Superenhancers activate the autophagy-related genes Beclin1 and LC3B to drive metastasis and drug resistance in osteosarcoma

Abstract

Metastasis and drug resistance are the leading causes of poor prognosis in patients with osteosarcoma. Identifying the relevant factors that drive metastasis and drug resistance is the key to improving the therapeutic outcome of osteosarcoma. Here, we reported that autophagy was highly activated in metastatic osteosarcoma. We found increased autophagolysosomes in metastatic osteosarcoma cell lines by using electron microscopy, Western blot, and immunofluorescence experiments. We further examined the expression of the autophagy-related genes Beclin1 and LC3B in 82 patients through immunohistochemistry and found that Beclin1 and LC3B were highly related to unfavorable prognosis of osteosarcoma. Knockdown of Beclin1 and LC3B reduced invasion, metastasis, and proliferation in metastatic osteosarcoma cells. In vitro and in vivo studies also demonstrated that inhibiting by 3-MA inhibited cell growth and metastasis. Moreover, we demonstrated that autophagy-related genes were activated by SEs and that the inhibition of SEs by JQ-1 decreased the metastasis of osteosarcoma. Overall, our findings highlighted the association of autophagy with osteosarcoma progression and shed new light on autophagy-targeting therapy for osteosarcoma.

Keywords

osteosarcoma / autophagy / metastasis / drug resistance / Beclin1 / LC3B

1 Introduction

Osteosarcoma is the most common primary malignant bone tumor in children and adolescents [1]. With the development of surgical techniques and chemotherapy, the survival rate of osteosarcoma has increased by nearly two decades. Studies show that the 5-year overall survival rate of osteosarcoma is approximately 70% [1–3]. However, the clinical outcome of osteosarcoma with metastasis and local recurrence remains unsatisfactory, and the current 5-year overall survival rate of metastatic or relapsed osteosarcoma is only approximately 20% [4–6]. Thus, identifying the factors contributing to the metastasis and local recurrence of osteosarcoma is key to improving the overall therapeutic outcome.

Autophagy is a common cellular catabolic degradation machinery in response to nutrient starvation or stress conditions [7,8]. The main function of this process is to maintain metabolic homeostasis through the selective degradation of unfolded or aggregated proteins and damaged or excess organelles [8]. Autophagy is a complicated regulatory process and is controlled by many proteins. Generally, autophagy is initiated by cellular stress and AMPK activation. The class III phosphatidylinositol 3-kinase (PI3K) VPS34, Beclin1, ATG14, and VPS15 help form autophagosomes [9–11]. The cytosolic form of microtubule-associated protein 1 light chain 3 LC3 (MAP1LC3), also known as LC3-I, conjugates to phosphatidylethanolamine and forms an LC3-phosphatidylethanolamine conjugate (LC3-II) [12,13]. LC3-II is recruited to the membranes of autophagosomes, which then fuse with lysosomes, form autophagolysosomes, and complete the degradation of proteins inside autophagolysosomes, including LC3 itself. Initiation of the autophagosome, fusion of lysosomes with autophagosomes, and degradation in autophagolysosomes induce autophagic flux [14].

Currently, the functions of autophagy in cancer remain controversial [15–17]. On one hand, autophagy is known as type II programmed cell death [18], and overactivated autophagy may trigger the death program and lead to cell death. On the other hand, emerging evidence has demonstrated that autophagy contributes greatly to the progression and drug resistance of malignant disease and that targeting autophagy contributes to cancer therapy [17]. Tumor cells usually undergo stressful conditions, such as hypoxia and limited nutrition [19,20]. Activated autophagy may help tumor cells overcome such situations. Radiotherapy and chemotherapy, as common neoadjuvant treatments for osteosarcoma, can also cause tumor-cell stress [21], such as disorders in protein folding, mitochondrial damage, and endoplasmic reticulum stress. Autophagy may contribute to maintaining the organization of cellular function and confer resistance to anticancer therapy.

