IFI16-dependent STING signaling is a crucial regulator of anti-HER2 immune response in HER2+ breast cancer.

Relapse to anti-HER2 monoclonal antibody (mAb) therapies, such as trastuzumab in HER2+ breast cancer (BC), is associated with residual disease progression due to resistance to therapy. Here, we identify interferon-γ inducible protein 16 (IFI16)-dependent STING signaling as a significant determinant of trastuzumab responses in HER2+ BC. We show that down-regulation of immune-regulated genes (IRG) is specifically associated with poor survival of HER2+, but not other BC subtypes. Among IRG, IFI16 is identified as a direct target of EZH2, the underexpression of which leads to deficient STING activation and downstream CXCL10/11 expression in response to trastuzumab treatment. Dual inhibition of EZH2 and histone deacetylase (HDAC) significantly activates IFI16-dependent immune responses to trastuzumab. Notably, a combination of a novel histone methylation inhibitor with an HDAC inhibitor induces complete tumor eradication and long-term T cell memory in a HER2+ BC mouse model. Our findings demonstrate an epigenetic regulatory mechanism suppressing the expression of the IFI16-CXCL10/11 signaling pathway that provides a survival advantage to HER2+ BC to confer resistance to trastuzumab treatment.

Adoptive cell transfer therapy with ex vivo primed peripheral lymphocytes in combination with anti-PDL1 therapy effectively inhibits triple-negative breast cancer growth and metastasis.

BACKGROUND: Adoptive cell transfer cancer immunotherapy holds promise for treating disseminated disease, yet generating sufficient numbers of lymphocytes with anti-cancer activity against diverse specificities remains a major challenge. We recently developed a novel procedure (ALECSAT) for selecting, expanding and maturating polyclonal lymphocytes from peripheral blood with the capacity to target malignant cells. METHODS: Immunodeficient mice were challenged with triple-negative breast cancer cell lines or patient-derived xenografts (PDX) and treated with allogeneic or autologous ALECSAT cells with and without anti-PDL1 therapy to assess the capacity of ALECSAT cells to inhibit primary tumor growth and metastasis. RESULTS: ALECSAT mono therapy inhibited metastasis, but did not inhibit primary tumor growth or prolong survival of tumor-bearing mice. In contrast, combined ALECSAT and anti-PDL1 therapy significantly inhibited primary tumor growth, nearly completely blocked metastasis, and prolonged survival of tumor-bearing mice. CONCLUSIONS: Combined ALECSAT and anti-PDL1 therapy results in favorable anti-cancer responses in both cell line-derived xenograft and autologous PDX models of advanced triple-negative breast cancer.

BID expression determines the apoptotic fate of cancer cells after abrogation of the spindle assembly checkpoint by AURKB or TTK inhibitors.

BACKGROUND: Drugs targeting the spindle assembly checkpoint (SAC), such as inhibitors of Aurora kinase B (AURKB) and dual specific protein kinase TTK, are in different stages of clinical development. However, cell response to SAC abrogation is poorly understood and there are no markers for patient selection. METHODS: A panel of 53 tumor cell lines of different origins was used. The effects of drugs were analyzed by MTT and flow cytometry. Copy number status was determined by FISH and Q-PCR; mRNA expression by nCounter and RT-Q-PCR and protein expression by Western blotting. CRISPR-Cas9 technology was used for gene knock-out (KO) and a doxycycline-inducible pTRIPZ vector for ectopic expression. Finally, in vivo experiments were performed by implanting cultured cells or fragments of tumors into immunodeficient mice. RESULTS: Tumor cells and patient-derived xenografts (PDXs) sensitive to AURKB and TTK inhibitors consistently showed high expression levels of BH3-interacting domain death agonist (BID), while cell lines and PDXs with low BID were uniformly resistant. Gene silencing rendered BID-overexpressing cells insensitive to SAC abrogation while ectopic BID expression in BID-low cells significantly increased sensitivity. SAC abrogation induced activation of CASP-2, leading to cleavage of CASP-3 and extensive cell death only in presence of high levels of BID. Finally, a prevalence study revealed high BID mRNA in 6% of human solid tumors. CONCLUSIONS: The fate of tumor cells after SAC abrogation is driven by an AURKB/ CASP-2 signaling mechanism, regulated by BID levels. Our results pave the way to clinically explore SAC-targeting drugs in tumors with high BID expression.

