Tumor Cell Dedifferentiation Research Articles

De- and re-differentiation of the melanocytic lineage

Terminally differentiated cells can be reprogrammed by the transient, ectopic overexpression of different sets of genes into induced pluripotent stem cells (iPSCs). This process not only has considerable implications for regenerative medicine but is also highly relevant to multiple stages of oncogenesis, including melanoma. In other settings, the de-differentiation of normal and tumor cells is also responsible for a phenotype switch which completely changes the cell fate. Conversely, iPSCs as well as embryonic stem cells (ESCs) can be differentiated in vitro toward specific lineages, for example melanocytes, which offer useful models to investigate the genetic and epigenetic mechanisms involved in cellular differentiation. Here, we summarize recent findings regarding the reprogramming and de-differentiation of melanocytic cells as well as the latest differentiation protocols of pluripotent stem cells into the melanocyte lineage.

Abstract B79: A first-in-human Phase 1 study of anti-cancer stem cell (CSC) agent OMP-54F28 (FZD8-Fc) targeting the WNT pathway in patients with advanced solid tumors.

Abstract Introduction: OMP-54F28 is a first-in-class recombinant fusion protein of the extracellular domain of the human frizzled (FZD) 8 receptor and a human IgG1 Fc fragment. OMP-54F28 binds Wnt ligands and thereby blocks signaling of the WNT/FZD signaling pathway, a key oncologic pathway in numerous tumor types that is implicated in tumor cell de-differentiation and cancer stem cell (CSC) function. In patient-derived xenograft models, OMP-54F28 inhibits growth of several tumor types (such as pancreatic, ovarian, hepatocellular, colorectal, and breast), reduces CSC frequency, promotes differentiation of tumor cells, and synergizes with chemotherapy. Methods: The objectives of this study are to determine the maximum tolerated dose, safety, pharmacokinetics (PK), pharmacodynamics (PD), and anti-tumor activity of OMP-54F28 as a single agent. A 3+3 design was used, and OMP-54F28 was given intravenously every 3 weeks. Results: 17 patients have been treated in 5 dose-escalation cohorts (0.5, 1, 2.5, 5 and 10 mg/kg). The most common related Grade 1 and 2 adverse events (AEs; >10% of patients) were decreased appetite, fatigue, hypocalcemia, nausea, dysgeusia, increased blood pressure, peripheral edema and vomiting. The only related Grade ≥3 AE was Grade 3 anemia in one patient at the 2.5 mg/kg dose level. OMP-54F28 clearance was dose-dependent, consistent with target-mediated drug disposition, with a half-life of approximately 4 days at 10 mg/kg. Exposure at the current highest dose level approximates the exposure in nonclinical models where optimal efficacy was observed. Two patients demonstrated doubling from baseline of β-C-terminal telopeptide (β-CTX), a marker of increased bone turnover, consistent with PD modulation of the WNT pathway in bone by OMP-54F28. One of these patients received zoledronic acid as per protocol (2nd patient declined), and β-CTX returned to baseline. Additional PD biomarker analyses are ongoing in hair follicles, blood cells and paired tumor biopsies. Best tumor response observed was stable disease in three patients (pancreatic cancer, renal cell carcinoma and desmoid tumor) for approximately 3, 3 and 2+ months, respectively. Conclusions: OMP-54F28 is well tolerated through 10 mg/kg. Dose escalation continues, and updated clinical, PK, and PD data will be presented. Bone biomarker data are consistent with modulation of the WNT pathway, and preliminary evidence of clinical activity has been noted in several patients. Three Phase 1b studies in solid tumor indications are being initiated to study OMP-54F28 in combination with chemotherapy and/or targeted agents. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B79. Citation Format: David C. Smith, Michael Gordon, Wells Messersmith, Rashmi Chugh, David Mendelson, Jakob Dupont, Robert Stagg, Ann M. Kapoun, Lu Xu, Rainer K. Brachmann, Antonio Jimeno. A first-in-human Phase 1 study of anti-cancer stem cell (CSC) agent OMP-54F28 (FZD8-Fc) targeting the WNT pathway in patients with advanced solid tumors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B79.

Abstract P6-04-01: Global analysis of breast cancer metastasis suggests cellular reprogramming is central to the endocrine resistant phenotype.