Several studies have revealed that autophagy is involved in osteosarcoma regulation, but the conclusion is controversial. On one hand, autophagy provides osteosarcoma cells with energy and nutrients in the stressful microenvironment of tumors, such as hypoxia and starvation. Additionally, autophagy is activated during chemotherapy, such as with cisplatin, in osteosarcoma [22]. Some studies have found that autophagy inhibition by rapamycin leads to autophagy-mediated cell death in osteosarcoma [23,24]. On the other hand, other studies have shown that autophagy induction, such as treatment with apatinib, also leads to cell death in osteosarcoma [25]. Apparently, the perturbation of autophagy, whether induction or decrement, leads to cell death of osteosarcoma, which may depend on the basal autophagic status of osteosarcoma cells. However, current studies on autophagy and osteosarcoma have focused mostly on drug innovation, and the basal status of autophagy in osteosarcoma and its relationship with disease progression and prognosis remain unclear.

In the present study, we reported that autophagy was activated in osteosarcoma and that metastatic osteosarcoma showed higher levels of autophagic flux. We also reported that high levels of autophagy were associated with poor prognosis of osteosarcoma and that autophagy inhibition sensitized osteosarcoma patients to chemotherapy. We further reported that osteosarcoma-related genes were activated by superenhancers and that the inhibition of these superenhancer decreased metastasis in osteosarcoma.

2 Materials and methods

2.1 Enrollment

Eighty-two patients with osteosarcoma were enrolled in this study. Tumor tissues were obtained after surgical removal. This study was approved by the ethics committee of Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine and was conducted according to the Declaration of Helsinki and its amendments. Informed consent of the patients was acquired.

2.2 Cell lines

U2OS, SaOS2, HOS, and 143B cells were provided by American Type Culture Collection. These cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) (04-001-1ACS, Biological Industries, Beit Haemek, Israel). NW5 was a primary osteosarcoma cell line established by our laboratory. In a typical procedure, three pieces of fresh osteosarcoma tumor tissue after surgical operation were cut and washed five times with PBS. Single-cell suspensions were obtained and transferred onto a 6 cm culture plate. Cells were cultured in DMEM (SH30022.01B, HyClone, Logan, UT, USA) supplemented with 10% fetal calf serum, amphotericin B, and penicillin/streptomycin. After five passages, the cell mix was seeded onto a 96-well plate to obtain a single clone. Six clones were collected, and metastatic ability was assessed. The cells with the highest metastatic ability were named NW5 cells.

2.3 Western blot

Cultured cells were washed with PBS for three times, and 1× 10⁵ cells were lysed in 100 μL 2× loading buffer (100 mmol/L Tris-Cl (pH6.8), 200 mmol/L DTT, 4% SDS, 20% glycerol, 0.1% bromphenol blue sodium salt). Whole-cell lysates were heated three times at 100 °C for 5 min. Whole-cell lysates were used for Western blot experiments. Antibodies against LC3B (CST3868S, Cell Signaling Technology, USA), Beclin1 (CST3738S, Cell Signaling Technology), GAPDH (SC25778, Santa Cruz Technology), and P-ULK1 (CST5869S, Cell Signaling Technology) were used.

2.4 siRNA-mediated knockdown of LC3B and Beclin1

siRNA targeting LC3B and Beclin1 was transduced into 143B cells with Lipofectamine 2000 (11668027, Thermo Fisher, USA) according to the manufacturer’s instructions. The sequences of the siRNAs were as follows: si-LC3B-sense, 5′-GCCCUCUACUGAUUGUUAATT-3′; si-LC3B-antisense, 5′- UUAACAAUCAGUAGAGGGCTT-3′; si-Beclin1-sense, 5′-CAGUUUGGCACAAUCAAUA-3′; and si-Beclin1-antisense, 5′-UAUUGAUUGUGCCAAACUG-3′.

