Background

The idea of using bacteria as a therapeutic for cancer has been around for about 150 years, beginning with William Coley, who gave cancer patients a mixture of heat-inactivated Streptococcus pyogenes and Serratia marcescens, known as “Coley’s toxin,” to destroy tumors. This idea gradually evolved into reprogramming bacteria to fight cancer using recombinant DNA. The two most popular vectors for bacterial cancer therapy are Salmonella and E. coli. E. coli that produced shRNA can knockdown an oncogene in colorectal cancer. The mechanism in which the bacteria silences the oncogene relies on transkingdom RNA interference, encoded in the bacteria's transkingdom RNAi plasmid (TRIP) (Xiang et al. 2006). The TRIP plasmid contains three major sections, the shRNA-producing gene, the invasin gene, and the hlyA gene. The shRNA-producing gene produces shRNA that corresponds to the mRNA sequence of the targeted oncogene. The invasin gene allows the bacteria to invade the cancer cell and get encapsulated in an endosome. The bacteria lyse inside the endosome, and the hlyA gene encodes for listeriolysin O, which produces holes (pores) in the endosome so that the shRNA produced by the bacteria can get out of the endosome to reach the mammalian cell’s cytoplasm. The released shRNA is then cleaved by the enzyme Dicer in the mammalian cell’s cytoplasm to become siRNA. The siRNA then associates with other proteins in the cytoplasm to form the RISC complex. The RISC complex then binds the corresponding target mRNA in the cytoplasm and cleaves it, silencing the gene that the mRNA encodes. This mechanism has been proven to be an effective inhibitor of a cancerous protein that has been very difficult to target with drugs.

In this project, we optimize and expand the applications of this mechanism. We will use quorum sensing as an additional safeguard to make sure that this mechanism only attacks cancer cells. With quorum sensing, this mechanism will only be activated when a certain bacterial cell density is reached. This density is only possible in very anaerobic environments, which is characteristic of tumors. Challenges to this include the natural anaerobic environment in the gut and whether the bacteria will proliferate in a healthy gut as well. To resolve this, we are working to put the quorum sensing circuit under the control of a nitric oxide promoter. Since nitric-oxide rich environments are only characteristic of areas of inflammation and are highly characteristic of cancer, having the quorum sensing circuit under the control of a nitric-oxide promoter that is only activated in nitric-oxide rich environments would prevent the bacteria from invading healthy gut cells even when quorum is reached. A synthetic alternative to this mechanism that would be simpler to test in the laboratory is to control the quorum circuit with a tet (tetracycline) on system where a tet repressor represses the expression of the invasion circuit when tetracycline and/or doxycycline is not present. When doxycycline/tetracycline is synthetically introduced to the environment, the tet repressor is repressed by the rtTA (reverse tetracycline-controlled transactivator), and transcription of the invasion circuit occurs. The doxycycline/tetracycline can only be introduced to cancer sites.

We are targeting cervical cancer and prostate cancer. We will determine the effectiveness of this mechanism in each of these cancers using proof-of-concept experiments by inhibiting eGFP. We will then target oncogenes in these cancers.