Targeting endogenous fatty acid synthesis stimulates the migration of ovarian cancer cells to adipocytes and promotes the transport of fatty acids from adipocytes to cancer cells.

Despite significant advances in oncology, 1 of 108 female patients succumb to ovarian cancer (OC) each year. Improved novel treatments against this aggressive disease would be a major improvement. The growth of OC cells has been demonstrated to be highly dependent on lipids. OC cells are abundantly present in the abdominal cavity and omentum, the main sites of OC expansion. Accordingly, it has been attempted not only to block the hyperactive synthesis of fatty acids (FAs) in cancer cells, but also to disrupt lipid supply. While either strategy has yielded promising results as monotherapy, the induction of resistance pathways diminishing the anticancer effects is yet conceivable. The endogenous regulation of lipid biosynthesis in OC has been extensively studied. However, the role of stromal cells in the modulation of the effects of anti­lipogenic drugs has not yet been well documented. The present study thus examined the interaction between OC cells and associated stromal cells, when de novo FA synthesis was blocked. It has recently been revealed by the authors that when FA are provided to OC cells in monoculture, the lipid deficiency induced by pharmacological inhibition of FA synthase (FASN), the key enzyme of endogenous FA synthesis, cannot be compensated through an increased FA uptake by OC cells. In the present study, OC cells were co­cultured with adipocytes preloaded with fluorescent FA and the effects of FASN­inhibition on OC homing to adipocytes and the transcellular delivery of fluorescent FA from adipocytes to OC cells were examined. The FASN inhibitors, G28UCM and Fasnall, stimulated the spontaneous migration of A2780 OC cells in a concentration­dependent manner and stimulated the transfer of FA from adipocytes to OC cells. Similar effects were observed with all types of adipocytes tested. The models applied in the present study demonstrated that co­cultured cancer­associated adipocytes may attenuate the anticancer effects of FASN inhibitors by attracting tumor cells and by supplying the cells with FA. This lipid­mediated dependency may provide a rationale for the design of new treatment approaches for the treatment of OC.

Vienna Cancer Stem Cell Club (VCSCC): 20 year jubilee and future perspectives.

INTRODUCTION: The Vienna Cancer Stem Cell Club (VCSCC) was launched by a group of scientists in Vienna in 2002. AREAS COVERED: Major aims of the VCSCC are to support research on cancer stem cells (CSC) in hematopoietic malignancies and to translate CSC-related markers and targets into clinical application. A primary focus of research in the VCSCC is the leukemic stem cell (LSC). Between 2013 and 2021, members of the VCSCC established a special research program on myeloproliferative neoplasms and since 2008, members of the VCSCC run the Ludwig Boltzmann Institute for Hematology and Oncology. In all these years, the VCSCC provided a robust intellectual platform for translational hematology and LSC research in Vienna. Furthermore, the VCSCC interacts with several national and international study groups and societies in the field. Representatives of the VCSCC also organized a number of international meetings and conferences on neoplastic stem cells, including LSC, in the past 15 years, and contributed to the definition and classification of CSC/LSC and related pre-malignant and malignant conditions. EXPERT OPINION: The VCSCC will continue to advance the field and to develop LSC-detecting and LSC-eradicating concepts through which diagnosis, prognostication, and therapy of blood cancer patients should improve.

A critical appraisal of the relative contribution of tissue architecture, genetics, epigenetics and cell metabolism to carcinogenesis.

Here we contrast several carcinogenesis models. The somatic-mutation-theory posits mutations as main causes of malignancy. However, inconsistencies led to alternative explanations. For example, the tissue-organization-field-theory considers disrupted tissue-architecture as main cause. Both models can be reconciled using systems-biology-approaches, according to which tumors hover in states of self-organized criticality between order and chaos, are emergent results of multiple deviations and are subject to general laws of nature: inevitable variation(mutation) explainable by increased entropy(second-law-of-thermodynamics) or indeterminate decoherence upon measurement of superposed quantum systems(quantum mechanics), followed by Darwinian-selection. Genomic expression is regulated by epigenetics. Both systems cooperate. So cancer is neither just a mutational nor an epigenetic problem. Rather, epigenetics links environmental cues to endogenous genetics engendering a regulatory machinery that encompasses specific cancer-metabolic-networks. Interestingly, mutations occur at all levels of this machinery (oncogenes/tumor-suppressors, epigenetic-modifiers, structure-genes, metabolic-genes). Therefore, in most cases, DNA mutations may be the initial and crucial cancer-promoting triggers.

