Secreted breast tumor interstitial fluid microRNAs and their target genes are associated with triple-negative breast cancer, tumor grade, and immune infiltration.

BACKGROUND: Studies on tumor-secreted microRNAs point to a functional role of these in cellular communication and reprogramming of the tumor microenvironment. Uptake of tumor-secreted microRNAs by neighboring cells may result in the silencing of mRNA targets and, in turn, modulation of the transcriptome. Studying miRNAs externalized from tumors could improve cancer patient diagnosis and disease monitoring and help to pinpoint which miRNA-gene interactions are central for tumor properties such as invasiveness and metastasis. METHODS: Using a bioinformatics approach, we analyzed the profiles of secreted tumor and normal interstitial fluid (IF) microRNAs, from women with breast cancer (BC). We carried out differential abundance analysis (DAA), to obtain miRNAs, which were enriched or depleted in IFs, from patients with different clinical traits. Subsequently, miRNA family enrichment analysis was performed to assess whether any families were over-represented in the specific sets. We identified dysregulated genes in tumor tissues from the same cohort of patients and constructed weighted gene co-expression networks, to extract sets of co-expressed genes and co-abundant miRNAs. Lastly, we integrated miRNAs and mRNAs to obtain interaction networks and supported our findings using prediction tools and cancer gene databases. RESULTS: Network analysis showed co-expressed genes and miRNA regulators, associated with tumor lymphocyte infiltration. All of the genes were involved in immune system processes, and many had previously been associated with cancer immunity. A subset of these, BTLA, CXCL13, IL7R, LAMP3, and LTB, was linked to the presence of tertiary lymphoid structures and high endothelial venules within tumors. Co-abundant tumor interstitial fluid miRNAs within this network, including miR-146a and miR-494, were annotated as negative regulators of immune-stimulatory responses. One co-expression network encompassed differences between BC subtypes. Genes differentially co-expressed between luminal B and triple-negative breast cancer (TNBC) were connected with sphingolipid metabolism and predicted to be co-regulated by miR-23a. Co-expressed genes and TIF miRNAs associated with tumor grade were BTRC, CHST1, miR-10a/b, miR-107, miR-301a, and miR-454. CONCLUSION: Integration of IF miRNAs and mRNAs unveiled networks associated with patient clinicopathological traits, and underlined molecular mechanisms, specific to BC sub-groups. Our results highlight the benefits of an integrative approach to biomarker discovery, placing secreted miRNAs within a biological context.

Topoisomerase I activity and sensitivity to camptothecin in breast cancer-derived cells: a comparative study.

BACKGROUND: Camptothecin (CPT) and its derivatives are currently used as second- or third-line treatment for patients with endocrine-resistant breast cancer (BC). These drugs convert nuclear enzyme DNA topoisomerase I (TOP1) to a cell poison with the potential to damage DNA by increasing the half-life of TOP1-DNA cleavage complexes (TOP1cc), ultimately resulting in cell death. In small and non-randomized trials for BC, researchers have observed extensive variation in CPT response rates, ranging from 14 to 64%. This variability may be due to the absence of reliable selective parameters for patient stratification. BC cell lines may serve as feasible models for generation of functional criteria that may be used to predict drug sensitivity for patient stratification and, thus, lead to more appropriate applications of CPT in clinical trials. However, no study published to date has included a comparison of multiple relevant parameters and CPT response across cell lines corresponding to specific BC subtypes. METHOD: We evaluated the levels and possible associations of seven parameters including the status of the TOP1 gene (i.e. amplification), TOP1 protein expression level, TOP1 activity and CPT susceptibility, activity of the tyrosyl-DNA phosphodiesterase 1 (TDP1), the cellular CPT response and the cellular growth rate across a representative panel of BC cell lines, which exemplifies three major BC subtypes: Luminal, HER2 and TNBC. RESULTS: In all BC cell lines analyzed (without regard to subtype classification), we observed a significant overall correlation between growth rate and CPT response. In cell lines derived from Luminal and HER2 subtypes, we observed a correlation between TOP1 gene copy number, TOP1 activity, and CPT response, although the data were too limited for statistical analyses. In cell lines representing Luminal and TNBC subtypes, we observed a direct correlation between TOP1 protein abundancy and levels of enzymatic activity. In all three subtypes (Luminal, HER2, and TNBC), TOP1 exhibits approximately the same susceptibility to CPT. Of the three subtypes examined, the TNBC-like cell lines exhibited the highest CPT sensitivity and were characterized by the fastest growth rate. This indicates that breast tumors belonging to the TNBC subtype, may benefit from treatment with CPT derivatives. CONCLUSION: TOP1 activity is not a marker for CPT sensitivity in breast cancer.

