LMTK3 escapes tumour suppressor miRNAs via sequestration of DDX5.

Lemur tyrosine kinase-3 (LMTK3) plays an important role in cancer progression and is associated with breast, lung, gastric and colorectal cancer. MicroRNAs (miRNAs) are small endogenous non-coding RNAs that typically repress target genes at post-transcriptional level and have an important role in tumorigenesis. By performing a miRNA expression profile, we identified a subset of miRNAs modulated by LMTK3. We show that LMTK3 induces miR-34a, miR-196-a2 and miR-182 levels by interacting with DEAD-box RNA helicase p68 (DDX5). LMTK3 binds via DDX5 to the pri-miRNA of these three mature miRNAs, thereby sequestrating them from further processing. Ectopic expression of miR-34a and miR-182 in LMTK3-overexpressing cell lines (MCF7-LMTK3 and MDA-MB-231-LMTK3) inhibits breast cancer proliferation, invasion and migration. Interestingly, miR-34a and miR-182 directly bind to the 3'UTR of LMTK3 mRNA and consequently inhibit both its stability and translation, acting as tumour suppressor-like miRNAs. In aggregate, we show that LMTK3 is involved in miRNA biogenesis through modulation of the Microprocessor complex, inducing miRNAs that target LMTK3 itself.

Integrated molecular analysis to investigate the role of microRNAs in pancreatic tumour growth and progression.

BACKGROUND: MicroRNAs (miRNAs) are small non-coding RNAs involved in the post-transcriptional regulation of mRNAs and are aberrantly expressed in cancer with important roles in tumorigenesis. A broad analysis of the combined effects of altered activities of miRNAs in pancreatic ductal adenocarcinoma (PDAC) has not been done, and how miRNAs might affect tumour progression or patient outcomes is unclear. METHODS: We combined data from miRNA and mRNA expression profiles from PDAC and normal pancreas samples (each n=9) and used bioinformatic analyses to identify a miRNA-mRNA regulatory network in PDAC. We validated our findings in PDAC cell-lines (PANC-1, MIA PaCa-2, LPc006, and LPc167), subcutaneous PDAC xenografts in mice, and laser capture microdissected PDACs from patients (n=91). We used this information to identify miRNAs that contributed most to tumorigenesis. FINDINGS: We identified three miRNAs (miR-21, miR-23a, and miR-27a) that acted as cooperative repressors of a network of tumour suppressor genes that included PDCD4, BTG2, and NEDD4L. Inhibition of miR-21, miR-23a, and miR-27a had synergistic effects in reducing proliferation of PDAC cells in culture and the growth of xenograft tumours. The level of inhibition was greater than that of silencing oncomiR-21 alone. In PDACs from patients, high levels of the combination of miR-21, miR-23a, and miR-27a was a strong independent predictor of short overall survival after surgical resection (hazard ratio 3·21, 95% CI 1·78-5·78). High expression of this combination was also associated with a more aggressive tumour phenotype: more microscopic tumour infiltration at resection margin and increased perineural invasion. INTERPRETATION: In an integrated data analysis, we identified functional miRNA-mRNA interactions that contribute to PDAC growth. These findings indicate that miRNAs act together to promote tumour progression and that future therapeutic strategies might require inhibition of several miRNAs. Furthermore, high tumour expression of the miR-21, miR-23a, and miR-27a combination could have potential use in the future as a prognostic signature for patients with PDAC. FUNDING: Peel Medical Research Trust, Alliance Family Foundation, Action Against Cancer, National Institute for Health Research, Association for International Cancer Research, Jason Boas Fellowship, Imperial Biomedical Research Centre, Rosetrees Trust, Joseph Ettedgui Charitable Foundation.

Growth arrest-specific transcript 5 associated snoRNA levels are related to p53 expression and DNA damage in colorectal cancer.

