Binbin Yue

Increased circulating Level of the survival Factor Gp88 (progranulin) in the serum of Breast cancer patients When compared to Healthy subjects

Introduction GP88 (PC-Cell Derived Growth Factor, progranulin) is a glycoprotein overexpressed in breast tumors and involved in their proliferation and survival. Since GP88 is secreted, an exploratory study was established to compare serum GP88 level between breast cancer patients (BC) and healthy volunteers (HV). Methods An IRB approved prospective study enrolled 189 stage 1–4 BC patients and 18 HV. GP88 serum concentration was determined by immunoassay. Results Serum GP88 level was 28.7+ 5.8 ng/ml in HV and increased to 40.7+ 16.0 ng/ml (P= 0.007) for stage 1-3 and 45.3 +23.3 ng/ml (P = 0.0007) for stage 4 BC patients. There was no correlation between the GP88 level and BC characteristics such as age, race, tumor grade, ER, PR and HER-2 expression. Conclusion These data suggest that serial testing of serum GP88 levels may have value as a circulating biomarker for detection, monitoring and follow up of BC.

GP88 (progranulin): a novel tissue and circulating biomarker for non–small cell lung carcinoma

GP88 (progranulin) is a growth and survival factor implicated in tumorigenesis and drug resistance. Previous studies showed that GP88 was expressed in breast cancer tissue in inverse correlation with survival. This study evaluates GP88 tissue expression in localized/locally advanced lung cancer and GP88 serum levels in advanced disease. GP88 expression was determined by immunohistochemistry in tumor tissue from non-small cell lung carcinoma (NSCLC) patients, 85 with localized (stage I-II), and 40 with locally advanced disease (stage IIIa) and correlated with clinical outcome. Serum GP88 levels from stage IIIb/IV patients, quantified by enzyme immunoassay were compared with GP88 levels from patients with chronic obstructive pulmonary disease and healthy individuals. GP88 was expressed in more than 80% adenocarcinoma and squamous cell carcinoma in contrast to normal lung or small cell lung cancer. There was a statistically significant inverse association of GP88 expression (GP88 immunohistochemistry score, 3+ versus < 3+) with survival for patients with localized resected NSCLC with hazard ratio (HR) = 2.28 (P = .0076) for disease-free survival and HR = 2.17 (P = .014) for overall survival. A statistically significant decrease in progression-free survival (HR = 2.9; P = .022) for GP88 scores of 3+ versus less than 3+ was observed for stage IIIa after chemoradiotherapy. In addition, serum GP88 was significantly elevated in stage IIIb/IV NSCLC compared with control subjects (49.9 ng/mL versus 28.4 ng/mL; P < .0001). This is the first study demonstrating GP88 tissue and serum expression as a prognostic biomarker in localized and advanced disease. Future research will determine utility of monitoring GP88 and the potential of GP88 expression as a predictive marker for anti-GP88 therapeutics.

GP88 (PC-Cell Derived Growth Factor, progranulin) stimulates proliferation and confers letrozole resistance to aromatase overexpressing breast cancer cells.

BackgroundAromatase inhibitors (AI) that inhibit breast cancer cell growth by blocking estrogen synthesis have become the treatment of choice for post-menopausal women with estrogen receptor positive (ER+) breast cancer. However, some patients display de novo or acquired resistance to AI. Interactions between estrogen and growth factor signaling pathways have been identified in estrogen-responsive cells as one possible reason for acquisition of resistance. Our laboratory has characterized an autocrine growth factor overexpressed in invasive ductal carcinoma named PC-Cell Derived Growth Factor (GP88), also known as progranulin. In the present study, we investigated the role GP88 on the acquisition of resistance to letrozole in ER+ breast cancer cellsMethodsWe used two aromatase overexpressing human breast cancer cell lines MCF-7-CA cells and AC1 cells and their letrozole resistant counterparts as study models. Effect of stimulating or inhibiting GP88 expression on proliferation, anchorage-independent growth, survival and letrozole responsiveness was examined.ResultsGP88 induced cell proliferation and conferred letrozole resistance in a time- and dose-dependent fashion. Conversely, naturally letrozole resistant breast cancer cells displayed a 10-fold increase in GP88 expression when compared to letrozole sensitive cells. GP88 overexpression, or exogenous addition blocked the inhibitory effect of letrozole on proliferation, and stimulated survival and soft agar colony formation. In letrozole resistant cells, silencing GP88 by siRNA inhibited cell proliferation and restored their sensitivity to letrozole.ConclusionOur findings provide information on the role of an alternate growth and survival factor on the acquisition of aromatase inhibitor resistance in ER+ breast cancer.