Gel-Based Proteomics of Clinical Samples Identifies Potential Serological Biomarkers for Early Detection of Colorectal Cancer.

The burden of colorectal cancer (CRC) is considerable-approximately 1.8 million people are diagnosed each year with CRC and of these about half will succumb to the disease. In the case of CRC, there is strong evidence that an early diagnosis leads to a better prognosis, with metastatic CRC having a 5-year survival that is only slightly greater than 10% compared with up to 90% for stage I CRC. Clearly, biomarkers for the early detection of CRC would have a major clinical impact. We implemented a coherent gel-based proteomics biomarker discovery platform for the identification of clinically useful biomarkers for the early detection of CRC. Potential protein biomarkers were identified by a 2D gel-based analysis of a cohort composed of 128 CRC and site-matched normal tissue biopsies. Potential biomarkers were prioritized and assays to quantitatively measure plasma expression of the candidate biomarkers were developed. Those biomarkers that fulfilled the preset criteria for technical validity were validated in a case-control set of plasma samples, including 70 patients with CRC, adenomas, or non-cancer diseases and healthy individuals in each group. We identified 63 consistently upregulated polypeptides (factor of four-fold or more) in our proteomics analysis. We selected 10 out of these 63 upregulated polypeptides, and established assays to measure the concentration of each one of the ten biomarkers in plasma samples. Biomarker levels were analyzed in plasma samples from healthy individuals, individuals with adenomas, CRC patients, and patients with non-cancer diseases and we identified one protein, tropomyosin 3 (Tpm3) that could discriminate CRC at a significant level (p = 0.0146). Our results suggest that at least one of the identified proteins, Tpm3, could be used as a biomarker in the early detection of CRC, and further studies should provide unequivocal evidence for the real-life clinical validity and usefulness of Tpm3.

Gaining insights into cancer biology through exploration of the cancer secretome using proteomic and bioinformatic tools.

INTRODUCTION: Tumor-associated proteins released by cancer cells and by tumor stroma cells, referred as 'cancer secretome', represent a valuable resource for discovery of potential cancer biomarkers. The last decade was marked by a great increase in number of studies focused on various aspects of cancer secretome including, composition and identification of components externalized by malignant cells and by the components of tumor microenvironment. Areas covered: Here, we provide an overview of achievements in the proteomic analysis of the cancer secretome, elicited through the tumor-associated interstitial fluid recovered from malignant tissues ex vivo or the protein component of conditioned media obtained from cultured cancer cells in vitro. We summarize various bioinformatic tools and approaches and critically appraise their outcomes, focusing on problems and challenges that arise when applied for the analysis of cancer secretomic databases. Expert commentary: Recent achievements in the omics- analysis of structural and metabolic aspects of altered cancer secretome contribute greatly to the various hallmarks of cancer including the identification of clinically significant biomarkers and potential targets for therapeutic intervention.

Proteomic profiling of triple-negative breast carcinomas in combination with a three-tier orthogonal technology approach identifies Mage-A4 as potential therapeutic target in estrogen receptor negative breast cancer.

Breast cancer is a very heterogeneous disease, encompassing several intrinsic subtypes with various morphological and molecular features, natural history and response to therapy. Currently, molecular targeted therapies are available for estrogen receptor (ER)(-) and human epidermal growth factor receptor 2 (Her2)-positive breast tumors. However, a significant proportion of primary breast cancers are negative for ER, progesterone receptor (PgR), and Her2, comprising the triple negative breast cancer (TNBC) group. Women with TNBC have a poor prognosis because of the aggressive nature of these tumors and current lack of suitable targeted therapies. As a consequence, the identification of novel relevant protein targets for this group of patients is of great importance. Using a systematic two dimensional (2D) gel-based proteomic profiling strategy, applied to the analysis of fresh TNBC tissue biopsies, in combination with a three-tier orthogonal technology (two dimensional PAGE/silver staining coupled with MS, two dimensional Western blotting, and immunohistochemistry) approach, we aimed to identify targetable protein markers that were present in a significant fraction of samples and that could define therapy-amenable sub-groups of TNBCs. We present here our results, including a large cumulative database of proteins based on the analysis of 78 TNBCs, and the identification and validation of one specific protein, Mage-A4, which was expressed in a significant fraction of TNBC and Her2-positive/ER negative lesions. The high level expression of Mage-A4 in the tumors studied allowed the detection of the protein in the tumor interstitial fluids as well as in sera. The existence of immunotherapeutics approaches specifically targeting this protein, or Mage-A protein family members, and the fact that we were able to detect its presence in serum suggest novel management options for TNBC and human epidermal growth factor receptor 2 positive/estrogen receptor negative patients bearing Mage-A4 positive tumors.

