Proteome Analysis for Downstream Targets of Oncogenic KRAS - the Potential Participation of CLIC4 in Carcinogenesis in the Lung

This study investigated the proteome modulated by oncogenic KRAS in immortalized airway epithelial cells. Chloride intracellular channel protein 4 (CLIC4), S100 proteins (S100A2 and S100A11), tropomyosin 2, cathepsin L1, integrinsα3, eukaryotic elongation factor 1, vimentin, and others were discriminated. We here focused on CLIC4 to investigate its potential involvement in carcinogenesis in the lung because previous studies suggested that some chloride channels and chloride channel regulators could function as tumor suppressors. CILC4 protein levels were reduced in some lung cancer cell lines. The restoration of CLIC4 in lung cancer cell lines in which CLIC4 expression was reduced attenuated their growth activity. The immunohistochemical expression of the CLIC4 protein was weaker in primary lung cancer cells than in non-tumorous airway epithelial cells and was occasionally undetectable in some tumors. CLIC4 protein levels were significantly lower in a subtype of mucinous ADC than in others, and were also significantly lower in KRAS-mutated ADC than in EGFR-mutated ADC. These results suggest that the alteration in CLIC4 could be involved in restrictedly the development of a specific fraction of lung adenocarcinomas. The potential benefit of the proteome modulated by oncogenic KRAS to lung cancer research has been demonstrated.


Introduction
Lung cancer is one of the most common causes of cancerrelated death in the developed world [1,2]. If primary tumors are successfully removed surgically removed, the incidence of recurrence remains high [1,2]. Although some lung tumors are sensitive to conventional chemotherapeutic agents or certain molecular targeting agents, many are not [3,4]. Thus, further understanding of the molecular basis of carcinogenesis in the lung is needed in order to develop novel therapeutic strategies.
Our previous studies identified important molecules involved in carcinogenesis in the lung through a comprehensive search for the downstream targets of oncogenic KRAS [3,5]. KRAS is known to transmit potential signals that cause opposing biological effects. Some downstream targets may be growth suppressors while others may be accelerators [3]. A disruption in the balance between these effects may occasionally result in a neoplastic transformation and also promote the progression of carcinogenesis.
Such downstream targets were previously shown to be involved in not only in the development of lung cancers with KRAS mutations, but also of those without KRAS mutations [3,5]. These findings indicated that investigating the downstream targets of oncogenic KRAS reveal the common important molecular basis of lung cancer.
The present study examined the post-translational expression profile (proteome) of oncogenic KRAS-transduced airway epithelial cells and identified some downstream molecules. We focused on CLIC4, a member of the chloride intracellular channel protein family [6][7][8], because previous studies suggested that some chloride channels and chloride channel regulators could function as tumor suppressors [5]. To verify the potential involvement of CLIC4 in carcinogenesis in the lung, we here examined lung cancer cell lines and primary human lung cancers for the expression of CLIC4, and analyzed the correlation between its expression levels and different clinicopathologic parameters.

Cell lines and culture
An immortalized human airway epithelial cell line (16HBE14o, Simian virus 40 (SV40)-transformed human bronchial epithelial cells) described by Cozens AL et al. (1994) [9] was kindly provided by Grunert DC (California Pacific Medical Center Research institute). A sub-clone of 16HBE14o cells, described as NHBE-T in this study, was used in the present study. Human lung cancer cell lines (A549, H358, H2087, H1819, H441, and H1299) and a human embryonic kidney cell line (HEK293T) were purchased from the American Type Culture Collection (ATCC, Manassas, VA). The human lung cancer cell lines, Lu135 and Lu139 were purchased from Riken Cell Bank (Tsukuba, Japan). PC9 and HARA were purchased from Immunobiological Laboratories Co. (Gunma, Japan). TKB5, TKB6, TKB7, TKB8, TKB8, TKB14, and TKB20 were established and gifted by Dr. H Kamma via Dr. T Yazawa (Kyorin University School of Medicine, Tokyo, Japan) [10]. The Ethics Committee of Yokohama City University approved the experimental protocol using these cell lines.
Vectors bearing a sense and antisense strand of cDNA were obtained. The pro-retrovirus vector pSINsi bearing a short hairpin RNA for the knockdown of CLIC4 was ordered from Takara Bio Inc.

