Milan R. Uskokovic

Rutgers, The State University of New Jersey, New Brunswick, New Jersey, United States

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Publications (261)1241.35 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Tumor-initiating cells (also known as cancer stem cells) are the subpopulation of cells shown to be responsible for tumor initiation, maintenance and recurrence. In breast cancer, CD44(+)/CD24(-/low) cells were identified as tumor-initiating cells. We previously reported that a Gemini vitamin D analog, 1,25-dihydroxy-20R-21(3-hydroxy-3-deuteromethyl-4,4,4-trideuterobutyl)-23-yne-26,27-hexafluoro-cholecalciferol (BXL0124), reduced CD44(+)/CD24(-/low) cells in MCF10DCIS basal-like breast cancer cells. Since Notch has been identified as one of the key signaling pathways involved in breast cancer stem cells, the effect of BXL0124 on the Notch signaling pathway was investigated in breast cancer. The CD44(+)/CD24(-/low) subpopulation of MCF10DCIS cells showed elevated Notch1 signaling and increased cell proliferation compared to the CD44(+)/CD24(high) subpopulation. Treatment with the Gemini vitamin D analog BXL0124 decreased the level of activated Notch1 receptor. In addition, mRNA and protein levels of the Notch ligands, Jagged-1, Jagged-2 and DLL1, were significantly reduced by treatment with BXL0124, which was followed by repression of c-Myc, a key downstream target of Notch signaling. Interestingly, HES1, a known downstream target of Notch signaling, was rapidly induced by treatment with BXL0124. The inhibitory effect of BXL0124 on Notch signaling was reversed by knockdown of HES1. Overexpression of HES1 inhibited Notch1 signaling and reduced the CD44(+)/CD24(-/low) subpopulation, confirming a role of HES1 in Notch1 signaling. In conclusion, the Gemini vitamin D analog, BXL0124, represses the tumor-initiating subpopulation by HES1-mediated inhibition of Notch1 signaling. The present study demonstrates BXL0124 as a potent inhibitor of Notch signaling to target tumor-initiating cells in basal-like breast cancer. This article is part of a Special Issue entitled "17th Vitamin D Workshop". Copyright © 2014. Published by Elsevier Ltd.
    The Journal of Steroid Biochemistry and Molecular Biology 12/2014; 148. DOI:10.1016/j.jsbmb.2014.12.013 · 4.05 Impact Factor
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    ABSTRACT: Identifies a potential stem cell population in basal-like breast cancer•Characterizes breast cancer stem cell markers in mammosphere cell culture•Assesses the effects of vitamin D compounds on putative breast cancer stem cells•Suggests a novel approach to therapeutically target putative cancer stem cells
    The Journal of Steroid Biochemistry and Molecular Biology 10/2014; 148. DOI:10.1016/j.jsbmb.2014.10.016 · 4.05 Impact Factor
  • Joseph Wahler · Hubert Maehr · Milan Uskokovic · Nanjoo Suh
    Cancer Research 10/2014; 74(19 Supplement):1236-1236. DOI:10.1158/1538-7445.AM2014-1236 · 9.28 Impact Factor
  • Cancer Research 10/2014; 74(19 Supplement):1248-1248. DOI:10.1158/1538-7445.AM2014-1248 · 9.28 Impact Factor
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    ABSTRACT: The potency of 25-hydroxyvitamin D3 (25(OH)D3 ) is increased by several fold through its metabolism into 1α,25-dihydroxyvitamin D3 (1α,25(OH)2 D3 ) by cytochrome P450 27B1 (CYP27B1). Thus, the pivotal role of 1α-hydroxylation in the activation of vitamin D compounds is well known. Here, we examined the metabolism of 25-hydroxy-16-ene-23-yne-vitamin D3 (25(OH)-16-ene-23-yne-D3 ), a synthetic analog of 25(OH)D3 in a cell-free system and demonstrated that 25(OH)-16-ene-23-yne-D3 is neither activated by cytochrome CYP27B1 nor inactivated by cytochrome P450 24A1 (CYP24A1). These findings were also confirmed in immortalized normal human prostate epithelial cells (PZ-HPV-7) which are known to express both CYP27B1 and CYP24A1, indicating that the structural modifications featured in 25(OH)-16-ene-23-yne-D3 enable the analog to resist the actions of both CYP27B1 and CYP24A1. To provide intelligible structure-function information, we also performed molecular docking analysis between the analog and CYP27B1. Furthermore, 25(OH)-16-ene-23-yne-D3 was found to suppress the growth of PZ-HPV-7 cells with a potency equivalent to 1α,25(OH)2 D3 . The