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Breast cancer; Heterodimerization Cáncer de mama; Heterodimerización Càncer de mama; Heterodimerització Anti-HER2 therapies have markedly improved prognosis of HER2-positive breast cancer. However, different mechanisms play a role in treatment resistance. Here, we identified AXL overexpression as an essential mechanism of trastuzumab resistance. AXL orchestrates epithelial-to-mesenchymal transition and heterodimerizes with HER2, leading to activation of PI3K/AKT and MAPK pathways in a ligand-independent manner. Genetic depletion and pharmacological inhibition of AXL restored trastuzumab response in vitro and in vivo. AXL inhibitor plus trastuzumab achieved complete regression in trastuzumab-resistant patient-derived xenograft models. Moreover, AXL expression in HER2-positive primary tumors was able to predict prognosis. Data from the PAMELA trial showed a change in AXL expression during neoadjuvant dual HER2 blockade, supporting its role in resistance. Therefore, our study highlights the importance of targeting AXL in combination with anti-HER2 drugs across HER2-amplified breast cancer patients with high AXL expression. Furthermore, it unveils the potential value of AXL as a druggable prognostic biomarker in HER2-positive breast cancer. A.A.-A., E.J.A., and F.B.-M. were supported by Asociación Española contra el Cáncer AECC (PRDVA18013LLUC to A.A.-A., POSTD211413AREN to E.J.A., and AECC_Postdoctoral17-1062 to F.B.-M.). A.M.-S. was funded by the Spanish Government (PFIS FI20/00188). J.Ar. is supported by Breast Cancer Research Foundation (BCRF-20-08), Instituto de Salud Carlos III Project reference number AC15/00062, and the EC under the framework of the ERA-NET TRANSCAN-2 initiative cofinanced by FEDER, Instituto de Salud Carlos III (CB16/12/00449 and PI19/01181), and Asociación Española Contra el Cáncer (AECC). A.P. was supported by Instituto de Salud Carlos III—PI19/01846, Breast Cancer Now—2018NOVPCC1294. P.E. and A.L. were funded by Instituto de Salud Carlos III and cofinanced by FEDER (PI18/01219 to P.E. and CB16/12/00481 to A.L.). J.M.C. was funded by Sociedad Española de Oncología Médica (Rio Hortega-SEOM) and Compromiso ADAMED.

Natural killer (NK) cells can orchestrate effective antitumor immunity. The presence of tumor-infiltrating NK cells in diagnostic biopsies predicts pathologic complete response (pCR) to HER2-specific therapeutic antibodies in patients with primary breast cancer. Here, we analyzed whether diversity in circulating NK cells might influence tumor infiltration and HER2-specific therapeutic antibody efficacy. We found that numbers of circulating CD57+ NK cells inversely correlated with pCR to HER2-specific antibody treatment in patients with primary breast cancer independently of age, traditional clinicopathologic factors, and CD16A 158F/V genotype. This association was uncoupled from the expression of other NK-cell receptors, the presence of adaptive NK cells, or changes in major T-cell subsets, reminiscent of cytomegalovirus-induced immunomodulation. NK-cell activation against trastuzumab-coated HER2+ breast cancer cells was comparable in patients with high and low proportions of CD57+ NK cells. However, circulating CD57+ NK cells displayed decreased CXCR3 expression and CD16A-induced IL2-dependent proliferation in vitro. Presence of CD57+ NK cells was reduced in breast tumor–associated infiltrates as compared with paired peripheral blood samples, suggesting deficient homing, proliferation, and/or survival of NK cells in the tumor niche. Indeed, numbers of circulating CD57+ were inversely related to tumor-infiltrating NK-cell numbers. Our data reveal that NK-cell differentiation influences their antitumor potential and that CD57+ NK cells may be a biomarker useful for tailoring HER2 antibody–based therapeutic strategies in breast cancer.

