Research Plans for the Ludwig-funded Research Program: The Ludwig Center for Molecular Oncology at MIT has been the driving force for establishing metastasis research into a major theme in the laboratory of Tyler Jacks. Having established a number of genetically-engineered mouse models of human cancer over the past twenty one years, the Jacks lab has begun to focus increasingly on using these models to understand the molecular and cellular factors that control metastatic spread. The majority of this work has utilized a mouse model of non-small cell lung cancer (NSCLC) initiated by mutation of the K-ras oncogene and the p53 tumor suppressor gene. As described in greater detail below, several of these projects are being pursued collaboratively with other members of the Ludwig Center at MIT. In addition, the Jacks laboratory is establishing collaborations with the groups from the Ludwig Center at the Dana-Farber/Harvard Cancer Center. The over-arching goals of this research is to translate the finding from mouse cancer models to human cancer with the hope of developing therapies aimed at inhibiting metastatic spread and others that designed to kill cancer cells at metastatic sites.
For many years, cancer research has been focused on understanding the mutational changes that occur within cancer cells. However, it is now recognized that tumors should be more appropriately viewed as heterogeneous collection of cells, admixtures of cancer cells and many other cell types that act both to inhibit and promote tumor progression. Despite the growing awareness that the stromal elements of a tumor contribute to cancer progression, a detailed understanding of how they function is currently lacking. In many cancer types, characterization of the cancer-associated stroma has revealed a surprising diversity of cell types and structural components present within tumors. These include endothelial cells, which are crucial for the formation of the neovasculature; inflammatory cells, like macrophages, which can be co-opted by cancer cells to secrete a variety of growth factors, chemokines, and proteases that facilitate tumor growth and invasion; cancer-associated fibroblasts, which have been recognized as critical contributors to neoplastic progression by stimulating angiogenesis and invasion; and T and B lymphocytes of the adaptive immune system. All of these stromal cells are embedded within a complex extracellular matrix (ECM) that not only provides the structural framework of the tumor but which also directly affects the growth and migration of cancer cells through the presence of pro-tumorigenic factors tethered to the matrix.
In order to functionally test the role of specific components of the microenvironment to tumor progression and metastasis, the Jacks laboratory has established a next-generation mouse lung cancer model with which they can control genetic manipulation of cancer cells and stromal cells separately. Developed with a combination of the Flp-FRT and Cre-LoxP recombinase systems, this model has been designed to allow the selective manipulation of specific genes, pathways or cell types within the stromal microenvironment in established tumors. For example, inspired by research in the Weinberg laboratory demonstrating the importance of stromal fibroblasts in tumor progression and metastasis, the Jacks lab is using this system to specifically ablate this cell type in autochthonous lung cancers at different stages of tumor development. They chose to use a Cre-inducible diphtheria toxin receptor-mediated ablation strategy to test how the depletion of this prominent stromal cell type affects tumorigenesis. Rosa26LSL-Dtr mice were purchased from Jackson labs and were crossed to KrasFSF-G12D/+;p53FRTed/FRTed mice. To specifically ablate CAFs, they crossed KrasFSF-G12D/+;p53FRTed/FRTed;Rosa26LSL-Dtr mice to mice expressing Cre under the control of the CAF-specific promoters smooth muscle actin (SMA; obtained from Dr. Raghu Kalluri, Beth Israel Deaconess Medical Center) and fibroblast specific protein 1 (FSP1; obtained from Dr. Harold Moses - Vanderbuilt-Ingram Cancer Center). After extensive breeding they have now generated KrasFSF-G12D/+;p53FRTed/FRTed;Rosa26LSL-Dtr/+;SMA-Cre and KrasFSF-G12D/+;p53FRTed/FRTed;Rosa26LSL-Dtr/+;FSP1-Cre cohorts. The Jacks lab is now ready to measure acute effects on the biology of both primary tumors and metastatic lesions by this perturbation as well as the frequency of metastatic spread in longer-term experiments. Future efforts will examine the consequences of mutation of specific genes, for example, those for growth factors and chemokines secreted by stromal fibroblasts. This system is equally well suited to manipulate the genes of macrophages (also under study in the Weinberg laboratory) and other tumor-infiltrating cell types, which will be investigated in time. These studies will provide proof-of-principal for small molecule and antibody-based therapies targeting these proteins and pathways. Successful pre-clinical validation of these targets in this model system will hasten the development of such therapies for use in humans.
