Tumour invasion and metastasis initiated by miRNA-10b in breast cancer

1. Tumour invasion and metastasis initiated by miRNA-10b in breast cancer Ma et al Erin White 9-18-08

2. miRNAs • miRNA regulate genes via imperfect complementarity to 3’ UTR of gene • Cleaved by Dicer from 70-100nt pre-miRNA hairpin in cytosol • Can be expressed in a tissue specific fashion

3. miRNAs expressed in breast cancer • miR-155 • miR-9 highly expressed in met. cancer • miR-10b • miR-335 • miR-206 low expression in met. cancer cells • miR-126

4. Cell Lines Used

5. miR-10b is highly expressed in metastatic breast cancer cells

6. miR-10b positively regulates cell migration and invasion Inhibition of miRNAs to determine regulation of migration/invasion. Comparison of normal mammary cells to tumorigenic but non-met cells.

7. miR-10b induces tumour invasion Tumors produced in NOD-SCID mice after 11 wks. Muscular H & E staining of tumors after 6 wks. Tumors expressing miR-10b are invasive. Vascular

8. miR-10b induces tumour invasion Ki-67 and MECA-32 staining of mammary tumors in NOD-SCID mice marking proliferation and epithelial cells. Invasion fronts of miR-10b express’g tumors show high levels of cell proliferation and angiogenesis

9. miR-10b induces distant metastasis Highly prolif. cells in lumina of vessels associated with miR-10b tumors. Micro-met in lung sections after 9 wks. Cytokeratin staining.

10. miR-10b induces distant metastasis Lungs from mice receiving miR-10b injexn after 11 wks.

11. Twist and Snai1 • Transcription factors • Twist is a metastasis-promoting gene • Epithelial-Mesenchymal transition (EMT) inducing factors • Authors think that Twist might regulate miR-10b expression • Snai1 used as a control in assays

12. miR-10b is regulated by Twist Expression of miR-10b in four mouse mammary tumour cell lines with Twist as regulator Putative transcription factors regulating miR-10b Putative binding sites for transcription regulation in miR-10b

13. miR-10b is regulated by Twist ChIP assay to determine where Twist binds miR-10b gene Overexpression of Twist leads to increase in motility and invasiveness 5-fold decrease by miR-10b inhibition

14. HOXD10 • Homeobox domain 10 • Previously implicated in suppression of cell migration and/or invasion • Expression lost in malignant breast cancer • Restored expression in MDA-MB-231 cells impairs migration and invasion in vitro and in vivo • 3’ UTR has partial complementarity to miR-10b

15. RHOC • Involved in cell migration and ECM remodelling • Identified as important player in metastasis • Robustly expressed in miR-10b-transduced cells • Repressed by HOXD10

16. miR-10b suppresses HOXD10, leading to induction of RHOC miR-10b does not cause degradation of HOXD10 mRNA Reduction in activity of 3’ UTR due to binding of miR-10b HOXD10 protein levels reduced by miR-10b expression as RHOC increases

17. miR-10b suppresses HOXD10, leading to induction of RHOC Constitutive expression of HOXD10 ablates cell migration and invasiveness Knockdown of RHOC reduces invasion and migration

18. miR-10b level is associated with the metastasis outcome in breast cancer patients

19. Conclusions • miR-10b is highly expressed in metastatic breast cancer cells • miR-10b induces tumour invasion • miR-10b induces distant metastasis • miR-10b is regulated by Twist transcription factor • miR-10b suppresses HOXD10

20. Model

21. Endogenous human miRNAs that suppress breast cancer metastasis Tavazoie et al Erin White 9-18-08

22. Cell Lines Used • Derivatives of MDA-MB-231 parent line • 231 cells poorly metastatic, high miR-335 expression • LM2 = highly metastatic to lung tissue • BoM2 = highly metastatic to bone • CN34 = cells derived from patient with metastatic breast cancer • CN34-LM1 • CN34-BoM1

23. ID of miRNAs that suppress lung and bone metastasis Lung sections after 8 wks showing decrease in number of metastastic foci miR-122a restoration did not result in signif. decrease in lung colonization Restoration of miR-335, -206, and -126 decreased lung colonization by five-fold

24. ID of miRNAs that suppress bone and lung metastasis Expression of miR-335, -206, and -126 decrease bone metastasis

25. ID of miRNAs that suppress lung and bone metastasis Mice injected with cells from pleural fluid of patient with met breast cancer miR-335, -206, and -126 reduced the ability of CN34-LM1 and CN34-BoM1 cells to metastasize to lung and bone Low levels of suppressor miRNAs shown in CN34 cells

26. miR-126 suppresses overall tumour growth and proliferation

27. miR-335 and miR-206 regulate migration, invasion, and morphology

28. Clinical association of miR-335, -126 with metastasis-free survival Median expression values of these miRNAs was eight-fold lower in patients that did relapse compared to those who did not relapse.

29. miR-335-regulated gene set includes SOX4 as direct target 3’ UTRs of all genes in set have complementarity to miR-335. Activity assayed via luciferase in LM2 or 231 cells Inhibition of miR-335 in 231 cells enhanced expression of gene set

30. SOX4 is a miR-335 direct target miR-335 suppresses activity of wt SOX4 3’ UTR but mutation abrogates suppression

31. miR-335 regulates metastasis and invasion through suppression of SOX4 and TNC Knockdown of SOX4 or TNC reduces the invasiveness of LM2 cells

32. Metastasis-free survival of patients expressing miR-335 six-gene signature Patients whose primary breast tumors were positive for metastasis signature had worse metastasis-free survival

33. Conclusions • miR-335, -206, and -126 are suppressors of metastasis • miR-335 and miR-206 regulate migration, invasion, and morphology • miR-126 suppresses overall tumour growth • miR-335 regulates a set of metastasis genes including SOX4 and TNC as direct targets