TGF-β signaling is a therapeutic target in advanced cancers. We identified tumor necrosis factor receptor-associated factor 4 (TRAF4) as a key component mediating pro-oncogenic TGF-β-induced SMAD ...and non-SMAD signaling. Upon TGF-β stimulation, TRAF4 is recruited to the active TGF-β receptor complex, where it antagonizes E3 ligase SMURF2 and facilitates the recruitment of deubiquitinase USP15 to the TGF-β type I receptor (TβRI). Both processes contribute to TβRI stabilization on the plasma membrane and thereby enhance TGF-β signaling. In addition, the TGF-β receptor-TRAF4 interaction triggers Lys 63-linked TRAF4 polyubiquitylation and subsequent activation of the TGF-β-activated kinase (TAK)1. TRAF4 is required for efficient TGF-β-induced migration, epithelial-to-mesenchymal transition, and breast cancer metastasis. Elevated TRAF4 expression correlated with increased levels of phosphorylated SMAD2 and phosphorylated TAK1 as well as poor prognosis among breast cancer patients. Our results demonstrate that TRAF4 can regulate the TGF-β pathway and is a key determinant in breast cancer pathogenesis.
•TRAF4 mediates efficient activation of TGF-β-initiated SMAD and non-SMAD signaling•TRAF4 targets SMURF2 degradation and TAK1 activation on TGF-β receptor complex•TRAF4 is needed for efficient TGF-β-induced EMT and metastasis•TRAF4 amplification correlates with SMAD/TAK1 activation and poor prognosis
Stroma tissue surrounding cancer cells plays an important role in tumor development and behavior. In colorectal cancer, it has been found that the amount of stroma within the primary tumor is of ...prognostic value. We therefore have evaluated the prognostic value of this tumor–stroma ratio for breast cancer. A cohort of 574 early breast cancer patients, primarily treated with surgery between 1985 and 1994 was analyzed for the tumor–stroma ratio. The percentage of stroma was visually estimated on Haematoxylin-Eosin (H&E) stained histological sections. Patients with more than 50% intra-tumor stroma were quantified as stroma rich and patients with less than 50% as stroma poor. For the total group of patients, stroma-rich tumors had a shorter relapse-free period (RFP) (
P
= 0.001) and overall survival (OS) (
P
= 0.025) compared to stroma-poor tumors. Tumor–stroma ratio was an independent prognostic parameter for the total group of patients (
P
< 0.001) and also in stratified analysis based on systemic treatment. Importantly, in the triple-negative cancer subpopulation, patients with stroma-rich tumors had a 2.92 times higher risk of relapse (
P
= 0.006) compared to those with stroma-poor tumors, independently of other clinico-pathological parameters. Five-year RFP-rates for triple-negative cancer patients with stroma-rich compared to stroma-poor tumors were 56 and 81%, respectively. Tumor–stroma ratio has proven to be an independent prognostic factor for RFP in breast cancer patients and especially in the triple-negative cancer subpopulation. Tumor–stroma ratio could be easily implemented in routine daily pathology diagnostics, as it is simple to determine, reproducible, and performed in quick time.
Cell surface NKG2D ligands (NKG2DL) bind to the activating NKG2D receptor present on NK cells and subsets of T cells, thus playing a role in initiating an immune response. We examined tumor ...expression and prognostic effect of NKG2DL in breast cancer patients.
Our study population (n = 677) consisted of all breast cancer patients primarily treated with surgery in our center between 1985 and 1994. Formalin-fixed paraffin-embedded tumor tissue was immunohistochemically stained with antibodies directed against MIC-A/MIC-B (MIC-AB), ULBP-1, ULBP-2, ULBP-3, ULBP-4, and ULBP-5.
