Breast cancer stem cells (CSCs) are thought to drive recurrence and metastasis. Their identity has been linked to the epithelial to mesenchymal transition (EMT) but remains highly controversial ...since--depending on the cell-line studied--either epithelial (E) or mesenchymal (M) markers, alone or together have been associated with stemness. Using distinct transcript expression signatures characterizing the three different E, M and hybrid E/M cell-types, our data support a novel model that links a mixed EM signature with stemness in 1) individual cells, 2) luminal and basal cell lines, 3) in vivo xenograft mouse models, and 4) in all breast cancer subtypes. In particular, we found that co-expression of E and M signatures was associated with poorest outcome in luminal and basal breast cancer patients as well as with enrichment for stem-like cells in both E and M breast cell-lines. This link between a mixed EM expression signature and stemness was explained by two findings: first, mixed cultures of E and M cells showed increased cooperation in mammosphere formation (indicative of stemness) compared to the more differentiated E and M cell-types. Second, single-cell qPCR analysis revealed that E and M genes could be co-expressed in the same cell. These hybrid E/M cells were generated by both E or M cells and had a combination of several stem-like traits since they displayed increased plasticity, self-renewal, mammosphere formation, and produced ALDH1+ progenies, while more differentiated M cells showed less plasticity and E cells showed less self-renewal. Thus, the hybrid E/M state reflecting stemness and its promotion by E-M cooperation offers a dual biological rationale for the robust association of the mixed EM signature with poor prognosis, independent of cellular origin. Together, our model explains previous paradoxical findings that breast CSCs appear to be M in luminal cell-lines but E in basal breast cancer cell-lines. Our results suggest that targeting E/M heterogeneity by eliminating hybrid E/M cells and cooperation between E and M cell-types could improve breast cancer patient survival independent of breast cancer-subtype.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The involvement of the death adaptor protein FADD and the apoptosis‐initiating caspase‐8 in CD95 and TRAIL death signalling has recently been demonstrated by the analysis of the native death‐inducing ...signalling complex (DISC) that forms upon ligand‐induced receptor cross‐linking. However, the role of caspase‐10, the other death‐effector‐domain‐containing caspase besides caspase‐8, in death receptor signalling has been controversial. Here we show that caspase‐10 is recruited not only to the native TRAIL DISC but also to the native CD95 DISC, and that FADD is necessary for its recruitment to and activation at these two protein complexes. With respect to the function of caspase‐10, we show that it is not required for apoptosis induction. In addition, caspase‐10 can not substitute for caspase‐8, as the defect in apoptosis induction observed in caspase‐8‐deficient cells could not be rescued by overexpression of caspase‐10. Finally, we demonstrate that caspase‐10 is cleaved during CD95‐induced apoptosis of activated T cells. These results show that caspase‐10 activation occurs in primary cells, but that its function differs from that of caspase‐8.
Abstract
Despite significant advances in cancer research the most aggressive breast cancers remain incurable. Better methods are necessary to predict efficacy of therapeutic regimes and are critical ...to improving clinical outcomes. Drug screens using conventional 2D and 3D in vitro tissue culture assays are relatively inexpensive, yet are often insufficient in predicting patient response to drugs. This is likely due to the inability of conventional tissue culture to recapitulate the complex tissue architecture and physiological cues required for cellular interactions. A particularly critical shortcoming of these models is the lack of tumor vasculature within the tissue. Although mouse models remain the gold standard for clinical drug development, they are expensive, of ethical concern, and importantly, do not reliably predict clinical outcome. Thus, promising drug candidates in preclinical trials often fail in expensive clinical studies. In response to these limitations, a number of groups
are beginning to develop microfluidic approaches to studying cancer biology.
Nortis provides microfluidic systems for the in vitro generation of living 3D perfused tissue microenvironments. At the heart of Nortis’ microfluidic chip is a small 25-ul culture chamber in which a lumenally perfused microvessel is formed by casting extracellular matrix around a thin glass fiber. Removal of the fiber creates a single tubular void in line with a fluidic circuit. With this method users can create complex microenvironments with perfusable vascular networks. The chip design also facilitates high-quality brightfield and fluorescence imaging, histology, and collection of fluids for downstream analysis.