Signaling Pathway of GP88 (Progranulin) in Breast Cancer Cells: Upregulation and Phosphorylation of c-myc by GP88/Progranulin in Her2-Overexpressing Breast Cancer Cells

Her2 is a receptor tyrosine kinase overexpressed in 25% of breast tumors. We have shown that the 88 kDa autocrine growth and survival factor GP88 (progranulin) stimulated Her2 phosphorylation and proliferation and conferred Herceptin resistance in Her2-overexpressing cells. Herein, we report that GP88 stimulates c-myc phosphorylation and upregulates c-myc levels in Her2-overexpressing cells. c-myc phosphorylation and upregulation by GP88 were not observed in non-Her2-overexpressing breast cancer cells. c-myc activation was inhibited upon treatment with ERK, PI3 kinase, and c-src pathway inhibitors, U0126, LY294002, and PP2. GP88 also stimulated c-src phosphorylation, a known upstream regulator of c-myc. Thus, we describe here a signaling pathway for GP88 in Her2-overexpressing cells, with GP88 stimulating Src phosphorylation, followed by phosphorylation and upregulation of c-myc. These data would suggest that targeting GP88 could provide a novel treatment approach in breast cancer.

Elevated serum levels of the growth factor GP88 are found in breast cancer patients when compared to healthy individuals.

CTRC-AACR San Antonio Breast Cancer Symposium: 2008 Abstracts Abstract #2006 Background: GP88 is an autocrine growth factor that plays a critical role in breast tumorigenesis. GP88 is expressed in human BC tumors in a positive correlation with their tumorigenicity. Increased GP88 expression is associated with resistance to anti-estrogen therapy in ER + cells and with herceptin resistance in Her-2 overexpressing breast tumors. Inhibition of GP88 expression in human breast adenocarcinoma inhibited tumor incidence and growth in nude mice. Immunohistochemical studies have shown that GP88 was expressed in invasive ductal carcinomas in correlation with the expression of poor prognosis markers whereas normal tissues and benign lesions were negative. High GP88 expression in tumor biopsies was accompanied by decreased disease-free survival. Since GP88 can be secreted, we have hypothesized that GP88 could be secreted in the circulation and found in serum. We examined whether GP88 could be found in the circulation and whether GP88 could be elevated in the sera of breast cancer patients when compared to healthy individuals. Methods: An IRB approved blood sampling study was conducted at the University of Maryland Breast Clinic to determine the serum level of GP88 in healthy volunteers (HV) and breast cancer patients (BC pts). Serum GP88 concentration was determined in triplicate by quantitative enzyme immunoassay. 189 BC pts were accrued. In addition, sera from 18 HV were obtained to establish a GP88 baseline in healthy volunteers. BC patient characteristics: Race: Caucasian- 91, African American-92, Asian-6; median age, 51 (range 29-86), stage I – 48, II - 52, III – 26, IV - 63. Results: Circulating GP88 was measurable in the serum. Median level of GP88 was 28.7 ng/ml (range 16.6-38.2) in HV; 40.7 ng/ml (range 6.4-100) in early stage (stage 1 –3) BC pts (p- value = 0.007) and 45.3 ng/ml (range 9.8 to 158.4) in stage 4 metastatic BC patients (p-value= 0.0007). Statistically significant increase in circulating GP88 level was found in early stages as well as in metastatic disease. Correlation studies with BC prognostic factors such as stage, tumor size, lymph node involvement, tumor grade and presence of ER and HER-2 will be presented. Conclusion: GP88 can be detected in the sera of HV and BC pts. Comparison between the two groups of subjects indicates that GP88 level is significantly higher in the sera of BC pts. These studies are important as they identify as a measurable circulating biomarker GP88 that is also a therapeutic target of malignant transformation or malignant progression of breast carcinoma (BC). Future studies will examine whether there is any correlation between the serum level of GP88 and therapeutic response to systemic therapy in breast cancer patients. This study was supported by grant from MIPS, the Avon Foundation and 1R43 CA 124179-01A1 from the National Institutes of Health. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2006.

Progranulin levels in blood in Alzheimer's disease and mild cognitive impairment

Changes in progranulin (GRN) expression have been hypothesized to alter risk for Alzheimers disease (AD). We investigated the relationship between GRN expression in peripheral blood and clinical diagnosis of AD and mild cognitive impairment (MCI).

