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.

Decreased levels of the cell-cycle inhibitor p27Kip1 protein: prognostic implications in primary breast cancer.

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.

E-Cadherin-dependent growth suppression is mediated by the cyclin-dependent kinase inhibitor p27(KIP1).

Recent studies have demonstrated the importance of E-cadherin, a homophilic cell-cell adhesion molecule, in contact inhibition of growth of normal epithelial cells. Many tumor cells also maintain strong intercellular adhesion, and are growth-inhibited by cell- cell contact, especially when grown in three-dimensional culture. To determine if E-cadherin could mediate contact-dependent growth inhibition of nonadherent EMT/6 mouse mammary carcinoma cells that lack E-cadherin, we transfected these cells with an exogenous E-cadherin expression vector. E-cadherin expression in EMT/6 cells resulted in tighter adhesion of multicellular spheroids and a reduced proliferative fraction in three-dimensional culture. In addition to increased cell-cell adhesion, E-cadherin expression also resulted in dephosphorylation of the retinoblastoma protein, an increase in the level of the cyclin-dependent kinase inhibitor p27(kip1) and a late reduction in cyclin D1 protein. Tightly adherent spheroids also showed increased levels of p27 bound to the cyclin E-cdk2 complex, and a reduction in cyclin E-cdk2 activity. Exposure to E-cadherin-neutralizing antibodies in three-dimensional culture simultaneously prevented adhesion and stimulated proliferation of E-cadherin transfectants as well as a panel of human colon, breast, and lung carcinoma cell lines that express functional E-cadherin. To test the importance of p27 in E-cadherin-dependent growth inhibition, we engineered E-cadherin-positive cells to express inducible p27. By forcing expression of p27 levels similar to those observed in aggregated cells, the stimulatory effect of E-cadherin-neutralizing antibodies on proliferation could be inhibited. This study demonstrates that E-cadherin, classically described as an invasion suppressor, is also a major growth suppressor, and its ability to inhibit proliferation involves upregulation of the cyclin-dependent kinase inhibitor p27.

A novel inhibitor of cyclin-Cdk activity detected in transforming growth factor beta-arrested epithelial cells.

Transforming growth factor beta (TGF-beta) is a potent inhibitor of epithelial cell growth. Cyclins E and A in association with Cdk2 have been shown to play a role in the G1-to-S phase transition in mammalian cells. We have studied the effects of TGF-beta-mediated growth arrest on G1/S cyclins E and A. Inhibition of cyclin A-associated kinase by TGF-beta is primarily due to a decrease in cyclin A mRNA and protein. By contrast, while TGF-beta inhibits accumulation of cyclin E mRNA, the reduction in cyclin E protein is minimal. Instead, we find that the activation of cyclin E-associated kinase that normally accompanies the G1-to-S phase transition is inhibited. A novel inhibitor of cyclin-Cdk complexes was detected in TGF-beta-treated cell lysates. Inhibition is mediated by a heat-stable protein that targets both Cdk2 and Cdc2 kinases. In G0-arrested cells, a similar inhibitor of Cdk2 kinase was detected. These data suggest the existence of an inhibitor of cyclin-dependent kinases induced under different conditions to mediate antiproliferative responses.

Transforming growth factor beta stabilizes p15INK4B protein, increases p15INK4B-cdk4 complexes, and inhibits cyclin D1-cdk4 association in human mammary epithelial cells.

