Open Access

High Tumoral KPNA2 Expression Is a Potential Biomarker for Poor Prognosis in Advanced Neuroblastoma Patients

SAYAKA OTAKE 1
KENJIRO OGUSHI 1
GENDENSUREN DORJKHORLOO 1
TAKEHIKO YOKOBORI 2
AKIRA NISHI 3
HIROYUKI KUWANO 1
HIROSHI SAEKI 1
  &  
KEN SHIRABE 1

1Department of General Surgical Science, Graduate School of Medicine, Gunma University, Maebashi, Japan

2Division of Gene Therapy Science, Gunma University, Initiative for Advanced Research (GIAR), Maebashi, Japan

3Department of Surgery, Gunma Children’s Medical Center, Shibukawa, Japan

Cancer Diagnosis & Prognosis Jan-Feb; 5(1): 1-7 DOI: 10.21873/cdp.10404
Received 27 September 2024 | Revised 19 February 2025 | Accepted 11 November 2024
Corresponding author
Takehiko Yokobori (ORCID: 0000-0003-3284-4796), MD, Ph.D., Division of Gene Therapy Science, Gunma University Initiative for Advanced Research (GIAR), 3-39-22 Showamachi, Maebashi 371-8511 Japan. Tel: +81 0272208022, Fax: +81 0272208230 bori45@gunma-u.ac.jp
pdf image icon

Abstract

Background/Aim: Karyopherin alpha 2 (KPNA2) has been reported to be associated with cancer aggressiveness and treatment resistance via transporting several cargo proteins into the nucleus, such as cancer-promoting E2F and DNA repair-related MRN complex. Recent studies have highlighted the KPNA2 functions in tumorigenesis and the progression of various cancers. However, the importance of KPNA2 expression has yet to be elucidated in clinical neuroblastoma patients. This study aimed to analyze the clinical impact of KPNA2 expression in neuroblastoma. Materials and Methods: KPNA2 expression in 81 resected neuroblastoma sections was examined using immuno-histochemical staining. The significance and prognostic value of tumoral KPNA2 expression were analyzed using our cohort and R2 database. Results: The KPNA2 was expressed in the nucleus of neuroblastoma cells. The expression level of nuclear KPNA2 was not associated with clinicopathological factors in neuroblastoma. Among our cohort (n=81), non-radically resected neuroblastoma patients (n=37) with high KPNA2 expression had poorer prognoses than those with low KPNA2 expression. The R2 database analysis validated that the high KPNA2 expression was related to the poor prognosis in the large-scale neuroblastoma cohort. Conclusion: KPNA2 expression evaluation in neuroblastoma is a promising indicator of prognosis in non-curative resected cases. KPNA2 targeting may be a promising therapeutic strategy against advanced neuroblastoma.
Keywords: Neuroblastoma, importin, prognostic markers

Neuroblastoma is one of the most common extracranial solid tumors in pediatric patients. Several critical factors, such as MYCN (proto-oncogene BHLH transcription factor) amplification and copy number variations, are essential for diagnosing high-risk neuroblastoma (1,2). These molecular markers have been pivotal in identifying high-risk patients who require more aggressive treatments (3). However, despite the implementation of multidisciplinary treatment strategies, the prognosis for high-risk neuroblastoma patients remains dismal. Therefore, developing new biomarkers is crucial for diagnosing tumor aggressiveness correctly and selecting a suitable therapeutic strategy against neuroblastoma.

Karyopherin subunit alpha 2 (KPNA2), a nuclear transport protein, has been implicated in cancer aggressiveness of various cancers through the nuclear translocation of several oncogenic proteins (4,5). Notably, KPNA2 facilitates the nuclear import of cancer-related proteins such as E2F, a crucial regulator of the cell cycle, and the MRN complex (MRE11-RAD50-NBS1), essential for DNA damage response and repair, suggesting the importance of KPNA2 in cancer proliferation and chemotherapy resistance (6). It has been reported that high KPNA2 expression in tumor tissues was associated with tumor aggressiveness, poor prognosis, and therapeutic resistance in several cancers (7-11). These findings indicate that KPNA2 may be a potential biomarker and therapeutic target in different types of primary tumors. However, the importance of the tumoral KPNA2 accumulation as a tumor promotion factor and prognostic biomarker has not been elucidated in clinical neuroblastoma samples.

