Periodic Report Summary 1 - IONTRAC (Ion Transport Proteins in Control of Cancer Cell Behaviour)
Pancreatic ductal adenocarcinoma (PDAC) has one of the worst prognoses of all cancers, with an overall 5-year survival rate of less than 5%. As PDAC is a disease of the elderly and given that the European population is aging, the incidence is very likely to increase. Pathophysiologically, PDAC is characterized by intense growth factor signaling leading to a massive stroma reaction (desmoplasia) which in turn causes local hypoxia and acidosis, resistance to apoptotic stimuli, as well as early local invasion and distant metastasis. Desmoplasia which is central to PDAC pathophysiology involves the mutual stimulation of PDAC cells and so-called pancreatic stellate cells (PSCs) from the pancreas stroma. The overall aim of the IonTraC project is to comprehensively test the hypotheses that proteins involved in ion transport ("transportome") are crucially involved in disease progression and thereby constitute novel diagnostic/therapeutic targets for PDAC. Thus, the project will be able to determine the concerted expression and function of ion channels and transporters required for the progression of PDAC, and to provide validated therapeutic and diagnostic concepts and tools that are based on transport proteins serving as novel drug targets and/or biomarkers.
Cancer is a major public health problem in the EU and a number of cancers including pancreatic cancer are still essentially incurable. Thus, there is an urgent need for new therapies and the demand of the academic and private sectors for researchers in oncology will remain high. Our training programme responds to this need. The fellows receive training in general experimental and clinical oncology, and in translating results from basic research into clinical practice. IonTraC fellows will also be trained in the (patho-) physiology of ion transport. This second branch of the training will enable them to work across disciplines since ion transport proteins are "key players" in many other disciplines, too.
The IonTraC project is divided into five scientific workpackages (WPs). WP1 establishes in vitro and in vivo models which will be refined through profiling of transportome expression and function in WP2. WP3 will provide a functional background on the role of the transportome in PDAC progression. WP4 and WP5 will assess the diagnostic and therapeutic potentials of the transportome. The scientific workpackages are complemented by an interdisciplinary training program on ion transport in and beyond oncology as well as by outreach and management activities. Recruitment of the fellows was accomplished by the end of the first year so that the current report covers 1 to 1.5 years of research within the project. All fellows (13 ESRs and 1 ER) contributed to multiple workpackages.
Initially, all fellows contributed to WP1, the establishment of the best experimental cell and animal models. We decided to use a panel of well established PDAC cell lines, primary cultures of PSCs and mouse models using orthotopic transplantation of PDAC cells. The experimental procedures including the choice of the extracellular matrix proteins, 3D culturing and co-culturing of PDAC cells and PSCs, environmental conditions such as pH, hypoxia and nutrient concentration as well as the orthotopic mouse models were optimized according to the needs of the respective projects. WP1 has been finalized so that we have completed the first two delivrables and the first milestone by establishing and validating our experimental models.
WP2 is devoted to revealing the expression profile of the transportome in PDAC cells and PSCs. We started with a "data mining" approach and explored existing data based on microarray analyses from cell lines and patient samples. Then the fellows investigated the expression of their respective target proteins in the experimental models using a combination of (q)PCR, Western blot, immunofluorescence, patch clamp, ionic imaging (Ca2+ and pH) and metabolic profiling. The combined effort of all groups provided a comprehensive overview of the PDAC transportome. Thus the expression of different TRP, K2P, KV, KCa, NaV, and TMEM16 channels, ORAI and STIM proteins, P2X receptors, as well as pH regulatory transport proteins and carboanhydrases has been validated on mRNA, protein and on a functional level. Profiling of the transportome was complemented by the analysis of expression of functionally associated proteins such as integrins, growth factor and chemokine receptors as well as other regulatory proteins. Notably, the expression profile differed among different PDAC cell lines. Moreover, it became apparent that it also depends in PDAC cells and in PSCs on environmental factors such as hypoxia. WP2 has almost been completed. We anticipate to have a complete list by month 30.
