A Crosstalk Imbalance Between p27 Kip1 and Its Interacting Molecules Enhances Breast Carcinogenesis

Introduction

Cell-cycle disruption is a hallmark of cancerous cells. ¹ p27 is a member of the cyclin-dependent kinase (CDK) inhibitor family, acting as a potent tumor suppressor in a variety of human cancers, and is a key cell-cycle regulator. ^{2 –4} To date, studies have shown that p27 is involved in cell differentiation, proliferation, apoptosis, cell-cell adhesion, and growth inhibition. ^5,6 Breast cancer is one of the most common cancers in women. Solid tumors have heterogeneous characteristics and exhibit variability in patient survival rates that are dependent on subtype. Moreover, a prolonged period of time exists during which relapse is possible but uncertain. There are currently several approved therapies for treating breast cancer, and the availability of clinical samples from large patient cohorts has contributed to the prolific research undertaken in the field of breast cancer. ⁷ Many studies have shown that the level of p27 expression decreases during carcinogenesis, correlating with poor prognosis. p27 is an independent prognostic factor in breast cancer, and inhibiting its deregulation has proved to be an effective target in breast cancer therapy. ^8,9 Moreover, there is considerable evidence that p27 inactivation is a fundamental step in the development of malignancies. ¹⁰

p27 and Tumor Progression

A hallmark of cancer is unrestricted cell growth and proliferation; a crucial genetic abnormality that occurs during tumor development is the loss of control at the G₁ to S transition, which is a period during which cells decide to either enter the cell cycle on mitogenic stimuli, or stay quiescent in response to antimitogenic or senescence signals. Hypophosphorylated pRb is pivotal in maintaining the quiescent state and controlling the G₁ to S checkpoint. Phosphorylation inactivates pRb, which allows a cell to enter the S phase. CDK family members, along with their corresponding cyclins expressed during the S phase, mediate the phosphorylation of several critical targets, including the Rb gene product, pRb. This CDK-mediated phosphorylation is inhibited by specific proteins called CDK inhibitors, or CDKIs, of which p27 is a family member. ^11,12

Most normal epithelial tissues, including breast, prostate, lung, and ovary, express high nuclear levels of the p27 protein. A variable loss of p27 has been observed in all human malignancies examined to date. ¹³ Even as a tumor suppressor, p27 does not fit the classic tumor suppressor paradigm in humans, as mutations in or the silencing of the p27 gene in human tumors are extremely rare. ¹⁴ However, a loss of or decrease in p27 protein levels is frequently observed in many human cancers. Current studies have reported that, in addition to cytosolic mislocalization, p27 expression levels decrease during breast cancer development and progression. A number of studies have characterized p27 as an independent prognostic factor in breast cancer. ^{15 –18}

p27 is regulated at both the expression level and subcellular localization. ¹² The absence or reduction of p27 expression has been associated with the aggressive behavior of human malignancies, including breast, gastric, prostate, colon, and lung carcinomas. ¹⁹ The exact molecular mechanisms responsible for the down-regulation and mislocalization of p27 remain unknown. However, phosphorylation has a high correlation with the mislocalization of p27 to the cytoplasm.

Phosphorylation of p27

Although transcriptional and noncovalent sequestration may pretranscriptionally regulate p27, the protein is primarily regulated post-transcriptionally at the level of both translation and stability. It has been suggested that two proteolytic pathways act in sequence during the cell cycle to control the abundance of p27. The first functions during the early and mid-G₁ phase, and is triggered by serum independently of Thr187 phosphorylation and CDK2. The second pathway works in the late G₁, S, and G₂ phases, and is dependent on both Thr187 phosphorylation and CDK2. The second pathway is well characterized and discussed in greater detail next, but the first pathway has not yet been completely elucidated. The p27 protein may also be degraded through a mitogen-activated protein kinase (MAPK)-dependent and CDK2-independent process. ^{20 –22}

In addition to Thr187, there are three additional phosphorylation sites, including Ser10, Thr157, and Thr198, which are involved in cellular localization. The phosphorylation at Ser10 stabilizes the p27 protein in G₁ and accounts for 75% of p27 phosphorylation. p27 is exported to the cytoplasm in a KPC1/2-induced ubiquitin-dependent degradation manner. Based on this mechanism, p27 is phosphorylated at Ser10, which promotes CRM1-dependent binding and nuclear export. This pathway operates in G₁ cells or cells stimulated to re-enter the cell cycle from a quiescent state. Phosphorylation at Thr157 by protein kinase B/Akt impairs the nuclear import of p27, but does not affect its stability in breast cancer and other cell types. ²³ Fujita et al. indicated that p27 phosphorylation at Thr198 by p90 ribosomal protein S6 kinases promotes cytoplasmic localization. Mutations in these phosphorylation sites may result in a more potent induction of apoptosis and the inhibition of cell growth for breast cancer gene therapy. The phosphorylation state of p27 is involved in signal transduction, apoptosis, and gene expression pathways. ²⁴ However, the specific mechanisms involved in the phosphorylation regulation of p27 expression remain elusive, and a better understanding of how this pathway interacts with its signals in both the cytoplasm and nucleus is needed.

