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Prostate cancer and bone cell interactions: implications for metastatic growth and therapy
Umeå University, Faculty of Medicine, Department of Radiation Sciences, Oncology.
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The skeleton is the most common site of prostate cancer bone metastasis, and at present, there are no curable treatments for these patients. To further understand what stimulates tumor cell growth in the bone microenvironment and to find suitable therapies, reliable model systems are needed. For this purpose, we have developed an in vitro co-culture system that can be used to study interactions between tumor cells and murine calvarial bones. To validate the model, we measured the release of collagen fragments and monitored changes in expression levels of genes normally expressed during active bone remodeling.

One of the major reasons why prostate cancer cells colonize bone is the abundance of tumor-stimulating factors, such as insulin-like growth factors (IGFs), present in this milieu. We found that the IGF-1 receptor (IGF-1R) was one of the most highly activated receptor tyrosine kinases in tumor cell lines stimulated with bone conditioned media. Since IGF-1 is known to be a strong survival factor for tumor cells, we hypothesized, that concurrent inhibition of IGF-1R signaling can enhance the effects of apoptosis-inducing therapies, such as castration. We used our co-culture model to target human prostate cancer cell lines, PC-3 and 22Rv1, with simvastatin (an inhibitor of the mevalonate pathway and an inducer of apoptosis), in combination with anti-IGF-1R therapy. Tumor cell viability declined with either one of the therapies used alone, and the effect was even more pronounced with the combined treatment. The hypothesis was also tested in rats that had been inoculated with rat prostate cancer cells, Dunning R3327-G, into the tibial bone, and treated with either anti-IGF-1R therapy, castration, or a combination of both therapies. Immunohistochemistry was used to evaluate therapeutic effects on tumor cell proliferation and apoptosis, as well as tumor cell effects on bone remodeling. The tumor cells were found to induce an osteoblastic response, both in vivo in rats, and in vitro using the co-culture model. Interestingly, the therapeutic response differed depending on whether tumor cells were located within the bone marrow cavity or if they had leaked out into the knee joint cavity, highlighting the role of the microenvironment on metastatic growth and therapeutic response. Therapies targeting the IGF-1R have been tested in clinical trials, unfortunately with disappointing results. By immunohistochemical evaluation of bone metastases from patients with castration-resistant prostate cancer, we found a large variance in IGF-1R staining within this group of patients. Hence, we postulate that the effects of anti-IGF-1R therapies could be more beneficial in patients with high tumoral IGF-1R-activity than in IGF-1R negative cases. We also believe that side effects, such as hyperglycemia, associated with anti-IGF-1R therapy, could be reduced if this treatment is administered only to selected patients and for shorter time periods.

In a separate study, using whole-genome expression data from bone metastases obtained from prostate cancer patients, we present evidence that a high activity of osteoblasts is coupled to a high activity of osteoclast. Moreover, we found that high bone remodeling activity is inversely related to tumor cell androgen receptor (AR) activity. The results from this study may be of importance when selecting therapy for patients with bone metastatic cancer, especially when bone-targeting therapies are considered, and could aid in the search for novel therapeutic targets.

In summary, we present an in vitro model for studies of the bidirectional interplay between prostate cancer cells and the bone microenvironment. We also demonstrate the importance of IGF-1 in prostate cancer bone metastases and suggest that inhibition of IGF-1R signaling can be used to treat prostate cancer as well as to enhance effects of other treatments such as androgen deprivation therapy. Furthermore, we emphasize the possibility of molecular tumor characterization when designing treatment plans for individual patients, thereby maximizing the therapeutic effects.

Place, publisher, year, edition, pages
Umeå: Umeå Universitet , 2017. , 65 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1886
National Category
Cancer and Oncology
Research subject
Oncology
Identifiers
URN: urn:nbn:se:umu:diva-131809ISBN: 978-91-7601-678-7 (print)OAI: oai:DiVA.org:umu-131809DiVA: diva2:1076296
Public defence
2017-03-17, Hörsal E04, Byggnad 6A, Norrland universitetssjukhus, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2017-02-24 Created: 2017-02-22 Last updated: 2017-03-16Bibliographically approved
List of papers
1. Establishment and validation of an in vitro co-culture model to study the interactions between bone and prostate cancer cells
Open this publication in new window or tab >>Establishment and validation of an in vitro co-culture model to study the interactions between bone and prostate cancer cells
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2009 (English)In: Clinical & experimental metastasis, ISSN 0262-0898, Vol. 26, no 8, 945-953 p.Article in journal (Refereed) Published
Abstract [en]

