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Simple Summary: Resistance to anti-HER2+ therapy remains the main clinical challenge in the management of the HER2+ breast cancer subtype. The objective of our study was to analyze the involvement of microRNAs in the resistance to trastuzumab. We identified miR-146a-5p as the major dysregulated microRNA among parental and trastuzumab-resistant HER2+ breast cancer cells. The gain- and loss-of-function of this miRNA modulates resistance to trastuzumab in vitro, and elevated levels of miR-146a-5p in the primary tumor have been associated with a poor prognosis. In addition, exosomes from trastuzumab-resistant cells contain high levels of miR-146a-5p, and may reduce the effect of trastuzumab on sensitive cancer cells, increasing the expression of epithelial-to-mesenchymal transition markers and the capacities for migration and angiogenesis. The results of this study demonstrate for the first time the involvement of miR-146a-5p in resistance to trastuzumab, and suggest that exosomes play an important role in this process. Trastuzumab treatment has significantly improved the prognosis of HER2-positive breast cancer patients. Despite this, resistance to therapy still remains the main clinical challenge. In order to evaluate the implication of microRNAs in the trastuzumab response, we performed a microRNA array in parental and acquired trastuzumab-resistant HER2-positive breast cancer cell lines. Our results identified miR-146a-5p as the main dysregulated microRNA. Interestingly, high miR-146a-5p expression in primary tumor tissue significantly correlated with shorter disease-free survival in HER2-positive breast cancer patients. The gain- and loss-of-function of miR-146a-5p modulated the response to trastuzumab. Furthermore, the overexpression of miR-146a-5p increased migration and angiogenesis, and promoted cell cycle progression by reducing CDKN1A expression. Exosomes from trastuzumab-resistant cells showed a high level of miR-146a-5p expression compared with the parental cells. In addition, the co-culture with resistant cells' exosomes was able to decrease in sensitivity and increase the migration capacities in trastuzumab-sensitive cells, as well as angiogenesis in HUVEC-2 cells. Collectively, these data support the role of miR-146a-5p in resistance to trastuzumab, and demonstrate that it can be transferred by exosomes conferring resistance properties to other cells. [ABSTRACT FROM AUTHOR]

Background: NF-κB signalling appears deregulated in breast tumours. The purpose of this study was to determine whether the non-canonical NF-κB pathway, is activated in oestrogen receptor positive (ER+) breast cancer, to identify any correlation between its activity and the clinico-pathological phenotype and to explore whether NF-κB2 and RelB subunits and/or any of their target genes might be used as a predictive marker.Methods: Two independent cohorts of ER+ early breast cancer patients treated with adjuvant endocrine therapy were included in the study. Activation of RelB and NF-κB2 subunits was determined in a training set of 121 patients by measuring DNA-binding activities in nuclear extracts from fresh frozen specimens by an ELISA-based assay. Samples of 15 ER- breast cancer patients were also included in the study. In a large validation cohort of 207 patients, nuclear immunostaining of RelB and NF-κB2 on formalin-fixed paraffin-embedded specimens was performed. Statistical correlation within clinico-pathological factors, disease-free survival (DFS) and overall survival (OS) was evaluated. Publicly available gene expression and survival data have been interrogated aimed to identify target genes.Results: Activation of NF-κB2 and RelB was found in 53.7 and 49.2% of the 121 ER+ tumours analysed, with similar levels to ER- breast tumours analysed in parallel for comparisons. In the validation cohort, we obtained a similar proportion of cases with activation of NF-κB2 and RelB (59.9 and 32.4%), with a 39.6% of co-activation. Multiplexing immunofluorescence in breast cancer tissue confirmed an inverse spatial distribution of ER with NF-κB2 and RelB nuclear expression in tumour cells. Interestingly, NF-κB2 and RelB mRNA expression was inversely correlated with ER gene (ESR1) levels (P<0.001, both) and its activation was significantly associated with worse DFS (P=0.005 and P=0.035, respectively) in ER+ breast cancer. Moreover, the co-activation of both subunits showed a stronger association with early relapse (P=0.002) and OS (P=0.001). Finally, higher expression of the non-canonical NF-κB target gene myoglobin was associated with a poor outcome in ER+ breast cancer (DFS, P<0.05).Conclusions: The non-canonical NF-κB pathway activation is inversely associated with oestrogen receptor expression in ER+ breast cancer and predicts poor survival in this subgroup. The myoglobin gene expression has been identified as a possible surrogate marker of the non-canonical NF-κB pathway activation in these tumours. [ABSTRACT FROM AUTHOR]