Abstract P1-03-06: Improvement in risk predictive value of Nottingham prognostic index by determining GP88 tumor tissue expression for estrogen receptor positive breast cancer patients

Background: The Nottingham Prognostic Index (NPI), which includes nodal status, tumor size and histological grade was established to provide predictive value information on post-surgery survival for primary breast cancer patients. Attempts to improve NPI9s performance have included addition of other biomarker expression and morphological features such as vascular invasion. In the present study, we investigated whether expression of the autocrine growth and survival factor GP88 (progranulin), known to be overexpressed in breast cancer, whereas it is negative in normal mammary tissue, would improve NPI9s predictive value. Methods: We examined the tumor tissue GP88 expression by immunohistochemistry (IHC) in formalin fixed paraffin embedded tissue sections from 508 cases of estrogen receptor positive (ER+) invasive ductal carcinoma (IDC) with known clinical outcomes (disease-free and overall survivals) and with known NPI. GP88 IHC tumor tissue expression was determined using an anti-GP88 antibody (clone 6B3) developed in our laboratory. GP88 expression was scored (0, 1+, 2+, 3+) by two board certified pathologists and classified into two IHC score groups of GP88 Results: Kaplan-Meier survival graphs categorized by NPI scores ( 5.4) and by GP88 expression ( Conclusions: The data suggest that measuring GP88 tumor tissue expression by IHC at time of diagnosis for breast cancer patients with primary ER+ IDC could provide recurrence prediction and survival information complementary to that provided by the determination of NPI alone and thus may be useful for risk management of patients diagnosed with breast cancer. Citation Format: Serrero G, Hawkins DM, Bejarano PA, Ioffe O, Tkaczuk KR, Elliott RE, Head JF, Phillips J, Godwin AK, Weaver J, Hicks D, Yue B. Improvement in risk predictive value of Nottingham prognostic index by determining GP88 tumor tissue expression for estrogen receptor positive breast cancer patients [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-03-06.

Abstract P3-05-12: Combination of anti-progranulin (GP88/PGRN) antibody and letrozole inhibits tumor formation of letrozole resistant breast cancer cell lines

The 88 kDa glycoprotein GP88 (Progranulin, PCDGF, acrogranin) is the largest member of the granulin/epithelin family of growth modulators identified as a driver of tumorigenesis. GP88 (PGRN) was also shown to be overexpressed in invasive ductal carcinoma (IDC)whereas it was negative in benign tumors and normal mammry epithelial tissue, thereby establishing GP88 as a therapeutic and diagnostic target in breast cancer (BC). Our laboratory has developed validated tools to measure GP88 in tumor biopsies and biological fluids as well as blocking its action. WE showed that GP88 was secreted and detected in the serum of BC patients at an increased level when compared to healthy subjects. Pathological studies with 530 cases of ER+ IDC with clinical outcomes showed that GP88 tumor expression was an independent prognostic indicator of recurrence in early stage BC patients. Training study followed by an independent validation study demonstrated that high GP88 tissue expression (GP88 3+) was associated with a 4-fold increase in risk of recurrence at 5 years. A neutralizing anti-GP88 antibody AG1 was expressed in a high yield CHO cell line was developed. The present study examined the effect of AG1 in letrozole resistant cell line AGLetR developed by long term selection in letrozole supplemented medium. This cell line showed decreased letrozole responsiveness in vivo and therefore constituted an excellent model for investigating letrozole resistance in vitro as well as in vivo. Here we report the results of studies investigating the effect of various doses of AG1 on LetR tumor development in combination with letrozole for AGLetR. We show that treatment with AG1 (10 mg/kg i.p.) in combination with letrozole was efficient to maintain long term responsiveness and inhibit tumor growth. Letrozole alone (5mg/kg) was unable to inhibit tumor growth and showed a doubling of tumor volume. Interestingly, long term combination treatment lead to tumor regression and inhibited tumor growth. These data suggest that inhibiting GP88 could provide a novel and alternative therapeutic strategy for patients with resistance to anti-estrogen therapy, being tamoxifen or letrozole. This works is supported by 2R44CA124179, HHSN 261201200060C, and HHSN2612014400C from NCI. Citation Format: Serrero G, Dong J, Yue B, Hicks D, Hayashi J. Combination of anti-progranulin (GP88/PGRN) antibody and letrozole inhibits tumor formation of letrozole resistant breast cancer cell lines. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-05-12.

