Upregulated ATG4B Predicts Poor Prognosis and Correlates with Angiogenesis in Osteosarcoma

doi:10.21203/rs.3.rs-4813346/v1

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Upregulated ATG4B Predicts Poor Prognosis and Correlates with Angiogenesis in Osteosarcoma

https://doi.org/10.21203/rs.3.rs-4813346/v1

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Background: Osteosarcoma (OS) is the most common primary bone cancer in children and adolescents. Patients with metastatic OS experience significantly poorer outcomes, largely due to resistance to chemotherapy. Between 35-45% of these patients do not respond to standard chemotherapeutic treatments, resulting in a very low 5-year survival rate of only 5-20%. This resistance often leads to treatment failures and unfavorable prognoses, highlighting the critical need for new therapeutic targets to improve treatment strategies. Autophagy-related gene 4 B (ATG4B) is a crucial cysteine protease for autophagosome formation. It is overexpressed and correlates with poor prognosis in various cancers. However, the relationship between ATG4B expression and angiogenesis in osteosarcoma remains unexplored. This study investigates the expression levels of ATG4B and VEGF in osteosarcoma and their correlation with clinicopathological data.

Materials and Methods: The study included 67 paraffin-embedded osteosarcoma tissue samples. ATG4B and VEGF expression levels were assessed via immunohistochemistry, and their associations with clinicopathological variables were statistically analyzed. Additionally, ATG4B gene expression in osteosarcoma was examined using GEO data sets from https://www.ncbi.nlm.nih.gov.

Results:The analysis showed that ATG4B and VEGF were expressed in 79.1% and 74.6% of the osteosarcoma samples, respectively. There was a significant positive correlation between ATG4B expression and tumor size, tumor stage, and histological response to neoadjuvant chemotherapy, with p-values of 0.013, 0.008, and 0.022, respectively. VEGF expression also significantly correlated with tumor size, tumor stage, and the presence of distant metastasis at diagnosis, with p-values of 0.022, 0.044, and 0.013, respectively. A notable positive correlation between ATG4B and VEGF expression levels was observed (p=0.002), supported by the GEO dataset analysis.

Conclusions: The results suggest that ATG4B acts as a tumor promoter in osteosarcoma, indicating its potential as a therapeutic target to inhibit tumor growth. Elevated ATG4B levels may also serve as a marker for poor prognosis. Additionally, VEGF overexpression is linked to a higher likelihood of pulmonary metastasis and worse overall prognosis. The positive correlation between ATG4B and VEGF suggests that the absence of both markers could be indicative of a better chemotherapy response, offering insights into potential new treatment approaches.

ATG4B

VEGF

Autophagy

Angiogenesis

Osteosarcoma

Osteosarcoma (OS) represents the predominant primary bone malignancy in children and adolescents, constituting 3–4% of all malignancies [1] and approximately 15% of bone malignancies [2]. It is a disastrous tumor characterized by brisk growth, local aggressiveness, and early pulmonary metastasis [3]. Till now, Patients with metastases face significantly poorer outcomes, largely attributed to chemotherapy drug resistance [4, 5]. A substantial portion of OS patients, ranging from 35–45%, do not respond to chemotherapeutic drugs, resulting in a mere 5–20% 5-year survival rate. Chemoresistance frequently leads to treatment failure and unfavorable prognoses [6, 7]. Consequently, there is a pressing need to explore novel therapeutic targets for the development of innovative treatment strategies in OS.

Autophagy, a conserved process that facilitates the degradation and recycling of altered cellular components and organelles, has been demonstrated to promote cell survival or cell death, underscoring its critical role in tumor development [8, 9]. The role of autophagy in various cancers remains a subject of debate; its impact on cancer is multifaceted and contingent upon the tumor's stage and type. While autophagy can function as a tumor suppressor, it may also act as a tumor promoter by enhancing the survival of cancer cells under conditions of nutrient deprivation stress [10, 11]. Moreover, numerous researchers have proved autophagy-mediated chemotherapy resistance during cancer treatment [12, 13].