Pharmacological Inhibition of Lipid Import and Transport Proteins in Ovarian Cancer.

Ovarian cancer (OC) is the most lethal gynecological malignancy with a 5-year survival rate of 49%. This is caused by late diagnosis when cells have already metastasized into the peritoneal cavity and to the omentum. OC progression is dependent on the availability of high-energy lipids/fatty acids (FA) provided by endogenous de novo biosynthesis and/or through import from the microenvironment. The blockade of these processes may thus represent powerful strategies against OC. While this has already been shown for inhibition of FA/lipid biosynthesis, evidence of the role of FA/lipid import/transport is still sparse. Therefore, we treated A2780 and SKOV3 OC cells with inhibitors of the lipid uptake proteins fatty acid translocase/cluster of differentiation 36 (FAT/CD36) and low-density lipoprotein (LDL) receptor (LDLR), as well as intracellular lipid transporters of the fatty acid-binding protein (FABP) family, fatty acid transport protein-2 (FATP2/SLC27A2), and ADP-ribosylation factor 6 (ARF6), which are overexpressed in OC. Proliferation was determined by formazan dye labeling/photometry and cell counting. Cell cycle analysis was performed by propidium iodide (PI) staining, and apoptosis was examined by annexin V/PI and active caspase 3 labeling and flow cytometry. RNA-seq data revealed altered stress and metabolism pathways. Overall, the small molecule inhibitors of lipid handling proteins BMS309403, HTS01037, NAV2729, SB-FI-26, and sulfosuccinimidyl oleate (SSO) caused a drug-specific, dose-/time-dependent inhibition of FA/LDL uptake, associated with reduced proliferation, cell cycle arrest, and apoptosis. Our findings indicate that OC cells are very sensitive to lipid deficiency. This dependency should be exploited for development of novel strategies against OC.

Cancer - A devastating disease, but also an eye-opener and window into the deep mysteries of life and its origins.

Although cancer is still the second leading cause of death worldwide, basic research has largely elucidated the underlying mechanisms that lead us deep into the laws of animate and inanimate nature. This review aims to demonstrate that the cancer process profoundly affects and reprograms fundamental principles and concepts of cellular life by harnessing the natural mechanisms of biological evolution. It is shown that mutation and selection - the drivers of cancer formation and progression - are mandatory consequences of Boltzmann's version of the second law of thermodynamics, which stipulates that entropy (or disorder) according to probability never decreases, followed by Darwinian evolution by filtering for the suitable geno- and karyotypes. Cancer research has shown that malignant cells can develop gradually or abruptly depending on the prevailing stress conditions. Similar principles were then observed in the evolution of species, referred to as micro- and macroevolution. Cancer cells can be related to phylogenetically older forms of life, and malignant transformation can be viewed as reverse (atavistic) evolution, accompanied by typical rearrangement of system information and loss of 'social' behavior. It becomes obvious that in nature no distinction is made between normal biology and pathobiology. Instead, everything follows the rules of natural evolution. This illustrates the depth of the cancer problem and may explain the serious difficulties faced in trying to eradicate cancer.

The Pharmacological or Genetic Blockade of Endogenous De Novo Fatty Acid Synthesis Does Not Increase the Uptake of Exogenous Lipids in Ovarian Cancer Cells.

