Eunjeong Yoo

The Endoplasmic Reticulum Stress Marker, Glucose-regulated Protein-78 (GRP78) in Visceral Adipocytes Predicts Endometrial Cancer Progression and Patient Survival

OBJECTIVE Currently, accurately identifying endometrial cancer patients at high risk for recurrence remains poor. To ascertain if changes in the endoplasmic reticulum (ER) stress marker, glucose-regulated-protein-78 (GRP78) can serve as a prognosticator in endometrial cancer, we examined GRP78 expression in patient samples to determine its association with clinical outcome. METHODS A retrospective cohort study was conducted in endometrial cancer patients. Archived specimens of visceral adipocytes and paired endometrial tumors were analyzed by immunohistochemistry for GRP78 and another ER stress marker, C/EBP homologous protein (CHOP). Expression of these markers was correlated with clinico-pathological information and outcomes. RESULTS GRP78 expression in visceral adipocytes was detected in 95% of the 179 endometrial cancer patients with analyzable visceral adipocytes. Within individual samples, 24% of adipocytes (range, 0-90%, interquartile range 18%-38%) exhibited GRP78 expression. High visceral adipocyte GRP78 expression positively correlated with advanced-stage disease (p=0.007) and deep myometrial invasion (p=0.004). High visceral adipocyte GRP78 expression was significantly associated with decreased disease-free survival (DFS) in multivariate analyses (hazard ratio 2.88, 95% CI 1.37-6.04, p=0.005). CHOP expression paralleled the GRP78 expression in adipocytes (r=0.55, p<0.001) and in the tumor (p=0.018). CONCLUSIONS Our study demonstrates that the ER stress markers, GRP78 and CHOP, are elevated in endometrial cancer patients. Furthermore, GRP78 expression levels in visceral adipocytes from these patients were significantly correlated to disease stage and patient survival. Our results demonstrate, for the first time, that the GRP78 levels in endometrial cancer patients may be a prognosticator and aid with clinical risk stratification and focused surveillance.

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

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.

Targeting the glucose-regulated protein-78 abrogates Pten-null driven AKT activation and endometrioid tumorigenesis

Rates of the most common gynecologic cancer, endometrioid adenocarcinoma (EAC), continue to rise, mirroring the global epidemic of obesity, a well-known EAC risk factor. Thus, identifying novel molecular targets to prevent and/or mitigate EAC is imperative. The prevalent Type 1 EAC commonly harbors loss of the tumor suppressor, Pten, leading to AKT activation. The major endoplasmic reticulum (ER) chaperone, GRP78, is a potent pro-survival protein to maintain ER homeostasis, and as a cell surface protein, is known to regulate the phosphatidylinositol 3-kinase (PI3K)/AKT pathway. To determine whether targeting GRP78 could suppress EAC development, we created a conditional knockout mouse model using progesterone receptor-Cre-recombinase to achieve Pten and Grp78 (cPtenf/fGrp78f/f) deletion in the endometrial epithelium. Mice with a single Pten (cPtenf/f) deletion developed well-differentiated EAC by 4 weeks. In contrast, no cPtenf/fGrp78f/f mice developed EAC, even after more than 8 months of observation. Histologic examination of uteri from cPtenf/fGrp78f/f mice also revealed no complex atypical hyperplasia, a well-established EAC precursor. These histologic observations among the cPtenf/fGrp78f/f murine uteri also corresponded to abrogation of AKT activation within the endometrium. We further observed that GRP78 co-localized with activated AKT on the surface of EAC, thus providing an opportunity for therapeutic targeting. Consistent with previous findings that cell surface GRP78 is an upstream regulator of PI3K/AKT signaling, we show here that in vivo short-term systemic treatment with a highly specific monoclonal antibody against GRP78 suppressed AKT activation and increased apoptosis in the cPtenf/f tumors. Collectively, these findings present GRP78-targeting therapy as an efficacious therapeutic option for EAC.

Abstract 534: Development and validation of sandwich ELISA to quantify circulating GRP78 as a cancer biomarker

