Prostate cancer is the second leading cause of cancer death in the Western world. While the overall 5-year survival of patients with primary prostate cancer reaches 98%, it drops to 30% when the cancer progresses to a metastatic disease (www.cancer.org
; American Cancer Society). Prostate cancer cells depend on androgens for their growth, thus androgen-deprivation therapy has been the mainstay for treatment of non-metastatic and early metastatic prostate cancer. However, all patients invariably progress during the course of their disease since prostate cancer cells adapt to the androgen-deprived environment becoming “castration-resistant”, which is considered the advanced stage of the disease [1
]. Several mechanisms are implicated in prostate cancer progression to advanced stage [1
]. Among them, deletion of the tumor suppressor Phosphatase and Tensin Homolog (PTEN) is frequently observed during cancer progression [2
]. Amplification of the androgen receptor (AR) gene is considered one of the main mechanisms driving transition to a castration-resistant phenotype [3
]. Current treatment for advanced prostate cancer relies on taxane-based therapy and on new generation antiandrogen drugs (abiraterone, enzalutamide) able to inhibit the activity of the AR [6
]. Besides the AR, estrogens and their receptors have also been implicated in prostate cancer initiation and progression but the translation into a potential therapeutic strategy still remains challenging [8
BReast CAncer susceptibility gene 2
) is a tumor suppressor gene that when mutated confers increased sensitivity to several cancer types, including prostate carcinoma [9
]. BRCA2 promotes repair of DNA double-strand breaks by homologous recombination (HR) [11
]. We have previously reported that silencing of BRCA2 expression promotes resistance to anoikis, i.e., apoptosis induced by detachment of epithelial cells from the extracellular matrix [12
], which is considered an important step in the metastatic cascade [13
]. Notably, the mechanism involved in modulation of apoptosis by BRCA2 was found to be evolutionarily conserved in yeast and humans [12
]. Prostate cancer patients with inherited BRCA2 mutations have a very aggressive disease, with higher grade, advanced stage at diagnosis and poor survival [14
], making a priority the development of novel therapeutic strategies for these patients. In addition, metastases derived from castration-resistant prostate cancers, besides showing AR amplification, display also frequent somatic deletions of the BRCA2
]. Several clinical trials are currently evaluating the use of targeted therapies such as poly-(ADP-ribose) polymerase (PARP) inhibitors in BRCA-associated prostate cancer [6
]. PARP is a DNA-binding protein that promotes repair of DNA single-strand breaks through the base excision and repair (BER) pathway. Suppression of the base excision and repair pathway by PARP inhibitors in BRCA2-deficient/HR-defective cancer cells has a synthetically lethal effect, because it generates an overwhelming genomic instability that fosters cancer cells to death [21
], thus making PARP inhibitors promising candidates for the treatment of BRCA2-deficient castration-resistant prostate cancers. However, BRCA2-mutated cancers often become resistant to PARP inhibitors via different mechanisms, including recovery of BRCA2 functionality due to secondary BRCA2 mutations [22
The thiopurine 6-thioguanine (6-TG) is a chemotherapeutic drug that induces DNA damage and is successfully used in the treatment of childhood acute lymphoblastic leukemia and other forms of leukemias [24
]. Previous studies on its efficacy in various solid tumors have shown infrequent but positive results and have suggested that combinations of 6-TG with other agents may enhance the antitumor effects [25
]. Recently, 6-TG was found to be a potent inhibitor of ubiquitin-specific protease 2, which plays a critical role in prostate tumor cell survival [27
]. Limited data are available on the effect of this drug on solid tumors also due to the toxicity that 6-TG may have on normal cells, this limiting its protracted use in therapy. So far, the potential antitumor effect of 6-TG has never been tested in castration-resistant prostate cancer cells.
