Reports of research work funded by grants prior to 2012
Malaghan Institute of Medical Research
Self-Renewal Properties of LAM cells
JW Baty and M Berridge
Lymphangioleiomyomatosis (LAM) is a progressive cellular disease characterized by excessive smooth muscle-like cells in the lungs of women in their childbearing years. It occurs as an isolated disorder with a prevalence of approximately 1-5 per million women, and also occurs in up to 40% of patients with tuberous sclerosis complex (TSC), which is an autosomal disorder affecting 1/5,800. Involvement of tumour suppressor genes, uncontrolled cellular proliferation, abnormal smooth muscle differentiation and disease progression, suggests that LAM is a cancer-like disease in which regulation of normal cell growth has been lost. The first goal of this project was to investigate the potential for LAM to be sustained by a population of cells with self-renewal properties as proposed in the cancer stem cell model. Primary early passage LAM and non LAM smooth muscle cells were obtained from Prof Judy Black and Dr Lyn Moir (Woolcock Institute, Sydney). Our work indicated that the LAM and non LAM lung cells were not able to form “spheres” typical of cancer stem cells even after 2 – 4 weeks of culture in stem cell-favouring media. However, the cells did increase expression of self-renewal genes that are associated with a stem cell phenotype. It remains unclear how upregulation of self-renewal genes relates to the inability of the cells to divide and it is possible that additional factors are required. These results suggest that upregulation of self-renewal genes is a general characteristic of lung smooth muscle cells grown under these conditions. Other approaches including HMB45 staining, gp100 gene expression, estrogen receptor expression, response to estradiol, and rapamycin sensitivity failed to distinguish between the LAM and non LAM cells. At least two scenarios may account for this: (1) the primary LAM cells have adapted to the culture conditions, perhaps with epigenetic changes that mask their abnormal phenotype; or (2) non LAM smooth muscle cells have been present in the original LAM samples and have overgrown the LAM cells in culture.
In subsequent work, we focused our attention on TSC2 knockout cells (TSC2 -/-). TSC2 is a key intracellular signalling protein that is inactivated by a two-hit mutation in LAM cells. Normal TSC2 forms a heterodimer with TSC1 that inhibits the activity of a central signalling hub called mTOR complex 1. However, a loss of function mutation in TSC2 leads to the constitutive activation of mTOR complex 1 and results in the unregulated cell growth observed in LAM. We acquired TSC2 wildtype (+/+), knockout (-/-) mouse embryonic fibroblasts (MEFs) from Assoc Prof Vera Krymskaya, University of Pennsylvania, and looked at the effect of targeted drug treatment on the cells, and also how the cells responded to specific conditions of stress.
Rapamycin and metformin
Constitutive activation of the mTOR complex 1 due to inactivated TSC2 can be overcome by treatment with the immuno-suppressant drug rapamycin. Rapamycin has been shown to restore homeostasis in cells with defective TSC gene function, and to be useful in treating LAM-related lung disease. Whereas rapamycin is thought to only target mTOR complex 1, the diabetes drug, metformin, has multiple intracellular targets including mTOR complex 1and mitochondrial respiratory chain complex 1. We treated the TSC2 +/+ and -/- MEFs with metformin and rapamycin and monitored their viability and proliferation over two days to see if combining the drugs might be more effective. TSC2 -/- MEFs were more sensitive to rapamycin treatment than TSC2 +/+ cells even at low concentrations (2 nM). However, there was no difference between the TSC2 -/- and +/+ MEFs with metformin treatment. A combination of the drugs was slightly more effective than either agent alone, but there was no specificity for TSC2 -/- MEFs.
A defining characteristic of TSC2 -/- cells is their ability to continue proliferating in the absence of serum in culture. To test our hypothesis that TSC2 -/- cells would also be resistant to other forms of stress such as oxidative stress we treated TSC2 +/+ and -/- MEFS with a range of hydrogen peroxide concentrations and monitored their viability and proliferation. Our results indicated that the TSC2-/- MEFs were indeed resistant to hydrogen peroxide at certain concentrations and suggest that TSC2 inactivation confers a growth advantage to cells under stress conditions.
Primary LAM and non LAM smooth muscle cells could not be distinguished by HMB45 staining, gp100 gene expression, estrogen receptor expression, response to estradiol or to the mTORC1 inhibitor, rapamycin, or by their stem cell properties. In contrast TSC2 deficient mouse embryonic fibroblasts were differentially sensitive to rapamycin and resistant to oxidative stress and serum-free cell culture. Taken together these results highlight the difficulty of using primary LAM cells in vitro as a model of LAM and suggest that changes might have occurred to the LAM cells in culture.