Androgen deprivation therapy (ADT) is a common treatment for advanced prostate cancer because it removes testosterone from the body, a hormone prostate cancer cells require for survival and proliferation. Patients often respond well to this treatment initially but nearly always end up progressing to castration-resistant prostate cancer (CRPC), a form that is aggressive and lethal. Importantly, CRPC regains androgen receptor (AR) signaling through several different mechanisms. For example, tumors can maintain locally high testosterone levels despite undetectable levels in the circulation though the mechanisms for how this occurs are not established. They may also use adrenal testosterone precursors such as andostenedione (AED) and dehydroepiandrosterone sulfate (DHEAS) or potentially synthesize testosterone de novo. The Nelson lab (Human Biology Division) challenged these hypotheses in a recent publication in the Prostate journal.
To follow the flux of testosterone between the extracellular media and the intracellular space, Dr. Kaipainen, first author of the study, and colleagues treated prostate cancer cells with tritium (H3)-labeled testosterone (H3-T). Measures of cellular radioactivity levels at different timepoints were used to quantify the amount of testosterone taken up from the media. Interestingly, the testosterone uptake profile was biphasic in both AR+ and AR- prostate cancer cells, with an immediate and rapid uptake followed by a slow accumulation of intracellular testoterone. The authors hypothesized that two distinct transport modalities could be responsible for the kinetic pattern. In order to determine the type of transport involved in the androgen uptake, the researchers performed a competition assay with cold (non-radioactive) testosterone (C-T). Whereas passive, non-specific transport of any metabolite cannot be competed, facilitated or active transports can be competed. Although increasing concentrations of C-T decreased the uptake of H3-T, a plateau was reached for the minimal amount of intracellular H3-T. These saturable competition assay results support the existence of two different transport modalities.
Active transporters are highly temperature dependent. However, no change in H3-T uptake rate was observed when the experiment was performed on ice or at 37°C. A minimal but persistent uptake of H3-T in formalin fixed prostate cancer cells confirmed that some passive diffusion was involved in the intracellular accumulation of testosterone. In addition, the authors demonstrated that maintaining a gradient of testosterone through the cell membrane was necessary to testosterone uptake, a hallmark of facilitated transport. Thus, facilitated and passive transports are the two modalities for testosterone entry in CRPC cells.
However, the origin of testosterone production under ADT was still unclear. Dr. Kaipainen and colleagues hypothesized that testosterone precursors AED and DHEA(S) could be an important source. Testosterone is synthesized from these two metabolites by AKR1C3. Through immunostaining experiments, they found that both epithelial cells and stromal cells expressed AKR1C3 in primary tumors. These results suggested that both compartments are capable of de novo testosterone synthesis and that paracrine testosterone signaling is possible in the prostate tumor microenvironment. Surprisingly, while tumor epithelial cells are the main source of AKR1C3 in lymph node and soft tissue metastases, stromal AKR1C3 dominates bone and liver metastases. Thus, production of testosterone by stromal cells may be a determinant mechanism driving different metastatic patterns.
This work reveals a new strategy that tumor cells adopt to survive hostile environments. Dr. Nelson summarizes: “The non-cancerous (stromal) cells in the prostate tumor make a minute amount of testosterone. To exploit this source of testosterone, prostate cancer cells use the mechanism of facilitated transport which is more efficient than free diffusion. But facilitated transport depends on a testosterone gradient, which is maintained by removal of excess intracellular testosterone via glucuronidation by UGT2B17 .” The fact that prostate cancer cells opt for another supply route for testosterone, rather than growing without it, strongly supports the complete addiction of these cells for this androgen signaling, and encourages the development of drugs aiming at disrupting testosterone metabolism. For instance, the work of the Nelson lab suggests that “removal of the enzyme UGT2B17, abolishes the gradient, hence diminishing the transport of testosterone to the cell” explains Dr. Nelson.
This work was supported by the Prostate Cancer Foundation and the National Institutes of Health.
Fred Hutch/UW Cancer Consortium members Drs. Nelson, Matsumoto, Morrissey, True and Mostaghel contributed to this research.
Kaipainen A, Zhang A, Gil da Costa RM, Lucas J, Marck B, Matsumoto AM, Morrissey C, True LD, Mostaghel EA, Nelson PS. Testosterone accumulation in prostate cancer cells is enhanced by facilitated diffusion. Prostate 79(13):1530-1542. doi: 10.1002/pros.23874