2.5 Immunofluorescence and immunochemistry (IHC) staining

IHC experiments were performed as previously described [26]. Immunofluorescence experiments were performed according to the manufacturer’s instructions for the Immunol Fluorescence Staining Kit (P0179, Beyotime, China). The IHC staining scores were calculated by multiplying the positive staining region (ranging from 0 to 4) and the positive levels (ranging from 0 to 4). Scores > 9 were considered as the highly expressed group, whereas scores < 9 were considered as the lowly expressed group.

2.6 Electron microscopy

Electron microscopy experiments were performed as previously described [27]. In a typical procedure, cells were fixed with 2% glutaraldehyde fixative solution for 2 h, washed, and used for electron microscopy analysis. A Philips CM120 electron microscopy device was used.

2.7 CCK-8 analysis

The CCK-8 analyses were performed according to the manufacturers’ instructions (CK04-500, Dojindo, Japan). OD values were determined with a Multiskan™ FC microplate photometer (Thermo Fisher) at 450 nm.

2.8 Wound-healing assays

Wound-healing assays were performed as previously described [28]. In a typical procedure, 143B cells were cultured in a 6-well plate in DMEM supplemented with 10% FBS. siRNAs targeting Beclin1, LC3B, or nontargeting siRNA were transfected into 143B cells with Lipofectamine 2000 (Invitrogen). The cells were scratched with a pipette tip after 18 h of transfection. Cell migration into the gap was imaged 24 h after scratching.

2.9 Transwell assays

Transwell assays were performed as previously described [28]. siRNA targeting Beclin1, LC3B, or nontargeting siRNA-transduced 143B cells was used for the Transwell assays. Matrigel (50 μL, BD) was added to the upper chamber of the Transwell plate before cell seeding. Cells (1 × 10⁵) cultured in DMEM supplemented with 2% FBS were seeded in the top chamber. DMEM supplemented with 10% FBS was added to the bottom chamber. The metastasized cells across the Matrigel were stained with crystal violet.

2.10 Xenograft experiment

The animal experiments were approved by the Institutional Animal Care and Use Committee of Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine and were conducted in accordance with institutional animal protocols. NW5 cells were stably transfected with luciferase expression plasmid by lentiviral transfection, and Luc-NW5 cells were obtained. Luc-NW5 cells were injected to NOD-SCID mice paratibially. Twenty days after xenografting, nice tumor-burdened mice were randomly divided into two groups and treated with 3-methyladenine (3-MA) or placebo. Tumor length and width were examined every five days post-injection by using a caliper. Tumor volume was calculated by length × (width)²/2.

2.11 Reanalysis of H3K27ac ChIP-seq in osteosarcoma cells

H3K27ac ChIP-seq data sets in osteosarcoma cells, including metastatic (LM7, MNNG, 143B, and MG63.3) and nonmetastatic (MG63, Saos-2, Hu09, and HOS) cells, were downloaded from the GEO database (GSE74230) [29]. Raw reads were mapped to the human genome (hg38) with bowtie2 [30]. H3K27ac peaks were called by MACS2 [31] and merged by bedtools [32]. H3K27ac signals of each cell under distinct peaks were calculated by homer2 [33]. Differential peaks were identified by fold-change and P value (t-test). Representative H3K27ac signals among the regulatory regions were visualized using the WashU browser.

2.12 Statistical analysis

All statistical analyses were performed with RStudio. For the survival analysis between two groups, log-rank tests were used. The Kaplan–Meier plot was generated with the Plot function in R. To compare differences between two groups such as autophagosome numbers, cell growth, and tumor sizes, two-tailed and unpaired Student’s t-tests were performed.

