| 1. |
- Boman, Karolina, et al.
(author)
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Membranous expression of podocalyxin-like protein is an independent factor of poor prognosis in urothelial bladder cancer
- 2013
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In: British Journal of Cancer. - : Springer Science and Business Media LLC. - 0007-0920 .- 1532-1827. ; 108:11, s. 2321-2328
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Journal article (peer-reviewed)abstract
- Background: Membranous expression of the anti-adhesive glycoprotein podocalyxin-like (PODXL) has previously been found to correlate with poor prognosis in several major cancer forms. Here we examined the prognostic impact of PODXL expression in urothelial bladder cancer. Methods: Immunohistochemical PODXL expression was examined in tissue microarrays with tumours from two independent cohorts of patients with urothelial bladder cancer: n = 100 (Cohort I) and n = 343 (Cohort II). The impact of PODXL expression on disease-specific survival (DSS; Cohort II), 5-year overall survival (OS; both cohorts) and 2-year progression-free survival (PFS; Cohort II) was assessed. Results: Membranous PODXL expression was significantly associated with more advanced tumour (T) stage and high-grade tumours in both cohorts, and a significantly reduced 5-year OS (unadjusted HR = 2.25 in Cohort I and 3.10 in Cohort II, adjusted HR = 2.05 in Cohort I and 2.18 in Cohort II) and DSS (unadjusted HR = 4.36, adjusted HR = 2.70). In patients with Ta and T1 tumours, membranous PODXL expression was an independent predictor of a reduced 2-year PFS (unadjusted HR = 6.19, adjusted HR = 4.60) and DSS (unadjusted HR = 8.34, adjusted HR = 7.16). Conclusion: Membranous PODXL expression is an independent risk factor for progressive disease and death in patients with urothelial bladder cancer.
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| 2. |
- Cepeda, D., et al.
(author)
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CDK-mediated activation of the SCFFBXO28 ubiquitin ligase promotes MYC-driven transcription and tumourigenesis and predicts poor survival in breast cancer
- 2013
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In: EMBO Molecular Medicine. - : EMBO. - 1757-4676 .- 1757-4684. ; 5:7, s. 999-1018
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Journal article (peer-reviewed)abstract
- SCF (Skp1/Cul1/F-box) ubiquitin ligases act as master regulators of cellular homeostasis by targeting key proteins for ubiquitylation. Here, we identified a hitherto uncharacterized F-box protein, FBXO28 that controls MYC-dependent transcription by non-proteolytic ubiquitylation. SCFFBXO28 activity and stability are regulated during the cell cycle by CDK1/2-mediated phosphorylation of FBXO28, which is required for its efficient ubiquitylation of MYC and downsteam enhancement of the MYC pathway. Depletion of FBXO28 or overexpression of an F-box mutant unable to support MYC ubiquitylation results in an impairment of MYC-driven transcription, transformation and tumourigenesis. Finally, in human breast cancer, high FBXO28 expression and phosphorylation are strong and independent predictors of poor outcome. In conclusion, our data suggest that SCFFBXO28 plays an important role in transmitting CDK activity to MYC function during the cell cycle, emphasizing the CDK-FBXO28-MYC axis as a potential molecular drug target in MYC-driven cancers, including breast cancer. FBXO28 is identified as part of a SCF complex acting as a regulator of tumor cell proliferation and an important modifier of MYC function. FBXO28 may be a new prognostic factor in breast cancer and a new potential drug target in MYC- driven tumors.
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| 3. |
- Larsson, Anna H, et al.
