No abstract

Recent studies showed that genetic aberrations in the gene, probably through the canonical WNT/β-catenin pathway, lead to the pathogenesis of uterine fibroids. However, a comprehensive analysis of the WNT pathway in-mutated and -wild-type fibroids has not been performed. The objective of this study was to determine the status of the WNT pathway in human fibroids. We performed Sanger sequencing to define the mutational status of fibroids and normal myometrium samples. qPCR arrays were carried out to determine the status of the WNT signaling pathway in -mutated and-wild-type fibroids. Liquid chromatography-mass spectrometry (LC-MS), Western blotting and immunohistochemistry were used to monitor the expression of β-catenin. We showed that β-catenin expression was increased in fibroids compared to the adjacent myometrium samples. However, β-catenin expression showed no correlation with mutation status. Of all the WNT signaling components, WNT inhibitors showed the greatest differences in expression between fibroids and controls., a WNT inhibitor, was identified as the most significantly upregulated gene in fibroids. We cultured primary fibroid cells on hydrogels of known stiffness to decipher the influence of biomechanical cues on β-catenin expression and revealed increased levels of β-catenin when cells were cultured on a stiffer surface. In conclusion, our data showed that β-catenin expression in fibroids occurs independently of mutations. Biomechanical changes upregulate β-catenin expression in fibroids, providing an attractive avenue for developing new treatments for this disease.

Background: Metastatic breast cancer remains incurable. Next-generation sequencing (NGS) offers the ability to identify actionable genomic alterations in tumours which may then be matched with targeted therapies, but the implementation and utility of this approach is not well defined for patients with metastatic breast cancer. Methods: We recruited patients with advanced breast cancer of any subtype for prospective targeted NGS of their most recent tumour samples, using a panel of 108 breast cancer-specific genes. Genes were classified as actionable or non-actionable using the European Society of Medical Oncology Scale for Clinical Actionability of Molecular Targets (ESCAT) guidelines. Results: Between February 2014 and May 2019, 322 patients were enrolled onto the study, with 72% (n = 234) of patients successfully sequenced (n = 357 samples). The majority (74%, n = 171) of sequenced patients were found to carry a potentially actionable alteration, the most common being a PIK3CA mutation. Forty-three percent (n = 74) of patients with actionable alterations were referred for a clinical trial or referred for confirmatory germline testing or had a change in therapy outside of clinical trials. We found alterations in AKT1, BRCA2, CHEK2, ESR1, FGFR1, KMT2C, NCOR1, PIK3CA and TSC2 to be significantly enriched in our metastatic population compared with primary breast cancers. Concordance between primary and metastatic samples for key driver genes (TP53, ERBB2 amplification) was > 75%. Additionally, we found that patients with a higher number of mutations had a significantly worse overall survival. Conclusion: Genomic profiling of patients with metastatic breast cancer can have clinical implications and should be considered in all suitable patients.

The central characteristic of uterine fibroids is excessive deposition of extracellular matrix (ECM), which contributes to fibroid growth and bulk-type symptoms. Despite this, very little is known about patterns of ECM protein expression in fibroids and whether these are influenced by the most common genetic anomalies, which relate to MED12. We performed extensive genetic and proteomic analyses of clinically annotated fibroids and adjacent normal myometrium to identify the composition and expression patterns of ECM proteins in MED12 mutation-positive and mutation-negative uterine fibroids. Genetic sequencing of tissue samples revealed MED12 alterations in 39 of 65 fibroids (60%) from 14 patients. Using isobaric tagged-based quantitative mass spectrometry on three selected patients (n = 9 fibroids), we observed a common set of upregulated (>1.5-fold) and downregulated (<0.66-fold) proteins in small, medium, and large fibroid samples of annotated MED12 status. These two sets of upregulated and downregulated proteins were the same in all patients, regardless of variations in fibroid size and MED12 status. We then focused on one of the significant upregulated ECM proteins and confirmed the differential expression of periostin using western blotting and immunohistochemical analysis. Our study defined the proteome of uterine fibroids and identified that increased ECM protein expression, in particular periostin, is a hallmark of uterine fibroids regardless of MED12 mutation status. This study sets the foundation for further investigations to analyze the mechanisms regulating ECM overexpression and the functional role of upregulated ECM proteins in leiomyogenesis.

Unopposed oestrogen is responsible for approximately 80% of all the endometrial cancers. The relationship between unopposed oestrogen and endometrial cancer was indicated by the increase in the number of endometrial cancer cases due to the widespread use of oestrogen replacement therapy. Approximately 30% of the endometrial cancer patients have mutations in the Wnt signalling pathway. How the unbalanced ratios of ovarian hormones and the mutations in Wnt signalling pathway interact to cause endometrial cancer is currently unclear. To study this, we have developed a uterine epithelial cell-specific inducible cre mouse model and used 3D in vitro culture of human endometrial cancer cell lines. We showed that activating mutations in the Wnt signalling pathway for a prolonged period leads to endometrial hyperplasia but not endometrial cancer. Interestingly, unopposed oestrogen and activating mutations in Wnt signalling together drive the progression of endometrial hyperplasia to endometrial cancer. We have provided evidence that progesterone can be used as a targeted therapy against endometrial cancer cases presented with the activating mutations in Wnt signalling pathway.