Abstract The development of breast cancer resistance to endocrine therapy results from altered cellular plasticity leading to the emergence of a hormone independent tumour. We have previously shown that the endocrine resistant phenotype is poorly differentiated and has greater metastatic potential when compared with an endocrine sensitive phenotype. The underlying mechanism of how an ER positive, endocrine sensitive tumour can turn on these adaptive mechanisms remains unresolved. We hypothesise that cellular reprogramming can occur as a result of long-term exposure to endocrine treatment which manifests clinically as tumour recurrence. Our aim was to identify the mechanism of breast cancer cellular reprogramming in a clinical context. In this study we adopted a global approach to define transcriptional networks significantly elevated in the breast cancer metastatic setting. Using RNAseq we determined the gene expression profile of three ER positive patients who developed tumour recurrence on tamoxifen treatment. RNAseq was performed on primary tumours, axillary node metastases and subsequent distant metastases. Following bioinformatic analysis, we defined the genes that were significantly elevated in the metastatic tumours. In the metastases, genes clustered away from those in the primary and node, with 209 genes uniquely elevated in the metastatic context, suggesting altered gene expression in response to endocrine therapy. Pathway analysis of genes significantly elevated in the metastatic setting included developmental pathways (WNT signalling and TGFbeta), growth factor signalling pathways (MAPkinase), cell adhesion molecules, junction adherens and pathways in cancer. Further analysis provided a list of clinically relevant transcriptional networks which may facilitate tumour cell de-differentiation. Transcription factors including Myc, SMAD2, ESR, TCF3, CUX1 and CLOCK were identified which potentially drive reprogramming in response to endocrine treatment. We interrogated this data to identify downstream targets of this ‘de-differentiation’ network for new predictive markers of response to endocrine treatment. Bioinformatic analysis identified PAWR, an inhibitor of progenitor cell expansion as a potential SMAD2 target. In a xenograft model of endocrine resistance, PAWR expression was found to be reduced in metastatic tissue from lung, liver and bone compared with the primary tumour. In human breast cancer patients PAWR significantly associated with a good response to endocrine treatment and luminal A status (p = 0.0008), establishing PAWR as a predictor of good response to endocrine therapies. We have identified a transcriptional network which may drive a de-differentiated phenotype following endocrine treatment. Data presented here proposes a mechanism by which apparently less aggressive Luminal A type tumours may fail endocrine treatment and develop subsequent recurrence. This represents a fundamental shift in how we approach the development of resistance to endocrine therapy, establishing a number of biomarkers which will allow tracking of response to endocrine therapy or allow accurate early prediction of those who will respond to treatment. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-04-01.

VEGF Exerts an Angiogenesis-Independent Function in Cancer Cells to Promote Their Malignant Progression

VEGF/vascular permeability factor (VEGF/VPF or VEGF-A) is a pivotal driver of cancer angiogenesis that is a central therapeutic target in the treatment of malignancy. However, little work has been devoted to investigating functions of VEGF that are independent of its proangiogenic activity. Here, we report that VEGF produced by tumor cells acts in an autocrine manner to promote cell growth through interaction with the VEGF receptor neuropilin-1 (NRP-1). Reducing VEGF expression by tumor cells induced a differentiated phenotype in vitro and inhibited tumor forming capacity in vivo, independent of effects on angiogenesis. Autocrine activation of tumor cell growth was dependent on signaling through NRP-1, and Ras was determined to be a critical effector signaling molecule downstream of NRP-1. Our findings define a novel function for VEGF in dedifferentiation of tumor cells expanding its role in cancer beyond its known proangiogenic function.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Progestin suppression of miR-29 potentiates dedifferentiation of breast cancer cells via KLF4

