi-drug resistance; not only to that drug but also to other drugs to which they have never been exposed, resulting in drug-resistant, recurrent cancer and metastasis [3-5]. It is well known that tumor formation is a multi-step process, largely comprising initiation, promotion, and progression. Tumor progression has been shown to be largely dependent on the overexpression of tumor-promoting genes (oncogenes, stimulators) and
4
dysfunction of tumor-suppressing genes (suppressors), with the balance being in favor of the former at each step [6]. Protein products of these oncogenes and tumor-suppressor genes function as regulatory signaling molecules during this process [6-8] (Fig. 1). Severe side effects could be induced when suppressing tumor stimulators, because these genes are also essential for normal development and homeostasis [9-12]. Therefore, enhancement of the amount or activity of a tumor suppressor provides a good alternative strategy to avoid side effects. Previous studies have identified intracellular tumor-suppressor genes (TSGs) such as p53, p16, PTEN, and RB, and their tumor-suppressing activities have been demonstrated in transgenic mice [13-16]. These genes were often identified in patients with head and neck squamous cell carcinoma (HNSCC) [17-20]. Since the products of these particular TSGs are intracellular signaling molecules, gene therapy to specifically increase intracellular TSGs in all tumor cells without affecting other normal cells would have been practically difficult. Furthermore, overexpression of the TSGs caused abnormalities in mice [21-23]. To identify promising molecules for HNSCC cancer therapy, the suppression of which would not lead to side effects, we screened for TSGs whose
5
products were extracellular molecules and found the chemokine CXCL14 to be a candidate molecule. Our recent work with transgenic mice overexpressing CXCL14 indicated that this chemokine is an extracellular multistep tumor suppressor [24]. In this short review, we describe our strategy for identifying this chemokine and the characteristics of the CXCL14 transgenic mice.
2. Discovery and functional analysis of CXCL14 2.1 Chemokine CXCL14 is a tumor suppressor To identify a promising candidate for chemotherapy without severe effects, we looked for a tumor-suppressor gene whose product functions in the extracellular space. We first cultured HSC-3 cells, derived from a HNSCC cell line, under serum-free conditions and treated them with epidermal growth factor (EGF), whose receptor is frequently over-activated in carcinoma cells, including HNSCC cells. CXC chemokine ligand 14 (CXCLl4), also known as breast and kidney expressed chemokine (BRAK), was found to be significantly down-regulated. Interestingly, the expression of CXCL14 was also shown to be reduced in tissues obtained from patients with HNSCC [25].
6
Chemokines (chemotactic cytokines) are a group of structurally-related proteins with molecular weights in the range of 8–12 k, and they have been reported to regulate the cellular trafficking of various types of leukocytes by interacting with a subset of G protein-coupled receptors [26]. Each chemokine is named based on the arrangement of the cysteine residues within it. Further, the two major subfamilies, defined by the presence of four conserved cysteine residues linked by two disulfide bonds, are the CC and CXC chemokines. These subfamilies are distinguished according to the position of the first two cysteine residues, which are adjacent to each other (CC subfamily) or separated by one amino acid (CXC subfamily). In the tumor microenvironment, chemokine expression acts to determine the distribution of immune cells, and thus controls the overall immune response to the tumor, which plays an integral role in the regulation of cancer progression and metastasis [27-30]. To further investigate whether CXCL14 had a tumor-suppressing effect in vivo, we prepared and cloned CXCL14 expression vector-transfected and mock vector-transfected tongue tumor-derived cells for xenotransplantation in mice. The rate of tumor formation in athymic nude mice or in severe combined immunodeficiency (SCID) mice following
7
xenotransplantation was significantly lower for the CXCL14-expressing cells than for the mock-transfected cells, though no differences were observed in the growth rates of these cells under in vitro culture conditions [31, 32]. These data indicate that CXCL14 expression in tumor cells functions to suppress the growth of these cells in vivo [31-33]. Next, in order to confirm whether CXCL14 would have a tumor-suppressing effect on cells of other tissue origins, we produced transgenic (Tg) mice expressing a 10-fold higher level of CXCL14 compared with the level produced by isogenic wild-type C57BL/6 (Wt) mice. Therefore, the Tg mice showed significantly less size increase in tumors formed by transplanted melanoma cells or Lewis lung carcinoma (LLC) cells compared with tumor-bearing Wt mice [24, 34].
