Role of mitochondria in cancer: G-quadruplex structures at fragile regions of the mitochondrial genome and a novel mode of BCL2 inhibition in cancer therapeutics

Abstract

Mitochondrial functions are supported by proteins encoded by both the mitochondrial genome and several nuclear-encoded proteins targeted to mitochondria. Mutations affecting mitochondrial functions lead to cellular reprogramming, resulting in enhanced proliferation and metastasis in cancers.

Common mitochondrial defects in cancers include:

Failure of oxidative phosphorylation, leading to increased glycolysis and anabolic pathways.

Defective mitochondrial apoptosis, resulting in accumulation of mutations and pro-metastatic retrograde signaling.

Thus, improper mitochondrial functioning and its therapeutic intervention are key areas in cancer progression and prevention.

Aim of the Study This study investigated:

Inhibition of the mitochondrial protein BCL2 as a strategy for cancer therapeutics.

The molecular basis of large-scale deletions in the mitochondrial genome, relevant to cancer progression.

BCL2 Inhibition and Cancer Therapy The BCL2 family of proteins regulate apoptosis at the mitochondrial outer membrane.

Overexpression of anti-apoptotic proteins like BCL2 imparts survival advantage and chemoresistance to cancer cells.

A novel inhibitor, Disarib, was identified and studied:

Highly selective for cell lines with elevated BCL2 expression.

IC50 values between 3-10 M in BCL2-high cell lines, ~3 times more effective than in low-BCL2 lines.

siRNA-mediated knockdown of BCL2 rendered cells resistant, while ectopic expression restored sensitivity.

Biophysical and in silico analyses confirmed specific binding to BCL2, not to its paralogue BCL-xL.

Disarib caused tumor regression in three mouse models with good tolerance and minimal toxicity.

Mechanism: competitive inhibition of BCL2-BAK complex formation, displacing BAK.

Less effective against BCL2-BAX complex.

Potentiated cytotoxic activity of paclitaxel in a synergistic manner.

Conclusion: Disarib is a promising anticancer tool for BCL2-dependent cancers, with potential in combination therapies.

Mitochondrial Genome Instability The mitochondrial genome lacks noncoding regions, making it highly vulnerable to mutations.

Patient samples show frequent aberrations: point mutations, insertions, and large-scale deletions.

Hypothesis: G-quadruplexes, non-B DNA structures prone to breakage, contribute to mitochondrial deletions associated with cancer.

G-Quadruplex Studies Bioinformatic tools (MITOMAP, QUADPARSER, Non-B DB) identified high-frequency deletion regions harboring putative G-quadruplex motifs.

Oligomers corresponding to these regions were synthesized and analyzed:

Gel mobility shift assays confirmed inter- and intramolecular G-quadruplex formation, some potassium-dependent.

Mutation analysis revealed involvement of four guanine stretches.

Circular dichroism indicated parallel strand orientation.

DMS footprinting identified specific guanine residues in quadruplex formation.

Functional assays:

G-quadruplexes stalled DNA polymerase progression on single- and double-stranded DNA.

Replication arrest was potassium-dependent.

Quadruplex motifs stalled transcription in ex vivo reporter assays.

Bisulfite modification assay provided direct evidence of G-quadruplex formation in mitochondrial DNA.

Structural modeling revealed intramolecular, parallel quadruplexes with three-plate stacking.

Conclusion: This study provides the first experimental evidence for G-quadruplex formation in mitochondrial DNA, offering a probable explanation for mitochondrial genomic instability associated with cancers.

Overall Conclusion This thesis demonstrates:

Disarib as a novel, selective BCL2 inhibitor with therapeutic potential.

Experimental evidence linking mitochondrial G-quadruplexes to genomic instability and cancer progression.

Together, these findings highlight mitochondrial dysfunction as both a therapeutic target and a mechanistic contributor to cancer biology.