Imaging tumour heterogeneity of the consequences of a PKCα-substrate interaction in breast cancer patients.
TL;DR: It is demonstrated that tissue imaging-derived parameters that pertain to or are a consequence of the PKC-ezrin interaction can be used for breast cancer prognostication, with inter-cohort reproducibility.
Abstract: Breast cancer heterogeneity demands that prognostic models must be biologically driven and recent clinical evidence indicates that future prognostic signatures need evaluation in the context of early compared with late metastatic risk prediction. In pre-clinical studies, we and others have shown that various protein–protein interactions, pertaining to the actin microfilament-associated proteins, ezrin and cofilin, mediate breast cancer cell migration, a prerequisite for cancer metastasis. Moreover, as a direct substrate for protein kinase Cα, ezrin has been shown to be a determinant of cancer metastasis for a variety of tumour types, besides breast cancer; and has been described as a pivotal regulator of metastasis by linking the plasma membrane to the actin cytoskeleton. In the present article, we demonstrate that our tissue imaging-derived parameters that pertain to or are a consequence of the PKC–ezrin interaction can be used for breast cancer prognostication, with inter-cohort reproducibility. The application of fluorescence lifetime imaging microscopy (FLIM) in formalin-fixed paraffin-embedded patient samples to probe protein proximity within the typically
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TL;DR: A novel role for Notch1 signaling in the regulation of MenaINV expression and transendothelial migration is indicated and mechanistic information essential to the use of therapeutic inhibitors of metastasis is provided.
Abstract: The process of intravasation involving transendothelial migration is a key step in metastatic spread. How the triple cell complex composed of a macrophage, Mena over-expressing tumor cell and endothelial cell, called the tumor microenvironment of metastasis (TMEM), facilitates tumor cell transendothelial migration is not completely understood. Previous work has shown that the physical contact between a macrophage and tumor cell results in the formation of invadopodia, actin-rich matrix degrading protrusions, important for tumor cell invasion and transendothelial migration and tumor cell dissemination. Herein, we show that the macrophage-induced invadopodium is formed through a Notch1/MenaINV signaling pathway in the tumor cell upon macrophage contact. This heterotypic tumor cell - macrophage interaction results in the upregulation of MenaINV through the activation of MENA transcription. Notch1 and MenaINV expression are required for tumor cell transendothelial migration, a necessary step during intravasation. Inhibition of the Notch signaling pathway blocked macrophage-induced invadopodium formation in vitro and the dissemination of tumor cells from the primary tumor in vivo. Our findings indicate a novel role for Notch1 signaling in the regulation of MenaINV expression and transendothelial migration and provide mechanistic information essential to the use of therapeutic inhibitors of metastasis.
79 citations
TL;DR: Prognostic assays based on proliferation based on Oncotype DX, MammaPrint DX and TMEM score combined with a prognostic derived from a signature of dissemination could provide a complementary and more personalized prognostic information for breast cancer patients.
Abstract: Gene expression profiling has yielded expression signatures from which prognostic tests can be derived to facilitate clinical decision making in breast cancer patients. Some of these signatures are based on profiling of whole tumor tissue (tissue signatures), which includes all tumor and stromal cells. Prognostic markers have also been derived from the profiling of metastasizing tumor cells, including circulating tumor cells (CTCs) and migratory-disseminating tumor cells within the primary tumor. The metastasis signatures based on CTCs and migratory-disseminating tumor cells have greater potential for unraveling cell biology insights and mechanistic underpinnings of tumor cell dissemination and metastasis. Of clinical interest is the promise that stratification of patients into high or low metastatic risk, as well as assessing the need for cytotoxic therapy, might be improved if prognostics derived from these two types of signatures are used in a combined way. The aim of this Cell Science at a Glance article and accompanying poster is to navigate through both types of signatures and their derived prognostics, as well as to highlight biological insights and clinical applications that could be derived from them, especially when they are used in combination.
56 citations
Cites background from "Imaging tumour heterogeneity of the..."
...Although the HIS contains additional prognostic markers that are currently being explored (Weitsman et al., 2014), the few that have already been investigated have provided an in-depth understanding of the process of tumor cell intravasation and dissemination, and more importantly have resulted in…...
