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A Novel Therapeutic Approach For Triple-Negative Breast cancer

A Novel Therapeutic Approach For Triple-Negative Breast cancer. TCMC The Commonwealth Medical College. Raj Kumar, Ph.D. Professor of Biochemistry The Commonwealth Medical College, Scranton, PA. TCMC The Commonwealth Medical College. Nothing to Disclose. TCMC The Commonwealth Medical College.

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A Novel Therapeutic Approach For Triple-Negative Breast cancer

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  1. A Novel Therapeutic Approach For Triple-Negative Breast cancer TCMCThe Commonwealth Medical College Raj Kumar, Ph.D. Professor of Biochemistry The Commonwealth Medical College, Scranton, PA. TCMCThe Commonwealth Medical College Nothing to Disclose

  2. TCMCThe Commonwealth Medical College Breast cancer is classified into clinical subtypes based upon receptor expression These subtypes dictate possible therapeutic options and vary in their prognosis Luminal: derived from the luminal cells ER+, PR+, HER+ Can use hormonal therapy Basal: derived from myoepithelial cells ER-, PR-, HER- No specific target for therapies

  3. TCMCThe Commonwealth Medical College TNBC accounts for 10-20% of all breast cancer, but much higher proportion of all breast cancer mortality. Younger age at diagnosis, high grade, large tumor size, aggressive relapse. High proliferation, poor differentiation, and aggressive clinical course with early relapse and decreased survival. There is no specific therapeutic target for TNBC, and therefore must use cytotoxic chemotherapeutics, surgery, radiation.

  4. TCMCThe Commonwealth Medical College Current Options for TNBC Standard course of treatment is very aggressive: surgery and radiation therapy combined with adjuvant and neo-adjuvant chemotherapy. Chemotherapy typically includes combinations of taxanes, anthracyclines, and oxazophorines. The search is on for specific therapeutic targets!!!

  5. TCMCThe Commonwealth Medical College It has previously been reported that because of the anti-apoptotic activity of glucocorticoid receptor (GR) in estrogen receptor (ER)-negative breast epithelial and cancer cells, high GR expression/activity in early-stage TNBC significantly correlates with chemotherapy resistance and increased recurrence. A recent study has shown that pre-treatment with mifepristone/RU486, a GR antagonist, potentiates the efficacy of chemotherapy in TNBC by inhibiting the anti-apoptotic signaling pathways of GR and increasing the cytotoxic efficiency of chemotherapy. These results suggest that blocking GR activity could be a useful strategy for increasing tumor cell apoptosis in TNBC. Clin. Cancer Res. 19, 6163–72, 2013

  6. TCMCThe Commonwealth Medical College Another recent study showed that a peptide containing LXXLL motif of steroid receptor coactivator-1 (SRC-1) can severely reduce the viability and proliferation of hormone-unresponsive breast cancer MDA-MB-231 cells. (Int. J. Mol. Sci. 2014, 15, 5680-5698) SRC-1 is known to function as a coactivator for steroid hormone receptors including GR. These results suggest that blocking GR/SRC-1 interaction may be an effective drug candidate in the treatment of TNBC.

  7. TCMCThe Commonwealth Medical College When viewing steroid receptors (e.g., GR) as therapeutic targets, the challenge is how to selectively control cell/tissue and gene specificity in a manner that affects only deleterious actions of receptor in diseased tissues without altering essential normal functions. One attractive, but limited, approach is the development of selective steroid receptor modulators (SRMs) that regulate a subset of the normal gene repertoire. SRMs have the clinically useful but enigmatic property of evoking anywhere from full agonist to full antagonist activity in a gene/tissue-dependent manner. Thus SRMs can display between 100% and 0% efficacy.

  8. COOH NH2 1 77 262 421 481 777 AF1 AF1C DBD LBD AF2 ? Nature 352 (1991) 497-505. Cell 110 (2002) 93-105 TCMCThe Commonwealth Medical College Human Glucocorticoid Receptor (GR) The “Holy Grail” for hormone therapies is to target full SR signaling in a receptor- and tissue/gene-selective manner to maximize therapeutic benefits and to minimize effects on other targets. However, due to available LBD/AF2 crystal structures, the current design of small molecule selective receptor modulators (SRMs) for clinical uses is primarily based on their modulation of AF2 activity, which often fail to inactivate AF1, and thereby missing the whole SR spectrum during endocrine-based therapies. This is despite the fact the most of cell/tissue-specific SRM activities are AF1-dpependent.

