Search

The potential role of hepatocytes versus hepatic progenitor cells (HPC) on the onset and pathogenesis of hepatocellular carcinoma (HCC) has not been fully clarified. Because the administration of 2‐acetylaminofluorene (2AAF) followed by a partial hepatectomy, selectively induces the HPC proliferation, we investigated the effects of chronic 2AAF administration on the HCC development caused by the chronic administration of the carcinogen diethylnitrosamine (DEN) for 16 weeks in the rat. DEN + 2AAF protocol impeded weight gain of animals but promoted prominent hepatomegaly and exacerbated liver alterations compared to DEN protocol alone. The tumor areas detected by γ‐glutamyl transferase, prostaglandin reductase‐1, and glutathione S‐transferase Pi−1 liver cancer markers increased up to 80% as early as 12 weeks of treatment, meaning 6 weeks earlier than DEN alone. This protocol also increased the number of Ki67‐positive cells and those of CD90 and CK19, two well‐known progenitor cell markers. Interestingly, microarray analysis revealed that DEN + 2AAF protocol differentially modified the global gene expression signature and induced the differential expression of 30 genes identified as HPC markers as early as 6 weeks of treatment. In conclusion, 2AAF induces the early appearance of HPC markers and as a result, accelerates the hepatocarcinogenesis induced by DEN in the rat. Thus, since 2AAF simultaneously administrated with DEN enriches HPC during hepatocarcinogenesis, we propose that DEN + 2AAF protocol might be a useful tool to investigate the cellular origin of HCC with progenitor features.

Ion channels and transporters are potential markers and therapeutic targets for several cancers. However, their expression during hepatocellular carcinoma (HCC) development remains unclear.To investigate the mRNA expression of Na(+), K(+) and Ca(2+) channels and ABC transporters during rat HCC development, as well as Abcc3 protein in human liver biopsies.Wistar rats were treated with diethylnitrosamine (DEN) and developed both cirrhosis (12 weeks of treatment) and either pre-neoplastic lesions (16 weeks of treatment) or multinodular HCC (16 weeks of treatment plus 2 weeks DEN-free). The mRNA expression of 12 ion channels and two ABC transporters was studied using real-time RT-PCR. Tumor-containing or tumor-free liver sections were isolated by laser-capture microdissection. Abcc3 protein expression was studied by immunohistochemistry in healthy, cirrhotic and HCC human biopsies.We observed expression changes in seven genes. Kcna3, Kcnn4, Kcnrg and Kcnj11 potassium channel mRNA expression reached peak values at the end of DEN treatment, while Scn2a1 sodium channel, Trpc6 calcium channel and Abcc3 transporter mRNA expression reached their highest levels in the presence of HCC (18 weeks). Whereas Kcnn4 and Scn2a1 channel expression was similar in non-tumor and tumor tissue, the Abcc3 transporter and Kcna3 potassium channels were preferentially overexpressed in the tumor sections. We observed differential Abcc3 protein subcellular localization and expression in human samples.The ion channel/transporter expression profile observed suggests that these genes are potential early markers or therapeutic targets of HCC. The differential localization of Abcc3 may be useful in the diagnosis of cirrhosis and HCC.

Artificial sweeteners are mainly used as substitutes for sucrose derivates. In this study, we analyzed if the chronic consumption of aspartame or acesulfame potassium at an early age, produces histological alterations, astrogliosis and decreased neuronal viability, in hippocampus, prefrontal cortex, amygdala and hypothalamus of male Wistar rats. A histological analysis was performed on male Wistar rats that consumed aspartame or acesulfame potassium during 90 days, initiating the consumption of sweeteners immediately after weaning. The evaluation of neuronal morphology in different areas of the brain was performed with hematoxylin - eosin staining. To measure astrogliosis and neuronal viability, we used the immunohistochemical technique, with the glial fibrillary acidic protein immunomodulators (GFAP) and with neuronal-specific enolase (NSE). The consumption of aspartame or acesulfame potassium promoted morphological changes of neurons including increased pyknotic nuclei and vacuolization in all the brain areas studied. In hippocampus, prefrontal cortex, amygdala and hypothalamus, astrogliosis and reduction of neural viability were observed in sweeteners consumers in comparison with the control group. Chronic consumption of ASP and ACK from early stages of development and during long periods, may promote neural modifications, astrogliosis and decrease neuronal viability in prefrontal cortex, amygdala, hippocampus, and hypothalamus.

