Tumor Biol. DOI 10.1007/s13277-013-0807-y

RESEARCH ARTICLE

Prognostic value of HMGB3 expression in patients with non-small cell lung cancer Ning Song & Bao Liu & Jian-Ling Wu & Rui-Fang Zhang & Lin Duan & Wen-Shu He & Cong-Min Zhang

Received: 28 March 2013 / Accepted: 12 April 2013 # International Society of Oncology and BioMarkers (ISOBM) 2013

Abstract HMGB3 overexpression has been reported in a variety of human cancers. However, the role of HMGB3 in human non-small cell lung cancer (NSCLC) remains unclear. In this study, the HMGB3 expression was examined at mRNA and protein levels by quantitative real-time reverse transcriptase–polymerase chain reaction (qRT-PCR), Western blotting, and immunohistochemistry in NSCLC tissues and adjacent non-cancerous tissues. Statistical analyses were applied to test the associations between HMGB3 expression, clinicopathologic factors, and prognosis. Western blotting and qRT-PCR showed that the expression levels of HMGB3 mRNA and protein were both significantly higher in NSCLC tissues than those in non-cancerous tissues. Immunohistochemistry analysis showed that HMGB3 expression was significantly correlated with tumor grade, tumor size, clinical stage, and lymph node metastases. The results of Kaplan– Meier analysis indicated that a high expression level of HMGB3 resulted in a significantly poor prognosis of NSCLC patients. Importantly, multivariate analysis showed that high HMGB3 expression was an independent prognostic factor for NSCLC patients. In sum, our data suggest that HMGB3 plays an important role in NSCLC progression, and that overexpression of HMGB3 in tumor tissues could be used as a potential prognostic marker for patients with NSCLC.

Ning Song and Bao Liu contributed equally to this paper. N. Song (*) : J.-L. Wu : R.-F. Zhang : L. Duan : W.-S. He : C.-M. Zhang Department of Respiratory Medicine, The Second Hospital of Hebei Medical University, No. 215, Heping Western Road, Shijiazhuang 050000 Hebei Province, China e-mail: [email protected] B. Liu Department of Respiratory Medicine, Henan Provincial People’s Hospital, Zhengzhou 450000, China

Keywords HMGB3 . NSCLC . Biomarker . Prognosis

Introduction Lung cancer is a leading cause of cancer death worldwide, and non-small cell lung cancer (NSCLC) accounts for more than 80 % of all lung cancer cases. Despite some advances in early detection and recent improvements in treatment, prognosis for patients with NSCLC remains poor [1]. The current challenge is to identify new therapeutic targets and strategies and to incorporate them into existing treatment regimens with the goal of improving therapeutic gain. Identifying reliable markers predictive of clinical outcome is also desirable to establish therapeutic strategies and select suitable treatment options for individual NSCLC patients. HMGB3 is an X-linked member of the high-mobility group (HMG) superfamily of HMG proteins and is classified into the HMG-Box subfamily containing HMGB1 and HMGB2 [2]. The HMG-box subfamily also plays an important role in DNA replication, transcription, recombination and repair, etc. [3, 4]. In adult vertebrates, HMGB3 mRNA was reported to be absent in most adult tissues [2, 5]. Recently, Nemeth et al. [6] reported that HMGB3 regulates the balance between hematopoietic stem cell self-renewal and differentiation. Also, it was known that HMGB3 is indispensible in promoting retention of self-renewing leukemia stem cells (LSCs) at the apex of cellular hierarchies in acute myeloid leukemia (AML). Petit et al. [7] detected that HMGB3 and NPU98 fusion protein forms a new oncogenic gene in leukemia. HMGB3 also participates in recurrence of acute lymphoid leukemia and it shows high expression in the progression phase of breast cancer [8]. In gastric adenocarcinoma, HMGB3 overexpression is associated with poor prognosis [9]. To the best of our knowledge, little has been uncovered regarding the involvement of HMGB3 genes in NSCLC. In this study, we investigated HMGB3 expression in

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NSCLC tissues and its correlation with clinicopathological features, including the survival of patients with NSCLC.

