Olivier Coqueret

New roles for p21 and p27 cell-cycle inhibitors: a function for each cell compartment?

Cell division relies on the activation of cyclins, which bind to cyclin-dependent kinases (CDKs) to induce cell-cycle progression towards S phase and later to initiate mitosis. Since uncontrolled cyclin-dependent kinase activity is often the cause of human cancer, their function is tightly regulated by cell-cycle inhibitors such as the p21 and p27 Cip/Kip proteins. Following anti-mitogenic signals or DNA damage, p21 and p27 bind to cyclin-CDK complexes to inhibit their catalytic activity and induce cell-cycle arrest. Interestingly, recent discoveries suggest that p21 and p27 might have new activities that are unrelated to their function as CDK inhibitors. The identification of new targets of Cip/Kip proteins as well as evidence of Cip/Kip cytoplasmic relocalization have revealed unexpected functions for these proteins in the control of CDK activation, in the regulation of apoptosis and in transcriptional activation. This article discusses recent insights into these possible additional functions of p21 and p27.

Linking cyclins to transcriptional control.

Cell cycle activation is coordinated by D-type cyclins which are rate limiting and essential for the progression through the G1 phase of the cell cycle. D-type cyclins bind to and activate the cyclin-dependent kinases Cdk4 and Cdk6, which in turn phosphorylate their downstream target, the retinoblastoma protein Rb. Upon Rb phosphorylation, the E2F transcription factors activate the expression of S-phase genes and thereby induce cell cycle progression. The raise of cyclin D levels in early G1 also serves to titrate Kip/Cip proteins away from cyclinE/Cdk2 complexes, further accelerating cell cycle progression. Therefore, cyclin D plays essential roles in the response to mitogens, transmitting their signal to the Rb/E2F pathway. Surprisingly, cyclin D1-deficient animals are viable and have developmental abnormalities limited to restricted tissues, such as retina, the nervous system and breast epithelium. This observation, combined with several other studies, have raised the possibility that cyclin D1 may have new activities that are unrelated to its function as a cdk regulatory subunit and as regulator of Rb. Effectively, cyclin D has been reported to have transcriptional functions since it interacts with several transcription factors to regulate their activity. Most often, this effect does not rely on the kinase function of Cdk4, indicating that this function is probably independent of cell cycle progression. Further extending its role in gene regulation, cyclin D interacts with histone acetylases such as P/CAF or NcoA/SRC1a but also with components of the transcriptional machinery such as TAF(II)250. Therefore, these studies suggest that the functions of cyclin D might need to be reevaluated. They have established a new cdk-independent role of cyclin D1 as a transcriptional regulator, indicating that cyclin D1 can act via two different mechanisms, as a cdk activator it regulates cell cycle progression and as a transcriptional regulator, it modulates the activity of transcription factors.

The STAT3 Transcription Factor Is a Target for the Myc and Riboblastoma Proteins on the Cdc25A Promoter

The STAT3 (signal transducer and activator of transcription) transcription factor functions as down-stream effector of growth factor signaling. Whereas STAT3 activation is transient in normal cells, constitutively activated forms of the transcription factor have been detected in several cancer cell lines and primary tumors. Through the up-regulation of cell cycle and survival genes, STAT3 plays important roles in cell growth, anti-apoptosis, and cell transformation yet the molecular basis for this behavior is poorly understood. In this study, we show that STAT3 and its transcriptional cofactors are recruited to the promoter of the Cdc25A gene to activate its expression. Using chromatin immunoprecipitations, we observed that Myc is recruited to this promoter following STAT3 DNA binding. Moreover, small interfering RNA-mediated knockdown of Myc specifically inhibits the STAT3-mediated activation of Cdc25A. Reduction in Myc protein level results in defective recruitment of the CREB-binding protein, Cdk9, and RNA polymerase complexes, indicating that Myc is necessary for STAT3 transcription. Surprisingly, the association of STAT3 with the Cdc25A promoter does not necessarily lead to transcriptional induction because this protein also functions as a transcriptional repressor of the Cdc25A gene. Following hydrogen peroxide stimulation, STAT3 forms a repressor complex with the retinoblastoma (Rb) tumor suppressor to occupy the Cdc25A promoter and block its induction. In coimmunoprecipitations and ChIP experiments, Rb was found to associate with STAT3 on DNA and we provide evidence that Rb binds directly to the transcription factor. Thus, we propose that Myc and STAT3 cooperate to induce the expression of Cdc25A and that their transcriptional activity is normally regulated by the Rb tumor suppressor gene.

Prediction of Recurrence and Survival for Triple-Negative Breast Cancer (TNBC) by a Protein Signature in Tissue Samples

To date, there is no available targeted therapy for patients who are diagnosed with triple-negative breast cancers (TNBC). The aim of this study was to identify a new specific target for specific treatments. Frozen primary tumors were collected from 83 adjuvant therapy-naive TNBC patients. These samples were used for global proteome profiling by iTRAQ-OFFGEL-LC-MS/MS approach in two series: a training cohort (n = 42) and a test set (n = 41). Patients who remains free of local or distant metastasis for a minimum of 5 years after surgery were classified in the no-relapse group; the others were in the relapse group. OPLS and Kaplan–Meier analyses were performed to select candidate markers, which were validated by immunohistochemistry. Three proteins were identified in the training set and validated in the test set by Kaplan–Meier method and immunohistochemistry (IHC): TrpRS as a good prognostic markers and DP and TSP1 as bad prognostic markers. We propose the establishment of an IHC test to calculate the score of TrpRS, DP, and TSP1 in TNBC tumors to evaluate the degree of aggressiveness of the tumors. Finally, we propose that DP and TSP1 could provide therapeutic targets for specific treatments.

