Cytoskeleton and Cancer - Research Units - Cytoskeleton and Cancer - Research themes
The role of LIMK2 in cancer metastasis and childhood cancers
Because of the similarity between LIMK1 and LIMK2 we would like to explore the possibility that LIMK2, like LIMK1 is also involved in metastasis. We propose to study the level of LIMK1 and LIMK2 in metastastic and non-metastatic breast and prostate cancer cell lines and in metastatic and non-metastatic human tumours. We will express LIMK2 in non-invasive cell lines and inhibit LIMK2 activity by expressing dominate-negative kinase dead LIMK2 and LIMK2-si-RNA. The invasiveness of these cell lines will be determined in vitro and in vivo. To study the role of LIMK2 in childhood cancers we chose to explore first its role in neuroblastoma. We have demonstrated the neuroblastoma cells resistant to vinblastin express high levels of LIMK2. We will explore the possibility that inhibition of LIMK2 will sensitize these cells to the drug.
The role of LIMK2 in obesity and type II diabetes
LIMK2 has two isoforms: LIMK2a and LIMK2b which differ by only 22 amino acids at their N-terminus. The difference between the functions of these two proteins is not yet known. One of the techniques used to establish the function of a protein is to generate mice that either overexpress or do not express this protein. We have generated a mouse devoid of the LIMK2a protein. These mice, known as LIMK2-/-, are obese and develop type II diabetes at 3 months of age. We are currently studying how the deletion of LIMK2a induces obesity in these mice.
The search for new LIMK2 substrates
There are data in the literature suggesting that LIMK2 may have additional substrate to cofilin. We are therefore exploring the possibility that twinfilin, a member of the cofilin family of actin binding proteins, is also a LIMK2 substrate. Furthermore, we are undertaking a search for LIMK2 substrates in the fat tissue of mice. We will phosphorylate proteins prepared from this tissue with active LIMK2, isolate phospho-proteins on affinity column and identify them by mass spectrometry. The aim of these experiments is to identify substrates important for keeping mice lean and inhibit the development of type II diabetes.
The role of Rnf6 in regulation of LIMK1 level in cells
We have demonstrated that overexpression of Rnf6 reduces the level of LIMK1 in the transfected cells. We will therefore study the effect of Rnf6, Rnf6 DN and Rnf6 siRNA overexpression on the level of LIMK1 in a variety of cell lines. We will compare LIMK1 levels in cells expressing Rnf6 mutants. To date 4 mutations in the Rnf6 gene were identified in human Esophageal Squamous Cell Carcinoma (ESCC), a very aggressive tumour with poor prognosis. We propose to generate constructs harbouring these mutations, express them in cell lines and compare the level of LIMK1 in the transfected cells with cells expressing wild type Rnf6 and Rnf6 siRNA. To demonstrate that these mutations are responsible for the aggressiveness of these tumours we will examine the invasiveness of cell lines stably expressing these Rnf6 mutants in vivo and in vitro.