19 research outputs found
Étude du rôle du récepteur ERa-36 dans la signalisation non génomique des oestrogènes
We study a novel splice variant of ERa, named ERa36, and its involvement in estrogen non genomic signaling. Unlike ERa, this variant has main cytoplasmic/plasma membrane localization and alternative splicing confers it with a unique, previously unidentified C-terminal domain. Interestingly, we found that ERa36 C-terminal domain contains a putative MAPK binding D-Domain for the serine/threonine kinase ERK2. This domain is a docking site for members of the MAPK family. Coupling in-vitro and in-cellulo approaches, we demonstrated that ERa36 binds specifically to ERK2 following estrogen, as well as clinical anti-estrogen (tamoxifen) stimulation.We demonstrated that ERa36 binding to ERK2 inhibits the latter’s dephosphorylation by the dual phosphatase MKP3, thereby leading to a sustained ERK activation. This mechanism had profound effects on ERK’s downstream molecular targets. In fact, pharmacological inhibition of the ERa36/ERK2 interaction abrogated the phosphorylation of Paxillin, which in turn led to a downregulation of CyclinD1 transcription.Futhermore, IHC analysis of ERa36 expression in 175 patient breast tumors revealed that its expression constituted an independent predictor of distant metastasis and influenced on overall survival. In conclusion, ERa36 expression could constitute a new biomarker in breast cancerNous avons étudié un nouveau varant d'épissage de ERa, nommé ERa36 et son implication dans la signalisation non génomique des œstrogènes. Contrairement à ERa, ce variant a une localisation majoritairement cytoplasmique et membranaire. Il possède une partie C-Terminale unique due à l'épissage alternatif. Nous avons découvert que le domaine C-Termonal de ERa36 contient un D-Domain, qui lui confère la capacité de se lier directement avec des protéines de la famille MAPK. En utilisant des approches in-vitro et in-cellulo, nous avons démontré que ERa36 se lie spécifiquement à la kinase ERK2 en réponse d'une stimulation ostrogénique ou anti-ostrogénique. Nous avons démontré que ERK2 lié à ERa36 lui conférait une résistance à la déphosphorylation par la phosphatase MKP3, conduisant ainsi à une activation soutenue de la voir ERK. Ce mécanisme a des effets profonds sur les cibles de ERK. En effet, l'inhibition pharmacologique de l'interaction ERa36/ERK2 diminue la phosphorylation de la Paxilline, qui a son tour conduit à une répression de la Cycline D1. En plus de ces observations, nous avons démontré, en étudiant l'expression de ERa36 par IHC dans 175 tumeurs de sein, que son expression était un facteur prédictif de métastases à distance et conduit à une diminution de la survie globale. Ce travail pourrait amener à dire que l'expression de ERa36 constitue un nouveau biomarqueur dans le cancer du sei
Analysis of HER2 genomic binding in breast cancer cells identifies a global role in direct gene regulation.
HER2 is a transmembrane receptor tyrosine kinase, which plays a key role in breast cancer due to a common genomic amplification. It is used as a marker to stratify patients in the clinic and is targeted by a number of drugs including Trastuzumab and Lapatinib. HER2 has previously been shown to translocate to the nucleus. In this study, we have explored the properties of nuclear HER2 by analysing the binding of this protein to the chromatin in two breast cancer cell lines. We find genome-wide re-programming of HER2 binding after treatment with the growth factor EGF and have identified a de novo motif at HER2 binding sites. Over 2,000 HER2 binding sites are found in both breast cancer cell lines after EGF treatment, and according to pathway analysis, these binding sites were enriched near genes involved in protein kinase activity and signal transduction. HER2 was shown to co-localise at a small subset of regions demarcated by H3K4me1, a hallmark of functional enhancer elements and HER2/H3K4me1 co-bound regions were enriched near EGF regulated genes providing evidence for their functional role as regulatory elements. A chromatin bound role for HER2 was verified by independent methods, including Proximity Ligation Assay (PLA), which confirmed a close association between HER2 and H3K4me1. Mass spectrometry analysis of the chromatin bound HER2 complex identified EGFR and STAT3 as interacting partners in the nucleus. These findings reveal a global role for HER2 as a chromatin-associated factor that binds to enhancer elements to elicit direct gene expression events in breast cancer cells
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A general approach for the site-selective modification of native proteins, enabling the generation of stable and functional antibody-drug conjugates.
