Malathy P.V. Shekhar

The Barbara Ann Karmanos Cancer Institute

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Pratima Nangia‐Makker

Wayne State University

Larry Tait

Florey Institute of Neuroscience and Mental Health

Brittany Haynes

National Center for Advancing Translational Sciences

Brigitte Gérard

The Barbara Ann Karmanos Cancer Institute

Andrew D. Westwell

Cardiff University

Matthew A. Sanders

Wayne State University

Hend Kothayer

Zagazig University

Guangzhao Mao

UNSW Sydney

Lisa Polin

The Barbara Ann Karmanos Cancer Institute

Robert J. Pauley

Wayne State University

Cooperative Institutions

Wayne State University

112

The Barbara Ann Karmanos Cancer Institute

Cardiff University

University of Michigan–Ann Arbor

Michigan United

Zagazig University

National Institutes of Health

University of Nebraska Medical Center

Prevent Cancer Foundation

Eugene Applebaum College of Pharmacy and Health Sciences

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Supplementary Methods and Materials from Rad6B Is a Positive Regulator of β-Catenin Stabilization

Malathy P.V. Shekhar Brigitte Gérard Robert J. Pauley Bart O. Williams Larry Tait

Supplementary Methods and Materials from Rad6B Is a Positive Regulator of β-Catenin Stabilization

Negative regulator

10.1158/0008-5472.22374488.v1

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Citations (0)

p53 and protein kinase C independent induction of growth arrest and apoptosis by bryostatin 1 in a highly metastatic mammary epithelial cell line: In vitro versus in vivo activity.

International Journal of Molecular Medicine (1998)

H Wang Ramzi M. Mohammad J Werdell Malathy P.V. Shekhar

We have evaluated the effects of bryostatin 1 on growth of a highly malignant p53-null mouse mammary tumor line, 4T1, and the mechanism by which bryostatin 1 inhibits in vitro growth and in vivo development of tumor and metastases from the orthotopic site. Bryostatin 1 at 20-400 nM concentrations inhibits growth of 4T1 cells by approximately 60% in two-day cultures. Inhibition of growth is associated with an increase in the number of cells undergoing apoptosis with concomitant elevation in the steady state levels of bax protein and drop in bcl-2 levels. The cytotoxic effect of bryostatin 1 on 4T1 cells occurs independently of p53, since there was no evidence of p53-mediated transcriptional activity in 4T1 cells following treatment with bryostatin 1.4T1 cells respond in vivo to bryostatin 1 therapy (75 microg/kg body weight). Intraperitoneal administration of bryostatin 1 inhibits both primary and secondary tumor growth by approximately 50%. However, although bryostatin 1 has a remarkable capacity to slow tumor growth and progression, it is unable to completely eradicate tumor growth and progression due to in vivo development of tumor resistance to bryostatin 1. Levels and cellular distribution of PKCalpha and delta do not correlate with the growth inhibitory effects of bryostatin 1 on 4T1 cells; however, reduction in cytosolic PKCalpha and delta without associated increase in membrane compartment appear to correlate with bryostatin-resistance. Our results suggest that the therapeutic effects of bryostatin 1 in our system do not involve alterations in levels and distribution of PKC but rather a direct upregulation of bax/ bcl-2 ratios that is independent of p53.

Bryostatin 1

10.3892/ijmm.1.6.915

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Citations (36)

Abstract 1662: Therapeutic relevance of the Rad6/translesion synthesis pathway in BRCA1-related triple-negative breast cancer cells

Cancer Research (2015)