Drugging the PI3K/AKT/mTOR Pathway in ER+ Breast Cancer.

The frequent activation of the PI3K/AKT/mTOR pathway and its crucial role in estrogen receptor-positive (ER+) breast cancer tumorigenesis and drug resistance has made it a highly attractive therapeutic target in this breast cancer subtype. Consequently, the number of new inhibitors in clinical development targeting this pathway has drastically increased. Among these, the PIK3CA isoform-specific inhibitor alpelisib and the pan-AKT inhibitor capivasertib were recently approved in combination with the estrogen receptor degrader fulvestrant for the treatment of ER+ advanced breast cancer after progression on an aromatase inhibitor. Nevertheless, the clinical development of multiple inhibitors of the PI3K/AKT/mTOR pathway, in parallel with the incorporation of CDK4/6 inhibitors into the standard of care treatment in ER+ advanced breast cancer, has led to a multitude of available therapeutic agents and many possible combined strategies which complicate personalizing treatment. Here, we review the role of the PI3K/AKT/mTOR pathway in ER+ advanced breast cancer, highlighting the genomic contexts in which the various inhibitors of this pathway may have superior activity. We also discuss selected trials with agents targeting the PI3K/AKT/mTOR and related pathways as well as the rationale supporting the clinical development of triple combination therapy targeting ER, CDK4/6 and PI3K/AKT/mTOR in ER+ advanced breast cancer.

Signaling pathways essential for triple-negative breast cancer stem-like cells.

Since the discovery of breast cancer stem cells (CSCs), a significant effort has been made to identify and characterize these cells. It is a generally believe that CSCs play an important role in cancer initiation, therapy resistance, and progression of triple-negative breast cancer (TNBC), an aggressive breast cancer subtype with poor prognosis. Thus, therapies targeting these cells would be a valuable addition to standard treatments that primarily target more differentiated, rapidly dividing TNBC cells. Although several cell surface and intracellular proteins have been described as biomarkers for CSCs, none of these are specific to this population of cells. Recent research is moving toward cellular signaling pathways as targets and biomarkers for CSCs. The WNT pathway, the nuclear factor-kappa B (NF-κB) pathway, and the cholesterol biosynthesis pathway have recently been identified to play a key role in proliferation, survival, and differentiation of CSCs, including those of breast cancer. In this review, we assess recent findings related to these three pathways in breast CSC, with particular focus on TNBC CSCs, and discuss how targeting these pathways, in combination with current standard of care, might prove effective and improve the prognosis of TNBC patients.

Aurora Kinase A and Bcl-xL Inhibition Suppresses Metastasis in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is a heterogeneous disease that accounts for 10-15% of all breast cancer cases. Within TNBC, the treatment of basal B is the most challenging due to its highly invasive potential, and thus treatments to suppress metastasis formation in this subgroup are urgently needed. However, the mechanisms underlying the metastatic ability of TNBC remain unclear. In the present study, we investigated the role of Aurora A and Bcl-xL in regulating basal B cell invasion. We found gene amplification and elevated protein expression in the basal B cells, which also showed increased invasiveness in vitro, compared to basal A cells. Chemical inhibition of Aurora A with alisertib and siRNA-mediated knockdown of BCL2L1 decreased the number of invading cells compared to non-treated cells in basal B cell lines. The analysis of the correlation between AURKA and BCL2L1 expression in TNBC and patient survival revealed significantly decreased relapse-free survival (n = 534, p = 0.012) and distant metastasis-free survival (n = 424, p = 0.017) in patients with primary tumors exhibiting a high combined expression of AURKA and BCL2L1. Together, our findings suggest that high levels of Aurora A and Bcl-xL promote metastasis, and inhibition of these proteins may suppress metastasis and improve patient survival in basal B TNBC.

