Intratumor cholesteryl ester accumulation is associated with human breast cancer proliferation and aggressive potential : a molecular and clinicopathological study

Background: The metabolic effect of intratumor cholesteryl ester (CE) in breast cancer remains poorly understood. The objective was to analyze the relationship between intratumor CE content and clinicopathological variables in human breast carcinomas. Methods: We classified 30 breast carcinoma samples into three subgroups: 10 luminal-A tumors (ER+/PR+/Her2-), 10 Her-2 tumors (ER-/PR-/Her2+), and 10 triple negative (TN) tumors (ER-/PR-/Her2-). We analyzed intratumor neutral CE, free cholesterol (FC) and triglyceride (TG) content by thin layer chromatography after lipid extraction. RNA and protein levels of lipid metabolism and invasion mediators were analyzed by real time PCR and Western blot analysis. Results: Group-wise comparisons, linear regression and logistic regression models showed a close association between CE-rich tumors and higher histologic grade, Ki-67 and tumor necrosis. CE-rich tumors displayed higher mRNA and protein levels of low-density lipoprotein receptor (LDLR) and scavenger receptor class B member 1 (SCARB1). An increased expression of acetyl-Coenzyme A acetyltransferase 1 (ACAT1) in CE-rich tumors was also reported. Conclusions: Intratumor CE accumulation is intimately linked to proliferation and aggressive potential of breast cancer tumors. Our data support the link between intratumor CE content and poor clinical outcome and open the door to new antitumor interventions

Circulating microRNAs in Early Breast Cancer Patients and Its Association With Lymph Node Metastases

MicroRNAs have emerged as important regulators of the metastatic process. In addition, circulating miRNAs appear to be surprisingly stable in peripheral blood making them ideal noninvasive biomarkers for disease diagnosis. Here, we performed a proof-of-principle study to investigate the expression profile of circulating miRNAs and their association with the metastatic lymph node status in early breast cancer patients. Sentinel lymph node status was detected by one-step nucleic acid (OSNA) analysis. We performed RNA-sequencing in 16 plasma samples and validated the results by qPCR. Gene Ontology term enrichment and KEGG pathway analyses were carried out using DAVID tools. We found16 differentially expressed miRNAs (q < 0.01) in patients with positive SLNs. Fourteen miRNAs were down-regulated (miR-339-5p, miR-133a-3p, miR-326, miR-331-3p, miR-369-3p, miR-328-3p, miR-26a-3p, miR-139-3p, miR-493-3p, miR-664a-5p, miR-146a-5p, miR-323b-3p, miR-1307-3p and miR-423-3p) and 2 were up-regulated (miR-101-3pand miR-144-3p). Hierarchical clustering using differentially expressed miRNAs clearly distinguished patients according to their lymph node status. Gene ontology analysis showed a significant enrichment of biological processes associated with the regulation of the epithelial mesenchymal transition, cell proliferation and transcriptional regulation. Our results suggest the potential role of several circulating miRNAs as surrogate markers of lymph node metastases in early breast cancer patients. Further validation in a larger cohort of patients will be necessary to confirm our results

ASPN and GJB2 are implicated in the mechanisms of invasion of ductal breast carcinomas

The mechanism of progression from ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC) remains largely unknown. We compared gene expression in tumors with simultaneous DCIS and IDC to decipher how diverse proteins participate in the local invasive process. Twenty frozen tumor specimens with concurrent, but separated, DCIS and IDC were mi-crodissected and evaluated. Total RNA was extracted and microarray analysis was performed using Affymetrix GeneChip® Human Gene 1.0 ST Arrays. Microarray data were validated by quantitative real time reverse transcription-PCR (qRT-PCR) and immunohistochemistry. Controls included seven pure in situ carcinomas, eight fragments from normal breast tissue, and a series of mouse breast carcinomas (MMTV-PyMT). Fifty-six genes were differentially expressed between DCIS and IDC samples. The genes upregulated in IDC samples, and probably associated with invasion, were related to the ep-ithelial-mesenchymal transition (ASPN, THBS2, FN1, SPARC, and COL11A1), cellular adhesion (GJB2), cell motility and progression (PLAUR, PLAU, BGN, ADAMTS16, and ENPP2), extracellular matrix degradation (MMP11, MMP13, and MMP14), and growth/proliferation (ST6GAL2). qRT-PCR confirmed the expression patterns of ASPN, GJB2, ENPP2, ST6GAL2, and TMBS10. Expression of the ASPN and GJB2 gene products was detected by immunohistochemistry in invasive carcinoma foci. The association of GJB2 protein expression with invasion was con-firmed by qRT-PCR in mouse tumors (P < 0.05). Conclusions: The upregulation of ASPN and GJB2 may play important roles in local invasion of breast ductal carcinomas