NKG2DL were frequently expressed by tumors (MIC-AB, 50% of the cases; ULBP-1, 90%; ULBP-2, 99%; ULBP-3, 100%; ULBP-4, 26%; ULBP-5, 90%) and often showed co-expression: MIC-AB and ULBP-4 (p = 0.043), ULBP-1 and ULBP-5 (p = 0.006), ULBP-4 and ULBP-5 (p < 0.001). MIC-AB (p = 0.001) and ULBP-2 (p = 0.006) expression resulted in a statistically significant longer relapse free period (RFP). Combined expression of these ligands showed to be an independent prognostic parameter for RFP (p < 0.001, HR 0.41). Combined expression of all ligands showed no associations with clinical outcome.
We demonstrated for the first time that NKG2DL are frequently expressed and often co-expressed in breast cancer. Expression of MIC-AB and ULBP-2 resulted in a statistically significant beneficial outcome concerning RFP with high discriminative power. Combination of all NKG2DL showed no additive or interactive effect of ligands on each other, suggesting that similar and co-operative functioning of all NKG2DL can not be assumed. Our observations suggest that among driving forces in breast cancer outcome are immune activation on one site and tumor immune escape on the other site.
The purpose of this study was to compare the expression and the prognostic effect of the breast cancer stem cell marker aldehyde dehydrogenase-1 (ALDH1) in young and elderly breast cancer patients.
...The study population (N = 574) consisted of all early breast cancer patients primarily treated with surgery in our center between 1985 and 1994. Median follow-up was 17.9 years (range: 0.1 to 23.5). Tissue microarray slides were immunohistochemically stained for ALDH1 expression and quantified by two independent observers who were blinded to clinical outcome. Assessment of the prognostic effect of ALDH1 expression was stratified according to age and systemic treatment.
Complete lack of expression of ALDH1 was found in 40% of tumors. With increasing age more tumors showed complete absence of ALDH1 expression (P < .001). In patients aged > 65 years, ALDH1 status was not associated with any clinical outcome. Conversely, in patients aged < 65 years, ALDH1 positivity was an independent risk factor of worse outcome for relapse free period (hazard ratio = 1.71 (95% CI, 1.09 to 2.68); P = .021) and relative survival (relative excess risks of death = 2.36 (95% CI, 1.22 to 3.68); P = .016). Ten-year relative survival risk was 57% in ALDH1-positive patients compared to 83% in ALDH1-negative patients.
ALDH1 expression and its prognostic effect are age-dependent. Our results support the hypothesis that breast cancer biology is different in elderly patients compared to their younger counterparts and emphasizes the importance of taking into consideration age-specific interactions in breast cancer research.
The tumor–stroma ratio (TSR) has previously been found to be a strong prognostic parameter in primary breast cancer tumors. Since the presence of tumor cells in lymph nodes is important for clinical ...decision making, the influence of TSR in the primary breast tumor combined with the TSR in tumor‐positive lymph nodes on prognosis was evaluated. Women with invasive breast cancer without distant metastasis who underwent an axillary lymph node dissection between 1985 and 1994 at the Leiden University Medical Center were retrospectively analyzed. TSR assessment was performed on hematoxylin and eosin stained tissue slides. In total, 87 (45.5%) primary tumors were scored as stroma‐low and 104 (54.5%) as stroma‐high. Patients with a high stromal percentage in the primary tumors had a statistically significant worse relapse free period (RFP) compared to stroma‐low tumors (HR 1.97, 95% CI 1.37–2.82, p < 0.001). A total number of 915 lymph nodes were assessed for TSR. In 101 (52.9%) patients, heterogeneity was observed between stroma percentage category in primary tumor and lymph nodes. The combination of TSR of the primary tumor combined with TSR of tumor‐positive lymph nodes strengthened each other as independent prognostic parameter for RFP (p = 0.019). Patients with primary tumor stroma‐low/lymph nodes stroma‐low tumors showed strongly improved RFP rates compared to patients with primary tumor stroma‐high/lymph node stroma‐high tumors with 10‐year percentages of 58 versus 8%, respectively. Assessing the TSR on tumor‐positive lymph nodes can provide additional prognostic information. Stromal activation strongly differs between primary tumors and lymph node metastasis.
What's new?
