Identification and characterisation of a novel p21 feedback loop in the regulation of p53

The p53 tumour-suppressor protein plays a critical role in the cellular response to environmental and intracellular stresses that threaten DNA integrity. Inactivation of p53 represents an important step during carcinogenesis and is associated with genomic instability and tumour development. A key transcriptional target of p53 is the cyclin-dependent kinase inhibitor, p21WAF1/CIP1 (hereafter referred to as p21), which mediates p53-dependent G1 arrest. The role of p21 in tumour development remains contentious. Early studies showed that p21 mutations are rare in human cancers however there is a growing list of human carcinomas that have aberrant p21 expression. p21-null animals also have elevated tumour incidence, but the mechanism underlying this is not yet defined.Our data identifies p21 as a component of a positive feedback loop that maintains the p53 transcriptional response. Three model systems were used to characterise this novel mechanism of p53 regulation. In the human colon carcinoma cell line HCT116 with targeted inactivation of p21, p53 stabilisation is uncoupled from its activity as a transcription factor and shows defects in the p53 response to DNA damage and double stranded RNA, indicating that a common mechanism prevents p53 activation by distinct stresses in the absence of p21. The p53 transcriptional programme in response to cellular damage can be reactivated after complementation of the p21 gene into the HCT116 p21-null cells. In B-cells from mice lacking the p21 gene, a striking loss of the p53-dependent transcription programme was identified using p53-specific microarray screens. Gene dosage effects indicate a progressive loss of p53 function in B-cells heterozygous or homozygous null for p21. Similarly, siRNA to p21 can attenuate the p53-dependent transcription response in normal human fibroblasts. In all three model systems, deletion of the p21 gene results in p53 nuclear export and eliminates the p53 transcriptional response. This data indicates that p53 has evolved a co-ordinated transcription mechanism to control its own function: a positive feedback loop maintained by p21 and a negative feedback loop maintained by MDM2, whose balance controls the specific activity of p53

Incorporation of aptamers in the terminal loop of shRNAs yields an effective and novel combinatorial targeting strategy.

Gene therapy by engineering patient's own blood cells to confer HIV resistance can potentially lead to a functional cure for AIDS. Toward this goal, we have previously developed an anti-HIV lentivirus vector that deploys a combination of shRNA, ribozyme and RNA decoy. To further improve this therapeutic vector against viral escape, we sought an additional reagent to target HIV integrase. Here, we report the development of a new strategy for selection and expression of aptamer for gene therapy. We developed a SELEX protocol (multi-tag SELEX) for selecting RNA aptamers against proteins with low solubility or stability, such as integrase. More importantly, we expressed these aptamers in vivo by incorporating them in the terminal loop of shRNAs. This novel strategy allowed efficient expression of the shRNA-aptamer fusions that targeted RNAs and proteins simultaneously. Expressed shRNA-aptamer fusions targeting HIV integrase or reverse transcriptase inhibited HIV replication in cell cultures. Viral inhibition was further enhanced by combining an anti-integrase aptamer with an anti-HIV Tat-Rev shRNA. This construct exhibited efficacy comparable to that of integrase inhibitor Raltegravir. Our strategy for the selection and expression of RNA aptamers can potentially extend to other gene therapy applications

Exosomes Derived from Radioresistant Breast Cancer Cells Promote Therapeutic Resistance in Naïve Recipient Cells

Radiation resistance is a significant challenge in the treatment of breast cancer in humans. Human breast cancer is commonly treated with surgery and adjuvant chemotherapy/radiotherapy, but recurrence and metastasis upon the development of therapy resistance results in treatment failure. Exosomes are extracellular vesicles secreted by most cell types and contain biologically active cargo that, when transferred to recipient cells, can influence the cells’ genome and proteome. We propose that exosomes secreted by radioresistant (RR) cells may be able to disseminate the RR phenotype throughout the tumour. Here, we isolated exosomes from the human breast cancer cell line, MDA-MB-231, and the canine mammary carcinoma cell line, REM134, and their RR counterparts to investigate the effects of exosomes derived from RR cells on non-RR recipient cells. Canine mammary cancer cells lines have previously been shown to be excellent translational models of human breast cancer. This is consistent with our current data showing that exosomes derived from RR cells can increase cell viability and colony formation in naïve recipient cells and increase chemotherapy and radiotherapy resistance, in both species. These results are consistent in cancer stem cell and non-cancer stem cell populations. Significantly, exosomes derived from RR cells increased the tumoursphere-forming ability of recipient cells compared to exosomes derived from non-RR cells. Our results show that exosomes are potential mediators of radiation resistance that could be therapeutically targeted

Melanocortin 1 receptor targeted imaging of melanoma with gold nanocages and positron emission tomography

Purpose: Melanoma is a lethal skin cancer with unmet clinical needs for targeted imaging and therapy. Nanoscale materials conjugated with targeting components have shown great potential to improve tumor delivery efficiency while minimizing undesirable side effects in vivo. Herein, we proposed to develop targeted nanoparticles for melanoma theranostics. Method: In this work, gold nanocages (AuNCs) were conjugated with α-melanocyte-stimulating hormone (α-MSH) peptide and radiolabeled with 64Cu for melanocortin 1 receptor-(MC1R) targeted positron emission tomography (PET) in a mouse B16/F10 melanoma model. Results: Their controlled synthesis and surface chemistry enabled well-defined structure and radiolabeling efficiency. In vivo pharmacokinetic evaluation demonstrated comparable organ distribution between the targeted and nontargeted AuNCs. However, micro-PET/computed tomography (CT) imaging demonstrated specific and improved tumor accumulation via MC1R-mediated delivery. By increasing the coverage density of α-MSH peptide on AuNCs, the tumor delivery efficiency was improved. Conclusion: The controlled synthesis, sensitive PET imaging, and optimal tumor targeting suggested the potential of targeted AuNCs for melanoma theranostics. </jats:sec

The class I PI3K/Akt pathway is critical for cancer cell survival in dogs and offers an opportunity for therapeutic intervention

<p>Abstract</p> <p>Background</p> <p>Using novel small-molecular inhibitors, we explored the feasibility of the class I PI3K/Akt/mTORC1 signaling pathway as a therapeutic target in canine oncology either by using pathway inhibitors alone, in combination or combined with conventional chemotherapeutic drugs <it>in vitro</it>.</p> <p>Results</p> <p>We demonstrate that growth and survival of the cell lines tested are predominantly dependent on class I PI3K/Akt signaling rather than mTORC1 signaling. In addition, the newly developed inhibitors ZSTK474 and KP372-1 which selectively target pan-class I PI3K and Akt, respectively, and Rapamycin which has been well-established as highly specific mTOR inhibitor, decrease viability of canine cancer cell lines. All inhibitors demonstrated inhibition of phosphorylation of pathway members. Annexin V staining demonstrated that KP372-1 is a potent inducer of apoptosis whereas ZSTK474 and Rapamycin are weaker inducers of apoptosis. Simultaneous inhibition of class I PI3K and mTORC1 by ZSTK474 combined with Rapamycin additively or synergistically reduced cell viability whereas responses to the PI3K pathway inhibitors in combination with conventional drug Doxorubicin were cell line-dependent.</p> <p>Conclusion</p> <p>This study highlighted the importance of class I PI3K/Akt axis signaling in canine tumour cells and identifies it as a promising therapeutic target.</p

Correction:Canine Mammary Cancer Stem Cells Are Radio- and Chemo- Resistant and Exhibit an Epithelial-Mesenchymal Transition Phenotype.

In the original article [...