Nature Medicine
8, 1076 - 1078 (2002)
doi:10.1038/nm1002-1076
Breast cancer banishes p27 from nucleusStacy W. Blain1
& Joan Massagué21 State University of New York Downstate Medical Center, Brooklyn, New York, USA 2 Memorial Sloan−Kettering Cancer Center and Howard Hughes Medical Institute, New York, New York, USA j-massague@ski.mskcc.org The cell-cycle inhibitor p27 is phosphorylated by the Akt kinase in breast cancer, according to three new studies. This phosphorylation keeps p27 in the cytoplasm and correlates with cancer aggressiveness (pages 1136−1144, 1145−1152 and 1153−1160.)Most genes known to control the cell cycle are mutated in cancer, some of them quite frequently. These include famous genes such as K-ras and p53. One notable exception is the cyclin-dependent kinase inhibitor p27Kip1. p27 is an inhibitor of the cell cycle, and thus a candidate tumor suppressor by birthright. But the homozygous loss or silencing of the p27 locus is extremely rare. Nonetheless, a decrease in p27 levels, due to p27 protein degradation occurs in roughly half of carcinomas and correlates with aggressive, high-grade tumors and poor prognosis1. And that isn't all. Certain carcinomas of the breast, thyroid, esophagus or colon contain normal levels of p27 but, strangely, the protein has shifted location in these cancers. Normally p27 resides in the nucleus, but in these tumors it can be found instead in the cytoplasm 2,
3,
4. How does this happen? And, what does it mean? Three studies in this issue by Lian et al., Shin et al. and Viglietto et al. shed light on the matter 2,
3,
4. They demonstrate that p27 phosphorylation by the oncogenically activated kinase Akt/PKB (protein kinase B; termed Akt in these studies) disables the nuclear localization capacity of p27. Thus barred from the nucleus, p27 can no longer rein in cell division, providing the tumor cell with a growth advantage and contributing to genome instability. Similar to the loss of p27, this accumulation of cytoplasmic, Akt-phosphorylated p27 correlates with tumor aggressiveness.
p27 is a direct inhibitor of cyclin-dependent kinase 2 (cdk2), one of the cdks responsible for the activation of E2F1 transcription factors that promote DNA replication5(Fig. 1). During periods of cell proliferation, p27 remains in storage by binding to cdk4 or cdk6 in a non-inhibitory fashion. Anti-proliferative signals, including transforming growth factor- (TGF-) and cell−cell contact, mobilize stored p27, enabling it to bind and inhibit cdk2 (ref. 6). This all must happen in the nucleus. Cdk2 is a nuclear protein, and cytoplasmic localization of p27 would effectively partition it away from this target. This partition is precisely what Akt seems to be accomplishing in cancer cells.
 | | Figure 1. p27 as a tumor-suppressor protein. | | |  | a, In normal epithelial cells, anti-mitogenic signals, such as TGF- or IL-6, act to inhibit growth. They do so by mobilizing p27 to inhibit cyclin E−cdk2. This prevents the activation of S phase−specific transcription factors, such as E2F, and cells become arrested in the G1 phase of the cell cycle. Thus, p27 helps to maintain homeostasis in epithelial cells. b, In many carcinoma cells, the Akt pathway is overstimulated. Overstimulation can occur through oncogenic activation of tyrosine kinase receptors, such as HER2, inactivation of Pten or activation of Akt itself. Activation allows Akt to phosphorylate p27 on a threonine residue in its NLS, impeding nuclear entry of p27. Thus relegated to the cytoplasm, p27 is unable to control cdk2, which can remain active in the nucleus even in the presence of anti-mitogenic signals. Unrestricted cdk2 would activate E2F and allow unchecked cell proliferation. Therefore, barring p27 from the nucleus has hypothetically the same effect as loss of the protein.
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Akt is a serine/threonine protein kinase that fosters cell proliferation, survival and motility. Akt is a central component of the phosphatidyl inositol 3'-kinase (PI3K) pathway7, a pathway replete with oncogenically relevant components8. The growth factor receptors HER2 and epidermal growth factor receptor (EGFR) activate PI3K, and they both figure prominently as oncogenes in numerous malignancies. Pten, a major tumor-suppressor, functions to reverse the accumulation of Akt-activating phospholipids produced by PI3K. Pten-inactivating mutations allow excessive activation of Akt. The upshot of these regulatory relationships is that Akt is commonly activated in many forms of cancer.
