E2F transcription factor-1 modulates expression of glutamine metabolic genes in mouse embryonic fibroblasts and uterine sarcoma cells

Metabolic reprogramming is considered as a hallmark of cancer and is clinically exploited as a novel target for therapy. The E2F transcription factor-1 (E2F1) regulates various cellular processes, including proliferative and metabolic pathways, and acts, depending on the cellular and molecular context, as an oncogene or tumor suppressor. The latter is evident by the observation that E2f1-knockout mice develop spontaneous tumors, including uterine sarcomas. This dual role warrants a detailed investigation of how E2F1 loss impacts metabolic pathways related to cancer progression. Our data indicate that E2F1 binds to the promoter of several glutamine metabolism-related genes. Interestingly, the expression of genes in the glutamine metabolic pathway were increased in mouse embryonic fibroblasts (MEFs) lacking E2F1. In addition, we confirm that E2f1 <sup>-/-</sup> MEFs are more efficient in metabolizing glutamine and producing glutamine-derived precursors for proliferation. Mechanistically, we observe a co-occupancy of E2F1 and MYC on glutamine metabolic promoters, increased MYC binding after E2F1 depletion and that silencing of MYC decreased the expression of glutamine-related genes in E2f1 <sup>-/-</sup> MEFs. Analyses of transcriptomic profiles in 29 different human cancers identified uterine sarcoma that showed a negative correlation between E2F1 and glutamine metabolic genes. CRISPR/Cas9 knockout of E2F1 in the uterine sarcoma cell line SK-UT-1 confirmed elevated glutamine metabolic gene expression, increased proliferation and increased MYC binding to glutamine-related promoters upon E2F1 loss. Together, our data suggest a crucial role of E2F1 in energy metabolism and metabolic adaptation in uterine sarcoma cells

Mathematical model of a telomerase transcriptional regulatory network developed by cell-based screening: analysis of inhibitor effects and telomerase expression mechanisms

Cancer cells depend on transcription of telomerase reverse transcriptase (TERT). Many transcription factors affect TERT, though regulation occurs in context of a broader network. Network effects on telomerase regulation have not been investigated, though deeper understanding of TERT transcription requires a systems view. However, control over individual interactions in complex networks is not easily achievable. Mathematical modelling provides an attractive approach for analysis of complex systems and some models may prove useful in systems pharmacology approaches to drug discovery. In this report, we used transfection screening to test interactions among 14 TERT regulatory transcription factors and their respective promoters in ovarian cancer cells. The results were used to generate a network model of TERT transcription and to implement a dynamic Boolean model whose steady states were analysed. Modelled effects of signal transduction inhibitors successfully predicted TERT repression by Src-family inhibitor SU6656 and lack of repression by ERK inhibitor FR180204, results confirmed by RT-QPCR analysis of endogenous TERT expression in treated cells. Modelled effects of GSK3 inhibitor 6-bromoindirubin-3′-oxime (BIO) predicted unstable TERT repression dependent on noise and expression of JUN, corresponding with observations from a previous study. MYC expression is critical in TERT activation in the model, consistent with its well known function in endogenous TERT regulation. Loss of MYC caused complete TERT suppression in our model, substantially rescued only by co-suppression of AR. Interestingly expression was easily rescued under modelled Ets-factor gain of function, as occurs in TERT promoter mutation. RNAi targeting AR, JUN, MXD1, SP3, or TP53, showed that AR suppression does rescue endogenous TERT expression following MYC knockdown in these cells and SP3 or TP53 siRNA also cause partial recovery. The model therefore successfully predicted several aspects of TERT regulation including previously unknown mechanisms. An extrapolation suggests that a dominant stimulatory system may programme TERT for transcriptional stability

Transduction of E2f-1 Tat Fusion Proteins into Primary Invasive Ductal Breast Carcinoma Cell Lines and Subsequent Effects on Gene Transcription

