Constitutive and Antibody-induced Internalization

Abridged from Cancer Research 57, 3629-3634, September 1, 1997.

Constitutive and Antibody-Induced Internalization of Prostate Specific Membrane Antigen

Liu H¹, Rajasekaran A.K.^1,2, Moy P., Xia Y., Kim S., Navarro V., Rahmati R., and Bander N.H.³

From the Departments of Urology (H.L., P.M., Y.X., S.K., V.N., R.R., N.H.B.) and Cell Biology (A.R.), New York Hospital-Cornell Medical Center, New York, NY., and the Ludwig Institute for Cancer Research, New York Branch (P.M., N.H.B.).

Abstract

Prostate specific membrane antigen (PSMA) is a cell surface glycoprotein expressed predominantly by prostate cancer cells. We have characterized four monoclonal antibodies that bind to the extracellular domain of PSMA (Liu et al., Cancer Res. 57;3629-3634, 1997). Here we report that viable LNCaP cells internalize these antibodies. Laser scanning confocal microscopy reveals that the internalized antibodies accumulate in endosomes and immunoelectron microscopy reveals that endocytosis of PSMA-antibody complex occur via clathrin coated pits. In addition, a quantitative cell surface biotinylation assay demonstrates that PSMA is constititively endocytosed in LNCaP cells and that anti-PSMA antibodies increases the rate of internalization of PSMA. These studies suggest that PSMA might function as a receptor mediating the internalization of a putative ligand. Availability of prostate specific internalizing antibodies should aid in developing novel therapeutic methods to target toxins, drugs or short-range isotopes to be delivered specifically to the interior of the prostate cancer cells.

Introduction

Prostate specific membrane antigen (PSMA) is the single most well-established, highly restricted prostate epithelial cell membrane antigen (1-8). In contrast to other highly restricted prostate-related antigens such as prostate specific antigen (PSA) and prostatic acid phosphatase (PAP), which are secretory proteins, PSMA is an integral cell membrane protein. The PSMA gene has been cloned, sequenced (9), and mapped to chromosome 11q14 (10). Among reasons for significant interest in PSMA is that it is ideal for in vivo prostate-specific targeting strategies. In addition to its prostate specificity (1-8), PSMA is expressed by a very high proportion of prostate cancers (PCa)(1,2,4,6,7), expression is further increased in higher grade cancers and in metastatic disease (4,6,7) and in hormone-refractory PCa (3,6,7). PSMA expression is modulated inversely by androgen levels (3,6). Furthermore, PSMA expression has been found in tumor, but not normal, vascular endothelium (7,11) further broadening its interest and potential applications.

PSMA has been shown to have peptidase (12) and folate hydrolase (13) activity. While sharing some homology with rat brain NAALADase (12) and the transferrin receptor (9), PSMA does not share the latterís internalization signal (9). The function of PSMA, with respect to prostate cancer and vascular endothelial cell biology, and the direct correlation between its expression and increasing PCa aggressiveness, remains intruiging and unclear.

Until recently, the only available mAb to PSMA was 7E11.C5 (14) which targets an epitope located within the short cytoplasmic tail of the molecule (15,16). As a result, 7E11 did not bind viable cells (11,14,16). We recently reported development of 4 IgG mAbs reacting with the external domain and defining 2 distinct epitopes of PSMA (PSMA_ext1 & _ext2; (11). As these mAbs are capable of binding viable PSMA-expressing cells, we have begun to utilize them in an effort to further understand the function of PSMA.

Results

Staining of viable cells and internalization of mAbs:

Immunofluorescence analysis of viable LNCaP cells incubated with mAbs J591, J533, E99 and J415 at 40C showed distinct plasma membrane staining (data not shown), while mAb 7E11 revealed no plasma membrane staining. Incubation of cells at 37oC with mAb J591 revealed labeling of both plasma membrane and intracellular vesicles (Fig. 1). After a 5 minute incubation at 37oC, the labeling was detected primarily on the plasma membrane (Fig. 1A). At 20 minutes, distinct staining of intracellular vesicles was apparent (Fig. 1B) and at 180 minutes, intense labeling was observed in the juxtanuclear region with sparse labeling throughout the cytoplasm (Fig. 1C). mAbs J415, J533 and E99 gave identical results (data not shown). 7E11 showed neither cell surface nor intracellular staining (data not shown) in these viable cells. These results indicate that mAbs to PSMAext are internalized by viable prostate cancer cells.