Abstract 3918: Circulating progranulin (GP88/PGRN) level correlates with survival in metastatic breast cancer patients

Current monitoring of therapy response in metastatic breast cancer (MBC) patients is dependent on expensive and time consuming imaging methods that have limited sensitivity to detect disease response in a timely manner. Understanding of real-time biological processes through measurement of circulating disease associated biomarkers may provide a clearer understanding of the disease state and thus aid real-time clinical management of MBC patients. Current biomarkers used in MBC include CA15-3, CA27.29 and CEA. While useful, they have limitations in providing clinicians with a reliable insight into real-time monitoring of disease processes. Thus, addition of new circulating biomarkers may improve the management of MBC patients. We characterized a target biomarker, the 88kDa glycoprotein Progranulin (GP88) expressed in tumor tissue and secreted in the circulation of BC patients. Biological studies have established GP88 as one of the critical drivers for tumor cell proliferation, survival, invasiveness and drug resistance. Clinical studies have demonstrated that elevated GP88 tissue levels are prognostic for poor outcome and that breast cancer patients have a statistically elevated GP88 serum level than healthy individuals. Using tissue and serum tests to detect and quantify GP88 could provide an ideal target for monitoring disease progression in BC patients undergoing therapy. In the present study, we examined whether GP88 serum levels were elevated in MBC patients and whether GP88 serum levels were correlated to patient survival. Under an IRB approved protocol, 92 MBC patients that met the inclusion criteria and were undergoing therapy at the UMGCC Breast Clinic were consented. Clinical and disease characteristics along with serum CA15-3 values were collected as part of the study. Serum samples were collected from each patient during therapy and subsequently the patients were monitored. The serum was stored at -80C until tested for GP88. The validated GP88 assay (AG 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3918.

Abstract OT2-4-01: A prospective study of glycoprotein 88 GP-88 blood test in healthy women with Gail model risk <=1.66 undergoing screening for breast cancer (BC) with mammography (MM)

Background: In the US, the majority of BC is diagnosed by screening XRAY mammography (MM) but up to ∼20% of BC are undetected by MM. The development of reliable, blood-based BC screening test to increase the sensitivity and specificity of currently existing BC screening methods such as MM. Rationale: GP88 is expressed & secreted by BC cells & is not expressed by normal mammary epithelial cells. Two retrospective randomized multi-site trials (one training study & one validating study with about 300 cases each) have demonstrated that elevated GP88 expression in ER+ IDC was statistically correlated with a 4-fold increase in the risk of 5-yr disease recurrence. Multivariate analysis showed that GP88 as a risk predictor was independent from PR expression, tumor size, grade, lymph node status & disease stage. The quantitative GP88 EIA developed to determine the amount of GP88 in biological fluids was developed at AG p=0.007). Additionally, a stratification of pts according to their clinical outcomes shows that pts having no evidence of disease (NED) have serum GP88 levels within the range of HV. These data suggest that pts with breast tumors express & secrete high levels of GP88. Objectives: 1. To determine prospectively GP-88 blood levels in HV at average risk of developing BC who are undergoing screening MM & in women with biopsy-confirmed BC. 2. To establish the statistical distribution of GP88 serum levels in these women by baseline BIRAD classification (1-6). 3. To determine if baseline GP88 level is predictive of change in BIRADS classification from baseline to 12-mos follow-up. 4. To determine if baseline GP88 level is predictive of the appearance of BC at 12 mos follow-up in HV who were cancer-free at baseline. Inclusion Criteria: Female, aged >=40 yrs old, presenting to UM Breast Center for screening MM or for diagnostic MM or diagnostic workup and/or biopsy due to BIRADS 0 MM at an outside facility Study procedures: Serum levels of GP88 in subjects with average BC risk factors will be measured prospectively at baseline; 3-6 mos & 6-12 mos. & correlated with BIRADS reading of the screening MM, BIRADS 1 or 2 (benign), BIRADS 3 (short term MM follow-up) & BIRADS 4 or 5 (suspicious and tissue diagnosis needed immediately). GP88 blood level will be correlated with pathologic results of breast biopsies performed on subjects with suspicious BIRADS (4 & 5) MM & final pathologically confirmed diagnosis of BC as BIRADS 6. Statistical Considerations The total number of pts will be 725 & screened up to 1400 subjects. Study is UM IRB approved & just started accruing. Funding is provided by Maryland Industry Partnership Grant (MIPS)& Avon Grant No. 02-2013-018. Contact: Ntait@umm.edu. Citation Format: Katherine HR Tkaczuk, Cristina Campassi, Susan Kesmodel, Emily Bellavance, John Olson, Elizabeth Nichols, Steven J Feigenberg, Binbin Yue, David Hicks, Ginette Serrero. A prospective study of glycoprotein 88 GP-88 blood test in healthy women with Gail model risk