Distinct mechanisms of resistance to fulvestrant treatment dictate level of ER independence and selective response to CDK inhibitors in metastatic breast cancer.

BACKGROUND: Resistance to endocrine treatment in metastatic breast cancer is a major clinical challenge. Clinical tools to predict both drug resistance and possible treatment combination approaches to overcome it are lacking. This unmet need is mainly due to the heterogeneity underlying both the mechanisms involved in resistance development and breast cancer itself. METHODS: To study the complexity of the mechanisms involved in the resistance to the selective estrogen receptor degrader (SERD) fulvestrant, we performed comprehensive biomarker analyses using several in vitro models that recapitulate the heterogeneity of developed resistance. We further corroborated our findings in tissue samples from patients treated with fulvestrant. RESULTS: We found that different in vitro models of fulvestrant resistance show variable stability in their phenotypes, which corresponded with distinct genomic alterations. Notably, the studied models presented adaptation at different cell cycle nodes to facilitate progression through the cell cycle and responded differently to CDK inhibitors. Cyclin E2 overexpression was identified as a biomarker of a persistent fulvestrant-resistant phenotype. Comparison of pre- and post-treatment paired tumor biopsies from patients treated with fulvestrant revealed an upregulation of cyclin E2 upon development of resistance. Moreover, overexpression of this cyclin was found to be a prognostic factor determining resistance to fulvestrant and shorter progression-free survival. CONCLUSIONS: These data highlight the complexity of estrogen receptor positive breast cancer and suggest that the development of diverse resistance mechanisms dictate levels of ER independence and potentially cross-resistance to CDK inhibitors.

DDX56 modulates post-transcriptional Wnt signaling through miRNAs and is associated with early recurrence in squamous cell lung carcinoma.

BACKGROUND: Early recurrence is a major obstacle to prolonged postoperative survival in squamous cell lung carcinoma (SqCLC). The molecular mechanisms underlying early SqCLC recurrence remain unclear, and effective prognostic biomarkers for predicting early recurrence are needed. METHODS: We analyzed primary tumor samples of 20 SqCLC patients using quantitative proteomics to identify differentially-expressed proteins in patients who experienced early versus late disease recurrence. The expression and prognostic significance of DDX56 was evaluated using a SqCLC tumor tissue microarray and further verified using different online databases. We performed in vitro and in vivo experiments to obtain detailed molecular insight into the functional role of DDX56 in SqCLC. RESULTS: We found that DDX56 exhibited increased expression in tumors of patients who experienced early versus late disease recurrence. Increased DDX56 expression in SqCLC tumors was subsequently confirmed as an independent prognostic factor of poor recurrence-free survival in independent SqCLC cohorts. Functionally, DDX56 promotes SqCLC cell growth and migration in vitro, and xenograft tumor progression in vivo. Mechanistically, DDX56 post-transcriptionally promotes expression of multiple Wnt signaling pathway-related genes, including CTNNB1, WNT2B, and represses a subset of miRNAs, including miR-378a-3p, a known suppressor of Wnt signaling. Detailed analysis revealed that DDX56 facilitated degradation of primary miR-378a, leading to down-regulation of mature miR-378a-3p and thus derepression of the target gene WNT2B. CONCLUSION: We identified DDX56 as a novel independent prognostic biomarker that exerts its oncogenic effects through miRNA-mediated post-transcriptional regulation of Wnt signaling genes to promote early SqCLC recurrence. DDX56 may assist in identifying SqCLC patients at increased risk of early recurrence and who could benefit from Wnt signaling-targeted therapies.

Remodeling and destabilization of chromosome 1 pericentromeric heterochromatin by SSX proteins.