Yeast is a useful model organism for studying tumorigenic mechanisms [28
] and for development of advanced technologies for drug discovery [29
]. In particular, in BRCA2-expressing yeast cells, a high increase in both intra- and inter-recombination events occurs, and the expression of selected BRCA2 variants differentially affects yeast recombination [30
], showing that BRCA2 function in homologous recombination-mediated DNA repair can be recapitulated in yeast. Thus, we first screened the effects of 6-TG and of its selected analogues on yeast cell growth and viability. We then investigated the effect of 6-TG alone and in combination with the taxane paclitaxel on normal immortalized and castration-resistant prostate cancer cells, and its dependence on BRCA2 expression. The effect of 6-TG treatment in BRCA2-knockdown prostate cancer cells before and after reconstitution of BRCA2 levels by ectopic expression was compared with treatment with olaparib, a Food and Drug Administration (FDA)-approved PARP inhibitor.
We report here that castration-resistant prostate cancer cells defective in BRCA2 are hypersensitive to 6-TG at levels comparable to olaparib treatment, and that this could be reversed by transient transfection of a vector expressing BRCA2. These results are consistent with a previous study showing that cancer cells, in which BRCA2
is silenced by shRNA, become sensitive to a compound structurally related to 6-TG and to 6-TG itself [38
]. Notably, yeast cells, that do not possess direct homologues of BRCA2
], show sensitivity to 6-TG, suggesting that the mechanism of action of 6-TG is evolutionary conserved in BRCA2-lacking cells.
We show that 6-TG inhibits cell proliferation both in normal and in cancer prostate cells but it promotes cell death by apoptosis only in cancer cells, suggesting a cancer-specific mechanism of 6-TG-induced apoptosis. We didn’t observe any difference in response to 6-TG between C4-2 (PTEN-null) and DU145 (PTEN-positive) prostate cancer cells, suggesting that the response is independent on PTEN status. Homologous recombination (HR) is implicated in the repair of 6-TG-induced double-strand DNA breaks (DSBs) [38
]. Since the castration-resistant prostate cancer cells assayed in this study have decreased BRCA2 levels compared to the normal PNT1A cells, it is possible that reduced repair of 6-TG-induced DSBs by HR contributes to activation of apoptosis in cancer cells. In addition, 6-TG-mediated toxicity in cancer cells has been also associated with mitochondrial-dependent overproduction of ROS [39
]. A previous study has shown that prostate cancer cells have higher ROS production compared with normal prostate cells through activation of the extramitochondrial source of ROS generator, NAD(P)H oxidase [40
]. It’s plausible that prostate cancer cells, having already high basal levels of ROS, would not be able to cope with the detrimental excess of ROS generated by 6-TG treatment, thus undergoing apoptosis. The enzyme methylthioadenosine phosphorylase (MTAP) plays a major role in the metabolism of polyamines and its deficiency has been implicated in the response of cancer cells to 6-TG. MTAP is frequently deleted in several cancers [41
]. A treatment strategy combining methylthioadenosine (MTA) with 6-TG has been proven successful in selectively killing MTAP-deficient tumor cells. In normal MTAP-proficient cells, MTA is cleaved by MTAP to 5-methylthioribose1-phosphate and to adenine. Adenine is phosphoribosylated by adenine phosphoribosyltransferase to form AMP, thus competitively inhibiting phosphoribosylation of 6-TG to a toxic nucleotide, and protecting normal cells while killing MTAP-deficient tumor cells [41
]. However, MTAP is very rarely mutated in prostate cancer as it plays an essential role in the homeostatic regulation of prostate cells’ metabolite pools [42
]. The castration-resistant prostate cancer cells used in this study, C4-2 and DU145, are MTAP-proficient [42
], thus the differential toxicity of 6-TG in cancer versus
normal prostate cells cannot be ascribed to a deficiency of MTAP in prostate cancer cells. Of note, we show that depletion of BRCA2, while increasing sensitivity to 6-TG in prostate cancer cells, did not affect the response in normal prostate cells, thus implying that other factors, together with BRCA2 levels, are important for modulating the sensitivity to 6-TG specifically in cancer cells. Notably, BRCA2 has been reported to protect against oxidative stress [43
]. The constitutively higher levels of ROS in cancer cells compared with normal cells may be one of the factor cooperating with BRCA2 signaling in promoting 6-TG-mediated apoptosis in cancer cells.