Materials and methods Patients and tissue samples The study was approved by the Research Ethics Committee of The Second Hospital of Hebei Medical University, Hebei, China. Informed consent was obtained from patients prior to surgery. One hundred fifty-five primary NSCLCs and corresponding normal tissues were collected by The Second Hospital of Hebei Medical University between 2005 and 2007. Patients did not receive chemotherapy or radiotherapy prior to surgery. Patient characteristics are shown in Table 1. Disease histology was determined in accordance to the criteria of the World Health Organization. Pathologic staging was performed in accordance to the current International Union

Table 1 Association between HMGB3 expression and various clinicopathological factors of NSCLC patients Variables

Histology Squamous cell carcinoma Adenocarcinoma Age <60 ≥60 Gender Male Female Smoking history No Yes Grade (G) G1 G2+G3 Tumor size (T) T1+T2 T3+T4 Clinical stage I–II III Lymph node metastases(N) N0 N1+N2

No. (n=155)

HMGB3 protein expression

p value

Low (n=72)

High (n=83)

76 79

34 38

42 41

0.675

52 103

26 46

26 57

0.529

94 61

43 29

51 32

0.827

58 97

27 45

31 52

0.985

94

50

44

0.037

61

22

39

60 95

36 36

24 59

0.007

62 93

41 31

21 62

<0.001

103 52

55 17

48 35

0.015

Against Cancer tumor–lymph node metastasis classification. Specimens were fixed in 10 % formalin and embedded in paraffin wax. For quantitative real-time reverse transcriptase– polymerase chain reaction (qRT-PCR) and Western blot analyses, 20 self-pairs of NSCLC specimens and corresponding normal tissues were immediately frozen in liquid nitrogen and stored at −70 °C until use. Quantitative real-time reverse transcriptase–polymerase chain reaction Total RNAs were purified from tissues using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA), and 2 μg RNA of each sample was reverse transcribed using Superscript RT kit (Invitrogen Life Technologies, Carlsbad, CA, USA). QRTPCR primers were designed with the assistance of the primer premier 5.0 software (PREMIER Biosoft International, Palo Alto CA, USA). Sequences of the primers are as follows: HMGB3, 5′-ATTCGGAATTCCGTATCTGGCCTTTTGAC3′ (forward), 5′-CGGTTACTCGGCTTACGCTTGGACTG-3′ (reverse), GAPDH, 5′-GACTCATGACCACAGTCCATGC-3′ (forward), 5′-AGAGGCAGGGATGATGTTCTG-3′ (reverse). We used the SYBR Green kit (Invitrogen Life Technologies, Carlsbad, CA, USA) to execute the amplification of the cDNA. The real-time PCR cycling parameters were performed as follows: denaturation at 95 °C for 15 s, annealing at 55 °C for 30 s, and extension at 72 °C for 30 s. The expression data were normalized to the geometric mean of housekeeping gene GAPDH to control the difference in expression levels and analyzed using the 2-Delta Delta C (T) method described by the previous report. Western blotting Tissues were lysed in lysis buffer containing protease inhibitor cocktail. Protein concentration was determined using a BioRad protein assay system (Bio-Rad, Hercules, CA, USA). Equivalent amounts of proteins were separated by SDSPAGE, and then transferred to polyvinylidene difluoride membranes (Bio-Rad). After being blocked in Tris-buffered saline (TBS) containing 5 % non-fatmilk, the membranes were incubated with specific primary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4 °C for 12 h and then with horseradish peroxidase-conjugated anti-rabbit antibody (Zhongshan, Beijing, China) for 2 h at room temperature. Signals were detected on X-ray film using the ECL detection system (Pierce, Rockford, IL, USA). The relative protein levels were calculated based on GAPDH as the loading control. Immunohistochemistry (IHC) All 155 tissue specimens were subjected to immunohistochemical analysis using the avidin–biotin–peroxidase

