Vetenskaplig slutrapport, AFA Försäkring

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Institutionen för Strålningsvetenskaper Avdelningen för Onkologi Emma Persson 2016-09-29 Sid 1 (5) Vetenskaplig slutrapport, AFA Försäkring Forskningsprojekt dnr 120338, Projekttitel:

1 Institutionen för Strålningsvetenskaper Avdelningen för Onkologi Emma Persson Sid 1 (5) Vetenskaplig slutrapport, AFA Försäkring Forskningsprojekt dnr , Projekttitel: Skelettmetastaserande cancer en dödlig cellkommunikation Scientific report findings, conclusions and future plans Development of clinically significant metastatic disease is one of the most common causes of death in cancer patients. Several cancer forms, including prostate, breast and lung cancer, develop metastases primarily in the skeleton. Progression to skeletal metastatic disease dramatically decreases both patient survival and quality of life. At present, there is no curative treatment available for patients with skeletal metastatic disease, resulting in palliative care being the only option for these patients. This clearly demonstrates the urgent need for increased knowledge on molecular factors important for both cancer progression and the cellular communication resulting in skeletal metastatic disease. The overall purpose of this project is to increase the knowledge on the molecular mechanisms and cell communication networks behind cancer disease progression and establishment of disseminated tumour cells (DTCs) of specific origins in the bone microenvironment. Below is a summary of the findings obtained within the different subprojects. Project 1: The biological importance of TGFβ for cancer progression and development of skeletal metastases The growth factor TGFβ is a well-known key player in cancer biology, and in prostate cancer (PCa) it regulates cell differentiation and promotes apoptosis or tumor progression, dependent on the cellular context. It is therefore of utmost importance to obtain further knowledge about the complex signaling pathways dictated by TGFβ. Within this project, the possible involvement of TGFβ in skeletal metastatic disease has been studied. Our focus has been on prostate cancer, and several PCa cell lines have been included in the study, comparing characteristics of tumor cells originating from primary tumor and skeletal metastases. Our findings indeed consolidate TGFβ as an important factor for metastatic potential in tumor cells. TGFβ released from osteoblasts, the bone-forming cells in the skeleton, increase the metastatic and self-renewal potential of PCa cells. This effect was shown to be mediated, at least in part, by a TRAF6-dependent non-canonical signaling pathway instead of the canonical intracellular signaling pathways involving

2 Sid 2 (5) Smad proteins. The metastatic potential of the PCa cells, as assessed by formation in 3D cultures of pro-metastatic protrusions on the cells, is also promoted by TGFβ. By comparison of tumor cells isolated from different tissue origins it was clear that tumor cells isolated from metastatic tissue respond with formation of a 10-fold higher number of protrusions when treated with bone cell-derived factors, whereas the tumor cells originating from a primary tumor did not respond to the bone cellderived factors at all in this assay. One possible explanation for the differences in responsiveness is identified differences in expression of TGFβ signaling components in the cell lines studied. For more details, see attached manuscript. In summary, we have established further the role of TGFβ as regulator of metastatic potential in prostate cancer tumor cells. The data obtained within this project constitutes a manuscript which was submitted to Nature Scientific Reports earlier this year, and we are currently supplementing the data after the comments from the referees. Our future aim is to verify our findings using animal models. Through local and national collaborations, we have access to tissue materials from both orthotopic and metastatic models. Using these materials, we can evaluate further the importance of TGFβ in disease progression and metastatic development. We plan to perform studies using our model with intratibial injection of tumor cells in the absence or presence of TGFβ signaling inhibitors to further study the importance of TGFβ signaling in the communication between tumor cells and different cell types in the bone tissue. In addition, our findings can be verified using our biobank of patientderived material from skeletal metastases, currently containing samples from over 200 patients with metastatic disease. In a longer perspective, the possible role of TGFβ and/or its signaling pathways as therapeutic targets should be evaluated. Project 2: The tumor cell recirculation concept search for factors important for cellular communication between tumor and support cells in primary and secondary sites The skeleton is a dynamic tissue that is constantly remodelled, and the general idea is that the unique microenvironment provided by the bone tissue attracts disseminated tumour cells (DTCs) to the skeleton. Previous studies have shown crucial roles of both homing of tumour cells to the bone and physical cell-cell contact for skeletal DTC establishment, but the knowledge on which communicative factors that are involved in this process is rather limited. Recently, the concept of recirculation of metastatic cells to primary tumours has been suggested to be an important factor for disease aggressiveness and metastatic potential. Increased knowledge on the mechanisms underlying initial skeletal homing, intraosseous establishment and recirculation of tumour cells would be an important step in the understanding and possible prevention of skeletal establishment of cancer cells. Within this project we have also studied the importance of specific factors for the communication between tumor and bone cells. The student project Molecular factors involved in the communication between non-small cell lung cancer (NSCLC) cells and bone cells by Charlotta Wallström showed that molecules released from osteoclasts, the bone-degrading cells, induce differentiation and a pro-migratory phenotype of