Rearrangement of the 1q12 pericentromeric heterochromatin and subsequent amplification of the 1q arm is commonly associated with cancer development and progression and may result from epigenetic deregulation. In many premalignant and malignant cells, loss of 1q12 satellite DNA methylation causes the deposition of polycomb factors and formation of large polycomb aggregates referred to as polycomb bodies. Here, we show that SSX proteins can destabilize 1q12 pericentromeric heterochromatin in melanoma cells when it is present in the context of polycomb bodies. We found that SSX proteins deplete polycomb bodies and promote the unfolding and derepression of 1q12 heterochromatin during replication. This further leads to segregation abnormalities during anaphase and generation of micronuclei. The structural rearrangement of 1q12 pericentromeric heterochromatin triggered by SSX2 is associated with loss of polycomb factors, but is not mediated by diminished polycomb repression. Instead, our studies suggest a direct effect of SSX proteins facilitated though a DNA/chromatin binding, zinc finger-like domain and a KRAB-like domain that may recruit chromatin modifiers or activate satellite transcription. Our results demonstrate a novel mechanism for generation of 1q12-associated genomic instability in cancer cells.

ARAP1 is an independent prognostic biomarker in older women with ovarian high-grade serous adenocarcinoma receiving first-line platinum-based antineoplastic therapy.

Background: Little is known about the biological factors influencing ovarian cancer (OC) patient outcome, especially in older patients who are often underrepresented in clinical trials. We examined alterations in the transcriptomic profile of primary high-grade serous carcinoma (HGSC) samples from older OC patients (>70 years) receiving first-line platinum-based treatment to identify potential biomarkers for prediction of response to this therapy.Material and methods: Tumor samples from 50 HGSC patients were identified from a retrospective cohort, analyzed by gene expression array. The protein expression of selected biomarkers was examined using immunohistochemistry (IHC).Results: Gene expression profiling revealed 81 genes with significantly altered expression in patients experiencing progression after first-line platinum-based treatment within 6 months versus those who progressed later than 12 months. Expression of ankyrin repeat and PH domain 1 (ARAP1) was significantly lower in the group with early versus late progression (p ≤ .01). Correlation between ARAP1 expression and outcome was further confirmed by IHC staining in the discovery cohort (χ2-test, p = .004) and in independent validation cohorts. The sensitivity of ARAP1 allowed identification of 64.7% of patients with early progression in the discovery population, with a specificity of 78.6% and a negative predictive value of 78.6%. Multivariate regression analysis identified ARAP1 as an independent prognostic factor.Conclusions: This hypothesis generating study suggests that low expression of ARAP1 is an independent prognostic biomarker of shorter RFS in older patients with HGSC receiving first-line platinum-based antineoplastic therapy, which could be used to identify patients who should receive more intensive treatment and closer surveillance.

CYPOR is a novel and independent prognostic biomarker of recurrence-free survival in triple-negative breast cancer patients.

Prognostic and predictive biomarkers of disease and treatment outcome are needed to ensure optimal treatment of patients with triple-negative breast cancer (TNBC). In a mass spectrometry-based global proteomic study of 44 formalin-fixed, paraffin-embedded (FFPE) primary TNBC tumors and 10 corresponding metastases, we found that Cytochrome P450 reductase (CYPOR) expression correlated with patient outcome. The correlation between CYPOR expression and outcome was further evaluated in a Danish cohort of 113 TNBC patients using immunohistochemistry and publicly available gene expression data from two cohorts of TNBC and basal-like breast cancer patients, respectively (N = 249 and N = 580). A significant correlation between high CYPOR gene expression and shorter recurrence-free survival (RFS), but not overall survival, was found in the cohort of 249 TNBC patients (p = 0.018, HR = 1.77, 95% CI 1.1-2.85), and this correlation was recapitulated in a cohort of 580 basal-like breast cancer patients (p = 0.018, HR = 1.4, 95% CI 1.06-1.86). High CYPOR protein expression was also associated with shorter RFS in the cohort of 113 TNBC patients (p = 0.017, HR = 2.73, 95% CI 1.20-6.19), particularly those who were lymph node tumor-negative (p = 0.029, HR = 5.22). Multivariate Cox regression analysis identified CYPOR as an independent prognostic factor for shorter RFS in TNBC patients (p = 0.032, HR = 2.19, 95% CI 1.07-4.47). Together, these data suggest high expression of CYPOR as an independent prognostic biomarker of shorter RFS, which could be used to identify patients who should receive more extensive adjuvant treatment and more aggressive surveillance.