Ovarian cancer(OC) is a serious threat to women worldwide. Peritoneal dissemination, ascites and omental metastasis are typical features for disease progression, which occurs in a micro-environment that is rich in high-energy lipids. OC cells require high amounts of lipids for survival and growth. Not only do they import lipids from the host, they also produce lipids de novo. Inhibitors of fatty acid(FA) synthase(FASN) - the rate-limiting enzyme of endogenous FA synthesis that is overexpressed in OC - induce growth-arrest and apoptosis, rendering them promising candidates for cancer drug development. However, cancer researchers have long hypothesized that the lipid deficiency caused by FASN inhibition can be circumvented by increasing the uptake of exogenous lipids from the host, which would confer resistance to FASN inhibitors. In contrast to a very recent report in colorectal cancer, we demonstrate in OC cells (A2780, OVCAR3, SKOV3) that neither FASN inhibitors (G28UCM, Fasnall) nor FASN-specific siRNAs can stimulate a relief pathway leading to enhanced uptake of extrinsic FAs or low density lipoproteins (LDLs). Instead, we observed that the growth-arrest due to FASN inhibition or FASN knock-down was associated with significant dose- and time-dependent reduction in the uptake of fluorescently labeled FAs and LDLs. Western blotting showed that the expression of the FA receptor CD36, the LDL receptor(LDLR) and the lipid transport proteins fatty acid binding proteins 1-9 (FABP1-9) was not affected by the treatment. Next, we compared experimental blockade of endogenous lipid production with physiologic depletion of exogenous lipids. Lipid-free media, similar to FASN inhibitors, caused growth-arrest. Although lipid-depleted cells have diminished amounts of CD36, LDLR and FABPs, they can still activate a restorative pathway that causes enhanced import of fluorophore-labeled FAs and LDLs. Overall, our data show that OC cells are strictly lipid-depend and exquisitely sensitive to FASN inhibitors, providing a strong rationale for developing anti-FASN strategies for clinical use against OC.

Degradation of BRD4 - a promising treatment approach not only for hematologic but also for solid cancer.

Bromodomain (BRD) and extra-terminal (BET) proteins are epigenetic readers that regulate gene expression and promote cancer evolution. Pharmacological inactivation of BRD4 has recently been introduced as a promising anti-neoplastic approach that targets MYC oncogene expression. However, resistance against BRD4-targeting drugs has been described. We compared the efficacy of the small-molecule-type BET BRD inhibitor JQ1 with the recently developed BET protein degraders dBET1 and dBET6 in colon, breast, melanoma, ovarian, lung and prostate cancer cell lines. As determined by qPCR, all BRD4 targeting drugs dose-dependently decreased MYC expression, with dBET6 introducing the strongest downregulation of MYC. This correlated with the anti-proliferative activity of these drugs, which was at least one order of magnitude higher for dBET6 (IC50 0.001-0.5 µM) than for dBET1 or JQ1 (IC50 0.5-5 µM). Interestingly, when combined with commonly used cytotoxic therapeutics, dBET6 was found to promote anti-neoplastic effects and to counteract chemoresistance in most cancer cell lines. Moreover, JQ1 and both BET degraders strongly downregulated baseline and interferon-gamma induced expression of the immune checkpoint molecule PD-L1 in all cancer cell lines. Together, our data suggest that dBET6 outperforms first-generation BRD4 targeting drugs like dBET1 and JQ1, and decreases chemoresistance and immune resistance of cancer.

Membrane disruption, but not metabolic rewiring, is the key mechanism of anticancer-action of FASN-inhibitors: a multi-omics analysis in ovarian cancer.

Fatty-acid(FA)-synthase(FASN) is a druggable lipogenic oncoprotein whose blockade causes metabolic disruption. Whether drug-induced metabolic perturbation is essential for anticancer drug-action, or is just a secondary-maybe even a defence response-is still unclear. To address this, SKOV3 and OVCAR3 ovarian cancer(OC) cell lines with clear cell and serous histology, two main OC subtypes, were exposed to FASN-inhibitor G28UCM. Growth-inhibition was compared with treatment-induced cell-metabolomes, lipidomes, proteomes and kinomes. SKOV3 and OVCAR3 were equally sensitive to low-dose G28UCM, but SKOV3 was more resistant than OVCAR3 to higher concentrations. Metabolite levels generally decreased upon treatment, but individual acylcarnitines, glycerophospholipids, sphingolipids, amino-acids, biogenic amines, and monosaccharides reacted differently. Drug-induced effects on central-carbon-metabolism and oxidative-phosphorylation (OXPHOS) were essentially different in the two cell lines, since drug-naïve SKOV3 are known to prefer glycolysis, while OVCAR3 favour OXPHOS. Moreover, drug-dependent increase of desaturases and polyunsaturated-fatty-acids (PUFAs) were more pronounced in SKOV3 and appear to correlate with G28UCM-tolerance. In contrast, expression and phosphorylation of proteins that control apoptosis, FA synthesis and membrane-related processes (beta-oxidation, membrane-maintenance, transport, translation, signalling and stress-response) were concordantly affected. Overall, membrane-disruption and second-messenger-silencing were crucial for anticancer drug-action, while metabolic-rewiring was only secondary and may support high-dose-FASN-inhibitor-tolerance. These findings may guide future anti-metabolic cancer intervention.

