Molecular Genetics of C. elegans Development
My lab is interested in the process of morphogenesis, the development of shape and form. What are the molecules that regulate the behaviour of cells as they change their shape, position and adhesiveness to generate their three-dimensional form during morphogenesis? The answer to this question will certainly add to our understanding of the role of cell adhesion and cell signaling during tissue inflammation and metastasis. We use the genetic model organism Caenorhabditis elegans to study simple examples of morphogenetic movements. The well defined anatomy of C. elegans will allow us to analyze these processes at a level of precision not easily attainable in other organisms. My lab uses genetic, molecular biology, biochemistry, and state of the art video microscopy techniques to elucidate the mechanisms by which tissues and organs are generated. In previous work we have shown that ephrin signaling is required for proper C. elegans morphogenesis.
Ephrin/Eph Receptor Tyrosine Kinase Signal Transduction in C. elegans Morphogenesis:
The Eph receptor tyrosine kinases and their ligands, the ephrins, are an exciting class of molecules that play a wide variety of roles in development, including axon guidance, blood vessel formation and cancer. C. elegans mutants that are defective in ephrin signaling have abnormal morphogenetic cell movements during embryogenesis and as a result usually die . The identification of ephrins and their receptor in an animal amenable to genetics makes it feasible to dissect the entire network of ephrin signaling in an organism. To understand the roles of Eph signaling during C. elegans development we will identify the downstream genes of the Eph receptor and how other genes might act redundantly or in parallel with Eph signaling during morphogenesis. These studies complement the approaches taken to understand Eph signaling in more complex animals and will expedite our understanding in the signal transduction pathways controlling morphogenesis. Further, Eph signaling has been linked to events of vertebrate neurogenesis, angiogenesis, and cancer, the latter of which is a prime candidate for anti-tumour therapies. It is expected that work done in model organisms will generate mechanistic information required to improve the efficacy of such treatments.
C. elegans and Cancer Research
"Ye have made your way from the worm to man, and much within you is still worm" Freidrich Nietzsche (1844-1900)
Eph RTKs, Cancer and My Fascination with Development:
I hope to convince you why our research is indeed an important aspect of cancer research. Keep in mind that cancer research is a multifaceted discipline ranging from clinical trials, drug development, epidemiology, and basic research. The latter is the heart of our continued efforts in cancer research. Specifically I will address the following 3 questions:
- How is the study of cell movements related to cancer and why we use the model organism C. elegans?
- What genes we study and their role in cancer? Specifically Eph RTKs, a gene called vab-1 in C. elegans.
- How can we use genetic approaches to identify components in this signaling pathway?
Why use C. elegans?
Animal development is an incredible feat of biological regulation. How are cells, tissues, and organs governed to reach their proper size, shape, and pattern? You can think about cancer as a developmental process, but a developmental process gone wrong. The philosophy of our research is that if we are going to fix something we have a better chance at fixing if we to know how it works in the first place. The Chin-Sang lab is interested in studying a cell and tissue behaviour called morphogenesis. That is, how cells change their shape and size and move during development. So what has all of this got to do with cancer? As I mentioned earlier cancer is a developmental process and currently there is a huge field of cancer research that is devoted to understanding how cells divide and multiply. What genes are turned on or off to tell the cell when to divide or when to stop dividing? We know that tumours arise because of unregulated or too much cell division. Correct? Yes, but this is only one aspect of cancer- in fact we can have benign tumours. What usually makes cancer life threatening is when a tumorous cell invades surrounding tissues or breaks away and travels to distant parts of the body to form yet another tumour- a process called metastasis. Well, surprisingly these cell movements during metastasis are very similar to cell movements seen in the developing embryos of all animals. So if we are going to understand metastasis we need to understand how cells naturally move.
We use this microscopic worm called Caenorhabditis elegans to study some very simple aspects of cell movements. C. elegans are primitive organism that share many biological characteristics of humans. What scientists have learned in the past 20 or so years is that very basic cellular machinery that control mechanisms such as cell division, movement and growth is highly conserved throughout evolution. This means that if you study single cell yeasts, worms, flies or a mouse, many of the genes that control cell growth and development in these organisms have direct counterparts in humans and therefore it has much significance to understanding basic cell biology in humans. The importance of the use of simple model organisms for human diseases is exemplified by the 2002 Nobel Prize in Physiology or Medicine which went to three of C. elegans researchers (Note: I should mention that it also went to yeast researchers in 2001 and fly researchers in 1995). In 2002 the Nobel prize was awarded to Sydney Brenner, Robert Horvitz and John Sulston who used C. elegans to give us significant understanding on genes that regulate program cell death or apoptosis—which has significant relevance to cancer as tumours cells seem to have lost their ability to respond to programmed cell death. The Chin-Sang lab's research objective is to use very similar genetic approaches as these Nobel laureates to understand the molecular mechanisms that control cell movements.
How do we do this?
We go about this by isolating and characterizing mutants that have defects in cell movements early in the developing embryo. You can imagine that cells must be positioned properly in the embryo to function properly. Cells such as neurons, muscles, gut, and epidermis must find their normal position in the embryo, as such if these movements are all messed up it usually leads to embryonic lethality. We cloned the genes responsible for some of these early cell movements and they encode an Eph Receptor Tyrosine Kinase and the ligands for this receptor. In Figure 1, the VAB-1 Receptor is shown in Blue and one of its ligands shown in Green are expressed on neuroblast the future nervous system. Mutations in either the receptor or ligand lead to abnormal neuroblasts movements during embryogenesis and as a result the embryo usually dies.
