Expression of stem cell biomarkers Bmi1 and KLF4 in osteosarcoma and its clinical significance

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Expression of stem cell biomarkers Bmi1 and KLF4 in osteosarcoma and its clinical significance

https://doi.org/10.21203/rs.3.rs-1648463/v1

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Objective

To observe the expression of osteosarcoma stem cell biomarkers Bmi1 and KLF4 in osteosarcoma tissues and explore their value in the diagnosis, treatment and prognosis of osteosarcoma.

Methods

Using retrospective research methods, 51 patients (28 males and 23 females) with osteosarcoma who were surgically resected and diagnosed by pathology in the Second Hospital of Shanxi Medical University from October 2009 to July 2019 were used as the experimental group. The age of cases varies from 10 to 67 (average 27.04) years old, and bone tissues adjacent to the tumor were taken from 10 samples as the normal control group. Immunohistochemical method was used to assess the expression levels of Bmi1 and KLF4 in 51 patients with osteosarcoma and 10 cases of paraneoplastic bone tissue specimens. Chi-square test was applied to analyze the relationship between the expression of Bmi1 and KLF4 and the clinical pathological data of patients. Correlation analysis was analyzed by the number of connections. The survival rate of patients was calculated by the Kaplan-Meier method. The log rank univariate analysis and Cox regression multivariate analysis were carried out to evaluate the prognostic value.

Results

The positive expression rates of Bmi1 and KLF4 in the osteosarcoma group were 78.43% (40/51) and 80.39% (41/51), respectively, and in the bone tissue group were both 3/10. The difference of the positive expression rates of Bmi1 and KLF4 in the osteosarcoma group and bone tissue group was statistically significant (P < 0.05). In osteosarcoma group, the expression levels of Bmi1 and KLF4 were positively correlated (R = 0.399, P < 0.01). Bmi1 protein-positive, KLF4 protein-positive, and Bmi1 and KLF4 protein double-positive expression were statistically significant in Enneking surgical staging, lung metastasis, and pathological typing (all P < 0.05), but there was no statistically significant difference between different ages, genders, local recurrences, and tumor sizes (all P > 0.05).

Conclusion

The expression of Bmi1 and KLF4 in osteosarcoma tissue was significantly higher than that in surrounding bone tissues, and their positive expression is a risk factor for the prognosis of patients with osteosarcoma.

osteosarcoma

biomarker

Bmi1

KLF4

clinical significance

Osteosarcoma (OS) is the most common primary malignant bone tumor, which usually occurs in adolescents with an incidence rate of 3 to 4 cases per 1 million people per year. OS is known for its high degree of malignancy, strong invasiveness, high recurrence and metastasis rate ^[1–3], which seriously endangers the health of young people. In recent years, with the development of neoadjuvant chemotherapy and surgical technology, great advances have been made in the treatment OS, but the local recurrence rate is still as high as 50%. In addition, 15%-20% of patients have experienced imaging visible at the time of initial diagnosis. Unfortunately, 50% of patients suffer from lung metastases during treatment, and 80% of patients with metastases die within 5 years ^{[4, 5]}.

Osteosarcoma stem cells (OSCs) are very small subsets of cells appeared in OS. The conventional treatment of OS does kill most of the malignant cells in OS, while it has little effect on OSCs appeared in OS. The remaining OSCs after treatment are sufficient to cause the recurrence and metastasis of OS. Clinical studies in animals and humans have shown that the survival of OSCs during chemotherapy is the main reason for the recurrence of OS ^[6].

It is reported that BMI1 is highly expressed in stem cells and some malignant tumors, and regulates the initiation and progression of tumor cells by controlling histone ubiquitination at multiple genomic sites ^{[7, 8]}. Studies have shown that KLF4 was overexpressed in OSCs ^[9], and inhibiting the expression of KLF4 can inhibit tumor growth. Therefore, Bmi1 and KLF4 are considered as biomarkers of OSCs, which can be used to isolate and verify OSCs, and are widely used in basic research of OS. However, in OS tissues, the research conclusions on the expression of KLF4 are not consistent. Additionally, there are few reports about the relation between Bmi1 and OS. In this study, the author detected the expression levels of Bmi1 and KLF4 protein in OS tissue by immunohistochemistry, and explored their clinical significance.