Supplementary Figures 1-6 and Tables 1-3

Differential DNA methylation patterns between docetaxel sensitive, resistant or residual disease TNBC PDX models

There is growing interest in delivering genomically informed cancer therapy. Our aim was to determine the concordance of genomic alterations between primary and recurrent breast cancer. Targeted next-generation sequencing was performed on formalin-fixed paraffin-embedded (FFPE) samples, profiling 3,320 exons of 182 cancer-related genes plus 37 introns from 14 genes often rearranged in cancer. Point mutations, indels, copy-number alterations (CNA), and select rearrangements were assessed in 74 tumors from 43 patients (36 primary and 38 recurrence/metastases). Alterations potentially targetable with established or investigational therapeutics were considered “actionable.” Alterations were detected in 55 genes (mean 3.95 alterations/sample, range 1–12), including mutations in PIK3CA, TP53, ARID1A, PTEN, AKT1, NF1, FBXW7, and FGFR3 and amplifications in MCL1, CCND1, FGFR1, MYC, IGF1R, MDM2, MDM4, AKT3, CDK4, and AKT2. In 33 matched primary and recurrent tumors, 97 of 112 (86.6%) somatic mutations were concordant. Of identified CNAs, 136 of 159 (85.5%) were concordant: 37 (23.3%) were concordant, but below the reporting threshold in one of the matched samples, and 23 (14.5%) discordant. There was an increased frequency of CDK4/MDM2 amplifications in recurrences, as well as gains and losses of other actionable alterations. Forty of 43 (93%) patients had actionable alterations that could inform targeted treatment options. In conclusion, deep genomic profiling of cancer-related genes reveals potentially actionable alterations in most patients with breast cancer. Overall there was high concordance between primary and recurrent tumors. Analysis of recurrent tumors before treatment may provide additional insights, as both gains and losses of targets are observed. Mol Cancer Ther; 13(5); 1382–9. ©2014 AACR.

XLSX - 13K, Table 1a. 182 genes across entire coding sequence Table 1b. 14 genes sequenced across selected introns.

XLSX - 22K, Substitution, insertion and deletion mutations/large structural alterations.

PDF - 138K, Adjuvant systemic treatments.

Excel file with all methylation related data

Supplementary Figure S1. MKP-1 protein abundance levels in MDA-MB-231 cells treated with docetaxel at progressively increasing times. Supplementary Figure S2. Cell growth in the presence of the docetaxel or doxorubicin of breast cancer cell lines overexpressing MKP-1. Supplementary Table S1. Clinical and molecular characteristics of a series of 350 patients with early breast cancer. Supplementary Figure S3. IHC detection of MKP-1 and p-JNK1/2 in a representative normal tissue sample. Supplementary Table S2. Univariate and multivariate Cox analyses in the cohort of 350 breast cancer patients (disease-free survival analysis). Supplementary Table S3. Clinical and molecular characteristics of 64 locally advanced breast cancer patients who received neoadjuvant taxane-based chemotherapy.Supplementary Table S4. MKP-1 and p-JNK1/2 marker determinations in locally advanced breast cancer patients who received neoadjuvant taxane-based chemotherapy.

MAPK phosphatase-1 (MKP-1) is overexpressed during malignant transformation of the breast in many patients, and it is usually associated with chemoresistance through interference with JNK-driven apoptotic pathways. Although the molecular settings of the mechanism have been documented, details about the contribution of MKP-1 to the failure of chemotherapeutic interventions are unclear. Transient overexpression of MKP-1 and treatment with JNK-modulating agents in breast carcinoma cells confirmed the mediation of MKP-1 in the resistance to taxanes and anthracyclines in breast cancer, through the inactivation of JNK1/2. We next assessed MKP-1 expression and JNK1/2 phosphorylation status in a large cohort of samples from 350 early breast cancer patients treated with adjuvant anthracycline–based chemotherapy. We detected that MKP-1 overexpression is a recurrent event predominantly linked to dephosphorylation of JNK1/2 with an adverse impact on relapse of the tumor and overall and disease-free survival. Moreover, MKP-1 and p-JNK1/2 determinations in 64 locally advanced breast cancer patients treated with neoadjuvant taxane–based chemotherapy showed an inverse correlation between MKP-1 overexpression (together with JNK1/2 inhibition) and the pathologic response of the tumors. Our results emphasize the importance of MKP-1 as a potential predictive biomarker for a subset of breast cancer patients with worse outcome and less susceptibility to treatment. Mol Cancer Ther; 15(11); 2780–90. ©2016 AACR.

PDF - 36K, Samples tested.

Figure legends for Supplementary Figures

PDF - 168K, Detailed analysis of matched primary and recurrent/metastatic breast tumors.

Supplementary Figure from mTOR Inhibition and T-DM1 in HER2-Positive Breast Cancer

Supplementary Figure 1 shows generation and cell-lineage characterization of primary CAF cultures derived from HER2-positive breast cancer patient biopsies. Moreover, it also shows NRG1 staining in two FFPE-associated patient samples from which primary CAFs were derived.

