Chondrolectin Is a Novel Diagnostic Biomarker and a Therapeutic Target For

Author Manuscript Published OnlineFirst on October 20, 2011; DOI: 10.1158/1078-0432.CCR-11-0619 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Chondrolectin is a novel diagnostic biomarker and a therapeutic target for

lung cancer

Ken Masuda1,4, Atsushi Takano1,2,3, Hideto Oshita1, Hirohiko Akiyama,5 Eiju

Tsuchiya, 6 Nobuoki Kohno4, Yusuke Nakamura1, Yataro Daigo1,2,3*

1Laboratory of Molecular Medicine, Human Genome Center, Institute of Medical

Science, The University of Tokyo, Tokyo 108-8639, Japan

2Department of Medical Oncology and 3Cancer Center, Shiga University of

Medical Science, Otsu 520-2192, Japan

4Department of Molecular and Internal Medicine, Graduate School of Biomedical

Sciences, Hiroshima University, Hiroshima 734-8551, Japan

5Department of Thoracic Surgery, Saitama Cancer Center, Saitama 362-0806,

Japan

6Kanagawa Cancer Center Research Institute, Yokohama 241-0815, Japan

*Request for reprints: [email protected]

Running title: CHODL as a Novel Cancer Biomarker

Key words: oncogenes, cancer antigen, biomarker, therapeutic target, lung

cancer

Downloaded from clincancerres.aacrjournals.org on September 30, 2021. © 2011 American Association for Cancer Research. Author Manuscript Published OnlineFirst on October 20, 2011; DOI: 10.1158/1078-0432.CCR-11-0619 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Statement of Clinical Relevance

Because there is a significant association of CHODL positivity with poor clinical

outcome of lung cancer patients, CHODL positivity in resected specimens could

be an index that provides valuable information to physicians in applying adjuvant

therapy and intensive follow-up to the patients who are likely to relapse. Since

CHODL encodes a cancer-testis antigen, it may be useful for screening of

HLA-restricted epitope peptides for cancer vaccine that can induce specific

immune responses by cytotoxic T cells against CHODL-positive cancers.

Since CHODL plays important roles in cancer proliferation and invasion,

targeting CHODL function by small molecule compounds could be a new

therapeutic strategy that is expected to have a powerful anti-cancer activity with

a minimal risk of adverse effects.

Abstract

Purpose: This study aims to identify molecules that might be useful as

diagnostic/prognostic biomarkers and as targets for the development of new

molecular therapies for lung cancer.

Experimental Design: We screened for genes that were highly transactivated in

a large proportion of 120 lung cancers by means of a cDNA microarray

representing 27,648 genes, and found chondrolectin (CHODL) as a candidate.

Tumor tissue microarray was applied to examine the expression of CHODL

protein and its clinicopathological significance in archival non-small cell lung

cancer (NSCLC) tissues from 295 patients. A role of CHODL in cancer cell

growth and/or survival was examined by small interfering RNA (siRNA)

experiments. Cellular invasive effect of CHODL on mammalian cells was

examined using Matrigel assays.

Results: Immunohistochemical staining revealed that strong positivity of

CHODL protein was associated with shorter survival of patients with NSCLC (P

= 0.0006), and multivariate analysis confirmed it to be an independent

prognostic factor. Treatment of lung cancer cells with siRNAs against CHODL

suppressed growth of the cancer cells. Furthermore, induction of exogenous

expression of CHODL conferred growth and invasive activity of mammalian cells.

Conclusions: CHODL is likely to be a prognostic biomarker in the clinic and

targeting CHODL might be a strategy for the development of anticancer drugs.

Introduction

Lung cancer is one of the most common cause of cancer death in the world, and

non-small cell lung cancer (NSCLC) accounts for nearly 80% of those cases (1,

2). In the last two decades several newly developed cytotoxic agents including

paclitaxel, docetaxel, gemcitabine, vinorelbine and irinotecan have emerged to

offer multiple therapeutic choices for patients with advanced NSCLC; however,

each of the new regimens can provide only modest improvements in survival

and quality of life compared with traditional cisplatin-based therapies (3).

Recently, molecular-targeted agents, including anti-EGFR or anti-VEGF

monoclonal antibody, cetuximab or bevacizumab, and small molecule inhibitors

of EGFR tyrosine kinase, such as gefitinib and erlotinib have been investigated

in the clinical trials and/or were approved for clinical use. These agents are

effective in the treatment of advanced NSCLC to a certain extent, but a

proportion of patients who could receive a survival benefit is still limited (4, 5).

Hence, new therapeutic strategies, such as the development of more selective

and effective molecular-targeted agents against lung cancer are eagerly

awaited.

Genome-wide cDNA microarray analysis of cancers enabled us to

obtain their comprehensive gene expression profiles and to compare the gene

expression levels with clinicopathological and biological information of cancers

(6). This approach is also useful to identify unknown molecules involved in the

carcinogenic pathway. Through the gene expression profile analysis of 120

lung cancers on a cDNA microarray consisting of 27,648 genes or expressed

sequence tags (ESTs) coupled with purification of cancer cell population by laser

microdissection, and subsequent comparison of the data with the expression

profile of 31 normal human tissues, we identified a number of potential molecular

targets for cancer diagnosis, treatment, and/or choice of therapy (6-11). To

verify the biological and clinicopathologic significance of the respective gene

products, we have also been performing high-throughput screening of

loss-of-function effects by means of the RNA interference technique as well as

tumor tissue microarray analysis of clinical lung cancer materials (12-41). This

systematic approach identified a set of molecules that appear to fall into the

category of cancer-testis antigens and whose up-regulation is a frequent and

important feature of the malignant nature of human cancer. These molecules

identified can be regarded as potential targets for the development of highly

sensitive and specific biomarkers as well as being useful in the development of

small-molecular compounds, antibodies, and cancer vaccines that could have a

more specific and efficient anti-cancer effect with minimal risk of adverse effects

(12-41). Among them, Chondrolectin (CHODL) was commonly overexpressed

in the great majority of lung cancers.