2.2 CXCL14 is an extracellular multistep tumor suppressor Cancer is a multistep process; therefore, in order to further confirm that CXCL14 is a promising molecular target for tumor suppression with no severe side effects, we investigated the effects of CXCL14 on tumorigenesis, growth of transplanted tumors, and tumor metastasis in CXCL14 transgenic (Tg) mice. CXCL14 decreased the rate of
8
azoxymethane (AOM)/dextran sodium sulfate (DSS)-induced colorectal carcinogenesis (Fig. 2 a), the growth of transplanted B16 melanoma (Fig. 2 b, d) or Lewis lung carcinoma (LLC; Fig. 2 c, e) tumors, and the number of metastatic nodules of B16 melanoma cells (Fig. 2 f, g) or Lewis lung carcinoma cells (LLC; Fig. 2 h) in the lungs of mice. In order to investigate if NK cells were involved in CXCL14 tumor suppression, we used anti-asialo-GM1 antibodies to deplete NK cells, and found that anti-asialo-GM1 antibodies injected into the mice before or after the injection of tumor cells attenuated the suppressing effects of CXCL14 on the tumor growth of B16 melanoma cells (Fig. 2 b, d) or Lewis lung carcinoma cells (LLC; Fig. 2 c, e), suggesting that NK cell activity plays an important role in CXCL14-mediated suppression of tumor growth. Further, the survival rates for Tg mice after tumor cell injection were significantly increased (Fig. 2 i). Since the Tg mice showed no obvious abnormality, and CXCL14 reportedly suppresses the growth of transplanted tumors of different cellular origins, such as squamous cell carcinoma [32-34], adenocarcinoma [24], melanoma [24, 34], and fibrosarcoma [35], and also suppresses the metastasis of melanoma and adenocarcinoma cells in the lungs of mice [24], we propose CXCL14 as a promising
9
extracellular multistep tumor suppressor with clinical potential for cancer suppression/prevention [24].
3. Mechanisms of expression of CXCL14 Epigenetic aberrations play an important role in the multiple steps of tumorigenesis. One type of epigenetic aberration is a large-scale change in DNA methylation in the genome [36]. Furthermore, hyper-methylation in the promoter region of CXCL14 is responsible for the loss of CXCL14 expression in different cancer cells, such as colorectal cancer cells [37], prostate cancer cells [38], gastric adenocarcinoma tissues [39], ulcerative colitis (UC) [40], hepatocellular carcinoma [41], human normal karyotype AML [42], and small intestinal-neuroendocrine tumors [43]. Another epigenetic aberration constitutes alterations in post-translational events [36], such as that CXCL14 protein was targeted for polyubiquitylation and proteasomal degradation in cancer cells [44].
3.1 Transcriptional regulation of the CXCL14 gene in HNSCC
10
In HNSCC cells, we found that EGF and reactive oxygen species (ROS) suppressed CXCL14 expression through the ERK1/2 MAP kinase signal pathway [33, 45], UV irradiation increases CXCL14 expression through the human p38 delta MAP kinase signal pathway [46], and calcium-calmodulin signaling enhances CXCL14 expression via the binding of SP1 to the CXCL14 promoter region [47].
3.2. Expression of the chemokine CXCL14 is a predictive biomarker for tumor suppression
Gefitinib (ZD1839, Iressa), an inhibitor specific for epidermal growth factor (EGF) receptor tyrosine kinase that acts by binding to the adenosine triphosphate (ATP)-binding site of the enzyme [48], is clinically administered to non-small-cell lung carcinoma patients with EGF receptor mutations. Recent studies have shown that the EGF receptor gene mutation is rare in squamous cell carcinoma in the esophageal and head and neck regions. Whether gefitinib is suitable for the treatment of HNSCC has been an issue. We found that EGF suppressed CXCL14 expression through the ERK1/2
11
MAP kinase signal pathway (Fig. 3, left panel) in HNSCC cells. Gefitinib suppressed tumor growth of xenografts of three HNSCC cell lines in athymic nude mice by restoring CXCL14 expression (Fig. 3, right panel) [33]. Fasudil is a potent Rho-kinase inhibitor and vasodilator that has been approved for use in Japan and China. It is used to prevent symptomatic vasospasm after subarachnoid hemorrhage [49, 50] and to promote axonal growth [51]. Recently, however, fasudil was found to inhibit tumor progression in human and rat tumor models [52]. We investigated the mechanism of tumor suppression by fasudil and found that fasudil suppressed fibrosarcoma growth by stimulating the secretion of CXCL14 [35, 53].
4. Conclusions: Towards molecular-targeting preventive medicine
Unlike other chemokines, CXCL14 has an amino acid sequence that has been highly conserved throughout evolution. We consider CXCL14 a very unique chemokine, one having an essential function in life (Table 1). Our data, obtained by examining transgenic mice overexpressing CXCL14, indicate that this chemokine is an
12
extracellular multistep tumor suppressor. CXCL14 suppresses not only the rate of colorectal carcinogenesis [24] but also the growth of transplanted tumors of different origins, such as HNSCC [32], B16 melanoma [24], Lewis lung carcinoma [24], and fibrosarcoma [35]. Moreover, the number of B16 melanoma and Lewis lung carcinoma metastatic nodules in the lungs of mice were decreased [24]. Furthermore, CXCL14 increases the survival rates of tumor-carrying animals. Because neither transgenic mice nor humans with high levels of CXCL14 show obvious abnormality, we propose that CXCL14 should be considered as a promising molecular target causing fewer side effects than other agents used for cancer suppression/prevention (Fig. 4).
Ethical approval This review cites papers that have already been published. Each had its own ethical approval and thus was documented in the original publications.
13
Conflict of interest The authors disclose no conflict of interests.
