Isthmus progenitor cells contribute to homeostatic cellular turnover and support regeneration following intestinal injury.

The currently accepted intestinal epithelial cell organization model proposes that Lgr5+ crypt-base columnar (CBC) cells represent the sole intestinal stem cell (ISC) compartment. However, previous studies have indicated that Lgr5+ cells are dispensable for intestinal regeneration, leading to two major hypotheses: one favoring the presence of a quiescent reserve ISC and the other calling for differentiated cell plasticity. To investigate these possibilities, we studied crypt epithelial cells in an unbiased fashion via high-resolution single-cell profiling. These studies, combined with in vivo lineage tracing, show that Lgr5 is not a specific ISC marker and that stemness potential exists beyond the crypt base and resides in the isthmus region, where undifferentiated cells participate in intestinal homeostasis and regeneration following irradiation (IR) injury. Our results provide an alternative model of intestinal epithelial cell organization, suggesting that stemness potential is not restricted to CBC cells, and neither de-differentiation nor reserve ISC are drivers of intestinal regeneration.

T Cell Fate at the Single-Cell Level.

T cell responses display two key characteristics. First, a small population of epitope-specific naive T cells expands by several orders of magnitude. Second, the T cells within this proliferating population take on diverse functional and phenotypic properties that determine their ability to exert effector functions and contribute to T cell memory. Recent technological advances in lineage tracing allow us for the first time to study these processes in vivo at single-cell resolution. Here, we summarize resulting data demonstrating that although epitope-specific T cell responses are reproducibly similar at the population level, expansion potential and diversification patterns of the offspring derived from individual T cells are highly variable during both primary and recall immune responses. In spite of this stochastic response variation, individual memory T cells can serve as adult stem cells that provide robust regeneration of an epitope-specific tissue through population averaging. We discuss the relevance of these findings for T cell memory formation and clinical immunotherapy.

The bone microenvironment invigorates metastatic seeds for further dissemination.

Metastasis has been considered as the terminal step of tumor progression. However, recent genomic studies suggest that many metastases are initiated by further spread of other metastases. Nevertheless, the corresponding pre-clinical models are lacking, and underlying mechanisms are elusive. Using several approaches, including parabiosis and an evolving barcode system, we demonstrated that the bone microenvironment facilitates breast and prostate cancer cells to further metastasize and establish multi-organ secondary metastases. We uncovered that this metastasis-promoting effect is driven by epigenetic reprogramming that confers stem cell-like properties on cancer cells disseminated from bone lesions. Furthermore, we discovered that enhanced EZH2 activity mediates the increased stemness and metastasis capacity. The same findings also apply to single cell-derived populations, indicating mechanisms distinct from clonal selection. Taken together, our work revealed an unappreciated role of the bone microenvironment in metastasis evolution and elucidated an epigenomic reprogramming process driving terminal-stage, multi-organ metastases.

Circulating Tumor Cell Clustering Shapes DNA Methylation to Enable Metastasis Seeding.

The ability of circulating tumor cells (CTCs) to form clusters has been linked to increased metastatic potential. Yet biological features and vulnerabilities of CTC clusters remain largely unknown. Here, we profile the DNA methylation landscape of single CTCs and CTC clusters from breast cancer patients and mouse models on a genome-wide scale. We find that binding sites for stemness- and proliferation-associated transcription factors are specifically hypomethylated in CTC clusters, including binding sites for OCT4, NANOG, SOX2, and SIN3A, paralleling embryonic stem cell biology. Among 2,486 FDA-approved compounds, we identify Na+/K+ ATPase inhibitors that enable the dissociation of CTC clusters into single cells, leading to DNA methylation remodeling at critical sites and metastasis suppression. Thus, our results link CTC clustering to specific changes in DNA methylation that promote stemness and metastasis and point to cluster-targeting compounds to suppress the spread of cancer.

Machine Learning Identifies Stemness Features Associated with Oncogenic Dedifferentiation.

Cancer progression involves the gradual loss of a differentiated phenotype and acquisition of progenitor and stem-cell-like features. Here, we provide novel stemness indices for assessing the degree of oncogenic dedifferentiation. We used an innovative one-class logistic regression (OCLR) machine-learning algorithm to extract transcriptomic and epigenetic feature sets derived from non-transformed pluripotent stem cells and their differentiated progeny. Using OCLR, we were able to identify previously undiscovered biological mechanisms associated with the dedifferentiated oncogenic state. Analyses of the tumor microenvironment revealed unanticipated correlation of cancer stemness with immune checkpoint expression and infiltrating immune cells. We found that the dedifferentiated oncogenic phenotype was generally most prominent in metastatic tumors. Application of our stemness indices to single-cell data revealed patterns of intra-tumor molecular heterogeneity. Finally, the indices allowed for the identification of novel targets and possible targeted therapies aimed at tumor differentiation.