Using this technology, we developed a vascularized breast cancer model. Vascular networks were created by tissue-engineering perfusable ‘parent’ endothelial vessels within a collagen matrix. The quiescent cell tubes were cultured for up to 60 days and could be induced to sprout directionally in response to growth factors. The sprouting vessels responded to flow, underwent anastomosis, and eventually established perfusable vascular networks. When co-cultured with tumor cells the sprouts invaded the tumor tissue yielding vascularized tumor microenvironments. The perfusable vasculature recapitulates a key feature of the tumor microenvironment, enables preclinical testing of drugs that target both tumor cells and vasculature, and allows controlled in vivo-like delivery of test agents. Using this model we tested different antiangiogenic therapeutics and were able to successfully demonstrate anti-angiogenic effects in our chips and with this, have a platform available to routinely test antiangiogenic and thus, novel anti-cancer drugs in a human in vitro tumor microenvironment.
Citation Format: Henning Mann, Anne Grosse-Wilde. A tissue-engineered vascularized tumor microenvironment for preclinical testing of anticancer therapeutics. abstract. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-045.
Many cancers harbor oncogenic mutations of KRAS. Effectors mediating cancer progression, invasion, and metastasis in KRAS-mutated cancers are only incompletely understood. Here we identify cancer ...cell-expressed murine TRAIL-R, whose main function ascribed so far has been the induction of apoptosis as a crucial mediator of KRAS-driven cancer progression, invasion, and metastasis and in vivo Rac-1 activation. Cancer cell-restricted genetic ablation of murine TRAIL-R in autochthonous KRAS-driven models of non-small-cell lung cancer (NSCLC) and pancreatic ductal adenocarcinoma (PDAC) reduces tumor growth, blunts metastasis, and prolongs survival by inhibiting cancer cell-autonomous migration, proliferation, and invasion. Consistent with this, high TRAIL-R2 expression correlates with invasion of human PDAC into lymph vessels and with shortened metastasis-free survival of KRAS-mutated colorectal cancer patients.
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•mTRAIL-R promotes KRAS-driven lung and pancreatic cancer growth and metastasis•Human TRAIL-R2 promotes tumor growth, migration, invasion, and metastasis•Endogenous mTRAIL-R constitutively activates Rac1 in vivo in tumors•TRAIL-R2 expression positively correlates with the onset of metastasis in patients
von Karstedt et al. show that mouse TRAIL-R and human TRAIL-R2, but not TRAIL-R1, are important for the progression, invasion, and metastasis of KRAS-mutant tumors through the regulation of Rac-1.
Abstract
Epithelial cells have an absolute requirement for surface adhesion for survival, and loss of adhesion activates apoptosis, a process named anoikis. During metastasis, cancerous epithelial ...cells are able to survive anoikis after they have spread from the primary site of the tumor. The mechanisms underlying anoikis survival are not well understood, but include cell lineage switching from epithelial to mesenchymal behavior, implying a role for stem cells. Several studies have shown that the mesenchymal population of tumorigenic breast cells contains stem cells, yet it appears that only a small yet unknown subpopulation of the mesenchymal cells is able to survive the strong selection pressure to survive anoikis in suspension and proliferate to form mammospheres. These studies have been complicated by the fact that heterogeneous cell lines consisting of epithelial and mesenchymal cells were analyzed, obscuring the role of small but possibly relevant subpopulations. Here, we were employing both single cell-derived homogeneous mesenchymal cell clones as well as novel microfluidics-based high throughput qRT-PCR analysis to unravel gene expression in epithelial and/or mesenchymal breast cancer cells during survival of anoikis. We show evidence that during culture of transformed mammary epithelial cells (HMLER) in suspension certain subpopulations disappear and then get repopulated by the surviving, proliferating and differentiating subpopulation in suspension. During this period in suspension critical for repopulation we find coordinate expression of both epithelial and mesenchymal markers, rapid upregulation of pluripotency factors (such as OCT4 and Nanog), as well as of several other genes that have previously been described to indicate poor prognosis for breast cancer survival. Those poor prognosis markers we also found elevated in replated and passaged anoikis-resistant HMLER cells as well as in HMLER-derived mesenchymal clones. By contrast expression of pluripotency factors was only restricted to the suspended state. In conclusion, here we show that in suspended breast epithelial cells there exists an epithelial/mesenchymal hybrid state likely to give rise to differentiated progenies, being able to adapt to the diverse challenging environments such as at secondary metastatic sites in cancer patients.