Measuring the amount of stroma in tumor‐ positive lymph nodes, not just only in the primary tumor, can better predict breast cancer relapse. In recent years, researchers have increasingly looked to the tumor microenvironment for prognostic clues. Here, the authors compared the prognostic value of the TSR in primary breast tumors alone with TSR from both primary tumor and tumor‐positive lymph nodes. Risk of relapse for patients with high amount of stroma at the primary tumor was 75%, while a combination of stroma‐high primary tumor and stroma‐high lymph nodes correlated with 92% relapse rate after 10 years of follow‐up.
Full-length tissue factor (flTF), the coagulation initiator, is overexpressed in breast cancer (BrCa), but associations between flTF expression and clinical outcome remain controversial. It is ...currently not known whether the soluble alternatively spliced TF form (asTF) is expressed in BrCa or impacts BrCa progression. We are unique in reporting that asTF, but not flTF, strongly associates with both tumor size and grade, and induces BrCa cell proliferation by binding to β1 integrins. asTF promotes oncogenic gene expression, anchorage-independent growth, and strongly up-regulates tumor expansion in a luminal BrCa model. In basal BrCa cells that constitutively express both TF isoforms, asTF blockade reduces tumor growth and proliferation in vivo. We propose that asTF plays a major role in BrCa progression acting as an autocrine factor that promotes tumor progression. Targeting asTF may comprise a previously unexplored therapeutic strategy in BrCa that stems tumor growth, yet does not impair normal hemostasis.
Epithelial mesenchymal transition (EMT) plays an important role in the development of metastases. One of the hallmarks of EMT is loss of E-cadherin and gain of N-cadherin expression, which are ...regulated by transcription factors, such as SNAIL, SLUG, and TWIST. We examined the prognostic value of these factors as well as E-cadherin and N-cadherin, in a well-described large cohort of breast cancer patients treated with primary surgery. Analyses were stratified by estrogen receptor (ER) status, because of its crucial role in the regulation of these transcription factors. SNAIL, SLUG, and TWIST expression were examined on a TMA containing 575 breast tumors using immunohistochemistry. Nuclear expression was quantified using a weighted histoscore and classified as high versus low expression, based on the median histoscore. High expression of SNAIL, SLUG, and TWIST was seen in 54, 50, and 50% of tumors, respectively. The level of SNAIL (
P
= 0.014) and TWIST (
P
= 0.006) expression was associated with a worse patient relapse-free period, specifically in patients with ER-positive tumors (interaction Cox proportional hazards
P
= 0.039). Combining both factors resulted in an independent prognostic factor with high discriminative power (both low versus either high: HR 1.15; both low versus both high HR 1.84;
P
= 0.010). Co-expression of SNAIL–TWIST was associated with low-E-cadherin and high-N-cadherin expression, especially in ER-positive tumors (
P
= 0.009), suggesting that, through interactions with ER, SNAIL and TWIST may regulate E- and N-cadherin expression, thereby inducing EMT. Our results are indicative that SNAIL and TWIST play a crucial role in EMT through regulation of E- and N-cadherin expression, exclusively in ER-positive breast cancer patients.
We hypothesized that T-cell immune interaction affects tumor development and thus clinical outcome. Therefore, we examined the clinical impact of human leukocyte antigen (HLA) class I tumor cell ...expression and regulatory T-cell (Treg) infiltration in breast cancer.
Our study population (N = 677) is consisted of all early breast cancer patients primarily treated with surgery in our center between 1985 and 1994. Formalin-fixed, paraffin-embedded tumor tissue was immunohistochemically stained using HCA2, HC10, and Foxp3 monoclonal antibodies.
HLA class I expression was evaluated by combining results from HCA2 and HC10 antibodies and classified into three groups: loss, downregulation, and expression. Remarkably, only in patients who received chemotherapy, both presence of Treg (P = 0.013) and higher HLA class I expression levels (P = 0.002) resulted in less relapses, independently of other variables. Treg and HLA class I were not of influence on clinical outcome in patients who did not receive chemotherapy.
We showed that HLA class I and Treg affect prognosis exclusively in chemotherapy-treated patients and are therefore one of the few predictive factors for chemotherapy response in early breast cancer patients. Chemotherapy may selectively eliminate Treg, thus enabling CTLs to kill tumor cells that have retained HLA class I expression. As a consequence, HLA class I and Treg can predict response to chemotherapy with high discriminative power. These markers could be applied in response prediction to chemotherapy in breast cancer patients.
In breast cancer, the prognostic impact of COX2 expression varies widely between studies. We examined the prognostic value of COX2 expression in a large cohort of breast cancer patients treated with ...primary surgery between 1985 and 1994 and explained the variable results of COX2 expression found in the literature. A tissue microarray was constructed of available tumour material, and ER, PgR, HER2, Ki67 and COX2 were examined by immunohistochemistry. Median follow-up was 19 years. Fifty-five percent (n = 369/677) of patients received no systemic treatment. COX2 was scored using a weighted histoscore. Analysis of COX2 expression in two groups based on the median (148; below vs. above) showed an increased hazard ratio (HR) of 1.35 (95% CI 1.05-1.75, P = 0.021) for disease-free survival (DFS) and of 1.39 (95% CI 1.03-1.82, P = 0.016) for overall survival (OS). However, COX2 did not remain independent in multivariate analysis. In patients with hormone receptor positive tumours, COX2 expression had a negative influence on outcome (low vs. high: DFS: HR 1.37, 95% CI 1.07-1.76, P = 0.013). This effect disappeared when endocrine therapy was administered (low vs. high: DFS: HR 0.93, 95% CI 0.51-1.70, P = 0.811) while it remained statistically significant when endocrine therapy was omitted (low vs. high: DFS: HR 1.48, 95% CI 1.12-1.94, P = 0.005). Our results show that COX2 plays a role in hormonal pathways. Our results can explain the results found in previously published studies.
Breast cancer is a heterogeneous disease with a highly variable clinical outcome in which both genetic and epigenetic changes have critical roles. We investigated tumor expression levels of ...histone-modifying enzymes LSD1, HDAC2 and SIRT1 in relation with patient survival and tumor relapse in a retrospective cohort of 460 breast cancer patients. Additionally, we correlated expression levels with tumor differentiation and tumor cell proliferation.
Immunohistochemical staining for LSD1, HDAC2 and SIRT1 was performed on tissue microarrays of tumor and corresponding normal formalin-fixed paraffin-embedded tissues from breast cancer patients. Median nuclear expression levels in tumor tissues were used to divide the patients into low and high expression categories. In combined expression analyses, patients were divided into four subgroups: 1, all enzymes below-median; 2, one enzyme above-median; 3, two enzymes above-median; 4, all three enzymes above-median. The Cox proportional hazard model was used for univariate and multivariate survival analyses. The Pearson Chi-square method was used to assess correlation of combined expression levels with tumor cell proliferation and tumor differentiation.
Expression of LSD1 and SIRT1, but not of HDAC2, was significantly increased in tumor tissues compared to their normal counterparts (both p < 0.001). Multivariate survival analyses identified SIRT1 as independent prognostic factor for relapse-free survival (RFS) with a hazard ratio (HR) of 1.34 (95% CI = 1.04-1.74, p = 0.02). For overall survival (OS), no significant differences were found when the individual enzymes were analyzed. Analyses of combined expression levels of the three histone-modifying enzymes correlated with OS (p = 0.03) and RFS (p = 0.006) with a HR of respectively 1.49 (95% CI = 1.07-2.08) and 1.68 (95% CI = 1.16-2.44) in multivariate analyses and were also related to tumor differentiation (p < 0.001) and tumor cell proliferation (p = 0.002).
When the combined expression levels were analyzed, high expression of LSD1, HDAC2 and SIRT1 showed shorter patient survival time and shorter time to tumor relapse and correlated with poor tumor differentiation and a high level of tumor cell proliferation. Expression of these histone-modifying enzymes might therefore be involved in breast cancer pathogenesis.
PDF