Breast carcinomas harbor specific genetic alterations that contribute to their immortalized state, including overexpression of telomerase and p53 tumor suppressor gene mutations. E2F-1 is a known transcriptional repressor of the catalytic subunit of telomerase, hTERT. In p53 mutated cells, E2F-1 possesses the ability to transactivate the p73 tumor suppressor pathway and p53-homologues involved in cell cycle arrest such as p21WAF1/CIP1. We investigated an alternative method of introducing the E2F-1 transcription factor via Tat-mediated protein transduction to effectively target and reversibly impact gene expression in breast cancer cell lines. Using affinity chromatography, the E2F-1 Tat fusion proteins were isolated and purified via FPLC and dialysis. Real-time RT-qPCR was utilized to assess the effects of E2F-1 Tat fusion protein treatment on gene expression of breast carcinomas. We investigated repression of hTERT and induction of p73 and p21WAF1/CIP1 genes in HCC1937 and HCC1599 primary invasive ductal breast cancer cell lines. Following cell treatment with E2F-1 Tat protein therapy, apoptotic activity was monitored via TUNEL assays.Findings and Conclusions: The E2F-1 Tat fusion proteins effectively transduced greater than 95% of cancer cells. Protein therapy with E2F-1/TatHA repressed expression of hTERT by 3.5-fold in HCC1937 cells and by 4.0-fold in HCC1599 cells versus control proteins. Treatment of HCC1937 and HCC1599 carcinoma cells with E2F-1 Tat fusion proteins resulted in greater than 2-fold induction of p73 gene expression. Upregulation of the p53 responsive gene, p21WAF1/CIP1, was observed in HCC1937 and HCC1599 cells following treatment with E2F-1 Tat proteins. A 3.3-fold induction of p21WAF1/CIP1 was observed in HCC1937 cells versus a smaller induction of 1.4-fold in HCC1599 cells, which was attributed to the presence of a BRCA1 mutation in HCC1599 cells. Following 24 hours of treatment with E2F-1 Tat fusion proteins, apoptotic activity was detected in approximately 10% of breast cancer cells versus control proteins. Overall, the E2F-1 Tat protein therapy proved to be a moderately effective repressor of hTERT and activator of both p73 and p21WAF1/CIP1 resulting in detectable apoptotic activity. This suggests a potential application of E2F-1 Tat protein therapy in cancer therapeutics to modulate gene expression in breast carcinomas.Department of Biochemistry and Molecular Biolog

Telomerase a prognostic marker an therapeutic target

Malignant glioma is the most common and aggressive form of tumours and is usually refractory to therapy. Telomerase and its altered activity, distinguishing cancer cells, is an attractive molecular target in glioma therapeutics. The aim of this thesis was to silence telomerase at the genetic level with a view to highlight the changes caused in the cancer proteome and identify the potential downstream pathways controlled by telomerase in tumour progression and maintenance. A comprehensive proteomic study utilizing 2D-DIGE and MALDI-TOF were used to assess the effect of inhibiting two different regulatory mechanisms of telomerase in glioma. RNAi was used to target hTERT and Hsp90α. Inhibition of telomerase activity resulted in down regulation of various cytoskeletal proteins with correlative evidence of the involvement of telomerase in regulating the expression of vimentin. Vimentin plays an important role in tumour metastasis and is used as an indicator of glioma metastasis. Inhibition of telomerase via sihTERT results in the down regulation of vimentin expression in glioma cell lines in a grade specific manner. While, 9 of 12 glioblastoma tissues (grade IV) showed vimentin to be highly expressed, its expression was absent in lower grades and normal tissues. This suggests that vimentin can be potentially used as a glioma progressive marker. This is the first study to report the potential involvement of telomerase in the regulation of vimentin expression. This study also identified that combination therapy, comprising siRNA targeted towards telomerase regulatory mechanisms and the natural product Epigallocatechin-3-gallate (ECGC), results in decreased cell viability producing comparable results to that of other chemotherapeutic drugs

