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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.

Incidence and mortality rates for prostate cancer are higher in African-American (AA) men than in European-American (EA) men, but the biologic basis for this disparity is unclear. We carried out a detailed analysis of gene expression changes in prostate cancer compared with their matched benign tissues in a cohort of AA men and compared them with existing data from EA men. In this manner, we identified MNX1 as a novel oncogene upregulated to a relatively greater degree in prostate cancer from AA men. Androgen and AKT signaling play a central role in the pathogenesis of prostate cancer and we found that both of these signaling pathways increased MNX1 expression. MNX1 in turn upregulated lipid synthesis by stimulating expression of SREBP1 and fatty acid synthetase. Our results define MNX1 as a novel targetable oncogene increased in AA prostate cancer that is associated with aggressive disease. Cancer Res; 76(21); 6290–8. ©2016 AACR.

Sclerotium rolfsii lectin (SRL) is a lectin isolated from fungus S. rolfsii and has high binding specificity toward the oncofetal Thomsen-Friedenreich carbohydrate antigen (Galβ1-3GalNAc-α-O-Ser/Thr, T or TF), which is expressed in more than 90% of human cancers. Our previous studies have shown that binding of SRL to human colon, breast and ovarian cancer cells induces cell apoptosis in vitro and suppresses tumor growth in vivo. This study investigated the SRL-mediated cell signaling in human colon cancer HT29 cells by mRNA and miRNA microarrays. It was found that SRL treatment results in altered expression of several hundred molecules including mitogen-activated protein kinase (MAPK) and c-JUN-associated, apoptosis-associated and cell cycle and DNA replication-associated signaling molecules. Pathway analysis using GeneSpring 12.6.1 revealed that SRL treatment induces changes of MAPK and c-JUN-associated signaling pathways as early as 2 h while changes of cell cycle, DNA replication and apoptosis pathways were significantly affected only after 24 h. A significant change of cell miRNA expression was also observed after 12 h treatment of the cells with SRL. These changes were further validated by quantitative real time polymerase chain reaction and immunoblotting. This study thus suggests that the presence of SRL affects multiple signaling pathways in cancer cells with early effects on cell proliferation pathways associated with MAPK and c-JUN, followed by miRNA-associated cell activity and apoptosis. This provides insight information into the molecular mechanism of the anticancer activity of this fungal lectin.

African American (AA) men have an approximately 60% higher incidence of prostate cancer (PCa) and have about two times greater risk of dying of the disease than their European American (EA) counterparts. Despite this staggeringly unequal burden of PCa incidence and outcome between AA and EA men, there is limited insight into the molecular mechanisms associated with this racial disparity. Using state-of-the-art mass spectrometry platform, we uncovered the very first metabolic and lipidomics landscape in PCa/benign adjacent tissue pairs and paired plasma and urine containing epidemiologic meta-data from ancestry-typed AA and EA men. A total of 190 polar and mid-polar metabolites and 495 lipids were measured in tissues and 183 metabolites and lipids were examined in the plasma and urine samples. AA PCa tissue and plasma signatures had unique alterations in metabolites and key enzymes associated with the methionine-homocysteine pathway, including adenosine and inosine levels, compared to corresponding tissues and plasma from EA case controls and EA PCa tissues. Intriguingly, methionine levels in AA PCa were also portrayed by distinct dietary practices in these patients. In addition, AA PCa tissues demonstrated unique patterns of bioenergetic metabolites and accumulated lipids reflecting impaired mitochondrial activity and TCA cycle. Further characterization of these first-in-the-field findings demonstrating reprogrammed metabolism in AA PCa relative to EA tumors could reveal insights into the biologic basis of PCa disparities and novel areas for therapeutic intervention. Citation Format: Stacy M. Lloyd*, Jie H. Gohlke*, Sumanta Basu*, Vasanta Putluri,Shaiju K Vareed, Rebeca San Martin, Thekkelnaycke Rajendiran, Tiffany A. Dorsey, Bandana Prasad, Rajni Sonavane, Uttam Rasaily, James Henderson, Balasubramanyam Karanam, Harene Venghatakrishnan, Salil Bhowmik, Alexander Zaslavsky,Nilanjan Guha, Rick Kittles, Stefan Ambs, Michael Ittmann,David Rowley, Ganesh Palapattu, Nagireddy Putluri,George Michailidis, Arun Sreekumar. Metabolomic landscape of African American prostate cancer: Insights into the biologic basis of the racial disparity [abstract]. In: Proceedings of the Tenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2017 Sep 25-28; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2018;27(7 Suppl):Abstract nr B92.

