Edited by: Zhe-Sheng Chen, St. John's University, United States
Reviewed by: Silvia Deaglio, University of Turin, Italy; Luca Falzone, University of Catania, Italy
*Correspondence: Hongyun Shi,
This article was submitted to Experimental Pharmacology and Drug Discovery, a section of the journal Frontiers in Pharmacology
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The current treatment for natural killer/T-cell lymphoma (NKTL) among advanced/relapsed patients is unsatisfying, thereby highlighting the need for novel therapeutic targets. B‐cell chronic lymphocytic leukemia/lymphoma 11 A (BCL11A), as a transcription factor, is oncogenic in several neoplasms. However, its function in NKTL remains unclear. Quantitative real-time polymerase chain reaction and Western blot analysis were used to measure the BCL11A expression levels among NKTL patients and in NKTL cell lines. Natural killer (NK) cells from healthy subjects were used as negative control. Transient transfection with small interfering RNA was used to knockdown the expression in NKTL cell lines. Samples and clinical histories were collected from 343 NKTL patients (divided into test and validation groups) to evaluate the clinical value of BCL11A expression level. The BCL11A expression was upregu\lated among NKTL patients and in NKTL cell lines. Reduced cell proliferation and increased apoptosis were observed after silencing BCL11A in NKTL cell lines. BCL11A expression level was correlated with RUNX3, c-MYC, and P53 in NKTL. Notably, a high BCL11A expression was correlated with unfavorable clinical characteristics and predicted poor outcomes in NKTL. In conclusion, BCL11A was overexpressed in NKTL, while its upregulation promoted tumor development. Therefore, BCL11A expression level may be a promising prognostic biomarker for NKTL.
Natural killer/T-cell lymphoma (NKTL) is an aggressive, rare disease that is more frequently observed in Asia and South America than in North America and Europe (
B‐cell chronic lymphocytic leukemia/lymphoma 11 a (BCL11A) is a C2H2-type zinc-finger transcription factor that plays an important role in pre–B-cell development, thymocyte maturation, and globin switching (
Accordingly, this study examined whether BCL11A is overexpressed in NKTL and explored its role in NKTL. The correlation of BCL11A with RUNX3, c-MYC, and P53 expression was also examined along with the clinical value of BCL11A expression level in NKTL.
Several NKTL cell lines, including SNK-1, NK-YS, HANK-1, and KHYG-1, were incubated at 37°C in a humidified atmosphere of 5% CO2.
Sixteen randomly selected NKTL patient samples were stained to assess the immunohistochemical expressions of BCL11A, RUNX3, c-MYC, and P53 proteins. Anti-human primary antibodies to RUNX3, BCL11A, MYC, and P53 (ab92336, ab242406, ab32072 and ab32389, Abcam, UK) were used in Immunohistochemistry (IHC) (1:500). HRP-conjugated secondary antibodies (1:250, Thermo Scientific) were used in IHC secondary staining. Antigen retrieval was performed at 120°C for 5 min by using a pressure cooker followed by an overnight incubation at 4°C. The appropriate positive tissue controls were used. The expression levels of BCL11A and RUNX3 were scored as percentages of the total tumor cell population per 1 mm core diameter (400×). The percentages of BCL11A and RUNX3 cells in three representative high-power fields of individual samples were analyzed. The positive expression for BCL11A and RUNX3 was defined as positive nuclear expression in at least 50% of the tumor cell population.
Samples from a retrospective cohort of 227 NKTL patients were stained to assess the immunohistochemical expression level of BCL11A. The intensities of BCL11A staining were scored from 0 to 4, with 0–1, 1–2, 2–3, and 3–4 indicating no, weak, medium, and strong staining, respectively. Individual samples were blindly evaluated by at least 2 pathologists, and scores of ≥ 2 and < 2 indicated high and low expressions, respectively.
Cell lines SNK-1, NK-YS, HANK-1, and KHYG-1 were transiently transfected with small interfering RNA (siRNA) (Santa Cruz Biotechnology) targeting BCL11A according to the manufacturer’s instructions. A total of 1 × 106 cells were seeded for the transient transfection. A nontargeting siRNA was used as control, and lipo2000 was used to assess the basal expression level of BCL11A. Protein and messenger RNA (mRNA) were extracted from the treated cell lines to evaluate the expression levels.
