Novel Cell Cycle Regulation in Breast Cancer Treatment Resistance

Classification of breast cancer relies on the presence or absence of estrogen receptor alpha (ERα) and progesterone receptor (PR) as well as the overexpression or amplification of the Her2/neu receptor. Targeted therapies against these proteins has increased the overall 5-year survival rate of breast cancer patients. However, a subset of breast cancer patients can acquire resistance or are initially unresponsive to these therapies. Understanding the molecular pathways that can cause resistance within the various types of breast cancer is of high priority. The cell cycle regulatory factor Speedy (Spy1) has been found to be upregulated in a variety of human cancers, including invasive mammary carcinomas, as well as being downstream of two important pathways in breast cancer initiation and progression; MAPK and c-Myc. My study sought to investigate the role of Spy1 downstream of ERα and to determine its role in regulating treatment response in the presence or absence of ERα. My work defines a novel positive feedback loop whereby Spy1 activates ERK1/2 in a MEK-independent fashion. This activation was further demonstrated to increase the ligand-independent activation of ERα, correlating with a decrease in tamoxifen sensitivity. We tested our findings using an in vivo zebrafish model, demonstrating elevated levels of Spy1 alter tamoxifen sensitivity. We further demonstrate significantly high levels of Spy1 within the triple negative group of breast cancers; which correlates with decreased sensitivity to chemotherapy as well as CDK inhibitor treatment. These data could define an efficient mechanism driving proliferation and resistance in select cancers and may represent a potent drugable target

The role of eIF4AIII and 4E-T in mRNA decay /

Translational control is crucial to balancing the cell's protein output and genetic expression. The substrate of the translational machinery---messenger RNA (mRNA)---is itself subject to regulation. The lifetime of an mRNA is limited and therefore mRNA decay is a critical step in the regulation of gene expression. Translation and mRNA decay are intimately related processes as they both handle the cell economy. mRNAs are generally in a balancing act between the translational and the repression/decay machinery, which ultimately decides the fate of an mRNA and its protein expression rate. In fact, translation affects the rate of mRNA decay. For instance, aberrant messages which contain a premature-termination codon (PTC) require ribosome scanning in order to read the message, discover the mistake, and essentially prompt its destruction. Here, a relationship between the nuclear translation-like factor---eIF4AIII, the nuclear import factor of cIF4E---4E-T, and mRNA decay was discovered. eIF4AIII is a nuclear protein that interacts physically or functionally with translation initiation factors eIF4G and eIF4B, respectively, and shares strikingly high identity with the initiation factors eIF4AI/II. This work demonstrates that eIF4AIII but not eIF4AI is required for nonsense-mediated decay (NMD). NMD is a surveillance mechanism in eukaryotes which degrades the mRNA when a PTC is present. NMD is a splicing and translation-dependent event in mammalians. We show eIF4AIII is deposited at the exon-exon junction during splicing, is a shuttling protein, and is necessary for NMD. At steady state, 4E-T is predominantly cytoplasmic and is concentrated in bodies that conspicuously resemble the recently described Processing bodies (P-bodies), which are sites of mRNA decay. We demonstrate that 4E-T colocalizes with mRNA decapping factors in bona fide P-bodies and that its binding partner, eIF4E, is tethered to P-bodies in a 4E-T dependent manner. Moreover, 4E-T controls mRNA half life. We demonstrate that 4E-T interaction with eIF4E represses translation, which is thought to be a prerequisite for targeting of mRNAs to P-bodies. Hence, analysis of prospective translation factors has led to elucidation of mRNA decay pathways

Targeting the Novel Cell Cycle Regulator, Spy1, for Treatment of Medulloblastoma

Targeting the Novel Cell Cycle Regulator, Spy1, for Treatment of Medulloblastoma Philip Habashy, Rosa M. Ferraiuolo, Dorota Lubanska, Lisa A. Porter Medulloblastoma (MB) is the most common malignant pediatric brain tumour. It occurs in 16-25% of cases, with higher incidence in children between the ages of 1 to 9 years. Current standard of care includes combination radiation, surgery and chemotherapy, this treatment relies on DNA damage to induce death of quickly growing cells. While effective for a small margin of patients the treatment is highly aggressive, is plagued with cytotoxicity and ultimately fails in many patients. One recent approach entering clinical development is the use of synthetic cyclin-dependent kinase inhibitors (CKIs). Finding new drugs and optimizing existing approaches for MB are of high importance. Our lab studies a cell cycle regulatory protein called Speedy (Spy1), which promotes cell proliferation, even during times of DNA damage produced by chemotherapeutic agents. Spy1 has been implicated in the maintenance and expansion of stem-like populations of tumour initiating cells known to be the most chemo-resistant among solid tumours. It is our hypothesis that Spy1 drives tumour initiating cells in MB and reducing the levels of Spy1 will increase sensitivity of aggressive MB to standard of care and CKI therapy. To address this hypothesis we have used patient-derived MB cells and have manipulated the levels of Spy1 using a lentiviral system. Using a high throughput platform these cells are injected into zebrafish prior to the establishment of the acquired immune system. We then determine the effect of CKI treatment on these in vivo tumours. To date our results show promise that this approach may sensitize, at least a subset of MB patients, to therapy. Our work may contribute toward optimizing the design of CKIs and the use in combination therapy. This project holds promise for improving survival and quality of life for MB patients

