Anna Gustafsson

Differential expression of Axl and Gas6 in renal cell carcinoma reflecting tumor advancement and survival

Purpose: Overexpression of the receptor tyrosine kinase Axl is implicated in several cancers. Therefore, we conducted this study to determine the expression of Axl and its ligand Gas6 in various renal cell carcinoma (RCC) types and in oncocytoma. Experimental Design: Real-time quantitative reverse transcription-PCR was used to quantify tumor mRNA levels for Axl and Gas6 in a cohort (n = 221) of RCC patients. Serum levels of soluble sAxl and Gas6 proteins were measured using specific ELISA assays (n = 282). The presence of Axl protein in tumor tissue was evaluated by immunohistochemistry (n = 294). Results were correlated to tumor-associated variables, clinical biochemical tests, and patient survival. Results: Tumor Axl mRNA levels correlated independently to survival when assessed against tumor stage and grade. In the study group, the median cancer-specific survival of all RCC patients during 307 months of follow-up was 55 months (confidence interval, ±40.4). The 25% of patients with lowest tumor Axl mRNA levels had significantly better survival than the rest (P = 0.0005), with 70% of the patients still alive at the end of follow-up. In contrast, in patients with medium-high Axl mRNA, only 25% were alive at the end of follow-up. Tumor Gas6 mRNA levels correlated to survival, tumor-associated variables, and disease severity as did serum levels of soluble sAxl and Gas6 protein. However, no correlation between Axl protein in tumor tissue and survival was found. Conclusions: Axl and Gas6 expression in RCC are associated with tumor advancement and patient survival. In particular, low tumor Axl mRNA levels independently correlated with improved survival.

Gas6 and the Receptor Tyrosine Kinase Axl in Clear Cell Renal Cell Carcinoma

Background The molecular biology of renal cell carcinoma (RCC) is complex and not fully understood. We have recently found that the expression of the receptor tyrosine kinase Axl in the RCC tumors independently correlates with survival of the patients. Principal Findings Here, we have investigated the role of Axl and its ligand Gas6, the vitamin-K dependent protein product of the growth arrest-specific gene 6, in clear cell RCC (ccRCC) derived cells. The Axl protein was highly expressed in ccRCC cells deficient in functional von Hippel-Lindau (VHL) protein, a tumor suppressor gene often inactivated in ccRCC. VHL reconstituted cells expressed decreased levels of Axl protein, but not Axl mRNA, suggesting VHL to regulate Axl expression. Gas6-mediated activation of Axl in ccRCC cells resulted in Axl phosphorylation, receptor down-regulation, decreased cell-viability and migratory capacity. No effects of the Gas6/Axl system could be detected on invasion. Moreover, in ccRCC tumor tissues, Axl was phosphorylated and Gas6 γ-carboxylated, suggesting these molecules to be active in vivo. Significance These results provide novel information regarding the complex function of the Gas6/Axl system in ccRCC.

Tensin2 reduces intracellular phosphatidylinositol 3,4,5-trisphosphate levels at the plasma membrane

Tensins are proposed cytoskeleton-regulating proteins. However, Tensin2 additionally inhibits Akt signalling and cell survival. Structural modelling of the Tensin2 phosphatase (PTPase) domain revealed an active site-like pocket receptive towards phosphoinositides. Tensin2-expressing HEK293 cells displayed negligible levels of plasma membrane phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) under confocal microscopy. However, mock-transfected cells, and Tensin2 cells harbouring a putative phosphatase-inactivating mutation, exhibited significant PtdIns(3,4,5)P(3) levels, which decreased upon phosphatidylinositol 3-kinase inhibition with LY294002. In contrast, wtTensin3, mock and mutant cells were identical in membrane PtdIns(3,4,5)P(3) and Akt phosphorylation. In vitro lipid PTPase activity was however undetectable in isolated recombinant PTPase domains of both Tensins, indicating a possible loss of structural stability when expressed in isolation. In summary, we provide evidence that Tensin2, in addition to regulating cytoskeletal dynamics, influences phosphoinositide-Akt signalling through its PTPase domain.

Strains caused by daily loading might be responsible for delayed healing of an incomplete atypical femoral fracture

Atypical femoral fractures are insufficiency fractures in the lateral femoral diaphysis or subtrochanteric region that mainly affect older patients on bisphosphonate therapy. Delayed healing is often seen in patients with incomplete fractures (cracks), and histology of bone biopsies shows mainly necrotic material inside the crack. We hypothesized that the magnitude of the strains produced in the soft tissue inside the crack during normal walk exceeds the limit for new bone formation, and thereby inhibit healing. A patient specific finite element model was developed, based on clinical CT images and high resolution μCT images of a biopsy from the crack site. Strain distributions in the femur and inside the crack were calculated for load cases representing normal walk. The models predicted large strains inside the crack, with strain levels above 10% in more than three quarters of the crack volume. According to two different tissue differentiation theories, bone would only form in less than 1-5% of the crack volume. This can explain the impaired healing generally seen in incomplete atypical fractures. Furthermore, the microgeometry of the crack highly influenced the strain distributions. Hence, a realistic microgeometry needs to be considered when modeling the crack. Histology of the biopsy showed signs of remodeling in the bone tissue adjacent to the fracture line, while the crack itself contained mainly necrotic material and signs of healing only in portions that seemed to have been widened by resorption. In conclusion, the poor healing capacity of incomplete atypical femoral fractures can be explained by biomechanical factors, and daily low impact activities are enough to cause strain magnitudes that prohibit bone formation.