The effects of transforming growth factor beta (TGF-beta) were studied in closely related human mammary epithelial cells (HMEC), both finite-life-span 184 cells and immortal derivatives, 184A1S, and 184A1L5R, which differ in their cell cycle responses to TGF-beta but express type I and type II TGF-beta receptors and retain TGF-beta induction of extracellular matrix. The arrest-resistant phenotype was not due to loss of cyclin-dependent kinase (cdk) inhibitors. TGF-beta was shown to regulate p15INK4B expression at at least two levels: mRNA accumulation and protein stability. In TGF-beta-arrested HMEC, there was not only an increase in p15 mRNA but also a major increase in p5INK4B protein stability. As cdk4- and cdk6-associated p15INK4B increased during TGF-beta arrest of sensitive cells, there was a loss of cyclin D1, p21Cip1, and p27Kip1 from these kinase complexes, and cyclin E-cdk2-associated p27Kip1 increased. In HMEC, p15INK4B complexes did not contain detectable cyclin. p15INK4B from both sensitive and resistant cells could displace in vitro cyclin D1, p21Cip1, and p27Kip1 from cdk4 isolated from sensitive cells. Cyclin D1 could not be displaced from cdk4 in the resistant 184A1L5R cell lysates. Thus, in TGF-beta 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-beta-mediated arrest. The importance of posttranslational regulation of p15INK4B by TGF-beta is underlined by the observation that in TGF-beta-resistant 184A1L5R, 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.

VEGFA links self-renewal and metastasis by inducing Sox2 to repress miR-452, driving Slug.

Cancer stem cells (CSC) appear to have increased metastatic potential, but mechanisms underlying this are poorly defined. Here we show that VEGFA induction of Sox2 promotes EMT and tumor metastasis. In breast lines and primary cancer culture, VEGFA rapidly upregulates SOX2 expression, leading to SNAI2 induction, EMT, increased invasion and metastasis. We show Sox2 downregulates miR-452, which acts as a novel metastasis suppressor to directly target the SNAI2 3'-untranslated region (3'-UTR). VEGFA stimulates Sox2- and Slug-dependent cell invasion. VEGFA increases lung metastasis in vivo, and this is abrogated by miR-452 overexpression. Furthermore, SNAI2 transduction rescues metastasis suppression by miR-452. Thus, in addition to its angiogenic action, VEGFA upregulates Sox2 to drive stem cell expansion, together with miR-452 loss and Slug upregulation, providing a novel mechanism whereby cancer stem cells acquire metastatic potential. Prior work showed EMT transcription factor overexpression upregulates CSC. Present work indicates that stemness and metastasis are a two-way street: Sox2, a major mediator of CSC self-renewal, also governs the metastatic process.

Targeting of RAGE-ligand signaling impairs breast cancer cell invasion and metastasis.

The receptor for advanced glycation end products (RAGE) is highly expressed in various cancers and is correlated with poorer outcome in breast and other cancers. Here we tested the role of targeting RAGE by multiple approaches in the tumor and tumor microenvironment, to inhibit the metastatic process. We first tested how RAGE impacts tumor cell-intrinsic mechanisms using either RAGE overexpression or knockdown with short hairpin RNAs (shRNAs). RAGE ectopic overexpression in breast cancer cells increased MEK-EMT (MEK-epithelial-to-mesenchymal transition) signaling, transwell invasion and soft agar colony formation, and in vivo promoted lung metastasis independent of tumor growth. RAGE knockdown with multiple independent shRNAs in breast cancer cells led to decreased transwell invasion and soft agar colony formation, without affecting proliferation. In vivo, targeting RAGE shRNA knockdown in human and mouse breast cancer cells, decreased orthotopic tumor growth, reduced tumor angiogenesis and recruitment of inflammatory cells, and markedly decreased metastasis to the lung and liver in multiple xenograft and syngeneic mouse models. To test the non-tumor cell microenvironment role of RAGE, we performed syngeneic studies with orthotopically injected breast cancer cells in wild-type and RAGE-knockout C57BL6 mice. RAGE-knockout mice displayed striking impairment of tumor cell growth compared with wild-type mice, along with decreased mitogen-activated protein kinase signaling, tumor angiogenesis and inflammatory cell recruitment. To test the combined inhibition of RAGE in both tumor cell-intrinsic and non-tumor cells of the microenvironment, we performed in vivo treatment of xenografted tumors with FPS-ZM1 (1 mg/kg, two times per week). Compared with vehicle, FPS-ZM1 inhibited primary tumor growth, inhibited tumor angiogenesis and inflammatory cell recruitment and, most importantly, prevented metastasis to the lung and liver. These data demonstrate that RAGE drives tumor progression and metastasis through distinct tumor cell-intrinsic and -extrinsic mechanisms, and may represent a novel and therapeutically viable approach for treating metastatic cancers.

