Estrogen Receptor Agonists, Antagonists, and Endocrine Resistance

Estrogen Receptor Agonist and Antagonist Mechanisms

• Estrogen Receptor (ER) Agonist vs. Antagonist:

  • The difference between agonist and antagonist action is most thoroughly studied for the estrogen receptor.

  • Agonist (Estradiol) Binding:

    • Estradiol binds to the Ligand Binding Domain (LBD).

    • It positions helix 12 (H12) in an active conformation.

    • This active H12 position is stabilized by Asp 351.

    • Asp 351 is located in the middle of the long H3 helix, opposite the N-terminal end of H12.

    • Three NH groups protruding from the helix end of H12 are about $3-4\mathring{A}$ away from the carboxylate groups of the acidic residue Asp 351, stabilizing its negative charges.

  • Antagonist Binding:

    • Antagonists possess side chains that remain in the entrance channel after ligand binding.

    • These side chains prevent the proper closure of H12.

    • A basic group at the end of the antagonist's side chain is likely in a positively charged state.

    • This basic group forms a salt bridge with Asp 351, disrupting the H12 stabilization achieved by agonists.

• Structural Basis of ER LBD Helix 12 Switch:

  

  • A study demonstrated the crucial role of H12 movement.

  • With agonist binding, H12 moves to open a groove, allowing for the binding of LxxLL peptide motifs found in coactivators.

  • With antagonist binding, H12 physically moves into the position where the LxxLL peptide would typically bind, thereby blocking coactivator interaction.

Estrogen Receptor Association with Coactivator Complex

• Primary Coactivators (SRCs):

  • Steroid Receptor Coactivators (SRCs) are primary coactivators interacting with Nuclear Receptors (NRs), including Estrogen Receptors (ERs) and Androgen Receptors (ARs).

• Secondary Coactivators:

  • SRCs, in turn, recruit multiple secondary coactivators, such as the histone acetyltransferases (HATs) p300/CBP.

• Transcriptional Activation:

  • These components form a minimal receptor-coactivator complex.

  • This complex can promote chromatin remodeling and facilitate transcriptional activation of target genes.

• Binding of ER with Coactivators:

  

  • Electron cryo-microscopy (cryo-EM) was used to study the quaternary structure of the NR and coactivator complex.

  • The complex (~$720$ kDa) included ER$\alpha$, SRC-3, p300, and CARM1, bound to a $700$bp Estrogen Response Element (ERE).

  • This complex exhibited transcriptional activity.

  • The resolution of the cryo-EM map (~25\mathring{A}}) was lower than typical crystal structures of individual domains like the LBD.

  • Key Findings:

    • Multiple interactions were found between SRC-3 and p300.

    • Four distinct regions of p300 interact with SRC-3.

    • This interaction induces a conformational change in p300, which increases its histone H3 acetylation activity.

    • The N-terminal domain (NTD), also known as Activation Function 1 (AF1), is located near the LBD (AF-2).

    • AF1 participates in SRC-3 recruitment alongside AF-2.

      

• Intrinsically Disordered Regions:

  • Both SRC and p300/CBP appear to contain intrinsically disordered regions.

  • These regions can convert to consistent structures upon interacting with other proteins, highlighting their dynamic nature in complex formation.

ER$\alpha$ Functions Out of the Ordinary

1. Action Independent of ERE (Indirect Genomic/Non-classical Pathway):

   • ER$\alpha$ can act as a co-regulator for other transcription factors, such as AP-1/c-Jun, ATF-2, NF-$\kappa$B, Sp1, and Sp3.

   • Sp-1 and AP-1 are observed to be dominant partners.

   • This pathway is significant because approximately $35\%$ of human primary estrogen-responsive genes lack ERE-like promoter sequences.

   • Caveat: Despite these ERE-independent actions, transgenic mice expressing ER$\alpha$ mutants lacking DNA-binding ability show phenotypes similar to ER$\alpha$-null mice (e.g., loss of fertility in both males and females).

   • This indicates that multiple key functions of ER$\alpha$ are still mediated via ERE.

2. Activation Without Ligand (Ligand-Independent Activation):

   

   • ER can be activated in the absence of its classical ligand, estrogen.

   • Mitogen-Activated Protein Kinase (MAPK) and Phosphoinositide 3-kinase (PI3K) Cascades:

     • Growth factors (e.g., EGF and IGF-1) activate these cascades.

     • These pathways phosphorylate cytoplasmic ER$\alpha$.

     • This phosphorylation leads to Estrogen-2 (E2)-independent nuclear import and subsequent ER$\alpha$ regulation of transcription.

   • Specific Pathways:

     • RTK/c-Src Pathway: c-Src, a Receptor Tyrosine Kinase (RTK), can activate ER via the PI3K/Akt pathway.

       • This is mediated by phosphorylation of ER at Serine 167 (S167).

       • mTOR also contributes to increased Akt activation.

       • Blocking this pathway is a potential therapeutic route for breast cancer.

     • Ras/Raf/MEK/ERK Signaling Pathway: Growth factors binding to RTK receptors activate this pathway.

       • This promotes the phosphorylation of Serine 118 (S118) in the AF-1 domain of ER.

       • Phosphorylation at S118 activates ER-target gene transcriptional activity.

   • NTD Phosphorylation: Phosphorylation in the N-terminal domain (NTD) generally promotes transactivation.

   • Calcium Activation: Ca$^2+$ can activate ER$\alpha$ via its LBD, inducing the expression of estrogen-responsive genes in the absence of ligand.

     

     • There are four potential calcium binding sites in the LBD.