Acknowledgments The authors appreciate the collaborators of the work cited in this review. The studies reviewed by the authors were supported in part by a Grant-in Aid from the High-Tech Research Center Project of the Ministry of Education, Culture, Sports, Science and Technology of Japan, by Grants-in Aid for Scientific Research from Japan Society for the Promotion of Science (Grant Numbers, 19592162, 22390353 and 24659843) to R. Hata.
14
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Legends for figures and a table.
Fig. 1. Multistep nature of tumor progression. Each step is determined by the balance between tumor stimulators (red) and tumor suppressors (blue). ANG, angiogenin; CTGF, connective tissue growth factor; CTNNB1, catenin (cadherin-associated protein), beta 1; CXCL12, chemokine (C-X-C motif) ligand 12; Endostatin, a fragment of type XVIII
20
collagen a1; FGF2, fibroblast growth factor 2 (basic); p16, cyclin-dependent kinase inhibitor 2A; p53, tumor protein p53; PTEN, phosphatase and tensin homolog; PEBP1, phosphatidylethanolamine binding protein 1; RECK, reversion-inducing-cysteine-rich protein with kazal motifs; VEGF, vascular endothelial growth factor; Raf, Raf proto-oncogene, serine/threonine kinase ; Ras, resistance to audiogenic seizures; Rb, retinoblastoma protein.
Fig. 2. Suppressed rate of carcinogenesis, decreases in tumor volume and lung metastasis, and increase in survival rate of CXCL14 transgenic mice carrying tumors. (a) Reduced incidence of AOM/DSS-induced colorectal carcinogenesis in CXCL14 Tg mice compared with that in Wt mice. The colon tissues were obtained, and the tumors were counted macroscopically (n=8). (b – e) B16 melanoma cells (b, d) or Lewis lung carcinoma cells (LLC; c, e) were inoculated (1 × 105 cells/site) into both sides of the dorso-lateral region of 8 each (B16) or 10 each (LLC) female Wt and Tg mice (homozygous line 20). Half of these mice were injected with anti-asialo-GM1 antibody (0.5 mg/200 μL/animal) three days before tumor cell inoculation and once a
21
week thereafter to deplete NK cells. The final volume of the tumors at day 25 is indicated in panels ‘‘d’’ and ‘‘e’’. R indicates the tumor size ratio between the two groups connected with a bracket. (f – h) B16 melanoma cells (f, g) or LLC cells (h) were injected (2x105 cells) into a tail vein of 12 each (B16) or 18 each (LLC) of Wt and CXCL14 Tg mice. After 18 days, the metastatic nodules in the lungs were counted (g, h). (i) Variable concentrations of B16-luc2LMT-3cells (3x103, 1x104, or 1x105 cells / 200 μL PBS) were injected into a tail vein of Wt and Tg mice, and the survival rate was determined at the indicated days following melanoma cell inoculation. The generalized Wilcoxon test was utilized. Data are means ± S.D. Student’s t-test. * P < 0.05, *** P < 0.001.
Fig. 3. Expression of the chemokine CXCL14 is a predictive biomarker for tumor suppression. In HNSCC cells, EGF suppresses CXCL14 expression through the ERK1/2 MAP kinase signal pathway (left panel). Gefitinib suppresses tumor growth of xenografts of three HNSCC cell lines in athymic nude mice through restoration of CXCL14 expression (right panel).
22
Fig. 4. CXCL14 is a multistep tumor suppressor. CXCL14 overexpression in HNSCC and/or fibrosarcoma cells suppresses the growth of tumor cell transplants. Cancer progression involves carcinogenesis, an increase in tumor size, and metastasis. In CXCL14 transgenic (Tg) mice, all of these steps are blocked. Table 1 CXCL14 is a chemokine with an amino acid sequence that has been highly conserved throughout evolution. The amino acid sequence 1-77 of human CXCL14 has 100% identity with those sequences of chimpanzee and dog, 99% identity with those of rat and cow, and 97% identity with that of mouse. This is similar to the case of beta-actin (human beta-actin amino acid sequence is 100% identical with those of chimpanzee, mouse, rat, and cow and 99% identical with the dog sequence, all with 375 amino acids). Such high amino acid sequence identity is not the case for all other chemokines, suggesting CXCL14 to be a very unique chemokine with an essential function for life. Letters in red are amino acid residues different from those of the human sequence.