Autophagy-related gene 4 (ATG4) serves as a cysteine protease crucial for autophagosome formation, comprising four homologues—ATG4A, ATG4B, ATG4C, and ATG4D. Particularly, ATG4B functions as an essential protease, facilitating the cleavage of proLC3 or lipidated LC3-II for proper autophagosome formation [14]. Previous investigations have revealed that ATG4B is overexpressed and correlates with poor prognosis in chronic myeloid leukemia, breast cancer, gastric cancer and oral cancer [15–18]. Additionally, accumulating studies suggest an oncogenic role for ATG4B in colorectal carcinoma and glioblastoma [19, 20]. Inhibition of ATG4B activity has been shown to reduce cancer cell viability and enhance the sensitivity of cancer cells to chemotherapeutic agents both in vitro and in vivo [21–23].

A few studies have explored of ATG4B in OS. Interestingly, OS Saos-2 cells lacking ATG4B exhibited autophagy defects and failed to develop tumors in mouse models. Moreover, the resistance of OS cell lines to doxorubicin, cisplatin, and methotrexate has been attributed to autophagy induction [23]. Given the potential of autophagy-based strategies in targeted tumor therapies and the importance of understanding the clinical relevance of ATG4B in OS patients, we conducted a comparative analysis of ATG4B protein levels using immunohistochemistry alongside clinical outcomes in OS patients.

Angiogenesis, a hallmark of tumors, plays a vital role in facilitating sustained tumor growth and dissemination [24]. This process is tightly regulated by a delicate balance between pro-angiogenic and anti-angiogenic factors. Among these factors, VEGF (vascular endothelial growth factor), a homodimeric protein, acts as a specific mitogen for endothelial cells and is often found to be overexpressed in various inflammatory diseases and tumors. Notably, the tumor microenvironment is largely influenced by the VEGF-VEGFR2 axis [25–27].

Emerging evidence sheds light on the crucial role of autophagy in endothelial cells (ECs). Moreover, genetic findings in recent years indicate that autophagy regulates pathological angiogenesis, a hallmark of solid tumors. In the hypoxic, nutrient-deprived, and pro-angiogenic tumor microenvironment (TME), heightened autophagy in blood vessels is emerging as a critical mechanism enabling endothelial cells to evade hypoxia-induced cell death [27]. However, the relation between ATG4B expression and angiogenesis in tumor cells is still not elucidated. Therefore, utilizing immunohistochemistry and gene microarray analysis, this study aimed to investigate the expression levels of ATG4B and VEGF in OS and their relation with clinicopathological data. Additionally, the study examined the correlations among the immunohistochemical markers.

2.1. Patients

This study included a total of 67 primary osteosarcoma (OS) patients, which were randomly selected. Those patients were sourced from the Pathology Department of Minia University Hospital and Minia Oncology Center in Egypt, spanning from the years 2016 to 2022. Clinical data and follow-up information were retrieved from the patients' medical records with the approval of the respective hospital districts. Furthermore, the study protocol received approval from the Ethics Committee of Minia University, Faculty of Medicine, Institutional Review Board (MUFMIRB). Patients included in the study underwent both chemotherapy and surgical treatment as part of their management regimen.

2.2. Histopathological Evaluation

Representative hematoxylin and eosin (H&E) stained slides for all cases were meticulously reviewed to validate the original diagnosis. Staging was performed following the Enneking staging system for malignant musculoskeletal tumors [28]. Additionally, the response to preoperative neoadjuvant chemotherapy was histologically evaluated based on the Huvos grading system [29]. Patients received neoadjuvant chemotherapy based on the SEOP-95 regimen, which comprised high-dose methotrexate, adriamycin, cisplatin, and ifosfamide. Overall survival was calculated in months from the date of diagnosis to the time of the tumor-related death or the last follow-up visit of the patient.

2.3. Expression of ATG4B in a publically available dataset. (Data set analysis)

The expression of ATG4B was investigated in OS samples from Gene Expression Omnibus database (GEO2R) data sets on https://www.ncbi.nlm.nih.gov. Gene expression values were extracted from every data set by comparing two groups e.g. OS and adjacent non tumor tissue then the value of expression of the gene in the microarray was calculated using GEO2R. The following data sets were investigated GSE126209 and GSE56001. The analysis was performed as described in previous research [30].

