Team:UT-Tokyo/CTCD/Content/Project

From 2014.igem.org

Cancer is one of the most common causes of death in the world, and the numerous people fight with this problem. The early detection of cancer is important for treatment, and the earlier cancer is detected, the better the prognosis of the patient.

Today definite diagnosis is commonly made through an invasive test (biopsy), but this test is painful for patients, so it is difficult to perform this test in routine health checks. So as an easy test, non-invasive tests are valuable.

Many biomarkers such as AFP or PSA are detected in non-invasive tests. These biomarkers, however, are sometimes detected even in the absence of cancer(Kilpelainen, T. P., et al. "False-positive screening results in the Finnish prostate cancer screening trial." British journal of cancer 102.3 (2010): 469-474.). Thus, these biomarkers are limited to the subsidiary use to confirm the cancer. Furthermore, as for some types of cancer such as breast cancer, a biomarker has yet to be found. For these reasons, other biomarkers which respond correctly to a wide range of cancers are required.

As a new biomarker , circulating tumor cells (CTCs) are attracting attention. Circulating tumor cells (CTCs) are tumor cells arising from primary cancer, flowing through blood vessels.

In the case of some kind of cancers, CTC makes it possible to detect cancer at earlier stage of cancer progression.(Nakagawa, Taku, et al. "Detection of circulating tumor cells in early-stage breast cancer metastasis to axillary lymph nodes." Clinical Cancer Research 13.14 (2007): 4105-4110.)
Furthermore, as CTCs are tumor cells themselves, if CTC is detected with high sensitivity and low false positive rates, it indicates the presence of cancer.

Most of CTC detection methods emphasized utilizing anti-EpCAM antibody, which is an adhesion protein used as a marker of cancers, and distinguishes CTCs from other cells. For example, CellSearch Assay is the FDA-approved CTC enumeration method, but has difficulty in detecting the low-concentration CTCs (Hou, Shuang, et al. "Capture and Stimulated Release of Circulating Tumor Cells on Polymer‐Grafted Silicon Nanostructures." Advanced materials 25.11 (2013): 1547-1551.). We aimed to develop a new approach for detection of low-concentration CTCs in order to find cancer at the early stages.

We focused on the fact that CTC is the cell itself.Therefore, we can apply synthetic biological approaches such as gene introduction to the blood sample. If an immunological approach is taken, only markers on the cell surface will be detected. If we introduce the genetic circuit into the CTC, we use intracellular information of the CTC such as RNAs, proteins and so on. Then We can decrease the false positive rate and increase the specificity.

Here we have developed a new detection method for CTCs focusing on miRNA profiles and the expression of EpCAM. It has increased specificity for CTCs by utilizing the two pieces of information. miRNA is a special short non-cording RNA and interferes with the translation of the complementary mRNA, interacting with some proteins and forming the RNA-induced silencing complex (RISC) in a wide range of species from virus to human. The circuit that we have developed has EGP-2 promoter, reporter and hematopoietic cell-specific miRNA (miR-142-3p/5p) binding sites. Two conditions, which are activation of EGP-2 promoter and an absence of blood cells-specific miRNA, are required for translation of the reporter. If we introduce this circuit into cells in blood, only in the presence of CTCs is the reporter produced (Fig.1).

<img src="" class ="figure" /> <legend style="margin-left:300px;">Fig.1 The outline of our method (BBa_K1461316, BBa_K1461322)</legend>

[1] Friedlander, Terence W., Gayatri Premasekharan, and Pamela L. Paris. "Looking back, to the future of circulating tumor cells." Pharmacology & therapeutics 142.3 (2014): 271-280.

<img src = "" class = "contTitle"/> <img src="" class = "figure" /> <legend style="margin-left:300px;">Fig.2 the construct of our circuit</legend>

EGP-2 promoter

EGP-2 promoter is the promoter of Epithelial cell adhesion molecule (EpCAM) protein.
EpCAM is a transmembrane glycoprotein which is expressed in epithelial cells and shows high level expression in various type of human epithelial carcinomas (van der Gun, Bernardina TF, et al. "EpCAM in carcinogenesis: the good, the bad or the ugly." Carcinogenesis 31.11 (2010): 1913-1921.). Using this promoter, cancer cells shows higher expression of reporter genes. EGP-2 promoter is originally about 3.5kb and therefore it is not convenient to make into a DNA construct. However, in the previous research, about 1.5 kb of EGP-2 promoter was shown to be sufficient for the specific expression in epithelial carcinomas (McLaughlin, Pamela MJ, et al. "Use of the EGP-2/Ep-CAM promoter for targeted expression of heterologous genes in carcinoma derived cell lines." Cancer gene therapy 11.9 (2004): 603-612.). In this project, We cloned the 1.5kb fragment of EGP-2 promoter, and made it useful as a biobrick parts ( BBa_K1461104).

miRNA-142

miRNA is a class of non-coding RNA. miRNA binds the miRNA-recognition element in the 3' untranslated region of the target mRNA leading to the degradation of the mRNA. miRNA shows tissue-specific expression pattern. In our project, we use miRNA-142, which is highly expressed only in hematopoietic cells (Landgraf, Pablo, et al. "A mammalian microRNA expression atlas based on small RNA library sequencing." Cell 129.7 (2007): 1401-1414.), to reduce the expression of the reporter gene in these cells. After miRNA-142 is transcribed, it is cleaved to miRNA-142-5p and miRNA-142-3p. Adding the recognition element of miRNA-142 in the 3' untranslated region of the reporter genes (BBa_K1461100, BBa_K1461101), leaky expression in non-carcinoma hematopoietic cells is expected to be reduced.

<img src="" class = "figure" /> <legend>Fig.3 When this construct is transfected to the epithelial cells like cancer cell, pEGP-2 promote the transcription of EGFP, and the mRNA is not degraded by miRNA because miR 142 is specific to hematopoietic cells.</legend> <img src="" class = "figure" /> <legend>Fig.4 When this construct is transfected to the hematopoietic cells, pEGP-2 does not work as a promoter, and mRNA is not transcribed. Even if the leak of the promoter makes few mRNA ,this mRNA is degraded by RNAi caused by miR 142.</legend>

The advantage of the CTC detection is detecting the tumor cell itself.
Much more information can be gained by detecting CTCs than by detecting conventional cancer biomarkers in the blood such as peptides. Therefore, our method can be applied to characterization of cancer. For example, if a fluorescent reporter is expressed only in CTCs, we can collect CTCs with FACS. By acquiring them, we can characterize the origin of the CTCs without biopsy.

In our project, we focused on miRNAs that are specific in hematopoietic cells. Most tissues have distinct expression profiles of miRNAs. Thus, by examining these profiles of the CTCs, we can identify the derivation of them. In addition, according to the oncogene causing malignant transformation, different symptom and prognosis are appeared. Therefore, if we can determine the molecular that causes malignant transformation, the molecular target therapy can be available. Moreover, we can clarify their metastatic potential or predict the prognosis.