23
Table. 1. CXCL14 is a chemokine with a highly conserved amino acid sequence throughout evolution Organis
Taxonom
NCBI
Ident
m
ic
Reference
ity
Classific
Sequence
With
ation
Sequence
Hum an
Human
Mammali
NP_004878
a
.2
Chimpa
Mammali
XP_527018
100
nzee
a
.2
%
Dog
Mammali
XP_005626
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PII: DOI: Reference:
S1349-0079(15)00108-5 http://dx.doi.org/10.1016/j.job.2015.08.003 JOB134
To appear in: Journal of Oral Biosciences Received date: 29 July 2015 Accepted date: 15 August 2015 Cite this article as: Xiaoyan Yang, Chihiro Miyamoto, Tetsu Akasaka, Kazuhito Izukuri, Yojiro Maehata, Takeharu Ikoma, Shigeyuki Ozawa and Ryu-Ichiro Hata, Chemokine CXCL14 is a multistep tumor suppressor, Journal of Oral Biosciences, http://dx.doi.org/10.1016/j.job.2015.08.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Chemokine CXCL14 is a multistep tumor suppressor Xiaoyan Yang
a,b
, Chihiro Miyamoto
a,b
, Tetsu Akasaka
a,b
a,b
, Kazuhito Izukuri
,
Yojiro Maehata a,b, Takeharu Ikoma c, Shigeyuki Ozawa c and Ryu-Ichiro Hataa,b,*
a
Oral Health Science Research Center, b Department of Oral Science, c Department of
Oral and Maxillofacial Surgery, Graduate School of Kanagawa Dental University, Yokosuka, 238-8580, Japan *Correspondence should be addressed to: Ryu-Ichiro Hata, Oral Health Science Research Center, Graduate School of Kanagawa Dental University, Yokosuka, 238-8580,
Japan,
Phone:
+81-46-822-9587;
Fax+81-46-822-9587;
E-mail:
[email protected]
Abstract Background The multistep nature of cancer that involves carcinogenesis, an increase in tumor size, and invasion and/or metastasis is well recognized. These respective steps depend on the
1
mutation of several genes, epigenetic changes in cells, and interactions between cancer cells and other cells in the microenvironment. To identify novel intercellular tumor suppressors, we screened for genes whose expression was down-regulated in oral cancer cells.
Highlight The chemokine, CXCL14, was down-regulated in oral carcinoma cells, and its up-regulation suppressed tumor cell growth in vivo, indicating that CXCL14 is a tumor growth suppressor. Moreover, to investigate whether CXCL14 suppresses tumors originating from the other cells in a paracrine or endocrine fashion, we generated CXCL14 transgenic (Tg) mice. The Tg mice exhibited a suppressed rate of carcinogenesis, decreased volume of transplanted tumors, and reduced pulmonary metastasis, as well as an increased survival rate following tumor cell injection, compared with wild-type mice. The CXCL14-expressing Tg mice showed no apparent abnormality when observed up to 2 years of age, and, interestingly, in a normal human population an individual was found to have a blood level of CXCL14 protein 10-fold
2
higher than the average but did not present any apparent abnormalities.
Conclusion These data indicate that CXCL14 expressed at high levels does not have severe side effects, and, thus, we propose CXCL14 as a promising molecular target for cancer suppression/prevention.
Key words: Chemokine; CXCL14; Tumor suppression; Multistep tumor suppressor; Molecular preventive medicine
3
1. Introduction: present problems of cancer therapy There has been a great deal of progress in the fight against cancer. However, cancer remains the leading cause of death worldwide. The major problems faced in cancer therapy are the side effects, including those of chemotherapy, as well as metastasis and the recurrence of drug-resistant cancers. For the past 30 years, dozens of oncogenes that stimulate cancer progression have been identified in cancer patients. Chemotherapy mostly targets the proteins encoded by these oncogenes, but such genes also play pivotal roles in normal development and homeostasis of our body; thus, anticancer drugs often suppress normal cell functions and induce side effects [1, 2]. There is, however, an additional side effect of chemotherapy: cancer cells that are exposed to one anticancer drug often develop a multi-drug resistance; not only to that drug but also to other drugs to which they have never been exposed, resulting in drug-resistant, recurrent cancer and metastasis [3-5]. It is well known that tumor formation is a multi-step process, largely comprising initiation, promotion, and progression. Tumor progression has been shown to be largely dependent on the overexpression of tumor-promoting genes (oncogenes, stimulators) and
4
dysfunction of tumor-suppressing genes (suppressors), with the balance being in favor of the former at each step [6]. Protein products of these oncogenes and tumor-suppressor genes function as regulatory signaling molecules during this process [6-8] (Fig. 1). Severe side effects could be induced when suppressing tumor stimulators, because these genes are also essential for normal development and homeostasis [9-12]. Therefore, enhancement of the amount or activity of a tumor suppressor provides a good alternative strategy to avoid side effects. Previous studies have identified intracellular tumor-suppressor genes (TSGs) such as p53, p16, PTEN, and RB, and their tumor-suppressing activities have been demonstrated in transgenic mice [13-16]. These genes were often identified in patients with head and neck squamous cell carcinoma (HNSCC) [17-20]. Since the products of these particular TSGs are intracellular signaling molecules, gene therapy to specifically increase intracellular TSGs in all tumor cells without affecting other normal cells would have been practically difficult. Furthermore, overexpression of the TSGs caused abnormalities in mice [21-23]. To identify promising molecules for HNSCC cancer therapy, the suppression of which would not lead to side effects, we screened for TSGs whose
5
products were extracellular molecules and found the chemokine CXCL14 to be a candidate molecule. Our recent work with transgenic mice overexpressing CXCL14 indicated that this chemokine is an extracellular multistep tumor suppressor [24]. In this short review, we describe our strategy for identifying this chemokine and the characteristics of the CXCL14 transgenic mice.
