The association of cdk4 with D-type cyclins to form functional kinase complexes is comparatively inefficient. This has led to the suggestion that assembly might be a regulated step. In this report we demonstrate that the CDK inhibitors pZl'^^'', p27^^^, and p57^^^^ all promote the association of cdk4 with the D-type cyclins. This effect is specific and does not occur with other cdk inhibitors or cdk-binding proteins. Both in vivo and in vitro, the abundance of assembled cdk4/cyclin D complex increases directly with increasing inhibitor levels. The promotion of assembly is not attributable to a simple cell cycle block and requires the function of both the cdk and cyclin-binding domains. Kinetic studies demonstrate that p21 and p27 lead to a 35-and 80-fold increase in K^, respectively, mostly because of a decrease in X^ff. At low concentrations, p21 promotes the assembly of active kinase complexes, whereas at higher concentrations, it inhibits activity. Moreover, immunodepletion experiments demonstrate that most of the active cdk4-associated kinase activity also associates with p21. To confirm these results in a natural setting, we examine the assembly of endogenous complexes in mammary epithelial cells after release from a GQ arrest. In agreement with our other data, cyclin Dl and p21 bind concomitantly to cdk4 during the in vivo assembly of cdk4/cyclin Dl complexes. This complex assembly occurs in parallel to an increase in cyclin Dl-associated kinase activity. Immunodepletion experiments demonstrate that most of the cellular cyclin Dl-associated kinase activity is also p21 associated. Finally, we find that all three CIP/KIP inhibitors target cdk4 and cyclin Dl to the nucleus. We suggest that in addition to their roles as inhibitors, the p21 family of proteins, originally identified as inhibitors, may also have roles as adaptor proteins that assemble and program kinase complexes for specific functions.

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.

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 Akt(T308DS473D)(PKB(DD)), 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.

Nutrients and bioenergetics are prerequisites for proliferation and survival of mammalian cells. We present evidence that the cyclin-dependent kinase inhibitor p27(Kip1), 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 (p27(T198A)) 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.

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 G 1 /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 p27 Kip1 . The identification of p21 Waf1/Cip1 and p27 Kip1 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 G 2 /M transition and its constitutive activation may lead to defects in DNA damage checkpoint control.

Breast cancer is the second leading cause of cancer death in North American women. There is considerable need for reliable prognostic markers to assist clinicians in making management decisions. Although a variety of factors have been tested, only tumor stage, grade, size, hormone receptor status, and S-phase fraction are used on a routine basis. The cell cycle is governed by a family of cyclin-dependent kinases (cdks), which are regulated by associated cyclins and by phosphorylation. p27Kip1, a cyclin-dependent kinase inhibitor, regulates progression from G1 into S phase by binding and inhibiting cyclin/cdks. p27Kip1 protein levels and/or activity are upregulated by growth inhibitory cytokines including transforming growth factor-beta (TGF-beta) and, thus, provide an important link between extracellular regulators and the cell cycle. Loss of p27Kip1, a negative cell-cycle regulator, may contribute to oncogenesis and tumor progression. However, p27Kip1 mutations in human tumors are extremely rare. We have demonstrated by immunohistochemistry that p27Kip1 protein levels are reduced in primary breast cancers and that this is associated with tumor progression in both in situ and invasive lesions. This was confirmed by western analysis, reflected in increased G1/S-phase cyclin-dependent kinase activities and shown to be regulated posttranscriptionally by in situ hybridization. Furthermore, on multivariate analysis, low p27Kip1 is a predictor of reduced disease-free survival. This simple and reliable immunohistochemical assay may become a routine part of breast cancer evaluation and may influence patient management.

SUMMARY 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 non-coding transcripts and transposable elements, stimulates the pattern recognition receptor RIG-I to activate STAT1-dependent anti-viral signaling. In parallel, stromal cells also activate NOTCH3 on BrCa cells. The paracrine anti-viral 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 anti-viral/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 cross-talk with BrCa cells by utilizing exosomes to instigate anti-viral signaling. This expands BrCa subpopulations adept at resisting therapy and re-initiating tumor growth.

Recent decades have seen an unprecedented rise in obesity and the health impact thereof is increasingly evident. In 2014, worldwide more than 1.9 billion adults were overweight (BMI 25–29.9kg/m2) and of these over 600 million were obese (BMI≥30kg/m2). While the association between obesity and risk of diabetes and coronary artery disease is widely known, the impact of obesity on cancer incidence, morbidity and mortality is not fully appreciated. Obesity is associated with a higher risk of developing breast cancer, particularly in postmenopausal women, and is associated with worse disease outcome for women of all ages. The first part of this review summarizes the relationships between obesity and breast cancer development and outcome in pre and postmenopausal women and in hormone receptor positive and negative disease. The second part of this review addresses hypothesized molecular mechanistic insights that may underlie the effects of obesity to increase local and circulating pro-inflammatory cytokines that promote tumor angiogenesis, and stimulate the most malignant cancer stem cell population to drive cancer growth, invasion and metastasis. Finally, we review observational studies showing that increased physical activity associates with lower breast cancer risk and better outcome. We discuss effects of recent lifestyle interventions to decrease sex steroids, insulin/IGF-1 pathway activation and inflammatory biomarkers associated with worse breast cancer outcome in obesity. Although many observational studies show that exercise with weight loss associates with improved breast cancer outcome, further prospective studies are needed to determine if weight reduction will lead to improved patient outcome. It is hoped that several ongoing lifestyle intervention trials, reviewed herein, will support the systematic incorporation of weight loss intervention strategies into breast cancer patient care.