Heterotypic CAF-tumor spheroids promote early peritoneal metastatis of ovarian cancer.

High-grade serous ovarian cancer (HGSOC) is hallmarked by early onset of peritoneal dissemination, which distinguishes it from low-grade serous ovarian cancer (LGSOC). Here, we describe the aggressive nature of HGSOC ascitic tumor cells (ATCs) characterized by integrin α5high (ITGA5high) ATCs, which are prone to forming heterotypic spheroids with fibroblasts. We term these aggregates as metastatic units (MUs) in HGSOC for their advantageous metastatic capacity and active involvement in early peritoneal dissemination. Intriguingly, fibroblasts inside MUs support ATC survival and guide their peritoneal invasion before becoming essential components of the tumor stroma in newly formed metastases. Cancer-associated fibroblasts (CAFs) recruit ITGA5high ATCs to form MUs, which further sustain ATC ITGA5 expression by EGF secretion. Notably, LGSOC is largely devoid of CAFs and the resultant MUs, which might explain its metastatic delay. These findings identify a specialized MU architecture that amplifies the tumor-stroma interaction and promotes transcoelomic metastasis in HGSOC, providing the basis for stromal fibroblast-oriented interventions in hampering OC peritoneal propagation.

Hitting two oncogenic machineries in cancer cells: cooperative effects of the multi-kinase inhibitor ponatinib and the BET bromodomain blockers JQ1 or dBET1 on human carcinoma cells.

In recent years, numerous new targeted drugs, including multi-kinase inhibitors and epigenetic modulators have been developed for cancer treatment. Ponatinib blocks a variety of tyrosine kinases including ABL and fibroblast growth factor receptor (FGFR), and the BET bromodomain (BRD) antagonists JQ1 and dBET1 impede MYC oncogene expression. Both drugs have demonstrated substantial anti-cancer efficacy against several hematological malignancies. Solid tumors, on the other hand, although frequently driven by FGFR and/or MYC, are often unresponsive to these drugs. This is due, at least in part, to compensatory feedback-loops in the kinome and transcription network of these tumors, which are activated in response to drug exposure. Therefore, we hypothesized that the combination of the multi-kinase inhibitor ponatinib with transcription modulators such as JQ1 or dBET1 might overcome this therapeutic recalcitrance. Using 3H-thymidine uptake, cell cycle analysis, and caspase-3 or Annexin V labeling, we demonstrate that single drugs induce moderate dose-dependent growth-inhibition and/or apoptosis in colon (HCT116, HT29), breast (MCF-7, SKBR3) and ovarian (A2780, SKOV3) cancer cells. Ponatinib elicited primarily apoptosis, while JQ1 and dBET1 caused G0/G1 cell cycle arrest and very mild cell death. Phospho-FGFR and MYC, major targets of ponatinib and BET inhibitors, were downregulated after treatment with single drugs. Remarkably, ponatinib was found to sensitize cells to BET antagonists by enhancing apoptotic cell death, and this effect was associated with downregulation of MYC. In summary, our data shows that ponatinib sensitizes colon, breast, and ovarian cancer cells to BET bromodomain inhibitors. Further studies are warranted to determine the clinical value of this phenomenon.

Interacting Cancer Machineries: Cell Signaling, Lipid Metabolism, and Epigenetics.

Cancer-specific perturbations of signaling, metabolism, and epigenetics can be a cause and/or consequence of malignant transformation. Evidence indicates that these regulatory systems interact with each other to form highly flexible and robust cybernetic networks that promote malignant growth and confer treatment resistance. Deciphering these plexuses using holistic approaches known from systems biology can be instructive for the future design of novel anticancer strategies. In this review, I discuss novel findings elucidating the multiple molecular interdependence among cancer-specific signaling, cell metabolism, and epigenetics to provide an insightful understanding of how major cancer machineries interact with each other during cancer development and progression, and how this knowledge may be used for future co-targeting strategies.