Breast cancer (BC) is the most common malignancy among the female population. Ablation Radiofrequency (RF) assisted with magnetic nanoparticles (MNPs) coupled to a monoclonal antibody anti-HER2 (Human epidermal growth factor receptor 2) has been proposed as an alternative therapeutic technique, the concept behind the technical proposal is to change the tissue electrical conductivity by the use of MNPs, however, the local biochemical and bioimpedance effects of MNPs in cancerous tissue remain unknown, as a first approach to explore such effects caused by MNPs, the present work was focused on to assess biochemically the bioconjugated “MNPs - anti-HER2” systemic infusion effect on tumor tissue through studying the protein dynamics of the Growth Factor Receptor-Bound Protein 2 (GRB2) and its implication on the HER2 signaling pathway as well as its correlation with changes in bioimpedance measurement. A breast cancer model in rats in three specific conditions was evaluated by tissue immunostaining and bioimpedance measurements. Normal breast tissue (Healthy), Breast Cancer tissue (BC) and Breast Cancer tissue with MNPs (BC+MNPs). The results show that GRB2 protein expression in BC+MNPs is null or similar to Healthy, both compared with respect to BC. In addition, bioimpedance measurements are similar in BC+MNPs and Healthy conditions, and different values are evident in BC, those findings are in agreement with GRB2 expression. It seems that systemic bioconjugate infusion inhibits HER-2 signaling pathway and changes in the electrical conductivity of the tumor tissue.

Prostaglandin reductase-1 (Ptgr1) is an alkenal/one oxidoreductase that is involved in the catabolism of eicosanoids and lipid peroxidation such as 4-hydroxynonenal (4-HNE). Recently, we reported that Ptgr1 is overexpressed in human clinical and experimentally induced samples of hepatocellular carcinoma (HCC). However, how the expression of this gene is regulated and its role in carcinogenesis are not yet known. Here, we studied parameters associated with antioxidant responses and the mechanisms underlying the induction of Ptgr1 expression by the activation of Nuclear Factor (erythroid-derived-2)-like-2 (NRF2). For these experiments, we used two protocols of induced hepatocarcinogenesis in rats. Furthermore, we determined the effect of PTGR1 on cell proliferation and resistance to oxidative stress in cell cultures of the epithelial liver cell line, C9. Ptgr1 was overexpressed during the early phase in altered hepatocyte foci, and this high level of expression was maintained in persistent nodules until tumors developed. Ptgr1 expression was regulated by NRF2, which bound to an antioxidant response element at -653bp in the rat Ptgr1 gene. The activation of NRF2 induced the activation of an antioxidant response that included effects on proteins such as glutamate-cysteine ligase, catalytic subunit, NAD(P)H dehydrogenase quinone-1 (NQO1) and glutathione-S-transferase-P (GSTP1). These effects may have produced a reduced status that was associated with a high proliferation rate in experimental tumors. Indeed, when Ptgr1 was stably expressed, we observed a reduction in the time required for proliferation and a protective effect against hydrogen peroxide- and 4-HNE-induced cell death. These data were consistent with data showing colocalization between PTGR1 and 4-HNE protein adducts in liver nodules. These findings suggest that Ptgr1 and antioxidant responses act as a metabolic adaptation and could contribute to proliferation and cell-death evasion in liver tumor cells. Furthermore, these data indicate that Ptgr1 could be used to design early diagnostic tools or targeted therapies for HCC.

