BRD4 Regulation of p53 in MCF-7 Breast Cancer Cells Study

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PhD THESIS DEFENSE 2024

Role of BRD4 in Regulation of p53 Expression in Breast Cancer Cells (MCF-7)

Establishing rMATS Pipeline for Alternative Splicing Analysis

CANDIDATE

[Student Name]

[Enrollment No.]

GUIDED BY

[Dr. Guide Name]

[Department of Biochemistry / Biotechnology]

[Institute Name]

[University Name]

[University Name] | [Institute Name] | [Student Name] | [Enrollment ID] | [Department]

p53

BRD4

Presentation Overview

Thesis Defense | BRD4 & p53 in Breast Cancer

[University Name] | [Institute Name] | [Student Name] | [Enrollment ID] | [Department]

01

🔬

Introduction

Breast Cancer Overview, Epidemiology

02

🧬

Molecular Biology

BRD4, p53, BET Family

03

🔍

Epigenetics

Histone Modifications, Super-Enhancers

04

💊

MZ1 PROTAC

Mechanism & Selectivity

05

🏥

MCF-7 Model

Cell Line & DNA Damage

06

⚗️

Methodology

Western Blot, RT-PCR, rMATS

07

📊

Results

BCA, Western Blot, qPCR Data

08

💬

Discussion

Findings & Significance

09

🎯

Conclusion

Future Scope & References

CHAPTER 1

Introduction

2.3 Million

New cases annually (GLOBOCAN 2021)

685,000

Deaths per year worldwide

Most common

Cancer in women globally

TP53 Mutation

50% of all cancers

BRD4 Upregulation

Epigenetic driver

TNBC Subtype

Most aggressive, no receptor targets

Cell Cycle Dysregulation

CDK pathway disruption

The interplay between BRD4 and p53 represents a critical therapeutic axis in breast cancer progression.

[University Name] | [Student Name] | [Enrollment ID]

EPIDEMIOLOGY

Global & Indian Burden of Breast Cancer

2.3M

685K

192,020

98,337

Source: GLOBOCAN 2022

Luminal A

Luminal B

HER2-enriched

TNBC / Basal-like

[University Name] | [Institute Name] | [Department]

Molecular Classification

Four Subtypes — Distinct Biology, Distinct Outcomes

LUMINAL A

~40%

Low Ki-67

TP53 Mut: <15%

Endocrine therapy responsive

BEST PROGNOSIS

LUMINAL B

~20%

TP53 Mut: 25%

Endocrine + Chemo

MODERATE PROGNOSIS

HER2-ENRICHED

~15%

TP53 Mut: 40%

Trastuzumab

MODERATE PROGNOSIS

TNBC / BASAL-LIKE

~15-20%

TP53 Mut: >82%

Chemo only (PARP/PD1)

POOR PROGNOSIS

Department of Oncology | Graduate Research Defense

MOLECULAR BIOLOGY

BRD4 & the BET Protein Family

BRD2

BRD3

BRD4

OVEREXPRESSED IN CANCER

BRDT

UNIVERSITY OF CALIFORNIA

DEPARTMENT OF MOLECULAR BIOLOGY

12

BRD4 Dual Function Pathway

Transcription regulation and oncogene activation mechanics

TUMOR SUPPRESSOR

p53: The Guardian of the Genome

TP53 — Most Frequently Mutated Gene in Human Cancer

TP53 mutated in ~50% of all cancers

>80% of TNBC | <15% of Luminal A

University Name | Institute Name | Department of Molecular Biology

MOLECULAR AXIS

BRD4–p53 Regulatory Interaction

How BRD4 Controls the Tumor Suppressor

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[University Name] | [Institute Name] | [Student Name] | [Enrollment ID] | [Department]

BRD4 occupancy required for baseline TP53 transcription (Zhou et al., 2022)

BRD4 Active

BRD4 reads H3K27ac at TP53 super-enhancer

Recruits transcriptional machinery

p53 mRNA transcribed

p53 protein expressed

Tumor suppression active

BRD4 Inhibited (JQ1)

BRD4 displaced from super-enhancer

Partial reduction in TP53 transcription

Some p53 protein reduction

BRD4 Degraded (MZ1)

Complete BRD4 elimination

Super-enhancer collapse

Post-translational p53 accumulation

Complex regulatory outcome

Nagarajan et al. (2022)

BRD4 regulates wild-type p53 at super-enhancers in luminal breast cancer

Zhou et al. (2022)

