iRFP (near-infrared fluorescent protein) imaging of subcutaneous and deep tissue tumours in mice highlights differences between imaging platforms.

BACKGROUND: In vivo imaging using fluorescence is used in cancer biology for the detection, measurement and monitoring of tumours. This can be achieved with the expression of fluorescent proteins such as iRFP, which emits light at a wavelength less attenuated in biological tissues compared to light emitted by other fluorescent proteins such as GFP or RFP. Imaging platforms capable of detecting fluorescent tumours in small animals have been developed but studies comparing the performance of these platforms are scarce. RESULTS: Through access to three platforms from Xenogen, Bruker and Li-Cor, we compared their ability to detect iRFP-expressing subcutaneous tumours as well as tumours localised deeper within the body of female NSG mice. Each platform was paired with proprietary software for image analyse, but the output depends on subjective decisions from the user. To more objectively compare platforms, we developed an 'in house' software-based approach which results in lower measured variability between mice. CONCLUSIONS: Our comparisons showed that all three platforms allowed for reliable detection and monitoring of subcutaneous iRFP tumour growth. The biggest differences between platforms became apparent when imaging deeper tumours with the Li-Cor platform detecting most tumours and showing the highest dynamic range.

The cytoskeleton adaptor protein ankyrin-1 is upregulated by p53 following DNA damage and alters cell migration.

The integrity of the genome is maintained by a host of surveillance and repair mechanisms that are pivotal for cellular function. The tumour suppressor protein p53 is a major component of the DNA damage response pathway and plays a vital role in the maintenance of cell-cycle checkpoints. Here we show that a microRNA, miR-486, and its host gene ankyrin-1 (ANK1) are induced by p53 following DNA damage. Strikingly, the cytoskeleton adaptor protein ankyrin-1 was induced over 80-fold following DNA damage. ANK1 is upregulated in response to a variety of DNA damage agents in a range of cell types. We demonstrate that miR-486-5p is involved in controlling G1/S transition following DNA damage, whereas the induction of the ankyrin-1 protein alters the structure of the actin cytoskeleton and sustains limited cell migration during DNA damage. Importantly, we found that higher ANK1 expression correlates with decreased survival in cancer patients. Thus, these observations highlight ANK1 as an important effector downstream of the p53 pathway.

Functional interplay between MDM2, p63/p73 and mutant p53.

Many cancers express mutant p53 proteins that have lost wild-type tumor suppressor activity and, in many cases, have acquired oncogenic functions that can contribute to tumor progression. These activities of mutant p53 reflect interactions with several other proteins, including the p53 family members p63 and p73. Mutations in p53 that affect protein conformation (such as R175H) show strong binding to p63 and p73, whereas p53 mutants that only mildly affect the conformation (such as R273H) bind less well. A previously described aggregation domain of mutant p53 is not required for p63 or p73 binding; indeed, mutations within this region lead to the acquisition of a mutant p53 phenotype-including a conformational shift, p63/p73 binding and the ability to promote invasion. The activity of wild-type p53 is regulated by an interaction with MDM2 and we have investigated the potential role of MDM2 in the mutant p53/p63/p73 interactions. Both mutant p53 and p73 bind MDM2 well, whereas p63 binds much more weakly. We found that MDM2 can inhibit p63 binding to p53R175H but enhances the weaker p53R273H/p73 interaction. These effects on the interactions are reflected in an ability of MDM2 to relieve the inhibition of p63 by p53R175H, but enhance the inhibition of p73 activity by p53R175H and R273H. We propose a model in which MDM2 competes with p63 for binding to p53R175H to restore p63 activity, but forms a trimeric complex with p73 and p53R273H to more strongly inhibit p73 function.

Functions of TAp63 and p53 in restraining the development of metastatic cancer.

Many tumours harbour mutations in the p53 tumour-suppressor gene that result in the expression of a mutant p53 protein. This mutant p53 protein has, in most cases, lost wild-type transcriptional activity and can also acquire novel functions in promoting invasion and metastasis. One of the mechanisms underlying these novel functions involves the ability of the mutant p53 to interfere with other transcription factors, including the p53 family protein TAp63. To investigate whether simultaneous depletion of both p53 and TAp63 can recapitulate the effect of mutant p53 expression in vivo, we used a mouse model of pancreatic cancer in which the expression of mutant p53 resulted in the rapid appearance of primary tumours and metastases. As shown previously, loss of one allele of wild-type (WT) p53 accelerated tumour development. A change of one WT p53 allele into mutant p53 did not further accelerate tumour development, but did promote the formation of metastasis. By contrast, loss of TAp63 did not significantly accelerate tumour development or metastasis. However, simultaneous depletion of p53 and TAp63 led to both rapid tumour development and metastatic potential, although the incidence of metastases remained lower than that seen in mutant p53-expressing tumours. TAp63/p53-null cells derived from these mice also showed an enhanced ability to scatter and invade in tissue culture as was observed in mutant p53 cells. These data suggest that depletion of TAp63 in a p53-null tumour can promote metastasis and recapitulate-to some extent-the consequences of mutant p53 expression.

Mutant p53 enhances MET trafficking and signalling to drive cell scattering and invasion.

Tumour-derived mutant p53 proteins promote invasion, in part, by enhancing Rab coupling protein (RCP)-dependent receptor recycling. Here we identified MET as an RCP-binding protein and showed that mutant p53 promoted MET recycling. Mutant p53-expressing cells were more sensitive to hepatocyte growth factor, the ligand for MET, leading to enhanced MET signalling, invasion and cell scattering that was dependent on both MET and RCP. In cells expressing the p53 family member TAp63, inhibition of TAp63 also lead to cell scattering and MET-dependent invasion. However, in cells that express very low levels of TAp63, the ability of mutant p53 to promote MET-dependent cell scattering was independent of TAp63. Taken together, our data show that mutant p53 can enhance MET signalling to promote cell scattering and invasion through both TAp63-dependent and -independent mechanisms. MET has a predominant role in metastatic progression and the identification of mechanisms through which mutations in p53 can drive MET signalling may help to identify and direct therapy.