The role of apoptosis repressor with a CARD domain (ARC) in the therapeutic resistance of renal cell carcinoma (RCC): the crucial role of ARC in the inhibition of extrinsic and intrinsic apoptotic signalling

Abstract

Background: Renal cell carcinomas (RCCs) display broad resistance against conventional radio- and chemotherapies, which is due at least in part to impairments in both extrinsic and intrinsic apoptotic pathways. One important anti-apoptotic factor that is strongly overexpressed in RCCs and known to inhibit both apoptotic pathways is ARC (apoptosis repressor with a CARD domain).

Methods: Expression and subcellular distribution of ARC in RCC tissue samples and RCC cell lines were determined by immunohistochemistry and fluorescent immunohistochemistry, respectively. Extrinsic and intrinsic apoptosis signalling were induced by TRAIL (TNF-related apoptosis-inducing ligand), ABT-263 or topotecan. ARC knock-down was performed in clearCa-12 cells using lentiviral transduction of pGIPZ. shRNAmir constructs. Extrinsic respectively intrinsic apoptosis were induced by TRAIL (TNF-related apoptosis-inducing ligand), ABT263 or topotecan. Potential synergistic effects were tested by pre-treatment with topotecan and subsequent treatment with ABT263. Activation of different caspases and mitochondrial depolarisation (JC-1 staining) were analysed by flow cytometry. Protein expression of Bcl-2 family members and ARC in RCC cell lines was measured by Western blotting. Statistical analysis was performed by Student's t-test.

Results: Regarding the extrinsic pathway, ARC knockdown strongly enhanced TRAIL-induced apoptosis by increasing the activation level of caspase-8. Regarding the intrinsic pathway, ARC, which was only weakly expressed in the nuclei of RCCs in vivo, exerted its anti-apoptotic effect by impairing mitochondrial activation rather than inhibiting p53. Topotecan- and ABT-263-induced apoptosis was strongly enhanced following ARC knockdown in RCC cell lines. In addition, topotecan pre-treatment enhanced ABT-263-induced apoptosis and this effect was amplified in ARC-knockdown cells.

Conclusion: Taken together, our results are the first to demonstrate the importance of ARC protein in the inhibition of both the extrinsic and intrinsic pathways of apoptosis in RCCs. In this context, ARC cooperates with anti-apoptotic Bcl-2 family members to exert its strong anti-apoptotic effects and is therefore an important factor not only in the therapeutic resistance but also in future therapy strategies (i.e., Bcl-2 inhibitors) in RCC. In sum, targeting of ARC may enhance the therapeutic response in combination therapy protocols.

Keywords: ABT-263; ARC; Apoptosis; Bcl-2 family; TRAIL; renal cell carcinoma (RCC).

Fig. 1

Expression of ARC in clear cell RCCs and RCC cell lines. a ARC was strongly overexpressed in the cytoplasm of clear cell RCCs (1) compared to non-neoplastic renal tissue (2), as determined by immunohistochemistry. Furthermore, nuclear ARC expression was only detectable in RCCs and not in non-neoplastic tissue. For semiquantitative analysis of ARC expression cytoplasmatic stainig was scored from 0 to 12 and nuclear expression was scored from 0 to 4. All values are expressed as the mean ± s.d. *p < 0.05. b ARC was also strongly expressed in the cytoplasm and nucleus of the RCC cell lines clearCa-6, −3 and −12. Nuclear and cytoplasmic distribution of ARC differed only slightly between the three RCC cell lines, as determined by fluorescent immunohistochemistry

Fig. 2

ARC and p53 in RCC cell lines. a For ARC knockdown by shRNA in clearCa-12 cells, three lentiviral shRNA vectors were tested, and the shRNA construct pGIPZ 1365 demonstrated the strongest ARC knockdown (approximately 90%) as determined by western blot. b Treatment of clearCa-12 cells with 10 μg/ml topotecan modulated gene expression of 4 out of 26 p53-regulated genes in control cells and 2 out of 26 p53-regulated genes in ARC-knockdown cells, as determined using the p53 Signaling Pathway RT² Profiler PCR Array (Qiagen, Hilden, Germany) after 12 h of topotecan treatment. Direct comparison of topotecan treated ARC knockdown and control clearCa-12 cells showed no significant differences with regard to the modulation of p53-regulated genes. Genes with a fold-increase in gene-expression greater than 4 are depicted as red points, and those with a fold-decrease greater than 4 are depicted as green points. c ARC knockdown did not change the subcellular localisation of p53 in RCCs. Fluorescent immunohistochemistry for p53 in clearCa-12 cells revealed that ARC knockdown did not change the amount of nuclear p53 in ARC knockdown cells compared to control cells transduced with non-silencing shRNA