EMT: 2016.

The significant parallels between cell plasticity during embryonic development and carcinoma progression have helped us understand the importance of the epithelial-mesenchymal transition (EMT) in human disease. Our expanding knowledge of EMT has led to a clarification of the EMT program as a set of multiple and dynamic transitional states between the epithelial and mesenchymal phenotypes, as opposed to a process involving a single binary decision. EMT and its intermediate states have recently been identified as crucial drivers of organ fibrosis and tumor progression, although there is some need for caution when interpreting its contribution to metastatic colonization. Here, we discuss the current state-of-the-art and latest findings regarding the concept of cellular plasticity and heterogeneity in EMT. We raise some of the questions pending and identify the challenges faced in this fast-moving field.

Central memory CD8+ T cells derive from stem-like Tcf7hi effector cells in the absence of cytotoxic differentiation.

Central memory CD8+ T cells (Tcm) control systemic secondary infections and can protect from chronic infection and cancer as a result of their stem-cell-like capacity to expand, differentiate, and self-renew. Central memory is generally thought to emerge following pathogen clearance and to form based on the de-differentiation of cytolytic effector cells. Here, we uncovered rare effector-phase CD8+ T cells expressing high amounts of the transcription factor Tcf7 (Tcf1) that showed no evidence of prior cytolytic differentiation and that displayed key hallmarks of Tcm cells. These effector-phase Tcf7hi cells quantitatively yielded Tcm cells based on lineage tracing. Mechanistically, Tcf1 counteracted the differentiation of Tcf7hi cells and sustained the expression of conserved adult stem-cell genes that were critical for CD8+ T cell stemness. The discovery of stem-cell-like CD8+ T cells during the effector response to acute infection provides an opportunity to optimize Tcm cell formation by vaccination.

USP21 deubiquitinase promotes pancreas cancer cell stemness via Wnt pathway activation.

The ubiquitin-specific protease (USP) family is the largest group of cysteine proteases. Cancer genomic analysis identified frequent amplification of USP21 (22%) in human pancreatic ductal adenocarcinoma (PDAC). USP21 overexpression correlates with human PDAC progression, and enforced expression of USP21 accelerates murine PDAC tumor growth and drives PanIN to PDAC progression in immortalized human pancreatic ductal cells. Conversely, depletion of USP21 impairs PDAC tumor growth. Mechanistically, USP21 deubiquitinates and stabilizes the TCF/LEF transcription factor TCF7, which promotes cancer cell stemness. Our work identifies and validates USP21 as a PDAC oncogene, providing a potential druggable target for this intractable disease.

CRIP1 suppresses BBOX1-mediated carnitine metabolism to promote stemness in hepatocellular carcinoma.

Carnitine metabolism is thought to be negatively correlated with the progression of hepatocellular carcinoma (HCC) and the specific molecular mechanism is yet to be fully elucidated. Here, we report that little characterized cysteine-rich protein 1 (CRIP1) is upregulated in HCC and associated with poor prognosis. Moreover, CRIP1 promoted HCC cancer stem-like properties by downregulating carnitine energy metabolism. Mechanistically, CRIP1 interacted with BBOX1 and the E3 ligase STUB1, promoting BBOX1 ubiquitination and proteasomal degradation, and leading to the downregulation of carnitine. BBOX1 ubiquitination at lysine 240 is required for CRIP1-mediated control of carnitine metabolism and cancer stem-like properties. Further, our data showed that acetylcarnitine downregulation in CRIP1-overexpressing cells decreased beta-catenin acetylation and promoted nuclear accumulation of beta-catenin, thus facilitating cancer stem-like properties. Clinically, patients with higher CRIP1 protein levels had lower BBOX1 levels but higher nuclear beta-catenin levels in HCC tissues. Together, our findings identify CRIP1 as novel upstream control factor for carnitine metabolism and cancer stem-like properties, suggesting targeting of the CRIP1/BBOX1/ß-catenin axis as a promising strategy for HCC treatment.

Hox genes are crucial regulators of periosteal stem cell identity.