Targeting Histone deacetylases (HDAC) for the treatment of soft tissue sarcoma

Targeting Histone deacetylases (HDAC) for the treatment of genetically complex soft tissue sarcoma Histone deactylase inhibitors (HDACi) are a new class of anticancer therapeutics; however, little is known about HDACi or the individual contribution of HDAC isoform activity in soft tissue sarcoma (STS). We investigated the potential efficacy of HDACi as monotherapy and in combination with chemotherapy in a panel of genetically complex STS. We found that HDACi combined with chemotherapy significantly induced anti-STS effects in vitro and in vivo. We then focused our study of HDACi in malignant peripheral nerve sheath tumor (MPNST), a subtype of highly aggressive, therapeutically resistant, and commonly fatal malignancies that occur in patients with neurofibromatosis type-1 (NF1) or sporadically. The therapeutic efficacy of HDACi was investigated in a panel of NF1-associated and sporadic MPNST cell lines. Our results demonstrate the NF1-assocaited cohort to be highly sensitive to HDACi while sporadic cell lines exhibited resistance. HDACi-induced productive autophagy was found to be a mode of resistance and inhibiting HDACi-induced autophagy significantly induced pro-apoptotic effects of HDACi in vitro and in vivo. HDACs are not a single enzyme consisting of 11 currently known isoforms. HDACis used in these studies inhibit a variety of these isoforms, namely class I HDACs which include HDAC1, 2, 3, and 8. Recently, HDAC8-specific inhibitors (HDAC8i) have been created and tested in various cancer cell lines. Lastly, the potential therapeutic efficacy of HDAC8i was investigated in human (NF1-associated and sporadic) and NF1-associated murine-derived MPNST. HDAC8i abrogated cell growth in human and murine-derived MPNST cells. Similar to the pattern noticed with pan-HDACis NF1-associated cells, especially murine-derived, were more sensitive to HDAC8i compared to human sporadic MPNST cell lines. S-phase arrest was observed in human and murine MPNST cells, independent of p53 mutational and NF1 status. HDAC8i induced apoptosis is all cell lines tested, with a more pronounced effects in human and murine-derived NF1-associated cells. Most importantly, HDAC8i abrogated murine-derived MPNST xenograft growth in vivo. Taken together, these findings support the evaluation of pan-HDACi and isoform-specific inhibitors as a novel therapy to treat MPNST, including in combination with autophagy blocking combination regimens in particular for patients with sporadic MPNST

The E2F1/DNMT1 Axis Represses AR in Both Normal and Malignant Prostate Epithelium.

The molecular events associated with the recurrence of castration resistant prostate cancer (CRPC) are of critical importance in prostate cancer research, as CRPC is associated with high morbidity and lethality. CRPC is associated with deregulated prostatic epithelium exhibiting decreased AR expression in over 30% of the cases and seems to mimic the proliferative AR-negative undifferentiated transit amplifying (T/A) cells of the developing prostate. In an effort to further characterize this proliferative and undifferentiated cell population, we evaluated possible mechanisms involved in AR gene repression. We have shown that E2F1, a known transcriptional activator, represses AR expression. To explore this mechanism further, we overexpressed E2F1 in prostate epithelial cells and found that AR levels decreased while a dominant negative E2F1 construct reversed the inhibitory effects on AR transcription. E2F1 activates the transcription of DNMT1, a protein that typically silences genes through DNA methylation, however, we found that DNMT1 repressed the AR gene in a DNA methylation independent fashion. We further explored the E2F1/DNMT1/AR regulatory axis in a CRPC mouse model. Heightened E2F1 expression was previously shown to be inversely correlated with AR expression during human prostate cancer progression to CRPC. We demonstrated that DNMT1 nuclear staining significantly increased from benign tissue to treatment resistant, metastatic prostate cancer in humans. Considering that abnormal levels of DNMT1 may methylate and repress AR, we evaluated tissue from CRPC mice injected with a DNA methylation inhibitor, 5-aza. A rise in AR positive tissue corresponded with a decrease in the amount of DNMT1 nuclear staining following treatment. The immunohistochemical data suggests that hypermethylation mediated repression of the AR gene by DNMT1 during the development of CRPC may represent an important etiological aspect of this disease. In summary, we have identified a mechanism of AR repression mediated by the E2F1/DNMT1 axis that results in methylation independent AR repression in proliferative, undifferentiated prostate epithelium. However, AR repression also identified in neoplastic cells appears to be dependent on DNA methylation during the emergence of CRPC. Our studies reveal novel epigenetic regulatory mechanisms involved in AR repression that may further elucidate the understanding of transcriptional regulation, particularly in CRPC.Ph.D.Cellular & Molecular BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/89826/1/vconrad_1.pd

Role of mouse PinX1 in maintaining the characteristics of mouse embryonic stem cells.

