Search

Pathological and Clinical Characteristics of Patients with Adrenocortical Carcinoma in the Training Set, Pathological and Clinical Characteristics of Patients with Adrenocortical Carcinoma in the Validation Set, Summary Findings of Features, Features Identified by Phase of Chromatography and Ionization

(A) MS-MS confirmation of identity of creatine riboside in comparison to commercial available and synthesized standards. (B) MS-MS confirmation of identity of L-tryptophan in comparison to commercial available and synthesized standards. (C) MS-MS confirmation of identity of Nε, Nε, Nε-trimethyllysine in comparison to commercial available and synthesized standards. (D) MS-MS confirmation of identity of 3-methylhistidine in comparison to commercial available and synthesized standards.

PCA plots showing discrimination between patients with mitotane ingestion (blue) and without mitotane ingestion (red) in reverse phase (RP) in both (A) ESI and (B) ESI- mode. Supervised OPLS-DA plots showing differences between the two groups in both (C) ESI and (D) ESI- mode. S-plot correlative analysis showing significant variables not correlated with patients with mitotane ingestion (highlighted in red) in both (E) ESI and (F) ESI- mode.

(A) Multivariate ROC curve analysis showing AUC with the combination of validated markers including creatine riboside and the four unidentified novel biomarkers. (B) Multivariate ROC curve analysis showing AUC with the combination creatine riboside and size of tumor.

Figure 3

Supplementary figures and tables

IMPACT II

PCA plots showing discrimination between patients with mitotane ingestion (blue) and without mitotane ingestion (red) in HILIC in both (A) ESI and (B) ESI- mode. Supervised OPLS-DA plots showing differences between the two groups in both (C) ESI and (D) ESI- mode. S-plot correlative analysis showing significant variables not correlated with patients with mitotane ingestion (highlighted in red) in both (E) ESI and (F) ESI- mode.

TCGA normal vs tumor raw data

Purpose: Adrenocortical carcinoma (ACC) is a rare and aggressive cancer, and no current effective therapy is available for locally advanced and metastatic ACC. Drug repurposing is an emerging approach for identifying new indications for existing drugs, especially for rare cancers such as ACC. The objective of this study was to use quantitative high-throughput screening to identify agents with antineoplastic activity against ACC.Experimental Design: A screening of 4,292 compounds was performed on three ACC cell lines: BD140A, SW-13, and NCI-H295R.Results: Twenty-one active compounds were identified, with an efficacy of >80% in all three cell lines. Of these, niclosamide showed higher efficacy and lower IC50 than established anti-ACC drugs. We then validated niclosamide-inhibited cellular proliferation in all three ACC cell lines. Next, we investigated the mechanism by which niclosamide inhibited ACC cell proliferation, and found that it induced caspase-dependent apoptosis and G1 cell-cycle arrest. Niclosamide also decreased cellular migration and reduced the level of mediators of epithelial-to-mesenchymal transition, such as N-cadherin and vimentin. Furthermore, niclosamide treatment resulted in decreased expression of β-catenin. We also evaluated the effect of niclosamide on energy metabolism in ACC cell lines and found it resulted in mitochondrial uncoupling. Niclosamide treatment inhibited ACC tumor growth with no observed toxicity in mice in vivo.Conclusions: Our findings suggest that niclosamide has anti-ACC activity through its inhibition of multiple altered cellular pathways and cellular metabolism in ACC. Our results provide a preclinical rationale for evaluating niclosamide therapy in a clinical trial for ACC. Clin Cancer Res; 22(14); 3458–66. ©2016 AACR.

Purpose: Adrenal incidentalomas must be differentiated from adrenocortical cancer (ACC). Currently, size, growth, and imaging characteristics determine the potential for malignancy but are imperfect. The aim was to evaluate whether urinary small molecules (Experimental Design: Preoperative fasting urine specimens from patients with ACC (n = 19) and benign adrenal tumors (n = 46) were analyzed by unbiased ultraperformance liquid chromatography/mass spectrometry. Creatinine-normalized features were analyzed by Progenesis, SIMCA, and unpaired t test adjusted by FDR. Features with an AUC >0.8 were identified through fragmentation patterns and database searches. All lead features were assessed in an independent set from patients with ACC (n = 11) and benign adrenal tumors (n = 46) and in a subset of tissue samples from patients with ACC (n = 15) and benign adrenal tumors (n = 15) in the training set.Results: Sixty-nine features were discovered and four known metabolites identified. Urinary creatine riboside was elevated 2.1-fold (P = 0.0001) in patients with ACC. L-tryptophan, Nϵ,Nϵ,Nϵ-trimethyl-L-lysine, and 3-methylhistidine were lower 0.33-fold (P < 0.0001), 0.56-fold (P < 0.0001), and 0.33-fold (P = 0.0003) in patients with ACC, respectively. Combined multivariate analysis of the four biomarkers showed an AUC of 0.89 [sensitivity 94.7% (confidence interval {CI}, 73.9%–99.1%), specificity 82.6% (CI, 68.6%–92.2%), PPV 69.2% (CI, 48.2%–85.6%), and NPV 97.4% (CI, 86.5%–99.6%)] for distinguishing ACC from benign tumors. Of the four, creatine riboside and four unknown features were validated. Creatine riboside, Nϵ,Nϵ,Nϵ-trimethyl-L-lysine, and two unknown features were elevated in ACC tumors.Conclusions: There are unique urinary metabolic features in patients with ACC with some metabolites present in patient tumor samples. Urinary creatine riboside can differentiate benign adrenal neoplasms from ACC. Clin Cancer Res; 23(17); 5302–10. ©2017 AACR.