The female hormone progesterone (P4) promotes the expansion of stem-like cancer cells in estrogen receptor (ER)- and progesterone receptor (PR)-positive breast tumors. The expanded tumor cells lose expression of ER and PR, express the tumor-initiating marker CD44, the progenitor marker cytokeratin 5 (CK5) and are more resistant to standard endocrine and chemotherapies. The mechanisms underlying this hormone-stimulated reprogramming have remained largely unknown. In the present study, we investigated the role of microRNAs in progestin-mediated expansion of this dedifferentiated tumor cell population. We demonstrate that P4 rapidly downregulates miR-29 family members, particularly in the CD44(+) cell population. Downregulation of miR-29 members potentiates the expansion of CK5(+) and CD44(+) cells in response to progestins, and results in increased stem-like properties in vitro and in vivo. We demonstrate that miR-29 directly targets Krüppel-like factor 4 (KLF4), a transcription factor required for the reprogramming of differentiated cells to pluripotent stem cells, and for the maintenance of breast cancer stem cells. These results reveal a novel mechanism, whereby progestins increase the stem cell-like population in hormone-responsive breast cancers, by decreasing miR-29 to augment PR-mediated upregulation of KLF4. Elucidating the mechanisms whereby hormones mediate the expansion of stem-like cells furthers our understanding of the progression of hormone-responsive breast cancers.

Abstract A68: Tumor cell dedifferentiation into endothelial cells and resistance to sunitinib in renal carcinoma models

Abstract Background: Acquired resistance to multikinase inhibitors stands as a major clinical challenge. Treatment with sunitinib was shown to strikingly reduce the tumor blood flow, which in turn induces cellular starvation, hypoxia, and necrosis. When angiogenesis inhibition is maintained, cancer cells and other cells in the stroma may adapt to recover from the primary stress to allow continued perfusion and tumor growth that is less reliant on VEGF/VEGFR signaling. Clinical evidence has suggested that resistance to VEGFR-targeted agents was mediated through changes in both cancer cells and the microenvironment. The aim of present study was to study in vitro and in vivo the molecular mechanisms of acquired resistance to sunitinib in RCC models. Materials and Methods: CAKI1 (VHL+) and 786-0 (VHL−) renal carcinoma cells were exposed to stepwise increasing doses of sunitinib for >6 months. Invasiveness of cells was determined by Matrigel invasion assay. qRT-PCR, western blot and IHC assays were used to assess a panel of genes and proteins. To compare the molecular mechanisms of sunitinib in vitro and in vivo, CAKI1 cells were engrafted in nude mice and treated continuously with a tumoristatic dose of 60 mg/kg oral sunitinib until progression. Results: CAKI-Suni, 786-Suni cells were 2-fold more resistant to sunitinib than parental cells. Several changes in gene expressions were detected in cells acquiring resistance to sunitinib. Protracted exposure to sunitinib led to a >3-fold increase of ErbB3 and CDH1 in CAKI-Suni and 786-Suni as compared to parental CAKI1 and 786-0 cells. Resistance to sunitinib was associated also with AKT and ERK activation. In addition, CAKI-Suni cells were at least 3-fold more invasive than their parental counterpart. In vivo, sunitinib induced significant tumor growth retardation with median PFS of 59 against 22 days for placebo (p<0.0001). After median of 35 days several tumors became progressive under sunitinib. IHC analysis of tumors revealed less necrosis and more blood vessels in sunitinib resistant as compared to sunitinib-sensitive tumors. Two-fold increased mRNA expression of VEGFA, CXCR4, CA9 and HIF1A and 4-fold decreased expression of E-cadherin were found in progressive tumors. CXCR4, vimentin and ErbB3 protein levels were higher in progressive tumors than in tumors responding to sunitinib. Using human and mouse primers for mRNA expression of CD31, we suggested that human cancer cells may differentiate into endothelial cells at progression. Tumor cells resistant to sunitinib situated in tumor vessels co-expressed both human renal cell carcinoma marker CD10 and CD31/Von Willebrand using immunostaining. Conclusions: Sustained exposure to sunitinib induces changes in gene expression, invasion properties and survival signaling pathways in RCC cells. In CAKI1 xenograft tumor cells that become insensitive to sunitinib may be found in tumor vessels and display markers of endothelial cells.