This study aimed to clarify the clinical significance and prognostic value of KPNA2 expression in clinical neuroblastoma tumors. Therefore, we evaluated the expression of tumoral KPNA2 in 81 neuroblastoma samples using immunohistochemical staining and analyzed the association of the KPNA2 expression levels with various clinicopathological parameters and survival. Moreover, the prognostic value of tumoral KPNA2 mRNA expression was validated using a database of large-cohort neuroblastoma cases.

Materials and Methods

Patients and samples. Tumor tissue samples were resected from 102 neuroblastoma-suspected cases who were subjected to surgical biopsy or surgery at Gunma University Hospital and Gunma Children’s Medical Center between 1991 and 2020. Preoperative chemotherapy was performed in eleven patients; therefore, they were excluded from further analysis. Among the patients, 81 were pathologically diagnosed with neuroblastoma, nine cases with ganglioneuroblastoma, and one case with ganglioneuroma (Figure 1). Among the 81 neuroblastoma samples, eight cases were MYCN-amplified, and 69 were non-MYCN-amplified. The MYCN status was not analyzed in four cases. In addition, tumors in 44 cases were radically resected, and tumors in 37 were not radically resected. The 81 neuroblastoma cases had the following postoperative complications, such as liver disorder, ileus, renal dysfunction, hearing disorder, chylothorax, sepsis, Horner’s syndrome, pseudomembranous enteritis, herpes zoster, and opsoclonus-myoclonus syndrome. This study follows the tenets of the Declaration of Helsinki and was approved by the Institutional Review Board of Gunma University Hospital (approval number: HS2024-169). Patient consent was obtained by using the opt-out method.

Immunohistochemistry. The staining procedure was carried according to standard methods, as described previously (12). In brief, the deparaffinized specimens (3.5 μm) were incubated in 0.3% hydrogen peroxide in 100% methanol for 30 min at room temperature to block the endogenous peroxidase activity. The antigen retrieval and blocking were performed using an Immunosaver solution (Nishin EM, Tokyo, Japan) and a protein block serum-free reagent (DAKO, Carpinteria, CA, USA). Then, tumor sections were incubated with anti-KPNA2 antibody (1:500; ab84440; Abcam, Cambridge, UK) and anti-Ki67 antibody (1:400; #9027; Cell Signaling Technology, Danvers, MA, USA) in a Dako REAL antibody diluent at 4˚C for 24 h. The primary antibodies were visualized using the Histofine Simple Stain MAX-PO (Multi) Kit (Nichirei, Tokyo, Japan) and 3,3-diaminobenzidine tetrahydrochloride (0.02% in 50 mM ammonium acetate–citrate acid buffer containing 0.005% hydrogen peroxide), and were lightly counterstained with hematoxylin. Negative control sections were not treated with the primary antibodies. Two experienced authors evaluated these slides blinded to the clinical data. The staining score was the average of the two researchers’ evaluations.

Evaluation of immunostaining. Immunostaining of KPNA2 was assessed as follows: The positive cell number of nuclear KPNA2 expression was calculated as the percentage of nuclear-stained cells for each section based on 400 neuroblastoma cells and was counted at the four representative positively stained sites in the slide. The neuroblastoma patients were classified as high KPNA2 or low KPNA2 groups based on the receiver operating characteristic (ROC)-derived cut-off value regarding poor prognosis. Accordingly, the KPNA2-positive cell number of ≥98 was defined as the high KPNA2 expression group, and <98 was defined as the low KPNA2 expression group. The immunoreactivity of Ki67 was evaluated as follows: The positive cell number of Ki67 in neuroblastoma tumors was calculated as the percentage of nuclear Ki67 stained cells for each section in 500 neuroblastoma cells. Nuclear Ki67 positive cell number of ≥88 was designated as the high Ki67 expression group, and <88 was designated as the low Ki67 expression group based on the ROC analysis regarding poor prognosis as previously described (12).

On-line microarray database search for KPNA2 mRNA expression in neuroblastoma. We used an online R2 database to validate the relevance of KPNA2 mRNA expression to overall survival in patients with neuroblastoma (R2 internal identifier: ps_avgpres_dgc2102a786_dgc2102).

Statistical analysis. Statistical differences were analyzed using Chi-square test. The ROC curve analyses were performed to define the cut-off values for KPNA2 and Ki67 scores to predict poor prognosis. Survival curves were calculated using the Kaplan–Meier method, and statistical differences between the curves were analyzed using the log-rank test. These statistical analyses were performed using JMP Pro 14 software (SAS Institute, Cary, NC, USA).

