Alteration of PAFAH1B1 in Human Lung Cancer and Its Roles in Tumor Progression and Poor Survival

BioFormosa（2010）45(2): 63-69

Fang-Yi Lo1,2, Hsiung-Ting Chen2, Han-Shui Hsu3,4, Tsu-Wei Wang1*, Guey-Jen Lee-Chen1* Yi-Ching Wang2,5* 1Department of Life Science, National Taiwan Normal University Taipei, Taiwan 2Department of Pharmacology, College of Medicine, National Cheng Kung University Tainan, Taiwan 3Institute of Emergency and Critical Care Medicine, National Yang Ming University Taipei, Taiwan 4Division of Thoracic Surgery, Taipei Veterans General Hospital Taipei, Taiwan 5Institute of Basic Medical Science, College of Medicine, National Cheng Kung University Tainan, Taiwan.

(Received: 7 April 2011, accepted: 6 May 2011)

ABSTRACT

Rationale and Objectives: Genomic DNA copy number variation is a hallmark of cancer. In our previous array-comparative genomic hybridization (array-CGH) study, we showed that PAFAH1B1 was amplified in lung cancer patients, suggesting that PAFAH1B1 is a potential oncogene in lung cancer. Methods: In this study, we have determined the mRNA and protein expression level of PAFAH1B1 in 91 lung cancer patients using quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC). Main Results: The PAFAH1B1 mRNA and protein overexpression frequency were 61.5% (56/91) and 56% (51/91) in lung cancer patients. The results indicated that mRNA and protein overexpression level of PAFAH1B1 was significantly associated with late stage (mRNA: P=0.001, protein: P=0.05) and poor survival in lung adenocarcinoma (P=0.049). Conclusions: The results revealed the roles of overexpressed PAFAH1B1 in tumor progression and poor survival in lung cancer.

Key words: PAFAH1B1, lung cancer, progression, oncogene

Introduction that PA FA H1 B 1 is a potential oncogene in lung cancer. Genomic DNA copy number variation is a The PA FA H1 B 1 (also named LIS1) gene was hallmark of cancer and can lead to alteration in first cloned in patients with Miller-Dieker expression and functions of genes residing within lissencephaly syndrome (MDLS), a disorder of the affected chromosomal region (Davies et al., neural development characterized by agyria and 2005; Schwab, 1999). In our previous study, we facial abnormalities, and classic lissencephaly (type generated a non-gapped array-comparative genomic I, LIS1), a disorder of isolated agyria (Ledbetter et hybridization (array-CGH) microarray representing al., 1992). PA FA H1 B 1 is composed 11 exons (Fig. 18 human chromosome imbalance hotspot regions. 1A) and encodes the 45K non-catalytic subunit of Our study involved a large cohort of cancer patients the brain isoform of platelet activating factor (PAF) including 40 Asian and 20 Caucasian lung cancer acetylhydrolase, a PAF-inactivating enzyme patients, and revealed one of the novel lung (Hattori et al., 1994; Lo Nigro et al., 1997). cancer-related genes, PA FA H 1 B 1 , which was PAFAH1B1 is a predicted microtubule-associated amplified in both Asian and Caucasian lung cancer protein with N-terminal coiled-coil domain, one patients (GEO: GSE21276). The results suggested Lissencephaly type-1-like homology (LisH) motif

*Corresponding author: Yi-Ching Wang; FAX: 886-6-2749296; E-mail: [email protected] Guey-Jen Lee-Chen; FAX: 886-2-29312904; E-mail: [email protected] Tsu-Wei Wang; FAX: 886-2-29312904; E-mail: [email protected] Fang-Yi Lo, Hsiung-Ting Chen, Han-Shui Hsu, Tsu-Wei Wang, Guey-Jen Lee-Chen, Yi-Ching Wang