(author)
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Overexpression of podocalyxin-like protein is an independent factor of poor prognosis in colorectal cancer
- 2011
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In: British Journal of Cancer. - : Springer Science and Business Media LLC. - 0007-0920 .- 1532-1827. ; 105:5, s. 666-672
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Journal article (peer-reviewed)abstract
- BACKGROUND: Podocalyxin-like 1 (PODXL) is a cell-adhesion glycoprotein and stem cell marker that has been associated with an aggressive tumour phenotype and poor prognosis in several forms of cancer. In this study, we investigated the prognostic impact of PODXL expression in colorectal cancer (CRC). METHODS: Using tissue microarrays and immunohistochemistry, PODXL expression was evaluated in 536 incident CRC cases from a prospective, population-based cohort study. Kaplan-Meier analysis and Cox proportional hazards modelling were used to assess the impact of PODXL expression on cancer-specific survival (CSS) and overall survival (OS). RESULTS: High PODXL expression was significantly associated with unfavourable clinicopathological characteristics, a shorter CSS (hazard ratio (HR) = 1.98; 95% confidence interval (CI) 1.38-2.84, P < 0.001) and 5-year OS (HR = 1.85; 95% CI 1.29-2.64, P = 0.001); the latter remaining significant in multivariate analysis (HR = 1.52; 95% CI 1.03-2.25, P = 0.036). In addition, in curatively resected stage III (T1-4, N1-2, M0) patients (n = 122) with tumours with high PODXL expression, a significant benefit from adjuvant chemotherapy was demonstrated (p(interaction) = 0.004 for CSS and 0.015 for 5-year OS in multivariate analysis). CONCLUSION: Podocalyxin-like 1 expression is an independent factor of poor prognosis in CRC. Our results also suggest that PODXL may be a useful marker to stratify patients for adjuvant chemotherapy.
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| 4. |
- Larsson, Anna H, et al.
(author)
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Podocalyxin-like protein expression in primary colorectal cancer and synchronous lymph node metastases
- 2013
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In: Diagnostic Pathology. - : Springer Science and Business Media LLC. - 1746-1596. ; 8:1, s. 109-
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Journal article (peer-reviewed)abstract
- Aims: Previous studies have shown that membranous expression of podocalyxin-like protein (PODXL) is associated with poor prognosis in colorectal cancer (CRC). In this study, we compared PODXL expression in primary CRC and synchronous lymph node metastases. We further analyzed whether its expression changed in rectal tumours after neoadjuvant radiation therapy. Methods and results: The studied cohort consists of 73 consecutive patients from the South-Swedish Colorectal Cancer Biobank. Immunohistochemical PODXL expression was examined on full-face sections from all primary tumours and all 140 available lymph node metastases from 31 cases. Membranous PODXL expression was denoted in 18/73 (24,7%) primary tumours, with a high concordance between primary and metastatic lesions. While all negative primary tumours had negative metastases, some PODXL positive primaries had a varying proportion of positive and negative metastatic lymph nodes. PODXL expression was also found to be mainly unaltered in pre- and post-irradiation surgically resected tumour specimens in rectal cancer patients (n=16). Conclusions: The findings in this study suggest that analysis of PODXL expression in the primary tumour is sufficient for its use as a prognostic and treatment predictive biomarker in CRC, also in patients with metastatic disease.
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| 5. |
- Larsson, Anna H., et al.
(author)
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Significant association and synergistic adverse prognostic effect of podocalyxin-like protein and epidermal growth factor receptor expression in colorectal cancer
- 2016
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In: Journal of Translational Medicine. - : BioMed Central. - 1479-5876. ; 14
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Journal article (peer-reviewed)abstract
- Background: Podocalyxin-like 1 (PODXL) is an anti-adhesive transmembrane protein that has been demonstrated to be an independent factor of poor prognosis in colorectal cancer (CRC). The gene encoding PODXL is located to chromosome 7, which also harbours the gene for the epidermal growth factor receptor (EGFR). The aim of this study was to examine the associations between PODXL and EGFR expression in CRC in vitro and in vivo. Methods: EGFR expression was analysed in tumours from three independent patient cohorts; cohort 1 (n = 533), cohort 2 (n = 259) and cohort 3 (n = 310), previously analysed for immunohistochemical PODXL expression and KRAS and BRAF mutations (cohort 1 and 3). Levels of EGFR and PODXL were determined by western blot in six different CRC cell lines. Results: High expression of PODXL was significantly associated with high EGFR expression (p < 0.001) in all three cohorts, and with BRAF mutation (p < 0.001) in cohort 1 and 3. High EGFR expression correlated with BRAF mutation (p < 0.001) in cohort 1. High EGFR expression was associated with adverse clinicopathological factors and independently predicted a reduced 5-year overall survival (OS) in cohort 1 (HR 1.77; 95 % CI 1.27-2.46), cohort 2 (HR 1.58; 95 % CI 1.05-2.38) and cohort 3 (HR 1.83; 95 % CI 1.19-2.81). The highest risk of death within 5 years was observed in patients with tumours displaying high expression of both EGFR and PODXL in cohort 1 and 3 (HR 1.97; 95 % CI 1.18-3.28 and HR 3.56; 95 % CI 1.75-7.22, respectively). Western blot analysis showed a uniform expression of PODXL and EGFR in all six examined CRC cell lines. Conclusions: The results from this study demonstrate that high expression of EGFR is an independent factor of poor prognosis in CRC. Moreover, strong links have been uncovered between expression of the recently proposed biomarker candidate PODXL with EGFR expression in CRC in vivo and in vitro, and with BRAF mutation in vivo. High expression of both PODXL and EGFR may also have a synergistic adverse effect on survival. These findings suggest a potential functional link in CRC between PODXL, EGFR and BRAF, all originating from chromosome 7, which may be highly relevant in the clinical setting and therefore merit future in-depth study.
