Effects of FGFR inhibitors TKI258, BGJ398 and AZD4547 on breast cancer cells in 2D, 3D and tissue explant cultures.

PURPOSE: Fibroblast growth factor receptors (FGFR) and pathways are important players in breast cancer (BC) development. They are commonly altered, and BCs exhibiting FGFR gene amplification are currently being studied for drug development. Here, we aimed to compare the effects of three FGFR inhibitors (FGFRis), i.e., non-selective TKI258 and selective BGJ398 and AZD4547, on different BC-derived cell lines (BCCs) and primary tissues. METHODS: The human BCCs MCF-7 and MDA-MB-231(SA) (wild-type FGFR) and MFM223 (amplified FGFR1 and FGFR2) were analyzed for FGFR expression using qRT-PCR, and the effects of FGFRis on FGFR signaling by Western blotting. The effects of FGFRis on proliferation, viability, migration and invasion of BCCs were assessed in 2D cultures using live-cell imaging, and in 3D cultures using phenotypic analysis of organoids. To study radio-sensitization, FGFRi treatment was combined with irradiation. Patient-derived BC samples were treated with FGFRis in explant cultures and immunostained for Ki67 and cleaved caspase 3. RESULTS: We found that all FGFRis tested decreased the growth and viability of BC cells in 2D and 3D cultures. BGJ398 and AZD4547 were found to be potent at low concentrations in FGFR-amplified MFM233 cells, whereas higher concentrations were required in non-amplified MCF7 and MDA-MB-231(SA) cells. TKI258 inhibited the migration and invasion, whereas BGJ398 and AZD4547 only inhibited the invasion of MDA-MB-231(SA) cells. FGFRi treatment of MCF7 and MFM223 cells enhanced the inhibitory effect of radiotherapy, but this effect was not observed in MDA-MB-231(SA) cells. FGFRi-treated primary BC explants with moderate FGFR levels showed a tendency towards decreased proliferation and increased apoptosis. CONCLUSIONS: Our results indicate that, besides targeting FGFR-amplified BCs with selective FGFRis, also BCs without FGFR amplification/activation may benefit from FGFRi-treatment. Combination with other treatment modalities, such as radiotherapy, may allow the use of FGFRis at relatively low concentrations and, thereby, contribute to better BC treatment outcomes.

Role of fibroblast growth factor receptors (FGFR) and FGFR like-1 (FGFRL1) in mesenchymal stromal cell differentiation to osteoblasts and adipocytes.

Fibroblast growth factors (FGF) and their receptors (FGFRs) regulate many developmental processes including differentiation of mesenchymal stromal cells (MSC). We developed two MSC lines capable of differentiating to osteoblasts and adipocytes and studied the role of FGFRs in this process. We identified FGFR2 and fibroblast growth factor receptor like-1 (FGFRL1) as possible actors in MSC differentiation with gene microarray and qRT-PCR. FGFR2 and FGFRL1 mRNA expression strongly increased during MSC differentiation to osteoblasts. FGF2 treatment, resulting in downregulation of FGFR2, or silencing FGFR2 expression with siRNAs inhibited osteoblast differentiation. During adipocyte differentiation expression of FGFR1 and FGFRL1 increased and was down-regulated by FGF2. FGFR1 knockdown inhibited adipocyte differentiation. Silencing FGFR2 and FGFR1 in MSCs was associated with decreased FGFRL1 expression in osteoblasts and adipocytes, respectively. Our results suggest that FGFR1 and FGFR2 regulate FGFRL1 expression. FGFRL1 may mediate or modulate FGFR regulation of MSC differentiation together with FGFR2 in osteoblastic and FGFR1 in adipocytic lineage.

Tactile/kinesthetic stimulation (TKS) increases tibial speed of sound and urinary osteocalcin (U-MidOC and unOC) in premature infants (29-32weeks PMA).

Preterm delivery (<37 weeks post-menstrual age) is associated with suboptimal bone mass. We hypothesized that tactile/kinesthetic stimulation (TKS), a form of infant massage that incorporates kinesthetic movement, would increase bone strength and markers of bone accretion in preterm infants. Preterm, AGA infants (29-32 weeks) were randomly assigned to TKS (N=20) or Control (N=20). Twice daily TKS was provided 6 days per week for 2 weeks. Control infants received the same care without TKS treatment. Treatment was masked to parents, health care providers, and study personnel. Baseline and week two measures were collected for tibial speed of sound (tSOS, m/sec), a surrogate for bone strength, by quantitative ultrasound (Sunlight8000) and urine markers of bone metabolism, pyridinium crosslinks and osteocalcin (U-MidOC and unOC). Infant characteristics at birth and study entry as well as energy/nutrient intake were similar between TKS and Control. TKS intervention attenuated the decrease in tSOS observed in Control infants (p<0.05). Urinary pyridinium crosslinks decreased over time in both TKS and CTL (p<0.005). TKS infants experienced greater increases in urinary osteocalcin (U-MidOC, p<0.001 and unOC, p<0.05). We conclude that TKS improves bone strength in premature infants by attenuating the decrease that normally follows preterm birth. Further, biomarkers of bone metabolism suggest a modification in bone turnover in TKS infants in favor of bone accretion. Taken together, we speculate that TKS improves bone mineralization.