Signal Transduction Pathway of p27

Signal transduction is the process by which an extracellular stimulus is converted to an intracellular signal that triggers a cascade or series of events, ultimately culminating in the production of an appropriate cellular response. ²⁵ The mechanisms controlling p27 abundance range from translational control observed in quiescent cells to proteolytic mechanisms operating at specific phases of the cell cycle or in specific subcellular compartments, such as the cytoplasm or nucleus. The concentration of p27 is thought to be predominantly regulated by the ubiquitin-dependent proteolytic pathway. The degradation of p27 is triggered by its phosphorylation on Thr187 by the cyclin E-CDK2 complex. The phosphorylation of Thr187 is required for the binding of p27 to Skp2, which is the F-box protein component of an SCF ubiquitin ligase (E3) complex, and this interaction, in turn, results in the polyubiquitination and degradation of p27. The control of p27 protein levels is affected by ubiquitin-dependent degradation in a ubiquitin- and Skp2-independent manner at G₁ as well as by JAB1-dependent degradation. The effect of p27 on the cell cycle is predominantly regulated by its stability and subcellular localization. ^{20 –22,26}

Ubiquitin-proteasome proteolysis is the major post-transcriptional pathway that is utilized for the regulation of p27. The removal of p27 CDK inhibitory activity in G₁, thus, appears to occur in at least three phases in this model system: sequestration in cyclin D-CDK complexes in early G₁, export/cytoplasmic degradation in mid-G₁, and Skp2-dependent degradation in the nuclear compartment beginning late in the G₁ phase. The phosphorylation of the p27 protein on Thr187 by CDK2 prepares the p27 protein for binding to the ubiquitin ligase SCF, which leads to 26S proteasome degradation. It has been proved that the Ras-MAPK pathway is the most likely signaling pathway involved in this process, occupying a central position in the network of interactive signaling cascades. The MAP kinases p42MAPK, or ERK1, and p44MAPK, or ERK2, are regulated as the central components of the growth-promoting signaling pathways. A dominant negative mutant of Grb2 and inhibitors of ERK1/2 can rescue the decrease in the p27 protein. In addition, ERK1/2 has been reported to regulate Thr160 phosphorylation of CDK2, which is required for activation of the kinase. Two types of dominant negative Ras proteins have been shown to block p27 deregulation due to increased CDK2-cyclinE activity. It has been observed that ERK2 can directly phosphorylate the p27 protein on Ser10. Overexpression of endogenous and exogenous Ras-MAPK decreases the half-life of p27 and reduces CDK2 activity. The inhibition of Ras-MAPK leads to an accumulation of p27 and decreased CDK2 activity. It is apparent that Ras-MAPK plays a role in the regulation of p27 protein levels. ^25,27

Previous studies have shown that PI3K-AKT regulates the expression of the p27 protein transcriptionally through forkhead transcription factors. The forkhead transcription factors activate the p27 gene, which leads to increased protein levels. Several studies have indicated that the inhibition of PI3K either by chemical inhibitors or by a dominant negative mutant of p85, which is the regulatory subunit of PI3K, can increase p27 and decrease p27-CDK2 activity. The expression of a dominant negative AKT can also increase p27 protein expression. In addition to the transcriptional suppression of p27 achieved by inhibiting forkhead transcription factors, the PI3K pathway is also able to post-transcriptionally regulate the localization and level of p27 through direct threonine phosphorylation of p27 by AKT. Several groups have demonstrated that AKT phosphorylates p27 at Thr157 in vitro and in vivo. Thr157 is in the nuclear localization motif of p27, and its phosphorylation causes p27 to exit the nucleus, which rescues CDK2 from both p27-induced inhibition and proteasomal degradation. In addition, AKT has been reported to be able to phosphorylate p27 at Thr198, which also causes the protein to localize to the cytoplasm. Clinically, the THR2-targeted antibody trastuzumab affects the phosphorylation of Thr157 and Thr198, which regulates the expression of p27 in the cytosol of breast cancer cells. ²⁸