Background: There are currently no biomarkers for early breast cancer patient populations at risk of bone metastasis. Identification of mediators of bone metastasis could be of clinical interest.Methods: A de novo unbiased screening approach based on selection of highly bone metastatic breast cancer cells in vivo was used to determine copy number aberrations (CNAs) associated with bone metastasis. The CNAs associated with bone metastasis were examined in independent primary breast cancer datasets with annotated clinical follow-up. The MAF gene encoded within the CNA associated with bone metastasis was subjected to gain and loss of function validation in breast cancer cells (MCF7, T47D, ZR-75, and 4T1), its downstream mechanism validated, and tested in clinical samples. A multivariable Cox cause-specific hazard model with competing events (death) was used to test the association between 16q23 or MAF and bone metastasis. All statistical tests were two-sided.Results: 16q23 gain CNA encoding the transcription factor MAF mediates breast cancer bone metastasis through the control of PTHrP. 16q23 gain (hazard ratio (HR) for bone metastasis = 14.5, 95% confidence interval (CI) = 6.4 to 32.9, P < .001) as well as MAF overexpression (HR for bone metastasis = 2.5, 95% CI = 1.7 to 3.8, P < .001) in primary breast tumors were specifically associated with risk of metastasis to bone but not to other organs.Conclusions: These results suggest that MAF is a mediator of breast cancer bone metastasis. 16q23 gain or MAF protein overexpression in tumors may help to select patients at risk of bone relapse. [ABSTRACT FROM AUTHOR]

Trastuzumab is the first-line targeted therapeutic drug for HER2-positive breast cancer, leading to improved overall survival. However, acquired resistance inevitably occurs. We aimed to identify, quantify, and assess the mechanisms of acquired resistance to trastuzumab. We established an acquired trastuzumab-resistant model in vitro from BT-474, a trastuzumab-sensitive, HER2-amplified breast-cancer cell line. A multi-omic strategy was implemented to obtain gene, proteome, and phosphoproteome signatures associated with acquired resistance to trastuzumab in HER2-positive breast cancer, followed by validation in human clinical samples. YAP1 dephosphorylation and TEAD2 overexpression were detected as significant alterations in the Hippo pathway in trastuzumab-resistant breast cancer. Because of the emerging role of these proteins as mediators of normal growth and tumorigenesis, we assessed the exogenous modulation of their activity, either by in vitro gene silencing or by pharmacological inhibition of the YAP1/TEAD complexes, both in vitro and in vivo. Moreover, we identified increased signaling through the Hippo pathway in human samples after progression following trastuzumab treatment. Finally, YAP1/TAZ nuclear accumulation in malignant cells in HER2 breast tumor was significantly associated with worse progression-free and overall survival in metastatic HER2-positive breast-cancer patients. Our results suggest the involvement of Hippo signaling in acquired trastuzumab resistance in breast cancer. Additionally, we provide novel evidence for a potential breast-cancer treatment strategy based on dual targeting of HER2 and Hippo pathway effectors, which may improve the antitumor activity of trastuzumab and help overcome resistance. [ABSTRACT FROM AUTHOR]

Aim Poly (ADP-ribose) polymerase (PARP) inhibitors have shown promising results in Breast Cancer (BRCA) deficient breast cancer, but not in molecularly unselected patient populations. Two lines of research in this field are needed: the identification of novel subsets of patients that could potentially benefit from PARP inhibitors and the discovery of suitable targeted therapies for combination strategies. Methods We tested PARP inhibition, alone or combined with the anti-HER2 antibody trastuzumab on HER2+ breast cancer. We used two PARP inhibitors in clinical development, olaparib and rucaparib, as well as genetic downmodulation of PARP-1 for in vitro studies. DNA damage was studied by the formation of γH2AX foci and comet assay. Finally, the in vivo anti-tumour effect of olaparib and trastuzumab was examined in nude mice subcutaneously implanted with BT474 cells. Results In a panel of four HER2 overexpressing breast cancer cell lines, both olaparib and rucaparib significantly decreased cell growth and enhanced anti-tumour effects of trastuzumab. Cells exposed to olaparib and trastuzumab had greater DNA damage than cells exposed to each agent alone. Mechanistic exploratory assays showed that trastuzumab downmodulated the homologous recombination protein proliferating cell nuclear antigen (PCNA). Combination treatment in the BT474 xenograft model resulted in enhanced growth inhibition, reduced tumour cell proliferation, and increased DNA damage and apoptosis. Conclusion Taken together, our results show that PARP inhibition has antitumour effects and increases trastuzumab activity in HER2 overexpressing breast cancer. These findings make this novel combination a promising strategy for clinical development. [ABSTRACT FROM AUTHOR]