Supplementary Figure 3 shows how trastuzumab plus pertuzumab, but not trastuzumab alone, overcome NRG1 enhanced pro-survival signaling in HER2-positive breast cancer cells and in ex vivo tumors.

Effects of T-DM1 on 750 cyclin B1 (CCNB1) mRNA expression in T751 DM1 sensitive and resistant breast cancer cells.

Supplementary Figure legend

Supplementary Table 1 shows main clinical-pathological characteristics of HER2-positive breast cancer patients whom primary CAF cultures were derived from.

Purpose: Trastuzumab-emtansine (T-DM1) is a standard treatment in advanced HER2-positive breast cancer. However, resistance inevitably occurs. We aimed to identify mechanisms of acquired T-DM1 resistance.Experimental Design: HER2-positive breast cancer cells (HCC1954, HCC1419, SKBR3, and BT474) were treated in a pulse-fashion with T-DM1 to induce a resistant phenotype. Cellular and molecular effects of T-DM1 in parental versus resistant cells were compared. CDK1 kinase activity and cyclin B1 expression were assayed under various conditions. Genetic modifications to up- or downregulate cyclin B1 were conducted. Effects of T-DM1 on cyclin B1 levels, proliferation, and apoptosis were assayed in human HER2-positive breast cancer explants.Results: We obtained three cell lines with different levels of acquired T-DM1 resistance (HCC1954/TDR, HCC1419/TDR, and SKBR3/TDR cells). HER2 remained amplified in the resistant cells. Binding to HER2 and intracellular uptake of T-DM1 were maintained in resistant cells. T-DM1 induced cyclin B1 accumulation in sensitive but not resistant cells. Cyclin B1 knockdown by siRNA in parental cells induced T-DM1 resistance, while increased levels of cyclin B1 by silencing cdc20 partially sensitized resistant cells. In a series of 18 HER2-positive breast cancer fresh explants, T-DM1 effects on proliferation and apoptosis paralleled cyclin B1 accumulation.Conclusions: Defective cyclin B1 induction by T-DM1 mediates acquired resistance in HER2-positive breast cancer cells. These results support the testing of cyclin B1 induction upon T-DM1 treatment as a pharmacodynamic predictor in HER2-positive breast cancer. Clin Cancer Res; 23(22); 7006–19. ©2017 AACR.

Supplementary Figure 2 shows how NRG1/CAFs, but not normal mammary fibroblasts, mediate trastuzumab resistance in HER2-positive breast cancer cells.

Representative immunohistochemistry images of control and 765 T-DM1 treated explant obtained from a liver metastasis.

Supplementary Figure 4 shows that NRG1 enhances pro-migratory capacity of breast cancer cells as well as the induction of early markers from the EMT process. Moreover, it also shows that trastuzumab plus pertuzumab, but not trastuzumab alone, are able to prevent NRG1-derived effects.

Supplementary Table 2 shows main clinical-pathological characteristics of HER2-positive breast cancer patient's cohort treated with neoadjuvant-based therapy and the association of NRG1 protein expression with the diverse clinical parameters analysed.

Breast cancer is the most frequent cancer in women worldwide. Despite the improvement in diagnosis and treatments, the rates of cancer relapse and resistance to therapies remain higher than desirable. Alterations in microRNAs have been linked to changes in critical processes related to cancer development and progression. Their involvement in resistance or sensitivity to breast cancer treatments has been documented by different in vivo and in vitro experiments. The most significant microRNAs implicated in modulating resistance to breast cancer therapies are summarized in this review. Resistance to therapy has been linked to cellular processes such as cell cycle, apoptosis, epithelial-to-mesenchymal transition, stemness phenotype, or receptor signaling pathways, and the role of microRNAs in their regulation has already been described. The modulation of specific microRNAs may modify treatment response and improve survival rates and cancer patients’ quality of life. As a result, a greater understanding of microRNAs, their targets, and the signaling pathways through which they act is needed. This information could be useful to design new therapeutic strategies, to reduce resistance to the available treatments, and to open the door to possible new clinical approaches.