3 Results

3.1 Autophagy-related genes were highly expressed in metastatic osteosarcoma

To determine the autophagic status of osteosarcoma and its relationship with malignancies, we first performed electron microscopy analysis, the gold standard for determining autophagosomes, in metastatic and nonmetastatic osteosarcoma cells. We observed that NW5 and 143B cells (identified as metastatic osteosarcoma in previous studies [29,34]) had more autophagosomes and more autophagolysosomes than U2OS cells (identified as nonmetastatic osteosarcoma in previous studies [29,34]) under steady status (Fig.1–1D). We also found that autophagosomes increased and autophagolysosomes decreased upon bafilomycin A1 (BafA1) treatment in 143B and NW5 cells. This finding indicated that BafA1 could indeed block autophagosome-lysosome fusion, as well as support high levels of autophagy and autophagic flux in metastatic osteosarcoma.

We subsequently examined the expression of two well-established autophagy-related genes, LC3B and Beclin1, in metastatic (143B and NW5) [29,34] and nonmetastatic (SaOS2, U2OS, and HOS) osteosarcoma cell lines [29,34]. LC3B and Beclin1 expression was much higher in metastatic osteosarcoma cells than in nonmetastatic ones (Fig.1 and S1). Considering that lower levels of autophagic markers may also be observed in the rapid degradation of the autolysosome, we further examined autophagic flux by blocking autophagosome–lysosome fusion by using BafA1. Consistent with the results of Western blot experiments, the basal expression of LC3B was much higher in metastatic (143B and NW5) than in nonmetastatic (U2OS and SaOS2) osteosarcoma cells (Fig.1). Moreover, upon treatment with the autophagosome-lysosome fusion inhibitor BafA1, stronger expression and colocalization of LC3B with lysosomes were observed in 143B and NW5 cells but not in U2OS cells or SaOS2 cells (Fig.1). This finding indicated strong autophagic flux and autophagy in metastatic osteosarcoma cells.

3.2 High levels of Beclin1 and LC3B were related to poor prognosis of osteosarcoma

To further investigate the correlation between autophagy and the clinical outcome of osteosarcoma, we performed IHC staining with antibodies against Beclin1 and LC3B in 82 osteosarcoma samples. We found that Beclin1 and LC3B were highly expressed in the majority of osteosarcoma cells, especially osteosarcomas with adverse outcomes (Fig.2 and 2B), suggesting that activated autophagy may be a malignancy factor of osteosarcoma.

The 82 osteosarcomas were further divided into groups according to their Beclin1 or LC3B expression level based on the IHC staining score, and overall survival and metastasis-free survival were examined. As shown in Fig.2 and 2D, high levels of LC3B (P < 0.0001, hazard ratio = 12.53, by log-rank test) and Beclin1 (P < 0.0001, hazard ratio = 6.471, by log-rank test) were significantly related to poor metastasis-free survival. This finding suggested that activated autophagy pathways may cause the metastasis of osteosarcoma cells. Additionally, high levels of LC3B (P < 0.0001, hazard ratio = 6.936, by log-rank test) and Beclin1 (P < 0.0001, hazard ratio = 3.130, by log-rank test) were also significantly related to poor overall survival in osteosarcoma (Fig.2 and 2F). Additionally, these observations strongly suggested that the autophagic factors Beclin1 and LC3B could be used as prognostic markers for osteosarcoma.

3.3 Knockdown of Beclin1 and LC3B or inhibition of autophagy could suppress cell growth and the invasion of osteosarcoma cells

To further investigate the function of autophagic factors in osteosarcoma metastasis, we knocked down the expression of LC3B and Beclin1 in 143B cells (a metastatic osteosarcoma cell line) and detected their effects on cell growth, cell migration, and metastasis. Cell-proliferation assays, wound-healing assays, and Transwell assays showed that LC3B and Beclin1 knockdown in 143B cells reduced cell growth, migration, and metastasis (Fig.3–3D).

We also used 3-MA, a type III phosphatidylinositol 3-kinase inhibitor that blocks autophagosome formation, to inhibit autophagy in osteosarcoma cells. Similar results were observed with LC3B and Beclin1 knockdown (Fig.3 and 3F).