Telomerase activity in prostate cancer, prostatic intraepithelial neoplasia, and benign prostatic epithelium.

Telomerase is a ribonucleoprotein that synthesizes telomeric DNA on chromosomal ends. Telomerase activation has been seen in many immortal cell lines and cancers. Telomerase activity was analyzed in prostate carcinoma; in coexistent prostatic intraepithelial neoplasia (PIN), benign prostatic hyperplasia (BPH), atrophy and normal tissue; and in benign prostate glands. Telomerase activity was detected in 80 of 87 (92%) prostate cancers. Forty-one matched samples (from a total of 32 cases) were available for comparative analysis. The presence of telomerase activity in adjacent PIN, BPH, and normal tissue was correlated with telomerase activity in the malignant epithelium. In these adjacent tissues, telomerase activity was found in 11 of 15 (73%) PINs, 13 of 26 (50%) BPHs, and 1 of 6 (16%) atrophy and 4 of 11 (36%) normal tissues. In contrast to the BPH tissue from cancer-bearing glands, all 16 BPH specimens from patients only diagnosed with BPH were telomerase activity negative. In cancer samples, there was no correlation between telomerase activity and Gleason grade or preoperation prostate-specific antigen level. Our data indicate that telomerase activity is present in most prostate cancers. The high rate of telomerase activity in the benign-appearing areas of these glands may be attributed either to the presence of occult cancer cells or to early molecular alterations of cancer that were histologically inapparent.

Reduced expression of the cell cycle inhibitor p27Kip1 in non-small cell lung carcinoma: a prognostic factor independent of Ras.

Levels of p27 have been found to have independent prognostic significance in a variety of tumors including breast, colon, prostate, ovary, and gastric carcinomas. We investigated p27 levels and determined ras mutational status in 136 non-small cell lung cancers. We found reduced levels of p27 in 86% of cases and showed a statistically significant inverse correlation between p27 levels and tumor grade. ras mutations were found exclusively in adenocarcinomas and showed no relationship to p27 levels. Clinical data on a subset of the patients studied indicated that all 16 patients who died of disease and 21 of 22 patients who relapsed had low p27 levels, whereas all patients with high p27 levels were alive at last follow up. These findings suggest that alteration in p27 levels plays an important role in lung tumor progression and that p27 levels may have independent prognostic significance in non-small cell lung cancer.

Loss of cyclin-dependent kinase inhibitor p27Kip1 is a novel prognostic factor in localized human prostate adenocarcinoma.

p27Kip1 is a cyclin-dependent kinase inhibitor that negatively regulates cell proliferation by mediating cell cycle arrest in G1. This study was undertaken to assess the prognostic value of p27Kip1 in localized human prostate cancer. Archival material from 113 radical prostatectomy specimens obtained between 1985 and 1993 was stained immunohistochemically for p27Kip1 protein using a commercially available antibody. Patient charts were reviewed for preoperative serum prostate-specific antigen, clinical and pathological staging, Gleason tumor grade, time to biochemical and clinical recurrence, and survival. Strong p27Kip1 staining was uniformly seen in benign prostatic epithelial components in all tumor sections. p27Kip1 staining was reduced in most prostate cancers and was variable in prostatic intraepithelial neoplasia. Decreased p27Kip1 staining (<25% of nuclei stained positive for p27Kip1) correlated with seminal vesicle involvement (P = 0.0032) and with higher Gleason grade (P = 0.0114). On univariate analysis, low p27Kip1 predicted an increased risk of treatment failure in the node-negative cohort (P = 0.0037) and in the subset who did not receive neoadjuvant hormonal therapy (P = 0.049). Low p27Kip1 expression was an independent predictor of treatment failure on multivariate analysis of lymph node negative prostate cancers following radical retropubic prostatectomy (n = 102; P = 0.047). Seminal vesicle involvement (P = 0.034) and positive surgical margins (P = 0.047) were also independent prognostic factors for disease recurrence. In patients who received preoperative neoadjuvant hormonal therapy, low p27Kip1 in the pathological specimen was an even stronger predictor of outcome than it was in the entire group (n = 23, P = 0.015).

