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OSTF1: A gene that enhances bone resorption by human breast cancer cells

OSTF1: A gene that enhances bone resorption by human breast cancer cells. University of Manitoba , Winnipeg Robert Shiu Barbara Iwasiow Alison Yarmill Anne Blanchard George Skliris Dr. Jiuyong Xi University of Texas Health Sciences Center, San Antonio Sakamuri Reddy.

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OSTF1: A gene that enhances bone resorption by human breast cancer cells

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  1. OSTF1: A gene that enhances bone resorption by human breast cancer cells • University of Manitoba, Winnipeg • Robert Shiu • Barbara Iwasiow • Alison Yarmill • Anne Blanchard • George Skliris • Dr. Jiuyong Xi • University of Texas Health Sciences Center, San Antonio • Sakamuri Reddy

  2. The Problem: Breast cancer metastasis & bone destruction

  3. Patients with osteolytic bone destruction • Pathologic fractures • Severe bone pain • Nerve compression • Immobility • Hypercalcemia, causing neurologicalsymptoms & cardiac arrhythmias

  4. OSTF1 Normal bone resorption Bone marrow cell TGFb, Ca++, PO4, collagen peptides bone ? Bisphosphonates osteoclast Humoral Cytokines ? Humoral Cytokines Parathyroid Hormone (PTH) osteoclast precursor RANKL Calcitonin osteoblast stromal cell Glucocorticoids Osteoprotegerin

  5. OSTF1 Viscious cycle of breast cancer induced bone resorption (GR Mundy) SD-208 (preclinical) breast cancer cell TGFb, ca++ bone ? Bisphosphonates osteoclast PTHrP & cytokines osteoclast precursor RANKL* osteoblast Denosumab (Phase III) stromal cell *TNF superfamily member

  6. Osteoclast Stimulating Factor (OSTF1) • RNA originally cloned from human bone marrow cells, but is expressed in many tissues and cell types • Non-secreted protein 28 kd localized to nucleus and cytoplasm • Mouse homolog SH3P2 or SH3D3 • Contains SH3 domain (binds proline-rich domain, PXXP) and ankyrin repeats • Binds Cbl via SH3 (forms OSTF1-Cbl-Src comlex), a key molecule in tyrosine kinase signaling and regulation • Binds SMN1 (survival of motor neuron 1 protein, mutated in spinal muscular dystrophy patients) that is involved in snRNP biogenesis & pre-mRNA splicing • Binds and co-localizes with actin filaments (via ankyrin repeats) • Only one known biologic function – stimulates osteolysis (bone resorption)

  7. Research objectives • Is OSTF1 expressed in human breast cancer cells? • Does OSTF1 affect the ability of breast cancer cells to destroy bone? • What is the mechanism of action of OSTF1?

  8. OSTF1 protein in human breast tumors MDA-MB-231 Tumor 1 Tumor 2 Tumor 3 Tumor 4 OSTF1 Actin

  9. OSTF1 protein in human breast tissue microarray (TMA) Tumor Normal

  10. OSTF1 protein in human breast tissue microarray (TMA) Tumor Normal Antiserum Normal serum

  11. Research objectives • Is OSTF1 expressed in human breast cancer cells? YES • Does OSTF1 affect the ability of breast cancer cells to destroy bone? • What is the mechanism of action of OSTF1?

  12. OSTF1 Knockdown of OSTF1 expression in MDA-MB-231 cells • highly metastatic and osteolytic • Express OSTF1 breast cancer cells MDA-MB-231 growth factors, ca++ RNAi bone osteoclast Cytokines (?) osteoclast precursor osteoblast RANKL stromal cell

  13. Stable knockdown of OSTF1 gene expression (RNAi) 972 GTAGAACAAGAAGATATTA Scrambled sequences GAAATAAGCATATAGGTAA TTACCTATATGCTTATTTC TAATATCTTCTTGTTCTAC GTAGAACAAGAAGATATTA CATCTTGTTCTTCTATAAT ATTATAGAAGAACAAGATG GUAGAACAAGAAGAUAUUA CAUCUUGUUCUUCUAUAAU

  14. OSTF1 knockdown by RNAi in MDA-MB-231 breast cancer cells Western Northern Actin OSTF1 SCR 972 972 898 SCR 866 435 257 RNAi sequence # 856 98 53 0 90 65 54 98 % Inhibition 3’ UTR coding

  15. OSTF1 knockdown does not alter cell growth 5% serum RNAi 3 Relative cell growth RNAscr 2 1 Serum-free 0 0 1 2 3 4 5 6 Days in culture 7 7 7 7 7 7

  16. 24h 12h Ca45 release from fetal mouse calvaria • Mouse killed • Fetal calvaria dissected, • halved, and pre-incubated % Released Ca45 72h D18 pregnant mouse Injected with Ca45 • Replaced with fresh medium • Test materials added to paired calvaria halves Calvaria cultured in BGJ/DMEM (80/20) with 12.5% Horse serum in atmosphere of O2/CO2/N2 (50/5/45)

  17. Ca45 release calculation & analysis • Two halves of calvaria from same embryo compared: • treatment 1 vs treatment 2 • For each half, % Ca45 released = dpm in medium / total dpm x 100 • Ratio = % released by treatment 1 / % released by treatment 2 *P <0.001 (Mann-Whitney Rank Sum Test)

  18. Research objectives • Is OSTF1 expressed in human breast cancer cells? YES • Does OSTF1 affect the ability of breast cancer cells to destroy bone? YES • What is the mechanism of action of OSTF1?

  19. OSTF1 Mechanism of OSTF1 breast cancer cells MDA-MB-231 growth factors, ca++ RNAi bone osteoclast Cytokines (?) osteoclast precursor osteoblast RANKL stromal cell

  20. Quantitative Realtime-PCR Fold change of gene expression = 2DCt(gene)-DCt(actin) =2(B-A)gene-(B-A)actin =28-1 = 27 = 128 A A B B Ct

  21. Fig. 6: OSTF1 knockdown decreases PTHrP but not IL8 expressionA Quantitative Realtime-PCR study Ct values ± SEM (n=6) *P < 0.001

  22. Cytokine & osteogenesis gene expression profiling(SupraArray Profiler™ PCR Array gene sets totalling 178 genes) Compared RNAi cells and control (RNAscr) cells: 1.E-03 Volcano Plot 1.E-02 p Value BMP6 0.05 0.05 1.E-01 1.E+00 -7 -5 -3 -1 1 3 5 7 9 DCt

  23. Down-regulated genes in OSTF1 knockdown cells

  24. Research objectives • Is OSTF1 expressed in human breast cancer cells? YES • Does OSTF1 affect the ability of breast cancer cells to destroy bone? YES • What is the mechanism of action of OSTF1? STIMULATES THE EXPRESSION OF HUMORAL CYTOKINES

  25. OSTF1 Viscious cycle of breast cancer induced bone resorption (GR Mundy) SD-208 (preclinical) breast cancer cell TGFb, ca++ bone Bisphosphonates osteoclast PTHrP , TGFa, BMP6, IL17B, TNF12 osteoclast precursor Therapeutic targets? RANKL* osteoblast Denosumab (Phase III) stromal cell

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