Estradiol impairs the antiproliferative and proapoptotic effect of Zoledronic acid in hormone sensitive breast cancer cells in vitro.

BACKGROUND: Zoledronic acid (ZA) has antiresorptive effects and protects from bone metastasis in women with early breast cancer. In addition, in postmenopausal women with endocrine responsive breast cancer ZA prolongs DFS. The exact mechanism is still unclear. We have therefore investigated the effect of increasing concentrations of ZA in breast cancer cell lines in the absence or presence of estradiol to mimic the hormonal environment in vitro. MATERIALS AND METHODS: Using assays for cell proliferation (EZ4U, BrdU) and cell death (Annexin/PI), we have analyzed the dose-dependent antiproliferative and pro-apoptotic effects of ZA in two hormone sensitive cell lines (MCF-7 and T47D) and a hormone insensitive, triple negative cell line (MDA-MB-231) in the presence of 0, 1 and 10 nM estradiol. RESULTS: In the absence of estradiol, ZA exerts dose-dependent antiproliferative and pro-apoptotic antitumor effects in both, hormone sensitive (MCF-7, T47D) and -insensitive (MDA-MB-231) breast cancer cell lines (p<0.0001). In the presence of estradiol, the antitumoral effect of ZA was significantly decreased only in the hormone sensitive MCF-7 and T47D cell lines (p = 0.0008 and p = 0.0008, respectively). CONCLUSION: We have demonstrated that estradiol impairs the antiproliferative and proapoptotic effect of ZA in hormone sensitive, but not in hormone insensitive breast cancer cell lines. Our findings provide a possible explanation for the differential effect of ZA on DFS in pre- and postmenopausal patients with hormone sensitive early breast cancer, which has been demonstrated clinically. We further hypothesize that endocrine insensitive tumors such as triple negative breast cancer (TNBC) should benefit from ZA irrespective of their menopausal status.

Long term storage in liquid nitrogen leads to only minor phenotypic and gene expression changes in the mammary carcinoma model cell line BT474.

BACKGROUND/AIM: Cancer cell lines are indispensible surrogate models in cancer research, as they can be used off-the-shelf, expanded to the desired extent, easily modified and exchanged between research groups for affirmation, reproduction or follow-up experiments.As malignant cells are prone to genomic instability, phenotypical changes may occur after certain passages in culture. Thus, cell lines have to be regularly authenticated to ensure data quality. In between experiments these cell lines are often stored in liquid nitrogen for extended time periods.Although freezing of cells is a necessary evil, little research is performed on how long-term storage affects cancer cell lines. Therefore, this study investigated the effects of a 28-year long liquid nitrogen storage period on BT474 cells with regard to phenotypical changes, differences in cell-surface receptor expression as well as cytokine and gene expressional variations. METHODS: Two batches of BT474 cells, one frozen in 1986, the other directly purchased from ATCC were investigated by light microscopy, cell growth analysis, flow cytometry and cytokine as well as whole-transcriptome expression profiling. RESULTS: The cell lines were morphologically indifferent and showed similar growth rates and similar cell-surface receptor expression. Transcriptome analysis revealed significant differences in only 26 of 40,716 investigated RefSeq transcripts with 4 of them being up-regulated and 22 down-regulated. CONCLUSION: This study demonstrates that even after very long periods of storage in liquid nitrogen, cancer cell lines display only minimal changes in their gene expression profiles. However, also such minor changes should be carefully assessed before continuation of experiments, especially if phenotypic alterations can be additionally observed.

Multi-level suppression of receptor-PI3K-mTORC1 by fatty acid synthase inhibitors is crucial for their efficacy against ovarian cancer cells.

