proximity-ligation-assay-allows-detection-localization-quantification

Proximity Ligation Assay Allows the Detection, Localization, and Quantification of Protein Arginine Methylation in Fixed Tissue

Arginine methylation is emerging as a key post-translational modification involved in a large range of biological processes. Its study in tissue is often ...

Université de Lyon

Centre de Recherche en Cancérologie de Lyon

Protein arginine methylation is a common post translational modification that regulates protein properties. This modification is carried out by a family of nine arginine methyltransferases (PRMTs). Arginine methylation has already been linked to tumourigenesis as overexpression of these enzymes was associated with various cancers, notably in breast cancers. Since the Jumonji Domain Containing 6 protein (JMJD6) possesses an arginine demethylase activity able to remove the methyl mark, we wanted to assess its potential role in breast tumourigenesis.

Role of JMJD6 in Breast Tumourigenesis.

Endocrine therapies targeting oestrogen signalling have significantly improved breast cancer management. However, their efficacy is limited by intrinsic and acquired resistance to treatment, which remains a major challenge for oestrogen receptor α (ERα)-positive tumours. Though many studies using in vitro models of endocrine resistance have identified putative actors of resistance, no consensus has been reached. We demonstrated previously that oestrogen non-genomic signalling, characterized by the formation of the ERα/Src/PI3K complex, is activated in aggressive breast cancers (BC). We wondered herein whether the activation of this pathway is also involved in resistance to endocrine therapies. We studied the interactions between ERα and Src or PI3K by proximity ligation assay (PLA) in in-vitro and in-vivo endocrine therapy-resistant breast cancer models. We reveal an increase in ERα/Src and ERα/PI3K interactions in patient-derived xenografts (PDXs) with acquired resistance to tamoxifen, as well as in tamoxifen-resistant MCF-7 cells compared to parental counterparts. Moreover, no interactions were observed in breast cancer cells resistant to other endocrine therapies. Finally, the use of a peptide inhibiting the ERα-Src interaction partially restored tamoxifen sensitivity in resistant cells, suggesting that such components could constitute promising targets to circumvent resistance to tamoxifen in BC.

Oestrogen Non-Genomic Signalling is Activated in Tamoxifen-Resistant Breast Cancer.

Arginine methylation is now recognized as a major contributor to proteome diversity and is, as such, involved in a large range of cellular processes. There is a growing need for assessing endogenous protein arginine methylation in cells. Besides the classical immunoprecipitation, in situ proximity ligation assay (PLA) is a useful technique allowing at the same time the detection, localization and quantification of arginine methylation of a given protein within a cellular context. Here, we described in depth a standard PLA protocol applied to the detection of arginine methylation in combination with RNA interference and specific methyltransferase inhibitors. We demonstrated that the glucocorticoid receptor is methylated by the arginine methyltransferase PRMT5 inside the nucleus of MCF-7 cells. In addition, the automated quantification of protein arginine methylation performed using Image J is reported. Hence, we demonstrated that PLA offers a novel approach to study protein arginine methylation and could be extended to other post-translational modifications when specific antibodies are available.

Using proximity ligation assay to detect protein arginine methylation.

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.

ERα-36 regulates progesterone receptor activity in breast cancer.

Endocrine therapies targeting estrogen signaling have significantly improved breast cancer (BC) patient survival, although 40% of ERα-positive BCs do not respond to those therapies. Aside from genomic signaling, estrogen triggers non-genomic pathways by forming a complex containing methylERα/Src/PI3K, a hallmark of aggressiveness and resistance to tamoxifen. We aimed to confirm the prognostic value of this complex and investigated whether its targeting could improve tumor response in vivo.

Analysis of genomic and non-genomic signaling of estrogen receptor in PDX models of breast cancer treated with a combination of the PI3K inhibitor alpelisib (BYL719) and fulvestrant.

Article	Year
Role of JMJD6 in Breast Tumourigenesis. PloS one\| PubMed ID: 25951181	2015
Oestrogen Non-Genomic Signalling is Activated in Tamoxifen-Resistant Breast Cancer. International journal of molecular sciences\| PubMed ID: 31195751	2019
Using proximity ligation assay to detect protein arginine methylation. Methods (San Diego, Calif.)\| PubMed ID: 31499160	2020
ERα-36 regulates progesterone receptor activity in breast cancer. Breast cancer research : BCR\| PubMed ID: 32429997	2020
Analysis of genomic and non-genomic signaling of estrogen receptor in PDX models of breast cancer treated with a combination of the PI3K inhibitor alpelisib (BYL719) and fulvestrant. Breast cancer research : BCR\| PubMed ID: 34020697	2021

Julien Jacquemetton

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