Retroviral-mediated gene transfer
pQCXIH/P-based expression plasmid vectors and the pCL10A1 retrovirus-packaging plasmid vector (IMGENEX, San Diego, CA) were cotransfected into HEK293T cells with Lipofectoamine 2000 reagent (Invitrogen, Carlsbad, CA), and conditioned medium was then recovered as a retroviral vector solution. The desired genes were transduced by incubating cells with the viral solution containing 10 mg/ml of polybrene (Sigma, St. Louis, MO). Cells stably expressing the desired genes were selected with 300 mg/ml of Hygromycin D or 5.0 mg/ml of Puromycin (Invitrogen) for 3 days. Pooled clones were used for biological analyses as follows.

Proteome analysis
Whole cell protein was labeled with Cy2, Cy3, or Cy5 using a protein-labeling kit (Amersham, Piscataway, NJ). Protein lysates were prepared from mock-transduced NHBE-T cells, KRAS G12transduced NHBE-T cells, and KRAS V12-transduced NHBE-T cells. Two-dimensional electrophoresis was performed using a horizontal electrophoresis system (Maltiphor II) (Amersham) according to the manufacturer's instructions. Briefly, an equal amount of a protein sample was subjected to first-dimensional isoelectric focusing, followed by second dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins on differentially expressed spots were identified by LC-MS/MS analysis. The details of this method have been described elsewhere [12,13] Western blotting Whole cell lysates were subjected to SDS-polyacrylamide gel electrophoresis, and transferred onto PVDF membranes (Amersham). The membranes were then incubated with nonfat dry milk in Tris-buffered saline containing Tween-20 (TBS-T) to block non-immunospecific protein binding, and then with a primary antibody against KRAS (Santa Cruz, Santa Cruz, CA), CLIC4 (Santa Cruz), Smad2/3 (Cell Signaling Technology, Danvers, MA), phosphorylated Smad2/3 (Cell Signaling Technology), caspase-3 (Cell Signaling Technology), or b-actin (Sigma). After washing with TBS-T, the membranes were incubated with animalmatched HRP-conjugated secondary antibodies (Amersham). Immunoreactivity was visualized with an enhanced chemiluminescence system (Amersham).

Primary Lung Cancer
All 235 tumors examined (180 adenocarcinoma (ADC), 39 squamous cell carcinomas (SQC), and 16 large cell carcinomas (LCC)) were removed by radical surgical resection at the Kanagawa Cardiovascular and Respiratory Center (Yokohama, Japan). The research plan was approved by the Ethics Committees of Yokohama City University and Kanagawa Prefectural Cardiovascular and Respiratory Center Hospital. Written informed consent for research use was obtained from all subjects providing materials. Histological types and disease stages were determined according to the International TNM Classification System (seventh edition of the UICC) [14]. Immunohistochemistry Tumor sections were cut from formalin-fixed, paraffin-embedded tissue blocks. Sections were deparaffinized, rehydrated, and incubated with 3% hydrogen peroxide, followed by 5% goat serum to block endogenous peroxidase activities and nonimmunospecific protein binding. Sections were boiled in citrate buffer (0.01 M, pH 6.0) for 15 minutes to retrieve masked epitopes and then incubated with a primary antibody against CLIC4 (Santa Cruz) or Ki-67 (DAKO, Ely, UK). Immunoreactivity was visualized using an Envision detection system (DAKO), and nuclei were counterstained with hematoxylin. CLIC4 immunohistochemical expression levels were evaluated by a scoring system as described in the Results section. The labeling index of Ki-67 was calculated as the proportion of positive nuclei cells by counting 500-1000 cancer cells. The Ki-67 labeling indices of ,10% and $10% were classified as low and high levels, according to the results of our previous study [4].

Colony formation assays
Cells (2.5610 4 ) were seeded onto a 10-cm culture dish (Iwaki, Tokyo, Japan), and grown for 10 days. Cells were fixed with methanol and Giemsa-stained, and colonies visible in scanned photographs were counted.

Growth curve assays
Cells (2.5610 5 ) were seeded onto a 10-cm culture dish, and grown to a semi-confluent state for the appropriate number of days. Cells were counted and 2.5610 5 cells were reseeded onto a 10-cm dish. Several passages were repeated in the same manner. The sum of population doublings at each point was calculated by the formula SPDL n = log 2 (count n /2.5610 5 )+SPDL n-1 .