Antibody-drug conjugates (ADCs) are a class of targeted therapeutics that utilize the specificity of antibodies to selectively deliver highly potent cytotoxins to target cells. Although recent years have witnessed significant interest in ADCs, problems remain with the standard linkage chemistries used for cytotoxin-antibody bioconjugation. These typically (1) generate unstable constructs, which may lead to premature cytotoxin release, (2) often give a wide variance in drug-antibody ratios (DAR) and (3) have poor control of attachment location on the antibody, resulting in a variable pharmacokinetic profile. Herein, we report a novel divinylpyrimidine (DVP) linker platform for selective bioconjugation via covalent re-bridging of reduced disulfide bonds on native antibodies. Model studies using the non-engineered trastuzumab antibody validate the utility of this linker platform for the generic generation of highly plasma-stable and functional antibody constructs that incorporate variable biologically relevant payloads (including cytotoxins) in an efficient and site-selective manner with precise control over DAR. DVP linkers were also used to efficiently re-bridge both monomeric and dimeric protein systems, demonstrating their potential utility for general protein modification, protein stabilisation or the development of other protein-conjugate therapeutics.AstraZeneca, Cambridge Trusts, EPSRC, BBSRC, Royal Society, MR
ERα-36 regulates progesterone receptor activity in breast cancer.
BACKGROUND: Alterations in estrogen and progesterone signaling, via their respective receptors, estrogen receptor alpha (ERα) and progesterone receptor (PR), respectively, are largely involved in the development of breast cancer (BC). The recent identification of ERα-36, a splice variant of ERα, has uncovered a new facet of this pathology. Although ERα-36 expression is associated with poor prognosis, metastasis development, and resistance to treatment, its predictive value has so far not been associated with a BC subtype and its mechanisms of action remain understudied. METHODS: To study ERα-36 expression in BC specimens, we performed immunochemical experiments. Next, the role of ERα-36 in progesterone signaling was investigated by generating KO clones using the CRISPR/CAS9 technology. PR signaling was also assessed by proximity ligation assay, Western blotting, RT-QPCR, and ChIP experiments. Finally, proliferation assays were performed with the IncuCyte technology and migration experiments using scratch assays. RESULTS: Here, we demonstrate that ERα-36 expression at the plasma membrane is correlated with a reduced disease-free survival in a cohort of 160 BC patients, particularly in PR-positive tumors, suggesting a crosstalk between ERα-36 and PR. Indeed, we show that ERα-36 interacts constitutively with PR in the nucleus of tumor cells. Moreover, it regulates PR expression and phosphorylation on key residues, impacting the biological effects of progesterone. CONCLUSIONS: ERα-36 is thus a regulator of PR signaling, interfering with its transcriptional activity and progesterone-induced anti-proliferative effects as well as migratory capacity. Hence, ERα-36 may constitute a new prognostic marker as well as a potential target in PR-positive BC
Study of the role of estrogen receptor variant, ERa36, in non genomic signaling and breast cancer
Nous avons étudié un nouveau varant d'épissage de ERa, nommé ERa36 et son implication dans la signalisation non génomique des œstrogènes. Contrairement à ERa, ce variant a une localisation majoritairement cytoplasmique et membranaire. Il possède une partie C-Terminale unique due à l'épissage alternatif. Nous avons découvert que le domaine C-Termonal de ERa36 contient un D-Domain, qui lui confère la capacité de se lier directement avec des protéines de la famille MAPK. En utilisant des approches in-vitro et in-cellulo, nous avons démontré que ERa36 se lie spécifiquement à la kinase ERK2 en réponse d'une stimulation ostrogénique ou anti-ostrogénique. Nous avons démontré que ERK2 lié à ERa36 lui conférait une résistance à la déphosphorylation par la phosphatase MKP3, conduisant ainsi à une activation soutenue de la voir ERK. Ce mécanisme a des effets profonds sur les cibles de ERK. En