In senescence, many studies have addressed the induction and maintenance of senescence in nontransformed cells, either through oncogene-induced senescence or replicative senescence. These tumor suppression processes are frequently disrupted or bypassed in cancer cells. While the idea of TIS as an alternative to cytotoxic treatment strategies was first proposed almost a decade ago [14
], a number of recent studies illustrate a quickly growing interest in this topic [13,20,23,24,33,44–46
]. However, little is known regarding the regulation of the senescence response in cancer cells.
One obstacle facing senescence research is a lack of specific senescence-inducing compounds. In the current study, we characterized the novel in vitro
and in vivo
senescence-inducing activity of AZQ, a compound recently identified in a high-throughput senescence screen of small molecules [24
]. Although this compound had been previously developed to treat neural and hematological malignancies, limited testing was performed in patients with solid tumors [25
]. Moreover, this compound was developed before any awareness of TIS as a tumor response. Our results show that the exposure of a panel of prostate cancer cell lines to 250-nM concentrations of AZQ effectively induces senescence in vitro
. We also demonstrate that low-dose AZQ administered over extended periods induces senescence with limited apoptosis in prostate tumor xenografts in vivo
. Tumor growth is significantly inhibited and survival is prolonged. These findings provide initial proof-of-principle for the potential of TIS in cancer treatment and prompt the investigation of other quinone and diaziquone analogs for TIS activity in cancer. Furthermore, AZQ provides a model compound for the investigation of mechanisms that regulate TIS.
In seeking to define pathways involved in TIS, we screened a panel of CDKIs and found that p27Kip1
expression increases during TIS in the prostate cancer cell lines investigated. p27Kip1
has tumor-suppressor functions [47
], and its targeted disruption leads to prostatic hyperplasia in mice [48
]. In human epithelial cancers, including prostate, p27Kip1
is commonly downregulated, and this loss of expression correlates with outcome [49
]. Disruption through deletion or mutation rarely occurs in human tumors [50,51
]. We found that, in the p53-deficient cell lines DU145 and PC3, p27Kip1
is the only CDKI that is induced. Increased p27Kip1
expression is a consistent feature of TIS in cancer cells induced through multiple agents and provides a potential marker of this process. Because p16Ink4a
plays a primary role in the replicative senescence of nontransformed primary prostate epithelia cells [38,41
], the expression of p27Kip1
may represent a backup mechanism for regulating cell cycle arrest and TIS in transformed cancer cells.
We found that the regulation of p27Kip1
in TIS occurs posttranslationally and is dependent on Skp2. Recently, Skp2 loss was found to restrict tumorigenesis in Skp2-/-
compound mutants [46
]. Our results demonstrate for the first time that Skp2 down-regulation is necessary for TIS. We found that Skp2 expression decreases after TIS induced through a variety of agents (). TIS can be stimulated through multiple mechanisms including DNA damage and demethylation [13
], induced by doxorubicin and 5-AZA, respectively. Decreased Skp2 is necessary for TIS induced by AZQ because the overexpression of Skp2 blocks growth inhibition after drug exposure in cell lines (). Our data suggest that Skp2, a gene frequently upregulated in prostate and other cancers [28
], has a major role in the senescence response in cancer cells. Furthermore, up-regulation of Skp2 may be involved in the resistance of senescence induction in cancer cells during treatment.
We also report the novel observation that decreased expression of Skp2 alone does not induce the full senescent phenotype that is observed with AZQ and other agents (). The regulation of replicative senescence and oncogene-induced senescence in nontumor cells has been the focus of multiple investigations [42
]. However, the regulation of senescence in cancer cells that lack the function of many tumor suppressors is not well defined. The extended down-regulation of Skp2 expression using stable shRNAs was not sufficient to induce senescence in any cell line. Inhibition of proliferation was more significant in LNCaP that expressed higher levels of Skp2 and markedly induced p27Kip1
. This lack of effect in other lines may be related to the lower basal expression of Skp2.
The failure of Skp2 to induce senescence suggests that other molecular pathways, possibly upstream of Skp2, are required for the full development of the senescent phenotype by TIS. For instance, the targeting of FOXM1, a transcription factor that regulates expression of Skp2 and other genes, induces senescence in gastric cancer cells [34
]. Another potential upstream mediator of TIS is myc
, whose oncogenic activity induces senescence in nontransformed cells and regulates p27Kip1
expression through Skp2 [52,53
]. Activities of these transcription factors in addition to Skp2 regulation may prove to be critical for complete senescence induction. In addition, other proteins are also reported to interact with p27Kip1
to regulate its stability, including ubiquitin ligases SIAH1/SIP and KPC [54,55
]. Recently, MLN4924, a compound that disrupts the activity of multiple ubiquitin ligases in addition to Skp2, was found to induce senescence in PC3 [46
]. TIS induction with AZQ, doxorubicin, and 5-AZA down-regulate Skp2 but clearly have the capacity to induce other signals including damage response signal transduction to generate a full senescence response in cancer. Skp2 represents one component of this response, and future study is required to identify other common pathways regulating TIS.
In summary, we conclude that AZQ induces senescence in prostate cancer cells in vitro and in xenograft tumors in vivo. The drug AZQ provides a benchmark by which future senescence-inducing compounds may be compared both in vitro and in tumor models. This drug also presents a tool with which the mechanisms regulating drug-induced senescence and the consequences of this response in tumors may be addressed. Decreased Skp2 and increased p27Kip1 are required for TIS induced by multiple drugs. Skp2 and p27Kip1 need further investigation as putative markers of the senescence response in treated and untreated tumor tissues. Furthermore, our results suggest that TIS in tumors might be more efficiently achieved by broad stress-inducing agents or by targeting regulators of upstream pathways.