It is the overall goal of this application to better define the differences between hepatic colorectal cancer metastases and the normal host tissues within which they reside and in the process of developing this understanding to develop reagents that specifically traffic to in vivo tumors. Recognizing and understanding these differences will help to create therapeutic strategies that are targeted to patients with metastatic colorectal cancer who as a group face an expected 5-year survival prognosis of less than 10% with current therapies.
The specific aims of this study are to: 1.To create RNA binding motifs through a novel in vivo selection process that specifically bind human intrahepatic colorectal cancers residing in immunodeficient mice and that possess the ability to traffic in vivo to the sites of tumor deposits upon systemic administration. 2. To define the protein targets of these tumor-specific RNA binding motifs and ascertain whether they possess binding characteristics consistent with RNA aptamers. 3.To determine if the identified RNA aptamers possess inhibitory actions on their protein target and/or whether they are capable of escorting therapeutic moieties to intrahepatic tumors. 4. Determine whether these same targets are also present in a broader spectrum of human colorectal metastases harvested by the applicant (BC) at the time of hepatic resection. To accomplish these aims, RNA aptamers will be generated through a novel selection strategy whereby mice bearing hepatic colorectal metastases will be injected via tail vein with a random library of RNA oligonucleotides. After a brief period of circulation, tumors are harvested and RNA retrieved, reverse transcribed, amplified, and transcribed back to RNA for repeat injection. This cycle is repeated until the population of RNA binding motifs is heavily enriched at which point cloning and sequencing is then performed. RNA aptamers created through this mechanism will then undergo in vitro binding assays to identify those with specific binding for tumor tissue. This process will be performed utilizing xenotransplants harvested from multiple patients in an effort to retrieve aptamers relevant to a broad spectrum of patients. Selections will also be carried out whereby different xenotransplants are utilized in alternate rounds. The resulting RNA binding motifs will be explored in their ability to traffic to the site of intrahepatic tumors. Through a ligand-mediated approach, the target of these aptamers will be isolated and sequenced. The ability of the RNA aptamers to inhibit the function of their target proteins and the proliferation of in vitro and in vivo tumors will be determined. In addition, the ability of RNA aptamer:bacterial toxin conjugates to effect cytotoxicity in vitro and in vivo will be determined. The relevance of the aptamers to a broad spectrum of patients with mCRC will be explored via cDNA arrays and IHC of resected tumors and through in vivo trafficking studies in mice bearing xenotransplants.
It is the purpose of the proposed research to better define the differences between human tumors (metastatic colorectal cancer) and normal tissue. Colorectal cancer is one of the leading causes of cancer death in the United States and rarely curable when spread (metastases) are detected. Defining the differences between cancer and normal tissues will allow for the further development of targeted therapeutic agents that are capable of inhibiting metastatic colorectal cancer while minimizing toxicity to normal tissues.