2.4. Immunohistochemistry

Immunohistochemistry was conducted on 4-µm tissue sections mounted on positively charged slides. The sections underwent deparaffinization and rehydration through xylene and graded ethanol solutions. To block endogenous peroxidase activity, the sections were treated with hydrogen peroxide for 20 minutes. Antigen retrieval was performed by treating sections with 0.1 mol/L citrate buffer (pH 6.0) in a 700-W microwave oven for 20 minutes. Subsequently, sections were incubated overnight at 4ºC with primary antibodies: ATG4B (dilution 1:100; A2981, Sigma-Aldrich, St. Louis, MO, USA) and monoclonal mouse VEGF antibody Ab-7 (clone VG1, ready to use, Thermo Fisher Scientific/ Lab vision corporation, USA). The reaction was visualized using the avidin-biotin detection kit with diaminobenzidine (DAB) as the chromogen. Finally, sections were counterstained with Mayer's hematoxylin.

For negative controls, nonspecific activity was evaluated by omitting the primary antibodies and incubating the slides with phosphate-buffered saline (PBS). Human normal kidney tissue served as a positive control for the ATG4B antibody, while endothelial cells of capillaries within the stained sections served as an internal positive control for the VEGF antibody.

2.5. Immunohistochemical scoring

All slides were reviewed and independently scored by two pathologists. The ATG4B immunoreactivity of the tumor cells was measured using a semi-quantitative estimation; a score was obtained by adding the values of the percentage and intensity of stained tumor cells. The intensity was graded as 0 negative; 1 weak; 2 moderate and 3 intense staining. The percentage of positive cells was graded from 0 to 3 (0 nil; 1: 1–25%; 2: 26 − 50%; 3: >51%). lowATG4B expression was defined if the score was between 0 and 4, while cases with scores 5 or 6 were considered high ATG4B expression [31]. Regarding VEGF immunoreactivity, scoring was done based on the percentage of positive tumor cells; high VEGF expression (> 30% of tumor cells) and low VEGF expression (≤ 30% of tumor cells) [30].

2.6. Statistical analysis

Chi-square and Fisher’s exact tests were employed to detect differences in categorical variables. Results were considered statistically significant when the p-value was < 0.05. Patient survival and differences were assessed using the Kaplan–Meier method. A multivariate Cox proportional hazards regression analysis was conducted to evaluate the specific impact of each variable on survival in the presence of other variables. Data analysis was performed using the Statistical Package for Social Sciences (SPSS) version 21 software.

3.1. Expression of ATG4B in a publically available dataset. (Data set analysis)

Comparing the expression of ATG4B in osteosarcoma tissue and surrounding non- tumor tissue, a significant increase in ATG4B gene expression was found in osteosarcoma tissue in two data sets GSE126209 and GSE56001 (P < 0.0001 and P = 0.0075 respectively) (Fig. 1).

3.2. Patient and tumor characteristics

In the current study, the mean age of patients was 14 years with a standard deviation of 1.9 years. The median age was 12 years, with a range of 9 to 18 years. Tumor size was categorized into two groups: ≤ 9cm and > 9cm, based on the median value. Among the patients, 56.7% (38 out of 67) received neoadjuvant chemotherapy. Among these patients, 13 were classified as poor responders (necrosis less than 90%), while 25 were classified as good responders (necrosis equal to or more than 90%). Detailed characteristics of the patients are provided in Table 1.