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| 6. |
- Larsson, Anna H, et al.
(author)
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Validation of podocalyxin-like protein as a biomarker of poor prognosis in colorectal cancer
- 2012
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In: BMC Cancer. - : Springer Science and Business Media LLC. - 1471-2407. ; 12, s. 282-
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Journal article (peer-reviewed)abstract
- Background: Podocalyxin-like 1 (PODXL) is a cell-adhesion glycoprotein and stem cell marker that has been associated with an aggressive tumour phenotype and adverse outcome in several cancer types. We recently demonstrated that overexpression of PODXL is an independent factor of poor prognosis in colorectal cancer (CRC). The aim of this study was to validate these results in two additional independent patient cohorts and to examine the correlation between PODXL mRNA and protein levels in a subset of tumours. Method: PODXL protein expression was analyzed by immunohistochemistry in tissue microarrays with tumour samples from a consecutive, retrospective cohort of 270 CRC patients (cohort 1) and a prospective cohort of 337 CRC patients (cohort 2). The expression of PODXL mRNA was measured by real-time quantitative PCR in a subgroup of 62 patients from cohort 2. Spearman's Rho and Chi-Square tests were used for analysis of correlations between PODXL expression and clinicopathological parameters. Kaplan Meier analysis and Cox proportional hazards modelling were applied to assess the relationship between PODXL expression and time to recurrence (TTR), disease free survival (DFS) and overall survival (OS). Results: High PODXL protein expression was significantly associated with unfavourable clinicopathological characteristics in both cohorts. In cohort 1, high PODXL expression was associated with a significantly shorter 5-year OS in both univariable (HR = 2.28; 95% CI 1.43-3.63, p = 0.001) and multivariable analysis (HR = 2.07; 95% CI 1.25-3.43, p = 0.005). In cohort 2, high PODXL expression was associated with a shorter TTR (HR = 2.93; 95% CI 1.26-6.82, p = 0.013) and DFS (HR = 2.44; 95% CI 1.32-4.54, p = 0.005), remaining significant in multivariable analysis, HR = 2.50; 95% CI 1.05-5.96, p = 0.038 for TTR and HR = 2.11; 95% CI 1.13-3.94, p = 0.019 for DFS. No significant correlation could be found between mRNA levels and protein expression of PODXL and there was no association between mRNA levels and clinicopathological parameters or survival. Conclusions: Here, we have validated the previously demonstrated association between immunohistochemical expression of PODXL and poor prognosis in CRC in two additional independent patient cohorts. The results further underline the potential utility of PODXL as a biomarker for more precise prognostication and treatment stratification of CRC patients.
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| 7. |
- Larsson, Karin, et al.
(author)
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Multiplexed PrEST immunization for high-throughput affinity proteomics
- 2006
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In: JIM - Journal of Immunological Methods. - : Elsevier BV. - 0022-1759 .- 1872-7905. ; 315:1-2, s. 110-120
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Journal article (peer-reviewed)abstract
- Monospecific antibodies dfdfdfdf (msAbs) generated through antigen specific purification of polyclonal antisera are valuable tools in proteome analyses. However, proteome wide generation of msAbs would require extensive immunization programs. Therefore, it would be desirable to develop efficient immunization and purification methods to reduce the number of animals needed for such antibody-based research. Here we describe a multiplex immunization strategy for generation of msAbs towards recombinantly produced human protein fragments, denoted PrESTs. Antisera from rabbits immunized with a mixture of two, three, five and up to ten different PrESTs have been purified by a two-step immunoaffinity-based protocol and the efficiency of the purification method was analyzed using a two-color protein array concept. The obtained results showed that almost 80% of the animals immunized with antigens composed of two or three different PrESTs yielded antibodies recognizing all the included PrESTs. Furthermore, the modified two-step purification method effectively eliminated all background binding and produced pure antibody pools against individual PrESTs. This indicates that the multiplexed PrEST immunization strategy described here could become useful for high-throughput antibody-based proteomics initiatives, thus significantly reducing the number of animals needed in addition to providing a more cost-efficient method for production of msAbs.
