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Article Open Access

BLID is a drug-responsive target of FOXO3a and multi-omics analysis reveals survival mechanisms and therapeutic vulnerabilities in BLID-deficient breast cancer cells

  • Authors:
    • Sivaramakrishna Yadavalli
    • Rong Hu
    • Antonina Rait
    • Rency Varghese
    • James Li
    • Ofer Eidelman
    • Xiaojun Zou
    • Habtom Ressom
    • Esther Chang
    • Meera Srivastava
    • Robert Clarke
    • Usha Kasid
  • View Affiliations / Copyright

    Affiliations: Georgetown Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC 20057, USA, Uniformed Services University of Health Sciences, Bethesda, MD 20814, USA
    Copyright: © Yadavalli et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 409
    |
    Published online on: June 24, 2025
       https://doi.org/10.3892/ol.2025.15155
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Abstract

BH-3 like motif containing inducer of cell death (BLID) is a known prognostic factor in breast cancer. The aim of the present study was to determine the significance of BLID in the outcomes of chemotherapy and mechanisms affected in BLID-deficient breast cancer cells. Reverse transcription-PCR, reverse transcription-quantitative PCR, dual-luciferase reporter and chromatin immunoprecipitation assays were used to determine the effects of drugs on BLID expression and binding of forkhead box protein O3a (FOXO3a) to the BLID promoter. RNA arrays, antibody microarrays and microRNA arrays were used to illuminate the omics features of BLID knockdown vs. isogenic control breast cancer cell lines. Kaplan-Meier plotter and receiver operating characteristic plotter tools were used to determine the prognostic and therapy response benefits of BLID expression using publicly available clinical datasets. BLID expression was induced in response to several chemotherapeutic drugs. Drug treatment resulted in increased binding of FOXO3a to the BLID promoter, and FOXO3a knockdown was associated with decreased expression of BLID. BLID depletion led to a decrease in the cytotoxicity of chemotherapeutic drugs. Through multi-omics profiling, three functionally distinct classes of effectors that were predominately influenced in BLID knockdown cells were identified: i) Genes and proteins associated with cell death and survival, including cellular inhibitor of apoptosis protein 2/baculoviral IAP repeat containing 3, phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit α/p110α and DFNA5/gasdermin E; ii) members of the aldo-ketoreductase family 1, specifically aldo-keto reductase family 1 member C3 implicated in drug metabolism; and iii) effectors of the interferon response, including IFNβ1, interferon-induced protein with tetratricopeptide repeats 2 and interferon-induced protein with tetratricopeptide repeats 3. Finally, higher BLID expression was associated with improved overall survival in several types of cancer and the response of breast cancer to anthracyclines. The results of the present study demonstrated that BLID is a target of FOXO3a, and BLID-deficiency in breast cancer cells was associated with modulation of cell death, survival and proliferation, chemoresistance, drug potency, and the interferon response. These findings highlighted BLID as a promising biomarker of drug response and offer a novel framework of integrative mechanisms of therapeutic resistance and disease progression.
View Figures

Figure 1

Effect of chemotherapeutic drugs on
BLID expression. Treatment with chemotherapeutics increased BLID
mRNA expression in (A) MCF-7 and (B) T47D breast cancer cells. Data
were normalized to GAPDH, and the fold change in BLID expression
was calculated relative to normalized vehicle control. Data are
presented as the mean ± SD of two to three replicates with two to
three experimental repeats per treatment group. All y-axes denote
BLID mRNA expression (fold change vs. vehicle). *P<0.05 (vs.
vehicle treatment at the time corresponding to the corresponding
experimental treatment time period). DXR, doxorubicin; DTX,
docetaxel; 5-FU, 5-fluorouracil; BLID, BH-3 like motif containing
inducer of cell death.