effet, l'inhibition pharmacologique de l'interaction ERa36/ERK2 diminue la phosphorylation de la Paxilline, qui a son tour conduit à une répression de la Cycline D1. En plus de ces observations, nous avons démontré, en étudiant l'expression de ERa36 par IHC dans 175 tumeurs de sein, que son expression était un facteur prédictif de métastases à distance et conduit à une diminution de la survie globale. Ce travail pourrait amener à dire que l'expression de ERa36 constitue un nouveau biomarqueur dans le cancer du seinWe study a novel splice variant of ERa, named ERa36, and its involvement in estrogen non genomic signaling. Unlike ERa, this variant has main cytoplasmic/plasma membrane localization and alternative splicing confers it with a unique, previously unidentified C-terminal domain. Interestingly, we found that ERa36 C-terminal domain contains a putative MAPK binding D-Domain for the serine/threonine kinase ERK2. This domain is a docking site for members of the MAPK family. Coupling in-vitro and in-cellulo approaches, we demonstrated that ERa36 binds specifically to ERK2 following estrogen, as well as clinical anti-estrogen (tamoxifen) stimulation.We demonstrated that ERa36 binding to ERK2 inhibits the latter’s dephosphorylation by the dual phosphatase MKP3, thereby leading to a sustained ERK activation. This mechanism had profound effects on ERK’s downstream molecular targets. In fact, pharmacological inhibition of the ERa36/ERK2 interaction abrogated the phosphorylation of Paxillin, which in turn led to a downregulation of CyclinD1 transcription.Futhermore, IHC analysis of ERa36 expression in 175 patient breast tumors revealed that its expression constituted an independent predictor of distant metastasis and influenced on overall survival. In conclusion, ERa36 expression could constitute a new biomarker in breast cance
Sulfatase-cleavable linkers for antibody-drug conjugates.
Antibody-drug conjugates (ADCs) are a class of targeted drug delivery agents combining the cell-selectivity of monoclonal antibodies (mAbs) and the cytotoxicity of small molecules. These two components are joined by a covalent linker, whose nature is critical to the efficacy and safety of the ADC. Enzyme-cleavable dipeptidic linkers have emerged as a particularly effective ADC linker type due to their ability to selectively release the payload in the lysosomes of target cells. However, these linkers have a number of drawbacks, including instability in rodent plasma and their inherently high hydrophobicity. Here we show that arylsulfate-containing ADC linkers are cleaved by lysosomal sulfatase enzymes to tracelessly release their payload, while circumventing the instability problems associated with dipeptide-linkers. When incorporated with trastuzumab and the highly potent monomethyl auristatin E (MMAE) payload, the arylsulfate-containing ADC 2 and ADC 3 were more cytotoxic than the non-cleavable ADC 4 against HER2-positive cells, while maintaining selectivity over HER2-negative cells. We propose that the stability, solubility and synthetic tractability of our arylsulfate linkers make them an attractive new motif for cleavable ADC linkers, with clear benefits over the widely used dipeptidic linkers
The arginine methyltransferase PRMT1 regulates IGF-1 signaling in breast cancer
International audienceAside from its well-known nuclear routes of signaling, estrogen also mediates its effects through cytoplasmic signaling. Estrogen signaling involves numerous posttranslational modifications of its receptor ERα, the best known being phosphorylation. Our research group previously showed that upon estrogen stimulation, ERα is methylated on residue R260 and forms the mERα/Src/PI3K complex, central to the rapid transduction of nongenomic estrogen signals. Regulation of ERα signaling via its phosphorylation by growth factors is well recognized, and we wondered whether they could also trigger ERα methylation (mERα). Here, we found that IGF-1 treatment of MCF-7 cells induced rapid ERα methylation by the arginine methyltransferase PRMT1 and triggered the binding of mERα to IGF-1R. Mechanistically, we showed that PRMT1 bound constitutively to IGF-1R and that PRMT1 became activated upon IGF-1 stimulation. Moreover, we found that expression or pharmacological inhibition of PRMT1 impaired mERα and IGF-1 signaling. Our findings were substantiated in a cohort of breast tumors in which IGF-1R expression was positively correlated with ERα/Src and ERα/PI3K expression, hallmarks of nongenomic estrogen signaling, reinforcing the link between IGF-1R and mERα. Altogether, these results provide a new insight into ERα and IGF-1R interference, and open novel perspectives for combining endocrine therapies with PRMT1 inhibitors in ERα-positive tumors