De novo pathway-based biomarker identification.

Gene expression profiles have been extensively discussed as an aid to guide the therapy by predicting disease outcome for the patients suffering from complex diseases, such as cancer. However, prediction models built upon single-gene (SG) features show poor stability and performance on independent datasets. Attempts to mitigate these drawbacks have led to the development of network-based approaches that integrate pathway information to produce meta-gene (MG) features. Also, MG approaches have only dealt with the two-class problem of good versus poor outcome prediction. Stratifying patients based on their molecular subtypes can provide a detailed view of the disease and lead to more personalized therapies. We propose and discuss a novel MG approach based on de novo pathways, which for the first time have been used as features in a multi-class setting to predict cancer subtypes. Comprehensive evaluation in a large cohort of breast cancer samples from The Cancer Genome Atlas (TCGA) revealed that MGs are considerably more stable than SG models, while also providing valuable insight into the cancer hallmarks that drive them. In addition, when tested on an independent benchmark non-TCGA dataset, MG features consistently outperformed SG models. We provide an easy-to-use web service at http://pathclass.compbio.sdu.dk where users can upload their own gene expression datasets from breast cancer studies and obtain the subtype predictions from all the classifiers.

SNAI2 upregulation is associated with an aggressive phenotype in fulvestrant-resistant breast cancer cells and is an indicator of poor response to endocrine therapy in estrogen receptor-positive metastatic breast cancer.

BACKGROUND: Endocrine resistance in estrogen receptor-positive (ER+) breast cancer is a major clinical problem and is associated with accelerated cancer cell growth, increased motility and acquisition of mesenchymal characteristics. However, the specific molecules and pathways involved in these altered features remain to be detailed, and may be promising therapeutic targets to overcome endocrine resistance. METHODS: In the present study, we evaluated altered expression of epithelial-mesenchymal transition (EMT) regulators in ER+ breast cancer cell models of tamoxifen or fulvestrant resistance, by gene expression profiling. We investigated the specific role of increased SNAI2 expression in fulvestrant-resistant cells by gene knockdown and treatment with a SNAIL-p53 binding inhibitor, and evaluated the effect on cell growth, migration and expression of EMT markers. Furthermore, we evaluated SNAI2 expression by immunohistochemical analysis in metastatic samples from two cohorts of patients with breast cancer treated with endocrine therapy in the advanced setting. RESULTS: SNAI2 was found to be significantly upregulated in all endocrine-resistant cells compared to parental cell lines, while no changes were observed in the expression of other EMT-associated transcription factors. SNAI2 knockdown with specific small interfering RNA (siRNA) converted the mesenchymal-like fulvestrant-resistant cells into an epithelial-like phenotype and reduced cell motility. Furthermore, inhibition of SNAI2 with specific siRNA or a SNAIL-p53 binding inhibitor reduced growth of cells resistant to fulvestrant treatment. Clinical evaluation of SNAI2 expression in two independent cohorts of patients with ER+ metastatic breast cancer treated with endocrine therapy in the advanced setting (N = 86 and N = 67) showed that high SNAI2 expression in the metastasis correlated significantly with shorter progression-free survival on endocrine treatment (p = 0.0003 and p = 0.004). CONCLUSIONS: Our results suggest that SNAI2 is a key regulator of the aggressive phenotype observed in endocrine-resistant breast cancer cells, an independent prognostic biomarker in ER+ advanced breast cancer treated with endocrine therapy, and may be a promising therapeutic target in combination with endocrine therapies in ER+ metastatic breast cancer exhibiting high SNAI2 levels.

