Investigators Who Have Received Career Development Awards through the Mayo Clinic Cancer Center's Prostate SPORE Program

Dr. Junjie Chen received a Career Development Award to study the role the tumor suppressor p53 binding protein 1 (53BP1) in DNA damage-signaling pathways. His mentor was Dr. Robert Jenkins. The tumor suppressor p53 binding protein 1 (53BP1) binds to the DNA-binding domain of p53 and enhances p53-mediated transcriptional activation. 53BP1 contains two breast cancer susceptibility gene 1 COOH terminus (BRCT) motifs, which are present in several proteins involved in DNA repair and/or DNA damage–signaling pathways. Dr. Chen investigated the potential role of 53BP1 in DNA damage–signaling pathways. He found that 53BP1 becomes hyperphosphorylated and forms discrete nuclear foci in response to DNA damage. These foci colocalize at all time points with phosphorylated H2AX (-H2AX), which has been previously demonstrated to localize at sites of DNA strand breaks. 53BP1 foci formation is not restricted to -radiation but is also detected in response to UV radiation as well as hydroxyurea, camptothecin, etoposide, and methylmethanesulfonate treatment. Several observations suggest that 53BP1 is regulated by ataxia telangiectasia mutated (ATM) after DNA damage. First, ATM-deficient cells show no 53BP1 hyperphosphorylation and reduced 53BP1 foci formation in response to -radiation compared with cells expressing wild-type ATM. Second, wortmannin treatment strongly inhibits -radiation–induced hyperphosphorylation and foci formation of 53BP1. Third, 53BP1 is readily phosphorylated by ATM in vitro. Taken together, these results suggest that 53BP1 is an ATM substrate that is involved early in the DNA damage–signaling pathways in mammalian cells. The results were published in the Journal of Cell Biology 2001 Apr 30;153(3):613-20.

These findings were used as preliminary data to secure funding from the NIH (R01CA 100109) on which Dr. Chen is the PI.

Dr. Wanguo Liu received a Career Development Award to study genetic mutations in associated with prostate cancer risk. His mentor was Dr. Steven Thibodeau. The DNA-damage-signaling pathway has been implicated in all human cancers. However, the genetic defects and the mechanisms of this pathway in prostate carcinogenesis remain poorly understood. In this study, Dr. Liu analyzed CHEK2, the upstream regulator of p53 in the DNA-damage-signaling pathway, in several groups of patients with prostate cancer. A total of 28 (4.8%) germline CHEK2 mutations (16 of which were unique) were found among 578 patients. Additional screening for CHEK2 mutations in 149 families with familial prostate cancer revealed 11 mutations (5 unique) in nine families. These mutations included two frameshift and three missense mutations. Importantly, 16 of 18 unique CHEK2 mutations identified in both sporadic and familial cases were not detected among 423 unaffected men, suggesting a pathological effect of CHEK2 mutations in prostate cancer development. Analyses of the two frameshift mutations in Epstein Barr virus-transformed cell lines, using reverse transcriptase polymerase chain reaction and western blot analysis, revealed abnormal splicing for one mutation and dramatic reduction of CHEK2 protein levels in both cases. Overall, these data suggest that mutations in CHEK2 may contribute to prostate cancer risk and that the DNA-damage-signaling pathway may play an important role in the development of prostate cancer. The results were published in the American Journal of Human Genetics. 72(2):270-80, 2003 Feb.

These findings were used as preliminary data to secure funding from the Department of Defense for two grants (FEDDAMD17-02-1-0093) and (FEDPC030340). Another grant from the NIH (R01CA 115555-1) is pending.

Dr. Kaustubh Datta received a Career Development Award to study the regulation of angiogenesis in prostate cancer. His mentor was Dr. Donald Tindall. Androgen ablation therapy is eventually followed by a more metastatic and androgen-refractory stage of prostate cancer. The detailed molecular mechanism of this gradual transition is not clearly understood. Recent reports correlate the high abundance of vascular endothelial growth factor-C (VEGF-C) to the lymph node metastasis seen in human prostate cancer. In this study, Dr. Datta showed that androgen ablation in LNCaP cells augment the transcriptional upregulation of VEGF-C and the down-regulation of the IGF-IR pathway, due to androgen withdrawal, is a potential mechanism for this observed VEGF-C transcription. Forkhead transcription factor FOXO-1, activated by SIRT-1, was identified as the downstream molecule within this pathway. Furthermore, the VEGF-C-induced increase of Bag-IL expression in LNCaP cells suggests that VEGF-C plays a role in the androgen-independent reactivation of the androgen receptor, resulting in androgen-refractory prostate cancer growth. The results were published in Oncogene. 2005 Aug 18;24(35):5510-20.

