Abstract p53 tumor suppressor undergoes mutational loss in majority of cancers contributing to tumor formation. Therapeutic strategies are aimed towards p53 overexpression in tumors or to identify targets that compensate for p53-functional loss. p63 & p73, share structural similarities to p53, making them excellent candidates for therapeutic compensation of p53. Unlike p53, p63 and p73 do not undergo mutational loss and their role in tumorigenesis is being delineated. p63 and p73 have two major isoforms, the transactivation (TA), with activities similar to p53 and the delta (Δ)N- isoform with oncogenic functions. Inhibition of TAp63 and TAp73 is observed in cancers as a consequence of overexpression of ΔN isoforms of p63 and p73. In disparity, recent studies report, tumor suppressive properties of ΔNp63 and ΔNp73 in activating genes involved in DNA repair and apoptosis. To define the functional roles of ΔNp63 and ΔNp73 in cancer, mouse models targeting the ΔN isoforms were generated. We observed that, ΔNp63+/- and ΔNp73−/− mice on a p53−/− background had lower thymic lymphoma incidence compared to the p53−/− mice. I found TAp63 and TAp73 up regulated in the double mutant mice that correspond with an increase in p53-downstream apoptotic (PUMA, Noxa, BAX) and cell cycle targets (p21, p16, PML). This suggests that ablation of ΔN isoforms mediate TAp63 and TAp73 up regulation inducing apoptosis or cell cycle arrest by activation of p53-downstream targets. To further demonstrate this, I ablated ΔNp63 and ΔNp73 in vivo in p53−/- mice thymic lymphoma by administering adenoviral-CRE specifically to the thymus. The CRE-treated mice had a significant thymic lymphoma regression within 3 weeks as imaged by MRI in comparison to the mock-treated mouse cohorts. Additionally, RNA-Seq analysis from CRE-treated versus untreated mice, has identified novel metabolic genes with apoptotic or cell-cycle functions. We further report, ΔNp63 and ΔNp73 to bind to promoter site of TAp63 and TAp73 by chromatin immunoprecipitation (ChIP). This supports the notion that ablation of ΔN isoforms of p63 and p73 restores the function of TAp63 and TAp73 thus compensating for p53-tumor suppressive function in vivo. To test, if ablation of ΔN isoforms reduces tumorigenesis in human cancers, ΔNp63 and ΔNp73 were knocked down in human cancer cell lines were p53 expression was ablated or mutated. TAp63 and TAp73 were upregulated in ΔNp63/ΔNp73 knock down human cancer cell lines. However, induction of apoptosis or cell-cycle arrest was observed in p53-deleted cancer cell lines in comparison to the p53-mutated cell lines. This highlights the co-repressive effect of mutant p53, preventing activation of TAp63/TAp73 downstream targets. Current work is aimed towards overcoming mutant p53 effect in these cancer cell lines. Thus, targeting the ΔNp63/ΔNp73 compensates for p53-functional loss mediating tumor suppression. Citation Format: Avinashnarayan Venkatanarayan, Deepavali Chakravarti, Xiaohua Su, Santosh Sandur, Lingzhi Liu, Eliot Fletcher Sananikone, Payal Raulji, Cristian Coarfa, William Norton, Preethi Gunaratne, Elsa Renee Flores. Deletion of ΔNp63 and ΔNp73 in p53 deficient mice results in TAp63 and TAp73 compensation of p53 tumor suppression in vivo. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2331. doi:10.1158/1538-7445.AM2013-2331
The discovery of microRNAs (miRNAs - small non-coding RNAs of ~22 nt) heralded a new and exciting era in biology. During this period miRNAs have gone from ignominy due to their origin mainly in 'junk DNA' to notoriety where they can be at once characterized as being all powerful (a single miRNA can target and potentially silence several hundred genes) and yet marginal (a given gene can be targeted by several miRNAs such that a given miRNA typically exerts a modest repression) [1-4]. The emerging paradox is exemplified by miRNAs that are prominently expressed in embryonic stem (ES) cells. The collective importance of miRNAs is firmly established by the fact that Dicer-/- mouse embryos die on day 7.5 due to defects in differentiation [5]. However, oppositely correlated expression that is expected of conventional repressors is increasingly being defied in multiple systems in relation to miRNA-mRNA target pairs. This is most evident in ES cells where miR-290-295 and 302 clusters the most abundant ES cell miRNAs, are both found to be driven by pluripotency genes Oct4, Nanog and Sox2 and are predicted to target Sox2 in 'incoherent feed-forward loops' [7]. Here the miRNAs are co-expressed and positively correlated with these targets that they repress suggesting that one of their primary roles is to fine tune gene expression rather than act as ON/OFF switches. On the other hand, let-7 family members that are notably low in ES cells and rapidly induced upon differentiation exhibit more conventional anticorrelated expression patterns with their targets [7, 8]. In an intricately designed auto-regulatory loop, LIN28, a key 'keeper' of the pluripotent state binds and represses the processing of let-7 (a key 'keeper' of the differentiated state) [9-11]. One of the let-7 family members, let-7g targets and represses LIN28 through four 3-UTR binding sites [12]. We propose that LIN28/let-7 pair has the potential to act as a 'toggle switch' that balances the decision to maintain pluripotency vs. differentiation. We also propose that the c-Myc/E2F driven miR17-92 cluster that together controls the G1 to S transition is fundamental for ES self-renewal and cell proliferation [13-18]. In that context it is no surprise that LIN28 and c-Myc (and therefore let-7 and miR-17-92 by association) and more recently Oct4/Sox2 regulated miR-302 has been shown to be among a handful of factors shown to be necessary and sufficient to convert differentiated cells to induced pluripotent stem (iPS) cells [19-29]. It is also no surprise that activation of miR-17-92 (OncomiRs) and down-regulation of let-7 (tumor suppressors) is a recurring theme in relation to cancers from multiple systems [30-48]. We speculate that the LIN28/let-7; c-MYC-E2F/miR-17-92 and Oct4/Sox2/miR-302-cyclin D1 networks are fundamental to properties of pluripotency and self-renewal associated with embryonic stem cells. We also speculate that ES cell miRNA-mRNA associations may also regulate tissue homeostasis and regeneration in the fully developed adult. Consequently, the appropriate regulation of LIN28/let-7; c-MYC-E2F/miR-17-92 and Oct4/Sox2/miR-302-cyclin D1 gene networks will be critical for the success of regenerative strategies that involve iPS cells. Perturbations in any of the key ES cell miRNA-mRNA networks maybe a hallmark of cancer stem cells (CSCs).