Heterogeneity of breast cancer can be classified according to different molecular signatures, including gene expression profiles that differentiate breast cancer into 5 clinically relevant subtypes: luminal A, luminal B, Her2-like, basal-like and claudin-low. Additionally the expression patterns of gene expression regulators like microRNAs can separate normal tissue from breast tumors, however there is currently limited information about the potential differences in microRNA expression profiles between the different tumor subtypes defined by gene expression. In order to determine microRNA expression profiles in breast tumors, and explore differences in the expression patterns of these molecules between different cancer subtypes, we analyzed the expression of 664 microRNAs with the TaqMan low-density array platform in 40 breast tumors from different subtypes. Tumor sub-typing was carried out with the PAM50 algorithm in expression data obtained with the Affymetrix Human Gene ST 1.0 array, obtaining 8 Luminal A tumors, 9 luminal B, 8 Her2-like and 3 Triple negative basal-like tumors, along with a set of microRNAs that distinguish each tumor subtype. Bioinformatic analysis of mRNA targets of the differentially expressed miRNAs, identified oncogenes like ERBB2, YY1, several MAP kinases, and known tumor-suppressors like FOXA1 and SMAD4. Pathway analysis identified that some biological process that are important in breast carcinogenesis are affected by the altered miRNA expression, including signaling through MAP kinases, RAS, programmed cell death and ERBB2-ERBB3 signaling. Given the clinical importance of the triple negative tumors, we included 48 additional triple-negative tumors defined by immunohistochemistry, to analyze the intra-heterogeneity of these phenotype based in the microRNA expression profiles. After the bioinformatics analysis with the algorithm Consensus Clustering we determined 6 sub-groups of triple negative tumors with different molecular, inmunophenotypical and clinical issues. We also compared the microRNA expression patterns between triple negative and other inmunophenotype (ER+, PR+, Her2+) tumors, detecting 56 differentially expressed microRNAs. Transcriptional targets of these microRNAs include genes involved in the carcinogenesis of triple negative tumors, like PARP1, or in the microRNA biogenesis machinery, like Dicer. Enrichment ontology analysis of the microRNAs differentially expressed in the triple negative tumors, detected pathways like p53 and focal adhesion whose role in cancer development, invasion and metastasis might be crucial; and MAPK, which has been related to recurrence of TN tumors. Immunochemistry analysis on the microRNA biogenesis machinery including Dicer and Argonaute2 revels a down-regulation (approximately 20% less) of both proteins, mainly in TN tumors. Our data identified the altered expression of microRNAs whose aberrant expression might have an important impact on cancer-related cellular pathways and whose role in breast cancer has not been previously described. microRNAs expression is also capable to discriminate between different tumor subtypes and to determine microRNAs that classifies triple negative tumors into different subgroups. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-10-12.

Breast carcinoma is the leading cause of cancer-related mortality in women worldwide with an estimated 1.38 million new cases and 458,000 deaths in 2008 alone1. This malignancy represents a heterogeneous group of tumours with characteristic molecular features, prognosis, and responses to available therapy2–4. Recurrent somatic alterations in breast cancer have been described including mutations and copy number alterations, notably ERBB2 amplifications, the first successful therapy target defined by a genomic aberration5. Prior DNA sequencing studies of breast cancer genomes have revealed additional candidate mutations and gene rearrangements 6–10. Here we report the whole-exome sequences of DNA from 103 human breast cancers of diverse subtypes from patients in Mexico and Vietnam compared to matched-normal DNA, together with whole-genome sequences of 22 breast cancer/normal pairs. Beyond confirming recurrent somatic mutations in PIK3CA11, TP536, AKT112, GATA313, and MAP3K110, we discovered recurrent mutations in the CBFB transcription factor gene and deletions of its partner RUNX1. Furthermore, we have identified a recurrent MAGI3-AKT3 fusion enriched in triple-negative breast cancer lacking estrogen and progesterone receptors and ERBB2 expression. The Magi3-Akt3 fusion leads to constitutive activation of Akt kinase, which is abolished by treatment with an ATP-competitive Akt small-molecule inhibitor.