The effect of oral glucose tolerance test on serum osteocalcin and bone turnover markers in young adults.

Osteocalcin (OC) is an osteoblast-derived protein implicated in the regulation of glucose tolerance and energy metabolism. This endocrine function has been suggested to be exerted via its undercarboxylated form, which has been shown to induce expression of adiponectin, insulin, and islet cell proliferation in mice. Furthermore, insulin has recently been shown to regulate the biological activity of OC in bone. Our aim was to explore the association between glucose and bone metabolism by evaluating the effect of a standard 75 g oral glucose tolerance test (OGTT) on serum OC, carboxylated OC (cOC) and bone-turnover markers (BTMs) C terminal telopeptide (ßCTX-I) and N terminal propeptide (PINP) of type I collagen and tartrate-resistant acid phosphatase 5b (TRACP5b). Serum samples collected at 0 and at 120 min were analyzed in a cohort of normoglycemic young adults (n = 23, mean age 23.6 years). During OGTT a significant decrease was observed in all BTMs (P < 0.001 for all variables). The median decreases from 0 to 120 min for OC, cOC, ßCTX-I, PINP, and TRACP5b were -32.1% (-37.9 to -19.6), -34.4% (-39.8 to -22.2), -61.4% (-68.5 to -53.0), -26.8% (-33.2 to -19.2), and -44.5% (-48.3 to -40.2), respectively. A strong association between the changes in OC and cOC was observed (r = 0.83, P < 0.001). The decrease in PINP was associated with changes in OC, whereas the changes in ßCTX-I and TRACP5b were not associated with decreases in OC or cOC. The observed OGTT-induced changes in bone-derived proteins were partially independent of each other and potentially mediated by different mechanisms.

Bone turnover markers are correlated with quantitative ultrasound of the calcaneus: 5-year longitudinal data.

SUMMARY: Associations between bone turnover markers and calcaneal ultrasound (quantitative ultrasound, QUS) were studied in a population-based sample of 810 elderly women. Baseline bone turnover markers correlated with baseline QUS as well as with 5-year prospective changes in QUS. INTRODUCTION: Bone turnover markers are associated with areal bone mineral density, but the knowledge on the association with QUS is limited. METHODS: Eight hundred ten women, all 75 years old, were investigated at baseline. Five hundred six completed a 5-year follow-up. Bone turnover markers and calcaneal QUS [speed of sound (SoS), broadband ultrasound attenuation (BUA), stiffness] were investigated at baseline. QUS was investigated at follow-up. RESULTS: All bone turnover markers were correlated with baseline QUS [standardized regression (Beta(std)) values from -0.07, p < 0.05 to -0.23, p < 0.001], with the exception of bone-specific alkaline phosphatase (S-Bone ALP) which was not correlated with BUA and stiffness index. When the correlations between baseline bone turnover markers and 5-year changes in QUS were analyzed, three serum osteocalcins were correlated with changes of SoS and stiffness index (Beta(std) = -0.11, p < 0.05 to -0.17, p < 0.001). Also S-CTX-I correlated with changes of SoS and stiffness index (Beta(std) = -0.10 and -0.09, respectively, p < 0.05). S-TRACP5b, urinary deoxypyridinoline/crea, and U-MidOC/crea correlated with changes of SoS (Beta(std) = -0.10 and p < 0.05 for all). S-Bone ALP did not correlate with change of QUS. None of the bone turnover markers correlated with changes of BUA. CONCLUSIONS: Bone turnover markers correlate with concomitantly assessed QUS as well as with longitudinal change in QUS.

Prediction of bone loss using biochemical markers of bone turnover.

UNLABELLED: The association between baseline levels of eleven bone turnover markers and 5-year rate of bone density change was prospectively studied in a population-based sample of 601 75-year-old women. Several bone formation and resorption markers as well as urinary osteocalcin were modestly correlated to rate of bone density change. INTRODUCTION: Prediction of bone loss by bone turnover markers (BTMs) has been investigated with conflicting results. There is limited information in the elderly. METHODS: Eleven bone turnover markers were analyzed in 75-year old women in the OPRA study (n = 601) and compared to the 5-year change of areal bone mineral density (aBMD) in seven skeletal regions. RESULTS: Annual aBMD change varied between +0.4% (spine) and -2.0% (femoral neck). Significant associations (p < 0.01) were found for four different serum osteocalcins (S-OCs) (standardized regression coefficient -0.20 to -0.22), urinary deoxypyridinoline (-0.19), serum TRACP5b (-0.19), serum CTX-I (-0.21), two of the three urinary osteocalcins (U-OCs) (-0.16) and aBMD change of the leg region (derived from the total body measurement). After adjustment for baseline aBMD, associations were found for all S-OCs (-0.11 to -0.16), two of the three U-OCs (-0.14 to -0.16) and aBMD change at the total hip, and for three of the four S-OCs (-0.14 to -0.15), S-TRACP5b (-0.11), two of the three U-OCs (-0.14 to -0.15) and aBMD change at the femoral neck. There were no significant results concerning aBMD change at the spine. CONCLUSION: This study indicates that BTMs are correlated with aBMD loss in some skeletal regions in elderly women.