Research in the Feldser Laboratory
One of the major goals of cancer biology is to understand the evolutionary process of tumorigenesis; from identification of the cell type of origin for a particular cancer to understanding how acquired mutations initiate the neoplastic process and ultimately lead to malignant disease. We employ an integrative approach utilizing quantitative molecular and biochemical techniques, effective gene engineering technologies, and powerful in vivo cancer models. Our objective is to uncover the oncogenic networks that are usurped by cancer cells to drive tumor progression as well as those tumor-suppressive pathways that are disabled during tumorigenesis. A detailed molecular understanding of the cellular state changes that occur both within the cancer cell itself and within cells in the tumor microenvironment will undoubtedly lead to better strategies to eradicate malignant cells.
The multistep process of tumorigenesis. Initiating mutagenic events convert single normal somatic cells to hyperplastic lesions. In the case of lung adenocarcinoma this is often due to oncogenic Kras mutations and mutation of a single copy of Kras is likely sufficient to initiate hyperplasia in certain cell types. Cancer evolves as a consequence of the selection of a diverse set of genetic drivers mutations, or progression events. In our own work, amplification of MAPK signaling that is readily acquired in the absence of p53 leads to tumor progression to an early carcinoma (Feldser et al. 2010, red cells in figure). It is likely that further mutations, such as loss of Nkx2.1 expression and up-regulation of Hmga2, are required to achieve an advanced primary tumor that has the capability to metastasize. Events that endow cells with the ability to leave the primary tumor site and seed new growths at distant sites are metastatic events. Much like in the human disease, in our lung adenocarcinoma model these metastases can be found in the draining lymph node, adrenal glands, liver, pleural cavity, and in other sites in the lung (Winslow et al. 2011).
Models of human lung cancer
Our laboratory exploits the outstanding genetic powers of the mouse in order to achieve our goal of deconstructing the multistep process that leads to the formation of lung cancer. In these models tumor initiation occurs in single cells that expand within the appropriate tissue microenvironment and undergo multiple cell state changes that ultimately result in the development of primary malignant carcinomas in the lung, some of which possess metastatic potential. We focus on adenocarcinoma and small cell carcinoma of the lung. These diseases represent two prevalent subtypes of human lung cancer that have disparate etiology but for which conventional genetically engineered mouse models exist.
Two models of lung cancer: Inhalation of viral particles that express cre recombinase initiate tumor formation in each model. Lung adenocarcinoma is initiated by activation of a latent oncogenic allele of Kras and deletion of p53 (Jackson et al. 2005). Small cell lung cancer is initiated by deletion of tumor suppressors p53 and Rb (Meuwissen et al. 2003).
XTR: A recombinase-based system for regulating gene function in a conditional and reversible manner
Synthetic biological tools that enable precise regulation of gene function within in vivo systems have enormous potential to discern gene function in diverse physiological settings. We have developed a synthetic gene switch that enables conditional inactivation, reports gene expression, and allows inducible restoration of virtually any gene of interest. Gene inactivation and reporter expression is achieved through Cre-mediated stable inversion of an integrated gene-trap-reporter, whereas inducible gene restoration is afforded by Flp-dependent deletion of the inverted gene trap. We term this allele system XTR to denote each of the allelic states and the associated expression patterns of the targeted gene: eXpressed (XTR), Trapped (TR), and Restored (R).(Robles-Oteiza et al. Nature Comm. 2016)
Distinct oncogenic thresholds to MAPK signaling.
In cancer, mutational activation of the Kras proto-oncogene engages the MAPK pathway, but little is understood about the cellular thresholds that govern MAPK-induced pathophysiological responses. We capitalized on the paradoxical ability of pharmacological inhibitors of wildtype Braf to amplify MAPK signaling specifically in the context of cells expressing oncogenic Kras in the lung. We found that amplification of MAPK signaling after oncogenic KrasG12D activation overcomes a tumor initiation threshold that exists in a subset of lung airway epithelial cells. Additionally, we found that a separate MAPK signal intensity threshold acts as a p53-activated checkpoint to limit lung adenoma progression to malignancy. Our data therefore highlight the presence of at least two separate thresholds to oncogenic MAPK signaling; those that govern cellular decisions to initiate tumorigenesis and those that drive the transition toward malignancy and activate p53 tumor suppression.
Amplified MAPK signaling downstream of KrasG12D expression initiates proliferation and drives transdifferentiation of airway epithelial cells toward an alveolar type II cell fate. KrasLSL-G12D/+;p53flox/flox;Rosa26LSL-YFP/+ mice infected with adenovirus expressing Cre restricted to CC10-expressing cells. Airway epithelium four weeks after recombination and amplification of MAPK signaling (Braf inhibition) showing hyperproliferation beginning to express the AT2 cell marker surfactant protein C (SPC). CC10 (cyan), SPC (red), YFP (yellow), DAPI (white). Cicchini et al. submitted.
Context dependent effects to p53 restoration
Our research has uncovered an association of amplified MAPK signaling and the carcinomatous state in lung adenocarcinoma. That this association is not observed in benign adenomatous precursor lesions suggests that acquisition of this aberrant molecular state may be causative for tumor progression. Concurrent with tumor progression these higher-grade tumors up-regulate the upstream activator of p53, the p19Arf tumor suppressor. This induction of the Arf protein is presumably a failed attempt to engage the p53 pathway. Using genetic alleles that allow p53 gene restoration these established cancers, we found that these Arf expressing carcinomatous cells were specifically sensitive to the tumor-suppressive effects of p53, whereas their precursor adenomatous counterparts were indifferent to p53 restoration.
Adenocarcinomas have high MAPK activity and express nucleolar Arf (Feldser et al. 2010). Immuno-fluorescence detection of activated pErk (red) and Arf (green).
Building on the lessons from our previous work, we are focused on elucidating the downstream functions of tumor-suppressor genes in lung cancer as well as the oncogenic Ras signaling in tumor initiation and progression. To this end, we have developed multiple novel tools and experimental systems to dissect tumor suppressor gene function, oncogene signaling, and the role of the tumor microenvironment in carcinogenesis. We plan to exploit these tools in a variety of contextual in vivo situations that are relevant to cancer biology in order to gain a more complete understanding of the etiology these diseases.