antiproliferative activity of 25(OH)-16-ene-23-yne-D3 was found to be vitamin D receptor (VDR)-dependent as it failed to inhibit the growth of mammary tumor cells derived from VDR-knockout mice. Furthermore, stable introduction of VDR into VDR-knockout cells restored the growth inhibition by 25(OH)-16-ene-23-yne-D3 . Thus, we identified 25-hydroxy-16-ene-23-yne-vitamin D3 as a novel non-1α-hydroxylated vitamin D analog which is equipotent to 1α,25(OH)2 D3 in its antiproliferative activity. We now propose that the low potency of the intrinsic VDR-mediated activities of 25(OH)D3 can be augmented to the level of 1α,25(OH)2 D3 without its activation through 1α-hydroxylation by CYP27B1, but by simply preventing its inactivation by CYP24A1. J. Cell. Biochem. 9999: XX-XX, 2014. © 2013 Wiley Periodicals, Inc.
    Journal of Cellular Biochemistry 08/2014; 115(8). DOI:10.1002/jcb.24789 · 3.37 Impact Factor
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    ABSTRACT: Ductal carcinoma in situ (DCIS) is a non-malignant lesion of the breast with the potential to progress to invasive ductal carcinoma (IDC). The disappearance and breakdown of the myoepithelial cell layer and basement membrane in DCIS have been identified as major events in the development of breast cancer. The MCF10DCIS.com cell line is a well-established model which recapitulates the progression of breast cancer from DCIS to IDC. We have previously reported that a novel Gemini vitamin D analog, 1α,25-dihydroxy-20R-21(3-hydroxy-3-deuteromethyl-4,4,4-trideuterobutyl)-23-yne-26,27-hexafluoro-cholecalciferol (BXL0124) is a potent inhibitor of the growth of MCF10DCIS.com xenografted tumors without hypercalcemic toxicity. In the present study, we utilized the MCF10DCIS.com in vivo model to assess the effects of BXL0124 on breast cancer progression from weeks 1 to 4. Upon DCIS progression to IDC from weeks 3 to 4, tumors lost the myoepithelial cell layer and basement membrane as shown by immunofluorescence staining with smooth muscle actin (SMA) and laminin 5, respectively. Administration of BXL0124 maintained the critical myoepithelial cell layer as well as basement membrane, and animals treated with BXL0124 showed a 43% reduction in tumor volume by week 4. BXL0124 treatment decreased cell proliferation and maintained vitamin D receptor (VDR) levels in tumors. In addition, the BXL0124 treatment reduced the mRNA levels of matrix metalloproteinases (MMPs) starting at week 3, contributing to the inhibition of invasive transition. Our results suggest that the maintenance of DCIS plays a significant role in the cancer preventive action of the Gemini vitamin D BXL0124 during the progression of breast lesions.
    Cancer Prevention Research 04/2014; 7(6). DOI:10.1158/1940-6207.CAPR-13-0362 · 5.27 Impact Factor
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    ABSTRACT: We have synthesized thirty-nine 1,25-dihydroxyvitamin D3 [1,25(OH)2 D3 ] analogs having 2 side-chains attached to carbon-20 (Gemini) with various modifications and compared their anticancer activities. Five structure-function rules emerged to identify analogs with enhanced anti-cancer activity. One of these active analogs, BXL-01-0126 was more potent than 1,25(OH)2 D3 in mediating 50% clonal inhibition of cancer cell growth. Murine studies found that BXL-01-0126 and 1,25(OH)2 D3 had nearly the same potency to raise serum calcium levels. Taken together, BXL-01-0126 as compared to 1,25(OH)2 D3 has greater anticancer potency, but similar toxicity causing hypercalcemia. We focused on the effect of these compounds on the stimulation of expression of human cathelicidin antimicrobial peptide (CAMP) whose gene has a vitamin D response element in its promoter. Expression of CAMP mRNA and protein increased in a dose-response fashion after exposure of AML cells to the Gemini analog, BXL-01-126 in vitro. A xenograft model of AML was developed using U937 AML cells injected into NSG immunodeficient mice. Administration of vitamin D3 compounds to these mice resulted in substantial levels of CAMP in the systemic circulation. This suggests a unique prophylactic treatment at diagnosis or during induction chemotherapy for AML patients, to provide them with protection against various microbial infections through CAMP induction. © 2013 Wiley Periodicals, Inc.
