Khoa Nguyen

AML-induced osteogenic differentiation in mesenchymal stromal cells supports leukemia growth

Genotypic and phenotypic alterations in the bone marrow (BM) microenvironment, in particular in osteoprogenitor cells, have been shown to support leukemogenesis. However, it is unclear how leukemia cells alter the BM microenvironment to create a hospitable niche. Here, we report that acute myeloid leukemia (AML) cells, but not normal CD34+ or CD33+ cells, induce osteogenic differentiation in mesenchymal stromal cells (MSCs). In addition, AML cells inhibited adipogenic differentiation of MSCs. Mechanistic studies identified that AML-derived BMPs activate Smad1/5 signaling to induce osteogenic differentiation in MSCs. Gene expression array analysis revealed that AML cells induce connective tissue growth factor (CTGF) expression in BM-MSCs irrespective of AML type. Overexpression of CTGF in a transgenic mouse model greatly enhanced leukemia engraftment in vivo. Together, our data suggest that AML cells induce a preosteoblast-rich niche in the BM that in turn enhances AML expansion.

IKK inhibition by BMS-345541 suppresses breast tumorigenesis and metastases by targeting GD2+ cancer stem cells

We have identified that the ganglioside GD2 is a marker for breast cancer stem cells (BCSCs), and that targeting the enzyme GD3 synthase (GD3S, which regulates GD2 biosynthesis) reduces breast tumorigenesis. The pathways regulating GD2 expression, and their anomalous functions in BCSC, are unclear. Proteomic analysis of GD2+ and GD2- cells from breast cancer cell lines revealed the activation of NFκB signaling in GD2+ cells. Dose- and time-dependent suppression of NFκB signaling by the small molecule inhibitor BMS-345541 reduced GD2+ cells by > 90%. Likewise, BMS-345541 inhibited BCSC GD3S expression, mammosphere formation, and cell migration/invasion in vitro. Breast tumor-bearing mice treated with BMS-345541 showed a statistically significant decrease in tumor volume and exhibited prolonged survival compared to control mice, with a median survival of 78 d for the BMS-345541-treated group vs. 58 d for the controls. Moreover, in an experimental metastases model, treatment with BMS-345541 reduced the lung metastases by > 5-fold. These data suggest that GD2 expression and function, and NFκB signaling, are related, and they control BCSCs tumorigenic characteristics. Thus, the suppression of NFκB signaling by BMS-345541 is a potentially important advance in controlling breast cancer growth and metastases.

Expression of ganglioside GD2, reprogram the lipid metabolism and EMT phenotype in bladder cancer

High-grade Bladder Cancer (BLCA) represents the most aggressive and treatment-resistant cancer that renders the patients with poor survival. However, only a few biomarkers have been identified for the detection and treatment of BLCA. Recent studies show that ganglioside GD2 can be used as cancer biomarker and/or therapeutic target for various cancers. Despite its potential relevance in cancer diagnosis and therapeutics, the role of GD2 is unknown in BLCA. Here, we report for the first time that high-grade BLCA tissues and cell lines have higher expression of GD2 compared to low-grade by high-resolution Mass Spectrometry. The muscle invasive UMUC3 cell line showed high GD2, mesenchymal phenotype, and cell proliferation. Besides, we have shown the cancer stem cells (CSC) property (CD44hiCD24lo) of GD2+ UMUC3 and J82 cells. Also, the evaluation of lipid metabolism in GD2+ BLCA cell lines revealed higher levels of Phosphatidylinositol (PI), Phosphatidic acid (PA), Cardiolipin (CL) and lower levels of Phosphatidylserine (PS), plasmenyl-phosphatidylethanolamines (pPE), plasmenyl-phosphocholines (pPC), sphingomyelins (SM), triglycerides (TGs) and N-Acetylneuraminic acid. These findings are significantly correlated with the tissues of BLCA patients. Based on this evidence, we propose that GD2 may be used as an effective diagnostic and therapeutic target for aggressive BLCA.