Results

Immunohistochemical staining of nuclear KPNA2 in clinical neuroblastoma tissues. The KPNA2 protein expression was mainly detected in the nucleus of neuroblasts and neuroblastoma cells (Figure 2). The positive rate of nuclear KPNA2 expression was higher in neuroblastoma patients (44.4%, 36/81 cases) than in ganglioneuroma (0%, 0/1 case) and ganglioneuroblastoma (33.3%, 3/9 cases) (Figure 1 and Figure 2). Of the 81 neuroblastoma-diagnosed cases, 58 (71.6%, 58/81 cases) and 23 (28.4%, 23/81 cases) were categorized as the low KPNA2 expression (Figure 2C) and high KPNA2 expression groups (Figure 2D), respectively (Table I).

Association of KPNA2 expression with the clinicopathological features in clinical neuroblastoma patients. The relationship between KPNA2 expression and clinicopathological characteristics is shown in Table I. Unexpectedly, the KPNA2 expression was not related to the factors in the total cohort (n=81) and radically resected cohort (n=44) (Table I, left and middle part). However, high nuclear KPNA2 expression in non-radically resected neuroblastoma patients (n=37) was significantly associated with elder disease onset age (p=0.014) and showed the tendency to be related to the MYCN amplification (p=0.054) and progression of International Neuroblastoma Pathology Classification (INPC) (p=0.062) and International Neuroblastoma Risk Group (INRG) staging system (p=0.057) (Table I, right part).

Prognostic significance of nuclear KPNA2 expression in neuroblastoma patients. Kaplan–Meier analysis in the total cohort (n=81) and radically resected neuroblastoma cohort (n=44) did not show a significant difference between high and low KPNA2 expression groups (Figure 3A, left and middle panel). In contrast to all neuroblastoma and radically resected cases, the high nuclear KPNA2 expression in non-radically resected neuroblastoma patients (n=37) was significantly associated with poor prognosis (p=0.0474, Figure 3A right panel).

This study used the R2 Genomics Analysis and Visualization Platform with prognostic data from 782 neuroblastoma patients to confirm the prognostic value of KPNA2 expression in a larger cohort (R2 internal identifier: ps_avgpres_dgc2102 a786_dgc2102). The results indicated that the neuroblastoma patients with high KPNA2 mRNA expression had poorer prognosis than those with low expression in not only all cases (n=782, p=8.87e-07, Figure 3B left panel) and non-MYCN-amplified cases (n=629, p=3.39e-04, Figure 3B middle panel) but also MYCN-amplified cases (n=153, p=0.054, Figure 3B right panel); suggesting the significant prognostic value of the KPNA2 expression in patients with neuroblastoma.

Discussion

In this study, we clarified that KPNA2 expression was detected in the nucleus of tumor cells and that high nuclear KPNA2 was associated with high-risk disease conditions and poor prognosis in non-radically resected patients with neuroblastoma. Moreover, the poor prognostic value of high KPNA2 in neuroblastoma was validated using public database analysis.

Numerous studies have indicated that the expression levels of KPNA2 in various tumors were higher than in non-cancerous tissues (5,13). As anticipated, the positive rate of nuclear KPNA2 expression in neuroblastoma tissues was higher than that in surrounding normal tissues, ganglioneuroma, and ganglioneuroblastoma (Figure 1 and Figure 2). It has been reported that KPNA2 expression is up-regulated by several cancer-promoting factors, such as E2F, the PI3K/AKT pathway, and miR-101 (5,14-16). Interestingly, the activation and dysregulation of E2F, AKT, and miR-101 were also found to be associated with the aggressiveness of neuroblastoma cells (17-20). Therefore, the expression level of KPNA2 in neuroblastoma is suggested to be regulated by factors such as E2F, the PI3K/AKT pathway, and miR-101, which are highly expressed and activated in neuroblastoma cells.

Non-radically resected neuroblastoma cases are mainly treated with systemic chemotherapy to prolong the prognosis (1). However, the results of this study showed that high KPNA2 expression was significantly associated with a poorer prognosis in such advanced patients who could not undergo radical resection. These findings suggest that KPNA2 expression may contribute to the treatment resistance to postoperative chemotherapy against residual neuroblastoma cells. In support of this hypothesis, it has been reported that KPNA2 transports the MRN complex into the nucleus, which is critical for DNA double-strand break repair; tumors that express high levels of KPNA2 are not only associated with cancer progression and poor prognosis but also show resistance to chemotherapy and radiation; and suppression of KPNA2 improves sensitivity to anticancer drugs (7,8,11,21,22). In clinical practice, KPNA2 expression in resected specimens is expected to be a biomarker predicting the risk of postoperative recurrence and treatment sensitivity. Moreover, the KPNA2 targeting strategy may be promising against refractory neuroblastoma by improving chemo-sensitivity. Further research is needed to test these promising hypotheses.