TGF-beta mediated G1 arrest in a human melanoma cell line lacking p15INK4B: evidence for cooperation between p21Cip1/WAF1 and p27Kip1.

We have studied TGF-beta mediated G1 arrest in WM35, an early stage human melanoma cell line. These cells have lost p15INK4B expression through loss of one chromosome 9 and rearrangement of the other. In asynchronously growing WM35, TGF-beta caused reductions in cyclin D1, cyclin A and cdk4 proteins and their associated kinase activities and an increase in both p21Cip1/WAF1 and p27Kip1. These findings were confirmed in cells released from quiescence in the presence of TGF-beta, in which TGF-beta inhibited or delayed the reduction in the cdk inhibitors that normally occurs in late G1. In contrast to observations in other cell types, there was an increased association of both p21Cip1/WAF1 and p27Kip1 with cyclin D1/cdk4 and with cyclin E/cdk2 during TGF-beta mediated arrest of asynchronously growing cells. Upregulation of p21Cip1/WAF1 preceded that of p27Kip1. Furthermore, p21Cip1/WAF1 and p27Kip1 were not present in the same cdk complexes but bound distinct populations of target cdk molecules. Both p21Cip1/WAF1 and p27Kip1 immunoprecipitates from asynchronously growing cells contained active kinase complexes. These KIP-associated kinase activities were reduced in TGF-beta arrested cells. It has been proposed that in TGF-beta arrested epithelial cells, up-regulation of p15INK4B and of p15INK4B binding to cdk4 serves to destabilize the association of p27Kip1 with cyclin D1/cdk4, promoting p27Kip1 binding and inhibition of cyclin E/cdk2. Our findings demonstrate that this is not a universal mechanism of G1 arrest by TGF-beta. In TGF-beta arrested WM35, which lack p15INK4B, the increased p21Cip1/WAF1 may serve a similar function to that of p15INK4B: initiating kinase inhibition and providing an additional mechanism to supplement the effect of p27Kip1 on G1 cyclin/cdks.

Germline p16INK4A mutation and protein dysfunction in a family with inherited melanoma.

The gene encoding the cell cycle inhibitor p16INK4A (also known as p16, MTS1, CDKN2 and INK4) has been mapped to human chromosome band 9p21, a region that also contains a putative melanoma susceptibility gene. Although germline mutations in the coding region of the p16INK4A gene have been detected in some families with inherited melanoma, many other families show no evidence of such mutations and hence the role of p16INK4A in the development of this tumor is still unclear. In this report, we describe a family with inherited melanoma in which a novel mutation in exon 2 of the p16INK4A gene segregates with the disease. The mutant gene encodes a protein with an in-frame deletion of two amino acids (Asp96 and Leu97). We show that the mutant protein is functionally abnormal: it is unable to bind cdk4 in vitro and does not inhibit colony formation in tertiary passage rat embryo fibroblasts. Moreover, in a metastatic lesion from one patient the wild type p16INK4A allele was deleted and the mutant allele retained. We conclude that family members carrying this germline mutation in the p16INK4A gene are predisposed to melanoma. By extension, these findings implicate the p16INK4A gene in the development of some cases of familial melanoma.

UVB radiation induces p21Cip1/WAF1 and mediates G1 and S phase checkpoints.

