Bone Metastasis in Breast Cancer: Molecular Pathogenesis

1. BBL Bone Biopathology Lab UnivAq Bone Metastasis in Breast Cancer: Molecular Pathogenesis Maurizio Longo Dip. Medicina Sperimentale, Università dell’Aquila Highlights in the Management of Breast Cancer Roma, Domus Sessoriana, Nov. 16-17 2006

2. Bone Metastasis: a Most Severe Complication of Breast Cancer • Up to 80% of patients with metastatic breast cancer will develop bone metastases • Long-term survivor patients with only bone metastasis undergo very poor life quality

3. Bone-Metastatic Disease of Breast Cancer: Important Symptoms • Hyper-calcemia • Pain, often disabling and resistant to palliation • Pathologic fracture • (Breast carcinomas account for >50% of the met. cases requiring orthopedic intervention)

4. Bone-Metastatic Disease of Breast Cancer: Clinical Features • Multifocal: at autopsy, nearly all patients demonstrate multiple, small “asymptomatic” bone metastases • Preferably located to the axial skeleton, together with active hemopoietic marrow • Indolent course (e.g. remarkable progression-free periods after treatment)

5. Osteolysis and Pathologic Fracture Lytic areas Mechanical properties are severely endangered also by “mixed” lesion (lytic/blastic)

6. Detachment from tumour • (dependent on E-M transition) • Transport in the bloodstream • (estimated <0.1% surviving this phase) • Adhesion to endothelial cells • (mostly random*) • Invasion of host tissue Target-independ. METASTASIS: a Multi-step Process (I) *Debated

7. METASTASIS: a Multi-step Process (II) • Target-dependent steps: • “Colonisation” of tissue • Triggering of growth • (cross-talk with the host tissue) • Independent growth of secondary tumour • (only autocrine and systemic regulation)

8. The Metastasis Process: “Classical” Description An “obsolete” model?

9. Main points presently challenged of the “classical” description: • Cell detachment as a late event • “Unbridled” growth of (micro)metastases • --> In fact, recent evidence obtained in in-vivo models shows: • single tumour cells in distant tissues during earliest development phases of metastatic tumours; • extremely slow development of many micrometastases.

10. Growth of micrometastases is a target-dependent process

11. The Bone Micro-environment: Main Cell Players The bone organ is also comprised of: marrow stromal cells, hemopoietic cells, adipocytes. (Fibrous tissue may also appear.)

12. Osteoblasts and Osteoclasts: Main Features • Osteoblasts (OBs) develop from mesenchimal cells • Osteoclasts (OCs) develop from the fusion of blood cells of the immune system, the monocytes • OBs are bone-residing cells • OC precursors are recruited from the bloodstream • OBs proliferate, OCs do not. The OC life-span is limited (apoptosis can only be delayed) • Both OBs and Ocs are polarised, very actively secreting cells: • OBs secrete bone matrix glycoproteins and collagen • OCs secrete HCl and lytic enzymes, and digest collagen

13. “Reversal” cells OBs OCs The Bone Remodeling Unit (I) Resorption cone. (Early osteoblasts do not adhere to the bone surface and proliferate actively.)

14. The Bone Remodeling Unit (II) Resorption is fast, apposition is slow, mineralisation slower. I.e. --> uncontrolled resorption can damage bone fast!

15. The resorption activity of osteoclasts is potentially very harmful and is finely regulated. What controls osteoclasts?

16. Bone Resorption by Osteoclasts: Multiple Control Levels • Systemic: hormones (incl. Vitamin D3) and metabolic signals • Local: • Secreted factors (chemokines, interleukins, cytokines) • Cell-cell interactions

17. Note well: at the local level, phisiological regulation relies crucially on: osteoblasts and their precursors.

18. Osteoclast control: Local Factors SDF-1 M-CSF RANKL OPG Chemotaxis Proliferation Fusion Differentiation Resorption Apoptosis Vitamin D3 IL-1, IL-6 Vitamin D3 PTH/PTHrP IL-6

19. Osteoclast control: Cell-cell Interaction OPG RANKL RANK c-Fms M-CSF

20. Key point: Bone met. cells are, or become*, highly responsive to many signals destined to bone cells. *Or both!

21. What survival and growth signals for bone cells do met. cells exploit ?

22. 1. Survival: Stem Cell Niches Micrometastasis cell originate from (actively) dividing tumour cells with unlimited division potential Despite this, micrometastasis show an indolent initial development ANALOGY: Stem cells are characterised by unlimited division potential BUT stem cells divide with extremely low frequency

23. The “Cancer Stem Cell” Hypothesis: Tumours originate from stem/progenitor cells of the host tissue Adenomas, sarcomas, etc. Normal tissues Carcinomas, etc.