Treatment for the HER2+ breast cancer subtype is still unsatisfactory, despite breakthroughs in research. The discovery of various new molecular mechanisms of transcription factors may help to make treatment regimens more effective. The transcription factor SALL4 has been related to aggressiveness and resistance therapy in cancer. Its molecular mechanisms and involvement in various signaling pathways are unknown in the HER2+ breast cancer subtype. In this study, we have evaluated the implication of SALL4 in the HER2+ subtype through its expression in patients’ samples and gain and loss of function in HER2+ cell lines. We found higher SALL4 expression in breast cancer tissues compared to healthy tissue. Interestingly, high SALL4 expression was associated with disease relapse and poor patient survival. In HER2+ cell lines, transient overexpression of SALL4 modulates PI3K/AKT signaling through regulating PTEN expression and BCL2, which increases cell survival and proliferation while reducing the efficacy of trastuzumab. SALL4 has also been observed to regulate the epithelial–mesenchymal transition and stemness features. SALL4 overexpression significantly reduced the epithelial markers E-cadherin, while it increased the mesenchymal markers β-catenin, vimentin and fibronectin. Furthermore, it has been also observed an increased expression of MYC, an essential transcription factor for regulating epithelial-mesenchymal transition and/or cancer stem cells. Our study demonstrates, for the first time, the importance of SALL4 in the HER2+ subtype and partial regulation of trastuzumab sensitivity. It provides a viable molecular mechanism-driven therapeutic strategy for an important subset of HER2-overexpressing patients whose malignancies are mediated by SALL4 expression.

BACKGROUND: MicroRNAs have emerged as new diagnostic and therapeutic biomarkers for breast cancer. Herein, we analyzed miR-99a-5p expression levels in primary tumours and plasma of breast cancer patients to evaluate its usefulness as a minimally invasive diagnostic biomarker. METHODS: MiR-99a-5p expression levels were determined by quantitative real-time PCR in three indepent cohorts of patients: I) Discovery cohort: breast cancer tissues (n=103) and healthy breast tissues (n=26). II) Testing cohort: plasma samples from 105 patients and 98 healthy donors. III) Validation cohort: plasma samples from 89 patients and 85 healthy donors. Receiver operating characteristic (ROC) curve analyses were applied to evaluate the diagnostic potential of miR-99a-5p expression levels in tissue and plasma samples. RESULTS: MiR-99a-5p was significantly downregulated in breast cancer tissues compared to healthy breast tissues (p < 0.0001), being able to discriminate BC from healthy breast tissues with an AUC of 0.85, 87.38% sensitivity, 76.92% specificity, and 85.27% accuracy. Conversely, miR-99a-5p levels were significantly higher in breast cancer patients than in healthy controls in plasma samples from both testing and validation cohorts (p < 0.0001). ROC curve analysis revealed that miR-99a-5p has good diagnostic potential, with an AUC of 0.76, 63.81% sensitivity, 79.59% specificity, and 71.43% accuracy. The value of circulating miR-99a-5p levels as a breast cancer biomarker was further validated in an independent cohort, where it was able to identify breast cancer with 57.30% sensitivity, 67.06% specificity, and 62.07% accuracy. Besides, we also confirmed that circulating miR-99a-5p levels were able to discriminate early breast cancer from healthy controls with a 66.67% accuracy, 68.80% sensitivity, 65.28% specificity, and an AUC of 0.69 (p < 0.0001). CONCLUSION: MiR-99a-5p’s deregulated expression distinguished healthy individuals from breast cancer patients in two different types of samples (tissues and plasma). Interestingly, plasma expression levels were significantly lower in healthy controls than in early-stage breast cancer patients. Our findings suggest that circulating miR-99a-5p as a novel promising non-invasive biomarker for breast cancer detection. Citation Format: Iris Garrido-Cano, Vera Constâncio, Anna Adam-Artigues, Ana Lameirinhas, Soraya Simón, Belen Ortega, María Teresa Martínez, Cristina Hernando, Begoña Bermejo, Ana Lluch, Paula Lopes, Rui Henrique, Carmen Jerónimo, Juan Miguel Cejalvo, Pilar Eroles. Circulating miR-99a-5p expression in plasma: A potential biomarker for early diagnosis of breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS2-31.

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.