High-fidelity chromosome segregation during mitosis requires kinetochores, protein complexes that assemble on centromeric DNA and mediate chromosome attachment to spindle microtubules. In budding yeast, phosphoinositide-specific phospholipase C (Plc1p encoded by PLC1 gene) is important for function of kinetochores. Deletion of PLC1 results in alterations in chromatin structure of centromeres, reduced binding of microtubules to minichromosomes, and a higher frequency of chromosome loss. The mechanism of Plc1p's involvement in kinetochore activity was not initially obvious; however, a testable hypothesis emerged with the discovery of the role of inositol polyphosphates (InsPs), produced by a Plc1p-dependent pathway, in the regulation of chromatin-remodeling complexes. In addition, the remodels structure of chromatin (RSC) chromatin-remodeling complex was found to associate with kinetochores and to affect centromeric chromatin structure. We report here that Plc1p and InsPs are required for recruitment of the RSC complex to kinetochores, which is important for establishing proper chromatin structure of centromeres and centromere proximal regions. Mutations in PLC1 and components of the RSC complex exhibit strong genetic interactions and display synthetic growth defect, altered nuclear morphology, and higher frequency of minichromosome loss. The results thus provide a mechanistic explanation for the previously elusive role of Plc1p and InsPs in kinetochore function.

Retinoblastoma is a pediatric eye cancer affecting children usually less than five years of age. Inactivation of both the copies of the RB1 gene in a child's retina initiates cancer formation. Whil...

Metabolic dysfunction or reprogramming is the underlying cause of many diseases including cancer. In this study, we looked into the transcriptomic and metabolomic profile of retinoblastoma (Rb), a pediatric eye cancer, from tumor and normal tissue, aqueous humor, vitreous humor and tear. The samples were collected from enucleated eyes of 9 subjects and 2 deceased controls, whose cause of death is not due to any eye related disease. The study was corroborated with cell line models of retinoblastoma, used for metabolic phenotyping. Combined pathway analysis of transcriptomics data from subject tissue samples and retinoblastoma cell line WERI-Rb-1 revealed a significant overlap in the differentially regulated genes belonging to both glycolysis and OXPHOS pathways. The results were correlated with Rb1 overexpressed and its respective control also. To better understand the implications of these two major pathways in the progression of retinoblastoma, metabolic study using Seahorse analyzer enables measurement of extracellular flux (oxygen consumption rate and extracellular acidification rate) to provide insight into mitochondrial function and glycolytic activity. Using a pharmacological approach, the contribution of glucose, glutamine and fatty acid oxidation pathways can be assessed. Overall the study demonstrates the value of using a multi-omics approach with functional metabolic profiling to understand the extent of metabolic dysfunction in retinoblastoma and also to identify implications of various biological pathways in Rb. Citation Format: Nilanjan Guha, Carolina Livi, Vishnu Suresh Babu, Lisa Winer, Deepak SA, Syed Lateef, Seetaraman Gundimeda, Arunkumar Padmanabhan, Brian Dranka, Arkasubhra Ghosh. Understanding metabolic dysfunction in retinoblastoma development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2515. doi:10.1158/1538-7445.AM2017-2515

