Abstract
Objective
Bcl-2-associated athanogene-3 (BAG-3) is a cytoplasmic multidomain protein member that belongs to the BAG family of co-chaperones that are known to be involved in several cellular processes, including control of apoptosis, autophagy, and cytoskeletal dynamics. Normal tissues show weak to negligible expression as it is downregulated. However, its expression is upregulated in various epithelial and hematological malignancies, including endometrial, cervical, and chronic lymphoid leukemia. It plays a vital role in tumor cell proliferation, metastasis, and therapy resistance; thus, its expression has been determined to be a valuable tool in developing targeted therapeutic strategies. Endometrial cancers are one of the most common female genital tract malignancies with high mortality. Therefore, in this study, we evaluated the BAG-3 protein immunoexpression in 33 cases of endometrial cancer and correlated its expression with prognostic determiners.
Materials and Methods
We performed a retrospective study of BAG-3 immunoexpression in 33 cases of endometrial carcinoma. Cytoplasmic and membranous staining was considered positive and its correlations with prognostic factors such as myometrial invasion, LVSI, and ovarian involvement were analyzed.
Results
BAG-3 immunopositivity was seen in 31 (93.9%) cases and two cases were found to be negative. Twenty-two (66.6%) showed strong (3 +) intensity, seven (21.2%) showed moderate, and two (6.1%) showed weak BAG-3 immunostaining. No significant correlation was seen between grade and intensity.
Conclusion
Inhibiting BAG-3 expression can induce apoptosis, thus promoting a new therapeutic target for the treatment of endometrial carcinoma.
Keywords: BAG-3, Endometrioid adenocarcinoma, Apoptosis, Carcinoma, Autophagy
Introduction
Cancer remains to be a global threat, as it is known to account for a high mortality rate. The commonly used cancer treatment methods include surgery, chemotherapy, and radiotherapy. The cancer cells possess a variety of defenses against treatments, thereby resulting in difficult treatments with high chances of recurrence. Current research has emphasized developing new therapeutic strategies by understanding cancer pathogenesis and various mechanisms adopted by the cancer cells to promote its growth [1].
Endometrial cancer (EAC) accounts for the majority of uterine corpus malignancies (> 80%) and is the sixth most diagnosed cancer in females and the second most reported female genital organ cancer globally following breast cancer. Prolonged unopposed exposure to estrogen has been determined to be a risk factor, including early menarche, late menopause, nulliparity, obesity, tamoxifen therapy, polycystic ovary syndrome, or estrogen-producing ovarian tumor. EACs have been earlier classified as types I or II based on clinical, endocrine, and epidemiological details where type I tumors were estrogen dependent and associated with endometrial hyperplasia and type II tumors were endometrial-associated estrogen-independent atrophy. They are also classified according to histopathological characteristics, with the most common subtypes, including endometrioid adenocarcinoma, serous carcinoma, carcinosarcoma, and clear-cell carcinoma. Molecular features, such as microsatellite instability, PTEN, PIK3CA, K-Ras, and β-catenin gene mutations, and small non-coding RNAs (sncRNAs) dysregulations, were described [2].
The cancer genome atlas research network has published a comprehensive molecular classification into four genomic classes as follows: (1) POLE (ultra-mutated) tumors, (2) microsatellite instable (MSI hypermutated), (3) copy-number low (endometrioid) tumors, and (4) copy-number high (serous-like) tumors [3, 4]. The majority of endometrial cancers (68%) are detected at an early stage (stages I–II) and reconsidered as low risk and are usually treatable; however, 32% of the patients have regional or distant metastasis at diagnosis with a significantly poor prognosis. The mechanisms and molecules that determine cell survival and therapy response/resistance in different EAC subtypes are yet to be fully clarified [5, 6].
Bcl-2-associated athanogene-3 (BAG-3) belongs to a family of co-chaperones that interacts with the ATPase domain of heat shock proteins (HSP70) [7]. BAG-3 is a 74 kDa cytoplasmic protein that is involved in several cellular processes, including cell proliferation, apoptosis, adhesion, and migration, which is downregulated in normal cells. Various studies have shown its upregulated expression in different epithelial, ovarian, and hematological malignancies that promote tumor invasion and metastasis. Therefore, BAG-3 expression inhibition could promote the efficiency of chemotherapy via different mechanisms [5].
Materials and Methods
Study Design
After receiving approval from the Institute Review Board bearing the number AIIMS/IEC/2016/233, the cases diagnosed as EACs over 4 years were retrieved from the department of pathology archives at our institute.
Inclusion Criteria
These included resection specimens and biopsies for endometrial adenocarcinomas.
Exclusion Criteria
Cases with insufficient biopsy material for immunohistochemical (IHC) studies or with inadequate clinical data were excluded from this study.
Methodology
This study primarily aims to determine the BAG-3 protein expression pattern in histopathologically identified cases of endometrial adenocarcinoma and the role of BAG-3 protein in histological tumor grade and invasiveness. Tissues were routinely fixed in 10% neutral buffered formalin and embedded in paraffin. Sections that are stained with hematoxylin and eosin (H&E) for histopathological evaluation were reviewed. Adenocarcinoma diagnosis and grading were based on histopathological features as described in the World Health Organization (WHO) classification of uterine corpus tumors [8]. BAG-3 IHC studies were performed on freshly cut 5-μ-thick formalin-fixed paraffin-embedded whole tumor tissue sections using a mouse monoclonal antibody against BAG-3 (St. John’s Laboratory, Polyclonal) at a dilution of 1:100 with overnight incubation. A universal labeled bond polymer refine detection kit was used as a detection system (Leica, United Kingdom). Antigen retrieval was performed using citrate buffer at pH 6.0 in a microwave oven.
Interpretation
Normal endometrial glands show faint to weak positivity for BAG-3, thus employed as an internal positive control for our cases. However, for superficial biopsies, sections of normal endometrial glands were run as positive controls. IHC was validated by two pathologists (AN and AB), who independently analyzed the sections and assigned the score. As per the cancer genome atlas [9], cytoplasmic expression of BAG-3 was considered positive and graded as the intensity and proportion of tumor cells displaying the protein expression. Intensity is scored as no staining in 0 and positive staining on a 3-point scale (1–3) as weak (1), moderate (2), and strong (3) expression. Proportion score was assigned as follows: 0 (0, no cells are positive), 1 (1–10%, cells are positive), 2 (11–30%, cells are positive), 3 (31–60%, cells are positive), and 4 (61–100%, cells are positive). Staining was scored as positive (intensity of staining 1–3) or negative (no staining, 0) (7).
Statistical Analysis
The statistical package for the social sciences software version 23 was used for statistical analysis. A p value of < 0.05 was considered significant.
Results
BAG-3 IHC expression was examined in 33 endometrial adenocarcinoma cases, all of the cases were of the endometrioid subtype (Figs. 1, 2). Of these, seven were endometrial biopsies, 25 were hysterectomies, and one was a cervical biopsy that shows EAC (Table 1). The mean age of the study participants was 60.2 ± 11.2 years (range, 25.0–99.0 years). The myometrial invasion was assessed in 25 hysterectomy specimens; of these, eight (32%) cases showed < 50% invasion, whereas > 50% invasion was observed in 17 (68%) cases. Additionally, 31 (93.9%) cases showed positive immunostaining for BAG3, and two cases were found to be negative (Table 2). Of the 31 cases, 22 (66.6%) showed strong (3 +) intensity, seven (21.2%) showed moderate, and two (6.1%) showed weak BAG-3 immunostaining (Figs. 3, 4). The proportion of tumor cells showing BAG-3 immunostaining determined was scored from 0 to 4, 26 (79%) cases were scored 4 while three (9%) cases were scored 3 and two (6%) cases scored 2 and 0 each. Twenty-one (81%) cases among the score showed strong expression of BAG-3, whereas four (15%) cases with moderate expression and 1 (4%) cases with weak expression. All score three cases showed moderate expression and score two cases showed strong (50%) and weak (50%) expression. The histopathological grade (the International Federation of Gynecology and Obstetrics [FIGO]) assessed in all the cases included 17 grade 1, eight grade 2, and eight grade 3 cases. Grade 1 tumors showed significant staining in 15 cases, whereas all grade 2 and 3 tumors had significant staining. However, no significant correlation was seen between grade and intensity of BAG-3 staining (Spearman’s rho = 0.007, p = 0.968) (Tables 3, 4). Three cases showed ovarian involvement with moderate to intense BAG-3 expression. Of the 25 hysterectomies, the lymphovascular invasion (LVI) was identified in three (12%) cases, which all showed strong BAG-3 immunopositivity. However, assessment of myometrial invasion, LVI, and FIGO score was limited to seven cases, which were superficial biopsies.
Fig. 1.
Photomicrograph of grade 1 EAC (A H&E × 200) and grade 3 EAC with areas of necrosis (B H&E × 40). EAC: endometrial cancer; H&E: hematoxylin and eosin
Fig. 2.
Photomicrograph of grade 1 EAC (A H&E × 100) with adjacent normal endometrial glands and weak BAG-3 staining seen in the normal endometrial glands (B IHC × 400). EAC: endometrial cancer; H&E: hematoxylin and eosin; BAG-3: Bcl-2-associated Athanogene-3; and IHC: immunohistochemical
Table 1.
Specimen type
| Specimen | Cases | Percentage (n = 33) (%) |
|---|---|---|
| Hysterectomy | 25 | 75.7 |
| Endometrial biopsy | 7 | 21.3 |
| Cervical biopsy | 1 | 3.0 |
n: total number of cases
Table 2.
BAG-3 staining intensity and proportion
| Cases | Percentage (%) | ||
|---|---|---|---|
| Intensity | No staining | 2 | 6.1 |
| Mild | 2 | 6.1 | |
| Moderate | 7 | 21.2 | |
| Intense | 22 | 66.6 | |
| Proportion | 4 (61–100%) | 26 | 79 |
| 3 (31–60%) | 3 | 9 | |
| 2 (11–30%) | 2 | 6 | |
| 0 (no cells are positive) | 2 | 6 | |
BAG-3: Bcl-2-associated Athanogene-3
Fig. 3.
Photomicrograph of EAC with strong diffuse BAG-3 staining (A IHC × 200) and no staining (B IHC × 200). EAC: endometrial cancer; BAG-3: Bcl-2-associated Athanogene-3; and IHC: Immunohistochemical
Fig. 4.
Photomicrograph of EAC with diffuse moderate BAG-3 staining (A IHC × 400) and weak BAG-3 staining (B IHC × 400). EAC: endometrial cancer; BAG-3: Bcl-2-associated Athanogene-3; and IHC: immunohistochemical
Table 3.
FIGO Grade and BAG-3 expression intensity correlation
| Significant staining (n = 33) | |||||
|---|---|---|---|---|---|
| No | Yes | ||||
| Cases | Percentage (%) | Cases | Percentage (%) | ||
| FIGO Grade | 1 | 2 | 11.8 | 15 | 88.2 |
| 2 | 0 | 0.0 | 8 | 100.0 | |
| 3 | 0 | 0.0 | 8 | 100.0 | |
FIGO: The International Federation of Gynecology and Obstetrics; BAG-3: Bcl-2-associated Athanogene-3; Grade: 1 (≤ 5%), 2 (6–50%), and 3 (> 50%) are solid, non-glandular, and non-squamous growth, respectively
Table 4.
Correlation between BAG-3 intensity and FIGO grade
| Intensity | |||
|---|---|---|---|
| Spearman’s rho | Grade | Correlation coefficient | 0.007 |
| Significant (two-tailed) | 0.968 | ||
| N (number of cases) | 33 | ||
BAG-3: Bcl-2-associated Athanogene-3; FIGO: The International Federation of Gynecology and Obstetrics
Discussion
BAG-3 protein is known to play an important role in cell survival as an anti-apoptotic protein with a well-established pro-survival role in many cancer types by stabilizing or enhancing Bcl-2 protein. Its activity has been identified in different tissue elements with its expression being weak and mainly expressed within the cell cytoplasm. Tumor cells are known to employ many defense mechanisms for their survival, one being anti-apoptotic protein overexpression, thereby promoting cell survival, growth, metastasis, and high chances of therapy resistance to currently employed chemotherapeutic agents. Recent results in ovarian, endometrial, and cervical cancers indicate that BAG-3 is involved in these tumors [5, 10, 11].
The WHO classifies uterine corpus tumors as epithelial carcinomas, mixed epithelial and mesenchymal or mesenchymal tumors, gestational trophoblastic diseases, and other malignant tumors. Endometrial tumors comprise histological types, including endometrioid, serous, and clear-cell variants. Molecular alterations are incorporated into the classification that plays a prognostic role. Mutations include PTEN, KRAS, CTNNB1, PIK3CA, microsatellite instability, HER2 amplification, and TP53. EAC pathogenesis has a strong association with the estrogen hormone [3].
Our study revealed that BAG-3 expression changes in tumors compared to normal cells. Additionally, BAG-3 positivity in normal endometrial glands and stroma according to the human protein atlas is weak to moderate cytoplasmic to membranous expression, and adjacent normal endometrial glands showed very low and mostly focal expression while increased and graded endometrial cancer cells expression as per percentage and intensity. BAG-3 protein overexpression was noted in tumor cells compared to adjacent normal endometrial tissue with significant staining seen in all grade 2 and 3 tumors and 88.2% in grade 1 tumors. However, no significant correlation was noted between grade and intensity. Additional histopathological parameters, such as the percentage of myometrial invasion, ovarian involvement, and lymphovascular invasion, play a vital role in EAC prognosis assessment; however, this present study revealed no significant correlation with BAG-3 expression. Different cancer cells, such as pancreatic cancer, prostate cancer, thyroid carcinoma, ovarian cancer, cervical cancer, and leukemic cells, demonstrate BAG-3 protein overexpression. Esposito et al. have examined the BAG-3 expression in 14 cases of endometrial cancer and noted BAG-3 overexpression with predominant cytoplasmic expression with predominance among lower-grade tumors in comparison to adjacent hyperplastic/normal endometrial glands, indicating its role in tumorigenesis. A similar overexpression pattern was also noted in other examined tumor types, explaining its pro-apoptotic role in carcinogenesis. However, aggressive behavior was seen in cervical cancer cases [11–13]. Wang et al. performed a bioinformatic analysis of clinicopathological and prognostic significance of BAG-3 expression in gynecological cancers, showed a downregulation of BAG-3 mRNA expression in strongly association with carcinogenic effect of breast and endometrial cancers and the histogenesis of cervical and endometrial cancer, thus can be used as a potential marker of the aggressiveness or prognosis of gynecological cancers [14].
Surgical therapy remains to be the gold standard for most tumors, as well as endometrial tumors, in association with chemotherapy and/or radiotherapy when indicated. However, limited effects are noted due to resistance and widespread disease. Hence, new therapeutic alternatives based on a better understanding of the underlying molecular pathogenesis are essential for targeted therapy, and BAG-3 expression has opened up many therapeutic avenues in this regard due to its pro-survival role in tumor cells [10].
Our study was limited due to the small sample size, which causes limited BAG-3 expression concerning myometrial invasion, lymphovascular space invasion, and FIGO grade for a better understanding of its correlation.
However, in our study, despite the small sample size, BAG-3 protein overexpression was seen in most of the EAC cases with significant staining patterns. Limited histological grade correlation explains its anti-apoptotic role in EAC tumorigenesis as compared to its expression in normal endometrial tissue. Thus, BAG-3 inhibition can induce apoptosis that promises a therapeutic target for this disease. Further studies with larger sample size and correlation with prognostic indicators including histological grade, LVSI, and amount of myometrial invasion can help in a better understanding of BAG-3 role in the pathogenesis of EAC and the development of a novel chemotherapeutic target.
Acknowledgment
Mr. Prem Shankar for applying immunohistochemistry on the cases.
Funding
The study was intramurally funded by All India Institute of Medical Sciences, Jodhpur, and the funding was used for acquisition of primary and secondary antibodies for the study.
Declarations
Conflict of interest
The authors declare no conflicts of interest.
Ethical Approval
Institutional ethical clearance was obtained.
Footnotes
Aasma Nalwa is an Additional Professor; Vinay N Gowda is a Senior Resident; Anand Bardia is a Senior Resident; Pratibha Singh is a Professor & Head; Meenakshi Rao is an Additional Professor; Poonam Elhence is a Professor & Head; Akhil D. Goel is an Additional Professor.
Publisher's Note
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