Figure 1. Schematic representation of PAFAH1B1 gene structure and predicted protein domain (not drawn to scale). (A) Exons are depicted as boxes and numbered with Roman numerals. Filled-in regions indicate translated portions of the gene. This schematic representation was modified from Lo Nigro et al (1997). (B) The SMART diagram of the predicted protein domains. PAFAH1B1 protein was predicted to contain one LisH domain, one coiled-coil domain, and eight repeat WD40 domains. domain and eight WD40-repeat domains (Fig. 1B). TRIzol reagent (Invitrogen, Carlsbad, CA). cDNA It was reported that PAFAH1B1 may enhance was generated using SuperScript reverse neuronal migration by acting as a transcriptase (Invitrogen). microtubule-associated protein (Cardoso et al., 2000). PAFAH1B1 was suggested to play some Quantitative reverse transcription polymerase roles in invasion of human neuroblastoma (Messi et chain reaction (qRT-PCR) al., 2008; Suzuki et al., 2007). qRT-PCR was used to measure the mRNA Here, we sought to extend our previous finding expression of four candidate genes in 91 NSCLC that PAFAH1B1 was amplified in lung cancer tumor and the corresponding normal samples. patients by evaluating the mRNA and protein qRT-PCR was conducted using the ABI 7900 expression level of PAFAH1B1 in 91 lung cancer Sequence Detection System (PE Applied patients. In addition, the roles of PAFAH1B1 in Biosystems). Total RNA (4 μg) from tumors and the tumor progression and poor survival in lung cancer corresponding normal tissues were reverse were examined. transcribed into cDNA and amplified using the SYBR Green PCR Master Mix (Roche) and specific Materials and methods primers for each candidate genes. The primer sequences for PA FA H1 B 1 are: forward ATG GGT Clinical samples preparation and DNA/RNA CGT AGC AAC AAA GG and reverse TCT TCA extraction TGC ATC GCT TGT TC. The annealing Tissues were collected after obtaining temperature was 58oC. The relative amount of appropriate institutional review board permission PA FA H1 B 1 amplification products was calculated and informed consent from the recruited patients. using the standard curve-based method and then Surgically resected tumor tissue and corresponding normalized to the relative amount of ß-actin as normal tissue were collected from 91 patients detected in the same run. The primer sequences for diagnosed with primary non-small cell lung cancer ß-actin are: forward GGC GGC ACC ACC ATG (NSCLC) admitted to Taipei Veterans General TAC CCT and reverse AGG GGC CGG ACT CGT Hospital, Taiwan. Histological classification was CAT ACT. The annealing temperature was 58 oC. determined according to the WHO classification Cutoff value for overexpression was the mean of system and the tumor-node-metastasis system. normal lung tissue expression. Information on the age, sex, tumor type, tumor stage and smoking history of the patients was Immunohistochemistry (IHC) obtained from hospital records. Total RNA from The protein expression level of PAFAH1B1 tumors and normal lung tissues was prepared using was evaluated by IHC in 91 NSCLC samples.

64 PAFAH1B1 Is a Metastasis-related Gene in Lung Cancer

Paraffin blocks of tumors were cut into 5-μm slices and then processed using standard deparaffinization and rehydration techniques. Polyclonal antibody against PAFAH1B1 (1:800; Novus, Littleton, CO) was used as the primary antibody to detect the protein expression. The evaluation of the IHC was conducted blindly without knowledge of the clinical and pathologic characteristics of the cases. The samples were graded high expression when >50% tumor cells were stained positive using adequate staining in surrounding normal stromal and epithelial cells. Figure 2. The mRNA expression level of PAFAH1B1 in

lung cancer. qRT-PCR was performed in normal and Statistical analysis tumor tissues from 91 lung cancer patients. The Y-axis is A two-tailed t test was used to determine the the mean mRNA expression ratio between candidate statistical significance of difference in mRNA gene and the internal control gene in all samples expression level of PA FA H1 B 1 in clinical samples. analyzed. The P values of comparison between normal Chi-square test was conducted to examine the (N) and tumor (T) samples for PAFAH1B1 mRNA association between overexpression of candidate expression is <0.0001 labeled with asterisk (***). genes and clinico-pathological parameters, Table 1. Association between mRNA expression level of including sex, age, tumor type, tumor stage, and PAFAH1B1 and lung cancer clinicopathological smoking. Survival curves were calculated according parameter. * to the Kaplan-Meier method, and comparison was performed using the log-rank test. A P < 0.05 was PAFAH1B1 mRNA considered to be statistically significant. Characteristics Total † Over-expression Normal n n (%) n (%) Results Overall 91 56 (61.5) 35 (38.5)

