The Role of PTEN in Chemoresistance Mediated by the HIF-1α/YY1 Axis in Pediatric Acute Lymphoblastic Leukemia

Abstract

Acute lymphoblastic leukemia (ALL) is the most common childhood cancer. Current chemotherapy treatment regimens have improved survival rates to approximately 80%; however, resistance development remains the primary cause of treatment failure, affecting around 20% of cases. Some studies indicate that loss of the phosphatase and tensin homolog (PTEN) leads to deregulation of the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway, increasing the expression of proteins involved in chemoresistance. PTEN loss results in deregulation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and induces hypoxia-inducible factor 1-alpha (HIF-1α) expression in various cancers. Additionally, it triggers upregulation of the Yin Yang 1 (YY1) transcription factor, leading to chemoresistance mediated by glycoprotein p-170 (Gp-170). The aim of this study was to investigate the role of the PTEN/NF-κB axis in YY1 regulation via HIF-1α and its involvement in ALL. A PTEN inhibitor was administered in RS4;11 cells, followed by the evaluation of PTEN, NF-κB, HIF-1α, YY1, and Gp-170 expression, along with chemoresistance assessment. PTEN, HIF-1α, and YY1 expression levels were assessed in the peripheral blood mononuclear cells (PBMC) from pediatric ALL patients. The results reveal that the inhibition of PTEN activity significantly increases the expression of pAkt and NF-κB, which is consistent with the increase in the expression of HIF-1α and YY1 in RS4;11 cells. In turn, this inhibition increases the expression of the glycoprotein Gp-170, affecting doxorubicin accumulation in the cells treated with the inhibitor. Samples from pediatric ALL patients exhibit PTEN expression and higher HIF-1α and YY1 expression compared to controls. PTEN/Akt/NF-κB axis plays a critical role in the regulation of YY1 through HIF-1α, and this mechanism contributes to Gp-170-mediated chemoresistance in pediatric ALL.

Keywords: Gp-170; HIF-1α; PTEN; YY1; leukemia.

Figure 1

SF1670 treatment decreases PTEN expression in RS4;11 cells. (A) Representative microphotograph of PTEN expression in RS4;11 cells treated with the inhibitor SF1670 at 1 µM. A decrease in the expression of PTEN is observed, although it is not statistically significant in RS4;11 cells treated with the inhibitor and an increase in the expression of pAkt, NF-κB, and PTEN (* p < 0.05; * p < 0.05) compared to untreated cells. (B) Expression of HIF-1α, YY1, and gp-170 in RS4 cells; 11 treated with the inhibitor, (HIF-1α * p < 0.05; YY1 * p < 0.05; Gp-170 * p < 0.05; untreated vs. inhibitor). 400× magnification.

Figure 2

Inhibition of PTEN with SF1670 promotes the expression of NF-κB, YY1, and Gp-170. (A) Western Blot representative of the amount of total protein in RS4;11 cells treated with SF1670; the results show differences in the expression of p-Akt and Gp-170 (** p < 0.01; *** p < 0.001), after treatment with the inhibitor of PTEN. (B) The expression of NF-κB and YY1 in the cell line used is modified after treatment with the inhibitor (* p < 0.05; ** p < 0.01); PTEN does not show changes in the level of protein expression when using the PTEN inhibitor, and HIF-1α has no significant changes. (C) Expression of mRNA of YY1 is increased in RS4;11 cells after SF1670 treatment (*** p = 0.001 treatment vs. untreated).

Figure 3

Inhibition of PTEN with SF1670 decreases cell death in RS4;11. (A) Western Blot analysis of active caspase-3 from RS4;11 cells treated with SF1670; the results show difference in expression after treatment (**** p < 0.0001). (B) Doxorubicin accumulation analysis after treatment with the PTEN inhibitor shows a change in the intracellular levels of the drug in RS4;11 cells (** p < 0.01). (C) Analysis of RS4;11 TUNEL positive cells treated with the inhibitor shows a decrease in TUNEL positive cells in the treatment with PTEN inhibitor and doxorubicin (** p < 0.01). 400× magnification.

Figure 4

Patients with ALL exhibit decreased expression of PTEN and elevated expression of pAkt, NF-κB, HIF-1α, and YY1. (A) PTEN expression in mononuclear cells from pediatric patients with ALL compared with cells from healthy controls showed lower expression of PTEN. An increase in the percentage of cells positive for pAkt, NF-κB, HIF-1, and YY1 was observed in cells from patients with ALL compared to healthy controls (PTEN **** p < 0.0001; pAkt **** p < 0.0001; NF-κB *** p < 0.001; HIF-1α **** p < 0.0001; YY1 ** p < 0.01; control vs. ALL, respectively) 600× magnification. (B) Correlation between the expressions of PTEN and HIF-1α in pediatric ALL, showing a negative correlation between the expression of PTEN and HIF-1α in cells from patients with ALL (Pearson’s test. r = 0.3387, p = 0.047). (C) Correlation between the expressions of PTEN and YY1 in pediatric ALL, showing a negative correlation between the expression of PTEN and YY1 in cells from patients with ALL (Pearson’s test. r = 0.2646, p = 0.0292).

Figure 5

Expression of PTEN, HIF-1α, and YY1 in the cellular immunophenotypes of pediatric ALL. The percentages of positive cells for PTEN, HIF-1α, and YY1 are shown in the different immunophenotypes of patients with ALL (n = 68) and control individuals (n = 50) in peripheral blood cells of each study subject. (A) Shows the expression of PTEN in the different immunophenotypes with a statistically significant increase in the percentage of positive PTEN cells in the samples of patients with ALL with immunophenotype Pro-B, Pre-B, B, and T vs. control (* p < 0.05; ** p < 0.01; *** p < 0.001; ** p < 0.01). (B) Indicates the expression of HIF-1α in the different immunophenotypes, with a statistically significant increase in the percentage of positive HIF-1α cells in the samples of patients with ALL with Pro-B, Pre-B, and B immunophenotype vs. control (*** p < 0.001; *** p < 0.001; *** p < 0.001). (C) Shows the expression of YY1 in the different immunophenotypes, with a statistically significant increase in the percentage of positive YY1 cells in the samples of patients with ALL with immunophenotype Pro-B vs. control (** p < 0.01). Comparison was made using unpaired two-tailed t-test.

Figure 6

Involvement of PTEN in chemoresistance mediated by the HIF-1α/YY1 axis in pediatric ALL. Inhibition of PTEN through SF1670 promotes the activation of Akt, which leads to the activation of NF-κB, this results in the translocation of transcription factors such as HIF-1α and YY1, which exert their activity on their genes target, importantly in the expression of the glycoprotein Gp-170, which expels drugs and promotes chemoresistance in pediatric ALL.