Background: AA men with PCa will die at a rate nearly 2.5 times higher than their EA counterparts. Presently, no mechanism has been described to explain the differences observed between AA and EA men with PCa; yet, many have interrogated the role of social and anthropometric data on PCa outcomes within these populations. Therefore, to obtain better insights into the molecular aspects of PCa disparities, we examined metabolomic and gene expression profiles of PCa and matched adjacent benign tissue from AA and EA men. Metabolomics is defined as the study of all the small molecule metabolites produced by cellular processes in the body. We hypothesize that racially distinct metabolic pathways may contribute to PCa health disparities and also delineate mechanistic biological pathways. Methodology: A total of 190 polar and mid-polar metabolites were measured using mass spectrometry across 50 and 28 PCa/benign tissue pairs from AA and EA men, respectively. Likewise, gene expression microarray analysis was performed on 48 and 21 PCa/benign tissue pairs from AA and EA men, and gene set enrichment (GSEA) analysis was performed. Ancestry informative markers were genotyped and ancestry estimates were determined. Metabolic profiles of AA and EA PCa and benign adjacent pairs were compared using paired t-tests. FDR corrected p-values were used to detect differential metabolites and genes. Biological validation was performed using tissue microarrays, qPCR, and western blots. Results: Unique biochemical alterations associated with AA tumors were identified. Relative to EA tumors, AA tumors had significant alterations in the cysteine/methionine pathway; specifically accumulations of adenosine were most prominent in AA PCa, as well as urine. Protein and transcript levels of Adenosine Deaminase (ADA), an enzyme that converts SAM-derived adenosine to inosine was significantly down regulated in AA PCa, a finding validated in both tissue and cell lines. GSEA analysis revealed that genes involved in the immune system were prominent among AA PCa compared to EA PCa. To this end, significant inverse correlations between ADA and CD4+, CD8+, and CD68+ levels were also identified. Conclusions: Taken together, this integromics approach generated data that alludes to the existence of an efficient immune escape mechanism in AA PCa, which may explain the disparity facing AA men with PCa. Citation Format: Stacy M. Lloyd, Jie Gohlke, Sumanta Basu, Salil Bhowmik, Vasanta Putluri, Kimal Rajapakshe, Cristian Coarfa, Michael Ittmann, Ganesh Palapattu, Nagireddy Putluri, George Michailidis, Arun Sreekumar. An Integromics Approach Identifies Immune Escape as a Potential Mechanism for Prostate Cancer Disparities. [abstract]. In: Proceedings of the Ninth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2016 Sep 25-28; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2017;26(2 Suppl):Abstract nr PR10.

Sclerotiumrolfsii, a soil borne plant pathogenic fungus secrete a developmental-stage specific lectin (SRL) which displays strong binding to TF antigen and its derivatives. Histochemical studies re...

Background: AA men with PCa will die at a rate nearly 2.5 times higher than their EA counterparts. Presently, no mechanism has been described to explain the differences observed between AA and EA men with PCa; yet, many have interrogated the role of social and anthropometric data on PCa outcomes within these populations. However, when these factors are statistically controlled, the differences in incidence, morbidity, and mortality between AA and EA PCa persist. These findings support the notion that biological factors are one of the major driving forces of PCa disparities. Therefore, to obtain better insights into the molecular aspects of PCa disparities, we examined metabolomic and gene expression profiles of PCa and matched adjacent benign tissue from AA and EA men. Metabolomics is defined as the study of all the small molecule metabolites produced by cellular processes in the body. We hypothesize that racially distinct metabolic pathways and the associated bioprocesses, may contribute to PCa health disparities Methodology: A total of 190 polar and mid-polar metabolites were measured using mass spectrometry across 50 and 28 PCa/benign tissue pairs from AA and EA men, respectively. Likewise, gene expression microarray analysis was performed on 48 and 21 PCa/benign tissue pairs from AA and EA men. Ancestry informative markers were genotyped and ancestry estimates were determined. Metabolic profiles of AA and EA PCa and benign adjacent pairs were compared using paired t-tests. FDR corrected p-values were used to detect differential metabolites and genes. Results: Unique biochemical alterations associated with AA tumors were identified. Pathways such as cysteine/methionine, arginine/proline and de novo purine biosynthesis were enhanced in AA PCa. Metabolites such as cystathione, cysteine, and S-Adenosylhomocysteine (SAH), are elevated in AA PCa compared to matched adjacent benign. Consistent with this, transcript and protein levels of DNA Methyl Transferase 1 (DNMT1) and Cystathione Beta Synthase (CBS) were elevated in AA PCa tissues and cell lines. Interestingly, protein and transcript levels of Adenosine Deaminase, an enzyme that converts SAM-derived adenosine to inosine was significantly down regulated in AA PCa, both in tissues and cell lines. Consistent with this, accumulated levels of adenosine were found in AA PCa. Conclusions: Taken together, the metabolomics data alludes to the existence of an efficient immune escape mechanism in AA PCa. Corroborating this, analysis of gene expression data from AA men with PCa also reveals enriched immune escape pathways. Citation Format: Stacy M. Lloyd, Jie Gohlke, Sumanta Basu, Salil Bhowmik, Vasanta Putluri, Rashmi Krishnapurnam, Kimal Rajapakshe, Cristian Coarfa, Nilanjan Guha, SA Deepak, Arunkumar Padmanabhan, Mohammed Sayeeduddin, Patricia Castro, Michael Ittmann, Ganesh Palapattu, Nagireddy Putluri, George Michailidis, Arun Sreekumar. An integromics approach identifies immune escape as a potential mechanism for prostate cancer disparities. [abstract]. In: Proceedings of the Eighth AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 13-16, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2016;25(3 Suppl):Abstract nr A65.