    International Journal of Cancer 01/2014; 134(1). DOI:10.1002/ijc.28328 · 5.01 Impact Factor
  • Cancer Research 08/2013; 73(8 Supplement):184-184. DOI:10.1158/1538-7445.AM2013-184 · 9.28 Impact Factor
  • Cancer Research 08/2013; 73(8 Supplement):180-180. DOI:10.1158/1538-7445.AM2013-180 · 9.28 Impact Factor
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    ABSTRACT: Human epidermal growth factor receptor 2 (HER2 or ErbB2), a member of ErbB receptor tyrosine kinases, is overexpressed in approximately 20 % of human breast cancer, and the ErbB2 signaling pathway is a critical therapeutic target for ErbB2-overexpressing breast cancer. We investigated the inhibitory effects of the Gemini vitamin D analog BXL0124, the synthetic triterpenoid CDDO-Im and the combination on the tumorigenesis of ErbB2-overexpressing breast cancer. MMTV-ErbB2/neu transgenic mice were treated with BXL0124, CDDO-Im or the combination from 3 months of age until the end of the experiment. Formation and growth of MMTV-ErbB2/neu mammary tumors were monitored every week, and all three treatments delayed the development of mammary tumors without significant toxicity. Decreased activation of ErbB2 as well as other ErbB receptors, ErbB1 and ErbB3, in MMTV-ErbB2/neu mammary tumors was shown by all treatments. Protein levels of downstream targets of the ErbB2 signaling pathway, including activated-Erk1/2, activated-Akt, c-Myc, CycD1 and Bcl2, were repressed by all three treatments, with the combination treatment exhibiting the strongest effects. To investigate therapeutic efficacy, the combination of BXL0124 and CDDO-Im was given to MMTV-ErbB2/neu mice after mammary tumors were established between 23-30 weeks of age. Short-term treatment with the combination did not show effects on tumor growth nor the ErbB2 signaling pathway. The present study demonstrates BXL0124, CDDO-Im and the combination as potential agents for prevention, but not treatment, against the tumorigenesis of ErbB2-overexpressing breast cancer.
    Cancer Prevention Research 07/2013; 6(9). DOI:10.1158/1940-6207.CAPR-13-0087 · 5.27 Impact Factor
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    ABSTRACT: CD44, a transmembrane glycoprotein, is a major receptor for extracellular proteins involved in invasion and metastasis of human cancers. We have previously demonstrated that the novel Gemini vitamin D analog BXL0124 [1α,25-dihydroxy-20R-21(3-hydroxy-3-deuteromethyl-4,4,4-trideuterobutyl)-23-yne-26,27-hexafluro-cholecalciferol] repressed CD44 expression in MCF10DCIS.com basal-like human breast cancer cells and inhibited MCF10DCIS xenograft tumor growth. In the present study, we investigated potential factors downstream of CD44 and the biological role of CD44 repression by BXL0124 in MCF10DCIS cells. The treatment with Gemini vitamin D BXL0124 decreased CD44 protein level, suppressed STAT3 signaling, and inhibited invasion and proliferation of MCF10DCIS cells. The interaction between CD44 and STAT3 was determined by co-immunoprecipitation. CD44 forms a complex with STAT3 and Janus kinase 2 (JAK2) to activate STAT3 signaling, which was inhibited by BXL0124 in MCF10DCIS cells. The role of CD44 in STAT3 signaling and invasion of MCF10DCIS cells was further determined by the knockdown of CD44 using small hairpin RNA in vitro and in vivo. MCF10DCIS cell invasion was markedly decreased by the knockdown of CD44 in vitro. The knockdown of CD44 also significantly decreased mRNA expression levels of invasion markers, matrix metalloproteinases (MMPs) and urokinase plasminogen activator (uPA), in MCF10DCIS cells. In MCF10DCIS xenograft tumors, CD44 knockdown decreased tumor size and weight as well as invasion markers. The present study identifies STAT3 as an important signaling molecule interacting with CD44 and demonstrates the essential role of CD44-STAT3 signaling in breast cancer invasion. It also suggests that repression of CD44-STAT3 signaling is a key molecular mechanism in the inhibition of breast cancer invasion by the Gemini vitamin D analog BXL0124.