Purpose: Mitochondrial glycerophosphate dehydrogenase (MGPDH) is the key enzyme connecting oxidative phosphorylation (OXPHOS) and glycolysis as well as a target of the antidiabetic drug metformin in the liver. There are no data on the expression and role of MGPDH as a metformin target in cancer. In this study, we evaluated MGPDH as a potential target of metformin in thyroid cancer and investigated its contribution in thyroid cancer metabolism.Experimental Design: We analyzed MGPDH expression in 253 thyroid cancer and normal tissues by immunostaining and examined its expression and localization in thyroid cancer–derived cell lines (FTC133, BCPAP) by confocal microscopy. The effects of metformin on MGPDH expression were determined by qRT-PCR and Western blot analysis. Seahorse analyzer was utilized to assess the effects of metformin on OXPHOS and glycolysis in thyroid cancer cells. We analyzed the effects of metformin on tumor growth and MGPDH expression in metastatic thyroid cancer mouse models.Results: We show for the first time that MGPDH is overexpressed in thyroid cancer compared with normal thyroid. We demonstrate that MGPDH regulates human thyroid cancer cell growth and OXPHOS rate in vitro. Metformin treatment is associated with downregulation of MGPDH expression and inhibition of OXPHOS in thyroid cancer in vitro. Cells characterized by high MGPDH expression are more sensitive to OXPHOS-inhibitory effects of metformin in vitro and growth-inhibitory effects of metformin in vitro and in vivo.Conclusions: Our study established MGPDH as a novel regulator of thyroid cancer growth and metabolism that can be effectively targeted by metformin. Clin Cancer Res; 24(16); 4030–43. ©2018 AACR.

Supplemental Table 1 shows compounds that are pan-active in all three adrenocortical carcinoma cell lines (NCI-H295R, BD140A and SW-13).

STR PROFILE

Comparison between patients with ACC and benign adrenal disease with respect to the four unknown features that were validated: (A) m/z 334.186, (B) m/z 306.153, (C) m/z 209.030, and (D) m/z 427.231.

Purpose: Mitochondrial glycerophosphate dehydrogenase (MGPDH) is the key enzyme connecting oxidative phosphorylation (OXPHOS) and glycolysis as well as a target of the antidiabetic drug metformin in the liver. There are no data on the expression and role of MGPDH as a metformin target in cancer. In this study, we evaluated MGPDH as a potential target of metformin in thyroid cancer and investigated its contribution in thyroid cancer metabolism. Experimental Design: We analyzed MGPDH expression in 253 thyroid cancer and normal tissues by immunostaining and examined its expression and localization in thyroid cancer–derived cell lines (FTC133, BCPAP) by confocal microscopy. The effects of metformin on MGPDH expression were determined by qRT-PCR and Western blot analysis. Seahorse analyzer was utilized to assess the effects of metformin on OXPHOS and glycolysis in thyroid cancer cells. We analyzed the effects of metformin on tumor growth and MGPDH expression in metastatic thyroid cancer mouse models. Results: We show for the first time that MGPDH is overexpressed in thyroid cancer compared with normal thyroid. We demonstrate that MGPDH regulates human thyroid cancer cell growth and OXPHOS rate in vitro. Metformin treatment is associated with downregulation of MGPDH expression and inhibition of OXPHOS in thyroid cancer in vitro. Cells characterized by high MGPDH expression are more sensitive to OXPHOS-inhibitory effects of metformin in vitro and growth-inhibitory effects of metformin in vitro and in vivo. Conclusions: Our study established MGPDH as a novel regulator of thyroid cancer growth and metabolism that can be effectively targeted by metformin. Clin Cancer Res; 24(16); 4030–43. ©2018 AACR.