BACKGROUND: The growth arrest-specific transcript 5 gene (GAS5) encodes a long noncoding RNA (lncRNA) and hosts a number of small nucleolar RNAs (snoRNAs) that have recently been implicated in multiple cellular processes and cancer. Here, we investigate the relationship between DNA damage, p53, and the GAS5 snoRNAs to gain further insight into the potential role of this locus in cell survival and oncogenesis both in vivo and in vitro. METHODS: We used quantitative techniques to analyse the effect of DNA damage on GAS5 snoRNA expression and to assess the relationship between p53 and the GAS5 snoRNAs in cancer cell lines and in normal, pre-malignant, and malignant human colorectal tissue and used biological techniques to suggest potential roles for these snoRNAs in the DNA damage response. RESULTS: GAS5-derived snoRNA expression was induced by DNA damage in a p53-dependent manner in colorectal cancer cell lines and their levels were not affected by DICER. Furthermore, p53 levels strongly correlated with GAS5-derived snoRNA expression in colorectal tissue. CONCLUSIONS: In aggregate, these data suggest that the GAS5-derived snoRNAs are under control of p53 and that they have an important role in mediating the p53 response to DNA damage, which may not relate to their function in the ribosome. We suggest that these snoRNAs are not processed by DICER to form smaller snoRNA-derived RNAs with microRNA (miRNA)-like functions, but their precise role requires further evaluation. Furthermore, since GAS5 host snoRNAs are often used as endogenous controls in qPCR quantifications we show that their use as housekeeping genes in DNA damage experiments can lead to inaccurate results.

Reduced dissemination of circulating tumor cells with no-touch isolation surgical technique in patients with pancreatic cancer.

Circulating tumor cells (CTCs) disseminate from the primary tumor and travel through the bloodstream and lymphatic system. The detection of and/or increase in the number of CTCs during a patient's clinical course may be a harbinger of forthcoming overt metastasis. We aimed to examine the impact of 2 different surgical techniques, standard (ST) pancreaticoduodenectomy (PD) and no-touch isolation (NT) PD, on tumor behavior and outcome in patients with pancreatic cancer by using CTCs as biomarkers. In this pilot study, patients were randomized to either ST-PD (n = 6) or NT-PD (n = 6). Intraoperatively, blood samples were taken from the portal vein for measurement of CTCs before and immediately after removal of the tumor. An increase in CTCs was seen in 5 of 6 patients (83%) with ST-PD but no patients with NT-PD (P = .003). In the ST-PD and NT-PD groups, median overall survival was 13.0 and 16.7 months, respectively (P = .33); there was no difference in disease-free survival (P = .42). The use of NT-PD significantly reduced the number of CTCs in the portal vein with no benefit in survival outcomes compared with ST-PD, although more extensive studies are required.

MicroRNAs cooperatively inhibit a network of tumor suppressor genes to promote pancreatic tumor growth and progression.

BACKGROUND & AIMS: There has not been a broad analysis of the combined effects of altered activities of microRNAs (miRNAs) in pancreatic ductal adenocarcinoma (PDAC) cells, and it is unclear how these might affect tumor progression or patient outcomes. METHODS: We combined data from miRNA and messenger RNA (mRNA) expression profiles and bioinformatic analyses to identify an miRNA-mRNA regulatory network in PDAC cell lines (PANC-1 and MIA PaCa-2) and in PDAC samples from patients. We used this information to identify miRNAs that contribute most to tumorigenesis. RESULTS: We identified 3 miRNAs (MIR21, MIR23A, and MIR27A) that acted as cooperative repressors of a network of tumor suppressor genes that included PDCD4, BTG2, and NEDD4L. Inhibition of MIR21, MIR23A, and MIR27A had synergistic effects in reducing proliferation of PDAC cells in culture and growth of xenograft tumors in mice. The level of inhibition was greater than that of inhibition of MIR21 alone. In 91 PDAC samples from patients, high levels of a combination of MIR21, MIR23A, and MIR27A were associated with shorter survival times after surgical resection. CONCLUSIONS: In an integrated data analysis, we identified functional miRNA-mRNA interactions that contribute to growth of PDACs. These findings indicate that miRNAs act together to promote tumor progression; therapeutic strategies might require inhibition of several miRNAs.

Is the detection of circulating tumor cells in locally advanced pancreatic cancer a useful prognostic marker?

Evaluation of: Bidard FC, Huguet F, Louvet C et al. Circulating tumor cells in locally advanced pancreatic adenocarcinoma: the ancillary CirCe 07 study to the LAP 07 trial. Ann. Oncol. 24(8), 2057-2061 (2013). Circulating tumor cells (CTCs) may be shed from the primary tumor and lead to metastatic disease. This evaluated article reports on CTCs in locally advanced pancreatic cancer (LAPC). By assessing CTCs from peripheral blood prior to any treatment and after 2 months of chemotherapy, 11% of patients had detectable CTCs. These patients had a poorer overall survival. With such low numbers of CTCs detected in LAPC patients, it is unclear whether CTCs can actually contribute toward tumor invasiveness and spread in such an aggressive cancer. Although this is a well-designed study, the small number of patients with detectable CTCs means that the statistical power is not great enough to make firm conclusions. Therefore, this expensive assay needs further investigation before being used a prognostic marker in patients with LAPC.

