original article

Wien Klin Wochenschr DOI 10.1007/s00508-016-1108-4

Dysregulated expression of homebox gene HOXA13 is correlated with the poor prognosis in bladder cancer Haiyi Hu · Yuebing Chen · Sheng Cheng · Gonghui Li · Zhigen Zhang

Received: 8 May 2016 / Accepted: 11 October 2016 © Springer-Verlag Wien 2016

Summary Background The homeobox (HOX) genes have been implicated playing important roles in many human cancers. HOXA13 is a member of HOX genes that encode transcription factors regulating embryonic development and cell fate. In the present study, we aimed to investigate the expression and prognostic significance of HOXA13 in bladder cancer. Methods Immunohistochemical staining was initially performed to screen the differentially expressed HOXA13 between bladder cancer tissues and paired adjacent non-cancerous tissues. Subsequent Western blotting analysis validation was conducted using tissue samples from patients with bladder cancer. Results The expression level of HOXA13 was significantly higher in bladder cancer tissues compared to that in adjacent non-cancerous tissues (P < 0.001). The χ2-test showed that expression of HOXA13 was positively correlated with lymphatic metastasis (P = 0.013), bladder tumor TNM stage (P = 0.002) and pathological grade (P < 0.001). Kaplan-Meier survival analysis revealed that patients with bladder cancer with high expression of HOXA13 had shorter overall survival time (P = 0.001) and disease-free survival time (P = 0.001) compared to patients with low expression of HOXA13. Multivariate analysis indicated that HOXA13 was an independent prognostic factor for overall survival for bladder cancer patients. Conclusions The results of our study show that high expression of HOXA13 is associated with the progression of bladder cancer and that HOXA13 might serve as a biomarker for prognosis of bladder cancer. H. Hu · Y. Chen () · S. Cheng · G. Li · Z. Zhang Department of Urology, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, 300016 Hangzhou, Zhejiang, China [email protected]

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Keywords HOXA13 · Bladder cancer · Prognosis · Survival

Introduction Bladder cancer is a common malignant disease of the urinary system, with an incidence of 6.61 in 100,000 admissions in 2012 in China [1]. Traditional surgery and chemotherapy are the mainstay of treatment. Unfortunately, there is no simple and effective way to predict bladder cancer progression and recurrence. The HOX genes are an evolutionarily highly conserved gene family with a common sequence element of 183 bp. The genes encode a series of transcription factors that play key roles in regulating embryonic development as well as in cell-cell and cell-extracellular matrix interactions. Anne et al. concluded that HOX genes were involved in differentiation, cell migration, cell growth, cell cycle, cellular identity, organogenesis and anterior-posterior patterning [2]. Human HOX genes are 39 genes organized into 4 groups termed HOXA, HOXB, HOXC, HOXD and are numbered from 1–13 within the groups. Increasing evidence indicates that expression of certain HOX genes is dysregulated in a variety of tumors including breast, leukemia, ovarian, lung and prostate cancers. Moreover, substantial evidence now indicates that dysregulated expression of HOX gene is closely related to the incidence and progress of genitourinary tumors [3, 4]. HOXA13 is a member of the HOX gene family which is located on chromosome 7 [5]. Studies show that HOXA13 is the most posterior of the HOX members and it is expressed in the genital tubercle during embryogenesis and thus plays an important role in the development of the external genitalia and urogenital tract. Although the abnormal expression of HOX families in bladder cancer tissue confirmed that these proteins might play a role in the development

Dysregulated expression of homebox gene HOXA13 is correlated with the poor prognosis in bladder cancer

original article

Table 1 Differential expression of HOXA13 between bladder cancer and corresponding paracarcinomatous tissues (cases) Tissues

Case number

HOXA13 expression

Positivity rate (%)

+~++

–

Bladder cancer tissues

106

78

28

73.6

Paracarcinomatous tissues

106

17

89

16.1

Table 2 HOXA13 expression status in relation to clinicopathological features in 106 bladder cancer patients Variable Gender

HOXA13 expression Low

High

χ2

P-value

–

–

0.088

0.766

Male

31

42

–

–

Female

13

20

–

–

Age (years)

–

–

0.658

0.417

<60

13

14

–

––

≥60

31

48

–

–

Smoking

–

–

0.018

0.893

No

24

33

–

–

Yes

20

29

–

–

Tumor size (cm)

