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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

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

DNA methylation signatures applied to breast cancer PDX models and comparasion with human normal mammary gland

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.

Excel file, with all expression related data and its correlation with methylation

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.

Global DNA methylation in docetaxel sensitive, resistant or residual disease TNBC PDX models

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.

Clinical significance of methylation signatures of resistance

Figure legends for Supplementary Figures

Methylation and gene expression changes in sensitive and resistant TNBC PDX tumors.

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

Clinical significance of methylation signatures of resistance

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.

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.

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.

BackgroundThere is growing interest in the possible effect of perioperative anesthetic management on the growth and spread of cancer. The impact of perioperative use of opioids on cancer recurrence remains controversial and an assessment cannot yet be established based on current publications. This study aimed to assess the differential expression of opioid receptors between healthy and tumor tissues in patients with stage II and III colorectal cancer undergoing elective surgery by immunohistochemistry (IHC).MethodsPropensity–score matched case–control study nested in a retrospective cohort of patients with stage II or III colorectal. The primary endpoint was the difference in µ–opioid receptor (MOR) expression measured by IHC between tumor and healthy tissue in subject with or without recurrence. Secondary endpoints were to evaluate the differences in Opioid Growth Factor Receptor (OGFR), cyclic adenosine monophosphate (cAMP) production and protein kinase A (PKA) in the matched sample and from a from samples of colorectal cancer stored in the Cancer Genome Atlas (TCGA) and Genotype Tissue Expression Project (GTEx).ResultsThere was a significant difference in MOR receptor (median 3 [intequartile range IQR: 1–3] and 0 [IQR: 0–2], PConclusionIn patients with stage II and III colorectal cancer, overall expression of MOR and OGFR was significantly increased but was not different between previously matched patients with or without recurrence. No differences were found in the analyzed metabolic pathway of cAMP–PKA: These results were confirmed by an in silico analysis of samples from the TCGA–GTEx database.

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.

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.

Polymorphisms in the PER3 gene have been associated with several human disease phenotypes, including sleep disorders and cancer. In particular, the long allele of a variable number of tandem repeat (VNTR) polymorphism has been previously linked to an increased risk of breast cancer. Here we carried out a combined germline and somatic genetic analysis of the role of the PER3

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.

[EN] We report herein an evaluation of the effect of several mesoporous silica nanoparticles (MSNs) on the cellular uptake and in vitro cytotoxicity in human cells. Bare MSNs and MSNs functionalized with polyethylene glycol or hyaluronic acid are employed to evaluate uptake efficiency and mechanisms of endocytosis in cancer (MDA-MB-231) and non-cancer (MCF10A) cells. Moreover, changes in viability, cell cycle, oxidative stress, and mitochondrial membrane potential are evaluated. Our results confirm that MSNs are internalized efficiently by human cells and that uptake mechanisms differ for cell types and particles. We also confirm that MSNs are biocompatible materials that do not induce ROS/RNS production, nor changes on mitochondrial membrane potential or cell cycle., The authors want to thank the Spanish Government RTI2018-100910-B-C41 (MCUI/AEI/FEDER, UE) and PI18/01219 (ISCIII), the Generalitat Valenciana (PROMETEO/2018/024 and ACIF/2016/030), and CIBER-BBN (CB07/01/2012) and CIBER-ONC (CB16/12/00481) for support.

[Purpose]: To characterize expression of neuregulin-1 (NRG1), an HER3 ligand, in HER2-positive breast cancer and its relation with the efficacy of trastuzumab with or without pertuzumab., [Experimental Design]: Characterization of NRG1 expression in tumor cell lines, in tumor specimens, and in cancer-associated fibroblasts (CAFs). Patient-derived CAFs were used to investigate NRG1 impact on the activity of trastuzumab with or without pertuzumab in HER2-positive breast cancer cells. The relationship between NRG1 expression and pathologic response to anti-HER2–based neoadjuvant therapy was assessed in a retrospective patient cohort and in the NeoSphere trial., [Results]: NRG1 was expressed in HER2-positive breast cancer–derived fibroblasts at significantly higher levels than in cancer cells. NRG1 and the conditioned media (CM) from CAFs phosphorylated HER3 and AKT in cancer cells and mediated trastuzumab resistance. Stable genetic depletion of NRG1 from CAFs overcame trastuzumab resistance. Pertuzumab effectively suppressed trastuzumab resistance mediated by either NRG1 or CAF's CM. NRG1 engaged an epithelial-to-mesenchymal transition that was prevented by trastuzumab and pertuzumab. In clinical samples, stromal and/or tumor cell expression of NRG1 determined by immunohistochemistry was uncommon (13.2%) yet significantly linked with residual disease following trastuzumab-based neoadjuvant therapy. In the NeoSphere trial, the magnitude of the difference of pathologic complete response rates favoring the pertuzumab arm was higher in the NRG1-high group., [Conclusions]: CAF-derived NRG1 mediates trastuzumab resistance through HER3/AKT, which might be reverted by pertuzumab. In patients with HER2-positive breast cancer, high expression of NRG1 was associated to poor response to trastuzumab, but not in combination with pertuzumab., This work is supported by ISCIII (CIBERONC CB16/12/00481, CB16/12/00241, PI18/00382, PI18/00006, PI18/01219 and by Generalitat de Catalunya (2017 SGR 507). S. Menendez is supported by Department de Salut, Generalitat de Catalunya (PERIS SLT006/17/00040). MARBiobanc is supported by ISCiii/FEDER (PT17/0015/0011) and by “Xarxa de Bancs de tumors” sponsored by Pla Director d’ Oncologia de Catalunya (XBTC) and Fundacion Jimenez Díaz Biobanks Platform by PT13/0010/0012 grant. Ministry of Economy and Competitiveness of Spain (BFU2015-71371-R) and the CRIS Cancer Foundation provides support to A. Pandiella. Work carried out in our laboratories receive support from the European Community through the Regional Development Funding Program (FEDER). J.C. Montero is funded by the ISCIII through a Miguel Servet program (CPII17/00015) and receives research support from the same institution (PI18/00796). J. Albanell is supported by Breast Cancer Research Foundation (BCRF20-08), Instituto de Salud Carlos III Project Reference number AC15/00062 and the EC under the framework of the ERA-NET TRANSCAN-2 initiative co-financed by FEDER, Instituto de Salud Carlos III (CB16/12/00449 and PI19/01181), and Asociacion Espanola Contra el Cáncer (AECC).