Study limitations. First, this study was performed using retrospectively collected tumor samples from only operated patients without preoperative treatments, not directly linked to the whole neuroblastoma patients. Second, the sample size was small in this study because pediatric neuroblastoma is a rare type of malignant tumor compared to adult solid cancer. Finally, functional analysis of KPNA2 was not performed using cell lines and animal experiment models.

In conclusion, this study showed that KPNA2 was strongly expressed in the nucleus of neuroblastoma cells, and high nuclear KPNA2 expression was significantly associated with poor prognosis in non-radically resected neuroblastoma patients. Our data suggested that assessing KPNA2 expression in neuroblastoma is a promising indicator of high-risk cases and that KPNA2 is a promising therapeutic candidate for patients with refractory advanced neuroblastoma.

Conflicts of Interest

The Authors declare no conflicts of interest relevant to this research.

Authors’ Contributions

SO, KO and GD contributed equally to this study. SO, KO, and AN collected the samples and analyzed the data. SO, KO, GD, TY, HK, HS, and KS analyzed and interpreted the data. SO, KO, GD, and TY drafted the manuscript. SO, KO, GD, TY, HS, and KS conceptualized the study. All the Authors have read and approved the final version of the manuscript.

Acknowledgements

The Authors thank Ms. Mariko Nakamura, Kao Abe, Yukiko Suto, Sayaka Okada, and Harumi Kanai for their assistance with this study. The work was supported by grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (JSPS; grant numbers: 23K24173). The work was also supported in part by the GIAR Grant.