Normal information

Paraffin-embedded OS tissue specimens were taken from October 2009 to July 2019. The paraffin specimens of 51 patients with OS who were pathologically diagnosed after surgical resection in the Department of Orthopedics at Second Affiliated Hospital of Shanxi Medical University were used as the experimental group, of which 10 bone tissues adjacent to the tumor (3 cm from the edge of the tumor shown by MRI, no tumor cells confirmed by pathological examination) served as control group. The diagnosis of the 51 patients with OS were certified by pathologists.

The inclusion criteria for this study are as follows: (1) limb osteosarcoma, (2) American Joint Cancer Committee (AJCC) stage II and III patients, (3) preoperative neoadjuvant chemotherapy and radical treatment arranged in our hospital Surgery, then adjuvant chemotherapy, (4) Incision biopsy specimens for immunohistochemistry and PAS staining. The exclusion criteria are as follows: (1) secondary osteosarcoma, (2) extraosseous osteosarcoma, (3) periosteum or paraosseous osteosarcoma, (4) skipping or metastatic disease at diagnosis, (5) no postoperative patient Re-examination, no follow-up information.

Among the 51 patients, 28 were males and 23 were females, their ages range from 10 to 67 (mean 27.04). There were 44 cases of ordinary osteosarcoma, 2 cases of chondroblastic osteosarcoma, 2 cases of telangiectatic osteosarcoma, 2 cases of giant cell osteosarcoma, and 1 case of low-grade central osteosarcoma. Ennecking staging: 34 cases in stage ⅡA, 13 cases in stage ⅡB, and 4 cases in stage Ⅲ. The patient's limb X-ray and chest CT were reviewed every 3 months after the operation, and the whole-body bone scan was reviewed every 6 months. The follow-up time of 51 patients range 4 to 78 months. During the follow-up period, 36 patients died, 37 patients suffered from metastasis or recurrence in situ (lung metastasis, pelvic metastasis, and multiple metastasis), 5 patients survived with tumor, and 10 patients survived without tumor. Record the clinical pathological variables including age, gender, the maximum diameter of the tumor on the initial magnetic resonance image, Ennecking stage, local recurrence, and lung metastasis. Follow up the clinical results were obtained from the day of surgery to the day of death or until March 2021. Survival time is defined as the time span from diagnosis to death caused by the primary tumor or the related reason.

Experimental reagents

Bmi1 antibody (dilute at 1:300, ab38295) and KLF4 antibody (dilute at 1:400, ab106629) were purchased from American Abcam. Hematoxylin and eosin staining kit and neutral glial fixation sheet were purchased from Shanghai Weiao Biotechnology Co., Ltd., streptavidin-biotin complex (SABC) immunohistochemical staining kit, Diaminobenzidine (DAB) color development kit and phosphate buffered saline (PBS) were purchased from Wuhan Bosite Bioengineering Co., Ltd. The biomarker goat anti-rabbit IgG was purchased from Beijing Zhongshan Bridge. 0.25% Tripsin-EDTA was purchased from Gibco, USA.

Experimental method

After the sections were made, HE staining was routinely performed firstly, then the cell morphology was observed using a microscope. Then immunohistochemical detection was performed, briefly, xylene dewaxing, ethanol solution rehydration, and EDTA antigen retrieval solution high pressure repair. Then the sections were blocked with PBS, and incubated with Bmi1, KLF4 antibody at 4 °C overnight. At the end of incubation, the sections were washed 3 times with PBS, and then incubated with biotin-labeled goat anti-rabbit IgG at 37 °C for 1 hour. Then, SABC was added, and incubated at 37 °C for 30 minutes. DAB solution was used to develop, hematoxylin solution was used to stain, hydrochloric acid alcohol was applied to turn blue, and the slide was mounted with neutral gum. The results of anti-Bmi1 and KLF4 staining were evaluated by Pannorama MIDI digital slide scanner and scanner control software (3DHISTECH, Ltd., Budapest, Hungary).

