SUPPLEMENTARY INFORMATION Knockdown of Malic Enzyme 2 Suppresses Lung Tumor Growth, Induces Differentiation and Impacts PI3K/AKT Signaling Jian-Guo Ren 1, Pankaj Seth 1, Clary B. Clish 2, Pawel K. Lorkiewicz 3,4, Richard M. Higashi 3,4, Andrew N. Lane 3,5, Teresa W.-M. Fan 3,4,5 and Vikas P. Sukhatme 1 * 1 Divisions of Interdisciplinary Medicine and Biotechnology, Hematology-Oncology and Nephrology, Beth Israel Deaconess Medical Center (BIDMC) and Harvard Medical School, 330 Brookline Avenue, Boston, MA 02215; and 2 Metabolite Profiling Initiative, The Broad Institute of MIT and Harvard, 7 Cambridge Center, Cambridge, MA 02142. 3 Center for Regulatory and Environmental Analytical Metabolomics, 4 Department of Chemistry, and 5 J. G. Brown Cancer Center, University of Louisville, Louisville, KY 40208. These authors made equal contributions. * Correspondence: Vikas P. Sukhatme MD PhD Beth Israel Deaconess Medical Center. 330 Brookline Avenue, Gryzmish 602, Boston, MA 02215 Tel: 617-667-9050 Fax: 617-667-7843 E-mail: vsukhatm@bidmc.harvard.edu Running title: ME2 modulates lung tumor growth
SUPPLEMENTAL TABLES AND FIGURE LEGENDS. Table S1. Characteristics of the study samples from tumor patients. Table S2. Characteristics of the study samples from lung tumor patients. Table S3. Characteristics of the study samples from melanoma patients. Figure S1. ME2 expression in solid tumor samples. Human cancer tissues lysed in RIPA buffer were purchased from Protein Biotechnologies and analyzed for ME2 and β-tubulin expression by Western blotting. Equal amounts of protein were loaded. A549 was used as a positive control and loaded in each membrane. The full-length blots are presented in Supplemental Figure S19. Figure S2. ME2 expression in lung cancer and the effects on Normal lung primaery, breast cancer and melanoma cell proliferation of independent shrnas targeting ME2. a, Human lung cancer tissues lysed in RIPA buffer were purchased from Protein Biotechnologies and analyzed for ME2 and β-tubulin expression by Western blotting. Equal amounts of protein were loaded. The blots showed here are from two different gels that have been run under the same experiment conditions and exposed on the same film. The full-length blots are presented in Supplemental Figure S20. b, Paired normal and tumor lung tissues from the same patient were analyzed for ME2, cyclophilin B and β- tubulin expression. Equal amounts of protein were loaded and the non-specific bands were used to show the similar loading. The full-length blots are presented in
Supplemental Figure S21. c, A representative image from the tissue array stained with ME2 antibody. d, Knockdown of ME2 in normal lung primary cells. Western blot analysis using an ME2 antibody of lysate from pools of normal lung primary epithelial cells transduced with two independent ME2 shrna lentiviruses, following 5 days growth without any selection. Data are representative of two independent experiments. The fulllength blots are presented in Supplemental Figure S22. e, The effects of knockdown of ME2 on cell death in normal lung primary cells. f, Western blot analysis using an ME2 antibody of lysate from pools of MCF-7 cells transduced with three independent ME2 shrna lentiviruses, following selection of puromycin for 10 days. Data are representative of at least three independent experiments. All three pools showed marked ME2 silencing. The full-length blots are presented in Supplemental Figure S23. g, Cell proliferation in MCF-7 cells transduced with the indicated shrna lentiviral constructs as described in f. Data are representative of three independent experiments. h, Western blot analysis using an ME2 antibody of lysate from pools of WM983B cells transduced with three independent ME2 shrna lentiviruses, following selection of puromycin for 10 days. Data are representative of at least three independent experiments. The full-length blots are presented in Supplemental Figure S24. i, Cell proliferation in WM983B cells transduced with the indicated shrna lentiviral constructs as described in h. Data are representative of three independent experiments. j, Western blot analysis using an ME2 antibody of lysate from pool of WM852 cells transduced with ME2 shrna lentiviruses, following selection of puromycin for 10 days. Data are representative of at least three independent experiments. The full-length blots are presented in Supplemental Figure S25.