In patients with trastuzumab-resistant HER2-positive breast cancer, the combination of everolimus (mTORC1 inhibitor) with trastuzumab failed to show a clinically significant benefit. However, the combination of mTOR inhibition and the antibody-drug conjugate (ADC) trastuzumab-emtansine (T-DM1) remains unexplored. We tested T-DM1 plus everolimus in a broad panel of HER2-positive breast cancer cell lines. The combination was superior to T-DM1 alone in four cell lines (HCC1954, SKBR3, EFM192A, and MDA-MB-36) and in two cultures from primary tumor cells derived from HER2-positive patient-derived xenografts (PDX), but not in BT474 cells. In the trastuzumab-resistant HCC1954 cell line, we characterized the effects of the combination using TAK-228 (mTORC1 and -2 inhibitor) and knockdown of the different mTOR complex components. T-DM1 did not affect mTOR downstream signaling nor induct autophagy. Importantly, mTOR inhibition increased intracellular T-DM1levels, leading to increased lysosomal accumulation of the compound. The increased efficacy of mTOR inhibition plus T-DM1 was abrogated by lysosome inhibitors (chloroquine and bafilomycin A1). Our experiments suggest that BT474 are less sensitive to T-DM1 due to lack of optimal lysosomal processing and intrinsic resistance to the DM1 moiety. Finally, we performed several in vivo experiments that corroborated the superior activity of T-DM1 and everolimus in HCC1954 and PDXderived mouse models. In summary, everolimus in combination with T-DM1 showed strong antitumor effects in HER2-positive breast cancer, both in vitro and in vivo. This effect might be related, at least partially, to mTOR-dependent lysosomal processing of T-DM1, a finding that might apply to other ADCs that require lysosomal processing., This work was supported by ISCIII (CIBERONC CB16/12/00481, CB16/12/00241, PI18/00382, PI18/00006, PI18/01219), Generalitat de Catalunya (2017 SGR 507). MINECO through gBFU2015-71371-R grant and the CRIS Cancer Foundation supported work in AP lab. D. Casadevall was supported by ISCIII (Rio Hortega Research Contract CM16/00023 and Juan Rodés Research Contract JR18/00003). F.J. Sánchez-Martín and S. Menéndez were supported by Department de Salut Generalitat de Catalunya (PERIS SLT002/16/00008 and PERIS SLT006/17/00040). M. Qin received financial support from the China Scholarship Council (CSC) for her doctoral fellowship. Work carried out in our laboratories receives support from the European Community through the Regional Development Funding Program (FEDER).

Purpose of Review Antagonists of mu-opioid receptor role in cancer progression remains to be elucidated. The objective of this review was to summarize the available evidence on antagonists of mu-opioid receptor effect on tumor progression and prognosis in different types of cancers and an evaluation of the available findings on their mechanism of action. Recent Findings We have found studies related to methylnaltrexone (MNTX) and naltrexone (NTX) usage in cancer outcomes-related setting. We found consistent preclinical evidence of a potential action of MNTX and NTX on cancer growth and spread mediated mainly by effect on the opioid growth factor receptor (OGFr) axis, which results in depressed cell replication. However, clinical results are scarce and limited to poor-quality evidence. Summary Further high-quality studies are warranted to study antagonists of mu-opioid receptor role as a therapeutic option in different types of cancer, especially in patients where the classical treatment causes unacceptable side effects.

The combination of trastuzumab plus pertuzumab plus docetaxel as a first-line therapy in patients with HER2-positive metastatic breast cancer has provided significant clinical benefits compared to trastuzumab plus docetaxel alone. However, despite the therapeutic success of existing therapies targeting HER2, tumours invariably relapse. Therefore, there is an urgent need to improve our understanding of the mechanisms governing resistance, so that specific therapeutic strategies can be developed to provide improved efficacy. It is well known that the tumour microenvironment (TME) has a significant impact on cancer behaviour. Cancer-associated fibroblasts (CAFs) are essential components of the tumour stroma that have been linked to acquired therapeutic resistance and poor prognosis in breast cancer. For this reason, it would be of interest to identify novel biomarkers in the tumour stroma that could emerge as therapeutic targets for the modulation of resistant phenotypes. Conditioned medium experiments carried out in our laboratory with CAFs derived from HER2-positive patients showed a significant capacity to promote resistance to trastuzumab plus pertuzumab therapies in two HER2-positive breast cancer cell lines (BCCLs), even in the presence of docetaxel. In order to elucidate the components of the CAF-conditioned medium that may be relevant in the promotion of BCCL resistance, we implemented a multiomics strategy to identify cytokines, transcription factors, kinases and miRNAs in the secretome that have specific targets in cancer cells. The combination of cytokine arrays, label-free LC-MS/MS quantification and miRNA analysis to explore the secretome of CAFs under treatment conditions revealed several up- and downregulated candidates. We discuss the potential role of some of the most interesting candidates in generating resistance in HER2-positive breast cancer.