Phosphatidylinositol phosphates are involved in signal transduction, cytoskeletal organization, and membrane trafficking. Inositol polyphosphates, produced from phosphatidylinositol phosphates by the phospholipase C-dependent pathway, regulate chromatin remodeling. We used genome-wide expression analysis to further investigate the roles of Plc1p (phosphoinositide-specific phospholipase C in Saccharomyces cerevisiae ) and inositol polyphosphates in transcriptional regulation. Plc1p contributes to the regulation of approximately 2% of yeast genes in cells grown in rich medium. Most of these genes are induced by nutrient limitation and other environmental stresses and are derepressed in plc1 Δ cells. Surprisingly, genes regulated by Plc1p do not correlate with gene sets regulated by Swi/Snf or RSC chromatin remodeling complexes but show correlation with genes controlled by Msn2p. Our results suggest that the increased expression of stress-responsive genes in plc1 Δ cells is mediated by decreased cyclic AMP synthesis and protein kinase A (PKA)-mediated phosphorylation of Msn2p and increased binding of Msn2p to stress-responsive promoters. Accordingly, plc1 Δ cells display other phenotypes characteristic of cells with decreased PKA activity. Our results are consistent with a model in which Plc1p acts together with the membrane receptor Gpr1p and associated G α protein Gpa2p in a pathway separate from Ras1p/Ras2p and converging on PKA.

In Saccharomyces cerevisiae, many osmotically inducible genes are regulated by the Sko1p-Ssn6p-Tup1p complex. On osmotic shock, the MAP kinase Hog1p associates with this complex, phosphorylates Sko1p, and converts it into an activator that subsequently recruits Swi/Snf and SAGA complexes. We have found that phospholipase C (Plc1p encoded by PLC1) is required for derepression of Sko1p-Ssn6p-Tup1p–controlled osmoinducible genes upon osmotic shock. Although plc1Δ mutation affects the assembly of the preinitiation complex after osmotic shock, it does not affect the recruitment of Hog1p and Swi/Snf complex at these promoters. However, Plc1p facilitates osmotic shock–induced recruitment of the SAGA complex. Like plc1Δ cells, SAGA mutants are osmosensitive and display compromised expression of osmotically inducible genes. The reduced binding of SAGA to Sko1p-Ssn6p-Tup1p–repressed promoters in plc1Δ cells does not correlate with reduced histone acetylation. However, SAGA functions at these promoters to facilitate recruitment of the TATA-binding protein. The results thus provide evidence that Plc1p and inositol polyphosphates affect derepression of Sko1p-Ssn6p-Tup1p–controlled genes by a mechanism that involves recruitment of the SAGA complex and TATA-binding protein.

