Sanjit Mukherjee1, Konrad Huppi1, Robert L. Walker1, Jack Zhu1, Marbin Pineda 1, Fan Yang1, James Purcell2, Lee J. Helman3 and Paul S. Meltzer1 1Genetics Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD, 2AbbVie Biotherapeutics, Redwood City, CA, 3Children’s Hospital Los Angeles, Los Angeles, CA.
- Submitter: Sanjit Mukherjee (Postdoc or fellow)
- email: sanjit.mukherjee@nih.gov
Background: Leucine-rich repeat containing 15 (LRRC15), a transmembrane protein, whose expression can be modulated by TGFβ, has been found to be highly expressed in multiple solid tumors (e.g. sarcoma, glioma and melanoma) (1) (2). LRRC15 has been nominated as a candidate target for immunotherapy with antibody drug conjugates in cancers, notably osteosarcomas that strongly express this surface marker (3). In addition, LRRC15 has been found to be significantly upregulated during osteogenic induction (4) and induction in mesenchymal stem cells (5). The present study explores the functional role of LRRC15 in osteosarcoma (OS) cells. Methods: We obtained the gene expression and chromatin activation profiles at the LRRC15 locus including H3K4me3, H3K27Ac, and RNA pol II and repressor marks with H3k27me3 for both high (SAOS2/HuO9) and low (HOS & U2OS) LRRC15 expressing OS cells. To delineate the role of LRRC15 in these cells, we generated inducible shRNA knockdown (KD) derivatives. We explored the cell-cell & cell-ECM interactions using cell adhesion to ECM array and migration (scratch assay), 3D colony formation (Matrigel) and spheroid formation assays. The effect of LRRC15 on intercellular communication through Gap junction (GJIC) was determined by the ‘Parachute assay’ using Dil & Calcein-AM blue as marker dyes. LRRC15 localization in spheroids were obtained using high resolution 3D imaging of the entire spheroids using light-sheet fluorescence microscopy, as well as immunohistochemical localization in cross-sections of spheroids. Results: We found a strong enrichment of activation marks around the LRRC15 locus in high LRRC15 expressing cells (SaOS2 and G292). In contrast, the LRRC15 locus in HOS and U2OS cells was enriched in H3K27Me3, suggesting epigenetic control of LRRC15 expression in this OS cell lines. LRRC15 expression and knockdown in different OS cells were confirmed by transcriptional, flow cytometric and western blot analysis. We found that expression of LRRC15 varies significantly among osteosarcoma (OS) cell-lines. We observed that LRRC15 expression can be induced with TGFβ in low expressing HOS & U2OS cells establishing the gene is still intact and poised for expression. Here we report that inducible KD of LRRC15 in high expressing OS cells significantly reduces migration, and 3D colony formation. An ECM screening array reveals that adhesion to Type 1collagen is significantly impaired when LRRC15 knocked down in high LRRC15 expressing cells (SaOS2 & HuO9). We observed compaction of spheroids over time was reduced in LRRC15 KD as well as disintegration degree of peripheral cells were much higher in high LRRC15 expressing cells. Lateral transfer of Calcein via gap junction was reduced in LRRC15 knockdown cells when compared to non-target cells. Immunolocalization through spheroid imaging and IHC revealed a significant reduction in LRRC15 in the cell surface or ECM in disintegrating LRRC15 KD spheroids. Conclusion: Taken together, these results suggest that LRRC15 plays a key role in cell-cell and cell-ECM interactions of OS cells providing insights relevant to biological function of LRRC15 in osteosarcoma cells. Reference:
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