Downregulation of microRNA-515-5p by the estrogen receptor modulates sphingosine kinase 1 and breast cancer cell proliferation.

Sphingosine kinase 1 (SK1) plays an important role in estrogen-dependent breast tumorigenesis, but its regulation is poorly understood. A subset of microRNAs (miRNA, miR) is regulated by estrogen and contributes to cellular proliferation and cancer progression. Here, we describe that miR-515-5p is transcriptionally repressed by estrogen receptor α (ERα) and functions as a tumor suppressor in breast cancer. Its downregulation enhances cell proliferation and estrogen-dependent SK1 activity, mediated by a reduction of miR-515-5p posttranscriptional repression. Enforced expression of miR-515-5p in breast cancer cells causes a reduction in SK1 activity, reduced cell proliferation, and the induction of caspase-dependent apoptosis. Conversely, opposing effects occur with miR-515-5p inhibition and by SK1 silencing. Notably, we show that estradiol (E2) treatment downregulates miR-515-5p levels, whereas the antiestrogen tamoxifen causes a decrease in SK1, which is rescued by silencing miR-515-5p. Analysis of chromatin immunoprecipitation sequencing (ChIP-Seq) data reveals that miR-515-5p suppression is mediated by a direct interaction of ERα within its promoter. RNA-sequencing (RNA-Seq) analysis of breast cancer cells after overexpressing miR-515-5p indicates that it partly modulates cell proliferation by regulating the Wnt pathway. The clinical implications of this novel regulatory system are shown as miR-515-5p is significantly downregulated in ER-positive (n = 146) compared with ER-negative (n = 98) breast cancers. Overall, we identify a new link between ERα, miR-515-5p, proliferation, and apoptosis in breast cancer tumorigenesis.

miR-23b regulates cytoskeletal remodeling, motility and metastasis by directly targeting multiple transcripts.

Uncontrolled cell proliferation and cytoskeletal remodeling are responsible for tumor development and ultimately metastasis. A number of studies have implicated microRNAs in the regulation of cancer cell invasion and migration. Here, we show that miR-23b regulates focal adhesion, cell spreading, cell-cell junctions and the formation of lamellipodia in breast cancer (BC), implicating a central role for it in cytoskeletal dynamics. Inhibition of miR-23b, using a specific sponge construct, leads to an increase of cell migration and metastatic spread in vivo, indicating it as a metastatic suppressor microRNA. Clinically, low miR-23b expression correlates with the development of metastases in BC patients. Mechanistically, miR-23b is able to directly inhibit a number of genes implicated in cytoskeletal remodeling in BC cells. Through intracellular signal transduction, growth factors activate the transcription factor AP-1, and we show that this in turn reduces miR-23b levels by direct binding to its promoter, releasing the pro-invasive genes from translational inhibition. In aggregate, miR-23b expression invokes a sophisticated interaction network that co-ordinates a wide range of cellular responses required to alter the cytoskeleton during cancer cell motility.

Altered expression of the miRNA processing endoribonuclease Dicer has prognostic significance in human cancers.

Several studies have implicated miRNAs in the initiation and progression of human cancers. Examining the biogenesis pathways that generate these important regulatory molecules has revealed new mechanisms for tumor development. Altered expression of the endoribonuclease Dicer in many tumors has given new hope to unraveling the complex relationship between miRNA processing and cancer. This may provide further insight into mechanisms for targeting multiple genes that are critical for the malignant phenotype of several cancers. The evaluated article demonstrates that Dicer is transcriptionally regulated by Sox4 and reduced levels of this transcription factor consequently leads to a reduction in expression, and therefore deregulation of cancer-related miRNAs in melanoma. Reduced Dicer expression in malignant melanoma is an independent predictor of poor survival. In this review, we assess the prognostic significance of Dicer expression in different tumor types.

Loss of miR-126 is crucial to pancreatic cancer progression.

