Challenges in Cancer Biology
Cancer is the second leading cause of death in the United States. Malignant tumors are difficult to treat due to multiple molecular and cellular aberrations and their ability to undergo metastasis. These challenges demand studies on cancer as a dynamic process, with an increased focus on understanding tumor initiation and progression mechanisms. Information from such studies will allow the detection of pre-cancerous lesions to move the first line of defense prior to full-blown malignancy.
A few key questions to be answered:
- Where and when does a tumor initiate?
- Do cancers originate from cancer stem cells, or from de-differentiated mature cells?
- What are the unique molecular and cellular features of tumor-initiating cells?
- How do tumor cells interact and co-evolve with their environment?
- How do tumor cells migrate to metastasize?
- How can we use the information to prevent and treat cancers?
|Left: Blood vessels (blue) infiltrate a tumor
Right: Mixed cell populations in a tumor. Green,
dividing cells; red, differentiated cells
The importance of using mouse models to study cancers.
Though cancers can be studied with many approaches, the answers to questions above require us to perform studies in native settings of cancers. Because one cannot study human patients unless they carry detectable tumors, it becomes very important to investigate these questions using mouse cancer models. One important event for human tumor initiation is the loss of heterozygosity (LOH) of tumor suppressor genes, which has been modeled in mice using gene knockout technique. However, current mouse genetic models have difficulties in controlling gene inactivation in a small number of cells to mimic the sporadic nature of the LOH events typical of human tumors. Even if gene inactivation can be limited to a few cells, it is very difficult to unambiguously distinguish them from surrounding cells for mechanistic analysis. Therefore, it becomes very important for cancer researchers to develop more physiologically relevant mouse models to address the critical questions raised above.
Use mouse genetic mosaic system to model cancers.
The research theme in the Zong lab is to use a mouse genetic mosaic system termed MADM (Mosaic Analysis with Double Markers) [Zong 2005 Cell] to probe into core cancer mechanisms. Rather than generating more TSG KO mouse models, MADM pushes the phenotypic analysis of currently available models to an unprecedented resolution [Muzumdar 2007 PNAS]. After recombining an available TSG KO allele onto a MADM-bearing chromosome, the MADM system generates sporadic mutant cells from a heterozygous mouse and uniquely labels the mutant cells with GFP via a single inter-chromosomal mitotic recombination event. Such a concurrent gene inactivation and cell labeling allows the study of the tumor initiation within hours of TSG loss. MADM also provides a perfect internal control, RFP labeled WT sibling cells of the GFP labeled mutant cells. Therefore, our lab will use the MADM system to model LOH of tumor suppressor genes (TSGs) and to study the detailed mechanisms of tumor initiation and progression.
How does MADM do the trick?
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- a) The basic components of MADM is a pair of chimeric fluorescent protein genes that are knocked-in at an equivalent locus of homologous chromosomes. The animal will be colorless because of the chimera proteins do not code functional proteins.
- b) MADM does not knock-out genes. A pre-existing mutated gene at the telomeric side needs to be recombined onto one of the MADM-bearing chromosomes. So our starting material is a heterozygous, colorless animal.
- c) MADM makes use of mitotic recombination to generate homozygous mutant cells. A single event of Cre-loxP mediated recombination will result in the reconstitution of fluorescent proteins and the movement of the mutant allele.
- d) Through Z segregation, MADM will generate a yellow, heterozygous cell that is not very informative. However, a X segregation will result in a green mutant cell, and a red sibling wildtype cell (a perfect internal control).
- e) All progeny from these colored cells will inherit the color, allowing MADM to be used as a lineage tracing tool. We hope to take advantage of this feature to locate cell-of-origin of cancers, and open the black box of tumor initiation and early progression mechanisms.
- f) Please see Zong et al, Cell 2005 for details.
Sparse cells labeled by MADM.
