Joyce M. Slingerland

PKB/Akt phosphorylates p27, impairs nuclear import of p27 and opposes p27-mediated G1 arrest.

Mechanisms linking mitogenic and growth inhibitory cytokine signaling and the cell cycle have not been fully elucidated in either cancer or in normal cells. Here we show that activation of protein kinase B (PKB)/Akt, contributes to resistance to antiproliferative signals and breast cancer progression in part by impairing the nuclear import and action of p27. Akt transfection caused cytoplasmic p27 accumulation and resistance to cytokine-mediated G1 arrest. The nuclear localization signal of p27 contains an Akt consensus site at threonine 157, and p27 phosphorylation by Akt impaired its nuclear import in vitro. Akt phosphorylated wild-type p27 but not p27T157A. In cells transfected with constitutively active AktT308DS473D (PKBDD), p27WT mislocalized to the cytoplasm, but p27T157A was nuclear. In cells with activated Akt, p27WT failed to cause G1 arrest, while the antiproliferative effect of p27T157A was not impaired. Cytoplasmic p27 was seen in 41% (52 of 128) of primary human breast cancers in conjunction with Akt activation and was correlated with a poor patient prognosis. Thus, we show a novel mechanism whereby Akt impairs p27 function that is associated with an aggressive phenotype in human breast cancer.NOTE: In the version of the article initially published online, the abstract contained one extraneous sentence. This error has been corrected in the HTML and PDF versions. The abstract will appear correctly in the forthcoming print issue.

The Cdk inhibitor p27 in human cancer: prognostic potential and relevance to anticancer therapy

The cyclin-dependent kinase (Cdk) inhibitor p27 (also known as KIP1) regulates cell proliferation, cell motility and apoptosis. Interestingly, the protein can exert both positive and negative functions on these processes. Diverse post-translational modifications determine the physiological role of p27. Phosphorylation regulates p27 binding to and inhibition of cyclin–Cdk complexes, its localization and its ubiquitin-mediated proteolysis. In cancers, p27 is inactivated through impaired synthesis, accelerated degradation and by mislocalization. Moreover, studies in several tumour types indicate that p27 expression levels have both prognostic and therapeutic implications.

Multiple roles of the PI3K/PKB (Akt) pathway in cell cycle progression.

As its role in tumor progression emerges, the PI3K/PKB (Akt) pathway presents an appealing cancer therapeutic target. Recent studies have investigated the mechanisms underlying the tumor-promoting effects of this pathway. PKB triggers a network that positively regulates G1/S cell cycle progression through inactivation of GSK3-beta, leading to increased cyclin D1, and inhibition of Forkhead family transcription factors and the tumor suppressor tuberin (TSC2), leading to reduction of p27Kip1. The identification of p21Waf1/Cip1 and p27Kip1 as novel substrates of PKB provided new insights into mechanisms whereby hyperactivation of this lipid signaling pathway may lead to cell cycle deregulation in human cancers. The PI3K pathway may also play a key role in the G2/M transition and its constitutive activation may lead to defects in DNA damage checkpoint control.

The energy sensing LKB1-AMPK pathway regulates p27kip1 phosphorylation mediating the decision to enter autophagy or apoptosis

Nutrients and bioenergetics are prerequisites for proliferation and survival of mammalian cells. We present evidence that the cyclin-dependent kinase inhibitor p27Kip1, is phosphorylated at Thr 198 downstream of the Peutz-Jeghers syndrome protein–AMP-activated protein kinase (LKB1–AMPK) energy-sensing pathway, thereby increasing p27 stability and directly linking sensing of nutrient concentration and bioenergetics to cell-cycle progression. Ectopic expression of wild-type and phosphomimetic Thr 198 to Asp 198 (T198D), but not unstable Thr 198 to Ala 198 (p27T198A) is sufficient to induce autophagy. Under stress conditions that activate the LKB1–AMPK pathway with subsequent induction of autophagy, p27 knockdown results in apoptosis. Thus LKB1–AMPK pathway-dependent phosphorylation of p27 at Thr 198 stabilizes p27 and permits cells to survive growth factor withdrawal and metabolic stress through autophagy. This may contribute to tumour-cell survival under conditions of growth factor deprivation, disrupted nutrient and energy metabolism, or during stress of chemotherapy.

Skp2 is oncogenic and overexpressed in human cancers

Skp2 is a member of the F-box family of substrate-recognition subunits of SCF ubiquitin–protein ligase complexes that has been implicated in the ubiquitin-mediated degradation of several key regulators of mammalian G1 progression, including the cyclin-dependent kinase inhibitor p27, a dosage-dependent tumor suppressor protein. In this study, we examined Skp2 and p27 protein expression by immunohistochemistry in normal oral epithelium and in different stages of malignant oral cancer progression, including dysplasia and oral squamous cell carcinoma. We found that increased levels of Skp2 protein are associated with reduced p27 in a subset of oral epithelial dysplasias and carcinomas compared with normal epithelial controls. Tumors with high Skp2 (>20% positive cells) expression invariably showed reduced or absent p27 and tumors with high p27 (>20% positive cells) expression rarely showed Skp2 positivity. Increased Skp2 protein levels were not always correlated with increased cell proliferation (assayed by Ki-67 staining), suggesting that alterations of Skp2 may contribute to the malignant phenotype without affecting proliferation. Skp2 protein overexpression may lead to accelerated p27 proteolysis and contribute to malignant progression from dysplasia to oral epithelial carcinoma. Moreover, we also demonstrate that Skp2 has oncogenic potential by showing that Skp2 cooperates with H-RasG12V to malignantly transform primary rodent fibroblasts as scored by colony formation in soft agar and tumor formation in nude mice. The observations that Skp2 can mediate transformation and is up-regulated during oral epithelial carcinogenesis support a role for Skp2 as a protooncogene in human tumors.

