Abstract
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.
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.
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.
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.
TOP1 activity is not a marker for CPT sensitivity in breast cancer.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Analysis of breast tumor interstitial fluids (TIF) may help identify protein biomarkers that are secreted in the circulation. Following extraction of breast tumor TIF, LC‐MS/MS TMT labeled ...proteomics, and bioinformatic analyses we identified a panel of secreted protein biomarkers. Experimental and computational validation using tissues and external datasets, respectively, indicated the potential of this biomarker panel for breast cancer subtype stratification.
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.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Bladder cancer associated protein (Blcap) expression is commonly down-regulated in invasive bladder cancer, and may have prognostic value given that its expression is negatively correlated with ...patient survival. We have previously investigated the expression patterns and cellular localization of Blcap in bladder cancer, where we found that about 20% of the lesions examined displayed strong nuclear expression of Blcap, and that this phenotype was associated with overall poor disease outcome. Here we report on the analysis of possible functional associations between nuclear expression of Blcap and canonical signaling pathways. We performed serial immunohistochemistry (IHC) analysis of bladder tissue samples, with serial sections stained with phospho-specific antibodies recognizing key signaling intermediates, such as P-Stat3, P-Akt, and P-Erk1/2, among others, in an immunophenotyping approach we have established and reported previously. Using this approach, we found that nuclear localization of Blcap was associated with expression of P-Stat3. A parallel analysis, cytokine profiling of bladder tumor interstitial fluids of samples expressing (or not) Blcap, showed interleukin (IL)-6, IL-8, and monocyte chemotactic protein 1 (MCP-1) to be correlated with nuclear expression of Blcap, independently supporting a role for Stat3 signaling in localization of Blcap. Multiple indirect immunofluorescence analysis of tissue biopsies confirmed that Blcap co-localized with Stat3. Furthermore, we could also demonstrate, using an in situ proximity ligation assay that Blcap and Stat3 are in close physical proximity of each other in bladder tissue, and that Blcap physically interacts with Stat3 as determined by co-immunoprecipitation of these proteins. Our data indicates that Blcap is a novel Stat3 interaction partner and suggests a role for Blcap in the Stat3-mediated progression of precancerous lesions to invasive tumors of the bladder.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
It has been hypothesized based on accumulated data that a class of small noncoding RNAs, termed microRNAs, are key factors in intercellular communication. Here, microRNAs present in interstitial ...breast tumor fluids have been analyzed to identify relevant markers for a diagnosis of breast cancer and to elucidate the cross‐talk that exists among cells in a tumor microenvironment. Matched tumor interstitial fluid samples (TIF, n = 60), normal interstitial fluid samples (NIF, n = 51), corresponding tumor tissue specimens (n = 54), and serum samples (n = 27) were collected from patients with breast cancer, and detectable microRNAs were analyzed and compared. In addition, serum data from 32 patients with breast cancer and 22 healthy controls were obtained for a validation study. To identify potential serum biomarkers of breast cancer, first the microRNA profiles of TIF and NIF samples were compared. A total of 266 microRNAs were present at higher level in the TIF samples as compared to normal counterparts. Sixty‐one of these microRNAs were present in > 75% of the serum samples and were subsequently tested in a validation set. Seven of the 61 microRNAs were associated with poor survival, while 23 were associated with the presence of immune cells and adipocytes. To our knowledge, these data demonstrate for the first time that profiling of microRNAs in TIF can identify novel biomarkers for the prognostic classification and detection of breast cancer. In addition, the present findings demonstrate that microRNAs may represent the cross‐talk that occurs between tumor cells and their surrounding stroma.
Tumor cells, tumor interstitial fluid (TIF), normal interstitial fluid, and serum all have a distinct microRNA profile. Specific microRNAs detected in TIF are associated with the presence of tumor‐infiltrating lymphocytes and adipocytes, reflecting the cross‐talk between the cells in stroma. Only a portion of these microRNAs are detected in serum, but are attractive candidates for biomarkers of breast cancer.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Proteomics provides powerful tools for the study of clinically relevant samples in the context of translational cancer research. Here we briefly review applications of gel-based proteomics for the ...study of bladder and lung cancer using fresh tissue biopsies. In general, these studies have emphasized the potential of the technology for biomarker discovery, as well as for addressing the issue of cancer heterogeneity.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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.