Table 1

Clinicopathological features of OS patients.
Clinicopathological Features (N = 67)	Number %
Age at diagnosis ≥ 14 years > 14 years	37 (55.2) 30 (44.8)
Gender Male Female	43 (64.2) 24 (35.8)
Location of tumor Extremities Axial	61(91) 6 (9)
Tumor Size ≤ 9cm > 9cm	43 (64.2) 24 (35.8)
Histological subtype Osteoblastic Chondroblastic Fibroblastic	37 (55.2) 26 (38.8) 6 (6)
Stage I II III	4 (6) 50 (74.6) 13 (19.4)
Distant Metastases at the onset of diagnosis No Yes	54 (80.6) 13 (19.4)
Histological response (n.=38) ≥ 90% < 90%	21(55.2) 17 (44.8)

3.3. Immunohistochemical results of ATG4B and VEGF

ATG4B protein expression was observed in the cytoplasm of tumor cells. Out of 67 OS patients tested, 53 of them (79.1%) exhibited high ATG4B immunoreactivity. Significant positive relations were identified between high ATG4B expression and tumor size, stage, and neoadjuvant chemotherapy histological response (with p-values of 0.013, 0.008, and 0.022, respectively) (Fig. 2a-c).

No significant relation was identified between the expression of ATG4B and patients' age, sex, or histological subtype. Moreover, no significant difference in ATG4B expression was noted based on the tumor's location or the presence of distant metastasis at the time of diagnosis. However, a higher incidence of high ATG4B expression was observed in axial osteosarcoma compared to extremity osteosarcoma (100% versus 77%), and in metastatic osteosarcoma compared to non-metastatic osteosarcoma (92.3% versus 75.9%).

Positive cytoplasmic VEGF immunoreactivity was observed in 50 out of 67 tumors (74.6%). The expression of VEGF was found to be significantly associated with tumor size, tumor stage and the presence of distant metastasis at the time of diagnosis (with p-values of 0.022, 0.044 and 0.013, respectively) (Fig. 2d-f). There was no significant association detected between VEGF expression and other clinicopathological features. The relationships of ATG4B and VEGF expression with different clinicopathological features were summarized in Table 2.

Table 2

Associations of ATG4B and VEGF with clinicopathological data.
Clinicopathalogical characteristics	N	ATG4B		P value	VEGF		P value
Clinicopathalogical characteristics	N	High	Low	P value	Positive	Negative	P value
Age at diagnosis ≥ 14 years > 14 years	37 30	28 (75.6%) 25 (83.4%)	9 (24.4%) 5 (16.6%)	0.40	29 (56.7%) 21 (70%)	16 (43.3%) 9 (30%)	0.67
Gender Male Female	43 24	32 (74.4%) 21 (87.5%)	11 (25.6%) 3 (12.5%)	0.20	30 (69.3%) 20 (83.3%)	13 (30.7%) 4 (16.7%)	0.22
Location of tumor Extremities Axial	61 6	47 (77%) 6 (100%)	14(23%) 0	0.18	44 (72.1%) 6 (100%)	17 (27.9%) 0	0.13
Tumor Size ≤ 9cm > 9cm	43 24	38 (88.4%) 15 (62.5%)	5 (11.6%) 9 (37.5%)	0.01	36 (83.7%) 14 (58.3%)	7 (16.3%) 10 (41.7%)	0.02
Histological subtype Osteoblastic Chondroblastic Fibroblastic	37 26 6	29 (78.3%) 20 (76.9%) 4 (66.7%)	8 (21.7%) 7 (23.1%) 2 (33.3%)	0.48	27 (72.9%) 18 (69.2%) 5 (83.3%)	10 (27.1%) 8 (30.8%) 1 (16.7%)	0.41
Stage I II III	4 50 13	1 (25%) 40 (80%) 12 (92.3%)	3 (75%) 10 (20%) 1 (7.7%)	0.01	2 (50%) 35(70%) 13 (100%)	2 (50%) 13 (30%) 0	0.04
Distant Metastases at the onset of diagnosis No Yes	54 13	41 (75.9%) 12 (92.3%)	13 (24.1%) 1 (6.7%)	0.19	38 (70.4%) 12 (92.3%)	14 (29.6%) 1 (6.7%)	0.01
Histological response (n.=38) ≥ 90% < 90%	21 17	15 (71.4%) 16 (94.1%)	6 (28.6%) 1 (5.9%)	0.04	14 (66.7%) 13 (76.5%)	7 (33.3%) 4 (23.5%)	0.50
Test of significance: Chi-square test and Fisher’s exact test, P-value < 0.05 is considered significant.