Tumor interstitial fluid - a treasure trove of cancer biomarkers.

Tumor interstitial fluid (TIF) is a proximal fluid that, in addition to the set of blood soluble phase-borne proteins, holds a subset of aberrantly externalized components, mainly proteins, released by tumor cells and tumor microenvironment through various mechanisms, which include classical secretion, non-classical secretion, secretion via exosomes and membrane protein shedding. Consequently, the interstitial aqueous phase of solid tumors is a highly promising resource for the discovery of molecules associated with pathological changes in tissues. Firstly, it allows one to delve deeper into the regulatory mechanisms and functions of secretion-related processes in tumor development. Secondly, the anomalous secretion of molecules that is innate to tumors and the tumor microenvironment, being associated with cancer progression, offers a valuable source for biomarker discovery and possible targets for therapeutic intervention. Here we provide an overview of the features of tumor-associated interstitial fluids, based on recent and updated information obtained mainly from our studies of breast cancer. Data from the study of interstitial fluids recovered from several other types of cancer are also discussed. This article is a part of a Special Issue entitled: The Updated Secretome.

Proteomic analysis of tissue samples in translational breast cancer research.

In the last decade, many proteomic technologies have been applied, with varying success, to the study of tissue samples of breast carcinoma for protein expression profiling in order to discover protein biomarkers/signatures suitable for: characterization and subtyping of tumors; early diagnosis, and both prognosis and prediction of outcome of chemotherapy. The purpose of this review is to critically appraise what has been achieved to date using proteomic technologies and to bring forward novel strategies - based on the analysis of clinically relevant samples - that promise to accelerate the translation of basic discoveries into the daily breast cancer clinical practice. In particular, we address major issues in experimental design by reviewing the strengths and weaknesses of current proteomic strategies in the context of the analysis of human breast tissue specimens.

TIMP-1 increases expression and phosphorylation of proteins associated with drug resistance in breast cancer cells.

Tissue inhibitor of metalloproteinase 1 (TIMP-1) is a protein with a potential biological role in drug resistance. To elucidate the unknown molecular mechanisms underlying the association between high TIMP-1 levels and increased chemotherapy resistance, we employed SILAC-based quantitative mass spectrometry to analyze global proteome and phosphoproteome differences of MCF-7 breast cancer cells expressing high or low levels of TIMP-1. In TIMP-1 high expressing cells, 312 proteins and 452 phosphorylation sites were up-regulated. Among these were the cancer drug targets topoisomerase 1, 2A, and 2B, which may explain the resistance phenotype to topoisomerase inhibitors that was observed in cells with high TIMP-1 levels. Pathway analysis showed an enrichment of proteins from functional categories such as apoptosis, cell cycle, DNA repair, transcription factors, drug targets and proteins associated with drug resistance or sensitivity, and drug transportation. The NetworKIN algorithm predicted the protein kinases CK2a, CDK1, PLK1, and ATM as likely candidates involved in the hyperphosphorylation of the topoisomerases. Up-regulation of protein and/or phosphorylation levels of topoisomerases in TIMP-1 high expressing cells may be part of the mechanisms by which TIMP-1 confers resistance to treatment with the widely used topoisomerase inhibitors in breast and colorectal cancer.

Non-metabolic functions of phosphofructokinase-1 orchestrate tumor cellular invasion and genome maintenance under bevacizumab therapy.