Abstract 984: High-level expression of YAP induces protumorigenic Notch signalling in human hepatocarcinogenesis

Abstract The evolutionary conserved Hippo-pathway negatively regulates organ size control by phosphorylation and cytoplasmic retention of the transcriptional co-activator yes-associated protein (YAP). Recent studies demonstrated that deletion of essential Hippo-pathway constituents (e.g., Mst-1/2 and WW45) or overexpression of YAP lead to the development of liver cancer. However, the underlying molecular tumor-supporting mechanisms in carcinogenesis have not been defined so far. Overexpression and nuclear accumulation of YAP in nearly 70% of all human hepatocellular carcinomas (HCC) significantly correlated with tumor cell proliferation and dedifferentiation. In human HCC cell lines, siRNA-mediated inhibition of YAP significantly reduced tumor cell viability, and migration/invasion. Based on transcriptomic profiling approaches, the Notch ligand Jagged-1 (Jag-1) was identified as YAP-dependent target gene in HCC cells and in primary murine hepatocytes of transgenic animals expressing constitutively active YAPS127A. Inhibition of YAP reduced the protein levels of Jag-1, cleaved Notch receptor (NICD), and Hes-1, while YAP overexpression increased the amounts of all factors. As detected for YAP knock down, transfection of gene-specific siRNA targeting Jag-1 diminished HCC cell viability and migration. Overexpression and concomitant inhibition of Jag-1 abolished Hes-1 expression and YAP-induced HCC cell viability. By applying different mutant isoforms of YAP (e.g., YAPS127A and YAP5SA-delta-C - dominant negative isoform), TEAD4 but not TEAD1 was identified as the transcription factor required for YAP-dependent regulation of Jag-1 and Hes-1. Furthermore, the WNT/beta-catenin pathway, a putative inducer of Jag-1, did not influence the YAP-dependent modulation of Jag-1. Knock down experiments revealed Mst-2 and Lats-2 as negative regulators of YAP activity and Jag-1/Hes-1 expression. The amounts of YAP, Jag-1, and Hes-1 transcripts as well as proteins significantly correlated with each other in human HCC tissues. Most importantly, increased concentrations of all factors significantly associated with poor prognosis of HCC patients. These data demonstrate that high-level expression of YAP in HCC cells induces tumor growth and tumor cell dissemination in part through activation of the Jag-1/Notch pathway in a TEAD4-dependent and beta-catenin-independent manner. This regulatory cross-talk between Hippo- and Notch-signalling defines a group of HCC patients with poor overall survival. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 984. doi:1538-7445.AM2012-984

The developing cancer stem-cell model: clinical challenges and opportunities

During the past decade, a stem-cell-like subset of cancer cells has been identified in many malignancies. These cells, referred to as cancer stem cells (CSCs), are of particular interest because they are believed to be the clonogenic core of the tumour and therefore represent the cell population that drives growth and progression. Many efforts have been made to design therapies that specifically target the CSC population, since this was predicted to be the crucial population to eliminate. However, recent insights have complicated the initial elegant model, by showing a dominant role for the tumour microenvironment in determining CSC characteristics within a malignancy. This is particularly important since dedifferentiation of non-tumorigenic tumour cells towards CSCs can occur, and therefore the CSC population in a neoplasm is expected to vary over time. Moreover, evidence suggests that not all tumours are driven by rare CSCs, but might instead contain a large population of tumorigenic cells. Even though these results suggest that specific targeting of the CSC population might not be a useful therapeutic strategy, research into the hierarchical cellular organisation of malignancies has provided many important new insights in the biology of tumours. In this Personal View, we highlight how the CSC concept is developing and influences our thinking on future treatment for solid tumours, and recommend ways to design clinical trials to assess drugs that target malignant disease in a rational fashion.

Acquired cancer stem cell phenotypes through Oct4-mediated dedifferentiation

There is enormous interest to target cancer stem cells (CSCs) for clinical treatment because these cells are highly tumorigenic and resistant to chemotherapy. Oct4 is expressed by CSC-like cells in different types of cancer. However, function of Oct4 in tumor cells is unclear. In this study, we showed that expression of Oct4 gene or transmembrane delivery of Oct4 protein promoted dedifferentiation of melanoma cells to CSC-like cells. The dedifferentiated melanoma cells showed significantly decreased expression of melanocytic markers and acquired the ability to form tumor spheroids. They showed markedly increased resistance to chemotherapeutic agents and hypoxic injury. In the subcutaneous xenograft and tail vein injection assays, these cells had significantly increased tumorigenic capacity. The dedifferentiated melanoma cells acquired features associated with CSCs such as multipotent differentiation capacity and expression of melanoma CSC markers such as ABCB5 and CD271. Mechanistically, Oct4 induced dedifferentiation was associated with increased expression of endogenous Oct4, Nanog and Klf4, and global gene expression changes that enriched for transcription factors. RNAi mediated knockdown of Oct4 in dedifferentiated cells led to diminished CSC phenotypes. Oct4 expression in melanoma was regulated by hypoxia and its expression was detected in a subpopulation of melanoma cells in clinical samples. Our data indicate that Oct4 is a positive regulator of tumor dedifferentiation. The results suggest that CSC phenotype is dynamic and may be acquired through dedifferentiation. Oct4 mediated tumor cell dedifferentiation may play an important role during tumor progression.