Receptor-PI3K-mTORC1 signaling and fatty acid synthase (FASN)-regulated lipid biosynthesis harbor numerous drug targets and are molecularly connected. We hypothesize that unraveling the mechanisms of pathway cross-talk will be useful for designing novel co-targeting strategies for ovarian cancer (OC). The impact of receptor-PI3K-mTORC1 onto FASN is already well-characterized. However, reverse actions-from FASN towards receptor-PI3K-mTORC1-are still elusive. We show that FASN-blockade impairs receptor-PI3K-mTORC1 signaling at multiple levels. Thin-layer chromatography and MALDI-MS/MS reveals that FASN-inhibitors (C75, G28UCM) augment polyunsaturated fatty acids and diminish signaling lipids diacylglycerol (DAG) and phosphatidylinositol 3,4,5-trisphosphate (PIP3) in OC cells (SKOV3, OVCAR-3, A2780, HOC-7). Western blotting and micropatterning demonstrate that FASN-blockers impair phosphorylation/expression of EGF-receptor/ERBB/HER and decrease GRB2-EGF-receptor recruitment leading to PI3K-AKT suppression. FASN-inhibitors activate stress response-genes HIF-1α-REDD1 (RTP801/DIG2/DDIT4) and AMPKα causing mTORC1- and S6-repression. We conclude that FASN-inhibitor-mediated blockade of receptor-PI3K-mTORC1 occurs due to a number of distinct but cooperating processes. Moreover, decrease of PI3K-mTORC1 abolishes cross-repression of MEK-ERK causing ERK activation. Consequently, the MEK-inhibitor selumetinib/AZD6244, in contrast to the PI3K/mTOR-inhibitor dactolisib/NVP-BEZ235, increases growth inhibition when given together with a FASN-blocker. We are the first to provide deep insight on how FASN-inhibition blocks ERBB-PI3K-mTORC1 activity at multiple molecular levels. Moreover, our data encourage therapeutic approaches using FASN-antagonists together with MEK-ERK-inhibitors.

Comparative transcriptome analysis links distinct peritoneal tumor spread types, miliary and non-miliary, with putative origin, tubes and ovaries, in high grade serous ovarian cancer.

High grade serous ovarian cancer (HGSOC) is characterized by extensive local, i.e. peritoneal, tumor spread, manifested in two different clinical presentations, miliary (many millet sized peritoneal implants) and non-miliary (few large exophytically growing peritoneal nodes), and an overall unfavorable outcome. HGSOC is thought to arise from fallopian tube secretory epithelial cells, via so called serous tubal intraepithelial carcinomas (STICs) but an ovarian origin was never ruled out for at least some cases. Comparative transcriptome analyses of isolated tumor cells from fresh HGSOC tissues and (immortalized) ovarian surface epithelial and fallopian tube secretory epithelial cell lines revealed a close relation between putative origin and tumor spread characteristic, i.e. miliary from tubes and non-miliary from ovaries. The latter were characterized by more mesenchymal cell characteristics, more adaptive tumor immune infiltration, and a favorable overall survival. Several molecular sub-classification systems (Crijns' overall survival signature, Yoshihara's subclasses, and a collagen-remodeling signature) seem to already indicate origin. Putative origin alone is a significant independent predictor for HGSOC outcome, validated in independent patient cohorts. Characteristics of both spread types could guide development of new targeted therapeutics, which are urgently needed.

HER Specific TKIs Exert Their Antineoplastic Effects on Breast Cancer Cell Lines through the Involvement of STAT5 and JNK.

BACKGROUND: HER-targeted tyrosine kinase inhibitors (TKIs) have demonstrated pro-apoptotic and antiproliferative effects in vitro and in vivo. The exact pathways through which TKIs exert their antineoplastic effects are, however, still not completely understood. METHODS: Using Milliplex assays, we have investigated the effects of the three panHER-TKIs lapatinib, canertinib and afatinib on signal transduction cascade activation in SKBR3, T47D and Jurkat neoplastic cell lines. The growth-inhibitory effect of blockade of HER and of JNK and STAT5 signaling was measured by proliferation- and apoptosis-assays using formazan dye labeling of viable cells, Western blotting for cleaved PARP-1 and immunolabeling for active caspase 3, respectively. RESULTS: All three HER-TKIs clearly inhibited proliferation and increased apoptosis in HER2 overexpressing SKBR3 cells, while their effect was less pronounced on HER2 moderately expressing T47D cells where they exerted only a weak antiproliferative and essentially no pro-apoptotic effect. Remarkably, phosphorylation/activation of JNK and STAT5A/B were inhibited by HER-TKIs only in the sensitive, but not in the resistant cells. In contrast, phosphorylation/activation of ERK/MAPK, STAT3, CREB, p70 S6 kinase, IkBa, and p38 were equally affected by HER-TKIs in both cell lines. Moreover, we demonstrated that direct pharmacological blockade of JNK and STAT5 abrogates cell growth in both HER-TKI-sensitive as well as -resistant breast cancer cells, respectively. CONCLUSION: We have shown that HER-TKIs exert a HER2 expression-dependent anti-cancer effect in breast cancer cell lines. This involves blockade of JNK and STAT5A/B signaling, which have been found to be required for in vitro growth of these cell lines.