CHODL, a human ortholog of mouse chondrolectin (chodl) encodes a

single-pass transmembrane protein with one carbohydrate recognition domain

(CRD) of C-type lectins (42). C-type lectin family is classified into 17 groups

according to their domain architecture. They are divided into two major types;

transmembrane C-type lectins and soluble ones. These lectins have a wide

variety of functions at cytoplasm, cytoplasmic membrane, and extracellular

space. Mouse chodl was mainly expressed in embryonic tissues, and its

expression level was tightly controlled during embryonic development. Chodl

transcripts were present at the stage of 7 days post-conception (dpc), and

elevated up to the stage of 15 dpc, followed by decrease at the stage of 17 dpc

(43). To date, the biological function of CHODL and its activation in human

cancers were not reported.

Here we report evidences that CHODL might play a significant role in

lung carcinogenesis, and is a potential diagnostic and prognostic marker for lung

cancer.

Materials and Methods

Cell lines and clinical tissue samples. The human lung-cancer cell lines

used in this study included five adenocarcinomas (ADCs; A549, LC319,

NCI-H1373, PC14, and NCI-H1781), five squamous cell carcinomas (SCCs;

LU61, NCI-H520, NCI-H1703, NCI-H2170, SK-MES-1), one large cell carcinoma

(LCC; LX1), and four small cell lung cancers (SCLCs; DMS114, DMS273, SBC-3,

and SBC-5). A human bronchial epithelial cell line (BEAS-2B) was used as a

control (Supplementary Table S1). All cells were grown in monolayers in

appropriate media supplemented with 10% fetal calf serum (FCS) and were

o maintained at 37 C in an atmosphere of humidified air with 5% CO2. Primary

lung cancers were obtained with informed consent along with adjacent normal

lung tissue samples from patients, as described previously (7, 11). All tumors

were staged on the basis of the pTNM pathological classification of the UICC

(International Union Against Cancer) (44). Formalin-fixed primary NSCLCs and

adjacent normal lung tissue samples used for immunostaining on tissue

microarrays had been obtained from 295 patients undergoing curative surgery at

Saitama Cancer Center (Saitama, Japan). To be eligible for this study, tumor

samples were selected from patients who fulfilled all of the following criteria: (a)

patients suffered from primary NSCLC with confirmed stage (only pT1 to pT3,

pN0 to pN2, and pM0), (b) patients underwent curative surgery, but did not

receive any preoperative treatment, (c) among them NSCLC patients with lymph

node metastasis positive (pN1, pN2) tumors were treated with platinum-based

adjuvant chemotherapies after surgery, whereas patients with pN0 did not

receive adjuvant chemotherapies, and (d) patients whose clinical follow-up data

were available. This study and the use of all clinical materials mentioned were

approved by individual institutional ethics committees.

Semi-quantitative RT-PCR analysis. Total RNA was extracted from cultured

cells and clinical tissues using Trizol reagent (Life Technologies, Inc.) according

to the manufacturer’s protocol. Extracted RNAs and normal human-tissue

polyA RNAs were treated with DNase I (Roche Diagnostics) and then

reverse-transcribed using oligo (dT) primer and SuperScript II reverse

transcriptase (Life Technologies, Inc.). Semi-quantitative RT-PCR experiments

were carried out with synthesized CHODL gene-specific primers

(5’-GGAAGGAAAGGAACTACGAAATC-3’ and

5’-GTTAAAAGGAGCACAGGGACATA-3’), or with beta-actin (ACTB)-specific

primers (5’-ATCAAGATCATTGCTCCTCCT-3’ and

5’-CTGCGCAAGTTAGGTTTTGT-3’) as an internal control. All PCR reactions

involved initial denaturation at 95oC for 2 min followed by 22 (for ACTB) or 30

cycles (for CHODL) of 95oC 30 s, 54-60oC for 30 s, and 72oC for 60 s on a

GeneAmp PCR system 9700 (Applied Biosystems).

Immunocytochemical analysis. Immunocytochemical analyses were done

as previously described (13), using 1 μg/mL of a goat polyclonal anti-CHODL

antibody (Catalog No. sc-54867; Santa Cruz Biotechnology) for detecting

endogenous CHODL and 4 μg/mL of a rabbit polyclonal anti-Calreticulin

antibody (Catalog No. SPA-600; StressGen) for detecting endogenous

carleticulin in the endoplasmic reticulum (ER) as a primary antibody. The cells

were incubated with these primary antibodies for 1 h at room temperature,

followed by incubation with Alexa 488–conjugated donkey anti-goat secondary

antibodies (Molecular Probe) and an Alexa 594–conjugated donkey anti-rabbit

secondary antibodies (Molecular Probe). Each stained specimen was mounted

with Vectashield (Vector Laboratories, Inc.) containing 4’,

6-diamidino-2-phenylindole and visualized with Spectral Confocal Scanning

Systems (TSC SP2 AOBS; Leica Microsystems). On immunocytochemical

analyses, we confirmed that the antibody was specific for CHODL protein, using

NSCLC cell lines that endogenously expressed CHODL as well as cell lines

derived from NSCLC or bronchial epithelia that did not express it. We also

confirmed the specificity of the antibody by immunostaining NSCLC cells that

were transfected with siRNAs against CHODL that could suppress CHODL

expression or control siRNAs that could not (Supplementary Fig. S1).