Result judgment

Determination of positive results: Bmi1 and KLF4 are positive cells with brown particles in the nucleus, respectively. Image analysis with Image J. Five fields of view were randomly selected under high magnification (x 400), and the average gray value (staining intensity) of positive cells and the percentage of positive area (stained area) were used as IHC measurement indicators. Finally, four scores were given: High positive (3+), Positive (2+), Low Positive (1+) and Negative (0). The final score of 0 ~ 1.5 and > 1.5 is defined as low expression and high expression, respectively.

Statistical Analysis

SPSS 22.0 and Graphpad prism 8.0 statistical software were used to analyze the data and draw, respectively. The categorical data was analyzed by χ2 test for comparison between groups. Survival analysis was calculated by Kaplan-Meier. Statistical differences in survival rate between each factor group were analyzed by Log-rank test. Multivariate survival analysis on survival data were performed by Cox proportional hazard regression model. P<0.05 was considered as statistically significant.

The patients were followed up by telephone or regular outpatient clinics. The follow-up date was from the day of surgery to March 1, 2021. The follow-up period ranges from 4 to 78 months. All the 51 patients in this group were followed-up with a median follow-up time of 38 months. Among the 51 patients, 36 cases died, 5 cases survived with tumors and 10 cases survived without tumors.

HE staining of OS tissue and adjacent bone tissue

HE staining showed that there was no peeling phenomenon in OS and adjacent bone tissues. (Figure 1A) In the OS group, the tumor cells were polygonal with large and deep stained nuclei, and more mitoses. (Figure 1B) Bone formation is appeared in the bone tissue group, the bone compact is lamellar, and bone cells can be seen in the lacuna.

Expression of Bmi1 protein and KLF4 protein in osteosarcoma tissue and adjacent bone tissue

Bmi1 and KLF4 can be seen under the microscope. As shown in Figure2 the expression is mainly located in the nucleus of OS cells, and both are negative expression (average IOD=0), low expression average (IOD≤1.5) and high expression (IOD>1.5). The positive expression rate of Bmi1 in OS tissue was 78.43% (40/51), which was significantly higher than the positive expression rate of 30% (3/10) in adjacent bone tissue. There were statistical differences between the two groups. The expression rate of KLF4 in osteosarcoma tissue in is 80.39% (41/51), which is significantly higher than the positive expression rate of 30% (3/10) in the bone tissue adjacent to the tumor. The difference between the two groups is statistically significant. （See Table 1 and Figure 2.）

Table1 Comparison of the expression of Bmi1 and KLF4

protein in the two groups

Group	Case	Bmi1 protein		KLF4 protein
		+ -		+ -
Adjacent bone tissue	10	3	7	1	7
Osteosarcoma group	51	40	11	41	10
χ²value		9.428 <0.05		10.561 <0.05
P value

The relationship between the expression levels of Bmi1 and KLF4 and the clinicopathological characteristics of patients with OS

The expression levels of Bmi1 and KLF4 protein in different clinicopathological factors of OS showed that it has nothing to do with the patient’s age and gender, but is closely related to tumor size. The expression of Bmi1 is closely related to lung metastasis regardless of Ennecking staging and local recurrence. However, the expression of KLF4 is closely associated with Ennecking staging and local recurrence, but not with lung metastasis. The difference was statistically significant (P<0.05). (See Table 2）

Table2 The relationship between the expression of Bmi1 and KLF4 and the clinicopathological characteristics of patients with osteosarcoma

Clinicopathological

Characteristics(case)

Bmi1expression level

X²

P value

KLF4 expression level

X²

P value

High

Low

High

Low

Age

≤18

>18

Gender

Male

Female

Tumor size

<5cm

≥5cm

Ennecking staging

Ennecking ⅡA

Ennecking ⅡB/Ⅲ

Lung metastasis

YES

Local recurrence

YES

0.749

1.000

0.043

0.263

0.038

0.479

0.171

0.680

0.021

0.002

0.185

0.005

Correlation between Bmi1 and KLF4 protein expression

Among 51 cases of OS, 40 cases were positive for Bmi1 protein and 11 cases were negative, 41 cases were positive for KLF4 protein, and 10 cases were negative. The expression of Bmi1 and KLF4 protein is positively correlated (R = 0.399, P <0.05). （See Table 3）