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method. Sections were deparaffinized in xylene and dehydrated using a graded alcohol series before endogenous peroxidase activity was blocked with 0.5 % H2O2 in methanol for 10 min. Nonspecific binding was blocked by incubating sections with 10 % normal goat serum in phosphatebuffered saline (PBS) for 1 h at room temperature. Without washing, sections were incubated with anti-HMGB3 (1:100; Abnova, Taipei, Taiwan) in PBS at 4 °C overnight in a moist box. Biotinylated goat anti-rabbit immunoglobulin G (1:400; Sigma, St. Louis, MO, USA) was incubated with the sections for 1 h at room temperature and detected with a streptavidin–peroxidase complex. The brown color indicative of peroxidase activity was developed by incubating sections with 0.1 % 3,3-diaminobenzidine (Sigma) in PBS with 0.05 % H2O2 for 5 min at room temperature. The tissue specimens were viewed separately by two pathologists under double-blinded conditions, where they had no prior knowledge of the clinical or clinicopathological status of the specimens. Expression of HMGB3 in the NSCLC specimens was evaluated by scanning the entire tissue specimen under low magnification (×40), and then confirmed under high magnification (×200 and×400). An immunoreactivity score system was applied as described elsewhere [10]. The percent of positive cells was scored as “0” (<5 %, negative), “1” (5–25 %, sporadic), “2” (25–50 %, focal), and “3” (>50 %, diffuse), respectively. The staining intensity was scored as “0” (no staining), “1” (weakly stained), “2” (moderately stained), and “3” (strongly stained), respectively. Both the percent of positive cells and cell staining intensity were decided in a double-blinded manner. The final HMGB3 immunostaining score was calculated using the percent of positive cell score×staining intensity score ranging 0–9. High HMGB3 expression level was defined as a total score≥4, and low HMGB3 expression level as a total score<4.

Fig. 2 Protein levels of HMGB3 in 20 paired NSCLC tissues by Western blotting. GAPDH was used as a loading control. T NSCLC tissues, N corresponding non-cancerous tissues

Statistical analysis The χ2 test was used to analyze the relationship between HMGB3 expression and clinicopathological characteristics. Survival curves were plotted using the Kaplan–Meier product-limit method, and differences between survival curves were tested using the log-rank test. Cox’s proportional hazards model was used to identify the factors that had a significant influence on survival. Statistical significance was set at p<0.05.

Results Overexpression of HMGB3 in human NSCLC To elucidate the role of HMGB3 in the initiation and progression of NSCLC, 20 pairs of NSCLC tissues and adjacent normal tissues were used for qRT-PCR and Western blotting analysis, respectively. We first analyzed its expression in NSCLC and adjacent non-cancerous tissues at the mRNA level. qRT-PCR analysis showed that HMGB3 expression was significantly upregulated in NSCLC tissues compared to that in adjacent non-cancerous tissues (Fig. 1). To investigate whether HMGB3 was also elevated at the protein level, Western blotting was performed on the same specimens which were used in the detection of HMGB3 mRNA. HMGB3 was similarly overexpressed at the protein level in NSCLC, as shown by Western blot analysis (Fig. 2). Relationship between HMGB3 protein expression and clinicopathological parameters According to the HMGB3 immunoreactive intensity, 72 (46.5 %) patients were classified as low-HMGB3 group

Fig. 1 mRNA levels of HMGB3 in 20 self-pairs of NSCLC specimens and corresponding normal tissues by qRT-PCR. HMGB3 expression in each sample was normalized to GAPDH. T NSCLC tissues, N corresponding non-cancerous tissues

Fig. 3 Immunohistochemistry of HMGB3 in NSCLC tissues. A high expression of HMGB3 in NSCLC tissues. B Low expression of HMGB3 in NSCLC tissues

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significant prognostic factors in the univariate analysis (Table 2). We also performed multivariate survival analysis by Cox proportional hazard model to test the effects of independent factors on survival. As presented in Table 2, multivariate analysis indicated that HMGB3 expression was one of the independent prognostic factors, along with tumor grade, tumor size, clinical stage, and lymph node metastases.

Discussion

Fig. 4 Kaplan–Meier survival curves of the patients with NSCLC according to the HMGB3 immunostaining results

and 83 (53.5 %) patients were classified as high-HMGB3 group (Fig. 3). As summarized in Table 1, HMGB3 expression was significantly correlated with tumor grade (p=0.037), tumor size (p=0.007), clinical stage (p<0.001), and lymph node metastases (p=0.015), but was not associated with histology, gender, age, and smoking history (all p>0.05). Correlation of HMGB3 expression with overall survival The prognostic value of HMGB3 for overall survival in NSCLC patients was evaluated by comparing the patients with high and low HMGB3 expression. According to the Kaplan–Meier survival analysis, patients with high HMGB3 expression had obviously lower overall survival rates than those with low HMGB3 expression (Fig. 4, p<0.001). Univariate and multivariate analyses were conducted using Cox proportional hazards model to examine the impact of HMGB3 expression and other clinicopathological parameters in NSCLC patients. HMGB3 expression, tumor grade, tumor size, clinical stage, and lymph node metastases were