Multicellular spheroids are excellent models for the analysis of cancer behavior. Just like small avascular tumors, they present a marked zonal heterogeneity which influences gene expression and thus, growth and response to chemotherapy. In the present paper, we sought to analyze the effects of three-dimensional culture in the expression and distribution of estrogen receptor alpha. Using MCF-7 breast cancer cells, we found that multicellular spheroids in estrogen-containing medium presented a paradoxical regulation of estrogen receptor alpha, with a decrease in protein expression and a marked increase in mRNA steady-state levels. Immunohistochemistry showed that only sparse cells in the periphery of the spheroid expressed estrogen receptor, in sharp contrast with progresterone receptor, which was more extensively expressed and HIF-alpha, which was expressed in the central core of the spheroid. This could mean that both hypoxia and ERA activation by estrogen participate in the expression heterogeneity of this hormone receptor in breast cancer These results are important to considerate in the analysis and interpretation of immunohistochemistry of ERA and downstream targets in samples of solid tumors.

In tumor cells the effectiveness of anti-neoplastic agents that cause cell death by induction of DNA damage is influenced by DNA repair activity. RAD50 protein plays key roles in DNA double strand breaks repair (DSBs), which is crucial to safeguard genome integrity and sustain tumor suppression. However, its role as a potential therapeutic target has not been addressed in breast cancer. Our aim in the present study was to analyze the expression of RAD50 protein in breast tumors, and evaluate the effects of RAD50-targeted inhibition on the cytotoxicity exerted by cisplatin and anthracycline and taxane-based therapies in breast cancer cells. Immunohistochemistry assays on tissue microarrays indicate that the strong staining intensity of RAD50 was reduced in 14% of breast carcinomas in comparison with normal tissues. Remarkably, RAD50 silencing by RNA interference significantly enhanced the cytotoxicity of cisplatin. Combinations of cisplatin with doxorubicin and paclitaxel drugs induced synergistic effects in early cell death of RAD50-deficient MCF-7, SKBR3, and T47D breast cancer cells. Furthermore, we found an increase in the number of DSBs, and delayed phosphorylation of histone H2AX after cisplatin treatment in RAD50-silenced cells. These cellular events were associated to a dramatical increase in the frequency of chromosomal aberrations and a decrease of cell number in metaphase. In conclusion, our data showed that RAD50 abrogation impairs DNA damage response and sensitizes breast cancer cells to cisplatin-combined therapies. We propose that the development and use of inhibitors to manipulate RAD50 levels might represent a promising strategy to sensitize breast cancer cells to DNA damaging agents.

γ-Glutamyl transferase (GGT) is useful as a marker in pathological conditions, including several types of cancer. We optimized the histochemical detection of GGT to assay the gene expression profiles of phenotype-specific cells selected by laser capture microdissection (LCM). For optimization, we used the livers of rats subjected to hepatocarcinogenesis. This model induced nodules of hepatocytes and tumors with GGT activity. To obtain sufficient high-quality RNA after histochemistry and LCM, we included an RNase inhibitor and air-dried the tissue sections. This optimization allowed the visualization of GGT activity in situ and a yield of 1.4 to 2.0 μg of total RNA from 15 to 18 mm 2 of microdissected tissue (20 µm thickness). The average RNA integrity number in GGT-positive tissue, determined by chip–capillary electrophoresis, was 6.9, and the 28S/18S ribosomal RNA (rRNA) ratio was 1.4. The RNAs were processed for the Rat Gene 1.0 ST Array (Affymetrix). Comparable quality control metrics, such as signal intensity and RNA degradation plots, were found between the LCM samples and non-LCM tissue. The increased expression of Ggt1 expected in GGT-positive tissue was confirmed by microarrays and quantitative reverse transcriptase polymerase chain reaction (qRT–PCR). This optimization provided a suitable method for whole-transcript analysis of GGT-positive tissue isolated using LCM.

Ethnopharmacological relevance Justicia spicigera is used for the empirical treatment of cervical cancer in Mexico. Recently, we showed that Justicia spicigera extracts exerted cytotoxic and antitumoral effects and the major component of this extract was kaempferitrin (KM). Materials and methods The cytotoxic and apoptotic effect of KM on human cancer cells and human nontumorigenic cells were evaluated using MTT and TUNEL assays, and Annexin V/Propidium iodide detection by flow cytometry. The effect of KM on cell cycle was analyzed by flow cytometry with propidium iodide. The apoptotic and cell cycle effects were also evaluated by western blot analysis. Also, different doses of KM were injected intraperitoneally daily into athymic mice bearing tumors of HeLa cells during 32 days. The growth and weight of tumors were measured. Results KM induces high cytotoxic effects in vitro and in vivo against HeLa cells. The general mechanisms by which KM induces cytotoxic effects include: cell cycle arrest in G1 phase and apoptosis via intrinsic pathway in a caspase dependent pathway. Also, KM exerts chemopreventive and antitumor effects. Conclusion KM exerts cytotoxic and antitumor effects against HeLa cells.

