Modeling cancer in the mouse
Over the past several years, we have been studying cancer and cancer-related processes with a focus on in vivo approaches. Using increasingly precise methods of genetic engineering in the mouse, our laboratory has generated a series of novel strains containing germline mutations in several genes implicated in human cancer. Using these strains (as well as some from others), we have directed single or combinations of mutations to tissues and cells of interest, successfully developing a number of powerful new models of human cancer. These models resemble the human disease both at the genetic and phenotypic levels.

The development of tumor cells from normal cells requires the sequential acquisition of mutations in several cellular genes. In general, two classes of genes are affected in tumor progression: genes that normally act to promote cell division (oncogenes) and genes that function to arrest or inhibit cell division (tumor-suppressor genes). Human familial cancer syndromes (in which affected individuals have a greatly increased risk of developing particular types of cancer) are often caused by the inheritance of a mutant allele of a tumor suppressor gene or an activated allele of an oncogene. Tumor suppressors are thought to regulate cell growth negatively, and they contribute to carcinogenesis when mutated or lost. Thus, individuals who carry only one functional copy of a given tumor suppressor gene are predisposed to cancer, because all of their cells are just one mutational event from lacking an important negative growth regulator.

Many tumors of epithelial origin in humans acquire mutations in the K-ras oncogene, including approximately 30% of non-small cell lung cancer (NSCLC), 50% of colon cancers, and 90% of pancreatic cancers. Our laboratory has been investigating the effects of K-ras mutation in the mouse. In order to control timing and tissue-specific activation of K-ras, we have developed a conditional, activatable allele of the K-ras oncogene, and have used it to generate various mouse models of human cancer. In the lung, activation of oncogenic K-ras leads to the development of atypical adenomatous hyperplasia, adenomas, and adenocarcinomas. Combining mutations in K-ras and p53 in the lung led to the development of more advanced tumors, which exhibited desmoplastic stroma, increased invasiveness and metastatic potential.
We have also developed several other mouse tumor models representing major human cancer types for which good pre-clinical models have been lacking. These include pancreatic cancer, ovarian cancer, soft tissue sarcoma, and invasive colon cancer. These models are being used for a wide variety of studies, including genomics, imaging, chemosensitivity and chemoresistance, microRNA biology, metastasis, putative tumor stem cells identification, and signaling pathway analysis.

Molecular Analysis of Tumor Progression
Using advanced gene targeting methods, generating mouse models of cancer that accurately reproduce the genetic alterations present in human tumors is now relatively straightforward. The challenge is to determine to what extent such models faithfully mimic human disease with respect to the underlying molecular mechanisms that accompany tumor progression. Working with the Golub lab at the Broad Institute, we have developed a method for comparing mouse models of cancer with human tumors using gene expression profiling. We applied this method to the analysis of our model of Kras-mediated lung cancer and found a good relationship to human lung adenocarcinoma, thereby validating the model (Sweet-Cordero et al., 2005). The K-ras lung cancer model has also been used in collaboration with the Golub laboratory to perform miRNA profiling experiments. The data from the model are consistent with human data, demonstrating a general down regulation of miRNA expression in tumors compared to normal tissue (Lu et al., 2005 and see below).

We have also developed a program in metastasis. Metastasis represents the cause of 90% of cancer-associated mortality, yet it is among the least well understood of the multiple processes of cancer pathogenesis. New insights suggest that cancer cells use biological programs that are normally operative during embryogenesis to become metastatic. There are two major subtypes of lung cancer in humans: non-small cell lung cancer (NSCLC), which comprises 80% of the total, and small cell lung cancer (SCLC), which comprises the remaining 20%. We are studying models of both diseases with a specific interest in understanding their metastatic spread. Soft tissue sarcomas are mesenchymal tumors that kill approximately 30% of patients because of lung metastasis. We have generated a mouse model of soft tissue sarcoma (Kirsch et al., 2007) in which primary sarcomas resemble human sarcomas at the genetic, histological, and the ultrastructural level. Like human sarcomas, these murine tumors metastasize to the lung, but not to lymph nodes. We are currently performing genomic analyses of primary tumors and metastases from the lung models and the sarcoma model. Information gained from these studies will be compared against data arising from the study of metastasis in humans, with the goal of understanding the genes and pathways that regulate metastatic spread.