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 LB-302. doi:1538-7445.AM2012-LB-302
Transforming growth factor-β (TGF-β) has been shown to induce apoptotic cell death in normal and transformed hepatocytes.
We recently identified tumor necrosis factor-related apoptosis-inducing ...ligand (TRAIL) as an important mediator of TGF-β–induced
apoptosis in hepatoma cells. In this study, we have further explored the mechanism by which TGF-β up-regulates TRAIL expression.
The 5′-flanking region of the TRAIL gene was isolated and characterized. Deletion mutants of the 5′-untranslated region of the TRAIL gene revealed a region comprising nucleotides −1950 to −1100 responsible for TRAIL induction following treatment with TGF-β.
Within this region, we have identified an activator protein-1 (AP-1) site indispensable for TGF-β–mediated induction of TRAIL.
Activation of this AP-1 site is mediated by a JunD·FosB heterodimer. Expression of DNSmad4, DNJunD, or DNFosB significantly
impairs TGF-β–mediated activation of the TRAIL promoter. Furthermore, with tRNA interference targeting Smad4, junD, FosB,
we could abolish TRAIL expression and, subsequently, TGF-β–induced TRAIL-mediated apoptosis in hepatoma cells. Our results
reveal a new AP-1 site within the TRAIL promoter functionally involved in TGF-β–induced TRAIL expression and apoptosis in
hepatomas and thus provide evidence for the underlying mechanism by which TGF-β might regulate cell death in liver cancer.
(Mol Cancer Res 2008;6(7):1169–77)
TRAIL is a promising anticancer agent due to its ability to selectively induce apoptosis in established tumor cell lines but not nontransformed cells. Herein, we demonstrate a role for the ...apoptosis-inducing TRAIL receptor (TRAIL-R) as a metastasis suppressor. Although mouse models employing tumor transplantation have shown that TRAIL can reduce tumor growth, autochthonous tumor models have generated conflicting results with respect to the physiological role of the TRAIL system during tumorigenesis. We used a multistage model of squamous cell carcinoma to examine the role of TRAIL-R throughout all steps of tumor development. DMBA/TPA-treated TRAIL-R-deficient mice showed neither an increase in number or growth rate of benign papillomas nor an increase in the rate of progression to squamous cell carcinoma. However, metastasis to lymph nodes was significantly enhanced, indicating a role for TRAIL-R specifically in the suppression of metastasis. We also found that adherent TRAIL-R-expressing skin carcinoma cells were TRAIL resistant in vitro but were sensitized to TRAIL upon detachment by inactivation of the ERK signaling pathway. As detachment from the primary tumor is an obligatory step in metastasis, this provides a possible mechanism by which TRAIL-R could inhibit metastasis. Hence, treatment of cancer patients with agonists of the apoptosis-inducing receptors for TRAIL may prove useful in reducing the incidence of metastasis.
Abstract
Targeting breast cancer stem cells promises to eliminate the root of metastasis and recurrence and thus to eradicate the ultimate cause of cancer-related death of patients. However, the ...identity of epithelial cancer stem-like is still elusive; cancer stem-like and metastasis initiating cells have been described to be either epithelial (E) or mesenchymal (M), and thus their identity appears to critically depend on the context of the analyzed cell line.