PET/CT in thyroid carcinoma

The diagnostic imaging procedures that have a role in detection of malignant thyroid tissue are radioiodine (131I) diagnostic whole-body scintigraphy (WBS), neck ultrasound, and CT and MRI for evaluation of the mediastinal area. Despite excellent morphologic characterization of metastatic nodal recurrences, MRI cannot reliably make a differentiation between benign and malignant lymph nodes. Although it detects enlarged metastatic lymph nodes, there are also many small nodal metastases that are usually missed. In one-third of patients with well differentiated thyroid carcinoma, there are carcinomas with dedifferentiated tumor cells: metastatic tissue may not concentrate radioiodine well; thus 131I-WBS is negative despite elevated thyroglobulin (Tg) levels. Although MRI helps in detection of these non-iodine avid metastases, FDG PET/CT can perform more effectively. Due to its high glycolytic rate, changes in glucose transport systems and hexokinase activity, [18F] fluorodeoxyglucose (FDG) accumulates in malignant tissue and is useful for identification of distant metastases in these patients. Iodine positive metastases are often negative with FDG-PET imaging while iodine negative metastases exhibit increased FDG-uptake. If a metastatic lesion is identified by FDG positron emission tomography/ computed tomography (PET/CT), the usual approach is to first send the patient to surgery for removal of neoplastic tissue, if possible. This is followed by re-treatment with 131I therapy after tumor redifferentiation with retinoic acid. In a limited number of patients, iodine negative thyroid cancer may express somatostatin receptors and radiopeptide therapy may be utilized. FDG PET/CT is a hybrid imaging diagnostic tool which helps in detection of non-iodine avid metastases. It has a role in exact localization of recurrences which will assist in the decision to remove the malignant tissue surgically.

Effects of 1α,25 dihydroxyvitamin D3 and testosterone on miRNA and mRNA expression in LNCaP cells

BackgroundThere is evidence from epidemiological and in vitro studies that the biological effects of testosterone (T) on cell cycle and survival are modulated by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) in prostate cancer. To investigate the cross talk between androgen- and vitamin D-mediated intracellular signaling pathways, the individual and combined effects of T and 1,25(OH)2D3 on global gene expression in LNCaP prostate cancer cells were assessed.ResultsStringent statistical analysis identifies a cohort of genes that lack one or both androgen response elements (AREs) or vitamin D response elements (VDREs) in their promoters, which are nevertheless differentially regulated by both steroids (either additively or synergistically). This suggests that mechanisms in addition to VDR- and AR-mediated transcription are responsible for the modulation of gene expression. Microarray analysis shows that fifteen miRNAs are also differentially regulated by 1,25(OH)2D3 and T. Among these miR-22, miR-29ab, miR-134, miR-1207-5p and miR-371-5p are up regulated, while miR-17 and miR-20a, members of the miR-17/92 cluster are down regulated. A number of genes implicated in cell cycle progression, lipid synthesis and accumulation and calcium homeostasis are among the mRNA targets of these miRNAs. Thus, in addition to their well characterized effects on transcription, mediated by either or both cognate nuclear receptors, 1,25(OH)2D3 and T regulate the steady state mRNA levels by modulating miRNA-mediated mRNA degradation, generating attenuation feedback loops that result in global changes in mRNA and protein levels. Changes in genes involved in calcium homeostasis may have specific clinical importance since the second messenger Ca2+ is known to modulate various cellular processes, including cell proliferation, cell death and cell motility, which affects prostate cancer tumor progression and responsiveness to therapy.ConclusionsThese data indicate that these two hormones combine to drive a differentiated phenotype, and reinforce the idea that the age dependent decline in both hormones results in the de-differentiation of prostate tumor cells, which results in increased proliferation, motility and invasion common to aggressive tumors. These studies also reinforce the potential importance of miRNAs in prostate cancer progression and therapeutic outcomes.