Through independent but complementary experiments, the authors of these three studies demonstrate how activated Akt bars p27 entry into the nucleus2,
3,
4. They show that Akt can directly phosphorylate p27 in vitro and cause p27 phosphorylation in vivo. Using phospho-peptide mapping, Shin et al. show that this phosphorylation occurs on a residue within the nuclear localization signal (NLS) of p27. Phosphorylation of NLS motifs is a known strategy by which Akt impairs nuclear localization of other substrates, presumably by blocking recognition of the NLS by the nuclear-import factor importin- . Using detergent-permeabilized cells, Lian et al. show that Akt activation directly blocks nuclear import of p27, rather than promoting its export from the nucleus. All three groups manipulated Akt in various ways to show that Akt activation banishes p27 from the nucleus. For example, diminishing Akt activation with the HER2 antagonist Herceptin, the PI3K blocker LY294002 or by overexpression of dominant negative Akt alleles, allows the return of p27 to the nucleus and cdk2 inhibition.
Viglietto et al. strengthen their case with a new tool. They generated an antibody that specifically recognizes the Akt-phosphorylated region, threonine 157 (T157), of p27 and found that it stains only the cytoplasm. Antibody staining disappears in cells treated with a PI3K blocker. When residue T157 is changed to alanine, the mutant p27 is exclusively nuclear and oblivious to Akt; overexpression of Akt prevents cell-cycle arrest by p27 but not by the T157A p27 mutant. Collectively, these reports indicate that activated Akt causes the cytoplasmic localization of p27, effectively allowing cdk2 to remain active.
In addition to defining a novel form of p27 regulation, these studies also suggest that p27 mislocalization is actively involved in the progression of breast cancer. Activated Akt is not detected in normal breast tissue, and the bulk of p27 is nuclear under these conditions. But the situation changes in cancer tissue. All three groups found cytoplasmic p27 in approximately 40% of p27-positive primary breast carcinomas, and this was strictly correlated with activated Akt. Using the antibody against phosphoT157, Viglietto et al. show that in tumors with low T157 phosphorylation, p27 is strictly nuclear and Akt is inactive. Conversely, in tumors with activated Akt, p27 is phosphorylated on T157 and mostly cytoplasmic.
Reduced p27 levels are known to correlate with tumor aggressiveness and poor patient survival. Here, Lian et al. look at p27 location as well in an analysis of 128 breast cancer cases. The patient group with high levels of p27 and strong nuclear localization had the highest survival rate, whereas the group with low p27 levels and cytoplasmic localization fared the worst. In both the high and the low p27 groups, cytoplasmic p27 localization always correlated with poor survival. Mechanistically, these observations suggest that p27 proteolysis and mislocalization may be separate events that disable p27 during tumor progression. From a clinical perspective, these results suggest that both p27 location and levels could be used in predicting disease and therapy outcomes.
Nuclear localization of p27, and consequent unregulated cdk2 activation, may make cancer cells unresponsive to antiproliferative signals. Lian et al. suggest that this could indeed be the case. They demonstrate that Akt activation renders cells resistant to growth inhibition by TGF- or interleukin-6 (IL-6). Furthermore, they were able to restore TGF- sensitivity by inhibiting the Akt pathway. Thus, targeting Akt could be an effective way to influence the sensitivity of a cancer cell to numerous growth-inhibitory agents, both endogenous and pharmacological.
The present findings notwithstanding, Akt can also act on other targets to promote cell proliferation. For example, using a mechanism similar to that proposed for p27, Akt can also bar nuclear entry of another cdk2 inhibitor, p21Cip1/Waf (ref. 9), and the forkhead transcription factor that prompts p27 expression10. What makes the effect of Akt on p27 so special is its apparent role in breast cancer progression, as described by the studies here. The new findings also corroborate the fact that the concomitant inactivation of one Pten allele and one p27 allele increases tumor incidence in a mouse model11. In contrast, the status of p21 in cancer is unclear12. It is conceivable that both p27 and p21 are phosphorylated and mislocalized in cancer. If so, their combined loss could provide an additional advantage to the cancer cell.
The new data raise questions about the mechanisms at work in normal epithelial cells. p27 is mostly nuclear in normal proliferating cells, even though Akt should be active under such conditions. However, most of the cells used in the three studies here are established cancer lines, which have overactive Akt along with other alterations. Is there something else besides a high level of Akt activity that is required for Akt-mediated phosphorylation of p27 in cancer?
The new data also raise some interesting possibilities for cancer therapies. p27 may not be a tumor-suppressor gene, but p27 now certainly seems to be a tumor-suppressor protein. From the standpoint of cancer therapy opportunities, there is an enormous difference in this distinction. Although it is extremely difficult to restore the function of a lost tumor-suppressor gene, it seems feasible (as shown here) to pharmacologically restore the function of a misplaced p27 tumor-suppressor protein. These exciting studies further our understanding of the role of p27 in cancer, and in doing so, they buttress the case for p27 as a prognostic indicator and the Akt pathway as a ready target for anti-cancer therapy.
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