2. Discovery and functional analysis of CXCL14 2.1 Chemokine CXCL14 is a tumor suppressor To identify a promising candidate for chemotherapy without severe effects, we looked for a tumor-suppressor gene whose product functions in the extracellular space. We first cultured HSC-3 cells, derived from a HNSCC cell line, under serum-free conditions and treated them with epidermal growth factor (EGF), whose receptor is frequently over-activated in carcinoma cells, including HNSCC cells. CXC chemokine ligand 14 (CXCLl4), also known as breast and kidney expressed chemokine (BRAK), was found to be significantly down-regulated. Interestingly, the expression of CXCL14 was also shown to be reduced in tissues obtained from patients with HNSCC [25].
6
Chemokines (chemotactic cytokines) are a group of structurally-related proteins with molecular weights in the range of 8–12 k, and they have been reported to regulate the cellular trafficking of various types of leukocytes by interacting with a subset of G protein-coupled receptors [26]. Each chemokine is named based on the arrangement of the cysteine residues within it. Further, the two major subfamilies, defined by the presence of four conserved cysteine residues linked by two disulfide bonds, are the CC and CXC chemokines. These subfamilies are distinguished according to the position of the first two cysteine residues, which are adjacent to each other (CC subfamily) or separated by one amino acid (CXC subfamily). In the tumor microenvironment, chemokine expression acts to determine the distribution of immune cells, and thus controls the overall immune response to the tumor, which plays an integral role in the regulation of cancer progression and metastasis [27-30]. To further investigate whether CXCL14 had a tumor-suppressing effect in vivo, we prepared and cloned CXCL14 expression vector-transfected and mock vector-transfected tongue tumor-derived cells for xenotransplantation in mice. The rate of tumor formation in athymic nude mice or in severe combined immunodeficiency (SCID) mice following
7
xenotransplantation was significantly lower for the CXCL14-expressing cells than for the mock-transfected cells, though no differences were observed in the growth rates of these cells under in vitro culture conditions [31, 32]. These data indicate that CXCL14 expression in tumor cells functions to suppress the growth of these cells in vivo [31-33]. Next, in order to confirm whether CXCL14 would have a tumor-suppressing effect on cells of other tissue origins, we produced transgenic (Tg) mice expressing a 10-fold higher level of CXCL14 compared with the level produced by isogenic wild-type C57BL/6 (Wt) mice. Therefore, the Tg mice showed significantly less size increase in tumors formed by transplanted melanoma cells or Lewis lung carcinoma (LLC) cells compared with tumor-bearing Wt mice [24, 34].
2.2 CXCL14 is an extracellular multistep tumor suppressor Cancer is a multistep process; therefore, in order to further confirm that CXCL14 is a promising molecular target for tumor suppression with no severe side effects, we investigated the effects of CXCL14 on tumorigenesis, growth of transplanted tumors, and tumor metastasis in CXCL14 transgenic (Tg) mice. CXCL14 decreased the rate of
8
azoxymethane (AOM)/dextran sodium sulfate (DSS)-induced colorectal carcinogenesis (Fig. 2 a), the growth of transplanted B16 melanoma (Fig. 2 b, d) or Lewis lung carcinoma (LLC; Fig. 2 c, e) tumors, and the number of metastatic nodules of B16 melanoma cells (Fig. 2 f, g) or Lewis lung carcinoma cells (LLC; Fig. 2 h) in the lungs of mice. In order to investigate if NK cells were involved in CXCL14 tumor suppression, we used anti-asialo-GM1 antibodies to deplete NK cells, and found that anti-asialo-GM1 antibodies injected into the mice before or after the injection of tumor cells attenuated the suppressing effects of CXCL14 on the tumor growth of B16 melanoma cells (Fig. 2 b, d) or Lewis lung carcinoma cells (LLC; Fig. 2 c, e), suggesting that NK cell activity plays an important role in CXCL14-mediated suppression of tumor growth. Further, the survival rates for Tg mice after tumor cell injection were significantly increased (Fig. 2 i). Since the Tg mice showed no obvious abnormality, and CXCL14 reportedly suppresses the growth of transplanted tumors of different cellular origins, such as squamous cell carcinoma [32-34], adenocarcinoma [24], melanoma [24, 34], and fibrosarcoma [35], and also suppresses the metastasis of melanoma and adenocarcinoma cells in the lungs of mice [24], we propose CXCL14 as a promising
9
extracellular multistep tumor suppressor with clinical potential for cancer suppression/prevention [24].
3. Mechanisms of expression of CXCL14 Epigenetic aberrations play an important role in the multiple steps of tumorigenesis. One type of epigenetic aberration is a large-scale change in DNA methylation in the genome [36]. Furthermore, hyper-methylation in the promoter region of CXCL14 is responsible for the loss of CXCL14 expression in different cancer cells, such as colorectal cancer cells [37], prostate cancer cells [38], gastric adenocarcinoma tissues [39], ulcerative colitis (UC) [40], hepatocellular carcinoma [41], human normal karyotype AML [42], and small intestinal-neuroendocrine tumors [43]. Another epigenetic aberration constitutes alterations in post-translational events [36], such as that CXCL14 protein was targeted for polyubiquitylation and proteasomal degradation in cancer cells [44].