In a search for effectors and targets of UVB signaling in mammalian cells, we screened a keratinocyte cDNA library with differentially subtracted UVB-enriched cDNA probes. One of the UVB induced cDNA clones proved to be the rat p21Cip1/WAF1 homologue. UVB irradiation caused a rise in p53 protein levels, in association with induction of p21Cip1/WAF1 and cyclin G expression. The effects of UVB irradiation induced p21Cip1/WAF1 on the cell cycle were examined. In contrast to gamma irradiation, which caused G2 arrest, UVB treatment of asynchronous neonatal rat keratinocytes (NK) led to a marked inhibition of replicative DNA synthesis and prolonged G1 and S phase arrests, persisting to 18-24 h, with recovery of cycling by 36 h post-UVB. G1 arrest was accompanied by inhibition of cyclin D-, E- and A-associated kinases. Kinase inhibition was not due to reduction in cyclin or cdk proteins. While the association of cyclin E with Cdk2 was moderately reduced, cyclin D1/Cdk4 and cyclin A/Cdk2 complexes were not disrupted. The activating threonine 160 phosphorylation of Cdk2 in cyclin complexes was not inhibited. An incremental binding of p21 with Cdk4 paralleled the inhibition of cyclin D1/Cdk4 kinase and a similar rise in Cdk2 binding to p21 was associated with inhibition of cyclin E and cyclin A dependent kinases. Furthermore, a rise in measurable p21Cip1/WAF1-Cdk2 inhibitory activity paralleled the loss of G1 cyclin-dependent kinase activity, supporting a role for p21Cip1/WAF1 in the UVB-induced checkpoints.

Interleukin-6 dependent induction of the cyclin dependent kinase inhibitor p21WAF1/CIP1 is lost during progression of human malignant melanoma.

Human melanoma cell lines derived from early stage primary tumors are particularly sensitive to growth arrest induced by interleukin-6 (IL-6). This response is lost in cell lines derived from advanced lesions, a phenomenon which may contribute to tumor aggressiveness. We sought to determine whether resistance to growth inhibition by IL-6 can be explained by oncogenic alterations in cell cycle regulators or relevant components of intracellular signaling. Our results show that IL-6 treatment of early stage melanoma cell lines caused G1 arrest, which could not be explained by changes in levels of G1 cyclins (D1, E), cdks (cdk4, cdk2) or by loss of cyclin/cdk complex formation. Instead, IL-6 caused a marked induction of the cdk inhibitor p21WAF1/CIP1 in three different IL-6 sensitive cell lines, two of which also showed a marked accumulation of the cdk inhibitor p27Kip1. In contrast, IL-6 failed to induce p21WAF1/CIP1 transcript and did not increase p21WAF1/CIP1 or p27kip1 proteins in any of the resistant lines. In fact, of five IL-6 resistant cell lines, only two expressed detectable levels of p21WAF1/CIP1 mRNA and protein, while in three other lines, p21WAF1/CIP1 was undetectable. IL-6 dependent upregulation of p21WAF1/CIP1 was associated with binding of both STAT3 and STAT1 to the p21WAF1/CIP1 promoter. Surprisingly, however, IL-6 stimulated STAT binding to this promoter in both sensitive and resistant cell lines (with one exception), suggesting that gross deregulation of this event is not the unifying cause of the defect in p21WAF1/CIP1 induction in IL-6 resistant cells. In somatic cell hybrids of IL-6 sensitive and resistant cell lines, the resistant phenotype was dominant and IL-6 failed to induce p21WAF1/CIP1. Thus, our results suggest that in early stage human melanoma cells, IL-6 induced growth inhibition involves induction of p21WAF1/CIP1 which is lost in the course of tumor progression presumably as a result of a dominant oncogenic event.

Cyclin E2: a novel CDK2 partner in the late G1 and S phases of the mammalian cell cycle.

We report here the cloning and characterization of human and mouse cyclin E2, which define a new subfamily within the vertebrate E-type cyclins, while all previously identified family-members belong to the cyclin El subfamily. Cyclin E2/CKD2 and cyclin E/CDK2 complexes phosphorylate histone H1 in vitro with similar specific activities and both are inhibited by p27Kip1. Cyclin E2 mRNA levels in human cells oscillate throughout the cell cycle and peak at the G1/S boundary, in parallel with the cyclin E mRNA. In cells, cyclin E2 is complexed with CDK2, p27 and p21. Like cyclin E, cyclin E2 is an unstable protein in vivo and is stabilized by proteasome inhibitors. Cyclin E2-associated kinase activity rises in late G1 and peaks very close to cyclin E activity. In two malignantly transformed cell lines, cyclin E2 activity is sustained throughout S phase, while cyclin E activity has already declined and cyclin A activity is only beginning to rise. We speculate that cyclin E2 is not simply redundant with cyclin E, but may regulate distinct rate-limiting pathway(s) in G1-S control.