Northern blot analysis. Human multiple tissue blots (23 normal tissues

including heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas,

spleen, thymus, prostate, testis, ovary, small intestine, colon, leukocyte, stomach,

thyroid, spinal cord, lymph node, trachea, adrenal gland, bone marrow; BD

Biosciences) were hybridized with a 32P-labeled PCR product of CHODL cDNA.

The cDNA probe of CHODL was prepared by reverse transcriptase-PCR

(RT-PCR) using primers CHODL-F (5’-GGTGCATAAACACTAATGCAGTC-3’)

and CHODL-R (5’-GTTAAAAGGAGCACAGGGACATA-3’). Prehybridization,

hybridization, and washing were performed according to the supplier's

recommendations. The blots were autoradiographed with intensifying screens

at −80°C for 7 days.

Tissue microarray and Immunohistochemistry. Tumor tissue microarrays

were constructed using 295 formalin-fixed primary lung cancers, as published

previously (45-47). The tissue area for sampling was selected based on visual

alignment with the corresponding HE-stained section on a slide. Three, four, or

five tissue cores (diameter 0.6 mm; height 3-4 mm) taken from a donor tumor

block were placed into a recipient paraffin block using a tissue microarrayer

(Beecher Instruments, Sun Prairie, WI). A core of normal tissue was punched

from each case, and 5 µm sections of the resulting microarray block were used

for immunohistochemical analysis.

To investigate the presence of CHODL protein in clinical samples that

had been embedded in paraffin blocks, we stained the sections in the following

manner. Briefly, 4 µg/ml of a goat polyclonal anti-human CHODL antibody

(Catalog No. sc-54867; Santa Cruz Biotechnology) was added after blocking of

endogenous peroxidase and proteins. The sections were incubated with

HRP-labeled anti-goat IgG as the secondary antibody. Substrate-chromogen

was added and the specimens were counterstained with hematoxylin. On

immunohistochemical analyses, we confirmed that the antibody was specific for

CHODL protein by antigen blocking assays using CHODL antigen peptides

(Catalog No. sc-54867P; Santa Cruz Biotechnology) that were used for

immunization of goats to produce polyclonal anti-human CHODL antibodies

(Supplementary Fig. S2). Three independent investigators assessed CHODL

positivity semi-quantitatively without prior knowledge of clinicopathological data.

The intensity of CHODL staining was evaluated using the following criteria:

strong positive (scored as 2+), brown staining in > 50% of tumor cells completely

obscuring cytoplasm; weak positive (1+), any lesser degree of brown staining

appreciable in tumor cell cytoplasm; and absent (scored as 0), no appreciable

staining in tumor cells. Cases were accepted as strongly positive if two or more

investigators independently defined them as such.

Statistical analysis. Statistical analyses were performed using the StatView

statistical program (SaS) to compare patient characteristics with responses to

therapy. Associations between clinicopathological variables and positivity for

CHODL were compared by Fisher's exact tests. Tumor-specific survival and

95% confidence intervals (CIs) were evaluated with the Kaplan-Meier method,

and differences between the two groups were evaluated with the log-rank test.

Factors associated with the prognosis were evaluated using Cox's

proportional-hazard regression model with a step-down procedure. Only those

variables with statistically significant results in univariate analysis were included

in a multivariate analysis. The criterion for removing a variable was the

likelihood ratio statistic, which was based on the maximum partial likelihood

estimate (default P value of 0.05 for removal from the model).

Western blot analysis. Cells were lysed in lysis buffer [50 mmol/L Tris-HCl

(pH 8.0), 150 mmol/L NaCl, 0.5% NP40, 0.5% sodium deoxycholate, and

Protease Inhibitor Cocktail Set III (EMD Biosciences, Inc.). Protein samples

were separated by SDS-polyacrylamide gels and electroblotted onto

Hybond-ECL nitrocellulose membranes (GE Healthcare Bio-Sciences). Blots

were incubated with a mouse monoclonal anti-myc antibody. Antigen-antibody

complexes were detected using secondary antibodies conjugated to horseradish

peroxidase (GE Healthcare Bio-Sciences). Protein bands were visualized by

enhanced chemiluminescence Western blotting detection reagents (GE

Healthcare Bio-Sciences).

RNA interference assay. To evaluate the biological functions of CHODL in

lung cancer cells, we used small interfering RNA (siRNA) duplexes against the

target genes. The target sequences of the synthetic oligonucleotides for RNA

interference were as follows: control 1 (si-LUC: siRNA against Photinus pyralis

luciferase gene), 5′-CGUACGCGGAAUACUUCGA-3′; control 2 (si-SCR: siRNA

against chloroplast Euglena gracilis gene coding for 5S and 16S (rRNAs),

5′-GCGCGCUUUGUAGGAUUCG-3′; si-CHODL-#A,

5′-AAAGUGGCAUGGAAGUAUA-3′; si-CHODL-#B,

5′-GGUAUAAUUCCCAAUCUAA-3′. Lung cancer cell lines, SBC-5 and

NCI-H2170 were plated onto 10 cm dishes (1.0 × 106 per dish), and transfected

with either of the siRNA oligonucleotides (100 nmol/L) using 24 µL of

Lipofectamine 2000 (Invitrogen) according to the manufacturers' instructions.

After 7 days of incubation, cell viability was assessed by 3-(4,

5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. The

number of colonies stained with Giemsa was also counted by colony formation

assay using colony counting software (ImageJ software 1.42, NIH).

Cell growth assay. COS-7, LC319 and BEAS-2B cells which scarcely

expressed endogenous CHODL were plated at densities of 1 × 106 cells/10 cm

dish and transfected with plasmids designed to express myc-tagged CHODL or

mock plasmids. Cells were selected in medium containing 0.4 mg/mL of

geneticin (Invitrogen) for 7 days, and cell viability was assessed by MTT assay

(cell counting kit-8 solution; Dojindo Laboratories).