Soft agar colony formation assay
Cells (1.25610 4 ) were grown in 1 ml of DMEM-based 0.3% agar (Agar Noble; Becton Dickinson, Sparks, MD) containing 10% FBS in a 3.5-cm culture dish (Iwaki) for 4 weeks. The agar was fixed with a buffered 4% paraformaldehyde solution, and colonies visible in scanned photographs were counted.

Statistical Analysis
Differences in the mean values of the immunohistochemical score for CLIC4 among the groups classified based on various clinicopathologic subjects were analyzed by one-way ANOVAs. Recurrence curves were plotted using the Kaplan-Meier method, and the absolute risk of recurrence at five years was estimated from these curves. Differences in the disease-free survival (DFS) span and rate were analyzed using the log-rank test. P values less than 0.05 were considered significant. Statistical analyses were

Protein expression profiling modulated by oncogenic KRAS
Two-dimensional electrophoresis (representative results are shown in Fig. 1A) discriminated 1662 spots. A comprehensive evaluation of the signal intensities of these spots revealed that oncogenic KRAS-transduced cells had different expression profiles to those of empty vector-and wild-type KRAS-transduced cells, as KRAS/V12-transduced cells were classified into a distant branch on a dendrogram described by hierarchical clustering analysis of the Ward's method (Fig. 1B). Proteins that were more than 2-fold higher or lower in oncogenic KRAS-transduced cells than in both mock-and wild-type KRAS-transduced cells are listed ( Table 1). The successful expression of the transduced KRAS gene was confirmed by Western blot analysis (Fig. 1C). Among the molecules listed, a focus was placed on VIM, CTSL1, and CLIC4, because they were repeatedly identified (Table 1). This finding was confirmed by Western blot analysis ( Fig. 2A). As mentioned in the Introduction section, previous studies have suggested that some chloride channels and chloride channel regulators may function as tumor suppressors [5,[15][16][17][18]. Thus, the present study focused on CLIC4, a member of the intracellular chloride channel family [6][7][8], to further investigate its potential involvement in carcinogenesis in the lung.

CLIC4 protein expression in lung cancer cell lines
Among the lung cancer cell lines examined, some cell lines (A549, TKB14, and H2087) had lower CLIC4 protein levels than those in immortalized non-cancerous airway cells (Fig. 3A).

CLIC4 protein expression in primary lung cancers
The CLIC4 protein was expressed in the cytoplasm and plasma membrane of airway epithelial cells in the bronchioles and alveoli (Fig. 3B). The level of CLIC4 protein expression was varied in neoplastic cells even in individual tumors. Some expressed CLIC4 at an equivalent level or more strongly than that of the bronchioles and alveoli (Fig. 3B), while expressed CLIC4 faintly or at an undetectable level (Fig. 3B). Expression levels were classified into negative (level 0), faint (level 1), modest (level 2), and strong (level 3). The faint level was defined as weaker than the level in the bronchioles and alveoli, but not negative. The modest level was defined as a level equivalent to that in the bronchioles and alveoli. The strong level was defined as an unequivocally stronger level than that in the bronchioles and alveoli. The immunohistochemical expression score was determined as an average level in the maximal tumor section (if 30%, 10%, 50%, and 10% of neoplastic cells in the maximal section of tumor were negative, faint, modest, and strong levels, respectively, the average level was calculated as ''1.4 = 0.360+0.161+0.562+0.161''). The average levels in all the tumors examined are shown (Fig. 3C). Neoplastic cells generally appeared to express CLIC4 at a lower level than that in bronchial and alveolar epithelial cells (Fig. 3C). No significant difference in CLIC4 levels was observed among histological subtypes. However, CLIC4 levels in ADCs were lower in the mucinous subtype and also decreased with the progression of the histological grade (Table 2). CLIC4 levels were higher in EGFRmutated ADCs than in KRAS-mutated ADCs ( Table 2).

