Our novel discovery that PPARγ upregulates RhoB in ATC cells was prompted by RS5444-mediated alterations in cell morphology coupled with supporting literature demonstrating that enhanced RhoB activity was associated with growth inhibition and elevated p21 (17
). RhoA and RhoC share ~86% amino acid homology with RhoB and also mediate actin stress fiber formation. Other RhoGTPases, which include Rac1 and Cdc42, have been found to promote oncogenesis, invasion and metastasis (30
). However, RhoB is antiproliferative and proapoptotic in cancer cells (32
), and overexpression of RhoB can inhibit cell migration, invasion, and metastasis (33
). In head and neck, lung and brain cancers, RhoB levels are decreased with tumor progression suggesting that silencing this pathway is critical to tumor survival and progression (34
). Our data demonstrate that RhoB mRNA and protein levels are low in ATC cell lines with upregulation upon exposure to RS5444 or FK228.
We found that RS5444 regulates RhoB at the transcriptional level with rapid upregulation of RhoB mRNA by 2 hrs of treatment and protein levels by 4 hrs. We identified two putative PPARγ response elements (−1302 to −1282; −1422 to −1402) in the RhoB promoter with 70−80% homology to a consensus PPARγ response element (PPRE). However, transient transfection of the RhoB promoter linked to a luciferase reporter (1876 bp promoter fragment) in DRO, KTC2 and KTC3 did not lead to induction of luciferase activity after 24 hr treatment with 10 nM RS5444 even though treatment with 1 ng/ml FK228 induced luciferase activity 3−9 fold (Figure S2
). Thus, the transcriptional regulation of RhoB by PPARγ may be regulated by another region of the RhoB gene or by a more complicated mechanism involving structured chromatin complexes not assayable using transfected RhoB promoter linked to luciferase. RhoB transcript levels have been observed to be transcriptionally suppressed by HDAC1 and found to be positively regulated when treated with an HDAC inhibitor (trapoxin A) via an inverted CCAAT element in the RhoB promoter 451 bp upstream of the transcriptional start site (27
). RhoB is an early response gene responding to environmental induction due to environment stresses, such as UV irradiation, which under this circumstance, is mediated by the RNA-binding protein HuR stabilizing RhoB mRNA (37
). Oppositely, oncogenic signals mediated by the Ras/PI3K/Akt pathway suppress RhoB expression (38
). Thus, while multiple mechanisms appear to regulate RhoB expression in cancer, the important consequence of our work is that RhoB can be re-expressed in ATC through the use of drugs, resulting in biologically active protein that opposes tumor growth.
We describe a novel mechanistic signaling pathway leading to tumor growth inhibition by two classes of drugs via a RhoB dependent mechanism that is upstream of p21. We show that PPARγ-mediated upregulation of p21 mRNA occurs through RhoB by using dominant negative RhoB and siRNA to silence RhoB. Two reports have identified a potential link between upregulation of p21 and RhoB (28
). In one report, RhoB-GG induced p21 in a p53-dependent manner, though this was dispensable because RhoB-GG still inhibited growth of p53-null cells that lacked p21 (28
). The authors concluded that RhoB-GG suppressed human tumor cell proliferation by more than one mechanism and that it promoted apoptosis as well as inhibited cell cycle transit. In the other report, ectopic expression of RhoB was demonstrated to upregulate p21 (17
). Our present study is the first to show that endogenous RhoB is directly responsible for regulation of p21 mRNA and protein levels by RS5444 and FK228 in p53-wild type (DRO and KTC2) as well as p53-mutant (ARO and KTC3) cells (39
In a recent report, ciglitazone and rosiglitazone increased apoptosis in several ATC cell lines (40
). The lack of induction of apoptosis by RS5444 (9
) is striking since expression of RhoB induces apoptosis in cancer cells in cell culture [reviewed in (32
)] and ectopic tumors grown in mice (41
). Even more surprising, DAPI staining revealed that treatment with FK228 also does not induce apoptosis in THJ-11T and THJ-16T with only slight apoptosis (~12%) in DRO (Figure S3A
). Using a LDH assay to examine overall cell death, FK228 treatment again only slightly induces cell death (~13%) in DRO with no effect on KTC2 and KTC3 cells (Figure S3B
). One study demonstrated that cyclin B1 is the target for suppression by RhoB causing apoptosis (43
). It is possible that both RS5444 and FK228 could stimulate an antiapoptotic pathway antagonizing the proapoptotic properties of RhoB that is independent of the regulatory role of RhoB in cell proliferation. Alternatively, it may be that induction of apoptosis by rosiglitazone may be PPARγ/RhoB-independent. One additional consideration is that cellular localization and post-translational modification (prenylation and sumoylation) of RhoB are critical regulators of its effects (32
). Site-directed mutagenesis studies demonstrated the requirement for palmitoylated cysteine 192 and prenylated cysteine 193 for the tumor suppressive and proapoptotic activities of RhoB (27
). Understanding these mechanisms could be important in the selection of the most potent PPARγ agonist or RhoB modulator for combinatorial therapy such as a Tzd and taxane against ATC (9
). Importantly, we found that chronic daily treatment of mice harboring ATC tumors with 0.025% RS5444 in the diet retains the ability to sustain elevated RhoB over 4 weeks of treatment (). This also suggests that RhoB and p21 may be ideal molecular markers of response to therapy and easily measured in biopsy tissue following treatment.
In summary, we have identified a novel growth inhibitory pathway regulated by PPARγ and identified a HDAC inhibitor that upregulates RhoB mRNA and protein in human ATC cells (). Elevated RhoB protein is necessary for these two drugs to then upregulate p21 mRNA and protein as well as inhibit cell proliferation (). Thus, RhoB is implicated as a critical signaling node that could be therapeutically targeted in ATC. Importantly, it is a target that can be upregulated by multiple classes of drugs. HMG CoA reductase inhibitors (statins) (44
), prenylation inhibitors (FTI, GGTI) (32
), HDAC inhibitors (FK228) and now PPARγ agonists upregulate RhoB in various cancers. Although the connection to RhoB has not been made in ATC, the aforementioned classes of drugs inhibit growth in ATC cells (45
) most likely acting in part through upregulation of functional RhoB. We are currently investigating the role of these drugs that mediate RhoB upregulation and growth inhibition in ATC. Post-translational modification and cellular localization of RhoB as a result of treatment with each of these drugs will also most likely dictate optimal antitumor activity (32
) and allow for rational selection of combinatorial therapy with maximum benefit to the patient.