Matrigel invasion assay. COS-7, LC319 and BEAS-2B cells transfected

either with plasmids expressing myc-tagged CHODL or with mock plasmids were

grown to near confluence in DMEM or RPMI containing 10% FCS. The cells

were harvested by trypsinization, washed in medium without addition of serum or

protease inhibitor, and suspended in medium at a concentration of 2 × 105/mL.

Before preparing the cell suspension, the dried layer of Matrigel matrix (Becton

Dickinson Labware) was rehydrated with medium for 2 h at room temperature.

Medium (0.75 mL) containing 10% FCS was added to each lower chamber in 24

well Matrigel invasion chambers, and 0.5 mL (1 × 105 cells) of cell suspension

was added to each insert of the upper chamber. The plates of inserts were

incubated for 22 h at 37°C, in which condition, no growth promoting effect by

exogenous CHODL expression was also confirmed by MTT assay. Then the

chambers were processed, and cells invading through the Matrigel were fixed

and stained by Giemsa as directed by the supplier (Becton Dickinson Labware).

Results

CHODL expression in lung cancers and normal tissues. To search for

novel target molecules for the development of therapeutic agents and/or

diagnostic biomarkers for NSCLC, we first screened genes that showed more

than a 5-fold higher level of expression in cancer cells than in normal cells, in

more than 50% of the lung cancers analyzed by cDNA microarray. Among

27,648 genes or ESTs screened, we identified that the expression of CHODL

transcript showed 5-folds or higher in 63% of NSCLCs compared with normal

lung (control). Moreover, our cDNA microarray database indicated that CHODL

was hardly detectable in normal tissues except testis. Therefore, we

determined to select CHODL as a good candidate for further analyses. We

confirmed its transactivation by semi-quantitative RT-PCR experiments in 7 of 15

additional lung cancer tissues and 10 of 15 lung cancer cell lines (Figs. 1A and

1B). To determine the subcellular localization of endogenous CHODL in lung

cancer cells, we performed immunocytochemical analysis using anti-CHODL

polyclonal antibodies; CHODL protein was localized in cytoplasm with granular

appearance in A549 and NCI-H2170 cells, which expressed endogenous

CHODL, but it was not detectable in CHODL-negative LC319 and BEAS-2B cells

(Fig. 1C). Furthermore, we confirmed that CHODL protein was mainly

co-localized with calreticulin that was used as an ER marker (Fig. 1D), but not

co-localized with Golgi and endosome markers (data not shown). We

confirmed that the positive signal by anti-CHODL antibody obtained in lung

cancer A549 cells was markedly reduced by suppression of CHODL expression

by siRNA against CHODL, which proved the specificity of antibody to CHODL

protein (Supplementary Fig. S1).

Northern blot analysis identified a 2.8-kb CHODL transcript to be highly

expressed in the testis; however it was hardly detectable in other normal tissues

examined (Fig. 2A). We subsequently examined expression of CHODL protein

in five normal tissues (heart, liver, lung, kidney, and testis), as well as lung

cancers using anti-CHODL antibody, and found that it was hardly detectable in

the former four tissues, whereas positive CHODL staining was detected at

cytoplasm of testis and lung cancer tissues (Fig. 2B). We confirmed that the

positive signal by anti-CHODL antibody obtained in lung cancer tissues was

markedly diminished by preincubation of the antibody with the CHODL antigen

peptide used for immunization of goat, which also independently indicated its

specificity to CHODL protein (Supplementary Fig. S2). The expression levels

of CHODL protein in lung cancer were significantly higher than those in testis.

We also evaluated CHODL staining in lung cancers and adjacent normal lung

tissues, and confirmed CHODL protein to be positively stained in the majority of

NSCLC tissues, but not in their corresponding normal lungs (Fig. 2C).

Furthermore, CHODL protein was hardly detectable in benign lung disease of

emphysema (Supplementary Fig. S3).

Association of CHODL protein expression with poor clinical outcome of

NSCLC patients. To verify the biological and clinicopathological significance of

CHODL, we also examined the expression of CHODL protein by means of tissue

microarrays containing lung cancer tissues from 295 NSCLC patients who

underwent surgical resection. We classified a pattern of CHODL expression on

the tissue array ranging from absent (scored as 0) to weak/strong positive

(scored as 1+ or 2+; Fig. 2D). As shown in Table 1A, the number of NSCLC

tissues scored as 2+, 1+, and 0 were 98 (33.2%), 131 (44.4%), and 66 (22.4%),

respectively. We then evaluated the association between CHODL status and

clinicopathologic variables among the 295 patients. The median survival time

of NSCLC patients with strong CHODL-staining (score 2+) was significantly

shorter than that with absent/weak CHODL expression (score0-1+) (P = 0.0006

by log-rank test; Fig. 2E). We also used univariate analysis to evaluate

associations between patient prognosis and other factors including age (≥65

versus 65>), gender (male versus female), histological classification

(non-adenocarcinoma versus adenocarcinoma), smoking status (current/former

smoker versus never smoker), pathological T stage (T2+T3 versus T1),

pathological N stage (N1+N2 versus N0), and CHODL positivity (strong positive

versus weak positive/absent) (Table 1B). Among those parameters, strong

CHODL-positivity, elderly, male gender, non-adenocarcinoma histology,

advanced pT stage and advanced pN stage were significantly associated with

poor prognosis. In multivariate analysis of prognostic factors, CHDOL-positivity,

age, larger tumor size and lymph node metastasis were significant and

independent prognostic factors for NSCLC patients.