microRNAs
We have been investigating the role of microRNAs (miRNAs) in cancer as well as other aspects of tumor development. MicroRNAs (miRNAs) are a recently discovered class of small noncoding RNAs that post-transcriptionally regulate the expression of target mRNA transcripts. Many miRNAs target mRNAs involved in processes aberrant in tumorigenesis, such as proliferation, survival, and differentiation. While previous work has shown a global decrease of mature miRNA expression in human cancers, it is unclear whether this global repression of miRNAs reflects the undifferentiated state of tumors or causally contributes to the transformed phenotype. We have demonstrated that impaired microRNA processing enhances cellular transformation and tumorigenesis (Kumar et al., 2007). Cancer cells expressing short hairpin RNAs (shRNAs) targeting three different components of the miRNA processing machinery showed a substantial decrease in steady-state miRNA levels and a more pronounced transformed phenotype. In animals, miRNA processing-impaired cells formed tumors with accelerated kinetics. We went on to study the let-7 miRNA family, which has been proposed to function in tumor suppression (reduced expression of let-7 family members is common in non-small cell lung cancer). We found that let-7 functionally inhibits non-small cell tumor development (Kumar et al., 2008). We are also studying miR-17~92, miR-106b~25, and miR-106a~363, a family of highly conserved miRNA clusters. Amplification and overexpression of miR-17 92 is observed in human cancers, and its oncogenic properties have been confirmed in a mouse model of B cell lymphoma. We have knocked out the miR-17~92 clusters in the mouse, revealing essential and overlapping functions (Ventura et al., 2008). Mice deficient for miR-17 92 die shortly after birth with lung hypoplasia and a ventricular septal defect. Absence of miR-17 92 also leads to increased levels of the proapoptotic protein Bim and inhibits B cell development at the pro-B to pre-B transition (Ventura et al., 2008). These results provide key insights into the physiologic functions of this family of microRNAs and suggest a link between the oncogenic properties of miR-17 92 and its functions during B lymphopoiesis and lung development.

Ras in Lung, Colon and Pancreatic Tumros (and its effect on putative stem cells)
Injury models have suggested that the lung contains anatomically and functionally distinct epithelial stem cell populations. We have isolated such a regional pulmonary stem cell population, termed bronchioalveolar stem cells (BASCs). These stem cells were enriched, propagated, and differentiated in vitro and found to be activated by the oncogenic protein K-ras (Kim et al., 2005). Our studies suggest that BASCs are a stem cell population that maintains the bronchiolar Clara cells and alveolar cells of the distal lung and that their transformed counterparts give rise to adenocarcinoma.

Kras is also commonly mutated in colon cancers, but mutations in Nras are rare. We have used genetically engineered mice to determine whether and how these related oncogenes regulate homeostasis and tumorigenesis in the colon. Expression of K-RasG12D in the colonic epithelium stimulated hyperproliferation in a Mek-dependent manner. N-RasG12D did not alter the growth properties of the epithelium, but was able to confer resistance to apoptosis. In the context of an Apc-mutant colonic tumor, activation of K-Ras led to defects in terminal differentiation and expansion of putative stem cells within the tumor epithelium (Haigis et al., 2008).

Finally, Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal of all human malignancies. Currently, there are no reliable means for early detection or effective treatment options. Pancreatic ductal adenocarcinoma has a particularly high incidence of activating K-RAS mutations, which are believed to be an initiating event. Identification and characterization of the cell of origin(s) of PDAC is essential for elucidating the pathways affected by oncogenic mutations early in tumor development, at a point when chemopreventative measures might still be effective. Insight into the complex aspects of PDAC biology should also result in improved detection and diagnosis of this devastating disease.

Chemo-sensitivity and - resistance in Lung and Ovarian Cancer
Lung cancer is the leading cause of cancer-related death worldwide with a five-year survival rate of 15%. A major obstacle to successful treatment is intrinsic and acquired resistance to chemotherapy. To improve cancer treatment, we need the ability to predict chemotherapy response and overcome mechanisms of resistance. However, the molecular mechanisms that govern tumor response to chemotherapy are poorly understood. The majority of studies investigating chemotherapy response have been performed with cell lines or xenograft models, which have often been ineffective in predicting response in humans. Using in vivo micro-CT imaging, we found that the majority of Kras driven-lung tumors respond to an initial dose of cisplatin. However, after prolonged therapy with cisplatin, most lung tumors acquire resistance to cisplatin (Oliver et al, in preparation). We are working toward the understanding the mechanism of resistance in these mice, using gene-set enrichment analysis and DNA copy number analysis (in collaboration with David Mu and Scott Powers at Cold Spring Harbor Laboratories).

