Carcinoma cells residing in an intermediate phenotypic state along the epithelial–mesenchymal (E–M) spectrum are associated with malignant phenotypes, such as invasiveness, tumor-initiating ability, ...and metastatic dissemination. Using the recently described CD104⁺/CD44hi antigen marker combination, we isolated highly tumorigenic breast cancer cells residing stably—both in vitro and in vivo—in an intermediate phenotypic state and coexpressing both epithelial (E) and mesenchymal (M) markers. We demonstrate that tumorigenicity depends on individual cells residing in this E/M hybrid state and cannot be phenocopied by mixing two cell populations that reside stably at the two ends of the spectrum, i.e., in the E and in the M state. Hence, residence in a specific intermediate state along the E–M spectrum rather than phenotypic plasticity appears critical to the expression of tumor-initiating capacity. Acquisition of this E/M hybrid state is facilitated by the differential expression of EMT-inducing transcription factors (EMT-TFs) and is accompanied by the expression of adult stem cell programs, notably, active canonical Wnt signaling. Furthermore, transition from the highly tumorigenic E/M state to a fully mesenchymal phenotype, achieved by constitutive ectopic expression of Zeb1, is sufficient to drive cells out of the E/M hybrid state into a highly mesenchymal state, which is accompanied by a substantial loss of tumorigenicity and a switch from canonical to noncanonical Wnt signaling. Identifying the gatekeepers of the various phenotypic states arrayed along the E–M spectrum is likely to prove useful in developing therapeutic approaches that operate by shifting cancer cells between distinct states along this spectrum.
The epithelial-to-mesenchymal transition (EMT) is a cell biological program that confers mesenchymal traits on carcinoma cells and drives their metastatic dissemination. It is unclear, however, ...whether the activation of EMT in carcinoma cells can change their susceptibility to immune attack. We demonstrate here that mammary tumor cells arising from more epithelial carcinoma cell lines expressed high levels of MHC-I, low levels of PD-L1, and contained within their stroma CD8
T cells and M1 (antitumor) macrophages. In contrast, tumors arising from more mesenchymal carcinoma cell lines exhibiting EMT markers expressed low levels of MHC-I, high levels of PD-L1, and contained within their stroma regulatory T cells, M2 (protumor) macrophages, and exhausted CD8
T cells. Moreover, the more mesenchymal carcinoma cells within a tumor retained the ability to protect their more epithelial counterparts from immune attack. Finally, epithelial tumors were more susceptible to elimination by immunotherapy than corresponding mesenchymal tumors. Our results identify immune cells and immunomodulatory markers that can be potentially targeted to enhance the susceptibility of immunosuppressive tumors to various therapeutic regimens.
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Ferroptosis-an iron-dependent, non-apoptotic cell death process-is involved in various degenerative diseases and represents a targetable susceptibility in certain cancers
. The ...ferroptosis-susceptible cell state can either pre-exist in cells that arise from certain lineages or be acquired during cell-state transitions
. However, precisely how susceptibility to ferroptosis is dynamically regulated remains poorly understood. Here we use genome-wide CRISPR-Cas9 suppressor screens to identify the oxidative organelles peroxisomes as critical contributors to ferroptosis sensitivity in human renal and ovarian carcinoma cells. Using lipidomic profiling we show that peroxisomes contribute to ferroptosis by synthesizing polyunsaturated ether phospholipids (PUFA-ePLs), which act as substrates for lipid peroxidation that, in turn, results in the induction of ferroptosis. Carcinoma cells that are initially sensitive to ferroptosis can switch to a ferroptosis-resistant state in vivo in mice, which is associated with extensive downregulation of PUFA-ePLs. We further find that the pro-ferroptotic role of PUFA-ePLs can be extended beyond neoplastic cells to other cell types, including neurons and cardiomyocytes. Together, our work reveals roles for the peroxisome-ether-phospholipid axis in driving susceptibility to and evasion from ferroptosis, highlights PUFA-ePL as a distinct functional lipid class that is dynamically regulated during cell-state transitions, and suggests multiple regulatory nodes for therapeutic interventions in diseases that involve ferroptosis.
The epithelial-to-mesenchymal transition enables carcinoma cells to acquire malignancy-associated traits and the properties of tumor-initiating cells (TICs). TICs have emerged in recent years as ...important targets for cancer therapy, owing to their ability to drive clinical relapse and enable metastasis. Here, we propose a strategy to eliminate mesenchymal TICs by inducing their conversion to more epithelial counterparts that have lost tumor-initiating ability. We report that increases in intracellular levels of the second messenger, adenosine 3',5'-monophosphate, and the subsequent activation of protein kinase A (PKA) induce a mesenchymal-to-epithelial transition (MET) in mesenchymal human mammary epithelial cells. PKA activation triggers epigenetic reprogramming of TICs by the histone demethylase PHF2, which promotes their differentiation and loss of tumor-initiating ability. This study provides proof-of-principle for inducing an MET as differentiation therapy for TICs and uncovers a role for PKA in enforcing and maintaining the epithelial state.