Expression and prognostic value of WISP‐1 in patients with endometrial endometrioid adenocarcinoma

It has been well established that the aberrant activation of the Wnt/β-catenin signal correlates to endometrial endometrioid adenocarcinoma (EEC). As an effector of the Wnt/β-catenin mediated antiapoptotic signal, the role of Wnt-induced secreted protein-1 (WISP-1) in EEC still remains unclear. We used immunohistochemistry and quantitative real-time RT-PCR to examine the expression of WISP-1, the estrogen receptor (ER) and progesterone receptor (PR) in 86 cases of EEC, with 20 cases of endometrial hyperplasia, 20 of proliferative endometrium and 20 of secretory endometrium used as the control group. We also correlated the expression of WISP-1 with various clinicopathologic factors and prognostic values in patients with EEC. A high expression of WISP-1 was observed in 26 of the 86 cases of EEC (30.2%). The expression rate of WISP-1 in EEC was significantly higher than that in secretory endometrium (P < 0.005). Histopathological grades and PR were associated with high WISP-1 expression (P = 0.002, P = 0.027, respectively). The estimated five-year survival rate of patients with low-to-moderate expression of WISP-1 was significantly higher than those with high WISP-1 expression (91.3% vs 65%, P = 0.011). Multivariate analysis revealed that high WISP-1 expression and positive lymphovascular space invasion were independent prognostic factors for survival. High expression of WISP-1 was related to tumor cell dedifferentiation and PR loss. WISP-1 might be a new molecular marker to predict the prognosis of patients with EEC.

Abstract 5172: Fluorescence labeling of cytokeratin-negative circulating tumor cells in peripheral blood: A new paradigm for the study of cancer progression

Abstract Introduction: The measurement of circulating tumor cells (CTCs) in blood generally requires either anti-EpCAM for capture, anti-cytokeratin (CK) for detection, or both. However, EpCAM and CK are absent in some tumor cells, and both may be down-regulated during neoplastic progression or during epithelial-to-mesenchymal transition (EMT). Here we show capture on a micro-fluidic system using single or multiple antibodies, and CTC detection with CEE-Enhanced (CE), a novel in situ staining method that fluorescently labels the capture antibodies bound to the CTCs. Methods: Buffy coat cells isolated from 8 mL of blood were pre-incubated with either anti-EpCAM alone or with a mixture of antibodies to epithelial and mesenchymal cell surface antigens. CTCs were captured on a micro-fluidic channel. Identification of CTCs was determined with anti-CK and with CE to detect cells with the capture antibodies bound to the cell surface antigens. All cells scored as positive for CK or CE were, by definition, CD45-negative and DAPI-positive. Results: Using anti-EpCAM alone for capture, significantly more CTCs were detected by CE staining than with anti-CK in breast and prostate cancers. This indicated that CK-negative CTCs were captured but not detected and that some EpCAM-positive CTCs were CK-negative. Results using capture antibody mixtures varied from sample to sample but gave up to 2-fold higher CK-positive cells than anti-EpCAM only, and as much as 4-fold higher CE-positive cells. Control blood from healthy donors was CK and CE-negative. All CK-positive cells co-stained with CE, as determined with different fluorescent labels. In a clinical study of stage IV breast cancer, CTCs were isolated with an antibody mixture and sequentially stained with CK and CE. Fifteen of 24 samples (63%) contained CK-positive cells (range 1-60 CTCs) while 24 of 24 samples (100%) contained additional CE-positive cells (range 1-41; median=11; Wilcoxon test, p=0.02). The modest correlation coefficient (r = 0.57, p=0.004) for the number of CE-positive and CK-positive cells in each sample suggests that one or more different phenotypes of CTCs were being detected. Amplified Her2 was detected by FISH in isolated CK-positive CTCs, and also in CK-negative, CE-positive CTCs from Her2-positive patients, indicating these were tumor cells. Conclusions: The CEE-Enhanced staining technology enables the detection of CK-negative CTCs. This novel detection method, based on the in situ labeling of antibodies used for capture, greatly expands single and multi-antibody approaches to the study of rare circulating cells. Specifically, this allows the exploration and study of circulating cells not expressing CK such as highly de-differentiated tumor cells, stem cells or those tumor cells undergoing EMT. The clinical significance of these CK-negative CTCs is under investigation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5172. doi:10.1158/1538-7445.AM2011-5172