BACKGROUND: Glioblastoma (GBM) is a highly lethal malignancy for which neoangiogenesis serves as a defining hallmark. The anti-VEGF antibody, bevacizumab, has been approved for the treatment of recurrent GBM, but resistance is universal. METHODS: We analyzed expression data of GBM patients treated with bevacizumab to discover potential resistance mechanisms. Patient-derived xenografts (PDXs) and cultures were interrogated for effects of phosphofructokinase-1, muscle isoform (PFKM) loss on tumor cell motility, migration, and invasion through genetic and pharmacologic targeting. RESULTS: We identified PFKM as a driver of bevacizumab resistance. PFKM functions dichotomize based on subcellular location: cytosolic PFKM interacted with KIF11, a tubular motor protein, to promote tumor invasion, whereas nuclear PFKM safeguarded genomic stability of tumor cells through interaction with NBS1. Leveraging differential transcriptional profiling, bupivacaine phenocopied genetic targeting of PFKM, and enhanced efficacy of bevacizumab in preclinical GBM models in vivo. CONCLUSION: PFKM drives novel molecular pathways in GBM, offering a translational path to a novel therapeutic paradigm.

Bladder cancer-associated protein, a potential prognostic biomarker in human bladder cancer.

It is becoming increasingly clear that no single marker will have the sensitivity and specificity necessary to be used on its own for diagnosis/prognosis of tumors. Interpatient and intratumor heterogeneity provides overwhelming odds against the existence of such an ideal marker. With this in mind, our laboratory has been applying a long term systematic approach to identify multiple biomarkers that can be used for clinical purposes. As a result of these studies, we have identified and reported several candidate biomarker proteins that are deregulated in bladder cancer. Following the conceptual biomarker development phases proposed by the Early Detection Research Network, we have taken some of the most promising candidate proteins into postdiscovery validation studies, and here we report on the characterization of one such biomarker, the bladder cancer-associated protein (BLCAP), formerly termed Bc10. To characterize BLCAP protein expression and cellular localization patterns in benign bladder urothelium and urothelial carcinomas (UCs), we used two independent sets of samples from different patient cohorts: a reference set consisting of 120 bladder specimens (formalin-fixed as well as frozen biopsies) and a validation set consisting of 2,108 retrospectively collected UCs with long term clinical follow-up. We could categorize the UCs examined into four groups based on levels of expression and subcellular localization of BLCAP protein and showed that loss of BLCAP expression is associated with tumor progression. The results indicated that increased expression of this protein confers an adverse patient outcome, suggesting that categorization of staining patterns for this protein may have prognostic value. Finally, we applied a combinatorial two-marker discriminator using BLCAP and adipocyte-type fatty acid-binding protein, another UC biomarker previously reported by us, and found that the combination of the two markers correlated more closely with grade and/or stage of disease than the individual markers. The implications of these results in biomarker discovery are discussed.

Functional Proteomic Profiling of Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is a subtype of breast cancer that comprises various disease entities, all of which share a set of common features: a lack of expression of the estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2, respectively. Because of their receptor status, conventional chemotherapy remains the main therapeutic option for TNBC patients. We employed a reverse phase protein array approach (RPPA), complemented by immunohistochemistry, to quantitatively profile the activation state of 84 actionable key signaling intermediates and phosphoproteins in a set of 44 TNBC samples. We performed supervised and unsupervised approaches to proteomic data analysis to identify groups of samples sharing common characteristics that could be amenable to existing therapies. We found the heterogenous activation of multiple pathways, with PI3 K/AKT/mTOR signaling being the most common event. Some specific individualized therapeutic possibilities include the expression of oncogenic KIT in association with cytokeratin 15 and Erk1/2 positive tumors, both of which may have clinical value.

High-throughput proteomics of breast cancer interstitial fluid: identification of tumor subtype-specific serologically relevant biomarkers.

Despite significant advancements in breast cancer (BC) research, clinicians lack robust serological protein markers for accurate diagnostics and tumor stratification. Tumor interstitial fluid (TIF) accumulates aberrantly externalized proteins within the local tumor space, which can potentially gain access to the circulatory system. As such, TIF may represent a valuable starting point for identifying relevant tumor-specific serological biomarkers. The aim of the study was to perform comprehensive proteomic profiling of TIF to identify proteins associated with BC tumor status and subtype. A liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of 35 TIFs of three main subtypes: luminal (19), Her2 (4), and triple-negative (TNBC) (12) resulted in the identification of > 8800 proteins. Unsupervised hierarchical clustering segregated the TIF proteome into two major clusters, luminal and TNBC/Her2 subgroups. High-grade tumors enriched with tumor infiltrating lymphocytes (TILs) were also stratified from low-grade tumors. A consensus analysis approach, including differential abundance analysis, selection operator regression, and random forest returned a minimal set of 24 proteins associated with BC subtypes, receptor status, and TIL scoring. Among them, a panel of 10 proteins, AGR3, BCAM, CELSR1, MIEN1, NAT1, PIP4K2B, SEC23B, THTPA, TMEM51, and ULBP2, was found to stratify the tumor subtype-specific TIFs. In particular, upregulation of BCAM and CELSR1 differentiates luminal subtypes, while upregulation of MIEN1 differentiates Her2 subtypes. Immunohistochemistry analysis showed a direct correlation between protein abundance in TIFs and intratumor expression levels for all 10 proteins. Sensitivity and specificity were estimated for this protein panel by using an independent, comprehensive breast tumor proteome dataset. The results of this analysis strongly support our data, with eight of the proteins potentially representing biomarkers for stratification of BC subtypes. Five of the most representative proteomics databases currently available were also used to estimate the potential for these selected proteins to serve as putative serological markers.