A fibre-reinforced poroviscoelastic model accurately describes the biomechanical behaviour of the rat achilles tendon.

Background Computational models of Achilles tendons can help understanding how healthy tendons are affected by repetitive loading and how the different tissue constituents contribute to the tendon’s biomechanical response. However, available models of Achilles tendon are limited in their description of the hierarchical multi-structural composition of the tissue. This study hypothesised that a poroviscoelastic fibre-reinforced model, previously successful in capturing cartilage biomechanical behaviour, can depict the biomechanical behaviour of the rat Achilles tendon found experimentally. Materials and Methods We developed a new material model of the Achilles tendon, which considers the tendon’s main constituents namely: water, proteoglycan matrix and collagen fibres. A hyperelastic formulation of the proteoglycan matrix enabled computations of large deformations of the tendon, and collagen fibres were modelled as viscoelastic. Specimen-specific finite element models were created of 9 rat Achilles tendons from an animal experiment and simulations were carried out following a repetitive tensile loading protocol. The material model parameters were calibrated against data from the rats by minimising the root mean squared error (RMS) between experimental force data and model output. Results and Conclusions All specimen models were successfully fitted to experimental data with high accuracy (RMS 0.42-1.02). Additional simulations predicted more compliant and soft tendon behaviour at reduced strain-rates compared to higher strain-rates that produce a stiff and brittle tendon response. Stress-relaxation simulations exhibited strain-dependent stress-relaxation behaviour where larger strains produced slower relaxation rates compared to smaller strain levels. Our simulations showed that the collagen fibres in the Achilles tendon are the main load-bearing component during tensile loading, where the orientation of the collagen fibres plays an important role for the tendon’s viscoelastic response. In conclusion, this model can capture the repetitive loading and unloading behaviour of intact and healthy Achilles tendons, which is a critical first step towards understanding tendon homeostasis and function as this biomechanical response changes in diseased tendons.

The Axl-Regulating Tumor Suppressor miR-34a Is Increased in ccRCC but Does Not Correlate with Axl mRNA or Axl Protein Levels

Background High expression of the receptor tyrosine kinase Axl is associated with poor prognosis in patients with Renal Cell Carcinoma (RCC), the most common malignancy of the kidney. The miR-34a has been shown to directly regulate Axl in cancer cells. The miR-34a is a mediator of p53-dependent tumor suppression, and low expression of miR-34a has been associated with worse prognosis in several cancers. Our aim was to elucidate whether miR-34a or the other members of the miR-34 family (miR-34b/c) regulate Axl in RCC. Methodology and Results Using western blot, flow cytometry, and RT-qPCR, we showed that Axl mRNA and protein are downregulated in 786-O cells by miR-34a and miR-34c but not by miR-34b. A luciferase reporter assay demonstrated direct interaction between the Axl 3’ UTR and miR-34a and miR-34c. The levels of miR-34a/b/c were measured in tumor tissue in a cohort of 198 RCC patients, and the levels of miR-34a were found to be upregulated in clear cell RCC (ccRCC) tumors, but not associated with patient outcome. Neither of the miR-34 family members correlated with Axl mRNA, soluble Axl protein in serum, nor with immunohistochemistry of Axl in tumor tissue. In addition, we measured mRNA levels of a known miR-34a target, HNF4A, and found the HNF4A levels to be decreased in ccRCC tumors, but unexpectedly correlated positively rather than negatively with miR-34a. Conclusions Although miR-34a and miR-34c can regulate Axl expression in vitro, our data indicates that the miR-34 family members are not the primary regulators of Axl expression in RCC.

Comparison of structural anisotropic soft tissue models for simulating Achilles tendon tensile behaviour

The incidence of tendon injury (tendinopathy) has increased over the past decades due to greater participation in sports and recreational activities. But little is known about the aetiology of tendon injuries because of our limited knowledge in the complex structure-function relationship in tendons. Computer models can capture the biomechanical behaviour of tendons and its structural components, which is essential for understanding the underlying mechanisms of tendon injuries. This study compares three structural constitutive material models for the Achilles tendon and discusses their application on different biomechanical simulations. The models have been previously used to describe cardiovascular tissue and articular cartilage, and one model is novel to this study. All three constitutive models captured the tensile behaviour of rat Achilles tendon (root mean square errors between models and experimental data are 0.50-0.64). They further showed that collagen fibres are the main load-bearing component and that the non-collagenous matrix plays a minor role in tension. By introducing anisotropic behaviour also in the non-fibrillar matrix, the new biphasic structural model was also able to capture fluid exudation during tension and high values of Poisson׳s ratio that is reported in tendon experiments.

