Overexpression of pyruvate dehydrogenase kinase 1 in retinoblastoma: A potential therapeutic opportunity for targeting vitreous seeds and hypoxic regions

Abstract

Pyruvate dehydrogenase kinase 1 (PDK1), a key enzyme implicated in metabolic reprogramming of tumors, is induced in several tumors including glioblastoma, breast cancer and melanoma. However, the role played by PDK1 is not studied in retinoblastoma (RB). In this study, we have evaluated the expression of PDK1 in RB clinical samples, and studied its inhibition as a strategy to decrease cell growth and migration. We show that PDK1 is specifically overexpressed in RB patient samples especially in vitreous seeds and hypoxic regions and cell lines compared to control retina using immunohistochemistry and real-time PCR. Our results further demonstrate that inhibition of PDK1 using small molecule inhibitors dichloroacetic acid (DCA) and dichloroacetophenone (DAP) resulted in reduced cell growth and increased apoptosis. We also confirm that combination treatment of DCA with chemotherapeutic agent carboplatin further enhanced the therapeutic efficacy compared to single drug treatment. In addition, we observed changes in glucose uptake, lactate and reactive oxygen species (ROS) levels as well as decreased cell migration in response to PDK1 inhibition. Additionally, we show that DCA treatment led to inhibition of PI3K/Akt pathway and reduction in PDK1 protein levels. Overall, our data suggest that targeting PDK1 could be a novel therapeutic strategy for RB.

Fig 1. Expression of PDK1 in retinoblastoma tissues.

(A) Immunohistochemistry (IHC) showing expression of PDK1 in retinoblastoma specimens. * Indicates vessel lumen. (B) IHC showing poor expression of PDK1 in uninvolved retina and strong expression in RB tumor tissue. Orange curly bracket indicates the uninvolved retina and red arrow denotes the expression of PDK1 in RB tumor region. (C) Relative mRNA expression of PDK1 was compared between retinoblastoma tissues and control retina. C1-C2, control retina; Y79 and Weri-Rb1, retinoblastoma cell lines; P1-P6, retinoblastoma tumor tissues.

Fig 2. PDK1 inhibition results in decreased retinoblastoma cell growth.

(A) Retinoblastoma cell lines (Y79 and Weri-Rb1) and patient derived cells (LRB1 and LRB2) were treated with different concentrations of DCA and cell growth was measured and compared to untreated cells. (B) RB cells were treated with increasing concentrations of DAP and cell growth was measured and compared to control cells. * Indicates significant difference (p<0.05) between control and treatment.

Fig 3. Combination activity of DCA and carboplatin on retinoblastoma cell growth.

Y79 and patient derived retinoblastoma cells (LRB1 and LRB2) were treated with DCA or carboplatin alone or combination of DCA and carboplatin at various concentrations. Cell growth was measured by MTS assay and combination indices (CI) were calculated. CI = 1 –additive effect; CI<1 –synergistic effect and CI>1 –antagonistic effect.

Fig 4. Inhibition of PDK1 induces apoptosis and decreases cell motility.

Y79 and patient derived retinoblastoma cells (LRB1 and LRB2) were treated with DCA and (A) apoptosis and (B, C and D) cell migration were measured. * Indicates significant changes (p<0.05) between control and treatment.

Fig 5. PDK1 inhibition alters metabolic parameters.

Y79 and retinoblastoma primary cells (LRB1and LRB2) were treated with DCA and different metabolic parameters were measured. (A) Glucose uptake was measured using 2-NBD glucose. (B) Reactive oxygen species (ROS) levels were measured using DCF-DA. (C) ROS levels were measured by flow cytometry in response to inhibition with DAP. (D) RB cells were treated with DCA and DCA+vitamin C and ROS levels were measured and compared to control cells. (E) RB cells were treated with DCA and DCA+vitamin C and cell viability was measured and compared to control cells. * Indicates significant difference (p<0.05) between control and treatment.

Fig 6. Effect of DCA treatment on PDK1 protein levels and PI3K/Akt pathway in RB.

(A) DCA treatment decreases protein levels of PDK1. (B) Treatment of RB cells with DAP results in reduced levels of PDK1 protein. (C) Treatment of RB cells with DCA leads to decreased PDK1 protein levels under hypoxia. (D) DCA alters the activity of PI3K/Akt pathway in RB cells.