k, Cell proliferation in WM852 cells transduced with the indicated shrna lentiviral constructs as described in j. Data are representative of three independent experiments. Figure S3. Tissue array analysis of ME2 expression in 8 types of human solid tumors. Figure S4. ME2 expression in human melanoma analyzed by tissue array and Western blot. a, Tissue array analysis of ME2 expression. b, ME2 levels in 3-paired primary and metastatic melanoma cell lines (primary melanoma cell lines from same patient). The full-length blots are presented in Supplemental Figure S26. Figure S5. DMM selectively kills tumor cells. a, DMM treatment induces ROS production in both normal and tumor lung cells. ROS was detected by flow cytometry using CM-H 2 DCFDA. b, DMM selectively kills lung tumor cells compared to normal lung epithelial cells and normal human fibroblasts. Cell death in A549 cells was analyzed by Annexin V reagent. Each histogram is representative of three experiments. Figure S6. DMM synergistically inhibits cisplatin-treated tumor growth. a, Cisplatin (CP) and DMM combination treatment synergistically inhibits A549 tumor growth. Approximately 5 x 10 6 control plko cells were subcutaneously implanted into female athymic nude mice and treated with DMM, cisplatin or DMM/cisplatin combination as described in Materials and Methods. Tumor sizes were measured and calculated. b, Direct injection of DMM into xenograft tumors causes tumor shrinkage and ulceration. c,
The combination of DMM and cisplatin treatment inhibits mucin-1 expression and increases expression of the differentiation markers E-cadherin and ZO-1 in vivo. Figure S7. Full length blots of Figure 1a. Figure S8. Full length blots of Figure 1c. Figure S9. Full length blots of Figure 1e. Figure S10. Full length blots of Figure 2b. Figure S11. Full length blots of Figure 3a. Figure S12. Full length blots of Figure 3c. Figure S13. Full length blots of Figure 3e. Figure S14. Full length blots of Figure 3g. Figure S15. Full length blots of Figure 4g. Figure S16. Full length blots of Figure 5d. Figure S17. Full length blots of Figure 6b.
Figure S18. Full length blots of Figure 6d. Figure S19. Full length blots of Figure S1. Figure S20. Full length of Figure S2a Figure S21. Full length blots of Figure S2b Figure S22. Full length blots of Figure S2d Figure S23. Full length blots of Figure S2f. Figure S24. Full length blots of Figure S2h. Figure S25. Full length blots of Figure S2j Figure S26. Full length blots of Figure S4b
Table S1 Samples Diagnosis Grade Stage TNM Sex Age Stomach Cancer: 2 Adenocarcinoma 3 II T3N0M0 M 52 3 Mucinous Adenocarcinoma n/a II n/a F 56 4 Infiltrating carcinoma n/a n/a n/a M 42 5 Infiltrating carcinoma n/a n/a n/a M 75 Larynx Cancer: 7 Squamous cell carcinoma 2 n/a n/a F 75 8 Squamous cell carcinoma 2 III T4N0Mx M 71 9 Squamous cell carcinoma 1 II T2N0Mx M 55 Uterus/Ovary Cancer: 10 Endometrial adenocarcinoma 1 IIIc T2N1M0 F 52 77 Endometrial adenocarcinoma 2 Ic T1N0M0 F 60 76 Endometrial adenocarcinoma 4 n/a n/a F 76 11 Uterine Leiomyosarcoma 1 n/a n/a F 40 Bladder Cancer: 12 Transitional cell carcinoma n/a I T1bNxM0 F 57 13 Papillary transitional cell carcinoma 1 II T2NxM0 M 56 14 Transitional cell carcinoma 2 II T2NxM0 M 64 15 Squamous cell carcinoma 3 II T2NxM0 M 54 16 Adenocarcinoma 3 II T2mNxM 0 M 74 Skin cancer: 79 Normal skin n/a n/a