Fig. 3

Expression of ARC and Bcl-2 family members and ABT263-induced apoptosis in RCC cell lines. a ARC as well as anti-apoptotic Bcl-2 family members were expressed in RCC cell lines, but expression intensity differed between these cell lines. Overall, Bcl-2, Bcl-xl and Mcl-1 exhibited the strongest expression, whereas Bcl-w showed weaker expression. Bcl-2 was not expressed in clearCa-3 cells and Bcl-w was not expressed in clearCa-12 cells. BCL-A1 could not be detected (not shown). The p53 mutational status of the cell lines were published by our group elsewhere [39, 40]. b RCC cell lines expressed pro-apoptotic Bcl-2 family members of all functional groups. Sensitisers (Bid, Bim), activators (Puma, Bad, Bok) and effectors (Bax, Bak) were detectable in all RCC cell lines, also with differences in the intensity of expression, with clear expression of PUMA only in clearCa-3, clearCa-6 and clearCa-11 and Bok only in clearCa-6, claerCa-7 and clearCa-11. c All RCC cell lines revealed sensitivity towards ABT263-induced cell death (10 and 20 μM ABT263), as determined by cell count. All values are expressed as the mean ± s.d. *p < 0.05. d In 3 arbitrarily selected cell lines (clearCa-3, −6 and −12) ABT263 (10 μM) induced mitochondrial apoptosis as determined by caspase-9 and mitochondrial activation (JC-1 stain). All values are expressed as the mean ± s.d. *p < 0.05

Fig. 4

Knockdown of ARC sensitises clearCa-12 towards TRAIL-, topotecan- and ABT263-induced apoptosis. ARC knockdown clearCa-12 or control clearCa-12 cells (non-silencing) were treated with (a) TRAIL (100 ng/ml), (b) topotecan (10 μg/ml), or (c) ABT263 (10 μM). ARC knockdown sensitised clearCa-12 cells against the extrinsic apoptotic pathway induced by TRAIL as determined by caspase-8 and caspase-3 activation. Topotecan and ABT263 induced mitochondrial apoptosis as determined by caspase-9 and caspase-3 activation respectively mitochondrial activation. Furthermore, ARC knockdown enhanced caspase-9 activation induced by TRAIL. All values are expressed as the mean ± s.d. *p < 0.05

Fig. 5

ARC knockdown further enhances topotecan-induced sensitisation towards ABT263-induced apoptosis. a ClearCa-3, −6 and −12 cells were treated with 0.1 μg/ml or 10 μg/ml topotecan for 24 h. Co-treatment with 10 μM ABT263 for the last 3 h of the incubation synergistically enhanced caspase-9 activation as well as mitochondrial depolarisation (JC-1) measured by flow cytometry, compared to single treatment with topotecan or ABT263. b ClearCa-12 cells transduced with non-silencing shRNA or ARC knockdown shRNA were treated with 10 μg/ml topotecan for 24 h, and 10 μM ABT263 was added for the final 3 h of incubation. ARC knockdown further enhanced caspase-9 activation as well as mitochondrial depolarisation following co-treatment compared to cells transduced with non-silencing shRNA. A synergistic effect (SYN) was determined as i_1,2 ≥ (i₁ + i₂) +20%, \where i_1,2 = effect of co-treatment, i₁ = effect of topotecan and i₂ = effect of ABT263

Fig. 6

ARC is not regulated by topotecan or UO126. a ClearCa-12 cells were treated with topotecan (10 μg/ml) for 12 h and 24 h. At the protein level (as determined by western blot), no change in ARC expression was observed. b ClearCa-12 cells were treated with the ERK1/2 inhibitor UO126 (50 μM) for 12 and 24 h. This treatment resulted in nearly the complete loss of ERK1/2 phosphorylation. However, no change in ARC expression could be demonstrated on protein level by western blot analysis