Cell-in-cell (CIC) structures are commonly seen in tumours. Their biological significance remains unclear, although they have been associated with more aggressive tumours. Here we report that mutant p53 promotes CIC via live cell engulfment. Engulfed cells physically interfere in cell divisions of host cells and for cells without p53 this leads to host cell death. In contrast, mutant p53 host cells survive, display aberrant divisions, multinucleation and tripolar mitoses. In xenograft studies, CIC-rich p53 mutant/null co-cultures show enhanced tumour growth. Furthermore, our results show that CIC is common within lung adenocarcinomas, is an independent predictor of poor outcome and disease recurrence, is associated with mutant p53 expression and correlated to measures of heterogeneity and genomic instability. These findings suggest that pro-tumorigenic entotic engulfment activity is associated with mutant p53 expression, and the two combined are a key factor in genomic instability.

The multiple enzymatic activities and functions of transglutaminase type 2 (TG2) may be attributed to alternative TG2 molecules produced by differential splicing of TG2 mRNA. Different RNA transcripts of the human TG2 gene (TGM2) have been identified, but the expression of TG2 multiple transcripts has never been systematically addressed. We have confirmed and rationalized the main TG2 variants and developed a screening assay for the detection of alternative splicing of TG2, based on real-time reverse-transcription PCR. We have quantified the multiple TG2 transcripts in a wide range of normal tissues and in cancer cell lines from four different sites of origin. Our data show a significant correlation in the expression of canonical and alternative TG2 isoforms in normal human tissue, but differences in alternative splicing of TG2 in cancer cell lines, suggesting that in cancer cells the alternative splicing of TG2 is a more active process.

Transgenic mouse models expressing mutant superoxide dismutase 1 (SOD1) have been critical in furthering our understanding of amyotrophic lateral sclerosis (ALS). However, such models generally overexpress the mutant protein, which may give rise to phenotypes not directly relevant to the disorder. Here, we have analysed a novel mouse model that has a point mutation in the endogenous mouse Sod1 gene; this mutation is identical to a pathological change in human familial ALS (fALS) which results in a D83G change in SOD1 protein. Homozgous Sod1D83G/D83G mice develop progressive degeneration of lower (LMN) and upper motor neurons, likely due to the same unknown toxic gain of function as occurs in human fALS cases, but intriguingly LMN cell death appears to stop in early adulthood and the mice do not become paralyzed. The D83 residue coordinates zinc binding, and the D83G mutation results in loss of dismutase activity and SOD1 protein instability. As a result, Sod1D83G/D83G mice also phenocopy the distal axonopathy and hepatocellular carcinoma found in Sod1 null mice (Sod1−/−). These unique mice allow us to further our understanding of ALS by separating the central motor neuron body degeneration and the peripheral effects from a fALS mutation expressed at endogenous levels.

Mutations in TARDBP, encoding Tar DNA binding protein-43 (TDP43), cause amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Attempts to model TDP43 dysfunction in mice have used knockouts or transgenic overexpressors, which have revealed the difficulties of manipulating TDP43, whose level is tightly controlled by auto-regulation. In a complementary approach, to create useful mouse models for the dissection of TDP43 function and pathology, we have identified a nonsense mutation in the endogenous mouse Tardbp gene through screening an N-ethyl-N-nitrosourea (ENU) mutant mouse archive. The mutation is predicted to cause a Q101X truncation in TDP43. We have characterised TardbpQ101X mice to investigate this mutation in perturbing TDP43 biology at endogenous expression levels. We found the TardbpQ101X mutation is homozygous embryonic lethal, highlighting the importance of TDP43 in early development. Heterozygotes (Tardbp+/Q101X) have abnormal levels of mutant transcript, but we find no evidence of the truncated protein and mice have similar full-length TDP43 protein levels as wildtype littermates. Nevertheless, Tardbp+/Q101X mice have abnormal alternative splicing of downstream gene targets, and limb-clasp and body tone phenotypes. Thus the nonsense mutation in Tardbp causes a mild loss-of-function phenotype and behavioural assessment suggests underlying neurological abnormalities. Due to the role of TDP43 in ALS, we investigated potential interactions with another known causative gene, mutant superoxide dismutase 1 (SOD1). Tardbp+/Q101X mice were crossed with the SOD1G93Adl transgenic mouse model of ALS. Behavioural and physiological assessment did not reveal modifying effects on the progression of ALS-like symptoms in the double mutant progeny from this cross. In summary, the TardbpQ101X mutant mice are a useful tool for the dissection of TDP43 protein regulation, effects on splicing, embryonic development and neuromuscular phenotypes. These mice are freely available to the community.

TP53 is the most frequently mutated gene in cancers. Mutations lead to loss of p53 expression or expression of a mutant protein. Mutant p53 proteins commonly lose wild-type function, but can also acquire novel functions in promoting metastasis and chemoresistance. Previously, we uncovered a role for Rab-coupling protein (RCP) in mutant p53-dependent invasion. RCP promotes endosomal recycling and signalling of integrins and receptor tyrosine kinases. In a screen to identify novel RCP-interacting proteins, we discovered P-glycoprotein (P-gp). Thus, we hypothesised that mutant p53 could promote chemoresistance through RCP-dependent recycling of P-gp. The interaction between RCP and P-gp was verified endogenously and loss of RCP or mutant p53 rendered cells more sensitive to cisplatin and etoposide. In mutant p53 cells we detected an RCP-dependent delivery of P-gp to the plasma membrane upon drug treatment and decreased retention of P-gp substrates. A co-localisation of P-gp and RCP was seen in mutant p53 cells, but not in p53-null cells upon chemotherapeutic exposure. In conclusion, mutant p53 expression enhanced co-localisation of P-gp and RCP to allow for rapid delivery of P-gp to the plasma membrane and increased resistance to chemotherapeutics.