Breast cancer is the neoplasia with the highest incidence in women worldwide. Proteomics approaches have accelerated the discovery of diagnostic and prognostic biomarkers. Here, we compared the proteomic profiles of breast tumors versus non-tumoral tissues in order to identify modulated proteins, which could represent potential markers associated to clinical features. By two-dimensional electrophoresis, we detected 28 differentially expressed proteins. Among these, 21 proteins were up-regulated and 7 were down-regulated in tumors (p0.05). Proteins were identified using LC/ESI-MS/MS tandem mass spectrometry. One protein was identified as glyoxalase 1 (GLO1), an enzyme involved in detoxification of methylglyoxal, a cytotoxic product of glycolysis. GLO1 overexpression was confirmed by western blot assays in paired normal and tumor breast tissues in clinical stages I-III, and by immunohistochemistry on tissue microarrays (TMA) comprising a cohort of 98 breast tumors and 20 healthy specimens. Results from TMA demonstrated that GLO1 is overexpressed in 79% of tumors. Interestingly, GLO1 up-regulation correlates with advanced tumor grade (p0.05). These findings demonstrate the association of GLO1 overexpression with tumor grade and pointed out for additional studies to establish the importance of GLO1 in breast cancer.

Heterogeneity of breast cancer, specifically in triple negative (TN) tumors represents a clinical challenge which deserves further research in order to understand tumor biology and improve treatment options. MicroRNAs are small non-coding RNAs that regulates gene expression and play crucial roles in breast carcinogenesis. In order to analyze the heterogeneity and identify different subgroups of TN tumors, we analyzed the genome-wide microRNA expression patterns of 1105 mature miRNAs in 98 FFPE TN and 15 additional tumors with other immunophenotypes, using the Affymetrix Gene Expression miRNA microarray v2.0. Bioinformatic analysis with the non-supervised Consensus Clustering algorithm defined 4 groups, each of them showing association with clinically relevant parameters like the presence of metastases and survival. Several cellular pathways like cell proliferation, programmed cell death and motility are enriched in each of the microRNA expression defined groups. Based on miRNAs expression profiles between TN tumors and other inmunophenotypes (N=15 tumors), 3 miRNAs (miR-125b-2-3p, miR-342-3p -both of them down-regulated in TN tumors- and miR-660 -over-expressed-) were chosen for further functional studies in cellular models to better define their relationship with the biology and phenotype of triple negative tumors. After transfections in TN cell lines (MDA MB 231 and 468) a genomic evaluation of their transcriptional targets was made with the Human Gene St 1.0 array (Affymetrix), this data, together with in silico analysis, defined at least 25 mRNAs that are regulated by those miRNAs. Through this approach, we found that different patterns of mRNA targets emerge for each cellular model, reflecting their distinctive molecular and cellular features. The transcriptional targets identified, are involved in different tumorigenic pathways, such as activation of immune responses, regulation of apoptosis, cell motility and adhesion. Some of these results have been validated by other methodologies like measurement of Ki67 for proliferation, Annexin V for apoptosis and cell cycle status. Our data identified the altered expression of microRNAs that reveals the existence of 4 subgroups with different prognostic values among TN tumors. Furthermore, we performed the characterization of 3 miRNAs with aberrant expression in TN tumors that regulate cancer-related mRNAs and whose role in TN breast cancer has not been previously described. Together, these findings confirm the participation of miRNAs as regulators of cancer programs in triple negative tumors. Citation Format: Sandra L. Romero-Cordoba, Rosa Rebollar-Vega, Valeria Quintanar-Jurado, Alfredo Hidalgo-Miranda, Sergio Rodriguez-Cuevas, Veronica Bautista-Pina, Antonio Maffuz-Aziz. miRNA profiles identify different subgroups of triple negative tumors and reveal novel miRNA-mRNA interactions in breast cancer tumorigenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4370. doi:10.1158/1538-7445.AM2014-4370

