Thomas C. Spelsberg, Ph.D.

Dr. Spelsberg's laboratory is engaged in studies of the mechanism of action of steroid hormones and the growth factor, TGFß in human bone and breast cancer tissues.

In project 1 (osteoblast biology) the actions of estrogen and SERMs in normal and transformed human osteoblast cells (for osteoporosis research) are being investigated. The latter includes immortalized fetal human osteoblast cell lines that were developed by this laboratory. These studies involve the estrogen effects on early gene- and late gene-expression, including those of bone matrix proteins, growth factors, and cytokines. We are currently examining the role of another form (i.e., the beta isoform) of the estrogen receptor (ERa) on breast cancer and bone cell functions and gene expression. We have compared the actions of ERa to those of ERß, as well as the interactions and antagonists that these receptors display when co-expressed in osteoblasts. Current studies are examining the molecular interactions of ERa and ERß at the gene transcription level, using chromatin immunoprecipitation assays and deletion constructs. The TIEG gene (described below) and RBP-1 (Rb binding protein) are the ERß and ERa specific genes respectively.

Project 2 (breast cancer and SERMs) This project is supported by the Breast Cancer Research Foundation and represents a collaboration with Dr. Jim Ingle, Mayo oncology. Similar to project 1, we are examining the molecular events involved in the repression of breast cancer cell growth by estrogens/SERMs (selective estrogen receptor modulators such as tamoxifen/raloxifene). These anti-estrogen therapies represent the best possible therapies to treat breast cancer. We have identified unique functions of these components, e.g., estrogen receptor isoforms and variants and co-regulators in these cancer cells, which could explain why these cells become resistant to tamoxifen therapy. Recently, we have been examining the functions of tamoxifen metabolites as inhibitors of breast cancer growth and have identified endoxifen as a primary "effector" and have shown that it acts more like a pure antagonist, ICI. Endoxifen actions appear unique among the other SERMs. We are examining the role/actions of the ERß isoform in SERM inhibition of breast cancer growth as well as the molecular changes which occur in the breast cancer resistance to SERMs. This research could lead to the development of better therapeutic SERMs, as well as a means to reverse this resistance to tamoxifen.

The third project addresses the actions and interactions of TGFß and estrogen on human osteoblast cells in culture. Our laboratory discovered a novel TGFß/estrogen inducible early gene (TIEG1) which is a member of the Kruppel-like, 3 zinc finger, family of transcription factors. TIEG1 was shown to be a Kruppel-like, nuclear, transcription factor, whose expression is rapidly induced upon TGFß treatment. A major goal is to identify functional roles for TIEG, including whether or not it mediates TGFß or estrogen actions in human osteoblasts, osteosarcomas, and other breast cancer cells. TIEG is a part of the TGFß signaling pathway, is a possible tumor suppressor gene and plays a role in cancer, especially with regard to TGFß-Smad pathway. In clinical studies with human tissues, TIEG shows a tissue and cell-type specificity. TIEG gene knockout mice have been generated to examine the developmental and biological functions of TIEG. These female TIEG null mice display bone loss (osteopenia) and more fragile bones compared to wild-type. The male TIEG null mice show no effect. The role of sex steroids in this gender specificity is underway.

Using yeast two hybrid systems, gene arrays, chromatin immunoprecipitation assays, viral expression vectors and si-and sh-RNAs, overexpressing cell lines, we are attempting to identify the signal pathway(s) and co-regulators which involve TIEG. To date, we have evidence that TIEG activates the TGFß-Smad 2, 3, 4 pathway by repressing Smad 7 gene transcription and by activating Smad 2 gene expression. The identity of the regulatory elements in the Smad 7 promoter which bind the TIEG protein is also underway. TIEG protein was recently shown to induce Runx2 gene expression and to interact with the Runx2 protein to regulate other genes. Further TIEG was shown to repress OPG and enhance RANKL expression, thus affecting osteoclastogenesis.

In a fourth project, we are collaborating with a former Mayo cardiology fellow, Dr. Rajamannan, now at Northwestern's Feinberg Medical Center, on the role of lipids and bone formation/calcification of heart valves. We are also collaborating with Dr. Rajamannan and Dr. Mike Ackermann, Mayo Cardiology, on the role of TGFß/TIEG as a cause of hypertrophic cardiomyopathy. We have discovered that our TIEG gene-deficient mice also develop hypertrophic cardiomyopathy (HCM) (large hearts) after puberty, but only in male hearts. The animals do not have any hypertension or other diseases. This is similar to the situation with young human males who die during athletics. We have further evidence that TIEG down-regulates a gene, pttg 1, a potent oncogene involved in enhancing cell proliferation. We hypothesize that the lack of TIEG increases pttg 1 in heart cells causing them to abnormally proliferate, causing an enlarged heart. In collaboration with Dr. Rajamannan and Dr. Mike Ackerman (Mayo Cardiology) we are examining whether TIEG might play a role in the human HCM disease.

Techniques used: The projects described above involve the measurement of growth factor/cytokine mRNA and protein levels using ELISA, RIA, as well as molecular biology techniques, e.g., Northern, Western, Southern, and Southwestern blotting, DNA gel shift assays, polymerase chain reaction, chromatin immunoprecipitation assays (ChIP) and double ChIP assays cloning, gene deletions and mutations, gene transfections, CAT assays, gene knockout and transgenic animals, yeast two hybrid systems, gene microarrays, adenoviral expression systems, silencing (si) RNAs, as well as biochemical techniques of immunohistochemistry, confocal microscopy, real time PCR, DNA/protein sequencing, HPLC, 2-dimensional gel electrophoresis and mass spectrometry.