Prominin-1 (CD133, AC133) and dipeptidyl-peptidase IV (CD26) are indicators of infinitive growth in colon cancer cells.

Advanced colorectal cancer is characterized by uncontrolled growth and resistance against anti-cancer agents, including ErbB inhibitors. Recent data suggest that cancer stem cells (CSC) are particularly resistant. These cells may reside within a CD133+ fraction of the malignant cells. Using HCT116 cells we explored the role of CD133 and other CSC markers in drug resistance in colon cancer cells. CD133+ cells outnumbered CD133- cells over time in long-term culture. Both populations displayed the KRAS mutation 38G > A and an almost identical target profile, including EGFR/ErbB1, ErbB2, and ErbB4. Microarray analyses and flow cytometry identified CD26 as additional CSC marker co-expressed on CD133+ cells. However, knock-down of CD133 or CD26 did not affect short-term growth of HCT116 cells, and both cell-populations were equally resistant to various targeted drugs except irreversible ErbB inhibitors, which blocked growth and ERK1/2 phosphorylation in CD133- cells more efficiently than in CD133+ cells. Moreover, the MEK inhibitor AS703026 was found to overcome resistance against ErbB blockers in CD133+ cells. Together, CD133 and CD26 are markers of long-term growth and resistance to ErbB blockers in HCT116 cells, which may be mediated by constitutive ERK activity.

Cancer stem cells in basic science and in translational oncology: can we translate into clinical application?

Since their description and identification in leukemias and solid tumors, cancer stem cells (CSC) have been the subject of intensive research in translational oncology. Indeed, recent advances have led to the identification of CSC markers, CSC targets, and the preclinical and clinical evaluation of the CSC-eradicating (curative) potential of various drugs. However, although diverse CSC markers and targets have been identified, several questions remain, such as the origin and evolution of CSC, mechanisms underlying resistance of CSC against various targeted drugs, and the biochemical basis and function of stroma cell-CSC interactions in the so-called 'stem cell niche.' Additional aspects that have to be taken into account when considering CSC elimination as primary treatment-goal are the genomic plasticity and extensive subclone formation of CSC. Notably, various cell fractions with different combinations of molecular aberrations and varying proliferative potential may display CSC function in a given neoplasm, and the related molecular complexity of the genome in CSC subsets is considered to contribute essentially to disease evolution and acquired drug resistance. In the current article, we discuss new developments in the field of CSC research and whether these new concepts can be exploited in clinical practice in the future.

Correlation of circular RNA abundance with proliferation--exemplified with colorectal and ovarian cancer, idiopathic lung fibrosis, and normal human tissues.

Circular RNAs are a recently (re-)discovered abundant RNA species with presumed function as miRNA sponges, thus part of the competing endogenous RNA network. We analysed the expression of circular and linear RNAs and proliferation in matched normal colon mucosa and tumour tissues. We predicted >1,800 circular RNAs and proved the existence of five randomly chosen examples using RT-qPCR. Interestingly, the ratio of circular to linear RNA isoforms was always lower in tumour compared to normal colon samples and even lower in colorectal cancer cell lines. Furthermore, this ratio correlated negatively with the proliferation index. The correlation of global circular RNA abundance (the circRNA index) and proliferation was validated in a non-cancerous proliferative disease, idiopathic pulmonary fibrosis, ovarian cancer cells compared to cultured normal ovarian epithelial cells, and 13 normal human tissues. We are the first to report a global reduction of circular RNA abundance in colorectal cancer cell lines and cancer compared to normal tissues and discovered a negative correlation of global circular RNA abundance and proliferation. This negative correlation seems to be a general principle in human tissues as validated with three different settings. Finally, we present a simple model how circular RNAs could accumulate in non-proliferating cells.