The oncogenic role of circPVT1 in head and neck squamous cell carcinoma is mediated through the mutant p53/YAP/TEAD transcription-competent complex

Background: Circular RNAs are a class of endogenous RNAs with various functions in eukaryotic cells. Worthy of note, circular RNAs play a critical role in cancer. Currently, nothing is known about their role in head and neck squamous cell carcinoma (HNSCC). The identification of circular RNAs in HNSCC might become useful for diagnostic and therapeutic strategies in HNSCC. Results: Using samples from 115 HNSCC patients, we find that circPVT1 is over-expressed in tumors compared to matched non-tumoral tissues, with particular enrichment in patients with TP53 mutations. circPVT1 up-and down-regulation determine, respectively, an increase and a reduction of the malignant phenotype in HNSCC cell lines. We show that circPVT1 expression is transcriptionally enhanced by the mut-p53/YAP/TEAD complex. circPVT1 acts as an oncogene modulating the expression of miR-497-5p and genes involved in the control of cell proliferation. Conclusions: This study shows the oncogenic role of circPVT1 in HNSCC, extending current knowledge about the role of circular RNAs in cancer

Silent Myocardial Ischemia and Late Ventricular Repolarization in the Genesis of Ventricular Fibrillation in Humans

Background We report a well-documented case of a patient, a 62-year-old man, with severe and asymptomatic left main coronary artery disease who had several episodes of silent myocardial ischemia and a syncopal attack during Holter recording. Methods and Results Ambulatory monitoring showed isolated giant U waves separated from the T wave 60 minutes before syncope that was due to reversible ventricular fibrillation lasting about 4 minutes and spontaneously reverting to asystole (7 seconds) and then to atrial fibrillation Conclusion Our experience suggests that myocardial ischemia may differently affect the repolarization times within the myocardium leading to widely disparate repolarization gradients that may represent the arrhythmogenic substrate for the occurrence of life-threatening ventricular tachyarrhythmias. A.N.E. 1999; 4(2):250–25

Chromatin conformation signatures of cellular differentiation

A suite of computer programs to identify genome-wide chromatin conformation signatures with 5C technology is reported

A role for the eIF4E-binding protein 4E-T in P-body formation and mRNA decay

4E-transporter (4E-T) is one of several proteins that bind the mRNA 5′cap-binding protein, eukaryotic initiation factor 4E (eIF4E), through a conserved binding motif. We previously showed that 4E-T is a nucleocytoplasmic shuttling protein, which mediates the import of eIF4E into the nucleus. At steady state, 4E-T is predominantly cytoplasmic and is concentrated in bodies that conspicuously resemble the recently described processing bodies (P-bodies), which are believed to be sites of mRNA decay. In this paper, we demonstrate that 4E-T colocalizes with mRNA decapping factors in bona fide P-bodies. Moreover, 4E-T controls mRNA half-life, because its depletion from cells using short interfering RNA increases mRNA stability. The 4E-T binding partner, eIF4E, also is localized in P-bodies. 4E-T interaction with eIF4E represses translation, which is believed to be a prerequisite for targeting of mRNAs to P-bodies. Collectively, these data suggest that 4E-T interaction with eIF4E is a priming event in inducing messenger ribonucleoprotein rearrangement and transition from translation to decay

The cyclin-like protein Spy1/RINGO promotes mammary transformation and is elevated in human breast cancer

Abstract Background Spy1 is a novel \u27cyclin-like\u27 activator of the G1/S transition capable of enhancing cell proliferation as well as inhibiting apoptosis. Spy1 protein levels are tightly regulated during normal mammary development and forced overexpression in mammary mouse models accelerates mammary tumorigenesis. Methods Using human tissue samples, cell culture models and in vivo analysis we study the implications of Spy1 as a mediator of mammary transformation and breast cancer proliferation. Results We demonstrate that this protein can facilitate transformation in a manner dependent upon the activation of the G2/M Cdk, Cdk1, and the subsequent inhibition of the anti-apoptotic regulator FOXO1. Importantly, we show for the first time that enhanced levels of Spy1 protein are found in a large number of human breast cancers and that knockdown of Spy1 impairs breast cancer cell proliferation. Conclusions Collectively, this work supports that Spy1 is a unique activator of Cdk1 in breast cancer cells and may represent a valuable drug target and/or a prognostic marker for subsets of breast cancers