Regulation of the Aurora-A gene following topoisomerase I inhibition: implication of the Myc transcription Factor

During the G2 phase of the cell cycle, the Aurora-A kinase plays an important role in centrosome maturation and progression to mitosis. In this study, we show in colorectal cell lines that Aurora-A expression is downregulated in response to topoisomerase I inhibition. Using chromatin immunoprecipitation assays, we have observed that the Myc transcription factor and its Max binding partner are associated with the Aurora-A promoter during the G2 phase of the cell cycle. RNA interference experiments indicated that Myc is involved in the regulation of the Aurora-A gene. Following topoisomerase I inhibition, the expression of Myc decreased whereas Mad was upregulated, and the association of Myc and Max with the promoter of the kinase was inhibited. In parallel, an increased association of Mad and Miz-1 was detected on DNA, associated with an inhibition of the recruitment of transcriptional coactivators. Interestingly, a gain of H3K9 trimethylation and HP1γ recruitment was observed on the Aurora-A promoter following sn38 treatment, suggesting that this promoter is located within SAHF foci following genotoxic treatment. Since Aurora-A is involved in centrosome maturation, we observed as expected that topoisomerase I inhibition prevented centrosome separation but did not affect their duplication. As a consequence, this led to G2 arrest and senescence induction.These results suggest a model by which the Aurora-A gene is inactivated by the G2 checkpoint following topoisomerase I inhibition. We therefore propose the hypothesis that the coordinated overexpression of Myc and Aurora-A, together with a downregulation of Mad and Miz-1 should be tested as a prognosis signature of poor responses to topoisomerase I inhibitors.

Olfactomedin‐4 is a candidate biomarker of solid gastric, colorectal, pancreatic, head and neck, and prostate cancers

Olfactomedin‐4 (OLFM4, OLM4) is a 72 kDa secreted glycoprotein belonging to the olfactomedin family. The OLFM4 gene expression is regulated by the transcription factors NF‐kappa B and AP‐1, and the OLM4 functions are poorly understood. OLM4 has been described as being able to interact with cell surface proteins such as lectins and concanavalin‐A suggesting that one function of OLM4 is to regulate cell adhesion and migration. OLM4 is a marker for intestinal stem cells and is expressed at the bottom of the intestinal crypts. Expression of OLM4 during tumor development showed that OLM4 expression is increased in the early stages of tumor initiation. As OLM4 is a secreted protein, it is a prime candidate for biomarker research for tumor detection or progression. Levels of circulating OLM4 were significantly higher in patients with gastric, colorectal, and pancreatic cancers than in healthy subjects.

Échappement tumoral lors de la chimiothérapie : un choix entre sénescence et apoptose dans des tumeurs hétérogènes

Understanding adaptive signaling pathways in response to chemotherapy is one of the main challenges of cancer treatment. Activated in response to DNA damage, cell cycle and mitotic checkpoints activate the p53-p21 and p16-Rb pathways and induce apoptosis or senescence. Since senescent cells survive and produce a secretome that influences neighbouring cells, it is not particularly clear whether these responses are equivalent and if tumor cells escape these two suppressive pathways to the same extent. Predicting escape is also complicated by the fact that cancer cells adapt to treatments by activating the epithelial-mesenchymal transition and by producing clones with cancer-initiating cells features. Dedifferentiation pathways used in stressful conditions reconstitute dividing and sometimes more aggressive populations in response to chemotherapy. These observations illustrate the importance of tumor heterogeneity and the adaptation capacities of different intra-tumoral subclones. Depending on their oncogenic profile, on their localisation within the tumor and on their interaction with stromal cells, these subclones are expected to have different responses and adaptation capacities to chemotherapy. A complete eradication will certainly rely on combination therapies that can kill at the same time the bulk of the sensitive tumor but can also prevent plasticity and the generation of persistent clones.

Circulating miRNAs as new activators of the JAK-STAT3 pathway.

Cell communication is well known to rely on direct contacts or on secreted factors that bind to receptors located on the surface of their target cells. In addition to this classical pathway, recent results have shown that cells produce microvesicles that contain functional DNA, RNA and proteins that can be directly transferred to recipient cells. This induces proliferation, differentiation or cell death to the same extent as classical soluble factors. New data obtained from the laboratory of Napoleone Ferrara show that these microvesicles also contain miRNAs that can induce angiogenic activities in neighboring endothelial cells. When secreted from cancer cells, these miRNA-loaded vesicles penetrate recipient cells where they activate the JAK-STAT pathway. This represents a new type of intercellular signaling and a new way of activating the STAT transcription factors that could be of interest for the design of cancer treatments.