VEGF drives cancer-initiating stem cells through VEGFR-2/Stat3 signaling to upregulate Myc and Sox2.

Vascular endothelial growth factor-A (VEGF), a potent angiogenic factor, is also implicated in self-renewal in several normal tissue types. VEGF has been shown to drive malignant stem cells but mechanisms thereof and tumor types affected are not fully characterized. Here, we show VEGF promotes breast and lung cancer stem cell (CSC) self-renewal via VEGF receptor-2 (VEGFR-2)/STAT3-mediated upregulation of Myc and Sox2. VEGF increased tumor spheres and aldehyde dehydrogenase activity, both proxies for stem cell function in vitro, in triple-negative breast cancer (TNBC) lines and dissociated primary cancers, and in lung cancer lines. VEGF exposure before injection increased breast cancer-initiating cell abundance in vivo yielding increased orthotopic tumors, and increased metastasis from orthotopic primaries and following tail vein injection without further VEGF treatment. VEGF rapidly stimulated VEGFR-2/JAK2/STAT3 binding and activated STAT3 to bind MYC and SOX2 promoters and induce their expression. VEGFR-2 knockdown or inhibition abrogated VEGF-mediated STAT3 activation, MYC and SOX2 induction and sphere formation. Notably, knockdown of either STAT3, MYC or SOX2 impaired VEGF-upregulation of pSTAT3, MYC and SOX2 expression and sphere formation. Each transcription factor, once upregulated, appears to promote sustained activation of the others, creating a feed-forward loop to drive self-renewal. Thus, in addition to angiogenic effects, VEGF promotes tumor-initiating cell self-renewal through VEGFR-2/STAT3 signaling. Analysis of primary breast and lung cancers (>1300 each) showed high VEGF expression, was prognostic of poor outcome and strongly associated with STAT3 and MYC expression, supporting the link between VEGF and CSC self-renewal. High-VEGF tumors may be most likely to escape anti-angiogenics by upregulating VEGF, driving CSC self-renewal to re-populate post-treatment. Our work highlights the need to better define VEGF-driven cancer subsets and supports further investigation of combined therapeutic blockade of VEGF or VEGFR-2 and JAK2/STAT3.

ERα, SKP2 and E2F-1 form a feed forward loop driving late ERα targets and G1 cell cycle progression.

Estrogen triggers transactivation coupled estrogen receptor α (ERα) proteolysis, but mechanisms thereof remain obscure. Present data link estrogen:ERα-driven transcription with cell cycle progression. Although liganded ERα induces many genes within 1-4 h, gene activation after 6 h is thought to be indirect. Here, we identify SKP2 as a late-acting coactivator that drives ERα targets to promote G1-to-S progression. Data support a model in which estrogen-activated cyclin E-CDK2 binds and phosphorylates ERαS341, to prime ERα-SCF(SKP2) binding via SKP2-L248QTLL252 in late G1. SKP2 activates ERα ubiquitylation and proteolysis. Putative late ERα targets were identified by expression profiling. SKP2 knockdown attenuated E2F-1 and BLM induction. SKP2 overexpression, but not coactivator motif mutant SKP2-L248QTAA252, enhanced estrogen-induced E2F-1 and BLM expression. SKP2 knockdown impaired estrogen-stimulated ERα, SKP2, SRC3 and RNA polymerase II recruitment to E2F-1 and BLM promoters. This work not only identifies these late-activated genes as bona fide ERα targets but describes a novel mechanism for their periodic activation. SKP2 serves as dual ERα E3 ligase/coactivator for late-activated target genes, revealing a novel mechanism whereby ERα/SCF(SKP2) transactivation of E2F-1 feeds forward to drive G1-to-S.