Abstract P6-02-01: Metabolic stress induces GD2 expression and cancer stem cell phenotype in triple negative breast cancer

Breast cancer stem cells (BCSCs) have been characterized as a fraction of cells in primary tumors that are drug resistant and have metastatic potential. Ganglioside GD2 has been shown by us and others as a marker for BCSCs. Furthermore, nutrient deprivation associated metabolic stress seen during tumor progression is reportedly associated with the cancer stem cell phenotype. We hypothesized that metabolic stress could induce spontaneous generation of GD2 + BCSCs during tumor progression. To test our hypothesis, we cultured breast cancer cell lines MDA-MB-231 and SUM159 at low seeding density and measured percentage and absolute number of GD2 + cells daily. Flow cytometry analysis revealed that the percentage of GD2 + cells increased from 4.5 ± 2.5 on day 2 to 15 ± 3.8% on day 5 in MDA MB-231 cells and from 8.5 ± 2.8% on day2 to 28 ± 6.2% on day 5 in SUM159 cells (both designated as triple-negative breast cancer, TNBC). To investigate this phenomenon in-vivo, we injected GFP + MDA-MB-231 cells in NSG mice mammary fat pads and examined GD2 expression in the implanted tumors weekly. Interestingly, we noticed that the percentage of GD2 + also increased from 12 ± 1.5% on week 1 to 30 ± 2.5% on week 6. Next, SUM159 cells were cultured in either nutrient rich (NR, i.e., 10% serum) or nutrient deprived (ND, 1% serum) for 4 days. We found that the percentage of GD2 + cells in NR medium at the end of 4 day culture was ~20% of the total cell population, whereas in ND medium was almost 50%. We then tested the effects of nutrient rich environment on GD2 expression by refreshing the media daily. Interestingly, cells that received fresh media had lower number of GD2 + cells (15 ± 1.5%) compared to cells cultured in the same medium for 4 days (33 ± 2.5%). Our data suggests that nutrient deprivation induces a stem cell phenotype in TNBC cells. Next, we performed global metabolic profiling (i.e., for a total of 300 biochemical metabolites) using a mass spectroscopy-based approach. We profiled SUM159 cells cultured with NR vs. ND medium (set-1); GD2 + vs GD2 - SUM159 cells (set -2); GD2 + vs GD2 - MDA-MB-231 cell (set-3). Metabolites associated with amino acid metabolism, in particular glutathione metabolism, including glutamyl-alanine, 5-oxy-proline, proline, glutamine, and glutathione itself were found to be most highly up-regulated in GD2 + compared to GD2 - cells and also in cells cultured in serum starved compared to serum rich conditions. Further analysis of these metabolites and their association with GD2 + cell signature raveled that gamma-glutamyl transferase (GGT5), was one of the most highly up-regulated (>150-fold) gene across all the groups. GGT is expressed on cell surface and transfers glutamyl group to amino acids, which then get transported across the membrane. In cancer, cells expressing GGT has been shown to be resistant to chemotherapeutic agents including cisplatin. Targeting glutathione metabolism could be future therapeutic strategy to inhibit BCSC growth in TNBC. Citation Format: Battula VL, Piyaranthna B, Nguyen K, Sun JC, Jin F, Coarfa C, Nagireddy P, Andreeff M. Metabolic stress induces GD2 expression and cancer stem cell phenotype in triple negative breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-02-01.

Quantitative mass spectrometry to interrogate proteomic heterogeneity in metastatic lung adenocarcinoma and validate a novel somatic mutation CDK12-G879V

Lung cancer is the leading cause of cancer death both in men and women. Tumor heterogeneity is an impediment to targeted treatment of all cancers, including lung cancer. Here, we sought to characterize changes in tumor proteome and phosphoproteome by longitudinal, prospective collection of tumor tissue of an exceptional responder lung adenocarcinoma patient who survived with metastatic lung adenocarcinoma for more than seven years with HER2-directed therapy in combination with chemotherapy. We employed “Super-SILAC” and TMT labeling strategies to quantify the proteome and phosphoproteome of a lung metastatic site and ten different metastatic progressive lymph nodes collected across a span of seven years, including five lymph nodes procured at autopsy. We identified specific signaling networks enriched in lung compared to the lymph node metastatic sites. We correlated the changes in protein abundance with changes in copy number alteration (CNA) and transcript expression. To further interrogate the mass spectrometry data, patient-specific database was built incorporating all the somatic variants identified by whole genome sequencing (WGS) of genomic DNA from the lung, one lymph node metastatic site and blood. An extensive validation pipeline was built for confirmation of variant peptides. We validated 360 spectra corresponding to 55 germline and 6 somatic variant peptides. Targeted MRM assays demonstrated expression of two novel variant somatic peptides, CDK12-G879V and FASN-R1439Q, with expression in lung and lymph node metastatic sites, respectively. CDK12 G879V mutation likely results in a nonfunctional CDK12 kinase and chemotherapy susceptibility in lung metastatic sites. Knockdown of CDK12 in lung adenocarcinoma cells results in increased chemotherapy sensitivity, explaining the complete resolution of the lung metastatic sites in this patient.

