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Legends for Supplementary Figures 1 and 2

Supplementary Figure 2. MNX1 Protein Overexpression and Knockdown A. Western blot of MNX1 protein levels in two different stable clones of DU145 cells transfected with MNX1 expression vector (OE1 and OE2) versus empty vector controls. β-Actin is a loading control. B. Western blot of MNX1 protein levels in LNCaP cells and stable knockdown cells. LNCaP is untransfected LNCaP and SCR is LNCaP with scrambled vector control. shA, shB and shC are three different MNX1 shRNA transfected stable clones. Note that shB did not knockdown MNX1 and was not used for further experiments. beta-Actin is a loading control.

Simple Summary Our study identified the differential expression and potential effects of microRNAs in retinoblastoma vs. pediatric retina and advanced vs. non-advanced tumors. We provide evidence of the epithelial-mesenchymal transition (EMT) and chemoresistance programs in advanced tumors, which were potentially attributed to miR-181a-5p. We analyzed the differential expression of relevant EMT- and chemoresistance-related proteins in advanced vs. non-advanced tumors and chemotherapy-adapted Y79 cells to assess whether EMT and chemoresistance mechanisms were linked. We further examined the possible role of TGF beta as a potential regulator of such differences and highlighted the role of miR-181a-5p in EMT- and chemoresistance-related gene expression and drug sensitivity. Advanced retinoblastoma (Rb) tumors display high metastatic spread to distant tissues, causing a potent threat to vision and life. Through transcriptomic profiling, we discovered key upregulated genes that belonged to the epithelial-mesenchymal transition (EMT) and chemotherapy resistance pathways in advanced Rb tumors. Through in vitro models, we further showed that Rb null tumor cells under prolonged chemo drug exposure, acquires a metastasis-like phenotype through the EMT program mediated by ZEB1 and SNAI2 and these cells further acquires chemotherapeutic resistance through cathepsin-L- and MDR1-mediated drug efflux mechanisms. Using a miRNA microarray, we identified miR-181a-5p as being significantly reduced in advanced Rb tumors, which was associated with an altered EMT and drug-resistance genes. We showed that enhancing miR-181a-5p levels in Rb null chemo-resistant sublines reduced the ZEB1 and SNAI2 levels and halted the mesenchymal transition switch, further reducing the drug resistance. We thus identified miR-181a-5p as a therapeutically exploitable target for EMT-triggered drug-resistant cancers that halted their invasion and migration and sensitized them to low-dose chemotherapy drugs.

Lack of retinoblastoma protein (Rb) causes aggressive intraocular retinal tumors in children. Recently, Rb tumors have been shown to have a distinctly altered metabolic phenotype, such as reduced expression of glycolytic pathway proteins alongside altered pyruvate and fatty acid levels. In this study, we demonstrate that loss of Hexokinase 1(HK1) in tumor cells rewires their metabolism allowing enhanced oxidative phosphorylation-dependent energy production. We show that rescuing HK1 or RB1 in these retinoblastoma cells reduced cancer hallmarks such as proliferation, invasion, spheroid formation and increased their sensitivity to chemotherapy drugs. Induction of HK1 was accompanied by a metabolic shift of the cells to glycolysis and a reduction in mitochondrial mass. Cytoplasmic HK1 bound Liver Kinase B1 (LKB1) and phosphorylated AMP-activated kinase-α (AMPKα Thr172), thereby reducing mitochondria-dependent energy production. We validated these findings in tumor samples from Rb patients compared to age-matched healthy retina. HK1 or RB1 expression in Rb-/- cells led to reduction in their respiratory capacity and glycolytic proton flux. HK1 overexpression reduced tumor burden in an intraocular tumor xenograft model. AMPKα activation by AICAR also enhanced the tumoricidal effects of chemotherapeutic drug topotecan in vivo. Therefore, enhancing HK1 or AMPKα activity can reprogram cancer metabolism and sensitize retinoblastoma tumors to lower doses of existing treatments, a potential therapeutic modality for retinoblastoma.