Table3 Correlation between Bmi1 and KLF4 protein expression in 51 cases of osteosarcoma tissues

Bmi1peotein	KLF4 protein		R value	P value
	+	-
+	32	8	0.399	0.0037
-	9	2

The relationship between Bmi1 and KLF4 protein expression levels and the prognosis of patients with OS

Kaplan-Meier survival analysis was applied to evaluate Bmi1 and KLF4 expression of 51 patients with OS and their prognosis. As shown in Figure 3, the overall survival rate of OS patients in the Bmi1/ KLF4 negative low expression group was higher than that of the Bmi1/KLF4 positive high expression group, and the difference was statistically significant (P<0.01).

A univariate survival analysis of 51 patients with OS showed that the factors affecting the prognosis of patients with OS include tumor size, Ennecking stage, lung metastasis, and the expression levels of Bmi1 and KLF4. Cox multivariate analysis showed that KLF4 expression, Ennecking staging, and lung metastasis were independent factors influencing the prognosis of patients with OS (P<0.05, Tables 4, 5).

Table4 Univariate postoperative survival analysis of patients with osteosarcoma

	Case	χ²	P value
Gender
Female	23	0.479	0.489
Male	28	0.479	0.489
Age
≤18	21	0.004	0.95
>18	30	0.004	0.95
Ennecking staging
Ennecking ⅡA	34	40.704	<0.001
Ennecking ⅡB/Ⅲ	17	40.704	<0.001
Tumor size
<5cm	20	7.263	0.007
≥5cm	31	7.263	0.007
Local recurrence
NO	35	0.206	0.65
YES	16	0.206	0.65
Lung metastasis
NO	18	15.133	<0.001
YES	33	15.133	<0.001
Bmi1 protein
Negative	11	13.793	0.001
Low express	8
High express	32
KLF4 protein
Negative	10	19.246	<0.001
Low express	12
High express	29

Table5 Cox multivariate postoperative survival analysis of patients with osteosarcoma

	Partial regression coefficient	Standard error	Wald χ²	P value	HR（95%CI）
Ennecking staging	1.518	0.539	11.929	0.005	4.565(1.586-13.141)
Lung metastasis	1.804	0.648	5.878	0.005	6.073(1.705-21.635)
Bmi1 positive	0.038	0.583	0.004	0.948	1.039(0.331-18.719)
KLF4 positive	1.763	0.595	8.767	0.003	5.828(1.815-18.719)

OS is the most common primary malignant bone tumor in humans, which is mainly characterized by local pain and early metastasis. Although surgery and chemotherapy is effective, the clinical prognosis is still poor. The existence of cancer stem cells (CSCs) is considered to be an important cause of tumor heterogeneity. Some scholars believe that OS also has a subset of cells that can maintain its own stem cell-like characteristics through self-renewal, which are called OSCs. Moreover, studies have found that OSCs have stronger tumorigenic ability and the potential to resist the chemotherapy killing ^{[10, 11]}.

BMI1 is one of the important members of PRC1 in the Polycomb family, and participates in chromatin monoubiquitination. BMI1 plays a key role in the self-renewal of stem cells ^{[12, 13]}. Studies have found that BMI1 expression was up-regulated in stem cells and some malignant tumors, and regulated tumor cell initiation and progression by controlling histone ubiquitination at multiple genomic sites ^{[7, 8]}. In this study, the results show that the expression rate of Bmi1 in OS is higher than that in adjacent bone tissues. The expression level of Bmi1 is closely correlated with tumor size and lung metastasis, and not with Ennecking staging and local recurrence, suggesting that Bmi1 can be used as a critical indicator of biological behavior of OS. In addition, the expression level of Bmi1 is related to lung metastasis and its prognosis in patients with OS.