Lau, Yuen Ting.Thesis (M.Phil.)--Chinese University of Hong Kong, 2011.Includes bibliographical references (leaves 156-163).Abstracts in English and Chinese.Abstract --- p.iAbstract in Chinese (摘要） --- p.iiiAcknowledgements --- p.ivTable of content --- p.VList of figures --- p.ixList of tables --- p.xiiiList of abbreviations --- p.xivChapter 1 --- INTRODUCTION --- p.PageChapter 1.1 --- Embryonic stem cells (ESCs) --- p.1Chapter 1.1.1 --- What are ESCs and the characteristics of ESCs --- p.1Chapter 1.1.2 --- Promising use of ESCs in drug development and regenerative medicine --- p.1Chapter 1.1.3 --- Maintenance of self-renewal and pluripotent properties of ESCs --- p.3Chapter 1.2 --- Cell cycle in ESCs --- p.5Chapter 1.2.1 --- Cell cycle --- p.5Chapter 1.2.2 --- Characteristics of cell cycle of ESCs --- p.6Chapter 1.3 --- Telomere --- p.8Chapter 1.3.1 --- Telomere structure and the telomeric proteins --- p.8Chapter 1.3.2 --- End replication problem --- p.10Chapter 1.3.3 --- Telomere dysfunction in cancer and cellular aging --- p.11Chapter 1.4 --- Telomerase --- p.12Chapter 1.4.1 --- Telomerase and stem cell characteristics --- p.13Chapter 1.4.1.1 --- Telomerase and cell proliferation --- p.13Chapter 1.4.1.2 --- Telomerase and stem cell differentiation --- p.14Chapter 1.4.2 --- Regulation of telomerase expression/ activity --- p.15Chapter 1.4.2.1 --- Regulation of telomerase at different levels --- p.15Chapter 1.4.2.2 --- Regulation of telomerase activity by cellular components in ESCs --- p.16Chapter 1.5 --- PinXl --- p.18Chapter 1.5.1 --- Expression of PinXl --- p.18Chapter 1.5.2 --- Effects of PinXl on the activities and the sub-cellular localization of telomerase --- p.19Chapter 1.5.3 --- Structure-function relationship of PinXl --- p.19Chapter 1.5.4 --- Effect of PinXl on the growth rate of normal and cancer cells --- p.21Chapter 1.5.5 --- Other functions of PinX 1 V --- p.22Chapter 1.5.6 --- Mouse homolog of PinXl and its function in mESCs --- p.23Chapter 1.6 --- Aims of this study --- p.24Chapter 2 --- METERIALS AND METHODS --- p.PageChapter 2.1 --- mESC culture and differentiation --- p.25Chapter 2.1.1 --- Cell line --- p.25Chapter 2.1.2 --- Irradiation of MEF --- p.25Chapter 2.1.3 --- mESC culture --- p.26Chapter 2.1.4 --- Differentiation of mESCs --- p.26Chapter 2.1.5 --- Establishment and' culture of feeder-free mESCs --- p.28Chapter 2.1.6 --- Culture of feeder-free mESCs --- p.28Chapter 2.2 --- Trypan Blue Exclusion Assay --- p.29Chapter 2.3 --- Sub-cloning --- p.29Chapter 2.3.1 --- Amplification of the insert gene by PCR --- p.29Chapter 2.3.2 --- Purification of PCR products --- p.31Chapter 2.3.3 --- Restriction enzyme digestion --- p.32Chapter 2.3.4 --- Ligation of digested insert and vector --- p.33Chapter 2.3.5 --- Transformation of ligation product into competent cells --- p.34Chapter 2.3.6 --- Confirmation of positive clone by colony PCR --- p.34Chapter 2.3.7 --- Small scale preparation of the recombinant plasmid DNA --- p.35Chapter 2.3.8 --- Confirmation of positive clone by restriction digestion --- p.36Chapter 2.3.9 --- DNA sequencing of the recombinant plasmid DNA --- p.36Chapter 2.3.10 --- Large scale preparation of the recombinant plasmid DNA --- p.37Chapter 2.4 --- Design of siRNA targeting mPinXl and mPinXlt --- p.38Chapter 2.5 --- Transient transfection --- p.38Chapter 2.6 --- Cloning of siRNA into shRNA insert in Lentiviral Vector pLVTHM --- p.39Chapter 2.7 --- Lentiviral vector-mediated gene transfer to mESCs --- p.42Chapter 2.7.1 --- Lentivirus packaging --- p.42Chapter 2.7.2 --- Checking of successful transduction by lentivirus in HEK cells --- p.43Chapter 2.7.3 --- Multiple transductions to mESCs --- p.43Chapter 2.7.4 --- Selection of positive clones --- p.44Chapter 2.7.5 --- Monoclonal establishment --- p.44Chapter 2.8 --- "Total RNA preparation, Reverse Transcription (RT) and Quantitative Polymerase Chain Reaction (qPCR)" --- p.45Chapter 2.9 --- Immunocytochemistry --- p.46Chapter 2.10 --- Western Blotting --- p.48Chapter 2.10.1 --- Total Protein Extraction vi --- p.48Chapter 2.10.2 --- Measurement of Protein Concentration --- p.48Chapter 2.10.3 --- SDS-PAGE and chemiluminescent detection --- p.49Chapter 2.11 --- Co-immunoprecipitation --- p.51Chapter 2.12 --- Telomere Repeat Amplification Protocol (TRAP) Assay --- p.52Chapter 2.13 --- Cell cycle analysis --- p.54Chapter 2.14 --- MTT assay --- p.54Chapter 2.15 --- Statistical analysis --- p.55Chapter 3 --- RESULTS --- p.PageChapter 3.1 --- mPinXlt was discovered in mESCs --- p.56Chapter 3.2 --- mPinXl and mPinXlt were expressed at transcriptional level in the inspected mouse tissues --- p.61Chapter 3.3 --- Expression of mPinXl and mPinXlt changed upon differentiation --- p.64Chapter 3.4 --- mPinXl and mPinXlt were both located in the nucleolus and the nucleoplasm in undifferentiated mESCs --- p.69Chapter 3.5 --- Co-immunoprecipitation (Co-IP) of mPinXl and mPinXlt with mTERT --- p.73Chapter 3.6 --- Transient knockdown of mPinXl in mESCs --- p.78Chapter 3.6.1 --- Knockdown of mPinXl decreased proliferation but did not change cell viability --- p.79Chapter 3.6.2 --- Knockdown of mPinXl decreased telomerase activity --- p.79Chapter 3.6.3 --- Knockdown of mPinXl did not change pluripotency --- p.80Chapter 3.6.4 --- Knockdown of mPinXl did not affect cell cycle progression --- p.80Chapter 3.7 --- Transient knockdown of mPinXlt using siRNA against mPinXlt in mESCs --- p.88Chapter 3.8 --- Transient over-expression of mPinXl and mPinXlt in mESCs --- p.90Chapter 3.8.1 --- Over-expression of mPinXl and mPinXlt decreased cell proliferation but didn't affect cell viability --- p.91Chapter 3.8.2 --- Over-expression of mPinXl increased telomerase activity --- p.92Chapter 3.8.3 --- Over-expression of mPinXl and mPinXlt did not affect pluripotency --- p.93Chapter 3.8.4 --- Over-expression of mPinXl and mPinXlt did not affect cell cycle progression --- p.93Chapter 3.9 --- Stable over-expression and knockdown of mPinXl and mPinXlt in mESCs --- p.103Chapter 3.9.1 --- Expression of mPinXl and mPinXlt at mRNA and protein levels in all over-expression stable cell lines --- p.108Chapter 3.9.2 --- Expression of mPinXl and mPinXlt at mRNA and protein levels in mPinXl knockdown stable cell lines --- p.113Chapter 3.9.3 --- Proliferation of all stable cell lines --- p.116Chapter 3.9.4 --- Telomerase activity of all stable cell lines --- p.121Chapter 3.9.5 --- Cell cycle distribution of all stable cell lines --- p.123Chapter 3.9.6 --- Pluripotency of all stable cell lines --- p.127Chapter 3.9.7 --- Differentiation of the stable cell lines --- p.130Chapter 3.9.7.1 --- Size of EBs formed from stable cell lines at Day 7 --- p.130Chapter 3.9.7.2 --- Beating curves of the stable cell lines derived EBs --- p.130Chapter 4 --- DISCUSSIONS --- p.PageChapter 4.1 --- mPinXlt gene was detected in mESCs --- p.137Chapter 4.2 --- "Presence of mPinXl and mPinXlt in mouse tissues, mESCs and their differentiation derivatives" --- p.138Chapter 4.3 --- Differences in expressions of mPinXl and mPinXlt in undifferentiated mESCs and their differentiation derivatives --- p.139Chapter 4.4 --- mPinXl and mPinXlt are pre-dominantly localized in the nucleolus --- p.141Chapter 4.5 --- mPinXl and mPinXlt interacted with mTERT --- p.143Chapter 4.6 --- "Transient knockdown of mPinXl slightly inhibited, while over-expression of mPinXl slightly promoted telomerase activity" --- p.143Chapter 4.7 --- Both transient knockdown and over-expression of mPinXl inhibited the growth of mESCs --- p.146Chapter 4.8 --- Both stable knockdown and over-expression of mPinXl did not affect cell proliferation and telomerase activity of mESCs --- p.148Chapter 4.9 --- Involvement of mPinXl and mPinXlt in the differentiation process of mESCs --- p.149Chapter 4.10 --- Regulation of mPinXl gene expression by mPinXlt --- p.151Chapter 4.11 --- Future perspectives --- p.152Chapter 5 --- CONCLUSION --- p.154Chapter 6 --- REFERENCES --- p.15