3.4. The relationship between ATG4B and VEGF

A statistically significant positive correlation was found between ATG4B and VEGF expression (p = 0.002), where a significant proportion of VEGF positive cases (65.6%) showed high ATG4B expression scores.

Moreover, the combined expression patterns of both ATG4B and VEGF regarding the histological response to chemotherapy in each tumor was studied, we found significant association between low ATG4B and negative VEGF expression (P = 0.02) and good histological response to chemotherapy, where all tumors showed low/negative expression for both markers, associated with good histological response to chemotherapy. (Table 3)

Table 3

Combined ATG4B and VEGF expression in association with histological response to chemotherapy
	≥ 90% necrosis	< 90% necrosis	P value
High ATG4B/ positive VEGF	12 (50%)	12 (50%)	0.025
Low ATG4B/ negative VEGF	4 (100%)	0
High ATG4B/ negative VEGF or Low ATG4B/ positive VEGF	9 (90%)	1 (10%)
Test of significance: Chi-square test, P-value < 0.05 is considered significant.

3.5. Follow-up and survival analysis

After a median follow-up of 5 years (range, 10 to 60 months), 4 patients (6%) were lost to follow-up during the study period. At the initial presentation, metastasis was detected in 13 patients (19.4%), while 21 patients (31.3%) experienced recurrent disease (3 local recurrences and 18 metastatic recurrences) during the follow-up period. By the data cut-off, 26 patients (38.8%) had died.

The 5-year overall survival (OS) rate was 68.6% and 16.7% for localized and metastatic OS patients, respectively. Using the Kaplan-Meier method, significant associations were found between shorter OS in osteosarcoma cases and high ATG4B expression (p = 0.01) (Fig. 3a), positive VEGF expression (p = 0.049) (Fig. 3b), larger tumor size (p = 0.034), advanced stage (p < 0.001), the presence of metastasis at onset of diagnosis (p < 0.001), and poor histological response to chemotherapy (p < 0.001).

Multivariate Cox analysis of all predictors with p-values < 0.05 in univariate analyses indicated that metastasis at the onset of diagnosis, poor response to chemotherapy, and advanced stage were significant independent predictors of survival (see Table 4).

Table 4

Summary of univariate and multivariate analysis for overall survival
Predictor factors	Univariate	(95% CI)	Multivariate	(95% CI)
Predictor factors	P values	(95% CI)	P values	(95% CI)
Age at diagnosis 14 years≥ > 14 years	0.62	(39.058–48.402)
Gender Male Female	0.41	(41.058–50.502)
Location of tumor Extremities Axial	0.48	(44.050-47.802)
Tumor Size ≤ 9cm > 9cm	0.03	(36.919–48.605)	0.39	(0.309–19.935)
Histological subtype Osteoblastic Chondroblastic Fibroblastic	0.72	(36.058–42.502)
Stage I II III	< 0.001	(16.058–34.324)	0.03	(0.035–0.845)
Distant Metastases at the onset of diagnosis No Yes	< 0.001	(40.170- 52.101)	0.03	(0.035–0.845)
Histological response (n.=38) ≥ 90% < 90%	< 0.001	(41.085–50.502)	0.02	(0.051–0.785)
High ATG4B expression	0.01	(37.161–48.605)	0.93	(0.000-1.335)
Positive VEGF expression	0.04	(37.919–49.605)	0.95	(0.000-6.592)
CI: confidence interval. Significant variables (P values < 0.05) are in bold.

Despite significant improvements made over the past few decades in the therapy of OS, the 5-year survival rate of patients remains low, typically ranging from 15–20% [32]. Moreover, patients with metastasis have an even worse prognosis. Therefore, the discovery of mechanisms enhancing the progression of OS gives novel prospects for the prognosis and the development of new therapeutic targets.

As mentioned earlier, autophagy may act as both a tumor suppressor and a tumor promoter [10, 11]. Among the ATG4 family, ATG4B serves as a functional protease that promotes autophagy activity [15]. Currently, there is limited knowledge about the role of ATG4B in the clinical outcomes of patients with OS. Hence, this study represents the first study to investigate the immunohistochemical expression of ATG4B in OS patients and its relation with clinicopathological parameters. Additionally, it aims to explore the correlation between ATG4B and VEGF expressions in OS tumor cells, shedding light on their potential interactions and implications for disease progression and treatment.

