Short-term hypoxia and nutrient deprivation stress induced shifts in p53 isoform expression in HepG2 tumourspheres.
The hypoxic, nutrient-deprived tumour microenvironment (TME), a hallmark of solid tumours, imposes cellular stress that can also paradoxically promote survival and resistance. While TP53 is the most frequently mutated gene in cancer, approximately 60% of hepatocellular carcinoma (HCC) cases retain wild-type TP53 (WTp53), suggesting its isoforms as potential tumorigenic modulators that override canonical tumour-suppressive functions. Therefore, this study aims to delineate p53 isoform profiles in response to short-term hypoxia and nutrient deprivation, recapitulating key stressors in the tumour biology.
We hereby established a 7-day HepG2 tumoursphere model by seeding 15,000 cells/well, which transitioned from normoxia (4 days) to a hypoxic (1% O2), low-serum (1% FBS) (HLS) condition (3 days). Expression of p53 isoforms and downstream targets was assessed.
The formation of HepG2 tumoursphere at different seeding densities determined the diameter, viability and proliferation profiles, with 15,000 cells/well producing optimal, viable tumourspheres with the highest yield. HLS conditions significantly reduced tumoursphere size, proliferation capacity and viability. Strikingly, multiplex long-amplicon ddPCR revealed substantial upregulation of FLp53α/Δ40p53α, Δ40p53α and Δ133p53α/Δ160p53α mRNA transcripts. While FLp53α/Δ40p53α remained dominant, Δ40p53α and Δ133p53α/Δ160p53α progressively increased, altering the balance among the isoforms. This shift correlated with enhanced expression of the pro-proliferative and survival markers (PCNA and BCL2) and reduced expression of the pro-apoptotic marker (BAX) and cell cycle inhibitor (CDKN1A), suggesting a potential functional role of these isoforms in promoting tumour cell adaptation under stress.
This study highlights stress-induced p53 isoform modulation as a potential survival mechanism in WTp53 HCC in response to TME stress, which warrants further exploration of isoform-specific p53 functions in understanding heterogeneity, resistance and cancer recurrence.
We hereby established a 7-day HepG2 tumoursphere model by seeding 15,000 cells/well, which transitioned from normoxia (4 days) to a hypoxic (1% O2), low-serum (1% FBS) (HLS) condition (3 days). Expression of p53 isoforms and downstream targets was assessed.
The formation of HepG2 tumoursphere at different seeding densities determined the diameter, viability and proliferation profiles, with 15,000 cells/well producing optimal, viable tumourspheres with the highest yield. HLS conditions significantly reduced tumoursphere size, proliferation capacity and viability. Strikingly, multiplex long-amplicon ddPCR revealed substantial upregulation of FLp53α/Δ40p53α, Δ40p53α and Δ133p53α/Δ160p53α mRNA transcripts. While FLp53α/Δ40p53α remained dominant, Δ40p53α and Δ133p53α/Δ160p53α progressively increased, altering the balance among the isoforms. This shift correlated with enhanced expression of the pro-proliferative and survival markers (PCNA and BCL2) and reduced expression of the pro-apoptotic marker (BAX) and cell cycle inhibitor (CDKN1A), suggesting a potential functional role of these isoforms in promoting tumour cell adaptation under stress.
This study highlights stress-induced p53 isoform modulation as a potential survival mechanism in WTp53 HCC in response to TME stress, which warrants further exploration of isoform-specific p53 functions in understanding heterogeneity, resistance and cancer recurrence.
Authors
Keoh Keoh, Ahmad Tarmizi Ahmad Tarmizi, Sawai Sawai, Oh Oh, Ramasamy Ramasamy
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