BRD4 required for baseline TP53 transcription in MCF-7 cells

DNA DAMAGE RESPONSE

Radiation & DNA Double-Strand Break Repair

2 Gy Ionizing Radiation

DNA Double-Strand Break (DSB)

MRN Complex (MRE11-RAD50-NBS1) detects DSB

ATM Kinase Recruited & Activated

DNA Repair Pathway

γH2AX foci formation at DSBs

Homologous Recombination (HR) via RAD51

BRD4 phosphorylated at Ser1117 by IKKα → coordinates DDR

p53 Pathway

p53 phosphorylated at Ser15/Ser20

MDM2 interaction disrupted

p53 stabilized & accumulated

p21/CDK arrest → G1/S checkpoint

BAX/PUMA → Apoptosis

MCF-7 Response: G2/M arrest (not apoptosis) — Radioresistant apoptotic phenotype

BRD4 degradation by MZ1 → ↓DNMT1 + ↓RAD51 → Radiosensitization (Foran et al., 2025)

BRD4 needed for baseline p53 levels → determines radiation response

Molecular Pathways in Radiation Pathology | Breast Cancer Research

EPIGENETICS

Histone Modification: Writers, Erasers & Readers

The Epigenetic Code in Breast Cancer

Ac marks (acetylation)

Me marks (methylation)

Ph marks (phosphorylation)

Ub marks (ubiquitination)

WRITERS

Deposit activating/repressive marks

HATs

CBP/p300, GCN5, PCAF

HMTs

EZH2, MLL, G9a

H3K27ac ★, H3K4me3

ERASERS

Remove histone marks

HDACs

HDAC1/2, SIRT1

HDMs

LSD1/KDM1A

HDAC1 reduced by BRD4-RAC1 inhibition

READERS

Recognize & bind marked histones

BRD4 (BET proteins)

Reads H3K27ac marks

Chromodomains

Read methylation

PHD fingers

Read methylation marks

★ BRD4 is the key oncogenic reader &rarr; THERAPEUTIC TARGET

H3K27ac

BRD4 docking, active super-enhancers

H3K27me3

Repressive, antagonizes BRD4

H3K9ac

Active transcription

H3K9me2/3

DSB repair sites

H4K16ac

Reduced at radiation DSBs

University Name | Epigenetics & Breast Cancer Research | 2024

Super-Enhancers & BRD4 Overexpression in Breast Cancer

TCGA Analysis: BRD4 as an Epigenetic Oncogene

BRD4 mRNA Expression Across Breast Cancer Subtypes (TCGA)

Normal Mammary

1.0×

Luminal A

1.8×

Luminal B

2.4×

HER2+

3.1×

TNBC / Basal-like

4.6×

4.6× overexpression uniquely in TNBC

High BRD4 = High tumor grade + Lymph node infiltration + Reduced survival

Potential prognostic biomarker for optimal risk stratification

Compact Chromatin (Repressed)

Open Chromatin with Super-Enhancer

MYC

BCL2

CCND1

TP53

BET Inhibitors (JQ1, MZ1) → BRD4 removed from super-enhancer → Preferential repression of oncogenes

BRD4 upregulation drives endocrine therapy resistance in ER+ breast cancer via super-enhancer reprogramming of ESR1

UNIV LOGO

INST LOGO

[University Name] | [Institute Name] | Data source: TCGA Breast Cancer (BRCA) Project

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EXPERIMENTAL MODEL

MCF-7: The Luminal A Breast Cancer Model

[University Name] | [Institute Name] | [Student Name] | [Enrollment ID] | [Department]

[UNIV]

[DEPT]

THERAPEUTIC TOOL

MZ1 — A PROTAC Degrader of BRD4

Induced Proximity → Ubiquitination → Proteasomal Degradation

[University Name] | [Department] | 2024

BRD4's post-translational regulation of p53 in ER+ breast cancer is poorly understood

No study examined BRD4 degradation (vs inhibition) effect on p53 in radiation context

rMATS alternative splicing analysis pipeline not established in lab for splicing modulator studies

To investigate the role of BRD4 in regulation of p53 expression in breast cancer cells (MCF-7) and to establish rMATS pipeline in lab to study splicing changes upon treatment with splicing modulators.