Cell lines SNK-1, NK-YS, HANK-1, and KHYG-1, which were transfected with BCL11A-targeting siRNA, nontargeting siRNA, and lipo2000, were examined to evaluate the apoptotic cell death rate
Cell lines SNK-1, NK-YS, HANK-1, and KHYG-1, which were transfected with BCL11A-targeting siRNA, nontargeting siRNA, and lipo2000, were examined to evaluate the cell proliferation rate by using the BrdU Cell Proliferation Assay Kit (EMD Chemicals, Gibbstown, NJ) according to the manufacturer’s instructions. A total of 1 × 106 cells were seeded for the transient transfection. Flow cytometric analysis was then performed to measure the cell proliferation rate.
Cell lines SNK-1, NK-YS, HANK-1, and KHYG-1, which were transfected with BCL11A-targeting siRNA, nontargeting siRNA, and lipo2000, were examined to evaluate the BCL11A mRNA expression level. A total of 1 × 106 cells were seeded for the transient transfection. The total RNA was prepared by using the miRNeasy Mini Kit (Thermo Fisher Scientific, USA) protocol including DNaseI treatment. A reverse transcription reaction was carried out by using the High-Capacity cDNA Reverse Transcription Kit System (Promega, USA). The real-time fluorescence monitoring of the PCR products was assayed with Taqman Gene Expression Master Mix (Promega, USA) and gene-specific Taqman probes by using the BioRad IQTM5 Multicolor Real-Time PCR Detection System (BioRad, USA). The relative mRNA levels were calculated using the ΔΔCT method by comparing the amount of endogenous GAPDH in the same sample.
Cell lines SNK-1, NK-YS, HANK-1, and KHYG-1, which were transfected with BCL11A-targeting siRNA, nontargeting siRNA, and lipo2000, were examined to evaluate the expression levels of BCL11A, RUNX3, MYC, and P53. A total of 1 × 106 cells were seeded for the transient transfection. The treated cell pellets were suspended in a lysis buffer with a cocktail of protease inhibitors (Thermo Fisher Scientific, USA). Protein detection
Refer to Methods in (
A total of 227 NKTL patients that were newly diagnosed in the Sun Yat-sen University Cancer Center between January 2008 and September 2015 were enrolled in this study to comprise the primary test group. In order to enhance the validity of the study, regarding the potential role of BCL11A expression level as a potential prognostic biomarker in NKTL, 116 NKTL patients were recruited from the first affiliated hospital of the Hainan Medical School during the same period to comprise an independent validation group. These patients were diagnosed according to the WHO classification of hematopoietic and lymphoid tumors, and their diagnosis was further confirmed by the positive EBV-encoded RNA results obtained
Two groups of RUNX3 expression levels were compared by conducting Student’s t-test, and the correlation between the BCL11A and RUNX3 expression levels was evaluated by conducting Spearman’s correlation test. Overall survival (OS) was defined as the time from diagnosis to death or last follow-up, whereas progression-free survival (PFS) was defined as the time from treatment to disease progression, death, or last follow-up. Clinical characteristics were compared by performing Mann–Whitney’s U test, chi-squared test, or Fisher’s exact test. The Kaplan–Meier method and log-rank test were used to analyze and compare the survival rates. Univariate and multivariate analyses were performed to evaluate the prognostic value of clinical characteristics, including BCL11A expression level. A two-sided P-value of <0.05 was considered statistically significant. The statistical analysis was performed by using SPSS 17.0.
Endogenous BCL11A expression in natural killer/T-cell lymphoma (NKTL) cells (SNK-1, NK-YS, HANK-1, and KHYG-1), NKTL patient samples and normal NK cells (NK01-NK02).
BCL11A inhibition in natural killer/T-cell lymphoma (NKTL) cells. BCL11A silenced NKTL cells (siBCL11A) showed deregulated cell proliferation and upregulated apoptosis.