PAFAH1B1 mRNA is overexpressed in lung cancer, Age ＜65 35 23 (65.7) 12 (34.3) and is significantly associated with late stage lung ≧65 56 33 (58.9) 23 (41.1) cancer patients. Sex Male 58 35 (60.3) 23 (39.7) To determine the mRNA expression level of Female 33 21 (63.6) 12 (36.4) the PAFAH1B1 gene in lung cancer, qRT-PCR of PA FA H1 B 1 was conducted with cDNA from tumors Smoker Yes 33 21 (63.3) 12 (36.4) No 29 16 (55.2) 13 (44.8) and corresponding normal tissues of 91 lung cancer Tumor type patients. The mean mRNA expression level of ADC 58 32 (55.2) 26 (44.8) PAFAH1B1 analyzed in the tumor tissues was SCC 31 22 (71) 9 (29) significantly higher than in the corresponding Tumor stage I + II 54 29 (53.7) 25 (46.3) normal tissues in lung cancer patients (P < 0.001) III + IV 35 26 (74.3)0.05 9 (25.7) (Fig. 2). The PA FA H1 B 1 mRNA overexpression * The P values with significance are shown as was defined using the mean mRNA expression of superscripts. normal lung tissue as a cutoff value. The † Total number of samples in some categories is less PAFAH1B1 mRNA overexpression frequency was than the overall number analyzed because clinical 61.5% (Table 1). To determine the statistical data or molecular data was not available for these significance of difference in mRNA expression samples. level of PA FA H1 B 1 in clinical samples. Chi-square test was conducted to examine the association significant correlation of PA FA H1 B 1 mRNA between mRNA overexpression of PAFA H1 B 1 and expressions was detected in these clinico- clinico-pathological parameters of 91 lung cancer pathological parameter, except that PAFAH1B1 patients, including age, sex, smoking, tumor type, mRNA overexpression was significantly associated and tumor stage. The results showed that no with late stage (P=0.05), suggesting that the

65 Fang-Yi Lo, Hsiung-Ting Chen, Han-Shui Hsu, Tsu-Wei Wang, Guey-Jen Lee-Chen, Yi-Ching Wang

alteration of PA FA H1 B 1 gene may be involved in tumor progression (Table 1).

PAFAH1B1 protein is overexpression in lung cancer, and is significantly associated with poor survival in lung adenocarcinoma. To determine PAFAH1B1 protein expression level in lung cancer patients, IHC was performed on paraffin blocks from 91 Asian patients (Fig. 3A). The frequency of PAFAH1B1 protein overexpression was 56% in lung cancer. The association between PAFAH1B1 protein overexpression and clinico-pathological parameters of 91 lung cancer patients was examined using the chi-square test, including age, sex, smoking, tumor type, and tumor stage. The results showed that no significant correlation of PAFAH1B1 protein expressions was detected in these clinico- Figure 3. (A) Representative IHC images of PAFAH1B1 pathological parameter, except that PAFAH1B1 protein expression in paraffin-embedded tissue from two protein overexpression was significantly associated lung cancer patients. A patient with protein with late stage lung cancer (P=0.001). The mRNA overexpression is shown in tumor cells that exhibit expression level of PAFAH1B1 showed significant brown color staining and is indicated by + symbol (left). correlation with PAFAH1B1 protein expression Another patient with normal expression is indicated by – level (P=0.045) (Table 2). Note that, Kaplan-Meier symbol (right). IHC images: 200X magnification. (B) Kaplan-Meier survival curves indicated that survival curves indicated that lung cancer patients overexpression of PAFAH1B1 protein showed poor with overexpression of PAFAH1B1 protein showed overall survival in lung adenocarcinoma patients. P value poor survival in adenocarcinoma (P < 0.049) (Fig. is 0.049 and labeled with asterisk (*). 3B). PAFAH1B1 in other cancers has never been Discussions examined. Here, we have determined the mRNA and In our previous array-CGH study, we found protein overexpression frequency of PAFAH1B1 to several candidate genes which were located in the be 61.5% and 56% in 91 Taiwanese lung cancer common chromosome alteration regions in both patients, respectively. The clinical correlation Asian and Caucasian, such as PA FA H1 B 1 gene on results indicated that mRNA and protein 17p13.3. In array-CGH data, PA FA H1 B 1 showed overexpression level of PAFAH1B1 was gain of copy number in 60% of Asian and 70% of significantly associated with late stage Caucasian patients. In addition, chromosome region (P=0.001~0.05) and poor survival in lung 17p13.3 harboring PA FA H1 B 1 gene demonstrated adenocarcinoma (P=0.049). Chi-square test was association with advanced tumor stage in conducted to examine the correlation between the array-CGH data (GEO: GSE21276). In previous mRNA and protein expression level of PAFAH1B1. reports, PAFAH1B1 was considered to function in In our results, the concordant group, such as microtubule movement by colocalization with positive of expression in both protein and mRNA NudE nuclear distribution gene E homolog-like 1 and negative of expression in both protein and (NDEL1) and cytoplasmic dynein heavy chain mRNA, was detected in 61.5% [(36+20)/91] of lung (CDHC) in neuron cell (Soukoulis et al., 2005; cancer patients. The mRNA expression level of Suzuki et al., 2007; Wynshaw-Boris, 2007). In PAFAH1B1 showed significant correlation with addition, decreased PAFAH1B1 protein expression PAFAH1B1 protein expression level (P=0.045). in human neuroblastoma cell lines led to reduced However, the discordant group, such as positive of cell migration and invasiveness (Aumais et al., protein expression without positive of mRNA 2001; Messi et al., 2008). However, the role of expression and negative of protein expression