–

–

0.281

0.596

<3

19

30

–

–

≥3

25

32

–

–

Number of tumors

–

–

1.515

0.218 –

1

28

32

–

>1

16

30

–

–

Lymph node metastasis

–

–

6.234

0.013

No

39

42

–

–

Yes

5

20

–

–

TNM stage

–

–

9.957

0.002

Ta-T1

37

34

–

–

T2-T4

7

28

–

–

Pathological grade

–

–

17.466

0.000

G1

20

7

–

–

G2

19

35

–

–

G3

5

20

–

–

of bladder cancer, the correlation between HOXA13 expression and prognosis in bladder cancer has not been previously reported. Therefore, in the present study, immunohistochemistry was used to examine the protein expression levels of HOXA13 in 106 samples of bladder cancer. Additionally, Western blotting analysis was also used to detect and validate protein expressions of HOXA13 in bladder cancer tissues. Furthermore, the survival analysis was tested by the Kaplan-Meier method. A multivariate survival analysis was performed for all parameters using the Cox regression model. All the abovementioned aimed to investigate the expression of HOXA13 in bladder cancer and its association with clinicopathological characteristics and prognosis.

Material and methods Patients and tissue samples The tumor and paracarcinomatous specimens were obtained from 106 patients (73 male, 33 female) with a definitive diagnosis of bladder cancer and underwent curative surgical resection surgery at the department of urology, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University (Hangzhou, China) between January 2012 and December 2014. Detailed pathological and clinical data, including age and gender, tumor size, number of tumor, smoking history, lymphatic metastasis, pathological grade, tumor-node-metastasis (TNM) stage were obtained from each patient’s medical records. The age of patients ranged from 28–77 years (mean 51 ± 16 years). Tumor differentiation and TNM classification were defined according to the American Joint Committee on Cancer and World Health Organization systems.

Dysregulated expression of homebox gene HOXA13 is correlated with the poor prognosis in bladder cancer

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original article

Fig. 1 Representative immunohistochemical staining of HOXA13 in bladder cancer and paracarcinomatous bladder tissues. HOXA13 expression showed mainly diffuse cytoplasmic staining in tumor and paracarcinomatous bladder tissues (arrow). Sections were collected from 6 different patients. a Negative HOXA13 expression in bladder cancer (400×). b Low HOXA13 expression in bladder cancer (400×). c High HOXA13 expression in bladder cancer (200×)

Immunohistochemistry and evaluation of immunostaining

Fig. 2 Differential expression of HOXA13 between bladder cancer and corresponding paracarcinomatous tissues. a Expression of HOXA13 in bladder tissues was detected by Western blot assay. b Densitometric analysis from the immunoblots given above is shown as a bar chart. 1, 2: paracarcinomatous tissues; 3, 4: bladder cancer tissues. **P < 0.01 compared with 1, ## P < 0.01 compared with 2

None of the patients had received direct or specific treatment before surgery. The specimens were fixed in formalin, embedded in paraffin for pathological analysis and confirmation of the diagnosis. Complete clinical follow-up data were obtained from the bladder cancer database of the Sir Run Run Shaw Hospital. This study was performed with approval of the human research ethics committee of School of Medicine, Zhejiang University and informed consent was obtained from each patient.

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Immunohistochemistry for HOXA13 was performed on each tumor specimen. The tumor samples (4 μm thick) were cut onto silanized glass slides consecutively. A two-step staining method was used to detect the protein expression. Briefly, after deparaffinization and dehydration, microwave antigen retrieval was performed for 5 min prior to peroxidase quenching with 0.3% hydrogen peroxide in methanol for 15 min at room temperature to block endogenous peroxidase activity and then washed in phosphate buffered saline (PBS 3 × 3 min). Sections were incubated at 4°C with a HOXA13 monoclonal antibody (1:400, Beijing Biosynthesis Biotechnology, Beijing, China) overnight, then incubated with Universal IgG antibody-HRP polymer (1:200, Santa Cruz Biotechnology, Santa Cruz, CA) for 15 min at 37°C followed by 3 washes (PBS 3 × 3 min). Finally, each section was treated with 50 μl diaminobenzidine (DAB) working solution at room temperature for 3–10 min, and then washed in PBS. All sections were counterstained with hematoxylin. For the negative control the primary antibody was replaced with PBS. The tumor expression of HOXA13 was semi-quantitatively assessed. The immunoreactivity of the protein was determined according to the intensity of immunostaining and the percentage of positive cells. The staining intensity was graded as follows: 0 none, 1 weak, 2 moderate and 3 strong. The proportion of positive cells was categorized as follows: 0: <1%, 1: 1–30%, 2: 30–70% and 3: >70%. The two scores were multiplied and the final immunostaining score was determined: scores 0, 1, 2 and 3 as low expression and scores 4, 6 and 9 as high expression. The immunohistochemical results were evaluated by two pathologists who were blinded to the clinical data and discrepancies were resolved by consensus.

