Low-molecular-weight cyclin E: the missing link between biology and clinical outcome

Cyclin E, a key mediator of transition during the G(1)/S cellular division phase, is deregulated in a wide variety of human cancers. Our group recently reported that overexpression and generation of low-molecular-weight (LMW) isoforms of cyclin E were associated with poor clinical outcome among breast cancer patients. However, the link between LMW cyclin E biology in mediating a tumorigenic phenotype and clinical outcome is unknown. To address this gap in knowledge, we assessed the role of LMW isoforms in breast cancer cells; we found that these forms of cyclin E induced genomic instability and resistance to p21, p27, and antiestrogens in breast cancer. These findings suggest that high levels of LMW isoforms of cyclin E not only can predict failure to endocrine therapy but also are true prognostic indicators because of their influence on cell proliferation and genetic instability

Parallel cyclin E and cyclin A expression in neoplastic lesions of the uterine cervix

Cyclin E levels are high during late G1 and early S-phase in normal cells. The cyclin E expression over the cell cycle in tumours is not fully known. The impact on patient outcome by high cyclin E levels during other parts of the cell cycle than late G1- and early S-phase is unknown. We set out to study the expression of cyclin E over the cell cycle in cervical carcinomas. Using immunofluorescence staining of cyclin A, digital microscopy, and digital image analysis, we determined which cells in a tissue section that were in S- or G2-phase. M-phase cells were detected by morphology. By simultaneously staining for cyclin E, we investigated the variation in cyclin E levels over the cell cycle in cervical carcinoma lesions. In a case–control study, in which each deceased patient was matched with a patient still alive and well after >5 years of follow-up, we found that the deceased patients had a considerably higher fraction of cyclin A-positive cells staining for cyclin E than the survivors (n=36). We conclude that parallel cyclin E and cyclin A expression is an indicator for poor outcome in cervical carcinomas. In addition, we investigated the expression pattern of cyclin E and cyclin A in consecutive biopsy samples from cervical carcinomas at different stages, as well as in human papillomavirus positive or negative adenocarcinomas in order to further study the cyclin E and cyclin A expression pattern in neoplastic lesions of the uterine cervix

siRNA-Based Targeting of Cyclin E Overexpression Inhibits Breast Cancer Cell Growth and Suppresses Tumor Development in Breast Cancer Mouse Model

Cyclin E is aberrantly expressed in many types of cancer including breast cancer. High levels of the full length as well as the low molecular weight isoforms of cyclin E are associated with poor prognosis of breast cancer patients. Notably, cyclin E overexpression is also correlated with triple-negative basal-like breast cancers, which lack specific therapeutic targets. In this study, we used siRNA to target cyclin E overexpression and assessed its ability to suppress breast cancer growth in nude mice. Our results revealed that cyclin E siRNA could effectively inhibit overexpression of both full length and low molecular weight isoforms of cyclin E. We found that depletion of cyclin E promoted apoptosis of cyclin E-overexpressing cells and blocked their proliferation and transformation phenotypes. Significantly, we further demonstrated that administration of cyclin E siRNA could inhibit breast tumor growth in nude mice. In addition, we found that cyclin E siRNA synergistically enhanced the cell killing effects of doxorubicin in cell culture and this combination greatly suppressed the tumor growth in mice. In conclusion, our results indicate that cyclin E, which is overexpressed in 30% of breast cancer, may serve as a novel and effective therapeutic target. More importantly, our study clearly demonstrates a very promising therapeutic potential of cyclin E siRNA for treating the cyclin E-overexpressing breast cancers, including the very malignant triple-negative breast cancers

High Distribution of CD40 and TRAF2 in Th40 T Cell Rafts Leads to Preferential Survival of this Auto-Aggressive Population in Autoimmunity