One of the most frequently-brought up question is the possibility of using MADM to model cancers - will MADM system be able to generate tumors at all? The well-accepted multi-hit hypothesis for cancers implicates that it takes 5-8 mutations to generate a full-blown tumor. Intuitively, MADM-induced sparse mutant cells, though mimics human cancer onset very well, would have less probability than full animal knockout to acquire mandatory hits/mutations to advance into full-blown tumors. Even if tumors could form, we would still face serious feasibility issues in both the penetrance and the latency of tumor formation. To circumvent these difficulties, we first engineered the MADM system onto mouse chromosome 11, on which a list of the most potent tumor suppressor genes such as p53 and NF1 reside. Second, we tailored our research projects based on a few well-established conventional mouse cancer models, in which p53 and/or NF1 play critical tumor suppression roles. At this time, our lab's research is focused on two areas:
Medulloblastoma is the most common malignant brain tumor in children, and resides in the cerebellum of affected patients. Current therapy involves cytotoxic agents, including radiation and chemotherapy. Though partially effective, these methods are not completely satisfactory because of their long-term detrimental effects, in particular on the normal development of young patients. Therefore, it is very important to understand the molecular mechanisms of medulloblastoma, which will allow the development of more specific and less toxic drugs to improve the treatment efficacy. Clinical and human genetic studies have indicated that multiple genetic alterations are involved in the formation of medulloblastoma. In particular, both a deregulation of Shh signaling pathway and a defective DNA repair machinery could contribute to the tumorigenesis.
Recently, we established a medulloblastoma model with perturbations in both pathways, namely MADM-induced mosaic p53 loss and a Ptc heterozygous mutation background. Using this genetic combination, we have successfully generated a MADM-based medulloblastoma model, with a 50% penetrance and a three-month latency. It demonstrated that not only tumors can be induced from mosaic TSG mutant mice, but also the latency of tumorigenesis could be surprisingly short with very high penetrance. More importantly, tumors identified from these brains are entirely green, despite of very low number of green cells in unaffected areas. This observation strongly suggests that a tumor induced by MADM was originated from the clonal expansion of a single mutant cell. Currently, we are further characterizing tumors to map out the molecular and cellular events for the medulloblastoma formation, and to explore the interactions between tumor cells and their environment critical for the tumorigenesis process.
Glioma is the most common type of brain tumors in humans. The malignant form of glioma is incurable due to its elusive tumor origin, highly diffusive nature and complicated tumor organization. The hope for better treatment lies within the identification of the cell of origin of glioma, the depiction of tumor-niche interactions, and the delineation of molecular changes during tumorigenesis. p53 loss acts as an initiating mutation in brain tumors, in particular glioma. However, the loss of p53 alone is not sufficient for glioma formation. NF1, another TSG relevant to glioma, is also located on the mouse chromosome 11. Recently, it was reported that the loss of both p53 and NF1 in mice could lead to glioma formation at a full penetrance. Our lab has established MADM-ready mice that can generate sporadic co-LOH of both p53 and NF1. We are underway to establish a mouse cancer model that would allow the clonal growth of glioma. With the unprecedented resolution provided by the MADM system, in combination with other molecular biology, biochemistry and pathology methods, we hope to address the following key questions:
- Identify the cell of origin for glioma.
- Map out the molecular changes during tumor initiation and progression.
- Analyze tumors at a clonal level.
- Understand tumor-niche interactions.
|MADM system generates the well-isolated clones in mouse brain via a drug-inducible Cre recombinase. (A) High dosage of Doxycycline (Dox) induces the labeling of cells in cortex with high density. (B) Very low dosage of Dox induces the very sparse labeling of cluster of cells in the same region with the clonal features.|
- Expand MADM-based cancer modeling to other tumor types.
- Expand the MADM system to study other critical tumor suppressor genes.
- Forward mutagenesis screening for oncogenes and tumor suppressor genes with genetic mosaic system (somatic and germline).
- Live imaging for tumor-niche interactions & metastasis studies. Establish cancer models for pre-clinical drug tests.