High aldehyde dehydrogenase and expression of cancer stem cell markers selects for breast cancer cells with enhanced malignant and metastatic ability

Cancer stem cells (CSCs) have recently been identified in leukaemia and solid tumours; however, the role of CSCs in metastasis remains poorly understood. This dearth of knowledge about CSCs and metastasis is due largely to technical challenges associated with the use of primary human cancer cells in pre-clinical models of metastasis. Therefore, the objective of this study was to develop suitable pre-clinical model systems for studying stem-like cells in breast cancer metastasis, and to test the hypothesis that stem-like cells play a key role in metastatic behaviour. We assessed four different human breast cancer cell lines (MDA-MB-435, MDA-MB-231, MDA-MB-468, MCF-7) for expression of prospective CSC markers CD44/CD24 and CD133, and for functional activity of aldehyde dehydrogenase (ALDH), an enzyme involved in stem cell self-protection. We then used fluorescence-activated cell sorting and functional assays to characterize differences in malignant/metastatic behaviour in vitro (proliferation, colony-forming ability, adhesion, migration, invasion) and in vivo (tumorigenicity and metastasis). Sub-populations of cells demonstrating stem-cell-like characteristics (high expression of CSC markers and/or high ALDH) were identified in all cell lines except MCF-7. When isolated and compared to ALDHlowCD44low/- cells, ALDHhiCD44+CD24- (MDA-MB-231) and ALDHhiCD44+CD133+ (MDA-MB-468) cells demonstrated increased growth (P \u3c 0.05), colony formation (P \u3c 0.05), adhesion (P \u3c 0.001), migration (P \u3c 0.001) and invasion (P \u3c 0.001). Furthermore, following tail vein or mammary fat pad injection of NOD/SCID/IL2 gamma receptor null mice, ALDHhiCD44+CD24- and ALDHhiCD44+CD133+ cells showed enhanced tumorigenicity and metastasis relative to ALDHlowCD44low/- cells (P \u3c 0.05). These novel results suggest that stem-like ALDHhiCD44+CD24- and ALDHhiCD44+CD133+ cells may be important mediators of breast cancer metastasis

Epithelial-to-mesenchymal transition leads to disease-stage differences in circulating tumor cell detection and metastasis in pre-clinical models of prostate cancer

Metastasis is the cause of most prostate cancer (PCa) deaths and has been associated with circulating tumor cells (CTCs). The presence of \u3e= 5 CTCs/7.5mL of blood is a poor prognosis indicator in metastatic PCa when assessed by the CellSearch (R) system, the gold standard clinical platform. However, similar to 35% of metastatic PCa patients assessed by CellSearch (R) have undetectable CTCs. We hypothesize that this is due to epithelial-to-mesenchymal transition (EMT) and subsequent loss of necessary CTC detection markers, with important implications for PCa metastasis. Two pre-clinical assays were developed to assess human CTCs in xenograft models; one comparable to CellSearch (R) (EpCAM-based) and one detecting CTCs semi-independent of EMT status via combined staining with EpCAM/HLA (human leukocyte antigen). In vivo differences in CTC generation, kinetics, metastasis and EMT status were determined using 4 PCa models with progressive epithelial (LNCaP, LNCaP-C42B) to mesenchymal (PC-3, PC-3M) phenotypes. Assay validation demonstrated that the CellSearch (R)-based assay failed to detect a significant number (similar to 40-50%) of mesenchymal CTCs. In vivo, PCa with an increasingly mesenchymal phenotype shed greater numbers of CTCs more quickly and with greater metastatic capacity than PCa with an epithelial phenotype. Notably, the CellSearch (R)-based assay captured the majority of CTCs shed during early-stage disease in vivo, and only after establishment of metastases were a significant number of undetectable CTCs present. This study provides important insight into the influence of EMT on CTC generation and subsequent metastasis, and highlights that novel technologies aimed at capturing mesenchymal CTCs may only be useful in the setting of advanced metastatic disease

TBX3 promotes progression of pre-invasive breast cancer cells by inducing EMT and directly up-regulating SLUG

