1. Bone module Summary of literature searches

2. Critique 1: The genes are not related to bone building but rather to angiogenesis. Relevant queries: –Are the genes relevant to bone and carlitage building? –Are the genes specifically expressed in bone? –Are the genes associated with angiogenesis? –Are the genes associated with atherosclerosis? –Are the genes associated with other vascular processes?

3. Bone related genes? 31 of 32 genes are bone/cartilage building –30 genes are related to bone/cartilage building – 1 gene is related to bone resorption

4. Bone specific genes? 14/32 genes are relatively specific to bone –6/14 are specific to bone or cartilage (including 3 critical TFs: Dlx3, Cart1, Sox9) –8/14 are present in bone or cartilage as well as a limited number of additional tissues: 2 in vascular tissue (MGP, FBN1) 2 in fibroblasts (OSF-2, Col12a1) 4 in other tissues (AEBP1 in adipocytes; HOXD13 in genital, Dlx5 in neurogenesis, SPP2 in liver) Note: These include 2 additional critical TFs: Dlx5 and Hoxd13

5. Angiogenesis related genes? 13/32 genes are associated with angiogenesis directly or indirectly –8/32 are angiogenic –3/32 are angiogenesis inhibitors –3/32 have only partial or indirect links to angiogenesis –There is no overlap between these 13 genes and the 14 “bone specific” ones (especially not the TFs) –Expect TIE, 10/13 [6 of the activators, all 3 inhibitors and 1 of the “partially related” genes] are secreted factors with pleiotropic functions

6. Atherosclerosis related genes 10/32 genes are related to atherosclerosis (calcification of vessels) [3 overlap with the 13/32 angiogenesis ones] –7 are activators of atherosclerosis –3 are inhibitors of atherosclerosis

7. Genes mentioned with relation to vasculature In addition to the atherosclerosis and angiogenesis genes, 3/32 genes were mentioned in general with respect to blood vessels.

8. Conclusion The great majority (31/32) of genes in the module are related to bone processes; many of them (14/32) are relatively specific to bone, including several important TFs –(Sox9, Dlx3, Cart1 which are specific and HoxD13 and Dlx5 which participate also only in genital and neural development). Only about a third (13/32) of the genes in the module were previously related to angiogenesis, –Only 8 promoted angiogenesis (the others are either inhibitory or only indirectly linked) –The 13 genes are primarily secreted factors with pleiotropic functions –3/13 are also related to atherosclerosis 10 additional genes in the module were associated with vasculature. –7 were related to atherosclerosis (which is related more to calcification and bone!), in addition to the 3 overlapping with the angiogenesis related group –3 were generally associated with vasculature Bottom line: The module is primarily a bone/cartilage module. This is the most common shared function, by far, and many are relatively specific to bone, including five important TFs A minority of the genes in the module may also be related to angiogenesis; these are primarily general factors. We cannot rule out a dual role for these genes in tumorigenesis, and believe that this may only enhance the importance of the module.

9. Module Module (32) Bone/Cartilage function (31) Specific tissue, including bone/cart (14) Unique to bone/cart (6) Angio associated (13) Angiogenic (8) Atherosclerosis (10) 3 5

10. Critique 2: The genes are not expressed in the cancer epithel but are primarily stromal Relevant queries: –Are the genes expressed in breast cancer epithel (RNA or protein in tissue sections or in cell lines)? –Are the genes expressed in breast cancer stroma (RNA or protein in tissue sections)? –Are the genes expressed in other cancer epithels (RNA or protein in tissue sections or in cell lines)? –Are the genes expressed in other cancer stroma (RNA or protein in tissue sections)?

11. Expression in BC epithelial cells 13 of 32 genes have direct evidence for expression in BC epithel or cell lines –5 are intracellular, including the TFs Sox9 and ETS2 –7 are secreted Partial or indirect evidence exists for 3 more genes

12. Expression in BC stroma 9/32 genes are expressed in BC stroma –5/9 are also expressed in BC epithelial cells –6/9 are secreted –3/9 are membranal receptors / adhesion molecules

13. Expression in other cancer epithelial cells 13/32 genes are reported in epithelial cells of other tumors –6/13 are also in BC epithel (including 2 that are also in BC stroma) –3/13 also have “partial evidence” in BC epithel –2/13 also have evidence in BC stroma (but not epithel) –Overall, only 2 “new” genes over BC findings –7 of the genes are intracellular or membranal; 6 are secreted

14. Expression in stroma of other tumors 6/32 genes were expressed in stroma from other tumors –3/6 also in BC stroma (1 of them also in BC epithel) –1/6 also in epithel of other tumors –Overall only 2 “new” genes to stroma list –3 are membranal; 4 are secreted

15. Conclusions 16 of the genes were shown in BC epithel or stroma (or both): 12 epithelial; 9 stromal There are 19 genes marked as epithel in BC or tumors in general. The stromal specific genes are primarily secreted. There are 12 genes marked as stroma in BC or other tumors. 8/12 are also shown to be epithelial in tumors. Overall, there is clear support for expression of most of the genes (for which we have prior evidence) in cancer epithelium. The few which are exclusively stromal are primarily secreted factors, lending support to paracrine signaling.

16. Module (32) Epithelial (19) BC (13) Stroma (12) BC stroma (9) 4 8 5 2 2 2 1 TF, Plasma membrane receptor, Extracellular 2, 1, 4 2, 3 1, 3 1, 1 1 2 2

17. BC (13) Specific tissue, including bone/ cart (14) Angio associated (13) Epithelial (19) 4 1 3 3 2 Stroma only (4) 1 2 Note, that angiogenesis related genes have probably been tested much more extensively in breast tissues, for obvious reasons Bone/cart unique Angio genesis 4 2 1 1 1 1 3