Here, we have studied the heterogeneous HMLER cell line to identify the relationships between epithelial and mesenchymal cells while they are differentiated and selected under suspension culture conditions. We find that CD24+/CD44- epithelial cells and CD44+/CD24- mesenchymal cells synergize in causing the proliferation of both sub-populations, increasing mammosphere production, and causing the emergence of aggressive motile cells when replated. We observed that when grown in suspension, isolated epithelial as well as isolated mesenchymal cell populations converged towards a hybrid E/M state characterized by coexpression of genes of both lineages, from population down to the single cell level. For epithelial populations convergence at the hybrid state during growth in suspension was thus achieved by proliferation of the most mesenchymal cells resembling an epithelial to mesenchymal transition at the population level. For mesenchymal populations, the hybrid state was reached by the reverse, MET, involving proliferation of the most epithelial cells in a context dependent manner.We use these findings to resolve which genes are predictive of poor outcome in primary human breast tumors. Here we show for the first time that high expression of mesenchymal genes in luminal B/epithelial tumors predicts poor outcome of patients, whereas expression of epithelial genes in basal/mesenchymal tumors predicts poor outcome and thus increased metastasis. Therefore, functional synergy between E and M cells can explain how the tumor context determines whether a minority of mesenchymal or epithelial cells can serve as metastatic stem cells.
Citation Format: Anne Grosse-Wilde, Aymeric d'Herouel, Rolf Kuestner, Gokhan Ertaylan, Alexander Skupin, Sui Huang, Adrian Ozinsky. Synergy between epithelial and mesenchymal cells in breast cancer: from mammospheres to predicting patient outcomes. abstract. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1918. doi:10.1158/1538-7445.AM2014-1918
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising candidate for cancer therapy, as it can induce apoptosis specifically in tumor cells but not in normal cells. Although ...earlier mouse tumor studies revealed a strong tissue dependency of TRAIL and its death receptor in suppressing primary tumorigenesis or experimental metastases, we recently found that TRAIL-R inhibits lymph node metastases without affecting primary tumor formation in a mouse model of multistage skin tumorigenesis. This finding uncouples the role of TRAIL in primary tumorigenesis from metastasis formation, likely by sensitization of previously TRAIL-resistant tumor cells upon detachment, an early step required for metastasis formation. Therefore, TRAIL-R is a novel metastasis suppressor, suggesting that TRAIL-related tumor therapy might be most effective in primary tumors and early metastatic cancers, before selection for TRAIL resistance occurs.
According to the sequential metastasis model, aggressive mesenchymal (M) metastasis-initiating cells (MICs) are generated by an epithelial-mesenchymal transition (EMT) which eventually is reversed by ...a mesenchymal-epithelial transition (MET) and outgrowth of life-threatening epithelial (E) macrometastases. Paradoxically, in breast cancer M signatures are linked with more favorable outcomes than E signatures, and M cells are often dispensable for metastasis in mouse models. Here we present evidence at the cellular and patient level for the cooperation metastasis model, according to which E cells are MICs, while M cells merely support E cell persistence through cooperation. We tracked the fates of co-cultured E and M clones and of fluorescent CDH1-promoter-driven cell lines reporting the E state derived from basal breast cancer HMLER cells. Cells were placed in suspension state and allowed to reattach and select an EMT cell fate. Flow cytometry, single cell and bulk gene expression analyses revealed that only pre-existing E cells generated E cells, mixed E/M populations, or stem-like hybrid E/M cells after suspension and that complete EMT manifest in M clones and CDH1-negative reporter cells resulted in loss of cell plasticity, suggesting full transdifferentiation. Mechanistically, E-M coculture experiments supported the persistence of pre-existing E cells where M cells inhibited EMT of E cells in a mutual cooperation via direct cell-cell contact. Consistently, M signatures were associated with more favorable patient outcomes compared to E signatures in breast cancer, specifically in basal breast cancer patients. These findings suggest a potential benefit of complete EMT for basal breast cancer patients.
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