Figure 2

Chemotherapeutics induce binding of
FOXO proteins to the BLID promoter in MCF-7 cells. Cells were
treated with (A) DXR or (B) PTX (+) or a vehicle control (−) as
shown, and ChIP-PCR was subsequently performed as shown in the
middle panels in (A and B). The left panels in (A and B) show input
PCR. The ChIP-PCR assay controls tested were chromatin
immunoprecipitated with RNA-Pol II antibody, mouse IgG or rabbit
IgG, and using GAPDH or BLID/S1 primers as shown in the right
panels in (A and B). (C) ChIP-qPCR. A total of 50 ng of the bound
DNA fraction or input DNA was used for qPCR. qPCR data were
normalized to input DNA. Data are presented as the mean ± SD of two
to three replicates with two to three experimental repeats per
treatment group. *P<0.05 (compared with vehicle treatment at the
time corresponding to the corresponding experimental treatment time
period). DXR, doxorubicin; PTX, paclitaxel; IP,
immunoprecipitation; ChIP-PCR, chromatin immunoprecipitation-PCR;
qPCR, quantitative PCR; RNA Pol-II, RNA polymerase II; prom.,
promoter; IgG, immunoglobulin G; BLID, BH-3 like motif containing
inducer of cell death.

Figure 3

BLID knockdown in breast cancer cells
decreases the sensitivity to chemotherapeutic drugs. (A) Knockdown
of BLID expression was confirmed using western blotting. Cells were
transfected with the indicated BLID shRNA lentiviral clone or Scr
and the cell lysates were analyzed by western blotting using
anti-BLID antibody (dilution, 1:10,000). The same blot was also
probed with anti-α-Tubulin (dilution, 1:1,000). Data were
semi-quantified as shown in the right panel. (B) Knockdown of BLID
expression using the sh47 lentiviral clone was further validated
using western blotting. The same blot was also probed with
anti-GAPDH antibody (dilution, 1:500). (C) Drug dose response.
Following BLID knockdown, cells were treated with DXR or vehicle
(DMSO) for 24 h and cell viability was assessed using an XTT cell
viability assay. The x-axis title of the top graph is identical to
that of the bottom graph. Data are presented as the mean ± SD from
two to three independent experiments with each data point
representing six wells. (D) Left panel: Drug response of MCF-7
cells stably expressing BLID shRNA (sh47) or control scrambled
shRNA. Cells were treated with 10 µM 5-FU, 50 nM PTX or 5 nM ETO
for 24 h and cell viability was measured. Solid bars, BLID shRNA;
empty bars, control scrambled shRNA. Right panel: Reverse
transcription-quantitative PCR analysis showing BLID knockdown in
BLID shRNA (sh47) vs. scrambled control shRNA-transfected MCF-7
cells. β-Actin served as the housekeeping gene. *P<0.05,
**P<0.01, n=3. shRNA/sh, short hairpin RNA; Scr, scrambled
control shRNA; Ctl, control; DXR, doxorubicin; 5-FU,
5-fluorouracil; PTX, paclitaxel; ETO, etoposide; BLID, BH-3 like
motif containing inducer of cell death.

Figure 4

RT-qPCR validation of changes in
expression of a subset of genes following BLID knockdown. RT-qPCR
analysis of several genes in (A) MCF-7 and (B) MDA-MB-231 cells.
β-Actin served as the internal control. (A) The y-axis title of the
middle graph is identical to the y-axis title of the left graph.
(B) The y-axis title of the middle-left graph is identical to the
y-axis title of the far-left graph. *P<0.05 and **P<0.01
(BLID shRNA vs. the corresponding Scr shRNA group), n=3. shRNA,
short hairpin RNA; Scr, scramble control; Ctl, control; RT-qPCR,
reverse transcription-quantitative PCR; BLID, BH-3 like motif
containing inducer of cell death.

Figure 5

Validation of changes in the
expression of a subset of genes in BLID-knockdown breast cancer
cells. (A) PI3K-p110α protein expression was increased in BLID
shRNA knockdown MCF-7 cells. (B) Left panel: cIAP2/BIRC3 protein
expression was increased and DFNA5 expression was decreased in the
BLID knockdown MDA-MB-231 cells in the absence or presence of Biri
(50 µM; 6 h). Right panel: Reverse transcription-quantitative PCR
analysis of BLID expression in MDA-MB-231 cells treated with BLID
siRNA or Ctl siRNA. Gene expression was normalized to β-actin as an
internal control. **P<0.01 vs. Ctl siRNA, n=3. shRNA, short
hairpin RNA; siRNA, small interfering RNA; Ctl, control; Biri,
birinapant; BLID, BH-3 like motif containing inducer of cell death;
cIAP, cellular inhibitor of apoptosis protein; XIAP, X-linked
inhibitor of apoptosis protein.