66 PAFAH1B1 Is a Metastasis-related Gene in Lung Cancer

Table 2. Association between protein expression level of References PAFAH1B1 and lung cancer clinicopathological parameter.* Aumais, J.P., Tunstead, J.R., McNeil, R.S., Schaar, B.T., McConnell, S.K., Lin, S.H., Clark, G.D., PAFAH1B1 protein Characteristics Total † Over-expression Normal and Yu-Lee, L.Y. (2001). NudC associates with n n (%) n (%) Lis1 and the dynein motor at the leading pole Overall 91 51 (56) 40(44) of neurons. J Neurosci 21, RC187. Cardoso, C., Leventer, R.J., Matsumoto, N., Kuc, Age ＜65 35 24 (68.6) 11 (31.4) ≧65 56 27 (48.2) 29 (51.8) J.A., Ramocki, M.B., Mewborn, S.K., Dudlicek, L.L., May, L.F., Mills, P.L., Das, S., Sex Male 58 30 (51.7) 28 (48.3) et al. (2000). The location and type of mutation Female 33 21 (63.6) 12(36.4) predict malformation severity in isolated Smoker Yes 33 16 (48.5) 17 (51.5) lissencephaly caused by abnormalities within No 29 17 (58.6) 12(41.4) the LIS1 gene. Hum Mol Genet 9, 3019-3028. Tumor type ADC 58 36 (62.1) 22(37.9) Davies, J.J., Wilson, I.M., and Lam, W.L. (2005). SCC 31 14 (45.2) 17(54.8) Array CGH technologies and their applications Tumor stage to cancer genomes. Chromosome Res 13, I + II 54 23 (42.6) 31(57.4) III + IV 35 27 (77.1)0.001 8 (22.9) 237-248. mRNA expression Hattori, M., Adachi, H., Tsujimoto, M., Arai, H., overexpression 56 36 (64.3)0.045 20 (35.7) and Inoue, K. (1994). Miller-Dieker normal expression 35 15 (42.9) 20(57.1) lissencephaly gene encodes a subunit of brain * The P values with significance are shown as superscripts. platelet-activating factor acetylhydrolase. † Total number of samples in some categories is less Nature 370, 216-218. than the overall number analyzed because clinical Ledbetter, S.A., Kuwano, A., Dobyns, W.B., and data or molecular data was not available for these Ledbetter, D.H. (1992). Microdeletions of samples. chromosome 17p13 as a cause of isolated lissencephaly. Am J Hum Genet 50, 182-189. without negative of mRNA expression, was Lo Nigro, C., Chong, C.S., Smith, A.C., Dobyns, detected in 38.5% [(15+20)/91] of lung cancer W.B., Carrozzo, R., and Ledbetter, D.H. patients. The discordant group may imply that other (1997). Point mutations and an intragenic mechanisms such as mutations of the gene, deletion in LIS1, the lissencephaly causative dysfunction of the regulatory proteins, or the gene in isolated lissencephaly sequence and unknown degeneration system of PAFAH1B1 could Miller-Dieker syndrome. Hum Mol Genet 6, be involved in the discordant expression of mRNA 157-164. and protein. Note that Tian et al. (2011) found that Messi, E., Florian, M.C., Caccia, C., Zanisi, M., and microRNA 302/367 regulated the mRNA and Maggi, R. (2008). Retinoic acid reduces protein expression of PAFAH1B1 through the human neuroblastoma cell migration and 3’UTR regions. Our previous array-CGH data invasiveness: effects on DCX, LIS1, suggested that PAFAH1B1 overexpression may via neurofilaments-68 and vimentin expression. gene amplification in lung cancer. In addition, BMC cancer 8, 30. PAFAH1B1 may be down-regulated by microRNA Schwab, M. (1999). Oncogene amplification in 302/367 (Tian et al., 2011). In conclusion, solid tumors. Semin Cancer Biol. 9, 319-325. overexpression of PAFAH1B1 correlates with late Soukoulis, V., Reddy, S., Pooley, R.D., Feng, Y., stage disease and poor survival, suggesting that Walsh, C.A., and Bader, D.M. (2005). PAFAH1B1 may serve as a prognostic biomarker of Cytoplasmic LEK1 is a regulator of non-small cell lung cancer metastasis. microtubule function through its interaction with the LIS1 pathway. Proc Natl Acad Sci U Acknowledgments S A. 102, 8549-8554. Suzuki, S.O., McKenney, R.J., Mawatari, S.Y., We thank Taipei Veterans General Hospital for Mizuguchi, M., Mikami, A., Iwaki, T., providing clinical samples. Goldman, J.E., Canoll, P., and Vallee, R.B.