24. The Hemopietic Stem Cell Niche in Bone (I) Niche-dependent differentiation in healthy bone

25. Stem Cell Niche in Bone: Overview of Present Knowledge • Formed by SNO cells, an osteoblastic sub-population • SNO cells are: • Spindle-shaped (poorly polarised / non-secreting) • N-Cadherin-positive • CD45-negative

26. Stem Cell Niche in Bone: Overview of Present Knowledge • Formed by SNO cells, an osteoblastic sub-population • SNO cells are: • Spindle-shaped (poorly polarised / non-secreting) • N-Cadherin-positive • CD45-negative • SNO cells maintain self-renewing long-term stem cells in a semi-quiescent state • Thus, activation of stem cells requires detachment from SNO cells

27. Stem Cell Niche in Bone: Overview of Present Knowledge • Formed by SNO cells, an osteoblastic sub-population • SNO cells are: • Spindle-shaped (poorly polarised / non-secreting) • N-Cadherin-positive • CD45-negative • SNO cells maintain self-renewing long-term stem cells in a semi-quiescent state • Activation of stem cells requires detachment from SNO cells • Detachment from the niche is favoured by various phisiological (e.g. chemotactic) and pathological signals

28. The Cancer Stem Cell Niche in Bone Hypothesis: Once in the bone marrow micro-environment, cancer stem cell may achieve long survival by interacting with stem-cell niches

29. The Cancer Stem Cell Niche in Bone Hypothesis: Once in the bone marrow micro-environment, cancer stem cell may achieve long survival by interacting with stem-cell niches On the other hand, the niche will prevent cancer stem cells from proliferate appreciably! <-- Explanation of metastasis indolence Additional signals can lead to further metastasis progression

30. The Hemopietic Stem Cell Niche in Bone (II) Abnormal niche-dep. differentiation in metastasis-bearing bone

31. 2. Growth: The Initial Development of Metastases or: «Spies in Enemy Headquarters»

32. How can an ex-epithelial cell manage to live well once free in the bone marrow? • Initial metastases (few cancer cells) cannot rely on autocrine growth-promoting (and anti-apoptosis) signals • Thus, only metastasis cells responding best to available growth signals will undergo positive selection • Of these, cells also capable of over-stimulating local bone resorption will be further favoured (due to creation of larger space for growth)

33. The Metastasis “Vicious Circle” (I)

34. The Metastasis “Vicious Circle” (II) Bone Matrix RankL-Rank IL-6, TNFα, M-CSF, PGE2 PTHrP, etc. OBs Pre-OCs Bone Resorption OCs Proliferation Survival Migration TGF- FGFs PDGF IGF-1 Cancer cells BMPs

35. Observation: Beside releasing same OC-genic factors as OBs, various bone met. cells also express bone-cell markers (e.g. BSP, OPN) and transcription factors (e.g. Runx2).

36. Observation: Beside releasing same OC-genic factors as OBs, various bone met. cells also express bone-cell markers (e.g. BSP, OPN) and transcription factors (e.g. Runx2). Just a coincidence?

37. How can an epithelial cell manage to live well in the bone marrow? • Initial metastases (few cancer cells) cannot rely on autocrine growth-promoting (and anti-apoptosis) signals • Thus, only metastasis cells responding best to available growth signals will undergo positive selection • Of these, cells also capable of over-stimulating local bone resorption will be further favoured (due to creation of larger space for growth)

38. Result: various metastasis cells will eventually “resemble”* the bone-residing cells governing bone resorption (i.e. OBs!) OR The Met. “Osteomimicry” Hypothesis *Pre-adaptation also possible

39. Met. Osteomimicry: Some Evidence • Lin DL, Tarnowski CP, Zhang J, Dai J, Rohn E, Patel AH, Morris MD, Keller ET. • Bone metastatic LNCaP-derivative C4-2B prostate cancer cell line mineralizes in vitro.Prostate. 2001 May 15;47(3):212-21. • Barnes GL, Javed A, Waller SM, Kamal MH, Hebert KE, Hassan MQ, Bellahcene A, Van Wijnen AJ, Young MF, Lian JB, Stein GS, Gerstenfeld LC. • Osteoblast-related transcription factors Runx2 (Cbfa1/AML3) and MSX2 mediate the expression of bone sialoprotein in human metastatic breast cancer cells.Cancer Res. 2003 May 15;63(10):2631-7. • Zhang JH, Tang J, Wang J, Ma W, Zheng W, Yoneda T, Chen J. • Over-expression of bone sialoprotein enhances bone metastasis of human breast cancer cells in a mouse model. Int J Oncol. 2003 Oct;23(4):1043-8. • Barnes GL, Hebert KE, Kamal M, Javed A, Einhorn TA, Lian JB, Stein GS, Gerstenfeld LC. • Fidelity of Runx2 activity in breast cancer cells is required for the generation of metastases-associated osteolytic disease.Cancer Res. 2004 Jul 1;64(13):4506-13.

40. Transcriptome Analysis: Genes more expressed in bone mets versus non-bone-mets: any bone-cell markers? YES!

41. Transcriptome Analysis: The Osteomimicry Signature

42. The Osteomimicry Signature: OB Genes* *Shown only cSrc-regulated genes

43. The Osteomimicry Signature: OC Genes

44. Hypothesis (I):The osteomimicry “strategies”* of bone metastasis cells relies not only on osteoblast-, but also on osteoclast-like features *Multiple osteomimetic pseudo-phenotypes possible

45. Hypothesis (II):Part of the observed pseudo-osteoclastic phenotype of bone met. cells might accomplish “micro-resorption”.

46. Hypothesis (II):Part of the observed pseudo-osteoclastic phenotype of bone met. cells might accomplish “micro-resorption”. Micro-resorption could be the trigger of the metastasis vicious circle.

47. BBL Bone Biopathology Lab UnivAq Thank You for your attention!