We also analyzed the effects of autophagy inhibition on tumor growth of osteosarcoma in vivo. We established a xenograft model of osteosarcoma using Luc-NW5 cells in NOD-SCID mice via paratibial injection. We found that treatment with the autophagy inhibitor 3-MA significantly reduced the tumor sizes (Fig.4 and 4B).

3.4 Targeting autophagy-sensitized osteosarcoma cells to chemotherapy

We then investigated autophagy in osteosarcoma and its correlation with chemotherapeutic outcome. We first determined the autophagy status post chemotherapy of osteosarcoma by electron microscopy. Results showed that chemotherapy with cisplatin and doxorubicin further increased autophagolysosomes in NW5 cells (Fig.5 and 5B), indicating that the autophagy level further increased upon chemotherapy.

We next knocked down LC3B and Beclin1 in NW5 cells with or without doxorubicin. We found that LC3B and Beclin1 knockdown enhanced the cell growth inhibition of doxorubicin (Fig.5 and 5D), indicating that autophagy inhibition could sensitize osteosarcoma to chemotherapy. Additionally, we treated NW5 cells with 3-MA, an autophagy inhibitor. We found that 3-MA treatment resulted in decreased IC50 of doxorubicin on NW5 cells, indicating that inhibition of autophagy could be synergistic with chemotherapy on the inhibition of osteosarcoma cell growth (Fig.5).

To further confirm whether the synergistic effects of 3-MA were attributed to autophagic regulation, we detected the expression of LC3B upon treatment with 3-MA and doxorubicin. Doxorubicin increased the expression of LC3B, whereas 3-MA decreased the expression of LC3B (Fig.5 and S2), supporting the notion that chemotherapy increased autophagy in osteosarcoma. We also found that the blockade of autophagosome-lysosome fusion by using BafA1 cannot reverse the 3-MA-induced decrease in LC3B, indicating that autophagic flux was blocked. Thus, the 3-MA-induced sensitization to chemotherapy was attributed to its regulation of autophagy. Together, these results indicated that autophagy inhibition partially inhibited the metastatic ability and enhanced the chemotherapeutic effect in osteosarcoma.

3.5 Enhancers of autophagy-related genes were highly activated in metastatic osteosarcoma

Next, we investigated the mechanisms underlying the activation of potential factors that contributed to osteosarcoma metastasis. We first analyzed the transcription features of these autophagy-related genes, such as BECN1, MAP1LC3B (LC3B), MAP1LC3A (LC3A), and ATG5. We found that H3K27ac signals were abundantly enriched at the regulatory regions of these cells (Fig.6). Given that abundant H3K27ac signals are reportedly associated with superenhancer regulation, superenhancers have been reported to be involved in the malignant transformation of many cancers. Thus, we further analyzed the superenhancer effect in metastatic and nonmetastatic osteosarcoma cells, including MG63, SaOS-2, Hu09, and HOS cells. Interestingly, we found that BECN1, MAP1LC3B, MAP1LC3A and ATG5 were all regulated by superenhancer in metastatic osteosarcoma cells but not in nonmetastatic osteosarcoma cells (Fig.6). These results indicated that transcriptional activation by superenhancer may be involved in overactivated autophagic features in osteosarcoma.

3.6 Inhibition of the superenhancer-inhibited cell invasion of osteosarcoma

Finally, to determine whether autophagy could be inhibited by superenhancers, we treated 143B and NW5 cells with JQ-1 (a BRD4 inhibitor) and found that the expression of BECN1, MAP1LC3B, MAP1LC3A, and ATG5 significantly decreased post-treatment (Fig.7). Invasion ability was also significantly reduced with JQ-1 treatment (Fig.7), further supporting the notion that autophagy played a vital role in osteosarcoma.

4 Discussion

Metastatic osteosarcoma is a life-threatening malignant disease in children and adolescents. Unveiling the factors contributing to metastatic and malignant transformation is required to improve the diagnosis and prediction of disease progression. Here, we reported that highly activated autophagy was the hallmark of metastatic osteosarcoma. We showed that higher levels of autophagic flux were associated with poor prognosis and metastasis of osteosarcoma. Targeting autophagy sensitized osteosarcoma to chemotherapy.