Growth-promoting effect of CHODL. Using siRNA oligonucleotides for

CHODL, we attempted to knock down the expression of endogenous CHODL in

lung cancer cell lines SBC-5 and NCI-H2170, which showed high levels of

endogenous CHODL expression. Two CHODL-specific siRNAs (si-CHODL-#A

and si-CHODL-#B) significantly suppressed expression of CHODL transcripts

compared with control siRNAs (si-LUC and si-SCR) (Fig. 3A). MTT and colony

formation assays revealed that reduction of CHODL expression by the two

siRNAs significantly suppressed the growth of SBC-5 and NCI-H2170 cells (Figs.

3B and 3C; Supplementary Fig. S4).

To further disclose a potential role of CHODL in tumorigenesis, we

prepared plasmids designed to express either CHODL (pcDNA3.1/myc-His

vector) or mock vector, and transfected them into COS-7, LC319 and BEAS-2B

cells which scarcely expressed endogenous CHODL. Cells that transiently

expressed exogenous CHODL revealed significant growth promotion compared

with the mock-transfected cells (Figs. 3D and 3E).

Enhanced cell invasion by CHODL. Since strong expression of CHODL in

NSCLC tissues was associated with poor prognosis of patients, we next

examined a possible role of CHODL in cellular invasion by Matrigel assays using

COS-7, LC319 and BEAS-2B cells. Transfection of CHODL cDNA into either of

the cells significantly enhanced their invasive activity (Figs. 4A-4C). This result

also suggests that CHODL could contribute to the more malignant phenotype of

cells.

Discussion

Several molecular-targeting drugs have been developed and proved their

efficacy in cancer therapy. However, the proportion of patients showing good

response is still limited. Therefore, further development of molecular-targeting

drugs for cancer is urgently awaited. We have screened diagnostic and

therapeutic target molecules by whole-genome gene expression profile analysis

as well as subsequent tissue microarray and functional analyses (7-41). Using

this approach, we have shown here that CHODL is frequently overexpressed in

clinical lung cancer samples and cell lines, and that its gene product plays

indispensable roles in the growth and progression of lung cancer cells.

CHODL appears to belong to C-type lectin-containing protein family that

is classified into 17 groups. They are involved in diverse processes including

cell recognition and communication, cell-cell adhesion, and extracellular

matrix-cell interactions (48). For example, of proteoglycans belong to soluble

C-type lectin family member without transmembrane domain, play an important

role in the structure and stability of the extracellular matrix (ECM) and the

interaction between the ECM and the cell (49). Some lectins are also important

in embryonic development and immune responses. They function as

recognition molecules in important processes in the immune system: for

example, the selectines, which belong to C-type lectin with a transmembrane

domain, function at cell surface in leukocyte-leukocyte and leukocyte-endothelial

interactions (49). Some transmembrane C-type lectins such as calnexin play

important roles as molecular chaperones like calreticulin in the ER. These

proteins bind to misfolded proteins and prevent them from being exported from

the ER to the Golgi apparatus (50). Since we confirmed that CHODL was partly

localized in the ER of cancer cells, it might have a similar function in control

system for some oncogenic proteins in the ER.

In this study, we demonstrated that CHODL was expressed only in testis

among the 23 normal tissues examined and was highly expressed in surgically

resected samples from NSCLC patients, indicating CHODL to be a typical

cancer-testis antigen. Clinicopathological evidences obtained through our

tissue microarray experiments indicated that NSCLC patients with strong

CHODL-positive tumors had shorter cancer-specific survival periods than those

with weak/absent expression tumors. Functional assays by knockdown of

CHODL expression with siRNA or exogenous expression of CHODL revealed

that CHODL was important for cellular growth and invasion. Although the

precise molecular mechanism underlying our observations is unknown, the

combined results strongly suggest that CHODL is likely to function as an

oncogenic protein in lung carcinogenesis and tumor progression. Elucidation of

the mechanism implied by these observations should reveal important new

information about cancer cell proliferation and cancer progression.

To date, several prognostic biomarkers for lung cancer were reported,

however, it is difficult to measure the malignant level of tumors in individual

patients using only a single marker. To quantitatively predict the prognosis of

NSCLC patients who had undergone curative surgery, we next attempted

combined assays as a pilot study using the positivity data of CHODL and the

available data of 2 other prognostic biomarkers for NSCLC (FGFR1OP and

DLX5) which had been previously identified by our group using the same set of

lung cancer tissues used in this study (23, 25). NSCLC patients were divided

into four groups; (group-1) all three markers were scored as strong positive in

each patient, (group-2) either of two markers were strong positive, (group-3)

either of one marker was strong positive, and (group-4) no marker was strong

positive. There is a significant correlation between the increased number of

strong positive markers and poorer prognosis of NSCLC patients

(Supplementary Fig. S5). Although further screening and validation analyses

that determine the best combination of promising prognostic biomarkers

providing the best separation of long survivors and short ones are necessary,

the results demonstrate that this type of diagnostic approach combined with

more quantitative scoring system of immunohistochemical staining may be a

useful and practical index in the clinic for application of adjuvant therapy to the

patients who are likely to have poor prognosis after surgery.

In summary, we have shown that over-expressed CHODL is likely to be

one of essential contributors to a growth-promoting pathway and to aggressive

features of lung cancers. CHODL is a possible prognostic biomarker in the

clinic.

Grant Support: This work was supported in part by Grant-in-Aid for Scientific

Research (B) and Grant-in-Aid for Scientific Research on Innovative Areas from

The Japan Society for the Promotion of Science. Y.D. is a member of Shiga

Cancer Treatment Project (Shiga Prefecture, Japan).