CD40-CD154 interactions have proven critical in autoimmunity, with the identification of CD4(lo)CD40(+) T cells (Th40 cells) as harboring an autoaggressive T cell population shedding new insights into those disease processes. Th40 cells are present at contained levels in non-autoimmune individuals but are significantly expanded in autoimmunity. Th40 cells are necessary and sufficient in transferring type 1 diabetes in mouse models. However, little is known about CD40 signaling in T cells and whether there are differences in that signaling and subsequent outcome depending on disease conditions. When CD40 is engaged, CD40 and TNF-receptor associated factors, TRAFs, become associated with lipid raft microdomains. Dysregulation of T cell homeostasis is emerging as a major contributor to autoimmune disease and thwarted apoptosis is key in breaking homeostasis.Cells were sorted into CD4(hi) and CD4(lo) (Th40 cells) then treated and assayed either as whole or fractionated cell lysates. Protein expression was assayed by western blot and Nf-kappaB DNA-binding activity by electrophoretic mobility shifts. We demonstrate here that autoimmune NOD Th40 cells have drastically exaggerated expression of CD40 on a per-cell-basis compared to non-autoimmune BALB/c. Immediately ex-vivo, untreated Th40 cells from NOD mice have high levels of CD40 and TRAF2 associated with the raft microdomain while Th40 cells from NOR and BALB/c mice do not. CD40 engagement of Th40 cells induces Nf-kappaB DNA-binding activity and anti-apoptotic Bcl-X(L) expression in all three mouse strains. However, only in NOD Th40 cells is anti-apoptotic cFLIP(p43) induced which leads to preferential survival and proliferation. Importantly, CD40 engagement rescues NOD Th40 cells from Fas-induced death.CD40 may act as a switch between life and death promoting signals and NOD Th40 cells are poised for survival via this switch. This may explain how they expand in autoimmunity to thwart T cell homeostasis

YopJ-Induced Caspase-1 Activation in Yersinia-Infected Macrophages: Independent of Apoptosis, Linked to Necrosis, Dispensable for Innate Host Defense

Yersinia outer protein J (YopJ) is a type III secretion system (T3SS) effector of pathogenic Yersinia (Yersinia pestis, Yersinia enterocolitica and Yersinia pseudotuberculosis) that is secreted into host cells. YopJ inhibits survival response pathways in macrophages, causing cell death. Allelic variation of YopJ is responsible for differential cytotoxicity in Yersinia strains. YopJ isoforms in Y. enterocolitica O:8 (YopP) and Y. pestis KIM (YopJKIM) strains have high cytotoxic activity. In addition, YopJKIM-induced macrophage death is associated with caspase-1 activation and interleukin-1β (IL-1β secretion. Here, the mechanism of YopJKIM-induced cell death, caspase-1 activation, and IL-1β secretion in primary murine macrophages was examined. Caspase-3/7 activity was low and the caspase-3 substrate poly (ADP-ribose) polymerase (PARP) was not cleaved in Y. pestis KIM5-infected macrophages. In addition, cytotoxicity and IL-1β secretion were not reduced in the presence of a caspase-8 inhibitor, or in B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax)/Bcl-2 homologous antagonist/killer (Bak) knockout macrophages, showing that YopJKIM-mediated cell death and caspase-1 activation occur independent of mitochondrial-directed apoptosis. KIM5-infected macrophages released high mobility group protein B1 (HMGB1), a marker of necrosis, and microscopic analysis revealed that necrotic cells contained active caspase-1, indicating that caspase-1 activation is associated with necrosis. Inhibitor studies showed that receptor interacting protein 1 (RIP1) kinase and reactive oxygen species (ROS) were not required for cytotoxicity or IL-β release in KIM5-infected macrophages. IL-1β secretion was reduced in the presence of cathepsin B inhibitors, suggesting that activation of caspase-1 requires cathepsin B activity. Ectopically-expressed YopP caused higher cytotoxicity and secretion of IL-1β in Y. pseudotuberculosis-infected macrophages than YopJKIM. Wild-type and congenic caspase 1 knockout C57BL/6 mice were equally susceptible to lethal infection with Y. pseudotuberculosis ectopically expressing YopP. These data suggest that YopJ-induced caspase-1 activation in Yersinia-infected macrophages is a downstream consequence of necrotic cell death and is dispensable for innate host resistance to a strain with enhanced cytotoxicity

Mouse models of breast cancer metastasis

Metastatic spread of cancer cells is the main cause of death of breast cancer patients, and elucidation of the molecular mechanisms underlying this process is a major focus in cancer research. The identification of appropriate therapeutic targets and proof-of-concept experimentation involves an increasing number of experimental mouse models, including spontaneous and chemically induced carcinogenesis, tumor transplantation, and transgenic and/or knockout mice. Here we give a progress report on how mouse models have contributed to our understanding of the molecular processes underlying breast cancer metastasis and on how such experimentation can open new avenues to the development of innovative cancer therapy

