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Role of heparanases
- 1. The Role of Heparanase in Cancer Yousef Layyous 2/5/2021 1
- 2. Agenda • Introduction to cancer disease (Onco 101) • The Extracellular Matrix and Heparanase • Supporting data • Targeted Heparanase • Function of Heparanase • Chemitherapy induced expression of Heparanase • Conclusions 2/5/2021 2
- 3. Introduction to Cancer • Cancer or Malignant Neoplasia • “state of autonomous cell division” 2/5/2021 3
- 4. Cancer Hallmarks 4 2/5/2021 1. Hanahan D., Weinberg RA. (2000) The Hallmarks of Cancer Cell.;100(1):57-70.
- 5. Conclusion from the hallmarks • Cancer is caused by more than one change in DNA regulation • Inflammation is similar to cancer • Treatment must be very specific. 5 2/5/2021
- 6. Classical vs Molecular Approach • Classical Approach Biopsy Tissue analysis Conclusion Treatment Tissue analysis: use of conventional staining methods (chemical affinity). Reviews histological architecture of tissue Treatment: Not highly specific. Targets cells. 6 2/5/2021
- 7. Classical vs Molecular Approach • Molecular Approach Biopsy Tissue analysis Conclusion Treatment Tissue analysis: use of immunohistochemistry, microarrays, cell culture. Reviews biomolecular markers, receptors, enzymes Treatment: Highly specific. Targets cancer cells and extracellular matrix. 7 2/5/2021
- 8. Causes of Cancer Classic approach • Smoking • Unhealthy lifestyle • Age • UV radiation • Virus Molecular approach • Somatic Mutations • Epigenetic changes • Gametic mutation • Chromatin rearrangement • Virus 8 2/5/2021
- 9. Disclaimer • I am not saying both approaches are mutually exclusive. Its just observing the same event from two different perspectives, which complement each other. 9 2/5/2021
- 10. The Extracellular Matrix (ECM) • Composed of proteins and glycosaminoglycans (ex. heparan sulfate). • Needed for: • Multicellularity • Mechanical support • Organization of cell architecture • Reservoir for biomolecular signals. • Examples: • Blood ECM is the plasma • ECM of Solid tissues is the connective tissue and basal membrane 10 2/5/2021
- 11. Heparanase (HPS) Cleaves heparan sulfate (HS), separating HS from Syndecan 1. Important role in metastasis and angiogenesis. Found in fibroblasts. Overexpression of HPS correlates to higher metastatic potential of tumour.
- 13. Function of Heparanase in Prostate Tumorigenesis: Potential for Therapy • Methods: • Cancer cell transfection • Mouse model • Microscopy 2/5/2021 13
- 14. Function of Heperanase in Prostate Tumorigenesis: Potential for Therapy • White = No staining • Grey = moderate staining • Black = high staining 2/5/2021 14
- 15. Transfected cells • Vo = vector only • Hpa= heparanase • Sp = secreted heparanase 2/5/2021 15
- 16. Mouse model • Blue = SP • Black = Vo • Red = Heparanase 16 2/5/2021
- 18. • Multiple myeloma cell culture and exposure to chemotherapeutical drugs. • Western blot • qPCR 2/5/2021 18
- 19. Different myeloma cell lines BTZ=(proteome) DEX=Dexamethasone (steroid) Dox= doxorubicin (alkylating) CHX = cycloheximide (protein synthesis) 2/5/2021 19
- 20. Drug resistant myeloma cells 2/5/2021 20 • RPMI 8226 = chemosensitive • LR5 = melphalan resistant • Dox IV = doxorubicin resistant
- 21. • In vivo study • Cell culture • Use of Chemotherapeuticals • Use of HPSE Inhibitor
- 22. Targeting heparanase overcomes chemoresistance and diminishes relapse in myeloma 2/5/2021 22
- 23. Mouse model 23
- 24. Conclusion 2/5/2021 24 • HPSE expression correlates to likelihood of metastasis • Very likely that mechanism involve reduced function of ECM. • Chemotherapy can induce HPSE expression • Targeting HPSE for future strategies
- 25. Biopharmaceutical science and the future of cancer 2/5/2021 25 • Personalized medicine and Cancer • Targeting cancer cell and ECM • Use of molecular biology and genomics for cancer diagnostics and treatment