Effect of CLIC4 on cell growth activity
The knockdown of CLIC4 in an immortalized non-cancerous cell line (NHBE-T) elevated growth activity because it increased clonogenicity and shortened the doubling time (Fig. 4).
The forced expression of CLIC4 reduced clonogenicity, prolonged the doubling time, and attenuated anchorage growth activity in some NSCLC cell lines in which the expression of CILC4 was weaker (A549 and H2087). Representative results from A549 cells are shown (Fig. 5A-E). The exogenous expression of CLIC4 was verified by Western blotting (Fig. 5F).
CLIC4 has been suggested to prolong the activation of Smad2/ 3 to induce apoptosis by inhibiting its dephosphorylation through a selective phosphatase (PPM1a) [19,20]. Thus, the potential involvement of this mechanism in the CLIC4-induced suppression of growth was investigated. However, the cleavage of caspase-3 and level of phosphorylated Smad2/3 was not modulated by the  forced expression of CLIC4 (Fig. 5G), which indicates that some other mechanisms may be controlling cell growth through CLIC4.

Association between CLIC4 expression and disease-freesurvival
A total of 144 patients were available for disease-free survival analysis. The averaged level of ,9.5% and $9.5% were classified as low and high, based on a receiver operating characteristic curve (area under the curve 0.615, 95% confidential interval 0.460-0.769). Seventy-five patients (52.1%) were low expressers, and 69 (47.9%) were high expressers. The incidence of recurrence was slightly higher in low expresser than in the high expressers, while no significant differences were observed in disease-free survival was not significant between the high and low expressers (five-year disease-free survival rate, 88.71% in the high expressers versus 81.70% in the low expressers, P = 0.1775) (Fig. 6).
CILC4 protein levels were reduced in some lung cancer cell lines (Fig. 3A). The knockdown of CLIC4 in an immortalized noncancerous cell line elevated growth activity (Fig. 4), and the restoration of CLIC4 in lung cancer cell lines in which CLIC4 expression was reduced attenuated their growth activity (Fig. 5), suggesting that CILC4 could be a tumor suppressor. Consistent with these results, the immunohistochemical expression of the CLIC4 protein was also weaker in primary lung cancer cells than in tumorous airway epithelial cells, and was occasionally  undetectable in some tumors (Fig. 3B). CLIC4 levels decreased with the progression of the histological grade among ADCs. The incidence of recurrence was slightly higher in low expressers than in high expressers. However, no significant differences were observed in disease-free survival (Fig. 6). These results indicate that CLIC4 may function as a tumor suppressor in the progression process of carcinogenesis of lung ADC. Moreover, CLIC4 levels were significantly lower in a subtype of mucinous ADC than in others, and were also significantly lower in KRAS-mutated ADCs than in EGFR-mutated ADCs ( Table 2). Mucinous ADC is known to be a subtype that is preferentially affected by KRAS mutations [32][33][34]. These results appear to be reasonable and suggest that an alteration in CLIC4 could be involved in restrictedly development of a specific fraction of ADCs. The different subtypes of ADCs may develop through the accumulation of subtype-specific molecular alterations.
CLIC4 belongs to the family of chloride intracellular channel proteins, which is composed of seven family members, p64, CLIC1 to CLIC5, and parchorin [19,35,36]. CLIC4 is multifunctional and is known to suppress cell growth through the transforming growth factor b (TGFb) signaling pathway and promote cellular differentiation through the protein kinase d (PKCd) signaling pathway [7,37,38]. Moreover, CLIC4 is a direct transcriptional target for p53 and c-Myc, and is essential for the induction of apoptosis [35]. Thus, CLIC4 has been suggested to have a suppressive effect on tumor development. Previous studies demonstrated that CLIC4 protein levels were reduced in neoplastic cells in several types of human malignancies including lung cancer [39]. This finding is compatible with the results of the present study, and supports our hypothesis that CLIC4 may be a tumor suppressor in carcinogenesis in the lung.
In summary, this study revealed the proteome modulated by oncogenic KRAS, and focused on CLIC4 to investigate its potential involvement in carcinogenesis in the lung. These results suggest that CLIC4 could be a tumor suppressor, and its downregulation may promote carcinogenesis in a specific fraction of lung cancers. The potential benefit of the proteome modulated by oncogenic KRAS to lung cancer research has been demonstrated. Figure 6. The relationship between CLIC4 levels and diseasefree survival in stage I lung adenocarcinomas was analyzed by the Log-rank test (P = 0.1775). Kaplan-Meier survival curves are shown (five-year disease-free survival rates were 81.70% and 88.71% in CLIC4 low-and high-expressers, respectively. doi:10.1371/journal.pone.0087193.g006