Undergraduate Research Proposal

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Project Title

Analyzing three genes that function non-autonomously to modulate oncogenic Ras activity

Project Summary

Ras is an important oncogene that affects cell proliferation and cancer progression in a range of cancers, including pancreatic, colorectal, and lung. While the oncogene is a driver for cancer growth, interactions with other, non-cancer cells that are part of the tumor (the tumor microenvironment, TME) can influence how Ras-active cancer cells proliferate. With the proposal, my goal is to identify genes that function in the TME to modulate the progression of cancer. This research is significant because it identifies possible genes for cancer diagnostic and treatment purposes.

 In the Chamberlin lab, Caenorhabditis elegans (C. elegans) are used as a simple model to discover non-autonomous regulators of Ras signaling. We have created genetically engineered C. elegans strains in which genes in the mesoderm can be subject to knock-down, and their effect on proliferating epithelial cells with activated let-60/Ras can be observed. With these knockdowns, genes were screened using RNAi, and three genes were found to stimulate epithelial cell proliferation using oncogenic let-60/Ras. This discovery is key to studying cancer genetics because these genes have human orthologs and point to a potential gene to knock down when analyzing human cancers.

The C. elegans system provides a model for the relationship between stromal factors and the cancer biology beyond the scope of the cancer cell. From the perspective of future cancer treatments, it is important to identify stromal factors that promote cancer progression. The eventual goal is to target these areas and prevents the spread of cancer to other organs. Stromal cells are important to consider when continuing cancer research because they are non-malignant cells that allow the tumor to grow due to the extracellular matrix environment.

Project Description

Background and Significance

Genetics plays a major role in tumor progression both within cancer cells and within non-cancer surrounding cells that compromise the tumor micro-environment (TME). The TME is an organized system in which different cell types create a niche of cell-to-cell communication that allows for cancer cells to proliferate, evolve, and survive. Within the TME environment and cell-to-cell communication, there is a downregulation of the immune system, upregulation of tumor invasion, and upregulation of adipocyte differentiation [4]. One example of a TME can be observed with stromal factors, as the stroma is made up of fibroblasts and pericytes, which are non-malignant. By having the ability to regulate the TME, this may allow for the decrease of tumorigenesis and spread of cancer.

Within cancer genetics, an important set of proteins are Ras. Ras is an important oncogene in many cancers, especially pancreatic cancer in which up to 95% of cancers have an oncogenic mutation in Ras [7]. Additionally, Ras is a GTPase that transmits signals within cells. From a biochemical perspective, Ras is activated by GDP and inactivated by GTP, therefore, when GTP is converted to GDP via the removal of a phosphate group, Ras is activated and can influence cells downstream [1]. With this reaction, the active Ras allows the cell to differentiate, survive, grow, and perform apoptosis.

Our lab has developed a simple model in the microscopic C. elegans that mimic the interaction between the tumor microenvironment and Ras-activated cancer cells. The motivation for this project is to identify specific genes that function non-autonomously to influence the Ras pathway.  The C. elegans genome contains a single Ras-related gene called let-60/Ras. let-60/Ras is a control that switches between vulval and hypodermal cell fates during C. elegans vulval induction [2]. When there is an activating mutation in let-60/Ras, more cells adopt vulval fate, and this presents as ectopic growth of the animal.

The overlying goal of this proposal is to distinguish the importance of studying mesodermal genes of C. elegans when researching cancer. C. elegans have long been an appropriate model for proliferation of oncogenic epithelial cells and the identification of identifying in vivo orthologs and functions. In C. elegans development, the vulva provides insight into cell regulation and cell proliferation responses. These cell modulatory systems that arise in the vulva will help evaluate the gene mutations that occur alongside the oncogenic Ras pathway.  

Preliminary Investigation and Data

Three genes isolated from the RNAi screen were selected for further study: D1007.4, W04A8.1, and phip-1. When each gene is knocked down, specifically in the mesodermal cells, it results in C. elegans with normal vulval development despite the fact that the let-60/Ras gene is still mutant (Figure 1). With the three genes chosen, it appears that there is a corresponding human ortholog that can be considered. With D1007.4, the human ortholog is GEMIN 6 (gem nuclear organelle associated protein 6). This ortholog is located on the striated muscle dense body in humans. With W04A8.1, the human ortholog is MCPH1 (microcephalin 1). In humans, W04A8.1 has RNA polymerase II CTD heptapeptide repeat phosphate activity. With phip-1, the human ortholog is the human ortholog is PHPT1 (phosphohistidine phosphatase 1). In humans, PHPT1 is expected to have protein histidine phosphatase activity.