Purpose: Adrenal incidentalomas must be differentiated from adrenocortical cancer (ACC). Currently, size, growth, and imaging characteristics determine the potential for malignancy but are imperfect. The aim was to evaluate whether urinary small molecules ( Experimental Design: Preoperative fasting urine specimens from patients with ACC (n = 19) and benign adrenal tumors (n = 46) were analyzed by unbiased ultraperformance liquid chromatography/mass spectrometry. Creatinine-normalized features were analyzed by Progenesis, SIMCA, and unpaired t test adjusted by FDR. Features with an AUC >0.8 were identified through fragmentation patterns and database searches. All lead features were assessed in an independent set from patients with ACC (n = 11) and benign adrenal tumors (n = 46) and in a subset of tissue samples from patients with ACC (n = 15) and benign adrenal tumors (n = 15) in the training set. Results: Sixty-nine features were discovered and four known metabolites identified. Urinary creatine riboside was elevated 2.1-fold (P = 0.0001) in patients with ACC. L-tryptophan, Nϵ,Nϵ,Nϵ-trimethyl-L-lysine, and 3-methylhistidine were lower 0.33-fold (P < 0.0001), 0.56-fold (P < 0.0001), and 0.33-fold (P = 0.0003) in patients with ACC, respectively. Combined multivariate analysis of the four biomarkers showed an AUC of 0.89 [sensitivity 94.7% (confidence interval {CI}, 73.9%–99.1%), specificity 82.6% (CI, 68.6%–92.2%), PPV 69.2% (CI, 48.2%–85.6%), and NPV 97.4% (CI, 86.5%–99.6%)] for distinguishing ACC from benign tumors. Of the four, creatine riboside and four unknown features were validated. Creatine riboside, Nϵ,Nϵ,Nϵ-trimethyl-L-lysine, and two unknown features were elevated in ACC tumors. Conclusions: There are unique urinary metabolic features in patients with ACC with some metabolites present in patient tumor samples. Urinary creatine riboside can differentiate benign adrenal neoplasms from ACC. Clin Cancer Res; 23(17); 5302–10. ©2017 AACR.

Purpose: Adrenocortical carcinoma (ACC) is a rare and aggressive cancer, and no current effective therapy is available for locally advanced and metastatic ACC. Drug repurposing is an emerging approach for identifying new indications for existing drugs, especially for rare cancers such as ACC. The objective of this study was to use quantitative high-throughput screening to identify agents with antineoplastic activity against ACC. Experimental Design: A screening of 4,292 compounds was performed on three ACC cell lines: BD140A, SW-13, and NCI-H295R. Results: Twenty-one active compounds were identified, with an efficacy of >80% in all three cell lines. Of these, niclosamide showed higher efficacy and lower IC50 than established anti-ACC drugs. We then validated niclosamide-inhibited cellular proliferation in all three ACC cell lines. Next, we investigated the mechanism by which niclosamide inhibited ACC cell proliferation, and found that it induced caspase-dependent apoptosis and G1 cell-cycle arrest. Niclosamide also decreased cellular migration and reduced the level of mediators of epithelial-to-mesenchymal transition, such as N-cadherin and vimentin. Furthermore, niclosamide treatment resulted in decreased expression of β-catenin. We also evaluated the effect of niclosamide on energy metabolism in ACC cell lines and found it resulted in mitochondrial uncoupling. Niclosamide treatment inhibited ACC tumor growth with no observed toxicity in mice in vivo. Conclusions: Our findings suggest that niclosamide has anti-ACC activity through its inhibition of multiple altered cellular pathways and cellular metabolism in ACC. Our results provide a preclinical rationale for evaluating niclosamide therapy in a clinical trial for ACC. Clin Cancer Res; 22(14); 3458–66. ©2016 AACR.

Purpose: Anaplastic thyroid cancer (ATC) is a rare but lethal malignancy without any effective therapy. The aim of this study is to use a high-throughput drug library screening to identify a novel therapeutic agent that targets dysregulated genes/pathways in ATC. ExperimentalDesign: We performed quantitative high-throughput screening (qHTS) in ATC cell lines using a compound library of 3,282 drugs. Dysregulated genes in ATC were analyzed using genome-wide expression analysis and immunohistochemistry in human ATC tissue samples and ATC cell lines. In vitro and in vivo studies were performed for determining drug activity, effectiveness of targeting, and the mechanism of action. Results: qHTS identified 100 active compounds in three ATC cell lines. One of the most active agents was the first-in-class survivin inhibitor YM155. Genome-wide expression analysis and immunohistochemistry showed overexpression of survivin in human ATC tissue samples, and survivin was highly expressed in all ATC cell lines tested. YM155 significantly inhibited ATC cellular proliferation. Mechanistically, YM155 inhibited survivin expression in ATC cells. Furthermore, YM155 treatment reduced claspin expression, which was associated with S-phase arrest in ATC cells. In vivo, YM155 significantly inhibited growth and metastases and prolonged survival. Conclusions: Our data show that YM155 is a promising anticancer agent for ATC and that its target, survivin, is overexpressed in ATC. Our findings support the use of YM155 in clinical trials as a therapeutic option in advanced and metastatic ATC. Clin Cancer Res; 21(18); 4123–32. ©2015 AACR.