This study illustrates a multi-omics approach combining transcriptomics and metabolomics datasets to study molecular events leading to progression of retinoblastoma. Retinoblastoma is a pediatric ocular cancer affecting children usually less than five years of age. It is a complex disease predisposed primarily by biallelic inactivating mutations in the RB1 gene. This gene has essential roles in cell cycle, differentiation, chromosome stability and is implicated in other functions. From a cohort of 9 patients undergoing enucleation of the affected eyes, we obtained tumor, aqueous humor, vitreous humor and tear samples. We obtained retina, aqueous humor and vitreous humor from enucleated eyes of 2 deceased pediatric controls, whose cause of death is not due to any eye related disease. The patients comprise of both high and low clinical and pathological risks. In the first stage of the study, we performed mRNA and miRNA gene expression using microarrays followed by pathway analysis to identify gene enrichment that would enable functional characterization of tumors. Differential expression analysis was carried out using moderated t-test with Benjamini Hochberg multiple testing correction. 108 (p≤0.05, fold change≥10) genes were found to be unique to patients possessing high risk of metastasis. Pathway analysis revealed key pathways that are known to be involved in the progression of retinoblastoma including cell cycle and Rap1 signaling pathway. Many of these responders were verified by RT-PCR and corroborated by immuno histochemistry studies. The study also revealed 18 novel miRNAs which had not been previously implicated in the disease, with a significant overlap of the miRNA target list with our mRNA expression data set. Metabolomics studies were performed using monophasic solvent extraction of aqueous, vitreous and tear samples. The extracted metabolites were analyzed on Accurate Mass QTOF mass spectrometer in positive and negative mode on a reverse phase C18 and HILIC columns. Data dependent MS/MS analysis was performed to confirm the compounds. Pathway analysis of the differentially expressed metabolites revealed enrichment of several pathways, including nucleotide metabolism and amino acids among others. Combined pathway analysis of metabolomics and transcriptomics data was performed in order to gain an understanding of the interrelationship between changes in gene expression and metabolomic profile. The results show overlap of key cellular pathways which can be mechanistically linked to disease progression. The study provide new biological insights that are made accessible by combining data from different biological and biochemical domains with a comprehensive integrated method. The information is useful not only to correlate expression markers with disease mechanism but also to better predict appropriate chemotherapy regimens and identify new mechanisms to treat even advanced stages of retinoblastoma. Citation Format: Nilanjan Guha, Deepak SA, Syed Lateef, Seetaraman Gundimeda, Arunkumar Padmanabhan, Carolina B Livi, Nigel Skinner, Arkasubhra Ghosh, Ashwin Mallipatna, Vishnu Suresh Babu, Arun Sreekumar. An integrated approach to identify functional signaling modules in retinoblastoma cancer progression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3753. doi:10.1158/1538-7445.AM2015-3753

The purpose of this investigation was to determine how polymyxin B stimulates the activity of mitochondrial glycerophosphate acyltransferase. Polymyxin B did not change the integrity of the mitochondrial outer membrane as judged by testing the latency (>80%) of cytochrome oxidase activity. The stimulation totally disappeared when polymyxin B-treated mitochondria were washed. The FA side chain in polymyxin B was unnecessary for stimulation, as the nonapeptide was as effective as the whole antibiotic. The stimulation by polymyxin B or the nonapeptide was observed only in the presence of BSA. Cytochrome c, when added to the incubation medium instead of albumin, did not stimulate the mitochondrial enzyme, but did produce a stimulatory effect of polymyxin B on the mitochondrial acyltransferase. As reported earlier for the bacterial and microsomal acyltransferase, other polycationic compounds such as spermine and spermidine stimulated mitochondrial glycerophosphate acyltransferase. The stimulation of the mitochondrial acyltransferase by spermine and spermidine also occurred only in the presence of BSA. The analysis of the products of esterification demonstrated the presence of more lysophosphatidic acid (LPA) in the polymyxin B- and polyamine-stimulated assays in comparison to their respective control. Furthermore, in comparison to the albumin-treated control, there was 60% more LPA present in the assay supernatant fractions of polymyxin B-treated samples. Our results suggest that polymyxin B stimulates the mitochondrial glycerophosphate acyltransferase activity by enhancing the extraction of more LPA from the mitochondria to the supernatant fraction.