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Mutation-Associated Phenotypic Heterogeneity in Novel and Canonical PIK3CA Helical and Kinase Domain Mutants
Arman Ali Ghodsinia1, J-Ann Marie T Lego1, Reynaldo L Garcia1
1Disease Molecular Biology and Epigenetics Laboratory, National Institute of Molecular Biology and Biotechnology, University of the Philippines Diliman, Quezon City 1101, Philippines.
Abstract:
Phosphatidylinositol 3-kinase, catalytic subunit alpha (PIK3CA) is an oncogene often mutated in colorectal cancer (CRC). The contribution of PIK3CA mutations in acquired resistance to anti-epidermal growth factor receptor (EGFR) therapy is well documented, but their prognostic and predictive value remain unclear. Domain- and exon-specific mutations are implicated in either favorable or poor prognoses, but there is paucity in the number of mutations characterized outside of the mutational hotspots. Here, two novel non-hotspot mutants-Q661K in exon 13 and C901R in exon 19-were characterized alongside the canonical exon 9 E545K and exon 20 H1047R mutants in NIH3T3 and HCT116 cells. Q661K and E545K both map to the helical domain, whereas C901R and H1047R map to the kinase domain. Results showed variable effects of Q661K and C901R on morphology, cellular proliferation, apoptosis resistance, and cytoskeletal reorganization, with both not having any effect on cellular migration. In comparison, E545K markedly promoted proliferation, survival, cytoskeletal reorganization, migration, and spheroid formation, whereas H1047R only enhanced the first three. In silico docking suggested these mutations negatively affect binding of the p85 alpha regulatory subunit to PIK3CA, thereby relieving PIK3CA inhibition. Altogether, these findings support intra-domain and mutation-specific variability in oncogenic readouts, with implications in degree of aggressiveness.
Insights
Novel PIK3CA mutations in colorectal cancer show varied effects on cell behavior. Understanding these specific PIK3CA alterations is crucial for predicting treatment response and patient prognosis.
Area of Science:
- Molecular Biology
- Oncology
- Biochemistry
Background:
- Phosphatidylinositol 3-kinase, catalytic subunit alpha (PIK3CA) is a key oncogene in colorectal cancer (CRC).
- PIK3CA mutations are linked to resistance against anti-epidermal growth factor receptor (EGFR) therapy, but their prognostic significance is not fully understood.
- Domain- and exon-specific mutations can influence patient outcomes, yet many mutations outside hotspots remain uncharacterized.
Purpose of the Study:
- To investigate the functional impact of two novel non-hotspot PIK3CA mutants (Q661K and C901R) compared to canonical mutants (E545K and H1047R).
- To assess the effects of these PIK3CA mutations on cellular processes including morphology, proliferation, apoptosis resistance, migration, and cytoskeletal organization.
- To explore the in silico binding interactions between mutated PIK3CA and its regulatory subunit.
Main Methods:
- Characterization of PIK3CA mutants (Q661K, C901R, E545K, H1047R) in NIH3T3 and HCT116 cell lines.
- Assessment of cellular phenotypes: morphology, proliferation, apoptosis resistance, migration, and spheroid formation.
- In silico molecular docking to analyze subunit binding affinity.
Main Results:
- Novel mutants Q661K and C901R exhibited variable effects on cell morphology, proliferation, and apoptosis resistance, with no impact on migration.
- Canonical mutant E545K significantly promoted proliferation, survival, cytoskeletal reorganization, migration, and spheroid formation.
- Canonical mutant H1047R enhanced proliferation, survival, and cytoskeletal reorganization but not migration or spheroid formation.
- In silico analysis suggested mutations impair the binding of the p85 alpha regulatory subunit to PIK3CA, reducing inhibition.
Conclusions:
- PIK3CA mutations display intra-domain and mutation-specific variability in their oncogenic effects.
- These findings highlight the importance of characterizing non-hotspot mutations for understanding colorectal cancer aggressiveness and therapeutic resistance.
- The differential impacts of PIK3CA mutations have implications for predicting prognosis and guiding treatment strategies in CRC.
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