BCL11A-targeting siRNA was used to knockdown BCL11A expression and to determine the function of BCL11A in NKTL. The effect of BCL11A knockdown on apoptosis and cell proliferation was subsequently assessed. Silencing BCL11A in NKTL cell lines reduced the BCL11A mRNA and protein expressions (
To further explore the role of BCL11A overexpression in NKTL, the protein expression levels of BCL11A and RUNX3 in 16 NKTL patients were measured
Expression of RUNX3 and BCL11A protein in 16 NKTL patient samples were examined using immunohistochemistry.
The protein expression of BCL11A may also be correlated with those of RUNX3, c-MYC, and P53 (
Correlation of BCL11A, RUNX3, c-MYC, and P53 protein expressing levels.
The BCL11A expression levels of NKTL patients in the test (227 patients) and validation (116 patients) groups were retrospectively examined to investigate the clinical value of BCL11A in NKTL. The clinical features according to BCL11A expression level are presented in
The clinical characteristics and treatment modalities of the test group of NKTL patients.
Parameters | Total |
BCL11A low expression |
BCL11A high expression |
|
---|---|---|---|---|
Overall | 227(100) | 126(55.5) | 101(44.5) | – |
Male gender | 154(67.8) | 85(67.5) | 69(68.3) | 0.891 |
Age >60 years | 38(16.7) | 24(19.0) | 14(13.9) | 0.298 |
ECOG score ≥2 | 42(18.5) | 19(15.1) | 23(22.8) | 0.138 |
Ann Arbor stage | ||||
I\II | 185(81.5) | 109(86.5) | 76(75.2) | 0.030 |
III\IV | 42(18.5) | 17(13.5) | 25(24.8) | |
B symptoms | 108(47.6) | 56(44.4) | 52(51.5) | 0.291 |
LDH > 245U/L | 77(33.9) | 34(27.0) | 43(42.6) | 0.014 |
Chemotherapy regimen | ||||
GELOX | 130(57.3) | 77(61.1) | 53(52.5) | 0.191 |
EPOCH | 97(42.7) | 49(38.9) | 48(47.5) | |
Treatment response | ||||
CR | 116(51.1) | 78(61.9) | 38(37.6) | <0.001 |
Non-CR | 111(48.9) | 48(38.1) | 63(62.4) |
NKTL, natural killer/T-cell lymphoma; BCL11A, B‐cell chronic lymphocytic leukemia/lymphoma 11 A; ECOG, Eastern Cooperative Oncology Group, LDH, lactate dehydrogenase, GELOX, gemcitabine, L-asparaginase, and oxaliplatin; EPOCH, cyclophosphamide, doxorubicin, vincristine, prednisone, and etoposide; CR, complete remission.
Univariate and multivariate analyses of prognostic factors in the test group of NKTL patients.
Parameters | Overall survival | Progression-free survival | ||||
---|---|---|---|---|---|---|
Univariate analysis | Multivariate analysis | Univariate analysis | Multivariate analysis | |||
P value | HR (95% CI) | P value | P value | HR (95% CI) | P value | |
Gender | ||||||
Male vs. female | 0.256 | 0.206 | ||||
Age | ||||||
>60 vs. ≤60years | 0.493 | 0.312 | ||||
ECOG scores | ||||||
≥2 vs. 0-1 | <0.001 | <0.001 | ||||
Stage | ||||||
III\IV vs. I\II | <0.001 | 4.29(2.73–6.73) | <0.001 | <0.001 | 4.09(2.66–6.30) | <0.001 |
B symptoms | ||||||
Yes vs. no | 0.106 | 0.110 | ||||
LDH | ||||||
Elevated vs. normal | <0.001 | <0.001 | ||||
Chemotherapy | ||||||
GELOX vs. EPOCH | 0.073 | 0.006 | 1.49(1.03–2.16) | 0.034 | ||
Treatment response | ||||||
Non-CR vs. CR | <0.001 | 1.62(1.02–2.57) | <0.001 | <0.001 | 1.62(1.08–2.41) | 0.019 |
BCL11A expression | ||||||
High vs. low | <0.001 | 2.62(1.65–4.16) | <0.001 | <0.001 | 2.23(1.50–3.30) | <0.001 |
NKTL, natural killer/T-cell lymphoma; HR, hazard ratio; CI, confidence interval; ECOG, Eastern Cooperative Oncology Group; LDH, lactate dehydrogenase; GELOX, gemcitabine, L-asparaginase, and oxaliplatin; EPOCH, cyclophosphamide, doxorubicin, vincristine, prednisone, and etoposide; CR, complete remission; BCL11A, B‐cell chronic lymphocytic leukemia/lymphoma 11 A.
