Osteosarcoma (OS) is the most common malignant bone tumor and the majority of recurrences are due to metastasis. However, the molecular mechanisms that regulate OS metastatic spread are largely unknown. In this study, we report that special AT-rich-binding protein 2 (SATB2) is highly expressed in OS cells and tumors. Short hairpin RNA-mediated knockdown of SATB2 (sh-SATB2) decreases migration and invasion of OS cells without affecting proliferation or viability. Microarray analysis identified genes that were differentially regulated by SATB2 including the actin-binding protein Epithelial Protein Lost In Neoplasm (EPLIN), which was upregulated in sh-SATB2 cells. Silencing EPLIN rescues the decreased invasion observed in sh-SATB2 cells. Pathway analyses of SATB2-regulated genes revealed enrichment of those involved in cytoskeleton dynamics, and increased stress fiber formation was detected in cells with SATB2 knockdown. Furthermore, sh-SATB2 cells exhibit increased RhoA, decreased Rac1 and increased phosphorylation of focal adhesion kinase (FAK) and paxillin. These findings identify SATB2 as a novel regulator of OS invasion, in part via effects on EPLIN and the cytoskeleton.

Survival for high-risk neuroblastoma remains poor and treatment for relapsed disease rarely leads to long-term cures. Large sequencing studies of neuroblastoma tumors from diagnosis have not identified common targetable driver mutations other than the 10% of tumors that harbor mutations in the anaplastic lymphoma kinase (ALK) gene. However, at neuroblastoma recurrence, more frequent mutations in genes in the RAS-MAPK pathway have been detected. The PTPN11-encoded tyrosine phosphatase SHP2 is an activator of the RAS pathway, and we and others have shown that pharmacologic inhibition of SHP2 suppresses the growth of various tumor types harboring KRAS mutations such as pancreatic and lung cancers. Here we report inhibition of growth and downstream RAS-MAPK signaling in neuroblastoma cells in response to treatment with the SHP2 inhibitors SHP099, II-B08, and RMC-4550. However, neuroblastoma cell lines harboring endogenous NRAS Q61K mutation (which is commonly detected at relapse) or isogenic neuroblastoma cells engineered to overexpress NRAS Q61K were distinctly resistant to SHP2 inhibitors. Combinations of SHP2 inhibitors with other RAS pathway inhibitors such as trametinib, vemurafenib, and ulixertinib were synergistic and reversed resistance to SHP2 inhibition in neuroblastoma in vitro and in vivo. These results suggest for the first time that combination therapies targeting SHP2 and other components of the RAS-MAPK pathway may be effective against conventional therapy-resistant relapsed neuroblastoma, including those that have acquired NRAS mutations.Significance: These findings suggest that conventional therapyresistant, relapsed neuroblastoma may be effectively treated via combined inhibition of SHP2 and MEK or ERK of the RAS-MAPK pathway.

Survival for high-risk neuroblastoma remains poor. Most patients who recur, present with metastatic disease, and few targetable pathways that govern spread to distant sites are currently known. We previously developed a metastatic mouse model to select cells with enhanced ability to spread to the bone and brain and identified a signature based on differentially expressed genes, which also predicted patient survival. To discover new neuroblastoma therapies, we utilized the Connectivity Map to identify compounds that can reverse this metastatic transcriptional signature and found calcipotriol, a vitamin D3 analog, to be a compound that selectively targets cell lines with enhanced metastatic potential. Calcipotriol treatment of enhanced metastatic, but not parental, cells reduces proliferation and survival via vitamin D receptor (VDR) signaling, increases the expression of RASSF2, a negative regulator of the Hippo signaling pathway, and reduces the levels of the Hippo pathway effectors YAP and TAZ. RASSF2 is required for the effects of calcipotriol and for the reduction of levels and nuclear localization of YAP/TAZ. Migration of the enhanced metastatic cells and YAP/TAZ levels are reduced after calcipotriol treatment and YAP overexpression reduces calcipotriol sensitivity. Furthermore, metastatic cells that overexpress VDR also showed lower tumor burden in vivo. Implications: This newly identified link between VDR signaling and the Hippo pathway could inform treatment strategies for metastatic neuroblastoma.

Supplementary Figure from Vitamin D Receptor Activation Attenuates Hippo Pathway Effectors and Cell Survival in Metastatic Neuroblastoma

Supplementary Figure from Vitamin D Receptor Activation Attenuates Hippo Pathway Effectors and Cell Survival in Metastatic Neuroblastoma

<p>Figure S1 - Sensitivity of NB cell lines to allosteric SHP2 inhibitors. Figure S2 - Sensitivity of NB cell lines to catalytic SHP2 inhibitors. Figure S3 - Sensitivity to SHP2 inhibitors in isogenic NB cells with exogenous PTPN11 mutations. Figure S4 - NRAS status determines sensitivity to SHP2 and MAPK inhibition. Figure S5 - MAPK inhibitors sensitize NB cells to SHP099 and II-B08. Figure S6 - SHP2 inhibitors are synergistic with trametinib and other MAPK inhibitors. Figure S7 - MAPK inhibitors synergize with SHP2 inhibitors in ALK-mutant NB cells. Figure S8 - Assessment of drug tolerability for SHP099 and trametinib in vivo. Figure S9 - Pre-clinical assessment of SHP099 plus trametinib in NRAS-mutant NB xenografts. Figure S10 - Pre-clinical assessment of SHP099 plus ulixertinib in NRAS-mutant NB xenografts.</p>

<p>Includes supplementary methodology for cell culture, chemicals, plasmids and transfections, GTP pulldown and immunoblotting analyses, and antibodies used for study.</p>

<p>Table S1 - Genetic background of neuroblastoma cell lines.</p>