Ovarian cancer is relatively chemotherapy sensitive with a response rate of ~80%. The most common first-line treatment regimens for ovarian cancer are platinum-based, with cisplatin and carboplatin being the most widely used. However, only ~20% of patients survive more than 5 years following treatment. The vast majority of patients acquire drug resistance and die from their disease. It is again critical to understand the molecular and cellular mechanisms of chemotherapy response and resistance in order to design more effective therapies for ovarian cancer patients.
We have generated a mouse model of endometroid ovarian cancer (Dinulescu et al., 2005). To monitor drug effects on tumor size over time, we explored two types of noninvasive bio-imaging: ultrasound imaging and luciferase-based bioluminescent imaging. We have generated LSL-KrasG12D/+/PTEN fl/fl that carry an additional allele, LSL-firefly luciferase. This allele was knocked into the ROSA26 locus, with expression of the enzyme luciferase behind the ubiquitous CAGGS (CMV-Chicken B-actin) promoter, which is prevented by the LoxP-stop-LoxP cassette (unpublished mouse, available from the MMHCC Mouse Repository at the NCI). In these mice, exposure of cells to Cre recombinase allows expression of activated K-ras, PTEN loss, and ubiquitous expression of the luciferase enzyme. Treatment of mice with the substrate, luciferin, allows noninvasive bioluminescent detection of tumor cells in vivo. Tumors and metastasis can be imaged by this method, allowing us to identify cisplatin sensitive and resistant tumors for genomic analysis. Mouse ovarian tumors are sensitive to cisplatin treatment similar to the human disease (Oliver et al, in preparation), but it is as yet unclear whether tumors from these mice acquire resistance after prolonged therapy, or whether a subset of tumors are inherently resistant.

p53 reactivation
We have also been studying whether the reactivation of p53 could have therapeutic effect in established tumors using a Cre-Lox based approach. We observed a dramatic response to p53 restoration in nearly all tumors tested to date (Ventura et al., 2007). Interestingly, the response to the reactivation of p53 differed between tumor types, with lymphomas undergoing apoptosis and sarcomas undergoing cell cycle arrest with features of senescence. This work indicates that the signaling pathways that impinge on p53 remain active in established tumors and provides support for efforts to activate this pathway in human cancer therapy.

Tumor Immunology
In the past few years, we begun to use mouse models to study tumor immunology. A long-standing goal of cancer immunotherapy is to use tumor-infiltrating T cells to fight cancer, yet many critical questions remain about the nature of these cells and how to best exploit them. The failure of infiltrating T cells to eliminate tumors has been attributed to tumor evasion via local immune suppression or tolerance induction; however, studies of immune-tumor interactions have predominantly utilized transplant models of cancer, which may have little relevance to the human disease. Thus, we have undertaken studies of tumor immune surveillance based on the autochthonous model of lung adenocarcinoma established in the Jacks lab (in which oncogenic K-ras is activated by Cre recombinase with temporal and spatial control to initiate tumor formation). A major strength of autochthonous mouse models of cancer is that tumors develop in animals with normal immune function. Therefore, tumor progression may be shaped by an interaction with the immune system. In order to study the response of T lymphocytes to tumor-specific antigens as well as the mechanisms of tumor evasion of the immune response, we have recently developed next-generation NSCLC models in which well-studied antigens are expressed specifically in developing tumor cells. Two strategies have been initiated toward this goal. One utilizes stably-integrating lentiviral vectors that express simultaneously both the antigens and Cre recombinase, while the other involves generation of a conditional knock-in allele of the antigens that can be activated concurrently with oncogenic K-ras following expression of Cre. In both systems, the antigenic peptides have been fused to a Firefly Luciferase gene, enabling us to monitor tumor size over time using in vivo bioluminescence-imaging technology. With these novel tools, we are studying the anti-tumor efficacy of tumor-reactive T cells and the mechanisms by which these cells are suppressed or tolerized during the course of tumor progression. We expect that an improved understanding of the regulatory processes that allow tumors to develop in the context of a functional immune system will be critical if immune-based therapies are eventually going to be effective in the treatment of cancer.