Publications

Scholarly Journals--Published

• Vinodh Radhakrishnan , Kameswaran Ravichandran , Chibuzo Eke , Amanda Ortiz-Vicil , Qianwei Tan , Marino De Leon and Daisy D. De León (2020) Methylation of a Newly Identified Region of the INS-IGF2 Gene Determines IGF2 Expression in Breast Cancer Tumors and in Breast Cancer Cells. Oncotarget, Vol.11 (No. 44) 3904-3920.    ABSTRACT IGF2 is essential in breast differentiation, lactation, tumor growth, and in breast cancer (BC) development and progression. This growth factor also inhibits apoptosis and promotes metastasis and chemoresistance, contributing to more aggressive tumors. We previously demonstrated that IGF2 protein levels are higher in BC tissues from African American women than in Caucasian women. We also showed that high IGF2 protein levels are expressed in normal breast tissues of African American women while little or no IGF2 was detected in tissues from Caucasian women. Others showed that decreased DNA methylation of the IGF2 gene leads to different BC clinical features. Thus, we designed this study to determine if differentially methylated regions of the IGF2 gene correspond to IGF2 protein expression in paired (Normal/Tumor) breast tissues and in BC cell lines. Methylation analysis was performed using Sodium Bisulphite Analysis and Methylation Sensitive Restriction Enzyme digestion methods. Our results show that a unique site in the INS-IGF2 region is hypermethylated in normal breast and hypomethylated in breast cancer. We designated this region the DVDMR. Furthermore, the methylation levels in the DVDMR significantly correlated with IGF2 protein levels. This novel DMR consists of 257bp localized in the INS-IGF2 gene. We propose that methylation of DVDMR represents a novel epigenetic biomarker that determines the levels of IGF2 protein expression in breast cancer. Since IGF2 promotes metastasis and chemoresistance, we propose that IGF2 levels contribute to BC aggressiveness. Validation of IGF2 as a biomarker will improve diagnosis and treatment of BC patients.  (11/2020) (link)
• Kalla-Singh, S., Tan, QW., Brito, C., De León, M., and De León, DD. (2011) Differential expression and signaling activation of insulin receptor isoforms A and B: A link between breast cancer and diabetes. Growth Factors, 29(6): 278-289. (09/2011)
• Richardson A. E., Hamilton, N., Davis, W., Brito C, Garberoglio., C. and De León D (2011) Insulin-like Growth Factor-2 (IGF-2) Activates Estrogen Receptor-? and -??via the IGF-1 and the Insulin Receptors in Breast Cancer Cells. Growth Factors, 29(2-3):82-93. (04/2011)
• Almaguel, F., Liu, J.W., Pacheco, F.J., De Leon, D.D,  Casiano, C.A., De Leon, M. (2010) Lipotoxicity Mediated Cell Dysfunction and Death Involves Lysosomal Membrane Permeabilization and Cathepsin L Activity. Brain Research. 1318, 133-143 (03/2010)
• Singh, K., Tan, Q.W. and De León D.D.  (2010) The Role of the Insulin-like growth factors I and II receptors in the Breast Cancer Survival Disparity among African-American Women. Growth Hormone & IGF Research 20, 245-254. (03/2010)
• Singh, K.S., Tan, Q.W., Brito, C., De León, M. and De León, D.D (2010) Differential Insulin-like Growth Factor II (IGF-II) expression: A potential role for breast cancer survival disparity. Growth Hormone & IGF Research. 20, 162-170. (01/2010)