Due to the lack of specific targets, cytotoxic chemotherapy still represents the common standard treatment for triple-negative breast patients. Despite the harmful effect of chemotherapy on tumor cells, there is evidence that treatment could modulate the tumor microenvironment in a way favoring the propagation of the tumor. In addition, the lymphangiogenesis process and its factors could be involved in this counter-therapeutic event. In our study, we have evaluated the expression of the main lymphangiogenic receptor VEGFR3 in two triple-negative breast cancer in vitro models, resistant or not to doxorubicin treatment. The expression of the receptor, at mRNA and protein levels, was higher in doxorubicin-resistant cells than in parental cells. In addition, we confirmed the upregulation of VEGFR3 levels after a short treatment with doxorubicin. Furthermore, VEGFR3 silencing reduced cell proliferation and migration capacities in both cell lines. Interestingly, high VEGFR3 expression was significantly positively correlated with worse survival in patients treated with chemotherapy. Furthermore, we have found that patients with high expression of VEGFR3 present shorter relapse-free survival than patients with low levels of the receptor. In conclusion, elevated VEGFR3 levels correlate with poor survival in patients and with reduced doxorubicin treatment efficacy in vitro. Our results suggest that the levels of this receptor could be a potential marker of meager doxorubicin response. Consequently, our results suggest that the combination of chemotherapy and VEGFR3 blockage could be a potentially useful therapeutic strategy for the treatment of triple-negative breast cancer.

BackgroundOpioid receptors are expressed not only by neural cells in the central nervous system, but also by many solid tumor cancer cells. Whether perioperative opioids given for analgesia after tumor resection surgery might inadvertently activate tumor cells, promoting recurrence or metastasis, remains controversial. We analysed large public gene repositories of solid tumors to investigate differences in opioid receptor expression between normal and tumor tissues and their association with long–term oncologic outcomes.MethodsWe investigated the normalized gene expression of µ, κ, δ opioid receptors (MOR, KOR, DOR), Opioid Growth Factor (OGFR), and Toll-Like 4 (TLR4) receptors in normal and tumor samples from twelve solid tumor types. We carried out mixed multivariable logistic and Cox regression analysis on whether there was an association between these receptors’ gene expression and the tissue where found, i.e., tumor or normal tissue. We also evaluated the association between tumor opioid receptor gene expression and patient disease–free interval (DFI) and overall survival (OS).ResultsWe retrieved 8,780 tissue samples, 5,852 from tumor and 2,928 from normal tissue, of which 2,252 were from the Genotype Tissue Expression Project (GTEx) and 672 from the Cancer Genome Atlas (TCGA) repository. The Odds Ratio (OR) [95%CI] for gene expression of the specific opioid receptors in the examined tumors varied: MOR: 0.74 [0.63–0.87], KOR: 1.27 [1.17–1.37], DOR: 1.66 [1.48–1.87], TLR4: 0.29 [0.26–0.32], OGFR: 2.39 [2.05–2.78]. After controlling all confounding variables, including age and cancer stage, there was no association between tumor opioid receptor expression and long–term oncologic outcomes.ConclusionOpioid receptor gene expression varies between different solid tumor types. There was no association between tumor opioid receptor expression and recurrence. Understanding the significance of opioid receptor expression on tumor cells remains elusive.

Estrogen receptor-positive (ER+) is the most common subtype of breast cancer. Endocrine therapy is the fundamental treatment against this entity, by directly or indirectly modifying estrogen production. Recent advances in novel compounds, such as cyclin-dependent kinase 4/6 inhibitors (CDK4/6i), or phosphoinositide 3-kinase (PI3K) inhibitors have improved progression-free survival and overall survival in these patients. However, some patients still develop endocrine resistance after or during endocrine treatment. Different underlying mechanisms have been identified as responsible for endocrine treatment resistance, where ESR1 gene mutations are one of the most studied, outstanding from others such as somatic alterations, microenvironment involvement and epigenetic changes. In this scenario, selective estrogen receptor degraders/downregulators (SERD) are one of the weapons currently in research and development against aromatase inhibitor- or tamoxifen-resistance. The first SERD to be developed and approved for ER+ breast cancer was fulvestrant, demonstrating also interesting activity in ESR1 mutated patients in the second line treatment setting. Recent investigational advances have allowed the development of new oral bioavailable SERDs. This review describes the evolution and ongoing studies in SERDs and new molecules against ER, with the hope that these novel drugs may improve our patients’ future landscape.