The tumor-suppressor miRNA 126 (miR-126) is downregulated in many tumors and has recently been placed at the heart of complex metastatic pathways. Hamada and colleagues have identified miR-126 as being downregulated in pancreatic ductal adenocarcinoma (PDAC) patient samples and cell lines. The protein ADAM9 has been implicated in the progression of various solid tumors including PDAC. ADAM9 is overexpressed in PDAC and also a direct target of miR-126. The miR-126/ADAM9 axis was subsequently established to control migration and invasion in PDAC, as well as reversal of epithelial-to-mesenchymal transition. miR-126 is also known to target other crucial oncogenes in PDAC such as KRAS and CRK. Replacing miR-126 in PDAC patients may be a novel strategy for preventing progression and metastasis.

Retinoblastoma protein determines aggressiveness in triple-negative breast cancer.

Retinoblastoma protein (RB) is one of the most important tumor suppressors and functions in multiple biological pathways that are deregulated during tumor initiation and progression. Epithelial-to-mesenchymal transition (EMT) is a reversible embryonic process by which epithelial cells lose cell-cell contact and polarity, and its aberrant activation can trigger tumor progression and metastasis. Previously, it has been shown that depletion of RB initiates EMT by downregulating the adhesion molecule E-cadherin. The evaluated article suggests that RB inactivation contributes to loss of cell cycle control and also leads to downregulation of the miR-200 family, thereby causing upregulation of ZEB expression and consequently EMT by downregulation of E-cadherin. RB inactivation could be a key event underlying the mesenchymal and aggressive phenotype of triple-negative breast cancer. Furthermore, exploring links between RB inactivation and EMT might reveal new therapeutic targets for triple-negative breast cancer.

The clinico-pathologic role of microRNAs miR-9 and miR-151-5p in breast cancer metastasis.

BACKGROUND: MicroRNAs (miRNAs) may function as suppressors or promoters of tumor metastasis according to their messenger RNA targets. Previous studies have suggested that miR-9 and miR-151-5p are associated with metastasis in breast cancer and hepatocellular carcinoma, respectively. We aimed to further establish the potential roles of miR-9 and miR-151-5p in tumor invasion and metastasis and investigate their use as biomarkers. METHODS: We used quantitative real-time PCR (qRT-PCR) to measure differences in miR-9 and miR-151-5p expression between primary breast tumors and their lymph-node metastases in 194 paired tumor samples from 97 patients. We also correlated expression levels with histologic data to investigate their utility as biomarkers. RESULTS: There were no significant differences in miR-9 expression between the primary tumors and lymph nodes; however, miR-151-5p expression was significantly lower in the lymph-node metastases than in their corresponding tumors (p < 0.05). miR-9 levels were elevated in primary breast tumors from patients diagnosed with higher-grade tumors (p < 0.05); however, no differences were observed in miR-151-5p levels between different grades of tumor. Interestingly, miR-9 levels were elevated in invasive lobular carcinomas (ILC) compared with invasive ductal carcinomas (IDC; p < 0.01). CONCLUSIONS: In aggregate, these data suggest that miR-151-5p upregulation may suppress metastasis in primary breast tumors. Both miRNAs may serve as useful biomarkers in future clinical trials in breast cancer.

miRNAs in breast cancer: ready for real time?

Over the past decade, major advances in our comprehension of breast cancer biology have led to improved diagnostic and prognostic techniques and the development of novel targeted therapies. However, the efficacy of new treatments remains limited by a combination of drug toxicity, resistance and persisting insufficiencies in our understanding of tumor-signaling pathways; furthermore, the reliability of identified biomarkers is contentious. Following their recent discovery, miRNAs have been established as critical regulators of gene expression, and their putative roles as oncogenes and tumor-suppressor genes has provided a potential new dimension to our clinical approach to breast cancer diagnosis and treatment. Their role as biomarkers and therapeutic targets is appealing; however, several barriers have limited our ability to translate this potential into a clinical reality. This review focuses on the currently accepted roles of miRNAs in breast cancer pathogenesis, and highlights the clinical challenges and breakthroughs in this field to date.

Whole blood stem cell reinfusion and escalated dose melphalan in castration-resistant prostate cancer: a phase 1 study.