• Vyas, S., Moretta, D., Almaguel, F., De León, M. and De Leon, D.D. (2008): Precursor IGF-II (proIGF-II) and mature IGF-II (mIGF-II) induce Bcl-2 and Bcl-XL Expression Through Different Signaling Pathways in Breast Cancer Cells. Growth Factors, 26(1):92-103. IGF-II plays a crucial role in fetal and cancer development by signaling through the IGF-I receptor. We have shown that inhibition of IGF-II by resveratrol (RSV) induced apoptosis and that proIGF-II (highly expressed in cancer) was more potent than mIGF-II in inhibiting this effect. Thus, we hypothesized that IGF-II differentially regulates the signaling cascade of the IGF-I receptor to stimulate the anti-apoptotic proteins Bcl-2 and Bcl-XL to prevent apoptosis. RSV treatment to breast cancer cells inhibited Bcl-2 and Bcl-XL expression and induced mitochondrial membrane depolarization. ProIGF-II was more potent than mIGF-II in: (1) activating the PI3/Akt pathway, (2) regulating Bcl-2 and Bcl-XL expression, and (3) inducing phosphorylation/nuclear translocation of Cyclic AMP-responsive element binding protein. Furthermore, IGF-II differentially regulated the intracellular translocation of Bcl-2 and Bcl-XL, a critical process in breast cancer progression to hormone-independence. Our study provides a novel mechanism of how proIGF-II promotes progression and chemoresistance in breast cancer development. (10/2008)
• Liu, R., Almaguel, F.,  De León, D.D. and De Leon, M. (2008) Expression of E-FABP in PC12 cells Increases neurite Extension during Differentiation: Involvement of n-3 and n-6 fatty acids Journal of Neurochemistry. 106, 2015–2029 (05/2008)
• Vyas, S., Moretta, D., Almaguel, F., De Leon, M. and De Leon, D.D. ProIGF-II Prevents Resveratrol Induced Cell Death by Regulating Survivin Cellular Localization and Mitochondrial Depolarization in Breast Cancer Cells. Growth Factors, 25(6):363-372. (10/2007)
• Vyas, S., Asmerom, Y., and De Leon, D.D. 2006 Insulin-like Growth Factor II mediates Resveratrol stimulatory effect on Cathepsin D in Breast Cancer Cells. Growth Factors, 24(1):79-87 (03/2006)
• Vyas, S., Asmerom, Y.,  and De Leon, D.D. 2006. Insulin-like Growth Factor II mediates Resveratrol stimulatory effect on Cathepsin D in Breast Cancer Cells. Growth Factors, 24(1):79-87 Cathepsin D (CD) is an enzyme that promotes breast cancer. CD is stored intracellularly; however, we demonstrated that IGF-II promotes CDsecretion in estrogen receptor positive (ERþ ) breast cancer cells. We also showed that resveratrol (RSV) stimulates IGF-II in ER(þ) breast cancer cells. Thus we designed this study to determine whether RSV regulates CD in MCF-7, T47D (ERþ ) breast cancer cells as well as in Hs578t (cancer) and MCF-10A (normal) ER2 cell lines. RSV (10 26 M) increased CD and IGF-II secretion in ERþ but not ER2 cells. RSV treatment (1024 M) inhibited CD in ERþ but not in ER2 cells. Transfection of ER2 cells with proIGF-II increased CD secretion. RSV (10 26 M) modulates CD secretion through IGF-II while RSV (10 24 M) inhibits CD in ERþ but not ER2 cells. Regulation of CD by RSV represents a novel mechanism by which RSV may protect against breast cancer. (03/2005)
• Vyas, S., Asmerom, Y., De Leon, D.D. 2005. Resveratrol Regulates IGF-II in MCF-7 Breast Cancer Cells (Endocrinology 146(10):4224-4233 IGF-II is a potent mitogen and inhibitor of apoptosis in breast cancer. Regulation of IGF-II is complex and includes inhibition by tumor suppressors, stimulation by oncogenes, and imprinting and hormonal regulation by estrogens. Resveratrol (RSV) is a phytoestrogen that displays estrogen-like agonistic and antagonistic activity. Recent studies have shown that RSV inhibits the growth of breast cancer cells and may represent a potent agent in chemopreventive therapy. Because 17 (01/2005)
• Faridi, J.S. and De León, D.D. 2004. Interactions of IGF-II in MCF-7 Breast Cancer Cells: Role of the IGF-II/M6P Receptor. Growth Factors 22 (3)  167-177. (10/2004)
