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Influence of ions, pH and crowding agents on structure and stability of PIM1 secondary structures
İrem Göller1, Saba Orouji1, Özgül Persil Çetinkol1
1Department of Chemistry, Middle East Technical University, Çankaya, 06800, Ankara, Turkey.
Abstract:
Triple Negative Breast Cancer (TNBC) is an aggressive subtype of breast cancer defined by the absence of ER, PR and HER-2 receptors. The lacking of these receptors limits the availability of the targeted therapy options. The recent identification of G-quadruplex-duplex hybrid (QDH) structures within PIM1 oncogenes, which are overexpressed in TNBC tumors, has highlighted their plausible potential as novel therapeutic targets for the treatment of TNBC. Herein, the effect of pH, ions and the molecular crowding on the selected PIM1 secondary structures was investigated in vitro using PAGE, UV-Vis and CD spectroscopy. The G-quadruplex (G4), i-motif (iM) and double helical DNA (dsDNA) structures formed by the selected PIM1 sequences were found to be stable under the investigated conditions and are likely to coexist specially under the acidic conditions. The lower pH was found to be stabilizing the iM structure. K+ ions exhibited a stronger stabilizing effect on G4 structures than Na+ ions. CD results suggested that molecular crowding alters the G4 structure in a cation-dependent manner, whereas the iM remained largely unaffected. Furthermore, the results demonstrated that molecular crowding stabilizes G-quadruplexes, but has a variable effect on i-motif and dsDNA stability based on their molecular weight. These findings may aid in modulating G4, iM, dsDNA, and QDH structures and the development of small molecules designed to target these structures, thereby contributing to future anticancer therapeutic strategies.
Insights
Triple Negative Breast Cancer (TNBC) is aggressive, lacking targeted therapies. PIM1 oncogene structures, like G-quadruplexes, show promise as new TNBC therapeutic targets, with stability influenced by pH, ions, and crowding.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- Triple Negative Breast Cancer (TNBC) lacks ER, PR, and HER-2 receptors, limiting targeted therapy options.
- PIM1 oncogenes are overexpressed in TNBC, and contain G-quadruplex-duplex hybrid (QDH) structures with therapeutic potential.
- Understanding DNA secondary structures in PIM1 is crucial for developing novel TNBC treatments.
Purpose of the Study:
- To investigate the in vitro effects of pH, ions (K+, Na+), and molecular crowding on PIM1 secondary structures (G-quadruplex, i-motif, dsDNA).
- To assess the stability and coexistence of these structures under various conditions relevant to the tumor microenvironment.
- To provide insights for designing small molecules targeting PIM1 structures for TNBC therapy.
Main Methods:
- Polyacrylamide Gel Electrophoresis (PAGE)
- UV-Vis Spectroscopy
- Circular Dichroism (CD) Spectroscopy
Main Results:
- G-quadruplex (G4), i-motif (iM), and double helical DNA (dsDNA) structures derived from PIM1 sequences are stable and can coexist, particularly in acidic conditions.
- Lower pH stabilizes iM structures; K+ ions stabilize G4 structures more than Na+ ions.
- Molecular crowding stabilizes G4 structures, alters G4 conformation cation-dependently, and affects iM and dsDNA stability based on molecular weight.
Conclusions:
- PIM1 secondary structures (G4, iM, dsDNA, QDH) exhibit stability under varying pH, ionic, and molecular crowding conditions.
- These findings support the potential of targeting PIM1 structures, including QDHs, for TNBC treatment.
- Modulating these DNA structures could lead to new anticancer therapeutic strategies against TNBC.
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