Retinoid X receptor γ up‐regulation is correlated with dedifferentiation of tumor cells and lymph node metastasis in papillary thyroid carcinoma

The expression of retinoid X receptor γ (RXRγ) and the clinicopathological parameters of total 69 patients with papillary thyroid carcinoma (PTC) larger than 1 cm were examined. The PTCs were classified into two groups according to the presence of loss of cellular polarity/cohesiveness (LOP/C). The expression of RXRγ mRNA was examined by reverse transcriptase polymerase chain reaction and quantitative real-time PCR. The RXRγ mRNA up-regulation was found to be positively correlated with extrathyroid invasion (r = 0.293, P = 0.019), advanced tumor stage (r = 0.318, P = 0.016) and lymph node metastasis (LNM) (r = 0.338, P = 0.005), as well as LOP/C (r = 0.345, P = 0.004), which was proposed as a histological characteristic of poor cellular differentiation. The RXRγ mRNA expression, as well as extrathyroid invasion, LOP/C and advanced tumor stage, was further confirmed to be one of the independent predictive factors (Odds ratio: 6.545; 95% confidence interval: 1.575-27.208) of LNM using multivariate analysis. These results suggest that RXRγ may play a role in the dedifferentiation and metastasis of PTC.

Epithelial mesenchymal transition and tumor budding in aggressive colorectal cancer: Tumor budding as oncotarget

Epithelial mesenchymal transition (EMT) is proposed as a critical mechanism for the acquisition of malignant phenotypes by epithelial cells. In colorectal cancer, tumor cells having undergone EMT are histologically represented by the presence of tumor buds defined as single cells or small clusters of de-differentiated tumor cells at the invasive front. Tumor budding is not a static, histological feature rather it represents a snap-shot of a dynamic process undertaken by an aggressive tumor with the potential to disseminate and metastasize. Strong, consistent evidence shows that tumor budding is a predictor of lymph node metastasis, distant metastatic disease, local recurrence, worse overall and disease-free survival time and an independent prognostic factor. Moreover, the International Union against Cancer (UICC) recognizes tumor budding as a highly relevant, additional prognostic parameter. The aim of this review is to summarize the evidence supporting the implementation of tumor budding into diagnostic pathology and patient management and additionally to illustrate its worthiness as a potential therapeutic target.

Prognostic significance of differentially expressed miRNAs in esophageal cancer

Altered microRNA (miRNA) expression has been found to promote carcinogenesis, but little is known about the role of miRNAs in esophageal cancer. In this study, we selected 10 miRNAs and analyzed their expression in 10 esophageal cancer cell lines and 158 tissue specimens using Northern blotting and in situ hybridization, respectively. We found that Let-7g, miR-21 and miR-195p were expressed in all 10 cell lines, miR-9 and miR-20a were not expressed in any of the cell lines, and miR-16-2, miR-30e, miR-34a, miR-126 and miR-200a were expressed in some of the cell lines but not others. In addition, transient transfection of miR-34a inhibited c-Met and cyclin D1 expression and esophageal cancer cell proliferation, whereas miR-16-2 suppressed RAR-β(2) expression and increased tumor cell proliferation. Furthermore, we found that miR-126 expression was associated with tumor cell dedifferentiation and lymph node metastasis, miR-16-2 was associated with lymph node metastasis, and miR-195p was associated with higher pathologic disease stages in patients with esophageal adenocarcinoma. Kaplan-Meier analysis showed that miR-16-2 expression and miR-30e expression were associated with shorter overall and disease-free survival in all esophageal cancer patients. In addition, miR-16-2, miR-30e and miR-200a expression were associated with shorter overall and disease-free survival in patients with esophageal adenocarcinoma; however, miR-16-2, miR-30e and miR-200a expression were not associated with overall or disease-free survival in squamous cell carcinoma patients. Our data indicate that further evaluation of miR-30e and miR-16-2 as prognostic biomarkers is warranted in patients with esophageal adenocarcinoma. In addition, the role of miR-34a in esophageal cancer also warrants further study.