• Faridi, J.S., Mohan, S.,  and De Leon, D.D. "Modulation of Cathepsin D Routing by IGF-II Involves IGF-II Binding to IGF-II/M6P Receptor in MCF-7 Breast Cancer Cells ." Growth Factors 22.3 (2004): 169-177. The IGF-II/M6P receptor targets cathepsin D to the lysosomes and it also binds IGF-II. Although the binding sites for IGF-II and cathepsin D are distinct, reciprocal interactions between the ligands have been observed. We have demonstrated that proIGF-II expression modulates routing of cathepsin D. To test the hypothesis that IGF-II modulation of cathepsin D routing in MCF-7 cells involves IGF-II binding to the IGF-II/M6P receptor, we expressed a mutant form of IGF-II (Arg 54 Arg55) that does not bind the IGF-II/M6P receptor and evaluated its effects on cathepsin D secretion. Northern blotting, Western and radioimmunoassay analyses confirmed that these cells express high levels of (Arg 54 Arg55) IGF-II mRNA and secretes high levels of IGF-II without modulating the secretion of cathepsin D. These data provide direct evidence that the IGF-II modulation of cathepsin D routing is IGF-II/M6P receptor mediated. (09/2004)
• Pottorf, W.J., De Leon, D.D., Hessinger, D.A., and Buchholz, J. 200: Effect of Aging on Function of SERCA Mediated Calcium Uptake and Expression of SERCA3 in Rat Cerebral Cortex.  Brain Res. 2001, 914 (1,2)57-65. (09/2001)
• De León, D.D., Issa, N.N, Nainani, S.B., and Asmerom, Y.  1999. Reversal of Cathepsin D Routing Modulation in MCF-7 Breast Cancer Cells expressing antisense Insulin- Like Growth Factor II (IGF-II) Endocrine Metabolism, 31:1-6. (06/1999)
• De León, D.D. and  Donovan, S.  1997.  Is Breast Cancer a potential side effect of GH treatment? Nature Medicine, 3 (10):12-13. (06/1997)
• De León, D.D. and Y. Asmerom 1997.  Quantification of Insulin-Like Growth Factor I (IGF-I) Without Interference by IGF Binding Proteins. Endocrinology 138(5)  2199-2202. (06/1997)
• De León, D.D., Terry C. Asmerom, Y. and Nissley, S.P.  1996. Insulin-Like Growth Factor II (IGF-II) Modulates the Routing of Cathepsin-D In MCF-7 Breast Cancer Cells. Endocrinology 137 (6):1851-1859. (06/1996)
• Kassem, M., Okasaki, R., De León, D.D., Harris, S.A., Spelsberg, T.C., Conover, C.A. and Riggs, B.L. 1996. Potential Mechanism for Estrogen-Mediated Decrease in Bone Formation: Estrogen increases Production of inhibitory Insulin-Like Growth Factor Binding Protein-4.  Proceedings of the Association of American Physicians 108 (2):155-164.   Using a recently developed human osteoblastic cell line (hFOB/ER9) with high levels (approximately 4,000 per nucleus) of estrogen receptors and the characteristic phenotype of the mature osteoblast, we tested the hypothesis that estrogen decreases bone formation by inhibiting the action of the insulin-like growth factor (IGF) paracrine/autocrine system. IGF-II, the predominant IGF produced by osteoblastic cells, was measurable in hFOB/ER9-conditioned medium (approximately 10 ng/mL) and its level did not change significantly after treatment with 17 beta-estradiol (E2) or anti-estrogens. Treatment with E2 at 0.1-100 nM decreased [3H]thymidine uptake to 53% of control (p < 0.001) in a dose-dependent fashion. The predominant IGF-binding proteins (IGFBPs) produced by hFOB/ER9 and by normal trabecular osteoblasts are IGFBP-3 and IGFBP-4, of which IGFBP-4 is consistently inhibitory of IGF action. Treatment with E2 at 0.01-10 nM for 48 h increased IGFBP-4 mRNA to 346% +/- 90% (mean +/- SE) of control (p < 0.05) and IGFBP-4 protein to 278% +/- 75% of control (p < 0.01) in a dose-dependent fashion but did not alter IGFBP-3 mRNA or protein. E2 treatment also attenuated IGF-dependent, IGFBP-4 specific proteolysis to approximately 50% of control. ICI 182,780, a pure anti-estrogen, completely blocked E2-mediated decreases in cell proliferation and increases in levels of IGFBP-4 mRNA and protein. Treatment of the hFOB/ER9 cells with recombinant human IGFBP-4 (200 ng/mL) decreased cell proliferation to 55% of control (p < 0.01). Thus, E2 acts on osteoblastic cells to increase availability of inhibitory IGFBP-4, by both increasing its production and decreasing its degradation, which may oppose the mitogenic effect of the IGFs on osteoblastic cells. This action may mediate, at least in part, the decreases in bone formation that are observed after estrogen treatment in vivo.  (03/1996)