Proteomic profiling of mammary carcinomas identifies C7orf24, a gamma-glutamyl cyclotransferase, as a potential cancer biomarker.

Breast cancer is the leading cause of cancer deaths in women today and is the most common cancer (excluding skin cancers) among women in the Western world. Although cancers detected by screening mammography are significantly smaller than nonscreening ones, noninvasive biomarkers for detection of breast cancer as early as possible are an urgent need as the risk of recurrence and subsequent death is closely related to the stage of the disease at the time of primary surgery. A set of 123 primary breast tumors and matched normal tissue was analyzed by two-dimensional (2D) gel electrophoresis, and a novel protein, C7orf24, was identified as being upregulated in cancer cells. Protein expression levels of C7orf24 were evaluated by immunohistochemical assays to qualify deregulation of this protein. Analysis of C7orf24 expression showed up-regulation in 36.4 and 23.4% of cases present in the discovery sample set (123 samples) and in an independent large TMA validation data set (2197 samples) of clinically annotated breast cancer specimens, respectively. Survival analysis showed that C7orf24 overexpression defines a subgroup of breast tumors with poor clinical outcome. Up-regulation of C7orf24 was also found in other cancer types. Four of these were investigated in greater detail, and we found that a proportion of tumors (58% in cervical, 38% in lung, 72% in colon, and 46% in breast cancer) expressed C7orf24 at levels exceeding those seen in normal samples. The observed overexpression of this protein in different types of cancer suggests deregulation of C7orf24 to be a general event in epithelial carcinogenesis, indicating that this protein may play an important role in cancer cell biology and thus constitute a novel therapeutic target. Furthermore, as C7orf24 is externalized to the tissue extracellular fluid and can be detected in serum, this protein also represents a potential serological marker.

Human BLCAP transcript: new editing events in normal and cancerous tissues.

Bladder cancer-associated protein (BLCAP) is a highly conserved protein among species, and it is considered a novel candidate tumor suppressor gene originally identified from human bladder carcinoma. However, little is known about the regulation or the function of this protein. Here, we show that the human BLCAP transcript undergoes multiple A-to-I editing events. Some of the new editing events alter the highly conserved amino terminus of the protein creating alternative protein isoforms by changing the genetically coded amino acids. We found that both ADAR1 and ADAR2-editing enzymes cooperate to edit this transcript and that different tissues displayed distinctive ratios of edited and unedited BLCAP transcripts. Moreover, we observed a general decrease in BLCAP-editing level in astrocytomas, bladder cancer and colorectal cancer when compared with the related normal tissues. The newly identified editing events, found to be downregulated in cancers, could be useful for future studies as a diagnostic tool to distinguish malignancies or epigenetic changes in different tumors.

A combined proteome and ultrastructural localization analysis of 14-3-3 proteins in transformed human amnion (AMA) cells: definition of a framework to study isoform-specific differences.