    PLoS ONE 01/2013; 8(1):e54020. DOI:10.1371/journal.pone.0054020 · 3.23 Impact Factor
  • Cancer Research 06/2012; 72(8 Supplement):929-929. DOI:10.1158/1538-7445.AM2012-929 · 9.28 Impact Factor
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    ABSTRACT: Derivatives of vitamin D(3) containing a second side-chain emanating at C-20 are known as gemini and act as vitamin D receptor agonists. Recently, two of these, namely Gemini-0072 and the epimeric Gemini-0097, were selected for further studies in view of their high biological activities and lack of hypercalcemic effects. We now show that the two analogs recruit coactivator SRC-1 better than the parental gemini and act as VDR superagonists. The crystal structures of complexes of zVDR with Gemini-0072 and Gemini-0097 indicate that these ligands induce an extra cavity within the ligand-binding pocket similar to gemini and that their superagonistic activity is due to an increased stabilization of helix H12.
    Medicinal Chemistry Communication 05/2011; 2(5):424-429. DOI:10.1039/C1MD00059D · 2.63 Impact Factor
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    ABSTRACT: CD44 is a multifunctional transmembrane protein involved in cell proliferation, angiogenesis, invasion, and metastasis. CD44 is identified as a cancer stem cell marker, and the CD44-positive breast cancer cells are enriched in residual breast cancer cell populations after conventional therapies, suggesting that CD44 may be an important target for cancer prevention and therapy. Therefore, we investigated for the inhibitory effect of a novel Gemini vitamin D analog, 1α,25-dihydroxy-20R-21(3-hydroxy-3-deuteromethyl-4,4,4-trideuterobutyl)-23-yne-26,27-hexafluoro-cholecalciferol (BXL0124), on mammary tumor growth and CD44 expression in MCF10DCIS.com human breast cancer in vitro and in vivo. MCF10DCIS.com cells were injected into mammary fat pads in immunodeficient mice, and BXL0124 was then administered intraperitoneally (0.1 μg/kg body weight) or orally (0.03 or 0.1 μg/kg body weight) 6 days a week for 5 weeks. At necropsy, mammary tumors and blood were collected for evaluating tumor growth, CD44 expression, and serum calcium level. BXL0124 suppressed mammary tumor growth and markedly decreased the expression of CD44 protein in MCF10DCIS xenograft tumors without causing hypercalcemic toxicity. BXL0124 also inhibited the expression of CD44 protein and mRNA as well as the transcriptional activity of the CD44 promoter in cultured MCF10DCIS.com cells. The repression of CD44 expression induced by BXL0124 was blocked by siRNA vitamin D receptor (VDR), indicating that the regulation of CD44 expression by BXL0124 is a VDR-dependent event. The novel Gemini vitamin D analog, BXL0124, represses CD44 expression in MCF10DCIS.com cells in vitro and in xenograft tumors, suggesting an inhibitory role of a Gemini vitamin D derivative on breast cancer stem cells.