Abstract 2875: Knockout of ST8SIA1 inhibits tumorigenesis in triple negative breast cancer by inducing PTEN and ganglioside GM1 mediated tumor growth arrest

We found that ganglioside GD2 is a biomarker for breast cancer stem cell (BCSCs) in triple negative breast cancer (TNBC) and that GD2 biosynthesis is tightly regulated by enzyme ST8SIA1 (GD3 synthase) in these cells. We have reported that ST8SIA1 is highly expressed in TNBC and its expression is highly correlated with TP53 mutations in primary tumors (based on TCGA data set). Here we hypothesized that ST8SIA1 has a functional role in BCSC mediated tumorigenesis in TNBC. To test this hypothesis, we deleted ST8SIA1 in SUM159 cells using CRISPR-Cas9 technology. As expected, deletion of ST8SIA1 in SUM159 cells reduced the absolute number of GD2+ cells from 17 ± 1.5% to 0.3 ± 0.1%. Although there was no significant difference in 2D cell growth, anchorage-independent growth by soft-agar and the mammosphere formation assays revealed a complete loss of colony formation in ST8SIA1-KO cells. Moreover, transplantation of ST8SIA1-KO- or Cas9 control- SUM159 cells in mammary fat pad of NSG mice revealed that ST8SIA1-KO cells failed to produce tumor formation even after a 15-wk after implantation. In addition, most of the cas9 control cell injected mice died within 4 wk after cell implantation whereas no deaths were observed in the ST8SIA1-KO cells as long as 100 d after tumor implantation. To investigate the mechanism involved in tumor growth inhibition associated with the loss of ST8SIA1 expression, we analyzed mRNA expression in ST8SIA1-KO- and Cas9 control- SUM159 cells by RNA sequencing. At p 2, we found 1502 genes down-regulated and 842 genes up-regulated in the ST8SIA1-KO-cells compared to controls. Ingenuity pathway analysis revealed that several stem cell-associated signaling pathways, including wnt, stat3, NFκB, nanog, and IL8, were down-regulated. Conversely, the potent tumor suppressor PTEN was induced in the ST8SIA1-KO-cells compared to controls. In addition, gene ontology by gene set enrichment analysis (GSEA) revealed inhibition of oncogenic signaling pathways including KRAS, p53, AKT, and RB. We have also identified a significant down-regulation of breast cancer-associated genes including BCL11A, PDGFRb, VCAM1, CXCR4, and wnt5a. Interestingly DKK1, which acts as an antagonist for wnt-β-catenin signaling, was up-regulated in the ST8SIA1-KO cells. These findings were validated by qRT-PCR, flow cytometry and western blot analysis. Ganglioside GM1 has been reported to have major in anti-tumor effect by inhibiting ERK signaling. Flow cytometry analysis revealed that ST8SIA-KO cells expressed ~10-fold higher GM1 compared to Cas9 control cells. In addition, pERK was down-regulated in ST8SIA-KO cells, suggesting a loss of tumorigenic potential of these cells. In conclusion, our data suggests that inhibition of ST8SIA1 in TNBC cells depletes BCSCs and inhibits tumorigenesis by down-regulating oncogenic pathways and up-regulation of PTEN and GM1 mediated signaling. Citation Format: Khoa Nguyen, Yuanqing Yan, Chandra Bartholomeusz, Naoto Ueno, Kim-Anh Do, Michael Andreeff, V. Lokesh Battula. Knockout of ST8SIA1 inhibits tumorigenesis in triple negative breast cancer by inducing PTEN and ganglioside GM1 mediated tumor growth arrest [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 2875. doi:10.1158/1538-7445.AM2017-2875

Abstract 2898: ST8SIA1 is up-regulated in triple negative breast cancer and its expression is positively correlated withTP53mutations and a cancer stem cell gene signature

We discovered ganglioside GD2 as a breast cancer stem cell (BCSCs) marker in triple negative breast cancer (TNBC). GD2 biosynthesis is tightly regulated by the enzyme ST8SIA1 (GD3 synthase). However, expression of ST8SIA and its association with different breast cancer sub-types is not known. Here we hypothesize that ST8SIA1 is up-regulated in TNBC or “basal-type” breast tumors and associated with stemness in primary breast tumors. To investigate ST8SIA1 expression in primary tumors, we analyzed RNAseq data from the cancer genome atlas (TCGA) data base, which includes data from 1105 primary and metastatic breast tumors as well as adjacent normal tissues. We found that ST8SIA1 expression varied widely among different breast tumors. Interestingly, “basal-type” tumors expressed the highest levels of ST8SIA1 compared to all other types of breast cancers including luminal-A or luminal-B or HER2-enriched tumors (p 2-fold higher in TP53-mutated compared to wild type tumors. The other positively correlated mutation was a nuclear envelop protein called the spectrin repeat containing protein (nuclear envelope 1 or SYNE1; p Citation Format: V. Lokesh Battula, Yuanqing Yan, Khoa Nguyen, Kim-Anh Do, Michael Andreeff. ST8SIA1 is up-regulated in triple negative breast cancer and its expression is positively correlated with TP53 mutations and a cancer stem cell gene signature [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 2898. doi:10.1158/1538-7445.AM2017-2898

