The onset of metastasis in organs such as the lung, bone and brain is a major cause of mortality in breast cancer patients. Many signalling pathways have been implicated in mediating progression to metastatic disease, including the transforming growth factor beta (TGFβ) signalling pathway. In many tissues TGFβ results in a growth inhibitory signal. This is mediated by transducers of the Smad family, which translocate to the nucleus and activate transcription. In tumour cells, however, TGFβ-dependent antiproliferative control is lost and cells acquire the ability to replicate in TGFβ-rich environments. Despite molecular and clinical evidence pointing to a role for TGFβ signalling in cancer progression and metastasis, it is unclear at which points of the metastatic process TGFβ signalling occurs and whether it is necessary and/or sufficient to elicit cancer cell motility.
To address these questions, MTln3E rat breast cancer cells were used as a relevant model system. When injected into the mammary fat pad of nude mice these cells form a primary tumour from which motile cells will depart to form metastasis in the lymph nodes and the lungs. To gain insight into TGFβ signalling in vivo, MTln3E cells were engineered to express either GFPSmad2 or a Smad3 responsive promoter driving the expression of ECFP (CAGA::ECFP). This allowed the monitoring of Smad-dependent TGFβ signalling in vivo using multiphoton confocal microscopy.
Our results indicate that TGFβ signalling, measured by cytoplasmic to nuclear translocation of GFPSmad2 and by activation of CAGA ECFP, does not occur ubiquitously within the primary tumour. In particular, cells that have acquired a motile phenotype display active TGFβ signalling. As nuclear localisation of GFPSmad2 and activation of CAGA ECFP are also observed in nonmotile cells, however, TGFβ signalling may be necessary but not sufficient to elicit cell motility in primary tumour cells. Furthermore, activation of TGFβ signalling in motile cells is transient, as lymph node metastasis display little activation of the pathway. In addition, we have uncovered a second role for TGFβ signalling in the metastatic process. After intravenous injection in mouse tail vein, TGFβ pretreated cells colonise the lungs more efficiently than untreated controls and this results from the ability of these cells to survive clearance from the lungs.
Together these data suggest that TGFβ signalling may positively influence two distinct steps of the metastatic cascade, first by enabling cells to become motile and second to enhance their survival during the lung colonisation.
Supported by Breast Cancer Campaign.