Evaluate effect of BRD4 degradation (MZ1) and ionizing radiation (2 Gy) on p53 PROTEIN expression in MCF-7 using Western blotting

Study effect of BRD4 degradation on p53 TRANSCRIPT expression using RT-PCR

Establish a functional rMATS pipeline for alternative splicing analysis

[University Name] | [Institute Name] | [Department]

Experimental Design

MCF-7 Cells | MZ1 PROTAC | Ionizing Radiation

MCF-7 (ER+, Wild-type p53) &nbsp;|&nbsp; DMEM + 10% FBS + 1% Pen/Strep &nbsp;|&nbsp; 37°C, 5% CO2 &nbsp;|&nbsp; 70-80% confluency

MZ1 Treatment

100 nM MZ1 (PROTAC BRD4 degrader)

Pre-treatment before radiation

Selective BRD4 degradation

8 Experimental Groups

Control

−MZ1 / −IR

MZ1 Only

+MZ1 / −IR

2Gy 30min

−MZ1 / +IR

MZ1+2Gy 30min

+MZ1 / +IR

2Gy 2hr

−MZ1 / +IR

MZ1+2Gy 2hr

+MZ1 / +IR

2Gy 4hr

−MZ1 / +IR

MZ1+2Gy 4hr

+MZ1 / +IR

Ionizing Radiation

2 Gy dose

Harvested at 3 time points: 30 min, 2 hr, 4 hr

Protein Analysis

Nuclear Protein Extraction

BCA Assay

Western Blot (p53 + α-tubulin)

Transcript Analysis

RNA Extraction

cDNA Synthesis

RT-PCR (p53/TBP)

Bioinformatics

rMATS Pipeline Setup

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[University Name] | [Institute Name] | [Student Name] | [Enrollment ID] | [Department]

METHODOLOGY

Nuclear Protein Extraction & BCA Protein Assay

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12

Western Blotting Protocol

SDS-PAGE → Transfer → Antibody Detection

Sample Preparation & SDS-PAGE

Denaturation at 95°C with 4× SDS loading dye + β-mercaptoethanol

12% resolving gel + 4% stacking gel

120V stacking → 100V resolving

Tris-glycine-SDS running buffer

PVDF Membrane Transfer

Wet transfer system

PVDF activated in methanol 15-20s

Transfer buffer: Methanol 20% + 1x Running buffer

Blocking

5% BSA in TBST — 1 hour RT

Prevents non-specific antibody binding

Antibody Incubation

Detection & Analysis

Li-Cor Odyssey Infrared Imaging System

Band densitometry: ImageJ software

Normalized to α-tubulin loading control

[University Name] | [Institute Name] | [Department]

MOLECULAR ANALYSIS

RNA Extraction & RT-PCR Protocol

RNA Extraction Flowchart (QIAGEN RNeasy Plus)

Cell Lysis

RLT buffer added to pellet, repeated pipetting, centrifuge 10,000 rpm/3 min

QIAshredder Column

Mechanical homogenization, reduce viscosity, remove debris

gDNA Eliminator Column

Removes genomic DNA contamination

Add 70% Ethanol

Promotes RNA binding to silica membrane

RNeasy Spin Column

RNA binds selectively, contaminants pass through

DNase Treatment

DNase + RDD buffer, 15 min RT, complete DNA digestion

Wash Steps

RW1 buffer washes, remove proteins

Elute with RNase-free water

Purified RNA collected

PURITY CHECK

Both samples: high purity, A260/280 ≈ 2.0 = RNA quality confirmed

cDNA SYNTHESIS

RT-PCR SETUP (SYBR Green)

PRIMERS

Analysis Method

[University Name] • [Institute Name] • Department of Biochemistry

rMATS Pipeline: Alternative Splicing Analysis

Offline Setup on WSL Ubuntu 20.04 | Differential Splicing Detection

Statistical framework for detecting differential alternative splicing from RNA-Seq data.

Compares splice junction reads between two conditions using replicate BAM files + GTF annotation.

Quality control of raw sequence data

Adapter removal & quality filtering

Splice-aware alignment to human genome

SAM → BAM conversion, sort & index

treated_bams.txt + control_bams.txt + GTF

5 distinct splicing event types

Ubuntu 20.04 (WSL)

Exon inclusion level difference

Statistical significance

Corrected for multiple testing

Pipeline Successfully Established

Framework ready for splicing modulator analysis

[University Name] | [Institute Name] | [Student Name] | Bioinformatics Core

RESULTS — SECTION 4.1 & 4.2

MCF-7 Cell Morphology & Protein Quantification

Adherent epithelial morphology confirmed

Polygonal to irregular shape — epithelial origin

Cobblestone arrangement — intact cell-cell adhesion

Active proliferation clusters visible

Cells suitable for downstream experiments

MZ1 + Radiation combinations show altered protein concentrations — treatment-dependent cellular responses confirmed