Abstract P5-07-02: ST8SIA1 regulates tumorigenesis in triple negative breast cancer

Recurrence in breast cancer is mainly due to metastases and drug resistance in a fraction of primary tumors cells which are also known as cancer stem cells or tumor initiating cells. We found that Ganglioside GD2 identifies breast cancer stem cell (BCSCs) in triple negative breast cancer (TNBC) and that GD2 biosynthesis is tightly regulated by enzyme ST8SIA1 (GD3 synthase) in GD2+ cells. We have reported that ST8SIA1 is highly expressed in TNBC and its expression is highly correlated with p53 mutations primary tumors (Yan et al, SABCS abstract 2016). Here we hypothesize that ST8SIA1 has a functional role in BCSC mediated tumorigenesis in TNBC. To test the hypothesis, we deleted ST8SIA1 in SUM159 cells using CRISPR-Cas9 technology. As expected, deletion of ST8SIA1 in SUM159 cells reduced GD2+ cells from 17±1.5% to 0.3±0.1%. However, cell proliferation assay revealed no significant difference between ST8SIA1-KO and Cas9 control cells. In contrast, in-vitro tumorigenesis by soft-agar assays revealed a complete loss of colony formation in ST8SIA1-KO cells, whereas Cas9 control cells produced 30±10 colonies out of 1000 cells plated. To investigate tumor initiation potential, ST8SIA1-KO- or Cas9 control- SUM159 cells were transplanted in mammary fat pad of NSG mice. Cas9 control cells produced tumors within 1-2 weeks and reached the maximum allowed size by IACUC (1.5cm) within 3-4 weeks. In contrast, ST8SIA1-KO cells failed to produce any tumors even 15 weeks after injections. In addition, survival analysis by log-rank test revealed that most of the cas9 control cell injected mice died within 4 weeks after cell implantation whereas no deaths were observed in ST8SIA1-KO cells injected mice even 100 days after tumor implantation. These data indicate that loss of ST8SIA1 in TNBC cells depletes GD2+ BCSCs and inhibits in-vitro and in-vivo tumorigenesis. To investigate gene expression changes due to loss of ST8SIA1 in CRISPR knockout cells, we analyzed mRNA expression in ST8SIA1-KO- and Cas9 control- SUM159 cells by RNAseq analysis (done in triplicates for each cell type). At p 2, we found 1502 genes down-regulated and 842 genes up-regulated in ST8SIA1-KO- compared to cas9 control- cells. Ingenuity pathway analysis revealed that several stem cell associated signaling pathway including, wnt, stat3, NFκB, nanog and IL8 whereas tumor suppressor PTEN and p38 MAPK signaling were activated in ST8SIA1-KO- compared to cas9 control- cells. In specific, proteins associated with stem cell function including NOTCH3, PDGFRB, PDGFRA, VCAM1, CXCR4, CXCL12, SOX2, wnt5a were down regulated in ST8SIA1-KO cells whereas DKK1 which acts as an antagonist for wnt-β-catenin signaling, was up-regulated in ST8SIA1-KO cells. These findings were validated by flow cytometry and western blot analysis using specific antibodies. In conclusion, our data suggests that deletion of ST8SIA1 in TNBC cells depletes BCSCs and inhibits tumorigenesis in-vitro and in-vivo. Development of specific inhibitors of ST8SIA1 could be of potential therapeutic value for patients with TNBC. Citation Format: Battula VL, Nguyen K, Sun JC, Dasgupta A, Bartholomeusz C, Andreeff M. ST8SIA1 regulates tumorigenesis in triple negative breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P5-07-02.