Endosomal localization of internalized antibodies:

To test whether internalized antibodies accumulate in endosomes, simultaneous uptake of mAbs and FITC-labeled transferrin (an endosomal marker) were carried out. LSCM revealed that internalized J591 (Fig. 2A) and transferrin (Fig. 2C) co-distributed to a large extent (Fig. 2E) indicating that internalized PSMA-antibody complex accumulates in the endosomes. Control experiments with 7E11 confirmed that this antibody is not internalized (Fig. 2B, D, and F).

Immunoelectron microscopy:

IEM of non-permeabilized LNCaP cells at 4oC revealed mAb J591 binding to the extracellular side of the plasma membrane (11). IEM of viable LNCaP cells incubated with mAb J591 at 37oC for 10 min showed accumulation of gold particles in clathrin-coated pits (Fig. 3A,B) and in vesicles close to the plasma membrane (Fig. 3C). After 2 hrs incubation at 37oC, vesicles containing gold beads were found in a juxtanuclear location (Fig. 3D). These findings indicate that mAb J591 internalization occurs via clathrin-coated pits in LNCaP cells.

Internalization of PSMA:

A cell-surface protein biotinylation assay was developed to test whether PSMA is internalized in the absence of antibody or whether antibody binding induces PSMA internalization. In this assay, a cleavable biotin analog (NHS-SS-biotin) was used to

label proteins exposed on the surface at 4oC. Return of surface biotinylated cells to 37^oC allows the internalization of appropriate cell surface proteins with their biotin tag. The NHS-SS-biotin label is removed from non-internalized cell surface proteins through cleavage of the disulphide linkage with glutathione (18), whereas internalized biotinylated proteins are protected from this cleavage. Appearance of biotin labeled protein that is resistant to glutathione reduction was taken as an indicator of internalization. As shown in Fig. 4A, biotinylated PSMA is sensitive to glutathione reduction after labeling cells at 4^oC (compare Lanes 1 and 2 in panel A). However, with progressively longer incubation periods at 37^oC, an increasing proportion of the labeled PSMA becomes increasingly resistant to reduction (Lanes 3-6, panel A). Quantitation of the blots revealed that 60% of the total cell surface PSMA was internalized (Fig. 4C) within 2 hours.
When the biotinylated cells were incubated with 1(g/ml of mAb J591 at 37^oC, a 3-fold increase in the rate of internalization of PSMA was observed (Fig. 4B compare Lanes 3 and 4 and Fig. 4D). Higher concentration of mAb J591 did not show any further increase in the internalization of PSMA (Fig. 4B lanes 5-7, Fig. 4D). Similar results were obtained with monovalent F(ab) fragments (data not shown).

Discussion:

The prostate-restricted nature of PSMA coupled with the direct association between the level of PSMA expression and increasingly aggressive disease (4) implies a potentially important role of PSMA in prostate cancer biology. The importance of understanding the function of PSMA is further stimulated by its expression by vascular endothelium specifically supplying cancers but not normal, resting endothelium (7,11). In the past, investigating PSMA function has been compromised as the sole antibody to PSMA reacted with a cytoplasmic epitope of the molecule and, therefore, bound only to cells which were permeabilized or dead (11,14, 16). Our recent development of mAbs to the extracellular domain of PSMA and their demonstrated ability to bind viable cells (11) has provided a means to study the function of PSMA.

In this study we demonstrate, by a combination of microscopical and biochemical techniques, that PSMA and mAbs to PSMAext are internalized by LNCaP cells. Confocal microscopy and immunoelectron microscopy reveal that PSMA-mAb complexes are endocytosed via clathrin coated pits (Fig. 2 and 3). A quantitative cell surface biotinylation assay demonstrates that PSMA is constitutively internalized in the absence of antibody binding. At 20 min, 15% of the total biotinylated surface PSMA is internalized (Fig. 4C). The proportion of surface PSMA internalized increases to 60% at 60 min and remains fairly constant at that level through 240 min when the assay was terminated. The stability of the labeled PSMA for a period of over 6 hr (data not shown) indicates that PSMA degradation during this period is minimal. Internalization of only 60% of the total labeled surface PSMA may be explained by recycling of internalized, biotinylated PSMA back to the cell surface (unpublished data) where it would be reduced and rendered undetectable in this assay.

Constitutive internalization of PSMA may reflect recycling of a structural protein through a plasma membrane location or may be mediated by binding of a ligand. While finding that anti-PSMA antibody significantly increases the rate of internalization of PSMA is consistent with the latter, ligand-receptor type function, it does not necessarily indicate that PSMA has a transport function. In the presence of mAb to PSMAext the rate of internalization of PSMA increased up to 3-fold in a dose dependent manner, reaching a maximum rate at an antibody concentration of 1-2 g/ml (Fig. 4). A similar increase in internalization rate has been shown for epidermal growth factor and its ligand (19).