• Siebler, T., Lopaczynski, W., Terry, C.,  Casella, S.J., Munson, P, De León, D.D., Phang, L.,Blakemore, K.J., McEnvoy, R., Kelley, R.I., and Nissley, P. 1995. Insulin-Like Growth Factor Receptor I Expression and Function in Fibroblasts from Two Patients with Deletion of the Distal Long Arm of Chromosome 15.  J. Clin Endocrinol. Metabol. 80 (12): 3447-3457. (12/1995)
• De León, D.D. 1995.  Prognostic Factors in Breast Cancer: What''''s New? Medical Society Bulletin. J. of San Bernardino County Medical Society. September; P-21-24. (09/1995)
• De León, M., Nahin, R.L., Molina ,C., De León, D.D., and Ruda, M.A.  1995 Comparison of c-Jun, JunB and JunD mRNA expression and protein in the rat dorsal root ganglia following sciatic nerve transection.  J. Neuroscience Res. 42:391-401. (06/1995)
• Durham, S.K., De León, D.D., Okasaki, R., Riggs, B.L.,  and Conover, C.A.  1995 Regulation of Insulin-Like growth factor binding protein-4 availability in normal human osteoblast-like cells: Role of endogenous insulin-like growth factors. J. of Clin Endocrinol and Metab. 80(1):104-110.     nsulin-like growth factor-binding protein-4 (IGFBP-4) is an important regulator of insulin-like growth factor-I (IGF-I) anabolic activity in bone. Although cultured human osteoblast-like (hOB) cells have been reported to secrete IGFBP-4, we could not detect IGFBP-4 protein in 8 of 27 individual donor-derived hOB-cell conditioned medium (hOB-CM) samples examined by Western ligand blotting. Nonetheless, this subset of hOB cells had normal IGFBP-4 messenger ribonucleic acid expression and protein secretion. Regulation of IGFBP-4 levels in hOB cultures appeared to occur extracellularly. hOB cells produce an IGFBP-4 proteinase that requires the presence of IGF for cleavage of the IGFBP-4 molecule into 2 fragments of approximately 18 and 14 kilodaltons. These fragments are not detected by Western ligand blotting. Our data indicate that elevated endogenous levels of IGF can activate IGFBP-4 proteolysis, because in hOB cultures lacking detectable IGFBP-4 protein 1) basal IGF messenger ribonucleic acid expression was increased; 2) IGF-II peptide levels were elevated; 3) IGF-neutralizing antibodies added to hOB-CM attenuated the proteolysis of exogenous IGFBP-4; and 4) recombinant human IGFBP-4 was proteolyzed into 2 immunoreactive fragments of approximately 18 and 14 kilodaltons during cell-free incubations in these hOB-CM without the addition of exogenous IGF. In conclusion, elevated IGF expression and secretion can contribute to enhanced proteolysis of endogenous and exogenous IGFBP-4 via a proteinase secreted by cultured hOB cells. Levels of endogenous IGF peptide may determine IGFBP-4 availability in the bone microenvironment and, thus, modulate the local cell response to IGF-I. (01/1995)
• Conover, C. and De León, D.D. 1994.  Acid-Activated Insulin-like growth factor binding protein-3 proteolysis in normal and transformed cells: Role of Cathepsin-D. J. Biol. Chem. 269:7076-7080. (06/1994)
• De León, D.D.,  Terry C. and Nissley, S.P.  1994.  Direct Detection of insulin-like growth factor II (IGF-II) by chemiluminescence without interference by IGF binding proteins. Endocrinology 134: 1960-1963. (06/1994)
• Barkley, M.S, De León, D.D. and Weste, R. 1993. Pheromonal Regulation of the mouse estrous cycle by a heterogenotypic male. J. Exp. Zool.  265:558-566. (06/1993)
• De León, D.D.  Powers, M., Wilson D.M., and Rosenfeld R.G. 1992. Effects of insulin-like growth factors (IGFs) and IGF receptor antibodies in human breast cancer cell proliferation. Growth factors, 6:327-336. (06/1992)