Dysregulated expression of homebox gene HOXA13 is correlated with the poor prognosis in bladder cancer

original article

Fig. 3 Kaplan-Meier analysis of overall survival (OS) and disease-free survival (DFS) of patients with bladder cancer. Kaplan-Meier analysis of OS and DFS of patients with bladder cancer (n = 106) was based on HOXA13 expression as positive (n = 78) and negative (n = 28). a OS curve of patients with bladder cancer based on HOXA13 expression. b DFS curve of patients with bladder cancer based on HOXA13 expression. The patients with positive HOXA13 expression showed significantly poorer DFS and OS rates than those with negative HOXA13 expression

Western blotting analysis

Statistical analysis

Frozen tumor tissues and corresponding non-cancerous tissues were homogenized in radio-immunoprecipitation assay (RIPA) buffer with 1 mM Phenylmethanesulfonyl fluoride (PMSF) at 4 °C for 30 min with ice vortex every 10 min, the homogenate was centrifuged at 12,000 g for 15 min and the protein concentrations of the supernatant were determined by BCA protein assay kit (Pierce, Waltham, MA). The supernatants obtained were diluted to 4 mg protein/ml and kept frozen at –80°C until use. A total of 50 µg of denatured protein was separated by 10% SDS-PAGE and transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA) in an icewater environment. The membranes were blocked with 5% non-fat milk solution for 2 h at room temperature and then incubated with primary antibodies of rabbit monoclonal anti-HOXA13 (1:500, Santa Cruz Biotechnology, Santa Cruz, CA) and mouse monoclonal anti-β-actin (1:400, Beijing Zhongshan Jinqiao, Beijing, China) at 4 °C overnight. After washing with tris-buffered saline Tween-20 (TBST), membranes were incubated with anti-rabbit or anti-mouse secondary antibodies conjugated with horseradish peroxidase (HRP) (1:20,000 Beijing Zhongshan Jingqiao) for 1 h at 37°C. The membrane was then developed using the enhanced chemiluminescence systems. Betaactin was used to confirm that an equal amount of protein was loaded in each lane. Band intensities were quantified with a computerized densitometer (Image J Launcher, Broken Symmetry Software, Bristol, UK).

The χ2-test was used to analyze the immunohistochemistry results. The Kaplan-Meier method and the log-rank test were applied to survival analysis. The Cox regression model was employed to determine the independent prognostic value and P < 0.05 was considered statistically significant. All statistical analyses were performed using the statistical package SPSS 17.0 (SPSS, Chicago, IL).

Results Immunohistochemical expression of HOXA13 proteins in bladder cancer tissues Immunohistochemistry showed that HOXA13 was mainly localized in the cytoplasm with varied staining intensity (Fig. 1). The positive rate of HOXA13 protein expression was 73.6% (78/106) and 16.1% (17/106) in adjacent non-cancerous samples (Table 1). The protein expression level of HOXA13 was significantly higher in bladder cancer tissues than the level in adjacent non-cancerous bladder tissues (P < 0.01).