Table 2 Univariate and multivariate analyses showing the overall survival rate for patients with NSCLC

RR relative risk, 95 % CI 95 % confidence interval

Variables

HMGB3 Histology Age Gender Smoking history Tumor grade Tumor size Clinical stage Lymph node metastases

Non-small cell lung cancer (NSCLC) is the most common type of lung cancer being 75–80 % of all cases [11]. The 5-year overall survival rate associated with NSCLC is only ∼15 %. Recently, molecular target therapy for tumor has been introduced into the clinical setting and the advent of targeted therapy for treatment of human cancers has added significantly to our armamentarium as we strive to prolong patient survival while minimizing toxicity [12]. However, the intrinsic or acquired resistance mechanisms limit their clinical efficacy. Thus, a better understanding of the molecular mechanisms involved in NSCLC progression should be helpful to identify novel therapeutic targets, or develop new modalities of NSCLC therapy. HMGB3 is an X-linked member of the high-mobility group (HMG) superfamily of HMG proteins and is classified into the HMG-Box subfamily containing HMGB1 and HMGB2 [2]. The HMG-box subfamily also plays an important role in DNA replication, transcription, recombination and repair, etc. [3, 4]. In adult vertebrates, HMGB3 mRNA was reported to be absent in most adult tissues [2, 5]. Recently, Nemeth et al. [6] reported that HMGB3 regulates the balance between hematopoietic stem cell self-renewal and differentiation. Also, it was known that HMGB3 is indispensible in promoting retention of self-renewing LSCs at the apex of cellular hierarchies in AML. Petit et al. [7] detected HMGB3 and NPU98 fusion protein forms a new

Univariate analysis

Multivariate analysis

RR

95 % CI

p value

RR

95 % CI

p value

1.542 1.321 1.214 1.183 1.192 1.201 1.193 1.298 1.291

0.672–2.194 0.713–1.829 0.683–2.028 0.591–2.128 0.693–1.982 0.682–2.193 0.619–1.893 0.592–2.193 0.672–2.084

<0.001 0.782 0.693 0.792 0.673 0.029 0.019 0.002 0.007

1.482 1.282 1.321 1.282 1.219 1.198 1.293 1.092 1.217

0.782–2.193 0.672–1.892 0.684–2.093 0.717–2.291 0.673–2.092 0.619–2.182 0.583–1.838 0.692–1.829 0.793–2.018

<0.001 0.672 0.628 0.762 0.771 0.031 0.002 0.001 0.009

Tumor Biol.

oncogenic gene in leukemia. HMGB3 also participates in recurrence of acute lymphoid leukemia and it shows high expression in the progression phase of breast cancer [8]. In gastric adenocarcinoma, HMGB3 overexpression is associated with poor prognosis [9]. In the present study, we analyzed HMGB3 protein expression in tumor tissue and assessed its prognostic significance for NSCLC. In the present study, we showed that HMGB3 expression determined by real-time quantitative PCR was significantly higher in NSCLC tissues than that in adjacent non-tumor tissues. These observations support the hypothesis that HMGB3 may function as an oncogene in NSCLC, and also suggest that HMGB3 may play an important role in the tumorigenesis of NSCLC. Furthermore, to investigate whether HMGB3 expression might be associated with the progression of NSCLC, the HMGB3 expression levels and the clinic pathologic characteristics of 155 patients with NSCLC were compared by immunohistochemistry. We found that high HMGB3 expression is significantly correlated with tumor grade, tumor size, clinical stage, and lymph node metastases. Kaplan–Meier survival analysis showed that high HMGB3 expression is negatively correlated with the overall survival of patients with NSCLC. More importantly, further analysis using the Cox regression model confirmed that the HMGB3 expression was an independent factor in predicting overall survival time for NSCLC patients. These findings provide evidence that HMGB3 could be regarded as a biomarker for predicting the outcome of NSCLC patients. In conclusion, our data suggest for the first time that HMGB3 overexpression is associated with advanced tumor progression and poor clinical outcome of NSCLC patients. HMGB3 might be a novel prognostic marker of NSCLC. Conflicts of interest None

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