Jun Hayashi

PC Cell-Derived Growth Factor Mediates Tamoxifen Resistance and Promotes Tumor Growth of Human Breast Cancer Cells

PC cell-derived growth factor, also known as progranulin, is an Mr 88,000 growth factor (referred as PCDGF/GP88) overexpressed in human breast cancer. Antisense inhibition of PCDGF/GP88 expression in MDA-MB-468 cells inhibited tumor formation in nude mice. In estrogen receptor-positive cells, PCDGF/GP88 was expressed in response to estradiol and shown to mediate its mitogenic effect. Pathologic studies indicated that PCDGF/GP88 was expressed in 80% of invasive ductal carcinomas in correlation with parameters of poor prognosis. In the present article, the relationship between PCDGF/GP88 expression and tamoxifen resistance was examined in MCF-7 cells. PCDGF/GP88 overexpression rendered MCF-7 cells able to proliferate in the absence of estrogen and in the presence of tamoxifen. The PCDGF/GP88-overexpressing cells formed tumors in ovariectomized nude mice in the absence of estradiol and in its presence, in contrast to MCF-7 cells. Tumor growth of the overexpressing cells was increased significantly when the mice were treated with tamoxifen. PCDGF/GP88 blocked tamoxifen-induced apoptosis by preventing down-regulation of bcl-2 expression and poly(ADP-ribose) polymerase cleavage. In addition, PCDGF/GP88-overexpressing cells presented higher level of the angiogenic factors vascular endothelial growth factor and angiopoietin-1 than MCF-7 control cells. Tamoxifen treatment additionally increased the level of vascular endothelial growth factor. These studies suggest that PCDGF/GP88 plays a critical role in breast cancer tumorigenesis and in the transition to estrogen independence and tamoxifen resistance, a hallmark of poor prognosis. On the basis of the in vivo studies, it is postulated that tamoxifen treatment of patients with estrogen receptor-positive breast tumors overexpressing PCDGF/GP88 could have adverse clinical consequences.

PC cell-derived growth factor confers resistance to dexamethasone and promotes tumorigenesis in human multiple myeloma.

Purpose: We have shown previously that the 88 kDa glycoprotein PC cell–derived growth factor (PCDGF/GP88) is expressed and acts as an autocrine growth factor in human multiple myeloma cells. The present study investigates whether PCDGF/GP88 expression in multiple myeloma cells leads to the development of resistance to dexamethasone, a conventional drug for multiple myeloma patients. Experimental Design: PCDGF functions and signaling pathways in dexamethasone-induced apoptosis were studied using a representative dexamethasone-sensitive multiple myeloma cell line ARP-1. The effect of PCDGF/GP88 was further confirmed in PCDGF/GP88–overexpressed ARP-1 cells. Results: Dexamethasone inhibits cell growth and induces apoptosis in a time- and dose-dependent fashion. Exogenous addition of PCDGF/GP88 to the ARP-1 cells prevented dexamethasone-induced apoptosis as examined by flow cytometry analysis and poly(ADP-ribose)polymerase cleavage assay. Signaling studies showed that mitogen-activated protein kinase, phosphatidylinositol 3-kinase, and nuclear factor-κB were involved in the antiapoptotic effect of PCDGF/GP88. Overexpression of PCDGF/GP88 in ARP-1 cells rendered the cells refractory to dexamethasone-mediated apoptosis, enhanced their ability to form colonies in soft agar, and to form tumors in vivo without any change in glucocorticoid receptor expression and function. Conclusion: These data suggest that expression of PCDGF/GP88 confers resistance to dexamethasone and increase tumorigenesis of multiple myeloma cells in mouse xenografts. Our data here also raises the possibility of PCDGF/GP88 as a potential therapeutic target for dexamethasone-resistant multiple myeloma.