In WP3 we analyse the function of the members of the PDAC transportome in order to determine to which pathophysiological processes they contribute during disease progression. So far we found that migration and invasion of PDAC cells and PSCs requires the activity of a number of transportome family members involved in Ca2+, pH and cell volume homeostasis. Similarly, voltage-gated K+ and Na+ channels turned out to be needed for efficient PDAC cell migration and invasion. Volume and Ca2+ regulatory channels also participate in the regulation of proliferation, apoptosis and drug resistance. Finally, we also identified transport proteins involved in the regulation of the PDAC cell metabolism. While these studies clearly reveal the importance of the transportome in pathophysiological processes underlying the aggressiveness of PDAC, they are still in a premature state because the exact cellular mechanisms have not yet been elucidated. For most groups this will be the focus of next year's work.
The analysis of the diagnostic potential of the PDAC transportome (WP4) is still in a very early state. However, in a way of proof-of-principle experiments we could verify the elevated expression of some of those transportome members identified in the data mining approach in patient tissue samples by means of immunofluorescence, too. Tools are now being developed for the use of these target proteins for multimodal whole animal imaging.
WP5 has also only just been started by performing initial experiments on the therapeutic potential of exploiting the necessitiy of K+ channels for the induction of apoptosis. Once WP3 has progressed further this novel knowledge will provide important input for designing transportome-based therapeutic strategies.
Training activities within WP6 proceed as planned. Thus far, the following network-wide training activities have been realized: two network meetings (organized by WWU and UOXF), two summer/winter schools ("Omics" in PDAC and beyond, organized by CAU; Ion transport in health and disease including a lecture on Preclinical drug discovery - Research at the border between industry and academia, organized by UCPH) and two workshops (Pancreas - from basic physiology to PDAC therapy, organized by WWU/ULi; scientific writing, organized by UOXF). In addition all fellows profit from multiple local training activities including first of all their project-specific training-through-research with secondments to partner laboratories but also language courses. Thus, WP6 is ongoing throughout the project lifetime.
The management activities are bundled in WP7. They are also ongoing throughout the project lifetime. The administrator supports the coordinator by maintaining the required infrastructure such as website, backoffice and by preparing meetings, financial accounting etc..
Outreach activites are covered by WP8. They include scientific publications, organization of and participation in scientific conferences/workshops or the submission of common grant proposals to international funding bodies. So far the IonTraC group already published three original papers and more than 10 reviews. In this context it is notable that two of the IonTraC members are guest editors of a Theme Issue of the Philosophical Transactions Series B devoted to ion transport in cancer. Almost all members of the IonTraC group contribute review articles to this issue which is to appear early in 2014. Moreover, our fellows presented their results in more than 20 abstracts/posters/oral communications at national and international meetings. The IonTraC itself project was introduced at several national and international conferences to a broader scientific audience. Furthermore, several international grant proposals in which IonTraC members teamed up with other scientists had been submitted. Thus, work within the IonTraC project has already created a considerable momentum. Several further publications are presently in preparation and will be submitted soon.
Cancer is a major public health problem in the EU and a number of cancers including pancreatic cancer are still essentially incurable. Thus, there is an urgent need for new therapies and the demand of the academic and private sectors for researchers in oncology will remain high. Our training programme responds to this need. The fellows receive training in general experimental and clinical oncology, and in translating results from basic research into clinical practice. IonTraC fellows will also be trained in the (patho-) physiology of ion transport. This second branch of the training will enable them to work across disciplines since ion transport proteins are "key players" in many other disciplines, too.
The IonTraC project is divided into five scientific workpackages (WPs). WP1 establishes in vitro and in vivo models which will be refined through profiling of transportome expression and function in WP2. WP3 will provide a functional background on the role of the transportome in PDAC progression. WP4 and WP5 will assess the diagnostic and therapeutic potentials of the transportome. The scientific workpackages are complemented by an interdisciplinary training program on ion transport in and beyond oncology as well as by outreach and management activities. Recruitment of the fellows was accomplished by the end of the first year so that the current report covers 1 to 1.5 years of research within the project. All fellows (13 ESRs and 1 ER) contributed to multiple workpackages.
Initially, all fellows contributed to WP1, the establishment of the best experimental cell and animal models. We decided to use a panel of well established PDAC cell lines, primary cultures of PSCs and mouse models using orthotopic transplantation of PDAC cells. The experimental procedures including the choice of the extracellular matrix proteins, 3D culturing and co-culturing of PDAC cells and PSCs, environmental conditions such as pH, hypoxia and nutrient concentration as well as the orthotopic mouse models were optimized according to the needs of the respective projects. WP1 has been finalized so that we have completed the first two delivrables and the first milestone by establishing and validating our experimental models.