Bone is the preferred site for prostate cancer (PCa) metastases. Once the tumor has established itself within the bone there is virtually no cure. To better understand the interactions between the PCa cells and bone environment in the metastatic process new model systems are needed. We have established a two-compartment in vitro co-culturing model that can be used to follow the trans-activation of bone and/or tumor cells. The model was validated using two PCa tumor cell lines (PC-3; lytic and LNCaP; mixed/osteoblastic) and one osteolytic inducing factor, 1,25-dihydroxyvitamin D(3) (D3). Results were in accordance with the expected bone phenotypes; PC-3 cells and D3 gave osteolytic gene expression profiles in calvariae, with up-regulation of genes needed for osteoclast differentiation, activation and function; Rankl, CathK, Trap and MMP-9, and down-regulation of genes associated with osteoblast differentiation and bone mineralization; Alp, Ocl and Dkk-1. LNCaP cells activated genes in the calvarial bones associated with osteoblast differentiation and mineralization, with marginal effects on osteolytic genes. The results were strengthened by similar changes in protein expression for a selection of the analyzed genes. Furthermore, the osteolytic gene expression profiles in calvarial bones co-cultured with PC-3 cells or with D3 were correlated with the actual ongoing resorptive process, as assessed by the release of collagen fragments from the calvariae. Our results show that the model can be used to follow tumor-induced bone remodeling, and by measuring changes in gene expression in the tumor cells we can also study how they respond to the bone microenvironment.

Keyword
Bone metastasis, Co-culture, Mouse calvaria, Osteoblastic, Osteolytic, Prostate cancer
National Category
Cancer and Oncology
Identifiers
urn:nbn:se:umu:diva-30073 (URN)10.1007/s10585-009-9285-4 (DOI)000271722400002 ()19728119 (PubMedID)
Available from: 2009-12-02 Created: 2009-12-02 Last updated: 2017-02-23Bibliographically approved
2. Inhibition of the insulin-like growth factor-1 receptor enhances effects of Simvastatin on prostate cancer cells in co-culture with bone
Open this publication in new window or tab >>Inhibition of the insulin-like growth factor-1 receptor enhances effects of Simvastatin on prostate cancer cells in co-culture with bone
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2013 (English)In: Cancer Microenvironment, ISSN 1875-2292, E-ISSN 1875-2284, Vol. 6, no 3, 231-240 p.Article in journal (Refereed) Published
Abstract [en]

Prostate cancer (PC) bone metastases show weak responses to conventional therapies. Bone matrix is rich in growth factors, with insulin-like growth factor-1 (IGF-1) being one of the most abundant. IGF-1 acts as a survival factor for tumor cells and we speculate that bone-derived IGF-1 counteracts effects of therapies aimed to target bone metastases and, consequently, that therapeutic effects could be enhanced if given in combination with IGF-1 receptor (IGF-1R) inhibitors. Simvastatin inhibits the mevalonate pathway and has been found to induce apoptosis of PC cells. The aims of this study were to confirm stimulating effects of bone-derived IGF-1 on PC cells and to test if IGF-1R inhibition enhances growth inhibitory effects of simvastatin on PC cells in a bone microenvironment. The PC-3 and 22Rv1 tumor cell lines showed significantly induced cell growth when co-cultured with neonatal mouse calvarial bones. The tumor cell IGF-1R was activated by calvariae-conditioned media and neutralization of bone-derived IGF-1 abolished the calvarium-induced PC-3 cell growth. Treatment of PC-3 and 22Rv1 cells with simvastatin, or the IGF-1R inhibitor NVP-AEW541, reduced tumor cell numbers and viability, and induced apoptosis. Combined simvastatin and NVP-AEW541 treatment resulted in enhanced growth inhibitory effects compared to either drug given alone. Effects of simvastatin involved down-regulation of IGF-1R in PC-3 and of constitutively active androgen receptor variants in 22Rv1 cells. In conclusion, we suggest that IGF-1 inhibition may be a way to strengthen effects of apoptosis-inducing therapies on PC bone metastases; a possibility that needs to be further tested in pre-clinical models.

Place, publisher, year, edition, pages
Springer, 2013
Keyword
Prostate cancer, Bone metastases, IGF-1R, Simvastatin, Cholesterol
National Category
Cancer and Oncology
Identifiers
urn:nbn:se:umu:diva-65834 (URN)10.1007/s12307-013-0129-z (DOI)23335094 (PubMedID)
Available from: 2013-02-12 Created: 2013-02-12 Last updated: 2017-02-23Bibliographically approved
3. Inhibition of the insulin-like growth factor-1 receptor potentiates acute effects of castration in a rat model for prostate cancer growth in bone
Open this publication in new window or tab >>Inhibition of the insulin-like growth factor-1 receptor potentiates acute effects of castration in a rat model for prostate cancer growth in bone
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2017 (English)In: Clinical and Experimental Metastasis, ISSN 0262-0898, E-ISSN 1573-7276, Vol. 34, no 3-4, 261-271 p.Article in journal (Other academic) Published
Abstract [en]