Brittany Haynes Hend Kothayer Andrew D. Westwell Malathy P.V. Shekhar

Abstract Triple negative breast cancers (TNBCs) comprise ∼15-20% of all breast cancers (BrCas), and their treatment is challenging because they are not treatable with ER, PgR or Her2/neu-targeted therapies. Since TNBCs and BRCA-mutated BrCas share many histopathologic features including aberrant DNA repair pathways, the concept of targeting DNA repair defects with agents such as platinum (Pt) compounds is applied for treatment of TNBC patients. Pt drugs induce DNA replication stalling interstrand DNA crosslinks (ICLs), the repair of which require concerted activities of nucleotide excision repair, Fanconi anemia (FA)/BRCA homologous recombination repair (HRR) and translesion synthesis (TLS) pathways. The therapeutic efficacies of these drugs are often limited by the cancer cell's enhanced ability to repair/tolerate these toxic DNA lesions. The TLS pathway a.k.a DNA damage tolerance pathway is critical for cell survival in the face of DNA damage. TLS is a crucial initial step in ICL repair as it synthesizes DNA across the lesion thus preparing the damaged DNA template for repair by the FA network and HR pathway, processes critical for ICL repair. The Rad6 gene is a principal component of the TLS pathway and previous work in our lab has shown its important role in BrCa development/progression and acquisition of cisplatin (CDDP) resistance. To understand the involvements of Rad6/TLS and FA/BRCA in CDDP-induced DNA damage repair, we evaluated CDDP sensitivities, CDDP-induced DNA damage responses and stalled replication restarts in wt-BRCA1 (MDA-MB-231, MDA-MB-468) and mut-BRCA1 (HCC1937, SUM1315) TNBCs. Data from MTT assays showed no correlation between CDDP sensitivity and BRCA1 status; however, pretreatment with our recently developed Rad6 small molecule inhibitor (SMI) enhanced CDDP sensitivity of all TNBC cell lines regardless of their BRCA1 status. Consistent with these data, immunoblot analysis showed that treatment with the Rad6 SMI attenuated the CDDP-induced PCNA ubiquitinations (a hall mark of Rad6/TLS activity), as well as the steady-state levels of FancD2 (a surrogate marker of FA pathway activation), pol eta (TLS polymerase), and Rad51 (critical for HRR) in both wt-BRCA1 and mut-BRCA1 TNBC cells. IdU/CldU labeling assays showed that Rad6 inhibition mitigated the restart of CDDP-induced stalled replication forks. Whereas Rad6 is expressed weakly in normal breast tissues and overexpressed in BrCas, immunohistochemical analysis showed strong nuclear staining for Rad6 in the normal ducts of mut-BRCA1 clinical breast tissues implicating an important role for Rad6 in mut-BRCA1 BrCas. Our data suggest an important role for the Rad6/TLS pathway in processing Pt-induced ICLs in both wt-BRCA1 and mut-BRCA1 TNBCs and the potential therapeutic value of inhibiting this pathway in TNBCs. Supported by NIH CA178117-01. Note: This abstract was not presented at the meeting. Citation Format: Brittany Haynes, Hend Kothayer, Andrew Westwell, Malathy Shekhar. Therapeutic relevance of the Rad6/translesion synthesis pathway in BRCA1-related triple-negative breast cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1662. doi:10.1158/1538-7445.AM2015-1662

Synthetic Lethality

Triple-negative breast cancer

XRCC1

10.1158/1538-7445.am2015-1662

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Design, synthesis and in vitro anticancer evaluation of 4,6-diamino-1,3,5-triazine-2-carbohydrazides and -carboxamides

Bioorganic & Medicinal Chemistry Letters (2013)

Hend Kothayer Abdalla A. El‐Shanawani Mansour E. Abu Kull Osama I. El‐Sabbagh Malathy P.V. Shekhar

Triazine

1,3,5-Triazine

IC50

10.1016/j.bmcl.2013.09.087

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Citations (31)

Rad6B Is a Positive Regulator of β-Catenin Stabilization

Cancer Research (2008)

Malathy P.V. Shekhar Brigitte Gérard Robert J. Pauley Bart O. Williams Larry Tait

Abstract Mutations in β-catenin or other Wnt pathway components that cause β-catenin accumulation occur rarely in breast cancer. However, there is some evidence of β-catenin protein accumulation in a subset of breast tumors. We have recently shown that Rad6B, an ubiquitin-conjugating enzyme, is a transcriptional target of β-catenin/TCF. Here, we show that forced Rad6B overexpression in MCF10A breast cells induces β-catenin accumulation, which despite being ubiquitinated is stable and transcriptionally active. A similar relationship between Rad6B, β-catenin ubiquitination, and transcriptional activity was found in WS-15 and MDA-MB-231 breast cancer cells, and mouse mammary tumor virus–Wnt-1 mammary tumor—derived cells, implicating Rad6B in physiologic regulation of β-catenin stability and activity. Ubiquitinated β-catenin was detectable in chromatin immunoprecipitations performed with β-catenin antibody in MDA-MB-231 but not MCF10A cells. Rad6B silencing caused suppression of β-catenin monoubiquitination and polyubiquitination, and transcriptional activity. These effects were accompanied by a reduction in intracellular β-catenin but with minimal effects on cell membrane–associated β-catenin. Measurement of β-catenin protein stability by cycloheximide treatment showed that Rad6B silencing specifically decreases the stability of high molecular β-catenin with minimal effect upon the 90-kDa nascent form. In vitro ubiquitination assays confirmed that Rad6B mediates β-catenin polyubiquitination, and ubiquitin chain extensions involve lysine 63 residues that are insensitive to 26S proteasome. These findings, combined with our previous data that Rad6B is a transcriptional target of β-catenin, reveal a positive regulatory feedback loop between Rad6B and β-catenin and a novel mechanism of β-catenin stabilization/activation in breast cancer cells. [Cancer Res 2008;68(6):1741–50]

