The treatment of advanced prostate cancer represents a significant challenge due to the accumulation of multiple mechanisms of drug resistance. Resistance to Docetaxel treatment includes changes in classical multiple drug resistant pathways, expression of different β-tublin isotypes, mutations in tumour suppressor proteins and altered expression of pro- and anti-apoptotic proteins [25
]. We have generated three models of docetaxel-resistance in prostate cancer cells which may mimic the situation in the clinical setting and used them to understand the complexity of mechanisms leading to treatment resistance. Understanding these pathways will be essential if we want to better impact on the outcome of these patients and identify additional co-treatments which will manipulate this resistance and develop personalised treatments. We demonstrated that all four sublines are resistant to increasing concentrations of Docetaxel as assessed by apoptosis and viability assays.
One of the main mechanisms of resistance to Docetaxel is the over expression of P-gp [4
] which would reduce intracellular concentrations of the drug through increased efflux. Chemoresistant PC-3 cells do not over express P-gp, while resistant DU145 cells exhibit both over expression of the protein and restored chemosensitivity with P-gp inhibition [5
]. This has been the basis for the development of cabazitaxel which can overcome P-gp over expression [27
] and is now used as a second line therapy for patients who fail Docetaxel treatments. In our study we demonstrated that the resistance in the PC-3 D8 and PC-3 D12 sublines was independent of P-gp over expression as there were no detectable levels and no effects of the P-gp inhibitor Elacridar on their resistance. However the DU-145 and 22RV1 cells expressed increasing levels of P-gp and Elacridar totally reversed the resistance in the 22RV1 and partially in the DU145 resistant sublines. Based on these results we focused on the PC-3 cells to understand the mechanisms of resistance independent of P-gp.
We next investigated cellular senescence as a possible mechanism of resistance. Premature cellular senescence, a phenomenon by which cells stop dividing and acquire a typical morphology and an altered gene expression profile in response to stress [3
], has recently been associated with chemotherapy resistance. We performed a ß-galactosidase assay to assess cellular senescence following docetaxel treatment. Although ß-galactosidase staining showed a modest increase in the percentage of senescent cells following docetaxel treatment, this increase was considered to be insufficient to explain the resistance of the PC-3 D8 and PC-3 D12 sublines to docetaxel.
Autophagy (macroautophagy), a well conserved mechanism by which cells adapt to stress such as starvation, has also been recently associated with resistance to cancer therapies [23
]. Therefore we investigated whether increased autophagy was involved in the resistance to docetaxel in the PC-3 D12 subline. Although western blotting showed increased expression of the autophagic marker LC3II in the PC-3 D12 compared to the PC-3 Ag cells following docetaxel treatment, this was only a modest effect. Interestingly, the resistant PC-3 D12 subline showed a higher baseline expression of LC3I, the precursor of LC3II, which warrants further investigation.
We next undertook to assess alterations in the apoptotic phenotype of the resistant cells in order to understand the mechanisms involved. Custom-designed LDA containing real-time PCR assays for all the key apoptotic genes including the IAPs, death receptors, death ligands, and signalling molecules as well as genes involved in cell cycle regulation, DNA damage and repair were developed. The LDA results showed a complex interplay between changes in the expression of both pro- and anti-apoptotic proteins. From our study, genes which were shown to be altered in both the docetaxel-resistant sublines (PC-3 D8 and PC-3 D12) included; FOXO1, NGFR, TRAF-1, Mcl-1, BIRC3, BIRC1, Bcl-2-A1, NOL3, Clusterin, BOK, NGFR, Fas, FasLG, TNF receptor member 11b and TRAIL.
Trougakos et al
demonstrated that secreted clusterin (sCLU) knockdown in human prostate cancer cells induces significant reduction of cellular growth and higher rates of spontaneous endogenous apoptosis [28
]. sCLU is a cytoprotective chaperone that stabilizes conformations of proteins during times of cellular stress, thereby inhibiting protein aggregation and precipitation [29
]. Additionally, sCLU can interact with and inhibit activated Bax, thereby inhibiting cytochrome C release and caspase activation [30
]. In prostate cancer, sCLU levels have previously been correlated with Gleason grade [31
]. A recent publication identified sCLU to be over-expressed in a docetaxel-resistant PC-3 subline and reported that knockdown of sClu chemosensitizes this cell line to taxane and mitoxantrone based chemotherapy both in vitro
and in vivo
Bcl-2 related protein A1 (Bcl2-A1) or BLF1 is a member of the Bcl-2 family. Although it is regulated differently from Bcl-2, it has similar anti-apoptotic activity. A recent publication demonstrated that Bcl2-A1 mediates resistance to doxorubicin in haematopoietic cell lines [33
Modur et al
found that FOXO1A was highly expressed in normal prostate. They also noted that in PTEN-deficient prostate carcinoma cell lines, FOXO1A was cytoplasmically sequestered and inactive and expression of TRAIL, a pro-apoptotic effector, was decreased. They determined that TRAIL is a direct target of FOXO1A, and they hypothesized that the loss of PTEN contributes to increased tumour cell survival through decreased transcriptional activity of FOXO1A and FOXO3A followed by decreased TRAIL expression and apoptosis [34
BNIP3L which is also elevated in both sublines, plays a pro-survival role in apoptosis. Yasuda et al
showed that in vitro
translated BNIP3L binds specifically and directly with GST fusion proteins of various Bcl-2 family anti-apoptosis proteins, suggesting that, like BNIP3, BNIP3L may antagonize the activity of Bcl-2 family anti-apoptosis proteins [35
]. In this setting, BNIP3L may be increased in our resistant sublines in an attempt to antagonize the very elevated levels of Bcl2-A1.
Genes down-regulated in both sublines include TRAIL, NGFR, TNF member 10D and ATR among others (see Tables , , and results). As PC-3 cells are highly resistant to TRAIL, it is unsurprising that levels of pro-apoptotic TRAIL are further decreased in the resistant sublines. Tumour necrosis factor superfamily member 10D (TNF member 10D) has been shown to act as a decoy receptor for TRAIL (TRAIL R4). Transient over-expression of TRAIL R4 in cells normally sensitive to TRAIL-mediated apoptosis conferred complete protection, and Degli-Esposti et al
suggested that one function of TRAIL R4 may be the inhibition of TRAIL cytotoxicity [36
]. As a large number of genes were either increased or decreased in the docetaxel-resistant sublines, targeting these individually may not lead to alterations in the resistant phenotype but understanding the central signalling pathways and transcription factors would represent a more appropriate therapeutic targeting approach.
NF-κB initiates the transcription of a wide variety of genes that code for angiogenic factors, cell adhesion molecules, anti-apoptotic factors, and cytokines, which are involved in cell survival, invasion, metastasis, and chemoresistance [37
]. In addition to its role in oncogenesis, NF-κB has been associated with chemoresistance in various models [38
]. Recent focus on inhibition of NF-κB activity has identified a sensitisation to apoptosis induced by various apoptotic triggers, including Docetaxel in multiple cell lines including PC-3 cells [41
]. Our low density array studies identified the up-regulation of a number of target genes of NF-κB (IAPs, IL-8, TNF receptor family members and FasLG). Based on these findings we focused our investigations towards the central role of NF-κB in mediating the resistant phenotype.
Despite a decrease in the baseline expression levels of the NF-κB subunits p52 and RelB and transcriptional activity, when the cells were treated with Docetaxel there was an increase in NF-κB transcriptional activity. This led us to the inhibition of NF-κB activity using the NF-κB inhibitor BAY11-7082 prior to treatment with Docetaxel which resulted in significant reversal of Docetaxel resistance.