Chromosome 17 Centromere Duplication and Responsiveness to Anthracycline-Based Neoadjuvant Chemotherapy in Breast Cancer

Human epidermal growth factor receptor 2 (HER2) and topoisomerase II alpha (TOP2A) genes have been proposed as predictive biomarkers of sensitivity to anthracycline chemotherapy. Recently, chromosome 17 centromere enumeration probe (CEP17) duplication has also been associated with increased responsiveness to anthracyclines. However, reports are conflicting and none of these tumor markers can yet be considered a clinically reliable predictor of response to anthracyclines. We studied the association of TOP2A gene alterations, HER2 gene amplification, and CEP17 duplication with response to anthracycline-based neoadjuvant chemotherapy in 140 patients with operable or locally advanced breast cancer. HER2 was tested by fluorescence in situ hybridization and TOP2A and CEP17 by chromogenic in situ hybridization. Thirteen patients (9.3%) achieved pathologic complete response (pCR). HER2 amplification was present in 24 (17.5%) of the tumors. TOP2A amplification occurred in seven tumors (5.1%). CEP17 duplication was detected in 13 patients (9.5%). CEP17 duplication correlated with a higher rate of pCR [odds ratio (OR) 6.55, 95% confidence interval (95% CI) 1.25-34.29, P =.026], and analysis of TOP2A amplification showed a trend bordering on statistical significance (OR 6.97, 95% CI 0.96-50.12, P =.054). TOP2A amplification and CEP17 duplication combined were strongly associated with pCR (OR 6.71, 95% CI 1.66-27.01, P =.007). HER2 amplification did not correlate with pCR. Our results suggest that CEP17 duplication predicts pCR to primary anthracycline-based chemotherapy. CEP17 duplication, TOP2A amplifications, and HER2 amplifications were not associated with prognosis

Mutations Associated with Acquired Resistance to PD-1 Blockade in Melanoma

BACKGROUND: Approximately 75% of objective responses to anti–programmed death 1 (PD-1) therapy in patients with melanoma are durable, lasting for years, but delayed relapses have been noted long after initial objective tumor regression despite continuous therapy. Mechanisms of immune escape in this context are unknown. METHODS: We analyzed biopsy samples from paired baseline and relapsing lesions in four patients with metastatic melanoma who had had an initial objective tumor regression in response to anti–PD-1 therapy (pembrolizumab) followed by disease progression months to years later. RESULTS: Whole-exome sequencing detected clonal selection and outgrowth of the acquired resistant tumors and, in two of the four patients, revealed resistance-associated loss-of-function mutations in the genes encoding interferon-receptor–associated Janus kinase 1 (JAK1) or Janus kinase 2 (JAK2), concurrent with deletion of the wild-type allele. A truncating mutation in the gene encoding the antigen-presenting protein beta-2-microglobulin (B2M) was identified in a third patient. JAK1 and JAK2 truncating mutations resulted in a lack of response to interferon gamma, including insensitivity to its antiproliferative effects on cancer cells. The B2M truncating mutation led to loss of surface expression of major histocompatibility complex class I. CONCLUSIONS: In this study, acquired resistance to PD-1 blockade immunotherapy in patients with melanoma was associated with defects in the pathways involved in interferon-receptor signaling and in antigen presentation. (Funded by the National Institutes of Health and others.

Novel mechanistic link between microtubule disruption and inhibition of tumor angiogenesis