Correlation of tissue HOXA13 expression with clinicopathological variables In order to evaluate the biological significance of HOXA13 in bladder cancer, we analyzed the associations between tissue HOXA13 expression and clinicopathological parameters, including age, gender, tumor size, number of tumor nodule, smoking, lymphatic metastasis, pathological grade and TNM stage in bladder cancer. As shown in Table 2, the increased

Dysregulated expression of homebox gene HOXA13 is correlated with the poor prognosis in bladder cancer

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original article

Table 3 Univariate analysis of overall survival (OS) and disease-free survival (DFS) Variable

OS

DFS

Mean survival (months)

95% CI

P

Mean survival (months)

95% CI

P

HOXA13

–

–

0.001

–

–

0.001

Low

62.630 ± 2.199

58.321–66.939

–

59.410 ± 3.100

53.333–65.486

–

High

47.744 ± 2.845

42.167–53.320

–

40.541 ± 3.533

33.617–47.465

–

Age (years)

–

–

0.516

–

–

0.455

<60

54.720 ± 2.456

49.907–59.533

–

49.758 ± 3.079

43.723–55.792

–

≥60

52.296 ± 3.530

45.378–59.214

–

45.455 ± 4.756

36.133–54.776

–

Gender

–

–

0.634

–

–

0.520

Male

54.587 ± 2.339

50.002–59.172

–

49.527 ± 2.955

43.736–55.318

–

Female

52.139 ± 3.974

44.350–59.927

–

45.063 ± 5.332

34.613–55.514

–

Tumor size (cm)

–

–

0.577

–

–

0.628

<3

54.789 ± 2.744

49.410–60.168

–

49.164 ± 3.634

42.041–56.287

–

≥3

52.720 ± 2.919

46.999–58.441

–

46.974 ± 3.612

39.895–54.052

–

Tumor nodule number

–

–

0.354

–

–

0.328

Single

55.693 ± 2.567

50.662–60.724

–

50.735 ± 3.302

44.263–57.207

–

Multiple

50.667 ± 3.083

44.623–56.710

–

44.378 ± 3.966

36.605–52.151

–

Smoking

–

–

0.940

–

–

0.955

No

53.784 ± 2.823

48.251–59.317

–

48.412 ± 3.620

41.317–55.506

–

Yes

54.200 ± 2.877

48.560–59.839

–

48.408 ± 3.713

41.131–55.686

–

LN metastasis

–

–

0.006

–

–

0.003

No

57.237 ± 2.107

53.107–61.367

–

52.660 ± 2.735

47.300–58.021

–

Yes

43.613 ± 4.567

34.663–52.564

–

34.720 ± 5.687

23.574–45.866

–

TNM stage

–

–

0.000

–

–

0.000

Ta-T1

61.469 ± 1.889

57.766–65.172

–

57.618 ± 2.610

52.502–62.735

–

T2-T4

38.922 ± 3.404

32.251–45.594

–

29.861 ± 4.145

21.737–37.985

–

Pathological grade

–

–

0.000

–

–

0.000

G1

63.947 ± 1.980

60.065–67.828

–

60.412 ± 2.884

54.760–66.064

–

G2

58.413 ± 2.937

52.656–64.171

–

53.548 ± 4.093

45.527–61.570

–

G3

28.172 ± 2.680

22.919–33.424

–

16.340 ± 2.371

11.693–20.987

–

HOXA13 expression was significantly associated with lymphatic metastasis (P = 0.013), higher tumor stage (P = 0.002) and higher grade (P < 0.001). In contrast, no significant correlation was found between HOXA13 expression and gender (P = 0.766), age (P = 0.417), smoking status (P = 0.893), tumor size (P = 0.596) or number of tumors (P = 0.218). These findings suggest that HOXA13 may play an important role in progression of bladder cancer.

Western blotting analysis of HOXA13 proteins in the bladder cancer tissues To further validate the relationship of HOXA13 expression with bladder cancer progress, the expression level of HOXA13 in the bladder cancer tissues and corresponding paracarcinomatous non-cancerous tissues was also detected by Western blotting assay and the results of Western blotting analysis were in accordance with the results of immunohistochemistry. The HOXA13 expression in the bladder cancer tissues is more evident than in corresponding paracarcinomatous non-cancerous tissues (P < 0.01) (Fig. 2).

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Relationship between HOXA13 expression and bladder cancer patient survival Kaplan-Meier survival analysis was plotted to compare the overall survival (OS) and disease-free survival time (DFS) according to HOXA13 expressions (Fig. 3). Patients with high expression of HOXA13 showed a more unfavorable prognosis than those with low expression (P =0.001 and P = 0.001, respectively) (Table 3). Multivariate survival analysis further revealed intratumoral HOXA13 expression (P = 0.047 and P = 0.027, respectively), tumor TNM stage (P = 0.010 and P = 0.010, respectively) and tumor differentiation (P < 0.001 and P < 0.001, respectively) as independent poor prognostic markers for OS and DFS (Table 4).