3.1 Transcriptional regulation of the CXCL14 gene in HNSCC
10
In HNSCC cells, we found that EGF and reactive oxygen species (ROS) suppressed CXCL14 expression through the ERK1/2 MAP kinase signal pathway [33, 45], UV irradiation increases CXCL14 expression through the human p38 delta MAP kinase signal pathway [46], and calcium-calmodulin signaling enhances CXCL14 expression via the binding of SP1 to the CXCL14 promoter region [47].
3.2. Expression of the chemokine CXCL14 is a predictive biomarker for tumor suppression
Gefitinib (ZD1839, Iressa), an inhibitor specific for epidermal growth factor (EGF) receptor tyrosine kinase that acts by binding to the adenosine triphosphate (ATP)-binding site of the enzyme [48], is clinically administered to non-small-cell lung carcinoma patients with EGF receptor mutations. Recent studies have shown that the EGF receptor gene mutation is rare in squamous cell carcinoma in the esophageal and head and neck regions. Whether gefitinib is suitable for the treatment of HNSCC has been an issue. We found that EGF suppressed CXCL14 expression through the ERK1/2
11
MAP kinase signal pathway (Fig. 3, left panel) in HNSCC cells. Gefitinib suppressed tumor growth of xenografts of three HNSCC cell lines in athymic nude mice by restoring CXCL14 expression (Fig. 3, right panel) [33]. Fasudil is a potent Rho-kinase inhibitor and vasodilator that has been approved for use in Japan and China. It is used to prevent symptomatic vasospasm after subarachnoid hemorrhage [49, 50] and to promote axonal growth [51]. Recently, however, fasudil was found to inhibit tumor progression in human and rat tumor models [52]. We investigated the mechanism of tumor suppression by fasudil and found that fasudil suppressed fibrosarcoma growth by stimulating the secretion of CXCL14 [35, 53].
4. Conclusions: Towards molecular-targeting preventive medicine
Unlike other chemokines, CXCL14 has an amino acid sequence that has been highly conserved throughout evolution. We consider CXCL14 a very unique chemokine, one having an essential function in life (Table 1). Our data, obtained by examining transgenic mice overexpressing CXCL14, indicate that this chemokine is an
12
extracellular multistep tumor suppressor. CXCL14 suppresses not only the rate of colorectal carcinogenesis [24] but also the growth of transplanted tumors of different origins, such as HNSCC [32], B16 melanoma [24], Lewis lung carcinoma [24], and fibrosarcoma [35]. Moreover, the number of B16 melanoma and Lewis lung carcinoma metastatic nodules in the lungs of mice were decreased [24]. Furthermore, CXCL14 increases the survival rates of tumor-carrying animals. Because neither transgenic mice nor humans with high levels of CXCL14 show obvious abnormality, we propose that CXCL14 should be considered as a promising molecular target causing fewer side effects than other agents used for cancer suppression/prevention (Fig. 4).
Ethical approval This review cites papers that have already been published. Each had its own ethical approval and thus was documented in the original publications.
13
Conflict of interest The authors disclose no conflict of interests.
Acknowledgments The authors appreciate the collaborators of the work cited in this review. The studies reviewed by the authors were supported in part by a Grant-in Aid from the High-Tech Research Center Project of the Ministry of Education, Culture, Sports, Science and Technology of Japan, by Grants-in Aid for Scientific Research from Japan Society for the Promotion of Science (Grant Numbers, 19592162, 22390353 and 24659843) to R. Hata.
14
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2012;420:217-22. [48] Lynch TJ, Bell DW, Sordella R, Gurubhagavatula S, Okimoto RA, Brannigan BW, Harris PL, Haserlat SM, Supko JG, Haluska FG, Louis DN, Christiani DC, Settleman J, Haber DA. Activating mutations in the epidermal
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non-small-cell lung cancer to gefitinib. N Engl J Med 2004;350:2129-39. [49] Iwabuchi S, Hayashi M, Yokouchi T, Sato K, Nakayama H, Harashina J, Iwama J, Ishii M, Hiramoto Y, Hirai N, Hirata Y, Saito N, Ito K, Kimura H, Aoki K. Prophylactic intra-arterial administration of fasudil hydrochloride 19
for vasospasm following subarachnoid haemorrhage. Acta Neurochir Suppl 2015;120:167-9. [50] Saito A, Inoue M, Kon H, Imaruoka S, Basaki K, Midorikawa H, Sasaki T, Nishijima M. Effectiveness of intraarterial administration of fasudil hydrochloride for preventing symptomatic vasospasm after subarachnoid hemorrhage. Acta Neurochir Suppl 2015;120:297-301. [51] Xiao WD, Yu AX, Liu DL. Fasudil hydrochloride could promote axonal growth through inhibiting the activity of ROCK. Int J Clin Exp Pathol 2014;7:5564-8. [52] Ying H, Biroc SL, Li WW, Alicke B, Xuan JA, Pagila R, Ohashi Y, Okada T, Kamata Y, Dinter H. The Rho kinase inhibitor fasudil inhibits tumor progression
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Legends for figures and a table.