Our results show that treatment of castration-resistant prostate cancer cells with 6-TG significantly decreased AR levels, independently on BRCA2 expression. These findings uncover the potential role of 6-TG in promoting cancer cell death by downregulating AR signaling, suggesting that 6-TG may function as a “novel” antiandrogen drug for prostate cancer. Olaparib treatment reduced AR levels only in BRCA2-knockdown prostate cancer cells, indicating that 6-TG and olaparib regulate AR through different molecular mechanisms, the first being BRCA2-independent and the second BRCA2-dependent. Future investigations using AR rescue experiments are warranted to assess the role of 6-TG- and olaparib-mediated regulation of AR expression in the induction of apoptosis in castration-resistant prostate cancer cells.
We show here that gain of BRCA2 expression in castration-resistant prostate cancer cells confers resistance to 6-TG- and olaparib-induced apoptosis but sensitivity to two 6-TG analogues, namely 2-N-6-BP and 2,6-DTP. Notably, normal prostate epithelial cells (expressing high levels of BRCA2) and BRCA2-defective castration-resistant prostate cancer cells are resistant to the two analogues, indicating that the mechanism involved in 2-N-6-BP- and 2,6-DTP-mediated apoptosis is effective only in cancer cells and requires BRCA2. Issaeva et al. [38
] have previously shown that BRCA2-defective cancer cells, including the Capan-1 pancreatic cancer cell line, retain sensitivity to 6-TG after BRCA2
genetic reversion. The discordance with our results may be due to the different cancer type as well as to the different genetic background. Indeed, Capan-1 are MTAP-deficient [44
], thus explaining their retained sensitivity to 6-TG after restoring BRCA2 expression. BRCA2 reversion conferred resistance of castration-resistant prostate cancer cells to olaparib, consistently with previous studies in breast and prostate cancer patients showing acquired resistance to olaparib in BRCA2-associated cancers due to BRCA2
reversion mutations [45
]. Intriguingly, we show that BRCA2-proficient castration-resistant cancer cells become resistant to paclitaxel treatment, suggesting that BRCA2
genetic reversion or high basal levels of BRCA2 in prostate cancer patients may predict resistance to taxane-based therapy. Future in vivo experiments in mouse models will assess the therapeutic potential of 6-TG in the treatment of BRCA2-deficient prostate cancers, and of 2-N-6-BP- and 2,6-DTP in the treatment of BRCA2-proficient, advanced prostate cancers.
In this context the comparison of results obtained with human and yeast cells is of interest. 6-TG is a non-competitive inhibitor of human ubiquitin-specific protease 2 (USP2) [27
], a member of the largest class of deubiquitinating (DUB) enzymes in yeast and humans playing an essential role in numerous cellular processes including cell cycle regulation and DNA repair [48
]. Allosteric binding of 6-TG to the enzyme involves covalent bonding interaction of its thiol group with a specific Cys and polar interaction of its amino group with a specific Gln residue, leading to the movement of a conserved Asp residue playing an essential role in catalysis [27
]. Since the USP domain fold is highly conserved despite low sequence similarity (for refs see [27
]), it is tempting to speculate that 2,6-DTP containing two thiols can inhibit a DUB enzyme conserved in yeast and humans, whereas 2-N-6-BP may exert its effect only on human enzymes. Indeed, both 6-TG analogues induce cell death in cells with an efficient DNA repair system. With this respect, it should be noted that Usp11 has been described as a DUB that exhibits pro-survival functions as part of the cellular response to DNA damage within the BRCA2 pathway, even though independently of BRCA2 deubiquitination [49
Overall, our study provides a pre-clinical rationale for further studies aimed at evaluating the use of 2-N-6-BP and 2,6-DTP for the treatment of BRCA2-proficient castration-resistant prostate cancers and suggests that 6-TG and the PARP inhibitor olaparib may have similar therapeutic potential in the treatment of BRCA2-deficient advanced prostate cancers.