Figure 6

BLID is a significant prognostic
biomarker in breast and liver cancer as shown by Kaplan-Meier
plotter analysis. BLID, BH-3 like motif containing inducer of cell
death; RNA-seq, RNA sequencing; FDR, false discovery rate; HR,
hazard ratio.

Figure 7

Kaplan-Meier analysis of the
relationship between BLID expression and overall survival
probability in patients with kidney and lung cancer. BLID, BH-3
like motif containing inducer of cell death; RNA-seq, RNA
sequencing; FDR, false discovery rate; HR, hazard ratio.

Figure 8

ROC plotter analysis of the
predictive response of breast cancer to anthracyclines. ROC
P=1.8×10−5. ROC, receiver operating characteristic; AUC,
area under the curve; TPR, true positive rate; TNR, true negative
rate; BLID, BH-3 like motif containing inducer of cell death.
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Copy and paste a formatted citation
Spandidos Publications style
Yadavalli S, Hu R, Rait A, Varghese R, Li J, Eidelman O, Zou X, Ressom H, Chang E, Srivastava M, Srivastava M, et al: BLID is a drug-responsive target of FOXO3a and multi-omics analysis reveals survival mechanisms and therapeutic vulnerabilities in BLID-deficient breast cancer cells. Oncol Lett 30: 409, 2025.
APA
Yadavalli, S., Hu, R., Rait, A., Varghese, R., Li, J., Eidelman, O. ... Kasid, U. (2025). BLID is a drug-responsive target of FOXO3a and multi-omics analysis reveals survival mechanisms and therapeutic vulnerabilities in BLID-deficient breast cancer cells. Oncology Letters, 30, 409. https://doi.org/10.3892/ol.2025.15155
MLA
Yadavalli, S., Hu, R., Rait, A., Varghese, R., Li, J., Eidelman, O., Zou, X., Ressom, H., Chang, E., Srivastava, M., Clarke, R., Kasid, U."BLID is a drug-responsive target of FOXO3a and multi-omics analysis reveals survival mechanisms and therapeutic vulnerabilities in BLID-deficient breast cancer cells". Oncology Letters 30.3 (2025): 409.
Chicago
Yadavalli, S., Hu, R., Rait, A., Varghese, R., Li, J., Eidelman, O., Zou, X., Ressom, H., Chang, E., Srivastava, M., Clarke, R., Kasid, U."BLID is a drug-responsive target of FOXO3a and multi-omics analysis reveals survival mechanisms and therapeutic vulnerabilities in BLID-deficient breast cancer cells". Oncology Letters 30, no. 3 (2025): 409. https://doi.org/10.3892/ol.2025.15155
Copy and paste a formatted citation
x
Spandidos Publications style
Yadavalli S, Hu R, Rait A, Varghese R, Li J, Eidelman O, Zou X, Ressom H, Chang E, Srivastava M, Srivastava M, et al: BLID is a drug-responsive target of FOXO3a and multi-omics analysis reveals survival mechanisms and therapeutic vulnerabilities in BLID-deficient breast cancer cells. Oncol Lett 30: 409, 2025.
APA
Yadavalli, S., Hu, R., Rait, A., Varghese, R., Li, J., Eidelman, O. ... Kasid, U. (2025). BLID is a drug-responsive target of FOXO3a and multi-omics analysis reveals survival mechanisms and therapeutic vulnerabilities in BLID-deficient breast cancer cells. Oncology Letters, 30, 409. https://doi.org/10.3892/ol.2025.15155
MLA
Yadavalli, S., Hu, R., Rait, A., Varghese, R., Li, J., Eidelman, O., Zou, X., Ressom, H., Chang, E., Srivastava, M., Clarke, R., Kasid, U."BLID is a drug-responsive target of FOXO3a and multi-omics analysis reveals survival mechanisms and therapeutic vulnerabilities in BLID-deficient breast cancer cells". Oncology Letters 30.3 (2025): 409.
Chicago
Yadavalli, S., Hu, R., Rait, A., Varghese, R., Li, J., Eidelman, O., Zou, X., Ressom, H., Chang, E., Srivastava, M., Clarke, R., Kasid, U."BLID is a drug-responsive target of FOXO3a and multi-omics analysis reveals survival mechanisms and therapeutic vulnerabilities in BLID-deficient breast cancer cells". Oncology Letters 30, no. 3 (2025): 409. https://doi.org/10.3892/ol.2025.15155
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