A RAC-GEF network critical for early intestinal tumourigenesis.

RAC1 activity is critical for intestinal homeostasis, and is required for hyperproliferation driven by loss of the tumour suppressor gene Apc in the murine intestine. To avoid the impact of direct targeting upon homeostasis, we reasoned that indirect targeting of RAC1 via RAC-GEFs might be effective. Transcriptional profiling of Apc deficient intestinal tissue identified Vav3 and Tiam1 as key targets. Deletion of these indicated that while TIAM1 deficiency could suppress Apc-driven hyperproliferation, it had no impact upon tumourigenesis, while VAV3 deficiency had no effect. Intriguingly, deletion of either gene resulted in upregulation of Vav2, with subsequent targeting of all three (Vav2-/- Vav3-/- Tiam1-/-), profoundly suppressing hyperproliferation, tumourigenesis and RAC1 activity, without impacting normal homeostasis. Critically, the observed RAC-GEF dependency was negated by oncogenic KRAS mutation. Together, these data demonstrate that while targeting RAC-GEF molecules may have therapeutic impact at early stages, this benefit may be lost in late stage disease.

Standardized experiments in mutant mice reveal behavioural similarity on 129S5 and C57BL/6J backgrounds.

Behavioural analysis of mice carrying engineered mutations is widely used to identify roles of specific genes in components of the mammalian behavioural repertoire. The reproducibility and robustness of phenotypic measures has become a concern that undermines the use of mouse genetic models for translational studies. Contributing factors include low individual study power, non-standardized behavioural testing, failure to address confounds and differences in genetic background of mutant mice. We have examined the importance of these factors using a statistically robust approach applied to behavioural data obtained from three mouse mutations on 129S5 and C57BL/6J backgrounds generated in a standardized battery of five behavioural assays. The largest confounding effect was sampling variation, which partially masked the genetic background effect. Our observations suggest that strong interaction of mutation with genetic background in mice in innate and learned behaviours is not necessarily to be expected. We found composite measures of innate and learned behaviour were similarly impacted by mutations across backgrounds. We determined that, for frequently used group sizes, a single retest of a significant result conforming to the commonly used P < 0.05 threshold results in a reproducibility of 60% between identical experiments. Reproducibility was reduced in the presence of strain differences. We also identified a P-value threshold that maximized reproducibility of mutant phenotypes across strains. This study illustrates the value of standardized approaches for quantitative assessment of behavioural phenotypes and highlights approaches that may improve the translational value of mouse behavioural studies.

In vivo functional analysis of Drosophila Gap1: involvement of Ca2+ and IP4 regulation.

Control of Ras activity is crucial for normal cellular behavior such as fate determination during development. Although several GTPase activating proteins (GAPs) have been shown to act as negative regulators of Ras, the mechanisms involved in regulating their activity in vivo are poorly understood. Here we report the structural requirements for Gap1 activity in cone cell fate decisions during Drosophila eye development. The Gap1 catalytic domain alone is not sufficient for in vivo activity, indicating a requirement for the additional domains. An inositol-1,3,4, 5-tetrakisphosphate (IP4)-sensitive extended PH domain is essential for Gap1 activity, while Ca2+-sensitive C2 domains and a glutamine-rich region contribute equally to full activity in vivo. Furthermore, we find a strong positive genetic interaction between Gap1 and phospholipase Cgamma (PLCgamma), an enzyme which generates inositol-1,4,5-trisphosphate, a precursor for IP4 and a second messenger for intracellular Ca2+ release. These results suggest that Gap1 activity in vivo is stimulated under conditions of elevated intracellular Ca2+ and IP4. Since receptor tyrosine kinases (RTKs) trigger an increase in intracellular Ca2+ and IP4 concentration through stimulation of PLCgamma, RTKs may stimulate not only activation of Ras but also its deactivation by Gap1, thereby moderating the strength and duration of the Ras signal.

The malignant capacity of skin tumours induced by expression of a mutant H-ras transgene depends on the cell type targeted.

BACKGROUND: . Pinpointing the cells from which tumours arise is a major challenge n tumour biology. Previous work has shown that the targeted expression of a mutant ras gene within the interfollicular cell compartment of mouse skin induces the formation of benign papillomas, but these do not spontaneously progress to malignancy. We have investigated the carcinogenic effects of expressing the same oncogene in a different population of epidermal cells. RESULTS: Expression of mutant ras from a truncated keratin 5 gene promoter, which directs expression to the follicular and interfollicular cells of newborn mice and the hair follicle cells of adults, stimulated the development of acanthotic areas in newborn mice. Within one week of birth, the acanthotic skin developed areas of carcinoma in situ and adult mice developed papillomas and keratoacanthomas, the latter having a high frequency of spontaneous malignant transformation to squamous and occasionally spindle carcinomas. The benign tumours that arose had several hallmarks of tumours at a high risk of malignant progression, including suprabasal cell proliferation and heterogeneous expression of keratin 13. In contrast to tumours induced by expressing mutant ras under the control of the keratin 10 or keratin 1 gene promoters, the formation of these lesions was not dependent on wounding or a tumour promoter. CONCLUSIONS: Benign tumours that are at a risk of malignant conversion are primarily derived from cells located within the hair follicle, and the nature of the cell in which tumour initiation occurs is a major determinant of malignant potential.