Periosteal stem and progenitor cells (PSPCs) are major contributors to bone maintenance and repair. Deciphering the molecular mechanisms that regulate their function is crucial for the successful generation and application of future therapeutics. Here, we pinpoint Hox transcription factors as necessary and sufficient for periosteal stem cell function. Hox genes are transcriptionally enriched in periosteal stem cells and their overexpression in more committed progenitors drives reprogramming to a naïve, self-renewing stem cell-like state. Crucially, individual Hox family members are expressed in a location-specific manner and their stem cell-promoting activity is only observed when the Hox gene is matched to the anatomical origin of the PSPC, demonstrating a role for the embryonic Hox code in adult stem cells. Finally, we demonstrate that Hoxa10 overexpression partially restores the age-related decline in fracture repair. Together, our data highlight the importance of Hox genes as key regulators of PSPC identity in skeletal homeostasis and repair.

Extended lifespan and improved genome stability in HepaRG-derived cell lines through reprogramming by high-density stress.

The characteristics and fate of cancer cells partly depend on their environmental stiffness, i.e., the local mechanical cues they face. HepaRG progenitors are liver carcinoma cells exhibiting transdifferentiation properties; however, the underlying mechanisms remain unknown. To evaluate the impact of external physical forces mimicking the tumor microenvironment, we seeded them at very high density for 20 h, keeping the cells round and unanchored to the substrate. Applied without corticoids, spatial confinement due to very high density induced reprogramming of HepaRG cells into stable replicative stem-like cells after replating at normal density. Redifferentiation of these stem-like cells into cells very similar to the original HepaRG cells was then achieved using the same stress but in the presence of corticoids. This demonstrates that the cells retained the memory required to run the complete hepatic differentiation program, after bypassing the Hayflick limit twice. We show that physical stress improved chromosome quality and genomic stability, through greater efficiency of DNA repair and restoration of telomerase activity, thus enabling cells to escape progression to a more aggressive cancer state. We also show the primary importance of high-density seeding, possibly triggering compressive stress, in these processes, rather than that of cell roundness or intracellular tensional signals. The HepaRG-derived lines established here considerably extend the lifespan and availability of this surrogate cell system for mature human hepatocytes. External physical stress is a promising way to create a variety of cell lines, and it paves the way for the development of strategies to improve cancer prognosis.

The PLOD2/succinate axis regulates the epithelial-mesenchymal plasticity and cancer cell stemness.

Aberrant accumulation of succinate has been detected in many cancers. However, the cellular function and regulation of succinate in cancer progression is not completely understood. Using stable isotope-resolved metabolomics analysis, we showed that the epithelial mesenchymal transition (EMT) was associated with profound changes in metabolites, including elevation of cytoplasmic succinate levels. The treatment with cell-permeable succinate induced mesenchymal phenotypes in mammary epithelial cells and enhanced cancer cell stemness. Chromatin immunoprecipitation and sequence analysis showed that elevated cytoplasmic succinate levels were sufficient to reduce global 5-hydroxymethylcytosinene (5hmC) accumulation and induce transcriptional repression of EMT-related genes. We showed that expression of procollagen-lysine,2-oxoglutarate 5-dioxygenase 2 (PLOD2) was associated with elevation of cytoplasmic succinate during the EMT process. Silencing of PLOD2 expression in breast cancer cells reduced succinate levels and inhibited cancer cell mesenchymal phenotypes and stemness, which was accompanied by elevated 5hmC levels in chromatin. Importantly, exogenous succinate rescued cancer cell stemness and 5hmC levels in PLOD2-silenced cells, suggesting that PLOD2 promotes cancer progression at least partially through succinate. These results reveal the previously unidentified function of succinate in enhancing cancer cell plasticity and stemness.

Multilayered gene control drives timely exit from the stem cell state in uncommitted progenitors during Drosophila asymmetric neural stem cell division.

Self-renewal genes maintain stem cells in an undifferentiated state by preventing the commitment to differentiate. Robust inactivation of self-renewal gene activity following asymmetric stem cell division allows uncommitted stem cell progeny to exit from an undifferentiated state and initiate the commitment to differentiate. Nonetheless, how self-renewal gene activity at mRNA and protein levels becomes synchronously terminated in uncommitted stem cell progeny is unclear. We demonstrate that a multilayered gene regulation system terminates self-renewal gene activity at all levels in uncommitted stem cell progeny in the fly neural stem cell lineage. We found that the RNA-binding protein Brain tumor (Brat) targets the transcripts of a self-renewal gene, deadpan (dpn), for decay by recruiting the deadenylation machinery to the 3' untranslated region (UTR). Furthermore, we identified a nuclear protein, Insensible, that complements Cullin-mediated proteolysis to robustly inactivate Dpn activity by limiting the level of active Dpn through protein sequestration. The synergy between post-transcriptional and transcriptional control of self-renewal genes drives timely exit from the stem cell state in uncommitted progenitors. Our proposed multilayered gene regulation system could be broadly applicable to the control of exit from stemness in all stem cell lineages.