In collaboration with the laboratories of Richard Hynes and Sangeeta Bhatia at MIT, the Jacks laboratory is studying the contributions of components of the ECM to metastatic progression. Working with the mouse NSCLC model developed in the Jacks lab, the Hynes group is using mass spec-based methods to characterize the elements of the ECM in metastatic primary tumors. In related studies, the Jacks and Bhatia labs used microarrays composed of ECM components to study the adhesive properties of lung cancer cell lines derived from metastatic tumors in this model. These descriptive studies were followed by functional experiments involving RNA interference (RNAi) and antibody inhibition in transplantation models of metastasis. They were able to identify cell extra-cellular matrix interactions that correlate with metastasis, namely that metastatic cells associate with fibronectin when combined with galectin-3, galectin-8 or laminin. These studies were published in Nature Communications at the end of 2012.
In addition to exploring the tumor microenvironment, the Jacks laboratory is interested in the cancer cell-specific changes that contribute to metastasis. Based on a gene expression profile that distinguishes metastatic primary lung tumors (as well as metastases) from non-metastatic primaries, the lineage-restricted transcription factor Nkx2-1 was the most significantly affected, being down-regulated in the metastatic cell lines by an average of 30 fold. Resourcing to a series of conditional mouse models to study the fate of normal lung epithelial cells and lung cancers upon loss of Nkx2-1, the Jacks lab used a series of techniques at the cutting-edge of genetic engineering. They found that Nkx2-1 deletion in normal and neoplastic lung caused loss of pulmonary identity and resulted in gastric transdifferentiation. Going beyond the histological analysis, they went on to study the mechanistic basis for this cell fate change. Using a series of loss-of-function and gain-of-function genetic experiments as well as chromatin immunoprecipitation-RNA sequencing analysis, they showed that Nkx2-1 normally sequesters the FoxA1/2 transcription factors away from their target genes involved in gut differentiation. Loss of Nkx2-1 allows FoxA1/2 to relocalize to these promoters and drive the gut transcriptional program in concert with other factors. Beyond their implications for normal developmental processes, these data show a surprising degree of plasticity of cancer cells and suggest that the emergence of fully undifferentiated, stem-like cells may require a step-wise conversion through layers of alternative differentiation states. This series of experiments was published in Molecular Cell.
Based on recent work in the Weinberg laboratory linking the epithelial-mesenchymal transition (EMT) to cellular plasticity and stem cell-like fates, the Jacks and Weinberg groups are collaborating to study the induction of EMT during lung tumor development. This project involves crossing an EMT reporter developed in the Weinberg lab to the Jacks lab’s lung tumor model. Cells undergoing an EMT will be fluorescently labeled and can be readily isolated and investigated further.
A second interesting gene that has emerged from expression profiling of cell lines from metastatic and non-metastatic tumors is the invadopodia-mediator Tks5. Importantly, although the overall transcription of Tks5 is similar between in the collection of cell lines tested, the metastatic cell lines express a unique isoform of the gene that is able to stimulate invadopodia formation. Invadopodia are actin-rich membrane protrusions that promote ECM degradation and cellular migration, two properties of metastatic cells. In collaboration with Frank Gertler from the Ludwig Center at MIT, the Jacks lab evidenced that Tks5 undergoes an isoform switch from a “short” form to a “long” form as tumors progress to metastasis in our lung cancer model. By using a variety of state-of-the-art techniques and approaches, both in cell culture models and, more powerfully, in whole animal studies, the Jacks lab was able to confirm the metastasis-promoting function of the long form of Tks5. Moreover, they showed that the short form acted to interfere with the function of the long form through a dominant-negative interaction. They have also demonstrated that the expression of the long form of Tks5 correlated with increased tumor progression and a worse prognosis for patients. Thus, in addition to making significant contributions to unraveling the molecular basis of Tks5 function in promoting metastasis, this research may provide an important prognostic marker for human cancer. This work was published in Genes and Development and made it to the cover of G&D July 2013 issue.
Finally, by using genome sequencing the Jacks lab characterized the somatic evolution of a genetically-engineered mouse model (GEMM) of small cell lung cancer (SCLC) initiated by loss of Trp53 and Rbl. They identified alterations in DNA copy number, complex genomic rearrangements, and demonstrated a low somatic point mutation frequency in the absence of tobacco mutagens. Alterations targeting the tumor suppressor Pten occurred in the majority of murine SCLC studied, and engineered Pten deletion accelerated murine SCLC and abrogated loss of Chrl9 in Trp53; Rbl; Pten compound mutant tumors. They found evidence for polyclonal and sequential metastatic spread of murine SCLC by comparative sequencing of families of related primary tumors and metastases. The Jacks lab proposes a temporal model of SCLC tumorigenesis with implications for human SCLC therapeutics and the nature of cancer genome evolution in GEMMs. This work resulted from collaborative efforts with the Getz laboratory in the Broad institute at MIT and the Massachusetts General Hospital.