n/a M 29 17 Squamous cell carcinoma 2 III T2N1M0 F 58 18 Basal cell carcinoma n/a n/a T3N0Mx F n/a 19 Keratinizing squamous cell carcinoma 3 II T2N0M0 F 39 20 Squamous cell carcinoma 2 n/a n/a F 32 21 Granular cell tumor n/a n/a n/a F 53 Keratinizing squamous cell 22 carcinoma n/a n/a T2N0M0 M 40 23 Malignant fibrous histiocytoma 3 n/a n/a M 46 24 Sebaceous adenocarcinoma n/a n/a n/a F 78 Lung Cancer: 25 Papillary Adenocarcinoma 2 IIIA T3N0M0 M 42 26 Adeno-Squamous Cell Carcinoma 2 I T2N0M0 M 62 27 Large Cell Carcinoma 3 III T3NxM0 M 26 28 Normal tissue n/a n/a n/a n/a n/a 29 Small Cell Carcinoma 3 IIB T2N1M0 M 73 30 Squamous Cell Carcinoma 2 I T2N0M0 M 45
31 Squamous Cell Carcinoma 3 n/a n/a M 37 32 Squamous Cell Carcinoma 2 I T1N0M0 M 58 33 Adenocarcinoma 2 II T2N0Mx F 51 34 Adenocarcinoma 3 n/a n/a F 70 35 Small Cell Carcinoma n/a II n/a F 56 36 Spindle Cell Carcinoma n/a I T2N0M0 F 25 Breast Cancer: 37 Ductal Carcinoma 2 IIIB T2N1M0 F 36 38 Ductal Carcinoma 2 IIB T2N1M0 F 44 39 Ductal Carcinoma 3 IIA T2N0M0 F 43 40 Ductal Carcinoma 3 IIB T2N1M0 F 39 41 Mucinous Carcinoma 1 II T2N1Mx F 51 42 Lobular Carcinoma n/a III T3N2M0 F 39 43 Metaplastic Carcinoma 3 IV T4N1bMx F 46 44 Intraductal Carcinoma 2 IIA T2N0M0 F 41 Colon Cancer: 45 Adenocarcinoma 2 I T2N0M0 M 77 46 Adenocarcinoma 1 II T3N0M0 M 44 47 Adenocarcinoma 2 II T3N0M0 M 49 48 Adenocarcinoma 3 II T3N0M0 M 52 49 Adenocarcinoma 3 II T4NxM0 M 67 50 Mucinous Adenocarcinoma 2 III T3N1M0 M 42 51 Adenocarcinoma 3 IIIA T2N1M0 M 68 52 Multipolyposis n/a n/a n/a M 40 53 Lymphoma n/a n/a n/a M 44 54 non-hodgkin Lymphoma n/a n/a n/a M 46 55 Leiomyosarcoma 2 I T2N0M0 M 65 56 Papillary Adenocarcinoma 2 III T3N0M0 F 56 Kidney Cancer: 57 Papillary renal cell carcinoma 1 n/a n/a M 56 58 Renal clear cell carcinoma 1 n/a n/a F 40 59 Renal cysts n/a n/a n/a n/a n/a 60 Renal clear cell carcinoma 3 III T2N0Mx M 57 61 Renal clear cell carcinoma 2 II n/a F 69 62 Adenocarcinoma 3 II T2N0M0 M 37 Liver Cancer: 63 Trabecular adenocarcinoma 2 n/a n/a M 39 64 Hepatocellular carcinoma 2 II T2N0Mx M 53 65 Hepatocellular carcinoma 3 IIIa T3N0Mx M 59 66 Hepatocellular carcinoma 3 IIIa T3N0M0 F 33 67 Hepatocellular carcinoma fibrolamellar 2 II T2N0Mx M 32 68 Gastrointestinal stromal carcinoma 1 II T3N0Mx F 34 81 Normal liver n/a n/a n/a M 50
Prostate Cancer: 69 Benign prostatic fibrosis n/a n/a n/a M 72 80 Normal prostate n/a n/a n/a M 40 70 Prostatic adenoma n/a n/a n/a M 74 71 Prostatic hyperplasia n/a n/a n/a M 65 72 Prostatic fibroma n/a n/a n/a M 68 Thyroid Cancer: 73 Benign colloid nodules n/a n/a n/a F 72 74 Papillary carcinoma n/a n/a n/a F 49 75 Papillary carcinoma 1 n/a n/a F 58 78 Medullary carcinoma 2 III T3N1Mx F 38 1 Papillary carcinoma 2 III T2N1M0 F 24 Brain tumpr 82 total protein normal brain - human n/a n/a n/a n/a n/a 84 total protein tumor brain - human n/a n/a n/a n/a n/a pancreatic tissue 83 total protein normal pancreas - human n/a n/a n/a n/a n/a 85 total protein pancreas tumor - human n/a n/a n/a n/a n/a
Table S2 Samples Diagnosis Grade Stage TNM Sex Age 1 Normal lung n/a n/a n/a M n/a 2 Adenocarcinoma 3 n/a n/a F 70 3 Large Cell Carcinoma 3 III T3N0Mx M 70 4 Spindle Cell Carcinoma n/a I T2N0M0 F 25 5 Small Cell Carcinoma n/a II n/a F 56 6 Squamous Cell Carcinoma 2 I T1N0M0 M 58 7 Squamous Cell Carcinoma 3 n/a n/a M 37 8 Adenocarcinoma 2 II T2N0Mx F 51 9 Squamous Cell Carcinoma 2 I T2N0M0 M 45 10 Small Cell Carcinoma 3 IIB T2N1M0 M 73 11 Large Cell Carcinoma 3 III T3NxM0 M 26 12 Adeno-Squamous Cell Carcinoma 2 I T2N0M0 M 62 13 Papillary Adenocarcinoma 2 IIIA T3N0M0 M 42