67 Fang-Yi Lo, Hsiung-Ting Chen, Han-Shui Hsu, Tsu-Wei Wang, Guey-Jen Lee-Chen, Yi-Ching Wang

(2007). Expression patterns of LIS1, dynein Regulation of lung endoderm progenitor cell and their interaction partners dynactin, NudE, behavior by miR302/367. Development 138, NudEL and NudC in human gliomas suggest 1235-1245. roles in invasion and proliferation. Acta Wynshaw-Boris, A. (2007). Lissencephaly and LIS1: Neuropathol. 113, 591-599. insights into the molecular mechanisms of Tian, Y., Zhang, Y., Hurd, L., Hannenhalli, S., Liu, neuronal migration and development. Clin F., Lu, M.M., and Morrisey, E.E. (2011). Genet 72, 296-304.

68 生物學報（2010）45(2)：63-69

肺癌病人 PAFAH1B1 基因變異參與癌症惡化

羅芳宜 1,2 陳湘婷 2 許瀚水 3,4 王慈蔚 1* 李桂楨 1* 王憶卿 2,5* 1 國立臺灣師範大學生命科學系 2 國立成功大學醫學院藥理所 3 國立陽明大學急重症醫學研究所 4 台北榮民總醫院胸腔外科 5 國立成功大學醫學院基礎醫學研究所

（收稿日期：2011.4.7，接受日期：2011.5.6）

摘要

目的：基因體結構變異為腫瘤形成的表徵之一。於本實驗室先前的研究，我們針對肺癌族群進行微矩陣比較基因體的雜交分析 (array-CGH)，其結果顯示 PAFAH1B1 基因具有基因體結構擴增的現象。因此，我們推測 PA FAH 1B1 基因為肺癌有潛力的致癌基因。材料與方法：本研究利用即時定量反轉錄 PCR (q-RT PCR) 及免疫組織染色法 (IHC) 對 91 位肺癌族群檢測 PA FAH1 B1 基因其 mRNA 及蛋白層次變異情形。結果：PA FA H 1 B 1 基因於 mRNA 層次的過度表現頻率為 61.5%，於蛋白層次過度表現頻率為 56%，且此基因於 mRNA 及蛋白層次的過度表現皆與病人的晚期具有相關性 (mRNA：P=0.001，蛋白層：P=0.05)，且屬於腺細胞癌的病人於蛋白層次的過度表現具有較差的預後 (P=0.049)。結論：其結果顯示 PA FAH1 B1 基因於肺癌族群具有過度表達的變異，且可能參與肺癌細胞轉移過程。

關鍵詞：PAFAH1B1、肺癌、癌症惡化、致癌基因

＊通信作者：王憶卿（Yi-Ching Wang）；FAX：886-6-2749296；E-mail：[email protected] 李桂楨（Guey-Jen Lee-Chen）；FAX：886-2-29312904；E-mail：[email protected] 王慈蔚（Tsu-Wei Wang）；FAX：886-2-29312904；E-mail：[email protected]