Preliminary findings from this study were used to secure funding from the Department of Defense with a grant (FEDW81XWH-04-1-0901-1)titled “Elucidation of the Molecular Mechanisms Underlying Lymph Node Metastasis in Prostate Cancer.”

Dr. Amy Tang received a Career Development Award to study the role of proteolysis in advanced prostate cancer. Her mentor was Dr. Charles Young. There is a need to understand the molecular events leading to the development of metastatic prostate cancer, and to identify new and effective therapy directed at these molecular alterations in order to successfully treat this lethal disease. Dr. Tang’s preliminary data showed that the human homologue of a highly conserved family of Drosophila RING domain E3 ubiquitin ligases - SEVENIN-ABSENTIA (SINA), SIAH is specifically and markedly upregulated in rapidly dividing cells in advanced prostate cancers. The increased SIAH expression seemed to correlate with high Gleason Score and aggressiveness of prostate cancer. Furthermore, she found that the proper RAS signaling is dependent on the SIAH-dependent proteolytic machinery as the proteolysis-resistant SIAH proteins block RAS-mediated foci formation. Therefore, she proposes to establish a correlation between the increased SIAH expression and the advanced stage of prostate cancer, and to determine whether the upregulation of SIAH proteins and the SIAH-dependent proteolysis could serve as good metastatic prostate cancer markers. Since the substrate specificity of ubiquitin-mediated proteolysis is primarily determined by the E3 ligases, results from this research should provide insights into the altered proteolysis in cancer and reveal SIAH substrates in metastatic prostate cancer cells. The information may lead to new avenues for prostate cancer diagnosis and proteosome/proteolysis-based therapeutic intervention.

Preliminary findings from this study were used to secure funding from the NIH (R01GM 69922-2) for a grant entitled “Regulated Proteolysis in RAS Signal Transduction.”

Dr. Alex Parker received a Career Development Award for the identification of clinical, pathologic and molecular predictors of prostate cancer recurrence in men undergoing salvage radiation therapy. His mentor will be Dr. James Cerhan. Salvage radiation therapy may cure patients who experience prostate cancer recurrence following radical prostatectomy. Currently, two important issues in the management of patients undergoing salvage radiation therapy are (1) a limited ability to accurately predict further recurrences and (2) the lack of knowledge regarding actual molecular alterations associated with an increased risk of recurrence. He will analyze data and biospecimens collected from over 300 patients undergoing salvage radiation therapy at Mayo Clinic in order to identify independent clinical and pathological predictors of recurrence. The end result will be an easy to use composite scoring system to help direct patient care and provide new insights to the biology of prostate cancer recurrence.

For this investigation, Dr. Parker will carry out the following specific aims:

1. Using data collected as part of the Mayo Clinic Salvage Radiation Database, he will identify all patients receiving salvage radiation therapy to treat a prostate cancer recurrence at Mayo Clinic between 1987-2003 (n=358). Further, he will use archived paraffin-embedded blocks from the original prostatectomy on each patient identified. Based on previous experience, he expects to obtain tumor blocks on 80-85% of the patients in the Salvage Radiation database.
   

   In collaboration with Dr. Alan Pollack at the University of Pennsylvania, Dr. Parker will determine protein expression levels of p53, Bax, Bcl-2, PTEN, Ki-67, COX, pAKT in tumor tissue samples from each patient identified in specific Aim #1.

2. In a univariate setting, he will use Kaplan-Meier and Cox regression analysis to determine the association of clinical and pathologic variables with risk of recurrence after salvage radiation therapy. Moreover, he will determine the association of expression of p53, Bax, Bcl-2, PTEN, Ki-67, COX, and pAKT with risk of recurrence after salvage radiation therapy.
3. He will use standard statistical approaches to select those variables (clinical, pathological and molecular) that are predictive of biochemical failure after salvage radiation therapy in a multivariate setting. From the final model generated by this process, he will develop a multivariate scoring algorithm based on the regression parameters associated with each covariate. He will then validate this final model internally using bootstrapping methodology.