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ERα-36 regulates progesterone receptor activity in breast cancer
Abstract: Background: Alterations in estrogen and progesterone signaling, via their respective receptors, estrogen receptor alpha (ERα) and progesterone receptor (PR), respectively, are largely involved in the development of breast cancer (BC). The recent identification of ERα-36, a splice variant of ERα, has uncovered a new facet of this pathology. Although ERα-36 expression is associated with poor prognosis, metastasis development, and resistance to treatment, its predictive value has so far not been associated with a BC subtype and its mechanisms of action remain understudied. Methods: To study ERα-36 expression in BC specimens, we performed immunochemical experiments. Next, the role of ERα-36 in progesterone signaling was investigated by generating KO clones using the CRISPR/CAS9 technology. PR signaling was also assessed by proximity ligation assay, Western blotting, RT-QPCR, and ChIP experiments. Finally, proliferation assays were performed with the IncuCyte technology and migration experiments using scratch assays. Results: Here, we demonstrate that ERα-36 expression at the plasma membrane is correlated with a reduced disease-free survival in a cohort of 160 BC patients, particularly in PR-positive tumors, suggesting a crosstalk between ERα-36 and PR. Indeed, we show that ERα-36 interacts constitutively with PR in the nucleus of tumor cells. Moreover, it regulates PR expression and phosphorylation on key residues, impacting the biological effects of progesterone. Conclusions: ERα-36 is thus a regulator of PR signaling, interfering with its transcriptional activity and progesterone-induced anti-proliferative effects as well as migratory capacity. Hence, ERα-36 may constitute a new prognostic marker as well as a potential target in PR-positive BC
ERα-36 regulates progesterone receptor activity in breast cancer
International audienceAbstract Background Alterations in estrogen and progesterone signaling, via their respective receptors, estrogen receptor alpha (ERα) and progesterone receptor (PR), respectively, are largely involved in the development of breast cancer (BC). The recent identification of ERα-36, a splice variant of ERα, has uncovered a new facet of this pathology. Although ERα-36 expression is associated with poor prognosis, metastasis development, and resistance to treatment, its predictive value has so far not been associated with a BC subtype and its mechanisms of action remain understudied. Methods To study ERα-36 expression in BC specimens, we performed immunochemical experiments. Next, the role of ERα-36 in progesterone signaling was investigated by generating KO clones using the CRISPR/CAS9 technology. PR signaling was also assessed by proximity ligation assay, Western blotting, RT-QPCR, and ChIP experiments. Finally, proliferation assays were performed with the IncuCyte technology and migration experiments using scratch assays. Results Here, we demonstrate that ERα-36 expression at the plasma membrane is correlated with a reduced disease-free survival in a cohort of 160 BC patients, particularly in PR-positive tumors, suggesting a crosstalk between ERα-36 and PR. Indeed, we show that ERα-36 interacts constitutively with PR in the nucleus of tumor cells. Moreover, it regulates PR expression and phosphorylation on key residues, impacting the biological effects of progesterone. Conclusions ERα-36 is thus a regulator of PR signaling, interfering with its transcriptional activity and progesterone-induced anti-proliferative effects as well as migratory capacity. Hence, ERα-36 may constitute a new prognostic marker as well as a potential target in PR-positive BC