Today, more than 55% of the world's breast cancer cases are diagnosed in low and middle-income countries and in 2020, more that 70% of the cases will come from the developing nations. In Mexico, breast cancer-specific mortality doubled during the past 20 years, representing the second-leading cause of death in women between 30 and 59 years and the leading cause of cancer related death in the female population. According to statistics, in Mexico a woman dies due to breast cancer every two hours. Even though breast cancer represents a major public health problem in the developing world, knowledge about the genetic and genomic structure of breast tumors in Mexican or Latin American populations is very limited. In the past four years, we have participated in the Slim Initiative of Genomic Medicine (SIGMA) Project, a collaboration between the Carlos Slim Institute of Health, the Broad Institute, and the National Institute of Genomic Medicine in Mexico city. The goal of the SIGMA project is to characterize the genomic basis of common diseases, including several types of cancer. This effort has focused on the application of whole genome and whole exome sequencing of human tumors. In the case of breast cancer, we have analyzed the whole genomes of 22 tumor/normal tissue pairs and the whole exomes of 103 tumor/normal tissues from Mexican and Vietnamese patients. Sequence analysis led to the novel identification of potential loss of function mutations of the CBFB transcription factor, and deletions of its partner RUNX1, an event which has never been previously reported in breast tumors or in any other epithelial tumor. Of clinical relevance, we also identified a somatic translocation involving MAGI3 and AKT3 in a triple negative breast tumor. Ectopic expression of the fusion transcrip leads to constitutive phosphorylation of downstream GSK and loss of contact inhibition. Most importantly, the activity of the fusion protein can be abrogated by an ATP-competitive small molecule inhibitor of AKT, potentially representing a new therapeutic avenue for these patients. In parallel with sequencing, we have also been working on the analysis of somatic DNA copy number aberrations, messenger RNA expression, and microRNA expression patterns in tumors from Mexican patients. Intrinsic breast cancer sub-typing in 125 tumors from Mexican patients showed that 13.6% of the tumors were basal-like, 16.8% were Her2-enriched, 24.8% Luminal A, 34.4% Luminal B and 10.4 normal-like. With microRNA expression, we have identified a group of microRNAs whose role in breast cancer has not been previously described and are currently analyzing differential microRNA expression across tumor sub-types, in particular triple negative tumors, where we have been able to identify at least three different tumor sub-groups based on microRNA expression patterns. Citation Format: Shantanu Banerji, Kristian Cibulskis, Claudia Rangel-Escareño, Kristin K. Brown, Scott L. Carter, Abbie M. Frederick, Michael S. Lawrence, Andrey Y. Sivachenko, Carrie Sougnez, Lihua Zou, Maria L. Cortes, Juan C. Fernandez-Lopez, Shouyong Peng, Kristin G. Ardlie, Daniel Auclair, Veronica Bautista-Piña, Fujiko Duke, Joshua Francis, Joonil Jung, Antonio Maffuz-Aziz, Robert C. Onofrio, Melissa Parkin, Nam H. Pho, Valeria Quintanar-Jurado, Alex H. Ramos, Rosa Rebollar-Vega, Sergio A. Rodríguez-Cuevas, Sandra L. Romero-Cordoba, Steven E. Schumacher, Nicolas Stransky, Kristin M. Thompson, Laura Uribe-Figueroa, Jose Baselga, Rameen Beroukhim, Kornelia Polyak, Dennis C. Sgroi, Andrea L. Richardson, Gerardo Jimenez-Sánchez, Eric S. Lander, Stacey B. Gabriel, Levi A. Garraway, Todd R. Golub, Jorge Meléndez-Zajgla, Alex Toker, Gad Getz, Matthew Meyerson, Alfredo Hidalgo-Miranda. Molecular profiling of breast cancer in Mexico: Identification of novel therapeutic targets through whole genome sequencing analysis. [abstract]. In: Proceedings of the Fifth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2012 Oct 27-30; San Diego, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2012;21(10 Suppl):Abstract nr PL07-01.