Neoplastic cells within individual carcinomas often exhibit considerable phenotypic heterogeneity in their epithelial versus mesenchymal-like cell states. Because carcinoma cells with mesenchymal ...features are often more resistant to therapy and may serve as a source of relapse, we sought to determine whether such cells could be further stratified into functionally distinct subtypes. Indeed, we find that a basal epithelial marker, integrin-β4 (ITGB4), can be used to enable stratification of mesenchymal-like triple-negative breast cancer (TNBC) cells that differ from one another in their relative tumorigenic abilities. Notably, we demonstrate that ITGB4⁺ cancer stem cell (CSC)-enriched mesenchymal cells reside in an intermediate epithelial/mesenchymal phenotypic state. Among patients with TNBC who received chemotherapy, elevated ITGB4 expression was associated with a worse 5-year probability of relapse-free survival. Mechanistically,we find that the ZEB1 (zinc finger E-box binding homeobox 1) transcription factor activity in highly mesenchymal SUM159 TNBC cells can repress expression of the epithelial transcription factor TAp63α (tumor protein 63 isoform 1), a protein that promotes ITGB4 expression. In addition, we demonstrate that ZEB1 and ITGB4 are important in modulating the histopathological phenotypes of tumors derived from mesenchymal TNBC cells. Hence, mesenchymal carcinoma cell populations are internally heterogeneous, and ITGB4 is a mechanistically driven prognostic biomarker that can be used to identify the more aggressive subtypes of mesenchymal carcinoma cells in TNBC. The ability to rapidly isolate and mechanistically interrogate the CSC-enriched, partially mesenchymal carcinoma cells should further enable identification of novel therapeutic opportunities to improve the prognosis for high-risk patients with TNBC.
Patients undergoing surgical resection of primary breast tumors confront a risk for metastatic recurrence that peaks sharply 12 to 18 months after surgery. The cause of early metastatic relapse in ...breast cancer has long been debated, with many ascribing these relapses to the natural progression of the disease. Others have proposed that some aspect of surgical tumor resection triggers the outgrowth of otherwise-dormant metastases, leading to the synchronous pattern of relapse. Clinical data cannot distinguish between these hypotheses, and previous experimental approaches have not provided clear answers. Such uncertainty hinders the development and application of therapeutic approaches that could potentially reduce early metastatic relapse. We describe an experimental model system that definitively links surgery and the subsequent wound-healing response to the outgrowth of tumor cells at distant anatomical sites. Specifically, we find that the systemic inflammatory response induced after surgery promotes the emergence of tumors whose growth was otherwise restricted by a tumor-specific T cell response. Furthermore, we demonstrate that perioperative anti-inflammatory treatment markedly reduces tumor outgrowth in this model, suggesting that similar approaches might substantially reduce early metastatic recurrence in breast cancer patients.
The recent discovery that normal and neoplastic epithelial cells re-enter the stem cell state raised the intriguing possibility that the aggressiveness of carcinomas derives not from their existing ...content of cancer stem cells (CSCs) but from their proclivity to generate new CSCs from non-CSC populations. Here, we demonstrate that non-CSCs of human basal breast cancers are plastic cell populations that readily switch from a non-CSC to CSC state. The observed cell plasticity is dependent on ZEB1, a key regulator of the epithelial-mesenchymal transition. We find that plastic non-CSCs maintain the ZEB1 promoter in a bivalent chromatin configuration, enabling them to respond readily to microenvironmental signals, such as TGFβ. In response, the ZEB1 promoter converts from a bivalent to active chromatin configuration, ZEB1 transcription increases, and non-CSCs subsequently enter the CSC state. Our findings support a dynamic model in which interconversions between low and high tumorigenic states occur frequently, thereby increasing tumorigenic and malignant potential.
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•Basal breast cancer non-CSCs are plastic populations that can generate CSCs de novo•ZEB1 drives non-CSC to CSC plasticity•Basal non-CSCs maintain ZEB1 in poised (bivalent) chromatin configuration•Poised ZEB1 promoter more readily responds to the microenvironment
Nonaggressive cells in basal, but not lumenal, breast cancers exist in a poised state, ready to switch to an aggressive cancer stem cell state in response to appropriate microenvironmental stimuli, a plasticity that may promote tumorigenicity in basal compared to lumenal breast cancers.
The epithelial-to-mesenchymal transition, which conveys epithelial (E) carcinoma cells to quasi-mesenchymal (qM) states, enables them to metastasize and acquire resistance to certain treatments. ...Murine tumors composed of qM mammary carcinoma cells assemble an immunosuppressive tumor microenvironment (TME) and develop resistance to anti-CTLA4 immune-checkpoint blockade (ICB) therapy, unlike their E counterparts. Importantly, minority populations of qM cells within a tumor can cross-protect their more E neighbors from immune attack. The underlying mechanisms of immunosuppression and cross-protection have been unclear. We demonstrate that abrogation of qM carcinoma cell-derived factors (CD73, CSF1, or SPP1) prevents the assembly of an immunosuppressive TME and sensitizes otherwise refractory qM tumors partially or completely to anti-CTLA4 ICB. Most strikingly, mixed tumors in which minority populations of carcinoma cells no longer express CD73 are now sensitized to anti-CTLA4 ICB. Finally, loss of CD73 also enhances the efficacy of anti-CTLA4 ICB during the process of metastatic colonization. SIGNIFICANCE: Minority populations of qM carcinoma cells, which likely reside in human breast carcinomas, can cross-protect their E neighbors from immune attack. Understanding the mechanisms by which qM carcinoma cells resist antitumor immune attack can help identify signaling channels that can be interrupted to potentiate the efficacy of checkpoint blockade immunotherapies.
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