References 1. Ahmedin J, Rebecca S, Elizabeth W, Yongping H, Jiaquan X, Michael J. T. Cancer Statistics, 2009. CA Cancer J Clin 2009; 59:225-49. 2. Miki D, Kubo M, Takahashi A, Yoon KA, Kim J, Lee GK, et al. Nat Genet 2010; 42:893-6. Variation in TP63 is associated with lung adenocarcinoma susceptibility in Japanese and Korean populations. 3. Schiller JH, Harrington D, Belani CP, Langer C, Sandler A, Krook J, et al. Eastern Cooperative Oncology Group. Comparison of four chemotherapy regimens for advanced non-small-cell lung cancer. N Engl J Med 2002; 346:92–8. 4. Dowell J, Minna JD, Kirkpatrick P. Erlotinib hydrochloride. Nat Rev Drug Discov 2005; 4:13–4. 5. Pal SK, Pegram M. Epidermal growth factor receptor and signal transduction: potential targets for anti-cancer therapy. Anticancer Drugs 2005; 16:483–94. 6. Daigo Y, Nakamura Y. From cancer genomics to thoracic oncology: Discovery of new biomarkers and therapeutic targets for lung and esophageal carcinoma. Gen Thorac Cardiovasc Surg 2008; 56:43–53. 7. Kikuchi T, Daigo Y, Katagiri T, Tsunoda T, Okada K, Kakiuchi S, et al. Expression profiles of non-small cell lung cancers on cDNA microarrays: identification of genes for prediction of lymph-node metastasis and sensitivity to anti-cancer drugs. Oncogene 2003; 22:2192-205. 8. Kakiuchi S, Daigo Y, Tsunoda T, Yano S, Sone S, Nakamura Y. Genome-wide analysis of organ-preferential metastasis of human small cell lung cancer in mice. Mol Cancer Res 2003; 1:485-99. 9. Kakiuchi S, Daigo Y, Ishikawa N, Furukawa C, Tsunoda T, Yano S, et al. Prediction of sensitivity of advanced non-small cell lung cancers to gefitinib (Iressa, ZD1839). Hum Mol Genet 2004; 13:3029-43. 10. Kikuchi T, Daigo Y, Ishikawa N, Katagiri T, Tsunoda T, Yoshida S, et al. Expression profiles of metastatic brain tumor from lung adenocarcinomas on cDNA microarray. Int J Oncol 2006; 28:799-805. 11. Taniwaki M, Daigo Y, Ishikawa N, Takano A, Tsunoda T, Yasui W, et al. Gene expression profiles of small-cell lung cancers: molecular signatures of lung cancer. Int J Oncol 2006; 29:567 -75. 12. Suzuki C, Daigo Y, Kikuchi T, Katagiri T, Nakamura Y. Identification of COX17 as a therapeutic target for non-small cell lung cancer. Cancer Res 2003; 63:7038–41. 13. Kato T, Daigo Y, Hayama S, Ishikawa N, Yamabuki T, Ito T, et al. A novel human tRNA-dihydrouridine synthase involved in pulmonary carcinogenesis. Cancer Res 2005; 65:5638–46. 14. Furukawa C, Daigo Y, Ishikawa N, Kato T, Ito T, Tsuchiya E, et al. Plakophilin 3 oncogene as prognostic marker and therapeutic target for lung cancer. Cancer Res 2005; 65:7102–10. 15. Suzuki C, Daigo Y, Ishikawa N, Kato T, Hayama S, Ito T, et al. ANLN plays a critical role in human lung carcinogenesis through the activation of RHOA and by involvement in the phosphoinositide 3-kinase/AKT pathway. Cancer Res 2005; 65:11314–25. 16. Ishikawa N, Daigo Y, Takano A, Taniwaki M, Kato T, Tanaka S, et al. Characterization of SEZ6L2 cell-surface protein as a novel prognostic marker for lung cancer. Cancer Sci 2006; 97:737–45. 17. Takahashi K, Furukawa C, Takano A, Ishikawa N, Kato T, Hayama S, et al. The neuromedin u-growth hormone secretagogue receptor 1b/neurotensin receptor 1 oncogenic signaling pathway as a therapeutic target for lung cancer. Cancer Res 2006; 66:9408–19. 18. Hayama S, Daigo Y, Kato T, Ishikawa N, Yamabuki T, Miyamoto M, et al.