PURPOSE: Nontaxane-based chemotherapeutic options in castrate-resistant prostate cancer (CRPC) are limited despite the long natural history of the disease. We carried out a phase 1 dose-escalation study of the alkylating agent melphalan with autologous stem cell transplantation, comparing rapid changes in circulating tumor cells (CTC) and prostate-specific antigen (PSA) as a measure of response. EXPERIMENTAL DESIGN: Cohorts of individuals with advanced CRPC received high-dose intravenous melphalan, and autologous blood was returned to patients during treatment. The efficacy endpoints were the PSA reduction rate, CTC response, survival parameters, toxicity and whether reinduction of endocrine sensitivity occurred. RESULTS: Twenty-four patients were recruited. Dose escalation was feasible with the highest dose cohort being reached. Of 23 individuals evaluable for response, 16 had a PSA response of more than 30%; of 11 patients with soft tissue disease, 4 achieved a partial response and 7 had stable disease. Patients with CTC counts that decreased to less than 5 within 2 weeks from the start of therapy had a longer overall survival (30.6 months vs. 15.3 months, P = 0.03) Treatment was associated with myelosuppression and frequent hospitalizations. In 20 patients after the study, hormone therapy was reintroduced when PSA increased again; response rates were high. CONCLUSIONS: Autologous transplantation following high-dose alkylating agent chemotherapy induces responses but proved toxic, although dose escalation proved possible. The possibility of using CTCs to identify responders at two weeks may be used to justify such an intensive approach. Many individuals went on to further respond to both docetaxel and hormonal therapy.

Monitoring early response to taxane therapy in advanced breast cancer with circulating tumor cells and [(18)F] 3´-deoxy-3´-fluorothymidine PET: a pilot study.

AIM: Early markers of response to chemotherapy, measured by blood markers and imaging, may ultimately lead to tailored therapies that avoid cumulative toxicity. MATERIALS & METHODS: We performed a small pilot study to compare early changes in levels of circulatory tumor cells (CTCs) with changes in tumor proliferation, using metabolic imaging with [(18)F] 3´-deoxy-3´-fluorothymidine PET (FLT-PET) in women with advanced breast cancer, before and during docetaxel therapy. RESULTS: In those individuals in whom we could detect CTCs, a decrease in CTC count correlated with a decrease in FLT-PET signal, within 2 weeks. CONCLUSION: Combined, these two technologies are likely to provide a powerful, albeit expensive, tool to assess immediate responses to therapy.

MicroRNAs targeting oncogenes are down-regulated in pancreatic malignant transformation from benign tumors.

BACKGROUND: MicroRNA (miRNA) expression profiles have been described in pancreatic ductal adenocarcinoma (PDAC), but these have not been compared with pre-malignant pancreatic tumors. We wished to compare the miRNA expression signatures in pancreatic benign cystic tumors (BCT) of low and high malignant potential with PDAC, in order to identify miRNAs deregulated during PDAC development. The mechanistic consequences of miRNA dysregulation were further evaluated. METHODS: Tissue samples were obtained at a tertiary pancreatic unit from individuals with BCT and PDAC. MiRNA profiling was performed using a custom microarray and results were validated using RT-qPCR prior to evaluation of miRNA targets. RESULTS: Widespread miRNA down-regulation was observed in PDAC compared to low malignant potential BCT. We show that amongst those miRNAs down-regulated, miR-16, miR-126 and let-7d regulate known PDAC oncogenes (targeting BCL2, CRK and KRAS respectively). Notably, miR-126 also directly targets the KRAS transcript at a "seedless" binding site within its 3'UTR. In clinical specimens, miR-126 was strongly down-regulated in PDAC tissues, with an associated elevation in KRAS and CRK proteins. Furthermore, miR-21, a known oncogenic miRNA in pancreatic and other cancers, was not elevated in PDAC compared to serous microcystic adenoma (SMCA), but in both groups it was up-regulated compared to normal pancreas, implicating early up-regulation during malignant change. CONCLUSIONS: Expression profiling revealed 21 miRNAs down-regulated in PDAC compared to SMCA, the most benign lesion that rarely progresses to invasive carcinoma. It appears that miR-21 up-regulation is an early event in the transformation from normal pancreatic tissue. MiRNA expression has the potential to distinguish PDAC from normal pancreas and BCT. Mechanistically the down-regulation of miR-16, miR-126 and let-7d promotes PDAC transformation by post-transcriptional up-regulation of crucial PDAC oncogenes. We show that miR-126 is able to directly target KRAS; re-expression has the potential as a therapeutic strategy against PDAC and other KRAS-driven cancers.