It is well established that many ligands and their transmembrane receptors are internalized via clathrin-coated pits (receptor-mediated endocytosis) (20). The formation of antigen-antibody complexes on the cell surface often results in internalization through a pathway closely resembling receptor-mediated endocytosis of peptide hormones, growth factors and other natural ligands (21). Based on our findings, we hypothesize that PSMA may have a transport function of an as yet unidentified ligand. The baseline internalization rate of PSMA may indicate that PSMA may internalize in the absence of ligand or, alternatively, that the PSMA ligand may be present in culture medium. Similarly, mAb or mAb fragments act as a surrogate ligand, inducing an increased rate of internalization. The internalization pattern seen in this study may have been influenced or modified by presence of mAb (22) and may not reflect the natural internalization pattern.

Targeting of most receptors to coated pits and their traffic through the endocytic compartment are thought to be mediated by a specific internalization motif in the cytoplasmic domain of the receptor (20). The first well characterized internalization motif of several receptors including the transferrin receptor, mannose-6-phosphate receptor, asialogylcoprotein receptor, polymeric immunoglobulin receptor and others are all tetrapeptides (Tyr-X-Arg-Phe) having an aromatic residue in the fourth position of the sequence (23). The cytoplasmic tail of PSMA lacks a sequence similar to the Tyr-X-Arg-Phe motif (9). Another signal is the di-leucine motif for which the only known requirement is the presence of two consecutive leucines or a leucine-isoleucine pair. The di-leucine motif has been shown to mediate internalization and targeting to endosomes and lysosomes (24). A di-leucine motif is present in the cytoplasmic tail of PSMA. Experiments are underway to confirm the dileucine internalization motif of PSMA. Interestingly, while PSMA is 85% homologous to a rat brain neuropeptidase (24), this homolgy is located primarily at the C-termini and declines to less than 50% homology at the N-termini. Furthermore, rat brain neuropeptidase lacks both the Tyr-X-Arg-Phe and di-leucine motifs (25) and presumably does not internalize. Therefore, the highly restricted expression of PSMA becomes increasingly prostate cancer-specific via different mechanisms. For example, normal and benign hyperplastic prostate epithelia express, at the mRNA level, predominantly the cytosolic PSM’ splice variant, without a significant membrane-expressed component whereas in PCa the membrane form predominates by 10-100-fold (26). Another form of functional specificity is demonstrated in rat brain astrocytes (25) where, although there is expression of an homologous neuropeptidase, this neuropeptidase is presumably not internalized as is PSMA in PCa cells.

The property of mAbs to PSMAext to be internalized in prostate cancer cells adds another dimension to their in vivo therapeutic potential. In addition to selective/specific binding to the prostate cancer cell surface, the mAb or fragment would be internalized into the targeted cells providing direct access to the neoplastic cell machinery. As such, this property opens up options such as the use of toxin or drug conjugates. Similarly, the juxtanuclear location of the internalized vesicles should increase the potency of mAb-alpha particle conjugates by improving the incident angle of the isotope and the target DNA.

Lastly, while mAb may function as a surrogate ligand, the question remains as to the identity of the putative natural ligand of PSMA. Troyer et al (16) noted a 40 Kd band which co-immunoprecipitated with PSMA and which they identified as S-glutamic oxalacetic transaminase (SGOT). We have not been able to demonstrate binding of SGOT to PSMA (data not shown). Further study will be required to define the putative natural ligand, which, in turn, may shed further light on the role of PSMA in cancer biology and tumor angiogenesis. The natural ligand, if similarly restricted in its tissue receptor binding profile, may substitute for mAb in a targeted therapy approach.

¹Equally contributed to this work

²Current Address: Department of Pathology and Laboratory Medicine,

University of California Los Angeles, Los Angeles, CA.

³ To whom requests should be addressed, at New York Hospital-Cornell Medical Center, Box 23, 525 East 68^th. Street, NY, NY 10021. e-mail: nhbander@mail.med.cornell.edu

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This work was supported by grants from CaP CURE, the David H. Koch Charitable Foundation, the Ronald P. and Susan E. Lynch Foundation, the Lawrence and Carol Zicklin Philanthropic Fund, the William T. Morris Foundation, the Bernice & Albert B. Cohen Philanthropic Fund through the Jewish Communal Fund, the John E. Wilson Research Fund, the Alissa Beth Bander Memorial Foundation, the Cornell Medical College Urological Oncology Research Fund and from BZL Biologics, Inc. NHB is a consultant to BZL Biologics, Inc. NHBís agreement with BZL is managed by Cornell University in accordance with its conflict of interest policies.

Acknowledgements: The authors thank Lee Cohen Gould for preparation of samples for electron microscopy and Lana Winter for expert administrative assistance.