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Page 4 of 12 Lorenzin et al. J Transl Genet Genom 2019;3:5. I https://doi.org/10.20517/jtgg.2019.01

technology-based characterization identified a mutational hot spot in the ligand-binding domain of the
[19]
gene , including four most frequently observed missense mutations, L702H, W742C, H875Y and T878A.
These retrospectively identified point mutations are rarely detected in primary tumors, indirectly suggesting
[22]
the emergence of these mutations as mechanisms to drive resistance to the therapy .
Recent reports based on the analysis of cell free DNA from CRPC patient’s plasma associate AR aberrations
(both amplification and mutations) with worse outcome and with resistance to abiraterone and/or
enzalutamide. These observations highlight the possibility of using plasma-based AR status as biomarker for
therapeutic management selection [37-40] .

Studies leveraging liquid biopsy to obtain prospectively collected patients’ circulating tumor DNA also
allowed for the evaluation of tumor clone dynamics and demonstrated the temporal association of AR
point mutations with clinical progression. Emergence of AR-L702H mutation was observed in patients
receiving exogenous glucocorticoid in combination with second generation AR antagonists (enzalutamide
or abiraterone), whereas H875Y and T878A AR mutations were detected at progression on abiraterone and
[41]
[39]
prednisolone . AR-L702H and T878A emerged at progression in patients treated with abiraterone . F877L
and other mutations were identified in a small number of patients analyzing cell free DNA and were found
in CRPC patients who had progressed while on ARN-509 or enzalutamide treatment [37,40,42] .

In vitro characterization of AR mutants driving resistance to antiandrogens was fostered by the discovery
[43]
of a mutated form of AR in the LNCaP cell line . Screening using a randomly mutagenized AR library
in prostate cancer cell lines under the selective pressure of enzalutamide linked the emergence of AR
[44]
mutations to the development of resistance . Mutations in the ligand-binding domain of AR resulted in
gain-of-function by modulation of the ligand-binding affinity with increased sensitivity to other androgenic
and nonandorgenic ligands, such as progesterone and glucocorticoid, which can remain at increased
levels in patients treated with abiraterone [45,46] , and conversion of direct antagonists into agonists (e.g.,
bicalutamide, flutamide). For instance, T878A and H875Y mutants are paradoxically activated by the anti-
androgens nilutamide and flutamide [43,47] . In this setting, the anti-androgens behave as an agonist leading
to the activation of AR target genes. AR L702H, but not wild type AR, is activated by prednisolone and this
[41]
activation at glucocorticoid concentrations found in patients is not inhibited by enzalutamide .

Expression of AR splicing variants (AR-Vs) has emerged as an additional mechanism driving resistance
to first and second generation AR-targeted therapies. These alternative AR-Vs lack the C-terminal ligand-
binding domain via truncation or exon skipping while retain the amino-terminal transactivation and
DNA-binding domains [48,49] . Profiling of several AR-Vs revealed recruitment of these isoforms to androgen
responsive elements in a ligand-independent manner, providing initial evidence for AR-Vs-sustained tumor
growth without the need of ligand/androgens [50,51] . AR-V7 is the best characterized isoform, showing nuclear
localization in the presence of partial nuclear localization signal, and androgen-independent activity in AR
transactivation reporter [49,52] .

In prostate cancer cell-based models, the high expression of AR-Vs compared to AR full length (AR-FL) is
associated with complex genomic rearrangements within the AR locus as exemplified by the 22Rv1 cell line
harboring AR gene structural rearrangements and expressing high levels of AR-V7 relative to AR-FL [53,54] .
Clinical data of CRPC patients also confirm that AR genomic structural rearrangements are responsible for
the expression of different AR-Vs, which are able to support constitutive and ligand-independent growth
[55]
of tumor cells even in presence of antiandrogens . In addition, redistribution of splicing factors and high
rate of AR gene transcription in response to ADT are also responsible for increased expression of AR-V7, as
[56]
shown in VCaP and in a LNCaP derivative cell line .
The prognostic value of AR-Vs detection, and of AR-V7 in particular, is still unclear. AR-Vs can be
detected in normal prostate tissue and early stage tumors, yet their expression seems to increase in CRPCs