The acquisition of cellular invasiveness by breast epithelial cells and subsequent transition from ductal carcinoma in situ (DCIS) to invasive breast cancer is a critical step in breast cancer progression. Little is known about the molecular dynamics governing this transition. We have previously shown that overexpression of the transcriptional regulator TBX3 in DCIS-like cells increases survival, growth, and invasiveness. To explore this mechanism further and assess direct transcriptional targets of TBX3 in a high-resolution, isoform-specific context, we conducted genome-wide chromatin-immunoprecipitation (ChIP) arrays coupled with transcriptomic analysis. We show that TBX3 regulates several epithelial–mesenchymal transition (EMT)-related genes, including SLUG and TWIST1. Importantly, we demonstrate that TBX3 is a direct regulator of SLUG expression, and SLUG expression is required for TBX3-induced migration and invasion. Assessing TBX3 by immunohistochemistry in early-stage (stage 0 and stage I) breast cancers revealed high expression in low-grade lesions. Within a second independent early-stage non-high-grade cohort, we observed an association between TBX3 level in the DCIS and size of the invasive focus. Additionally, there was a positive correlation between TBX3 and SLUG, and TBX3 and TWIST1 in the invasive carcinoma. Pathway analysis revealed altered expression of several proteases and their inhibitors, consistent with the ability to degrade basement membrane in vivo. These findings strongly suggest the involvement of TBX3 in the promotion of invasiveness and progression of early-stage pre-invasive breast cancer to invasive carcinoma through the low-grade molecular pathway. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland

Deletion of the thrombin cleavage domain of osteopontin mediates breast cancer cell adhesion, proteolytic activity, tumorgenicity, and metastasis

<p>Abstract</p> <p>Background</p> <p>Osteopontin (OPN) is a secreted phosphoprotein often overexpressed at high levels in the blood and primary tumors of breast cancer patients. OPN contains two integrin-binding sites and a thrombin cleavage domain located in close proximity to each other.</p> <p>Methods</p> <p>To study the role of the thrombin cleavage site of OPN, MDA-MB-468 human breast cancer cells were stably transfected with either wildtype OPN (468-OPN), mutant OPN lacking the thrombin cleavage domain (468-ΔTC) or an empty vector (468-CON) and assessed for <it>in vitro </it>and <it>in vivo </it>functional differences in malignant/metastatic behavior.</p> <p>Results</p> <p>All three cell lines were found to equivalently express thrombin, tissue factor, CD44, αvβ5 integrin and β1 integrin. Relative to 468-OPN and 468-CON cells, 468-ΔTC cells expressing OPN with a deleted thrombin cleavage domain demonstrated decreased cell adhesion (p < 0.001), decreased mRNA expression of MCAM, maspin and TRAIL (p < 0.01), and increased uPA expression and activity (p < 0.01) <it>in vitro</it>. Furthermore, injection of 468-ΔTC cells into the mammary fat pad of nude mice resulted in decreased primary tumor latency time (p < 0.01) and increased primary tumor growth and lymph node metastatic burden (p < 0.001) compared to 468-OPN and 468-CON cells.</p> <p>Conclusions</p> <p>The results presented here suggest that expression of thrombin-uncleavable OPN imparts an early tumor formation advantage as well as a metastatic advantage for breast cancer cells, possibly due to increased proteolytic activity and decreased adhesion and apoptosis. Clarification of the mechanisms responsible for these observations and the translation of this knowledge into the clinic could ultimately provide new therapeutic opportunities for combating breast cancer.</p

Triple-Negative Primary Breast Tumors Induce Supportive Premetastatic Changes in the Extracellular Matrix and Soluble Components of the Lung Microenvironment

The lung is one of the deadliest sites of breast cancer metastasis, particularly in patients with triple-negative (TN) disease. We hypothesized that the presence of a TN primary breast tumor induces changes in the extracellular matrix (ECM) and soluble components of the lung microenvironment that support metastatic behavior. SUM159 (TN) and MCF7 (luminal A) breast cancer cells were injected into mice, and primary breast tumors were established prior to assessing metastatic niche changes. We observed increased CD117+ hematopoietic progenitor cells in the bone marrow of SUM159 mice versus MCF7 or control mice (p < 0.05). Relative to mice bearing MCF7 tumors and non-tumor controls, mice bearing SUM159 tumors demonstrated enhanced expression of ECM proteins in the lung (fibronectin, tenascin-c and periostin), with similar changes observed in lung fibroblasts treated with extracellular vesicles (EVs) from TN breast cancer cells (p < 0.05). Exposure to lung-conditioned media (LCM) from SUM159 tumor-bearing mice resulted in increased migration/proliferation of both SUM159 and MCF7 cells relative to the control (p < 0.05). In contrast, LCM from MCF-7 tumor-bearing mice had no such effect. LCM from SUM159 tumor-bearing mice contained 16 unique proteins relative to other LCM conditions, including the metastasis-associated proteins CCL7, FGFR4, GM-CSF, MMP3, thrombospondin-1 and VEGF. These findings suggest for the first time that the TN breast cancer molecular subtype may be an important determinant of premetastatic changes to both the ECM and soluble components of the lung, potentially mediated via breast cancer-derived EVs.Pharmaceutical Sciences, Faculty ofNon UBCReviewedFacult

