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Supplementary Figure S6. Localization of miR-135 and miR-203 binding sites within the human Runx2 3'UTR.

Supplementary Figure Legends. Legends for Supplementary Figures S1-S7.

Supplementary Figure S1. Runx2 and Smad5 expression in primary breast tumors.

Supplementary Figure S3. Delivery of miR-135 and miR-203 reduce chemotactic migration of MDA-MB-231 cells.

Supplementary Figure S5. Expression of exogenous miR-135 and miR-203 in orthotopically transplanted breast cancer.

Supplementary Figure S2. Runx2 and Runx2-targeting miRNAs are regulated in metastatic breast cancer cells and bone metastases.

Supplementary Figure S7. miR-135 and miR-203 reduce breast cancer metastasis to lung.

Supplementary Figure S4. Delivery of miR-135 and miR-203 decrease the expression of CCL-7 and CXCL-12.

Background: Bone is the main metastatic site for breast cancer. Single disseminated tumor cells (DTCs), the seeds of metastases detected in the bone marrow, can be found still years after the primary diagnosis. We have shown that the presence of DTCs in the bone marrow at both the time of diagnosis and after chemotherapy is an independent predictor of poor prognosis in primary non-metastatic breast cancer. We identified retinoic acid-induced 2 (RAI2) as a novel metastasis suppressor gene significantly associated with both positive DTC status and poor prognosis, especially among hormone-receptor positive breast cancer patients. RAI2 sustains differentiation of ER-positive tumor cells, whereas loss of RAI2 induces hormone independent growth and activation of AKT signalling as an important mediator of dormancy control. Methods: Xenograft models were established by injecting orthotopically RAI2 KO (CRISPR/Cas9 mediated RAI2 depletion) and parental KPL1 cells in immune deficient SCID/J mice. A transgenic RAI2 KO (RAIKO) mouse was established with the CRISPR/Cas9 system. The in vitro effect of RAI2 on the bone marrow cells was studied with osteoblast and osteoclast differentiation assays either with conditioned media from parental and RAI2 KO (MCF7 and KPL1) cells or with extracellular vesicles (EVs) isolated by ultracentrifugation from the supernatant fractions. miRNA content of the EVs from the different cell line models was assessed by next generation sequencing (NGS). NGS data was validated in plasma from a cohort of bone and brain metastatic breast cancer patients, non-metastatic breast cancer patients and in healthy donors. Results: Xenograft experiments in SCID/J mice showed an increased early tumor dissemination (increased numbers of CTCs and DTCs) when RAI2 KO cells were injected orthotopically versus the parental cell line, whereas the RAIKO mouse model showed that RAI2 is not involved in initial tumor development. Besides, both conditioned media as well as the EV fraction from RAI2 KO cells significantly increase osteoclast differentiation. Five miRNA candidates were identified to be involved in the RAI2-mediated crosstalk between tumor and bone cells. Validation assays on clinical samples identified miR-135a-5p to be lower expressed on EVs from bone metastatic patients compared to heathy donors and non-metastatic patients. Conclusions: RAI2 is a novel inhibitor of breast cancer tumor cells dissemination in the bone marrow thereby attenuating the vicious cycle of tumor crosstalk with the bone microenvironment. Citation Format: Thais Pereira Veiga, Stefan Werner, Desiree Loreth, Michael Horn, Melanie Groninger, Moritz Grabe, Anke Baranowsky, Hanna Taipaleenmäki, Laura Brylka, Jolanthe Kropidlowski, Thorsten Schinke, Klaus Pantel, Harriet Wikman. RAI2 is a novel bone metastasis-associated gene, mediating a vicious cross talk between breast cancer cells and osteoclasts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1295.