- 26. References 2/5/2021 26 1. Rhonda Kalin (2019) Cancer Drug Pharmacology Table. BC Cancer Agency. British Columbia 2. O’Dowd G. , Bell S., Wright S. (2019) Wheater's Pathology: a Text, Atlas and Review of Histopathology. Pgs.71-91Elsevier. Amsterdam 3. A.D. Bradshaw (2016) Encyclopedia of Cell Biology The Extracellular Matrix Vol. 2, pgs. 694-703. https://doi.org/10.1016/B978-0-12-394447-4.20067-9 4. Lerner, Immanuel ; Baraz, Leah ; Pikarsky, Eli ; Meirovitz, Amichay ; Edovitsky, Evgeny ; Peretz, Tamar ; Vlodavsky, Israel ; Elkin, Michael (2008) Function of Heparanase in Prostate Tumorigenesis: Potential for Therapy. Clin Cancer Res, 14 (3) 668-676. DOI: 10.1158/1078-0432.CCR-07-1866 5. Ramani, Vishnu C ; Vlodavsky, Israel ; Ng, Mary ; Zhang, Yi ; Barbieri, Paola ; Noseda, Alessandro ; Sanderson, Ralph D (2016) Chemotherapy induces expression and release of heparanase leading to changes associated with an aggressive tumor phenotype. Matrix Biol. 55 (1), 22-34. DOI: 10.1016/j.matbio.2016.03.006 6. Ramani Vischnu. ; Zhan, Fenghuang ; He, Jianbo ; Barbieri, Paola ; Noseda, Alessandro ; Tricot, Guido ; Sanderson, Ralph D. (2015) Targeting heparinase overcomes chemoresistance and diminishes relapse in myeloma. Oncotarget. 7 (2) 1598-1607.DOI: 10.18632/oncotarget.6408 7. Hanahan D., Weinberg RA. (2000) The Hallmarks of Cancer Cell.;100(1):57-70. 8. Goldberg, R., Meirovitz, A., Hirshoren, N., Bulvik, R., Binder, A., Rubinstein, A. M., & Elkin, M. (2013). Versatile role of heparanase in inflammation. Matrix Biology, 32(5), 234–240. doi: 10.1016/j.matbio.2013.02.00
- 27. THE END Image from https://www.quantamagazine.org/unexpected-diversity-found-in-16-new-lab-mouse-genomes-20181001/ 27 2/5/2021
Editor's Notes
- Neoplasia is the used by pathologist No need of external stimulus.
- Eosin= pink and acidic, affinity for basic Hematoxilin = purple and basic, affinity for acidic Von Kossa = affinity for mineralized tissue Ect ect ect
- Affinity for receptors, for enzymes for biomolecules of interest
- P < 0.0001 n = 171 for overexpression of heparanase in cancer vs non cancerous tissue. Immunostaning for anti-hep in prostate tissue. Left non cancer, right cancer Gleason score
- Cell culture and transfection. VO vector only; sp secreted heap chimera of chicken signal peptide and hep; hp heap A lysate and B conditioned medium, labelled with 35S-labeled heparan sulfate degradation fragments Each experiment done three times. A and b validates hep secretion by sp cells. C = adherence to bovine aorta epithelia cells in resuspenden cell culture. Paper said that it confirms increased adherence, but I consider it to variable to draw conclusion. D = Response to chemoattractant (chemokines).
- 22 days = palatable tumours in sp 46 days growth. Vo=black, sp= blue hpa = red A. Acceleration of tumor formation correlates to HPSE expression. A . Right image = no bone tissue, mainly tumor sp; left = small tumor with tissue, hpa. FOR B, Osteoclast activity was reviewed. Significant mainly for sp and lesser ext in hpa. Not in vo.
- Lung tissue metastasis from tibial tumor implantation.
- Multiple myeloma cells
- . (A) Heparanase protein was assessed by western blotting of total cell extracts from different myeloma cell lines after 14 h treatment with BTZ (25 nM), Dex (10 μM) or doxorubicin (Dox, 5 μM). At these concentrations the drugs inhibited cell proliferation to a similar extent. (B) Heparanase transcript level in myeloma cells was assessed by real time PCR after 8 h treatment with BTZ (50 nM), CFZ (100 nM), Mel (20 μM). Transcript levels in vehicle treated cells served as control, *p < 0.05 versus vehicle treated cell C) HPSE protein levels as revealed by western blotting after treatment with BTZ (25 nM) alone for 14 h or with BTZ after 2 h pretreatment with cycloheximide (eukaryotic protein synthesis inhibitor) (CHX, 30 μg/ml).
- Heparanase expression is elevated in drug resistant myeloma cells. (A) Western blotting for heparanase (HPSE) and syndecan-1 (SDC1) in chemosensitive (RPMI-8226) cells and chemoresistant RPMI-8226 clones (Dox1V—doxorubicin resistant clone, LR5—melphalan resistant clone). (B) Heparanase transcript levels were determined by real time PCR and normalized against 18S rRNA levels in the above cell lines, *p < 0.05 versus RPMI-8226. (C) Level of soluble HGF in conditioned media from RPMI-8226 and LR5 cells as determined by ELISA, *p < 0.01 versus RPMI-8226. Syndecan1 transmembrane protein which supports heparan sulfate. Increased HPSE = decreases size of SDC1.
- Heparanase increases survival Baseline = diagnostic, before treatment Post-ind = 5 weeks after first treatment 2.5 months after first transplant and second treatment = post 1 Post 2nd = after second transplant and third treatment.
- A and B MTT assay to review apoptosis and cellular death. Oxydoreductase to asses cell viability. C ELISA to quantify Kappa. Kappa chain correlates to cancerous cells. The lower the less cancer CAG very aggressive cells.