As just described, the phosphorylation of Thr187 helps degrade p27, but Ser10 phosphorylation increases its stability. Nuclear protein human kinase-interacting stathmin (hKIS) phosphorylates Ser10; this phosphorylation, however, does not stabilize p27, but instead promotes its export to the cytoplasm. Ser10 can be phosphorylated by various kinases, which determines the phosphorylation outcome during G₀ and G₁. Ser10 phosphorylation by the mitogen-induced kinase hKIS leads to the translocation of the protein from the nucleus to the cytosol, where p27 cannot inhibit CDK2 and is also susceptible to proteolysis. hKIS overexpression overcomes the growth arrest induced by p27, whereas the depletion of hKIS with siRNA inhibits Ser10 phosphorylation and enhances growth arrest. ^29,30

Other known molecules are involved in this specific signal transduction pathway as well, including TGF-β, IL-6, and STAT3. The inhibition at any level of this signal transduction pathway or the modification of the phosphorylation state of these molecules can regulate p27 function. Targeted drugs that interfere with the signal transduction cascade in blocking tumor cell proliferation seem promising in cancer therapy. ³¹ However, in order to establish screens for identifying the key inhibitors of p27 in tumor cells and to understand the exact mechanisms involved in the mislocalization and down-regulation of expression, a profile of p27 interacting partners should first be elucidated.

Interacting Protein Profiling of p27

Cellular proliferation can occur at different rates and is dependent on extracellular factors and genetic profiles, which determine the differentiation status and the eventual transformation grade. The proliferation rate is regulated by the proteins that are involved in cell-cycle control, specifically cyclins and their associated kinases. The fluctuating activities of several Cdk/Cyclin complexes establish the progression of the cell cycle through each phase (G₁, S, G₂, and M). The composition and regulation of Cdks vary according to cell type and environment. ³² In addition to cyclins and their associated kinases, the CDKIs are the third group of proteins involved in cell-cycle progression. Among them, p27 plays a crucial role in linking extracellular growth-regulatory signals to the progression or exit from the cell cycle. CDK2 is the predominant kinase that allows progression through the G₁/S phase and subsequent replication events. ² p27 is a CDKI that binds to CDK2 to prevent its premature activation. ⁶ The protein can also directly bind to a CDK-activating kinase through its C-terminal domain. ³¹

p27 has been reported to interact with a number of intracellular and extracellular molecules, including CRM1, Jab1, SKP2, Ras, Spy1, and TGF-α. ^{33 –38} The overexpression of Jab1/CSN5 elicits nuclear export/degradation of p27, suggesting that the relocalization of these molecules is required before degradation. Recent studies have further characterized the mechanisms underlying p27 export. Jab1/CSN5 promotes the export and degradation of p27 through a CRM1-dependent mechanism, possibly involving interactions of p27 with the adaptor protein Grb2 in the cytoplasm. The export of p27 to the cytoplasm after growth factor stimulation may also inhibit Grb2/SOS interactions and serve to limit Ras effector functions.

Several additional molecules are specific for breast cancer, such as the estrogen receptor, progesterone receptor, and ErbB-2. Gompel et al. ³⁹ showed that E2 increases the level of the various cyclins involved in cell-cycle progression and decreases the cyclin kinase inhibitors p27. Progesterone up-regulates p27 expression, and induction is required for the growth-inhibitory action of progesterone. ^40,41 Research also has shown that a combined expression of high/low p27(kip1) and low/high Ki-67 values showed 100% specificity and sensitivity in predicting long-term versus short-term survivors. ⁴² Moreover, ErbB-2 induces mammary tumors. Experiments conducted on cultured cells have shown that p27 can inhibit ErbB-2 signaling. It has also been suggested that p27 may be the downstream target of HER-2/neu oncogenic signals in promoting tumor development, and p27 is a potential target for HER-2/neu-associated tumors. ⁴³ Furthermore, our meta-analysis indicated that p27 is an independent prognostic factor for breast cancer. ⁴⁴ With continued efforts in cancer research, new therapeutic molecules will undoubtedly be discovered; however, only full characterization and assessment will elucidate their potential clinical utility.

Conclusions

In conclusion, we hypothesize that crosstalk between p27 and other molecules in this signaling pathway plays an indispensable role in cell proliferation and differentiation. The imbalance of this crosstalk may lead to the malignancy of breast cancer. Furthermore, understanding the molecular profile of p27 could lead us into a contemporary era of tailored and individualized breast cancer treatment.