Dec 17, 2021 Uncategorized



background  : Discuss

objectives : Discuss

Objective 1: determine single-agent IC50 concentrations for Fulvestrant, CDK4/6 inhibitor (abemaciclib), Rac1 inhibitor (GYS32661) in wild type (WT) and mutant ESR1 (Y527S, D538G) MCF7 cell line, and determine effects on ER-mediated gene expression in the mutant cells

  • Skills involved: cell culture, drug dilution, proliferation assays, PCR + RTqPCR

*Sub objective:  determine Rac1 activity in WT, ESR1 mutant cell lines treated +/- IC50 of agents

Objective 2: Determine the combinatorial efficacy of fulvestrant +/- CDk4/6 inhibitor +/- Rac1 inhibitor in WT and mutant ESR1 MCF7 cells on cell proliferation, 2D colony formation, 3D colony formation

  1. No estrogen control
  2. Estrogen (E2) stimulation
  3. Fulvestrant alone (+E2)
  4. CDK4/6inhibitor alone (abemaciclib)  (+E2)
  5. Rac1 inhibitor alone (GYS32661) (+E2)
  6. Fulv + CDK4/6i (+E2)
  7. Fulv + Rac1i (+E2)
  8. CDK4/6i + Rac1i (+E2)
  9. Fulv + CDK4/6i + Rac1 I (+E2)

literature search : See references (last 10 years)

draft methodology:

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5.            Lei, J.T., Gou, X., Seker, S. & Ellis, M.J. ESR1 alterations and metastasis in estrogen receptor positive breast cancer. J Cancer Metastasis Treat 5(2019).

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12.          Guarducci, C., et al. Mechanisms of Resistance to CDK4/6 Inhibitors in Breast Cancer and Potential Biomarkers of Response. Breast Care (Basel) 12, 304-308 (2017).

13.          Jamieson, C., Lui, C., Brocardo, M.G., Martino-Echarri, E. & Henderson, B.R. Rac1 augments Wnt signaling by stimulating beta-catenin-lymphoid enhancer factor-1 complex assembly independent of beta-catenin nuclear import. J Cell Sci 128, 3933-3946 (2015).

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17.          Fanning, S.W., et al. Estrogen receptor alpha somatic mutations Y537S and D538G confer breast cancer endocrine resistance by stabilizing the activating function-2 binding conformation. Elife 5(2016).

18.          Harrod, A., et al. Genomic modelling of the ESR1 Y537S mutation for evaluating function and new therapeutic approaches for metastatic breast cancer. Oncogene 36, 2286-2296 (2017).

19.          Jeselsohn, R., et al. Allele-Specific Chromatin Recruitment and Therapeutic Vulnerabilities of ESR1 Activating Mutations. Cancer Cell 33, 173-186 e175 (2018).

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23.          Heasman, S.J. & Ridley, A.J. Mammalian Rho GTPases: new insights into their functions from in vivo studies. Nat Rev Mol Cell Biol 9, 690-701 (2008).

24.          Burridge, K. & Wennerberg, K. Rho and Rac take center stage. Cell 116, 167-179 (2004).

25.          Rathinam, R., Berrier, A. & Alahari, S.K. Role of Rho GTPases and their regulators in cancer progression. Front Biosci (Landmark Ed) 16, 2561-2571 (2011).

26.          Wang, S.E., Shin, I., Wu, F.Y., Friedman, D.B. & Arteaga, C.L. HER2/Neu (ErbB2) signaling to Rac1-Pak1 is temporally and spatially modulated by transforming growth factor beta. Cancer Res 66, 9591-9600 (2006).

27.          Zhu, G., et al. An EGFR/PI3K/AKT axis promotes accumulation of the Rac1-GEF Tiam1 that is critical in EGFR-driven tumorigenesis. Oncogene 34, 5971-5982 (2015).

28.          Goka, E.T. & Lippman, M.E. Loss of the E3 ubiquitin ligase HACE1 results in enhanced Rac1 signaling contributing to breast cancer progression. Oncogene 34, 5395-5405 (2015).

29.          Akunuru, S., Palumbo, J., Zhai, Q.J. & Zheng, Y. Rac1 targeting suppresses human non-small cell lung adenocarcinoma cancer stem cell activity. PLoS One 6, e16951 (2011).

30.          Khosravi-Far, R., Solski, P.A., Clark, G.J., Kinch, M.S. & Der, C.J. Activation of Rac1, RhoA, and mitogen-activated protein kinases is required for Ras transformation. Mol Cell Biol 15, 6443-6453 (1995).

31.          Cai, D., et al. AND-34/BCAR3, a GDP exchange factor whose overexpression confers antiestrogen resistance, activates Rac, PAK1, and the cyclin D1 promoter. Cancer Res 63, 6802-6808 (2003).

32.          Felekkis, K.N., et al. AND-34 activates phosphatidylinositol 3-kinase and induces anti-estrogen resistance in a SH2 and GDP exchange factor-like domain-dependent manner. Mol Cancer Res 3, 32-41 (2005).

33.          Formisano, L., et al. Aberrant FGFR signaling mediates resistance to CDK4/6 inhibitors in ER+ breast cancer. Nature communications 10, 1373 (2019).

34.          Wander, S.A., et al. The Genomic Landscape of Intrinsic and Acquired Resistance to Cyclin-Dependent Kinase 4/6 Inhibitors in Patients with Hormone Receptor-Positive Metastatic Breast Cancer. Cancer discovery 10, 1174-1193 (2020).

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36.          Knight-Krajewski, S., et al. Deregulation of the Rho GTPase, Rac1, suppresses cyclin-dependent kinase inhibitor p21(CIP1) levels in androgen-independent human prostate cancer cells. Oncogene 23, 5513-5522 (2004).

37.          Michaelson, D., et al. Rac1 accumulates in the nucleus during the G2 phase of the cell cycle and promotes cell division. The Journal of cell biology 181, 485-496 (2008).

38.          May, M., Schelle, I., Brakebusch, C., Rottner, K. & Genth, H. Rac1-dependent recruitment of PAK2 to G2 phase centrosomes and their roles in the regulation of mitotic entry. Cell Cycle 13, 2211-2221 (2014).

39.          Gao, Y., Dickerson, J.B., Guo, F., Zheng, J. & Zheng, Y. Rational design and characterization of a Rac GTPase-specific small molecule inhibitor. Proc Natl Acad Sci U S A 101, 7618-7623 (2004).

40.          Gastonguay, A., et al. The role of Rac1 in the regulation of NF-kappaB activity, cell proliferation, and cell migration in non-small cell lung carcinoma. Cancer Biol Ther 13, 647-656 (2012).

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