Promotion of cytoplasmic mislocalization of p27 by Helicobacter pylori in gastric cancer.

The cyclin-dependent kinase (CDK) inhibitor p27 has an important role in cell cycle regulation. Reduced expression of p27 is commonly associated with poor prognosis in many malignancies, including gastric cancer. Cytoplasmic p27 mislocalization may be an additional indicator of high-grade tumors and poor prognosis in cancer. As chronic infection by Helicobacter pylori is the most important risk factor for gastric cancer development, we evaluated the effects of H. pylori on p27 expression and localization in gastric cancer cells. Co-culture of gastric cells with H. pylori induced cytoplasmic p27 expression and reduced nuclear p27 expression in vitro. Cytoplasmic p27 expression was associated with and dependent upon phosphorylation of p27 at T157 and T198: wild-type p27 accumulated in the cytoplasm, but non-phosphorylatable mutants affecting T157 or T198 were nuclear in H. pylori-infected cells. These post-translational p27 changes were secondary to activation of cellular phosphoinositide-3 kinase (PI3K) and AKT signaling pathways, and dependent upon a functional H. pylori cag pathogenicity island. We investigated the clinical significance of cytoplasmic p27 mislocalization in 164 cases of resected gastric cancer in tissue microarrays. In 97 cases (59%), cytoplasmic p27 mislocalization was observed, and this was associated with increased mortality in multivariate analysis. These results show that H. pylori infection induces AKT/PI3K-mediated phosphorylation of p27 at T157 and T198 to cause cytoplasmic p27 mislocalization in gastric cancer, and that p27 mislocalization is an adverse prognostic feature in gastric cancer. This is the first demonstration of the translocation of a specific bacterial virulence factor that post-translationally regulates a host cell CDK inhibitor. This is of particular significance, because p27 has both tumor-suppressive and oncogenic activities, depending upon its subcellular localization. Cytoplasmic mislocalization of p27 induced by H. pylori may be an important mechanistic link between H. pylori infection and gastric carcinogenesis.

Transforming activity of mutant human p53 alleles.

Mutant forms of the p53 gene have been shown to cooperate with an activated ras gene in transforming primary cells in culture. The aberrant proteins encoded by p53 mutants are thought to act in a dominant negative manner in these assays. In vivo data, however, reveal that where p53 has undergone genetic change in tumors, both alleles have been affected. We previously identified a case of human acute myelogenous leukemia (AML) in which both alleles of the p53 gene had undergone independent missense mutations (at codons 135 cys to ser and 246 met to val). In these blasts, p53 mutations appear to be acting recessively. We have assayed the transforming potential of these p53 mutations, as well as that of another mutation at codon 273, also identified in a human neoplasm. Both mutations from the AML blasts (codon 135 and codon 246) confer transforming ability on the mutant protein. While transformation assays may define functionally different subsets of p53 mutations, the overexpression phenotype of mutants in this assay may not accurately reflect the pathological effects of p53 mutations in vivo.

PTEN deficiency: a role in mammary carcinogenesis.

The PTEN gene is often mutated in primary human tumors and cell lines, but the low rate of somatic PTEN mutation in human breast cancer has led to debate over the role of this tumor suppressor in this disease. The involvement of PTEN in human mammary oncogenesis has been implicated from studies showing that germline PTEN mutation in Cowden disease predisposes to breast cancer, the frequent loss of heterozygosity at the PTEN locus, and reduced PTEN protein levels in sporadic breast cancers. To assay the potential contribution of PTEN loss in breast tumor promotion, Li et al. [1] crossed Pten heterozygous mice with mouse mammary tumor virus-Wnt-1 transgenic (Wnt-1 TG, Pten+/-) mice. Mammary ductal carcinoma developed earlier in Wnt-1 TG, Pten+/- mice than in mice bearing either genetic change alone, and showed frequent loss of the remaining wild-type PTEN allele. These data indicate a role for PTEN in breast tumorigenesis in an in vivo model.