A cloned rat thymic epithelial cell line established from serum-free selective culture

SummaryA serum-free system has been developed for selective growth and long-term culture of rat thymic epithelial cells. The growth media is a modification of McKeehans WAJC 404, plus insulin, cholera toxin, dexamethasone, and epidermal growth factor. Cultures have been continuously passaged and maintained for over 6 mo., and a cloned cell line, TEA3A1, has been established. These cells are epithelial, judging by morphology and ultrastructure, and are positive for A2B5 and thymosin α markers for thymic endocrine cells.

Thymocytes stimulate metabolism of arachidonic acid in rat thymic epithelial cells

Arachidonic acid metabolites play an important role in the development of T cells in the thymus. In the normal animal, prostaglandin levels in the thymus are significantly higher than in plasma. Herein, we have studied the regulation of arachidonic acid metabolism using the thymic endocrine epithelial cell line, TEA3A1, and thymocytes. We have found that TEA3A1 cells, but not thymocytes, produced prostaglandins and thromboxanes. We have also found that thymocytes could stimulate the production of arachidonic acid metabolites in TEA3A1 cells when both cells were cocultured. The strongest stimulation was observed when TEA3A1 cells were cocultured with thymocyte subpopulations either negative for OX8 or W3/25 functional surface markers (either double-negative or single-positive thymocytes). Furthermore, cell-cell contact seems to be absolutely required for the activation of arachidonic acid metabolism in TEA3A1 cells. The study presented here describes the existence of a novel regulatory mechanism of arachidonic acid metabolism which may play an important role in the development of T cells in the thymus.

Control of angiotensinogen production by H4 rat hepatoma cells in serum-free culture

SummaryA serum-free, hormone and attachment factor supplemented culture for rat H4 hepatoma cells was established. In the defined medium (Dulbeccos Modified Eagles +Hams F12+insulin, transferrin, fibronectin liver cell growth factor, and sodium selenite), H4 cells grew equally well as in 10% fetal bovine serum supplemented medium. H4 cells in either defined or serum-containing culture conditions produce transferrin but not albumin or alpha-fetoprotein. In this paper we have studied the effect of various hormones and pressor peptides on the production of angiotensinogen by H4 cells cultured in defined conditions. Only glucocorticoid hormone had a significant effect on the production of angiotensinogen, whereas other hormones previously reported to exert their effect on angiotensinogen production had little or no effect.

EGF receptors on TEA3A1 endocrine thymic epithelial cells

Thymic endocrine epithelial cell line TEA3A1 can be maintained and passaged in a serum-free WAJC404A medium supplemented with insulin, transferrin, dexamethasone and EGF. EGF not only promotes the growth of these cells but also regulates the activation of phospholipase A2 enzyme activity. The binding of [125I]EGF to the TEA3A1 cells is temperature and time dependent, saturable and can be blocked by excess unlabelled EGF. Two classes of EGF receptors are found on these cells. One with Kd of 5 X 10(-11)M (approximately 3000 sites/cell) and the other with Kd of 5 X 10(-9)M (approximately 30,000 sites/cell). The resynthesis of EGF receptor in TEA3A1 cells after down-regulation requires about 24 hrs and can be blocked by both actinomycin D and cycloheximide.

Culture and characterization of thymic epithelium from autoimmune NZB and NZB/W mice

Autoimmune NZB and NZB/W mice display early abnormalities in thymus histology, T cell development, and mature T cell function. Abnormalities in the subcapsular/medullary thymic epithelium (TE) can also be inferred from the early disappearance of thymulin from NZB. It has also been reported that NZB thymic epithelial cells do not grow in culture conditions that support the growth of these cells from other strains of mice. In order to study the contribution of TE to the abnormal T cell development and function in NZB and NZB/W mice, we have devised a culture system which supports the growth of TE cells from these mice. The method involves the use of culture vessels coated with extracellular matrix produced by a rat thymic epithelial cell line. TEA3A1, and selective low-calcium, low-serum medium. In addition TEA3A1 cells have been used as an antigen to generate monoclonal antibodies specific for subcapsular/medullary TE. These antibodies, as well as others already available, have been used to show that the culture conditions described here select for cells displaying subcapsular/medullary TE markers, whereas markers for cortical TE and macrophages are absent.