[University Name] | [Institute Name] | [Student Name] | [Enrollment ID] | [Department]

Western Blot Analysis: p53 Protein Expression

BRD4 Degradation (MZ1) Consistently Elevates p53 Levels

p53

BRD4

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RESULTS — 4.4 & 4.5

RT-PCR: TP53 Transcript Analysis & Agarose Gel

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RNA Quality Summary

1472.44 ng/µL | A260/280 = 2.094

1272.32 ng/µL | A260/280 = 2.103

p53

TBP

26.32

26.48

22.72

22.31

ΔCq = 3.60

ΔCq = 4.17

ΔΔCq = 0.56

Fold Change = 0.68

Fold change ≈ 0.68 — NOT significantly different from 1.0 (unity).

MZ1 does NOT alter TP53 mRNA levels.

No visible change in band intensity between control and MZ-1 samples → corroborates RT-PCR finding

MZ1 does NOT affect TP53 at transcriptional level → BRD4 regulates p53 POST-TRANSCRIPTIONALLY / POST-TRANSLATIONALLY (protein stability mechanism)

[University Name] | [Institute Name] | [Student Name] | [Enrollment ID] | [Department]

rMATS Pipeline: Alternative Splicing Output

Successfully Established Framework for Splicing Analysis

LOGO

LOGO

Bioinformatics Pipeline | Splicing Analysis | Transcriptomics

Application Context

Key Laboratory Milestone

This establishes the first rMATS workflow in the laboratory — enabling future transcriptome-wide splicing studies in cancer.

DISCUSSION

Interpretation of Findings

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Key Finding 1: MZ1 Elevates p53 Protein

BRD4 degradation by MZ1 → Consistent p53 protein upregulation across all conditions

Strongest effect at 2hr post-2Gy radiation

Mechanism proposed: BRD4 regulates MDM2-p53 axis OR directly stabilizes p53 protein post-translationally

Supporting literature: Nagarajan et al. (2022), Zhou et al. (2022)

Key Finding 2: No Transcriptional Change

TP53 mRNA levels unchanged by MZ1 (fold change = 0.68, near unity)

BRD4 does NOT regulate p53 at transcriptional level in these conditions

Mechanistic implication: BRD4 may regulate p53 protein STABILITY via MDM2 degradation pathway or direct protein interaction

Paradigm: Protein-level ≠ mRNA-level regulation

MCF-7 Model Significance

Wild-type p53 — clean model for epigenetic regulation study

Luminal A → 40% of all breast cancers → high clinical relevance

Intact MDM2-p53 feedback loop allows clean interpretation

Results free from gain-of-function mutant p53 confound (unlike MDA-MB-231)

rMATS Pipeline Achievement

First rMATS workflow established in laboratory

Successfully processes RNA-Seq → BAM → Differential splicing events

Framework ready for: splicing modulator experiments, spliceome characterization

Foundation for future transcriptome-wide studies

Integrative Model: BRD4-p53 Regulation & Radiation Response

BRD4 Active

p53 Protein Regulated at Post-translational Level

Normal p53 Function

MZ1 Treatment

BRD4 Degraded

Altered p53 Protein Stability

Enhanced p53 Accumulation

Radiation

p53 Stabilized via ATM

Combined Effect Amplified with MZ1

[University Name] | [Institute Name] | [Student Name] | [Enrollment ID] | [Department]

BRD4 as a Post-Translational Regulator of p53 in Breast Cancer

Experimental model successfully established

MCF-7 cells cultured, nuclear protein isolation, BCA quantification, Western blotting, RNA extraction, RT-PCR all performed effectively — reliable data generated

BRD4 Degradation Increases p53 Protein

MZ1 treatment leads to increased p53 protein intensity in MCF-7 nuclear extracts → BRD4 plays a positive regulatory role in p53 protein expression

Enhanced effect observed under 2Gy radiation — peak at 2hr post-irradiation

BRD4 Does NOT Affect TP53 Transcription

qPCR fold change ≈ 0.68 (near unity) → MZ1 does not alter TP53 mRNA levels

BRD4 regulates p53 POST-TRANSCRIPTIONALLY or POST-TRANSLATIONALLY

rMATS Pipeline Successfully Established

First rMATS framework in the laboratory — bioinformatics infrastructure for future splicing studies validated

Therapeutic Implication

BRD4 may be a therapeutic target for enhancing radiosensitivity — particularly in tumors with aberrant p53 expression