Consultable des del TDXTítol obtingut de la portada digitalitzadaL'angiogènesis, el desenvolupament de nous vasos sanguinis a partir de vasculatura preexistent, és un procés complex que involucra múltiples productes gènics expressats per diferents tipus cel·lulars, i tots ells contribueixen a una seqüència integrada de fenòmens. L'angiogènesis és necessària per tal de permetre el creixement tumoral més enllà d'una certa mida. La hipòxia, un fenomen inherent en els tumors, és un dels factors principals que desencadena el procés angiogènic. En concordança amb el fet que la hipòxia juga un paper clau en tot el procés, un nombre elevat de gens involucrats en diferents processos de l'angiogènesis, incloent el factor de creixement de l'endoteli vascular (VEGF), responen de manera independent a la hipòxia. El principal mecanisme de resposta dels tumors a nivells reduïts d'oxigen és a través de l'activació del factor induïble per hipòxia-1 (HIF-1). HIF-1 és un heterodímer format per dues subunitats; el factor sensible a la hipòxia HIF-1, i el factor no regulable HIF-1. En presència d'oxigen, HIF-1 és hidroxilat i això permet la seva interacció amb la proteïna supressora de tumors Von Hippel Lindau (VHL) que produirà la seva subseqüent i ràpida degradació a través del proteasoma. En absència d'oxigen, HIF-1 és estabilitzat i és transportat cap al nucli cel·lular on heterodimeritza amb HIF-1, unint-se llavors als llocs de resposta a la hipòxia (HRE) i activant la transcripció de nombrosos gens que juguen un paper fonamental en la supervivència tumoral, l'adaptació metabòlica i l'angiogènesis. Per tant, el desenvolupament d'agents capaços d'inhibir l'angiogènesis ha esdevingut una important modalitat terapèutica. Fruit dels bons resultats en estudis preclínics, diversos agents antiangiogènics sols o en combinació amb teràpies convencionals estan en fase de desenvolupament clínic. Entre aquests agents trobem el 2-Metoxiestradiol (2ME2), un metabolit natural de l'estradiol, actualment en avaluació clínica com a inhibidor de l'angiogènesis tumoral. Diversos estudis han demostrat que el 2ME2 és una molècula petita, mot ben tolerada i que posseeix activitats antitumorals i antiangiogèniques en diferents models in vivo. In vitro, el 2ME2 és capaç d'unir-se a la tubulina al lloc d'unió a la colchicina i despolimeritzar els microtúbuls en interfase, produint arrest en mitosi i la posterior mort cel·lular en línies cel·lulars tumorals. A més a més, s'ha mostrat l'abilitat de diferents agents amb capacitat per inhibir la funció dels microtúbuls, sobretot d'aquells que s'uneixen al lloc d'unió a la colchicina, per fer desaparèixer ràpidament la vasculatura tumoral. Tot i això, encara que s'han proposat diferents mecanismes d'acció per al 2ME2, el seu mecanisme d'acció encara no està ben definit. La nostra hipòtesi és que el 2ME2 inhibeix l'angiogènesis tumoral mitjançant la inhibició del factor de transcripció HIF-1 un cop ja ha exercit els seus efectes sobre el citoesquelet de microtúbuls. Així doncs, l'objectiu del nostre estudi va ser I) caracteritzar el mecanisme a nivell molecular pel qual el 2ME2 inhibeix el HIF-1 i l'angiogènesis, i II) investigar si hi ha cap relació causa-efecte entre les propietats antiangiogèniques i anti-tubulina del 2ME2. El nostre treball demostra que el 2ME2 inhibeix l'angiogènesis tumoral mitjançant la inhibició dels nivells proteics del factor HIF-1 així com la seva activitat transcripcional, incloent la transcripció del VEGF, en diverses línies cel·lulars. El 2ME2 no indueix la degradació proteica a través del proteosoma ni tampoc redueix els nivells d'ARNm del HIF-1, sinó que més aviat inhibeix la síntesi proteica de novo. Notablement, hem mostrat que el 2ME2 inhibeix el factor HIF-1 amb posterioritat a la disrupció del citoesquelet de microtúbuls aportant sòlides proves que afavoreixen una relació funcional entre els efectes anti-angiogènics i la disrupció dels microtúbuls. Aquests efectes no són una propietat única del 2ME2 sinó que és compartida per altres agents que actuen sobre els microtúbuls, com ara el taxol i la vincristina, suggerint un mecanisme d'acció semblant per a tots aquests agents. Finalment, la nostra investigació demostra per primera vegada que el 2ME2 inhibeix l'angiogènesis en dosis que són eficaces despolimeritzant els microtúbuls in vivo. Així doncs, el nostre treball aporta la primera demostració d'una associació mecanística entre la disrupció del citoesquelet de microtúbuls i la inhibició de l'angiogènesis a través del factor de transcripció HIF-1 i dóna suport a la hipòtesis que els agents que actuen sobre la tubulina inhibeixen tant el creixement tumoral com la vascularització tumoral amb posterioritat a la disrupció del citoesquelet de microtúbuls. Una caracterització més exhaustiva dels senyals que tenen lloc a nivell molecular entre la disrupció del citoesquelet de microtúbuls i l'angiogènesis mediada pel factor de transcripció HIF-1, molt possiblement doni lloc a la identificació de noves dianes per al desenvolupament de noves estratègies terapèutiques.Angiogenesis, the development of new vessels from preexisting vasculature, is a complex process involving multiple gene products expressed by different cell types, all contributing to an integrated sequence of events. Angiogenesis is required to support tumor growth beyond a certain threshold size. Inherent tumor hypoxia is one of the major factors triggering angiogenesis and consistent with a major role for hypoxia in the overall process, a large number of genes involved in different steps of angiogenesis, including vascular endothelial growth factor (VEGF), are independently responsive to hypoxia. The primary mechanism of tumor response to reduced oxygen levels is via activation of hypoxia inducible-factors-1 (HIF-1). HIF-1 is a heterodimer consisting of two subunits; the labile hypoxic responsive factor, HIF-1, and the non-regulated factor HIF-1β. In the presence of oxygen, HIF-1a is hydroxylated, allowing its interaction with the tumor suppressor protein Von Hippel Lindau (VHL) and its subsequent and rapid degradation by the proteasome. Following hypoxic stabilization, HIF-1 is translocated to the nucleus. There it heterodimerizes with HIF-1 and binds to hypoxia-response elements (HREs), activating the transcription of numerous genes important for cancer survival, metabolic adaptation to hypoxia and angiogenesis. Therefore, the development of agents that inhibit angiogenesis are attractive therapeutic options. Based on successful preclinical data, several anti-angiogenic agents alone or in combination with conventional therapies are now in clinical trials. Among these agents is 2-methoxyestradiol (2ME2), a natural occurring derivative of estradiol, currently undergoing clinical evaluation as an inhibitor of tumor angiogenesis. 2ME2 has been shown to be a well-tolerated small molecule posses antitumor and antiangiogenic activity in different in vivo models. In vitro, 2ME2 has been shown to compete with colchicine for tubulin binding and to disrupt interphase microtubules leading to mitotic arrest and cell death in cultured cancer cells. Furthermore, the ability of microtubule-targeting agents, especially those binding to the colchicine site, has been shown to rapidly shut down existing tumor vasculature. Although several mechanism have been proposed for 2ME2 activity, its mechanism of action still remains unclear. Our hypothesis is 2ME2 inhibits tumor angiogenesis by targeting the HIF pathway downstream of its effects on the microtubule cytoskeleton. Therefore, in this study we seek to I) characterize the molecular mechanism by which 2ME2 inhibits HIF-1 and angiogenesis, and II) Investigate whether there is cause-effect relationship between 2ME2's antiangiogenic and anti-tubulin properties. Our work demonstrated that 2ME2 inhibits tumor angiogenesis by effectively inhibiting HIF-1α levels and transcriptional activity, including transcription of VEGF, in a variety of human cancer cell lines. 2ME2 does not induce proteasomal degradation of HIF-1 nor does it reduces HIF-1 mRNA levels, but rather inhibits the de novo HIF-1 protein synthesis. Notably, we showed that 2ME2 inhibits HIF-1α downstream of disruption of the microtubule cytoskeleton, providing solid proof of a functional relationship between the antitubulin and antiangiogenic effects of 2ME2. These effects of 2ME2 are not unique but rather shared by other microtubule-targeting drugs, such as taxol or vincristine, suggesting a common mechanism of action for all microtubule-targeting drugs. Finally, our findings demonstrate for the first time that 2ME2 inhibits angiogenesis at doses are efficacious disrupting microtubules in vivo. Herein, our work provides the first demonstration of a mechanistic link between disruption of the microtubule cytoskeleton and inhibition of tumor angiogenesis via the HIF-pathway and supports the hypothesis that tubulin-targeting drugs inhibit both tumor cell growth and tumor vascularization following disruption of the microtubule-cytoskeleton. The further characterization of the molecular signal linking the disruption of the microtubule-cytoskeleton with the downregulation of the HIF-mediated angiogenesis, are likely to identify novel targets for the development of new anticancer therapies