Fig. 1. Multistep nature of tumor progression. Each step is determined by the balance between tumor stimulators (red) and tumor suppressors (blue). ANG, angiogenin; CTGF, connective tissue growth factor; CTNNB1, catenin (cadherin-associated protein), beta 1; CXCL12, chemokine (C-X-C motif) ligand 12; Endostatin, a fragment of type XVIII
20
collagen a1; FGF2, fibroblast growth factor 2 (basic); p16, cyclin-dependent kinase inhibitor 2A; p53, tumor protein p53; PTEN, phosphatase and tensin homolog; PEBP1, phosphatidylethanolamine binding protein 1; RECK, reversion-inducing-cysteine-rich protein with kazal motifs; VEGF, vascular endothelial growth factor; Raf, Raf proto-oncogene, serine/threonine kinase ; Ras, resistance to audiogenic seizures; Rb, retinoblastoma protein.
Fig. 2. Suppressed rate of carcinogenesis, decreases in tumor volume and lung metastasis, and increase in survival rate of CXCL14 transgenic mice carrying tumors. (a) Reduced incidence of AOM/DSS-induced colorectal carcinogenesis in CXCL14 Tg mice compared with that in Wt mice. The colon tissues were obtained, and the tumors were counted macroscopically (n=8). (b – e) B16 melanoma cells (b, d) or Lewis lung carcinoma cells (LLC; c, e) were inoculated (1 × 105 cells/site) into both sides of the dorso-lateral region of 8 each (B16) or 10 each (LLC) female Wt and Tg mice (homozygous line 20). Half of these mice were injected with anti-asialo-GM1 antibody (0.5 mg/200 μL/animal) three days before tumor cell inoculation and once a
21
week thereafter to deplete NK cells. The final volume of the tumors at day 25 is indicated in panels ‘‘d’’ and ‘‘e’’. R indicates the tumor size ratio between the two groups connected with a bracket. (f – h) B16 melanoma cells (f, g) or LLC cells (h) were injected (2x105 cells) into a tail vein of 12 each (B16) or 18 each (LLC) of Wt and CXCL14 Tg mice. After 18 days, the metastatic nodules in the lungs were counted (g, h). (i) Variable concentrations of B16-luc2LMT-3cells (3x103, 1x104, or 1x105 cells / 200 μL PBS) were injected into a tail vein of Wt and Tg mice, and the survival rate was determined at the indicated days following melanoma cell inoculation. The generalized Wilcoxon test was utilized. Data are means ± S.D. Student’s t-test. * P < 0.05, *** P < 0.001.
Fig. 3. Expression of the chemokine CXCL14 is a predictive biomarker for tumor suppression. In HNSCC cells, EGF suppresses CXCL14 expression through the ERK1/2 MAP kinase signal pathway (left panel). Gefitinib suppresses tumor growth of xenografts of three HNSCC cell lines in athymic nude mice through restoration of CXCL14 expression (right panel).
22
Fig. 4. CXCL14 is a multistep tumor suppressor. CXCL14 overexpression in HNSCC and/or fibrosarcoma cells suppresses the growth of tumor cell transplants. Cancer progression involves carcinogenesis, an increase in tumor size, and metastasis. In CXCL14 transgenic (Tg) mice, all of these steps are blocked. Table 1 CXCL14 is a chemokine with an amino acid sequence that has been highly conserved throughout evolution. The amino acid sequence 1-77 of human CXCL14 has 100% identity with those sequences of chimpanzee and dog, 99% identity with those of rat and cow, and 97% identity with that of mouse. This is similar to the case of beta-actin (human beta-actin amino acid sequence is 100% identical with those of chimpanzee, mouse, rat, and cow and 99% identical with the dog sequence, all with 375 amino acids). Such high amino acid sequence identity is not the case for all other chemokines, suggesting CXCL14 to be a very unique chemokine with an essential function for life. Letters in red are amino acid residues different from those of the human sequence.