Table S3 No. Age Sex Organ Pathology diagnosis TNM Type 1 80 F Skin Malignant melanoma of head and neck T4N1M0 Malignant 2 60 M Skin Malignant melanoma of left buttocks T4N1M0 Malignant 3 16 F Skin Malignant melanoma of back T4N0M0 Malignant 4 74 M Skin Malignant melanoma of left foot T4N0M0 Malignant 5 55 M Skin Malignant melanoma of sole T4N0M0 Malignant 6 51 M Skin Malignant melanoma of shoulder T4N0M0 Malignant 7 63 F Skin Malignant melanoma of left foot T4N0M0 Malignant 8 46 F Skin Malignant melanoma of thigh T4N0M0 Malignant 9 32 F Skin Malignant melanoma (sparse) of right leg T4N0M0 Malignant 10 42 M Skin Malignant melanoma of left heel T3N2M1 Malignant 11 42 M Skin Malignant melanoma of right sole T2N0M0 Malignant 12 50 M Skin Malignant melanoma of left shoulder T4N0M0 Malignant 13 41 M Skin Malignant melanoma of left leg T3N0M0 Malignant 14 88 F Skin Malignant melanoma of left sole T4N0M0 Malignant 15 57 M Skin Malignant melanoma of left shoulder T4N0M0 Malignant 16 54 F Skin Malignant melanoma of left sole T4N0M0 Malignant 17 42 F Skin Malignant melanoma of left thumb T4N0M0 Malignant 18 58 F Skin Malignant melanoma of left buttocks T4N0M0 Malignant 19 38 M Skin Malignant melanoma of right abdominal wall T4N1M0 Malignant 20 52 F Skin Malignant melanoma of left heel T3N0M0 Malignant 21 40 M Skin Malignant melanoma of right chest wall T4N0M0 Malignant 22 59 F Skin Malignant melanoma of anus T4N0M0 Malignant 23 77 F Skin Malignant melanoma of left sole T1N0M0 Malignant 24 62 F Skin Malignant melanoma of right thumb T4N0M0 Malignant 25 66 F Skin Malignant melanoma of chest wall T4N0M0 Malignant 26 25 M Skin Malignant melanoma of heel T4N0M0 Malignant 27 38 F Skin Malignant melanoma of right toe T4N0M0 Malignant 28 34 F Vulva Malignant melanoma T4N0M0 Malignant 29 52 M Skin Malignant melanoma of right heel T4N1M1 Malignant 30 51 M Skin Malignant melanoma of left buttocks T4N1M0 Malignant 31 36 M Skin Malignant melanoma of back T3N0M0 Malignant 32 45 F Skin Malignant melanoma of left thigh T2N0M0 Malignant 33 51 M Skin Malignant melanoma of left oxter T4N0M0 Malignant 34 61 F Skin Malignant melanoma of left foot T4N0M0 Malignant 35 66 M Skin Malignant melanoma of right thigh T4N0M0 Malignant 36 55 M Skin Malignant melanoma (sparse) of left buttocks T4N0M0 Malignant 37 65 M Skin Malignant melanoma of scalp T4N0M0 Malignant 38 45 M Skin Malignant melanoma of perianal T4N0M0 Malignant 39 73 F Skin Malignant melanoma of right thumb T3N0M0 Malignant 40 41 F Vulva Malignant melanoma T4N0M0 Malignant 41 72 F Vulva Malignant melanoma T1N0M0 Malignant 42 72 F Skin Malignant melanoma of right cheek T4N0M0 Malignant 43 46 F Skin Malignant melanoma (sparse) with necrosis of right thumb T4N0M0 Malignant 44 47 F Skin Malignant melanoma of right upper arm T2N0M0 Malignant 45 53 M Skin Malignant melanoma of right sole T4N0M0 Malignant 46 56 F Skin Malignant melanoma of left leg T3N0M0 Malignant 47 70 M Skin Malignant melanoma of right sole T4N0M0 Malignant 48 65 M Skin Malignant melanoma with necrosis of right thumb T4N0M0 Malignant 49 76 M Skin Malignant melanoma of right medial malleolus T4N0M0 Malignant 