Simple Summary Hyperactivation of the PI3K/AKT/mTOR cell signalling pathway is an important and well-described mechanism of trastuzumab resistance in HER2-positive breast cancer. In cell-line models of acquired trastuzumab resistance generated in our laboratory, we demonstrate this type of activation, which is independent of HER2-mediated regulation. We investigate whether the use of specific mTOR inhibitors, a PI3K/AKT/mTOR pathway effector, could lead to decreased activity of the pathway, influencing trastuzumab resistance. We demonstrate that TAK-228, a mTORC1 and mTORC2 inhibitor, can reverse resistance and increasing response to trastuzumab in models of primary and acquired resistance. The use of anti-HER2 therapies has significantly improved clinical outcome in patients with HER2-positive breast cancer, yet a substantial proportion of patients acquire resistance after a period of treatment. The PI3K/AKT/mTOR pathway is a good target for drug development, due to its involvement in HER2-mediated signalling and in the emergence of resistance to anti-HER2 therapies, such as trastuzumab. This study evaluates the activity of three different PI3K/AKT/mTOR inhibitors, i.e., BEZ235, everolimus and TAK-228 in vitro, in a panel of HER2-positive breast cancer cell lines with primary and acquired resistance to trastuzumab. We assess the antiproliferative effect and PI3K/AKT/mTOR inhibitory capability of BEZ235, everolimus and TAK-228 alone, and in combination with trastuzumab. Dual blockade with trastuzumab and TAK-228 was superior in reversing the acquired resistance in all the cell lines. Subsequently, we analyse the effects of TAK-228 in combination with trastuzumab on the cell cycle and found a significant increase in G0/G1 arrest in most cell lines. Likewise, the combination of both drugs induced a significant increase in apoptosis. Collectively, these experiments support the combination of trastuzumab with PI3K/AKT/mTOR inhibitors as a potential strategy for inhibiting the proliferation of HER2-positive breast cancer cell lines that show resistance to trastuzumab.

The bromodomain-containing protein 4 (BRD4), a member of the bromodomain and extra-terminal domain (BET) protein family, has emerged in the last years as a promising molecular target in many tumors including breast cancer. The triple negative breast cancer (TNBC) represents the molecular subtype with the worst prognosis and a current therapeutic challenge, and TNBC cells have been reported to show a preferential sensitivity to BET inhibitors. Interestingly, BRD4 phosphorylation (pBRD4) was found as an alteration that confers resistance to BET inhibition and PP2A proposed as the phosphatase responsible to regulate pBRD4 levels. However, the potential clinical significance of pBRD4, as well as its potential correlation with the PP2A pathway in TNBC, remains to be investigated. Here, we evaluated the expression levels of pBRD4 in a series of 132 TNBC patients. We found high pBRD4 levels in 34.1% of cases (45/132), and this alteration was found to be associated with the development of patient recurrences (p = 0.007). Interestingly, BRD4 hyperphosphorylation predicted significantly shorter overall (p &lt, 0.001) and event-free survival (p &lt, 0.001). Moreover, multivariate analyses were performed to confirm its independent prognostic impact in our cohort. In conclusion, our findings show that BRD4 hyperphosphorylation is an alteration associated with PP2A inhibition that defines a subgroup of TNBC patients with unfavorable prognosis, suggesting the potential clinical and therapeutic usefulness of the PP2A/BRD4 axis as a novel molecular target to overcome resistance to treatments based on BRD4 inhibition.

Circulating microRNAs have emerged as potential diagnostic biomarkers. The deregulation of the microRNA miR-99a-5p has been previously described as an effective biomarker of early breast cancer. Herein, we present a new nanoporous anodic alumina (NAA)-based biosensor that can detect plasma miR-99a-5p with high sensitivity and selectivity. NAA pores are loaded with rhodamine B and capped with a specific oligonucleotide that is able to block cargo release until the target is present. In the presence of miR-99a-5p, the capping oligonucleotide recognizes the miR-99a-5p sequence and displaces it allowing the release of the encapsulated dye. This method is able to successfully distinguish healthy controls from breast cancer patients, even at early stages with high efficiency, showing the presented system as a promising tool for breast cancer detection.