23
multiple myeloma (MM), clinical studies show patients with low p27 expression have a shorter survival period compared to those with high p27 expression on high-dose chemotherapy.72 SKP2 is overexpressed in several human cancers, giving a poor
prognosis in gastric, colon, prostate and breast cancers. Xenografts of breast cancer cells with high SKP2 levels in mice grow faster than xenografts expressing low SKP2 levels. In mouse knock-out models, disruption of SKP2 alone does not induce cellular
senescence, but SKP2-null environments or siRNA silencing of SKP2 does induce senescence and inhibits cell growth in melanoma, oral cancer, glioblastoma and lung cancer. SKP2-null mice are still viable and do not show an increased incidence of cancer. This evidence suggests that pharmacological inhibitors of SKP2 activity would be of therapeutic value for cancer therapy.73 Several protein regulators of SKP2 have been discovered, many of which are down-regulated or inactivated in cancer cells (Figure 6). Their mechanisms of action on the activity of SKP2 are discussed below:
1. PI3K/Akt
Phosphatidylinositol-3-kinases (PI3Ks) regulate many cellular functions including cell growth, proliferation, differentiation and signal transduction. Akt is an important regulator of cell growth and survival, which is able to inhibit or promote apoptosis through the regulation of several signaling pathways including Bcl-2-Associated-Death (BAD), caspase-9 and mTOR. Studies in which the PI3K/Akt pathway has been
disrupted report reduced SKP2 protein expression, coupled with increased p27 levels.62
2. PTEN
Phosphatase and tensin homolog (PTEN) is a tumour suppressor protein which
functions as a negative regulator of the PI3K/Akt pathway and is frequently inactivated in several human cancers, e.g. prostate cancer. PI3K/Akt signaling is up-regulated by 30-50% in prostate cancer cells, often due to PTEN inactivation, but restoration of PTEN activity inhibits the growth of PTEN-null prostate cancer xenografts in mice.62 PTEN regulates the oncogenic activity of SKP2 in prostate cancer cells. DU145 cells with down-regulated PTEN function showed a PTEN/Akt-dependent regulation of SKP2 activity, with an inverse correlation observed between PTEN levels and SKP2 expression.62
24
3. Androgen and AR
The androgen receptor (AR) is a ligand-activated transcription factor which, upon binding to androgen, undergoes phosphorylation and relocates to the nucleus where it interacts with DNA, initiating gene transcription and subsequently promoting cell growth. The AR has been implicated in all stages of prostate carcinogenesis including initiation, proliferation and treatment resistance, partly through regulation of SKP2 by inhibiting its degradation.62 SKP2 in turn serves as an effector of the AR by promoting cell proliferation independent of AR activity. Androgen expression has been shown to promote G1 cell cycle arrest in prostate cancer cells through reduction of SKP2 expression and induction of p27 in an AR-dependent manner.62
Figure 6: The cross-communication between the PI3K/Akt, PTEN and AR pathways and their combined
regulation of SKP2 activity.62
4. MYCN
MYCN is a member of the MYC family of transcription factors and is frequently amplified in human neuroblastomas.74, 75 Half of neuroblastomas are considered high risk (survival rates under 40%) and all instances with amplified MYCN fall into this
25
category, being associated with rapid disease progression and metastasis.74, 76 SKP2 levels are higher in neuroblastomas where MYCN is amplified than when it is not. In one study, SKP2 expression was 2- to 9.5-fold higher in MYCN-amplified cancer cells than in cells showing no MYCN amplification and this SKP2 activation was
independent of mitogenic signalling.76 This evidence shows that MYCN is another important regulator of SKP2 activity in neuroblastomas.
Currently, the only marketed drug that targets the ubiquitin-proteasome system (UPS) is bortezomib (Velcade; Millenium Pharmaceuticals) (15),46 which is used to treat
multiple myeloma (MM).Myeloma cells are up to 1000-fold more susceptible to apoptosis in the presence of 15 than normal cells in vitro, due to the inhibition of nuclear factor-κB (NF-κB), a transcription factor crucial to the pathogenesis of many inflammatory and neoplastic diseases.46, 77
The boronic acid group in 15 interacts irreversibly with and inhibits the action of the 26S proteasome (Figure 7), which is responsible for the degradation of multiple pre- ubiquitinated targets, including p27.46 In normal cells, NF-κB, which exists as a dimer of p50 and p65, is bound to an inhibitory protein, IκB, which keeps it inactive. In cancer cells, the 26S proteasome activates NF-κB by catalysing the destruction of IκB and the generation of p50 from its inactive precursor p105. NF-κB then enters the nucleus where it helps the cancer cells to survive and proliferate. Inhibition of the 26S proteasome keeps NF-κB inactivated by IκB and blocks cell proliferation.
26
Figure 7: The mechanism of action of 15. By inhibiting the 26S proteasome (red cross), and therefore the
activation of NF-κB (orange crosses), 15 suppresses the growth of myeloma cells; adapted from46
Bortezomib has numerous side effects, including peripheral neuropathy, as the result of targeting a proteasome involved in the regulation of many proteins with diverse cellular functions.46 Targeting the F-box protein of an SCF E3 ligase is an attractive therapeutic approach, as the F-box protein defines E3 ligase selectivity and each E3 ligase has fewer target proteins than the 26S proteasome. This means that targeting a specific E3 ligase pathway would have reduced patient toxicity.
27