KLF4 is a member of the Kruppel-like zinc finger transcription factor family. It participates in the regulation of important life processes such as cell proliferation, differentiation, and apoptosis by regulating the transcriptional activation or inhibition of target genes. KLF4 has different functions of oncogene or tumor suppressor gene in cells with different genetic backgrounds ^[14]. Recent researches found that KLF4 promoted the proliferation and metastasis of OS cells by regulating CRYAB ^[16], and was overexpressed in OSCs ^[16]. Additionally, KLF4 promotes the growth of OS and improve the resistance of chemotherapy drugs by regulating HMGB1 (high-mobility group box 1) ^[17], indicating that it may play a key role in OSCs growth of OS and the resistance of chemotherapy drugs. Therefore, the pluripotency of OSCs and formation of osteosarcoma may be inhibited by inhibiting the gene KLF4 ^[18]. KLF4 is also an important factor in maintaining the pluripotency of stem cells. KLF4 plays a key regulatory role in the process of adriamycin-induced osteosarcoma cell transformation to OSCs. Studies have found that after inhibiting KLF4, the ratio of CD133 cells induced by adriamycin is significantly reduced, and the expression levels of CD133, AB-CG2, ALDHlAl, etc. are also significantly down-regulated ^[19]. Another study further confirmed the importance of KLF4 in OSCs ^[18], this study also found that osteosarcoma cells with overexpressed KLF4 had some characteristics of OSCs such as increased spheroidizing ability, high expression of stem-related genes CD133, ABCG2 and ALDHlAl, chemotherapeutic drug resistance, etc. AS shown in our study, KLF4 was significantly highly expressed in patients with OS, with a positive rate of 80.39%. The expression level of KLF4 was closely related to Ennecking staging and local recurrence, but not significantly related to lung metastasis, suggesting that KLF4 can be used as an important reflection indicator of OS biology. In addition, we found that the expression level of KLF4 is related to the prognosis of patients with OS.

It is obvious that Bmi1 and KLF4 are co-expressed in osteosarcoma, and the two are positively correlated. However, whether there is a synergistic carcinogenic effect between Bmi1 and KLF4 needs to be further confirmed by conducting cell and animal experiments. This research is a retrospective study, lacking randomized controls, and there is a possibility of selection bias. And the characteristics and metastasis mechanism of Bmi1 and KLF4 positive cells still need to be further studied.

In summary, the expression levels of Bmi1 and KLF4 are related to lung metastasis of patients, which are independent factors affecting the prognosis of patients with OS and can be used as one of the monitoring indicators of lung metastasis and prognosis of patients with OS.

All data generated or analysed during this study are included in this published article and its supplementary information files.

Ethics Statement

Ethics approval was obtained from the ethics review committee of the Second Hospital of Shanxi Medical University ((2021) YX NO.098). The study was in Accordance with the Declaration of Helsinki. All participants and their legal guardians gave written informed consent to participate in the study.

Conflict of Interest

This research was conducted in the absence of any commercial or financial relationships.

Author Contributions

SWC, ZZW, and PCL designed the study. TYW and SWC supervised the study. SWC, TYW and ZZW performed the experiments. WXJ and SMN analyzed the data. SWC and ZZW interpreted the results and wrote the manuscript. All authors contributed to the article and approved the submitted version.

Funding

This study was funded by grants from the National Natural Science Foundation of China (nos. 81772867)

This study was funded by grants from the Youth Science Fund Project of China (nos.81902273)

This study was funded by grants from the Youth Fund of the Second Hospital of Shanxi Medical University of China (nos.202002-4)

This study was funded by grants from the Key plan of Shanxi Province of China (nos.201903D321156)

This study was funded by grants from the Natural Science Foundation of Shanxi Province (nos.201801D121325)

Acknowledgements

Thanks for the experimental conditions provided by Shanxi Key Laboratory of Bone and Soft Tissue Injury Repair for the experimental conditions.

