JHU/CCR Fellowship in Molecular Targets and Drug Discovery Technologies Project Details

Project Sponsor/Mentor:	Curtis Harris
Title:	Lab/Branch Chief
Address:	37 Convent Dr., Rm 3068A Betheda MD, 20892
Telephone:	301-496-2048
Fax:	301-496-0497
Email:	Curtis_Harris@nih.gov
Sponsoring Laboratory/Branch:	Laboratory of Human Carcinogenesis

Project Title:	MicroRNAs as therapeutic targets for cancer
Target(s) of Interest:	microRNAs

Project Synopsis:
Last year in the United States, 215,000 new lung cancer and 150,000 new colon cancer cases were diagnosed. These diseases represent a significant health burden as they are the top two causes for cancer-related mortality in US. Approximately 162,000 lung cancer-related deaths and 50,000 colon cancer-related deaths occur annually. While current adjuvant treatment modalities significantly improve survival outcome for patients for both cancer types, identifying additional therapeutic strategies may reduce the burden of both of these diseases. We are interested in determining if specific microRNAs have potential as therapeutic targets for cancer. MicroRNAs are small, 20-22 nucleotide, noncoding RNA molecules that regulate the translation of specific genes. Since their discovery microRNAs have been found to regulate a variety of cellular processes including apoptosis, differentiation and cell proliferation. Altered expression of specific microRNAs has been linked to cancer. MicroRNA expression levels are altered in all tumor types examined. Experimental manipulation of specific microRNAs modulates tumor development in mouse-model systems demonstrating that microRNAs have potential to be causal factors in carcinogenesis and possible therapeutic targets. Deletions of microRNA-15 and microRNA-16 are frequently the only genetic abnormality present in chronic lymphocytic leukemia, thereby demonstrating that deletion of these microRNAs is likely sufficient to cause this disease in humans. Due to the small size and known sequences of microRNAs, they are relatively easy to design mimics for or inhibitors against specific microRNAs. Modified, anti-microRNA oligonucleotides have been successfully used to inhibit microRNAs in non-human primates to demonstrate the plausibility of targeting microRNAs as a therapeutic strategy. We propose to test if anti-microRNA therapeutics can work on their own or synergistically with 5-fluorouracil (5FU) as an anti-tumor therapy for colon cancer cells. We will perform similar studies in non-small cell lung cancer cell lines to determine if anti-microRNA therapeutics and work synergistically with EGFR-inhibitors. We will begin our studies with the oncogenic microRNA, miR-21. We have found that human colon tumors with elevated levels of microRNA miR-21 are more aggressive and lead to shorter survival times in sporadic colon adenocarcinoma patients in two independent cohorts (Schetter et al, JAMA 2008). In that study, patients with tumors expressing high levels of miR-21 are also less responsive to 5-fluorouricil treatment. MiR-21 is a microRNA that when expressed at high levels, can inhibit apoptosis in vitro. We propose that high levels of miR-21 in tumors may prevent 5-fluorouracil induced apoptosis and provide an explanation for why tumors expressing high levels of miR-21 have higher resistance to 5-fluorouracil chemotherapy. Therefore, anti-miR-21 therapeutics may enhance the effectiveness of 5-florouracil to treat colon tumors. We have found similar results in lung cancer where elevated miR-21 expression was associated with poor prognosis in non-small cell lung adenocarcinoma patients (Yanaihara et al, Cancer Cell, 2007). Additionally, we have demonstrated that miR-21 inhibitors enhance apoptosis caused by EGFR inhibitors in non-small cell lung cancer cell lines indicating that anti-miR-21 therapeutics may be act synergistically with EGFR inhibitors to treat lung tumors (Seike et al, PNAS, 2009). We propose to continue these studies to test if miR-21 inhibitors can sensitize cells to chemotherapeutic agents. These hypotheses will first be tested in vitro, using several colon and lung cancer cell lines. If successful, we will begin separate studies using xenograft models of human colon cancer cell lines and human non-small lung cancer cell lines in mice using anti-miR-21 therapeutics. We will also explore whether inhibition of other oncogenic microRNAs or mimics of tumor suppressor microRNAs can work on their own or synergistically with current chemotherapies. In addition to targeting miR-21, we will also select additional microRNAs to target in similar ways and determine if targeting any of these microRNAs can work synergistically with 5FU or anti-EGFR inhibitors. The specific Aims of this project are as follows: Aim 1: Determine if over-expression miR-21 or inhibition of miR-21 cause colon cancer cell lines to become resistant or more sensitive to 5-flourouracil. Aim 2: Determine if over-expression miR-21 or inhibition of miR-21 cause lung cancer cell lines to become resistant or more sensitive to EGFR inhibitors. Aim 3: Select other candidate microRNAs to test if inhibition or overexpression can work synergistically with 5FU or EGFR inhibitors.
Fellow Research Plan:
The main research goal will be to determine if specific microRNAs may be suitable drug targets for lung cancer and colon cancer therapy. During a fellows training, they will gain expertise in several laboratory techniques including: cell culture, cell transfection, RNA isolation, quantitative RT-PCR, cell proliferation assays, cell invasion assays, apoptosis assays, FLOW cytometry and western blot techniques. They will also be trained how to perform the appropriate statistical tests to identify significant results for each of the biological assays they are performing. The fellow will participate in monthly lab meetings to gain experience presenting and critically evaluating scientific works. They will also attend weekly journal clubs to discuss recent published works relating to cancer. The fellow will be given the opportunity to present their work as a poster at a scientific meeting. If the results support moving these experiments into mouse xenograft systems, the fellow will be given the opportunity to participate in the follow-up studies as well.