Lines of evidence have demonstrated that targeting autophagy inhibits the cell growth of osteosarcoma, but little is reported on the relationship between autophagy and metastasis of osteosarcoma. Previous studies testing the effects of autophagy inhibitors on osteosarcoma cells have demonstrated that targeting autophagy with spautin-1 (Beclin1 inhibitor) [35], geldanamycin (Hsp90 inhibitor) [36], and rapamycin (mTOR inhibitor) [24] inhibits the growth of osteosarcoma cells. Additionally, factors contributing to drug resistance in osteosarcomas such as HSP90AA1 and GFRA1 regulate resistance by promoting autophagy. Thus, enhanced autophagy is a negative signal for osteosarcoma therapy and a positive factor for osteosarcoma progression. Herein, we found that targeting autophagy by 3-MA could suppress tumor growth in vivo, although some limitations of this approach remain, including the following: (1) the method for tracking tumor size may be inaccurate due to the depth of the tumor site and the implications of nontumor tissues (such as skin, muscle, and bone), which should be improved in the future; and (2) the effect of 3-MA on metastasis must be explored. We further reported that targeting autophagy by 3-MA enhanced the effects of chemotherapy in metastatic osteosarcoma cells. Thus, targeting autophagy can also be effective in metastatic osteosarcoma.

Beclin1 is an autophagy-related gene homologous to ATG6 [10,37]. It is a subunit of the phosphatidylinositol-3-kinase complex and is responsible for the formation of phosphatidylinositol 3-phosphate [10]. It has been intensively studied as a key factor in autophagy and the development of tumors [38]. The induction of Beclin1 can reportedly inhibit tumorigenesis via autophagy [39]. Recently, higher levels of Beclin1 have been shown to contribute to drug resistance in malignant diseases through its regulation of autophagy. Studies on osteosarcoma have also demonstrated that increased levels of Beclin1 contribute to protective autophagy in response to paclitaxel-mediated chemotherapy [40]. Downregulation of Beclin1 has further been shown to increase the sensitivity of gemcitabine-induced cell death [41]. Lines of evidence have demonstrated that a high level of Beclin1 is correlated with favorable prognosis in breast [42] and gastric [43] cancers, whereas a higher level of LC3 is correlated with poorer prognosis in breast cancer [44]. Herein, we reported that Beclin1 and LC3 were highly expressed in osteosarcoma with poor prognosis, indicating that the functions of Beclin1 and LC3 could be versatile. Increased levels of Beclin1 and LC3 resulting in autophagy should be one of the key factors determining the metastasis and poor prognosis of osteosarcoma.

Superenhancers are cis-regulatory elements and regulators of cell identity. They are characterized by abundant BRD4 and MED1 binding and H3K27ac signals. Lines of evidence have unveiled the contribution of superenhancer to malignant transformations. A superenhancer drives the high expression of oncogenes such as MYC [45–47], HOXB8 [48], and CCAT1 [49] in malignancies. Small chemical compounds targeting BRD4 such as JQ-1 can decrease BRD4 activity and are recognized as inhibitors of superenhancers [50,51]. Previous studies have reported that JQ-1 can suppress the proliferation of U2OS and 143B cells by inhibiting the MYC oncogene [52]. Herein, we reported that autophagy-related genes were regulated and activated by superenhancer, and that JQ-1 decreased the expression of these genes. Additionally, JQ-1 inhibited osteosarcoma metastasis. To date, targeting superenhancers in osteosarcoma is rarely reported. Accordingly, we suggested that the inhibition of superenhancers may decrease osteosarcoma metastasis by reducing the expression of autophagy-related gene.

Our study highlighted that autophagy contributed to metastasis and poor prognosis of osteosarcoma. Functional studies indicated that targeting autophagy may also be a potential approach to metastatic-osteosarcoma therapy, and more evidence is needed in future studies.

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