Telomere biology in the freshwater planarian Schmidtea mediterranea

Freshwater planarian Schmidtea mediterranea is an emerging model for studying in vivo gene functions and regulation in native cell niches. The obligate asexual strain of this species reproduces by fission, in which succession of soma occurs without passing through the germline. To achieve this somatic immortality the somatic stem cells need to overcome the end replication problem. Therefore it can be hypothesised that somatic telomere maintenance in asexual S. mediterranea must possess a germ-like property, with which age-related erosions can be adequately repaired. In this PhD project, the telomere repeat unit in S. mediterranea was confirmed to be the vertebrate-like TTAGGG. Attrition of whole body telomere length was found in ageing sexual worms and also in asexual worms which had not gone through recent fission events. Opposite telomere length dynamics were observed in regenerated samples of the two strains, with erosion in the sexuals and reset in the asexuals. The telomere maintenance was found to increase during regeneration in both strains, with a higher level of increase in asexual worms. A homolog of the telomerase reverse transcriptase subunit, Smed_Tert, was identified and characterised in this organism. High level of Smed_Tert expression was seen in germ cells in mature sexual worms and adult stem cells in asexual worms. Knockdown of Smed_Tert expression by RNA interference caused progressive telomere erosion, however effects on cell proliferation and viability have not been observed in knockdown samples. Four alternate splice isoforms of Smed_Tert were identified. The enhanced telomerase activity during regeneration correlates with a proportional increase in the full-length isoform and a decrease in isoforms with a truncated TRBD domain, suggesting a dominant negative regulation of telomerase by alternative splicing. Significant increase in the expression of the full-length isoform was seen in regenerating asexual samples but not in sexual strains, which correlates with their telomere length dynamics. It is hoped that the comparative studies between the sexual and asexual strains can improve our understanding of how soma can evolve to become an effective inheritable unit