An interface damage model that captures crack propagation at the microscale in cortical bone using XFEM

Reliable tools for fracture risk assessment are necessary to handle the challenge with an aging population and the increasing occurrence of bone fractures. As it is currently difficult to measure local damage parameters experimentally, computational models could be used to provide insight into how cortical bone microstructure and material properties contribute to the fracture resistance. In this study, a model for crack propagation in 2D at the microscale in cortical bone was developed using the extended finite element method (XFEM). By combining the maximum principal strain criterion with an additional interface damage formulation in the cement line, the model could capture crack deflections at the osteon boundaries as observed in experiments. The model was used to analyze how the Haversian canal and the interface strength of the cement line affected the crack trajectory in models depicting osteons with three different orientations in 2D. Weak cement line interfaces were found to reorient the propagating cracks while models with strong interfaces predicted crack trajectories that penetrated the cement line and propagated through the osteons. The presented model is a promising tool that could be used to analyze how local, age-related material changes influence the crack trajectory and fracture resistance in cortical bone.

Linking multiscale deformation to microstructure in cortical bone using in situ loading, digital image correlation and synchrotron X-ray scattering

The incidence of fragility fractures is expected to increase in the near future due to an aging population. Therefore, improved tools for fracture prediction are required to treat and prevent these injuries efficiently. For such tools to succeed, a better understanding of the deformation mechanisms in bone over different length scales is needed. In this study, an experimental setup including mechanical tensile testing in combination with digital image correlation (DIC) and small/wide angle X-ray scattering (SAXS/WAXS) was used to study deformation at multiple length scales in bovine cortical bone. Furthermore, micro-CT imaging provided detailed information about tissue microstructure. The combination of these techniques enabled measurements of local deformations at the tissue- and nanoscales. The orientation of the microstructure relative to the tensile loading was found to influence the strain magnitude on all length scales. Strains in the collagen fibers were 2-3 times as high as the strains found in the mineral crystals for samples with microstructure oriented parallel to the loading. The local tissue strain at fracture was found to be around 0.5%, independent of tissue orientation. However, the maximum force and the irregularity of the crack path were higher when the load was applied parallel to the tissue orientation. This study clearly shows the potential of combining these different experimental techniques concurrently with mechanical testing to gain a better understanding of bone damage and fracture over multiple length scales in cortical bone. STATEMENT OF SIGNIFICANCE To understand the pathophysiology of bone, it is important to improve our knowledge about the deformation and fracture mechanisms in bone. In this study, we combine several recently available experimental techniques with mechanical loading to investigate the deformation mechanisms in compact bone tissue on several length scales simultaneously. The experimental setup included mechanical tensile testing in combination with digital image correlation, microCT imaging, and small/wide angle X-ray scattering. The combination of techniques enabled measurements of local deformations at the tissue- and nanoscales. The study clearly shows the potential of combining different experimental techniques concurrently with mechanical testing to gain a better understanding of structure-property-function relationships in bone tissue.

Gas6-Axl signaling in presence of Sunitinib is enhanced, diversified and sustained in renal tumor cells, resulting in tumor-progressive advantages

ABSTRACT Clear Cell Renal Cell Carcinoma (CCRCC) is a lethal cancer with bad prognosis due to development of chemoresistance and recurrence of more aggressive tumors. Investigation of Gas6‐mediated Axl signaling in CCRCC and endothelial cells reveals a Sunitinib resistant Gas6‐Axl signaling that is sustained and enhanced and specifically triggers downstream AKT and PRAS40 activation in an intensified manner. Gas6‐induced Axl signaling in presence of Sunitinib is also diversified displaying onset of Axl‐dependent EGFR and METR activation and activation of classical MAPK pathways. Gas6+Sunitinib‐adapted CCRCC cells present increased viability and decreased apoptosis and enhanced production of the multi‐tumorigenic Osteopontin (OPN) and of one of its activator matrix metalloproteinase‐7. Axl activity is necessary for CCRCC cell sphere formation and the ability of the cells to attach after non‐adhesive growth. In addition, Gas6+Sunitinib‐adapted CCRCC cells displayed enhanced migration and sphere formation, both mechanisms being Axl and OPN dependent. Altogether, this suggests that Sunitinib while targeting endothelial cells and tumor angiogenesis, simultaneously provides protumorigenic effects due to a constitutively, intensified and divergent Gas6‐Axl system. Implications: Gas6‐mediated Axl signaling, which is enhanced and diversified in the presence of Sunitinib possibly contributes to acquired chemoresistance, recurrence of aggressive disease and metastasis of CCRCC tumors. Therefore, combinatorial Axl‐targeted therapy might be beneficial for CCRCC patients intended for Sunitinib treatment. HIGHLIGHTSAxl is constitutively activated in Sunitinib‐treated CCRCC cells.Sunitinib increases and diversifies Gas6‐Axl signaling.Sunitinib enhances Axl‐dependent migration, sphere‐formation and OPN secretion.Repopulation of CCRCC cells is dependent on Axl activity.Axl is a highly interesting drug target in CCRCC.