The role of autophagy in OS remains unclear; a dual role was suggested to be in OS [33]. downregulation of autophagy-related genes such as ATG7 has been shown to inhibit cell proliferation in OS [34]. On the contrary, Parlayan et al. [35] suggested that activation of autophagy could inhibit the proliferation of OS cells by regulating BECN1 expression. These contrasting findings highlight the complexity of autophagy's involvement in OS.

In the current study, ATG4B was significantly upregulated in OS compared to normal osteoblasts in the GSE12865 dataset. Additionally, our results align with studies conducted on OS cell lines, which demonstrated that OS Saos-2 cells lacking ATG4B failed to form tumors in mouse models. Moreover, researchers have developed an ATG4B antagonist that effectively suppresses tumor growth and induces tumor regression in OS xenografts [23]. These findings suggest that ATG4B may act as a tumor promoter in OS, and targeting ATG4B to inhibit autophagy could be a promising strategy for inhibiting tumor growth in OS.

Our findings revealed a significant association between ATG4B overexpression and adverse prognostic factors in OS, including increased tumor size and advanced stage. These results are consistent with several studies across various tumor types, such as colorectal, breast, gastric carcinoma, chronic myeloid leukemia, glioblastoma and pancreatic, which have demonstrated that higher ATG4B levels are indicative of poor prognosis and lower survival rates [16-20- 36]. These data strongly supports the hypothesis that ATG4B plays a pivotal role in tumor growth and progression, making it a potential target for therapeutic intervention in various cancers, including OS.

Interestingly, we observed a significant association between high ATG4B expression and chemoresistance in OS. In recent years, the relationship between autophagy and the chemoresistance of tumor cells has gained significant attention. Our findings suggest that ATG4B may play a role in the development of chemoresistance and that its overexpression could potentially serve as a predictor of poor response to neoadjuvant chemotherapy in OS. Notably, recent studies by Tan et al. and Pu et al. have demonstrated that the induction of protective autophagy by chemotherapy enhances the chemotherapeutic resistance of tumor cells [37, 38]. It was suggested that autophagy mediates chemoresistance to conventional anti-OS agents through high mobility group box 1 protein (HMGB1). HMGB1 is a highly conserved nuclear protein, bounds to the autophagy regulator Beclin1 [39]. HMGB1 is overexpressed in OS tissue and its downregulation minimizes tumor development and may hinder tumor metastasis [40]. It was known that HMGB1 also regulates the formation of the Beclin1-PI3K class 3 complex that facilitates autophagic progression [39]. Therefore, autophagy may have a protective effect on OS cells against chemotherapeutic agents. Overall, our results provide further insight into the potential role of ATG4B in OS chemoresistance and highlight the complex interplay between autophagy and chemotherapy response in this disease.

VEGF has undeniable role in metastasis, thus numerous studies have extensively investigated its expression in OS. Consistent with our findings, these studies have reported that OS cells exhibit overexpression of VEGF. Additionally, we found that higher levels of VEGF have been significantly associated with larger tumor size, advanced stage and the presence of distant metastasis at the time of diagnosis. These findings are supported by several reports indicating that VEGF overexpression is indicative of poor prognosis and lower survival rates, further supporting the hypothesis that VEGF is crucial for tumor growth and progression [30, 41–43].