A pre-B acute lymphoblastic leukemia cell line model reveals the mechanism of thalidomide therapy-related B-cell leukemogenesis.

Lenalidomide, a thalidomide derivative, is prescribed as maintenance therapy for multiple myeloma (MM). Patients with MM receiving lenalidomide were found to develop a distinct therapy-related B cell acute lymphoblastic leukemia (B-ALL). However, the molecular mechanism by which lenalidomide drives B-ALL is unknown. We show that thalidomide treatment of B cell lines increased CD34 expression and fibronectin adhesion. This resembled the effects of Ikzf1 loss of function mutations in B-ALL. IKZF1 is a transcription factor that can act as both a transcriptional activator and a repressor depending upon the target loci. In our experiments, thalidomide-induced degradation of IKZF1 increased the expression of its transcriptional repression targets Itga5 and CD34 explaining the increased adhesion and stemness. Strikingly, withdrawal of thalidomide lead to the mis-localization of IKZF1 to the cytoplasm. Moreover, chromatin immunoprecipitation data showed a long-term effect of thalidomide treatment on IKZF1 target loci. This included decreased chromatin occupancy at early B cell factor 1 (EBF1) and Spi1 (PU.1). Consequently, B-cell lineage specifying transcription factors including Pax5, Spi1 and EBF1 were downregulated even after 7 days of thalidomide withdrawal. Our study thus provides a molecular mechanism of thalidomide-induced B-ALL whereby thalidomide alters the chromatin occupancy of IKZF1 at key B-cell lineage transcription factors leading to a persistent block in B-cell differentiation.

Cell fate simulation reveals cancer cell features in the tumor microenvironment.

To elucidate the dynamic evolution of cancer cell characteristics within the tumor microenvironment (TME), we developed an integrative approach combining single-cell tracking, cell fate simulation, and three-dimensional (3D) TME modeling. We began our investigation by analyzing the spatiotemporal behavior of individual cancer cells in cultured pancreatic (MiaPaCa2) and cervical (HeLa) cancer cell lines, with a focus on the α2-6 sialic acid (α2-6Sia) modification on glycans, which is associated with cell stemness. Our findings revealed that MiaPaCa2 cells exhibited significantly higher levels of α2-6Sia modification, correlating with enhanced reproductive capabilities, whereas HeLa cells showed less prevalence of this modification. To accommodate the in vivo variability of α2-6Sia levels, we employed a cell fate simulation algorithm that digitally generates cell populations based on our observed data while varying the level of sialylation, thereby simulating cell growth patterns. Subsequently, we performed a 3D TME simulation with these deduced cell populations, considering the microenvironment that could impact cancer cell growth. Immune cell landscape information derived from 193 cervical and 172 pancreatic cancer cases was used to estimate the degree of the positive or negative impact. Our analysis suggests that the deduced cells generated based on the characteristics of MiaPaCa2 cells are less influenced by the immune cell landscape within the TME compared to those of HeLa cells, highlighting that the fate of cancer cells is shaped by both the surrounding immune landscape and the intrinsic characteristics of the cancer cells.

Deciphering cellular plasticity in pancreatic cancer for effective treatments.

Cellular plasticity and therapy resistance are critical features of pancreatic cancer, a highly aggressive and fatal disease. The pancreas, a vital organ that produces digestive enzymes and hormones, is often affected by two main types of cancer: the pre-dominant ductal adenocarcinoma and the less common neuroendocrine tumors. These cancers are difficult to treat due to their complex biology characterized by cellular plasticity leading to therapy resistance. Cellular plasticity refers to the capability of cancer cells to change and adapt to different microenvironments within the body which includes acinar-ductal metaplasia, epithelial to mesenchymal/epigenetic/metabolic plasticity, as well as stemness. This plasticity allows heterogeneity of cancer cells, metastasis, and evasion of host's immune system and develops resistance to radiation, chemotherapy, and targeted therapy. To overcome this resistance, extensive research is ongoing exploring the intrinsic and extrinsic factors through cellular reprogramming, chemosensitization, targeting metabolic, key survival pathways, etc. In this review, we discussed the mechanisms of cellular plasticity involving cellular adaptation and tumor microenvironment and provided a comprehensive understanding of its role in therapy resistance and ways to overcome it.

Direct interaction of ß-catenin with nuclear ESM1 supports stemness of metastatic prostate cancer.