Activation of CDCA1-KNTC2, members of centromere protein complex, involved in pulmonary carcinogenesis. Cancer Res 2006; 66:10339–48. 19. Kato T, Hayama S, Yamabuki Y, Ishikawa N, Miyamoto M, Ito T, et al. Increased expression of IGF-II mRNA-binding protein 1 is associated with the tumor progression in patients with lung cancer. Clin Cancer Res 2007; 13:434–42. 20. Suzuki C, Takahashi K, Hayama S, Ishikawa N, Kato T, Ito T, et al. Identification of Myc-associated protein with JmjC domain as a novel therapeutic target oncogene for lung cancer. Mol Cancer Ther 2007; 6:542–51. 21. Hayama S, Daigo Y, Yamabuki T, Hirata D, Kato T, Miyamoto M, et al. Phosphorylation and activation of cell division cycle associated 8 by aurora kinase B plays a significant role in human lung carcinogenesis. Cancer Res 2007; 67:4113–22. 22. Taniwaki M, Takano A, Ishikawa N, Yasui W, Inai K, Nishimura H, et al. Activation of KIF4A as a Prognostic Biomarker and Therapeutic Target for Lung Cancer. Clin Cancer Res 2007; 13:6624–31. 23. Mano Y, Takahashi K, Ishikawa N, Takano A, Yasui W, Inai K, et al. Fibroblast growth factor receptor 1 oncogene partner as a novel prognostic biomarker and therapeutic target for lung cancer. Cancer Sci 2007; 98:1902–13. 24. Kato T, Sato N, Hayama S, Yamabuki T, Ito T, Miyamoto M, et al. Activation of holliday junction recognizing protein involved in the chromosomal stability and immortality of cancer cells. Cancer Res 2007; 67:8544–53. 25. Kato T, Sato N, Takano A, Miyamoto M, Nishimura H, Tsuchiya E, et al. Activation of Placenta Specific Transcription Factor Distal-less Homeobox 5 Predicts Clinical Outcome in Primary Lung Cancer Patients. Clin Cancer Res 2008; 14:2363–70. 26. Dunleavy EM, Roche D, Tagami H, Lacoste N, Ray-Gallet D, Nakamura Y, et al. HJURP is a Cell-Cycle-Dependent Maintenance and Deposition Factor of CENP-A at Centromeres. Cell 2009; 137:485–97. 27. Hirata D, Yamabuki T, Miki D, Ito T, Tsuchiya E, Fujita M, et al. Involvement of Epithelial Cell Transforming Sequence 2 (ECT2) Oncoantigen in Lung and Esophageal Cancer Progression. Clin Cancer Res 2009; 15:256–66. 28. Sato N, Koinuma J, Fujita M, Hosokawa M, Ito T, Tsuchiya E, et al. Activation of WD repeat and high-mobility group box DNA binding protein 1 in pulmonary and esophageal carcinogenesis. Clin Cancer Res. 2010; 16: 226–39. 29. Sato N, Koinuma J, Ito T, Tsuchiya E, Kondo S, Nakamura Y, et al. Activation of an oncogenic TBC1D7 (TBC1 domain family, member 7) protein in pulmonary carcinogenesis. Genes Chromosomes Cancer. 2010; 49: 353–67. 30. Nguyen MH, Koinuma J, Ueda K, Ito T, Tsuchiya E, Nakamura Y, et al. Phosphorylation and activation of cell division cycle associated 5 by mitogen-activated protein kinase play a crucial role in human lung carcinogenesis. Cancer Res. 2010; 70: 5337–47. 31. Ishikawa N, Daigo Y, Yasui W, Inai K, Nishimura H, Tsuchiya E, et al. ADAM8 as a novel serological and histochemical marker for lung cancer. Clin Cancer Res 2004; 10:8363–70. 32. Ishikawa N, Daigo Y, Takano A, Taniwaki M, Kato T, Hayama S, et al. Increases of amphiregulin and transforming growth factor-alpha in serum as predictors of poor response to gefitinib among patients with advanced non-small cell lung cancers. Cancer Res 2005; 65:9176–84. 33. Yamabuki T, Takano A, Hayama S, Ishikawa N, Kato T, Miyamoto M, et al. Dickkopf-1 as a novel serologic and prognostic biomarker for lung and esophageal carcinomas. Cancer Res 2007; 67:2517–25.

34. Ishikawa N, Takano A, Yasui W, Inai K, Nishimura H, Ito H, et al. Cancer-testis antigen lymphocyte antigen 6 complex locus K is a serologic biomarker and a therapeutic target for lung and esophageal carcinomas. Cancer Res 2007; 67: 11601–11. 35. Takano A, Ishikawa N, Nishino R, Masuda K, Yasui W, Inai K, et al. Identification of nectin-4 oncoprotein as a diagnostic and therapeutic target for lung cancer. Cancer Res 2009; 69: 6694–703. 36. Sato N, Yamabuki T, Takano A, Koinuma J, Aragaki M, Masuda K, et al. Wnt inhibitor Dickkopf-1 as a target for passive cancer immunotherapy. Cancer Res 2010; 70: 5326–36. 37. Suda T, Tsunoda T, Daigo Y, Nakamura Y, Tahara H. Identification of human leukocyte antigen-A24-restricted epitope peptides derived from gene products upregulated in lung and esophageal cancers as novel targets for immunotherapy. Cancer Sci 2007; 98:1803–8. 38. Mizukami Y, Kono K, Daigo Y, Takano A, Tsunoda T, Kawaguchi Y, et al. Detection of novel cancer-testis antigen-specific T-cell responses in TIL, regional lymph nodes, and PBL in patients with esophageal squamous cell carcinoma. Cancer Sci 2008; 99: 1448–54. 39. Harao M, Hirata S, Irie A, Senju S, Nakatsura T, Komori H, et al. HLA-A2-restricted CTL epitopes of a novel lung cancer-associated cancer testis antigen, cell division cycle associated 1, can induce tumor-reactive CTL. Int J Cancer 2008; 123: 2616–25. 40. Kono K, Mizukami Y, Daigo Y, Takano A, Masuda K, Yoshida K, et al. Vaccination with multiple peptides derived from novel cancer-testis antigens can induce specific T-cell responses and clinical responses in advanced esophageal cancer. Cancer Sci. 2009; 100: 1502–9. 41. Tomita Y, Imai K, Senju S, Irie A, Inoue M, Hayashida Y, et al. A novel tumor-associated antigen, cell division cycle 45-like can induce cytotoxic T lymphocytes reactive to tumor cells. Cancer Sci. 2011; 102(4): 697-705 42. Weng L, Smits P, Wauters J, Merregaert J. Molecular cloning and characterization of human chondrolectin, a novel type I transmembrane protein homologous to C-type lectins. Genomics. 2002 Jul; 80(1): 62-7. 43. Weng, L, Hübner, R, Claessens A, Smits P, Wauters J, Tylzanowski P, et al. Isolation and characterization of a novel C-type lectin, Chondrolectin (Chodl), which is predominantly expressed in muscle cells. Gene 2003; 308:21-9. 44. Sobin LH, Witterkind C, editors. TNM Classification of Malignant Tumours. 6th ed New York: Whiley-Liss; 2002. 45. Chin SF, Daigo Y, Huang HE, Iyer NG, Callagy G, Kranjac T, et al. A simple and reliable pretreatment protocol facilitates fluorescent in situ hybridization on tissue microarrays of paraffin wax embedded tumour samples. Mol Pathol 2003; 56:275-9. 46. Callagy G, Cattaneo E, Daigo Y, Happerfield L, Bobrow LG, Pharoah PD, et al. Molecular classification of breast carcinomas using tissue microarrays. Diagn Mol Pathol 2003; 12:27-34. 47. Callagy G, Pharoah P, Chin SF, Sangan T, Daigo Y, Jackson L, et al. Identification and validation of prognostic markers in breast cancer with the complementary use of array-CGH and tissue microarrays. J Pathol 2005; 205:388-96. 48. Zelensky AN, Gready JE. The C-type lectin-like domain superfamily. FEBS Lett 2005; 272:6179-217. 49. Drickamer K, Taylor ME. Biology of animal lectins. Annu Rev Cell Biol 1993; 9:237-64. 50. Ellgaard L, Helenius A. Quality control in the endoplasmic reticulum. Nat Rev 2003; 4:181-91.