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| 8. |
- Larsson, Karin, et al.
(author)
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Novel antigen design for the generation of antibodies to G-protein-coupled receptors
- 2011
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In: JIM - Journal of Immunological Methods. - : Elsevier BV. - 0022-1759 .- 1872-7905. ; 370:1-2, s. 14-23
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Journal article (peer-reviewed)abstract
- Antibodies are important tools for the study of G-protein-coupled receptors, key proteins in cellular signaling. Due to their large hydrophobic membrane spanning regions and often very short loops exposed on the surface of the cells, generation of antibodies able to recognize the receptors in the endogenous environment has been difficult. Here, we describe an antigen-design method where the extracellular loops and N-terminus are combined to a single antigen for generation of antibodies specific to three selected GPCRs: NPY5R, B2ARN and GLP1R. The design strategy enabled straightforward antigen production and antibody generation. Binding of the antibodies to intact receptors was analyzed using flow cytometry and immunofluorescence based confocal microscopy on A-431 cells overexpressing the respective GPCR. The antibody-antigen interactions were characterized using epitope mapping, and the antibodies were applied in immunohistochemical staining of human tissues. Most of the antibodies showed specific binding to their respective overexpressing cell line but not to the non-transfected cells, thus indicating binding to their respective target receptor. The epitope mapping showed that sub-populations within the purified antibody pool recognized different regions of the antigen. Hence, the genetic combination of several different epitopes enables efficient generation of specific antibodies with potential use in several applications for the study of endogenous receptors.
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| 9. |
- Nilsson, Peter, et al.
(author)
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Towards a human proteome atlas : high-throughput generation of mono-specific antibodies for tissue profiling
- 2005
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In: Proteomics. - : Wiley. - 1615-9853 .- 1615-9861. ; 5:17, s. 4327-4337
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Journal article (peer-reviewed)abstract
- A great need exists for the systematic generation of specific antibodies to explore the human proteome. Here, we show that antibodies specific to human proteins can be generated in a high-throughput manner involving stringent affinity purification using recombinant protein epitope signature tags (PrESTs) as immunogens and affinity-ligands. The specificity of the generated affinity reagents, here called mono-specific antibodies (msAb), were validated with a novel protein microarray assay. The success rate for 464 antibodies generated towards human proteins was more than 90% as judged by the protein array assay. The antibodies were used for parallel profiling of patient biopsies using tissue microarrays generated from 48 human tissues. Comparative analysis with well-characterized monoclonal antibodies showed identical or similar specificity and expression patterns. The results suggest that a comprehensive atlas containing extensive protein expression and subcellular localization data of the human proteome can be generated in an efficient manner with mono-specific antibodies.
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| 10. |
- Uhlén, Mathias, et al.
(author)
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A human protein atlas for normal and cancer tissues based on antibody proteomics
- 2005
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In: Molecular & Cellular Proteomics. - 1535-9476 .- 1535-9484. ; 4:12, s. 1920-1932
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Journal article (peer-reviewed)abstract
- Antibody-based proteomics provides a powerful approach for the functional study of the human proteome involving the systematic generation of protein-specific affinity reagents. We used this strategy to construct a comprehensive, antibody-based protein atlas for expression and localization profiles in 48 normal human tissues and 20 different cancers. Here we report a new publicly available database containing, in the first version, similar to 400,000 high resolution images corresponding to more than 700 antibodies toward human proteins. Each image has been annotated by a certified pathologist to provide a knowledge base for functional studies and to allow queries about protein profiles in normal and disease tissues. Our results suggest it should be possible to extend this analysis to the majority of all human proteins thus providing a valuable tool for medical and biological research.
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