Polymorphisms in the PER3 gene have been associated with several human disease phenotypes, including sleep disorders and cancer. In particular, the long allele of a variable number of tandem repeat (VNTR) polymorphism has been previously linked to an increased risk of breast cancer. Here we carried out a combined germline and somatic genetic analysis of the role of the PER3VNRT polymorphism in breast cancer. The combined data from 8284 individuals showed a non-significant trend towards increased breast cancer risk in the 5-repeat allele homozygous carriers (OR = 1.17, 95% CI: 0.97–1.42). We observed allelic imbalance at the PER3 locus in matched blood and tumor DNA samples, showing a significant retention of the long variant (risk) allele in tumor samples, and a preferential loss of the short repetition allele (p = 0.0005). Gene co-expression analysis in healthy and tumoral breast tissue samples uncovered significant associations between PER3 expression levels with those from genes which belong to several cancer-associated pathways. Finally, relapse-free survival (RFS) analysis showed that low expression levels of PER3 were linked to a significant lower RSF in luminal A (p = 3 × 10−12) but not in the rest of breast cancer subtypes.

Together with its reported ability to modulate AKT phosphorylation (p-AKT) status in several tumor types, the oncoprotein CIP2A has been described to induce breast cancer progression and drug resistance. However, the clinical and therapeutic relevance of the CIP2A/AKT interplay in breast cancer remains to be fully clarified. Here, we found high p-AKT levels in 80 out of 220 cases (36.4%), which were associated with negative estrogen receptor expression (p = 0.049) and CIP2A overexpression (p < 0.001). Interestingly, p-AKT determined substantially shorter overall (p = 0.002) and progression-free survival (p = 0.003), and multivariate analyses showed its CIP2A-independent prognostic value. Moreover, its clinical relevance was further confirmed in the triple negative and HER2-positive subgroups after stratifying our series by molecular subtype. Functionally, we confirmed in vitro the role of CIP2A as a regulator of p-AKT levels in breast cancer cell lines, and the importance of the CIP2A/AKT axis was also validated in vivo. Finally, p-AKT also showed a higher predictive value of response to doxorubicin than CIP2A in ex vivo analyses. In conclusion, our findings suggest that CIP2A overexpression is a key contributing event to AKT phosphorylation and highlights the CIP2A/AKT axis as a promising therapeutic target in breast cancer. However, our observations highlight the existence of alternative mechanisms that regulate AKT signaling in a subgroup of breast tumors without altered CIP2A expression that determines its independent value as a marker of poor outcome in this disease.

Angiogenesis process contributes to the pathogenesis of B-cell chronic lymphocytic leukemia (B-CLL) being the levels of VEGFA and bFGF higher in patients than in healthy controls. Our aim was to evaluate the implication of angiogenesis factors genetic variants in the predisposition to B-CLL and their association with clinical factors and survival. We performed a population-based case-control study in 224 Spanish B-CLL patients and 476 healthy randomly selected controls to evaluate susceptibility to developing B-CLL. Six polymorphisms were evaluated: rs1109324, rs1547651, rs3025039 (+936 C>T), rs833052 of the VEGFA gene, rs1449683 (c.233C>T) of the bFGF gene and (−710 C>T) of the VEGFR1 gene. The association between clinical parameters and patient outcome was analyzed. Carriers of the CT/TT variants of rs3025039 showed a significant protective effect against developing B-CLL. The CT/TT variants of rs1449683 show a tendency towards the development of the disease and the same variants associated significantly with higher genetic risk and with reduced disease free survival. Moreover, the association persisted in the early-stage disease subgroup. Our study provides evidence of the protective effect of the T/- rs3025039 VEGFA variant against B-CLL development and the association of CT/TT variants of the rs1449683 bFGF gene with genetic risk and an adverse survival.

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., The present work was supported by grants from the Spanish Ministry of Economy and Competitiveness (MINECO) with European Regional Development Fund (ERDF) funding through the Institute of Health Carlos III (AES Program, grants PI15/00934, PI18/00382 and PI18/00006; CIBERONC, Biomedical Research Networking Centre for Cancer; Biobanks Platform, PT13/0010/0012; ProteoRed, PRB2-ISCIII, PT13/0001); and the Community of Madrid (S2010/BMD-2344). P.G.-A. was supported by a Fundación Conchita Rábago de Jiménez Díaz grant. P.M. was supported by the ISCIII Miguel Servet Program (CP16/00116).