Figure legends Figure 1. CHODL expression in lung cancers. A, Expression of CHODL in clinical lung cancers, examined by semi-quantitative RT-PCR. B, Expression of CHODL in lung-cancer cell lines, examined by semi-quantitative RT-PCR. C, Subcellular localization of endogenous CHODL protein in lung cancer cells. CHODL was stained in the cytoplasm of cells with granular appearance in CHODL-positive A549 and NCI-H2170 cells, while CHODL was not stained in LC319 and BEAS-2B cells that did not express endogenous CHODL. D, Co-localization of endogenous CHODL with calreticulin as endoplasmic reticulum marker in A549 cells.

Figure 2. Expression of CHODL in normal tissues and lung tumors. A, Northern blot analysis of the CHODL transcript in 23 normal adult human tissues. B, Immunohistochemical evaluation of CHODL protein using anti–CHODL antibody in lung ADC, lung SCC, lung LCC and five normal tissues. C, Immunohistochemical staining of CHODL protein using anti-CHODL antibody in four representative paired lung tumors and adjacent normal lung tissues. D, Immunohistochemical evaluation of CHODL expression on tissue microarrays (X100). Examples are shown of strong, weak, absent, and normal lung tissue. E, Kaplan-Meier analysis of tumor-specific survival in patients with NSCLCs according to CHODL expression (P = 0.0006; Log-rank test). Number of cases at risk is shown in Supplementary Table S2.

Figure 3. Growth effect of CHODL expression. A-C, Response of SBC-5 and NCI-H2170 cells to si-CHODL#A, si-CHODL#B or control siRNAs (si-LUC or si-SCR). A, The level of CHODL mRNA expression detected by semi-quantitative RT-PCR in cells transfected with either control siRNAs or si-CHODLs. B, The effect of siRNA against CHODL on cell viability, evaluated by MTT assay. C, The effect of siRNA against CHODL on colony formation, evaluated by colony formation assays. All assays were performed thrice and in triplicate wells. D-E, Growth promoting effects of CHODL. D, Transient expression of CHODL in COS-7, LC319 and BEAS-2B cells, detected

by western blot analysis. E, Effect of CHODL on growth of COS-7, LC319 and BEAS-2B cells. At each time point, cell viability was evaluated by MTT assay.

Figure 4. Enhancement of cellular invasiveness by CHODL. A, Transient expression of CHODL in COS-7, LC319 and BEAS-2B cells, detected by western blot analysis. B and C, Assays demonstrating the invasive nature of COS-7, LC319 and BEAS-2B cells in Matrigel matrix after transfection with expression plasmids for human CHODL. The relative number of cells migrating through the Matrigel-coated filters (B) and Giemsa staining (C; magnification, X100). Assays were done thrice and in triplicate wells.

Table 1A. Association between CHODL-status in NSCLC and patients' characteristics (n=295) CHODL CHODL CHODL P -value Total strong weak absent strong vs. weak or absent n=295 n=98 n=131 n=66 Gender Male 209 75 87 47 0.1372 Female 86 23 44 19 Age(years) ＜65 130 45 55 30 0.7091 >=65 165 53 76 36 Histological type ADC 176 62 79 35 SCC 85 24 43 18 0.3818* Others 34 12 9 13 Smoking status Never 78 23 41 14 0.484 Smoker 217 75 90 52 pT factor T1 120 33 53 34 0.1018 T2+T3 175 65 78 32 pN factor N0 193 57 88 48 0.0699 N1+N2 102 41 43 18 ADC, adenocarcinama, SCC, squamous cell carcinoma Others, large cell carcinoma(LCC) plus adenosquamous cell carcinoma(ASC) *ADC versus non-ADC

Table 1B. Cox's proportional hazards model analysis of prognostic factors in patients with NSCLCs Variables Hazards ratio 95%Cl Unfavorable/Favorable P -value Univariate analysis CHODL 1.841 1.294-2.620 Strong/Weak or absent 0.0007* Age(years) 1.699 1.188-2.431 65=

Variables Hazards ratio 95%Cl Unfavorable/Favorable P -value Multivariate analysis CHODL 1.660 1.161-2.372 Strong/Weak or absent 0.0054* Age(years) 1.869 1.303-2.680 65=

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Chondrolectin is a novel diagnostic biomarker and a therapeutic target for lung cancer

Ken Masuda, Atsushi Takano, Hideto Oshita, et al.

Clin Cancer Res Published OnlineFirst October 20, 2011.

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