    Molecular pharmacology 03/2011; 79(3):360-7. DOI:10.1124/mol.110.068403 · 4.12 Impact Factor
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    ABSTRACT: We examined the metabolism of two synthetic analogs of 1α,25-dihydroxyvitamin D₃ (1), namely 1α,25-dihydroxy-16-ene-23-yne-vitamin D₃ (2) and 1α,25-dihydroxy-16-ene-23-yne-26,27-dimethyl-vitamin D₃ (4) using rat cytochrome P450 24A1 (CYP24A1) in a reconstituted system. We noted that 2 is metabolized into a single metabolite identified as C26-hydroxy-2 while 4 is metabolized into two metabolites, identified as C26-hydroxy-4 and C26a-hydroxy-4. The structural modification of adding methyl groups to the side chain of 1 as in 4 is also featured in another analog, 1α,25-dihydroxy-22,24-diene-24,26,27-trihomo-vitamin D₃ (6). In a previous study, 6 was shown to be metabolized exactly like 4, however, the enzyme responsible for its metabolism was found to be not CYP24A1. To gain a better insight into the structural determinants for substrate recognition of different analogs, we performed an in silico docking analysis using the crystal structure of rat CYP24A1 that had been solved for the substrate-free open form. Whereas analogs 2 and 4 docked similar to 1, 6 showed altered interactions for both the A-ring and side chain, despite prototypical recognition of the CD-ring. These findings hint that CYP24A1 metabolizes selectively different analogs of 1, based on their ability to generate discrete recognition cues required to close the enzyme and trigger the catalytic mechanism.
    Archives of Biochemistry and Biophysics 02/2011; 509(1):33-43. DOI:10.1016/j.abb.2011.02.004 · 3.04 Impact Factor
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    ABSTRACT: ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.
    ChemInform 12/2010; 22(52). DOI:10.1002/chin.199152163
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    ABSTRACT: ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.
    ChemInform 12/2010; 24(51). DOI:10.1002/chin.199351072
  • G. PIZZOLATO · P. M. WOVKULICH · M. R. USKOKOVIC · A. NORMAN
    [Show abstract] [Hide abstract]
    ABSTRACT: ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.
    ChemInform 12/2010; 26(50). DOI:10.1002/chin.199550259
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    ABSTRACT: ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.
    ChemInform 11/2010; 28(48). DOI:10.1002/chin.199748324
  • [Show abstract] [Hide abstract]
    ABSTRACT: ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.
    ChemInform 09/2010; 23(38). DOI:10.1002/chin.199238091

Publication Stats

6k Citations
1,241.35 Total Impact Points

Institutions

  • 2006–2014
    • Rutgers, The State University of New Jersey
      • Department of Chemical Biology
      New Brunswick, New Jersey, United States
    • University of Birmingham
      Birmingham, England, United Kingdom
  • 2010
    • Wayne State University
      • Department of Chemistry
      Detroit, Michigan, United States
  • 1990–2010
    • University of California, Riverside
      • Division of Biomedical Sciences
      Riverside, California, United States
  • 2008
    • University of Leeds
      Leeds, England, United Kingdom
  • 1993–2008
    • Rutgers New Jersey Medical School
      • Department of Pathology and Laboratory Medicine
      Newark, New Jersey, United States
    • Case Western Reserve University
      Cleveland, Ohio, United States
  • 2005
    • Boston Medical Center
      Boston, Massachusetts, United States
  • 2003
    • Queen Elizabeth Hospital Birmingham
      Birmingham, England, United Kingdom
  • 2001
    • Shinshu University
      • Department of Pediatrics
      Shonai, Nagano, Japan
  • 1995–2001
    • Cedars-Sinai Medical Center
      • • Division of Hematology and Oncology
      • • Cedars Sinai Medical Center
      Los Angeles, CA, United States
  • 1992–2000
    • Alpert Medical School - Brown University
      • Department of Pediatrics
      Providence, Rhode Island, United States
  • 1999
    • University of Vienna
      Wien, Vienna, Austria
  • 1996
    • American University Washington D.C.
      Washington, Washington, D.C., United States
  • 1991
    • Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center
      Torrance, California, United States
  • 1983
    • University of Texas at Dallas
      • Chemistry
      Richardson, Texas, United States
  • 1982
    • University of Texas Health Science Center at Tyler
      Tyler, Texas, United States
  • 1981
    • Iowa State University
      • Department of Animal Science
      Ames, Iowa, United States