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Identification of important regulators of metastasis in pancreatic cancer

Final Report Summary - CANCER METASTASIS (Identification of important regulators of metastasis in pancreatic cancer)

Cancer is a significant heath issue responsible for approximately 13% of deaths worldwide. Cancer is caused by activating mutations in oncogenes such as Ras and inactivation of tumor suppressors such as p53. Cancer may arise in any tissue in the body although it occurs more frequently in certain tissues such as breast, prostate and lung. In many cases the cancer will, at a later stage of the disease, spread to other tissues in the body in a process known as metastasis. This disseminated form of cancer is very difficult to effectively treat and contributes significantly to cancer related mortality. Thus there is a need for scientific research into the roles of mutated genes in the inception and maintenance of primary cancer and also research into the processes responsible for the increased virulence of advanced metastatic cancer.

This project is focused on identification of regulators of pancreatic cancer metastasis. Pancreatic cancer has one of the poorest prognosis of all cancers. Within Europe approximately 95,000 people are diagnosed with pancreatic cancer per year while a similar number die from this disease each year. This form of cancer has been intensively studied for several years and many of the genetic and molecular changes associated with pancreatic cancer have been uncovered. For example, K-Ras is mutated in greater than 90% of pancreatic adenocarcinomas while p53, Cdkn2A and Smad4 are often inactivated. However, thus far, translation of current knowledge into therapeutic benefit has not been achieved. One of the main factors contributing to this is the late diagnosis of pancreatic cancer at a stage when metastasis has already occurred.

We have performed an shRNA screen to highlight genes involved in the regulation of metastasis. We have defined a target gene list based on previous pancreatic cancer research describing frequently mutated genes in pancreatic cancer, frequently up-regulated genes in pancreatic cancer and genes regulated by oncogenic K-Ras. To complete the screen, we infected cells with shRNA targeted against these genes and injected the cells intravenously to facilitate tumor cell growth at a secondary site, the lungs. We then compared the abundance of shRNA in cells at the beginning of the experiment with the abundance of shRNA in lung tumors at the end of the experiment. Enriched shRNA abundance indicates that gene knockdown provides advantages to metastatic pancreatic tumors while depleted shRNA abundance indicates that the genes are required for optimal survival and growth in the lung tissue. We have identified 18 genes whose shRNA were dramatically enriched or depleted in lung tumors. Furthermore, the shRNA against these genes are modulated to a greater extent in lung tumors than cells grown in vitro for the same period of time. This indicates that the identified genes are required specifically for metastatic colonization within the lung tissue environment rather than intrinsically required for the growth and survival of our pancreatic tumor cells.

We have validated 5 of the hit genes identified in our screen and shown that individual knockdown of these genes using 2 different shRNAs modulates the ability of pancreatic cancer cells to form tumors within the lungs. We have been investigating the functions of these genes which regulate diverse cellular processes. One gene is involved in receptor signaling, which will have an impact on the survival and proliferation of pancreatic cells in the lungs and also in communication with other cell types within the lung environment. Another target gene regulates transcriptional activation, which will allow the cancer cells to make widespread transcriptional expression changes towards a more metastatic phenotype. Other genes are involved in endocytosis and actin dynamics which impacts on signaling via many important pathways in the cell and may modulate survival, cell morphology, migration and invasion within the lung tissue. We have now identified some of the pathways modulated downstream of gene knockdown. We are continuing to investigate these genes with the aim to therapeutically target these proteins or downstream pathways to reduce metastatic tumor formation.

Our research has been important for increased understanding of the molecular basis of invasive/aggressive cancer. As a subset pancreatic patients present with local metastasis to the regional lymph nodes, but have not yet developed metastasis in distant organs, identification of genes or processes that are important for more widespread metastasis may provide real therapeutic benefit for these patients. Our screen has highlighted a number of genes that regulate the survival and colonisation of pancreatic cancer cells in the lungs. Our study builds on previous knowledge derived from mutation and gene expression analysis and has helped to determine which of the many genes deregulated in pancreatic cancer cells contributes to metastasis progression. This research will provide new avenues for investigation in the therapeutic treatment of metastatic pancreatic cancer. Furthermore, the knowledge gained here investigating pancreatic cancer metastasis may also be applied to metastasis of other cancer types and enhances our understanding of the biological basis of metastasis in general.