Rationale for BRD4-targeted therapy combined with radiotherapy to overcome resistance

These findings highlight a previously underappreciated role of BRD4 in post-translational p53 regulation in hormone-responsive breast cancer

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PhD Defense | Conclusion | BRD4 and p53 Regulation

FUTURE DIRECTIONS

Future Work & Research Scope

Mechanistic Validation

Co-immunoprecipitation (Co-IP) to confirm BRD4-MDM2 interaction

Ubiquitination assays — confirm BRD4 role in p53 proteasomal regulation

CHX chase experiment — p53 protein stability assay

Functional Assays

Apoptosis detection (Annexin V/PI)

Clonogenic survival assay post-MZ1+radiation

Cell cycle analysis (flow cytometry)

Validate protein changes → biological outcomes

ChIP-seq Analysis

ChIP-seq for BRD4 and H3K27ac at TP53 locus

Confirm BRD4 super-enhancer occupancy at TP53

Map epigenetic changes post-MZ1 treatment

Extension to TNBC

Study BRD4-p53 axis in MDA-MB-231 (mutant p53 R280K)

Compare regulation between wt-p53 (MCF-7) vs mutp53 (MDA-MB-231)

Determine subtype-specificity of BRD4-p53 regulation

In Vivo & Clinical Translation

MZ1 + tamoxifen combination in MCF-7 xenograft models

MZ1 + ionizing radiation in vivo — test radiosensitization

Clinical relevance: BRD4 as predictive biomarker for radiation response

Establishing BRD4 as a radiosensitization target could revolutionize treatment of ER+ breast cancer patients resistant to endocrine therapy

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Slide 24

References

Key Citations Supporting This Research

37 references

GLOBOCAN 2022 | WHO 2019 | WCRF 2025

[University Name] | [Institute Name] | [Student Name] | [Enrollment ID] | [Department]

1.

Ali et al. (2021) — Co-targeting BRD4 and RAC1.

Int J Biol Sci, 17(14), 3760–3779.

2.

Dillon et al. (2023) — BRD4 inhibition in ER+ breast cancer.

Cancers, 15(16), 4066.

3.

Frezzo et al. (2023) — BRD4-p53 signalling axis.

Int J Mol Sci, 24(7), 6201.

4.

Gadd et al. (2017) — PROTAC structural basis.

Nature Chem Biol, 13(5), 514–521.

5.

Kotekar & Bhatt (2023) — BRD4 and MYC.

FEBS J, 290(20), 4820–4842.

6.

Liu et al. (2022) — BRD4 post-translational modifications.

Front Oncol, 12, 847701.

7.

Ma et al. (2022) — MZ1 in AML.

Cancer Biol Ther, 23(1), 1–15.

8.

Nagarajan et al. (2022) — BRD4 and wt-p53 in luminal cancer.

Nucleic Acids Res, 50(11).

9.

Naeimzadeh et al. (2024) — Mutant p53 in TNBC.

Cell Commun Signal, 22, 484.

10.

Qian et al. (2023) — Super-enhancers and BRD4.

Cell Death Discov, 9(1), 470.

11.

Wan et al. (2022) — BRD4 super-enhancer in breast cancer.

PNAS, 119(6).

12.

Wang et al. (2023) — BET proteins: biology and therapy.

Signal Transduct, 8, 420.

13.

Yousuf & Khan (2025) — MDM2-p53 in breast cancer.

Oncology Res, 33(4).

14.

Zengerle et al. (2015) — MZ1 selective BRD4 degradation.

ACS Chem Biol, 10(8).

15.

Zhou et al. (2022) — BRD4 and mutant p53 in TNBC.

Int J Mol Sci, 23(23).

16.

Arnold et al. (2022) — Global breast cancer burden.

The Breast, 66, 15–23.

17.

Essmann et al. (2004) — MCF-7 radiation apoptosis resistance.

Cancer Res, 64(19).

18.

Floyd et al. (2013) — BRD4 insulates chromatin from DDR.

Nature, 498(7453).

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Thank You

Questions & Discussion

Research Summary

🔬

BRD4 positively regulates p53 protein in MCF-7 cells

🧬

Regulation is post-translational, not transcriptional

⚗️

rMATS pipeline successfully established

[Student Name]

[student.email@university.edu]

[Department of Biochemistry / Biotechnology]

[Institute Name | University Name]

[Dr. Guide Name]

[Department | Institute | Year]

[University Name] | [Institute Name] | [Enrollment ID] | [Department]