Figure 1: RNAi knockdowns of D1007.4, W04A8.1, or phip-1 suppresses the proliferation defect observed in worms with an oncogenic mutation in let-60/Ras (control).  

Specific Aims

Aim 1: Validate RNAi effect in chimeric animals from the confirmed genotype for pre-made chimeras. I will generate strains that permit the RNAi to be evaluated in chimeric animals, only capable of mediating RNAi in the mesodermal lineage, to compare these results to the strains using cell-specific promoters.

Aim 2: Generate null mutants for three genes, D1007.4, W04A8.1, and phip-1, to validate the function. I will use CRISPR-mediated genome editing to generate deletion alleles of each gene to provide a genetic confirmation of results obtained using RNAi.

Aim 3: Evaluate the phenotype for each mutant and test for suppression of let-60/Ras. I will construct double mutants between the null mutants and the activated let 60/Ras mutant to ask whether or not they are genetic suppressors.

Experiment Design Methods

Aim 1:

A genetic cross was carried out to introduce mutant rde-1 (RNAi-defective) into a strain with activated let-60/Ras and germline over-expression of GPR-1, used to generate chimeric animals, and another to introduce a fluorescent marker (hjSi20) into a strain mutant for rrf-3. The genotype of these animals was confirmed (Figure 2).

These strains will be used to generate chimeric animals to be tested for RNAi knockdown in the P1-derived cells following the protocol of Artiles et al. (2019) [6].  According to Artiles, the P1-derived cells gives rise to germline, which allow me to make mutations to C. elegans for rrf-3 in chimeric animal.

Figure 2: Example of strain mutant for rrf-3 on Gel

Aim 2:

sgRNA plasmids will be generated using PCR, to insert a gene-specific sequence that can be expressed in animals, using the plasmids of Dickinson et al. [3].

These plasmids will be screened from insertion of the gene-specific DNA into the plasmid using PCR. Positive clones will be sequenced. A preliminary example of positive clones is show in Figure 3.   

These clones will be injected into the gonad of animals, and offspring will be screened using PCR.

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Figure 3: Positive sgRNA plasmid clones

Aim 3:

Genetic mutants will be crossed with let-60/Ras strains, and homozygous mutants identified using PCR. Once strains with gene deletion are identified, I will breed the animals to establish homozygous strains. Animals from these strains will then be crossed with the let-60/Ras strain, heterozygotes will be allowed to self-cross, and double homozygotes will be selected and confirmed by genotyping. These double mutants will be evaluated for vulval development (as in Figure 1) to ask whether the genetic mutation suppresses over-proliferation as does the RNAi knockdown.  

Bibliography

[1] Murugan AK, Grieco M, Tsuchida N. RAS mutations in human cancers: Roles in precision medicine. Seminars in Cancer Biology. 2019;59:23-35. doi:10.1016/j.semcancer.2019.06.007.

[2] Lu J, Dentler WL, Lundquist EA. FLI-1 Flightless-1 and LET-60 Ras control germ line morphogenesis in C. elegans. BMC Developmental Biology. 2008;8:54. doi:10.1186/1471-213X-8-54.

[3] Dickinson DJ, Ward JD, Reiner DJ, Goldstein B. Engineering the Caenorhabditis elegans genome using Cas9-triggered homologous recombination. Nature Methods. 2013;10(10):1028-1034. doi:10.1038/nmeth.2641.

[4] Conti I, Varano G, Simioni C, et al. miRNAs as Influencers of Cell–Cell Communication in Tumor Microenvironment. Cells (2073-4409). 2020;9(1):1-28. doi:10.3390/cells9010220.

[5] Wang Z Editor, Gauthier K, Rocheleau CE, Walker JM. S editor. C. elegans Vulva Induction: An In Vivo Model to Study Epidermal Growth Factor Receptor Signaling and Trafficking. ErbB Receptor Signaling: Methods and Protocols. 2017:43. doi:10.1007/978-1-4939-7219-7_3.

[6] Artiles KL, Fire AZ, Frokjaer-Jensen C. Assessment and Maintenance of Unigametic Germline Inheritance for C. elegans. Developmental Cell. 2019;(6). doi:10.1016/j.devcel.2019.01.020.

[7] Fernández-Medarde A, Santos E. Ras in cancer and developmental diseases. Genes Cancer. 2011;2(3):344–358. doi:10.1177/1947601911411084.

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