Abstract P5-07-03: GD2-mediated FAK signaling regulates breast cancer stem cell function in triple negative breast cancer

Ganglioside GD2 identifies breast cancer stem cells (BCSCs, Battula et al., JCI, 2012) and expression of GD2 is tightly regulated by GD3 synthase (GD3S). GD3S is highly expressed in GD2 + cells and inhibition of GD3S inhibits tumor formation and metastasis of breast cancer cells. However, the mechanism of GD2-mediated regulation of BCSC function is not known. Here we hypothesize that GD2 regulates signaling pathways involved in cell adhesion, migration and invasion of breast cancer cells. To identify these signaling pathways, antibody micro-arrays were used with 850 validated antibodies specific to total or phosphorylated proteins. Interestingly, focal adhesion kinase (FAK) was the most significantly phosphorylated protein in GD2 + compared to GD2 - cells (S910 and S722). In addition, expression of FAK downstream mediators including Csk, PKCq, PKCl/I, Pyk2, and p38MAPK, was up-regulated in GD2 + compared to GD2 - cells. Western blot analysis of FACS sorted SUM159 cells also revealed increased phosphorylation of FAK >80% at Y397 and >25% at Y861 in GD2 + compared to GD2 - cells. FAK downstream targets including paxillin, p130 Cas, pERK were also up-regulated in GD2 + cells compared to GD2 - cells indicating definitive activation of FAK signaling in GD2 + BCSCs. To investigate the functional role of GD2 in FAK mediated functions, we genetically deleted GD3S using the CRISPR knock-out system in SUM159 cells. only 20% in parental cells was observed. GD3S-KO cells grew 5-10% slower in cell culture mostly because of the reduction (15±5%) in adherence. Trans-well assays revealed 3-5 fold reduction in migration and invasion of GD3S-KO compared to parental cells. These data indicate that GD2 and GD3S are not only the markers of BCSCs but also regulators of their function. Finally, we tested the effect of FAK inhibitor (PF-573228) against GD2 + BCSCs and GD3S-KO SUM159 cells. PF-573228 treatment decreased the number of mammospheres generated by GD2 + cells 3-4 fold in a dose dependent manner (100nM-1µm). In addition, treatment of PF-573228, inhibited migration and invasion of GD2 + cells 2 and 3 fold, respectively. However, treatment with PF-573228 on GD3S-KO cells further reduced their ability to migrate and invade by over 70% compared to untreated cells. In addition, GD3S-KO cells failed to form any mammospheres when cultured under low adherence conditions (p + cells but that FAK inhibition alone may not be sufficient to inhibit BCSC function. Combined FAK and GD3S inhibition may exert highly synergistic effects against BCSCs. Citation Format: Nguyen K, Sun JC, Hortobagyi GN, Andreeff M, Battula VL. GD2-mediated FAK signaling regulates breast cancer stem cell function in triple negative breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P5-07-03.

Abstract P6-07-26: ST8SIA1 is over-expressed in triple negative breast cancer and associated with p53 mutations

Cancer stem cells constitute a small fraction of cells present in primary tumors, are highly tumorigenic, drug resistant and cause metastases. We discovered Ganglioside GD2 as a breast cancer stem cell (BCSCs) marker in triple negative breast cancer (TNBC). GD2 biosynthesis is tightly regulated by the enzyme ST8SIA1 (GD3 synthase). However, expression of ST8SIA and its association with different breast cancer sub-types is not known. Here we hypothesize that ST8SIA1 is up-regulated in TNBC or basal-type breast tumors and associated with stemness in primary breast tumors. Frist, we examined ST8SIA1 protein expression in breast cancer cell lines (n=12) and found up-regulation of ST8SIA1 in basal compared to luminal-type breast cancer cells. To investigate ST8SIA19s expression in primary tumors, we analyzed RNAseq data from the cancer genome atlas (TCGA) data base which has expression data from 1105 primary and metastatic breast tumors as well as adjacent normal tissues. We found that ST8SIA1 expression varies widely among different breast tumors and that 9basal9 type tumors express highest levels of ST8SIA1 compared to all other types of breast cancers including luminal-A or luminal-B or HER2 enriched (p To investigate the association between ST8SIA1expression and the most commonly mutated genes in breast cancer, we chose the top 20 most frequently mutated genes in TCGA dataset and examined their correlation with ST8SIA1 mRNA expression. We found that, among the top 20 mutations, p53 has a very strong positive correlation with ST8SIA1 expression (p 2 fold higher in p53 mutated tumors compared to p53 wild type tumors. The other positively correlated mutation was a nuclear envelop protein called Spectrin Repeat Containing, Nuclear Envelope 1 (SYNE1; p Citation Format: Yan Y, Nguyen K, Do K-A, Ueno N, Andreeff M, Battula VL. ST8SIA1 is over-expressed in triple negative breast cancer and associated with p53 mutations [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-07-26.