p53-Bad* in a Hepatocellular Carcinoma Mouse Model.

Recent advances in liver cancer treatments have not changed the fact that the majority of patients will not survive the disease. In order to advance future liver cancer treatments, this work presents an exploration of various iterations of the liver cancer specific AFP promoter as well as the gene construct p53-Bad*. p53-Bad* is a mitochondrially targeted re-engineered p53 therapy that has shown previous success in a zebrafish HCC model. Both the most promising AFP promoter and p53-Bad* were packaged in an adenoviral delivery system and tested in vitro in liver cancer cell lines. Finally, mixed results for adenoviral p53-Bad* in vivo are presented, and this work suggests future modifications to study parameters in order to further explore the potential of p53-Bad* as a potential liver cancer therapeutic.

The Challenges and Prospects of p53-Based Therapies in Ovarian Cancer.

It has been well established that mutations in the tumor suppressor gene, p53, occur readily in a vast majority of cancer tumors, including ovarian cancer. Typically diagnosed in stages three or four, ovarian cancer is the fifth leading cause of death in women, despite accounting for only 2.5% of all female malignancies. The overall 5-year survival rate for ovarian cancer is around 47%; however, this drops to an abysmal 29% for the most common type of ovarian cancer, high-grade serous ovarian carcinoma (HGSOC). HGSOC has upwards of 96% of cases expressing mutations in p53. Therefore, wild-type (WT) p53 and p53-based therapies have been explored as treatment options via a plethora of drug delivery vehicles including nanoparticles, viruses, polymers, and liposomes. However, previous p53 therapeutics have faced many challenges, which have resulted in their limited translational success to date. This review highlights a selection of these historical p53-targeted therapeutics for ovarian cancer, why they failed, and what the future could hold for a new generation of this class of therapies.

p53-Bad* Fusion Gene Therapy Induces Apoptosis In Vitro and Reduces Zebrafish Tumor Burden in Hepatocellular Carcinoma.

With few curative treatments and a global yearly death rate of over 800,000, hepatocellular carcinoma (HCC) desperately needs new therapies. Although wild-type p53 gene therapy has been shown to be safe in HCC patients, it has not shown enough efficacy to merit approval. This work aims to show how p53 can be re-engineered through fusion to the pro-apoptotic BH3 protein Bcl-2 antagonist of cell death (Bad) to improve anti-HCC activity and potentially lead to a novel HCC therapeutic, p53-Bad*. p53-Bad* is a fusion of p53 and Bad, with two mutations, S112A and S136A. We determined mitochondrial localization of p53-Bad* in liver cancer cell lines with varying p53 mutation statuses via fluorescence microscopy. We defined the apoptotic activity of p53-Bad* in four liver cancer cell lines using flow cytometry. To determine the effects of p53-Bad* in vivo, we generated and analyzed transgenic zebrafish expressing hepatocyte-specific p53-Bad*. p53-Bad* localized to the mitochondria regardless of the p53 mutation status and demonstrated superior apoptotic activity over WT p53 in early, middle, and late apoptosis assays. Tumor burden in zebrafish HCC was reduced by p53-Bad* as measured by the liver-to-body mass ratio and histopathology. p53-Bad* induced significant apoptosis in zebrafish HCC as measured by TUNEL staining but did not induce apoptosis in non-HCC fish. p53-Bad* can induce apoptosis in a panel of liver cancer cell lines with varying p53 mutation statuses and induce apoptosis/reduce HCC tumor burden in vivo in zebrafish. p53-Bad* warrants further investigation as a potential new HCC therapeutic.

Advances in delivery vectors for gene therapy in liver cancer.

Hepatocellular carcinoma (HCC) is the third most common cause of cancer death globally, mainly due to lack of effective treatments - a problem that gene therapy is poised to solve. Successful gene therapy requires safe and efficient delivery vectors, and recent advances in both viral and nonviral vectors have made an important impact on HCC gene therapy delivery. This review explores how adenoviral, retroviral and adeno-associated viral vectors have been modified to increase safety and delivery capacity, highlighting studies and clinical trials using these vectors for HCC gene therapy. Nanoparticles, liposomes, exosomes and virosomes are also featured in their roles as HCC gene delivery vectors. Finally, new discoveries in gene editing technology and their impacts on HCC gene therapy are discussed.

p53-Bad: A Novel Tumor Suppressor/Proapoptotic Factor Hybrid Directed to the Mitochondria for Ovarian Cancer Gene Therapy.