  9. TCMCThe Commonwealth Medical College The AF1/NTD of all the SHRs studied so far have been found to exit in an intrinsically disordered (ID) conformation. Experiments showing intrinsically disordered AF1 conformation HSQC NMR spectrum of 15N labeled AF1 Deuterium/hydrogen exchange /mass (HDXM) Similar results are also obtained using several other biophysical methods such as CD and FTIR

  10. TCMCThe Commonwealth Medical College Conformational flexibilityinIDs allows it to interact with binding partners with high specificity and low affinity that means on one hand these provide specific interactions at the same time allow them to be easily dispersed when need arises, an important function of steroid receptors to turn on or turn off signals of target genes as required. ID proteins normally undergo “disorder-to-order” transition upon interactions with their target molecule(s). Like C-terminal AF-2, AF1 interacts with several specific cofactor proteins including several coactivators Question: Does GR AF1 adopt a functionally active ordered structure when bound to a binding partner protein such that its interaction with SRC-1 is facilitated?

  11. Interaction of TBP to agonist-bound PR alters LBD conformation Cell-Structure 22, 961–973, July 8, 2014

  12. Interaction of TBP to antagonist-bound PR alters NTD/AF1 conformation Color bar (% of deuterium uptake difference) Cell- Structure 22, 961–973, July 8, 2014

  13. A therapeutic Model for Endocrine-related cancers TCMCThe Commonwealth Medical College Structure 22, 961–973, July 8, 2014

  14. TCMCThe Commonwealth Medical College TBP SRC-1 Other binding partner protein Ordered GR AF1 GR AF1 and TBP Disordered Multi-protein assembly involved in GR-mediated gene regulation

  15. AF1 DBD LBD AF1c 77 262 421 481 777 1 GRa DAF1 GRa 1 500 GR500 DAF1GR500 Constructs of GR TCMCThe Commonwealth Medical College DAF1cGR500

  16. TCMCThe Commonwealth Medical College CFP-GR500 + YFP-TBP After PB Before PB CFP-DAF1_GR500 YFP-TBP Before PB AfterPB Before PB CFP-GR500 + YFP-TBP+pTAL Before PB After PB FRET analysis showing the GR AF1:TBP interaction in CV-1 cells Copik et al., Mol. Endocrinol. 2006

  17. Kinetics of binding between GR AF1 and TBP by Surface Plasmon resonance (SPR) method using BIACORE 130 110 90 Fc2 70 Response (RU) 50 30 10 Fc1 -10 -50 0 50 100 150 200 250 300 Time (s) [AF1] (μM) 295 6.4 245 3.2 195 KD = 0.46 µM 145 1.6 Response (RU) 95 0.8 0.4 45 0.2 0.0 -5 290 330 370 410 450 490 530 570 610 650 Time (s)

  18. Far-UV CD spectra of AF1:TBP complex shows that AF1 adopts ordered structure when bound to TBP

  19. TCMCThe Commonwealth Medical College Far-UV CD spectra of TBP at various concentrations showing no structural changing happening in TBP

  20. TCMCThe Commonwealth Medical College TBP-binding/folding induced conformation in AF1 facilitates AF1’s interaction with SRC-1 AF1+NE AF1+TBP+NE NE IP = SRC-1 and blot = AF1 antibody 1 2 3 4 5 *

  21. TCMCThe Commonwealth Medical College Synergistic effects of TBP and SRC-1 on GR AF1 activity. GRE-SEAP

  22. TCMCThe Commonwealth Medical College Western blots showing higher levels of TBP and SRC-1 expression in MDA-MB-231 cells compared to MCF-7 cells. MCF-7 MDA-MB-231 Nuclear GR C Dex C Dex MCF-7 MDA-MB-231 TBP SRC-1

  23. TBP-binding to GR AF1 enhances its interaction with SRC-1 in NDA MB 231 cell nuclear extracts (NE) MDA-MB-231 NE AF1 + NE AF1 + TBPc + NE TBPc + NE NE IP: SRC-1 IB: GR FoldIncrease

  24. TCMCThe Commonwealth Medical College Dex treatment results into higher cell viability of MDA MB-231 cells Control Dex treated WST1-Absorbance

  25. TCMCThe Commonwealth Medical College TBP Drug SRC-1 AF1/NTD Other binding partner protein Tissue specific gene regulation and a drug target for TNBC

  26. TCMCThe Commonwealth Medical College Acknowledgements Anna S. Garza Alicja J. Copik Shagufta Khan Jun Ling Christian Carbe Santina Possanza S. Stoney Simons, Jr., NIDDK, National Institutes of Health, Bethesda, MD. Patrick R. Griffin, The Scripps Research Institute, Jupiter, FL. Dean P. Edwards, Baylor College of Medicine, Houston, TX. Iain J. McEwan, University of Aberdeen, Aberdeen, Scotland, UK. E. Brad Thompson, University of Houston, Houston, TX Ravi Jasuja, Harvard Medical School, Boston, MA

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