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. 2013 Jul;133(1):21-30.
doi: 10.1002/ijc.27994. Epub 2013 Feb 8.

AKT inhibition mitigates GRP78 (glucose-regulated protein) expression and contribution to chemoresistance in endometrial cancers

Michael J Gray  1 , Paulette Mhawech-Fauceglia, Eunjeong Yoo, Wangrong Yang, Eijean Wu, Amy S Lee, Yvonne G Lin
Affiliations

AKT inhibition mitigates GRP78 (glucose-regulated protein) expression and contribution to chemoresistance in endometrial cancers

Michael J Gray et al. Int J Cancer. 2013 Jul.

Abstract

Overexpression of the unfolded protein response master regulator GRP78 is associated with poor prognosis and therapeutic resistance in numerous human cancers, yet its role in endometrial cancers (EC) is undefined. To better understand the contribution of GRP78 to EC, we examined its expression levels in EC patient samples and EC cell lines. We demonstrate that GRP78 overexpression occurs more frequently in EC tissues compared with that found in normal endometrium, and that GRP78 expression occurs in most EC cell lines examined. Functional analysis demonstrated that GRP78 is inducible by cisplatin in EC cells, and siRNA knockdown of GRP78 augments chemotherapy-mediated cell death. Examination of AKT and GRP78 expression demonstrated that inhibition of AKT activity by MK2206 blocks GRP78 expression in EC cells. SiRNA studies also revealed that knockdown of GRP78 reduces but does not abrogate AKT activity, demonstrating that GRP78 is required for optimal AKT activity. In the presence of MK2206, siRNA knockdown of GRP78 does not augment AKT mediated survival in response to cisplatin treatment, suggesting that GRP78's antiapoptosis functions are part of the AKT survival pathway. Targeted therapies that reduce GRP78 expression or activity in cancers may serve to increase the effectiveness of current therapies for EC patients.

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Conflict of interest statement

Conflict of Interest Disclosure: The authors listed above have no commercial or financial relationships relevant to the subject of this manuscript

Figures

Figure 1
Figure 1. Assessment of GRP78 expression in human endometrial tissues and cell lines
A, GRP78 patient tissue analysis. Graphical representation of normal endometrium and EC tissues samples strained for GRP78 expression. Samples where strong GRP78 detection occurred were considered positive. B, Western blot analysis of GRP78 and pTEN expression in nine endometrial cancer cell lines. Vinculin was used as an internal loading control. C, Western blot time course analysis of the ability of cisplatin (1 uM) to induce GRP78 expression in the EC cell lines Ishikawa, ECC-1, AN3PCA, and ARK1. Vinculin was used as an internal loading control. Thapsigargin, a known GRP78 inducer was used as a positive control (TG).
Figure 2
Figure 2. Effect GRP78 knockdown on cisplatin mediated cytotoxicity and apoptosis in human endometrial cancer cells
A, Western blot analysis of GRP78 levels in lentivirus infected siRNA controls (siCon) and siRNA-GRP78 knockdown cells (siGRP78) in Ishikawa (left) and AN3CA cells (right). Cells were grown for the same time length as those in EC50 studies to ensure extended knockdown. B, EC50 assay examining the effectiveness of cisplatin, cisplatin+SiCon and cisplatin + siGRP78 cytotoxicity in Ishikawa (left) and AN3CA cells (right) lentiviral infected cells. C, Western blot analysis of apoptotic regulatory proteins in Ishikawa (left) and AN3CA cells (right). Cells were transient transfected with control siRNA scrambled (siCon) or siRNA-GRP78 specific sequences (siGRP78) and levels of GRP78, PARP, cleaved PARP, Caspase-3, and cleaved caspase-3 determined after treating with cisplatin (1uM) for the times indicated. Vinculin was used as an internal loading control.
Figure 3
Figure 3. Requirement for AKT activity in GRP78 expression in endometrial cancer cells
A, Western blot analysis of cisplatin (1uM) treated Ishikawa cells (left) and AN3CA cells (right) for levels of GRP78, phospho-AKT, and total AKT, and with graphical representation of image analysis of changes in pAKT activity. Vinculin was used as an internal loading control. B, Analysis of Ishikawa cells (left) and AN3CA cells (right) pretreated with or without the AKT inhibitor MK2206 (50nM) followed by cisplatin treatment (1uM). Levels of GRP78, phospho-AKT, total AKT, and phospho-ERK 1/2 were determined by western analysis. Changes in GRP78 levels accompanying AKT inhibition represented by graphical representation of image analysis for GRP78 are displayed. Vinculin was used as an internal loading control.
Figure 4
Figure 4. Effect of GRP78 knockdown on AKT and ERK 1/2 signaling in endometrial cancer cells
A, Western analysis of siRNA controls (siCon) and GRP78 knockdowns (siGRP78) transiently transfected as described and treated with and without cisplatin for 24, 48 and 72 hours. Ishikawa cells (upper) and AN3CA cells (lower) were probed for levels of GRP78, phospho-AKT, phospho-ERK, and total AKT. Vinculin was used as an internal loading control.
Figure 5
Figure 5. Effect of AKT inhibition and GRP78 knockdown on cisplatin mediated cytotoxicity
A, Western analysis examining the dose response MK2206 on AKT and GRP78 in Ishikawa cells. B EC50 assays examining the cytotoxic effects of cisplatin on Ishikawa as described in materials and methods. Treatment groups were parental control (Cisplatin), or cisplatin with siRNA control (Cisplatin+siCon), or cisplatin+GRP78 knockdown (Cisplatin +siGRP78) with or without combination with MK2206 (5nM, 50nM). Graph is representative of one of five independent experiments. C, Western analysis examining the dose response MK2206 on AKT and GRP78 in AN3CA cells. B EC50 assays examining the cytotoxic effects of cisplatin on AN3CA as described in materials and methods. Treatment groups were parental control (Cisplatin), or cisplatin with siRNA control (Cisplatin+siCon), or cisplatin+GRP78 knockdown (Cisplatin +siGRP78) with or without combination with MK2206 (5nM, 50nM). Graph is representative of one of five independent experiments.
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