Humanised in vitro co-culture demonstrates CRPC growth depends on adrenal androgensJ.M. Moll1, W.J. Teubel1, J. Kumagai1, I. Hickson2, R. Graeser2, G.W. Jenster1, W.M. van Weerden1

1Department of Urology, Erasmus MC Cancer Institute, Rotterdam, Netherlands, 2Janssen Pharmaceutica, Beerse, Belgium, corresponding author: j.moll@erasmusmc.nl

32

Results

Materials and methods

Introduction Concept Conclusions

Acknowledgements

References

Conversion and de novo androgen synthesis:With the testes no longer in situ (surgical castration) or a blockade of testosterone production (chemical castration), PC needs a new source of androgens to stimulate its AR.

In our model, de novo synthesis means that a prostate cancer cell has all the enzymes needed to produce testosterone from the basic steroid substrate: cholesterol. Addding the first intermediates, pregnenolone (preg) and progesterone (prog) - which can be metabolised into testosterone via CYP17, AKR1C3 and HSD3B1 activity - should induce growth.

Conversion means that PC is only able to convert the adrenal androgens DHEA and androstenedione (adione) into testosterone via AKR1C3 and HSD3B1. If testosterone synthesis via conversion is the main source of androgens in CRPC, only DHEA and adione will induce growth, whereas preg and prog will not.

Abiraterone is a specific inhibitor of CYP17A1 that blocks the synthesis of DHEA and adione from preg and prog. MDV3100 is a second-line antiandrogen that blocks the AR from binding its ligand.

predectThe research leading to these results has received support from the Innovative Medicines Initiative Joint Undertaking under grant agreement n° 115188, resources of which are composed of financial contribution from the European Union’s Seventh Framework Programme (FP7/2007-2013) and EFPIA companies in kind contribution. Development of CRPC clones was supported by TI-Pharma Project T3-107

1. Hofland, J., et al., Evidence of limited contributions for intratumoral steroidogenesis in prostate cancer. Cancer Res, 2010. 70(3): p. 1256-64.

A rising PSA is one of the hallmarks of castration resistant prostate cancer (CRPC). With PSA being transcribed under an androgen receptor (AR)-specific promotor, this indicates the AR is still active despite low serum testosterone levels. Among other mechanisms of AR-activation, intratumoural androgen synthesis, either via conversion of adrenal androgens or de novo synthesis from cholesterol, is a mechanism of castration resistance. We have previously demonstrated that clinical CRPC samples only expressed markers for conversion of adrenal androgens and not for de novo synthesis1.

In this study, we compared growth stimulation of hormone-naïve prostate cancer (PC) and CRPC cell lines by adrenal androgens (conversion) or androgen precursors (de novo synthesis) and tested a novel co-culture model to mimic adrenal stimulation of (CR)PC.

•VCaP and DuCaP CRPC lines were generated by long-term culturing in androgen depleted medium with or without the antiandrogens bicalutamide or flutamide.

•To test cell growth, VCaP and DuCaP cells and their CRPC sublines were cultured in the presence of androgen precursors (pregnenolone and progesterone) or adrenal androgens (DHEA and androstenedione) at levels found in men.

•Cultures were treated with vehicle, the CYP17A1-inhibitor Abiraterone (0.1 mM) or the anti-androgen MDV3100 (1 mM). •Cell proliferation was assessed by MTT-assay on day 9 with each experiment performed in triplicate, except for those

marked with * (n=1).•To establish a co-culture system, VCaP cells were cultured with human adrenal (H295R) cells in separate compartments

between which only medium could diffuse freely, and treated with vehicle, Abiraterone (1 mM) or DHT (0.1 nM).

Left: Both hormone naïve and CRPC clones of VCaP use conversion.In VCaP parental cells, androgen precursors did not induce growth. Adrenal androgens induced growth similar to that of DHT (optimal stimulus), which could not be inhibited by Abiraterone, but was blocked by treatment with MDV3100.In our CRPC clones (DCC-E, BIC-B and FLU-D), only DHEA and ADIONE stimulated growth, indicating that even CRPC cells do not rely on de novo synthesis.

Adrenal androgens synthesised extratumourally stimulate PC cell growth. Co-culture with human adrenal (H295R) cells stimulated VCaP cell growth. Inhibition of CYP17 activity using 1 mM Abiraterone blocked H295R-induced cell growth, but not proliferation of VCaP alone, indicating Abiraterone mainly acts in the adrenals and not in PC cells. Stimulous with DHT induced VCaP cell growth alone, showing they still responded to androgens as a positive control.

In VCaP and DuCaP cells and their CRPC sublines, the substrate for conversion of adrenal androgens induced growth, while the substrate for de novo androgen synthesis did not. These data support our observations that growth of (CR)PC relies more on conversion of adrenal androgens than on de novo androgen synthesis.

Co-culturing PC cells with human adrenal cells using a 2-compartment system is a relevant model to test CRPC growth stimulation in vitro, demonstrating Abiraterone efficacy. We are currently generating a humanised xenograft model by inoculating PC and H295R cells simultaneously in nude mice to more accurately reflect human CRPC.

pregnenoloneprogesterone

DHEAandrostenedione

DHT

Androgen ReceptorCell growth, PSA

ARDHT

cholesterol

testosterone

STARCYP11A1

CYP17A1

AKR1C3HS3B1

SRD5A

pregnenoloneprogesterone

Prostate cancer cell

cholesterol

DHT

DHT

Adrenal glandDHEA

a-dione

testosterone

AKR1C3HS3B1

SRD5A

DHT

AR

Prostate cancer cell

de novo conversion

DHEAa-dione

MDV

ABI

DuCaP parental(hormone-naïve)

DuCaP BIC-B(Bicalutamide resistant)

DuCaP BIC-H*(Bicalutamide resistant) VCaP parental ± H295R in vitro

Medium conditions: DCC = steroid stripped medium; PREC = DCC + 1.5 nM pregnenolone and 0.5 nM progesterone; DHEA = DCC + 10 nM DHEA; ADIONE = DCC + 2.5 nM androstenedione; ADRENAL = DCC + 10 nM DHEA and 2.5 nM androstenedione; DHT = DCC + 0.1 nM DHT

VCaP parental(hormone-naïve)

1. In hormone naïve and castration resistant VCaP clones, physiological levels of adrenal androgens induced growth while precursors preg and prog did not

2. Adrenal androgens, but not androgen precursors stimulated DuCaP CRPC proliferation

3. Co-culture of VCaP with H295R cells stimulated VCaP cell growth and is a good model for testing steriod synthesis inhibition in prostate cancer treatment in vitro

VCaP DCC-E(castration resistant)

VCaP FLU-D(Flutamide resistant)

VCaP BIC-B(Bicalutamide resistant)

vector (DMSO) Abiraterone 10-7M MDV3100 10-6M

Cholesterol

CYP17A1

AKR1C3HSD3B1SRD5A

pregprog

DHEAadione

DHT AR

STARCYP11A1

Prostate cancer cell

Adrenal cell

ABI

Top-left: both parental DuCaP cells did not use conversion or de novo synthesis and CRPC DuCaP used only conversion.In parental DuCaP cells, only DHT induced cell growth. In DuCaP CRPC clones BIC-H and BIC-B, only DHEA and ADIONE stimulated growth while androgen precursors did not, indicating that in these CRPC cells, proliferation does not rely on de novo synthesis.

Medium conditions: as with VCaP (top figures)vector (DMSO) Abiraterone 10-7M MDV3100 10-6M