Wnt/ß-catenin signaling is frequently activated in advanced prostate cancer and contributes to therapy resistance and metastasis. However, activating mutations in the Wnt/ß-catenin pathway are not common in prostate cancer, suggesting alternative regulations may exist. Here, we report that the expression of endothelial cell-specific molecule 1 (ESM1), a secretory proteoglycan, is positively associated with prostate cancer stemness and progression by promoting Wnt/ß-catenin signaling. Elevated ESM1 expression correlates with poor overall survival and metastasis. Accumulation of nuclear ESM1, instead of cytosolic or secretory ESM1, supports prostate cancer stemness by interacting with the ARM domain of ß-catenin to stabilize ß-catenin-TCF4 complex and facilitate the transactivation of Wnt/ß-catenin signaling targets. Accordingly, activated ß-catenin in turn mediates the nuclear entry of ESM1. Our results establish the significance of mislocalized ESM1 in driving metastasis in prostate cancer by coordinating the Wnt/ß-catenin pathway, with implications for its potential use as a diagnostic or prognostic biomarker and as a candidate therapeutic target in prostate cancer.

Cell softness regulates tumorigenicity and stemness of cancer cells.

Identifying and sorting highly tumorigenic and metastatic tumor cells from a heterogeneous cell population is a daunting challenge. Here, we show that microfluidic devices can be used to sort marker-based heterogeneous cancer stem cells (CSC) into mechanically stiff and soft subpopulations. The isolated soft tumor cells (< 400 Pa) but not the stiff ones (> 700 Pa) can form a tumor in immunocompetent mice with 100 cells per inoculation. Notably, only the soft, but not the stiff cells, isolated from CD133+ , ALDH+ , or side population CSCs, are able to form a tumor with only 100 cells in NOD-SCID or immunocompetent mice. The Wnt signaling protein BCL9L is upregulated in soft tumor cells and regulates their stemness and tumorigenicity. Clinically, BCL9L expression is correlated with a worse prognosis. Our findings suggest that the intrinsic softness is a unique marker of highly tumorigenic and metastatic tumor cells.

Dynamic EMT: a multi-tool for tumor progression.

The process of epithelial-mesenchymal transition (EMT) is fundamental for embryonic morphogenesis. Cells undergoing it lose epithelial characteristics and integrity, acquire mesenchymal features, and become motile. In cancer, this program is hijacked to confer essential changes in morphology and motility that fuel invasion. In addition, EMT is increasingly understood to orchestrate a large variety of complementary cancer features, such as tumor cell stemness, tumorigenicity, resistance to therapy and adaptation to changes in the microenvironment. In this review, we summarize recent findings related to these various classical and non-classical functions, and introduce EMT as a true tumorigenic multi-tool, involved in many aspects of cancer. We suggest that therapeutic targeting of the EMT process will-if acknowledging these complexities-be a possibility to concurrently interfere with tumor progression on many levels.

Modulation of osteoblastogenesis by NRF2: NRF2 activation suppresses osteogenic differentiation and enhances mineralization in human bone marrow-derived mesenchymal stromal cells.

Mesenchymal stromal stem cells (MSCs) or skeletal stem cells (SSCs) play a major role in tissue repair due to their robust ability to differentiate into osteoblasts, chondrocytes, and adipocytes. Complex cell signaling cascades tightly regulate this differentiation. In osteogenic differentiation, Runt-related transcription factor 2 (RUNX2) and ALP activity are essential. Furthermore, during the latter stages of osteogenic differentiation, mineral formation mediated by the osteoblast occurs with the secretion of a collagenous extracellular matrix and calcium deposition. Activation of nuclear factor erythroid 2-related factor 2 (NRF2), an important transcription factor against oxidative stress, inhibits osteogenic differentiation and mineralization via modulation of RUNX2 function; however, the exact role of NRF2 in osteoblastogenesis remains unclear. Here, we demonstrate that NRF2 activation in human bone marrow-derived stromal cells (HBMSCs) suppressed osteogenic differentiation. NRF2 activation increased the expression of STRO-1 and KITLG (stem cell markers), indicating NRF2 protects HBMSCs stemness against osteogenic differentiation. In contrast, NRF2 activation enhanced mineralization, which is typically linked to osteogenic differentiation. We determined that these divergent results were due in part to the modulation of cellular calcium flux genes by NRF2 activation. The current findings demonstrate a dual role for NRF2 as a HBMSC maintenance factor as well as a central factor in mineralization, with implications therein for elucidation of bone formation and cellular Ca2+ kinetics, dystrophic calcification and, potentially, application in the modulation of bone formation.