The 14-3-3 proteins constitute a family of highly conserved and broadly expressed multifunctional polypeptides that are involved in a variety of important cellular processes that include cell cycle progression, growth, differentiation, and apoptosis. Although the exact cellular function(s) of 14-3-3 proteins is not fully elucidated, as a rule these proteins act by binding to protein ligands, thus regulating their activity; so far more than 300 cellular proteins have been reported to interact with 14-3-3 proteins. Binding to cognate interacting partners is isoform-specific, but redundancy also exists as several binding peptides can be recognized by all isoforms, and some functions can be carried out by any isoform indistinctly. Moreover by interacting with different ligands in a spatially and temporally regulated fashion the same isoform can play multiple possibly even opposing roles where the resultant cellular outcome will be determined by the integration of the various effects. Although there is a large body of literature on specific aspects of 14-3-3 biology, not much is known on the coordinated aspects of 14-3-3 isoform expression, post-translational modifications, and subcellular localization. To address the question of isoform-specific differences, we carried out a comparative analysis of the patterns of expression, phosphorylation, and subcellular localization of the 14-3-3 beta, epsilon, sigma, tau, and zeta protein isoforms in transformed human amnion (AMA) cells. To validate as well as broaden our observations we analyzed the occurrence of the various isoforms in a large number of established cell lines and mammary and urothelial tissue specimens. Given the systematic approach we undertook and our application of isoform-discriminating technologies to the analysis of various cellular systems, we expect the data presented in this study to serve as an enabling resource for researchers working with 14-3-3 proteins.

15-prostaglandin dehydrogenase expression alone or in combination with ACSM1 defines a subgroup of the apocrine molecular subtype of breast carcinoma.

Established histopathological criteria divide invasive breast carcinomas into defined groups. Ductal of no specific type and lobular are the two major subtypes accounting for around 75 and 15% of all cases, respectively. The remaining 10% include rarer types such as tubular, cribriform, mucinous, papillary, medullary, metaplastic, and apocrine breast carcinomas. Molecular profiling technologies, on the other hand, subdivide breast tumors into five subtypes, basal-like, luminal A, luminal B, normal breast tissue-like, and ERBB2-positive, that have different prognostic characteristics. An additional subclass termed "molecular apocrine" has recently been described, but these lesions did not exhibit all the histopathological features of classical invasive apocrine carcinomas (IACs). IACs make up 0.5-3% of the invasive ductal carcinomas, and despite the fact that they are morphologically distinct from other breast lesions, there are presently no standard molecular criteria available for their diagnosis and as a result no precise information as to their prognosis. Toward this goal our laboratories have embarked in a systematic proteomics endeavor aimed at identifying biomarkers that may characterize and subtype these lesions as well as targets that may lead to the development of novel targeted therapies and chemoprevention strategies. By comparing the protein expression profiles of apocrine macrocysts and non-malignant breast epithelial tissue we have previously reported the identification of a few proteins that are specifically expressed by benign apocrine lesions as well as by the few IACs that were available to us at the time. Here we reiterate our strategy to reveal apocrine cell markers and present novel data, based on the analysis of a considerably larger number of samples, establishing that IACs correspond to a distinct molecular subtype of breast carcinomas characterized by the expression of 15-prostaglandin dehydrogenase alone or in combination with a novel form of acyl-CoA synthetase medium-chain family member 1 (ACSM1). Moreover we show that 15-prostaglandin dehydrogenase is not expressed by other breast cancer types as determined by gel-based proteomics and immunohistochemistry analysis and that antibodies against this protein can identify IACs in an unbiased manner in a large breast cancer tissue microarray making them potentially useful as a diagnostic aid.

Different Camptothecin Sensitivities in Subpopulations of Colon Cancer Cells Correlate with Expression of Different Phospho-Isoforms of Topoisomerase I with Different Activities.

The heterogeneity of tumor cells and the potential existence of rare cells with reduced chemotherapeutic response is expected to play a pivotal role in the development of drug resistant cancers. Herein, we utilized the colon cancer cell lines, Caco2 and DLD1, to investigate heterogeneity of topoisomerase 1 (TOP1) activity in different cell subpopulations, and the consequences for the chemotherapeutic response towards the TOP1 targeting drug, camptothecin. The cell lines consisted of two subpopulations: one (the stem-cell-like cells) divided asymmetrically, was camptothecin resistant, had a differently phosphorylated TOP1 and a lower Casein Kinase II (CKII) activity than the camptothecin sensitive non-stem-cell-like cells. The tumor suppressor p14ARF had a different effect in the two cell subpopulations. In the stem-cell-like cells, p14ARF suppressed TOP1 activity and downregulation of this factor increased the sensitivity towards camptothecin. It had the opposite effect in non-stem-cell-like cells. Since it is only the stem-cell-like cells that have tumorigenic activity our results point towards new considerations for future cancer therapy. Moreover, the data underscore the importance of considering cell-to-cell variations in the analysis of molecular processes in cell lines.

Noninvasive profiling of serum cytokines in breast cancer patients and clinicopathological characteristics.