Discussion The HOX genes are a family of transcription factors implicated in the regulation of cell proliferation, tissue differentiation and embryonic development [5]. HOX genes are relatively small molecules consisting

Dysregulated expression of homebox gene HOXA13 is correlated with the poor prognosis in bladder cancer

original article

Table 4 Multivariate Cox regression analysis of overall survival and disease-free survival Variable

Overall survival

Disease-free survival

RR

95% CI

P-value

RR

(95% CI)

P-value

Age (<60, ≥60 years)

1.286

0.571–2.894

0.544

0.756

0.347–1.645

0.481

Gender (male, female)

1.045

0.488–2.237

0.910

0.955

0.462–1.971

0.900

Smoker (no, yes)

0.842

0.405–1.752

0.645

0.717

0.352–1.459

0.358

Tumor size (<3, ≥3 cm)

1.115

0.529–2.352

0.775

0.839

0.402–1.750

0.640

Number of tumors (single, multiple)

0.977

1.504–1.896

0.946

0.867

0.454–1.654

0.665

LN metastasis (present, absent)

1.526

0. 638–3.651

0.342

1.272

0.548–2.952

0.575

TNM stage (Ta-T1, T2-T4)

0.319

0.134–0.764

0.010

0.302

0.121–0.751

0.010

Pathological grade (G1, G2, G3)

0.084

0.036–0.198

0.000

0.100

0.044–0.226

0.000

HOXA13 expression (low, high)

0.453

0.207–0.989

0.047

0.423

0.198–0.907

0.027

RR risk ratio, CI confidence interval, LN lymph node

of less than 100 amino acids, all which involve the cell development [6]. The HOX genes are classified into four categories according to the gene sequence and its location in the chromosome [7]. Studies in recent years have suggested that dysregulated expression of HOX played a vital role in tumor progression in lung [8], colon [9], thyroid [10], ovarian [11], liver [12] and prostate cancers [13]. HOXA13, a member of HOX genes family, plays an important role in extraembryonic vascularization [14]. Additionally, HOXA13 dysregulated expression in hepatocellular carcinoma [15], gastric cancer [16], pancreatic cancer [17], glioma [18] and esophageal squamous cell carcinoma [19] was disclosed by multiple studies. Recently, some studies reported that HOXA13 expression was detected in the urine of patients with bladder cancer [20–22]; however, the expression level of HOXA13 in the bladder cancer tissue has not appeared in these study papers. In the present study, we detected expression levels of HOXA13 in bladder cancer patients who underwent surgery and analyzed potential associations between expression levels of HOXA13 in bladder cancer tissues and clinicopathological characteristics of the tumor. We found that HOXA13 expression elevated was associated with lymphatic metastasis, tumor TNM stage and tumor differentiation, while the χ2-test showed that expression levels of HOXA13 in bladder cancer tissues were significantly higher than the corresponding paracarcinomatous non-cancerous tissues. Further evaluation elucidated that OS and DFS were better in bladder cancer patients with low expression levels of HOXA13 than those patients with elevated HOXA13 levels. Both Kaplan-Meier and multivariate analyses showed that the enhanced expression level of HOXA13 was the independent predictor of a poor prognosis for OS and DFS. The relationship between HOXA13 expression level and patient survival was also examined. It turns out that the increased expression level of HOXA13 was associated with the dismal outcome of bladder cancer. In summary, our study indicated that HOXA13 may be a malignant phenotypic feature of bladder cancer

cells. In addition, our study first reported HOXA13 protein expression as a new independent prognostic marker in bladder cancer. Shortcomings of this study include the small sample size and larger sample sizes are required to further validate the relationship between HOXA13 expression and prognosis. Conflict of interest H. Haiyi, C. Yuebing, C. Sheng, L. Gonghui, and Z. Zhigen declare that they have no competing interests.

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Dysregulated expression of homebox gene HOXA13 is correlated with the poor prognosis in bladder cancer

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Dysregulated expression of homebox gene HOXA13 is correlated with the poor prognosis in bladder cancer