microRNA expression signatures can differentiate normal and breast cancer tissues and can define specific clinico-pathological phenotypes in breast tumors. In order to further evaluate the microRNA expression profile in breast cancer, we analyzed the expression of 667 microRNAs in 29 tumors and 21 adjacent normal tissues using TaqMan Low-density arrays. 130 miRNAs showed significant differential expression (adjusted P value = 0.05, Fold Change = 2) in breast tumors compared to the normal adjacent tissue. Importantly, the role of 43 of these microRNAs has not been previously reported in breast cancer, including several evolutionary conserved microRNA*, showing similar expression rates to that of their corresponding leading strand. The expression of 14 microRNAs was replicated in an independent set of 55 tumors. Bioinformatic analysis of mRNA targets of the altered miRNAs, identified oncogenes like ERBB2, YY1, several MAP kinases, and known tumor-suppressors like FOXA1 and SMAD4. Pathway analysis identified that some biological process which are important in breast carcinogenesis are affected by the altered microRNA expression, including signaling through MAP kinases and TP53 pathways, as well as biological processes like cell death and communication, focal adhesion and ERBB2-ERBB3 signaling. Our data identified the altered expression of several microRNAs whose aberrant expression might have an important impact on cancer-related cellular pathways and whose role in breast cancer has not been previously described.

Breast cancer can be classified according to the gene expression signatures of the tumors into four clinically relevant sub-types, luminal A, luminal B, Her2-like and basal-like. Additionally microRNA expression patterns can separate normal tissue from breast tumors, but there is currently limited information about the potential differences in microRNA expression profiles between the different tumor subtypes, defined by gene expression. In order to determine the microRNA expression profiles in breast tumors, and explore the differences in the expression patterns of these molecules between different cancer subtypes, we analyzed the expression of 664 microRNAs with the TaqMan low-density array platform in 40 breast tumors from different subtypes, and in 21 adjacent normal breast tissues. Tumor sub-typing was carried out with the PAM50 algorithm in expression data obtained with the Affymetrix Human Gene ST 1.0 array, obtaining 8 Luminal A tumors, 9 luminal B, 8 Her2-like and 3 Triple negative basal-like tumors. Given the low number of basal-like tumors, for the microRNA expression analysis, we included 12 additional triple-negative tumors defined by immunohistochemistry. Finally, we analyzed the expression of DICER and Ago2, involved in the biogenesis of microRNAs in the breast tumors. 131 microRNAs showed significant differential expression (adjusted P value=0.05, Fold Change=2) in breast tumors compared to the normal adjacent tissue. The role of 25% of these microRNAs has not been previously reported in breast cancer. 10 microRNAs showed differential expression between basal/triple negative tumors compared to hormone receptor and HER2 positive tumors. Transcriptional targets of these microRNAs include genes involved in the carcinogenesis of triple negative tumors, like PARP1, or in the microRNA biogenesis machinery, like DICER. Enrichment ontology analysis of the microRNAs differentially expressed in the TN tumors, detected pathways like p53 and focal adhesion whose role in cancer development, invasion and metastasis might be crucial; and MAPK, which has been related to recurrence of TN tumors. Regarding the expression of Ago2 and DICER, we detected down-regulation (approximately 20% less) of both proteins, mainly in TN tumors. Down-regulation in the triple negative tumors was further validated at the mRNA level by RT-qPCR assays. Our data identified the altered expression of several microRNAs whose aberrant expression might have an important impact on cancer-related cellular pathways and whose role in breast cancer has not been previously described. microRNAs expression is also capable to discriminate between different tumor subtypes and the expression of proteins involved in microRNA biogenesis might play a role in breast carcinogenesis, specifically in the triple negative subtype. Citation Format: Sandra L. Romero-Cordoba, Rosa G. Rebollar-Vega, Valeria Quintanar-Jurado, Veronica Bautista-Pina, Sergio Rodriguez-Cuevas, Antonio Maffuz-Aziz, Alfredo Hidalgo-Miranda. Differences in microRNA expression patterns in breast cancer subtypes [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer; 2012 Jan 8-11; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(2 Suppl):Abstract nr A25.