Costello syndrome (CS) may be caused by activating mutations in codon 12/13 of the HRAS proto-oncogene. HRAS p.Gly12Val mutations have the highest transforming activity, are very frequent in cancers, but very rare in CS, where they are reported to cause a severe, early lethal, phenotype. We identified an unusual, new germline p.Gly12Val mutation, c.35_36GC>TG, in a 12-year-old boy with attenuated CS. Analysis of his HRAS cDNA showed high levels of exon 2 skipping. Using wild type and mutant HRAS minigenes, we confirmed that c.35_36GC>TG results in exon 2 skipping by simultaneously disrupting the function of a critical Exonic Splicing Enhancer (ESE) and creation of an Exonic Splicing Silencer (ESS). We show that this vulnerability of HRAS exon 2 is caused by a weak 3’ splice site, which makes exon 2 inclusion dependent on binding of splicing stimulatory proteins, like SRSF2, to the critical ESE. Because the majority of cancer- and CS- causing mutations are located here, they affect splicing differently. Therefore, our results also demonstrate that the phenotype in CS and somatic cancers is not only determined by the different transforming potentials of mutant HRAS proteins, but also by the efficiency of exon 2 inclusion resulting from the different HRAS mutations. Finally, we show that a splice switching oligonucleotide (SSO) that blocks access to the critical ESE causes exon 2 skipping and halts proliferation of cancer cells. This unravels a potential for development of new anti-cancer therapies based on SSO-mediated HRAS exon 2 skipping.

Fas ligand (FasL) is expressed on some cancers and may play a role in the immune evasion of the tumour. We used immuno-histochemistry to study the expression of Fas and FasL in tissue samples from breast cancer patients, as well as normal breast tissue. Our results show that Fas and FasL are co-expressed both in normal tissue and in breast tumours. Fas and FasL mRNA were expressed in fresh normal and malignant breast tissue, as well as cultured breast epithelium and breast cancer cell lines. Flow cytometry analysis of live cells failed to detect FasL on the surface of normal or malignant breast cells; however, both stained positive for FasL after permeabilization. Fas was detected on the surface of normal breast cells and T47D and MCF-10A cell lines but only intracellularly in other breast cell lines tested. Neither normal breast epithelium nor breast cell lines induced Fas-dependent apoptosis in Jurkat cells. Finally, 20 tumour samples were stained for apoptosis. Few apoptotic cells were detected and there was no increase in apoptotic cells on the borders between tumour cells and lymphocytes. We conclude that FasL is expressed intracellularly in both normal and malignant breast epithelium and unlikely to be important for the immune evasion of breast tumours. © 2000 Cancer Research Campaign http://www.bjcancer.com

Background: In the year 2020 breast cancer was the most common form of cancer. Roughly 70% of breast cancers are estrogen receptor positive. MicroRNA-190b has previously been reported to be up-regulated in estrogen receptor positive breast cancers. Our group has previously demonstrated that microRNA-190b is hypomethylated in ER+ breast cancers, potentially leading to its upregulation. Results: In this study, using data from the Cancer Genome Atlas, we confirm that microRNA-190b is overexpressed in breast cancer via differential expression analysis and show that high expression of microRNA-190b results in more favorable outcomes in Luminal A patients (HR=0.29, 95% CI 0.12-0.71, P value=0.0063). MicroRNA190b target analysis, using immunoprecipitation of biotin labelled microRNA-190b, followed by RNA sequencing, identified RFWD3 as one of microRNA-190b’s regulatory targets in estrogen receptor positive breast cancer. Survival analysis of RFWD3 showed that elevated levels result in poorer overall survival in Luminal A breast cancer patients (HR = 2.22, 95% CI 1.33-3.71, P = 0.002). Gene ontology analysis of our sequencing results indicate that miR-190b may have a role in breast cancer development and/or tumorigenesis and that it may be a suitable tool in characterization between the estrogen receptor positive subtypes, Luminal A and Luminal B.Conclusions: We show that miR-190b targets RFWD3 in ER+ breast cancers leading to lower RFWD3 protein expression. Low levels of RFWD3 are associated with better outcomes in Luminal A breast cancer patients but not in Luminal B patients.