Clinical trials involving p53 gene therapy for ovarian cancer failed due to the dominant negative inhibition of wild-type p53 and multiple genetic aberrations in ovarian cancer. To overcome this problem, we have designed a more potent chimeric gene fusion, called p53-Bad, that combines p53 with the mitochondrial pro-apoptotic factor Bad. Unlike wild-type p53, which acts as a nuclear transcription factor, this novel p53-Bad construct has multiple unique mechanisms of action including a direct and rapid apoptotic effect at the mitochondria. The mitochondrial localization, transcription activity, and apoptotic activity of the constructs were tested. The results suggest that p53 can be effectively targeted to the mitochondria by controlling the phosphorylation of pro-apoptotic Bad, which can only localize to the mitochondria when Ser-112 and Ser-136 of Bad are unphosphorylated. By introducing S112A and S136A mutations, p53-Bad fusion cannot be phosphorylated at these two sites and always localizes to the mitochondria. p53-Bad constructs also have superior activity over p53 and Bad alone. The apoptotic activity is consistent in many ovarian cancer cell lines regardless of the endogenous p53 status. Both p53 and the BH3 domain of Bad contribute to the superior activity of p53-Bad. Our data suggests that p53-Bad fusions are capable of inducing apoptosis and should be further pursued for gene therapy for ovarian cancer.

Mitochondrially targeted p53 or DBD subdomain is superior to wild type p53 in ovarian cancer cells even with strong dominant negative mutant p53.

BACKGROUND: While tumor suppressor p53 functions primarily as a transcription factor in the nucleus, cellular stress can cause p53 to translocate to the mitochondria and directly trigger a rapid apoptotic response. We have previously shown that fusing p53 (or its DNA binding domain, DBD, alone) to the mitochondrial targeting signal (MTS) from Bak or Bax can target p53 to the mitochondria and induce apoptosis in gynecological cancer cell lines including cervical cancer cells (HeLa; wt p53), ovarian cancer cells (SKOV-3; p53 267del non-expressing), and breast cancer cells (T47D; L194F p53 mutation). However, p53 with Bak or Bax MTSs have not been previously tested in cancers with strong dominant negative (DN) mutant p53 which are capable of inactivating wt p53 by homo-oligomerization. Since p53-Bak or Bax MTS constructs act as monomers, they are not subject to DN inhibition. For this study, the utility of p53-Bak or p53-Bax MTS constructs was tested for ovarian cancers which are known to have varying p53 statuses, including a strong DN contact mutant p53 (Ovcar-3 cells), a p53 DN structural mutant (Kuramochi cells), and a p53 wild type, low expressing cells (ID8). RESULTS: Our mitochondrial p53 constructs were tested for their ability to localize to the mitochondria in both mutant non-expressing p53 (Skov-3) and p53 structural mutant (Kuramochi) cell lines using fluorescence microscopy and a nuclear transcriptional activity assay. The apoptotic activity of these mitochondrial constructs was determined using a mitochondrial outer membrane depolarization assay (TMRE), caspase assay, and a late stage cell death assay (7-AAD). We also tested the possibility of using our constructs with paclitaxel, the current standard of care in ovarian cancer treatment. Our data indicates that our mitochondrial p53 constructs are able to effectively localize to the mitochondria in cancer cells with structural mutant p53 and induce apoptosis in many ovarian cancer cell lines with different p53 statuses. These constructs can also be used in combination with paclitaxel for an increased apoptotic effect. CONCLUSIONS: The results suggest that targeting p53 to mitochondria can be a new strategy for ovarian cancer treatment.

Delivery of drugs and macromolecules to the mitochondria for cancer therapy.

Mitochondria are organelles that have pivotal functions in producing the energy necessary for life and executing the cell death pathway. Targeting drugs and macromolecules to the mitochondria may provide an effective means of inducing cell death for cancer therapy, and has been actively pursued in the last decade. This review will provide a brief overview of mitochondrial structure and function, how it relates to cancer, and importantly, will discuss different strategies of mitochondrial delivery including delivery using small molecules, peptides, genes encoding proteins and MTSs, and targeting polymers/nanoparticles with payloads to the mitochondria. The advantages and disadvantages for each strategy will be discussed. Specific examples using the latest strategies for mitochondrial targeting will be evaluated, as well as potential opportunities for specific mitochondrial compartment localization, which may lead to improvements in mitochondrial therapeutics. Future perspectives in mitochondrial targeting of drugs and macromolecules will be discussed. Currently this is an under-explored area that is prime for new discoveries in cancer therapeutics.