Integrated analysis of miRNA and mRNA profiles enables target acquisition in human cancers.

miRNAs play a role in post-transcriptional gene regulation by translational repression and/or mRNA degradation in a very tissue-specific manner. In order to understand the function of a miRNA, it is best to identify the genes that it regulates. Putative mRNA targets of miRNAs identified from seed sequence matches are available using computational algorithms in various web-based databases. However, these tend to have high false-positive rates and, owing to a whole-genome approach, cannot identify tissue/tumor specificity of regulation. The evaluated article presents a large amount of data analyzing global RNA expression in breast cancer and examines whether miRNAs are prognostic due to their effects on mRNA targets. This valuable and important resource of combined miRNA and mRNA expression in breast cancer and its subtypes has been summarized. Many studies have now investigated the integrated analysis of miRNA:mRNA profiles in human malignancies, the goal as always being to identify novel biomarkers and therapeutic targets for each tumor.

microRNAs as markers of survival and chemoresistance in pancreatic ductal adenocarcinoma.

microRNAs (miRs) are a recently recognized class of noncoding short RNAs, 17-25 nucleotides in length, that play a role in post-transcriptional gene regulation by translational repression and/or mRNA degradation. Various miRs have been highlighted in pancreatic cancer development and metastasis, and as potential clinical diagnostic/prognostic biomarkers. Recently, studies have indicated that miRs are responsible for resistance to chemotherapeutic agents. The miR-10b has been identified as a 'metastamiR' in various tumor types, notably breast cancer, but data surrounding its relevance in pancreatic ductal adenocarcinoma has been sparse. The evaluated article presents data indicating that miR-10b is upregulated in pancreatic ductal adenocarcinoma and can be used as a diagnostic marker in endoscopic ultrasound-guided fine-needle aspiration biopsies of suspicious pancreatic lesions. In addition, miR-10b may be able to guide neoadjuvant gemcitabine-based chemoradiotherapy and predict metastatic-free survival and overall survival.

[18F]-3'Deoxy-3'-fluorothymidine positron emission tomography and breast cancer response to docetaxel.

PURPOSE: To establish biomarkers indicating clinical response to taxanes, we determined whether early changes in [(18)F]-3'deoxy-3'-fluorothymidine positron emission tomography (FLT-PET) can predict benefit from docetaxel therapy in breast cancer. EXPERIMENTAL DESIGN: This was a prospective unblinded study in 20 patients with American Joint Committee on Cancer (AJCC) stage II-IV breast cancer unresponsive to first-line chemotherapy or progressing on previous therapy. Individuals underwent a baseline dynamic FLT-PET scan followed by a scan 2 weeks after initiating the first or second cycle of docetaxel. PET variables were compared with anatomic response midtherapy (after 3 cycles). RESULTS: Average and maximum tumor standardized uptake values at 60 minutes (SUV(60,av) and SUV(60,max)) normalized to body surface area ranged between 1.7 and 17.0 and 5.6 and 26.9 × 10(-5) m(2)/mL, respectively. Docetaxel treatment resulted in a significant decrease in FLT uptake (P = 0.0003 for SUV(60,av) and P = 0.0002 for SUV(60,max)). Reduction in tumor SUV(60,av) was associated with target lesion size changes midtherapy (Pearson R for SUV(60,av) = 0.64; P = 0.004) and predicted midtherapy target lesion response (0.85 sensitivity and 0.80 specificity). Decreases in SUV(60,av) in responders were due, at least in part, to reduced net intracellular trapping of FLT (rate constant, K(i)). Docetaxel significantly reduced K(i) by 51.1% (±28.4%, P = 0.0009). CONCLUSION: Changes in tumor proliferation assessed by FLT-PET early after initiating docetaxel chemotherapy can predict lesion response midtherapy with good sensitivity warranting prospective trials to assess the ability to stop therapy in the event of non-FLT-PET response.

Determination of cut-offs for circulating tumor cell measurement in metastatic cancer.

The metastatic transformation of epithelial tumors progresses through various steps leading to the generation of circulating tumor cells (CTCs). Measurement of CTCs in the peripheral blood is being increasingly recognized as a promising tool in breast oncology. Several studies have evaluated the prognostic significance of CTCs in newly diagnosed metastatic breast cancer (MBC) patients. The IC 2006-04 was a high-powered, prospective, multicenter, observational study conceived to assess CTC changes in women with MBC treated with first-line chemotherapy. Levels ≥ 5 CTCs/7.5 ml blood at baseline and before the second cycle of treatment were independent prognostic factors associated with shorter progression-free and overall survival. This study provides further level II evidence for the clinical and prognostic value of CTCs in MBC, confirming data from earlier small studies. It also provides proof that CTCs should be investigated in ongoing interventional trials to see if better patient outcomes can be attained by altering treatment based on CTC levels.