50 54 F Skin Malignant melanoma of left heel T4N0M0 Malignant 51 49 M Skin Malignant melanoma of left foot T4N2M0 Malignant 52 55 M Skin Malignant melanoma of right buttocks T4N0M0 Malignant 53 74 M Skin Malignant melanoma of left sole T4N0M0 Malignant 54 65 M Skin Malignant melanoma of right sole T2N0M0 Malignant 55 31 M Skin Malignant melanoma of scalp T4N0M0 Malignant 56 41 F Skin Malignant melanoma of scalp T4N0M0 Malignant 57 63 F Lymph node Metastatic malignant melanoma of right groin - Metastasis 58 46 M Lymph node Metastatic malignant melanoma of right groin - Metastasis 59 55 F Lymph node Metastatic malignant melanoma with necrosis of left groin - Metastasis 60 44 M Lymph node Metastatic malignant melanoma of right oxter - Metastasis 61 72 M Lymph node Metastatic malignant melanoma of right groin - Metastasis
62 47 F Lymph node Metastatic malignant melanoma of right oxter - Metastasis 63 63 M Lymph node Metastatic malignant melanoma of right neck - Metastasis 64 40 F Lymph node Metastatic malignant melanoma of right groin - Metastasis 65 70 M Lymph node Metastatic malignant melanoma of right oxter - Metastasis 66 56 M Lymph node Metastatic malignant melanoma of left preauricula - Metastasis 67 68 M Lymph node Metastatic malignant melanoma of right neck - Metastasis 68 72 F Lymph node Metastatic malignant melanoma of left groin - Metastasis 69 41 F Lymph node Metastatic malignant melanoma of left groin - Metastasis 70 61 F Lymph node Metastatic malignant melanoma of right groin - Metastasis 71 42 F Lymph node Metastatic malignant melanoma of groin - Metastasis 72 43 F Lymph node Metastatic malignant melanoma of groin - Metastasis 73 56 F Lymph node Metastatic malignant melanoma of right groin - Metastasis 74 56 F Lymph node Metastatic malignant melanoma of groin - Metastasis 75 49 F Lymph node Metastatic malignant melanoma of neck - Metastasis 76 41 F Lymph node Metastatic malignant melanoma of left groin - Metastasis 77 45 M Skin Pigmented mole (hyperplasia of squamous epithelium ) - Benign 78 29 F Skin Atypical melanophoric nevus of left shoulder - Benign 79 36 F Skin Intradermal nevus of left abdominal wall - Benign 80 35 F Skin Intradermal nevus of chest wall - Benign 81 23 F Skin Compound nevus of right waist - Benign 82 25 M Skin Compound nevus of left leg - Benign 83 20 M Skin Intradermal nevus of left shoulder - Benign 84 19 M Skin Intradermal nevus of right cheek - Benign 85 10 F Skin Atypical melanophoric nevus of right dorsum of foot - Benign 86 2 F Skin Intradermal nevus of frontal region - Benign 87 6 M Skin Compound nevus of left face - Benign 88 5 Mon. M Skin Intradermal nevus of face - Benign 89 25 M Skin Intradermal nevus (sparse) of scalp - Benign 90 46 M Skin Intradermal nevus of back - Benign 91 2 F Skin Intradermal nevus of left leg - Benign 92 42 M Skin Intradermal nevus of face - Benign 93 11 M Skin Intradermal nevus of left thigh - Benign 94 30 F Skin Compound nevus of right buttocks - Benign 95 62 M Skin Malignant transformation of junctional nevus of abdominal wall - Benign 96 39 M Skin Compound nevus of scalp - Benign 97 7 F Skin Junctional nevus of right forearm - Benign 98 50 M Skin Compound nevus of upper arm - Benign 99 53 M Skin Sebaceous nevus (hyperplasia of squamous epithelium) of face - Benign 100 32 M Skin Sebaceous nevus of left elbow - Benign 101 Tissue marker