The resistance of the tumor cells toward apoptosis is a hallmark in many tumors. Combination therapy targeting apoptotic pathways (i.e., Bcl-2 inhibition) is a promising strategy and is currently evaluated in many clinical trials [1]. Despite marked efforts in therapeutic strategies the resistance remains a major problem in cancer therapy. There is a growing interest to find molecular targets by which apoptosis can be selectively induced in tumor cells. One of the promising targets is Bcl-2, against which some inhibitiors have already been approved (i.e., ABT-263, ABT-199) [23]. Bcl-2 inhibitors showed enhanced efficiacy in combination with conventional chemotherapeutic drugs (i.e., paclitaxel, 5-FU, topotecan) but with these combinations still not all of the tumor cells can be reached [23, 24]. For this reason, we focused on ARC protein, which was expressed in all RCC cell lines and tumor samples, we have investigated so far.
This resistance towards mitochondrial apoptosis has mainly been attributed to the anti-apoptotic members of the Bcl-2 family [27, 28, 36, 37]. Cancer cells can evade apoptosis by the up-regulation of the pro-survival Bcl-2 family proteins such as Bcl-2, Bcl-xl, and Mcl-1 [26]. Controversely, Mcl-1 can be up-regulated by ABT-263 which contributes to ABT-263 resistance in cancer cells. ABT-263 increases Mcl-1 stability, but the inhibition of ERK, JNK or Akt activity can sensitise the cancer cells to ABT-263 [38].
The current study was the first to show that ARC, which is overexpressed especially in the cytoplasm of RCCs, strongly participates in this mitochondrial resistance. Therefore, ARC is a functionally relevant anti-apoptotic factor in RCCs acting upstream of the Bcl-2 family members and supporting anti-apoptotic Bcl-2 family members in preventing apoptosis. ARC overexpression could be detected in a couple of cancer types and cancer cell lines and in colorectal cancer cells its expression level is correlated inversely to apoptosis in response to chemotherapy [3,4,5,6,7, 9].
However, our previous study of ARC expression in RCCs did not systematically analyse the subcellular localisation of ARC in RCCs. Here, we refined our analysis of ARC expression in RCCs in vivo with regard to its cellular distribution and found that RCCs express ARC mainly in the cytoplasm, whereas nuclear expression was observed in a much smaller proportion of tumour cells in vivo. ARC is also strongly expressed in the cytoplasm and nucleus of RCC cell lines, with nuclear and cytoplasmic distribution of ARC differing only slightly between three tested cell lines. These results are consistent with the findings of other groups demonstrating strong ARC expression not only in the cytoplasm but also in the nuclei of multiple cancer cell lines [5].
ARC inhibits apoptosis on multiple levels and thereby acts as an upstream apoptosis inhibitor regulating the extrinsic and intrinsic apoptotic pathways in different solid tumours. Regarding the intrinsic pathway of apoptosis, ARC has been reported to prevent p53 tetramerisation [32], inhibit caspase-2 activation [3], and bind pro-apoptotic Bcl-2 family members [18, 19]. With regard to the extrinsic pathway, ARC interacts with caspase-8 [3]. However, the exact role of ARC in the inhibition of apoptosis in RCCs has not been evaluated. Here, we demonstrated that nuclear ARC expression was of only minor importance for the regulation of p53-induced apoptosis in RCCs, as RT-PCR array analysis revealed that knockdown of ARC did not influence the regulation of p53 target genes. Furthermore, the overall regulation of p53 target genes following treatment with a high concentration of topotecan was weak, with only 4 of 26 p53-target genes regulated. These results show that p53 activity is strongly impaired in RCCs [33] and this impaired p53 activation was not due to ARC expression. In contrast to observations made in breast cancer cell lines, ARC knockdown in RCC cells did not result in p53 translocation to the nucleus [14, 32].
In regard to the extrinsic apoptotic pathway, our results demonstrate that ARC plays an important role in the inhibition of TRAIL-induced apoptosis in RCCs, in concordance with other solid tumours. Consequently, TRAIL-mediated caspase-8 and −3 activation were significantly enhanced by ARC-knockdown. Furthermore, ARC-knockdown slightly enhanced mitochondrial apoptosis, providing an initial clue that ARC may also participate in protecting the mitochondria of RCCs against apoptotic stimuli. We conclude that, ARC prevents activation of the apoptotic initiator caspase-8 as well as activation of the mitochondrial amplification loop.