Transcriptional Repression of SKP2 Is Impaired in MYCN-Amplified Neuroblastoma

The cell cycle regulator, SKP2, is overexpressed in various cancers and plays a key role in p27 degradation, which is involved in tumor cell dedifferentiation. Little is known about the mechanisms leading to impaired SKP2 transcriptional control in tumor cells. We used neuroblastoma as a model to study SKP2 regulation because SKP2 transcript levels gradually increase with aggressiveness of neuroblastoma subtypes. The highest SKP2 levels are found in neuroblastomas with amplified MYCN. Accordingly, we found 5.5-fold (range, 2-9.5) higher SKP2 core promoter activity in MYCN-amplified cells. Higher SKP2 core promoter activity in MYCN-amplified cells is mediated through a defined region at the transcriptional start site. This region includes a specific E2F-binding site that makes SKP2 activation largely independent of mitogenic signals integrated through the SP1/ELK-1 site. We show by chromatin immunoprecipitation that SKP2 activation through the transcriptional start site in MYCN-amplified cells is associated with the low abundance of pRB-E2F1 complexes bound to the SKP2 promoter. Transcriptional control of SKP2 through this regulatory mechanism can be reestablished in MYCN-amplified cells by restoring pRB activity using selective small compound inhibitors of CDK4. In contrast, doxorubicin or nutlin-3 treatment-both leading to p53-p21 activation-or CDK2 inhibition had no effect on SKP2 regulation in MYCN-amplified cells. Together, this implies that deregulated MYCN protein levels in MYCN-amplified neuroblastoma cells activate SKP2 through CDK4 induction, abrogating repressive pRB-E2F1 complexes bound to the SKP2 promoter.

Abstract 568: Transcriptional repression of SKP2 is impaired in MYCN-amplified neuroblastoma

Abstract The cell cycle regulator, SKP2, is overexpressed in various cancers, and plays a key role in p27 degradation, which is involved in tumor cell dedifferentiation. Little is known about the mechanisms leading to impaired SKP2 transcriptional control in tumor cells. We used neuroblastoma as a model to study SKP2 regulation because SKP2 transcript levels gradually increase with aggressiveness of neuroblastoma subtypes. Highest SKP2 levels are found in neuroblastomas with amplified MYCN. Accordingly, we found 5.5-fold (range 2-9.5) higher SKP2 core promoter activity in MYCN-amplified cells. Higher SKP2 core promoter activity in MYCN-amplified cells is mediated through a defined region at the transcriptional start site (TSSR). This region includes a specific E2F-binding site that makes SKP2 activation largely independent of mitogenic signals integrated through the SP1/ELK-1 site. We demonstrate by chromatin immunoprecipitation that SKP2 activation through the TSSR in MYCN-amplified cells is associated with low abundance of pRB-E2F1 complexes bound to the SKP2 promoter. Transcriptional control of SKP2 via this regulatory mechanism can be re-established in MYCN-amplified cells by restoring pRB activity using selective small compound inhibitors of CDK4. In contrast, doxorubicin or nutlin-3 treatment - both leading to p53-p21 activation - or CDK2 inhibition had no effect on SKP2 regulation in MYCN-amplified cells. Together, this implies that deregulated MYCN protein levels in MYCN-amplified neuroblastoma cells activate SKP2 through CDK4 induction, abrogating repressive pRB-E2F1 complexes bound to the SKP2 promoter. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 568.

Does telomerase reverse transcriptase induce functional de-differentiation of human endothelial cells?

By counteracting the shortening of chromosome telomeres, telomerase reverse transcriptase (hTERT) prevents senescence and age-related cell death. Embryonic cells display a high telomerase activity that declines rapidly with cell differentiation. Conversely, de-differentiated tumor cells tend to re-express telomerase. In view of the controversial data on the reciprocal correlation between cell proliferation and differentiation, we questioned whether telomerase overexpression and the resulting immortalization would affect the functional phenotype of human endothelial cells. Our comparative analysis addressed (1) distinct cell adhesion to different ECM-proteins analyzed on miniaturized multisubstrate arrays (MSA), (2) protein expression of diverse markers, (3) the uptake of DiI-Ac-LDL, (4) the inflammatory response based on upregulation of ICAM-1, (5) tube formation, and (6) the barrier properties of cell monolayers in transfilter cultures. Our results, based on some 40 data sets, demonstrate that immortalization of primary endothelial cells by hTERT maintains the typical endothelial characteristics without any sign of functional de-differentiation.