References

1 Nakagawara A Li Y Izumi H Muramori K Inada H & Nishi M Neuroblastoma. Jpn J Clin Oncol. 48(3) 214 - 241 2018. DOI: 10.1093/jjco/hyx176
2 Maris JM Recent advances in neuroblastoma. N Engl J Med. 362(23) 2202 - 2211 2010. DOI: 10.1056/NEJMra0804577
3 Chung C Boterberg T Lucas J Panoff J Valteau-Couanet D Hero B Bagatell R & Hill-Kayser CE Neuroblastoma. Pediatr Blood Cancer 68 Suppl 2(Suppl. 2) e28473 2021. DOI: 10.1002/pbc.28473
4 Yang X Wang H Zhang L Yao S Dai J Wen G An J Jin H Du Q Hu Y Zheng L Chen X Yi Z & Tuo B Novel roles of karyopherin subunit alpha 2 in hepatocellular carcinoma. Biomed Pharmacother. 163 114792 2023. DOI: 10.1016/j.biopha.2023.114792
5 Han Y & Wang X The emerging roles of KPNA2 in cancer. Life Sci. 241 117140 2020. DOI: 10.1016/j.lfs.2019.117140
6 Teng SC Wu KJ Tseng SF Wong CW & Kao L Importin KPNA2, NBS1, DNA repair and tumorigenesis. J Mol Hist. 37(5-7) 293 - 299 2006. DOI: 10.1007/s10735-006-9032-y
7 Tsukagoshi M Araki K Yokobori T Altan B Suzuki H Kubo N Watanabe A Ishii N Hosouchi Y Nishiyama M Shirabe K & Kuwano H Overexpression of karyopherin-α2 in cholangiocarcinoma correlates with poor prognosis and gemcitabine sensitivity via nuclear translocation of DNA repair proteins. Oncotarget. 8(26) 42159 - 42172 2017. DOI: 10.18632/oncotarget.15020
8 Altan B Yokobori T Mochiki E Ohno T Ogata K Ogawa A Yanai M Kobayashi T Luvsandagva B Asao T & Kuwano H Nuclear karyopherin-α2 expression in primary lesions and metastatic lymph nodes was associated with poor prognosis and progression in gastric cancer. Carcinogenesis. 34(10) 2314 - 2321 2013. DOI: 10.1093/carcin/bgt214
9 Kubo N Araki K Altan B Hoshino K Ishii N Tsukagoshi M Igarashi T Watanabe A Kato T Hirai K Yokobori T Saito F Suzuki H Kuwano H & Shirabe K Enhanced karyopherin-α2 expression is associated with carcinogenesis in patients with intraductal papillary mucinous neoplasms. Pancreatology. 17(4) 611 - 616 2017. DOI: 10.1016/j.pan.2017.04.011
10 Saito F Araki K Yokobori T Ishii N Tsukagoshi M Watanabe A Kubo N Altan B Shirabe K & Kuwano H High expression of karyopherin-α2 and stathmin 1 is associated with proliferation potency and transformation in the bile duct and gall bladder epithelia in the cases of pancreaticobiliary maljunction. J Surg Oncol. 114(4) 462 - 468 2016. DOI: 10.1002/jso.24330
11 Takada T Tsutsumi S Takahashi R Ohsone K Tatsuki H Suto T Kato T Fujii T Yokobori T & Kuwano H KPNA2 over-expression is a potential marker of prognosis and therapeutic sensitivity in colorectal cancer patients. J Surg Oncol. 113(2) 213 - 217 2016. DOI: 10.1002/jso.24114
12 Ogushi K Yokobori T Nobusawa S Shirakura T Hirato J Erkhem-Ochir B Okami H Dorjkhorloo G Nishi A Suzuki M Otake S Saeki H & Shirabe K High tumoral STMN1 expression is associated with malignant potential and poor prognosis in patients with neuroblastoma. Cancers (Basel). 15(18) 4482 2023. DOI: 10.3390/cancers15184482
13 Zhou LN Tan Y Li P Zeng P Chen MB Tian Y & Zhu YQ Prognostic value of increased KPNA2 expression in some solid tumors: A systematic review and meta-analysis. Oncotarget. 8(1) 303 - 314 2017. DOI: 10.18632/oncotarget.13863
14 van der Watt PJ Ngarande E & Leaner VD Overexpression of Kpnβ1 and Kpnα2 importin proteins in cancer derives from deregulated E2F activity. PLoS One. 6(11) e27723 2011. DOI: 10.1371/journal.pone.0027723
15 Huang L Zhou Y Cao XP Lin JX Zhang L Huang ST & Zheng M KPNA2 promotes migration and invasion in epithelial ovarian cancer cells by inducing epithelial-mesenchymal transition via Akt/GSK-3β/Snail activation. J Cancer. 9(1) 157 - 165 2018. DOI: 10.7150/jca.20879
16 Dong L Jiang H Qiu T Xu Y Chen E Huang A & Ying K MiR-101-3p targets KPNA2 to inhibit the progression of lung squamous cell carcinoma cell lines. Histol Histopathol. 38(10) 1169 - 1178 2023. DOI: 10.14670/HH-18-573
17 Strieder V & Lutz W E2F proteins regulate MYCN expression in neuroblastomas. J Biol Chem. 278(5) 2983 - 2989 2003. DOI: 10.1074/jbc.M207596200
18 Wang H Wang X Xu L & Zhang J Prognostic analysis of E2F transcription factors E2F1 and E2F3 in four independent pediatric neuroblastoma cohorts. BMC Pediatr. 22(1) 376 2022. DOI: 10.1186/s12887-022-03424-w
19 King D Yeomanson D & Bryant HE PI3King the lock: targeting the PI3K/Akt/mTOR pathway as a novel therapeutic strategy in neuroblastoma. J Pediatr Hematol Oncol. 37(4) 245 - 251 2015. DOI: 10.1097/MPH.0000000000000329
20 Buechner J Tømte E Haug BH Henriksen JR Løkke C Flægstad T & Einvik C Tumour-suppressor microRNAs let-7 and mir-101 target the proto-oncogene MYCN and inhibit cell proliferation in MYCN-amplified neuroblastoma. Br J Cancer. 105(2) 296 - 303 2011. DOI: 10.1038/bjc.2011.220
21 Tseng SF Chang CY Wu KJ & Teng SC Importin KPNA2 is required for proper nuclear localization and multiple functions of NBS1. J Biol Chem. 280(47) 39594 - 39600 2005. DOI: 10.1074/jbc.M508425200
22 Sakai M Sohda M Miyazaki T Suzuki S Sano A Tanaka N Inose T Nakajima M Kata H & Kuwano H Significance of Karyopherin-α 2 (KPNA2) expression in esophageal squamous cell carcinoma. Anticancer Res. 30(3) 851 - 856 2010.