Impact of cyclin E overexpression on Smad3 activity in breast cancer cell lines

Smad3, a component of the TGFβ signaling pathway, contributes to G1 arrest in breast cancer cells. Overexpression of the cell cycle mitogen, cyclin E, is associated with poor prognosis in breast cancer, and cyclin E/CDK2 mediated phosphorylation of Smad3 has been linked with inhibition of Smad3 activity. We hypothesized that the biological aggressiveness of cyclin E overexpressing breast cancer cells would be associated with CDK2 phosphorylation and inhibition of the tumor suppressant action of Smad3. Expression constructs containing empty vector, wild-type (WT) Smad3 or Smad3 with CDK phosphorylation site mutations were co-transfected with a Smad3-responsive reporter construct into parental, vector control (A1) or cyclin E overexpressing (EL1) MCF7 cells. Smad3 function was evaluated by luciferase reporter assay and mRNA analysis. The impact of a Cdk2 inhibitor and cdk2 siRNA on Smad3 activity was also assessed. Cells expressing Smad3 containing mutations of the CDK phosphorylation sites had higher p15 and p21 and lower c-myc mRNA levels, as well as higher Smad3-responsive reporter activity, compared with controls or cells expressing WT Smad3. Transfection of cdk2 siRNA resulted in a significant increase in Smad3-responsive reporter activity compared with control siRNA; reporter activity was also increased after the treatment with a Cdk2 inhibitor. Thus, cyclin E-mediated inhibition of Smad3 is regulated by CDK2 phosphorylation of the Smad3 protein in MCF7 cells. Inhibition of CDK2 may lead to restoration of Smad3 tumor suppressor activity in breast cancer cells, and may represent a potential treatment approach for cyclin E overexpressing breast cancers

The structure of cyclin E1/CDK2: implications for CDK2 activation and CDK2-independent roles

Cyclin E, an activator of phospho-CDK2 (pCDK2), is important for cell cycle progression in metazoans and is frequently overexpressed in cancer cells. It is essential for entry to the cell cycle from G0 quiescent phase, for the assembly of prereplication complexes and for endoreduplication in megakaryotes and giant trophoblast cells. We report the crystal structure of pCDK2 in complex with a truncated cyclin E1 (residues 81–363) at 2.25 Å resolution. The N-terminal cyclin box fold of cyclin E1 is similar to that of cyclin A and promotes identical changes in pCDK2 that lead to kinase activation. The C-terminal cyclin box fold shows significant differences from cyclin A. It makes additional interactions with pCDK2, especially in the region of the activation segment, and contributes to CDK2-independent binding sites of cyclin E. Kinetic analysis with model peptide substrates show a 1.6-fold increase in k(cat) for pCDK2/cyclin E1 (81–363) over k(cat) of pCDK2/cyclin E (full length) and pCDK2/cyclin A. The structural and kinetic results indicate no inherent substrate discrimination between pCDK2/cyclin E and pCDK2/cyclin A with model substrates

Reduced spermatogonial proliferation and decreased fertility in mice overexpressing cyclin E in spermatogonia

Cyclin E is a key component of the cell cycle regulatory machinery, contributing to the activation of Cdk2 and the control of cell cycle progression at several stages. Cyclin E expression is tightly regulated, by periodic transcription and ubiquitin-mediated degradation. Overexpression of cyclin E has been associated with tumor development and poor prognosis in several tumor types, including germ cell tumors and both cyclin E and its partner Cdk2 are required for normal spermatogenesis. Here we have generated and characterized transgenic mice overexpressing a cyclin E mutant protein, resistant to ubiquitin-mediated proteolysis, in testicular germ cells, under the control of the human EF-1alpha promoter. The transgenic mice develop normally and live a normal life span, with no signs of testicular tumor development. The transgenic mice display however reduced fertility and testicular atrophy, due to reduced spermatogonial proliferation as a consequence of deregulated cyclin E levels. Overall our results show that deregulation of cyclin E expression contribute to infertility, due to inability of the spermatogonial cells to start the mitotic cycles prior to entering meiosis