Elucidation of Altered Pathways in Tumor-Initiating Cells of Triple-Negative Breast Cancer: A Useful Cell Model System for Drug Screening.

A limited number of cancer cells within a tumor are thought to have self-renewing and tumor-initiating capabilities that produce the remaining cancer cells in a heterogeneous tumor mass. Elucidation of central pathways preferentially used by tumor-initiating cells/cancer stem cells (CSCs) may allow their exploitation as potential cancer therapy targets. We used single cell cloning to isolate and characterize four isogenic cell clones from a triple-negative breast cancer cell line; two exhibited mesenchymal-like and two epithelial-like characteristics. Within these pairs, one, but not the other, resulted in tumors in immunodeficient NOD/Shi-scid/IL-2 Rγ null mice and efficiently formed mammospheres. Quantitative proteomics and phosphoproteomics were used to map signaling pathways associated with the tumor-initiating ability. Signaling associated with apoptosis was suppressed in tumor-initiating versus nontumorigenic counterparts with pro-apoptotic proteins, such as Bcl2-associated agonist of cell death (BAD), FAS-associated death domain protein (FADD), and myeloid differentiation primary response protein (MYD88), downregulated in tumor-initiating epithelial-like cells. Functional studies confirmed significantly lower apoptosis in tumor-initiating versus nontumorigenic cells. Moreover, central pathways, including ß-catenin and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB)-related signaling, exhibited increased activation in the tumor-initiating cells. To evaluate the CSC model as a tool for drug screening, we assessed the effect of separately blocking NF-κB and Wnt/ß-catenin signaling and found markedly reduced mammosphere formation, particularly for tumor-initiating cells. Similar reduction was also observed using patient-derived primary cancer cells. Furthermore, blocking NF-κB signaling in mice transplanted with tumor-initiating cells significantly reduced tumor outgrowth. Our study demonstrates that suppressed apoptosis, activation of pathways associated with cell viability, and CSCs are the major differences between tumor-initiating and nontumorigenic cells independent of their epithelial-like/mesenchymal-like phenotype. These altered pathways may provide targets for future drug development to eliminate CSCs, and the cell model may be a useful tool in such drug screenings. Stem Cells 2017;35:1898-1912.

One-step FPLC-size-exclusion chromatography procedure for purification of rDMBT1 6 kb with increased biological activity.

Deleted in Malignant Brain Tumor 1 (DMBT1, alias SAG or gp340) is a pattern recognition receptor involved in immune defense, cell polarization, differentiation and regeneration. To investigate the role of the protein in physiological and pathological processes, the protein has often been isolated from saliva or produced in vitro and purified by a multistep affinity purification procedure using bacteria, followed by FPLC. Here, we compared a simple, one-step FPLC-SEC protocol for purification of recombinant DMBT1 6 kb, with that of the standard bacteria affinity purification-based protocol. Our data suggest that our FPLC-SEC protocol yields DMBT1 in a more native conformation.

KeyPathwayMinerWeb: online multi-omics network enrichment.

We present KeyPathwayMinerWeb, the first online platform for de novo pathway enrichment analysis directly in the browser. Given a biological interaction network (e.g. protein-protein interactions) and a series of molecular profiles derived from one or multiple OMICS studies (gene expression, for instance), KeyPathwayMiner extracts connected sub-networks containing a high number of active or differentially regulated genes (proteins, metabolites) in the molecular profiles. The web interface at (http://keypathwayminer.compbio.sdu.dk) implements all core functionalities of the KeyPathwayMiner tool set such as data integration, input of background knowledge, batch runs for parameter optimization and visualization of extracted pathways. In addition to an intuitive web interface, we also implemented a RESTful API that now enables other online developers to integrate network enrichment as a web service into their own platforms.