Our results also indicated that ARC plays an important role in the impairment of intrinsic apoptosis: mitochondrial activation was suppressed and controlled by ARC. In contrast, ARC-knockdown sensitised RCC cell lines to mitochondrial apoptosis induced by topotecan and/or Bcl-2 antagonist ABT-263. In case of ARC knockdown, assembly of death-inducing signaling complex (DISC) will be facilitated and spontaneous Bax activation will be triggered resulting in apoptosis [8, 16]. In conclusion, our results suggest that ARC expression in RCCs plays a major role in therapy resistance, even if a targeted drug (i.e., Bcl-2 inhibitor) is given.
The increase in mitochondrial apoptosis upon ARC knockdown was an important finding, as we and others have previously demonstrated that altered mitochondrial activation is crucial for the therapy resistance observed in RCCs [27, 28, 36, 37, 39].
Membrane binded Bcl-2 and Bcl-xl inhibit the release of many apoptotic proteins from mitochondria (i.e., cytochrome c, pro-caspase 3, and apoptosis inducing factor). Bcl-2, which is overexpressed in most RCCs, contributes to tumor development and progression. In addition, Bcl-2 overexpression is correlated with a low apoptosis rate of tumor cells [1]. ARC functions as an anti-apoptotic regulator upstream of Bcl-2 family members by interacting with and thereby reducing the availability of pro-apoptotic binding partners of the Bcl-2 family, including Puma, Bax and Bad. Thus, ARC plays a pivotal role in fine-tuning the apoptotic machinery.
In addition to Puma, Bax, and Bad, all RCC cell lines expressed pro-apoptotic Bcl-2 family members, including Bid, Bim, Bok, and Bak. Although expression levels of these proteins differed between the tested cell lines, members of all functional groups (sensitisers, activators, and effectors) were detectable in each cell line, and therefore, we termed RCC cell lines “primed for death” according to the model of Deng et al. [34]. In summary, these findings provided rationale for the sensitivity of RCCs towards Bcl-2 inhibitors such as ABT263. In accordance with previous observations on ABT-737 and RCCs [28], these “primed for death” cells were all sensitive to ABT-263-induced apoptosis, albeit only to a certain degree. This limited sensitivity of our RCC cell lines towards ABT-263 showed some correlation to the expression profile of anti-apoptotic Bcl-2 family members. In all cell lines evaluated, Mcl-1, which is not inhibited by ABT263 [26], was detected at the protein level. The ABT-263 binding partners Bcl-2, Bcl-w and Bcl-xl were also expressed in these cell lines, with the exception of Bcl-2 in clearCa-3 and Bcl-w in clearCa-12.
Taken together, our findings regarding ARC and Bcl-2 family member expression suggest that the limited sensitivity of RCCs to Bcl-2 inhibitors, which is commonly explained by the presence or absence of pro- and anti-apoptotic Bcl-2 family members themselves [27, 28, 36, 37], also depends on other factors such as ARC interacting with these Bcl-2 family members. These additional factors can modulate the sensitivity of cells towards Bcl-2 antagonists, thereby supporting the role of anti-apoptotic Bcl-2 inhibitors in protecting the mitochondria from apoptotic signals. Moreover, the downregulation of ARC expression likely increased the availability of pro-apoptotic binding partners, including Bax, Bad and Puma, at the mitochondria and thereby enhanced mitochondrial apoptosis.
Due to its important role in inhibiting extrinsic and intrinsic apoptosis, we sought to downregulate the expression of ARC in our RCC cell lines using chemical compounds to evaluate new approaches for targeting ARC overexpression therapeutically. However, our attempts to modulate ARC expression by mechanisms previously described in other cell lines were not successful in RCC cells; neither topotecan, as a classic chemotherapeutic compound [19], nor the ERK-inhibitor UO126 [35] were able to downregulate ARC expression, although both mechanisms were previously described to inhibit ARC gene expression in myocardial and colon cancer cell lines, respectively. Thus, the precise cellular mechanism responsible for the strong ARC expression in RCCs needs to be determined in further experiments.
Next, we tried to further enhance ABT-263-induced apoptosis in RCCs by a pre-treatment with topotecan. Although the synergistic enhancement of ABT263-induced apoptosis by topotecan was strongest in clearCa-6, a synergistic enhancement of mitochondrial apoptosis could also be observed in clearCa-3 and clearCa-12.
As a result, both strategies – indirectly increasing the availability of pro-apoptotic Bcl-2 family members by ARC knockdown as well as topotecan pre-treatment – led to an increased sensitivity of the RCC cell lines towards ABT263-induced apoptosis.
Therefore, it was reasonable to attempt a combination of these strategies to further enhance anti-Bcl-2 treatment sensitivity. In fact, this strategy synergistically enhanced ABT263-induced apoptosis in all cell lines, which suggests that ARC supports the function of anti-apoptotic Bcl-2 family members in preventing mitochondrial apoptosis in RCC cell lines.