• Rosenfeld, R.G., Lamson G., Pham, H., Oh, Y. Conover, C., De León,D.D., Donovan, S.M., Ocrant, I., Giudice, L. 1991. Insulin-like growth factor binding proteins. Recent Progress in Hormone Research. 46:99-163. (06/1991)
• De León, D.D., Wilson, D.M., Bakker B., Lamson, G. and Rosenfeld, R.G. 1990. Insulin like growth factor IGF-Binding proteins in human breast cancer cells: Relationship to hIGFBP-2 and hIGFBP-3. J. Clin. Endocrinol. and Metabol. 71 (2), 530-532. (06/1990)
• De León, D.D., Zelinsky-Wooten, M.B. and Barkley, M.S. 1990. Hormonal basis of variation in oestrous cyclicity in selected strains of mice. Journal of Reproduction and Fertility 89, 117-126. (06/1990)
• Lamson, G., Pham, H., Oh, Y., De Leon, D.D., Donovan, S., Schwander, J., Rosenfeld, R.G.: Expression of the 31 kD (hBP-31) and 30 kD (rBP-30) IGF binding proteins in human and rat tissues. In: Drop S, Hintz RL (eds) Insulin-like Growth Factor Binding Proteins, Excerpta Medica, Amsterdam, 1989, pp 143-148 (06/1989)
• Wilson, D.M., De Leon, D.D., Bakker, B., Lamson, G., Rosenfeld, R.G: IGFs and IGF binding proteins in human breast cancer cell lines. In: Drop S, Hintz RL (eds) Insulin-like Growth Factor Binding Proteins, Excerpta Medica, Amsterdam, 1989, pp 241-246. (06/1989)
• De León, D.D., Wilson, D.M., Bakker B., Lamsom, G., Hintz, R.L. and Rosenfeld R. 1989. Characterization of insulin-like growth factor (IGF) binding proteins from human breast cancer cells. Mol. Endocrinol. 3:567-574. (06/1989)
• De León, D.D., Bakker, B., Wilson, D.M., Hintz, R.L. and Rosenfeld, R. 1988. Demonstration of Insulin-like growth factor (IGF-I and -II) receptors and binding protein in human breast cancer cells lines. Biochem and Biophys. Res. Commun. 152:398-405. (06/1988)
• De León, D.D. and Barkley, M.S. 1987. Male and female genotype mediate pheromonal regulation of the mouse estrous cycle. Biology of Reproduction 37:1066-1074. (05/1987)

Scholarly Journals--Accepted

• Expression of Intratumoral IGF-II Is Regulated by the Gene Imprinting Status in Triple Negative Breast Cancer from Vietnamese Patients.  Vinodh Kumar Radhakrishnan , Lorraine Christine Hernandez  Kendra Anderson 1, Qianwei Tan , Marino De León , Daisy D De León  Abstract African American women suffer higher incidence and mortality of triple negative breast cancer (TNBC) than Caucasian women. TNBC is very aggressive, causing the worst clinical outcome. We previously demonstrated that tumors from these patients express high IGF-II and exhibit high activation of the IGF signaling pathways. IGF-II gene expression is imprinted (monoallelic), promotes tumor progression, and metastasis and regulates Survivin, a TNBC prognostic marker. Since BC mortality has increased among young Vietnamese women, we analyzed 48 (paired) TNBC samples from Vietnamese patients to assess IGF-II expression. We analyzed all samples by qrtPCR for identification of IGF-II heterozygosity and to determine allelic expression of the IGF-II gene. We also analyzed the tissues for proIGF-II and Survivin by RT-PCR and Western blotting. A total of 28 samples displayed IGF-II heterozygosity of which 78% were biallelic. Tumors with biallelic IGF-II gene expression exhibited the highest levels of proIGF-II and Survivin. Although 100% of these tissues corresponding normal samples were biallelic, they expressed significantly lower levels of or no proIGF-II and Survivin. Thus, IGF-II biallelic gene expression is differentially regulated in normal versus tumor tissues. We propose that intratumoral proIGF-II is dependent on the IGF-II gene imprinting status and it will promote a more aggressive TNBC. (10/2018) (link)
• Jian, T. and De León, D.D. (2010). Expression of proIGF-II but not IGF-II in human breast cancer cells line MCF-7 induces loss of estrogen requirement for tumor growth in nude and scid mice. (Accepted with revision, IGF and GH Journal).   (09/2011)