Glucose regulated protein 78 kDa (GRP78), also referred to as immunoglobulin heavy chain binding protein (BiP), is a chaperone protein belonging to HSP70 protein family. GRP78 resides primarily in the lumen of the endoplasmic reticulum (ER) and plays an important role in protein folding, by targeting misfolded proteins, controlling the activation of transmembrane ER stress sensor and Ca 2+ homeostasis. It contains the KDEL sequence (Lys-Asp-Glu-Leu) on its C-terminus, which is responsible for its retention in the ER through binding onto KDEL receptor. Some ER proteins escape the ER retention when they associate with other proteins and thus block the KDEL domains. Several in vitro studies have shown that GRP78 is found on the cancer cell surface. In clinical studies, increased expression of GRP78 has been correlated with chemoresistance in breast, colon, lung, prostate and endometrial cancers. However, quantification of GRP78 in the circulation has not been described as a diagnostic marker in cancer patients. Although circulating autoantibodies against GRP78 have been detected at high levels in the sera from cancer patients, these assays are difficult to perform by ELISA due to presence of immune complex bounded GRP78 in sera. We have developed and validated an ELISA-based assay able to quantify the free levels of GRP78 with a lower level quantification (LLQ) of 25 ng/mL. Since GRP78 is highly protein bound, acid dissociation using glycine demonstrated a linear range from 12.5 to 1600 ng/mL. The actual amounts of GRP 78 in plasma were measured using sandwich ELISA using TMB substrate, where the absorbance was measured at 450 nm. Acid dissociated plasma at pH 3.0 was able to liberated GRP78 where with no interfering protein was detected. Standard curve with a range 25-1600 ng/mL of GRP78 was linear (R 2 >0.98). To validate this ELISA method, interday and intraday variability testing were performed and demonstrated that the assays were linear, robust, and reproducible, over their respective concentration ranges and R 2 ≥0.96. To determine the utility of this assay, circulating GRP78 in plasma was determined in breast cancer and compared to non-cancer volunteers. Breast cancer patients had significantly higher circulating GRP78 when compared to healthy volunteers, where the circulating levels correlated with disease staging. To further confirm the utility of this assay, circulating levels of GRP78 was also evaluated in patients with recurrent cervical cancer undergoing chemotherapy. This study showed a correlation with chemotherapy responsiveness. These studies demonstrated that a new technique to accurately and precisely quantify GRP78 in plasma may be a potential biomarker for the presence of tumor and tumor burden, and thus may be used to monitor for treatment effectiveness. This finding represents a new approach to quantify GRP78 levels in plasma that can be used in the clinic as a biomarker to guide cancer diagnosis. Citation Format: Eunjeong Yoo, Yvonne Lin, Nicos Petasis, Augustin Garcia, Stan Louie, Isaac Asante, Eugene Zhou, Song Ah Chae. Development and validation of sandwich ELISA to quantify circulating GRP78 as a cancer biomarker. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 534. doi:10.1158/1538-7445.AM2015-534

Simultaneous quantitation of folates, flavins and B6 metabolites in human plasma by LC–MS/MS assay: Applications in colorectal cancer

HIGHLIGHTSAn LC–MS/MS assay to quantify vitamin B metabolites has been developed and validated.Assay facilitates the simultaneous determination of concentrations of folates, flavins and B6 metabolites using 50&mgr;L of plasma.The method was applied on plasma samples obtained from healthy volunteers and colorectal cancer patients.The method can be used to probe the alteration in the metabolites of one‐ carbon metabolism in various disease conditions. ABSTRACT An analytical method using electrospray ionization and high‐ performance liquid chromatography/tandem mass spectrometry (LC/ESI–MS/MS) was developed to quantify the vitamin B metabolites found in the folate one‐carbon metabolism, using 50&mgr;L of human plasma. Analytes in plasma were extracted using protein precipitation after being stabilized in 1% ascorbic acid. The analytes were separated using a Kinetex 2.6&mgr;m Pentafluorophenyl (2.1×30mm) column utilizing a gradient mobile phase system of 0.1% formic acid in water and 100% acetonitrile in a 13.2min run. The MS detector run using a positive multiple reaction monitoring with parameters optimized for each analyte’s ion pair. The assay was selective and linear for all analytes at defined dynamic ranges. The recoveries were generally above 80% except for the folate metabolites whose recoveries dipped possibly due to the drying process. The inter‐day precision (%coefficient of variation) and accuracy (%calculated concentration of the nominal concentrations) for six replicates of all quality control samples were ≤14% and within 12.2%, respectively. The lower limit of quantification ranged from 0.2 to 3.9nM. No significant instability was observed after repeated freezing and thawing or in processed samples. The LC–MS/MS assay was found applicable for sensitive, accurate and precise quantitation of vitamin B metabolites in plasma of healthy volunteers and colorectal cancer patients.

Endoplasmic reticulum stress in complex atypical hyperplasia as a possible predictor of occult carcinoma and progestin response

Glucose-regulated protein (GRP)-78, the key regulator of endoplasmic reticulum (ER) stress, is associated with endometrial cancer (EC) development and progression. However, its role in the continuum from complex atypical hyperplasia (CAH) to EC is unknown and the focus of this study. METHODS 252 formalin-fixed, paraffin-embedded endometrial biopsies from patients with CAH diagnosed between 2003 and 2011 were evaluated for GRP78 expression by immunohistochemistry. Expression was also evaluated in subsequent biopsies from those patients treated with progestins. Differences in GRP78 expression were assessed using standard statistical methods. RESULTS GRP78 expression was undetectable in 45(18%) patients with CAH, while 120(48%) CAH cases showed moderate/strong expression. Among women who ultimately underwent hysterectomy for CAH (n=134), 54(40%) had occult EC while 57(43%) had persistent CAH. Those with occult EC upon hysterectomy had significantly stronger GRP78 expression than those who did not have occult EC (p=0.007). Greater GRP78 expression within CAH remained independently associated with the presence of an occult EC (p=0.017). Thirty-four of 54 (63%) patients with occult EC had moderate/strong GRP78 expression compared to 36 of 80 (45%) patients with persistent CAH, benign or non-atypical hyperplastic endometrium. In those treated with progestins, samples with persistent CAH and EC were more likely to have high levels of GRP78 expression in the initial biopsies than those who responded (p=0.014). CONCLUSIONS Increased GRP78 expression in untreated CAH correlates with the presence of an occult EC. In addition, CAH specimens with greater GRP78 expression may identify patients who are less likely to respond to progestin therapy.