23
Table. 1. CXCL14 is a chemokine with a highly conserved amino acid sequence throughout evolution Organis
Taxonom
NCBI
Ident
m
ic
Reference
ity
Classific
Sequence
With
ation
Sequence
Hum an
Human
Mammali
NP_004878
a
.2
Chimpa
Mammali
XP_527018
100
nzee
a
.2
%
Dog
Mammali
XP_005626
100
a
617.1
%
Norway
Mammali
NP_001013
99%
rat
a
155.1
Cattle
Mammali
NP_001029
a
582.1
Mammali
NP_062514
a
.2
Amphibia
NP_001107
Mouse
Western clawed
SKCKCSRKGPKIRYSDVKKLEMKPKYPHCEEKMVIITTKSVSRYRGQEHCLH PKLQSTKRFIKWYNAWNEKRRVYEE SKCKCSRKGPKIRYSDVKKLEMKPKYPHCEEKMVIITTKSVSRYRGQEHCLH PKLQSTKRFIKWYNAWNEKRRVYEE SKCKCSRKGPKIRYSDVKKLEMKPKYPHCEEKMVIITTKSVSRYRGQEHCLH PKLQSTKRFIKWYNAWNEKRRVYEE SKCKCSRKGPKIRYSDVKKLEMKPKYPHCEEKMVIITTKSMSRYRGQEHCLH PKLQSTKRFIKWYNAWNEKRRVYEE
99%
SKCKCSRKGPKIRYSDVKKLEMKPKYPHCEEKMVIITTKSMSRYRGQEHCLH PKLQSTKRFIKWYNAWNEKRRVYEE
97%
SKCKCSRKGPKIRYSDVKKLEMKPKYPHCEEKMVIVTTKSMSRYRGQEHCLH PKLQSTKRFIKWYNAWNEKRRVYEE
70%
714.1
SKCKCSRKGPKIRFTDVQKLEIKPKYPYCEERMIIVTMQNVSRFRGQQYCLH PKLHSTKKFLKWYTIWKDKNRVYED
frog Chicken
Aves
NP_990043
62%
.1 Zebrafis
Actinopt
NP_571702
h
erygii
.1
VKCKCSRKGPKIRFSNVRKLEIKPRYPFCVEEMIIVTLWTKVRGEQQ-HCLN PKRQNTVRLLKWYRVWKEKGRVYEE
60%
YKCRCTRKGPKIRYIDVQKLEIKPKHPYCQEKMIFVTMENVSRFKGQEYCLH PRLQSTRNLVKWFKIWKDKHRTFEA
24
Intracellular$
Cell Membrane$ Raf$
p53
Rb$
PTEN$
Ras$
p16$
1g
109
PEBP1
VEGF$
!"#$%&'("
RECK$
CTGF$
FGF2$
ANG$
CXCL12$
CTNNB1$
Suppressor Stimulator
!"#$
1mg
106
Progression$
Suppressor Stimulator
Initiation$
Promotion$
Suppressor Stimulator
Extracellular$
Figure
1kg
Malignant tumor$
1012 Death$
b Tumor volume ( mm3 x 103 )
Number of tumors
6
***
5 4 3 2 1
Wt + Anti-asialo-GM1 Tg + Anti-asialo-GM1 Wt + PBS Tg + PBS
8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0 0
0
Wt
c
B16 melanoma cells
Tumor volume ( mm3 x 103 )
Figure a
5
Tg
10
15
20
2.0 1.5 1.0 0.5 0
0
e
6.0
*
R=6.7
* R=1.4
2.0 0 Wt
Tg
Wt
+ PBS
f
R=13
4.0
0
2.5 1.5 1.0 0.5 0
***
R=2.8
***
Wt
Number of nodules
Number of nodules
120 100 80 60 40
Survival rate (%)
80
60
40
20
0 60
70
80
90
Days after injection (days)
: Wt
1 X105 (n =14)
: Wt
1 X104 (n =15)
: Wt
3 X103 (n =16)
: Tg
1 X105 (n =14)
: Tg
1 X104 (n =14)
: Tg
3 X103 (n =16)
P < 0.0001
P < 0.005
100 80 60 40 20
Wt
100
50
Wt
Tg
+ Anti-asialo GM1 Ab
0 Tg
i
40
Tg
***
140
Wt
30
***
Lewis lung carcinoma cells
0
20
R=11
h
20
10
25
R=5.5
***
0
20
R=1.4
+ PBS
+ Anti-asialo GM1 Ab
B16 melanoma cells
Tg + PBS
***
2.0
Tg
g
Wt + PBS
10 15
Lewis lung carcinoma cells
Tumor volume ( mm3 x 103 )
Tumor volume ( mm3 x 103 )
8.0
*
***
5
Days after transplantation
B16 melanoma cells
R=2.8
Wt + Anti-asialo-GM1 Tg + Anti-asialo-GM1 Wt + PBS Tg + PBS
2.5
25
Days after transplantation
d
Lewis lung carcinoma cells
P < 0.005
100
Tg
AKT
!" !"
EGF EGFR
MEK
RAF
RAS
Intracellular
Cell Membrane
Extracellular
CXCL14
ERK1/2
Proliferation
Survival
Figure
Proliferation
ERK1/2
MEK
RAF
! ! !
RAS
CXCL14
!
EGF EGFR
!" !"
Gefitinib
!" !"
oge
CXCL14 Tumor Growth
CXCL14 Carcinogenesis
Multi-steps
14 L C CX
sis sta a t Me
Metastasis
CXCL14
Melanoma & Lewis Lung Carcinoma
Tumor Suppressor
106
1mg
HNSCC & Fibrosarcoma Cells
14 L C CX
Colon Carcinoma
14 L C CX
ci n Car
is nes
th row G r o Tum
Transgenic Mice
Tumor Cells Transplantation
Figure
1g
109