Enzalutamide Enhances PSMA Expression of PSMA-Low Prostate Cancer

Abstract

Prostate-specific membrane antigen (PSMA)-directed radioligand therapy (RLT) prolongs overall survival in men with metastatic castration-resistant prostate cancer (mCRPC). However, men with low PSMA expression are excluded from RLT. We explored the effect of androgen receptor blockade with enzalutamide on PSMA expression. Assessment of PSMA and androgen receptor (AR) expression on the human PC cell lines 22Rv1, C4-2, and LNCaP by immunohistochemistry and flow cytometry revealed low (22Rv1) and high (C4-2 and LNCaP) PSMA expression, and high, comparable AR positivity. Treatment with enzalutamide increased PSMA levels in 22Rv1, C4-2, and LNCaP (2.2/2.3/2.6-fold, p = 0.0005/0.03/0.046) after one week compared to DMSO-treated controls as assessed by flow cytometry. NOD/Scid mice bearing 22Rv1 tumors were treated with enzalutamide for two weeks. Positron emission tomography/computed tomography (PET/CT) demonstrated higher tumor uptake of 68Ga-PSMA after enzalutamide treatment (p = 0.004). Similarly, a clinical case with low baseline PSMA avidity demonstrated increased uptake of 68Ga-PSMA after enzalutamide on PET/CT and post-therapeutic 177Lu-PSMA scintigraphy in a patient with mCRPC. Enzalutamide induced PSMA expression in the 22Rv1 xenograft model and in an mCRPC patient, both with low baseline tumoral PSMA levels. Therefore, enzalutamide pre-treatment might render patients with low PSMA expression eligible for 177Lu-PSMA RLT.

Keywords: 22Rv1; C4-2; CT; LNCaP; PET; PSMA; androgen receptor blockade; enzalutamide; prostate cancer; radioligand therapy.

Conflict of interest statement

Wolfgang P. Fendler was a consultant for BTG, and he received fees from RadioMedix, Bayer, and Parexel outside of the submitted work. Katharina Lückerath reports consulting activities for Sofie Biosciences/iTheranostics, and funding from AMGEN outside of the submitted work. Boris Hadaschik reports personal fees and non-financial support from AstraZeneca, Amgen, Bayer, BMS and Janssen, personal fees from ABX, Lightpoint medical, Inc, and Pfizer, and grant funding from German Research Foundation, all outside the submitted work. Tibor Szarvas received fees from BRAHMS and Janssen outside of the present study. Ken Herrmann reports personal fees from Bayer, personal fees and other from Sofie Biosciences, personal fees from SIRTEX, non-financial support from ABX, personal fees from Adacap, personal fees from Curium, personal fees from Endocyte, grants and personal fees from BTG, personal fees from IPSEN, personal fees from Siemens Healthineers, personal fees from GE Healthcare, personal fees from Amgen, personal fees from Novartis, personal fees from ymabs, personal fees from Aktis Oncology, personal fees from Theragnostics, personal fees from Pharma15, outside the submitted work. Henning Reis is on the advisory board of Bristol-Myers Squibb, received honoraria from Roche and Bristol-Myers Squibb, received travel support from Philips, Roche, and Bristol-Myers Squibb, received grants from Bristol-Myers Squibb and holds shares of Bayer. The other authors declare no conflict of interest.

Figures

Figure 1

PSMA and AR in 22Rv1, C4-2, and LNCaP prostate cancer cell lines by PET and IHC. (A) Exemplary 68Ga-PSMA-11-PET/CT maximum intensity projection (MIP) images of NOD/Scid mice bearing tumors (white arrows) of 22Rv1, C4-2, or LNCaP cell lines. Average tumor uptake (%IA/g) per gram tissue is stated below the image. (B) Representative images of PSMA IHC and bar plots for % positivity (mean ± SD, n = 3). (C) Representative images of AR IHC and bar plots for % positivity (mean ± SD, n = 3). Scale = 20 µm. Abbreviations: PSMA, prostate-specific membrane antigen; %IA/g, % injected activity per gram tissue; AR, androgen receptor. Statistics: ordinary one-way ANOVA with Sidak’s multiple comparisons test; **** p ≤ 0.0001; ns = non significant.

Figure 2

In vitro PSMA expression after enzalutamide treatment over time measured by flow cytometry. (A) Representative histograms of unstained and PSMA-stained DMSO-treated or enzalutamide-treated 22Rv1, C4-2 and LNCaP cells. Cells were treated for one, two, or three weeks. (B) Enzalutamide treatment significantly increased PSMA levels in 22Rv1, C4-2, and LNCaP cells after one week compared to DMSO-treated controls as assessed by flow cytometry; PSMA levels remained increased in all three cell lines two and three weeks after treatment initiation. Data are shown as geometric mean normalized to unstained control. Statistics: unpaired t-test with Welch’s correction; * p ≤ 0.05; ** p ≤ 0.01.

Figure 3

In vivo PSMA levels before and after treatment with enzalutamide by PET/CT. (A) 22Rv1 tumor cells were injected into the shoulder region of male, 6–10-week-old NOD/Scid mice. As soon as tumors were palpable (approx. 2–3 weeks after inoculation), baseline 68Ga-PSMA-11 PET/CT was performed and enzalutamide treatment (10 mg/kg daily by oral gavage (p.o.), 5 days a week for 2 weeks) was started. After 2 weeks of enzalutamide, follow-up 68Ga-PSMA-11 PET/CT was performed. (B) 68Ga-PSMA uptake per gram tissue in 22Rv1 tumors increased significantly after two weeks of treatment with enzalutamide. PVE-corrected tumor size before and after enzalutamide is shown in (C). Measurements of bloodpool (D), salivary glands (E), kidneys (F) and liver (G) served as controls and did not show a significant change in 68Ga-PSMA uptake. Abbreviations: %IA/g, % injected activity per gram tissue. Statistics: Wilcoxon matched-pairs signed-rank test. * p ≤ 0.05, ** p ≤ 0.01.

Figure 4

Enhanced PSMA levels under enzalutamide in a patient with low-PSMA nodal metastases. Images from an 81-year-old mCRPC patient are shown. At baseline, a few lymph node metastases with low 68Ga-PSMA-11 uptake on PET/CT are present (A, arrows). Patient started mCRPC treatment with enzalutamide. A follow-up 68Ga-PSMA-11 PET/CT scan after five months showed progressive lymph node disease and increased 68Ga-PSMA uptake (B, arrows). Subsequently, two cycles of 7.4 GBq 177Lu-PSMA radioligand therapy (RLT) were administered with decreasing uptake on the post-therapeutic scintigraphies (C–first cycle, D–second cycle, arrows). Follow-up 68Ga-PSMA-11 PET/CT was consistent with treatment response and demonstrated size decrease of tumor lesions along with reduced PSMA-ligand uptake (E, arrows). Abbreviations: SUV, standardized uptake value.