NADH-Cytochrome b5 Reductase 3 Promotes Colonization and Metastasis Formation and Is a Prognostic Marker of Disease-Free and Overall Survival in Estrogen Receptor-Negative Breast Cancer.

Metastasis is the main cause of cancer-related deaths and remains the most significant challenge to management of the disease. Metastases are established through a complex multistep process involving intracellular signaling pathways. To gain insight to proteins central to specific steps in metastasis formation, we used a metastasis cell line model that allows investigation of extravasation and colonization of circulating cancer cells to lungs in mice. Using stable isotopic labeling by amino acids in cell culture and subcellular fractionation, the nuclear, cytosol, and mitochondria proteomes were analyzed by LC-MS/MS, identifying a number of proteins that exhibited altered expression in isogenic metastatic versus nonmetastatic cancer cell lines, including NADH-cytochrome b5 reductase 3 (CYB5R3), l-lactate dehydrogenase A (LDHA), Niemann-pick c1 protein (NPC1), and nucleolar RNA helicase 2 (NRH2). The altered expression levels were validated at the protein and transcriptional levels, and analysis of breast cancer biopsies from two cohorts of patients demonstrated a significant correlation between high CYB5R3 expression and poor disease-free and overall survival in patients with estrogen receptor-negative tumors (DFS: p = .02, OS: p = .04). CYB5R3 gene knock-down using siRNA in metastasizing cells led to significantly decreased tumor burden in lungs when injected intravenously in immunodeficient mice. The cellular effects of CYB5R3 knock-down showed signaling alterations associated with extravasation, TGFß and HIFα pathways, and apoptosis. The decreased apoptosis of CYB5R3 knock-down metastatic cancer cell lines was confirmed in functional assays. Our study reveals a central role of CYB5R3 in extravasation/colonization of cancer cells and demonstrates the ability of our quantitative, comparative proteomic approach to identify key proteins of specific important biological processes that may also prove useful as potential biomarkers of clinical relevance. MS data are available via ProteomeXchange with identifier PXD001391.

The stepwise evolution of the exome during acquisition of docetaxel resistance in breast cancer cells.

BACKGROUND: Resistance to taxane-based therapy in breast cancer patients is a major clinical problem that may be addressed through insight of the genomic alterations leading to taxane resistance in breast cancer cells. In the current study we used whole exome sequencing to discover somatic genomic alterations, evolving across evolutionary stages during the acquisition of docetaxel resistance in breast cancer cell lines. RESULTS: Two human breast cancer in vitro models (MCF-7 and MDA-MB-231) of the step-wise acquisition of docetaxel resistance were developed by exposing cells to 18 gradually increasing concentrations of docetaxel. Whole exome sequencing performed at five successive stages during this process was used to identify single point mutational events, insertions/deletions and copy number alterations associated with the acquisition of docetaxel resistance. Acquired coding variation undergoing positive selection and harboring characteristics likely to be functional were further prioritized using network-based approaches. A number of genomic changes were found to be undergoing evolutionary selection, some of which were likely to be functional. Of the five stages of progression toward resistance, most resistance relevant genomic variation appeared to arise midway towards fully resistant cells corresponding to passage 31 (5 nM docetaxel) for MDA-MB-231 and passage 16 (1.2 nM docetaxel) for MCF-7, and where the cells also exhibited a period of reduced growth rate or arrest, respectively. MCF-7 cell acquired several copy number gains on chromosome 7, including ABC transporter genes, including ABCB1 and ABCB4, as well as DMTF1, CLDN12, CROT, and SRI. For MDA-MB-231 numerous copy number losses on chromosome X involving more than 30 genes was observed. Of these genes, CASK, POLA1, PRDX4, MED14 and PIGA were highly prioritized by the applied network-based gene ranking approach. At higher docetaxel concentration MCF-7 subclones exhibited a copy number loss in E2F4, and the gene encoding this important transcription factor was down-regulated in MCF-7 resistant cells. CONCLUSIONS: Our study of the evolution of acquired docetaxel resistance identified several genomic changes that might explain development of docetaxel resistance. Interestingly, the most relevant resistance-associated changes appeared to originate midway through the evolution towards fully resistant cell lines. Our data suggest that no single genomic event sufficiently predicts resistance to docetaxel, but require genomic alterations affecting multiple pathways that in concert establish the final resistance stage.