Impact of the cyclin-dependent kinase inhibitor p27Kip1 on resistance of tumor cells to anticancer agents.

A low proliferating fraction in solid tumors limits the effectiveness of cell cycle–dependent chemotherapeutic agents. To understand the molecular basis of such “kinetic” resistance we cultured tumor cells as multicellular spheroids and examined levels of p27kip1, a cyclin–dependent kinase inhibitor known to be upregulated by intercellular contact in normal cells. When transferred from monolayer to three–dimensional culture, a consistent upregulation (up to 15–fold) of p27 protein was observed in a panel of mouse and human carcinoma cell lines. Antisense–oligonucleotide–mediated downregulation of p27 in EMT–6 mammary tumor cell spheroids reduced intercellular adhesion, increased cell proliferation, sensitized tumor cells to 4–hydroperoxycyclophosphamide, and restored drug– or radiation–induced cell–cycle perturbations repressed in spheroid culture. Our results implicate p27 as a regulator of drug resistance in solid tumors and suggest that tumor–targeted p27 antagonists may be useful chemosensitizers in conjunction with conventional anticancer therapy.

Next-generation mTOR inhibitors in clinical oncology: how pathway complexity informs therapeutic strategy

Mammalian target of rapamycin (mTOR) is a PI3K-related kinase that regulates cell growth, proliferation, and survival via mTOR complex 1 (mTORC1) and mTORC2. The mTOR pathway is often aberrantly activated in cancers. While hypoxia, nutrient deprivation, and DNA damage restrain mTORC1 activity, multiple genetic events constitutively activate mTOR in cancers. Here we provide a brief overview of the signaling pathways up- and downstream of mTORC1 and -2, and discuss the insights into therapeutic anticancer targets - both those that have been tried in the clinic with limited success and those currently under clinical development - that knowledge of these pathways gives us.

p27 Phosphorylation by Src Regulates Inhibition of Cyclin E-Cdk2

The kinase inhibitor p27Kip1 regulates the G1 cell cycle phase. Here, we present data indicating that the oncogenic kinase Src regulates p27 stability through phosphorylation of p27 at tyrosine 74 and tyrosine 88. Src inhibitors increase cellular p27 stability, and Src overexpression accelerates p27 proteolysis. Src-phosphorylated p27 is shown to inhibit cyclin E-Cdk2 poorly in vitro, and Src transfection reduces p27-cyclin E-Cdk2 complexes. Our data indicate that phosphorylation by Src impairs the Cdk2 inhibitory action of p27 and reduces its steady-state binding to cyclin E-Cdk2 to facilitate cyclin E-Cdk2-dependent p27 proteolysis. Furthermore, we find that Src-activated breast cancer lines show reduced p27 and observe a correlation between Src activation and reduced nuclear p27 in 482 primary human breast cancers. Importantly, we report that in tamoxifen-resistant breast cancer cell lines, Src inhibition can increase p27 levels and restore tamoxifen sensitivity. These data provide a new rationale for Src inhibitors in cancer therapy.

Exosome Transfer from Stromal to Breast Cancer Cells Regulates Therapy Resistance Pathways

Stromal communication with cancer cells can influence treatment response. We show that stromal and breast cancer (BrCa) cells utilize paracrine and juxtacrine signaling to drive chemotherapy and radiation resistance. Upon heterotypic interaction, exosomes are transferred from stromal to BrCa cells. RNA within exosomes, which are largely noncoding transcripts and transposable elements, stimulates the pattern recognition receptor RIG-I to activate STAT1-dependent antiviral signaling. In parallel, stromal cells also activate NOTCH3 on BrCa cells. The paracrine antiviral and juxtacrine NOTCH3 pathways converge as STAT1 facilitates transcriptional responses to NOTCH3 and expands therapy-resistant tumor-initiating cells. Primary human and/or mouse BrCa analysis support the role of antiviral/NOTCH3 pathways in NOTCH signaling and stroma-mediated resistance, which is abrogated by combination therapy with gamma secretase inhibitors. Thus, stromal cells orchestrate an intricate crosstalk with BrCa cells by utilizing exosomes to instigate antiviral signaling. This expands BrCa subpopulations adept at resisting therapy and reinitiating tumor growth.

Overexpression of Akt converts radial growth melanoma to vertical growth melanoma

Melanoma is the cancer with the highest increase in incidence, and transformation of radial growth to vertical growth (i.e., noninvasive to invasive) melanoma is required for invasive disease and metastasis. We have previously shown that p42/p44 MAP kinase is activated in radial growth melanoma, suggesting that further signaling events are required for vertical growth melanoma. The molecular events that accompany this transformation are not well understood. Akt, a signaling molecule downstream of PI3K, was introduced into the radial growth WM35 melanoma in order to test whether Akt overexpression is sufficient to accomplish this transformation. Overexpression of Akt led to upregulation of VEGF, increased production of superoxide ROS, and the switch to a more pronounced glycolytic metabolism. Subcutaneous implantation of WM35 cells overexpressing Akt led to rapidly growing tumors in vivo, while vector control cells did not form tumors. We demonstrated that Akt was associated with malignant transformation of melanoma through at least 2 mechanisms. First, Akt may stabilize cells with extensive mitochondrial DNA mutation, which can generate ROS. Second, Akt can induce expression of the ROS-generating enzyme NOX4. Akt thus serves as a molecular switch that increases angiogenesis and the generation of superoxide, fostering more aggressive tumor behavior. Targeting Akt and ROS may be of therapeutic importance in treatment of advanced melanoma.