Beta-catenin

Deubiquitinating enzyme

10.1158/0008-5472.can-07-2111

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Citations (55)

Novel Inhibitors of Rad6 Ubiquitin Conjugating Enzyme: Design, Synthesis, Identification, and Functional Characterization

Molecular Cancer Therapeutics (2013)

Matthew A. Sanders Ghali Brahemi Pratima Nangia‐Makker Vitaly Balan Matteo Morelli

Abstract Protein ubiquitination is important for cell signaling, DNA repair, and proteasomal degradation, and it is not surprising that alterations in ubiquitination occur frequently in cancer. Ubiquitin-conjugating enzymes (E2) mediate ubiquitination by selective interactions with ubiquitin-activating (E1) and ubiquitin ligase (E3) enzymes, and thus selective E2 small molecule inhibitor (SMI) will provide specificity unattainable with proteasome inhibitors. Here we describe synthesis and functional characterization of the first SMIs of human E2 Rad6B, a fundamental component of translesion synthesis DNA repair. A pharmacophore model for consensus E2 ubiquitin-binding sites was generated for virtual screening to identify E2 inhibitor candidates. Twelve triazine (TZ) analogs screened in silico by molecular docking to the Rad6B X-ray structure were verified by their effect on Rad6B ubiquitination of histone H2A. TZs #8 and 9 docked to the Rad6B catalytic site with highest complementarity. TZs #1, 2, 8, and 9 inhibited Rad6B-ubiquitin thioester formation and subsequent ubiquitin transfer to histone H2A. SMI #9 inhibition of Rad6 was selective as BCA2 ubiquitination by E2 UbcH5 was unaffected by SMI #9. SMI #9 more potently inhibited proliferation, colony formation, and migration than SMI #8, and induced MDA-MB-231 breast cancer cell G2–M arrest and apoptosis. Ubiquitination assays using Rad6 immunoprecipitated from SMI #8- or 9-treated cells confirmed inhibition of endogenous Rad6 activity. Consistent with our previous data showing Rad6B-mediated polyubiquitination stabilizes β-catenin, MDA-MB-231 treatment with SMIs #8 or 9 decreased β-catenin protein levels. Together these results describe identification of the first Rad6 SMIs. Mol Cancer Ther; 12(4); 373–83. ©2013 AACR.

Ubiquitin-conjugating enzyme

Deubiquitinating enzyme

F-box protein

10.1158/1535-7163.mct-12-0793

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Citations (58)

Tamoxifen Suppresses Histologic Progression to Atypia and DCIS in MCFIOAT Xenografts, a Model of Early Human Breast Cancer

Breast Cancer Research and Treatment (2001)

Daniel W. Visscher Pratima Nangia‐Makker Gloria H. Heppner Malathy P.V. Shekhar

Antiestrogen

Carcinoma in situ

Atypia

Atypical Hyperplasia

Tumor progression

Proliferation index

10.1023/a:1006490000659

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Citations (12)

A Review of Endocrine Therapy in Early-stage Breast Cancer

American Journal of Clinical Oncology (2023)

Jailan A. Elayoubi Jie Chi Amr A. Mahmoud Abdurahman Alloghbi Hadeel Assad

Endocrine therapy (ET) is the standard of care for hormone receptor-positive early-stage breast cancer in the adjuvant setting. However, response to ET can vary across patient subgroups. Historically, hormone receptor expression and clinical stage are the main predictors of the benefit of ET. A “window of opportunity” trials has raised significant interest in recent years as a means of assessing the sensitivity of a patient’s cancer to short-term neoadjuvant ET, which provides important prognostic information, and helps in decision-making regarding treatment options in a time-efficient and cost-efficient manner. In the era of genomics, molecular profiling has led to the discovery and evaluation of the prognostic and predictive abilities of new molecular profiles. To realize the goal of personalized medicine, we are in urgent need to explore reliable biomarkers or genomic signatures to accurately predict the clinical response and long-term outcomes associated with ET. Validation of these biomarkers as reliable surrogate endpoints can also lead to a revolution in the clinical trial designs, and potentially avoid the need for repeated tissue biopsies in the surveillance of disease response. The clinical potential of tumor genomic profiling marks the beginning of a new era of precision medicine in breast cancer treatment.

Personalized Medicine

Neoadjuvant Therapy

Surrogate endpoint

10.1097/coc.0000000000000993

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Supplementary Methods from Novel Inhibitors of Rad6 Ubiquitin Conjugating Enzyme: Design, Synthesis, Identification, and Functional Characterization

Matthew A. Sanders Ghali Brahemi Pratima Nangia‐Makker Vitaly Balan Matteo Morelli

Identification

Ubiquitin-conjugating enzyme

10.1158/1535-7163.22497676.v1

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