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.

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.

The effects of transforming growth factor ␤ (TGF-␤) were studied in closely related human mammary epithelial cells (HMEC), both finite-life-span 184 cells and immortal derivatives, 184A1 S , and 184A1L5 R , which differ in their cell cycle responses to TGF-␤ but express type I and type II TGF-␤ receptors and retain TGF-␤ induction of extracellular matrix. The arrest-resistant phenotype was not due to loss of cyclin-dependent kinase (cdk) inhibitors. TGF-␤ was shown to regulate p15INK4B expression at at least two levels: mRNA accumulation and protein stability. In TGF-␤-arrested HMEC, there was not only an increase in p15 mRNA but also a major increase in p15 INK4B protein stability. As cdk4-and cdk6-associated p15 INK4B increased during TGF-␤ arrest of sensitive cells, there was a loss of cyclin D1, p21 Cip1 , and p27Kip1 from these kinase complexes, and cyclin E-cdk2-associated p27Kip1 increased. In HMEC, p15 INK4B complexes did not contain detectable cyclin. p15 INK4Bfrom both sensitive and resistant cells could displace in vitro cyclin D1, p21 Cip1, and p27 Kip1 from cdk4 isolated from sensitive cells. Cyclin D1 could not be displaced from cdk4 in the resistant 184A1L5 R cell lysates. Thus, in TGF-␤ arrest, p15INK4B may displace already associated cyclin D1 from cdks and prevent new cyclin D1-cdk complexes from forming. Furthermore, p27Kip1 binding shifts from cdk4 to cyclin E-cdk2 during TGF-␤-mediated arrest. The importance of posttranslational regulation of p15INK4B by TGF-␤ is underlined by the observation that in TGF-␤-resistant 184A1L5 R , although the p15 transcript increased, p15INK4B protein was not stabilized and did not accumulate, and cyclin D1-cdk association and kinase activation were not inhibited.Cell cycle transitions are governed by a family of cyclindependent kinases (cdks), whose activity is regulated by association with positive effectors, the cyclins (41, 61), with negative regulators, the cdk inhibitors (62), and by phosphorylation (45,66). The cdks control a series of biochemical pathways, or checkpoints, which integrate extracellular mitogenic and growth-inhibitory signals, monitor chromosome integrity, and coordinate cell cycle transitions (23, 42). Passage through G 1 into S phase is regulated by the activities of cyclin D-, cyclin E-, and cyclin A-associated kinases. D-type cdks play a major role in phosphorylation of the retinoblastoma protein (pRb) (61), which is required for G 1 -to-S phase transition.Two different families of cdk-inhibitory proteins have been identified (for reviews see references 58 and 62). The kinase inhibitor protein (KIP) family is composed of three members, p21 (also identified as Cip1, Waf1, and Sdi1), p27 (Kip1), and p57 (Kip2). p21 (21, 73) is a transcriptional target of p53 (9) and plays a role in senescence (44) and differentiation (17,49,64) and in the coordination of DNA damage repair with cell cycle arrest (6,13,34,52,72). p27Kip1 was identified as an inhibitory activity in cells arrested by TGF-␤ (see below) (31,55,56,65) and was cloned independe...

Medicine is poised to undergo a digital transformation. High-throughput platforms are creating terabytes of genomic, transcriptomic, proteomic, and metabolomic data. The challenge is to interpret these data in a meaningful manner - to uncover relationships that are not readily apparent between molecular profiles and states of health or disease. This will require the development of novel data pipelines and computational tools. The combined analysis of multi-dimensional data is referred to as 'panomics'. The ultimate hope of integrative panomics is that it will lead to the discovery and application of novel markers and targeted therapeutics that drive forward a new era of 'precision medicine' where inter-individual variation is accounted for in the treatment of patients.

p27Kip1 is an important effector of G1 arrest by transforming growth factor β (TGF-β). Investigations in a human mammary epithelial cell (HMEC) model, including cells that are sensitive (184S) and resistant (184A1L5R) to G1 arrest by TGF-β, revealed aberrant p27 regulation in the resistant cells. Cyclin E1-cyclin-dependent kinase 2 (cdk2) and cyclin A-cdk2 activities were increased, and p27-associated kinase activity was detected in 184A1L5R cells. p27 from 184A1L5R cells was localized to both nucleus and cytoplasm, showed an altered profile of phosphoisoforms, and had a reduced ability to bind and inhibit cyclin E1-cdk2 in vitro when compared to p27 from the sensitive 184S cells. In proliferating 184A1L5R cells, more p27 was associated with cyclin D1-cdk4 complexes than in 184S. While TGF-β inhibited the formation of cyclin D1-cdk4-p27 complexes in 184S cells, it did not inhibit the assembly of cyclin D1-cdk4-p27 complexes in the resistant 184A1L5R cells. p27 phosphorylation changed during cell cycle progression, with cyclin E1-bound p27 in G0 showing a different phosphorylation pattern from that of cyclin D1-bound p27 in mid-G1. These data suggest a model in which TGF-β modulates p27 phosphorylation from its cyclin D1-bound assembly phosphoform to an alternate form that binds tightly to inhibit cyclin E1-cdk2. Altered phosphorylation of p27 in the resistant 184A1L5R cells may favor the binding of p27 to cyclin D1-cdk4 and prevent its accumulation in cyclin E1-cdk2 in response to TGF-β.