Keratin 34betaE12/keratin7 expression is a prognostic factor of cancer-specific and overall survival in patients with early stage non-small cell lung cancer.

BACKGROUND: Carcinomas and their metastases often retain the keratin patterns of their epithelial origin, and are therefore useful as lineage-specific markers in diagnostic pathology. Recently, it has become clear that intermediate filaments composed by keratins play a role in modulation of cell proliferation, migration, and possibly cancer invasion, factors impacting prognosis in early stage non-small cell lung cancer (NSCLC). MATERIAL AND METHODS: Tumor tissue from a retrospective Danish cohort of 177 patients with completely resected NSCLC, stage I-IIIA tumors, were analyzed for keratin 7 (K7) and keratin 34ßE12 expression by immunohistochemistry and validated in a comparable independent Norwegian cohort of 276 stage I-IIIA NSCLC patients. RESULTS: Based on keratin 34ßE12/K7 expression, three subgroups with significantly different median cancer-specific survival rates were identified (34ßE12+/K7+, 168 months vs. 34ßE12+/K7+, 73 months vs. 34ßE12-/K7+, 30 months; p = 0.0004). In multivariate analysis, stage II-IIIA (HR 2.9), 34ßE12+/K7+ (HR 1.90) and 34ßE12-/K7+ (HR 3.7), were prognostic factors of poor cancer-specific survival (CSS) (p < 0.001). Validation in the Norwegian cohort confirmed that stage II-IIIA (HR 2.3), 34ßE12+/K7+ (HR 1.6), and 34ßE12-/K7+ (HR 2.0) were prognostic factors of poor CSS (p < 0.05). Multivariate Cox proportional-hazard analysis demonstrated that 34ßE12+/K7 + and 34ßE12+/K7 + status was significantly associated with poor overall survival (p < 0.05). CONCLUSION: Keratin 34ßE12/K7 expression is a prognostic parameter in resected early stage NSCLC that allows identification of high-risk NSCLC patients with poor cancer-specific and overall survival.

S100A14 is a novel independent prognostic biomarker in the triple-negative breast cancer subtype.

Triple-negative breast cancer (TNBC) represents a heterogeneous subgroup with generally poor outcome and lack of an effective targeted therapy. Prognostic or predictive biomarkers to guide treatment decisions for this group of patients are needed. To evaluate the potential of S100A14 protein as a novel biomarker in TNBC, the protein expression of S100A14 was correlated with clinical outcomes in a Pilot Sample set and a Danish cohort of predominantly TNBC patients. Kaplan-Meier analysis identified a prognostic impact of S100A14 on disease-free survival and overall survival, showing that tumors with high S100A14 protein expression levels were significantly correlated with poor outcome in TNBC patients (p = 0.017; p = 0.038), particularly those in the basal-like subgroup (p = 0.006; p = 0.037). Importantly, TNBC patients with high S100A14 expression, but tumor-negative axillary lymph nodes (N-), had equally poor outcomes as those with tumor-positive axillary lymph nodes (N+), while TNBC/N- patients with low S100A14 expression had a significantly better disease free survival (p = 0.013). Multivariate analysis revealed that S100A14 is an independent prognostic factor for TNBC patients (p = 0.024; p = 0.05). At the cellular level, S100A14 was found to be expressed in epithelial-like, but not in mesenchymal-like, TNBC cells in vitro. S100A14 is an independent prognostic factor in TNBC and a novel potential therapeutic target in TNBC.