Alterations in androgen deprivation enhanced prostate-specific membrane antigen (PSMA) expression in prostate cancer cells as a target for diagnostics and therapy

B Meller, F Bremmer, C O Sahlmann, S Hijazi, C Bouter, L Trojan, J Meller, P Thelen, B Meller, F Bremmer, C O Sahlmann, S Hijazi, C Bouter, L Trojan, J Meller, P Thelen

Abstract

Background: Prostate-specific membrane antigen (PSMA) is a promising target for diagnostics and therapy of prostate carcinoma (PCa). Based on the hypothesis that PSMA expression can be modulated by variations in androgen deprivation therapy (ADT), we investigated the binding of a PSMA-directed radiopharmaceutical in vitro in order to get an insight of the interactions between altered premedication and PSMA expression before repetitive PSMA-directed PET/CT for therapy response and targeted therapy implementation.

Methods: The human castration-resistant PCa cell line VCaP (CRPC) was treated with either 1 nmol/L testosterone (T) over 20 passages yielding the androgen-sensitive cell line (revCRPC) or with 5 μmol/L abiraterone acetate (AA) generating the abiraterone-tolerant subtype CRPCAA. In these cell lines, T and AA were varied by either supply or withdrawal of T and AA. PSMA expression of the three cell culture models was detected by Western blot and immunohistochemical staining. For quantitative measurement of tracer uptake, 0.3 nmol/L (68)Ga-labelled PSMA-HBED-CC peptide (100-300 kBq/ml) was added to different treated parallel cultures (n = 9 each). Time-dependent uptake per 10(6) cells of each culture was calculated and evaluated. PSMA mRNA expression was investigated by qPCR.

Results: PSMA expression increased dependently on intensified ADT in all three basic cell lines. (68)Ga-PSMA-HBED-CC uptake almost doubled during 3 h in all cell lines (p < 0.01). Compared to the basic cells, pre-incubation with abiraterone for 48 h resulted in a significant increased uptake in CRPC (p < 0.001). In revCRPC, 48-h AA pre-incubation resulted in an eightfold higher uptake after 3 h (p < 0.001). Additional withdrawal of external testosterone increased the uptake up to tenfold (p < 0.01). The increase of PSMA expression upon ADT and AA treatments was confirmed by qPCR and Western blot data. Furthermore, in CRPCAA, 48-h AA withdrawal increased the uptake up to fivefold (p < 0.01).

Conclusions: The investigated three PCa cell culture subtypes represent a serial preclinical model of androgen deprivation therapy as a proxy for clinical situations with differing basal PSMA expression. The uptake of PSMA-binding tracers could be stimulated by therapeutic effective short-term variation in premedication in all stages of ADT response. These complex interactions have to be considered in the interpretation of diagnostic imaging using PSMA ligands as well as in the optimal timing of PSMA-based therapies.

Keywords: 68Ga peptide; Abiraterone acetate; Androgen deprivation; Androgen receptor; Prostate cancer; Prostate-specific membrane antigen.

Figures

Fig. 1
Fig. 1
Western blot total protein extraction of the three cell lines subtypes with different levels of androgen deprivation sensitivity (revCRPC, CRPC, CRPCAA). Displayed is total PSMA protein at 100 kDa and the housekeeping gene α-tubulin (a). Relative density was determined by a chemiluminescent substrate (b)
Fig. 2
Fig. 2
Immunohistological staining of PSMA in the three investigated cell lines subtypes. Cytoplasmic PSMA (brown signal) in revCRPC (a ×20, b ×60), CRPC (c ×20, d ×60) and CRPCAA (e ×20, f ×60)
Fig. 3
Fig. 3
Time-dependent uptake of 68Ga-PSMA-HBED-CC in androgen-sensitive (revCRPC), castration-resistant prostate cancer cells (CRPC) and abiraterone-insensitive cells (CRPCAA). Displayed is the percentage uptake of applied radioactivity per 106 cells (mean ± SD; n = 9 cultures)
Fig. 4
Fig. 4
Membrane binding and internalisation of 68Ga-PSMA-HBED-CC after 5 h of incubation in androgen-sensitive (revCRPC), castration-resistant prostate cancer cells (CRPC) and abiraterone-insensitive cells (CRPCAA) as well as in revCRPC after 48 h ADT with testosterone (T) withdrawal and 5 μM abiraterone acetate (AA). Values are expressed as percentage of applied radioactivity bound to 106 cells. Mean ± SD (n = 3 cultures)
Fig. 5
Fig. 5
Time-dependent binding of 68Ga-PSMA-HBED-CC in (a) castration-resistant prostate cancer (CRPC) cells with and without coincubation with abiraterone acetate (AA, 5 μmol/L) and testosterone (T, 1 nmol/L) and (b) in androgen-sensitive revCRPC cells with and without AA-treatment (5 μmol/L) or additional withdrawal of testosterone (−T) from the medium. Displayed is the percentage uptake of applied radioactivity per 106 cells (mean ± SD; n = 9 cultures)
Fig. 6
Fig. 6
Time-dependent changes of PSMA mRNA expression, androgen receptor splice variant 7 (AR-V7) and ribosomal protein L13a (RPL) housekeeping gene in (a) CRPC cells under abiraterone acetate (AA) treatment and (b) revCRPC cells under AA treatment and additional androgen withdrawal from the medium
Fig. 7
Fig. 7
Time-dependent changes of 68Ga-PSMA-HBED-CC uptake 1 and 3 h after application in abiraterone-tolerant cells 48 h (CRPCAA) after abiraterone acetate (AA) withdrawal with or without additional application of 1 nmol/L testosterone (T). Displayed is the percentage uptake of applied radioactivity per 106 cells (mean ± SD; n = 9 cultures)

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