WP2 is devoted to revealing the expression profile of the transportome in PDAC cells and PSCs. We started with a "data mining" approach and explored existing data based on microarray analyses from cell lines and patient samples. Then the fellows investigated the expression of their respective target proteins in the experimental models using a combination of (q)PCR, Western blot, immunofluorescence, patch clamp, ionic imaging (Ca2+ and pH) and metabolic profiling. The combined effort of all groups provided a comprehensive overview of the PDAC transportome. Thus the expression of different TRP, K2P, KV, KCa, NaV, and TMEM16 channels, ORAI and STIM proteins, P2X receptors, as well as pH regulatory transport proteins and carboanhydrases has been validated on mRNA, protein and on a functional level. Profiling of the transportome was complemented by the analysis of expression of functionally associated proteins such as integrins, growth factor and chemokine receptors as well as other regulatory proteins. Notably, the expression profile differed among different PDAC cell lines. Moreover, it became apparent that it also depends in PDAC cells and in PSCs on environmental factors such as hypoxia. WP2 has almost been completed. We anticipate to have a complete list by month 30.
In WP3 we analyse the function of the members of the PDAC transportome in order to determine to which pathophysiological processes they contribute during disease progression. So far we found that migration and invasion of PDAC cells and PSCs requires the activity of a number of transportome family members involved in Ca2+, pH and cell volume homeostasis. Similarly, voltage-gated K+ and Na+ channels turned out to be needed for efficient PDAC cell migration and invasion. Volume and Ca2+ regulatory channels also participate in the regulation of proliferation, apoptosis and drug resistance. Finally, we also identified transport proteins involved in the regulation of the PDAC cell metabolism. While these studies clearly reveal the importance of the transportome in pathophysiological processes underlying the aggressiveness of PDAC, they are still in a premature state because the exact cellular mechanisms have not yet been elucidated. For most groups this will be the focus of next year's work.
The analysis of the diagnostic potential of the PDAC transportome (WP4) is still in a very early state. However, in a way of proof-of-principle experiments we could verify the elevated expression of some of those transportome members identified in the data mining approach in patient tissue samples by means of immunofluorescence, too. Tools are now being developed for the use of these target proteins for multimodal whole animal imaging.
WP5 has also only just been started by performing initial experiments on the therapeutic potential of exploiting the necessitiy of K+ channels for the induction of apoptosis. Once WP3 has progressed further this novel knowledge will provide important input for designing transportome-based therapeutic strategies.
Training activities within WP6 proceed as planned. Thus far, the following network-wide training activities have been realized: two network meetings (organized by WWU and UOXF), two summer/winter schools ("Omics" in PDAC and beyond, organized by CAU; Ion transport in health and disease including a lecture on Preclinical drug discovery - Research at the border between industry and academia, organized by UCPH) and two workshops (Pancreas - from basic physiology to PDAC therapy, organized by WWU/ULi; scientific writing, organized by UOXF). In addition all fellows profit from multiple local training activities including first of all their project-specific training-through-research with secondments to partner laboratories but also language courses. Thus, WP6 is ongoing throughout the project lifetime.
The management activities are bundled in WP7. They are also ongoing throughout the project lifetime. The administrator supports the coordinator by maintaining the required infrastructure such as website, backoffice and by preparing meetings, financial accounting etc..
Outreach activites are covered by WP8. They include scientific publications, organization of and participation in scientific conferences/workshops or the submission of common grant proposals to international funding bodies. So far the IonTraC group already published three original papers and more than 10 reviews. In this context it is notable that two of the IonTraC members are guest editors of a Theme Issue of the Philosophical Transactions Series B devoted to ion transport in cancer. Almost all members of the IonTraC group contribute review articles to this issue which is to appear early in 2014. Moreover, our fellows presented their results in more than 20 abstracts/posters/oral communications at national and international meetings. The IonTraC itself project was introduced at several national and international conferences to a broader scientific audience. Furthermore, several international grant proposals in which IonTraC members teamed up with other scientists had been submitted. Thus, work within the IonTraC project has already created a considerable momentum. Several further publications are presently in preparation and will be submitted soon.