3 Sid 3 (5) lung cancer cells. In addition, expression in the tumor cells of molecules important for homing to the skeleton was also increased. Another factor that was upregulated in the lung cancer cells was the cytokine IL-6, previously shown to promote aggressive tumor growth. The student project by Olga Shnyra further investigated the importance of IL-6 signaling for metastatic potential of tumor cells, with focus on prostate cancer. The findings within this project further strengthen the role of IL-6-type cytokines as promoters of disease progression and metastatic potential. For evaluation of the effects by IL-6-type cytokine signaling, both established drugs and other pathway inhibitors were used, including inhibitors for the transcription factor STAT3. The data revealed that inhibition of IL-6 signaling decreases both migration and self-renewal potential of PCa tumor cells, with the STAT3 inhibitors being the most efficient inhibitors of the effects studied. As a result of the findings described above obtained using cell culture systems, we have continued our work using STAT3 inhibitors, to further evaluate the importance of this signaling pathway for metastatic disease. During the last year, we have initiated studies using our animal model based on tibia injections of tumor cells. Interestingly, the tumors treated with STAT3 inhibitors are significantly smaller and the pathological effects on the bone tissue are reduced, further strengthening the importance of IL-6-STAT3 signaling for the development of skeletal metastatic disease. For studies on recirculation of tumor cells from the metastatic site to the primary organ, we did several attempts during the first project year where we injected cell from the mouse PCa cell line TRAMP-C1 orthotopically in mouse prostate after a priming period when the cells had been cultured in the absence or presence of whole bone marrow from mouse for 24 hours. The aim was to study whether the cells that had interacted with bone cells would have a more aggressive behavior. Unfortunately, we had a low tumor take in these experiments, and we didn t get enough tumors to be able to draw robust conclusions from the studies. However, what we could see was that in several animals injected with the TRAMP-C1 tumor cells that had been cultured in the presence of bone marrow, there was a local spread of tumor cells in the abdominal area, suggesting a higher metastatic potential in these cells. This is an interesting finding that encourages us to analyze the tumors further, and to try to repeat these experiments using another animal model instead of the TRAMP-C1 cell line. In collaboration with Dr. Sofia Halin and Prof. Anders Bergh (Medicinsk Biovetenskap, Umeå Univ.) we also have access to a rat-based model. Using this system, rat PCa cells have been injected in tibia of Copenhagen rats. Tumor cells injected in the tibia has after 8-12 weeks been isolated and established as boneprimed cell lines. These cells have subsequently been injected in the prostate of new rats to compare tumor aggressiveness in the cells compared to the original cell line. Interestingly, the bone-primed cells grow much faster and the tumor volume is almost twenty times larger in tumors formed by the bone-primed tumor cells (Halin et al., manuscript in preparation). These findings strengthen the theory about the tumor recirculation concept, suggesting that tumor cells recirculating to the organ of origin contribute to disease progression.

4 Sid 4 (5) In addition to the already collected materials we also plan to develop a system where we perform double injections, using luciferase/gfp-expressing tumor cells for tibia injections and cells without luciferase/gfp expression for injection in the primary organ (prostate, breast or lung). Possible re-circulation of tumor cells from the tibial bone to primary sites will be studied using non-invasive detection of bioluminescence using IVIS camera. Project 3: The role of tumor cell-derived exosomes in the interplay between tumor cells and different types of cells in the bone microenvironment During the last years, the role of tumor cell-derived microvesicles in cancer development and progression has been in major focus. A number of recent reports have also suggested a role for microvesicles in communication between tumor cells and different cell types in the tumor stroma. The aim of this project was to study whether exosomes secreted from tumor cells affect bone cell formation and activity. The first manuscript (attached) shows that exosomes from the murine PCa cell line TRAMP-C1 efficiently decrease both the formation and activity of bone-degrading osteoclasts. These data are well in line with the fact that prostate cancer metastases are characterized as sclerotic/osteoblastic metastases with a local increase in bone mass. In contrast, exosomes from normal fibroblast did not have any effects on osteoclast formation. Our future plans for this project are to investigate whether exosomes from other types of cancer also affect bone cell formation and activity. Scientific progress manuscripts and reports The findings obtained within this projects have resulted in two manuscript submitted to journals, as well as two student reports which we are currently working on to convert to full manuscripts, with expected submission to journals in Karlsson T, Lundholm M, Widmark A, Persson E. Tumor cell-derived exosomes from the prostate cancer cell line TRAMP-C1 impair osteoclast formation and differentiation. Resubmitted to PLoS ONE Karlsson T, Sundar R, Widmark A, Landström M, Persson E. Osteoblast-derived factors increase metastatic potential in human prostate cancer cells, an effect partially mediated by non-canonical, TRAF6-dependent TGFβ signaling. Submitted to Nature Scientific Reports Charlotta Wallström, 10 th semester thesis project, Medical school, Umeå University, 2013: Molecular factors involved in the communication between non-small cell lung cancer (NSCLC) cells and bone cells

5 Sid 5 (5) Olga Shnyra, 10 th semester thesis project, Medical school, Umeå University, 2014: The importance of IL-6-type cytokines for prostate cancer progression and disease aggressiveness National and international meetings We have presented data obtained within this project at the following national and international scientific meetings: The 3 rd Swedish Cancer Meeting, Karolinska Institutet, Stockholm, 2-3/9-14. Terese Karlsson, poster presentation Osteoblast-derived factors increase metastatic potential in human prostate cancer cells. The Swedish Prostate Cancer Research Meeting, Rosersbergs slott, Stockholm, 24-25/ Terese Karlsson, oral presentation Osteoblastderived factors increase metastatic potential in human prostate cancer cells. 4th Joint Meeting of ECTS and IBMS, Rotterdam, The Netherlands, 25-28/4-15. Parallell programs for ECTS (European Calcified Tissue Society), IBMS (International Bone and Mineral Society) and CABS (Cancer and Bone Society). Terese Karlsson, poster presentation Osteoblast-derived factors increase metastatic potential in human prostate cancer cells, and Emma Persson, poster presentation Prostate cancer cell-derived exosomes impair osteoclast formation and differentiation. 43 rd Annual European Calcified Tissue Society Congress, Rome, Italy, 14-17/5-16. Terese Karlsson, poster presentation Osteoblast-derived factors increase metastatic potential in human prostate cancer cells. Contact information Project leader Researcher Emma Persson, Dept. of Radiation Sciences, Section for Oncology, Umeå University. emma.e.persson@umu.se, Tel:

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