3. Membrane Estrogen Receptors:

   • ERs can exist as plasma membrane-associated receptors, mediating rapid, non-genomic (independent of gene expression) pathways.

   • Membrane Localization:

     • Membrane localization of ER is mediated by Heat Shock Protein 27 (HSP27).

     • It associates with caveolae lipid rafts in the membrane through interactions with caveolin-1, Src, and striatin.

     • Asp-His-His-Cys (DHHC) domain palmitoyl acyltransferases (DHHC-7 and -21) palmitoylate ER$\alpha$ and promote its plasma membrane localization.

     • Approximately $5\%$ of total ER$\alpha$ is localized to the plasma membrane.

   • Signaling Pathways from Membrane ERs:

     • Estrogen-bound membrane-localized ER directly interacts with RTKs (e.g., the p85 regulatory subunit of PI3K, Src, and Shc).

     • These interactions activate PI3K/Akt/mTOR as well as RAS/RAF/MEK1/2-ERK1/2 signaling pathways.

     • These kinase pathways promote cell survival and proliferation.

     • They also phosphorylate ER and its coregulators, leading to the activation of nuclear genomic transcription.

4. Other Functions (Other Crazy Stuff):

   • Control of Mitochondria: ER$\alpha$ localizes in the mitochondria and mediates the transcription of mitochondrial DNA (mtDNA) following E2 stimulation.

     • This is possible due to ERE-like sequences found in the D-loop of mtDNA.

   • mRNA Binding and Regulation of Translation: ER$\alpha$ binds to many mRNAs, primarily at the $3^{\prime}$ Untranslated Region (UTR), to regulate translation.

     • It interacts with proteins that regulate RNA splicing, stability, and XPB1 (involved in unfolded protein response).

Endocrine Resistance

• Loss of ER Expression:

  

  • Approximately $20\%$ of breast cancer patients treated with endocrine therapy eventually lose ER expression over time.

  • Loss of ER is problematic because tumor growth becomes independent of ER, making the endocrine pathway ineffective for control.

• Control of ER Expression (ESR1 Gene):

  • Methylation: ER$\alpha$ has at least nine promoters and CpG islands for the A, B, and C promoters of ESR1, which are highly methylated in ER-negative breast cancer cases.

    • Methylation status is negatively correlated with ER$\alpha$ expression and resistance to endocrine treatment in breast cancer cells.

  • Histone Acetylation: Histone acetylation is involved in gene control.

    • Histone deacetylase (HDAC) inhibitors, such as trichostatin A, have been shown to re-express ER$\alpha$ in ER-negative breast cancer cells.

    • DNA demethylating agents and HDAC inhibitors restored tamoxifen sensitivity in ER-negative breast cancer cells.

  • RNA Stabilization:

    • AU-rich elements (AREs), typically AUUUA motifs, in the $3^{\prime}$-UTR of ESR1 mRNA are critical for its stabilization.

    • ESR1 mRNA contains $13$ AUUUA motifs.

    • The ARE-binding protein HuR binds to ESR1 mRNA to stabilize it.

    • More than ten types of microRNAs (miRNAs) directly bind to ESR1 mRNA and repress its expression.

• ER Post-Translational Modification (PTM):

  

  • ER can be extensively modified, including phosphorylation, methylation, acetylation, SUMOylation, glycosylation, ubiquitination, palmitoylation, and isomerization.

  • ER has at least $20$ known phosphorylation sites.

  • These PTMs can control ER stabilization or activation.

  • Calcineurin Influence: Calcineurin, a Ca$^{2+}$-dependent serine/threonine phosphatase, stabilizes and activates ER$\alpha$.

    • High calcineurin expression is associated with shorter recurrence-free survival in endocrine-treated ER-positive breast cancer patients.

      

  • Ubiquitination:

    

    • Fulvestrant, a Selective Estrogen Receptor Downregulator (SERD), exerts its tumor suppression by inhibiting ER$\alpha$ through antagonistic action AND by promoting ER$\alpha$ degradation via the ubiquitin proteasome system.

    • E3 ligases that polyubiquitinate ER$\alpha$ (e.g., E6AP, CHIP, SKP2) generally promote its degradation.

    • E3 ligases that monoubiquitinate ER$\alpha$ (e.g., BRCA1/BARD1, RNF8, RNF31, and TRIM11) tend to stabilize ER$\alpha$.

      

    • A detailed understanding of ER$\alpha$ ubiquitination sites is not fully achieved, as ubiquitination is not always site-specific (e.g., mutation of a primary target lysine can result in ubiquitination of neighboring lysines).

    • K302 and K303 are listed as major monoubiquitination sites for BRCA1/BARD1 and major polyubiquitination sites triggered by fulvestrant.

• ER Mutation (Hot Spot Mutations):

  • ER mutations are commonly found within the LBD.

  • The most common hot spot mutations are Y537S, Y537N, and D538G.

  • These mutations control the agonist state of the ER LBD, affecting conformational changes and protein stability.

  • Mutations at these residues alter the conformational dynamics of the loop connecting H11 and H12 in the ER LBD, leading to a stabilized agonist state that is unaffected by antagonists.

  • Y537 and D538 ER mutants are phosphorylated on S118 by the TFIIH kinase, cyclin-dependent kinase (CDK)7, in an estrogen-independent manner.

  • This phosphorylation may lead to endocrine resistance by increasing transcriptional activity.

• Influence of Coregulators:

  • Cancer likelihood may increase if the balance or expression of coregulators is altered, affecting ER signaling and therapeutic response.