AKT is the downstream target of GRP78 in mediating cisplatin resistance in ER stress-tolerant human lung cancer cells

Thoracic Surgery Department, West China Hospital, Sichuan University School of Medicine, Chengdu Sichuan Province 610041, China.
Lung cancer (Amsterdam, Netherlands) (Impact Factor: 3.96). 03/2011; 71(3):291-7. DOI: 10.1016/j.lungcan.2010.06.004
Source: PubMed


Cisplatin [cis-diaminodichloroplatinum (II) (CDDP)] is the cornerstone of lung cancer chemotherapy. However, its efficacy is limited due to the development of drug resistance in cancer cells. This study was designed to uncover the mechanisms under CDDP resistance in lung cancer cells involving endoplasmic reticulum (ER) stress tolerance-induced and GRP78-dependant Akt activation. In this study we established ER stress-tolerant (ERST) human lung cancer lines H460et and A549et. We found that the ERST Lung cancer cells are resistant to CDDP treatment. We further showed that, compared to the parental cell lines, H460et and A549et show significantly increased GRP78 and phospho(p)-Akt levels. And phosphorylation of Akt, which can be regulated by GRP78, is essential to the ERST-associated CDDP resistance. Our findings identify a new mechanism of regulating Akt activity and a new mechanism through which CDDP resistance is formed in lung cancer cells.

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    • "We identified glucose-regulated protein 78 (GRP78), also known as BiP or HSPA5, with relatively low abundance. This protein also functions as a molecular chaperone in the ER, like GRP94 and calreticulin, and GRP78 expression levels have been correlated with cisplatin resistance in certain cancers [49], [50]. Overexpression and/or cisplatin-induced up-regulation of GRP78 significantly reduce the antineoplastic efficacy of cisplatin. "
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    ABSTRACT: Cisplatin is widely used as an antineoplastic drug, but its ototoxic and nephrotoxic side-effects, as well as the inherent or acquired resistance of some cancers to cisplatin, remain significant clinical problems. Cisplatin's selectivity in killing rapidly proliferating cancer cells is largely dependent on covalent binding to DNA via cisplatin's chloride sites that had been aquated. We hypothesized that cisplatin's toxicity in slowly proliferating or terminally differentiated cells is primarily due to drug-protein interactions, instead of drug-DNA binding. To identify proteins that bind to cisplatin, we synthesized two different platinum-agarose conjugates, one with two amino groups and another with two chlorides attached to platinum that are available for protein binding, and conducted pull-down assays using cochlear and kidney cells. Mass spectrometric analysis on protein bands after gel electrophoresis and Coomassie blue staining identified several proteins, including myosin IIA, glucose-regulated protein 94 (GRP94), heat shock protein 90 (HSP90), calreticulin, valosin containing protein (VCP), and ribosomal protein L5, as cisplatin-binding proteins. Future studies on the interaction of these proteins with cisplatin will elucidate whether these drug-protein interactions are involved in ototoxicity and nephrotoxicity, or contribute to tumor sensitivity or resistance to cisplatin treatment.
    PLoS ONE 06/2013; 8(6):e66220. DOI:10.1371/journal.pone.0066220 · 3.23 Impact Factor
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    • "In addition, another study showed that the phosphorylation of Akt was decreased in astrocytoma after transfection with GRP78 siRNA [29]. Lin et al. (2011) recently reported that Akt was a downstream target of GRP78 in intervening cisplatin resistance in ER-stress-tolerant human lung cancer cells [30]. These findings indicated that the GRP78-directed Akt pathway plays an important role in cancer survival. "
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    ABSTRACT: The 78-kDa glucose-regulated protein (GRP78) is induced in the cancer microenvironment and can be considered as a novel predictor of responsiveness to chemotherapy in many cancers. In this study, we found that intracellular reactive oxygen species (ROS) and nuclear factor erythroid 2-related factor 2 (Nrf2) nuclear translocation were higher in GRP78 knockdown DLD-1 colon cancer cells compared with scrambled control cells. Treatment with epirubicin in GRP78 knockdown DLD-1 cells enhanced apoptosis and was associated with decreased production of intracellular ROS. In addition, apoptosis was increased by the antioxidants propyl gallate (PG) and dithiothreitol (DTT) in epirubicin-treated scrambled control cells. Epirubicin-treated GRP78 knockdown cells resulted in more inactivated Akt pathway members, such as phosphorylated Akt and GSK-3β, as well as downstream targets of β-catenin expression. Knockdown of Nrf2 with small interfering RNA (siRNA) increased apoptosis in epirubicin-treated GRP78 knockdown cells, which suggested that Nrf2 may be a primary defense mechanism in GRP78 knockdown cells. We also demonstrated that epirubicin-treated GRP78 knockdown cells could decrease survival pathway signaling through the redox activation of protein phosphatase 2A (PP2A), which is a serine/threonine phosphatase that negatively regulates the Akt pathway. Our results indicate that epirubicin decreased the intracellular ROS in GRP78 knockdown cells, which decreased survival signaling through both the Akt pathway and the activation of PP2A. Together, these mechanisms contributed to the enhanced level of epirubicin-induced apoptosis that was observed in the GRP78 knockdown cells.
    PLoS ONE 04/2012; 7(4):e35123. DOI:10.1371/journal.pone.0035123 · 3.23 Impact Factor
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    • "The ER-specific chaperone, GRP78, has recently been reported to regulate AKT phosphorylation at the Ser473 residue [22], [23]. Additionally, it is also known that ER stress induces expression of GRP78 in the severity-dependent manner [12], [28], and so this appears a good candidate for further investigation. "
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    ABSTRACT: Endoplasmic reticulum (ER) stress is a common factor in the pathophysiology of diverse human diseases that are characterised by contrasting cellular behaviours, from proliferation in cancer to apoptosis in neurodegenerative disorders. Coincidently, dysregulation of AKT/PKB activity, which is the central regulator of cell growth, proliferation and survival, is often associated with the same diseases. Here, we demonstrate that ER stress modulates AKT substrate specificity in a severity-dependent manner, as shown by phospho-specific antibodies against known AKT targets. ER stress also reduces both total and phosphorylated AKT in a severity-dependent manner, without affecting activity of the upstream kinase PDK1. Normalisation to total AKT revealed that under ER stress phosphorylation of Thr308 is suppressed while that of Ser473 is increased. ER stress induces GRP78, and siRNA-mediated knock-down of GRP78 enhances phosphorylation at Ser473 by 3.6 fold, but not at Thr308. Substrate specificity is again altered. An in-situ proximity ligation assay revealed a physical interaction between GRP78 and AKT at the plasma membrane of cells following induction of ER stress. Staining was weak in cells with normal nuclear morphology but stronger in those displaying rounded, condensed nuclei. Co-immunoprecipitation of GRP78 and P-AKT(Ser473) confirmed the immuno-complex consists of non-phosphorylated AKT (Ser473 and Thr308). The interaction is likely specific as AKT did not bind to all molecular chaperones, and GRP78 did not bind to p70 S6 kinase. These findings provide one mechanistic explanation for how ER stress contributes to human pathologies demonstrating contrasting cell fates via modulation of AKT signalling.
    PLoS ONE 03/2011; 6(3):e17894. DOI:10.1371/journal.pone.0017894 · 3.23 Impact Factor
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