In breast cancer, there is a significant degree of molecular diversity among tumors. Multiple perturbations in signal transduction pathways impinge on transcriptional networks that in turn dictate malignant transformation and metastatic progression. Detailed knowledge of the sequence-specific transcription factors that become activated or repressed within a tumor and comparison of their relative levels of expression in cancer versus normal tissue should therefore provide insight into disease mechanisms, improving patient stratification and facilitating personalized treatment. While high-throughput tandem mass spectrometry methods for global proteome profiling have been developed, existing approaches have limited sensitivity and are often unable to detect low-abundance transcription factors in a complex biological specimen like a biopsy or tumor cell extract. To this end, we have undertaken a systematic comparative evaluation of three MS/MS methods for the ability to detect reference transcription factors spiked in known amounts into a cell-free breast cancer nuclear extract: Data-Dependent Acquisition (DDA), wherein precursor ion intensity dictates selection for fragmentation; Targeted Peptide Monitoring (TPM), a directed approach using successive isolation and fragmentation of predefined m/ z ratios; and Multiple Reaction Monitoring (MRM), in which specific precursor ion to product ion transitions are selectively monitored. Through a series of controlled, parallel benchmarking experiments, we have determined the relative figures-of-merit of each approach, and have established that prior knowledge of signature proteotypic peptides markedly improves overall detection sensitivity, reliability, and quantification.

Protein expression becomes altered in breast epithelium during malignant transformation. Knowledge of these perturbations should provide insight into the molecular basis of breast cancer, as well as reveal possible new therapeutic targets. To this end, we have performed an extensive comparative proteomic survey of global protein expression patterns in proliferating MCF-7 breast cancer cells and normal human mammary epithelial cells using gel-free shotgun tandem mass spectrometry. Pathophysiological alterations associated with the malignant breast cancer phenotype were detected, including differences in the apparent levels of key regulators of the cell cycle, signal transduction, apoptosis, transcriptional regulation, and cell metabolism.

Replicative senescence may be an important tumor suppressive mechanism for human cells. We investigated the mechanism of cell cycle arrest at senescence in human mammary epithelial cells (HMECs) that have undergone a period of`self-selection', and as a consequence exhibit diminished p16 INK4A levels. As HMECs approached senescence, the proportion of cells with a 2N DNA content increased and that in S phase decreased progressively. Cyclin D1-cdk4, cyclin E-cdk2 and cyclin A-cdk2 activities were not abruptly inhibited, but rather diminished steadily with increasing population age. In contrast to observations in ®broblast, p21Cip1 was not increased at senescence in HMECs. There was no increase in p27Kip1 levels nor in KIP association with targets cdks. While p15 INK4B and its binding to both cdk4 and cdk6 increased with increasing passage, some cyclin D1-bound cdk4 and cdk6 persisted in senescent cells, whose inhibition could not be attributed to p15 INK4B. The inhibition of cyclin E-cdk2 in senescent HMECs was accompanied by increased inhibitory phosphorylation of cdk2, in association with a progressive loss of Cdc25A. Recombinant Cdc25A strongly reactivated cyclin E-cdk2 from senescent HMECs suggesting that reduction of Cdc25A contributes to cyclin E-cdk2 inhibition and G1 arrest at senescence. Although ectopic expression of Cdc25A failed to extend the lifespan of HMECs, the exogenous Cdc25A appeared to lack activity in these cells, since it neither shortened the G1-to-S phase interval nor activated cyclin E-cdk2. In contrast, in the breast cancer-derived MCF-7 line, Cdc25A overexpression increased both cyclin E-cdk2 activity and the S phase fraction. Thus, mechanisms leading to HMEC immortalization may involve not only the re-induction of Cdc25A expression, but also activation of this phosphatase. Oncogene (2000) 19, 5314 ± 5323.