The Parkinson’s disease-associated protein alpha-synuclein (α-syn) is often highly expressed in melanoma. α-Syn appears to exert a pro-survival function in melanoma cells, possibly by maintaining autophagy homeostasis. The long-term goal of our work is to determine the mechanism by which α-syn confers survival in melanoma cells. Herein, we used CRISPR (clustered regularly interspaced short palindromic repeats) to disrupt SNCA, the gene that codes for α-syn, in the melanoma cell line SK-Mel-28. Western blotting and real-time-qPCR verified that we generated four α-syn knockout (KO) clones. Three clones showed no expression of α-syn, whereas one had about 50% less α-syn than the control cells. DNA sequencing is consistent with three of the clones having both alleles of SNCA knocked out, whereas one clone has only one allele knocked out. α-Syn KO clones were tested in a variety of assays to ascertain the function of α-syn. First, the percentage of cells with exofacial phosphatidylserine (PS) was determined by annexin V-FITC/PI staining in conjunction with fluorescence-activated cell sorting. On average, there was a statistically significant 50% increase in exofacial PS (annexin V+/PI-) in three of the four α-syn KO clones compared to control cells. Exofacial PS was increased in the α-syn KO clones without a concomitant increase in apoptotic cells (annexin V+/PI+). Second, the clones were tested for growth in vivo using SCID mice. All four clones showed a ~65% reduction in the rate of tumor growth, followed over 12 weeks. Third, immunohistochemical staining of excised tumors from the SCID mice showed that the proliferation marker Ki-67 was significantly (p< 0.0001) lower in the tumors generated from the KO clones compared to tumors from control cells. In conclusion, given that α-syn avidly binds to PS, one possibility is that α-syn is like molecular Velcro that binds to PS molecules embedded in the inner leaflet, and this binding helps ATP-driven flippases keep PS in the inner leaflet. Loss α-syn then increases exofacial PS, which is a marker of senescence. Our working model is that α-syn is an anti-senescence protein, and that the α-syn KO clones grow slower than control cells in vivo because the clones have entered a senescent state. Citation Format: Sahar Shekoohi, Santhanasabapathy Rajasekaran, Shu Yang, Sureshbabu Nagarajan, Dhaval Patel, Xiuping Yu, Stephan N. Witt. Knocking out the gene (SNCA) that codes for the Parkinson’s disease-related protein alpha-synuclein in SK-Mel-28 melanoma cells significantly retards tumor growth in SCID mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 896.

Unequivocally, cumulative mutations are required for the development of cancer and many sources of mutagenic activity contribute to tumorigenesis. Recent evidence has implicated that a member of cytidine deaminase family, APOBEC3B (Apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3B or A3B) is enriched in the genomes of many human cancers such as cervical, bladder, lung, head and neck and breast cancers (Burns et al., Nature, 2013). APOBEC3B deaminate cytosines in the host genome to generate C-T mutations. Here we report for the first time that APOBEC3B is overexpressed with adrenocortical carcinoma (ACC), a very aggressive and lethal malignancy. We found that APOBEC3B is significantly overexpressed in primary and metastatic ACC as compared to normal adrenal and cortical adenomas by qPCR, immunofluorescence. We observed that γH2AX, a marker of DNA double-strand breaks co-localizes with the expression of APOBEC3B in patients with primary and metastatic ACC. Also ACC tumor samples with high APOBEC3B expression had high chromosomal gain/loss particularly in chromosome 4 and 8 compared to tumor samples with low APOBEC3B expression. To identify the cellular function of APOBEC3B, we performed transient knock-down studies in three ACC cell lines and found that it reduces cellular proliferation and induces cell cycle arrest. Furthermore, we observed that knockdown of APOBEC3B inhibits MAPK signaling by downregulating phospho ERK1/2 levels. Interestingly, we found that tumors with high APOBEC3B expression levels had twice as many TP53 inactivating mutations as compared to tumors with low levels of APOBEC3B. In order to understand the regulation of APOBEC3B expression, we analyzed the methylation status, copy number variation and potential targeting miRNAs of APOBEC3B from genome wide array platform but found no associations in human ACC samples. Therefore, we did a functional, knockdown screen of 90 cancer-associated transcription factors and found that GATA3 showed positive regulation of APOBEC3B. Our data provide novel insights into the function and regulation of APOBEC3B expression beyond its known mutagenic ability. Citation Format: Sudheer Kumar Gara, Yi Liu-Chittenden, Shweta Kotian, Dhaval Patel, Electron Kebebew. Novel insights into the cellular function and gene regulation of a master mutator, APOBEC3B. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3578.