To better understand how does pathological angiogenesis heighten autophagic flux? In solid tumors, including OS, angiogenesis is stimulated to provide the necessary nutrients for tumor cell energy needs and growth. The anoxic TME stabilizes hypoxia-inducible factors (HIFs), particularly HIF-1α or HIF-2α, which regulate VEGF, inducing angiogenesis [44]. The imbalance between pro- and anti-angiogenic signaling in the TME, primarily mediated by the VEGF/VEGFR2 axis, fuels the pathological angiogenesis [45]. This results in unpleasant TME conditions characterized by an abundance of VEGF, nutrient deprivation, aberrant blood flow, which consequently, drive uncontrolled vessel sprouting and affect the vessel maturation and function. The newly formed vessels are structurally weak, liable to collapse by rapidly proliferating tumor cells, and they do not respect a structured hierarchy. As a consequence, there is insufficient perfusion of oxygen and nutrient-rich blood, leading to nutrient deprivation, hypoxia, inflammation, and acidity within the TME, all of which foster tumor growth and dissemination. Endothelial cells embedded in the TME experience stressful conditions, leading to magnified autophagic flux [46]. This heightened autophagy is a cellular response to cope with stress and promote cell survival. Given these considerations, the positive correlation between ATG4B and VEGF in OS tumor cells is not surprising. ATG4B, as a key regulator of autophagy, may be upregulated in response to the stressful conditions induced by pathological angiogenesis, contributing to the survival and aggressive behavior of OS cells in the TME.

Apart from the impact of angiogenesis on hypoxia, acidity, and increased interstitial fluid pressure contributing to drug resistance, the abnormal vascular structure of OS also hampers the delivery of anticancer drugs [47]. Chemotherapeutic agents must traverse blood vessel walls and penetrate tumor tissues to reach cancer cells, resulting in asymmetrical distribution within the tumor. Consequently, the efficacy of the drug is limited [48]. Clinical trials of targeted anti-angiogenic drugs in OS patients have demonstrated that those with a low vascularization phenotype exhibit higher overall and relapse-free survival rates. Furthermore, patients with a low vascularization phenotype show a better response to neoadjuvant chemotherapy compared to other patient groups [49–51]. These findings underscore the importance of addressing the abnormal vascular structure and associated challenges in drug delivery when developing therapeutic strategies for OS.

The relationship between autophagy and carcinoma is indeed complex, indicating that autophagy plays a dual role in tumorigenesis and tumor development [52]. Increasing evidence suggests that autophagy can help tumor cells cope with intracellular and environmental stresses, such as hypoxia or nutrient shortage, thereby favoring tumor progression and resistance to anti-cancer therapy in OS tumor cells. Overall, the role of autophagy in tumor development is complex and context-dependent, with both pro-tumorigenic and anti-tumorigenic effects depending on the stage of tumor progression and the specific cellular and environmental conditions [53–55]. Understanding the dual role of autophagy in tumor development is crucial for developing effective therapeutic strategies targeting autophagy pathways in cancer treatment.

In our study, several key findings were observed: Firstly, ATG4B expression was significantly up-regulated in OS patients. Secondly, high levels of ATG4B were associated with poor prognostic factors and chemotherapy resistance in OS patients. Thirdly, VEGF was found to be overexpressed in OS, and this overexpression was significantly associated with adverse prognostic factors. Fourthly, a positive correlation was detected between ATG4B and VEGF expression in tumor cells, lack of both markers could be a predictor of good response to chemotherapy. Further research is warranted to elucidate the underlying mechanisms and potential therapeutic implications of these findings.

Ethics approval and consent to participate

The study was conducted according to the guidelines of the Declaration of Helsinki. This project was approved by the Ethics Committee of Minia University, Faculty of Medicine, Institutional Review Board ( MUFMIRB).

Funding:

No funding was received.

Author Contribution

Study design, data acquisition, analysis and drafting of manuscript: Khalil EI, Mohamed FA and Kamal RM. Statistical analysis: Khalil EI.

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Upregulated ATG4B Predicts Poor Prognosis and Correlates with Angiogenesis in Osteosarcoma

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Abstract

Figures

1. Introduction

2. Materials and Methods

2.1. Patients

2.2. Histopathological Evaluation

2.3. Expression of ATG4B in a publically available dataset. (Data set analysis)

2.4. Immunohistochemistry

2.5. Immunohistochemical scoring

2.6. Statistical analysis

3. Results

3.1. Expression of ATG4B in a publically available dataset. (Data set analysis)

3.2. Patient and tumor characteristics

3.3. Immunohistochemical results of ATG4B and VEGF

3.4. The relationship between ATG4B and VEGF

3.5. Follow-up and survival analysis

4. Discussion

5. Conclusions

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