Clinical cancer proteomics aims at the identification of markers for early detection and predictive purposes, as well as to
provide novel targets for drug discovery and therapeutic intervention. ...Proteomics-based analysis of traditional sources of
biomarkers, such as serum, plasma, or tissue lyzates, has resulted in a wealth of information and the finding of several potential
tumor biomarkers. However, many of these markers have shown limited usefulness in a clinical setting, underscoring the need
for new clinically relevant sources. Here we present a novel and highly promising source of biomarkers, the tumor interstitial
fluid (TIF) that perfuses the breast tumor microenvironment. We collected TIFs from small pieces of freshly dissected invasive
breast carcinomas and analyzed them by two-dimensional polyacrylamide gel electrophoresis in combination with matrix-assisted
laser desorption/ionization time-of-flight mass spectrometry, Western immunoblotting, as well as by cytokine-specific antibody
arrays. This approach provided for the first time a snapshot of the protein components of the TIF, which we show consists
of more than one thousand proteinsâeither secreted, shed by membrane vesicles, or externalized due to cell deathâproduced
by the complex network of cell types that make up the tumor microenvironment. So far, we have identified 267 primary translation
products including, but not limited to, proteins involved in cell proliferation, invasion, angiogenesis, metastasis, inflammation,
protein synthesis, energy metabolism, oxidative stress, the actin cytoskeleton assembly, protein folding, and transport. As
expected, the TIF contained several classical serum proteins. Considering that the protein composition of the TIF reflects
the physiological and pathological state of the tissue, it should provide a new and potentially rich resource for diagnostic
biomarker discovery and for identifying more selective targets for therapeutic intervention.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Particular N‐glycan structures are known to be associated with breast malignancies by coordinating various regulatory events within the tumor and corresponding microenvironment, thus implying that ...N‐glycan patterns may be used for cancer stratification and as predictive or prognostic biomarkers. However, the association between N‐glycans secreted by breast tumor and corresponding clinical relevance remain to be elucidated. We profiled N‐glycans by HILIC UPLC across a discovery dataset composed of tumor interstitial fluids (TIF, n = 85), paired normal interstitial fluids (NIF, n = 54) and serum samples (n = 28) followed by independent evaluation, with the ultimate goal of identifying tumor‐related N‐glycan patterns in blood of patients with breast cancer. The segregation of N‐linked oligosaccharides revealed 33 compositions, which exhibited differential abundances between TIF and NIF. TIFs were depleted of bisecting N‐glycans, which are known to play essential roles in tumor suppression. An increased level of simple high mannose N‐glycans in TIF strongly correlated with the presence of tumor infiltrating lymphocytes within tumor. At the same time, a low level of highly complex N‐glycans in TIF inversely correlated with the presence of infiltrating lymphocytes within tumor. Survival analysis showed that patients exhibiting increased TIF abundance of GP24 had better outcomes, whereas low levels of GP10, GP23, GP38, and coreF were associated with poor prognosis. Levels of GP1, GP8, GP9, GP14, GP23, GP28, GP37, GP38, and coreF were significantly correlated between TIF and paired serum samples. Cross‐validation analysis using an independent serum dataset supported the observed correlation between TIF and serum, for five of nine N‐glycan groups: GP8, GP9, GP14, GP23, and coreF. Collectively, our results imply that profiling of N‐glycans from proximal breast tumor fluids is a promising strategy for determining tumor‐derived glyco‐signature(s) in the blood. N‐glycans structures validated in our study may serve as novel biomarkers to improve the diagnostic and prognostic stratification of patients with breast cancer.
Using quantitative glycome profiling of breast tumor interstitial fluid and matched serum, we identified N‐glycan pattern that (a) segregates tumor and normal interstitual fluids, (b) correlates with the presence of immune cells within tumor, (c) can be predictive for overall survival outcome of patients with breast cancer, and (d) can be detected in the serum of patients with breast cancer.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
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.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
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