TBX3 promotes progression of pre‐invasive breast cancer cells by inducing EMT and directly up‐regulating SLUG

The acquisition of cellular invasiveness by breast epithelial cells and subsequent transition from ductal carcinoma in situ (DCIS) to invasive breast cancer is a critical step in breast cancer progression. Little is known about the molecular dynamics governing this transition. We have previously shown that overexpression of the transcriptional regulator TBX3 in DCIS-like cells increases survival, growth, and invasiveness. To explore this mechanism further and assess direct transcriptional targets of TBX3 in a high-resolution, isoform-specific context, we conducted genome-wide chromatin-immunoprecipitation (ChIP) arrays coupled with transcriptomic analysis. We show that TBX3 regulates several epithelial–mesenchymal transition (EMT)-related genes, including SLUG and TWIST1. Importantly, we demonstrate that TBX3 is a direct regulator of SLUG expression, and SLUG expression is required for TBX3-induced migration and invasion. Assessing TBX3 by immunohistochemistry in early-stage (stage 0 and stage I) breast cancers revealed high expression in low-grade lesions. Within a second independent early-stage non-high-grade cohort, we observed an association between TBX3 level in the DCIS and size of the invasive focus. Additionally, there was a positive correlation between TBX3 and SLUG, and TBX3 and TWIST1 in the invasive carcinoma. Pathway analysis revealed altered expression of several proteases and their inhibitors, consistent with the ability to degrade basement membrane in vivo. These findings strongly suggest the involvement of TBX3 in the promotion of invasiveness and progression of early-stage pre-invasive breast cancer to invasive carcinoma through the low-grade molecular pathway. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland

Assessment of osteopontin in early breast cancer: correlative study in a randomised clinical trial

Abstract Introduction Osteopontin (OPN) is a malignancy-associated glycoprotein that contributes functionally to tumor aggressiveness. In metastatic breast cancer, we previously demonstrated that elevated OPN in primary tumor and blood was associated with poor prognosis. Methods We measured OPN in plasma by ELISA, and in tumors by immunohistochemistry, in 624 (94%) and 462 (69%), respectively, of 667 postmenopausal women with hormone responsive early breast cancer treated by surgery followed by adjuvant treatment with tamoxifen +/− octreotide in a randomized trial (NCIC CTG MA.14; National Cancer Institute of Canada Clinical Trials Group Mammary.14). Results Plasma OPN was measured in 2,540 samples; 688 at baseline and 1,852 collected during follow-up. Mean baseline plasma OPN was 46 ng/ml (range 22.6 to 290) which did not differ from normal levels. Mean percentage OPN tumor cell positivity was 33.9 (95% CI: 30.2 to 37.9). There was no correlation between plasma and tumor OPN values. In multivariate analysis, neither was associated with event-free survival (EFS), relapse-free survival (RFS), overall survival (OS), bone RFS or non-bone RFS. An exploratory analysis in patients with recurrence showed higher mean OPN plasma levels 60.7 ng/ml (23.9 to 543) in the recurrence period compared with baseline levels. Conclusions The hypothesis that OPN tumor expression would have independent prognostic value in early breast cancer was not supported by multivariate analysis of this study population. Plasma OPN levels in women with hormone responsive early breast cancer in the MA.14 trial were not elevated and there was no evidence for prognostic value of plasma OPN in this defined group of patients. However, our finding of elevated mean OPN plasma level around the time of recurrence warrants further study. Trial registration NCT00002864, http://clinicaltrials.gov/show/NCT0000286

Invadopodia Are Required for Cancer Cell Extravasation and Are a Therapeutic Target for Metastasis

Tumor cell extravasation is a key step during cancer metastasis, yet the precise mechanisms that regulate this dynamic process are unclear. We utilized a high-resolution time-lapse intravital imaging approach to visualize the dynamics of cancer cell extravasation in vivo. During intravascular migration, cancer cells form protrusive structures identified as invadopodia by their enrichment of MT1-MMP, cortactin, Tks4, and importantly Tks5, which localizes exclusively to invadopodia. Cancer cells extend invadopodia through the endothelium into the extravascular stroma prior to their extravasation at endothelial junctions. Genetic or pharmacological inhibition of invadopodia initiation (cortactin), maturation (Tks5), or function (Tks4) resulted in an abrogation of cancer cell extravasation and metastatic colony formation in an experimental mouse lung metastasis model. This provides direct evidence of a functional role for invadopodia during cancer cell extravasation and distant metastasis and reveals an opportunity for therapeutic intervention in this clinically important process