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...Although the HIS contains additional prognostic markers that are currently being explored (Weitsman et al., 2014), the few that have already been investigated have provided an in-depth understanding of the process of tumor cell intravasation and dissemination, and more importantly have resulted in the development of clinically useful predictive markers of metastasis called TMEM and Mena....
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TL;DR: The current available methodologies to measure HER family receptors are presented and the clinical implications of target quantification are discussed.
Abstract: The clinical success of trastuzumab in breast cancer taught us that appropriate tumor evaluation is mandatory for the correct identification of patients eligible for targeted therapies. Although HER2 protein expression by immunohistochemistry (IHC) and gene amplification by fluorescence in situ hybridization (FISH) assays are routinely used to select patients to receive trastuzumab, both assays only partially predict response to the drug. In the case of epidermal growth factor receptor (EGFR), the link between the presence of the receptor or its amplification and response to anti-EGFR therapies could not be demonstrated. Even less is known for HER3 and HER4, mainly due to lack of robust and validated assays detecting these proteins. It is becoming evident that, besides FISH and IHC, we need better assays to quantify HER receptors and categorize the patients for individualized treatments. Here, we present the current available methodologies to measure HER family receptors and discuss the clinical implications of target quantification.
41 citations
Cites methods from "Imaging tumour heterogeneity of the..."
...extended this method to measure endogenous proteinprotein interactions in archived pathological material [71]....
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TL;DR: Analysis of 131 tissue microarray cores demonstrated that the extent of HER2-HER3 dimer formation as measured by Förster Resonance Energy Transfer determined through FLIM predicts the likelihood of metastatic relapse up to 10 years after surgery, and Interestingly there was no correlation between the level of Her2 protein expressed and Her2- HER3 heterodimer formation.
Abstract: Overexpression of HER2 is an important prognostic marker, and the only predictive biomarker of response to HER2-targeted therapies in invasive breast cancer. HER2-HER3 dimer has been shown to drive proliferation and tumor progression, and targeting of this dimer with pertuzumab alongside chemotherapy and trastuzumab, has shown significant clinical utility. The purpose of this study was to accurately quantify HER2-HER3 dimerisation in formalin fixed paraffin embedded (FFPE) breast cancer tissue as a novel prognostic biomarker.FFPE tissues were obtained from patients included in the METABRIC (Molecular Taxonomy of Breast Cancer International Consortium) study. HER2-HER3 dimerisation was quantified using an improved fluorescence lifetime imaging microscopy (FLIM) histology-based analysis. Analysis of 131 tissue microarray cores demonstrated that the extent of HER2-HER3 dimer formation as measured by Forster Resonance Energy Transfer (FRET) determined through FLIM predicts the likelihood of metastatic relapse up to 10 years after surgery (hazard ratio 3.91 (1.61-9.5), p = 0.003) independently of HER2 expression, in a multivariate model. Interestingly there was no correlation between the level of HER2 protein expressed and HER2-HER3 heterodimer formation. We used a mathematical model that takes into account the complex interactions in a network of all four HER proteins to explain this counterintuitive finding.Future utility of this technique may highlight a group of patients who do not overexpress HER2 protein but are nevertheless dependent on the HER2-HER3 heterodimer as driver of proliferation. This assay could, if validated in a group of patients treated with, for instance pertuzumab, be used as a predictive biomarker to predict for response to such targeted therapies.
28 citations
TL;DR: The combined use of HER dimer imaging and conventional mutation analyses will be able to identify a small subclass of patients (>10%) who will have better prognosis following chemotherapy and confirm its utility in predicting the outcome of anti-EGFR treatment.
Abstract: BACKGROUND: The phase 3 MRC COIN trial showed no statistically significant benefit from adding the EGFR-target cetuximab to oxaliplatin-based chemotherapy in first-line treatment of advanced colorectal cancer. This study exploits additional information on HER2-HER3 dimerization to achieve patient stratification and reveal previously hidden subgroups of patients who had differing disease progression and treatment response. METHODS: HER2-HER3 dimerization was quantified by 'FLIM Histology' in primary tumor samples from 550 COIN trial patients receiving oxaliplatin and fluoropyrimidine chemotherapy +/-cetuximab. Bayesian latent class analysis (LCA) and covariate reduction was performed to analyze the effects of HER2-HER3 dimer, RAS mutation and cetuximab on progression-free survival (PFS) and overall survival (OS). All statistical tests were two-sided. RESULTS: LCA on a cohort of 398 patients revealed two patient subclasses with differing prognoses (median OS: 1624 days [95%CI=1466-1816] vs 461 [95%CI=431-504]): Class 1 (15.6%) showed a benefit from cetuximab in OS (HR = 0.43 [95%CI=0.25-0.76]; p = 0.004). Class 2 showed an association of increased HER2-HER3 with better OS (HR = 0.64 [95%CI=0.44-0.94]; p = 0.02). A class prediction signature was formed and tested on an independent validation cohort (N = 152) validating the prognostic utility of the dimer assay. Similar subclasses were also discovered in full trial dataset (N = 1,630) based on 10 baseline clinicopathological and genetic covariates. CONCLUSIONS: Our work suggests that the combined use of HER dimer imaging and conventional mutation analyses will be able to identify a small subclass of patients (>10%) who will have better prognosis following chemotherapy. A larger prospective cohort will be required to confirm its utility in predicting the outcome of anti-EGFR treatment.
13 citations
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TL;DR: It is demonstrated that a high basal level of active cofilin stored by binding to PIP(2), as well as the highly enriched local milieu of F-actin at the cell edge, is essential to capture the EGF-induced barbed-end amplification observed experimentally.
31 citations
TL;DR: It is shown that ezrin, which resides primarily in the apical membrane in normal breast epithelium, relocalizes primarily to the cytoplasm in >80 % of traditional (T3) invasive ductal LABC tumors, and may represent a novel marker for large malignant tumor size, reflecting the unique biology of LABC.
Abstract: Locally advanced breast cancer (LABC) was initially characterized as a large primary tumor (≥5 cm), associated with or without skin or chest-wall involvement, fixed axillary lymph nodes, or disease spread to the ipsilateral internal mammary or supraclavicular nodes. Since 2002, LABC has been reclassified to include smaller stage IIB tumors (2 to 80 % of traditional (T3) invasive ductal LABC tumors (≥5 cm). Cytoplasmic ezrin is very strongly associated with a single characteristic in breast cancer—large tumor size. In contrast, in large non-malignant fibroadenomas, ezrin staining was similar to that of normal breast epithelium. Small (T1, 1 cm) invasive ductal carcinomas displayed largely apical membrane and perinuclear ezrin localization with weak cytoplasmic staining. Cytoplasmic ezrin localization was also associated with positive lymph node status, but no other clinical–pathological features, including hormone receptor status, histological or nuclear grade of tumor cell. The cytoplasmic relocalization of ezrin may therefore represent a novel marker for large malignant tumor size, reflecting the unique biology of LABC.
22 citations
TL;DR: It is concluded that JNKs are essential for induced cell invasion by COX-2, but not tamoxifen resistance, in ERα-positive breast cancer cells.
Abstract: Elevated cyclooxygenase-2 (COX-2) expression in breast tumors is associated with a lower survival rate in patients with estrogen receptor α (ERα)-positive tumors. We hypothesized that COX-2 reduces the survival rate of breast cancer patients with ERα-positive tumors since COX-2 increases the invasiveness of ERα-positive breast tumors and decreases tumor sensitivity to tamoxifen. Previously, we demonstrated that COX-2 stimulates the activity of protein kinase C (PKC) to increase the invasiveness of ERα-positive MCF-7 breast cancer cells and to decrease the sensitivity of MCF-7 cells to tamoxifen. High levels of COX-2 are associated with the activation of the mitogen-activated protein kinase (MAPK) family and the Akt kinase. However, it is not known whether these kinases mediate COX-2-induced invasive activity and tamoxifen resistance. In the present study, we report that COX-2 utilizes PKC to enhance the phosphorylation of Jun N-terminal kinases (JNKs), but not that of other MAPK family members or Akt. Inhibition aimed at JNKs reduced COX-2-induced invasion but not COX-2-induced tamoxifen resistance. We conclude that JNKs are essential for induced cell invasion by COX-2, but not tamoxifen resistance, in ERα-positive breast cancer cells.
10 citations