Understanding the role of TWIST1 in colorectal cancer progression and metastasis

Project Summary: Although almost all colorectal cancer (CRC) mortality is due to metastasis, there are currently no drugs to prevent or halt metastasis. A better understanding of the underlying biology of CRC progression and metastasis could lead to the next generation of therapeutics aimed at blocking steps in the metastatic cascade. Previous research

provided a framework to understand tumor metastasis through sequential stages of cancer cell invasion into the basement membrane, migration into vasculature, circulation, extravasation, and colonization of distant organs. Each step is heavily dependent on diverse cell-cell communications and the microenvironment, which has not

been well recapitulated in previous in vitro and in vivo model systems to study CRC metastasis. However, the recent development of CRISPR-engineered 3D colorectal cancer organoids (CRCOs) and colonoscopy-guided orthotopic CRC transplantation models have provided a more physiological model system to study metastasis.

The CRCOs are precisely engineered with oncogenic mutations found in the majority of human CRC, such as mutations in the Apc, Kras, and P53 genes (AKP). Moreover, these engineered CRCOs are transplanted into the murine colon, where they grow in their native environment and metastasize to the liver: the most common

site of CRC metastasis in humans. Previous research using these model systems identified Twist1 as significantly upregulated in CRC liver metastases compared to the primary tumor. Twist1 is a well-known transcription factor involved in promoting epithelial-mesenchymal transition (EMT). EMT is when epithelial cells

lose their cell adhesions and acquire a more motile mesenchymal phenotype. It is unclear what role Twist1 plays in CRC progression and metastasis. Aim 1 will verify that Twist1 is upregulated in liver metastases compared to the primary tumor from AKP CRCOs in orthotopically transplanted mice using immunohistochemistry,

RNAscope, and RT-qPCR. Aim 1 will also assess whether Twist1 is necessary for liver metastasis formation through CRISPR-Cas9-mediated loss in AKP organoids and a diphtheria toxin receptor cell-elimination model. Moreover, to evaluate if overexpression of Twist1 increases metastatic potential, the AKP CRCO will be

engineered to overexpress Twist1 using complementary DNA. Thus, Aim 1 will determine whether Twist1 is required for CRC metastasis. The role of TWIST1 in human CRC progression and metastasis will be the focus of Aim 2. Aim 2 will screen human CRC tissue microarrays for expression of TWIST1 and other EMT

markers. TWIST1 in each tissue will be quantified and correlated with patient tumor site, stage, genotype, and outcome. Moreover, to validate TWIST1 is more highly expressed in human CRC metastases, a human CRCO will be transplanted into immunodeficient mice and assayed for expression of TWIST1 by immunohistochemistry,

RNAscope, and RT-qPCR. Such knowledge of TWIST1’s role in CRC metastasis can illuminate targets for the first metastasis-specific therapies and improve the field’s understanding of EMT and metastasis.