Survival curves of natural killer/T-cell lymphoma (NKTL) patients with high and low expression level of BCL11A.
NKTL is a rare and aggressive disease with poor outcomes, especially among patients with advanced/relapsed disease. Therefore, novel therapeutic targets are urgently needed. Several reports have shown that BCL11A is oncogenic in B‐cell lymphoma, B‐cell chronic lymphoblastic leukemia, and several solid tumors (
The proto-oncogene BCL11A is located on chromosome 2p16 and is involved in some hematological and solid neoplasms. In B-NHL and HD, BCL11A is involved in tumourigenesis through the gains and amplifications of chromosomes 2p13 and t(2;14) (
The clinical value of BCL11A in solid tumors has elicited increasing interest in recent years. Several studies showed that the upregulation of BCL11A contributes to cancer development through various pathways in certain solid tumors. For instance,
The results of this study show that BCL11A was overexpressed in NKTL and that the inhibition of BCL11A reduced cell proliferation and increased the apoptosis of NKTL cell lines, thereby suggesting that BCL11A may be an oncogene in NKTL. These results also reveal the essential role of BCL11A in NKTL. The underlying mechanism may be related to the lymphopoiesis of NK/T cells or various oncogenic pathways in solid tumors. Therefore, further studies on this topic are warranted.
To further understand the role of BCL11A overexpression in NKTL, this study examined the correlation of BCL11A expression with RUNX3, c-MYC, and P53 expression levels. Results show that BCL11A expression may be associated with the expression of RUNX3, c-MYC, and P53 in NKTL cells. A recent study on NKTL (
The clinical values of BCL11A expression level in NKTL were also analyzed in this study. NKTL patients were recruited and divided into test and validation groups to examine the prognostic value of BCL11A expression level. The results from the validation group were consistent with those from the test group, thereby enhancing their validity. A high BCL11A expression was shown to be correlated with late-stage and elevated LDH level. The univariate and multivariate analyses identified high BCL11A expression, late stage, and non-CR response as independent unfavorable prognostic factors for OS and PFS among NKTL patients. These findings highlight the functional importance of BCL11A upregulation in NKTL and explain the correlation of high BCL11A with late-stage, elevated LDH levels and worsened prognosis. These findings are also consistent with those of previous reports (
BCL11A was overexpressed in the NKTL cell lines and patient samples, and its inhibition reduced cell proliferation and increased apoptosis. The expression of BCL11A may be correlated with those of RUNX3, c-MYC, and P53. Notably, a high BCL11A expression was correlated with unfavorable clinical features and predicted inferior survival and progression. These results highlight the essential role of BCL11A in the development of NKTL and indicate that its expression may be a promising prognostic biomarker for NKTL. However, this study has several limitations. For instance, while the results reveal that BCL11A was correlated with RUNX3, c-MYC, and P53, such correlation was only tested among a small number of NKTL patients. Nevertheless, these results remain promising despite the small sample. Moreover, the underlying mechanism of such correlation in NKTL remains unclear and warrants further investigation.
The data that support the findings of this work are available from the corresponding authors upon reasonable request.
All procedures involving human participants were performed in accordance to the ethical standards of the ethics committee and the institutional review board of the Sun Yat-Sen University Cancer Centre and to the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Informed consent was obtained from all participants.
HS designed and performed the research. CL analyzed the data and wrote the paper. WF performed the research and analyzed the data. JY, JS, and AP performed the research. HW designed the research and wrote the paper.
This study was funded by the National Natural Science Foundation of China (Contract/Grant Number: 81700148).
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
We thank the patients, their families, and all investigators, including the physicians, nurses, and laboratory technicians, for their contributions to this study.
The Supplementary Material for this article can be found online at: