Clinicopathological Significance and Expression Pattern of Bcl2 in Breast Cancer: A Comprehensive in silico and in vitro Study

Abstract

B-cell lymphoma/leukemia gene-2 (Bcl-2) is the primary proto-oncogene that has been shown to work by preventing apoptosis/programmed cell death. Bcl-2 combines a variety of cell-generated signals associated to the survival and death of cells. In glioma, lung, and breast cancer, Bcl-2 over-expression has been linked to an increase in invasion and migration. Many treatment regimens that target Bcl2 have been established and approved, and thus increasing the survival rates of the patients. The primary goal of this research was to recognize new therapeutic compounds that target Bcl2 and assess Bcl2 expression pattern in BC patients. We used various bioinformatic tools as well as several in vitro assays to look out the expression and inhibition of Bcl2 in BC. Our study depicted that Bcl2 had a strong connection with tumour stroma, notably with suppressor cells originating from myeloid tissues. Moreover, in vitro and in silico research identified Paclitaxel as a promising natural substance that targets Bcl2. Overall, this work shows that Bcl2 overexpression accelerates the development of BC, and that targeting Bcl2 in combination with other drugs will dramatically improve BC patient's response to treatment and prevent the emergence of drug resistance.

Keywords: Apoptosis; B cell lymphoma gene 2; Breast Cancer; Chemoresistance; Expression pattern; Metastasis; Oncogene.

Graphical abstract

Fig. 1

Expression of Bcl2 in several malignancies using box plot model form TIMER Database. Bcl2 is vastly upregulated in most of the cancers, especially breast cancer where it shows highest expression with respect to other tumors.

Fig. 2

Bcl2 expression is linked with several clinicopathological features. The high expression of Bcl2 is associated with HER2^-, ER⁺, PR⁺, Nodal negative, SBR1, NPI1, wild type P53, and non TNBC status.

Fig. 3

Gene-Gene interaction of Bcl2 with other genes show that Bcl2 is highly correlated with Bcl2L11, FKBP8, BBC3 and other genes. Correlation of Bcl2 with Bcl2L11 with a R value of 0.4 using GEPIA.

Fig. 4

Expression profile of Bcl2 in BC patients. High expression was seen in Patients with primary tumor in the age group of 61–80. The expression was seen higher in Luminal BC patients and that too in the race of caucassian group.

Fig. 5

Pathway status in association with expression of Bcl2 across pan-cancer. The Bcl2 expression was examined for modulation in signaling pathways through, A Wnt signaling, B mTOR pathway, and C p53/Rb-related pathway (p-value < 0.01).

Fig. 6

Expression pattern of Bcl2 in BC. A: Primary breast tumors. B: Metastatic BC. Bcl2 is expressed more in primary as related to metastatic tumors.

Fig. 7

A) PPI interaction of Bcl2 with other proteins. B) Top ten hub genes obtained from PPI network using cytoscape.

Fig. 8

GO and KEGG analysis of Bcl2 in breast cancer.

Fig. 9

Molecular surface view of the Bcl2 with paclitaxel bound in deep cavity. 2D interaction exhibiting the interactions between ligand and protein and dotted lines exhibiting interactions.

Fig. 10

RMSD plots of Bcl2 + paclitaxel, where black plot is of protein and red plot is for ligand. (B) RMSF of Cα backbone of Bcl2 bound to Paclitaxel-ligand, (C) Radius of gyration (Rg) of Cα backbone of Bcl2 bound to Paclitaxel-ligand (D) Solvent accessible surface area of Bcl2 bound to Paclitaxel-ligand. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 11

Anti-proliferative effect of paclitaxel on BC cell lines; A) MDA-MB 231 BC cells, C) 4 T1 Cells. The cells were exposed with increased concentrations of PTX. After 72 h, the cell viability was determined by MTT test.

Fig. 12

In MDA-MB 231 cells, paclitaxel reduces the colony formation in dose dependent manner. There is a decrease in colony number with increase in the concentration of paclitaxel. Well A treated with no drug shows highest number of colonies, while as well E receiving highest concentration of paclitaxel (c4 = 2.9 nM) shows lowest number of colonies in comparision to other wells.

Fig. 13

Florescence intensity of ROS levels in MDA-MB 231 BCE cells. The Ros levels are highest in control well and lowest in the highly concentrated paclitaxel well. Therefore, suggesting that Paclitaxel decreases the ROS concentration in MDA-MB 231 BC cells.