What are Eph Receptors and what do they have to do with Cancer?
Eph RTK in cancer regulation:
These molecules are found on the cell's surface and belong to a large family called the Receptor Tyrosine Kinases or RTKs. The RTKs are a well-established molecule in oncogenesis as overexpression of certain RTKs can lead to tumour formation. Hence, many of these RTKs and their ligands serve as cancer drug targets (e.g.Trastuzumab/ Herceptin ®, imatinib mesylate/ Gleevec® , gefitinib/ Iressa™ , and Avastin™.) The class that we are particularly interested in are the Eph RTKs. They are the largest family of vertebrate RTK- you and I have 14 different Eph receptors in our body. This large number has in part complicated our understanding of how these RTKs signal. However, in C. elegans there is only one receptor making it easier to elucidate the entire pathway in an organism.
So what do we know about Eph RTKs and Cancer?
There is considerable evidence for the roles for Eph RTKs in both metastasis and tumour formation. These receptors and ligands are frequently overexpressed in a wide variety of cancers. For example EphA2 is overexpressed in 40% of all Breast Cancers. I should stress the role of Eph receptors in cancer regulation is not that clear. The downstream signaling mechanisms are different from most other RTKs. But these RTKs have sparked a great deal of interest in cancer researcher because they have been shown to regulate many types of cancers. As such, the Eph RTKs, their ligands, and effectors represent targets for cancer drug development. However, before drug development proceeds we need to fill in significant gaps in our understanding of how these receptors signal. What molecules do the cells use to communicate with each other? Recently we showed that the VAB-1 Eph RTK inhibits the worm version of the human tumour suppressor gene called PTEN.
The Research strategy:
Classical Genetic Analysis:
C. elegans has become a choice genetic model organism for many researchers. Because of its fast life cycle, and small cell number we can genetically manipulate these organisms and observe the consequences at a single cell resolution. Various "genetics tricks" can be used to identify the genes that encode the molecules that enable cells to communicate with each other during development. I only mention 2 below and focus on the logic rather than the details:
The first approach is called Synthetic lethal Screens:
The logic is as follows: Say you have two genes A and B. A mutation in A does not lead to a phenotype. In fact it is believed that most genes when mutated do not lead to a phenotype. If you have a mutation in gene B you may not get a phenotype for the same reason. But when you make a double combination, that is, two mutations A and B you get a phenotype- in this case death. This is what geneticists call synthetic lethality and it tells you genes and A and B have functional relationship. The easiest way to look at this is that gene A and gene B function in a parallel or redundant pathway. That is, bothA and B are required for some essential developmental function “X”. Knock out A by mutation and it is still ok because B is present, knock out B and it is still ok because A is present. But the double combination leads to a phenotype –lethality.
There is another genetic method called suppressor analysis, which is essentially the opposite of synthetic lethality or enhancement. That is, you start with a phenotype and you identify mutations in other genes that restore the phenotype back to wild type or normal.
Suppose you have a regulatory pathway (See Figure 3): A turns off B , B turns off C and C turns on or Activates X. “X" is an arbitrary developmental process. When A is on this leaves B off, which in turn lets C come on therefore X is ON. Therefore the net effect of A is to turn on X. If there was a loss of function mutation in A such that A is off this allows B to be on which in turn turns off C which can’t activate X leading to a phenotype. If the only function of gene A is to turn off gene B then a suppressor of mutant A would include loss of function mutations in B as this mutation bypasses the need for gene A. Other potential suppressors include gain of function activating mutations in C or X. This type of suppressor approach is what geneticists call suppression by epistasis.
Biochemical Approaches and Yeast 2 Hybrid Screens:
Although researchers like to use phrases like "the awesome power of genetics" keep in mind that genetic approaches are powerful, however they are not all powerful. We are also using molecular and biochemical approaches such as affinity chromatography and yeast 2 hybrid screens to identify components that physically associate with the VAB-1 Eph RTK.
This combined approach of genetics and biochemistry is an effective strategy in deciphering molecular pathways controlling development. Using genetic approaches we have isolated suppressors and synthetic lethal genes that may work with ephrin signaling in C. elegans. From our biochemical work we have identified two new proteins that associate with the Tyrosine Kinase of VAB-1. Both genes have been shown to regulate cancers in humans. Taken the data as a whole, that Eph RTKs are involved in cancer regulation and that we had identified physical interactions and genetic interactions with know tumour suppressor genes as well as genes involved in promoting cancer we believe that we are on our way to understanding how the Eph RTK signal transduction mechanism regulates cancer in humans.
Reverse Genetics: We live in a time when the entire DNA sequence of many organisms is known- The era of Genomics and Proteomics. This information has shaped the way researchers think about understanding how genes and their products (proteins) function at the cellular level and at the organism level. Yes, we too are interested in the growing field of reverse genetics and the Chin-Sang lab has planned many experiments using this technology.
Current Project Focus
Suppressors of vab-1 and daf-18 mutations
Identification and Characterization of New Genes Involved in Morphogenesis
What is the role of Eph Signaling in the adult nervous System ?
Regulators of DAF-18/PTEN levels
Yeast Two-Hybrid Screens and TAP-TAG approaches
Genome wide systematic RNAi screens