Angelini, Andrea, et al. "The role of 18F-FDG PET/CT in the detection of osteosarcoma recurrence." European Journal of Nuclear Medicine and Molecular Imaging 44.10 (2017): 1712–1720. DOI: 10.1007/s00259-017-3698-0
Wenjian Wang, Xiuchun Yu, Jia Han, et al. "A retrospective analysis of 1593 cases of osteosarcoma epidemiology and treatment." Chinese Journal of Orthopaedics 18 (2018): 1097–1107. DOI: 10.3760/cma.j.issn.0253-2352.2018.18.002
Klein, Michael J., and Gene P. Siegal. "Osteosarcoma: anatomic and histologic variants." American journal of clinical pathology 125.4 (2006): 555–581. DOI: 10.1309/UC6K-QHLD-9LV2-KENN
Choi, Lisa E., et al. "Analysis of outcomes in extraskeletal osteosarcoma: a review of fifty-three cases." The Journal of Bone and Joint Surgery. American Volume 96.1 (2014): e2. DOI: 10.2106/JBJS.M.00339
Jingli Shi, Danhua Shen et al. "Clinicopathological characteristics of primary osteosarcoma in adults: a report of 98 patients over 30 years of age." Tumor 35.8 (2016): 883. DOI: CNKI:SUN:ZZLL.0.2015-08-008
Brown, Hannah K., Marta Tellez-Gabriel, and Dominique Heymann. "Cancer stem cells in osteosarcoma." Cancer letters 386 (2017): 189–195. DOI: 10.1016/j.canlet.2016.11.019
Bednar, Filip, et al. "Bmi1 is required for the initiation of pancreatic cancer through an Ink4a-independent mechanism." Carcinogenesis 36.7 (2015): 730–738. DOI: 10.1093/carcin/bgv058
Arora, Mansi, et al. "RING1A and BMI1 bookmark active genes via ubiquitination of chromatin-associated proteins." Nucleic acids research 44.5 (2016): 2136–2144. DOI: 10.1093/nar/gkv1223
Martins-Neves, Sara R., et al. "Osteosarcoma Stem Cells Have Active Wnt/β‐catenin and Overexpress SOX2 and KLF4." Journal of cellular physiology 231.4 (2016): 876–886. DOI: 10.1002/jcp.25179
Yan, Guang-Ning, Yang-Fan Lv, and Qiao-Nan Guo. "Advances in osteosarcoma stem cell research and opportunities for novel therapeutic targets." Cancer letters 370.2 (2016): 268–274. DOI: 10.1016/j.canlet.2015.11.003
Martins-Neves, Sara R., et al. "Therapeutic implications of an enriched cancer stem-like cell population in a human osteosarcoma cell line." BMC cancer 12.1 (2012): 1–16. DOI: 10.1186/1471-2407-12-139
Jung, Yunjoon, and Jan A Nolta. "BMI1 regulation of self-renewal and multipotency in human mesenchymal stem cells." Current stem cell research & therapy 11.2 (2016): 131–140. DOI: 10.2174/1574888x1102160107171432
López-Arribillaga, Erika, et al. "Bmi1 regulates murine intestinal stem cell proliferation and self-renewal downstream of Notch." Development 142.1 (2015): 41–50. DOI: 10.1242/dev.107714
Rowland, Benjamin D., René Bernards, and Daniel S. Peeper. "The KLF4 tumour suppressor is a transcriptional repressor of p53 that acts as a context-dependent oncogene." Nature cell biology 7.11 (2005): 1074–1082. DOI: 10.1038/ncb1314
Zhang, Lu, et al. "Krüppel-like factor 4 promotes human osteosarcoma growth and metastasis via regulating CRYAB expression." Oncotarget 7.21 (2016): 30990. DOI: 10.18632/oncotarget.8824
Martins-Neves, Sara R., et al. "Osteosarcoma Stem Cells Have Active Wnt/β‐catenin and Overexpress SOX2 and KLF4." Journal of cellular physiology 231.4 (2016): 876–886. DOI: 10.1002/jcp.25179
Huang, Jun, et al. "Krüppel-like factor 4 promotes high‐mobility group box 1‐induced chemotherapy resistance in osteosarcoma cells." Cancer science 107.3 (2016): 242–249. DOI: 10.1111/cas.12864
Qi, Xiao-tian, et al. "KLF4 functions as an oncogene in promoting cancer stem cell-like characteristics in osteosarcoma cells." Acta Pharmacologica Sinica 40.4 (2019): 546–555. DOI: 10.1038/s41401-018-0050-6
Li, Yangling, et al. "Inhibition of KLF4 by statins reverses adriamycin-induced metastasis and cancer stemness in osteosarcoma cells." Stem cell reports 8.6 (2017): 1617–1629. DOI: 10.1016/j.stemcr.2017.04.025

No competing interests reported.

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Expression of stem cell biomarkers Bmi1 and KLF4 in osteosarcoma and its clinical significance

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