Abstract Background: Epithelial ovarian cancer is the 5th leading cause of cancer death in women. Our objective was to identify key genetic events important for the pathogenesis of this lethal disease. Methods: Levels of microRNA (miRNA) expression were examined in specimens of primary papillary serous carcinomas, normal ovary and distal fallopian tube using Next Generation Sequencing and a custom expression array. Chromosomal gains and losses were also examined by CGH. SYBR Green reagents were used to measure relative expression of target gene expression by quantitative real-time PCR. Functional impact of altered miRNA expression was tested using standard MTT and Caspase 3/7 assays to measure proliferation and apoptosis (Promega). Key outcome demographics were coded and correlated with miRNA and gene expression by Kaplan-Meier analysis. Results: A total of 140 miRNAs were differentially expressed when papillary serous ovarian cancers were compared to either fimbrae of normal fallopian or normal ovary. Of these, 36 miRNAs were found to correlate with either overall survival, disease free interval (DFI) or platinum sensitivity. Nineteen (19) of these clinically significant miRNAs mapped to single primate-specific genomic locus located at 19q13.41. This locus spanned 125 Kb of non-coding DNA and encoded a total of 44 miRNAs, most all of which showed significant copy number variation in papillary serous ovarian cancers (n = 178) and showed copy losses in the majority of tumors. Using established algorithms for target prediction, we found that this miRNA cluster collectively targeted more than 2800 distinct genes. Key loci included gene products implicated in the epithelial-to-mesenchymal transition (Snail, Slug) as well as both the G1-S and G2-M cell cycle checkpoints (MYCN and Wee1). Transfection of established ovarian cancer cell lines with individual 19q13.41 miRNAs significantly reduced expression of Snail, Slug, Wee1, resulting in altered proliferation and apoptosis. Conclusions: Altered expression of 19q13.41 cluster miRNAs correlate with significant clinical outcomes for women with papillary serous ovarian cancers. These miRNAs appear to play a key role in regulating the expression of gene products critical for the ongoing proliferation and metastasis of ovarian cancer. Future work will focus on dissecting the role of individual 19q13.41 miRNAs in ovarian and other cancers as well as validating the novel nanoparticle-based strategies we have developed for therapeutic miRNA delivery. Supported by NIH TCGA and the Ovarian Cancer Research Foundation (OCRF) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2029.
Background Over 96% of high‐grade ovarian carcinomas and 50% of all cancers are characterized by alterations in the p53 gene. Therapeutic strategies to restore and/or reactivate the p53 pathway have been challenging. By contrast, p63 , which shares many of the downstream targets and functions of p53 , is rarely mutated in cancer. Methods A novel strategy is presented for circumventing alterations in p53 by inducing the tumor‐suppressor isoform TAp63 (transactivation domain of tumor protein p63 ) through its direct downstream target, microRNA‐130b (miR‐130b), which is epigenetically silenced and/or downregulated in chemoresistant ovarian cancer. Results Treatment with miR‐130b resulted in: 1) decreased migration/invasion in HEYA8 cells ( p53 wild‐type) and disruption of multicellular spheroids in OVCAR8 cells ( p53 ‐mutant) in vitro, 2) sensitization of HEYA8 and OVCAR8 cells to cisplatin (CDDP) in vitro and in vivo, and 3) transcriptional activation of TAp63 and the B‐cell lymphoma ( Bcl )‐inhibitor B‐cell lymphoma 2‐like protein 11 ( BIM ). Overexpression of TAp63 was sufficient to decrease cell viability, suggesting that it is a critical downstream effector of miR‐130b. In vivo, combined miR‐130b plus CDDP exhibited greater therapeutic efficacy than miR‐130b or CDDP alone. Mice that carried OVCAR8 xenograft tumors and were injected with miR‐130b in 1,2‐dioleoyl‐sn‐glycero‐3‐phosphatidylcholine (DOPC) liposomes had a significant decrease in tumor burden at rates similar to those observed in CDDP‐treated mice, and 20% of DOPC–miR‐130b plus CDDP‐treated mice were living tumor free. Systemic injections of scL–miR‐130b plus CDDP in a clinically tested, tumor‐targeted nanocomplex (scL) improved survival in 60% and complete remissions in 40% of mice that carried HEYA8 xenografts. Conclusions The miR‐130b/ TAp63 axis is proposed as a new druggable pathway that has the potential to uncover broad‐spectrum therapeutic options for the majority of p53 ‐altered cancers.
Supplementary Table 4 from Molecular Profiling Uncovers a p53-Associated Role for MicroRNA-31 in Inhibiting the Proliferation of Serous Ovarian Carcinomas and Other Cancers
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