Cancers elicit an immune response by modifying the microenvironment. The immune system plays a pivotal role in cancer recognition and eradication. While the potential clinical value of infiltrating lymphocytes at the tumor site has been assessed in breast cancer, circulating cytokines - the molecules coordinating and fine-tuning immune response - are still poorly characterized. Using two breast cancer cohorts (MicMa, n = 131, DCTB, n = 28) and the multiplex Luminex platform, we measured the levels of 27 cytokines in the serum of breast cancer patients prior to treatment. We investigated the cytokine levels in relation to clinicopathological characteristics and in perspective of the tumor infiltrating immune cells predicted from the bulk mRNA expression data. Unsupervised clustering analysis of the serum cytokine levels in the MicMa cohort identified a cluster of pro-inflammatory, pro-angiogenic, and Th2-related cytokines which was associated with poor prognosis. Notably high levels of platelet derived growth factor BB (PDGF) reflected a more aggressive tumor phenotype and larger tumor size. A significant positive correlation between serum levels of interferon gamma-induced protein 10 (IP10) and its mRNA expression at the tumor site suggested that tumor-IP10-production may outflow to the bloodstream. High IP10 serum levels were associated with a worse prognosis. Finally, we found serum levels of both PDGF and IP10 associated with enrichment scores of specific tumor infiltrating immune cells. Our study suggests that monitoring cytokine circulating levels in breast cancer could be used to characterize breast cancers and the immune composition of their microenvironment through readily available biological material.

Release of transcriptional repression via ErbB2-induced, SUMO-directed phosphorylation of myeloid zinc finger-1 serine 27 activates lysosome redistribution and invasion.

HER2/ErbB2 activation turns on transcriptional processes that induce local invasion and lead to systemic metastasis. The early transcriptional changes needed for ErbB2-induced invasion are poorly understood. Here, we link ErbB2 activation to invasion via ErbB2-induced, SUMO-directed phosphorylation of a single serine residue, S27, of the transcription factor myeloid zinc finger-1 (MZF1). Utilizing an antibody against MZF1-pS27, we show that the phosphorylation of S27 correlates significantly (p < 0.0001) with high-level expression of ErbB2 in primary invasive breast tumors. Phosphorylation of MZF1-S27 is an early response to ErbB2 activation and results in increased transcriptional activity of MZF1. It is needed for the ErbB2-induced expression of MZF1 target genes CTSB and PRKCA, and invasion of single-cells from ErbB2-expressing breast cancer spheroids. The phosphorylation of MZF1-S27 is preceded by poly-SUMOylation of K23, which can make S27 accessible to efficient phosphorylation by PAK4. Based on our results, we suggest for an activation mechanism where phosphorylation of MZF1-S27 triggers MZF1 dissociation from its transcriptional repressors such as the CCCTC-binding factor (CTCF). Our findings increase understanding of the regulation of invasive signaling in breast cancer by uncovering a detailed biological mechanism of how ErbB2 activation can rapidly lead to its invasion-promoting target gene expression and invasion.

Omics-based profiling of carcinoma of the breast and matched regional lymph node metastasis.

Axillary lymph node (ALN) status is currently used as an important clinical indicator of breast cancer prognosis. However, the molecular mechanisms underlying lymph node metastasis are poorly understood and the relationship between ALN metastasis and the primary tumor remains unclear. In an effort to reveal structural changes in the genome and related protein responses that may drive regional metastatic progression we have analyzed matched pairs of primary breast tumors and ALN metastases both at the genomic and proteomic levels using comparative genomic hybridization (CGH) array, quantitative high-resolution 2-D PAGE in combination with MS, and immunohistochemistry (IHC). Array CGH revealed a remarkable similarity in genomic aberration profiles between the matched primary tumors and the ALN metastases. Quantitative profiling of 135 known proteins also revealed striking similarities in their overall expression patterns, although we observed distinct changes in the levels of individual proteins in some sample pairs. The remarkable similarities of the overall genomic and proteomic profiles between primary tumors and matched ALN metastases are taken to suggest that, in general, key biological characteristics of the primary breast tumor are maintained in the corresponding lymph node metastases. Given that the omics-based technologies are oblivious to changes that only occur in minor cellular subsets we validated the proteomic data using IHC, which provides protein expression information with a valuable topological component. Besides confirming the omics-derived data, the IHC analysis revealed that in two cases the ALN metastases may have been derived from a distinct minor cell subpopulation present in the primary tumor rather than from the bulk of it.