Prostate cancer (PCa) patients with bone metastases are primarily treated with androgen deprivation therapy (ADT). Less pronounced ADT effects are seen in metastases than in primary tumors. To test if acute effects of ADT was enhanced by concurrent inhibition of pro-survival insulin-like growth factor 1 (IGF-1), rats were inoculated with Dunning R3327-G tumor cells into the tibial bone marrow cavity and established tumors were treated with castration in combination with IGF-1 receptor (IGF-1R) inhibitor NVP-AEW541, or by each treatment alone. Dunning R3327-G cells were stimulated by androgens and IGF-1 in vitro. In rat tibia, Dunning R3327-G cells induced bone remodeling, identified through increased immunoreactivity of osteoblast and osteoclast markers. Tumor cells occasionally grew outside the tibia, and proliferation and apoptotic rates a few days after treatment were evaluated by scoring BrdU- and caspase-3-positive tumor cells inside and outside the bone marrow cavity, separately. Apoptosis was significantly induced outside, but unaffected inside, the tibial bone by either castration or NVP-AEW541, and the maximum increase (2.7-fold) was obtained by the combined treatment. Proliferation was significantly reduced by NVP-AEW541, independently of growth site, although the maximum decrease (24%) was observed when NVP-AEW541 was combined with castration. Tumor cell IGF-1R immunoreactivity was evaluated in clinical PCa bone metastases (n = 61), and positive staining was observed in most cases (74%). In conclusion, IGF-1R inhibition may be evaluated in combination with ADT in patients with metastatic PCa, or in combination with therapies for the subsequent development of castration-resistant disease, although diverse responses could be anticipated depending on metastasis site.

Place, publisher, year, edition, pages
Springer, 2017
Keyword
Bone metastasis, IGF-1R, Apoptosis, Proliferation, Immune response, RUNX2, TRAP
National Category
Cancer and Oncology
Research subject
Oncology
Identifiers
urn:nbn:se:umu:diva-131804 (URN)10.1007/s10585-017-9848-8 (DOI)000401996600008 ()28447314 (PubMedID)
Note

Special Issue.

Originally published in thesis in manuscript form.

Available from: 2017-02-22 Created: 2017-02-22 Last updated: 2017-06-26Bibliographically approved
4. Bone remodeling in relation to androgen receptor activity in prostate cancer bone metastases
Open this publication in new window or tab >>Bone remodeling in relation to androgen receptor activity in prostate cancer bone metastases
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Prostate cancer often metastasizes to bone and the metastases are generally classified as osteoblastic, although a mixed osteoblastic/osteolytic bone response may exist. The present study aimed to characterize the bone remodeling activity in clinical bone metastasis samples, with the overall hypothesis that diversities exist that may be of importance for clinical response to current therapies. Specifically, we aimed to study bone remodeling activity in relation to tumor cell androgen receptor (AR) activity. Metastasis tissue obtained from treatment-naïve (n=11) and castration-resistant (n=28) patients during surgery for spinal cord compression was characterized using whole-genome expression analysis followed by multivariate modeling and functional enrichment analysis as well as by histological evaluation. By analyzing expression levels of a predefined set of markers representing osteoclasts (ACP5, CTSK, MMP9), osteoblasts (ALPL, BGLAP, RUNX2) and osteocytes (SOST), we found high osteoblast activity to be coupled to a high osteoclast activity. Immunohistochemistry verified a significant correlation between RUNX2 positive osteoblasts and TRAP (ACP5) positive osteoclasts lining metastatic bone surfaces in close contact to tumor cells. No difference in bone remodeling activity was seen between treatment naïve and castration-resistant patients, while the bone remodeling activity was inversely correlated to AR activity within the tissue (measured as expression of the AR, FOXA1, HOXB13, KLK2, KLK3, NKX3-1, STEAP2, and TMPRSS2) and patient serum PSA levels. Ontology analysis suggested enriched BMP signaling in metastases with high bone remodeling activity and, accordingly, BMP4 mRNA expression was significantly higher in bone metastases with than without ongoing bone formation, as determined from histological evaluation of van Gieson-stained sections. In conclusion, we have observed diversities in bone remodeling activity among clinical samples of prostate cancer bone metastases that may be of importance when selecting therapy for patients with bone metastatic cancer, especially when bone-targeting therapies are considered. The importance of the BMP signaling system for the development of sclerotic metastasis lesion deserve further exploration.

National Category
Cancer and Oncology
Research subject
Oncology
Identifiers
urn:nbn:se:umu:diva-131806 (URN)
Available from: 2017-02-22 Created: 2017-02-22 Last updated: 2017-02-23

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