A tribute to Dr. Gordon Hisashi Sato (December 24, 1927–March 31, 2017)

Gordon H. Sato, an innovator in mammalian tissue culture and integrated cellular physiology, passed away in 2017. In tribute to Dr. Sato, In Vitro Cellular and Developmental Biology—Animal presents a collection of invited remembrances from six colleagues whose associations with Dr. Sato spanned more than 40 years. Dr. Sato was a past president of the Tissue Culture Association (now the Society for In Vitro Biology), editor-in-chief of In Vitro Cellular and Developmental Biology (1987–1991), and the recipient of the lifetime achievement award from the Society for In Vitro Biology (2002). He was elected to the US National Academy of Sciences in 1984.

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 4514A: Antibody to Progranulin (Anti-GP88) potentiates tamoxifen and letrozole effect in estrogen receptor positive breast cancer cells

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA GP88 (Progranulin, PCDGF, acrogranin), the largest member of the granulin/epithelin family was originally characterized in our laboratory as a driver of tumorigenesis. Published evidence showed that 1) GP88 expression increases with tumorigenesis; 2) in ER+ breast cancer (BC) ells, GP88 stimulates proliferation and its overexpression confers estrogen independence and resistance to several anti-estrogens and aromatase inhibitor; 3) Antisense inhibition of GP88 expression inhibited proliferation in vitro and in vivo; 4) In Her-2 overexpressing BC, GP88 stimulated Her-2 phosphorylation and conferred trastuzumab resistance; 5) GP88 is expressed in 80% invasive ductal carcinoma (IDC) and 60% of ductal carcinoma whereas it is negative in lobular carcinoma and normal mammary epithelial cells; 6) GP88 is secreted and detected in the serum of breast cancer patients at an increased level when compared to healthy subjects. As GP88 represents an ideal therapeutic and diagnostic target in breast cancer, we have developed tools to measure GP88 in tumor biopsies and biological fluids as well as blocking its action. Pathological studies with 530 cases of ER+ IDC showed that GP88 tumor expression measured by immunohistochemistry was an independent prognostic indicator of recurrence in early stage breast cancer 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. Since GP88 shows not only diagnostic but also therapeutic potentials, we developed a neutralizing anti-GP88 antibody AG1 that inhibits GP88 biological effect (proliferation and migration) in a dose-dependent fashion in vitro. AG1 was expressed in a high yield CHO cell line and formulated. Here we examined the effect of AG1 on responsiveness of breast cancer cells to two drugs commonly used in ER+ patients tamoxifen and letrozole. For these studies, we have developed from a tamoxifen sensitive and from a letrozole sensitive cell ER+ BC cell lines, tamoxifen resistant (TamR) and letrozole- resistant (LetR) cell lines by long term in vitro cell culture selection. Here we report the results of studies investigating the effect of various doses of AG1 on TamR and LetR tumor development alone or in combination with the anti-estrogen drugs. We show that treatment with AG1 (5 mg/kg and 10 mg/kg i.p.) in ombination with tamoxifen or letrozole increased the effect of tamoxifen and letrozole alone, maintained long term responsiveness to tamoxifen or letrozole and inhibited tumor growth. Analysis of anti-GP88 mode of action was examined. These data suggest that inhibiting GP88 provides a novel and alternative therapeutic strategy for patients with resistance to anti-estrogen therapy, being tamoxifen or letrozole. Support: 2R44CA124179 and HHSN 261201200060C from NCI and 02-2013-018 from the Avon Foundation. Citation Format: Ginette Serrero, Jianping Dong, Jorge Marquez, Binbin Yue, Jun Hayashi. Antibody to Progranulin (Anti-GP88) potentiates tamoxifen and letrozole effect in estrogen receptor positive breast cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4514A. doi:10.1158/1538-7445.AM2014-4514A