Circulating miRNA Expression Is Inversely Correlated with Tumor Tissue or Sentinel Lymph Nodes in Estrogen Receptor-Positive Early Breast Cancer Patients

The deregulation of microRNAs (miRNAs) is associated with the various steps of the metastatic process. In addition, circulating miRNAs are remarkably stable in peripheral blood, making them ideal noninvasive biomarkers for disease diagnosis. Here, we performed a proof-of-principle study to determine whether tumor-tissue-derived miRNAs are traceable to plasma in ER-positive early breast cancer patients. We performed RNA-sequencing on 30 patients for whom plasma, sentinel lymph nodes (SLNs) and tumor tissue were available. We carried out differential expression, gene ontology and enrichment analyses. Our results show that circulating miRNAs are inversely expressed compared with tumor tissue or SLNs obtained from the same patients. Our differential expression analysis shows the overall downregulation of circulating miRNAs. However, the expression of miR-643a-3p and miR-223 was up-regulated in patients with positive SLNs. Furthermore, gene ontology analysis showed the significant enrichment of biological processes associated with the regulation of epithelial cell proliferation and transcriptional regulation commonly involved in the promotion of metastases. Our results suggest the potential role of several circulating miRNAs as surrogate markers of lymph node metastases in early breast cancer patients. Further preclinical and clinical studies are required to understand the biological significance of the most significant miRNAs and to validate our results in a larger cohort of patients